2019 Book LacticAcidBacteria

Wei Chen Editor
Lactic Acid
Bacteria
Omics and Functional Evaluation
Lactic Acid Bacteria
Wei Chen
Editor
Lactic Acid Bacteria

Omics and Functional Evaluation
Editor
Wei Chen
Jiangnan University
Wuxi, China
The print edition is not for sale in The Mainland of China. Customers from The Mainland
of China please order the print book from: Science Press.
ISBN 978-981-13-7831-7 ISBN 978-981-13-7832-4 (eBook)

https://doi.org/10.1007/978-981-13-7832-4
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Contents
1 Introduction�� 1
Fengwei Tian
2 Genetic Operation System of Lactic Acid Bacteria
and Its Applications �� 35
Haiqin Chen, Chen Chen, Chunqing Ai, Chengcheng Ren, and He
Gao
3 Comparative Genomic Analyses of Lactic Acid Bacteria�� 77
Wei Chen and Hongchao Wang
4 Transcriptomics of Lactic Acid Bacteria �� 97
Zhennan Gu and Guozhong Zhao
5 Proteomics of Lactic Acid Bacteria�� 131
Yue Xiao, Yanjun Tong, and Wei Chen
6 Metabolomics of Lactic Acid Bacteria �� 167
Wanqiang Wu and Nan Zhao
7 Functional Evaluation Model for Lactic Acid Bacteria�� 183
Qixiao Zhai and Wei Chen
8 Lactic Acid Bacteria and Gut Health �� 239
Haitao Li and Zhifeng Fang
9 Lactic Acid Bacteria and Host Immunity�� 261
Linlin Wang, Zhao He, Peijun Tian, and Gang Wang
10 Commercial Strains of Lactic Acid Bacteria
with Health Benefits�� 297
Xin Tang and Jichun Zhao
11 Safety Evaluation of Lactic Acid Bacteria �� 371
Wei Chen, Leilei Yu, and Ying Shi
v
Chapter 1
Introduction
Fengwei Tian
1.1 ection I Overview and Definition of Lactic Acid

S
Bacteria
1.1.1 Overview of Lactic Acid Bacteria
Lactic acid bacteria (LAB) is a general term for a class of bacteria that use the
metabolism of carbohydrates in the external environment to produce lactic acid.
Lactic acid bacteria are widely distributed in nature and exist in a variety of habitats.
Extremely rich in biodiversity, they are closely related to human production and life
and have important social and economic value. They are valuable biological
resources for human beings. The utilization of lactic acid bacteria by human beings
has a very long history. According to reliable archaeological evidence, it can be
dated back to more than 10,000 years ago. In the long historical process, the utiliza-
tion of lactic acid bacteria has made outstanding contributions to the development
and practice of human society. In the great development of human natural science
and engineering science in the late nineteenth century and early twentieth century,
along with the rapid development of biological science, microbiology, and other
related disciplines, the science and technology of lactic acid bacteria also achieved
unprecedented development. On the one hand, in terms of basic science, lactic acid
fermentation is adopted as a model metabolic method. Lactic acid bacteria are mode
research organisms of basic biological sciences such as microbiology, biochemistry,
genetics, and molecular biology, which have important theoretical research signifi-
cance. On the other hand, in practical application, lactic acid bacteria have a wide
range of application significance in industrial biomanufacturing, food production
and processing, high-efficiency agriculture, high-efficiency livestock breeding,
F. Tian (*)
Jiangnan University, Wuxi, China
e-mail: [email protected]
© Springer Nature Singapore Pte Ltd. and Science Press 2019 1

W. Chen (ed.), Lactic Acid Bacteria,
https://doi.org/10.1007/978-981-13-7832-4_1
2 F. Tian
Fig. 1.1 Number of incomplete statistics for science and technology of lactic acid bacteria related
research publications
medical health, and other important fields closely related to human beings. In recent
years, the application of new technologies and new methods such as microbiome,
proteomics, bioinformatics, and big data analysis technology has further revealed
the great importance of lactic acid bacteria in related fields and attracted the
researchers from different research fields. In Fig. 1.1, the number of publications on
the research of lactic acid bacteria science and technology has grown significantly
during the last 10 years of the twentieth century to the first 10 years of the twenty-
first century. It has become a major research and application development hotspot of
modern science and application technology.
1.1.2 The Definition and Membership of Lactic Acid Bacteria
Lactic acid bacteria are an important class of prokaryotic microorganisms. However,

lactic acid bacteria do not have a clear definition, nor is it a taxonomic term of
microbiology and bacteriology. Different researchers have different understandings
and definitions of lactic acid bacteria and their range in different development peri-
ods. In the early twentieth century, when the lactic acid bacteria science and tech-
nology system began to form, the lactic acid bacteria mainly refer to the bacteria
that can acidify the milk (milk-souring bacteria) (Beijerinck 1901; Orla-Jensen
1919). Later, different researchers successively proposed different definitions and
1 Introduction 3
descriptions of lactobacillus from the aspects of cell morphology characteristics and

physiological metabolic reactions. For example, Kandler (1983) believed that
Lactobacillus was “a kind of gram-positive, non-spore-producing, micro-aerobic
bacteria whose main products of carbohydrate fermentation was lactic acid.” Wood
and Holzapfel (1995) defined Lactobacillus as “a kind of bacterial group that
metabolizes carbohydrates and takes lactic acid as the only or main metabolite.”
Carr et al. (2002) considered that lactic acid bacteria are a class of heterogeneous
bacteria that are Gram-positive, do not form spores, are negatively contacted with
enzymes, and are metabolites of lactic acid. Axelsson et al. (2004) considered that
lactic acid bacteria are a type of bacillus and cocci which are Gram-positive, do not
form spores, do not breathe, and ferment carbohydrates with lactic acid as the main
product. It can be seen from these definitions that lactic acid bacteria do not have a
clear definition and boundary but are a general term for a class of bacteria with simi-
lar physiological characteristics. Many definitions suggest that lactic acid bacteria
do not form spores; however, some spore-forming bacteria such as Sporolactobacillus
and Bacillus bacteria can also form lactic acid by homolactic fermentation.
Therefore, we believe that the production of lactic acid by metabolism is the most
important core metabolic characteristic of lactic acid bacteria. Lactic acid bacteria
are a general term for a variety of heterogeneous bacteria with different taxonomic
status and morphological physiology, which are positive in Gram staining, generally
do not produce spores, usually do not produce energy by breathing, and mainly
metabolize carbohydrates to produce lactic acid. At the beginning of the research of
lactic acid bacteria, mainly Lactobacillus, Leuconostoc, Pediococcus, and
Streptococcus bacteria formed the core of lactic acid bacteria, and later with the
discovery of more lactic acid bacteria and the evolution and correction of bacterial
classification, the current lactic acid bacteria have already included about 41 genera
(Bergey’s Manual Trust 2015). Among them, lactic acid bacteria with important
scientific and application value are mainly concentrated in Lactococcus,
Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, and Aerococcus,
Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus, and
Weissella for 12 genera. Bifidobacterium adopts a bifidus metabolic pathway differ-
ent from that of general lactic acid bacteria. Although it belongs to the Actinomycete
class in microbial taxonomy, Bifidobacterium bacteria generates lactic acid and ace-
tic acid through heterolactic fermentation. Therefore, relevant researchers tradition-
ally believe that Bifidobacterium belongs to lactic acid bacteria. In addition, there
are some human and animal pathogenic bacteria, such as Listeria bacteria, which
are in line with the description of the characteristics of lactic acid bacteria and also
belong to the generalized member of lactic acid bacteria family. The species involved
in lactic acid bacteria are described in Chap. 2 of this book.
4 F. Tian
1.2 ection II Development History and Applications

S
of the Science and Technology of Lactic Acid Bacteria
1.2.1 tage Division of Development Course and Applications

S
of the Science and Technology of Lactic Acid Bacteria
In the long history of human civilization, lactic acid bacteria have been widely used
for a long time. However, until modern times, with the development of science and
technology systems, the development of science and technology systems of lactic
acid bacteria has been formed and developed. The development course of scientific
research and technical application of lactic acid bacteria can be divided into five
stages: The first stage, the original stage of lactic acid bacteria development (from
prehistoric period to 1857 in which Pasteur discovered lactic acid bacteria and asso-
ciated it with lactic acid fermentation); the second stage, the initial stage of the
formation of lactic acid bacteria science and technology application system (from
1857 to 1919 in which Orla-Jensen established lactic acid bacteria system in 1919);
the third stage, the formal formation and development stage of the lactic acid bacte-
ria science and technology application system (from 1919 to 1960); the fourth stage,
the mature stage of the lactic acid bacteria science and technology application sys-
tem (from 1960 to 2001 in which the sequencing of the strain of lactic acid bacteria
is completed ); and the fifth stage, the gradual development stage of the lactic acid
bacteria science and technology application system (the whole genome sequencing
of the first strain of lactic acid bacteria since 2001). Figure 1.2 shows some mile-
stones in the history of lactic acid bacteria science and technology. The important
representative events in the development of lactic acid bacteria research and tech-
nology application are shown in Table 1.1.
1.2.2 evelopment Course of the Practical Application

D
of Lactic Acid Bacteria
The practical application of lactic acid bacteria can also be divided into two stages:
the first stage is the unconscious use of lactic acid bacteria derived from empirical
knowledge; the second stage is the practical application of lactic acid bacteria based
on science.
1
Introduction
Fig. 1.2 Important events in the development of science and technology of lactic acid bacteria.
5
6 F. Tian
Table 1.1 Development history of practice application and technology research of lactic acid
bacteria
Time Events and comments
8000 BC to North African residents began to consume natural acidified fermented milk and
5000 BC cheese and other dairy products (Dunne et al. 2012)
6000 BC to Ancient India mastered the production process of yogurt (dahi), cheese, and sour
4000 BC cream
5000 BC to Ancient Egyptians used pottery to make natural yogurt and mastered the
3000 BC production process of different series of fermented dairy products (Farnworth
2008a)
3000 BC Thracians made yogurt products by natural fermentation of goat milk (prokish or
kvasenomliako) (Chomakow 1973; Tamang and Kailasapathy 2010)
2000 BC to Hellenes and Chaldaic began to make cheese, and natural fermented sour cream
1300 BC appeared in Mesopotamia
1000 BC Chinese people started making kimchi (salted fermented vegetables) (Chen Gong
2011)
76 BC In ancient Rome, Pliny described a method for making white cabbage into sour
kimchi using a ceramic container (McElhatton and Marshall 2007)
540 in Anno The book of Qimin Yaoshu recorded the production method of yogurt and soaked
Domini AD vegetables (Qimin Yaoshu, volume ninth, li lao eighty-fifth)
800 AD Turks started making yogurt
1000 AD In the mountains of North Caucasus, goat milk was placed in the sheepskin sac
and naturally fermented to form kefir fermented milk
1145 The prehistory books such as Samkuksaki recorded that the production of kimchi
in the Korean peninsula was made by fermenting vegetables in stone containers
1719 Levinhoek discovered microbes found in many natural samples, including feces
and oral food debris
1780 Carl Wilhelm Scheele, Swedish medical chemist, first isolated and identified
lactic acid (acid of milk) from sour milk and described its chemical properties
(Scheele 1780)
1847 C. Blondeau determined that lactic acid was a product of the fermentation
process of a microorganism (then considered to be a certain fungus Penicillium)
(Blondeau 1847)
1857 French scientist Pasteur discovered lactic acid bacteria (then called yeast lactis)
under the microscope for the first time in the study of rancidity in wine drinks,
linking lactic acid production to microorganisms (Pasteur 1857)
1873 For the first time, a series of dilution methods were used to separate the pure
culture strain of lactic acid bacteria, Streptococcus lactis, named Lactococcus
lactis, from lactated milk (Lister 1873)
1880 Danish Chr. D. A. Hansen began to produce commercial lactic acid bacteria
starter
1881 French scientist Fremy produced lactic acid by fermentation. In 1881, the first
industrial fermentation of lactic acid was carried out in the United States
(Ghaffar et al. 2014)
1884 Hueppe used Bacterium acidilactici to name the bacteria that made the milk sour,
and for the first time, the name “yogurt bacteria” was named “lactic acid
bacteria” (Milth 1884)
(continued)
1 Introduction 7
Table 1.1 (continued)

1885 Cantani A G attempted to use lactic acid bacteria to produce antagonistic
substances for bacterial biotherapy against Mycobacterium tuberculosis (Cantani
1885; Florey 1945)
1892 German doctor Albert Döderlein first described a bacterium (Doderlein bacillus)
derived from vaginal secretions of healthy pregnant women, which antagonizes
staphylococci by producing lactic acid (Lash and Kaplan 1926)
1899 Henry Tissier, a doctor from the Pasteur Institute, isolated a Bacillus bifidus from
breast-fed healthy baby feces and supplemented this food with a Mycobacterium
to treat intestinal flora imbalance (Tissier 1906)
1895–1910 Different Lactobacillus members such as Lactobacillus delbrueckii,
Lactobacillus casei, Streptococcus, and Bifidobacterium were isolated and
discovered successively
1900 Ernst Moro, an Austrian pediatrician, discovered Bacillus acidophilus n. spec.,
which is now Lactobacillus acidophilus (Moro 1900)
1901 The Dutchman Martinus Willem Beijerinck promoted the understanding of the
production of lactic acid bacteria in kefir and yogurt and proposed the term
Lactobacillus (genus Lactobacillus Beijerinck 1901) as the official name of lactic
acid bacteria (van Iterson et al. 2013)
1901 Cahn (1901) studied bacilli in infant feces and studied the ecology of infant feces
1905 Stamen Grigoroff, a physiologist and microbiologist from Bulgaria, isolated a
Bacillus bulgaricus from yogurt at the age of 27, namely, Bacillus bulgaricus
(Grigoroff 1905)
1905 Pasteur Institute’s Metchnikoff published the Longevity: An Optimistic Study
based on his observations of diet and health during his travels in Bulgaria,
proposing the hypothesis that the intake of fermented yogurt contributes to health
and longevity (Metchnikoff and Mitchell 1908)
1911 Loudon M. Douglas published the book The Bacillus of Long Life, which further
discussed the role of bacilli in yogurt and yogurt in health and longevity
(Douglas 1911)
1915 Daviel Newman first used intravesical infusion of lactobacillus culture to reduce
the probability of urinary tract infection in women with cystitis, laying the
foundation for the clinical application of lactic acid bacteria (Newman 1915)
1919 The Danish bacteriologist Orla-Jensen firstly classified lactic acid bacteria based
on culture and physiological and biochemical characteristics systematically. For
the first time, he wrote a monograph on lactic acid bacteria, marking the initial
formation of lactic acid bacteria science and technology system (Orla-Jensen
1919)
1917 Lactobacillus drug lactasin (lactasin, “Biofermin”) named active Lactobacillus
powder preparation (including Enterococcus faecalis) put on the market as a drug
1919 Dr. Isaac Carasso used strains introduced in the Balkans and strains isolated in
the Mei Laboratory as a starter to produce yogurt in Barcelona and sold it
through pharmacies
1920 Yale University’s Rettger et al. proved that B. bulgaricus, which is derived from
yogurt, cannot survive in the gut of model animals and humans, thus questioning
Michnikov’s hypothesis and promoting further exploration of lactic acid bacteria
and health
(continued)
8 F. Tian

1921~1922 Rettger et al. reported the clinical efficacy of Lactobacillus acidophilus-
fermented yogurt, especially for digestion (Cheplin and Rettger 1920; Rettger
and Cheplin 1921)
1928 Roger and Whittler of the US Department of Agriculture’s Dairy Industry Bureau
firstly reported Lactococcus lactis-produced antibacterial peptide, nisin (Rogers
and Whittier 1928)
1930~1935 Minoru Shirota (Da Tianyu) isolated a Lactobacillus casei Shirota and in 1935
realized the commercial production of Lactobacillus casei-fermented lactic acid
bacteria beverage
1935 Eggerth and Gagnon used anaerobic separation techniques to isolate intestinal
anaerobic bacteria such as Bacteroides and Eubacterium (Eggerth and Gagnon
1933; Eggerth 1935)
1942 Five strains of lactic acid bacteria were isolated from sour milk by Tang Tenghan
et al., and one strain with high lactic acid production ability was obtained. The
acid production rate reached 88.44%, and the industrial production of lactic acid
products was realized
1940~1970 Common media for lactic acid bacteria culture, such as MRS medium, SL
medium, M17 medium, and BL medium, were developed successively, which
promoted the development and research of lactic acid bacteria culture technology
1945 The development of antibiotic application, aseptic animal model technology, and
anaerobic microbial culture technology has promoted the development of lactic
acid bacteria and intestinal microecology
1970 Tomotari Mitsuoka began the study of intestinal microflora, established the
classical analytical method of intestinal microflora, and made a systematic
analysis of intestinal microflora
1953–2001 The concept and related products of probiotics represented by lactic acid bacteria
were gradually formed, developed and maturity.
1962 Three glycopeptides with anticancer activity were isolated from the cell wall of
Lactobacillus bulgaricus. The antitumor effect of lactic acid bacteria was
reported for the first time. The research on the function of lactic acid bacteria is
deeper
1970 Woese established a bacterial identification method based on 16S rRNA
oligonucleotide sequence analysis and constructed a life tree of phylogenetic of
prokaryotic microorganisms and promoted the development of bacterial
taxonomy
1974 CHR-HANSEN Company introduced direct vat set starter to the market (DVS®
cultures or Direct Vat Set), significantly improving the production conditions of
fermented dairy products
1983 LGG (Lactobacillus rhamnosus) isolated from healthy human has the
characteristics of strong activity and gastric acid resistance and can be colonized
in the intestines for up to 2 weeks (Gorbach and Goldin 1989)
1984 CHR-HANSEN Company introduced probiotics of Bifidobacterium BB-12
1987 Zhang Hao and Xu Benfa started work on the development and application of
bifidobacteria in China
(continued)
1 Introduction 9

1989 In 1989, the US Food and Drug Administration (FDA) and the Association of
American Feed Control Officials (AAFCO) published a list of 42 microbial
strains that can be directly fed and generally considered safe, involving 30
species of lactic acid bacteria
1999 Document No. 105 issued by the Ministry of Agriculture of the People’s
Republic of China, Catalogue of Allowable Feed Additives, covered 12 kinds of
microbial additives, of which 7 are lactic acid bacteria
2001 The Food and Agriculture Organization of the United Nations (FAO) and the
World Health Organization (WHO) have introduced the definition of probiotics,
“a type of living microorganism that has a beneficial effect on the host after
sufficient intake”
2001 The Ministry of Health of the People’s Republic of China promulgated the
“Regulations on the Evaluation of Probiotics for Health Foods” and published
the “List of Probiotics for Health Foods” (Wei Fa Jian Fa [2001] No. 84), giving
the official definition of probiotics in China: “Microecological preparations that
promote the ecological balance of the gut microbiota and have a beneficial effect
on the human body”
2001 France’s Alexander Bolotin et al. published the complete genome-wide sequence
of the first strain of lactic acid bacteria (Lactococcus lactis ssp. lactis IL1403),
marking the study of lactic acid bacteria into the era of omics
2002 In the Cities of London, Ontario, and Canada, the FAO/WHO Joint Expert
Working Group drafted Guidelines for the Evaluation of Probiotic in Food
2003 Document No. 84 of the Ministry of Health of the People’s Republic of China
approved Lactobacillus reuteri as a probiotic strain that can be used in health
foods, making the number of probiotic bacteria increase to ten species for health
food
2003 Notice 318 of the Ministry of Agriculture of the People’s Republic of China
announced that 14 kinds of microorganisms are allowed to be used in feed, of
which 10 are lactic acid bacteria
2008 The World Gastroenterology Organisation (WGO) identifies the specific and
possible functions of lactic acid bacteria
1.2.2.1 nconscious Use of Lactic Acid Bacteria Derived from Empirical

U
Knowledge
In the practice of human society, lactic acid bacteria are mainly used in the produc-
tion of food and their products for disease prevention and treatment. The traditional
fermented foods with lactic acid fermentation as the main features include various
types of fermented dairy products, fermented cereals, and fermented fruit and veg-
etable products. These fermented foods appear in the historical development of
ancient civilizations in all different regions, reflecting an important role of lactic
acid bacteria in the practice of human society and economic development.
10 F. Tian
ifferent Types of Lactic Acid Bacteria-Fermented Dairy Products Are

D
Important Practical Applications of Lactic Acid Bacteria
Fermented dairy products made by fermentation of lactic acid bacteria have a long
history. As early as prehistoric times, more than 8000 years ago, residents living in
the Sahara area of North Africa began to raise animals such as cows and sheep and
began to consume dairy products such as milk, naturally acidified fermented milk,
and cheese (Dunne et al. 2012). It is the earliest record of human use of lactic acid
bacteria supported by archaeological evidence. In ancient India, from 6000 to 4000
years ago, humans mastered the production process of yogurt, cheese, and sour
cream and produced lactic acid bacteria-fermented dairy products such as dahi,
chakka, and cheese. These fermented dairy products are widely recorded in Buddhist
works and Sanskrit literature. For example, as the Great Nirvana Sutra records,
“best Tihu (the scream part of the dairy products) makes best effect. If you can take
it, all diseases will be removed, for every medicine contains in it.” “From the cow to
get the milk, from the milk to make the cheese, from the cheese to make the raw
cake, from the raw cake to make the boiled cake, from the boiled cake to get the
“Tihu”, and Tihu is the best.” Nirvana Sutra, volume 14, records “just as we get
cheese from milk and milk is thin while cheese is thick.” Besides, yogurt is listed
among the “eight good things” in Tibetan Buddhism. Today, fermented dairy prod-
ucts are still the main food for people in these areas. Furthermore, the content of
lactic acid bacteria-fermented yogurt is also recorded in the chapter of Genesis of
the Holy Bible. The Jewish ancestor Patriarch Abraham treated three angels with
yogurt and sweet milk and by doing so, they get longevity.
In the 5000 BC to 3000 BC, the ancient Egyptians began to make natural yogurt
with earthenware vessels and mastered the different varieties of production process
of fermented dairy products including Zabadi/Zabady yogurt, Laban zeer/Khad
cheese, and Kishk/Karish cheese. In 3000 BC, the Thracian people living in the cur-
rent Bulgarian nomads found that goat’s milk poured on the skin often became sour
under the effect of temperature and body temperature and formed a good odor and
mouthfeel and poured the sour milk into the boiled milk to make a yogurt product
(prokish or kvasenomliako), which later evolved into a modern yogurt product; in
the ancient Thrace language, “yog” means thick, and “urt” means milk.
Ancient Greece and Babylon began making cheese as early as 1550 BC. Cheese
has become an important part of the diet of the people at that time. In 750 BC,
ancient Rome began to make cheese. Around 1300 BC, fermented butter was pro-
duced in the Babylon region of Mesopotamia. Around 800 AD, Turkey began to
produce yogurt. It is said that yogurt is a tribute to the ancient Islamic saints, so it is
called yogurt. From a linguistic point of view, yogurt is imported into English from
the Turkish (Turkey Dictionary) language. Around 1000 AD, people living in the
mountains of the North Caucasus put goat’s milk in a sheepskin sac and formed a
kefir after natural fermentation. Kefir is an alcoholic fermented milk by yeast and
lactic acid bacteria. The product is considered to be a magical food given to people
by the Prophet Muhammad of Islam.
1 Introduction 11
There are also many records of lactic acid bacteria-fermented dairy products in
China. Qimin Yaoshu is a book written by Jia Sixie, a famous agronomist in the
Northern Wei Dynasty. (Qimin Yaoshu was finished around the middle of the sixth
century.) In the Volume 6: 57th of sheep breeding mentioned the production method
of lactic acid bacteria-fermented milk, “as for the methods of cheese, cattle and goat
milk are both available, and they also can be mixed.” “Cow’s or goat’s milk is both
available. The milk should be boiled for four or five times, and it should be filtered
into a jar with a bag. The cheese is as warm as the temperature of the human body,
and a half spoon of fragrant should be added to one liter of the boiled milk. Then stir
vigorously to spread the added fragrant cheese in the boiled milk and to get the
mature cheese the next day.” This is the earliest written record of using lactic acid
bacteria to produce fermented dairy products in China. Later, the method of making
fermented cheese was also briefly mentioned in the Compendium of Materia
Medica.
ermentation of Fruits and Vegetables and Fermented Grains by Lactic Acid

F
Bacteria Is a Practical Application of Lactic Acid Bacteria
Fermented dairy products are a typical example of human use of lactic acid bacteria.
In addition, people have also explored the use of lactic acid bacteria to make other
types of fermented foods, such as fermented vegetables and fermented grains. In
China, as early as the third century BC, people began to unconsciously use lactic
acid bacteria to make fermented vegetables. Chinese (salted) kimchi began more
than 3000 years ago during the Shang and Zhou dynasties. “There are radishes and
miscellaneous melons in the fields. Peeled the radish and marinated the melon,
which is dedicated to the ancestor. The pickled melon is the Chinese sauerkraut.”
Zhou Li Tian Guan records that “the next soup will not have five flavors, and salt
dishes will be added, and a soup is a soup made with meat or pickles.” Later, the
fermented vegetable production process originated in China was introduced to other
parts of Asia and even Europe with Genghis Khan’s expedition in Eurasia. The
ancient Roman historian and naturalist Pliny first described the use of ceramic con-
tainers and advocated a method of making white cabbage into sour kimchi and
promoting the use of fermented dairy products to treat gastroenteritis in his book
Natural History (McElhatton and Marshall 2007). In 1145, in the Korean historical
work Samkuksaki, it was mentioned that the Korean peninsula had begun to ferment
vegetables with stone containers to make kimchi.
In addition to fruits and vegetables, humans also unconsciously use lactic acid
bacteria for the production of grain fermented foods. As early as 7000 BC, cereal
crops such as wheat and barley were used as human staples. Human beings have
also begun to unconsciously use the life metabolic activities of microorganisms
such as ethanol fermentation of yeast, lactic acid fermentation of lactic acid bacte-
ria, and acetic acid fermentation of acetic acid bacteria to produce all kinds of
12 F. Tian
fermented cereals, including sour bread, beer, steamed bread, and so on. Around
3000 BC, the ancient Egyptians discovered that after the dough was put on for a
period of time, it would inflate and form a wine and sour taste. After roasting, a soft
and delicious fermented wheat food was obtained. Therefore, the processing tech-
nology of bread was grasped. In China, the important fermented cereal food involved
in the action of lactic acid bacteria is steamed buns. The history of steamed buns can
be traced back to at least the Warring States Period. Shi wu gan zhu records “The
king of qin made steamed cakes”; Xiao Zixian recorded in the qi Shu the use of
“mianqi cakes” at the time of Temple sacrifices. “Mianqi cakes” can be seen as the
prototype of steamed buns. In Africa, the working people use lactic acid bacteria to
ferment sour porridge, which can be used as a weaning food for young children. It
is still widely eaten in some countries in Africa today.
ong-Term Practical Application Makes People Realize that Lactic Acid

L
Bacteria-Fermented Products Have Good Disease Prevention and Treatment
Effects
In the long-term production practice, people have gradually realized that lactic acid
bacteria-fermented products have good disease prevention and treatment effects. In
the first century AD, the ancient Roman historian and naturalist Pliny advocated the
use of fermented dairy products to treat gastroenteritis and related diseases (Natural
History). In the fifteenth century, the first record of European functions related to
fermented dairy products originated from France: Francis the First suffered from a
serious gastrointestinal disease (probably is dysentery); when the French doctors
were helpless, the king of the king, Suleiman the First, sent a doctor who used fer-
mented goat milk to cure his disease. In China, there are also some records of using
lactic acid bacteria fermentation products for disease treatment. In the fourth vol-
ume of The Secret History of the Mongols, sour milk for the treatment of Genghis
Khan’s injury is mentioned: “Genghis Khan’s neck was injured. He felt so thirsty
but he couldn’t find horse milk in the carriage except a bucket of cheese. He ordered
others to find water to dilute the cheese and then drank the dilute cheese for three
times. After it Genghis Khan said, ‘I feel better, and my life has been saved’” (Zhang
Heping 1994). Li Shizhen once mentioned in his Compendium of Materia Medica,
“Cheese, can detoxify, quench thirst, remove the heat in the chest, treat heat sores
and muscle sores on the body, stop the thirst and heat, and can be laxative, enhance
the color and spirit” (Heping Zhang 1994).
1.2.2.2 Practical Application of Lactic Acid Bacteria Based on Science
With the development of microbiology represented by the research of Pasteur and

Koch, the application of lactic acid bacteria in social practice has also been greatly
developed, thus entering the second stage of its development. Lactic acid bacteria
from different species are widely used in industrial production practices such as
1 Introduction 13
food, light industry, medicine, and feed, that is, conscious and rational application
based on the science of lactic acid bacteria. The representative events at this stage
are as follows.
ommercial Production of Yogurt and Other Lactic Acid Bacteria-Fermented

C
Foods
In 1919, in view of the digestive and intestinal diseases of young children at that
time, inspired by the works of Mechnikov, Isaac Carasso (1874–1939), a Spanish
Jewish businessman and doctor, used strains introduced in the Balkans and isolated
in the Mei Laboratory as a starter to produce yogurt products and sold in pharmacies
to help treating children diarrhea. The Danone yogurt business is the predecessor of
Danone Group, which now is the world’s largest dairy producer. After Koch, with
the understanding of microbial pure culture and the application of related technolo-
gies, around 1890, Germany, the United States, and Denmark began to use lactic
acid bacteria starter culture for the industrial production of yogurt and cheese,
which greatly improved lactic acid bacteria fermentation products like the yogurt
and cheese production. Besides, it is also one of the important events in the history
of modern biological industrial technology, laying the basis for the development of
the industrial microbiology and modern biological engineering technology. Entering
the 1980s, with the application of the freeze-drying technology in the preparation of
lactic acid bacteria starter, a highly active directed vat set lactic acid bacteria starter
appeared, which avoided the cumbersome preparation process of the starter and
greatly promoted the development of lactic acid bacteria-related products.
Application of Lactic Acid Bacteria Fermentation Products
In addition to the lactic acid bacteria starter, useful metabolites of lactic acid bacte-
ria have also been developed. As early as 1891, the United States began industrial
production of lactic acid. In 1895, Germany’s Boehringer Ingelheim succeeded in
industrializing the production of lactic acid by fermentation. In 1901, in Japan,
Lactobacillus delbrueckii was inoculated with sweet potato as a raw material to
produce lactic acid by industrial fermentation of lactic acid bacteria. In addition,
another important event in the development and utilization of useful metabolites of
lactic acid bacteria is the commercial production of nisin, a biological preservative
for foods using lactic acid bacteria. Nisin is a polypeptide-type bacteriocin pro-
duced by Lactococcus lactis and has a broad and good inhibitory effect on Gram-
positive bacteria. It was commercialized by the British company Aplin & Barrett in
1959 (Hall 1963) and was approved by FAO/WHO as a food additive in 1961. A
high-yield mutant strain of nisin was also selected from the Institute of Microbiology
of the Chinese Academy of Sciences. In 1994, it was commercialized at Zhejiang
Yinxiang Bioengineering Co., Ltd.
14 F. Tian
Drugs and Medical Applications of Lactic Acid Bacteria
In the medical application of lactic acid bacteria, Cantani (1885) proposed microbial-
based bacterial therapy based on the observed in vivo antagonism. In 1915, Daviel
Newman used lactobacillus to treat bladder infection for the first time because of its
anti-infection ability based on lactic acid produced by lactobacillus, laying a foun-
dation for its clinical application in medicine. As early as 1917, Takeda
Pharmaceutical Co., Ltd. of Japan began to use Enterococcus faecalis to produce
lactobacillus preparations for treatment of gastrointestinal diseases. China began to
use fecal bacillus to produce lactobacillus preparations such as lactase in the 1950s.
In addition, there are drugs such as Lactobacillus Lactobacillin produced by
Lactobacillus acidophilus. In addition to treatment for digestive diseases, there are
also some lactic acid bacteria preparations for the treatment of reproductive tract
infections in women.
1.2.3 he Development of Scientific Research on Lactic Acid

T
Bacteria
Although lactic acid bacteria have been widely used in human society practice, the
scientific and technological research on lactic acid bacteria has been developed sys-
tematically with the development of modern science. Several important nodes and
landmark events in the development of lactic acid bacteria research are shown in
Table 1.1. The following will focus on the history of important events in the devel-
opment of lactic acid bacteria science and technology.
1.2.3.1 Pasteur’s Contribution to Lactic Acid Bacteria
The founder of modern microbiology who has made great contributions to the
development of lactic acid bacteria and the development of microbiology is the
French chemist and microbiologist Louis Pasteur (1822–1895). Prior to Pasteur, the
Dutch amateur scientist Antonie van Leeuwenhoek, who liked to sample and
observe, used a self-made microscope to observe a variety of samples, such as feces
and food residue attached to the teeth, and found that they existed in different envi-
ronments. The kind of microbes (micro-movements) opened the door to the world
of wonderful microscopic organisms but did not specify the role of microorganisms;
Sweden’s Scheler discovered the lactic acid found in milk. However, neither of
these individuals linked microbes to the formation of lactic acid. The chance of
discovering the formation of lactic acid by lactic acid bacteria finally fell on a well-
prepared and well-known person who entered the kingdom of science. Pasteur
insisted on the perfect combination of pure science and practice and insisted on
experimentation as a method and the means to carry out scientific discovery and
solve the basic strategies of practical problems and made great contributions to the
1 Introduction 15
development of related science. For lactic acid bacteria, Pasteur’s contribution is

linking lactic acid formation to microorganisms for the first time. From 1854 to
1856, ethanol fermentation and brewing were the main industries in the Lille region
where Pasteur lived. M. Bigo, the father of one of Pasteur’s students, worked in the
wine industry. He found that in the production of alcoholic beverage from beetroot,
the problem of rancidity which formed acid liquid rather than ethanol was easy to
appear. Pasteur analyzed and compared the normal sample by field sampling and
microscope observation technique. Two kinds of microorganism were found. One is
yeast which exists in the normal fermentation sample and can make the beet juice
carry on the normal ethanol fermentation. The other is the rod-shaped “lactic acid
yeast” (lactic yeast), which is present in the acidic fermentation sample. It is the
existence and activity of the “lactic acid yeast” that makes the fermentation of sugar
in the beetroot to the direction of producing lactic acid. It is Pasteur’s research that
stems from the practice of production that makes lactic acid-forming microorgan-
isms enter the human eye for the first time. In practice, Pasteur told Bigo that if the
rod-shaped lactic acid yeast was found in the fermentation broth, it would be dis-
carded. This extremely simple method solved the problem faced by the brewing
industry at that time. It can be said that it saved the wine industry at that time. In
terms of basic theory, according to this study, Pasteur submitted a title entitled
“Report on Lactic Acid Fermentation” to the Science Society of Lille (English
translation titled Report of the Lactic Acid Fermentation, German translation titled
Mémoiresur la fermentation Appeléelactique), published in the French journal
Chemical Progress exhibition (Pasteur 1857, 1995). The scientific discovery of “liv-
ing microbes for lactic acid fermentation” marks the birth and beginning of modern
microbiology and has become a milestone in the history of microbiology (Pasteur
1857).
1.2.3.2 Discovery of Some Important Lactic Acid Bacteria
As early as 1847, C. Blondeau studied the fermentation process of lactic acid,

butyric acid, acetic acid, and urea and determined that lactic acid is the end product
of the fermentation process of a certain microorganism (then he considered to be a
fungus Penicillium). In 1857, after Pasteur discovered the relationship between
microbes and lactic acid fermentation, under the impetus of pure bacterial culture
technology, during the period from the end of the nineteenth century to the begin-
ning of the twentieth century, different scholars discovered and isolated various
lactic acid bacteria including important members such as Lactobacillus,
Bifidobacterium, Streptococcus, Leuconostoc, etc.
First, in 1873, Joseph Lister first used a series of dilutions to acidify cow’s milk
in an attempt to confirm Pasteur’s theory. The first pure culture strain of lactic acid
bacteria, Streptococcus lactis, was isolated from the milk, and Liszt named it
Bacterium lactis (Streptococcus Lactis, the current Lactococcus lactis subsp.)
(Lister 1873). Lactobacillus is the most abundant strain of lactic acid bacteria.
16 F. Tian
The Dutch microbe scientist and botanist Martinus Willem Beijerinck proposed the
term Lactobacillus as the official form of Lactobacillus genus (genus Lactobacillus;
Beijerinck 1901) (van Iterson et al. 2013). In 1896, Leichmann and Lafar discov-
ered and isolated Lactobacillus delbrueckii from sour grain mash, and L-lactic acid
was the main metabolite. The Austrian pediatrician Ernst Moro discovers Bacillus
acidophilus n. spec., which is named Lactobacillus acidophilus now, while studying
infant digestive physiology (Moro 1900); In 1899, Henry Tissier, a pediatrician at
the Pasteur Institute in Paris, France, isolated a dominant Y-shaped bacterium from
the feces of breast-fed healthy infants and named it Bacillus bifidus (Tissier 1906).
In 1905, a 27-year-old Bulgarian physiologist and microbiologist Stamen Grigoroff
(1878–1945) isolated Lactobacillus bulgaricus (Grigoroff 1905) from acidified
milk. In 1906, Andrewes and Horder isolated Enterococcus faecalis from endocar-
ditis patients (then named Streptococcus faecalis); Orla-Jensen isolated Lactobacillus
casei from cheese in 1916.
Through the discovery of these lactic acid bacteria, it is recognized that lactic
acid bacteria represent a very important physiological metabolic group of bacteria
in nature and it is necessary to systematically classify lactic acid bacteria, which
laid the foundation for Orla-Jensen to establish a systematic lactic acid bacteria
system.
1.2.3.3 stablishment of the Basis and Application System of Lactic Acid

E
Bacteria
In addition to his contribution to basic biology, bacterial taxonomy, Orla-Jensen’s

contribution to lactic acid bacteria science and technology is that he established a
complete system of lactic acid bacteria, laying the foundation for the development
of lactic acid bacteria technology and applications. Orla-Jensen has been adhering
to a highly rigorous scientific attitude. After 10 years of unremitting experimenta-
tion, observation, and analysis, he systematically described a variety of lactic acid
bacteria and determined the most stable indicator system that can be used as a clas-
sification standard for lactic acid bacteria and wrote the book Dairy Bacteriology in
1919. In addition, he conducted in-depth research on the theoretical and practical
applications related to lactic acid bacteria. In his doctoral thesis (1904), he studied
the types and formation of volatile fatty acids in cheese, and in his later years, he
developed lactic acid bacteria nutrition. In his later years, he carried out research on
nutritional requirements of lactic acid bacteria and published some pioneer papers
on nutritional physiology of lactic acid bacteria (Orla-Jensen et al. 1936; Orla-
Jensen and Snog-kjaer 1940). In addition, Orla-Jensen is also concerned with dairy
hygiene. The methylene blue reduction test was developed as a method of milk
grading, which is still widely used today, and the use of lactic acid bacteria to make
silage was explored. It can be said that the work of Orla-Jensen laid the foundation
for the sustainable development of lactic acid bacteria science and technology in
both basic theory and practical application.
1 Introduction 17
1.2.3.4 nalysis of Basic Physiological Metabolism of Lactic Acid

A
Bacteria
The physiological metabolism of lactic acid bacteria mainly involves nutrition

physiology, cell membrane transport, environmental physiology (interaction
between environmental factors and lactic acid bacteria), carbohydrate metabolism,
and protein metabolism. Since the beginning of the twentieth century, the people’s
understanding of lactic acid bacteria has made great progress. However, limited to
the level of understanding at that time, the understanding of the physiology of lactic
acid bacteria was mainly limited to the apparent physiological characteristics such
as acid production and gas generation (Orla-Jensen 1919). Although Eduard
Buchner et al. in Germany discovered the phenomenon of ethanol fermentation of
sucrose by cell-free yeast extract as early as 1897, its research really paid attention
to it after winning the Nobel Prize in 1907 (Buchner 1907). Beginning to understand
the physiological metabolic activity of microorganisms at the subcellular level
marks the birth of the modern “enzymology theory” of life sciences and also pro-
motes the study of physiological metabolism of lactic acid bacteria at the subcellu-
lar level and enzyme molecular level. In terms of carbohydrate metabolism, after
almost 50 years of exploration from the Buchner period (1897) to the 1940s, people
realized the whole details of the glycolysis pathway (EMP pathway, Embden-
Meyerhof-Parnas pathway), which is a basic metabolic pathway shared by different
types of life. Regarding lactic acid bacteria, in theory, Kluyver and Donker pro-
posed the terms of homofermentation and heterofermentation in 1924. After that,
key enzymes related to carbohydrate metabolism of lactic acid bacteria and key
enzymes derived from physiological metabolism of different lactic acid bacteria,
such as lactate dehydrogenase, were successively isolated, identified, and character-
ized (Stephenson 1928; Garvie 1980), and related metabolic pathway networks
have been discovered and confirmed. A model of lactic acid fermentation based on
glycolytic pathway for carbohydrate metabolism of lactic acid bacteria was devel-
oped. The classical model of heterogeneous lactic acid fermentation (Nelson and
Werkman 1935; DeMoss et al. 1951) based on pentose phosphate pathway and the
bifid fermentation model (de Vries and Stouthamer 1968) based on fructose-6-
phosphate phosphoketolase (F6PPK). By the 1960s and 1970s, the reaction mecha-
nism of carbohydrate metabolism in lactic acid bacteria was well-understood.
In addition to carbohydrate metabolism, microbial nutrition is also the main con-
tent of lactic acid bacteria physiological metabolism research. Orla-Jensen is a pio-
neer in the study of microbial nutrition in lactic acid bacteria (Orla-Jensen et al.
1936; Orla-Jensen and Snog-kjaer 1940). This work began in the mid-1930s with
microbial nutrition studies of lactic acid bacteria. The development mainly occurred
in the 1940s and 1950s. Lactobacillus is a typical growth factor heterotrophic bac-
terium, and its nutritional requirements are very demanding. At first, people only
realized the nutritional requirements of lactic acid bacteria at different raw material
level (Davis 1939). Later, people gradually realized the nutritional requirements of
lactic acid bacteria from the compound level. During this period, the key nutritional
18 F. Tian
factors affecting the growth and metabolism of lactic acid bacteria were gradually
discovered. These nutritional factors mainly included fatty acids-like growth fac-
tors, amino acid-like growth factors, vitamin-like growth factors, base-like growth
factors, and bifido cytokines that specifically promote the growth of Bifidobacteria,
etc. (Snell 1945; Kitay and Snell 1950; Tittsler et al. 1952). To sum up, people began
to form a systematic understanding of the nutritional requirements of lactic acid
bacteria. At the same time, the culture technology of lactic acid bacteria has also
been developed, and many scholars have found different types of mediums (such as
the MRS medium, the M17 medium, and the SL medium) suitable for lactic acid
bacteria culture and culture techniques (like anaerobic culture technology, etc.),
which strongly promoted the development of lactic acid bacteria science and appli-
cation technology.
Solute transport and environmental physiology are important aspects of micro-
bial physiology. Microbial environmental physiology mainly refers to the interac-
tion of environmental factors such as pH and temperature with microbial cells.
Environmental factors such as pH, oxygen, osmotic pressure, and nutrients play an
important role in the metabolic process of lactic acid bacteria (Thompson 1987).
The metabolic modes and product types of lactic acid bacteria change significantly
under different environmental conditions. For example, oxygen causes Lactococcus
lactis to change from homogenous lactic acid fermentation mode to heterotypic
lactic acid fermentation mode (Thomas et al. 1979). Hemoglobin in the external
environment can transform the metabolic patterns of certain lactic acid bacteria
such as Enterococcus faecalis, Lactococcus, and Leuconostoc from fermentation to
respiration, which is also an important part of the physiological metabolism research
of lactic acid bacteria (Bryan-Jones and Whittenbury 1969; Sijpesteijn 1970).
Microbial cell solute transport is an important physiological process in which
cells absorb nutrients from the external environment by a specific means or mecha-
nism and transport them into the cytoplasm through transmembrane. The research
work in this area is mainly carried out around Lactococcus lactis. It is the main
content of research on lactic acid bacteria in the 1970s and 1990s. We mainly car-
ried out some research on the basic laws of transport and absorption of important
solutes such as carbohydrates, amino acids, protein short-peptides, and some anions/
cations of lactic acid bacteria and analyzed the solute transport involving lactic acid
bacteria. We have analyzed some mechanisms of action involving lysate transport
and energy metabolism in lactic acid bacteria, such as proton potential (PMF) co-
transport, precursor-product reverse transport, and group translocation based on
phosphotransferase systems.
1.2.3.5 Molecular Biology Period of Lactic Acid Bacteria Research
With the analysis of the double-helix structure of DNA molecules in the 1960s, the
people’s understanding of biological sciences and life phenomena has entered a new
period of molecular biology, which has a greater impact on the classification of
lactic acid bacteria (as mentioned above). It also greatly promotes people’s
1 Introduction 19
understanding of the metabolic process of lactic acid bacteria. People began to try
to reveal the mechanism of physiological metabolism and product formation and
genetic regulation model of lactic acid bacteria at the nucleic acid molecular level
and found the material basis of life activity phenomenon of lactic acid bacteria such
as plasmids, chromosomes, and so on. Genetic transfer patterns such as conjuga-
tion, transduction, transformation, transfection, protoplast fusion, and transposon
were found in different lactic acid bacteria. The gene cloning and expression sys-
tems of Lactococcus and Lactobacillus were established. The application of molec-
ular biology theory and technology in the field of lactic acid bacteria not only
enables people to understand the life metabolism of lactic acid bacteria at the
molecular level but also provides the theoretical basis and powerful tools for bioen-
gineering and utilization of lactic acid bacteria. The understanding and application
have reached an unprecedented height.
1.2.3.6 xploration and Application of the Function of Lactic Acid

E
Bacteria
Before the development of lactic acid bacteria science and technology, people have
gradually realized the beneficial functions of lactic acid bacteria and their products
on humans and animals in production practice. For example, ancient Roman natu-
ralists advocate the use of fermented dairy products to treat gastrointestinal dis-
eases. The fermented yogurt made with goat’s milk in the century treated the
diarrhea disease of the French emperor Francis I. In the Chinese Yuan Dynasty, Hui
Sihui mentioned in the yinshanzhengyao that the “cow cheese” obtained by fermen-
tation of lactic acid bacteria has “sweet, non-toxic, stop the thirst and heat, get rid
of the heat in the chest ” and other functions. In the Ming Dynasty, Li Shizhen
recorded in the fiftieth volume of Materia Medica of the Compendium of that
“cheese” was used to “be laxative, eliminate swelling, enhance the color and spirit.”
However, the scientific excavation and rational understanding of the beneficial func-
tions of lactic acid bacteria began in the late nineteenth and early twentieth centu-
ries, when people have noticed that certain lactic acid bacteria may have functions
to promote health and disease treatment. Cantani and Döderlein proposed bio-
antagonism based on lactic acid bacteria to attempt biotherapy for different infec-
tious bacteria (Cantani 1885; Döderlein 1892). Henry Tissier, a pediatrician at the
Pasteur Institute in Paris, France, isolated a dominant Y-shaped bacterium from
breast-fed healthy baby feces and named it Bacillus bifidus. Tissier noted that
breast-fed infants have a relatively low probability of developing diarrheal disease
and found that supplementing this Bacillus bifidus in food can treat infantile diar-
rhea due to intestinal flora imbalance (hyperproliferation of hydrolyzed bacteria)
(Tissier 1906). Yale University’s Rettger reported the clinical efficacy of
Lactobacillus acidophilus yogurt in 1920 and 1921, especially for digestion
(Cheplin and Rettger 1920; Rettger and Cheplin 1921).
However, the work of really promoting the discovery of lactic acid bacteria
comes from the theoretical hypothesis of lactic acid bacteria and human health pro-
20 F. Tian
posed by several scientists represented by Metchnikoff, which believes that lactic

acid bacteria and their fermented products are beneficial to human health. These
theoretical hypotheses have greatly promoted the development of lactic acid bacte-
ria science and technology. Meichenikov (1845–1916) was a Russian-born Jewish
zoologist. He shared a Nobel Prize in Physiology or Medicine with Paul Ehrlich in
1908 for discovering the macrophage immune defense mechanism that exists in
innate immunity. Known as the “Father of innate immunity,” his work and writings
have had a tremendous impact on immunology, lactic acid bacteria, and health,
respectively. The sensible scientist who had committed suicide twice because of his
two wives who died of illness (death) had traveled to Bulgaria during his tenure at
the Pasteur Institute in France. Meichenikov, meanwhile, noticed that there were
many long-lived centenarians in Bulgaria. The main diet of these long-lived people
is a fermented yogurt (yahourth), made by so-called Bulgarian Bacillus (isolated
from fermented yogurt by Bulgarian scientists). Meichenikov believes that intesti-
nal bacteria can cause intestinal damage and is harmful to the body. By ingesting
fermented yogurt containing lactic acid bacteria, it can inhibit the occurrence of
intestinal decay and make people prolong life. Based on this, he published a far-
reaching book in 1908, The Prolongation of Life: Optimistic Studies (Metchnikoff
and Mitchell 1908), which first proposed the role of lactic acid bacteria in human
health in the form of speculation and hypothesis through epidemiological investiga-
tions. In 1911, the British dairy nutritionist Loudon M. Douglas (1864–1944) pub-
lished The Bacillus of Long Life, which treats yogurt as a natural therapy. Eating
yogurt can alleviate the toxic factors produced by the human body and promote
human health (Douglas 1911).
The theoretical contribution of Mitsuoka (1990) is as follows: Mitsuoka from the
University of Tokyo, Japan, in the 1960s established an analytical method based on
selective culture of intestinal flora species (Mitsuoka et al. 1965). To understand the
composition and relative proportions of intestinal bacteria, he tried to correlate the
functions of different members, which promoted people to explore and understand
the healthy beneficial function of lactic acid bacteria. In theory, Mr. Mitsuoka pro-
posed a concept of health that the intestinal tract and gastrointestinal dysfunction
are the root cause of different diseases of the body and gastrointestinal health is the
basis and core of the body’s health. The beneficial effects of Lactobacillus and
Bifidobacterium were pointed out; and Bifidobacterium was considered to be an
important index to characterize the health degree of the body. The microecological
balance of gastrointestinal tract by dietary supplementation of Bifidobacterium or
by dietary regulation to increase the level of Bifidobacterium is helpful to prevent
and alleviate a series of diseases and the aging process (Mitsuoka 1978,1990).
Although these theories do not have sufficient and definitive scientific evidence,
they have greatly promoted the in-depth exploration of the relationship between
lactic acid bacteria and human health.
In the twentieth century, people used various methods and techniques of func-
tion excavation and evaluation to deeply explore the beneficial physiological func-
tions of lactic acid bacteria. The position of lactic acid bacteria also expanded from
the intestinal tract to all aspects of the body, extending from the initial beneficial
1 Introduction 21
function of the intestine to systemic metabolism, neurodevelopment, mental cogni-

tion, and so on. In 2009, the World Gastroenterology Organisation (WGO) pub-
lished guidelines for the use of probiotics and prebiotics. For the first time,
international authoritative medical organizations have identified the functions and
possible functions of lactic acid bacteria (Farnworth 2008b; World Gastroenterology
Organisation 2009). Nowadays, with the development of the National Human
Microbiome Project, people are more aware that the intestinal flora, including lactic
acid bacteria, plays an important role in the physiological metabolism and the
occurrence and development of diseases. The further research on the physiological
function of lactic acid bacteria will continue.
1.3 ection III Application of Lactic Acid Bacteria in Human

S
Social Practice
In the previous section, we briefly reviewed the development of lactic acid bacteria
science and technology. Regardless of the history of development or the current
situation, lactic acid bacteria play an important role in human social practice. This
section describes the social practice of lactic acid bacteria from the following
aspects.
1.3.1 Lactic Acid Bacteria and Food Manufacturing Industry
Fermented food is the precious wealth given by nature to mankind, and it is also an
important part of human main food. The preparation of fermented food involves
many microorganisms recognized as safe (GRAS) and edible directly. Lactic acid
bacteria (lab) is one of the most important microorganisms in food production.
Different countries and regions have their own characteristics of traditional lactic
acid fermented food based on lactic acid fermentation principle. On the one hand,
lactic acid bacteria as the main fermentation agent, through lactic acid fermentation
to form short-chain fatty acids acidification and the accompanying role of biological
metabolism, improves the sensory characteristics of food matrix profile. The nutri-
tional value of fermented products was improved by releasing amino acids and
forming vitamins. The lactic acid bacteria bio-antagonism substances such as short-
chain fatty acids, bacteriocins, and hydrogen peroxide have significantly improved
the preservation and safety of the products and are typical models for the natural
green processing of foods. On the other hand, lactic acid bacteria are an important
component of functional probiotics and, as a supplemental starter, can impart spe-
cific health-promoting functional properties to the fermented product. In addition,
some antagonistic lactic acid bacteria can also be directly used as biopreservatives
for commercial use in food processing, manufacturing, and biological control of
pathogenic bacteria in food. In recent years, with people’s attention to health and the
22 F. Tian
need for functional properties of products, lactic acid bacteria fermentation prod-
ucts with specific functional properties have received widespread attention.
Although there are still a series of related legislative management issues, lactic acid
bacteria-fermented foods have achieved rapid development. The food produced by
lactic acid bacteria fermentation includes fermented dairy products, fermented meat
products, fermented aquatic products, fermented cereal foods, fermented fruits and
vegetables products, edible lactobacillus preparations, and so on. According to
incomplete statistics, at present, there are as many as 120 kinds of food products
concerning lactic acid bacteria fermentation (involving primary fermentation and
secondary fermentation) (Steinkraus 1995; Reed and Nagodawithana 1996; Hui
et al. 2004). Lactic acid bacteria-fermented food has become an important part of
food biomanufacturing. The economic output value of related products has reached
about 50 billion US dollars, which is an important component of human social and
economic activities.
1.3.2 actic Acid Bacteria and Farming and Aquaculture

L
Industry
Lactic acid bacteria are also widely used in the field of modern agriculture.
Firstly, lactic acid bacteria (lactic acid bacteria), as the important microorgan-
isms of direct feeding microorganisms (direct-fed microbial, DFM), are widely
used in the field of feed. Feed lactobacillus preparation can improve the growth
efficiency and disease resistance of farmed animals by inhibiting the growth of
pathogenic bacteria, regulating the micro-ecological balance of animal intestines,
forming vitamins and beneficial enzymes, and so on. At the same time, beneficial
lactic acid bacteria and other direct feed microorganisms can also replace or reduce
the extensive use of growth-promoting antibiotics in animal breeding, which is an
effective solution to the growing problem of microbial drug resistance. Lactic acid
bacteria can be used as a microbial inoculant for silage production, inhibiting patho-
genic bacteria and toxin-producing fungi in feed by bio-metabolism of lactic acid
bacteria, improving the palatability of animal feed, and improving the nutritional
value and safety of feed. Lactic acid bacteria can also be used as an effective micro-
organism (EM) preparation for the production of fermented feeds using cereals,
agricultural product processing by-products, and crude plant biomass. Lactic acid
bacteria have prominent inhibitory effects on some aquaculture species such as
prawns, and some lactic acid bacteria preparations such as Pediococcus can improve
the ability of aquaculture species to resist aquatic pathogenic bacteria and viral
infections. In addition, lactic acid bacteria are endosymbiotic bacteria and epiphytes
commonly found on plants. The bacteriocin produced by lactic acid bacteria plays
an important role in the rhizosphere micro-ecological balance of plants. The lactic
acid bacteria produce some antibacterial compounds through lactic acid fermenta-
tion and have inhibitory effects on plant pathogenic bacteria such as Xanthomonas,
Erwinia, and Pseudomonas. The lactic acid bacteria inoculant has a certain protec-
1 Introduction 23
tive effect on plant tissues. Based on this, lactic acid bacteria can be used as prepara-
tions of biological fertilizers and biocontrol agents. For the pathogenic bacteria of
crops, lactic acid bacteria can be used as an inoculant to achieve biological control
of certain crop pathogenic bacteria such as Fusarium and have biosuppressive
effects on certain mold-forming mycotoxins. Lactic acid bacteria can also be used
in the fermentation of bio-fertilizer and the safe treatment of fertilizer, which has a
good application prospect in the field of eco-agricultural planting.
1.3.3 Lactic Acid Bacteria and Medical Health Industry
Lactic acid bacteria can give food a rich and beneficial effect. People have long
noticed the application of lactic acid bacteria and their products in medicine and
health, including dysfunction, disease prevention and treatment, and specific health
promotion. First, despite the lack of a clear mechanism of action, based on extensive
evidence-based medical evidence, lactic acid bacteria live/dead preparations have a
good therapeutic and recovery effect on dysfunction and disease of the digestive
system of the gastrointestinal tract. Some commonly used lactic acid bacteria include
Enterococcus, Lactobacillus, Bifidobacterium, and Streptococcus. These lactic acid
bacteria preparations can effectively correct the gastrointestinal flora disorder caused
by various factors such as bad diet, antibiotic use, food-borne pathogen infection,
radiotherapy and chemotherapy, surgery, etc. and inhibit the production and absorp-
tion of intestinal toxic factors. They have been clinically proven and widely used.
Secondly, based on the biological antagonism of lactic acid bacteria producing lactic
acid to reduce pH, hydrogen peroxide, and bacteriocin, lactobacillus preparations
made from specific lactic acid bacteria are used to control infection of body system
or specific body parts. For example, lactobacillus preparations (Lactobacillus del-
brueckii, Lactobacillus rhamnosus, etc.) were used to control the imbalance of
reproductive tract flora and inflammatory infections in women, and Lactobacillus
bulgaricus cream was used to treat skin burns. Lactobacillus acidophilus and
Lactobacillus bulgaricus freeze-dried capsules were used for the treatment and pre-
vention of oral infection and dental caries. Thirdly, antibiotics play an important role
in the treatment and control of infectious diseases. However, the excessive use of
antibiotics has also brought about the growing problem of microbial resistance,
because of the great potential of beneficial lactic acid bacteria and probiotics for
infection control and disease treatment. Due to the great potential of beneficial lactic
acid bacteria and probiotics for infection control and disease treatment, after antibi-
otics, biotherapy based on beneficial lactic acid bacteria and probiotics is expected
to be an effective alternative medicine program for infectious diseases, which has
been widely concerned by people. Finally, different levels of model research and
clinical practice have proved that lactic acid bacteria and its preparation have a good
health promotion effect by strengthening the biological barrier of the body, synthe-
sizing of essential vitamins, promoting the absorption of nutrients, and having the
beneficial regulation of specific immunity and non-specific immunity.
24 F. Tian
In summary, it has been confirmed that certain lactic acid bacteria and probiotic
strains have outstanding clinical application effects, including alleviating lactose
intolerance and treating diarrhea caused by different causes. There are also many
studies to prove that lactic acid bacteria have potential disease treatment effects,
such as antitumor, alleviating hypertension, adjuvant treatment for Helicobacter
pylori infection, regulating blood lipids and lowering cholesterol levels, preventing
diabetes, etc., but more clinical validation is needed. In recent years, some studies
on microbiology have also shown that beneficial lactic acid bacteria, probiotic bac-
teria, and certain gastrointestinal functional bacteria can exert important interven-
tion effects on the whole body through the brain-gut axis. In addition to the digestive
system, lactic acid bacteria and probiotics also have important effects on mental,
emotional control, nerves, and endocrine. It can be foreseen that with the deepening
of research, functional lactic acid bacteria and probiotics have very broad applica-
tion prospects in the medical and health industry.
1.3.4 actic Acid Bacteria and Industrial Manufacturing

L
Industry
In addition to its wide application in the food and pharmaceutical fields, the applica-
tion of lactic acid bacteria in the industrial sector mainly includes biofermentation
of lactic acid, biorefinery of biomass, and bioengineering of various valuable prod-
ucts using lactic acid bacteria as a cell factory.
Lactic acid, chemically known as 2-hydroxypropionic acid, is a natural organic
acid having two optical isomers, D and L, in the form of a colorless or yellow trans-
parent syrupy liquid. Lactic acid is an important industrial raw material with many
important uses. It is widely used as a fine chemical in food, chemical, medical,
pharmaceutical, textile, agricultural, and environmental protection fields. The use of
lactic acid bacteria for lactic acid fermentation to prepare lactic acid is one of the
main methods of industrial lactic acid production. After saccharification of starchy
raw materials, Lactobacilli are mainly inoculated for fermentation production. The
strains currently used for industrial lactic acid fermentation production mainly
include certain strains of Lactobacillus delbrueckii subsp. bulgaricus, Bacillus
coagulans, and Lactobacillus sp. Biorefinery is a bioprocessing method that pro-
duces agricultural chemicals, fuels, and bio-based materials from agricultural waste,
plant-based starch, and lignocellulose. It can be seen from this definition that biore-
finery is a very broad concept in the field of bioengineering, covering biotransfor-
mation with microbes as the main body and core. Lactic acid bacteria are one of the
suitable microbial groups for biorefinery. The fermentation of lactic acid also
belongs to the content of biorefinery. In addition, the metabolic pathway of “cell
factories” of lactic acid bacteria was genetically modified to produce a variety of
high value-added metabolites, such as sugar alcohol, vitamins, functional exopoly-
1 Introduction 25
saccharide, γ-aminobutyric acid, food flavor substances, short-chain organic acids,

conjugated linoleic acid, active peptides, and nutritional drugs (nutraceutics).
1.4 ection IV Lactic Acid Bacteria Science and Technology

S
System and Development Frontier
1.4.1 omposition of Lactic Acid Bacteria Science

C
and Technology System
Around the basic science and application of lactic acid bacteria, a system of science
and technology involving microbiology, fermentation engineering, food manufac-
turing, nutrition, and health science has been formed, although it is somewhat far-
fetched and intersects with each other in many fields, such as microbiology,
fermentation engineering, food manufacturing, nutrition and health science, and so
on.
However, we can still generalize the connotation and scope of “lactic acid bacte-
ria science and technology”: “lactic acid bacteria science and technology” is a
whole set of research on lactic acid-producing bacteria at the level of cells, mole-
cules, or specific ecological groups as well as the basic laws of life activities.
Besides, it explores the interaction among lactic acid bacteria and non-lactic acid
bacteria, and other organisms such as plants, animals, and humans, and then, applied
it to science and technology in the fields of industrial fermentation, food manufac-
turing, agriculture, animal husbandry, medicine, health, bioengineering, and envi-
ronmental protection. Specifically, the science and technology of lactic acid bacteria
include the biological basis of lactic acid bacteria, mainly related to the basic micro-
life activities of lactic acid bacteria, with emphasis on the basis. The application
technology of lactic acid bacteria includes industrial fermentation of lactic acid bac-
teria, food bio-production based on lactic acid fermentation and biotransformation,
etc. These parts are relatively independent of each other, but there is a tight or sparse
relationship.
1.4.2 evelopment Frontier in Research and Application

D
of Lactic Acid bacteria
From the history of the development of lactic acid bacteria, lactic acid bacteria is a
multidisciplinary cross-field; its hot issues and development front are also involved
in a number of different fields. Some new theories, techniques, and methods, such
as histology and systems biology, provide a powerful tool for the study of lactic acid
26 F. Tian
bacteria and accumulate a great deal of information about the biological behavior of
lactic acid bacteria, which also makes the scientific understanding and application
of lactic acid bacteria more in depth. Here, based on the editor’s own understanding,
the development frontiers of lactic acid bacteria research and application are briefly
reviewed, which is inevitably biased and generalized, only for readers’ reference.
1.4.2.1 Research on Basic Microbiology of Lactic Acid Bacteria
Under the guidance of macrogenomic information of environmental microorganisms,

we should further optimize and improve the culture methods and techniques suitable
for lactic acid bacteria, isolate and culture new lactic acid bacteria from specific natu-
ral habitats, and explore the biodiversity resources of lactic acid bacteria. We should
perfect and enrich the existing resource system of lactic acid bacteria, enrich and
perfect the theory and technology of lactic acid bacteria taxonomy, continue to dis-
cover the information molecules with taxonomic significance, and make the taxo-
nomic classification and intraspecific classification of lactic acid bacteria more
reasonable and further straighten out the phylogeny of Lactobacillus. We use modern
omics techniques such as metagenomics, genomes, transcriptomes, proteomics, and
metabolomes to carry out the physiology, carbohydrate metabolism, organic acid
metabolism, protein metabolism, environmental physiological response and adapta-
tion, genetic and genetic recombination of lactic acid bacteria, as well as the activity
process and detailed regulation mechanisms based on metabolite-controlled protein
A (CcpA) and transcription factor (TF). Besides, we carried out a global phenotypic
characterization analysis of lactic acid bacteria microbial cells to further improve
people’s understanding of the phenomenon of apparent life activities of lactic acid
bacteria, by doing so to form a systematic understanding of lactic acid bacteria.
1.4.2.2 igh-Efficiency Biomanufacturing Research of Lactic Acid

H
Bacteria Cell Factory
Lactic acid bacteria are ideal model organisms for biomanufacturing and biorefin-
ery. At present, the main research on biomanufacturing and biorefinery by using
lactic acid bacteria as a cell factory focuses on the construction of gene efficient
expression elements and gene efficient expression systems, modification of meta-
bolic pathways, and reconstruction of new metabolic pathways. First of all, in the
context of understanding the genomic information, we use bioinformatics methods
and techniques to further explore the genetic structural elements and regulatory ele-
ments of valuable lactic acid bacteria that carry specific biological traits, and to
construct and improve exogenous gene efficient expression systems (such as food-
grade expression systems) and surface display systems for valuable lactic acid
1 Introduction 27
bacteria for specific applications such as bio-manufacturing, food, and medicine.

Besides, we explore the use of lactic acid bacteria as a carrier to produce and pre-
pare biological products such as biological vaccines, and use metabolic engineering
techniques, genome editing techniques, synthetic biology techniques, etc. to mod-
ify, transform, and create metabolic pathways of valuable lactic acid bacteria and to
realize valuable products such as organic acids, functional sugar alcohols, vitamins,
fatty acids, and flavor substances based on biomass raw materials.
1.4.2.3 tudy on Environmental Physiology and Cell Response of Lactic

S
Acid Bacteria for Practical Application
Lactic acid bacteria are the most commonly used industrial fermentation microor-
ganisms. Food biomanufacturing and other bioengineering applications are subject
to a variety of stresses from bioprocessing conditions and external physical, chemi-
cal, and biological environmental factors, such as acid, oxygen, starvation, low tem-
perature, osmotic pressure, dryness, and bacteriophage, which affect the
physiological function and activity of lactic acid bacteria cells. For the purpose of
improving and guiding the biomanufacturing of lactic acid bacteria, the character-
ization and molecular mechanism exploration and optimization research of bio-
processing conditions, external environmental factors, and lactic acid bacteria are a
prominent hotspot in the application field of lactic acid bacteria, including research
on the effects of environmental factors on the functional properties of lactic acid
bacteria and the physiological stress mechanism of lactic acid bacteria to various
environmental factors. Metabolic engineering techniques and strategies were used
to modify the signal transduction network, transcription, key enzymes, and meta-
bolic pathways in environmental response of lactic acid bacteria cells. We use meta-
bolic engineering techniques and strategies to directionally modify important
regulatory points at the level of signal transduction networks, transcription, key
enzymes, and metabolic pathways of lactic acid cell environmental response, and to
improve the resistance ability of lactic acid bacteria to environmental stress during
biological manufacturing and processing. Besides, from the perspective of cell
physiology and metabolic damage, we systematically studied the physiological
damage and functional weakening in the whole process of lactic acid bacteria starter
preparation, determined the key factors affecting the reduction of lactic acid bacte-
ria activity and weakening the function, and carried out targeted protection strategy
research to improve lactic acid bacteria ability to resist environmental stress.
28 F. Tian
1.4.2.4 tudy on the Interaction Between Lactic Acid Bacteria and Other
S
Life Species and the Function Mining and Development of Lactic
Acid Bacteria
Species in different forms of natural ecosystems depend on each other, and there are
complex interactions between them. The relationship between lactic acid bacteria
and other life species is a prominent hotspot in the research of lactic acid bacteria in
recent years. Related life species involve humans, animals, and plants, and related
fields include medical health, animal science, and plant science. People began to
explore the specific interaction mode and regulation mechanism between lactic acid
bacteria and other organisms at different micro and macro levels, such as molecular,
cell, tissue, organ, system metabolic network, population, and the effects of this
interaction. Taking lactic acid bacteria and humans as examples, in the early twen-
tieth century, people have noticed the role of lactic acid bacteria in the health of the
body. With the advancement of the Human Genome Project and the Human
Microbiome Project, it was discovered that human symbiotic microbiota, including
lactic acid bacteria, may play a huge role in the body’s physiological metabolism,
health maintenance, and disease development. Specifically, people began to explore
the relationship between lactic acid bacteria (and other human commensal microor-
ganisms) and hosts by using different levels of research models such as model in
vitro, cell models, tissue model in vivo, animal models, and population experiments.
Microecological balance, gastrointestinal health, physiological metabolism,
immune balance, neuropsychological, aging, and the effects of the overall body
system homeostasis and intervention effects were explored and analyzed. The func-
tion of lactic acid bacteria was excavated in order to form a systematic theoretical
system of the interaction between lactic acid bacteria and the body and to optimize
the practical application based on lactic acid bacteria. Studies on the interaction
between lactic acid bacteria and other living species such as animals are similar to
that between lactic acid bacteria and human beings.
1.4.2.5 he Development of Lactic Acid Bacteria for Food and Animal

T
Use with Vitality and Function as Their Core Features, as well
as the Preparation and Application of Starter Cultures
As mentioned above, lactic acid bacteria as a starter (starter culture) have a high
value of industrial applications, of which the key is the vitality and function of lactic
acid bacteria. Therefore, on the basis of the development of microbiology and func-
tional exploitation of lactic acid bacteria, the research on the preparation and appli-
cation of lactic acid bacteria-fermented food and animal feed with the core
characteristics of vitality and function is a research hotspot in the field of applica-
tion technology of lactic acid bacteria. For the preparation and application of fer-
mented food such as cheese, fermented milk, fermented vegetables, and fermented
1 Introduction 29
grain food, natural or recombinant lactobacillus strains that can significantly

improve product quality, flavor, manufacturing process, and food safety were devel-
oped by using classical and new genetic breeding technologies. On the basis of
understanding the environmental physiology of lactic acid bacteria, the preparation
technology and related starter products of lactic acid bacteria with high activity and
suitable for industrial application were developed. The application and development
of new starter product of lactic acid bacteria in different food base materials, such
as fruits and vegetables, cereals, milk, meat, aquatic products, are also being
developed.
1.5 Section V Organization Structure of the Book
Lactobacillus science and technology is a rapidly growing field. The author’s

research team tried the basic biology of lactic acid bacteria (involving system clas-
sification, ecological distribution, physiological metabolism, genetic recombina-
tion, phage), experimental methods of lactic acid bacteria (involving separation and
identification, culture preservation, genetic manipulation, omics analysis), lactic
acid bacteria environmental physiology and ecology (involving acid, osmotic pres-
sure, temperature, oxidation, phage, bile salts, hunger, and other environmental
stresses and cellular responses), lactic acid bacteria bioengineering (involving
organic acids, extracellular polysaccharides, vitamins, bacteriocins, sugar alcohols,
substance, functional fatty acids, drugs, and biological products), lactic acid bacte-
ria function mining (involving functional mining methodology, intestinal health,
immunity, food safety, typical cases, lactic acid bacteria safety evaluation, etc.),
lactic acid bacteria industrial applications (involving fermented milk, fermented
fruits and vegetables, fermented grains, alcoholic beverages, starter preparation,
pharmaceutical preparations, animal feed), lactic acid bacteria-related regulations,
and other major levels to carry out the organization of the entire book and strived to
present new theories, technologies, functions, and applications of lactic acid bacte-
ria science and technology to readers based on the existing knowledge system of
lactic acid bacteria. The organization structure and relationship of the whole book
are shown in Fig. 1.3. The first is introduction; the second is basic biology of lactic
acid bacteria; the third is histology of lactic acid bacteria; the fourth is environmen-
tal physiology of lactic acid bacteria; the fifth is bioengineering of lactic acid bacte-
ria; the sixth is functional excavation and evaluation of lactic acid bacteria; the
seventh is the industrial application of lactic acid bacteria; the eighth is the regula-
tion and management of lactic acid bacteria; and the ninth is the experimental meth-
odology of lactic acid bacteria.
30
F. Tian
Fig. 1.3 The organization and chapters of the book

1 Introduction 31
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Chapter 2
Genetic Operation System of Lactic Acid
Bacteria and Its Applications
Haiqin Chen, Chen Chen, Chunqing Ai, Chengcheng Ren, and He Gao
Lactic acid bacteria (LAB), a class of commonly existing microorganisms in nature,

are important components of gut commensal microflora in humans and animals.
Previous studies suggested that LAB exerted specific physiological and biochemi-
cal functions on the host such as improving intestinal microbial balance, immuno-
modulation, inhibiting tumor growth, lowering cholesterol levels, as well as
regulating blood pressure and are therefore widely used in food manufacturing and
functional food development. Due to the continuous development of modern molec-
ular biology techniques, studies regarding exploiting LAB as expression hosts in
addition to fermentation starter cultures and probiotics have received increasing
attention from both academia and industry. In the 1980s, some researchers initiated
molecular genetic research for LAB. They characterized lactose metabolism-related
genes and proteins in LAB and established preliminary DNA delivery systems for
LAB. Over the past decades, owing to the advances in modern DNA sequencing and
gene characterization techniques, structures and functions of LAB genomes and
plasmid-related genes have been further elucidated, which lays a solid theoretical
foundation for the further development of LAB-based gene expression systems
(Bolotin et al. 2001; Altermann et al. 2005).
H. Chen (*) · C. Ren · H. Gao

e-mail: [email protected]; [email protected]; [email protected]
C. Chen
Shanghai Institute of Technology, Shanghai, China
C. Ai
Dalian Polytechnic University, Dalian, China

https://doi.org/10.1007/978-981-13-7832-4_2
36 H. Chen et al.
2.1 LAB-Associated Gene Expression Systems
LAB expression systems were developed lately as compared to traditional

Escherichia coli, Bacillus, and yeast expression systems. Generally, there’re still
many limitations in LAB-based gene expression systems such as low gene expres-
sion efficiency, complicated procedures, and low transformation efficiency.
Nevertheless, genetic engineering of LAB offers remarkable advantages over other
traditional expression systems due to the intrinsic properties of LAB strains, thereby
exhibiting great potential for applications in antigen screening, protease expression,
and immunotherapy. The advantages of LAB-based expression systems are listed
below: (1) LAB are safe and edible food-grade microorganisms granted by WHO
and FAO and have been applied in food products for thousands of years; (2) LAB
are inherent enteric microflora in humans and animals and play a key role in the
establishment and maintenance of host immune system; (3) some LAB strains
adhere tightly to the gut mucosa; and (4) LAB culture supernatants can be directly
consumed without the necessity of purifying expressed heterologous proteins.
LAB-based gene expression systems comprise host strains, expression vectors, and
heterologous genes. This chapter will briefly describe host strains and cloning vec-
tors in LAB-based gene expression systems in terms of their compositions and
features.
2.1.1 Host Strains in Expression Systems for LAB
LAB strains of different genera or species and even different strains within the same
species differed greatly in terms of their biochemical, ecological, and molecular
immune properties (Meijerink et al. 2012; Ai et al. 2015). This strain diversity cre-
ates extensive host strain options in LAB but also causes difficulty of selecting
proper strains as expression hosts since high-level production of heterologous pro-
teins in a specific strain-based expression system cannot always be achieved in other
strains (Rigaux et al. 2009).
Currently, several genera including Lactococcus, Enterococcus, and Lactobacillus
have been most widely applied as host strains (Table 2.1) owing to their extraordi-
nary features such as good resistance to the harsh conditions in the digestive tract,
low immunogenicity, relatively high electro-transformation efficiency, and long
retention time in the gastrointestinal tract. Due to the progress in molecular genetics
research on LAB, LAB as food-grade delivery vehicles have received increasing
attentions. In order to further facilitate the screening of recombinant LAB strains,
host strains can be modified based on specific requirements such as lacF-deficient
strains and strains with nisRK integrated into their chromosomes.
2 Genetic Operation System of Lactic Acid Bacteria and Its Applications 37
Table 2.1 Host strains commonly used in LAB

Expression
Host strain vector References
Lactobacillus pGIT032 Corthesy et al. (2005)
plantarum
Lactococcus lactis pTREX1, Lee et al. (2001), Mannam et al. (2004), and
pP16pip Robinson et al. (2004)
Lactobacillus pNZ123 Kim et al. (2005)
acidophilus
Lactobacillus casei pPGS1 Lee et al. (2005)
Lactobacillus pFBYC04 Biet et al. (1998)
cremoris
Lactobacillus pNZ124 Scheppler et al. (2002)
johnsonii
Enterococcus faecalis hBD2-Cy3 Kandaswamy et al. (2013)
Lactobacillus sakei pSIP, pMG36c Jimenez et al. (2015)
Lactobacillus pCI Oliveira et al. (2006)
helveticus
Lactobacillus reuteri pNIES Wu and Chung (2007)
Lactobacillus pG Liu et al. (2011)
pentosus
2.1.2 Vectors in LAB-Based Gene Expression System
2.1.2.1 Plasmids in LAB
Since Chassy and Flickinger (1987) detected plasmids in LAB, researchers have
further studied plasmids that are present in LAB. The distribution of plasmids has
been suggested to be highly uneven and strain-specific in LAB. More plasmids were
found in LAB strains such as Lactobacillus reuteri, Lactobacillus helveticus, and
Lactobacillus acidophilus when compared with strains belonging to other species.
Moreover, the size (1–150 kb) and amounts of plasmids differed greatly in different
LAB strains (Chassy et al. 1976; Vescovo et al. 1981; McKay and Baldwin 1990).
Overall, plasmids in LAB are characterized by the following features: (1) Most
plasmids in LAB are cryptic plasmids, and only a few of them are associated with
the host’s specific phenotypes such as bacteriocin synthesis, sugar metabolism, and
antibiotic resistance (Smiley and Fryder 1978; Fortina et al. 1993). (2) The copy
number of the LAB plasmids is correlated with their size. Smaller plasmids are
replicated at a higher number. (3) Plasmids from LAB, which have a broad host
range, can replicate in a wide range of host bacteria.
Since most of plasmids from LAB are cryptic, their functions on gene transcrip-
tion, translation, and protein secretion are still not completely clear, which might be
one of causative factors for delayed development in LAB molecular biologics study.
Nevertheless, owing to the advances in genetic engineering techniques, vectors for
cloning, expression, and integration in LAB were successively developed through
38 H. Chen et al.
studying the regulatory elements within plasmids isolated from LAB. At present,
cloning and expression vectors are the most extensively applied vectors in LAB.
2.1.2.2 Cloning Vectors for LAB
Genetic cloning techniques, one of the effective means of resolving the complexity
of genetics, allow isolation of single desired genes and construction of new LAB
starter strains carrying these isolated genes. Cloning vectors are used for amplifica-
tion and propagation of foreign DNA inserts and have certain copy numbers within
host cells. Cloning vectors have the following essential features: (1) suitable cloning
sites, into which foreign DNA can be inserted, (2) autonomous replication or repli-
cation along with chromosomal DNA once plasmids are integrated into the host
chromosome, and (3) selectable markers which facilitate the selection of trans-
formed cells harboring DNA insert-containing plasmids. To date, various types of
cloning vectors including plasmids, viral vectors/bacteriophages, and artificial vec-
tors that incorporate segments from the plasmids, bacteriophage, or genomic DNA
have been constructed. These artificial vectors might only have an origin of replica-
tion but not promoters for expression.
Even though Escherichia coli is the most frequently applied host in molecular
cloning, other microorganisms sometimes are also used as hosts. Thus, shuttle vec-
tors containing a second replication origin, which assures their replication in other
types of microorganisms (e.g. LAB), are needed. The enterococcal plasmid
pAMβ1, a θ-type-replicating plasmid with a broad host range, is the first plasmid
used for constructing LAB cloning vectors. It is also one of the prototype cloning
vectors for lactococci and lactobacilli. Several plasmids such as pIL252 and
pIL253, which are based on pAMβ1, were established. Other common plasmid
replicons such as pWV01 and pSH71 can propagate and replicate in various LAB
strains. Moreover, there are other low-copy and high-copy derivative plasmids
such as pGK1 and pGK12.
In order to screen the right transformants, one or multiple resistance genes are
ligated into vectors as selectable markers. Since a large number of wild-type LAB
strains are resistant to ampicillin, kanamycin, and tetracycline, erythromycin and
chloramphenicol resistance genes serve as the common selection markers. Of note,
since the transfer and dissemination of antibiotic resistance genes in the environ-
ment are potentially deleterious to the environmental ecosystem, resistance genes
are not suitable selection markers in the food industry. Therefore, several food-
grade expression systems for LAB based on the sugar utilization, sensitivity to PH
and temperature, and bacteriocin resistance of host strains have been developed.
Apart from single-component cloning vectors, some researchers developed sev-
eral two-component cloning systems. Emond et al. (2001) constructed a two-
component food-grade cloning vector pVEC1, which is a pCD4 derivative and
carries the functional pCD4 replicon. Another pCD4-derived plasmid pCOM1 was
also constructed as a companion vector. Plasmid pCOM1, in which an erythromycin
resistance gene serves as the dominant selection marker, is deficient of repB gene.
After the selection of recombinant strains carrying both plasmids, recombinant bac-
terial cells will lose plasmid pCOM1 when grown under antibiotic-free conditions
owing to the incompatibility between pVEC1 and pCOM1. By using this cloning
system, Lactococcus lactis MG1363 and industrial strains were shown to exhibit
stable phage-resistant phenotype.
2.1.2.3 Expression Vectors for LAB
To ensure effective expression of genes of interest, expression vectors must have

elements for constitutive or inducible protein expression besides the basic elements
in cloning vectors such as origins of replication and heterologous genes. In terms of
the cellular location of expressed proteins, there’re three types of protein expression
in LAB as described below:
1. Cytoplasmic expression: it can effectively protect produced protein from the
external environment. But cell lysis is required for intracellular protein release.
2. Secreted expression: the secretion of recombinant protein into extracellular
medium is directed by signal peptide.
3. Cell surface displaying of target protein: it can be achieved by anchoring recom-
binant proteins to the cell wall.
In recent years, a large number of expression vectors in LAB have been constructed
for different applications in the fields of food, medicine, and life sciences.
2.1.3 Intracellular Expression Systems for LAB
Intracellular expression of heterologous proteins in LAB is achieved by inserting

foreign genes into LAB expression vectors without signal peptides and introducing
recombined vectors into LAB hosts via electroporation. Plasmids for intracellular
expression in LAB comprise promoters, multiple cloning sites for insertion of for-
eign genes, terminators, selection markers, and replicons. Thus far, most commonly
applied promoters in LAB expression systems are lacA, lacR, lacF, T7, xylA, lacS,
nisA/nisZ, and nisF (Kleerebezem et al. 1997). Based on the carried resistance
gene, expression vectors in LAB can be divided into antibiotic resistance-based vec-
tors and food-grade expression vectors. Traditional expression vectors for LAB
carry one or multiple antibiotic resistance-encoding genes such as erythromycin and
chloramphenicol resistance genes, and transformants are selected via antibiotic
selection pressure. The plasmids pNZ8037 and pNZ8048 carrying chloramphenicol
resistance genes were developed on the basis of the food-grade NICE system and
are standard expression vectors for L. lactis. These two plasmids contain the pSH71
replicon and nisin-inducible promoter nisA and can be utilized as expression vectors
in both Escherichia coli and L. lactis. To overcome plasmid pNZ8048-induced low-
level expression in LAB, plasmids pNZ8148 and pNZ8150 carrying
40 H. Chen et al.
chloramphenicol resistance genes were constructed based on pNZ8048. These two

upgraded plasmids are capable to drive highly efficient expressions in LAB.
2.1.4 Secreted Expression Systems for LAB
2.1.4.1 Features of Secretion Vectors in LAB
Exploiting LAB as expression hosts or cell factories to produce antigenic proteins,

pharmaceutical molecules and other functional factors have become a new research
filed on LAB. To achieve secretory expression of protein in LAB, heterologous
genes are inserted expression vectors with signal peptides, which direct the secre-
tion of intracellular recombinant proteins to extracellular environment after trans-
forming LAB hosts with recombinant vectors via electroporation. Signal peptides,
a vital determining factor for secretory expression, are peptides residing on recom-
binant precursor proteins, which can be targeted to the secretory pathway by signal
peptides. Through the cleavage of signal peptidase, signal peptides are separated
from the mature proteins, which are translocated to extracellular compartment after-
ward (Nielsen et al. 1997).
Compared with intracellular expression, secreted expression holds the advantage
of synthesizing heterologous proteins in an active form without aggregation within
the host cells, which effectively prevents the loss of proteins during protein recov-
ery. Moreover, secreted recombinant proteins can be separated from other cellular
proteins, thus simplifying the downstream purification procedures for target pro-
teins. However, not all proteins are suitable for secretory expression, especially for
those naturally nonsecreted proteins, whose secretion is determined by multiple
factors such as their size, structure, charges, and signal peptide. For one specific
protein, different signal peptides can result in differential secretion efficiencies
(Meazza et al. 1997). Zhang et al. (2010) showed that modifying signal peptide
structure pronouncedly improved the secretion efficiency of recombinant protein in
LAB. Therefore, selection of proper signal peptides is of great importance for secre-
tory expression system for LAB or other hosts.
2.1.4.2 Secretion Systems Based on Usp45 Signal Peptide
At present, the signal peptide of Usp45 protein isolated from L. lactis is the most
extensively applied signal peptide for secretory expression in LAB and was identi-
fied in the genome of L. lactis MG1363 by van Asseldonk et al. (1990). LAB secre-
tion systems can efficiently recognize Usp45 signal peptide, which resulted in
enhanced secretion of heterologous proteins such as antigens (Ribeiro et al. 2002;
Bermudez-Humaran et al. 2003b; Zhang et al. 2011) and antibodies (Bermudez-
Humaran et al. 2003a; Zhang et al. 2010). To date, extensive studies have suggested
that enhanced heterologous protein expressions via secretory expression are closely
correlated with the abilities of signal peptides to stabilize variable proteins, inhibit
the proteolysis by intracellular proteases, and improve protein secretion efficiency.
In the study by Enouf et al. (2001), bovine rotavirus NSP4 protein was intracellu-
larly and extracellularly expressed in L. lactis, but mature NSP4 protein could not
be efficiently secreted. In addition to the Usp45-NSP4 precursor and mature NSP4
protein, two degradation products of mature NSP4 protein were also detected in the
intracellular compartment of the NSP4-secreting LAB strain. This indicates that
mature NSP4 protein might be partially degraded by intracellular proteases, which
could be inhibited by infusion with the Usp45 signal peptide.
2.1.4.3 Secretion Systems Based on the Signal Peptide of S-Layer Protein
Signal peptide sequences exert crucial influences on the secretion efficiency of het-
erologous proteins in LAB. Due to the complex relation between protein secretion
and signal peptides, it is not realistic to apply one specific signal peptide for the
secretory expression of all different types of heterologous proteins in a specific
LAB strain. Thus, searching for novel signal peptides to establish a diverse signal
peptide library is an effective approach to elevate protein secretion expression levels
in LAB. S-layer protein, whose relative molecular weights are in the range from
40,000 to 200,000, is a layer of bioactive macromolecules present on the cell wall
surface of many bacteria and archaea. Most S-layer proteins consist of a single spe-
cies of protein or glycoprotein. S-layer proteins account for 10–15% of the total
bacterial proteins, and the gene expression machinery for their synthesis and secre-
tion is quite strong.
Many researchers consider abundant signal peptide options as one of the effective
means to enhance the secretion efficiency in LAB. S-layer protein-encoding genes
are highly efficiently expressed and secreted in LAB, which are closely linked to the
high transcription efficiency of their promoters and high secretion efficiency of their
signal peptides. Based on this characteristic of S-layer proteins in LAB, their one
major application in LAB is to develop highly efficient (secretion) expression sys-
tems for LAB (Zhang et al. 2010). Kahala and Palva (1999) introduced the promoter
of a S-layer protein SIpA in two different LAB hosts and found that this promoter
significantly enhanced beta-glucuronidase (gusA) and aminopeptidase N (pepN)
expression in L lactis and L. plantarum, respectively. Expression levels of gusA and
pepN account for 15% and 28% of the total cellular proteins in L. lactis and L. plan-
tarum, respectively. In addition, Sibakov et al. (1991) increased secretion of
β-lactamase in a recombinant L. lactis strain by using the strong promoter and signal
peptide of lactobacilli S-layer protein-encoding gene. Even though promoter and
signal peptide have been suggested to be closely related to the expression and secre-
tion efficiency of heterologous proteins in LAB, the characteristics of the expression
hosts also affect the transcription efficiency of S-layer protein promoter. It was shown
that using the same S-layer protein promoter yielded differential protein expression
efficiencies in different LAB hosts (Kahala and Palva 1999), which further facilitates
the research on signal peptide sequences of LAB expression systems.
42 H. Chen et al.
2.1.4.4 Secretion Systems Based on Other Types of Signal Peptides
In addition to LAB-originated signal peptides, signal peptides derived from other

bacteria have also been applied to construct secretion expression vectors for LAB. In
a study by Le Loir et al. (2001), staphylococcal nuclease (nuc) was secreted by
recombinant L. lactis with the native signal peptide of nuc gene, whose secretion
efficiency was lower than when applying the Usp45 signal peptide. This might be
attributed to the effects of signal peptides on protein conformation (Kajava et al.
2000). In addition to screening suitable native signal peptides, many researchers
focused on modifying native signal peptide sequences to elevate the secretion effi-
ciency of recombinant proteins in LAB. In L. lactis, insertion of a nine-residue
synthetic propeptide (LEISSTCDA) after the Usp45 signal peptide sequence
achieved a secretion efficiency of up to 80% for nuc, the yield of which was upregu-
lated 2–4-folds (Le Loir et al. 2005).
2.1.5 he Surface Expression System of Lactic Acid Bacteria

T
(LAB)
2.1.5.1 S-Layer Protein Expression System
In 1985, G. P. Smith took the advantage of major coat protein P3 of the filamentous
bacteriophage to establish a molecular genetic system for phage, which plays vari-
ous important roles in some fields, such as interactions between protein-protein and
DNA-protein, analysis of antigen peptides, protein directed evolution, and signal
transduction. However, the nature characteristics of the phage make it hard to
express high molecular weight (HMW) protein, which limits the application of the
phage expression system. To solve the above problem, some researchers did some
efforts to develop new system by using bacteria that can fully express heterologous
HMW protein. Based on the safety feature of LAB, its expression system received
lots of attention and exhibited a high potential value in live vaccines, diagnosis,
enzyme immobilization, and so on.
Recent studies indicate that some proteins that anchor on the cell surface of LAB
play key roles in cell adhesion, immune responses, signal transduction, and other
life activities. Based on the structural characteristics of surface protein, some were
used to establish new expression system. It was apparent that the monomolecular
crystalline array of proteinaceous subunit, which was termed as S-layer, was con-
sidered as one of the most common surface structures on bacteria. Sequence analy-
sis shows that S-layer protein of LAB has two conserved domains (N-terminal
secretion signal peptide and C-terminal anchoring peptide) and an intermediate
variable region that participates in the protein refolding and crystallization process.
Based on the above structure property of S-layer protein, the expression gene of
heterologous protein can be inserted into S-layer protein coding gene, and then it
was expressed on the cell surface of LAB following the S-layer protein expression.
There is a growing body of evidence showing that bacterial S-layer protein expres-
sion system could exert great application potential in the field of microbiology,
molecular biology, immunology, and biological catalysis. Due to its safety property,
the expression system in LAB has received more attention from different study
fields, such as recombinant vaccines, bacterial adhesion, and antibody.
2.1.5.2 Cell Wall Anchoring Expression System
To date, some enzymes that anchor surface proteins to the cell wall are proven to be
used as cell wall anchoring domain of heterologous protein in the surface expres-
sion system based on LAB, especially cell wall hydrolases and the aggregation
factor. Due to the difference in anchor position, the surface expression system can
be divided into cell wall anchoring form, cell membrane anchoring form, and sur-
face layer-associated proteins anchoring form. According to the bonding form
between the anchoring protein and cell, the surface expression system is divided
into covalent bond and non-covalent bond formation. On account of the binding site
between heterologous protein and anchoring protein, it can be divided into two
main forms: N-terminal and C-terminal formation. For the former, the expressed
heterologous protein locates in the middle of the signal peptide and the anchoring
region, such as the M6 protein from Streptococcus pyogenes, protein A of
Staphylococcus aureus, and other surface protein in the Gram-positive bacteria. For
the latter, the expressed protein locates the downstream of the anchoring region,
such as proteinase PrtP from L. lactis, lysozymes from Bacillus phages, extracel-
lular hydrolase from Lactobacillus strain, and peptidoglycan hydrolase of
Enterococcus strain.
The LPXTG motif contained a hydrophobic domain and a positively charged
tail, which was termed as a marker sequence of proteins which was anchored to the
bacterial cell wall (Kuczkowska et al. 2015). After translocation, LPXTG motif
would be firstly cleaved and then cross-linked at the threonine residue to a nucleo-
phile, i.e., an active amino group of the peptidoglycan stems peptide or the lysine
residue of the pilin motif (Kuczkowska et al. 2015). García-Mantrana et al. (2016)
showed that two phytases from bifidobacteria could be cloned in L. casei under the
control of a nisin-inducible promoter, and they were able to produce, export, and
anchor to the cell wall. Kuczkowska et al. (2015) indicated that recombinant L.
plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag-derived antigen
causes increased recruitment of T cells, in which the heterologous proteins were
expressed in the cell surface.
LysM is widely distributed in more than 4000 proteins in both prokaryotes and
eukaryotes, and this protein was firstly discovered in lysozyme of Bacillus phage
φ29 acting as a C-terminal repeat comprising of 44 amino acids with seven amino
acids inserted (Buist, Kok et al. 1995). The best-characterized LysM containing pro-
tein is the N-acetylglucosaminidase AcmA of L. lactis, which is required for cell
separation and cell lysis during the stationary phase of L. lactis (García-Mantrana
et al. 2016). The composition of Lysm domain family is highly abundant, which
44 H. Chen et al.
could be attributed to gene diversity, varied amount, and combinational diversity

with other protein domain, and thus some of them were used as an anchor to display
heterologous proteins on the surfaces of LAB. Hu et al. (2010) showed that a Novel
LysM domain was isolated from L. fermentum bacteriophage endolysin and used as
an anchor to display protein in the surfaces of LAB. To date, there is a growing body
of evidence indicating that LAB could be used as a potential vehicle although some
disadvantages need to be solved, such as thicker cell walls, less heterologous pro-
tein production, and so on.
2.1.6 Applications of LAB-Based Gene Expression System
2.1.6.1 LAB as Vaccine Delivery Vehicles
(1) Applications of LAB vaccine delivery system

In recent years, due to the progress in developing LAB as mucosal vaccine deliv-
ery vectors, the application of recombinant LAB-based vaccines has been exten-
sively expanded. Vaccines based on genetically engineered LAB strains have been
exploited for disease management or as nutritional supplements. Herein, we will
describe in detail the current status of preclinical laboratory research regarding
diverse applications of recombinant LAB vaccines.
1. Prevention and treatment of infectious diseases
At the early stage, the primary recombinant LAB-based mucosal vaccines against
infectious diseases were genetically modified LAB strains expressing key antigen
proteins (or fragments). Recombinant LAB vaccines are applied in two distinctive
forms: immunoprophylaxis and immunotherapy. These two immunization forms
differ in their timing of intervention. For immunoprophylaxis animals are adminis-
tered with recombinant LAB strains prior to establishing experimental disease mod-
els, while for immunotherapy immunization animals administered with recombinant
vaccines are carried out after animal models are developed. LAB vaccines against
infections will be described below based on the types of pathogenic microorganisms
involved.
(A) Bacterial and fungal infections
Thus far, a large number of genetically modified LAB strains producing crucial
antigens (fragments) of common human or animal pathogens have been success-
fully constructed and confirmed to confer immune protection against infections in
various animal infection models (Wells et al. 1993; Norton et al. 1997; Maassen
et al. 1999; Grangette and Muller-Alouf 2001; Robinson et al. 1997, 2004; Cheun
et al. 2004; Corthésy et al. 2005; Hanniffy et al. 2007).
In order to optimize the immune efficacy of recombinant vaccines, researchers
attempted to construct recombinant LAB strains expressing different antigen proteins
or fragments derived from one specific pathogenic microorganism for immunization
(Lee et al. 2001; Wu and Chung 2007; Wei et al. 2010; Hongying et al. 2014). Wu and
Chung (2007) constructed a recombinant Lactobacillus reuteri strain producing the
fusion protein of heat-stable enterotoxin and heat-labile enterotoxin B of enterotoxi-
genic Escherichia coli (ETEC) as a mucosal vaccine against ETEC infections. Wei
et al. (2010) exploited Lactobacillus casei as a carrier for fimbriae protein of ETEC
K99 in mucosal immunization.
Through certain evolved strategies, pathogens are capable to evade the host
immune defense system so that they can continuously colonize, proliferate, and
migrate in the hosts to cause infections. These strategies including specific cell sur-
face components (e.g., surface capsules and pili) and synthesis of virulence factors
(e.g., toxins and proteases) are unitized by pathogens to interfere or subvert host
immune surveillance. Thus, in order to promote the recognition of specific patho-
gens by the host immune system and to trigger specific defensive immune responses,
it is of vital importance to select essential virulence factor of pathogens as the target
of vaccine development.
Besides, developing recombinant LAB strains as mucosal delivery vectors for
antibody fragments to enhance host immunity against pathogens is another effective
strategy against infections. Beninati et al. (2000) constructed recombinant
Streptococcus gordonii strains expressing a microbicidal single-chain antibody
(H6), which were found to inhibit Candida albicans-induced vaginal inflammation
via vaginal immunization.
(B) Viral infections
So far, researchers have successfully constructed genetically modified LAB
strains against many common pathogenic viruses such as rotavirus, hepatitis B
virus, and influenza virus and evaluated their efficacy in experimental animal mod-
els of viral infection (Xin et al. 2003; Ho et al. 2005; Perez et al. 2005; Lee et al.
2005; Poo et al. 2006; Lei et al. 2011; Liu et al. 2011; Zhang et al. 2011). It is prom-
ising that Xin et al. (2003) engineered a recombinant L. lactis strain to express and
anchor the envelope protein of the human immunodeficiency virus (HIV) on its cell
surface. They found that oral immunization of mice with this genetically modified
LAB strain induced HIV-specific cellular (high level of IFN-γ-secreting lympho-
cytes in the spleen and intestinal lymph node) and humoral immune responses (high
level of HIV-specific serum IgG and fecal IgA antibodies). Furthermore, expressed
HIV antigen fragments were shown to be presented to T lymphocytes by dendritic
cells, thereby mounting adaptive immune reactions (Xin et al. 2003).
(C) Parasitic infections
Parasites, another major group of infectious organisms, can cause severe organ
or tissue damage in the hosts. However, due to the difficulty in their in vitro
cultivation, the development of anti-parasitic vaccines has been greatly hindered.
Therefore, synthesis of recombinant parasite antigens using molecular biology tech-
niques for immunization is of great significance for facilitating anti-parasitic vac-
cines development. Thus far, there have been some promising results with regard to
applying LAB as vaccine vehicles against parasitic infections (Zhang et al. 2005;
46 H. Chen et al.
Ramasamy et al. 2006; Lee et al. 2009; Yam et al. 2011). Zhang et al. (2005) geneti-
cally modified Lactococcus lactis to express Plasmodium yoelii antigen fragment
MSP-119 and orally immunized two strains of mice (BALB/c and C57BL/6) with
this recombinant strain. They observed that this recombinant LAB strain effectively
enhanced host immunity against malaria parasites in both strains of mice.
2. Inflammatory bowel disease (IBD) management
IBD is group of chronic, recurrent gastrointestinal inflammatory disorders caused
by intestinal dysfunction. IBD is often concomitant with parenteral complications
and needs long-term therapy, thus leading to a marked decline in IBD patients’ qual-
ity of life. In spite of the similar clinical symptoms such as abdominal pain and
diarrhea, IBD should be discriminated from pathogen-induced gastroenteritis.
Ulcerative colitis (UC) and Crohn’s disease (CD) are two main forms of IBD. In
recent years, the prevalence of IBD has been increasing worldwide; effective IBD
prevention and treatment are desperately needed. So far, researchers have carried
out a range of studies on mucosal vaccination with genetically engineered LAB
against IBD based on differential IBD pathogenesis and therapeutic directions and
obtained some promising results. The first attempt to apply genetically modified
LAB strains against IBD was performed by Steidler and Hans (2000). They con-
structed a recombinant L. lactis strain expressing anti-inflammatory cytokine inter-
leukin-10 (IL-10). Intragastric administration with this recombinant strain
pronouncedly attenuated dextran sulfate sodium (DSS)-induced colitis in mice by
50% and also effectively suppressed the development of IL-10 gene deficiency-
caused colitis in mice. More encouragingly, an “upgraded” recombinant IL-10-
expressing L. lactis strain has been tested in a phase I clinical trial and demonstrated
to be safe in CD patients and biologically contained (Braat et al. 2006). This is also
the first clinical trial performed with genetically modified LAB (Braat et al. 2006),
which greatly promotes research on recombinant LAB vaccines.
Apart from IL-10, some other anti-inflammatory molecules such as trefoil factor
(TFF) and neuropeptide α-melanocyte-stimulating hormone (α-MSH) have also
been expressed in genetically modified LAB strains for controlling IBD. TFF are
small polypeptides with beneficial effects on mucosal protection and repair, while
α-MSH is a neuroendocrine peptide with anti-inflammatory properties (Ren et al.
2005; Zhu et al. 2014). Vandenbroucke et al. (2004) observed pronounced allevia-
tion of inducible acute colitis and spontaneous chronic colitis in mice after intragas-
tric administration with TFF-expressing L. lactis. Yoon et al. (2008) found that oral
immunization of mice with a α-MSH-secreting L. casei strain significantly amelio-
rated DSS-induced acute colitis in mice.
Neutralizing the inflammatory cytokine tumor necrosis factor (TNF)-α is another
effective therapeutic strategy for IBD. In accordance with this strategy, several
recombinant mucosal vaccines based on genetically modified LAB have been
developed. A study by Vandenbroucke et al. (2009) confirmed the protective effi-
cacy of recombinant L. lactis secreting anti-TNF-α nanobodies on both DSS- and
IL-10 deficiency-induced colitis in mice after oral immunization. Furthermore, the
affibody against TNF-α has been surface displayed in L. lactis and recombinant
affibody was shown to exhibit TNF-α-binding capability. These results indicate that
this affibody-expressing strain has a potential in binding intestinal TNF-α and might
be used to mitigate gut inflammation in IBD (Ravnikar et al. 2010).
Since oxidative stress plays a key role in the pathogenesis of inflammatory disor-
ders, antioxidant enzymes become another type of potential IBD therapeutic agents.
Thus far, researchers have engineered LAB strains as mucosal delivery vectors for
antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) to
achieve the immunomodulation of IBD (Carroll et al. 2007; LeBlanc et al. 2011).
LeBlanc et al. (2011) constructed recombinant L. casei expressing SOD or CAT and
assessed their protective efficacy in mice with trinitrobenzenesulfonic acid (TNBS)-
induced CD. It was observed that mice that received these SOD or CAT-producing
LAB strains displayed less weight loss, less bacterial translocation to the liver, and
alleviated colonic tissue damage.
Although the precise pathogenesis of IBD is still obscure, it has been proposed
that insufficient innate antigenic stimulation can result in immune dysfunction,
which triggers excessive immune reactions against innocuous intestinal antigens
and thereby leads to inflammation and tissue damage. Therefore, utilization of anti-
inflammatory agents to suppress excessive inflammatory responses in IBD patients
is a major clinical treatment option for IBD. A study conducted by Foligne et al.
(2007) offers new therapeutic targets for IBD management. It is known that some
pathogens can effectively circumvent the host immune surveillance, stimulate anti-
inflammatory cytokine production, and resist the host immune responses against
them. Based on this theory, the low-calcium response V (LcrV) protein, one viru-
lence factor of enteropathogenic Yersinia pseudotuberculosis was expressed in
genetically engineered L. lactis (Foligne et al. 2007). The protective properties of
this recombinant strain were demonstrated in TNBS- and DSS-induced murine coli-
tis models. It was shown that this strain effectively induced IL-10 secretion and
dampened colonic inflammation. Intriguingly, the protective effects of this recom-
binant strain in mice with TNBS-induced colitis were IL-10-dependent. Moreover,
similar protective efficacy in TNBS-induced colitis model was observed by immu-
nization with this LcrV-expressing L. lactis strain or an IL-10-expressing L. lactis
strain (Foligne et al. 2007).
3. Management of allergic disorders
Type I allergy, also known as immediate hypersensitivity reactions, is an IgE-
mediated, immune disorder characterized by Th2-skewed immune responses.
Clinical symptoms of type I allergy occur immediately upon allergen exposure.
Food allergy, dust mite allergy, and pollen allergy are all immediate hypersensitivity
disorders. In recent years, there has been accelerating incidence of type I allergy
worldwide. However, there are still great difficulties in its effective clinical
prevention or treatment. Traditional type I hypersensitivity treatment is allergen-
specific immunotherapy, in which immune tolerance to specific allergens is devel-
oped. In the traditional desensitization therapy, patients are exposed to or injected
with allergen extracts via non-mucosal routes for administration such as subcutane-
ous injection. Due to the limitation in the availability of highly pure allergen extracts,
48 H. Chen et al.
severe adverse reactions often occur in allergic patients receiving traditional desen-
sitization therapy. Therefore, genetic engineering of LAB strains for producing and
mucosal delivering recombinant allergens can effectively circumvent the aforemen-
tioned problems. The generally recognized safe status and immunomodulatory
properties of LAB render them ideal vectors for presenting allergens to the host
mucosal surface. So far, a number of allergen-expressing recombinant LAB strains
have been successfully applied for type I allergy management (Adel-Patient et al.
2005; Charng et al. 2006; Cortes-Perez et al. 2007; Huibregtse et al. 2007; Rigaux
et al. 2009; Schwarzer et al. 2011).
Allergic reactions are triggered upon the binding of allergen-specific IgE anti-
bodies to the IgE binding epitopes on allergens. Allergen T cell epitopes is the
molecular basis for the recognition of allergens by T cells, thereby conferring
immunomodulatory effects. The progress in identifying allergen epitopes in recent
years greatly facilitates the development of recombinant hypoallergenic allergens or
T cell epitope peptide-based allergy therapies. T cell epitope peptides have been
successfully applied to modulate cat and insect venom (Bohle 2006). To summarize,
it is promising to apply recombinant LAB strains expressing novel allergen mutants
as mucosal vaccines for the prophylaxis and alleviation of allergic diseases.
In recent years, some researchers have applied cytokine-expressing genetically
engineered LAB against allergy, indicating a new research direction for managing
allergic diseases (Frossard et al. 2007). Frossard et al. (2007) found that oral admin-
istration of mice with an IL-10 expressing recombinant L. lactis strain effectively
inhibited allergic reactions and reduced serum Th2-type antibody (IgE and IgG1)
production. Cytokine-expressing recombinant LAB strains were also used to boost
the immunomodulatory effects of allergen (or allergen fragments)-expressing LAB
in allergy management. Cortes-Perez and Ah-Leung (2007) showed that an IL-12-
producing recombinant L. lactis strain enhanced the protective efficacy of a geneti-
cally engineered LAB strain expressing cow’s milk allergen in a cow’s milk allergy
mouse model.
Cross-linking of allergen-specific IgE antibodies with cell surface receptors on
basophils or mast cells is a prerequisite for triggering the release of inflammatory
mediators such as leukotriene and histamine and mounting allergic reactions.
Therefore, using humanized anti-human IgE antibodies to bind free IgE to prevent
the cross-linking of IgE with mast cells is an effective approach for allergy preven-
tion or alleviation. Based on this, Scheppler et al. (2005) genetically modified
L. johnsonii to express and anchor either an IgE mimotopes or an anti-idiotypic
single-chain fragment variable (scFv) mimicking an IgE epitope on the cell surface.
This recombinant stain was shown to be recognized by anti-human IgE monoclonal
antibody and induce systemic IgG antibodies against human IgE through both sys-
temic and mucosal immunization. These results indicate that these recombinant
strains can potentially induce anti-IgE response, preventing or relieving allergic
symptoms.
(2) Clinical applications of recombinant LAB as vaccines

As stated above, preclinical studies on recombinant LAB-based mucosal vaccines
have achieved remarkable results, promoting their clinical research. Nevertheless, there
are still some concerns regarding the clinical effectiveness and safety of recombinant
LAB-based vaccines, which hinders the launch of their clinical trials. Steidler and Hans
(2000) confirmed the remarkable protective efficacy of mucosal immunization of an
IL-10-expressing genetically engineered L. lactis strain in two different mouse models
of colitis. This research group developed a thymidine-deficient expression system
(ActoBiotics™) for L. lactis to facilitate its clinical research. The thymidylate syn-
thase-encoding gene thyA in L. lactis chromosome was replaced with the human IL-10
gene so that this modified recombinant strain cannot survive under thymidine- or thy-
mine-free conditions. This modification prevents the release of this genetically engi-
neered strain into the environment, thus eliminating the biosafety concerns toward this
strain raised by its potential replication and pervasion (Bahey-El-Din 2012). Based on
this thyA-deficient expression system, Braat et al. (2006) launched the first phase I
clinical trial on recombinant LAB-based mucosal vaccines. After obtaining promising
results in CD patients during its phase I clinical trial, its phase IIA clinical trial has also
been conducted. Although the phase IIA clinical trial confirmed its biosafety status, it
was not effective on mucosal repair (Bermúdez-Humarán et al. 2011).
Clinical studies have also been performed with another genetically engineered
LAB strain (AG013), which was constructed to express human TFF1 based on
ActoBiotics™ expression system. Its phase Ib trial demonstrated its safety and tol-
erability in participants as well as its efficacy in improving chemotherapy-induced
ulcerative oral mucositis in patients with locally advanced head and neck cancer
(Limaye et al. 2013).
2.1.6.2 Recombinant LAB in Enzyme Preparations
In addition to being developed as mucosal vaccines, genetically engineered LAB

also exhibit great potential in the production of catalytically active enzymes.
Relevant studies have also made some progress. We will describe their applications
below based on their specific purposes.
Some researchers constructed enzyme-expressing LAB strains to regulate rele-
vant biochemical reactions in food manufacturing, thereby improving food attri-
butes such as flavor and texture. Yao et al. (2010) developed a recombinant L. lactis
NZ9000 strain, which carries a secreted expression plasmid inserted with the bovine
trypsin gene. Recombinant precursor protein with signal peptide was detected in the
protoplast fraction of recombinant bacteria. The recombinant bovine trypsin was
also shown to be biologically active. It avoids the biosafety risk from extracting
bovine trypsin from cow pancreas, strengthens the proteolytic systems in LAB, and
enhances bioactive peptide production during the manufacturing of fermented dairy
products, thereby boosting the potential health benefits of fermented dairy products
(Yao et al. 2010). Alpha-amylase has also been expressed in different cellular loca-
tions of genetically engineered LAB. Native α-amylase gene was introduced and
50 H. Chen et al.
expressed in two L. plantarum host strains (strain WCFS1 and a food-grade strain
TGL02) with a secretion efficacy of 90% (Kanpiengjai et al. 2015). Notably, the
properties of native wild-type α-amylase such as broad pH tolerability and maltose-
producing activity were also found in secreted α-amylase by the recombinant
TGL02 strain (Kanpiengjai et al. 2015). Furthermore, alpha-amylase was also effec-
tively expressed on the cell surface of L. casei by using the Bacillus subtilis anchor
protein PgsA (Narita et al. 2006). This α-amylase-displaying strain was shown to
exhibit strong hydrolytic ability on soluble starch and to hydrolyze 36.3 g/L of sol-
uble starch, yielding 21.8 g/L of lactic acid within 24 h (Narita et al. 2006).
Another application of enzyme-expressing recombinant LAB is to deliver recom-
binant enzymes with biocatalytic activities to host mucosa in order to modulate the
host immune functions. Furthermore, significant progress has been made in related
research in this field, which has been drawing great attention.
Oxidative stress is known to cause damage in cells or tissues, thereby triggering
inflammatory diseases and accelerating host aging. Antioxidants have been a popu-
lar research topic in multiple fields in recent years owing to their capabilities of
eliminating oxygen free radicals. Thus far, in the field of recombinant LAB-based
mucosal vaccines, LAB have been genetically engineered to express antioxidant
enzymes such as SOD and CAT, and their potential in managing gut inflammatory
disorders has also been demonstrated in animal studies. Carroll et al. (2007)
observed that genetically modified L. gasseri expressing Streptococcus thermophilus-
derived SOD significantly alleviated intestinal inflammation in IL-10-deficient
mice. Moreover, genetically engineered LAB (e.g., L. plantarum and L. casei) as
delivery vectors for SOD or CAT were shown to prevent or mitigate TNBS- or DSS-
induced gut inflammation in mice or rats (Rochat et al. 2007; Watterlot et al. 2010;
LeBlanc et al. 2011).
In addition to antioxidant enzymes, some researchers also developed recombi-
nant LAB as mucosal delivery vehicles for certain enzymes to improve clinical
symptoms induced by the deficiency of these enzymes. Drouault et al. (2002)
applied a Staphylococcus hyicus lipase-expressing L. lactis strain to improve pan-
creatic insufficiency-elicited defective lipid metabolism in pigs.
2.1.6.3 Recombinant LAB in Metabolic Regulation
In recent years, researchers have directionally modified or regulated certain meta-

bolic pathways in LAB using genetic and biochemical engineering techniques,
thereby altering the substrate utilization spectra of LAB, facilitating their substrate
utilization, and directionally augmenting the production of desired metabolites. To
a certain extent, these studies have greatly improved the production efficiency of
LAB in the food industry.
Diacetyl, a natural by-product during dairy and alcoholic fermentations, has
been widely exploited as a flavoring agent in the food industry due to its strong but-
tery flavor. LAB can utilize citric acid that is minorly present in milk to synthesize
an intermediate α-acetolactate (α-AL), which is subsequently converted to diacetyl
via oxidative decarboxylation. In order to enhance diacetyl production by LAB,

researchers have attempted to regulate the production of crucial enzymes involved
in diacetyl synthesis in LAB (Platteeuw and Hugenholtz 1995; Hugenholtz and
Kleerebezem 2000). A lactate dehydrogenase (LDH)-deficient L. lactis NZ2700
strain was genetically modified to overexpress α-ALS, achieving a high production
of the diacetyl analog acetoin with lactose as substrates. However, no high diacetyl
production was found by this strain. Afterward, Hugenholtz and Kleerebezem
(2000) constructed a genetically engineered α-AL decarboxylase (ALDB)-deficient
L. lactis strain overexpressing NADH oxidase. This recombinant strain cannot con-
vert α-AL to acetoin due to its α-ALDB deficiency. Moreover, high expression of
NADH oxidase in this strain imparts it efficient synthesis of diacetyl.
Apart from diacetyl, regulating L-alanine synthesis is another classic case of uti-
lizing genetic engineering tools for metabolic engineering in LAB. By genetically
modifying a L-LDH- and alanine racemase-deficient L. lactis strain to express the
alanine dehydrogenase (L-AlaDH) gene from B. sphaericus, Hols and Kleerebezem
(1999) successfully altered the carbon flux in the sugar metabolism of wild-type L.
lactis from homolactic fermentation to homoalanine fermentation. Besides, by using
the LDH promoter from S. thermophilus, the B.subtilis AlaDH was overexpressed in
recombinant L. lactis NZ9000, whose alanine production was upregulated 26 folds
as compared to the untransformed L. lactis strain (Ye et al. 2010).
To date, a large number of studies have suggested the beneficial effects of LAB-
produced exopolysaccharide (EPS) during milk fermentation on the hosts. EPS
from LAB have been substantially applied as thickeners in the food industry because
of its specific textural properties (Tong et al. 2015). However, limited production
levels of EPS in many LAB strains hinder their industrial application. Therefore,
some research teams have successfully applied genetic engineering tools to enhance
EPS production by LAB (Levander and Svensson 2002; Boels et al. 2003; Svensson
et al. 2005). By regulating the expression levels of key enzymes in the central path-
ways of carbohydrate metabolism, EPS expression levels were elevated in S. ther-
mophilus (Levander and Svensson 2002; Svensson et al. 2005).
2.2 Food-Grade Expression System for Lactic Acid Bacteria
2.2.1 Basic Requirements of Food-Grade Expression System
The food-grade expression system is an expression system that maximizes the pro-
duction of food and food-related products. Food-grade genetic expression system
must contain the following characteristics: first, genetic expression vectors in the
system are food-grade, consisting of DNA from known safe microbes and cannot
contain non-food-grade DNA fragments. The lactic acid bacteria (LAB) vectors that
are currently used generally carry one or more antibiotic (such as erythromycin,
chloramphenicol) resistance genes to maintain a certain selection pressure. However,
these resistance factors will drift, and if these carriers are used in daily life, they will
52 H. Chen et al.
have serious consequences for biosafety. Therefore, the use of food-grade selection
markers that are harmless to the human body in place of antibiotic resistance mark-
ers is one of the effective means to solve this problem. Second, the expression host
must be a safe, well-characterized, and stable food-grade microorganism.
Lactobacillus, Lactococcus lactis, and Bifidobacteria are generally regarded as safe
(GRAS) food-grade microorganisms. In addition, the host bacteria also need to be
stable enough in food or in the body. Third, the inducer used in the expression sys-
tem is also food-grade, such as sucrose, lactose, pyrimidine, nisin, etc.
2.2.2 Selective Marker of Food-Grade
Food-grade vectors of lactic acid bacteria require the vector is free of non-food-
grade functional fragments. According to the difference of screening methods,
selective marker of food-grade about LAB can be divided into two categories: com-
plementary selection markers and dominant selection markers. A complementary
selection marker requires a deletion mutation in the host chromosome, and then the
vector’s selective marker is used to compensate for the deletion mutation, thereby
restoring the host to a certain characteristic. Complementary markers often use
genes that encode important proteins involved in metabolic transformation. The
drawback of defective marker is that it can only be used for specific vectors – the
host system. Dominant selection markers are mainly used to provide new pheno-
typic characteristics by taking advantage of the characteristics of the host bacteria
themselves, and do not depend on the expression genes of the host. Therefore, such
markers can be applied to other bacteria of the same genus or even to other lactoba-
cillus. However, there are not enough food-grade dominant selection markers
applied at present, mainly due to the following: (1) the selective process is compli-
cated; and (2) sometimes the labeling system is too large because it needs to contain
several genes.
Selective markers of food-grade that have been applied to LAB can be classified
into four categories: saccharide utilization selective markers, auxotrophic
complementary selection markers, bacteriocin resistance markers, and heavy metal
resistance markers.
2.2.2.1 Saccharide Utilization Selective Markers
Sugar is an important raw material for industrial fermentation. Different LAB are
different in using the types and efficiencies of sugar. Most LAB generally cannot
use xylose, inulin, sucrose, and melibiose. Therefore, cloning genes associated with
the utilization of these sugars into non-fermenting strains give them the ability to
ferment a certain sugar.
The current research on lactose operons is the most in-depth. In the lactose
operon, the lacF gene encodes the key enzyme IIA of the lactose phosphotransfer-
ase system in L. lactis. First, the complete lactose operon was integrated into the
chromosome of L. lactis, and then the lacF deletion was made by double crossover
homologous recombination to construct a lac− type receptor strain. At the same
time, the LAB replicon pSH71, the promoter P32, and the lacF gene were used to
construct the vector pFI846. When a plasmid containing the lacF gene is introduced
into the lac− strain, the bacteria will restore the lac+ phenotype, and positive colo-
nies can be screened on plate medium containing bromocresol purple (Platteeuw
et al. 1996). Platteeuw et al. (1996) also constructed a food-grade vector by using
the lacF gene as a selective marker, which includes the lacA promoter and the tran-
scription terminator of the aminopeptidase N gene pep N. It enhances the stability
of the vector, and the gusA gene was successfully expressed using this vector.
Domestic Hesong et al. (2010) cloned the β-galactosidase gene from the L. aci-
dophilus genome and expressed it in L.lactis. Positive clones were screened by
5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal). The recombinant L.
lactis was passaged for 60 generations by X-gal color development screening
method, and the β-galactosidase enzyme activity and specific activity were mea-
sured. The results showed that positive clones could be successfully screened by
expressing active β-galactosidase and X-gal color development. The β-galactosidase
specific activity assay was performed on recombinant L. lactis after 60 generations
by X-gal. There was no significant difference compared with the second generation
of X-gal screening (P = 0.592 > 0.05) and no significant difference compared with
the erythromycin screening (P = 0.882 > 0.05). Thus, the β-galactosidase gene has
good activity and stability as a screening marker.
Posno and Heuvelmans (1991) cloned the xyl gene for xylose ferment of the L.
pentosus MD353 into the E. coli-lactobacillus shuttle vector pLP3537 to obtain a
recombinant pLP3537-xyl plasmid. Further, the plasmid was transferred to L. casei
ATCC393 which could not utilize xylose. As a result, the transformant obtained the
ability to utilize xylose, thereby obtaining xylose fermentation as a food-grade
selective marker of Lactobacillus.
2.2.2.2 Auxotrophic Complementary Selection Markers
The auxotrophic complementary selection marker is a commonly used method for

constructing food-grade expression systems. In bacteria, some products encoded
by genes can catalyze the basic metabolic reactions of bacteria. When these genes
are deleted or mutated, the bacteria will not be able to synthesize the corresponding
products, thus causing the bacteria to fail to grow normally in the original growth
environment. Only by supplementing the corresponding substrate will the bacteria
return to its original phenotype, which is the auxotrophy of the bacteria. These
genes are cloned into a plasmid and introduced into an auxotrophic strain, which is
complementary to the host bacteria, and the bacteria can restore a certain charac-
teristic. Therefore, you can choose according to this feature. Food-grade expres-
sion vectors have been successfully constructed with using the thyA, arl, thr, supB,
and supD genes.
54 H. Chen et al.
The thyA gene encodes a thymidylate synthase, which plays a key role in DNA
synthesis. If the gene is missing, the strain cannot grow on the basic medium. The
thyA gene is a safe food-grade selection marker that can be used to construct food-
grade expression systems. At the same time, the content of thymine or thymidine in
dairy products and the gut of animals (including humans) is very small, providing
favorable conditions for the use of such carriers. Ross and O’Gara (1990) first con-
structed a food-grade vector with the thyA gene as a screening marker. However,
since LAB lacking thy A gene were not constructed, the application of the gene in
LAB was limited. Wang Chunfeng et al. (2001) of China Agricultural University
obtained recombinant vectors by replacing the erythromycin resistance gene on
plasmid pW425e with thyA. At the same time, thyA-deficient Lactobacillus aci-
dophilus was screened as a recipient strain, and a food-grade carrier receptor system
with thyA as a screening marker was obtained. The Eimeria tenella SO7 gene was
introduced into the vector and expressed in LAB and E. coli. Xiong Yanwen et al.
(2004) constructed a vector pSH91 applied to L. lactis. With a total length of 2337
bases, it is a food-grade vector composed of selective marker thyA gene, replicator
of pWV01, and polyclonal site of plasmid pUC18. After transforming the vector
into thyA-deficient L. lactis, the host bacteria returned to the wild type.
Alanine is a structural component of the Gram-positive bacteria wall, and
D-alanine is an indispensable component of the bacterial cell wall peptidoglycan.
When it is absent, the cell wall synthesis is blocked, causing the bacteria to die.
D-Alanine is usually not contained in the raw materials of industrial fermentation,
and L-alanine in the medium needs to be converted into D-alanine. Alanine race-
mase catalyzes the conversion of D-alanine and L-alanine. Therefore, the alr gene
can serve as a food-grade complementary marker. Bron and Benchimol (2002) first
used homologous recombination to mutate the alr gene on the chromosome to con-
struct a mutant strain of the alr gene, providing a receptor strain. Bron and Benchimol
(2002) obtained a mutant strain of L. plantarum and L. lactis arl gene as a recipient
strain by using the deletion mutation. After introducing the vector of cloned alr gene
into the host bacteria, the two alr gene mutant host bacteria can grow on the medium
without D-alanine. Lu Wenwei (2014) established a selective marker for the alanine
racemase gene (alr). First, the alanine racemase gene of L. lactis NZ9000 and
L. casei BL23 was knocked out by the temperature-sensitive plasmid pG+host9 and
the suicide integration vector pRV300, respectively. The host cell itself became
D-Ala auxotrophy, and then the alanine racemase gene acts as a complementary
selection marker to achieve functional complementation.
In the process of prokaryotic ruthenium synthesis, if the related genes are mete-
orite mutation, the strain will be purine auxotrophy. The product encoded by the
nonsense suppressor gene supB can make up for the auxotrophy. Similarly, when
the gene in the pyrimidine synthesis pathway undergoes an amber mutation, the
nonsense suppressor gene supD gene product can compensate for this defect. Thus,
food-grade vectors can be constructed using supB and supD as selective markers.
Sorensen and Larsen (2000) constructed a new food-grade expression vector
pFG200 based on the vector pFG1. The pFG200 expression vector uses supD as a
selection marker and a pyrimidine auxotrophic strain as a host strain. Moreover, the
pFG200 vector has no significant effect on the growth rate of the host bacteria and
the acidification rate of the milk. However, a major drawback of such complemen-
tary systems is that specific mutations must be introduced into each mature host in
a food-grade manner prior to application of the complementary plasmid.
2.2.2.3 Bacteriocin Resistance Markers
Since bacteriocin is a food-grade product, its resistance genes and immune genes
can be used as food-grade selection markers. The bacteriocin resistance genes that
have been utilized now include the nisin resistance gene (nsr), the immune gene (nis
I), and the immunogenic gene of Lactobacillus F (laf I). A strain containing the
nisin resistance gene (nsr) or the immunogenic gene Nis I can grow normally on a
substrate containing a certain concentration of nisin. Therefore, the nisin resistance
gene or immune gene is an ideal food-grade selection marker. The nsr gene contains
an open reading frame of 957 bases that encodes a protein of 318 amino acids. Von
Wright et al. (1990) inserted a fragment containing the nsr gene in the cloning vec-
tor pVS34 and eliminated the chloramphenicol resistance gene originally carried in
the vector and constructed a food-grade vector with NSR as a selective marker.
Hughes and Mc Kay (1992) constructed a food-grade cloning vector pFM011 with
NSR as a selection marker and cloned a sequence encoding bacteriophage resis-
tance in this vector to obtain a plasmid with both nisin resistance and phage resis-
tance, named pFM012. The vector was introduced into L. lactis LM0230, which has
phage resistance. This indicates that the nsr gene can be used to directly screen
transformants, thereby replacing traditional antibiotic resistance markers.
The laf I gene is an immune gene of Lactobacillus F produced by L. johnsonii
VPI 11088. Studies have shown that if laf I is destroyed, the strain is sensitive to
Lactobacillus F. The vector pTRK434 carrying laf I was introduced into the
Lactobacillus F-sensitive strain Lactobacillus johnsonii. The host bacteria restored
the immunity of the bacteriocin and increased the immune tolerance by 64-fold
compared with the non-transformant cells. Allison and Klaenhammer (1996)
transformed the plasmid pTRK434 containing the laf I gene into the fermentative
lactobacillus NCDO 1750. The transformant was selected using a medium contain-
ing Lactobacillus F to achieve selective labeling using laf I.
2.2.2.4 Selection Markers for Heavy Metal Resistance
Some plasmids of LAB contain resistance genes of metal ions such as cadmium
(Cd) and copper (Cu), and food-grade carriers have been successfully constructed
using these resistance genes. Liu et al. (1996) isolated a copper resistance gene
(cuR) from L. lactis plasmid pND306 to construct a food-grade vector pND968.
Wong et al. (2003) linked the cadmium ion resistance genes cadA and cadC to the
S. thermophilus vector pND913 and removed the non-food-grade fragments to
56 H. Chen et al.
obtain the food-grade cloning vector pND919, which was successfully applied to
the food-grade expression of S. thermophilus.
In addition, a dual plasmid selection marker system has great potential for use in
food microbial applications and has been developed and utilized by researchers. The
system comprises two plasmids, one is a vector carrying a functional replicon and
the foreign gene to be expressed, but no selection marker, and the other is a con-
comitant plasmid, and the plasmid system with the antibiotic resistance selection
marker is also applied to the food-grade selectable marker of LAB. Emond et al.
(2001) successfully constructed a dual plasmid system in which two plasmids dis-
sociate the major antibiotic markers of the vector plasmid. The vector consists
entirely of L.lactis and complements the phenotypic characteristics of the associ-
ated plasmid. After transformation screening, the associated plasmid is readily
removed in antibiotic-free medium and remains highly stable without selective
pressure.
2.2.3 Food-Grade Inducer
In the process of cloning and expression of LAB, certain inducers are required. In
food expression systems, it is required that the inducer must be food-grade and
edible for humans, such as lactose, sucrose, nisin, and the like. Among them, nisin
belongs to the wool sulfur bacteriocin, which is produced by L. lactis. The mature
nisin molecule contains 34 amino acids with a molecular weight of 3510 Da. Nisin
is a polypeptide substance, which is non-toxic and does not produce antigens in
humans. After consumed, nisin can be inactivated by protease action in the digestive
tract, so it will not change the intestinal flora structure. Nisin has been accepted as
a food additive by the FAO/WHO expert committee in 1969 and is now used in
more than 50 countries around the world. These properties of nisin determine that it
can act as an inducer to induce the production of heterologous proteins in food-
grade expression systems without any toxic effects on the human body and thus is a
food-grade inducer. In addition to nisin, mutant derivatives of nisin and certain nisin
analogs can also be used as inducers to induce the nisA promoter and can even be
induced by the fermentation supernatant of nisin or its homologs or even nisin-
producing bacteria. The using concentration of nisin is significantly lower than its
minimum inhibitory concentration (MIC), ranging from 0.01 to 10 ng/mL. This
concentration does not inhibit the growth of microorganisms, even if the host does
not contain the NisI and NisFEG immune systems. In the process of inducing
expression, the inducing agent needs to be added in the log phase, and the expres-
sion level can be controlled within a power range of 1000 times, and there is a linear
dose-response relationship between the induced concentration and the protein pro-
duction level in this range. The highest production rate arrives 2 h after induction,
and the yield of the target protein can reach 60% of the soluble protein.
2.2.4 ood-Grade Expression System of LAB and Its

F
Application
2.2.4.1 NICE System
Eleven genes related to nisin biosynthesis are clustered into a DNA fragment about
14 kb in the order of nisA/Z, nisB, nisT, nisC, nisI, nisP, nisR, nisK, nisF, nisE, and
nisG, in which nisR and nisK are two components of the regulatory system and the
promoter nisA and nisF can be induced by nisin. In 1995, the Dutch Dairy Research
Institute (now NIZO Food Research Institute) invented the food-grade expression
system NICE (nisin-controlled gene expression system) by studying the self-
regulating biosynthesis mechanism of nisin. Based on the promoter nisA of the
nisin biosynthesis gene cluster and the two-component regulatory system gene
nisRK, this system regulates gene expression through the induction of nisin. A valid
nisin-induced NICE system consists of three parts: (1) Gram-positive bacteria con-
taining the nisRK gene as host bacteria; (2) nisin, nisin analogs, or nisin mutants as
inducers; (3) nisA or plasmid for the nisF promoter.
(1) The working mechanism of the NICE system
In the process of nisin autonomic regulation in NICE system, the histidine kinase
NisK acts as a sensor for nisin, and the NisR protein acts as a regulator of transcrip-
tion, which activates the transcription of the target gene. Once the extracellular nisin
exists, nisin binds to the receptor NisK. Subsequently, NisK transfers the phosphate
group to NisR by autophosphorylation, and the activated NisR induces the nisin
operon at the nisA promoter, which induces a regulatory process shown in Fig. 2.1.
Fig. 2.1 NICE system and its mechanism

58 H. Chen et al.
(2) Host
Gram-positive bacteria, including Lactobacillus, Streptococcus, Enterococcus,
Bacillus, Leuconostoc, and particularly any of the NisR and NisK proteins that can
express a certain level in Lactococcus can be used as a host for the NICE system. So
far, researchers have built two series of host cells: (1) L. lactis that can produce
nisin, such as L. lactis FI5876 and NZ9700; they are all modified from wild bacteria
by plasmid elimination or phage elimination. Other examples include that L. lactis
NZ9800 is obtained by reducing the four bases of the nisA gene; FI7332 integrates
the erythromycin resistance gene into the nisA gene of FI5876. (2) Host cells that
cannot produce nisin. These host cells integrate the nisRK gene into the genome,
such as the commonly used host strains L. lactis NZ900 and L. lactis NZ3900. For
nisin-producing bacteria, nisin not only induces its own production but also induces
the expression of its target gene. For non-nisin-producing bacteria, nisin only
induces the expression of the target gene. In addition to integrating nisK and nisR
into the genome, the researchers designed multiple plasmids such as pNZ9520 and
pNZ9530 to apply the NICE system to other hosts based on the plasmid pAMβ1 for
wide host applications.
(3) Plasmid vector
The plasmid vector requires a promoter comprising nisA or nisF for nisin-induced
expression, and, to date, more than 20 related plasmid vectors have been constructed
for expression of Gram-positive bacteria, even E. coli. Commonly used plasmid vec-
tors are divided into five categories: One type is a transcriptional fusion vector, such
as the common pNZ8020. The vector comprises a promoter of nisA, a NisR binding
site, and the ribosome binding site. The polyclonal site is located behind the pro-
moter region, and the gene expression needs its own initiation codon. The second
type is a translational fusion vector, such as the commonly used pNZ8048. After the
Nco I restriction endonuclease is inserted into the promoter region, since it contains
ATG as a starting site, the inserted target gene can achieve high transcription effi-
ciency and ultimately guarantee the expression level of target protein. Studies have
shown that the use of this type of fusion vector to express β-glucosidase, its expres-
sion activity is six times higher than that of the transcriptional fusion vector. In the
third mode, the promoter of Nisin A and the target gene were ligated in vitro and
then cloned into the target vector together. The vector was used to successfully
express germicidin PA-1 and Escherichia coli V. The fourth mode is a two-plasmid
NICE system consisting of two compatible plasmids: one plasmid carries the nisRK
regulatory gene and the other carries the target gene under the control of the nisA
promoter. Its inducibility is the same as in L. lactis, but the time to reach the highest
protein production level is significantly longer than L. lactis. The fifth mode is a vec-
tor for secretion expression, such as pNZ8110, which uses a signal peptide secreting
the protein Usp45 for secretion expression of the protein of interest.
(4) Application of NICE system
The NICE system has the characteristics of high-efficiency expression and the
value of practical application. It can be applied to the high-efficiency expression of
important proteins in biotechnology, such as bacteria, viruses and eukaryotic anti-

gens, cytokines, bacteriocins, and membrane proteins in various LAB. In the cellu-
lar metabolic pathway, it is also possible to precisely modulate the activity of key
enzymes and control the production of metabolites of interest.
1. Overexpression of protein
Using the NICE system, proteins from different sources can be overexpressed to
help people study the properties of proteins or enzymes. At the same time, because
it is derived from Gram-positive bacteria, there is no inclusion body when express-
ing proteins with close relationship. For example, de Ruyter and Kuipers (1996)
used the NICE system to homologously express the pepN gene, and its protein
expression reached 50% of that of soluble cells, and no inclusion bodies were
produced.
2. Expression of membrane protein
Under normal physiological conditions, the natural expression level of mem-
brane proteins is relatively low, and a large number of its heterologous expression
has become a restrictive condition for the study of function and structure. Combined
with the difficulty of purification and crystallization of membrane proteins, heter-
ologous expression of membrane proteins has become a bottleneck in membrane
protein-related research due to its hydrophobic properties and toxicity to host cells.
The NICE system has certain advantages for overexpressing membrane proteins:
(1) the proteolytic ability of L. lactis is weak; (2) the nisA promoter is a strong and
rigorous promoter, which can make the expression of membrane proteins under
reasonable control; and (3) the expressed membrane protein can be dissolved by a
surfactant. The membrane proteins of many prokaryotic cells have been expressed
by the NICE system, such as ABC transporter, MFS transporter, peptide transporter,
etc., and the expression level can reach 10% of membrane protein. Bernaudat and
Frelet-Barrand (2011) expressed several eukaryotic membrane proteins derived
from Arabidopsis thaliana in L.lactis, including polycopper oxidase and naphtho-
quinone oxidoreductase. Some scholars have tried to promote the expression of
eukaryotic membrane proteins through systematic transformation, such as the addi-
tion of fusion proteins, but the related research is generally relatively difficult and
lagging.
3. Secretory protein expression and surface display system
Food-grade lactobacillus is used in the production of protein in industrial fer-
mentation but is also a suitable candidate for the delivery of heterologous proteins
in food or the digestive tract. Antigens, vaccines, drugs, and the like can be expressed
in LAB by secreting protein expression and surface display systems. Enouf et al.
(2001) expressed the recombinant immunogen protein NSP4 using the NICE sys-
tem. The results showed that the NICE system can produce rNSP4 protein with
antigen and immunogenic properties and has the same immunogenic properties as
viral proteins. Bermudez-Humaran and Langella (2002) used this system to pro-
duce IL-12. Experiments show that the activity of IL-12 produced by the system is
60 H. Chen et al.
similar to that of commercial IL-12, which can be used in mass production and
in vivo application of IL-12.
(1) Other bacteriocin-induced food-grade expression systems
In addition to the NICE system, some other LAB have a similar mechanism for
bacteriocin production. But unlike the NICE system, the main function of peptide
secretion is not the bacteriocins but the expression of the whole operating unit stim-
ulated by external hormones. Sorvig et al. found a bacterial production operon in L.
sakei and used it to construct a series of expression vectors. β-Glucuronidase and
aminopeptidase were used as expression genes, respectively, and the expression
effects of different vectors were compared. It was found that using pSH71 vector
and sakacin P promoter, and using sakacin P as the inducer, the expression level of
proglucuronidase was ten times that of the wild-type strain. However, the expres-
sion level of aminopeptidase accounted for half of the intracellular soluble protein
(Sorvig and Gronqvist 2003; Sorvig and Mathiesen 2005). Nguyen and Nguyen
(2012a, b) induced the expression of β-galactosidase (LacZ) of L. bulgaricus DSM
20081 in L. plantarum using the plasmids pSIP403 and pSIP409 constructed above.
By adding the corresponding inducing peptide IP-673, the expression level of LacZ
reached 53,000 U/L, accounting for 63% of intracellular soluble protein. In the
same year, the author used these systems to express the chitosanase of B. lichenifor-
mis in L. plantarum with an expression level of 5 mg recombinant protein per liter
of medium. Therefore, the above system is very valuable for the expression of LAB,
particularly the expression of Lactobacillus proteins.
(2) Sugar-induced food-grade expression system
Through genome-wide sequencing of a large number of LAB, it can be known
that the LAB genome contains a plurality of sugar metabolism-related gene clusters,
which can ensure that LAB grow on a medium containing various sugars as a car-
bon source. In the sugar-induced expression system of lactobacillus, the sugar selec-
tive marker is mainly used. As a food-grade inducer, sugar can be used as a marker
for non-antibiotic resistance. Since the lactose operon is well studied, most
expression systems use lactose as a selective marker to induce gene expression.
Many lactose-induced food-grade expression systems were constructed by lacA
promoter. Eaton et al. (1993) introduced the reported gene luxAB behind the pro-
moter lacA and expressed L.lactis as the host bacteria. When lactose was used as the
carbon source, the activity of the promoter was increased by seven times. Platteeuw
et al. (1996) constructed a vector containing the lac A promoter with the lacF gene
as a selection marker and inserted a transcription terminator of pep N into the vector
to increase the stability of the vector and cloned DNA. The β-glucuronidase gene
(gusA) was cloned downstream of the lac A promoter and introduced into the
plasmid-free L. lactis NZ3000 receptor. The strain could be grown in lactose-
containing medium, and the gus A gene was highly expressed with lactose induc-
tion. The food-grade plasmids pLEB590 and pLEB600 with L casei as host bacteria
and pSH71 replicator and repA gene as the skeleton, respectively, carrying P45 and
PpepR promoter, and screening with lactose selection markers. The proline imino-
peptidase gene pep I was successfully expressed with these two plasmids (Takala
and Saris 2002). Payne and MacCormick (1996) first inserted the lactose operon
into the chromosome of L. lactis MG5267 and then integrated the lysin gene derived
from Listeria phage LM-4 into the lac promoter, and its expression was controlled
by the lac promoter. And ultimately the expression of the LM-4 lysin gene is con-
trolled by lactose.
In addition, there are carbon sources such as xylose sucrose as selection markers.
Using D-xylose as a selective marker, the e-lactobacillus lactobacillus shuttle vector
pLP3537 was cloned and expressed the xylose reductase gene cluster xyl on the
chromosome of L. pentosaceus, which enabled the recombinant lactobacillus to
have the ability to utilize D-xylose. The possibility of using xylose as a selective
marker was confirmed. Lokman et al. (1991) linked the gene of chloramphenicol
acetyltransferase to the downstream of xylose promoter and introduced it into
Lactobacillus pentosaceus, which were cultured in the medium containing xylose
and glucose, respectively. The results showed that the activity of chloramphenicol
acetyltransferase was 60~80 times that of glucose culture under the induction of
xylose. Xylose-inducible expression system (XIES) is a sugar-dependent expres-
sion system. By using xylose-inducible promoter PxylT, intracellular or secreted
proteins can be produced. The first use of this expression system was the heterolo-
gous expression of the S. aureus nuclease gene (intracellular and extracellular
expression using the signal peptide of the Usp45 protein) in L. lactis NCDO2118.
When the system is induced with an appropriate inducer, such as xylose or glucose,
a large amount of the target protein can be expressed. The system can be a good
complement to the NICE system, as its induction is tightly regulated. At the same
time, when the system is used for protein secretion expression, the secreted protein
is not observed to decompose (de Azevedo and Karczewski 2012).
Leenhouts and Bolhuis (1998) constructed a food-grade expression system with
sucrose as the selective marker by the integration of lactobacillus. The gene scrA/
scrB encoding sucrose and hydrolase was cloned into lactococcal plasmid pWV01.
By using the copy of this plasmid, lactobacillus expression vector pINT124
pINT125 with sucrose as the screening marker was successfully constructed.
Mahmoud and Sameh (2011) used this expression vector to express the bacteriocin
gene pctA in L. lactis.
(3) Zinc-induced expression system
Llull and Poquet (2004) designed a new expression system PZNZitR-driven het-
erogeneous expression system based on the promoter of zit operator. The zit operon
is primarily involved in zinc regulation. When zinc is absent, such as when the cell
is starving, the operator’s transcriptional repressor leaves its repressor site, and
RNA polymerase can bind to the PZN promoter to induce subsequent protein
expression. By expressing two target proteins, it was shown that this expression
system is highly inducible, but the amount of protein obtained is lower than that of
the NICE system.
Recently, a new zinc-induced expression system, Zirex, can induce protein
expression in L. lactis. The promoter is induced by zinc with a concentration below
62 H. Chen et al.
toxicity, and the SczA repressor regulates the activation of PczcD and leads to the
induced expression of subsequent fluorescent proteins. In addition, the NICE sys-
tem and the Zilex system are combined to produce different proteins at different
times in one microorganism.
(4) Environmentally induced food-grade expression system
When LAB express proteins in vitro, they can induce protein expression by add-
ing specific inducers. However, in vivo, the relevant inducers cannot be supple-
mented to the microorganisms at the appropriate concentration to achieve the
induced concentration. To compensate for this deficiency, researchers have succes-
sively studied the stress-inducible expression system (SICE) and P170, which
induce the expression of specific proteins in the gut.
Benbouziane and Ribelles (2013) found a stress-inducible expression system in
L. lactis and studied it. The system is controlled by the pGroESL promoter and
expresses the target protein only under specific stress conditions such as high salt,
high temperature, and low pH. In addition, the system has great potential for the
expression and transport of vaccine proteins at specific sites in the body for the
treatment of inflammatory bowel disease (IBD) and human papillomavirus.
Another major feature of LAB is the production of lactic acid during growth.
Based on this feature, a pH-induced expression system can be constructed. Madsen
et al. (1999) found that the promoter P170 regulated by pH value was induced in the
culture environment with a pH value of 6.5~6.0 and the bacteria entering the stable
period. Through genetic manipulation, the deletion of 72 bases at the front end of
P170 mrna does not affect the promoter regulation, but increases the pH induction
effect of the promoter by 150 times and the gene expression level accordingly.
Temperature is also an important factor influencing protein expression. Nauta
et al. (1997) designed a thermo-unstable Rro repressor variant Rrol2 using lac Z as
a reporter gene to construct a temperature-controlled expression system. When the
temperature was increased from 24 to 42 °C, the amount of β-glucosidase expres-
sion was increased by 500 times.
2.3 Gene Knockout System in Lactic Acid Bacteria
2.3.1 he Mechanisms and Characteristics of Gene Knockout

T
Vector
A gene knockout is a genetic technique in which foreign gene is inserted into site-
specific position, and its purpose is to make the target gene have a directed change.
This technique conquered the defect of random mutation (blindness and contin-
gency) and is an ideal method for genetically modified organisms. Compared with
other organisms, the development of gene knockout in LAB is relatively low speed
owing to thicker cell wall, low conversion rate, and less vectors. The gene knockout
technique can be used to modulate the metabolic responses, decrease ferment cost,
and enhance the production and purity of the aimed product. Moreover, it could be
used to study the structure and function of genes. So far, the most common ways
used in gene knockout depend on a traditional homologous recombination, and a
few studies adopt means of site-specific recombination, homologous recombination
involving single-stranded DNA substrate, and CRISPR/Cas system.
2.3.1.1 Gene Knockout Based on Homologous Recombination
The theoretical basis of classic gene knockout technique is based on homologous

recombination. Homologous recombination is a type of genetic recombination in
which nucleotide sequences are exchanged between two similar or identical mole-
cules of DNA. Based on the above theory, the targeted gene deletion, insertion, and
missense mutation could be reached via homologous recombination between a
recombinant vector and host genome in the host cell.
Considering the difference in the number of homologous recombination, the
gene knockout can be simply divided into two types: one-time homologous recom-
bination and twice homologous recombination. Leenhouts and Bolhuis (1998)
showed that the targeted gene of L. lactis was inactivated via insertion of foreign
gene by the single-crossover integration system. In addition, prolyl dipeptidyl ami-
nopeptidase gene was inactivated in the chromosomal of L. helveticus via recombi-
nation between the pepXP-derived repeat (Bhowmik and Steele 1993). Compared
with one-time homologous recombination, two recombination reactions were
observed in the latter, and it caused two different results, in which the targeted gene
was completely deleted in one result and the gene was disrupted in other. Ferain
et al. (1996) demonstrated that two lactate dehydrogenases played a major impact
on peptidoglycan precursor synthesis in L. plantarum via two recombination reac-
tions. L. citreum is an important lactic acid bacterium in fermented foods, but dex-
tran production often causes undesired ropiness. To prevent this side effect, a
dextransucrase knockout mutant was constructed by Jin and Li (2014), and it indi-
cated that L. citreum dextransucrase not only synthesizes dextran for cell protection
but also provides fructose as an important carbon source for cell growth.
2.3.2 ene Knockout Based on Improved Homologous

G
Recombination
2.3.2.1 omologous Recombination with a Counterselectable Selection
H
Marker
Homologous recombination can be used for gene disruption, but gene targeting is
inefficient because of low homologous recombination frequency in the secondary
reaction, which leads to some difficulty in screening the mutants. To solve the above
64 H. Chen et al.
Plasmid
Up Down
Up gene X Down
First homologous recombination
Up Down Up gene X Down

Second homologous recombination
Up Down Up Down
h h
Up gene X Down Up gene X Down
Up gene X Down Up Down

wild-type strain gene X mutant strain
Fig. 2.2 Gene deletion through two-hybrid system
problem, the use of a reasonable counterselectable selection marker is helpful in

screening the correct mutant (Fig. 2.2). It means that the mutant containing this
counterselectable selection marker can be died in an exact selection condition, and
the mutant without this selection marker is able to survive in the same condition.
The use of selection marker could reach the aim of rapid screening of exact mutant
without introducing new gene.
The fourth intermediate in the pathway was identified to be orotate, which was
also confirmed that it could not be utilized as a pyrimidine source by most L. lactic
strains due to lack of the special transporter (Kilstrup, Hammer et al. 2005).
Furthermore, the gene responsible for utilizing orotate as a sole pyrimidine source
by an auxotrophic mutant has been already identified to be oroP, which also lead to
its sensitivity to its toxic analog 5-fluoroorotate. Based on the above property, a new
selection vector pCS1966 was constructed by Solem and Defoor (2008), considered
as an efficient tool for special strain construction, which was proper for sequence-
specific integration based on homologous recombination and also that in a bacterio-
phage attachment site.
In addition, another counterselectable selection marker gene that was always
used in LAB is the upp gene that encodes uracil phosphoribosyltransferase results
in resistance to 5-fluorouracil (Martinussen and Hammer 1994). The main draw-
backs of using the upp is that this gene is found in almost every organism and that
5-fluorouracil may be toxic even in a upp mutant (Martinussen and Hammer 1995).
Previous study showed that the upp gene deletion did not affect pyrimidine metabo-
lism and biological characteristics. In 2009, a 3.0-kb pOR-based counterselectable
integration vector, which beared a upp expression cassette, called pTRK935, was
constructed by Goh et al., and this tool was identified to be very valuable to deter-
mine how L. acidophilus performed its probiotic function (Goh and Azcarate-Peril
2009). Furthermore, a temperature-sensitive replicon called pWV01 was also

adopted by Song and Cui (2014) in order to construct two integration plasmids with
chloramphenicol-resistant using upp gene as a counterselective marker for L. casei
ATCC393 and L. lactis MG1363. Many results have showed that no significant dif-
ference existed between the wild-type and mutant lactic acid bacteria except for
5-FU resistance through determining the genetic stability, growth curve, carbon uti-
lization, and also scanning electronic microscopy.
Although the upp gene was widely used in the screening of resistant strains,
some disadvantages still exist in the gene knockout process. Because the upp gene
widely exists in some bacteria and may participate in some important metabolic
responses, the upp gene deletion in the host cell could cause some influence in the
growth.
2.3.2.2 omologous Recombination with a Temperature-Sensitive Type

H
System
The replicon of a thermosensitive plasmid is a thermosensitive replicon, which can

make the plasmid replicate at lower temperature and shut off at elevated tempera-
ture. This genetic system for homologous recombination allows the thermosensitive
plasmid massive replication at lower temperature and increases the transformation
frequency. Maguin et al. (1992) isolated a replication-thermosensitive mutant
pVE6002 and used it for the construction of a thermosensitive plasmid. The result
showed that its transposition frequencies were about 1%, which were higher than
wild type. Biswas et al. (1993) constructed a thermosensitive broad host range
rolling-circle plasmid, pG+ host5, which contains a pBR322 replicon for propaga-
tion in E. coli at 37 °C, and developed a protocol for gene replacement. Cultures
were first maintained at 37 °C to select for a bacterial population enriched for plas-
mid integrants; activation of the integrated rolling-circle plasmid by a temperature
shift to 28 °C resulted in efficient plasmid excision by homologous recombination
and replacement of a chromosomal gene by the plasmid-carried modified copy.
These results show that gene replacement can be obtained at an extremely high
efficiency by making use of the thermosensitive rolling-circle nature of the delivery
vector. Atiles et al. (2000) disrupted IlvE activity in L. lactis LM0230 via a thermo-
sensitive plasmid, indicating that IlvE is the only enzyme capable of synthesis of Ile
and Val from their biosynthetic precursors. To study the role of propanediol dehy-
drogenases pdh30 during glycerol metabolism, the gene disruption mutant L. brevis
INIA ESI38::pORI28-pdh30 was constructed by Langa and Arques (2015) by site-
specific integration of plasmid pORI28 and the temperature-sensitive plasmid
pVE6007. HPLC analysis of the glycerol fermentation products showed an involve-
ment of the pdh30 in the 3-hydroxypropionic acid (3-HP) biosynthesis. The
temperature-sensitive plasmid is a valuable tool in the gene knockout system, and it
could be used in some Gram-positive bacteria. However, this genetic system is
unstable, and its high-temperature condition also limits its application in LAB.
66 H. Chen et al.
2.3.3 ene Knockout System Based on Site-Specific

G
Recombination
It is known for us that site-specific recombination possesses significant conservative

characteristics, and this recombination would occur in these DNA strands with seg-
ments possessing at least a certain degree of sequence homology. A recombinase
enzyme and also the corresponding recombination sites are enough for some certain
systems to carry out all the reactions; however, many accessory proteins and/or the
corresponding sites would be necessary. Cre/loxp and TP901–1/att family are con-
sidered as the two most common site-specific systems according to the amino acid
sequence homology and mechanistic relatedness.
2.3.3.1 cre/loxp Recombination System
cre/loxp recombination system just included a single enzyme, called cre recombi-
nase, recombining a pair of short sequences, also known as lox sequence, in which
the enzyme and the lox site are separated from bacteriophage P1. There are 4 sub-
units and 2 domains (C-terminal and N-terminal domains) among Cre protein with
343 amino acids in total. The structure of C-terminal domain is similar to that of
integrase family protein in lambda phage (Sternberg and Hamilton 1981). LoxP is a
special site on bacteriophage P1, which consisted 34 bp, including two sets of 13-bp
symmetric sequences with asymmetric 8-bp sequence in the middle of the sequence,
in which the symmetric 13-bp structure is palindromic and the middle 8-bp sequence
is not. This special structure provided a certain direction for loxP. Generally, the
loxP site existed in pairs when genetic manipulation was performed. The behavior
of floxed sequence depended on the orientation of loxP sites, while it will be excised
with loxP site in the same direction and it will be inverted with loxP sites in the
opposite orientation. The Cre recombinase can not only recognize wild LoxP
sequence but also recognize the mutant LoxP sequence in which a set of symmetric
13-bp sequences or asymmetric 8-bp sequences have slight changes, which further
expand its application range. Lambert and Bongers (2007) constructed an effective
mutagenesis vector that contains Cre/Lox cassette and disrupted bile salt hydrolase
bsh gene and α-galactosidase melA gene in L. plantarum WCFS1. Remus and
Kranenburg (2012) studied the effect of four clusters of genes on surface polysac-
charide production by using the Cre/LoxP system.
The role of selection marker gene is easy to screen the exact mutants, but its
existence could also cause some unnecessary changes in the expression of its
upstream and downstream genes in the genome. Considering the characteristics of
the Cre/LoxP system, it is always used to remove the market gene in the genome.
Zhu and Zhao (2014) constructed the thymidylate synthase gene (thyA)-deficient
strain derived from L. lactis NZ9000 using the Cre-loxP recombination system and
used it as a food-grade selection marker for L. lactis in food and industry
applications.
2.3.3.2 TP901-1/att Recombination System
TP901-1/att recombination system can stably replicate, and site-specific integrate

their DNA into the host chromosome, which is constructed by integrating TP901-1,
lactococcal temperate bacteriophage, into the chromosome of Lactococcus subspe-
cies. The phage attachment site (attP) and the chromosomal attachment site (attB)
can be integrated into new hybrid sites: attL and attR. Zhu and Zhao (2014) used the
integration elements encoded by the temperate lactococcal bacteriophage TP901-1
to obtain chromosomal single-copy transcriptional fusions in L. lactis. A genetic
tool special for repetitive, marker-free, and site-specific integration was constructed
by Petersen and Martinussen (2013) in L. lactis. In this tool, a vector with nonrepli-
cating plasmid, called pKV6, included a phage attP, useful to be integrated into a
bacterial attB. i pKV6 would be integrated into the chromosome of the host with
being flanked by attL and attR hybrid attachment sites in high frequency just when
the corresponding vector was transformed into L. lactis with the ability to express
the phage TP901-1. loxP recombinants would be selected based on the 5-fluo-
roorotic acid just when a plasmid responsible for expressing cre recombinase lacks
the replicating ability in L. lactis. The gene involved in controlling xylose utilization
was usefully adopted to be integrated into the chromosome of L. lactis strain MG
1363 in two steps in order to determine whether the constructed system would per-
form its function.
2.3.3.3 Linear Transformation Knockout System
Linear DNA, containing the mutated or deleted gene flanked by homologous regions
of the chromosome, is transformed into recombination-proficient strains, which is
one of the hot spots on study of gene knockout method. It’ s reported that a recom-
binant E. coli strain was constructed through replacing the RecBCD function with
phageλ’s Red function via recombining the chromosome with short linear DNA
fragments at a greatly elevated rate by Murphy (1998), with at least 70-fold higher
than that by arecBC, sbcBC, or recD strain. The rate is at least 70-fold higher than
that exhibited by arecBC sbcBC or recD strain. Compared with double crossover
homologous recombination, the homologous arm of the above method is about
35~50 bp, and it has a high recombination efficiency. However, this method is
always used in Gram-negative bacteria, and few studies were found in LAB.
Recently, single-stranded DNA recombination is always used in the gene knock-
out system in LAB. The single-stranded DNA binding proteins produced by redβ
and redT gene are always used as recombinases that can promote annealing of com-
plementary DNA strands. Van Pijkeren and Britton (2012) constructed the plasmid
pJP042 and pJP005 to assess the ability of the L. reuteri RecT protein to support
recombineering. It showed that the intrinsic vancomycin resistance of L. reuteri
was significantly lower in the mutations, and the minimum inhibitory concentration
of vancomycin was reduced from >256 to 1.5 mu g/mL by creating a single AA
change in the d-Ala-d-Ala ligase enzyme. Compared with classic double crossover
68 H. Chen et al.
homologous recombination, ssDNA recombineering exhibits more advantages,

such as easy operation, high recombination efficiency, not restricted by restriction
enzyme cutting site, shorter homologous arms, and acquisition by direct PCR.
2.3.4 New Gene Knockout Method
With the development of molecular biology, some new gene knockout methods
have attracted lots of attention, such as RNA interference, transcription activator-
like effector nuclease technique, zinc finger nuclease gene targeting technique,
CRISPR/Cas system, and so on. Among them, CRISPR/Cas system could be used
in the gene knockout of lactic acid bacteria in the future. The exact mechanism of
CRISPR/Cas system is that CRISPR-derived RNA form a compound of tracrRNA/
crRNA with trans-activating RNA, which guides the Cas 9 nuclease cut the DNA
target specified by the guide RNA. By designing the above two RNA, it can form a
leading indicator short guide RNA that guide Cas nuclease cut the targeted DNA. As
a RNA-oriented dsDNA binding protein, Cas9 nuclease is one of the first known
unifying factors that collectively recognize RNA, DNA, and protein. The com-
pounds of protein and Cas9 nuclease-null can specifically bind any DNA sequence
together with moderate amount of sgRNA. The terminal of sgRNA can bind with
the target DNA without affecting the combination between Cas9 and the target
DNA. Therefore, Cas9 can deliver any compounds of protein and RNA to any DNA
sequence (Liu and Xu 2015).
CRISPR-Cas9 selection system has been identified to be the most ideal system
used to edit some genes in lactic acid bacterium for its high efficiency (Oh and van
Pijkeren 2014), in which the efficiency in recovering subtle changes in the genome
would range from 90% to 100%. CRISPR-Cas9 has been successfully used in
recombineering, for example, the codon saturation mutagenesis in L. reuteri chro-
mosome and also identification of such low-efficiency events as oligonucleotide-
mediated chromosome deletion. CRISPR-Cas 9 would be also useful in identifying
the recombinant bacterial cells with low recombineering efficiency and also elimi-
nating the need for ssDNA recombineering optimization procedures.
2.3.5 The Common Vectors and Their Application
The gene knockout can be used to modulate the metabolic flux, interfere with the
production of unnecessary secondary metabolism, reduce ferment cost, and raise
the production and purity of target product. In addition, it can also be used to study
the structure and function of genome of LAB and its beneficial mechanisms.
Increasing reports have focused on the peptidoglycan-degrading enzyme involved
in a range of bacterial processes and also the interaction between the host and the
microbe; however, the function of this enzyme in lactobacilli is still unknown.
Systematic gene deletion system has been adopted by Rolain and Bernard (2012) in
exploring the functional role of the peptidoglycan hydrolase (PGH) complement
located in the genome of L. plantarum. The role of N-acetylglucosaminidase Acm2
and NplC/P60 D, L-endopeptidase LytA, as key determinants in the morphology of
L. plantarum has been shown in that study. eps gene cluster has been reported in L.
johnsonii requiring for the biosynthesis of homopolymeric exopolysaccharides
(EPS)-1 and heteropolymeric EPS-2 which was useful in constructing a capsular
layer. epsA is the first gene of the cluster with putative function as transcriptional
regulator, and the function has been identified by the result that deletion of epsA
gene would result in complete loss of the ability of growing the EPS-1 and EPS-2 on
the cell surface, and this ability would be fully restored just when this gene was
complemented. All these results showed that epsA gene could regulate the EPS pro-
duction positively. Reutericyclin is a unique antimicrobial tetramic acid produced by
some strains of L. reuteri, but its synthesized mechanism is still unclear. Lin and
Lohans (2015) showed that deletions of rtcNRPS or rtcPhlABC in L. reuteri
TMW1.656 abrogated reutericyclin production but did not affect reutericyclin resis-
tance. To raise the production of diacetyl in LAB, Platteeuw and Hugenholtz (1995)
cloned the als gene for alpha-acetolactate synthase of L. lactis MG1363 in a multi-
copy plasmid under the control of the inducible L. lactis lacA promoter. Furthermore,
the effect of alpha-acetolactate synthase overproduction on the formation of end
products in various L. lactis strains was also determined under different fermenta-
tion conditions. These metabolic engineering studies suggest that more than 80% of
the lactose can be converted via the activity of the overproduced alpha-acetolactate
synthase in L. lactis.
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Chapter 3
Comparative Genomic Analyses of Lactic
Acid Bacteria
Wei Chen and Hongchao Wang
3.1 Interspecific Evolution of the Lactobacillus Genome
Genetic diversity is the sum of the genetic variation between different species and
within the same population. The genetic information of an organism is stored in the
DNA sequences of chromosomes and organelle genomes. Lactobacillus keeps its
traits stable by accurately duplicating its DNA and passing on the genetic informa-
tion generation by generation. However, many factors can affect the accuracy of
DNA replication, including external factors and internal factors, which together
lead to different levels of genetic variation. These accumulated genetic variations
enrich the genetic diversity. Genetic polymorphism is not only the result of evolu-
tion, but also the premise of evolution.
3.1.1 volution of Lactobacillus Based on Comparison of 16S

E
rRNA Genes
In bacterial classification, 16S rDNA sequence analysis is a necessary and most

commonly used identification and analysis method, and is now the most basic detec-
tion index for studying bacterial evolution. Prokaryotes mainly contain 23S rRNA,
16s rRNA and 5S rRNA, which contain about 2900 bases, 1540 bases and 120 bases
respectively. Since the 16S rRNA sequence is the most conserved, it has become the
most fundamental molecular indicator for the study of bacterial classification, iden-
tification and evolution. Besides highly conserved regions, 16S rRNA sequences
also contain relatively variable regions, and the homology of conserved regions is
W. Chen (*) · H. Wang (*)

e-mail: [email protected]; [email protected]

https://doi.org/10.1007/978-981-13-7832-4_3
78 W. Chen and H. Wang
the basis to determine the classification status of bacteria. This foundation reflects
the relationship between bacteria, while the specific sequences of the variable
regions reveal the differences between bacteria. By comparing the highly conserved
and relatively variable 16S rDNA sequences, the taxonomic position of bacteria and
the evolutionary relationship between bacteria can be determined. Different bacte-
rial classification results in changes in the sequence of the 16s rRNA variable region,
but the constant region is basically conserved, so this method can not only reflect the
differences between different species, but also make it relatively easy to obtain the
sequence by sequencing technology, which has been accepted by scientists. After
isolation, purification and culture of lactic acid bacteria, total DNA and ribosomal
RNA were extracted. Then16S rDNA sequence was amplified by polymerase chain
reaction (PCR) through probe hybridization or 16S rRNA specific primer.
The sequence information of 16S rDNA gene was compared with the sequence
data in the database to determine its position in the evolutionary tree, and then the
species of lactobacillus was identified. However, the public database contains public
and unpublished sequence information (Olsen et al. 1991; Cole et al. 2009), also
contains a lot of poor quality and inaccurate sequence information, and even incor-
rect comments, which will have a negative impact on the application of the method.
Since the 1880 s, 16 s rRNA sequence analysis as the reference of bacterial taxon-
omy was gradually applied to classification and classification greatly influenced the
professional term. The variable region analysis of 16S rRNA sequences has been
widely used in the classification study of lactobacillus, which has led to the classifi-
cation of some undefined taxonomic groups based on phenotypic characteristic sys-
tems, and also found many homologous mutant lactobacillus groups (Holzapfel
et al. 2001). Zheng Huajun (2010) constructed a phylogenetic tree of 11 strains of 9
strains of lactobacillus, and found that the 16S rRNA sequence could not distin-
guish the sequences of different strains between the same lactobacillus, and all spe-
cies were divided into 3 groups of lactobacillus, lactobacillus and streptococcus
(Zheng Huajun 2010). Due to differences in mutation rate and gene level transfer,
differences in 16S rRNA gene sequences cannot accurately reflect the phylogenetic
relationship (Bakker et al. 2007). So scientists hope to reconstruct phylogenetic
trees by encoding gene sequences.
3.1.2 volution of Lactobacillus Based on Butler Gene

E
Comparison
In past studies, scientists have used protein-coding genes as phylogenetic markers to

classify and evolve lactobacilli. Single-locus sequence analysis (SLSA) of protein-
coding genes can obtain higher taxonomic resolution and analysis speed is higher
than 16S rRNA gene analysis. Steward-type gene sequence analysis is an important
component of lactobacillus SLSA method (Bruyne et al. 2009; Ehrmann et al.
2009), which is superior to 16S rRNA gene sequence analysis in terms of taxonomic
3 Comparative Genomic Analyses of Lactic Acid Bacteria 79
resolution. When the genetic sequence of sodA(manganese superoxide dismutase)

was analyzed, the Streptococcus infantarius subsp. Coli in the genus Streptococcus
was reclassified as a new species Streptococcus lutetiensis, while other groups of
Streptococcus pasteurianus (Poyart et al. 2002) were proposed. However, subse-
quent studies by Schlegel et al. (2000) proved that neither streptococcus Paris nor
parabiosa were new species. Obviously, the difference between the inside and out-
side of the protein-coding gene can identify the phylogenetic relationship between
the individual species and the strain. However, the level of gene transfer will change
the development of the system, so there are many defects in the analysis of indi-
vidual genes. (Konstantinidis et al. 2006). At least five housekeeping genes from
different chromosomal loci and widely distributed in taxonomic groups are ana-
lyzed to provide sufficient information for identification of bacterial species from
related groups (Stackebrandt et al. 2002). Zeigler (2003) argued that the “set” type
prediction of candidate genes is a limited data set. He also suggested that the five
genes actually corresponded to or even exceeded the DNA hybridization capacity
required by the distribution-related bacterial strains for other species (Zeigler 2003).
3.1.3 volution of Lactobacillus Based on Whole-Genome

E
Comparison
The phylogenetic relationships of lactobacillus which are analyzed by single or sev-

eral genes cannot accurately reflect the taxonomic evolution. The study of genomics
based on whole genome sequence analysis can lay a solid foundation for the study
of lactobacillus classification and evolution relationship. (Makarova et al. (2006a)
for the first time conducted comparative genomics study on 9 different strains of
lactobacillus by using the method of interspecific comparative genomics analysis,
and found that the lactobacillus showed gene acquisition and deletion in the evolu-
tionary process. When lactobacillus is in a nutrient-rich environment, the protein-
coding genes related to nutrient substance metabolism in the genome will be missing
in different circumstances (Makarova et al. 2006a). It is because of the continuous
development of this mutation that the nutritional deficient strain is finally formed.
Gao (2014), Zhong (2015) compared 138 strains of Streptococcus pneumoniae gene
groups to illustrate the evolution of different kinds of streptococcus. Phylogenetic
trees were constructed based on the protein sequences encoded by 278 core genes.
The results showed that two genetic lineages existed in the evolution process of
streptococcus, and the evolution path of each group was the same as that of genus.
Zheng Huajun (2010) found that the three genera of lactobacillus, lactobacillus and
streptococcus had similar classification conditions in the evolutionary tree and 16S
rRNA sequence tree, but their topological structures were significantly different. At
the species level, the differentiation of the three genera was no longer as clear as that
of the 16S rRNA sequence tree. And the evolutionary tree based on the method of
comparative genomics shows that there are significant differences between different
strains from the same subspecies (Zheng Huajun 2010). In traditional research, sci-
entists use 16S rRNA sequences to construct phylogenetic trees to understand the
evolutionary relationships between lactic acid bacteria. However, the 16S rRNA
gene sequence analysis is difficult to distinguish lactobacillus which is closely
related to evolution. The evolution of lactobacillus can be explained by studying the
number of variation at genomic level through phylogenetic analysis. The 16S rRNA
system development tree is the basic research method to understand the basic evo-
lutionary relationship between lactic acid bacteria. Genomic phylogenetic tree and
16S rRNA phylogenetic tree are considered as references and supplements. Sun
et al. (2015) constructed a phylogenetic tree by comparing the protein nucleic acid
sequences of 16 strains of lactic acid bacteria with 452 genera of lactic acid bacteria
in the phylum, in which the evolution of all model strains can be divided into two
large branches. The results showed that lactobacillus genus is a paraphyletic taxa
(paraphyletic), which evolved continuously by a ancestors. In other words, the fac-
ultative heterozygous lactobacillus was differentiated into obligate homozygous
and heterozygous fermentation to adapt to its living environment. At the same time,
the progenitor of this genus was the lactobacillus mode strain belonging to faculta-
tive heterozygous fermentation, while the progenitor was the lactobacillus mode
strain belonging to strictly homozygous fermentation and strictly heterotypic fer-
mentation. The animal lactobacillus in the first branch of evolution had niche move-
ment, while the animal lactobacillus in the second branch of evolution had parallel
evolution in the animal habitat and other habitats (Zhong Zhi 2015). Zhong Zhi
et al. (2015) constructed a comparative genomic phylogenetic tree based on the
analysis of 37 strains of Enterococcus. By comparing and analyzing the phyloge-
netic tree of 16S rRNA sequences, the whole topology is more similar, but there are
large differences in deep branches. Based on the core genome, the guiding value of
the branch nodes of most phylogenetic trees is 100%, and the phylogenetic tree can
more accurately reflect the phylogenetic position of the strain. The analysis also
revealed the relationship between evolution and the environment in which entero-
cocci may have first been hosted by plants and birds, then passed on to mammals,
and accelerated evolution in the environment (Zhong Zhi 2015). The differences in
the branches of the enterococcus genome show that the differentiation of enterococ-
cus is accompanied by different levels of gene transfer. Strains in the younger
branch had larger genomes and more genes than those in the older branch, suggest-
ing that gene acquisition may have dominated the evolution of the strain, and that its
environment at the time may have determined the direction of evolution (Zhong Zhi
2015). The above results indicate that genome-wide sequence analysis can truly
reflect the phylogenetic relationship and taxonomic status of strains, and is a power-
ful tool for studying the classification and evolution of lactic acid bacteria.
3.2 The Microevolution About the Lactic Acid Bacteria
Microevolution focus on character impacted by gene and genovariation influenced

by selective pressure, which is the evolution of the same or similar species and a
process that caused intraspecific differentiation (Song et al. 2015). Microevolution
includes gene mutation, gene recombination, gene transfer and natural selection
(Siezen et al. 2008). Gene mutations include nucleotide substitution, transcriptional
deletion, recombination, and gene conversion, which are the absolute driving forces
of evolution. Genetic recombination can't change the frequency of allele, but it can
increase inheritance change in a certain extent. Genetic shift pay a important role in
fixation of mutants with neutral mutations in small populations. The positive selec-
tion of natural selection weed out the genovariation, however the balance seleciton
can increase the genovariation (Siezen et al. 2008).The heritable variation and heri-
table basis is the research emphasis in biology, that can retrospect the rule of their
development process. The analysis of microevolution research the phylogeny and
the evolution process in molecular level, it is the heritable basis which can reveal the
biological function and the evolutionary mechanism about lactic acid bacteria,
which offer the theoretical basis which can guide how to apply the lactic acid bac-
teria among the food industry.
3.2.1 I ntraspecies Evolution of Lactic Acid Bacteria Based

on DNA Fingerprinting
We only can research the microevolution of the lactic acid bacteria with the aid of
the conserved sequence dut to they are prokaryote. We can explain the possible
microevolution process about the lactic acid bacteria by the distribution of the vari-
ant population, which rely on the difference population structure of different strain
by the high-resolution mark. There are many DNA finger-print analysis technology
and analysis methods base on DNA sequence which can reveal the change
ofunknown population structure , such as randomly amplified polymorphic DNA,
RAPD (Solieri and Giudici 2010), restriction fragment length polymorphism, RFLP
(Yu et al. 2011), amplified fragment length polymorphism, AFLP (Tanigawa and
Watanabe 2011) and pulsed-field gel slectrophoresis, PFGE etc. (Picozzi et al. 2010;
Sawadogo-Lingani et al. 2007).
RAPD is the molecular marker technology which can classify and authenticate
unknown strain, which it adopt random primer and as a template base on genome
DNA and get DNA fragment by PCR and analysis the finger-print produced by gel
electrophores (Doria et al. 2013). This method is convenient and efficient and
already proceed the classification and evolution research about lactobacillus and
Bifidobacterium successfully. RFLP and fluorescence labeling often use jointly, it
detect the size of DNA fragment which is Restriction Enzyme Digestion DNA
amplify product, but the recognition and cutting site of restriction enzyme will
change because of replacement、deficiency and recombination among the individ-
ual allele, which can cause the difference length of restriction fragment among the
different genotype (Bokulich and Mills 2012). This method has stable analysis
result and can be used for the classification and recognition of a bulk of samples,
however it's consumption of time and labor is more and has lower sensitivity because
the transportation of cytomembrane and the Southern hybridization is necessary for
RFLP. AFLP is a kind of molecular marker based on PCR. At present has been
widely used in construction of genetic map, genetic diversity analysis, important
gene location, molecular marker assisted breeding and classification of biological
systems, etc. (Pothakos et al. 2014). Its drawback is that the cost is higher, and
stronger technical, because its are dominant marker, is not conducive to analysis of
population genetic structure. PFGE technique separable 10 KB ~ 10 MB of large
molecules of DNA, its principle is by changing electric field direction, make the
DNA molecules in the agarose gel swimming party to subsequently corresponding
change, small molecules with DNA electric field conversion can quickly change the
direction of movement, although large molecules and DNA in pulse field for use
under the will to move, but more difficult in the gel. Different sizes of DNA mole-
cules in gel will present different mobility, DNA band spectrum to a certain extent,
reflects the DNA content and the molecular size, can be used as the basis for clas-
sification. PFGE is a kind of important for separation of oita sub quality linear DNA
electrophoresis, is currently the polymorphism of lactic acid bacteria research one
of the widely used methods. PFGE stable results, high resolution, good repeatability
and easy standardization etc, and therefore are bacteria molecular classification of
the “gold standard” (Gonzalez-Arenzana et al. 2014). The ideal DNA molecular
typing technology has the following characteristics: the genome contains multiple
polymorphic sites and has a characteristic distribution; it can generate multiple
independent and reliable molecular markers; high resolution, simple, fast, economi-
cal and efficient; DNA samples Less demand; correlation with phenotypic charac-
teristics; no need for genomic DNA sequence information (Zhou Ning 2012).
Although there are many methods for taxonomic identification and polymorphism
research, no molecular typing technology can fully satisfy the taxonomic identifica-
tion of all lactic acid bacteria due to the difference of genomic information of lactic
acid bacteria and genotyping techniques in genome abundance, polymorphism
detection, specific loci, repeatability and technical requirements. Therefore, scien-
tists need to determine the research method according to the purpose of the
experiment.
Yang Jixia (2013) obtained the fingerprints of 39 strains of bacteria from yak
milk by RAPD technology. The clustering of strains showed that the strains of dif-
ferent strains clustered into one or more groups. Meanwhile, it also shows that there
is a certain correlation between strains and regions. (Yang Jixia 2013) For example,
the strains from Yunnan and Tibet are mostly distributed in a group, while the strains
from Xinjiang are far away. This may be related to their geographical location, and
also reflects that there is a certain relationship between strains and their original
origin. Psoni et al. (2007) analyzed the diversity of 40 Lactococcus lactis strains
isolated from Greek goat cheese by PFGE, and compared them with RAPD and
plasmid typing. The results showed that the 40 strains were abundant in polymor-
phism, which were consistent with those obtained by RAPD and plasmid typing
(Zhou Ning 2012). Dimitrov et al. (2008) identified and analyzed the polymorphism
of lactic acid bacteria isolated from healthy human body by AFLP, PFGE and
RAPD. The results show that the RAPD technology is the easiest and quickest to
operate, but the resolution is general. PFGE method is more stable and has high
resolution, but it takes a long time. AFLP technology has the highest resolution in
strain typing, relatively simple operation, and strong stability and resolution (Zhou
Ning 2012). Because the above techniques are not practical in identification of
closely related strains, limited by laboratory conditions and difficult to achieve data
exchange and comparison, a more convenient, reliable and higher resolution method
is needed for identification of closely related strains.
Genomic short sequences, distributed on different sites of bacterial genome and
separated at different distances, present differences in strain and species levels. Rep-
PCR takes the bacterial genome DNA as template, and uses genomic short sequence
as primer to carry out PCR, amplifying DNA sequences among repeated sequences.
After gel electrophoresis, the amplified products can form a series of bands, that is,
DNA fingerprint. The fingerprints of different strains of bacteria or subspecies are
specific, so the identification and diversity of bacteria can be studied (Zhou Ning
2012). Rep-PCR is more and more widely used in the classification, identification
and polymorphism of lactic acid bacteria because of its simple operation, high reso-
lution, economy, rapidity and applicability. de Urraza et al. (2000) identified 37
thermophilic lactic acid bacteria isolated from dairy products by Rep-PCR and ana-
lyzed their diversity. Adıguzel et al. (2009) used Rep-PCR to analyze 76 strains of
lactic acid bacteria isolated from fermented sausages in Turkey and identified them
at the level of species and strains. Gao Yang (2013) used Rep-PCR to analyze 24
strains of lactic acid bacteria in the intestines of cold water fish from different
sources and found that the fingerprints of low-temperature resistant lactic acid bac-
teria produced more bands, which can reflect the differences in genome levels of
different strains. The dendrogram of fingerprint cluster analysis showed that all
strains could be divided into six groups at 70% similarity level. At the same time,
the genetic differences between Lactococcus and Enterococcus at species and strain
level could be clearly seen from the dendrogram (Gao Yang 2013).

on DNA Sequence Differences
In order to study bacterial genotypes and microevolution more deeply, a series of

methods based on DNA sequence difference analysis technology emerged as the
times require. The 16S rRNA intergenic spacer region(ISR) (Sun 2006) based on a
single gene and the nucleotide sequence analysis based on partial fragments of
multiple housekeeping genes were early used for genotyping of lactic acid bacteria
(Yu et al. 2012), such as multiple-locus variable-number tandem-repeat
analysis(MLVA) (Chiou et al. 2010) and multilocus sequence typing (MLST)
(Picozzi et al. 2010; Maiden et al. 2013). MLST, once the most mature and stan-
dardized analytical method, has been widely used in the research of genetic diver-
sity, population structure and micro-evolution of lactic acid bacteria because of its
high resolution, simplicity, and reproducibility.
Maiden et al. (1998) improved the multilocus enzyme electrophoresis (MLEE)
to obtain a new molecular typing method, MLST. By studying 11 housekeeping
genes of 107 strains of Neisseria meningitidis, they analyzed about 470 base pairs
of each gene, and compared the diversity of alleles. At the same time, they took each
group of different alleles as a genotype to construct allele profile. Finally, the phy-
logenetic tree was constructed by clustering analysis, and it was found that each
unique genotype corresponded to a sequence typing ST (Maiden et al. 1998). MLST
technology can be used to compare ST to find correlations between different strains,
and strains with high correlation have the same ST or ST with only a few individual
loci and STs of unrelated strains differ in at least 3 or more loci (Taylor et al. 1999).
MLST technology can also be used to analyze the genetic relationship between
strains from different sources, especially to monitor gene recombination events
between different populations. Therefore, it can make up for the shortcomings of
traditional typing methods when analyzing the evolution relationship of strains and
judging isolates. With the rapid development and wide application of sequencing
technology, MLST technology is being widely used in laboratories all over the
world (Maiden et al. 1998). The technology can be standardized, the results are easy
to verify and preserve, and the genetic information of strains can be transmitted
through the Internet.
The selection of housekeeping genes is the key to the multi-site sequence typing
method. The housekeeping genes that exist in almost all prokaryotes are highly
conserved, and there will be differences between the strains. Konstantinidis et al.
(2006) confirmed that 3 genes were the least number to be used in MLSA to predict
gene horizontal transfer (Konstantinidis et al. 2006). Rivas et al. (2004) selected five
housekeeping genes from 18 different strains of Leuconostoc cerevisiae and used
MLST technology for multi-site sequence typing analysis for the first time. The
results showed that MLST could effectively identify Leuconostoc alcoholicus iso-
lates and gene recombination was an important reason for the increase of genetic
diversity of Leuconostoc alcoholicus (De et al. 2005). Cai et al. (2007) analyzed the
alleles of 40 Lactobacillus casei strains by MLST technology. The results showed
that the specific living environment caused the specific genetic evolution of the
strain, and the genetic characteristics were closely related to the living environment.
At the same time, it was pointed out that the evolution process of lactic acid bacteria
under the action of different natural environments was completely traceable
(Tanganurat et al. 2009). Calmin et al. (2008) studied five genes (recA,rplB, pyrG,
leuS and mle) of Pediococcus parvulus and Pediococcus damnosus in wine using
MLST technology and found that MLST effectively completed the molecular typ-
ing of these two cocci, which laid the foundation for screening wine starter(Calmin
et al. 2008). In 2008, scientists used MLST technology to study Lactobacillus

plantarum isolated from fermented fruits and vegetables, and completed analysis of
genetic diversity and strain evolution, and found that carbon plays an important role
in the evolution of L. plantarum (Tanganurat et al. 2009).
Diancourt et al. (2007) studied the population structure of Lactobacillus casei
using MLST technology. Seven housekeeping genes were selected to distinguish 12
alleles and 31 ST types. Only ST1 appeared in 17 Lactobacillus casei, and each of
the other strains represented 1 ST type alone. This indicated that MLST technology
was effective in typing Lactobacillus casei (Diancourt et al. 2007). Dan et al. (2014)
used MLST technology to analyze the population structure of 50 strains of L. lactis
isolated from natural fermented dairy products from China and Mongolia. The
results showed that there was a lateral transfer of genes in Leuconostoc cerevisiae
and two subgroups with high similarity were formed (Tong et al. 2013). Sun Zhihong
(2014) selected eight housekeeping genes of 305 Lactobacillus deltoides Bulgaria
subspecies for MLST study, which were divided into 121 ST types and consisted of
14 homologous complexes (Sun Zhihong 2014). Five possible major ancestor
groups were predicted, and it was inferred that the Bulgarian subspecies of
Lactobacillus delbrueckii isolated from Mongolia were closer to the earliest ances-
tor group. At the same time, the progenitor strain of Lactobacillus delbrueckii
Bulgaria subspecies was the closest to Mongolian isolates, showing a trend of
spreading from Mongolia to Russia and then to China, which indicated that differ-
ent sequence types are directly related to their isolation sources and isolation sites
(Zhong Zhi 2015).
Delorme et al. (2009) applied MLST technology to illustrate the evolution of
Streptococcus thermophilus, and found that the relationship between S. thermophi-
lus and Streptococcus salivarius was the closest. The genetic relationship between
the strains of S. thermophilus was analyzed and it was found that ST3 is the most
primitive ST type, which is the ancestral type. It has also been found that some
genes have metastasized and lost during the evolution of S. thermophilus (Delorme
et al. 2009). Yu Jie (2013) performed MLST analysis on 10 target genes in 260
strains of Streptococcus thermophilus isolated from different countries and found
that these strains were divided into 119 ST types, with the largest number of ST-29
strains, followed by ST-57 and ST-106. Six strains isolated from Russia were
divided into four different ST types, 106 strains of Streptococcus thermophilus iso-
lated from China were divided into 50 different ST types, and 148 strains of
Streptococcus thermophilus isolated from Mongolia were divided into 71 different
ST type. The ST types of Streptococcus thermophilus isolates isolated from differ-
ent regions are different. The number of alleles of 10 target genes is 7–17, which
may be the result of natural environment selection (Yu Jie 2013). There was a sig-
nificant linkage disequilibrium in these strain populations, and there was no genetic
recombination between the alleles of the regional strains. Isolation of strains from
the same region will form corresponding clonal complexes, indicating that the evo-
lution of Streptococcus thermophilus in different environments is entirely different.
The main factors affecting evolution are environmental climate and geographical
location, and the milk system may not be a major factor.
Therefore, MLST technology is a powerful tool to study the genetic evolution of

lactic acid bacteria. However, because MLST site carry very limited genetic
information and the genetic evolution relationship revealed by MLST loci is limited
by population structure and population information, it is difficult to analyze
microevolution.

on Genome Resequencing
Due to the continuous development of high-throughput sequencing technology,

DNA sequence difference analysis technology based on genome-wide information
has been continuously applied to the study of microevolution of bacterial popula-
tions. This technology has the advantages of low cost, high throughput and short
time-consuming, which provides a strong support for the study of microevolution of
lactic acid bacteria (Li et al. 2009). Genomic sequencing of different individuals for
species of known genomic sequences is called genomic resequencing technology,
which analyzes genetic evolution by detecting differences in genes or structures of
populations or individuals. The principle of this technique is to find single nucleo-
tide polymorphic loci, insertion/deletion loci, structural variation loci and copy
number variation by comparing the known genome sequences with the results of
re-sequencing, so as to find genetic differences to analyze genetic evolution. The
continuous completion of lactic acid bacteria genome sequencing not only clarifies
the biological metabolic pathway, but also provides a rich reference sequence for
studying the evolution of lactic acid bacteria at the genome-wide level. More and
more scientists are planning to study the species differentiation and population
inheritance of lactic acid bacteria at the genome-wide level. Whole genome sequenc-
ing can comprehensively analyze the genetic information of the genome and sys-
tematically clarify the complexity and diversity of the genome, which can provide
powerful technical support for the study of micro-evolution of lactic acid bacteria.
Cai et al. (2009a) used the Lactobacillus casei ATCC 334 genome as a reference
for comparative genomic hybridization analysis of 21 genomes of Lactobacillus
casei isolated from human, plant and cheese from different regions. The analysis
showed that the Lactobacillus casei strain isolated from different environments had
a special functional gene compared with the reference strain, indicating that the
strain had different degrees of gene insertion/deletion when adapting to the environ-
ment, and this result coincided with MLST analysis. The same results not only
confirmed the accuracy of MLST technology, but also proved that genome rese-
quencing is an effective technical means to study the micro-evolution of lactic acid
bacteria (Cai et al. 2009a). Smokvina et al. (2013) completed the genome re-
sequencing of 34 strains of Lactobacillus casei with reference to the Lactobacillus
casei Zhang genome sequence, and systematically analyzed the differences in sugar
metabolism between different strains of Lactobacillus casei. The results showed
that Lactobacillus casei contains multiple phosphoenolpyruvate transport systems

(PTS), which allows these strains to utilize more sugars and adapt to different
environments. It was also found that Lactobacillus casei isolates from milk source
encoded relatively few PTS, which may be due to the adaptation of Lactobacillus
casei to the nutritional environment of milk and the abandonment of part of the
transport system that had not been used for a long time in the evolution process,
indicating that Lactobacillus casei has a oriented micro-evolutionary process in
order to adapt to different environments (Smokvina et al. 2013). The above results
fully confirm that the genome re-sequencing technology can completely reflect the
genetic information of the genome, so as to fully reveal the complexity and diversity
of the genome, analyze the inheritance and evolution of the lactic acid bacteria
population. In addition, genome re-sequencing technology can also provide a pow-
erful technical means for clarifying the microevolution of lactic acid bacteria and
revealing the influence of environment on the evolution of lactic acid bacteria.
The study of microevolution of bacteria can deeply analyze the influence of envi-
ronment on the evolution of microbial population. The genetic background and evo-
lution of lactic acid bacteria are the primary basis for the research and development
of lactic acid bacteria and their application in the food industry. By studying the
micro-evolution of lactic acid bacteria, we can confirm the relatives and evolution-
ary relationship among various genus of lactic acid bacteria, which is conducive to
the reconstruction of phylogenetic tree, and provide a solid theoretical basis for
establishing a more accurate and rapid classification and identification system of
lactic acid bacteria. Although scientists have been discovering the probiotic proper-
ties of lactic acid bacteria in recent years, many unknown functions of lactic acid
bacteria still need to be explored urgently. Therefore, the study of the microevolu-
tion mechanism of lactic acid bacteria can provide molecular support for the analy-
sis of the formation and evolution of its biological functions. In-depth discovery of
key genes with probiotic characteristics and basic research on related genes can
further exploit the resources of lactic acid bacteria.
Among the methods described above, the 16S rRNA gene sequence of a single
gene cannot accurately reflect the evolution of lactic acid bacteria in nature. Nucleic
acid sequence analysis based on multiple housekeeping loci has many advantages,
but it is limited to individual loci in the whole genome, which will miss important
genetic information and result in more errors between the constructed population
structure and the real structure. Scholars can analyze the structure and composition
of Lactobacillus genome by exploring the complete genome data in depth, thus
providing new ideas for studying the evolution mechanism of lactobacillus.
3.3 Core Genome and Pan Genome of Lactic Acid Bacteria
With the development of microbial genome sequencing technology, new genes are
always found in different strains of the same strain after genome sequencing.
Therefore, the whole genetic information of a strain cannot be represented by the
genome information of a single strain. Medini et al. 2005 put forward the concept of
core genome (Medini et al. 2005), which called the genes existing in all strains as
the core genome of the strain. Pan genome refers to the entire genome of a species,
including the core genome (Lefebure and Stanhope 2007). The core genome is
essential to maintain the basic survival and phenotype of the strain, which retained
in evolution because of the high selective pressure. The diversity of pan-genome can
explain the difference of genome genetic information among different strains (Wang
Yajun 2012). Bacteria with low pan-genomic diversity live in stable environments,
while bacteria with high pan-genomic diversity have strong adaptability to the envi-
ronment. According to the genome, genes and the degree of variability are different.
Pan genomes and core genomes are commonly used to identify gene families rather
than single genes. Once a genome is added, the gene family is redefined and the
number of gene families in the core genome and pangenome is recalculated.
3.3.1 The Core Genome and Pan Genome of Lactobacillus
Cai et al. (2009b) analyzed the microarray hybridization of Lactobacillus casei

ATCC344, which indicated that 1941 core genes existed in 2678 predicted genes of
all 21 strains analyzed (Cai et al. 2009b). The comparative genome analysis of 11
strains of 9 Lactobacillus species showed that it was difficult to identify homology
at the nucleic acid level, so it was necessary to compare them at the protein level.
When a protein whose matching length exceeds 40% of the minimum protein length
and whose homology is also over 40% is defined as a homologous protein, the num-
ber of core genes decreased gradually with the increase of strains. Only 529 core
genes were found in 11 strains of Lactobacillus, mainly in energy metabolism,
transport and binding protein, transcription, cell process, replication and protein
synthesis (Zheng Huajun 2010). The core genome and pangenome analysis of 66
Lactobacillus showed that there were 16 663 gene families and 365 gene families in
the pangenome and core genome of 37 Lactobacillus genus, respectively. When the
genome of Lactococcus was increased, the number of pangenomes increased greatly
(18,232), because the increased Lactococcus gene did not exist in Lactococcus,
while the core genome decreased substantially. The pangenome of 66 Lactobacillus
genomes contains 29,247 gene families, while the core genome contains only 261
gene families. Lactobacillus is a large genus, containing a wide variety of species,and
the difference between core genome and pan genome can be used as a criterion for
determining intraspecific differences (Schillinger and Endo 2014). For Lactobacillus,
comparative genomic analysis of 37 Lactobacillus strains from 17 species showed
that the difference was 16,298 gene families (16 663 pangenomes subtracted 365
core genomes) (Schillinger and Endo 2014), which was much larger than the differ-
ence observed in 27 genomic analysis of 7 Vibrio strains (Vesth et al. 2010). Sun
et al. (2015) sequenced the genome of 149 Lactobacillus model strains and obtained
the accurate genome map of the strain. The core gene family was determined based
on the principle that the amino acid similarity of coding proteins was more than
50% and it was found that the genome of each model strain encoded an average of
2200 genes, and the core genome contained 72 core genes which encoded proteins
related to cell-based growth and replication. The pangenome of these strains is
expanding with the increase of genome number, which contains more than 70,000
genes, while the core genome is decreasing with the increase of genome number
(Sun et al. 2015). (Siezen et al. (2010) compared the microarray data of 42 strains
of fermented food with Lactobacillus plantarum WCFS1,discovering that the core
genome of 2049 genes existed in all strains and 121 genes had no homology com-
pared with other sequenced lactic acid bacteria (Siezen et al. 2010).Through com-
parative genomics analysis of Lactobacillus bulgaricus, it was found that the 265 KB
gene sequence was unique to 2038 strains of genome. Among the three strains, 1301
genes are highly homologous at the nucleic acid level, which is the core gene of
Lactobacillus bulgaricus, while the pangenome contains 2199 genes.112 genes in
the core genome are identical in nucleic acid sequence, 214 genes only have syn-
onymous mutations in coding region, and the remaining genes have differences in
coding protein level. The ancestors of Lactobacillus bulgaricus may have about
2200 genes, losing 15–30% in the course of evolution and the main source of
genomic variability is the slow spread of core genes (Zheng Huajun 2010). Makarova
and Koonin (2007) identified the core genomes of 12 sequenced genomes of
Lactobacillus with 567 gene clusters in the direct homologous group. Major genes
in the core genome are associated with replication, transcription and translation, but
about 100 genes have not been identified. An interesting phenomenon is that there
are no two core genomes that are directly homologous outside the genus
Lactobacillus. One of these unique genes encodes proteins that contain the peptido-
glycan-binding LysM domain, and the other contains no identified domain, located
in the conserved genome region and containing two enzymes associated with tRNA
4-thiourea nucleoside modification (Makarova and Koonin 2007).
3.3.2 The Core Genome and Pan Genome of Enterococcus
Enterococci are normal constituents of the gastrointestinal flora of humans and

other animals (Willems and van Schaik 2009; Willems et al. 2011). Enterococcus
faecalis is the most common enterococcal species recovered from infections. Data
show that from 1990 to 2010, infections with E. faecalis have been on the rise in the
United States, Europe, and South America (Boyd et al. 2002; Leavis et al. 2006;
Hidron et al. 2009; Willems and van Schaik 2009). The comparative genomic analy-
sis of Enterococcus from different sources showed that their genomic sequences
were quite different. The genomic differences of Enterococcus strains could be ana-
lyzed by studying the pan-genome and core genome.
Enterococcus faecium has a large genome, strong adaptability to the environ-
ment, and easy to obtain the sequence of foreign genes. Scientists have found that
only about 50% of the genes in the Enterococcus genome belong to the core genome
and share it with other genomes. This indicates that there are more specific genes in
E. faecium strains from different sources, while the other 50% genes are included in
the pangenome, which also indicates that the genome of E. faecium from different
sources is more diverse. Wang Yajun (2012) made comparative genomics analysis
of 28 strains of E. faecium,discovering that the pangenome of E. faecium increased
with the addition of new strains and new gene families were added to the pange-
nome of each new strain.The pan genome and core genome contain 5743 genes and
1440 genes respectively.Only about half of the genes in E. faecium genome belong
to the core genome, which indicates that there are more specific genes in
Enterococcus faecium strains from different sources (Wang Yajun 2012). Qin et al.
(2012) studied the comparative genome of 22 strains of E. faecium and found that
the pangenome and core genome contained 3169 genes and 1652 genes, respec-
tively (Qin et al. 2012). The estimated number of core genomes is similar to that
reported by Leavis et al. (2007) by microarray method (Leavis et al. 2007). Zhong
Zhi (2015) conducted a comparative genomics study on 37 Enterococcus sp. strains
in order to analyze the genetic relationship and species evolution of Enterococcus.
The pan genome of Enterococcus contains 29,545 genes, and the core genome con-
tains 605 genes.Enterococcus genome contains an average of 3062 genes, with only
20% of the core genome and 28% of the smallest Enterococcus genome, indicating
that the diversity of Enterococcus genome is very strong.Most of the genes in the
core genome are essential for maintaining life and reproduction, and are essential
components of the Enterococcal genome (Zhong Zhi 2015). In addition to the core
genome and pan-genome, some strain-specific genes have been found in the genome
of Enterococcus, which only exist in one strain of Enterococcus. Through compara-
tive analysis of 37 strains of Enterococcus, 368 strain-specific genes were found,
with an average of less than 10 per strain. Compared with the large pan-genome, the
strain-specific gene is only 1% of the pan-genome, indicating that although the
genome of Enterococcus is more complex and diverse, the gene exchange between
species within the genus is much more than that between strains within the genus
and those outside the genus (Zhong Zhi 2015).
3.3.3 The Core Genome and Pan Genome of Streptococcus
The human small-intestinal microbiota is characterised by relatively large and

dynamic Streptococcus populations. Lefebure and Stanhope (2007) analyzed the
pangenome of 1898 genes and the core genome of 1487 genes of Streptococcus
thermophilus based on two completed genome sequences and one preliminary com-
pleted genome sequence (Lefebure and Stanhope 2007). The comparison of all
complete and preliminary genomic sequences of Streptococcus showed that the
pangenome of Streptococcus could exceed 6000 genes. Rasmussen et al. (2008)
analyzed three complete genome sequences and gene information on GenBank, and
constructed a pangenome of more than 2200 genes for S.thermophilus. By molecu-
lar hybridization of DNA from 47 strains, the core genomes of 1271 genes can be
identified. However, these data suggest that adding additional strains to the analysis
will reduce the number of genes in the core genome (Rasmussen et al. 2008).
Tettelin et al. (2005) sequenced the whole genome of 8 strains of Streptococcus
lactis and found that there was a core genome in 1800 genes, and the pangenome of
each strain increased to a critical value of about 33 genes (Tettelin et al. 2005).
Similar data were also found in Streptococcus pyogenes. Specific aspects of the
environmental interaction-potential and the metabolic capacity of 6 small-intestinal
Streptococcus strains were investigated through analysis of their genome sequences.
The results of classification of isolates showed that three different strains of
Streptococcus mutans were isolated from one ileal sample, which belonged to
Streptococcus mitis group, Streptococcus bovis group and Streptococcus salivarius
group. Compared with 58 strains of Streptococcus pneumoniae in the public data-
base, the pangenome and core genome of S.pneumoniae were composed of 12,403
genes and 574 genes, respectively.
3.3.4 The Core Genome and Pan Genome of Bifidobacterium
Bifidobacteria are commonly found as part of the microbiota in the human gastroin-
testinal tract (GIT) (Ventura et al. 2007), where their presence has been positively
correlated with the health status of their host (Ventura et al. 2009a, b). Bifidobacteria
have been claimed to elicit several health-promoting or probiotic effects, such as
strengthening of the intestinal barrier, modulation of the immune response and
exclusion of pathogens (Marco et al. 2006; O’Hara and Shanahan 2007). Whole-
genome sequencing efforts have revolutionized the study of bifidobacterial genetics
and physiology. Unfortunately, the sequence of a single genome does not provide
information on bifidobacterial genetic diversity and on how genetic variability sup-
ports improved adaptation of these bacteria to the environment of the human gastro-
intestinal tract (GIT). Lukjancenko et al. (2012) completed the comparative genome
analysis of nine Bifidobacterium strains, and determined that the pan-genome of
Bifidobacterium contains 5000 genes, and the core genome contains 506 genes.
These conserved core genes can encode most of the core housekeeping functions,
such as replication, transcription, translation, and adaptation and interaction with
specific environments, such as sugar metabolism, cell biosynthesis and signal trans-
duction. The difference in the number of genes between pan-genome and core
genome indicates that there are some genetic differences between strains. The study
of the functions of specific genes and conservative genes will provide a strong basis
for the study of the commonness and characteristics of Bifidobacterium adapting to
intestinal environment (Lukjancenko et al. 2012). In order to study the complexity
of Bifidobacterium genome, Bottacini et al. (2010) carried out comparative genome
analysis of bifidobacterium and determined the total number of genes of
Bifidobacterium pangenome and core genome. The genome analysis of 14 strains of
Bifidobacterium showed that the total number of genes in the core genome and
pangenome was 521 and 5125 respectively, which was twice the average number of
genes in the Bifidobacterium genome for the number of Bifidobacterium genomes
is limited and the whole genome of Bifidobacterium cannot be fully understood

(Makarova et al. 2006b). After completing the comparative genomic analysis of
Bifidobacterium longum JDM301 and other bifidobacteria, Wei Yanxia (2012)
found that the core genome of Bifidobacterium was composed of 1265 genes,
playing an important role in amino acid transport and metabolism, carbohydrate
transport and metabolism, ribosome structure and synthesis, DNA replication,
recombination and repair. 21% of the core protein was associated with carbohy-
drate, amino acid transport and metabolism. (Wei Yanxia 2012)
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Chapter 4
Transcriptomics of Lactic Acid Bacteria
Zhennan Gu and Guozhong Zhao
4.1 Summary of Transcriptomics
4.1.1 Concept of Transcriptomics
Transcriptomics is a frontier subject in the post-genomics era and an important part

of functional genomics, the study of the species, structure, function, and transcrip-
tion regulation that occurred in the cells (the study of gene expression on RNA
level). The purpose to study transcriptome is to understand all the genetic bases of
organisms, including the expression regulation system, as well as the function and
interaction of proteins expressed. Transcriptome is a bridge to link genetic informa-
tion of the genome with the biological function of proteome. Thereby, regulation on
transcriptional level is the most widely studied and most important method of
organism regulation currently. Transcriptomics is one of the most important tools
for large-scale analysis of biological gene expression processes at present and is
more efficient compared to the analysis on genome level (genomics). Different with
genomics, transcriptomics is also influenced by time phase and cell environment.
Because of its capacity to study any moment of cell life cycle, transcriptomics can
not only be successfully used to interpret the functional components of the genome,
or to predict molecular components, but also be applied to study biological pro-
cesses and the pathogenesis of disease.
There are two definitions in the transcriptome, which are distinguished from
broad sense and narrow sense, respectively. The definition of transcriptome in broad
Z. Gu (*)
G. Zhao
Tianjin University of Science & Technology, Tianjin, China

https://doi.org/10.1007/978-981-13-7832-4_4
98 Z. Gu and G. Zhao
sense refers to species. A particular case is the total RNA transcribed from all the
genetic information of the cell or tissue of the species, including the RNA and non-
coding RNA encoding the protein of this species (such as rRNA, tRNA, snoRNA,
snRNA, and microRNA). These RNAs have a wide range of functions and play an
extremely important role in the growth and development of species and various
biological processes (Lewin 2004). Based on the above definition of the transcrip-
tomics, we know that the transcriptome has its specific time and space constraints.
Thereby, the transcriptome is different in different growth conditions and different
growth stages in its life stage in the same cell or a tissue.
This difference is not only reflected in the difference in the type and number of
genes expressed but also in the same cell at different times and in different physio-
logical states. At the same time, the existence of mRNA splice variants is also a
significant difference between the transcriptome and the genome, that is, the same
mRNA precursor is processed into a plurality of normal mRNA isoforms with dif-
ferent exon compositions, so that the gene regulates regulation. Expression products
are more diverse and complex. In addition, the transcriptome has an extremely com-
plex regulatory network, including the regulation of genes and genes between genes
and proteins, and the microenvironment of cells, the different physiological and
biochemical reactions of cells, and cells. The signal transduction pathway in the
body constitutes a complex transcriptional regulatory network mechanism in the
body. Under the guidance of the principles of the genetic center, the genetic infor-
mation of cells is precisely regulated by genes, which can be transcribed from
mRNA into mRNA, and then translated to reach proteins, which ultimately deter-
mines the structure and function of the species cells. The biological macromolecules
associate and coordinate different cells to further constitute tissues, organs, and
biological individuals. Therefore, RNA is a bridge between gene-linked proteins,
and the identity of all expressed genes and their transcription levels are collectively
referred to as transcription (Wang et al. 2009; Costa et al. 2010).
4.1.2 ignificance and Current Status of Transcriptomics

S
Study
The study of gene sequences and gene structures on the genome of a species is only
part of the genomics study, and genomics research alone cannot explain all the mys-
teries in the life system. The functions of gene sequences, the life processes involved,
the way of expression regulation, and the gene expression levels of specific genes
under different environmental conditions need to be resolved. Transcriptomics is an
important technology based on genomics. Transcriptomics can study the structure
and function of specific genes at the overall level of the species and reveal the mech-
anisms of specific biological processes and diseases. Functional genomics studies
of various genes and their transcriptional expression products can provide new ideas
4 Transcriptomics of Lactic Acid Bacteria 99
for food microbial fermentation and resource development and improvement and
provide new methods for humans to solve health, food, energy, and environmental
problems (Velculescu et al. 1997).
Through the study of transcriptomics, information on the expression of specific
genes under specific conditions can be found to speculate on the special function of
the unknown gene, thereby revealing the regulatory mechanism of the specific gene.
The molecular tag of a specific gene expression profile can not only discriminate the
gene phenotype of the cell but also be used for the diagnosis of major diseases.
Transcriptomics research can be applied to production practices to efficiently mine
key genes and enhance their function. Through the analysis of transcriptome dif-
ferential expression profiles of specific genes, it is possible to predict the adverse
factors of fermentation in the production process in more detail.
4.2 Methodologies of Transcriptomics
4.2.1 Hybridization-Based Microarray (EST)
The concept of EST is derived from the computer chip, also know as a microarray.
Gene chip is a gene-based technique developed in recent years, which is used to
rapidly detect differential gene expression, large-scale genome expression profiling,
and differential identification of disease-causing genes, or research on disease-
related genes (Schena et al. 1998). The principle of gene chip technology is using a
series of techniques (photoconductive chemical synthesis, solid surface chemical
synthesis, and photolithography) to synthesize many oligonucleotide probes
(including cDNA, EST, or gene-specific oligonucleotides) on solid supports (such
as silicon wafers, polypropylene, glass sheets, and nylon membranes), which are
then hybridized to the first-strand cDNAs generated by reverse transcription of
mRNA derived from different cells, tissues, or organs, and the cDNA is radioac-
tively isotoped (P32 or P33) or fluorescent (cy3 or cy5) labeled, and the gene expres-
sion levels of target material were detected and analyzed using autoradiography and
laser confocal technology.
Gene chip technology analyzes gene expression and has the characteristics of
high information quantity, high capacity, high accuracy, etc. It is the most com-
monly used technique in transcriptomics research. Its applications include gene
expression detection, mutation detection, genomic polymorphism analysis, gene
library mapping, and hybridization sequencing. However, there are also many prob-
lems that hinder its development, such as high cost, complicated technique, low
detection sensitivity, poor repeatability, narrow analysis range, etc., mainly found in
probe synthesis, probe immobilization, molecular labeling, reading, and efficient
and reasonable analysis of large amount of data in the later stage.
4.2.2 Sequencing-Based Transcriptome Analysis
4.2.2.1 Expressed Sequence Tag (EST)
The expressed sequence tag (EST) technique originated from the Human Genome
Project. The first report on the application of EST technology by Adams and col-
leagues proposed the EST concept in 1991 (Adams et al. 1991). They first randomly
picked 609 clones from a cDNA library of human brain tissue and then sequenced
to obtain a set of expressed sequence tags and finally aligned the sequence homol-
ogy between the database and the expressed sequence tags and concluded that only
36 of the expressed sequence tags represent known genes and another 337 represent
unknown genes. Some very short sequences, ranging from 300 to 500 bp, generated
from cDNA libraries, the expressed sequence tags, are essentially small stretches of
cDNA fragments corresponding to mRNA. This small segment of genes represents
a specific recognition site on the genome and is expressed in cells, tissues, or indi-
viduals at a particular tissue or developmental stage. It is useful by using each tag to
represent a gene to examine the type and amount of transcripts as a whole. A suffi-
cient number of ESTs from a certain tissue can represent the expression of the gene
of the tissue. The number of ESTs can reflect the expression level of a gene. The
more copies of a gene, the more abundant its expression and the corresponding
ESTs that can be detected. Therefore, it is possible to understand the expression and
abundance of genes in the organisms using the technology of expressing sequence
tag (Ewing and Green 2000; Nagaraj et al. 2007).
4.2.2.2 Sander Sequencing (SAGE)
The serial analysis of gene expression (SAGE) is a rapid detection technique for the
detection of mRNA and gene expression levels in cells (Velculescu et al. 1995). It is
well known that the types and amounts of mRNA are very large in different cells,
and it is very difficult to completely sequence and measure them individually.
However, if a short nucleotide sequence can be used to represent each mRNA, the
workload will be reduced dramatically. There are two reasons for SAGE: First is a
nine- to ten-base sequence containing enough information. A short nucleotide
sequence tag can uniquely identify a transcript. For example, the human genome
can only encode about 80,000 transcripts, and a nine-base sequence can resolve
262,144 different transcripts (49). So theoretically it can be said that a nine-base tag
can represent a transcript sequence uniquely. Second, when the nine-base tags in a
clone are sequenced and the data from these short sequence nucleotide sequences
are input into a computer for processing in a continuous data format, thousands of
mRNA transcripts can be analyzed. So the 9 bp sequence length was originally
selected for construction of nucleotide tags.
SAGE technology is characterized by its high throughput, fastness, and high
sensitivity, which contribute to its wide applications in various fields (Boon et al.
2002). When the nature of the gene is not known in advance, it can also study the
biological phenomenon caused by the transcriptional changes of the cell and can
complete almost all mRNA detection in the cell in a short time, obtain its copy
number, and discover the potential unknown gene. This is the biggest advantage of
SAGE technology. In addition, this technique can also be used to distinguish differ-
ent variants and antisense transcripts of transcripts, providing a very large data plat-
form for the study of transcriptomes. SAGE technology provides a powerful tool for
the study of quantitative classification and expression of genes in various states by
identifying mRNA transcripts with specific short sequence tags on the transcript
(CDNA). Multiple tags are randomly ligated into a large number of concatemers
and cloned into vectors, and then each clone was sequenced. The initial promotion
of SAGE technology has certain difficulties, mainly because of some defects in the
previous SAGE method, including difficulty in label identification, large workload,
complicated technical process, and huge cost. However, with the development of
science and technology, this technology has been widely used in many countries
and regions. Nowadays, the development of SAGE method is toward to simplify the
operation steps, reduce the sample size, increase the label length, and improve the
specificity between the tag and the target gene and other aspects (Ye et al. 2000).
4.2.2.3 Massively Parallel Signature Sequencing (MPSS)
In 2000, American scientist Brenner et al. invented a new gene expression analysis tech-
nology based on gene microarray—massively parallel signature sequencing (MPSS)—
which is an efficient and rapid detection method (Brenner et al. 2000). The technology
has been improved on the basis of SAGE, adding a universal linker to the cDNA, which
is a pioneer in the development of next-generation sequencing technology.
The principle of MPSS: There is a sequence tag site in the base sequence of cDNA
that can specifically recognize transcript information, and the length is 10–20b, so
the tag sequence is linked together with an unknown long contiguous base sequence.
The analysis of the base sequence can be performed on the basis of molecular clon-
ing. The measured gene expression level is a digital expression system implemented
on the basis of quantitatively determining the expression level of the corresponding
transcript. First, a base sequence is detected at one end of the mRNA, that is, a tag
sequence containing 10–20 bases; secondly, the copy number of the tag sequence is
calculated, that is, the copy number of the mRNA; and finally, corresponding gene
expression levels are calculated by the number of copies of the mRNA. MPSS tech-
nology can be used for detection and analysis regardless of whether the gene sequence
is known or not and regardless of the level of gene expression. Moreover, this method
utilizes powerful statistical techniques to provide an unprecedented depth of analysis
for discovering functional relationships between genes (2008).
4.2.2.4 High-Throughput RNA-Seq Technology
With the development of next-generation sequencing platforms, high-throughput

RNA-Seq technology has been widely used in transcriptomics research in recent
years based on deep sequencing technology. RNA-Seq, also known as whole
transcriptome sequencing, uses a second-generation high-throughput sequencing

technology to sequence cDNA libraries that are reverse transcribed from total RNA
in tissues or cells and align the sequencing sequences with reference sequences,
obtain information quickly and comprehensively on the entire transcript of a tissue
or cell under certain conditions, and provide epoch-making new technologies for
transcriptomics research. High-throughput RNA-Seq technology opened a new era
of genome-wide transcriptomics research. The advent of RNA-Seq has altered our
understanding of the variety and complexity of transcriptomes, as well as a way to
more accurately evaluate transcripts and their homologs.
Accurate and comprehensive information on transcripts of the sample is the most
versatile feature of the high-throughput RNA-Seq technology, in addition to its high
detection sensitivity, low background signal, accurate accuracy, and good repeat-
ability. The sample consumption is small, the experimental operation is relatively
simple, and the detection cost is relatively low. The advantages of RNA-Seq technol-
ogy are significant compared to the several other sequencing techniques described
above. First, RNA-Seq technology not only detects the overall transcriptional level
of multiple species at the single nucleotide level but also finds unknown and rare
transcripts and can identify and analyze gene fusions and alternative splicing; sec-
ond, RNA-Seq can be performed on different platforms (e.g., Illumina’s genomic
analysis platform) for different experimental purposes. Of course, the biggest advan-
tage of RNA-Seq technology is that it can accurately identify and screen different
tissues or cells in the same condition (time) or different conditions (time) in a short
time with only a small amount of samples. All differential transcripts are transcribed,
as well as comprehensive information annotation and analysis of the genomes cor-
responding to these transcripts (Wang et al. 2009; Robinson and Oshlack 2010).
4.3 pplication of Transcriptomics in the Study

A
of Environmental Stress Responses of LAB
During the growth cycle of lactic acid bacteria, the pH will decrease with the change
of its growth environment or the gradual consumption of nutrients, resulting in the
accumulation of its metabolites. At this time, the survival and growth of lactic acid
bacteria will be affected by acid stress, salt stress, oxygen stress, and hunger stress.
Therefore, understanding of the stress-resistant mechanism of lactic acid bacteria
will be helpful in relieving the damage caused by stress, thereby improving the
growth efficiency of lactic acid bacteria. Transcriptomics is a good tool for this
purpose. It can study the transcription and transcriptional regulation of lactic acid
bacteria under different environmental conditions. Studying the gene expression
levels of lactic acid bacteria in different stresses from the RNA level is helpful for
comprehensive understanding of various stress response mechanisms. So, it is
important, yet hard, to find the anti-stress measures to reduce the effects of stress
factors on the survival, growth, and metabolism of lactic acid bacteria.
4.3.1 Heat Shock Response
The heat stress response is a stress response in which microorganisms resist high
temperatures in a normal physiological temperature environment. Although lactic
acid bacteria are not subject to heat stress in the normal intestinal environment,
lactic acid bacteria suffer from some high temperature stress during the production
of many products, such as spray drying, pasteurization, etc. (Chen et al. 2013). After
being stimulated by lactic acid bacteria, lactic acid bacteria must regulate their
expression and metabolic pathways through their own defense systems and produce
a series of heat shock proteins to reduce or eliminate the damage caused by high
temperature stress. Heat stress response is often accompanied by transient induction
of specific proteins and changes in cellular physiological structure, thereby enhanc-
ing the resilience of microbial cells to the thermal environment.
Bifidobacterium is a strain that naturally resides in the human intestine and has
been applied widely in various functional foods. However, during the production
and processing of these functional foods, Bifidobacterium is often exposed to a high
temperature environment, and the heat stress will have a greater impact on its growth
and accumulation of its metabolites. Therefore, in order to better understand the
response of Bifidobacterium under high temperature stress in depth, Rezzonico et al.
used microarray technology to study the transcriptome of Bifidobacterium NCC2705
under stress condition of different high temperatures (50 °C treatment for 3, 7, and
12 min). The level has been studied in depth. The study showed that the expression
of Bifidobacterium genome was significantly changed under high temperature treat-
ment conditions and 46% of the gene expression levels were changed. Under the
high temperature stress, the metabolic activity of Bifidobacterium was reduced and
the high temperature stress stimulating factor was increased. At the same time, sev-
eral genes with unknown functions were significantly induced, while the expression
levels of three of these genes have remained stable under the high temperature stress
condition from other strains, which is sufficient to demonstrate their multifaceted
role in high temperature stress response. The final results indicate that the induction
of the gene encoding the bifidobacteria and SmpB protein may be related to the high
temperature stress response of bifidobacteria (Rezzonico et al. 2007).
It was showed by studies on heat stress of Bifidobacterium longum NCC2705
that 46% of genes had changes on transcriptional levels and the metabolic activity-
related genes of Bifidobacterium longum are downregulated. Genes with significant
changes in expression levels include chaperone genes DnaJ, DnaK, GroEL, and
GroES, transcriptional activator ClgR, heat shock transcription factor HspR, cold
shock protein CspA, transcriptional repressors Lex4 and HreA, endopeptidase ClpB,
DNA repair protein RecA, etc. (Rezzonico et al. 2007). The Bifidobacterium breve
UCC2003 genome contains a clpB gene belonging to the Clp proteolytic system,
similar to the proteolytic system-related gene clpB in actinomycete which induces
heat stress and osmotic stress in Bifidobacterium breve (Ventura et al. 2005).
When the growth temperature of Streptococcus thermophilus changed from 42 to
50 °C, the expression level of 196 genes (10.4% of total genes) changed, of which
102 were upregulated and 94 were downregulated. The expression of heat stress-
related genes such as dnaK, groESL, and clpL is elevated. The gene expression of
transcriptional regulators such as the HrcA, CtsR, Fur, MarR, and MerR family
protein changed as well. The expressions of genes related to signal transduction,
cell wall, ion homeostasis, ABC transporter, and restriction modification system are
also induced. There are many alterations of gene expression from a number of genes
coding for the proteins with unknown functions (Li et al. 2011).
4.3.2 Cold Shock Response
The preparation of the lactic acid bacteria starter is often carried out by vacuum
freezing-drying, but the lactic acid bacteria are largely killed at a low temperature of
4 °C (Marceau et al. 2004). Therefore, to study the effect of cold stress on lactic acid
bacteria during the preparation of lactic acid bacteria fermenting agent has become
more important. When lactic acid bacteria grow below their optimal growth tem-
perature, many physiological and morphological changes occur, such as the effect
of low temperature on the cell membrane, low temperature affecting the activity of
related enzymes, low temperature causing changes in fatty acids, and low tempera-
ture stress on gene expression. Different types of cold-induced proteins produced by
different lactic acid bacteria are different. Under cold stress conditions, lactic acid
bacteria are affected by two major problems, decreased cell membrane fluidity and
disruption of DNA and RNA second structural stability, and further affect their
transport systems and DNA transcription, translation, and repair. Some cold stress
proteins are also induced by heat shock, and many pressure-related proteins and
HSP proteins are inhibited under cold stress (Dominguez and O’Sullivan 2013).
The cspL, cspP, and cspC genes of Lactobacillus plantarum are associated with
cold stress. Under low temperature conditions, overexpression of cspL gene can accel-
erate the growth of Lactobacillus plantarum. cspP can reduce the lag phase of
Lactobacillus plantarum in the fresh medium. cspC can increase the tolerance of
Lactobacillus plantarum to cold stress (Derzelle et al. 2003). Li et al. found that low
temperature caused a decrease in the enzyme activity of the lactic acid bacteria and a
delay in the action of the enzyme, which may cause a change in the metabolites of the
lactic acid bacteria. Growth of lactic acid bacteria under low temperature stress can
also reduce the sensitivity of certain metabolic regulation processes, leading to meta-
bolic imbalances and even growth arrest. Although freezing has little effect on hexo-
kinase and pyruvate kinase, it has a significant effect on lactate dehydrogenase. During
the freezing process, the expression of the strain lactate dehydrogenase gene was
inhibited, which was a major factor in the damage of lactic acid bacteria (Li 2011).
Propionibacterium faecalis is a safe probiotic identified by the US Food and
Drug Administration. It is usually used to produce Swiss cheese. When matured at
room temperature of 30 °C, the cheese was immediately transferred to a 4 °C cold
storage and stored for about 9 days. During this low temperature period of 9 days,
Propionibacterium freudenreichii did not die, but continued to survive. Dalmasso
et al. used transcriptomics, proteomics, and RT-qPCR methods to explore and study
the survival rate and metabolism of the probiotics at low temperatures (Dalmasso
et al. 2012). A transcriptome microarray analysis of 565 genes in this strain revealed
that 25% of the genes had differences in gene expression at 30 and 4 °C. At 4 °C,
gene expression in the cell structure of Propionibacterium freudenreichii decreased.
In the process of starch and glycogen synthesis, in the link of lactic acid, alanine,
and serine to pyruvate, the related genes are overexpressed. Therefore, it is con-
cluded that Propionibacterium freudenreichii plays an important role in the forma-
tion of cheese flavor. Even at low temperatures, P. faecalis still maintains good
cellular metabolic activity. It has been reported that the cold stress of L. lactis is
often induced by lower temperatures in a transient way (Panoff et al. 1994).
4.3.3 Acid Stress Response
Lactic acid bacteria produce a large amount of organic acids during their growth.
With the accumulation of various organic acids, lactic acid bacteria are in a low pH
acidic environment, causing most of the growth and metabolism of lactic acid bac-
teria to be carried out in this low pH acidic environment. Thus, the lactic acid bac-
teria have an acid stress response. The acid stress response mechanism of lactic acid
bacteria is a complicated network of regulation system, involving many aspects,
among which the more important regulation includes the regulation of gene and
protein expression (Wu et al. 2007). Acid stress often induces various stress response
mechanisms of lactic acid bacteria, including regulating the homeostasis of pH in
lactic acid bacteria cells, inducing expression of stress-regulated proteins, and
maintaining various physiological functions of cell membranes (Wu et al. 2012a, b).
Broadbent et al. (2010) used a combination of transcriptomics and physiological
analysis to analyze the mechanism of acid tolerance in Lactobacillus casei
ATCC334. In physiological studies, cell membrane fatty acids such as saturated
fatty acids and cyclopropane fatty acids were significantly higher in acid-adapted
cells than in control cells. It was also found that the percentages of C14:0, C16;1n(9),
C16:0, and C19:0(1lC) in the acid-adapted cells were significantly higher than those in
the control group, while the percentages of C18:1n(9) and C18:1n(11) were significantly
decreased (Broadbent et al. 2010). The transcriptome analyses showed that acid
adaptation induced a stress response by comparing the acid-adapted group (pH 4.5
for 20 min) with the control group (pH 6.0 growth condition), which promoted the
acid-resistant response of cells, including fermentation of malic acid and lactic acid
and accumulation of intracellular histidine. The microarray dataset shows that in an
acidic environment at pH 2.5, the induction of chemical stress, by adding malic acid
or 30 mmoL histidine, can increase the survival rate of C. sinensis by nearly 100-
fold. Organic acid lactate is the main fermentation product of Lactobacillus planta-
rum. Lactic acid in free state has a greater inhibitory effect on living cells. In general,
organic acid stress is difficult to study because its toxicity is highly dependent on its
environment, including free acid and pH. Pieterse studied the lactic acid stress
mechanism of Lactobacillus plantarum by culturing Lactobacillus plantarum in
different concentrations of lactate and different pH and osmolality to observe the
changes in the transcriptome triggered by stress factors of different lactic acids

(Pieterse et al. 2005). Microarray analyses showed that the genes encoding transke-
tolase (lp_3538) and transaldolase (lp_3539) were upregulated under the lactic acid
stress conditions and both of them were inevitably associated with the pentose phos-
phate cycle. At the same time, the expression of the gene encoding phosphotransfer-
ase (lp_3531) was increased by fivefolds; the genes encoding phosphoenolpyruvate
synthase (lp_l9l2) and phosphoenolpyruvate hydroxylase (lp_3418) were signifi-
cantly increased as well. Both enzymes are essential enzymes on the pathway for
the conversion of pyruvate to oxaloacetate. The subsequent conversion to malic acid
by malate dehydrogenase can produce NAD+, and this pathway is further activated
by downregulation of both pyruvate hydroxylase gene (lp_2136) and pyruvate
kinase gene (lp_l897).
Koponen et al. used a combination of transcriptomics and proteomics to study
the phosphorylation and protein expression of Lactobacillus rhamnosus LGG after
acid stress with two physiological conditions most relevant to their physiology were
selected, pH 48 and pH 58 (Koponen et al. 2012). The results from both microarray
transcriptomics and proteomics were consistent. The results of transcriptomics
revealed that the most important enzyme against strains of acid stress is F0F1-
ATPase. F0F1-ATPase can maintain a relatively stable pH in the lactic acid bacteria
producing cells and pump out the protons (H+) from inside of the cells consuming
the ATP energy in the cells. The study found that the expression of F0F1-ATPase
gene was upregulated under acidic conditions in LGG strains, while the expression
levels of a large number of genes related to nucleic acid biosynthesis and protein
synthesis were dramatically reduced. It was also proved that LGG strain can regu-
late pyruvate metabolism under the influence of pH. In addition, pH changes lead to
changes in the protein phosphorylation of the strain.
Based on the pH change during the fermentation of skim milk with Streptococcus
thermophilus, Streptococcus thermophilus at different fermentation time points
were collected, and 1962 genes were compared by transcriptomics. The first time
period was used as a control group, and the expression level of 115 genes in the
second time period was changed. Among them, 65 genes were upregulated and 50
genes were downregulated. In the third period, the expression levels of 114 genes
changed, of which 33 genes were upregulated and 81 genes were downregulated. In
the fourth time period, the expression levels of 125 genes changed, of which 43
genes were upregulated and 82 genes were downregulated. Through the analysis of
different expressions of genes, it was found that ptsG plays a certain role in the
utilization of glucose by Streptococcus thermophilus and may also be involved in
the regulation of certain metabolism. Its encoded protein plays an important role in
acid tolerance. Two-component systems, Nudix family hydrolase and κRE family
regulators, also play important regulatory roles in acid tolerance (Li 2014).
Fernandez et al. used a combination of proteomics and transcriptomics tech-
niques to reveal the cellular changes in Lactobacillus bulgaricus during acid adapta-
tion (Fernandez et al. 2008). Under acid-stimulated conditions, two proteins
associated with carbohydrate and pyruvate metabolism, PoxI and Fba, were induced
by Lactobacillus bulgaricus. Fatty acid biosynthesis-related genes accC, fabH, and
fabl were also induced. The chaperone protein genes grpE, groES, groEL, hrcA,
dnaK, dnaJ, clpE, clpL, and clpP were induced by acid, while the synthesis of ClpC
involved in stress response was inhibited. It is clear that when the Lactobacillus
bulgaricus is subjected to acid stress, the pyruvate metabolism changes, and the
biosynthesis of fatty acids is further strengthened, thereby affecting the fluidity of
the cell membrane and improving the acid resistance of Lactobacillus bulgaricus.
4.3.4 Oxidative Stress Response
The effect of oxygen on the physiological metabolism of lactic acid bacteria has two
sides. The biomass of lactic acid bacteria grown under aerobic conditions is higher
than that of lactic acid bacteria grown under anaerobic conditions, because in the
case of aerobic conditions, the energy released by conversion of lactic acid to acetic
acid can be utilized after the end of glucose consumption. However, in the conver-
sion process of lactic acid and acetic acid, H2O2 is produced under the action of
NADH oxidase and pyruvate oxidase (Fu 2013). H2O2 is a non-free radical oxygen
derivative of glucose reactive oxygen species (ROS). These reactive oxygen species,
non-radical oxygen derivatives, can cause some degree of damage to the organism
(Messner and Imlay 1999). Reactive oxygen species can destroy the biofilm, protein,
and iron-dependent enzymes of lactic acid bacteria cells and also cause DNA oxida-
tive damage and metabolic dysfunction and expression disorder. The reduction of
reactive oxygen species will reduce the damage to cells, and reducing such damage
is one of the key factors affecting the biomass and the survival of lactic acid bacteria.
The regulation mechanism of lactic acid bacteria on oxidative stress is mainly
divided into two types, namely, scavenging reactive oxygen species and body pro-
tection mechanisms to reduce the negative effects caused by oxidative stress.
Zuo and colleagues used physicochemical structural analysis and transcriptomics
techniques to study the oxidative stress response mechanism of Bifidobacterium
longum subsp. longum BBMN68 (Zuo 2014). The morphological and surface prop-
erties under oxidative stress showed that the aggregation and surface h ydrophobicity
of Bifidobacterium were stronger with the increase of oxidative stress and time and
polyphosphate particles were gradually produced in the cells. Bifidobacterium can
adapt to oxidative stress by altering the structure of its cell surface and simultane-
ously produce polyphosphate as a molecular chaperone to protect its protein. In
addition, they also used RNA-Seq technology to determine the expression profile of
Bifidobacterium longum subtype BBMN68 under oxidative stress conditions and
analyzed the functions of differential genes and the metabolic pathways involved in
these genes by bioinformatics. The results showed that the response of
Bifidobacterium longum subtype BBMN68 to oxidative stress is a comprehensive
and complex process involving the whole genome and 20% of genes had more than
two times difference in the level of gene expression. Responses to oxidative stress
include a range of defense and adaptive mechanisms, such as promoting the degra-
dation of reactive oxygen species and maintaining redox balance, inducing general
stress response and adaptive adjustment of physiological processes such as protein
repair and central metabolic pathways. By overexpressing the differentially
expressed genes in the oxidative stress response of Bifidobacterium longum

BBMN68, the genes of interest, such as nfB1, nfnB2, trxB1, nox, and ahpC, were
functionally verified. The results indicate that AhpC is the main enzyme in
Bifidobacterium that clears endogenous peroxides produced by aerobic metabolism.
AhpC also interacts with other antioxidant stress genes to promote or activate dif-
ferent electron acceptors, such as thioredoxin (Trx), or other NADPH-based ROS
clearance pathways to cope with oxidative stress. By recombinant co-expression of
LpKatL and SSodA4 in Bifidobacterium, the accumulation of peroxide in the cells
was greatly reduced, the nucleic acid is protected from free radical damage, and the
viability of Bifidobacterium cells was greatly improved under lethal oxidative stress
conditions. It is worth noting that the introduction of LpKatL and StSodAa effec-
tively alleviated the stress of the cell’s own antioxidant system, including TrxB1 and
HenN involved in reactive oxygen scavenging, Fe-S protein complex, Fe2+ chelated
Dps protein, protein repair associated DnaK and Lpd2, etc.
Serrano et al. (2007) used transcriptomics techniques to study the role of thiore-
doxin (TRX) in the anti-oxidative stress of Lactobacillus plantarum WCFS1
(Serrano et al. 2007). Sulfuroreductin is an important disulfide oxidoreductase that
catalyzes a series of redox reactions in cells. trxBI-encoded thioredoxin reductase is
an important enzyme expressed by Lactobacillus plantarum WCFS in response to
oxidative stress, which can help the cells to resist the damage caused by oxidative
stress. It was found that the activity of thioredoxin reductase activity in the trxBI
gene-overexpressing strain was three times higher than that of the wild-type strain.
At the same time, as the intracellular thioredoxin reductase activity is increased, a
series of antioxidant stress reactions occur. The transcriptome analysis of wild-type
and txB gene-overexpressing strains under oxidative stress conditions showed that
267 genes were upregulated due to oxygen stress and the production of a large num-
ber of thioredoxin reductase. In addition, the expression of 27 genes in the trxBI-
overexpressing strain was also confirmed to be significantly changed by gene
expression analysis. The overexpression of trxBI gene can activate repair DNA
repair, stress response, and sulfur and sulfur-containing amino acid related amino
acids biosynthesis pathways. There are 16 genes that respond to the large-scale
production of thioredoxin reductase and oxidative stress, including genes related to
sputum metabolism, energy metabolism (gapB), stress-related response (groEL,
npr2), and manganese transport (mntH2). All of the above results indicate that thio-
redoxin reductase encoded by trxBI can increase the tolerance of Lactobacillus
plantarum to oxidative stress, and this response simultaneously induces transcrip-
tion of 16 genes involved in anti-oxidative stress.
4.3.5 Osmotic Stress Response
During the fermentation and preservation of lactic acid bacteria-fermented foods,

lactic acid bacteria are often in an environment of high salt concentration. However,
the high concentration salt causes the change of osmotic pressure to trigger the
water inside of the cells flow to the outside and the cytoplasmic separation which
leads to bacteria cells to stop growing or even die. Therefore, the ability to adapt to
salt stress is a very important factor for lactic acid bacteria to survive and grow in
natural living environment and industrial production. Lactic acid bacteria use many
strategies to cope with changes in external osmotic pressure to maintain normal
osmotic pressure within the cell.
Some outer membrane proteins of lactic acid bacteria can directly or indirectly
regulate the permeability of cell membranes to salt ions to participate in the regula-
tion of osmotic pressure. The balance of Na+ on lactic acid bacteria cell membrane
is achieved by the efflux of Na+ via the N+a/H+ reverse transporter on the plasma
membrane. In addition to the efflux of Na+, it usually increases its absorption of K+
to restore the balance of Na+/K+, thereby restoring the relationship between water
and ions in the lactic acid bacteria cells. K+ can promote the recovery of morphol-
ogy and osmotic pressure of lactic acid bacteria cells and function as an enzyme
activator and messenger of gene expression (Glaasker et al. 1996). Lactic acid bac-
teria are Gram-positive bacteria, but their ion balance is very similar to Gram-
negative bacteria, such as E. coli (Calderon et al. 2004). The primary response of E.
coli to external osmotic pressure is to absorb a large amount of K+ from the outside
and then absorb the compatible soluble solutes to protect its osmosis. Some scholars
have studied the salt adaptation of Halophilic tetrazolium and Phytophthora sphaer-
oides and found that there is no significant relationship between intracellular K+
concentration and changes in extracellular salt concentration (Glaasker et al. 1996;
Robert et al. 2000). Thus, K+ plays a relatively small role in the osmotic balance of
lactic acid bacteria. A large amount of compatible solutes that accumulate in cells
may be the main strategy for lactic acid bacteria to cope with the increase of exter-
nal osmotic pressure (Le Marrec 2011).
Under the condition of hyperosmotic concentration, decomposing metabolic
compatible solutes are inhibited in the lactic acid bacteria cells by intracellular
enzymes. A high concentration of compatible solutes can be accumulated in bacte-
ria cells, thereby exerting the function of osmotic protection. At present, researchers
have found that compatible solutes that can accumulate in bacterial cells are mainly
amino acid derivatives, amino acids, small peptides, methylamines, and their sul-
fonic acid analogs, sulfates, and polyols (Roberts 2005). Many species of lactic acid
bacteria, such as Lactobacillus casei, Lactococcus lactis, and Lactobacillus planta-
rum, are able to absorb certain compatible solute from the outside of the cell to
protect the cells (Hutkins et al. 1987; Molenaar et al. 1993; Kets and de Bont 1994;
Glaasker et al. 1996).
Many lactic acid bacteria cells, after salt stress, respond to increased osmotic
pressure by altering the lipid composition of their cell membranes. Changes in the
lipid composition of the cell membrane, particularly the ability to modify changes
in the lipid head group, can affect the activity of the compatible solute transporter.
Studies have shown that when salt stress occurs, the most important change in the
fatty acid composition of L. lactis NCDO 763 cell membrane is the increase in the
content of cyclopropanol fatty acid, and the ratio of unsaturated fatty acid to satu-
rated fatty acid in the cell membrane does not change. In addition, the amount of
cardiolipin has also changed when the osmotic pressure in the environment has
increased, and this change is also one of the most important factors for bacteria to
adapt to salt stress (Romantsov et al. 2009).
Studies have shown that when the extracellular osmotic pressure of lactic acid
bacteria increases, the expression levels of some genes in lactic acid bacteria will
change accordingly. These genes do not participate in the absorption of compatible
solute, such as genes encoding universal stress response proteins. These universal
stress response proteins include molecular chaperones and some proteases such as
DnaK, GroEL, and GroES. Through transcriptomics and proteomics studies, it has
been found that lactic acid bacteria induce increased expression of stress-responsive
proteins under conditions of high osmotic pressure (Flahaut et al. 1996).
Transcriptomics studies have shown that the high salt environment greatly affects
the transition between citric acid and succinic acid, hindering the progress of the
lactic acid degradation cascade. Citric acid is more involved in the transport process
between cell membranes in response to the effects of high concentrations of osmotic
pressure. It also affects some of the functions of cell membranes including the syn-
thesis of teichoic acid (Bron et al. 2006). Under the induction of high concentration
of salt, the expression level of genes involved in carbohydrate metabolism in lactic
acid bacteria cells will also change accordingly. For example, when Lactobacillus
rhamnosus is subjected to 3.5% salt stress, glycolysis-related enzymes were all sig-
nificantly altered, such as glycolytic enzymes glyceraldehyde-3-phosphate dehy-
drogenase, lactate dehydrogenase, enolase, phosphoglycerate kinase, and triose
phosphate isomerase (Prasad et al. 2003). Lactobacillus sakei showed a downward
trend in the expression of fructose kinase converting fructose-6-phosphate to
fructose-1,6-phosphate in a growth environment with a salt concentration of 4%
(Marceau et al. 2004). It can be explained that when the lactic acid bacteria respond
to changes in the external osmotic pressure, their own metabolic regulation has not
stopped, and corresponding changes have occurred.
Zhao analyzed the functional role of glycine betaine in salt tolerance of
Lactobacillus plantarum ST-III and the salt tolerance response of Lactobacillus
plantarum ST-III using RNA-Seq transcriptomics sequencing technology (Zhao
2014). The salt-tolerant gene cluster ProU of Lactobacillus plantarum ST-III was
verified functionally. The results showed that the transcription level of Lactobacillus
plantarum ST-III was affected to some extent by the amount of salt added, including
the balance of inorganic ions, carbohydrate transport and metabolism, amino acid
transport and metabolism, cell wall/membrane/encapsulated biosynthesis and DNA
replication, recombination, repair, etc. Compared to those cultured at low salt (2%
NaCl) concentration, when cultured with Lactobacillus plantarum ST-III at high
salt concentration (6% NaCl), more compatible solute transporters were found, and
the genes coding for inorganic ion transporters and DNA repair have changed. In
addition, in three samples with different salt concentrations (control, 2% NaCl, 6%
NaCl), a total of 146 ncRNAs were predicted, of which 33, 54, and 10 ncRNAs were
in the control group, 2% NaCl and 6% NaCl, respectively. Of the 146 ncRNAs, 37
ncRNA target genes were successfully predicted. When glycine betaine was present
in the environment, the expression level of the compatible solute transporter did not
change significantly. Carbohydrate transport and metabolism, ribosomal structure

and synthesis, cell wall/membrane/membrane biosynthesis, and inorganic ion trans-
port and metabolism are all altered by the addition of glycine betaine.
4.3.6 Bile Stress Response
Bile is the second stress other than acid that lactic acid bacteria face when entering the
human gastrointestinal tract. It is composed of a mixture of electrolytes, bile salts,
phospholipids, cholesterol, bilirubin, and proteins. Bile can be secreted into the duo-
denum to promote the emulsification and absorption of fat-soluble nutrients (Begley
et al. 2005). The presence of a certain proportion of bile can cause stress on the lactic
acid bacteria cells, resulting in DNA damage, protein structure changes, and pro-
longed protein transport time. Lactic acid bacteria cells play an important role in bile
salt stress by lipid composition and membrane egg yolk and cell membrane function.
The tolerance of intestinal commensal microorganisms to bile salt exposure is an
important feature of their survival and colonization of the intestinal environment.
Ruiz et al. studied the bile salt protection system of Bifidobacterium breve UCC2003
by means of transcriptomics and found a large number of bile salt-inducing genes
with specific functions, such as the Bbr_0838, Bbr_0832, and Bbr_1756 genes that
assist the transporter MFS superfamily, and three genes that direct the ABC trans-
porter Bbr_0406–0407, Bbr_l804–1805, and Bbr_1826–182 (Ruiz et al. 2012).
Bron et al. applied gene hybridization microarray technology to study the whole
gene transcription of Lactobacillus plantarum WCFS1 under 0.1% porcine bile salt
stimulation conditions (Bron et al. 2006). Transcriptome measurements revealed
that 28 genes were upregulated and 62 genes were downregulated in the presence of
bile salts. Among them, seven genes involved in oxidative stress and acid stress are
also involved in the bile salt stress response of Lactobacillus plantarum WCFS1,
including glutathione reductase gene and glutamate decarboxylase gene. It was also
found that 14 genes localized on the cell membrane changed, including the dlt
operon, F0F1-ATPase enzyme, etc.
Koskenniemi et al. combined transcriptomics and proteomics to study the
response of Lactobacillus rhamnosus LGG under different concentrations of bile
salt (Koskenniemi et al. 2011). The results showed that under the influence of 0.2%
of bovine bile salts, there were 316 genes that changed significantly at the transcrip-
tional level, while the expression of 42 proteins changed at the protein level, includ-
ing proteins in the cell and on the cell surface. Among them, changes of 14 proteins
correspond to the genes changes at transcriptional level. These results indicate that
under normal bile salt stress, common stress responses and cell membrane-related
functionalities occur in a variety of ways, including effects on fatty acid composi-
tion, cell surface charge, and extracellular polysaccharide thickness. These changes
may enhance the ability of cell membranes to resist bile salt stress. It is worth noting
that there is a significant decrease in the protein that catalyzes the synthesis of extra-
cellular polysaccharides, whereas a protein specifically designed to remove bile
salts from cells is upregulated. All these indicate that cell can alleviate bile-triggered
damage by regulating the protein expression.
Lactobacillus is constantly secreting bile salt hydrolase, and whether bile salt
hydrolase affects bile salt tolerance requires further investigation. Most of the bile
salt hydrolase gene bsh1 of Lactobacillus salivarius is located on the mitochondria
of the strain, and small number of Lactobacillus salivarius has an additional bile
salt hydrolase gene bsh2 on the chromosome. The bsh2 gene tends to have a higher
level of bile salt tolerance. By exposing Lactobacillus salivarius to bile and bile salt
medium, respectively, transcriptomics was used to study the gene expression level;
it was found that the expression of proteins related to pressure and promoting sub-
stance outflow changes significantly, while the gene expression of bile salt hydro-
lase has no significant change. Thus, the changes in bile salt hydrolase of
Lactobacillus salivarius do not determine the tolerance level of bile salt tolerance,
and other biological effects may exist in bile salt hydrolase (Fang et al. 2009).
4.4 pplication of Transcriptomics in the Analyses of LAB

A
Diversity of Fermented Food
Lactic acid bacteria-fermented food usually refers to a microbial fermentation pro-

cess mainly consisting of lactic acid bacteria, which degrades macromolecules such
as proteins and carbohydrates into small molecules, and the main metabolite is lac-
tic acid, with a taste of acidic, aromatic, and fresh flavor. As one of the main micro-
bial groups of traditional lactic acid-fermented foods, lactic acid bacteria have the
isotypic and heterotypic lactic acid fermentation. In the isotypic lactic acid fermen-
tation process, carbohydrate products are degraded into the lactic acid only. In addi-
tion to lactic acid, volatile compounds such as alcohols and aldehydes are also
produced in the heterotypic lactic acid fermentation process, giving the unique taste
and aroma of fermented foods. Meanwhile, the growth of lactic acid bacteria via
interspecific competition forms an acidic environment and produces antagonistic
metabolites, which preferably inhibits spoilage microorganisms and pathogenic
microorganisms in the product. Therefore, lactic acid bacteria play an important
role in the flavor and safety of the product. Traditional lactic acid-fermented foods
are inseparable from the important role of lactic acid bacteria. In our daily life, there
are many types of foods fermented by lactic acid bacteria, which can be divided into
fermented vegetables, fermented seasonings, fermented sourdoughs, fermented
dairy products, fermented meat products, etc. (Miao et al. 2015). Changes of micro-
flora in fermented foods can be studied by transcriptomics techniques, which can
systematically analyze the metabolic changes and responses of the entire microbial
community. The following is an introduction to the application of transcriptomics
from the perspective of different fermented foods.
4.4.1 Fermented Vegetables
The lactic acid bacteria-fermented vegetables are mainly sauerkraut, Sichuan kim-
chi, and kimchi. These fermented vegetables are not only crispy, delicious, and
appetizing but also have the effect of promoting digestion and other special effects.
For example, sauerkraut contains a large amount of cellulose, minerals, and organic
compounds which are indispensable for human metabolism, among which are lactic
acid, choline, acetylcholine, vitamin C, vitamin B12, and so on. Sauerkraut is not
only unique in flavor but also has physiological functions such as regulating choles-
terol, regulating blood balance, and preventing atherosclerosis.
The main fermentation strain of sauerkraut is lactic acid bacteria. Studies have
shown that in the long fermentation process, Leuconostoc is the dominant bacteria in
the early fermentation of sauerkraut in Northeast China, and then the acid is produced
by Lactobacillus acidophilus, Lactobacillus plantarum, and fermented Lactobacillus.
The final fermentation process is mainly completed by Lactobacillus plantarum. It is
clearly that the dominant bacterium in the early stage of sauerkraut fermentation is
Leuconostoc and the dominant bacterium in the middle and late stages of fermenta-
tion is Phytophthora sinensis (Wu et al. 2014). The metagenomic analysis was used
to study the distribution of microbial flora and the diversity of bacteria in Sichuan
kimchi fermentation process. The study found that the microbial flora in Sichuan
kimchi was mainly lactic acid bacteria. Weissella can reach 74.5% at the beginning of
fermentation, while in the later stage, it is maintained at about 10%, and the dominant
bacteria shit to Lactobacillus, and its content can reach 80%–85%. It is proved that in
the fermentation process of Sichuan kimchi, Weissella is only a starter and the key
bacterium in the fermentation process is Lactobacillus (Tong et al. 2015).
Kimchi is a classical Korean fermented vegetable, with about 23 lactic acid bacte-
ria involved in the fermentation of Korean kimchi (Nam et al. 2009; Jung et al. 2013).
Jung et al. used RNA-Seq sequencing macrotranscriptome methods to study the struc-
tural micro-changes and genetic characteristics of microbial community and meta-
bolic changes 29d fermentation of Korean kimchi. A total of 701,556 reading lengths
were obtained by 454 sequencing from kimchi samples, with an average length of
438 bp. 16S rRNA sequencing analyses showed that the kimchi microbial community
was mainly composed of three genera, namely, Leuconostoc, Lactobacillus, and
Weissella (Jung et al. 2011). The coverage of transcriptome sequencing data also
showed that Leuconostoc mesenterica subspecies ATCC8293 and Lactobacillus sake
23K were highly expressed, suggesting the importance of these two strains in Korean
kimchi. Leuconostoc mesenterica was the most active in the early stage of fermenta-
tion, while Lactobacillus sake and Weissella koreensis were more active in the late
stage of fermentation (Bokulich et al. 2016). In addition, some bacteriophage DNA
sequences were found, which proved that the strains in fermentation process had been
contaminated by bacteriophages. In conclusion, the application of transcriptome to
explore the evolution of microbial communities in fermented vegetables is of practi-
cal significance for the development of industrial fermented vegetables.
4.4.2 Fermented Condiments
The most common traditional fermentation condiments, such as brewing soy sauce,
vinegar, tofu (fermented bean curd), soy sauce, etc., are rich in lactic acid bacteria
in the fermentation process, which play an important role in its quality and taste.
Lactic acid bacteria can ferment sugars to produce lactic acid, citric acid, and other
organic acids, which may improve the flavor of condiments and soften the taste. At
the same time, organic acids can esterify with ethanol produced by ethanol fermen-
tation to produce esters and other flavor substances. Therefore, it is speculated that
Lactobacillus may be related to the formation of organic acids and ester flavor sub-
stances and may contribute to the formation of flavor in fermented condiments.
Secondly, Lactobacillus produces acid in the ethanol fermentation stage, which can
produce synergistic bacteriostasis with ethanol to prevent contamination during the
ethanol fermentation stage. The lactic acid bacteria in soy sauce are mainly
Lactobacillus plantarum, Leuconostoc mesogenes, Lactobacillus brevis,
Pediococcus lactis, and Tetracoccus (Zhang et al. 2014). The study of Lactobacillus
in vinegar using macrogenomic method found that Lactobacillus, Staphylococcus,
and Lactococcus were the main species of Lactobacillus (Wang et al. 2016).
Tetracoccus halophilis and salt-tolerant Lactobacillus such as Campylobacter and
Lactobacillus casei are the main fermentation strains of tofu (Han et al. 2001).
Cheonggukjang is a local delicacy in South Korea. It smells like a corpse, so it
gets its name. This sauce is thick and tastes delicious. Transcriptomic studies found
that the dominant bacteria in this food were Bacillus spp. and Lactobacillus spp.
(Nam et al. 2012). Duan et al. used macrotranscriptome and 16S rRNA sequencing
to explore the correlation between flavor formation and flora structure during shrimp
paste fermentation (Duan et al. 2016). They found that Tetracoccus accounted for
95.1% of the total population. After searching the Nr database, 520 of 588 tran-
scripts matched the transcripts of Tetracoccus halophilis. The citric acid cycle and
oxidative phosphorylation of Tetracoccus halophilis were activated, but the lactate
dehydrogenase gene was not expressed. It was proved that Tetracoccus halophilis
mainly undergoes aerobic metabolism during the fermentation of shrimp paste.
Amino acid metabolism, peptidase production, and gene expression of dipentene
and pinene degradation pathway in Tetracoccus halophilis are also very active.
4.4.3 Fermented Sourdough
The traditional sourdough dough is a flour product obtained by microbial fermenta-

tion of wet flour and is a fermenting strain which is necessary in the process of making
steamed bread. The main fermentation bacteria in steamed bread fermentation process
come from microorganisms in sourdough, which play a very important role in fermen-
tation of pasta. That is to say, the fermentation process of sourdough is a process in
which microorganisms decompose the protein and carbohydrate components in the
dough and interact to produce flavor substances such as alcohol, phenol, aldehyde,
ester, and so on. The fermentation process not only improves the texture of dough,
forms unique flavor and taste, but also forms an acidic environment by microbial fer-
mentation and prevents food spoilage caused by fungi or bacteria contamination.
Sourdough is an extremely complex micro-ecological system. Studies have
shown that there are no less than 50 kinds of lactic acid bacteria and about 20 kinds
of yeasts, mainly Lactobacillus, Saccharomyces, and Candida in sourdough (De
Vuyst and Neysens 2005). Zota et al. isolated 41 strains of lactic acid bacteria from
the traditional sourdough “Cornetto” in Southern Italy, including Phytophthora,
Leuconostoc, Lactobacillus plantarum, Lactobacillus curvatus, pentose
Lactobacillus, and Weissella sinensis (Zotta et al. 2008). Metagenome analysis of
Mexican fermented corn dough “pozol” found that it contains 14 species of bacte-
ria, including Lactococcus lactis, Streptococcus suis, Lactobacillus plantarum,
Lactobacillus casei, Lactobacillus alimentarius, Lactobacillus delbrueckii,
Clostridium, etc. (Escalante et al. 2001).
Macrotranscriptome analysis of traditional “fermentation starter” found that
Lactobacillus plantarum and Lactobacillus fermentum are the most important lactic
acid bacteria, followed by Pediococcus pentosaceus. Lactococcus lactis acts early
in the fermentation of the entire flora ecosystem (Weckx et al. 2010). Wu and col-
leagues isolated and identified yeast and lactic acid bacteria from sourdough in the
western part of Inner Mongolia, China. A total of 85 yeast strains and l08 lactic acid
bacteria strains were isolated from 28 sourdough samples, including the following:
37 strains of Lactobacillus plantarum, 14 strains of Leuconostoc citreum, 10 strains
of Weissella sinensis, 8 strains of Lactobacillus hominis, 7 strains of Weissella fae-
calis, and 7 strains of Lactobacillus helveticus (Wusiriguleng 2011).
4.4.4 Fermented Dairy Products
Lactic acid bacteria-fermented dairy products are mainly divided into acidic fer-
mented milk (yogurt, sour camel milk), alcoholic fermented milk (kumiss, posset),
cheese, and sour cream. Dairy products fermented using lactic acid bacteria have
the functions of improving the quality of dairy products, improving the flavor,
enhancing the healthcare function, and extending the shelf life of products and are
well received by people all over the world.
Sun et al. isolated and identified lactic acid bacteria in traditional sour camel
milk produced by herders from Xinjiang and Mongolia, China (Sun et al. 2006).
The lactic acid bacteria isolated from four parts of camel milk include four strains
of Lactobacillus helveticus, two strains each of L. casei subsp. pseudoplantarum
and Lactobacillus bulgaricus, and one strain each of Pediococcus lactis, Bacterium
flexuosus, Pediococcus urinaeequi, and Enterococcus faecalis. The separation and
identification of the kumiss fermenting bacteria were carried out with the horse milk
wine from pastoral area in Xilin Gol, China, as the source of separation (Ma 2005).
A total of 47 strains were isolated and belonged to 18 genera including Lactobacillus,
Enterococcus, Leuconostoc, and Lactococcus. The leading strain of cheese fermen-

tation is lactic acid bacteria, which decomposes lactose into lactic acid, thereby
lowering the pH of the fermentation environment, inhibiting the growth and repro-
duction of spoilage bacteria to extend the preservation time of the cheese. Ma et al.
isolated and identified lactic acid bacteria in cheese products from different pastoral
areas in Xinjiang, China, and obtained 104 strains of lactic acid bacteria, of which
82 strains belonged to the genus Lactobacillus, 12 belonged to the genus
Enterococcus, and 10 belonged to the genus Weissella. These lactic acid bacteria are
Lactobacillus casei, Lactobacillus plantarum, Lactobacillus licheniformis,
Lactobacillus militaris, Weissella sinensis, and Escherichia coli (Ma et al. 2011).
Lactococcus lactis is widely used in the production of cheese. Lactococcus lactis
faces different stress environments at every stage of cheese ripening. Cretenet et al.
applied transcriptomics to study the expression of Lactococcus lactis in different
processes of cheese making (Cretenet et al. 2011). The first step of cheese fermenta-
tion process is to produce lactic acid by Lactococcus lactis, and the pH is continu-
ously lowered. Under acid stress, F0F1-ATPase acts on the elimination of cytoplasmic
proton. Both the arginine deiminase and the citrate decarboxylase pathway are
involved in pH homeostasis and energy production. At the 8 h time point of cheese
fermentation, its pH was about 5.9. As showed by expression profiling, only three
genes in the F0F1-ATPase system (seven in total) were overexpressed in the process
of cheese fermentation. The genes atpG and atpD were overexpressed at 8 h time
point of fermentation, and atpD and atpF were overexpressed at day 7 of fermenta-
tion. Citrate metabolism-related genes citC, citD, citE, and citF have transient over-
expression at 8 h time point of fermentation. The transcriptomics analysis showed
that the transcription of genes related to the F0F1-ATPase pathway did not change
significantly after 8 h of Lactococcus lactis fermentation, which proved that the
F0F1-ATPase pathway is not the acid stress pathway in Lactococcus lactis. The
expression of arginine deiminase pathway activators ahrC, αrcB, and arcC1
increased, while the expression of arginine reverse transport system-related genes
arcD1 and arcD2 decreased. The arginine deiminase pathway is involved in the pH
stress response of Lactococcus lactis.
Bisanz et al. is the first to use the RNA-Seq technique for transcriptomics analy-
sis of two yogurts with different flavors of strawberry and vanilla and at different
time points of fermentation (Bisanz et al. 2014). They used the ABI SOLID4
sequencing platform to sequence mRNA, yielding 48,658,804 read reads and 37.2
time coverage per sample, but the sequence coverage of Lactococcus lactis is less
than 39%. At the same time, they also compared the abundance of mRNA with high
sequencing coverage in yogurt with different flavors and at different times. By clus-
tering analysis of protein adjacent clusters (COG) of genes with RPKM values
≥200 and RPKM values ≥1000, the functions of the gene can be found in each
microorganism. Genes functionally related to carbohydrate transport and metabo-
lism, amino acid transport and metabolism protein translation, and amino acid
transport and metabolism are all highly expressed. Statistics analysis showed that
the high abundance genes in most microorganisms can be classified into the func-
tional area of “transportation,” except Streptococcus thermophilus. Streptococcus
thermophilus exhibits a high abundance in both carbohydrate transport and meta-

bolic domains, including the beta-galactosidase gene annotated as “Lacz,” which
primarily functions as a lactose-degrading function. There is also higher expression
in Lactobacillus delbrueckii subsp. bulgaricus, but it is reversed in the
Bifidobacterium animalis subsp. lactis. Analyzed using the Kyoto Gene and
Genomic Encyclopedia (KEGG) pathway, genes involved in the carbon transport
metabolic process and the glycolysis process are highly expressed in Streptococcus
thermophilus, while the genes associated with energy production and transfer are
highly expressed in Bifidobacterium lactate subspecies in animals. In addition, the
KEGG analysis also showed that these highly expressed genes contain six F0F1-
ATPase genes that play an important role in the tolerance of the bile salts and acids.
Among the mRNA transcripts expressed from all microorganisms in different sam-
ples, the protein metabolism and specific ribosomal protein components are the
most representative; further analysis revealed that prokaryotic bacteria can adapt to
this fermentation environment.
The research group from Inner Mongolia Agricultural University, China, also
used RNA-Seq transcriptomics to compare the growth of Lactobacillus casei in
milk and soymilk. When Lactobacillus casei Zhang was grown in cow’s milk, the
expressions of 84 genes were significantly changed in the stable growth phase of pH
4.5 and the logarithmic growth phase of pH 5.2. Fifty-nine of them were signifi-
cantly upregulated, and 40.5% of these genes are functionally associated with car-
bohydrate and energy metabolism. The major upregulated genes are associated with
the PTC system and the pentose phosphate pathway. In the soymilk, there are 63
genes which had significant change in the expression at the stable growth period of
pH 4.5 and at the logarithmic growth phase of pH 5.2, and expression of 162 genes
changed significantly at the logarithmic growth phase of pH 5.2 and at the hysteretic
growth phase of pH 6.4. Among them, 48.6% of the genes were upregulated in the
logarithmic growth phase, 48.8% of the genes were upregulated in the stable growth
phase, and all of them were related to the transport and metabolism of amino acids.
The function of main upregulated genes is associated with proteolytic enzyme sys-
tem (extracellular proteases, oligopeptide transport systems, and intracellular pepti-
dases), amino acid (glutamic acid, lysine, and methionine), and nucleotide (purine
and pyrimidine) metabolism. Particularly, the active expression of the genes related
to proteolytic enzyme system makes Lactobacillus casei Zhang able to decompose
soy protein in soymilk, providing sufficient amino acids and nucleotides for its
growth, and may contribute to the better growth of Lactobacillus casei Zhang in
soymilk than in cow’s milk (Wang 2012).
Burenqiqige and colleagues studied the community structure and the expression
of functional gene in sour horse milk samples from different fermentation periods
(Burenqiqige 2014). Followed by transcriptomics metagenomics analyses, they
adjusted and optimized the community structure and community function, which
provides a theoretical basis for the safety, clinical application, and improvement of
traditional fermentation of sour horse milk. The selected sour horse milk samples
from the fermentation of six different stages were subjected to the metagenomic
analyses of bacterial community structure succession during the fermentation pro-
cess. Sour horse milk macrotranscriptome libraries were constructed from the sam-
ples of three different fermentation stages and subjected to RNA-Seq high-throughput
sequencing to obtain functional gene expression during fermentation. The main
results are as follows: a high-quality short sequence of 12.2 Gb from sour horse milk
samples was obtained, and the number of Lactobacillus increased with the fermenta-
tion time and then decreased, while the number of Lactococcus has been increasing
with the progress of fermentation. At the level of phylum, Proteobacteria and
Firmicutes are the dominant bacteria in the acid horse milk, and genus Lactobacillus
and the genus Lactococcus are the dominant genera. In this study, 64,410 merge-
Unigenes were assembled, of which 5939 Unigenes could be directly linked to cor-
responding protein coding region (CDS) and sequence orientation, the remaining
Unigenes were analyzed by software, and 2230 of them were predicted as the new
protein coding sequence. The functional classification of merge-Unigene showed that
the fermentation process was closely related to bacterial cell processing, catalytic
ability, and metabolic process. The results of COG functional classification showed
that the genes involved in amino acid transcription and metabolism play a key regula-
tory role at gene replication, recombination, and repair during the fermentation pro-
cess of sour horse milk. According to the analysis of KEGG metabolic pathway, the
metabolic pathway, the secondary metabolism of biosynthesis, and the microbial
metabolic pathway under complex environment are the main signaling pathways in
the fermentation process. The functional enrichment analysis of GO (differentially
expressed genes) shows that the metabolic process, cell processing, and metabolic
processing of organic substances are the main biological processes from the early to
the middle stages of fermentation. During this period, the expressions of genes
related to some molecular functions are relatively active, such as organic cyclic com-
pound binding, heterocyclic compound binding, and small molecule compound bind-
ing. Gene related to biological processes, such as cell processing, cellular
macromolecular metabolism, and single organism cell processing, plays a decisive
role during the mid-to-end fermentation period. The expressions of genes associated
with molecular structure activity, ribosome structure, and protein binding are very
active as well at this period. KEGG-based enrichment analysis of differentially
expressed gene pathways shows that the ribosome pathway is the dominant meta-
bolic pathway in the early to middle stages of fermentation, while metabolic pathway
of lipid metabolism, aminobenzoate degradation, steroid biosynthesis, mRNA moni-
toring, etc. play a major role in the middle to late stages of fermentation.
The current yogurt on the market is dominantly fermented by the Streptococcus
thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. These two strains
stimulate each other’s growth by mutually utilizing their metabolites such as folic
acid and carbon dioxide. By a combinatorial analysis of transcriptomics and related
metabolite assays, Sieuwerts and colleagues found the relevance between the growth
and metabolism of these two strains, mainly involving the metabolism of sputum,
amino acids, and long-chain fatty acids (Sieuwerts et al. 2010). Formic acid, folic
acid, and fatty acids are mainly produced by Streptococcus thermophiles, while the
amino acids required for the growth of both strains are provided by protein hydroly-
sis activity of the Lactobacillus delbrueckii subsp. bulgaricus. However, observed
from the expression levels of amino acid-related genes, the amino acids generated in
the mixed system, such as sulfur-containing amino acids and branched-chain amino
acids, are not sufficient to support the growth for both strains. Genes related to iron
absorption and epithelial production in Streptococcus thermophilus are also affected
by the mixed system, and their expression levels are higher than those in the culture
with single strain. These studies provide us a better understanding of the ecology
and interaction mechanism of mixed culture of strains in fermented foods.
4.4.5 Fermented Meat Products
Lactic acid bacteria-fermented meat products are more commonly found in China,
such as cured meat, sour meat, sausage, bacon, plate duck, salted fish, etc.
Appropriate pickling will give meat products a richer taste and good color and con-
ducive to the meat preservation and storage. Lactic acid bacteria play a very impor-
tant role in the fermentation of meat products, providing an acidic environment to
prevent the growth of spoilage bacteria, promoting color development, inhibiting
the formation of toxins, and enhancing the nutritional value (Pan 1997).
Lactic acid bacteria are the dominant bacteria in fermented sausages and play a
vital role in the fermentation, preservation, and flavor formation of sausages. Three
kinds of lactobacilli were isolated from the fermented sour meat in the area of Dong
minority, China, including Pediococcus pentosaceus, Lactobacillus delbrueckii,
and Lactobacillus citrifolia (Zhang et al. 2008). Drosinos et al. isolated Lactobacillus
plantar, Lactobacillus curvatus, and Lactobacillus sakei from Greek fermented
sausages (Drosinos et al. 2005). Lactobacillus sakei strain 23K is a psychrophilic
bacterium that is widely used in fermented meat products (Chaillou et al. 2005). In
order to understand the characteristic gene expression of Lactobacillus sakei in the
living environment of meat products, Xu et al. applied gene microarray transcrip-
tome technology to study the transcriptional expression under different culture con-
ditions, and the transcripts from a total of 551 genes were detected. Compared to the
expression of the strain grown in the blank medium, the expressions of the genes
involved in the hydrolysis of the peptide chain were upregulated in various degrees
in the medium containing the muscle fibers and the sarcoplasm (Sun et al. 2015).
The expression of the gene oppB and oppC, which is related to the amino acid and
polypeptide transport systems, was upregulated. Except the glnA and meK genes,
most of the genes involved in the metabolism of peptides, amino acids, and related
molecules are overexpressed in media containing muscle fibers and sarcoplasm.
The stress-related genes were not induced to express in the medium containing
muscle fiber.
Lactic acid bacteria are involved in the fermentation of meat products, which is
critical to the improvement of the taste of meat products, but a small number of
lactic acid bacteria can cause the corruption of meat products. Lactococcus is a
psychrophilic bacterium that is a spoilage organism causing meat products to decay
during storage and produce oily and acidic odors during storage at low temperature.
Studies have shown that the different growth environments of Lactococcus can
cause great differences in degree of spoilage. By transcriptomic analysis of lactoba-
cillus glucose metabolism at different time points, it was found that glucose was
continuously consumed over time and gene expression of lactobacillus carbohy-
drate and glycerol metabolic pathways was upregulated gradually. At the same time,
the expression of pyruvate metabolic pathway-related genes associated with the
production of spoilage substances is abnormal. Through the transcriptomics
approach, it was observed that Lactococcus can maintain its survival through the
upregulation of related gene expression and the activation of metabolic pathway at
the genetic level (Andreevskaya et al. 2015). Leuconostoc gelidum subsp. gasicomi-
tatum is also a harmful microorganism that can cause the decay of meat products,
often giving the meat a spoiled smell of butter. Gas chromatography-mass spec-
trometry (GCMS) combined with transcriptomics can be used to study the source of
buttery taste of this strain under the growth conditions of different carbon sources
(Jaaskelainen et al. 2015).
4.5 pplication of Transcriptomics in Study of Interactions

A
Between LAB and Their Hosts
The human body is a natural place where the microbial community can effectively
reside and a large number of microorganisms parasitized to varying degrees in
human body, including the mouth, digestive tract, respiratory tract, reproductive
tract, and skin. Human and microbes are interdependent and maintain a dynamic
balance. If this balance is broken, the human body may be faced with illness. For
example, intestinal symbiotic microorganisms have a great influence on the physi-
ological and pathological state of the host. Intestinal microbes can help the host to
metabolize and absorb nutrients more efficiently and to resist the attack of foreign
microorganisms or viruses. Comprehensive study of the relationship between
human hosts and microorganisms will allow us to further understand the occurrence
and treatment of human diseases (Hu et al. 2012). Although lactic acid bacteria have
a small population in the human microbial community, most studies have shown
that lactic acid bacteria play a positive role in the formation of human microbial
communities and various functions of the human body.
4.5.1 Gut Environment
Intestinal microbes provide the body with an important innate adaptation to the
immune system and simultaneously regulate intestinal metabolism and immune bal-
ance. Probiotic lactic acid bacteria can regulate intestinal homeostasis and immune
response. The transcriptomics study of living tissue suggested that the proto-onco-
gene BCL9, protein kinase ERK3, proto-oncogene JUN, and poly(ADP-ribose)
polymerase (PARP) 14 play an important role in the downstream signaling pathway

in the immune response and can be signaled through interferon and transcriptional
activation regulator IRF to regulate the production of cytokine Th1 (van Baarlen
et al. 2009). Van Baarlen et al. used microarray transcriptomics to determine the
gene expression of duodenal mucosa tissue after intervention with Lactobacillus
plantarum and found that NF-κB regulatory pathway gene expression changes
greatly, which is beneficial to human health by inducing human immune tolerance.
Researchers applied hybrid microarray transcriptome to study the gene expres-
sion of Lactobacillus johnsonii in different locations in the intestine after oral sup-
plementation of Lactobacillus johnsonii. The gene expression patterns of
Lactobacillus johnsonii in different locations in the intestine are different. Alteration
in gene expression of Lactobacillus johnsonii in the stomach was the most obvious,
with 786 gene changes. Followed by the cecal and jejunal, there were 391 and 296
gene expression changes, respectively. Only 26 genes changed their expression in
the colon. Among these expression-changed genes, besides the genes related to
energy transport, most of them are related to sugar PTS transport, such the genes
specifically expressed in the cecum, galactosamine PTS transport gene, and fruc-
tose, glucose, and fiber II sugar PTS transport-related genes in the jejunum. These
genes display different expression patterns at different digestion sites (Denou et al.
2007). Marco et al. also studied the expression of functional genes by hybrid micro-
array transcriptomics after oral ingestion of Lactobacillus plantarum and found that
Lactobacillus plantarum, which is clustered in the cecum, has a functional gene
related to carbohydrate transport and metabolism (Marco et al. 2009). When oral
supplementing Lactobacillus plantarum with different diets (Chinese or western
style), it was found that the growth rate of Lactobacillus plantarum under western-
style diet was lower and carbohydrate metabolism was altered. Kleerebezem and
Vaughan compared the activity of bifidobacteria in infant and adult gut by macro-
transcriptome sequencing (Kleerebezem and Vaughan 2009). The sequencing
results showed that the expression of genes associated with oligosaccharide metab-
olism and vitamin-producing and trans-aldehyde enzyme had significant change.
There is a very close correlation between gut microbes and human health.
Prebiotics in the human ileum can selectively promote the growth of probiotics in
the human gut to significantly improve intestinal flora balance. For example, both
oligofructose and galactooligosaccharide have the effect of increasing the number
of anaerobic bacteria in the human intestinal tract and can promote the growth and
reproduction of bifidobacteria and lactic acid bacteria and inhibit the growth of
pathogenic bacteria such as Enterobacter (Li 2012). Fructooligosaccharide is a
commonly used prebiotic. Chen et al. used RNA-Seq transcriptomics sequencing
technology to study the molecular mechanism of Lactobacillus plantarum ST-IIII
using fructooligosaccharides by using glucose as control. Sequencing results
showed that there were 363 gene expression changes, of which 324 genes were
upregulated and 39 genes were downregulated. It was also found that two 75 kb and
4.5 kb gene clusters of Lactobacillus plantarum ST-III may be involved in the utili-
zation of fructooligosaccharides. In order to more efficiently use fructooligosac-
charides, the gene expression of fatty acid synthesis is downregulated to change the
fatty acid composition and improve the fluidity of the cell membrane. By knockout
experiments to remove the gene encoding the β-glucosidase (SacA) and PTS trans-
port system (SacPTS1 and SacPTS2), it was found that fructooligosaccharides were
transported to inside of cells by the PTS system and subsequently hydrolyzed to
monosaccharides by SacA in Lactobacillus plantarum ST-III (Chen 2014).
Probiotics promote intestinal mucosal repair and maintain intestinal balance.
Probiotics are also often used in combination with antibiotics to treat intestinal
infections. Lactobacillus has been used clinically to prevent and treat intestinal dis-
eases in some infants. Kumar et al. used transcriptomics to understand the coloniza-
tion of the strain in neonatal duodenum and ileum within 7 days after ingestion of
L. rhamnosus LGG and Lactobacillus acidophilus (Kumar et al. 2014). Probiotic
colonization in the gut and the benefits were determined by up- and downregulation
of gene expression related to immune function, as well as metabolism of small
molecular substances (such as vitamins and minerals) and macromolecular sub-
stances (such as carbohydrates, proteins, lipids, etc.). Compared with the level of
transcription, the immune regulation and carbohydrate metabolism pathways are
active when supplemented with LGG, while the energy and lipid metabolism are
more active when supplemented with Lactobacillus acidophilus.
4.5.2 Oral Environment
There are many microbial populations in the human oral environment, and it is an
extremely complex micro-ecological system. The interaction between microbes in
the mouth is closely related to the health condition of the human oral cavity.
Microorganisms in the mouth have two living states, one is in a free state of the
flowing saliva, and the other is on the surface of the organ, such as back of the
tongue, cheek mucosa, dorsal tongue, crown plaque, and gingival sulcus. Excessive
growth of pathogenic bacteria in the oral cavity or microorganisms that are unfavor-
able to the oral environment can cause problems such as dental caries, periodontal
disease, and bad breath. Currently, probiotic therapy is gradually applied to the field
of stomatology, and its advantages in the prevention and treatment of oral diseases
are becoming more and more obvious.
Lactic acid bacteria have a good preventive effect on dental caries and periodon-
tal disease. Studies have shown that lactic acid bacteria isolated from the mouth of
healthy people have the effect on inhibiting the activity of cariogenic bacteria and
inhibiting the proliferation of periodontal bacteria. Yang screened 32 strains of
Gram-positive bacilli from human saliva and plaque samples (Yang 2013). Two
strains of lactic acid bacteria isolated from plaque samples are Lactobacillus fer-
mentum Y29 and Lactobacillus brevis BBE-Y52. Lactobacillus brevis BBEY52 is a
good oral probiotic, since it is able to synthesize hydrogen peroxide, and its acid
production capability is also weaker than that of Lactobacillus salivarius and
Streptococcus mutans, a dental caries pathogen. It was found that L. brevis BBE-
Y52 has the ability to self-aggregate and co-aggregate with other oral microorgan-
isms to form biofilm and adhere to oral epidermal cells, which is beneficial to its
reproduction and survival in the oral environment. When L. brevis BBE-Y52 co-
cultivated with human peripheral blood stimulated by S. mutans, the ratio of anti-
inflammatory cytokine I-10 to pro-inflammatory cytokine IL-12p70 increased. This
result suggests that L. brevis BBE-Y52 has the anti-inflammation activity in vitro
and has potential for maintaining oral health as probiotic.
A follow-up survey of children aged 1–6 found that children who had long-term
intake of milk with R. rhamnosus had a lower probability of caries, probably due to
the presence of R. rhamnosus in oral cavity inhibited the growth of caries pathogen
S. mutans, reduced its amount, and prevented the occurrence of dental caries (Nase
et al. 2001). The similar studies found that intake of Lactobacillus reuteri had the
same control effect on dental caries (Caglar et al. 2006). Lactobacillus reuteri also
has a certain effect on the treatment of periodontal disease. Krasse et al. found that
the treatment with L. reuteri effectively reduced the clinical indicators of periodon-
tal disease and reduced the deposition of bacterial plaque and relieved periodontal
disease to a certain extent in a double-blind experiment on patients with moderate
or higher gingivitis (Krasse et al. 2006). Hatakka et al. found that probiotics also
play a role in the prevention and treatment of bad breath and oral candidiasis in the
elderly (Hatakka et al. 2007).
4.5.3 Vaginal Environment
The female reproductive tract is colonized with a large number of bacteria of differ-
ent species, which plays a very important role in human health. Ling and colleagues
studied the differences in vaginal microbial community diversity between bacterial
vaginosis patients and healthy women from China by PCR-DGGE and 454 pyrose-
quencing. The study showed that there were far more bacterial strains in the vagina
than in the healthy control group. Phylum Firmicutes accounts for the majority of
vaginal flora in healthy women, of which the dominant flora includes lactic acid
bacteria, mainly Lactobacillus, used to maintain the normal range of pH in the
vagina and to inhibit the growth of potential pathogens. Micrococcus varians and
Lactobacillus work together to maintain the health and balance of the vaginal micro-
ecology. Lactobacillus inert is a dominant bacterium of the genus Lactobacillus in
healthy women. It is considered to be a typical bacterium of the normal vaginal
microbiota and a sensitive marker of vaginal microbial community changes. During
the development of bacterial vaginal diseases, the copy number of inert Lactobacillus
decreased by two to three orders of magnitude, even undetectable in pathogenic
samples. In addition, other low-abundance lactobacilli, such as Lactobacillus curly
and Lactobacillus jensenii, have been found (Ling 2012).
Kohler et al. found that mixed bacteria of Lactobacillus rhamnosus GR-I and
Lactobacillus reuteri RC-14 completely inhibited the growth of pathogenic Candida
albicans, which is prone to genital tract infections, and proposed the mechanism of
interference exerted by probiotics. This result is important for studying how to
maintain a healthy reproductive tract environment. Experiments have shown that the
low pH environment caused by lactic acid plays an important role in inhibiting the
growth of fungi. Finally, Candida albicans lost its metabolic activity and subse-
quently died. Transcriptomics was used to analyze the expression of stress-related
genes in Candida albicans in this study. The genes involved in lactic acid utilization
were effectively stimulated during the early stage and co-culture with lactic acid
bacteria, such as cytochrome C oxidoreductase gene CYB2, lactic acid transport-
related genes, stress-related genes, etc., and elucidated the fluconazole drug toler-
ance in the absence of lactic acid bacteria intervention (Kohler et al. 2012).
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Chapter 5
Proteomics of Lactic Acid Bacteria
Yue Xiao, Yanjun Tong, and Wei Chen
5.1 Introduction to Proteomics
Protein is the direct mediator of gene functions. The biological processes in protein
level including dynamic modification, processing, transportation and localization,
and structure formation cannot be predicted from gene content. The expression of
mRNA cannot directly reflect the expression of the corresponding protein. Therefore,
proteomics rather than genomics can provide direct evidence for the “true” occur-
rence of life. In the mid-1990s, proteomics research, as a newly merged discipline,
initiated benefiting from the development on human genome project. The proteomes
are of diversity and variability, wherein the compositions and abundances of protein
pool are different in the different cells within the same organism. Meanwhile, the
proteomes are also variable under different phases and conditions in the same cell.
Therefore, proteomics can provide an effective means for research on complexity of
protein functions during life process from dynamic and comprehensive perspectives.
5.1.1 Introduction to Proteomics
5.1.1.1 The Conception of Proteomics
The concept of proteome was first proposed by Marc Wilkins, which refers to a
whole set of proteins expressed and modified by a genome. Sometimes it also means
a combination of proteins expressed by a cell at any given time and environment.
Proteomics mainly focuses on studying the existence and activity of all proteins in
cells. Proteome changes with tissues or even the environment. Proteomics mostly
Y. Xiao · Y. Tong · W. Chen (*)

https://doi.org/10.1007/978-981-13-7832-4_5
132 Y. Xiao et al.
concentrates on the dynamic description of gene regulation and thus quantitatively

determines gene expression in protein level. Proteomics essentially refers to the
study of protein characteristics at the large-scale level, including the expression
levels of protein, posttranslational modifications, protein-protein interactions, etc.,
providing an overall and comprehensive understanding of the process of cell metab-
olism and disease occurrence at the protein level.
5.1.1.2 The Significance and Development Status of Proteomics
With the implementation and advancement of the Human Genome Project, more
than 150 genome projects (including the human genome project) have been pub-
lished. Scientists are more concerned about the genome encoding the structure and
function of proteins. The research of life science has entered the post-genome era.
During the era, the main research objects of life sciences are functional genomics,
including structural genomics and proteomics. Although the genomes of several
species are now sequenced, the function of more than half of the genes in these
genomes is currently unknown. Genomics, transcriptomics, and proteomics, respec-
tively, study life activities from three levels of DNA, mRNA, and protein. From
DNA to mRNA, and then to proteins, there are three levels of regulation, namely,
transcriptional control, translational control, and posttranslational control. There is
no one-to-one correlation between mRNA expression levels and protein levels, and
transcriptional level regulation cannot wholly represent protein expression levels.
In addition, complex posttranslational modifications of proteins, subcellular
localization or migration of proteins, protein-protein interactions, etc. can hardly be
judged from mRNA levels. Protein is the executor of physiological function and also
directly shows life phenomenon. The study of protein structure and function will
directly clarify how mechanism of life changes under different physiological or path-
ological conditions. In order to explore the existence and activity of proteins, such as
posttranslational modifications, protein-protein interactions, and protein conforma-
tion, directly researching on proteins plays an important role. Therefore, using large-
scale, high-throughput and high-sensitivity proteomics techniques to study proteins
at a global, dynamic, and network level by globally studying the expression profiles
and functional maps of all proteins at different times and spaces contributes to a
comprehensive and in-depth understanding of the complex activities of life.
Proteomics research is developing rapidly; both theoretical basis and technical
methods are constantly improved. In recent years, research techniques of proteomics
have been applied to various fields of life science, such as cell biology and neurobi-
ology and so on. In terms of research object, it covers the range of prokaryotic
microorganisms, eukaryotic microorganisms, plants and animals, etc., and it involves
various important biological phenomena, such as signal transduction, cell differen-
tiation, and protein folding. In the future development, the research field of pro-
teomics will be more extensive. In terms of proteomics research techniques, research
methods of proteomics will have multiple technologies coexisting, each with its
advantages and limitations, and it is difficult to form a comparatively consistent
5 Proteomics of Lactic Acid Bacteria 133
approach like genomics research. Except for developing new methods, it should put
more emphasis on the integration and complementarity of the various methods to
accommodate the different characteristics of different proteins. In addition, the
intersection of proteomics and other disciplines will become increasingly significant
and important. This crossover is the source of living water for new technologies and
new methods, especially proteomics and other large-scale sciences (such as genom-
ics, bioinformatics, etc.). The intersection of the omics biotechnology research
methods presented by the system biology research model will become an exciting
new frontier for the future life sciences.
5.1.2 Proteomics Classification
5.1.2.1 Comparative Proteomics
Comparative proteomics is an important part of proteomics, which concerns on

researching the identification of expression differences in protein level within the same
biological system under different conditions or times, for example, the comparison
between cells under normal state and harsh environmental stress, which is generally
referred to as differential proteomics or comparative proteomics. By searching and
screening differential protein components present in different protein expression pro-
files of different samples, it helps to study changes in protein components and reveal
the progress and nature of cellular physiological states in different environments,
which are also useful for researching the response of cells or tissues to external stress
stimuli and cellular regulation mechanisms and qualitative, quantitative, and func-
tional analysis of certain key proteins. In comparative proteomics, qualitative analysis
was performed using the positional and density ratios of the colored spots in the two-
dimensional gel electrophoresis to analyze the differential expression of the proteins.
5.1.2.2 Structural Proteomics
Structural proteomics focuses on studying proteins in an active conformation,

depicting the three-dimensional structure of a protein or protein complex. The
methods for determining the three-dimensional structure of proteins mainly include
X-ray crystal diffraction pattern method, nuclear magnetic resonance method, and
circular dichroism spectroscopy.
5.1.2.3 Functional Proteomics
Functional proteomics is a broad term that includes many specific protein methods.
The physiological functions of all proteins in the cell are determined by analyzing
the interaction between proteins, the three-dimensional structure of the protein, the
134 Y. Xiao et al.
cellular localization of the protein, and the posttranslational modification of the

protein. Functional proteomics focuses on the identification and classification of
protein functions, protein interactions, and protein activities from a global perspec-
tive. The research methods of functional proteomics involve high-throughput multi-
dimensional methods including molecular biology, biochemistry, and bioinformatics
analysis.
5.1.2.4 Posttranslational Modification Proteomics
Most of proteins in eukaryotes undergo posttranslational modification, and phos-

phorylation and glycosylation are the two major modifications of protein transla-
tion. All posttranslational modifications are accompanied by an increase or decrease
in molecular weight. Therefore, mass spectrometry has become a rational tool for
posttranslational modification proteomics identification and characterization.
5.1.2.5 Interaction Proteomics
Interacting proteomics concerns on the interaction between genetic and physical

proteins and the interaction between proteins and nucleic acids or small molecules.
Analysis of protein interactions can not only provide functional information about
the protein itself but also provide information about the role of proteins in metabolic
pathways, regulatory networks, and complexes; research methods for interacting
proteomics require different technology platforms to provide different information,
which is closely linked to functional proteomics.
5.2 Technology of Proteomics Research
5.2.1 Protein Separation Technology

5.2.1.1 Two-Dimensional Gel Electrophoresis (2-DE)
DE was put forward by Farrel in 1975. It can expand the complex protein mixture
on two-dimensional planes based on the isoelectric point of protein (the first direc-
tion) and the relative molecular mass (the second direction). The gel is stained with
Coomassie brilliant blue, silver, or fluorescent substance after electrophoresis, and
then the electrophoresis images are analyzed using related software. Up to 10,000
proteins can be separated in a 2-DE. The protein spots are cut off from the gel, sub-
sequently, hydrolyzed, digested, identified, and sequenced, which are usually man-
ual and time-consuming. In addition, many factors make protein detection difficult:
(a) low-copy-number proteins; (b) isoelectric focusing – the isoelectric point (pI)
determined by 2-DE is 3 ~ 10, some extremely acid or alkali protein cannot be
detected; (c) protein molecular weight – the relative molecular mass range of pro-
teins detected by 2-DE is 10 ~ 200 kDa, extremely large (> 200 kDa) or small
(< 10 kDa) protein cannot be separated; (d) proteins (including some important
membrane proteins) which are difficult to dissolve in conventional separation.
Although these factors limit the establishment of a good 2-DE method, 2-DE is still
being used as a core technique for proteomics research. Analyze the increase or
decrease of the intensity of protein spots in the gel to determine the differential
expression of proteins in comparison with the protein 2-DE under normal growth
environment and pressure conditions. The procedures of 2-DE generally include
sample preparation, isoelectric focusing (the first direction), SDS polyacrylamide
gel electrophoresis (SDS-PAGE) separation (the second direction), gel staining,
image acquisition and analysis, protein identification, and so on.
5.2.1.2 Differential Gel Electrophoresis (DIGE)
DIGE can separate multiple samples labeled by different fluorescence simultane-

ously in the same gel based on traditional 2-DE combined with multiple fluorome-
try. As different fluorescein-labeled samples have different excitation wavelengths,
multiple samples can be separated and analyzed in the same gel. DIGE can be used
to compare the distinction between different samples as well as avoid systematic
errors effectively among different gels. There are certain limitations in the DIGE
when selecting fluorochromes used for labeling protein sample. The isoelectric
point of protein with fluorochromes should not be changed, and the molecular
weight changes should be as small as possible after labeling. Furthermore, the
molecular weight changes induced by different fluorochromes should be consistent.
Cy2, Cy3, and Cy5 are the frequently used fluorochromes at present.
5.2.1.3 Isotope-Coded Affinity Tag (ICAT)
ICAT is a new technique proposed by Gygi et al. (1999) for protein separation and
analysis. Label sample proteins containing Cys with ICAT reagents selectively
before hydrolyzed by protease. And then samples are identified by mass spectrom-
etry after purified by affinity chromatography. According to the intensity level of a
pair of peptide ions labeled by different ICAT reagents in the mass spectrogram, the
relative abundance of the corresponding protein in the original sample can be quan-
titatively analyzed, and the difference of protein expression level between two sam-
ples can be compared accurately. The identification of protein can be achieved by
determining peptides using tandem mass spectrometry.
The key of ICAT is the application of ICAT reagents, which are generally com-
posed of sulfhydryl-specific reaction groups, connecting parts (eight hydrogen or
heavy hydrogen atoms), and biotin. And it is divided into light chain reagent and
heavy chain reagent. ICAT separates and purifies proteins at the peptide level, which
can solve the solubility problem of membrane proteins; furthermore, it can reduce
136 Y. Xiao et al.
the complexity of protein mixtures by selectively labeling peptide containing Cys.

ICAT cannot be used for analyzing proteins without Cys, and ICAT reagent is a
large modifier relative to the small peptide (the molecular weight of the ICAT
reagent is approximately 0.5 kDa), which increases the complexity of protein
analysis.
5.2.1.4 Isobaric Tags for Relative and Absolute Quantification (iTRAQ)
iTRAQ was developed by Applied Biosystems, Incorporated, which is one of the

most powerful methods for differential protein quantitative analysis with the highest
flux and the smallest systematic error. At present, four (or eight) different isotopic
coding reagents are used in iTRAQ to label the amino acid groups of polypeptides
specifically and then analyze them by tandem mass spectrometry.
The procedures of iTRAQ generally include extraction of proteins, elimination
of proteins with high abundance, determination of protein concentration, reduction
and alkylation, trypsin cleavage, labeling by iTRAQ reagent, and mixing of sam-
ples. The mixed samples were separated by online or offline liquid chromatography,
detected by first-order mass spectrometry (MS) subsequently. The detected parent
ions are dissociated by high-energy collision, and the obtained ions are tested by
second-order MS. Comparing the relative molecular mass of different fragment ions
obtained through the abovementioned detections with known databases can deter-
mine the corresponding protein precursors.
The combination of iTRAQ and LC-MS/MS is widely used in quantitative study
of proteomics at present. In the iTRAQ experiment, 2~8 samples can be labeled at
the same time simply and efficiently. In addition, the iTRAQ reagent can label all
enzymatic fragments in biological samples, including posttranslational modified
fragments, without damaging some important structural information in proteins.
5.2.1.5 Multidimensional HPLC (MD-HPLC)
MD-HPLC is a chromatographic technique of continuously using concentrated liq-

uid chromatography to separate complex components to a greater degree. MD-HPLC
is an effective method for the separation of complex samples, which belong to
chromatography-chromatography coupling technology.
Two-dimensional liquid chromatography (2D-LC) is one of the most commonly
used MD-HPLC. The separation and purification system of 2D-LC is composed of
two tandem chromatographic columns with different separation mechanism and
independent of each other. The samples are separated by one-dimensional
chromatographic column and then purified by two-dimensional chromatographic
column. As usual, 2D-LC separates and analyzes samples according to the differ-
ences of their molecular weight, isoelectric point, hydrophilicity, or special inter-
molecular forces. The common 2D-LC techniques include ion exchange
chromatography- reversed-
phase liquid chromatography (IEC-RPLC), affinity
chromatography-reversed-phase liquid chromatography, molecular sieve chroma-

tography-reversed-phase liquid chromatography, chromatofocusing-reversed-phase
liquid chromatography, and so on.
5.2.2 Protein Identification Technology

5.2.2.1 Edman Degradation
Edman degradation is a method to determine the primary structure of protein which

mainly analyzes amino acid residue from the N-terminal of protein or polypeptide. The
N-terminal amino acid residue is modified by phenylisothiocyanate; then the modi-
fied amino acid residue (phenylthiohydantoin amino acid) is cut from the polypep-
tide chain and identified by chromatography. The remaining polypeptide chain (one
less residue) is recycled for the next cycle of degradation. In this way, each amino
acid residue in the polypeptide chain can be identified by the analysis phenylthiohy-
dantoin amino acids through an Edman and HPLP cycle. Edman degradation is gener-
ally divided into coupling, cutting, extraction, transformation, and identification.
5.2.2.2 Mass Spectrometry (MS)
MS determines the type of protein by measuring the quality of the protein. Aston
created the first velocity-focused MS in 1919, which became a milestone in the
development of MS. With the development of ion optics theory, MS has been
improved and its application range has been expanded. The application of MS in the
field of life science has formed a unique food MS technology. Matrix-assisted laser
desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and
electrospray ionization mass spectrometry (ESI-MS) are commonly used in protein
identification. And MALDI-TOF-MS uses laser pulse to gasify and dry the samples
in the crystal matrix so that the samples are charged, and the flight time of different
samples with different nucleocytoplasmic ratio is different in the time-of-flight MS
composed of accelerating electric field and magnetic field. We can obtain the spec-
tral lines of different nucleocytoplasmic ratio by using the detection system, which
form the peptide mass fingerprint. ESI-MS is often used for the separation and iden-
tification of complex proteins through ionizing the sample with high electric field.
5.2.3 Image Acquisition and Bioinformatics
The application of imaging technology in proteomics enables the rapid develop-

ment of proteomics study. Proteomics imaging can not only distinguish the charac-
teristics and composition of proteome which is invisible to the naked eye but also
138 Y. Xiao et al.
analyze the differences between various proteome samples, making proteome anal-
ysis more intuitive. Proteomics imaging mainly includes 2-DE imaging, LC-MS
imaging, molecular scanner imaging, and tissue MS imaging in vivo. In addition, a
variety of analysis software such as PDQuest, ImageMaster 2D Elite, Biolmage
Investigator can be used to collect and compare data of gel images.
5.2.4 Interaction Technology
5.2.4.1 Yeast Two-Hybrid System
Yeast two-hybrid system was established by Fields and Song in 1989. It was used to
study the transcriptional regulation of eukaryotic genes initially. During continuous
improvement and development, it has become a simple and effective technique to
study the interaction of proteins. More importantly, it is applied to the discovery of
new unknown proteins interacting with known proteins.
Yeast two-hybrid system is a highly sensitive technique for studying the relation-
ship between proteins that carried out in the eukaryotic model yeast to study the
interaction of proteins in cells. The weak and instantaneous interaction between
proteins can also be detected sensitively by reporting gene expression products.
Yeast two-hybrid system can be used to study the interaction between proteins
encoded by either mammalian genome or higher plant genome. Therefore, yeast
two-hybrid system is widely used in many research fields.
5.2.4.2 Co-immunoprecipitation
Co-immunoprecipitation is based on the specific interaction of antigen-antibody,

which is used to study the physiological interaction between proteins in intact cells.
Many interactions among proteins in the cell non-denatured lysate are preserved.
The basic principle of immunoprecipitation is perceived that the antibodies added to
the lysate form specific immune complexes with the known antigens, and if there
are proteins interacting with the antigens, they will be participating in this forma-
tion. Then the interaction between antigen and other proteins can be verified by
SDS-PAGE, imprinting, or mass spectrometry.
The object of study of co-immunoprecipitation is close to the physiological envi-
ronment of organism, and it is carried out in the natural state of protein interaction,
which can avoid the influence of human factors. However, co-immunoprecipitation
also has some limitations, that is, the target protein needs to reach a certain concen-
tration in order to form precipitation with the antibody, the technique is only suit-
able for the study of the target protein with high expression level, and its sensitivity
is not as high as the affinity chromatography. In addition, co-immunoprecipitation
can only detect soluble protein components and is not suitable for analyzing the
interactions between insoluble macromolecular proteins.
5.2.4.3 Protein Chip
Protein chip, which is evolved from gene chip, is a high-throughput protein analy-
sis technology, also known as protein array or protein microarray, and the research
object of chip technology is protein. The principle of protein chip is as follows:
firstly, the solid phase carrier is treated by special chemical method, and then the
known molecules (antibodies, antigens, enzymes, cytokines) are immobilized on
it; secondly, capture the proteins those can specifically bound to known molecules
according to the biological characteristics of them; finally, analyze the proteins by
laser scanning confocal microscope, atomic force microscope, mass spectrome-
try, or surface plasmon resonance detection techniques to high-throughput deter-
mine of protein-protein interactions or interactions between proteins and small
molecules.
At present, according to the application fields of protein chip, it can be divided
into analytical protein chip and functional protein chip. Analytical protein chip is
mainly used to identify proteins. Functional protein chip is mainly used to study the
interaction between proteins and other molecules.
5.3 pplication of Proteomics in the Study of Lactic Acid

A
Bacteria
5.3.1 pplication of Proteomics in Environmental Adaptation

A
The response of microorganisms to environmental changes is well documented

(van de Guchte et al. 2002; de Angelis and Gobbetti 2004; zhao et al. 2014). Lactic
acid bacteria can be affected by harsh environments in the gastrointestinal tract or
in food processing. In the food industry, stable starter with good food processing
adaptability is generally used. For probiotic strains, the highlight of the research is
their adaptability to harsh environments in order to obtain better protection sys-
tems for strains against harsh environments. In the changeable environment of
temperature, osmotic pressure, or oxidation level, proteomics can be used to ana-
lyze the response mechanism of lactic acid bacteria to harsh environments. In
addition, genomics, transcriptomics, and metabolomics can be combined to pro-
vide new clues to reveal physiological changes in lactic acid bacteria under envi-
ronmental stress. Lactobacillus can induce protein expression to change in stress
response to external stress. The application of proteomics technology, through
revealing the dynamic change of protein expression pattern from proteome level,
helps to elucidate the molecular mechanism of the regulation of lactobacillus
stress response.
140 Y. Xiao et al.
5.3.1.1 Heat Shock Response
High temperature (usually refers to 40~65 °C) can remove the non-covalent interac-
tion in the protein molecules or molecular and lead to its degeneration. Heat shock
stress response is induced by a group of heat shock proteins (HSP), and heat shock
proteins are not commonly used as stress response proteins. After Lactobacillus
casei are heat-treated, the expression levels of chaperone proteins such as GroEL
increased, and the rate of synthesis of total and secreted proteins also changed
(Haddaji et al. 2015). Lactococcus lactis growing at high temperatures has at least a
twofold increase in the expression of its chaperone protein GroESL, compared to
low temperatures (Chen et al. 2015). DnaK, GroEL, and GroES as companion pro-
teins can assist in the refolding of new proteins or denatured proteins (Guisbert and
Morimoto 2013). The kinetics of heat shock protein expression is changed during
heat shock stress reaction. During the initial phase of heat shock stress response,
both DnaK and GroEL were induced to express immediately, while the remaining
heat shock proteins were subsequently expressed. Proteomic analysis showed that
Lactobacillus paracasei NFBC 338 overexpressed its chaperone protein GroEL in
response to heat shock stress. In addition, studies have shown that overexpression of
the chaperone protein GroES and GroEL in Lactococcus lactis and Lactobacillus
paracasei transformants can increase the resistance of lactobacillus to solvents,
especially butanol. This cross-protective effect may be associated with changes in
cell membrane fluidity and membrane protein modification caused by solvent and
thermal pressure (Desmond et al. 2004). Similar heat stress induced by heat shock
proteins has also been reported in other lactobacilli (Fiocco et al. 2007; Parente
et al. 2010).
Heat treatment was performed on Lactobacillus plantarum DPC2 at the middle
stage of exponential growth and the stable stage, and then bidirectional gel electro-
phoresis was performed on the bacteria after heat treatment. The results revealed
that there were 31 and 18 differentially expressed proteins in the strains with
medium growth stage and 18 differentially expressed proteins in stable stage of heat
treatment. And the researchers believe that the heat shock stress response of plant
lactobacillus is a complex process, which involves the activity of chaperone pro-
teins, ribosome stability, and temperature sensitivity (DE Angelis et al. 2004).
Similar protein modifications may occur under different stress conditions, suggest-
ing that many proteins are involved in response to different environmental stresses
(Mills et al. 2011).
5.3.1.2 Acid Shock Response
An important feature of lactic acid bacteria is that saccharides can be converted to

lactic acid by fermentation. Lactic acid can act as a preservative to inhibit the pro-
duction of bacteria during fermentation and contribute to the development of the
overall flavor and texture of the fermented product. Lactic acid bacteria have differ-
ent response mechanisms in response to lactic acid stress during food processing
and low pH environment in gastric juice. At present, the mechanism of resistance to

acid stress in bacteria has been reported (Corcoran et al. 2008).
A variety of lactic acid bacteria have been currently reported to protect against
low pH. Proteomic analysis of Lactobacillus brevis NCL912 under acid stress
revealed 25 differential proteins, mainly pressure stress proteins, DNA repair-
related proteins, protein synthesis-related proteins, and proteins involved in the gly-
colytic pathway, and 18 differential proteins were significantly upregulated, such as
UspA, 50S ribosomal protein L10, and NADP-dependent glyceraldehyde-3-
phosphate dehydrogenase (NADP-GAPDH). These differential proteins play an
important role in the resistance of strains to acid stress (Huang et al. 2011). A study
has recorded that changes in the protein profile of L. plantarum 423 when exposed
to pH 2.5 by using a gel-free nanoLC-MS/MS proteomics approach; the proteins
involved in the synthesis of biomacromolecules were significantly down-regulated;
proteins involved in fatty acid synthesis like FabZ1, FabZ2, FabH2, FabG1, AccD2,
AccA2, and AcpA2 were significantly downregulated, and the expression levels of
Ddl, MurE1, and GlmU in peptidoglycan synthesis were also downregulated under
acid stress. In the acid stress environment, the proteins involved in carbon metabo-
lism also have certain changes. These altered carbon metabolism pathway proteins
have certain effects on cell energy production and intracellular oxidation-reduction
potential. In addition, the accumulation of alkaline substances in cells also has a
certain effect on the environment against acid stress. Proteomic analysis of
Lactobacillus casei Zhang strains in different H systems revealed 33 differential
proteins in which proteins involved in their carbon source metabolism were signifi-
cantly upregulated. These significantly upregulated proteins play an important role
in the energy supply of Lactobacillus casei Zhang strain against acid stress (Wu
et al. 2011). In addition, certain mechanisms can resist acid stress as well as other
environmental stresses. By proteomic analysis of Lactobacillus reuteri under acid
stress, it was found that the expression level of F0F1-ATP synthase was upregu-
lated, while the protein involved in nucleotide and protein synthesis showed a
downward trend, F0F1-ATP synthase can utilize both protons in the cellular envi-
ronment to synthesize ATP and ATP hydrolysis to export protons out of the cell in
order to protect against external acid stress (Koponen et al. 2012). Stress proteins
that are resistant to low-pH environments are also involved in the protection of bile
salts (Lee et al. 2008). Low pH and bile salts are two unfavorable conditions for
lactic acid bacteria to pass through the gastrointestinal tract. Therefore, they can
resist bile salt stress while resisting low pH.
Proteomics studies of the response mechanism of Bifidobacterium longum to
low-pH environments have revealed that low-pH environments had a certain effect
on proteins involved in the glycolytic pathway such as alpha-1,4-glucosidase, glu-
cose phosphate mutase, and UDP-glucose-4 isomerase, the expression levels of
these enzymes were upregulated, and they were involved in the utilization of com-
plex carbohydrates and the fructose-6-phosphate pathway. In addition, higher NH4+
concentrations in the cells can alleviate the intracellular low-pH environment.
Therefore, Bifidobacterium longum responds to the acidic environment by altering
the glycolysis flux and regulating its intracellular pH, whereas bile salt hydrolase
142 Y. Xiao et al.
exhibits a downward trend in acidic environments (Sánchez et al. 2007a). In addi-

tion, Jin et al. (2012) studied the acid tolerance mechanism of Bifidobacterium
longum using gene expression technology. The cell wall integrity and cell mem-
brane permeability of Bifidobacterium longum in the acid environment were
changed to prevent H+ from entering the cell.
The acid adaptation of Lactobacillus delbrueckii subsp. bulgaricus was studied
by proteomics and transcriptomics (Fernandez et al. 2008), and it was found that
many chaperone proteins were expressed during acid stress, such as GroES, GroEL,
HrcA, GrpE, DnaK, DnaJ, ClpE, ClpP, and ClpL, and upregulation of chaperone
expression can assist in the correct folding of intracellular proteins of Lactobacillus
delbrueckii subsp. bulgaricus under acid stress or promote the hydrolysis of mis-
folded proteins. The expression of genes involved in fatty acid synthesis was upreg-
ulated, such as fabH, accC, and fabI; the expression of related genes involved in the
synthesis of isoprenoid compounds was inhibited, such as mvaC and mvaS. Studies
have shown that during acid stress process, Lactobacillus delbrueckii subsp. bul-
garicus subspecies reconstituted pyruvate to fatty acids to alter cell membrane fluid-
ity. However, during the acid stress of some lactic acid bacteria, there were no
significant changes in pressure stress proteins such as GroEL, GroES, DnaK, and
GrpE, which may be due to the fact that the lactic acid bacteria had a certain adapt-
ability to low-pH environments (Koponen et al. 2012). At present, proteomics and
transcriptomics studies on the acid adaptation of Lactobacillus delbrueckii subsp.
bulgaricus found that the protein expression associated with the glycolytic pathway
is upregulated to promote optimal use of carbohydrates and provide for cell growth
and provide energy for strain growing. And it also revealed that the expression level
of its transcriptional regulator Ldb0677 was also upregulated, and the change in its
expression significantly increased the acid tolerance of the strain (Zhai et al. 2014).
5.3.1.3 Cold Shock
Lactic acid bacteria are often in a low-temperature environment during the initial
stages of production and storage or during the food processing stage. It is well
known that low temperature causes a decrease in cell viability and damage to cell
membranes (such as changes in cell membrane morphology and fluidity) and has a
certain effect on replication, transcription, and translation (Passot et al. 2012;
Louesdon et al. 2015). The effect of low temperature on the bacteria also affects the
technical performance of the bacteria, such as bacterial viability or acidification
ability, and ultimately affects product quality.
At present, research on the low-temperature tolerance of lactic acid bacteria is
gradually carried out. It is reported that many bacteria increased their viability in a
frozen environment by applying moderate or different pressures in advance
(Mendoza et al. 2014). Researchers used multidisciplinary approaches, including
comparisons between proteomes. Studies have reported that moderate cooling tem-
perature (26 °C) can improve the low-temperature tolerance of Lactobacillus aci-
dophilus RD758 (Wang et al. 2005). Lactobacillus acidophilus RD758 cells will be
obtained after cold treatment at 26 °C for 8 h optimal low-temperature tolerance.

Under this treatment condition, the ratio of unsaturated fatty acids and saturated
fatty acids increases and can induce the expression of ATP-dependent ClpP protein,
as well as pyruvate kinase and glycoprotein endopeptidase. The ATP-dependent
ClpP protein is required for cell growth under stress conditions and can participate
in proteolysis due to misfolding or breakage due to cold stress.
In an in-depth study, it has been revealed that the cryotolerance of L. bulgaricus
CFL1 has been improved during the 30-min acidification at pH 5.15, which slightly
minimized the acid shock after acidification (Streit et al. 2007). Further analysis of
the proteomic analysis of the bacterial stress of Lactobacillus brevis in Bulgaria
showed that 21 differential proteins were involved in energy metabolism, nucleotide
and protein synthesis, and stress response. However, these differential proteins can
be attributed to increased cold tolerance of the cells after acid stress, changes in
saturated cyclic fatty acid concentrations, and decreased cell membrane fluidity
(Streit et al. 2008).
5.3.1.4 Oxidative Stress Response
Lactic acid bacteria produce certain oxidative stress reactions during fermentation,
drying, storage, or oxidizing substances in the intestines or food. Reactive oxygen
species such as hydrogen peroxide, superoxide anion, or hydroxyl radicals can react
with lipids, proteins, or DNA, which leads to serious consequences for bacterial
growth (Miyoshi et al. 2003; Dijkstra et al. 2014).
When reactive oxygen species (ROS) exceeds a certain level, oxidative stress can
activate the corresponding defense mechanism in bacteria. Arena et al. (2006) used
proteomics to study the oxidative stress response of Streptococcus thermophilus.
Two-dimensional gel electrophoresis, MALDI-TOF-MS analysis, and one-way gel
electrophoresis-liquid chromatography-coupled electrospray ionization mass spec-
trometry were used to analyze differentially expressed proteins under different
stresses. Studies have shown that hydrogen peroxide can promote the expression of
general stress proteins, especially some proteins that can resist oxidative damage,
such as NADH oxidase, manganese superoxide dismutase, iron-sulfur protein, glu-
tathione reductase, Suf B, and Suf C, etc., and it was also found that the expression
levels of related proteins involved in energy metabolism were reduced.
5.3.1.5 Osmotic Stress Response
The substance which causes osmotic stress to lactic acid bacteria is generally a salt
substance added during fermentation or food pickling, such as sodium chloride,
potassium chloride etc., or a saccharide substance. Belfiore et al. (2013) performed
proteomic analysis of Lactobacillus sakei CRL1756 strain under salt stress and
found that the expression levels of 18 differential proteins were upregulated while
the expression levels of some proteins involved in the glycolytic pathway (such as
144 Y. Xiao et al.
Fba, Pgk, Gpm5, and Tpi) were downregulated and the expression levels of stress
proteins and proteins involved in nucleotide synthesis were upregulated. At the
same time, it was pointed out that the addition of glycine-betaine could alleviate
osmotic stress and promote the growth of cells.
Proteomics studies have shown that many genes of Bifidobacterium longum were
regulated by osmotic pressure, in which the expression of the chaperone protein was
upregulated in heat stress and osmotic stress and the expression level of proteins
involved in the metabolism of amino acid-related, nucleotide-related, and glycolysis-
related was also upregulated (Sánchez et al. 2005). In addition, the expression of
proteolytic enzymes of Lactococcus lactis and phospho-glucose transferase system
(PTS system) was inhibited in osmotic stress, while in other stress responses, such
as heat shock stress response or the acid stress response, the expression levels of
these proteins were upregulated (Xie et al. 2004).
The researchers used proteomics to analyze the stress response of Lactobacillus
sakei to osmotic pressure at low temperatures and then constructed protein gene
mutants with upregulated or downregulated expression (Marceau et al. 2004). The
study found that at least six proteins were involved in the environmental stress
response of Lactobacillus sakei in meat processing. The phosphofructokinase
mutant had a reduced survival rate under low ambient temperature of 4 °C or 4%
sodium chloride; likewise, methionine sulfoxide reductase A (MSRA), universal
stress protein (USP family protein), and Alkaline shock proteins (ASP family pro-
teins) were involved in the survival of Lactobacillus sakei at low temperatures.
Moreover, functional genomics derived from the Lactobacillus acidophilus
NCFM genome sketch sequence showed that cell division proteins (Cdp A) were
involved in stress responses in various environments, such as the observed S-layer
proteins and cell wall-associated proteins (Altermann et al. 2004). In addition,
Machado et al. (2004) found that the ratio of saturated/unsaturated fatty acids and
cell membrane cyclopropane fatty acid concentrations in Lactobacillus casei ATCC
393 was changed in 1 mol/L sodium chloride. To overcome the limitations of pro-
teomics in cell membrane and cell wall studies, it is necessary to use a variety of
combinatorial methods to study responses under a variety of stress conditions.
5.3.1.6 Biliary Salt Stress Response
Bacterial bile salt tolerance is an important feature of its survival as it passes through
the gastrointestinal tract. Bile salts are secreted into the intestine during food diges-
tion, and their main function is to emulsify and promote the absorption of fat.
However, bile salts have a bactericidal effect by destroying the cell membrane struc-
ture of the cells, thereby studying the misfolding of proteins and the oxidative dam-
age of DNA. Hamon et al. (2011) used comparative proteomics techniques to
analyze the protein differences between Lactobacillus casei gallbladder-tolerant
strains and bile salt-sensitive strains, and found that differential proteins are mainly
involved in cell membrane-modifying proteins (e.g., NagA, RmlC), cytoprotective
proteins, and detox proteins (e.g., ClpL and OpuA), as well as proteins involved in
metabolism (e.g., Eno, Pky, Pta, etc.). In addition, studies have reported that biliary
efflux can be promoted by regulating the membrane protein composition of the cell
membrane to increase the bile salt tolerance of the strain (Elkins et al. 2001).
Lactobacillus johnsonii PF01 is a strain currently reported to be more tolerant to
bile salts. By proteomic analysis of Lactobacillus johnsonii PF01 strain exposed to
0.1%, 0.2%, and 0.3% bile salts, 215 differential proteins were found, of which 94
proteins were upregulated and 121 proteins are downregulated; these differential
proteins were mainly involved in pressure stress proteins, cell division, transcription
and translation, cell wall synthesis, carbohydrate metabolism, amino acid synthesis,
etc. In addition, bile salt hydrolase can catalyze the early dissociation of bile salts to
reduce the toxicity of bile salts. Under different concentrations of bile salt stress, the
expression of bile salt hydrolase of Lactobacillus johnsonii PF01 was significantly
upregulated (Lee et al. 2012).
Proteomics method was used to study the difference of protein expression of
Bifidobacterium longum under bile salt stress. Forty-four differential proteins were
identified by mass spectrometry. And it was found that these differential proteins
were involved in various aspects of bacterial metabolism by bioinformatics analy-
sis. The response mechanism of bifidobacteria against bile salt stress was proposed:
bile salt hydrolase, bile salt pumping transporter, and some membrane proteins,
such as membrane proteins, which prevent bile salt invasion; general stress response,
glucose metabolism, nucleotides. Bile salt adaptation mechanisms such as synthe-
sis, amino acid synthesis, and transmembrane transport (An et al. 2014). In addition,
bile salts had a certain effect on intracellular redox state-related proteins, and the
result was associated with intracellular NADH and FAD levels (Sánchez et al.
2007b).
5.3.1.7 High-Pressure Stress Response
Nowadays, ultrahigh pressure is increasingly used as a new food preservation

method due to its nonthermal properties and inhibition of harmful microorganisms
and less impact on the sensory quality of food (Rastogi et al. 2007). Juices, oysters,
and cooked meat products are often used for ultrahigh-pressure preservation.
Ultrahigh pressure can affect deadly morphological, biochemical, and genetic
changes in living cells (Wang Sui-lou et al. 2006). The inactivation of bacteria
caused by ultrahigh-pressure process is well documented. The effectiveness of
ultra-high pressure is associated with not only the pressure level and duration but
also the temperature, pH, osmotic pressure, and other factors in the ultra-high pres-
sure process. Some natural pressure-tolerant microorganisms, such as those grown
in deep-sea environments, can also grow at a pressure of 94 MPa (Abe and Horikoshi
2001).
Hörmann et al. (2006) analyzed the proteomics of Lactobacillus sanfranciscen-
sis DSM 20451T under pressure of 80 MPa and found 16 differential proteins, in
which the differential protein similar to Clp protease was closely related to stress.
Two-dimensional gel electrophoresis analysis of low-temperature stress, high tem-
146 Y. Xiao et al.
perature stress, salt stress, acid stress, and starvation stress showed that the pro-
teomics of high-pressure stress strains were similar to low-temperature stress or
NaCl salt stress, and there were 11 identical differential proteins included. It is
believed that there was no specific pressure stress response in the Lactobacillus
sanfranciscensis DSM 20451T. After treatment at 400 MPa, the recovery of the
morphology of two strains of Lactobacillus sakei, an Enterococcus and a Listeria
monocytogenes strain, was strain dependent. Different strains produced different
proteins to resist the stress of ultrahigh-pressure stress, showing different ultrahigh-
pressure tolerance. Transcription factors, proteins involved in protein synthesis, and
proteins involved in energy metabolism were induced to be expressed among the
four species. However, the specificity of some stress proteins in two strains of
Lactobacillus sakei was induced to be expressed. The differential proteins in
Enterococcus were generally involved in energy metabolism, and the differential
proteins of monocyte-producing strains are mostly stress proteins and proteases.
Listeria strains showed the strongest hyper-high-pressure sensitivity, while
Enterococcus was the weakest in hyper-high pressure (Jofré et al. 2007).
The study of the lactic acid bacteria proteome can provide an effective way to
truly understand the life-operating mechanism of lactic acid bacteria, thereby
improving the viability of probiotics under stress conditions and improving the pro-
cess properties, which are beneficial to the better utilization of probiotics by lactic
acid bacteria.
5.3.2 Application of Proteomics in Fermented Food Research
5.3.2.1 Sourdough
There exists a typical complex food ecosystem in the sourdough, and the interaction
of symbiotic Lactobacillus has an impact on species behavior and performance
(Rul and Monnet 2015; Ventimiglia et al. 2015). The molecular mechanism of the
interaction between lactic acid bacteria in sourdoughs was studied using proteomics
techniques (di Cagno et al. 2009; Shevchenko et al. 2014). When Lactobacillus
plantarum DC400 or Lactobacillus brevis CR13 were co-cultured to the late sta-
tionary phase, compared with co-culture growth of Lactobacillus plantarum DC400,
Lactobacillus brevis CR13, or Lactobacillus reuteri A7, Lactobacillus sanfrancis-
censis CB1 has the largest number of dead or damaged cells in a solo medium (di
Cagno et al. 2007). After two-dimensional gel electrophoresis analysis, the expres-
sion level of the induced expression protein of Lactobacillus brevis CB1 was sig-
nificantly increased in the co-culture system with Lactobacillus plantarum DC400
or Lactobacillus brevis CR13. Nineteen overexpressed proteins had been identified,
which were primarily involved in the glycolytic pathway and stress response. On
the one hand, when Lactobacillus sanfranciscensis CB1 and Lactobacillus brevis
CR13 were co-cultured, particularly with Lactobacillus plantarum DC400, dihy-
drofuranosyl-5-ethyl and 5-pentyl were considered to be signal molecules. On the
other hand, when Lactobacillus plantarum DC400 was co-cultured with

Lactobacillus sanfranciscensis DPPMA174 or with Lactobacillus reuteri A7, the
cell viability was not affected (di Cagno et al. 2009). However, two-dimensional gel
electrophoresis analysis showed that the protein expression level under co-culture
conditions was increased in the mid-log phase to the early stationary phase of
Lactobacillus plantarum DC400. Induced expression of the polypeptide had been
identified and was generally involved in energy metabolism, stress response, quo-
rum sensing (adenosylmethionine synthase MetK), and elongation factor Tu (EF-
Tu). When the Lactobacillus plantarum DC400 was co-cultured with other lactic
acid bacteria, the self-inducing polypeptide Pln A can be expressed (di Cagno et al.
2010). When co-cultured with Lactobacillus plantarum DC400, it inhibited the sur-
vival rate of Lactobacillus sanfranciscensis DC400 and Pediococcus pentosaceus.
Compared with the single culture of Lactobacillus sanfranciscensis, its proteomic
group showed 58 differentially expressed proteins when co-cultured with
Lactobacillus plantarum DC400, of which 47 proteins were upregulated and 11
proteins were downregulated.
Similarly, proteomics techniques were used to study the physiological changes
of Streptococcus thermophilus LMG18311 in milk fermentation (Herve-Jimenez
et al. 2008), and proteomics techniques were used to study the fermentation of
yogurt, Streptococcus thermophilus, and bacterium Bulgarian subspecies co-cul-
ture (Herve-Jimenez et al. 2009). During the milk fermentation process,
Streptococcus thermophilus LMG18311 had two distinct growth stages, the second
growth stage of which was significantly different from that of culture alone or co-
culture with Lactobacillus delbrueckii subsp. bulgaricus ATCC11842. When cul-
tured alone, Streptococcus thermophilus LMG18311 had poor growth conditions,
and when co-cultured with Lactobacillus delbrueckii subsp. bulgaricus.
ATCC11842, it could overcome this unfavorable growth condition. The proteomic
analysis of S. thermophilus LMG18311 cultured and co-cultured showed 27 dif-
ferential proteins in two stages of growth, of which 13 proteins were downregulated
and 14 proteins were upregulated. These differential proteins were mainly involved
in the biosynthesis of amino acids, the metabolism of carbon and purine-pyrimi-
dine, the stress regulator RR05, and other proteins of unknown function. Proteins
involved in amino acid synthesis are p rimarily involved in the metabolism of cyste-
ine and methionine. The conversion from homocysteine to methionine and the pro-
teins involved in the cysteine metabolic
pathway were upregulated. Streptococcus
thermophilus LMG18311, when cultured alone or co-cultured, induces the expres-
sion of amino acid and polypeptide transporters and sulfur-containing amino acids.
When co-cultured with S. thermophilus LMG18311 and Lactobacillus brevisii sub-
species, the sulfur-containing amino acid biosynthesis pathway was controlled by
the interaction between Lactobacillus brevisii subsp. bulgaricus ATCC11842 and
S. thermophilus LMG18311. In addition, at the later stage of the growth phase of S.
thermophilus, the expression level of the stress regulator RR05 [is part of the two-
component regulatory factor (2CRS)] was decreased, indicating that S. thermophi-
lus is rapid or normal growth requires the presence of 2CRS.
148 Y. Xiao et al.
5.3.2.2 Fermented Dairy Products
Proteomics technology can study the interaction between fermented dairy products
and microbial proteins, such as in different cheeses, as well as different protein
systems and complex microbial ecosystems. In addition, the main component of
cheese comes from milk, including casein, whey protein, peptides, fats, minerals,
and organic acids (Manso et al. 2005). The typical texture and flavor characteristics
of cheese are mainly derived from protein hydrolysis. Proteomics technology can
delve into the degradation of global casein to the hydrolysis of peptides and amino
acids. Some researchers used high-performance liquid chromatography (HPLC)
online and mass spectrometry to analyze the hydrolytic activity and specificity of
different proteases (Gagnaire et al. 2001). The initial requirement of proteomics
technology was to isolate water-soluble proteins from insoluble cheese and then
separate and purify them to reduce sample complexity (Gagnaire et al. 2004). For
the proteomic analysis of Swiss cheese, the bacterial ecosystem of Swiss cheese is
mainly Streptococcus thermophilus, Lactobacillus helveticus, Lactococcus lactis,
and Streptococcus salivarius. Its protein component analysis identified 62 differen-
tial protein spots, and its differential proteins were mainly involved in hydrolysis,
glycolysis pathway, stress response, and DNA and RNA repair and redox
reactions.
5.3.2.3 pplication of Lactic Acid Bacteria to Produce Aromatic

A
Substances
Fermentation and drying can extend the shelf life and enhance the flavor and nutri-
tional quality of the product. As the main starter, lactic acid bacteria can reduce the
time of fermentation, improve the sensory properties of the product, and inhibit the
growth of pathogenic bacteria and spoilage bacteria. What’s more, Lactobacillus is
also regarded as beneficial microorganisms, which promotes the fermentation in
milk-derived products.
In terms of sensory properties, fermentation products such as cheese or sausage
are ultimately acidified and dehydrated. The lactic acid bacteria carbohydrate fer-
mentation products are mainly lactic acid, especially D-lactic acid. The lactic acid
produced by fermentation reduces the fermentation pH to 5.0, resulting in protein
coagulation and promoting the hardness of the product. At the same time, acidifica-
tion can also reduce the water retention capacity of the fermented food and indi-
rectly contributes to color development. Aromatic qualities such as taste, aroma, and
scent are the most popular characteristics of food. In addition, organic acids pro-
duced during fermentation, such as lactic acid and acetic acid, can also inhibit
pathogens and spoilage bacteria in food.
The aromatic substances of the fermented products are mainly derived from the
metabolism of raw materials such as sugars, proteins, and lipids (Steele et al. 2013).
Aromatic substances are strain dependent, and initial culture conditions have a large
effect on the final flavor characteristics of the product (Kieronczyk et al. 2003).
Food fermentation is usually carried out by mixed culture fermentation of strains of

different species to enhance the flavor characteristics of the fermented food. The
fermentation kinetics of the flora is critical to the fermentation performance. In
early studies, the classical molecular biology method was used to analyze the
changes of the flora in the mixed culture food fermentation. The availability of
genomic sequences and advances in functional genomic technology have facilitated
research between food microbes and food microbial metabolic activities. In this
sense, Sieuwerts et al. (2008) used transcriptomics and proteomics to illustrate the
interaction between microorganisms.
Functional genomics can provide a basis for metabolomics research. From the
entire genome sequence analysis, certain lactic acid bacteria can form aromatic pre-
cursors (Flahaut et al. 2013). In addition, in situ proteomics methods have been used
in research and development in fermentation systems such as milk or cheese. Yvon
et al. (2008) studied the enzymatic activity, proteomics, and metabolomics of aro-
matic substances in Lactococcus lactis subsp. lactis cell extracts during cheese fer-
mentation. Proteomics is relatively less affected, and there were significant
differences in bacterial metabolites produced after 7 days of acid stress and starva-
tion stress. In addition, in mixed culture, proteomics methods are applied to identify
proteins involved in cheese ripening. The proteins of Emmental cheese model were
separated by two-dimensional gel electrophoresis and identified by MALDI-
TOF-MS or de novo sequencing mass spectrometry.Among these differential pro-
teins, about 20 are from Streptococcus thermophilus, 17 are from Lactobacillus
helveticus, and 8 are from the genus Propionibacterium. Some of these differential
proteins were involved in the sensory properties of cheese, such as PepN, PepE,
PepO, and prolyl amino acid enzymes derived from Lactobacillus helveticus. These
studies have shown that proteomics can identify proteins associated with the pro-
duction of aromatic substances in fermented foods.
5.3.3 he Application of Proteomics in the Quorum Sensing

T
Biomes play an important role in the environment through their metabolic diversity
and evolutionary capabilities. Bacteria have a highly complex mechanism for col-
lecting, processing, and transforming environmental information. The diversity of
the ecological niche depends on its ability to sense the external environment and
adapt to the regulation of specific gene expression, generally including microbial
synthesis, release detection, and hormonal small-molecule response. These small
molecules participate in the process called “growth induction” (di Cagno et al.
2011; Popat et al. 2015). Among the Gram-negative strains, quorum sensing is
mainly regulated by two-component LuxI/LuxR and acetyl homoserine lactone, and
Gram-positive bacteria are characterized by a self-inducing polypeptide (AIP) as a
specific community signal. It is speculated that bacteria can effectively transmit and
150 Y. Xiao et al.
receive signals, but the information of these signals is used for a certain purpose
rather than for statistical purposes, which may explain that some organisms have
multiple signaling systems. In the literature reports, language and behavior are often
used to describe “group sensing” (von Bodman et al. 2008). The language or cross
talk among bacteria or between bacteria and animal or plant hosts determines the
outcome of the action (such as beneficial or pathogenic effects). Currently, research
focuses on understanding and interpreting language signals between biomes
(Castillo 2015). Given the large number of extracellular metabolites and the com-
plexity of known quorum sensing signals, genomics and transcriptomics are the
primary methods for studying quorum sensing mechanisms. Genomics provides the
basis for comparative proteomics and functional proteomics. Proteomics shows the
global nature of the proteins expressed in the genome and provides new and simple
insights into bacterial behavior in quorum sensing phenomena. Proteomics can illu-
minate a large number of linguistic signals between biomes through regulatory
mechanisms between different microbial populations. As a follow-up study of tran-
scriptomics, proteomics can perform protein detection, cellular functional entities,
and posttranslational modifications that cannot be predicted by mRNA expression
analysis. In general, quorum sensing can be controlled through the operation of the
posttranslational mechanism.
Probiotics can confer a certain health benefit to the body when given a suffi-
cient amount of probiotics (Bouchard et al. 2015), which must meet two condi-
tions: one is the close interaction between the microorganism and the host, and the
other is the microorganism has to have the ability to adapt to the environment. The
mechanisms by which probiotics regulate health promotion are (1) inhibiting the
growth of pathogens and repairing microbial balance through interactions between
microorganisms, (2) enhancing the barrier function of epithelial cells, and (3)
regulating immune responses (Bastani et al. 2012; Bermudez-Brito et al. 2012;
Hemaiswarya et al. 2013). Quorum sensing molecules show good biological
activity and far exceed their transmission among bacteria, such as the acyl-homo-
serine lactones (AHL) synthesized by Pseudomonas aeruginosa, which has func-
tions of antibacterial, pharmacological, and immunomodulatory activities (Diggle
et al. 2007). One of the important mechanisms is based on the two-component
system, in which transmembrane domain of histidine protein kinase (HPK)
receives signals and the bacteria react correspondingly by sensing the changes of
the external environment through the two-component system. Therefore, the
microflora density colonized in the intestinal tract has high density and diversity.
Studies have speculated that the coordinated adaptation process of intestinal tract
includes the competition and cooperation of nutrients and adhesion sites (Kaper
and Sperandio 2005).
Interactions between probiotics and human gut are mediated by proteins which
were exposed to bacteria’s surface (Remus et al. 2013; Jensen et al. 2014). Therefore,
these proteins are particularly important in assessing the prebiotic properties of cer-
tain bacteria. Surface layer proteins can be divided into covalent and non-covalent
proteins. Covalent proteins include lipoproteins that bind to cell membranes through
N-terminal LXXC sequences (lipid box sequences) as well as proteins that bind to
S-layer through C-terminal PPXTG sequences. Non-covalent proteins include
transmembrane proteins with specific C-terminal repeat sequences or glycine-
tryptophan sequences that bind to lipophosphoric acid or phosphoric acid, as well as
S-layer proteins that bind to cell wall matrix through specific SL domain polysac-
charides. Due to the complexity of cell membrane and the different mechanisms of
binding between proteins and membrane proteins, the related research is
challenging.
In terms of time, Bifidobacterium was first colonized in the intestinal tract.
Bifidobacterium is considered to be the main bacteria that promote health and can
regulate the immune response and maintain the integrity of the gastrointestinal bar-
rier (Leahy et al. 2005; Amund et al. 2014). The factors involved in the interaction
between Bifidobacterium are relatively unfamiliar. It is well known that
S-Ribosylhomocysteinase (LuxS protein) plays a key role in the quorum sensing of
Bifidobacterium longum NCC2705 (Yuan et al. 2007; Wei et al. 2014). Ruiz et al.
studied the physiological characteristics of Bifidobacterium longum NCIMB8809
and Bifidobacterium brevis NCIMB8807 in co-culture conditions (2009). The phys-
iological properties of bifidobacteria were analyzed by two-dimensional gel electro-
phoresis and mass spectrometry under co-culture conditions, it was found that 16
differential proteins belong to Bifidobacterium longum NCIMB8809 and
Bifidobacterium breve NCIMB8807, and 10 of them were up-regulated by ribo-
somal protein expression (Ruiz et al. 2009). Among these 10 differential proteins, 5
are from Bifidobacterium longum and the remaining are from Bifidobacterium
breve. Ribosomal proteins are essential for ribosome assembly and its stability and
can sense changes in the external environment (Rutherford and Bassler 2012). The
expression of transcriptional regulation factor clgR which regulates the expression
of clpC gene and clpP operon was upregulated in Bifidobacterium breve (Lindner
et al. 2007; Milani et al. 2016). Similarly, the expression of glycosyltransferase
which involves peptidoglycan biosynthesis and cell division was upregulated
(Mohammadi et al. 2007). Bifidobacterium brevis may respond to the inhibition of
Bifidobacterium longum by enhancing cell wall biosynthesis. The expression of
three different hydrolytic products of fructose-6-phosphotransferase was upregu-
lated in Bifidobacterium longum. Strains perceive each other and regulate carbohy-
drate metabolism to increase their competitive power.
Yuan et al. (2007) studied the proteomics of Bifidobacterium longum NCC2705
cultured in vitro and in vivo. The expression of 14 proteins was upregulated under
the growth condition of imitating gastrointestinal tract, 4 of which were associated
with membrane fluidity. All the proteins involved in adaptation of Bifidobacterium
longum NCC2705 in gastrointestinal environment are mainly related to stress
response and translation, for example, the extension factor Tu (EF-Tu) can act as an
adhesion factor for Bifidobacterium and promote the attachment of strain. Bile salt
hydrolase (BSH) can promote the interaction between bacteria and gastrointestinal
tract, and more importantly, it can be used as the bacterial stress protein which can
resist the harmful compounds of gastrointestinal tract. LuxS protein can change the
152 Y. Xiao et al.
membrane fluidity, and phosphorylated LuxS protein can act as the mechanism of
interspecies signal interference by enterobacter. In the case of higher bacteria con-
centration and more species in human gastrointestinal tract, the concentration of
signal factor Al-2 is also higher. Two different mechanisms have been proposed for
Bifidobacterium longum NCC2705. The first mechanism is that when a large num-
ber of bacteria are present in the gastrointestinal tract, these signals can be used to
identify the presence of Al-2. The second mechanism is to interfere with the signal
transmission ability of other bacteria through distancing or destroying the Al-2 syn-
thesized by other bacteria.
The secretomics of Bifidobacterium longum NCIMB8809 have been reported,
and its environmental perception information (Sánchez et al. 2008) has been
obtained. The secretomics study found 17 differential proteins, of which 14 pro-
teins have been identified. The identified proteins include two solute binding pro-
tein ABC transporters: one is phosphate binding transporter, belonging to the ABC
transport system, and the other is the cell wall synthase, which is involved in cell
division-specific transpeptidase formed by the diaphragm, the cell wall hydrolase
that invades related homologous proteins, and the protein that catalyzes cell wall
turnover.
Gastrointestinal mucosa adhesion is a common mechanism involving both small
molecules and protein compounds in the process of adhesion (Bron et al. 2013; Gao
et al. 2011; Glenting et al. 2013). Proteomics technique was used to study the cell
aggregation process of Lactobacillus crispatus M247. Compared with the mutant
strain without cell aggregation phenotype, the expression of elongation factor Tu
was upregulated in wild strain, which indicated that Tu was involved in the adhe-
sion process of strain (Siciliano et al. 2008). Lactobacillus reuteri RC-14 alters the
virulence of Staphylococcus aureus by secreting intercellular signaling molecules
(Laughton et al. 2006). In co-culture of two strains, Lactobacillus reuteri RC-14
can secrete one or more molecules to inhibit the expression of staphylococcal
exotoxin.
In addition, bacteria can transmit information with host by quorum sensing sig-
nals, which can be called cross-cutting intercellular signal transduction, and the
signal transduction is almost analyzed by transcriptome technique (Wang et al.
2008; Maeda et al. 2012; Jiang et al. 2013). At present, only the effects of Candida
albicans and farnesol quorum sensing signaling molecules (Scheper et al. 2008;
Décanis et al. 2011) or Lactobacillus plantarum DC400 and pheromone PlnA (di
Cagno et al. 2010) were studied by proteomics. In particular, PlnA can increase the
viability of Caco-2/TC7 cells (human colon cancer cells). Caco-2 cells are usually
used to simulate intestinal mucosa in vitro. Under the condition of culture, caco-2
cells have certain characteristics of morphology and intestinal cell function, mainly
including tight intercellular connections and judging integrity of Caco-2 cells by
measuring transepithelial electrical resistance (TEER). Compared to the negative
test (isolated culture), PlnA significantly increased TEER levels (Yan et al. 2007).
In addition, PlnA signaling molecules can block the damage of interferon γ to
Caco-2/TC7 cells and eliminate the inhibition of cytokines.
5.3.4 pplication of Proteomics in Energy Metabolism

A
of Lactobacillus
Lactobacillus originated about 3 billion years ago during the earth transition period
from anaerobic to aerobic. During its evolution, it did not have the ability to biosyn-
thesis heme (Carr et al. 2002). Porphyrin rings present in cytochrome, peroxidase,
and catalase have respiration (including aerobic and anaerobic respiration) and
detoxification of oxygen free radicals (Levering et al. 2012). Lactobacillus requires
the addition of heme to obtain energy through respiration. The ATP obtained by
fermentation is not enough to support the rapid growth of Lactobacillus, so it is
necessary to change the strategy to obtain energy. There are three main energy pro-
duction pathways in Lactobacillus: amino acid decarboxylation, malic acid decar-
boxylation, and arginine deaminase pathway (ADI pathway). These pathways not
only provide energy but also play a role in pH regulation (Luo et al. 2012). Lactic
acid fermentation can lead to lactic acid accumulation, decarboxylation of amino
acid causes decrease of pH, and the ADI pathway can produce some ammonia.
Different from strict anaerobic bacteria, Lactobacillus can tolerate oxygen with cer-
tain concentration (Brioukhanov and Netrusov 2007). Due to the lack of heme in
Lactobacillus, the defense mechanism of Lactobacillus against oxygen is mainly
based on the accumulation of metals: in fact, some Lactobacillus can accumulate
large amounts of manganese (up to 25mmol/L), selenium, and zinc in cells.
Manganese can act as an operating system similar to manganese superoxide dis-
mutase, selenium can act as selenocysteine system to remove free radicals, and zinc
can form oxygen free radical ion trap (de Angelis and Gobbetti 2004; Vandenplas
et al. 2007; Hosseini Nezhad et al. 2015). These mechanisms make the anaerobic
Lactobacillus have the oxygen tolerance, and Lactobacillus can be used as probiot-
ics to reduce the metal ions in the gastrointestinal tract. Some heterozygous
Lactobacillus can transform oxygen molecules into water through short electron
transport chains (NADH-flavin-O2) (Brooijmans et al. 2007). In this system, acetic
acid is generated by acetyl phosphate, and NADH is oxidized to form NAD+, which
is accompanied by formation of ATP (Carr et al. 2002).
Amino acid decarboxylation can be catalyzed by pyridoxal phosphate or pyruvyl-
dependent decarboxylase (either soluble or membrane bound) (Fernández-Pérez
et al. 2014; Alcántara et al. 2016). In addition to aspartic acid and glutamic acid, all
amino acid can produce alkali compounds called biogenic amines. Although the
decarboxylation of amino acids can reduce the acidity of the system, most of the
amines (tyramine, histamine, and β-phenylethylamine) have different degree of
pathogenicity to the central nervous system or blood vessels (Liu et al. 2013). In
addition, putrescine and cadaverine will affect the sensory properties of food
(Alvarez and Moreno-Arribas 2014). From different perspectives, the decarboxyl-
ation of glutamic acid and aspartic acid generates aminobutyric acid and β-alanine.
The acidity of aminobutyric acid and β-alanine is lower than their precursors, but
they are still acidic compounds and can increase the buffer capacity of pH slightly.
As aminobutyric acid can regulate smooth muscle and central nervous system
154 Y. Xiao et al.
(Inoue et al. 2003), probiotic which can produce aminobutyric acid can be used for
health products. The decarboxylation of acids and amino acids is accompanied by
the electron reverse transport system (Wolken et al. 2006), which can produce PMF
(PMF can synthesis ATP by F0F1-ATP synthase) and neutralize the intracellular
acidic environment. In most Lactobacillus strains, the decarboxylase gene and the
reverse transporter gene are located in the same operon (Capitani et al. 2003). Due
to the acidified environment, some proteins can change their own decarboxylase
conformation to obtain catalytic activity. Therefore, these pathways are comple-
mentary in lactic acid fermentation, and lactic acid accumulation can inhibit the
decrease of pH.
There is ADI pathway which can generate energy in bacteria especially in
Lactobacillus (Brandsma et al. 2012). Arginine deaminase and arginine/ornithine
transporter were the first hypothetical “reverse” urea cycle in which 1 mole of argi-
nine was converted to form 1 mole of ornithine and 2 moles of NH3. However,
activities of arginase and urease were not detected in arginine medium extracts. On
the contrary, three different enzymes were obtained by isolation and purification:
the arginine deaminase (ADI), ornithine transcarbamylase (OTC), and carbamate
kinase (CK). In lactobacillus, these three enzymes are located in the same operator.
1 mol of arginine deimino group forms citrulline, and at the same time, it can pro-
duce 2 mol of NH3 and 1 mol of ATP. Citrulline is formed by carbamoylation to
form ornithine.
Malic acid decarboxylation reaction is accomplished by one-step catalysis of
malolactic enzyme or through the catalysis of malolactic enzyme to form pyruvate
intermediates, which are reduced to form lactic acid (Landete et al. 2013). Malolactic
enzyme and malic enzyme both contain binding sites of Mn2+ and NAD+, and they
are very similar in phylogenetic process. In industry, these decarboxylation reac-
tions are called malolactic fermentation (MLF), which transforms dicarboxylic acid
(malic acid) into monocarboxylic acid (lactic acid), which is more common in wine-
making process and can increase the overall acidity of red wine.
5.3.4.1 Factors Affecting Alternative Energy Access
The complex factors of Lactobacillus are different in each growth phase based on
culture conditions, nutrient availability, strain characteristics, and auxotroph.
According to the analysis of amines and proteomics, there was a positive correlation
between the histamine accumulation and the excessive expression of histidine
decarboxylase (HDC) in Lactobacillus in stable phase (Pessione et al. 2005), and a
similar result was found in Lactococcus lactis NCDO2118 (Mazzoli et al. 2010). In
the contrary, the expression of HDC gene in Lactobacillus hilgardii ISE 5211 in
logarithmic growth phase was analyzed by Western blotting (Landete et al. 2006).
Meanwhile, Mazzoli et al. researched HDC and ornithine decarboxylase in logarith-
mic growth phase, respectively (Mazzoli et al. 2009). PH, carbon source, ATP con-
sumption, and nitrogen source can make certain effects to stable phase. In the whole
fermentation process, there are different forms of mechanism, and different factors
play an important role in different growth phase of bacteria. According to the litera-
ture, both the ADI and MLF pathways of the Lactobacillus sanguices and
S. cerevisiae occur in the growth phase of the bacterial index (de Angelis et al. 2002).
Carbohydrates can affect the decarboxylation pathway of amino acids and the
output of Oenococcus oeni histamine in the absence of carbohydrates. It is reported
that glucose does not affect the catalysis of HDC in Oenococcus oeni, and the bio-
synthesis controlled HDC operon was proposed (Coton et al. 2010). In addition,
Lactobacillus activates the HDC pathway during the stable phase when glucose is
depleted, and glucose and fructose inhibit the expression of HDC operon in
Lactobacillus hilgardii, Oenococcus oeni, and Pediococcus parvulus. Moreno-
Arribas et al. reported that in the presence of fructose or fructose and L-malic acid,
the accumulation of tyramine reached the maximum (Moreno-Arribas et al. 2003).
The accumulation of tyramine reached the maximum during the exponential phase
of Enterococcus faecalis DISAV1022 (Pessione et al. 2009). The effect of carbohy-
drates on the decarboxylation pathway of amino acids has not been fully
elucidated.
For amino acid decarboxylation, the ability of tyrosine decarboxylation of
Lactobacillus brevis to form tyramine was increased tenfold and the activity of TDC
in MRS medium was increased twofold (García-Ruiz et al. 2011). In the chemically
defined medium (CDM) without tyrosine and phenylalanine, the tyramine produc-
tion of Enterococcus faecalis DISAV1022 was only slightly increased, and the
2.5 mmol/L tyramine was obtained from CDM supplemented with 2.5 mmol/L
tyrosine and 14 mmol/L phenylalanine (Pessione et al. 2009).The biosynthesis of
tyrosine decarboxylase was found by studying the membrane proteomics of
Enterococcus faecalis DISAV1022 strain. In Oenococcus oeni, the accumulation of
cadaverine was accompanied by the increase of lysine, ornithine increased the accu-
mulation of putrescine, and 100% of ornithine was converted to putrescine (Guerrini
et al. 2002). It has been proven that histidine can induce histamine accumulation in
Lactobacillus 30a and Lactobacillus hilgardii ISE5211. The increase of histamine
was related to the biosynthesis of histidine in HDC operon, and the constitutive
expression level of HDC was significantly increased. In addition, Landete et al. used
Northern blotting to investigate Lactobacillus hilgardii, Oenococcus oeni, and
Pediococcus parvulus and found that histidine had the same inductive effect on
HDC operon (2007). In contrast, histidine can feedback inhibit the expression of
HDC operon and the catalytic activity of HDC. The experimental study of CDM in
Lactococcus lactis NCDO 2118 showed that the amount of biogenic amine-
aminobutyric acid produced by glutamic acid decarboxylation was very small,
which indicated there was no amino acid precursor. It is possible that glutamine is
converted to glutamate in CDM, and glutamate can be decarboxylated to form ami-
nobutyric acid (Mazzoli et al. 2010). Although the differential expression of gluta-
mate decarboxylase has not been demonstrated by proteomics or transcriptomics
techniques, the enhancement of aminobutyric acid biosynthesis can catalyze the
regulation of glutamate decarboxylation. Therefore, when the constitutive expres-
sion level of the amino acid decarboxylase is low, or is completely induced to
156 Y. Xiao et al.
express, the precursor amino acid has a core role, which can activate the biosynthe-
sis of the amino acid decarboxylase.
In Lactococcus lactis, ADI enzyme and OTC enzyme are induced by arginine in
the ADI pathway, while CK is constitutively expressed. In other dairy strains, such
as Lactobacillus buchneri and Enterococcus faecalis and so on, the expression of
ADI, OTC, and CK is induced by arginine, which is similar to the strain in wine. In
contrast, ADI, OTC, and CK are all constitutive expressions in Oenococcus, and
transcriptome analysis shows that arginine can enhance the transcription of ADI,
OTC, and CK encoding genes (Tonon et al. 2001). In addition, it was found that the
reverse transporter arcD involved in arginine/ornithine exchange was constitutively
expressed through real-time quantitative PCR. Arginine can promote the formation
of putrescine in Oenococcus oeni. Agmatine is formed through arginine decarbox-
ylation, and then agmatine is converted to putrescine. Not all ornithine is used for
reverse transport in the ADI pathway, and some of ornithine is decarboxylated to
form putrescine. Arginine has a certain influence on the ADI pathway of Lactobacillus
ft., and it was used in rye and wheat bread industry to produce bread by sourdough
and Italian cake. The products of trypsin hydrolysates were analyzed by two-
dimensional gel electrophoresis and peptide sequencing, and the 17mmol/L argi-
nine could increase the expression of ADI, OTC, and CK by tenfold, fourfold, and
twofold, respectively (DE Angelis et al. 2002).
5.3.4.2 Interrelationship of Energy Generation Pathways in Lactobacillus
The effects of carbohydrate and acid compounds (including L-malic acid) on the
decarboxylation of ADI/MLF and amino acids have been reported commonly, but
the interactions between these production pathways have been studied rarely.
Researchers have studied Lactobacillus that produce amines in foods to control the
production of biogenic amines. Comparative proteomics is often chosen as the
method for the determination of enzyme expression and can help to understand the
complex networks of different metabolic pathways existing in bacterial biological
systems.
1. Proteomics of the effect of amino acids on glucose metabolism
At present, a large number of studies have focused on the regulation of carbohy-
drates to amino acid metabolism, while there are few reports on the effects of amino
acids on glycolysis pathway (Embden-Meyerhof) and pentose phosphate pathway.
Adding histidine and ornithine to the growth medium of Lactococcus lactis could
inhibit the synthesis of Embden-Meyerhof pathway enzyme and glyceraldehyde
3-phosphate dehydrogenase II, compared with the control group, and the expression
of both enzymes decreased by sevenfold (Pessione et al. 2005). The results indi-
cated that glucose metabolism can be regulated by other energy production path-
ways. On the contrary, the research of Lactobacillus hilgardii ISE5211 by
heterolactic fermentation in wine showed that there was no effect on the glucose
metabolism during the growth phase under the condition of excessive histidine
(Mazzoli et al. 2009; Lamberti et al. 2011). Transcriptomics and proteomic analysis
of homofermentative Lactococcus lactis NCDO 2118 producing aminobutyric acid

showed that glycolysis and glutamic acid decarboxylation were carried out at the
same time, with or without glutamic acid (Oliveira et al. 2014). Comparison of pro-
teomics and transcriptomics analysis showed that the expression of phosphoglycer-
ate mutase, 6-phosphofructokinase, and glyceraldehyde-3-phosphate dehydrogenase
was upregulated, but the expression of pyruvate kinase, phosphoglycerate kinase,
and fructose diphosphate aldolase was downregulated. Whether it is activating or
inhibiting the glucose metabolism pathway, glutamic acid has a fine-tuning effect
on the glucose metabolism pathway. On the contrary, Lactobacillus hilgardii, which
has adapted to unfavorable growth conditions (low pH and high ethanol concentra-
tion), can grow in the pH range of 3.8–5.8 in wine. In addition, since Lactobacillus
hilgardii is a kind of heterotypic lactic acid fermentation, the ethanol can make
contribution to balance the production of lactic acid. In this case, the production of
histamine can replenish energy demand through heterolactic fermentation (one mol-
ecule of ATP can be produced in each cycle) (Dias et al. 2015).
In addition, in Lactococcus lactis and Lactobacillus, there is a unique photoregu-
lation phenomenon. The maximum yield of biogenic amines was obtained in the
stable growth phase, indicating that the amino acid decarboxylation reaction was
rapid, and pH had no effect on the biosynthesis of biogenic amines (Mazzoli et al.
2010). When the main carbon sources (such as glucose) are depleted, ornithine,
histidine, or glutamic acid decarboxylation reactions are performed to replenish the
energy through the electron reverse transporter. It is worth mentioning that gluta-
mate will be decarboxylated to aminobutyric acid which is still acid, causing only a
little alkalinization. Thus, in these cases, amino acid decarboxylation seems to solve
energy problems. Tyramine and β-phenylethylamine have a strong buffer capacity,
and proteomics and metabonomics research can be used to further study the effects
of pH on the production of biogenic amines.
2. Proteomics on the effects of amino acids on ADI pathway and amino acid
decarboxylation
The ADI pathway was first found in Enterococcus faecalis, and tyrosine and
phenylalanine cannot regulate it (Barcelona-Andrés et al. 2002). Compared with
Lactococcus lactis, Lactobacillus hilgardii, and Oenococcus oeni, Enterococcus
faecalis has the strongest acid resistance. Therefore, the ADI pathway and the amino
acid decarboxylation reaction are carried out simultaneously to produce enough
alkaline substances (ammonia, tyramine, β-phenylethylamine) to maintain the opti-
mal growth pH and ensure efficient glycolysis pathways that provide sufficient
energy. During the production process of aminobutyric acid by Lactococcus lactis
NCDO 2118, there is a slight negative regulatory mechanism in the mRNA and
protein levels (Mazzoli et al. 2010). In contrast, the biosynthesis level of aminobu-
tyric acid increased in arginine-containing media, which may be due to the oxida-
tive deamination of ornithine (the metabolite of arginine) to form glutamic
semialdehyde which is easy to produce glutamate by oxidation. Therefore, the two
energy metabolic pathways of arginine deamination and glutamate decarboxylation
may be simultaneous, but there are specific regulatory effects. Considering the opti-
mal growth pH of Lactococcus lactis, the transformation of glutamic acid into ami-
158 Y. Xiao et al.
nobutyric acid (which can produce a small amount of alkaline substances) may be
more inclined to utilize ADI pathway to produce ammonia (Mazzoli et al. 2009). In
Lactobacillus hilgardii ISE5211, histidine was not transformed into histamine by
ADI pathway. The proteomics results showed that histidine could inhibit the expres-
sion of ADI enzyme. In this case, arginine in the medium inhibited the decarboxyl-
ation of histidine (Mazzoli et al. 2009). In addition, SDS-PAGE result showed that
HDC was greatly affected by arginine. There was a competitive relation between
ADI pathway and histidine decarboxylation in Lactobacillus hilgardii ISE5211.
Comparative proteomics can provide some information under the condition of
excessive amino acids regardless of the interaction between energy sources. The
interaction between stress chaperone and protease has been reported gradually
(Dougan et al. 2002). Chaperone can not only help the folding of new protein but
also prevent the misfolding of protein and help the refolding of damaged proteins
under stress conditions. In addition, chaperone can cooperate with protease to
destroy the irreversible damage protein, and chaperone can obtain protease proper-
ties by activating proteolytic sites (Moliere and Turgay 2009). Therefore, it has been
proven that stress response and proteolysis are closely related. In the study of
Enterococcus faecalis DISAV1022 grown in tyrosine and phenylalanine, it was
found that stress protein DnaJ and Gls24, protease, glutamyl aminopeptidase (Pep
A), and some ABC transporters like glycine-betaine-carnitine-choline-binding pro-
tein were overexpressed. Glycine-betaine-carnitine-choline-binding protein is the
first protective mechanism against osmotic pressure. Lactobacillus can respond to
hypertonic or hypotonic stress by accumulating or releasing osmotic agents (beta-
ine) (Konings 2006). In contrast, when Lactococcus lactis NCDO 2118 grew in the
condition of glutamate excessively, the expression of glutamyl aminopeptidase, Clp
P protease, and other stress proteins (such as superoxide dismutase and the Cts R)
was downregulation detected by proteomics and transcriptomics analysis. And Cts
R regulates the expression of Clp gene under stress conditions in Lactococcus lactis
and Oenococcus oeni (Grandvalet et al. 2005).
The regulation of productivity pathway on protein network in Lactobacillus
(homolactic fermentation, heterolactic fermentation, lactic acid fermentation, amino
acid decarboxylation, and arginine deamination pathway) is complex. At the same
time, the cellular response of Lactobacillus that is exposed to different substrates is
not only species-specific, but also strain-specific, and there is a certain hierarchical
structure of energy utilization. Transcriptomics and proteomics techniques can pro-
vide information about energy metabolism pathways.
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Chapter 6
Metabolomics of Lactic Acid Bacteria
Wanqiang Wu and Nan Zhao
6.1 Overview of Metabolomics
6.1.1 Metabolomics
Metabonomics/metabolomics, developed in the late 1990s, is a science that deals

with the types, quantities, and changes of metabolites (molecular weight <1000) of
living organisms after different interferences (such as genetic engineering and
changes in growth environment), and it is an important part of systems biology.
According to different research purposes, metabolomics is classified as a nontar-
get metabolic profiling and metabolite target analysis (Link et al. 2012). The former
focuses on getting as many metabolites as possible in one sample treatment and then
carries out comprehensive qualitative and semiquantitative analysis. The latter
adopts accurate and sensitive qualitative and quantitative analysis techniques for
selected target metabolites. Target metabolites usually refer to a group of similar
substances, a collection of all intermediates in a metabolic pathway, or markers of
multiple metabolic pathways. According to the nature of the samples, they were
divided into metabolic fingerprinting and metabolic footprint analysis. The former
generally studies endometabolomes; the latter studies extracellular metabolites
(exometabolome) (Mashego et al. 2007).
After obtaining the information of metabolites through a series of analytical
techniques, it is the core purpose of metabonomics to establish the relationship
between these substances and the metabolic network in organisms. At present, there
are KEGG, BioCyc (including EcoCyc and MetaCyc databases), BRENDA, and
W. Wu (*)
N. Zhao
Sichuan Academy of Agricultural Sciences, Chengdu, China
https://doi.org/10.1007/978-981-13-7832-4_6
168 W. Wu and N. Zhao
WIT databases, which have their own characteristics. When conducting metabolite
pathway analysis, appropriate databases can be selected according to needs. Weiss
and Kim (2012) argue that genomics and transcriptome predict what may happen,
proteomics predict what will happen, and metabolomics indicate what has already
happened. Because of the need of bioinformatics, the combination of genomics,
transcriptome, proteomics, and metabonomics can provide a more comprehensive
understanding of the overall regulation of organisms and bring overall regulation
and guidance to production and scientific research.
6.1.2 pplication of Metabonomics in the Field of Microbial

A
Research
Metabonomics, as a new science and technology, has been more and more valuable
in microbiology research, such as metabonomics and bioengineering (flux analysis,
flux-genome combination, dynamic analysis), identification of microbial species,
microbial environmental stress, single-cell level metabonomics analysis and intesti-
nal flora research, and so forth.
6.1.2.1 Metabonomics Analysis of Biological Transformation Effect
Metabolic flux analysis (MFA) is a basic method for metabolic network analysis,
including stoichiometric MFA, isotope labeled unsteady 13C-MFA (Antoniewicz
2015). Significant changes in metabolic flux indicate that the effect of biotechno-
logical transformation of strains is explained and validated from the overall point of
view of metabolic network.
6.1.2.2 Metabolic Flux Analysis Guided Molecular Modification
By analyzing and calculating the metabolic flux, we can reveal the specific flow
direction of intracellular metabolites and characterize the metabolic capacity net-
work of cells. In the meanwhile, we can not only discover the effect of molecular
modification on cellular metabolic status but also accurately describe the impor-
tance of some metabolic pathways and identify the control nodes of cellular meta-
bolic pathways.
6 Metabolomics of Lactic Acid Bacteria 169
6.1.2.3 Optimization Control of Fermentation Process
Fermentation process optimization is an effective means to improve the level of

microbial beneficial substance products. Combining with metabolomics analysis
method, fermentation process can be regulated purposefully according to the cor-
responding metabolic situation.
6.1.2.4 Metabolic Characteristics Analysis
Different microorganisms have their unique metabolites and pathways. Combining

metabolomics technology to study the metabolic characteristics of microorganisms
will provide abundant information for industrial production and basic research.
6.1.2.5 Environmental Stress
Metabonomics can reflect the response of organisms to gene and environmental

changes. The ability of microorganisms to adapt to the environment or the utiliza-
tion of different substances can be studied by giving different environmental stimuli
to microorganisms.
6.1.2.6 Identification of Microbial Species
Microbial metabonomics directly reflects the actual metabolic characteristics of

organisms and the influence of genes and environment on them. It can quickly and
accurately identify different kinds of microorganisms by analyzing the difference of
microbial metabolism.
6.2 Metabolomics Research Technology
6.2.1 Platform for Metabolite Analysis
At present, GC-MS, LC-MS, and NMR are the most commonly used analytical
methods in metabonomics.
6.2.1.1 Gas Chromatography-Mass Spectrometry
Gas chromatography-mass spectrometry is also called as GC-MS. When GC-MS is

separated, the sample must be gasified first, and the capillary column is programmed
to warm up. According to the volatility of the substance, the retention time in the
capillary is different to achieve the purpose of separation.
6.2.1.2 Liquid Chromatography-Mass Spectrometry
The volatility and thermal stability of samples are not required by liquid chromatog-
raphy, and derivatization is not required. Simple pretreatment (such as enrichment
and filtration) can be used to detect the volatility of samples, which has a wider
application range.
6.2.1.3 Nuclear Magnetic Resonance
At present, hydrogen spectrum (1H-NMR), carbon spectrum (13C-NMR), and phos-

phorus spectrum (31P-NMR) are commonly used.
6.2.2 Data Analysis
For the target material, only the raw data detected by the instrument are obtained.
For nontarget substances, the main multidimensional analysis methods are unsuper-
vised and supervised. The unsupervised methods include cluster analysis (HCA)
and principal component analysis (PCA). The supervised methods include (orthog-
onal) partial least squares discriminant analysis ((O) PLS-DA), artificial neural net-
work (ANN), and support vector machine (SVM) (Putri et al. 2013).
6.3 pplication of Metabolomics in the Environmental Stress

A
Research of Lactic Acid Bacteria
Whether in the food industry or as a probiotic preparation, lactic acid bacteria would
go through the digestive tract of the host with a series of environmental stresses
which include the changes of energy substances, extreme pH, temperature and high
osmotic pressure, oxidative stress, and so on. The gene regulation induced by exter-
nal environmental stress factors not only affects the physiological state and proper-
ties of the cells but also indirectly affects the formation of their metabolic substances,
and thereby affects their fermentation characteristics and physiological functions.
Since the bacterial stress response under environmental stress may lead to the
change in its metabolic pathway, the comparison of the metabolite differences of
microorganisms under different environmental stress states and the further clarifica-
tion of differential metabolic pathways of different metabolites are essential strate-
gies to reveal the stress strategy of microorganisms under microbial stress states.
The utilization of metabolomics technology can comprehensively and dynamically
compare the change of microorganisms under different environmental stresses and
find out the change of corresponding metabolic pathways and can explore the toler-
ance and adaptation mechanism of lactic acid bacteria at the molecular level.
There are relatively few reports on metabolomics in the study of environmental
stress mechanisms in lactic acid bacteria. Lippert and Galinski (1992) found that the
synthesis of trehalose by the bacteria as a compatible solute is resistant to environ-
mental stress by lactic acid bacteria. In recent years, some scholars have found that
environmental stress metabolism changes of lactic acid bacteria affect the flavor
substances of fermented foods. Settachaimongkon et al. (2015) used metabolomics
to study the changes of metabolites under the sublethal pressure of lactic acid bac-
teria and found that the metabolites of lactic acid bacteria under stress state changed
significantly, which further dramatically affected the sensory quality of the fer-
mented yogurt. Settachaimongkon et al. (2016) measured the volatile and nonvola-
tile substances of Lactobacillus plantarum WCFS1 under the pressure of high salt
and low acid and found the potential effect of environmental stress on the metabo-
lites of Lactobacillus plantarum WCFS1, some of which were related to flavor. In
summary, the application of metabolomics in environmental stress research is a
relatively new technology platform compared to transcriptomics and proteomics.
Therefore, the technology still has great potential to explore the mechanism of
stress of lactic acid bacteria under different environmental stresses and the impact
of stress on production.
6.4 pplication of Metabolomics in the Study of Fermented

A
Foods of Lactic Acid Bacteria
As a common strain in fermented foods, lactic acid bacteria were involved in the
production of many traditional fermented foods, such as dairy products, soy prod-
ucts, and some pickled foods. The metabolism of the lactic acid bacteria was closely
related to the flavor, aroma, texture, and nutrition of fermented food. With the devel-
opment of metabolomics technology, metabolomics has been widely used in various
studies such as the fermentation of lactic acid bacteria and the formation of charac-
teristic flavor substances.
Considering the metabolites including organic acids, amino acids, oligosaccha-
rides, small peptides, and some aromatic substances produced in the fermentation of
lactic acid bacteria, it is important to reveal the relationship between these metabo-
lites and the environment with the purpose of improving the quality of fermented
foods and optimizing the fermentation process. Due to its comprehensiveness and
high resolution, metabolomics can comprehensively evaluate and discover subtle
differences between samples, thus providing qualitative and quantitative data for the
fermentation of lactic acid bacteria, the optimization of fermentation process, and
the improvement of product quality.
6.4.1 pplication of Metabolomics in Fermented Dairy

A
Products
The application of lactic acid bacteria in fermented dairy products is very wide,
including yogurt, cheese, and kefir and so on. In those fermented dairy products,
Lactococcus, Lactobacillus, Pediococcus, Enterococcus, and Leuconostoc are com-
monly used, which can metabolize lipids, sugars, and proteins in milk and gradually
degrade the above substances into small molecule compounds, providing a unique
taste and flavor to fermented dairy products. In recent years, in order to more pre-
cisely control the fermentation process of lactic acid bacteria, metabolomics tech-
nology has been widely used in the research of fermented dairy products, aiming to
find out the relationship between the texture, taste, and nutrition of lactic acid bac-
teria and fermented dairy products. Mazzei and Piccolo (2012) used NMR technol-
ogy to study the flavor and nutritional characteristics of the famous mozzarella
cheese with special flavor. It was found that mozzarella cheese produced more iso-
butanol, lactic acid, and acetic acid than the common cheese during the fermenta-
tion process, and metabolomics research can clearly identify the mozzarella cheese
produced in specific region. Piras et al. (2013) used metabolomics to study the role
of lactic acid bacteria in the ripening process of traditional Italian Fiore Sardo
cheese. As a result, it was found that the lactic acid bacteria formed by natural fer-
mentation had a significant difference in flavor compared with the commercial lac-
tic acid bacteria starter, and the commercial lactic acid bacteria starter could not
produce the designated product of origin protection certified by the EU agricultural
product inspection agency. Ochi et al. (2012) adopted GC-TOF-MS to detect the
hydrophilic small molecule metabolites of 13 cheeses and linked this quantitative
result to the sensory quality of cheese, providing a material target for future sensory
quality optimization. Rodrigues and Antony (2011) used NMR techniques to com-
prehensively evaluate the fingerprint of prebiotic-containing cheese and obtained a
metabolite map capable of determining the maturity of the cheese.
Settachaimongkon et al. (2014) showed that in co-inoculation of nonprotein
hydrolyzing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bul-
garicus in bovine milk, both strains can achieve higher growth, and milk acidifica-
tion speed is faster. The content of the flavor metabolite of the lactic acid bacteria is
also high. The study also showed that the metabolite profile of microorganisms
during fermentation can determine the typing and quality of coagulated yogurt.
Palomo et al. (2014) studied the characteristics of metabolites of lactic acid bacteria
in selenium-enriched yogurt, indicating that selenium is distributed in selenocyste-

ine, thioredoxin, glutaredoxin, albumin, β-lactoglobulin, and lactoperoxidase.
Selenium may be related to the expression of heat shock protein and chaperone.
With the wide application of metabolomics, the study on the identification of bio-
markers and specific metabolic profiles of several special cheeses, as well as the
relationship between these substances and lactic acid bacteria, will be more in-depth
and comprehensive, which provided theoretical bases for the scientific assessment
and deep utilization of lactic acid bacteria fermented foods.
6.4.2 pplication of Metabolomics in Fermented Vegetable

A
Products
The lactic acid bacteria commonly found in fermented vegetables are

Lactobacillus. The metabolism of lactic acid bacteria provides organic amino
acids, small peptides, and some aromatic substances to fermented vegetables. In
addition, lactic acid bacteria also have the effect of inhibiting bacteria and pre-
venting vegetable spoilage during vegetable fermentation. Jung et al. (2012)
combined the nuclear magnetic resonance technique with high- throughput
sequencing technology to study the effects of Leuconostoc mesogenes as a starter
on the microbial flora and main flavor substances in kimchi fermentation process.
The results showed that Leuconostoc mesophila not only had an effect on the
bacterial phase change during the fermentation of kimchi but also had an effect
on the production of some of the main flavor substances of the fermented vegeta-
bles. Subsequently, another scholar also studied the role of red pepper powder in
kimchi through the combination of nuclear magnetic resonance technology and
high-throughput sequencing technology. The results showed that the addition of
red pepper powder during kimchi fermentation prolonged the fermentation lag
phase. Moreover, the abundance of Weissella was significantly higher than that of
the other two lactic acid bacteria (Larixella and Lactobacillus) (Jeong et al.
2013). Zhao et al. (2016) obtained the iconic taste substances in traditional old
brine pickles based on the global metabolomics strategy and established the rela-
tionship between these target flavor substances and lactic acid bacteria groups by
means of targeted metabolomics.
6.4.3 pplication of Metabolomics in Fermented Soybean

A
Products
Fermented soy products are traditional foods that are fermented by microorganisms
based on soybeans or black beans. It has a long history in Asian countries such as
China, South Korea, and Japan. Fermented soy products are considered to be highly
nutritious and therefore more and more popular. The lactic acid bacteria involved in
the fermentation process of soybean paste mainly include Lactobacillus plantarum,
Lactobacillus brevis, Lactobacillus sakei, Lactobacillus fermentum, and Tetra-
cocci, which produce important flavor substances such as lactic acid, amino acids,
monosaccharides, and amino acids through the metabolic activities of these lactic
acid bacteria.
Kim et al. (2012) conducted a global metabolomics study on the fermentation
process of fermented soybean products by GC-TOF-MS and CE-TOF-MS. The
results revealed the global metabolic pathway associated with the metabolism of
sugar and amino acids of the soybean products. Kang et al. (2011) monitored the
process of the accumulation of marker flavors in meju fermentation using UPLC-Q-
TOF MS, which provided a theoretical basis for the control of meju fermentation.
Namgung et al. (2010) monitored the dynamic changes of metabolites in the fer-
mentation process of Korean traditional soybean paste. The results showed that the
eight amino acids including alanine increased significantly after fermentation in the
fermentation process, five kinds of fatty acids including palmitic acid are high in the
whole fermentation process, and organic acid metabolites mainly include carbonic
acid, citric acid, and lactic acid. Lee et al. (2014) used metabolomics to examine the
monosaccharides, amino acids, and fatty acids, as well as isoflavones and soy sapo-
nins, in different fermentation stages of doenjang, including cooking, drying, fer-
mentation, salt soaking, and post-ripening.
6.4.4 pplication of Metabolomics in Lactic Acid Bacteria

A
Fermented Drink
Lactic acid bacteria also play an important role in the fermentation of some fer-
mented beverages and wines. Lee et al. (2009) used Oenococcus oeni for Meoru
(Vitis coignetiae) wine malolactic fermentation (MLF), PCA, and orthogonal par-
tial least squares discriminant analysis (OPLS-DA) results. There are no significant
differences in the O. oeni primary products of different strains, but some important
volatile flavors in secondary metabolites (such as 2-phenylethanol, isoamyl alcohol,
2-butanol, and ethyl octanoate) have significant differences, and these substances
are closely related to the quality of the wine. In recent years, many researchers have
used metabolites to study the effects of the interaction between yeast and lactic acid
bacteria on the fermentation process of wine. Son et al. (2009) showed that the addi-
tion of lactic acid bacteria to the fermentation system of Bayesian yeast resulted in
a decrease in the content of malic acid and citric acid and an increase in lactic acid
content. The increase in succinic acid content indicated the inhibition effect of MLF
by S. bayanus. Lópezrituerto et al. (2012) studied the metabolite characteristics of
ethanol fermentation and MLF metabolic process with nine commercial wine sam-
ples. The NMR combined with interval extended canonical variate analysis (iECVA)
method was used as the screening method. At last, they identified that isoamyl alco-
hol and isobutanol are biomarkers to distinguish the authenticity of La Rioja wines.
6.5 pplication of Metabolomics in the Study of Interaction

A
Between Lactic Acid Bacteria and Host
6.5.1 valuation of Probiotic Function of Lactic Acid Bacteria

E
Based on Metabolomics
In addition to imparting flavor to fermented foods, lactic acid bacteria also provide
fermented foods with many probiotic properties. In recent years, the health effects
of lactic acid bacteria on regulating the balance of human intestinal flora have been
paid more and more attention by researchers at home and abroad. Metabolomics is
used to monitor the dynamics of animal and human metabolites and to explore lactic
acid bacteria and intestinal flora. The role of interaction in the development of meta-
bolic diseases such as obesity, colon cancer, and diabetes has become a new research
hotspot. Intestinal flora has been valued by researchers as an important part of the
human body, and intestinal microbes are increasingly found to be associated with
many chronic diseases (Nicholson et al. 2005). As a normal intestinal flora, lactic
acid bacteria can improve the health of the host by regulating the structure and
metabolism of the intestinal flora. Martin et al. (2008) showed that lactic acid bac-
teria can regulate the structure and function of intestinal microbes to affect the meta-
bolic products of the host gut, such as short-chain fatty acids (citric acid, acetic acid,
and butyric acid), succinic acid, lactic acid, and dimethylamine in liver extracts,
choline in plasma extracts, and choline, acetic acid, and bile acids in stool extracts,
thereby improving the health of the host. Hong et al. (2010) used nuclear magnetic
resonance (NMR) techniques to evaluate the effects of lactic acid bacteria on colitis
mice. The results showed that probiotics not only upregulated cytokine expression
but also promoted short-chain fatty acids (butyric acid, acetic acid, and propionic
acid) and amino acids in plasma (isoleucine, valine, alanine, lysine, tyrosine),
nucleotides (uracil and hypoxanthine), and lactic acid content. The physiologic cor-
relation of intestinal flora changes under the action of lactic acid bacteria can be
directly detected by metabolomics, which provides an effective tool for further elu-
cidating the probiotic mechanism of lactic acid bacteria.
6.5.2 pplication of Metabolomics in the Evaluation

A
of Probiotic Effects of Fermented Foods Based on Lactic
Acid Bacteria
As the main fermentation strain of many fermented foods, lactic acid bacteria give
many probiotic properties to fermented foods. As a comprehensive material investi-
gation method, metabolomics is used to clarify the probiotic effect evaluation of
lactic acid bacteria fermented foods for the host. Zheng et al. (2015) used metabo-
lomics to make a more comprehensive and detailed evaluation of the probiotic
properties of cheese. By comparing the effects of milk and cheese on human

metabolism, it was found that intaking of cheese significantly decreased the levels
of citric acid, creatine, and creatinine in the urine, but significantly increased the
levels of the metabolites related to intestinal microbes (such as butyric acid, hip-
puric acid, and malonate). Correlation analysis found that intaking of cheese regu-
lated blood cholesterol levels by altering microbial metabolism and lipid
metabolism, which revealed the probiotic properties of cheese through metabolo-
mics studies.
There are many fermented foods based on lactic acid bacteria, and most of them
have a long history. Most of these fermented foods are considered to be beneficial
to the health of the host, but many have no specific evidence and no specific evalu-
ation indicators. Therefore, the comprehensive evaluation of the probiotic charac-
teristics of fermented foods based on metabolomics provides a theoretical basis for
the development of probiotic fermented foods.
6.6 pplication of Metabolomics in Lactic Acid Bacteria

A
Fermentation Engineering
6.6.1 verview of Lactic Acid Bacteria Fermentation

O
Engineering
Lactic acid bacteria are not only used as fermenting agents in fermented foods but
also as an important industrial strain, which is used in the production of various
natural products, such as Leuconostoc mesenteroides produce glucan and L-lactic
acid, Lactobacillus plantarum produce conjugated linoleic acid and some natural
preservatives such as nisin and extracellular polysaccharides. Through bioengineer-
ing, to increase the yield of target products of lactic acid bacteria, on the one hand,
we are able to obtain more end products, such as Leuconostoc mesenteroides, which
are widely used in the production of products such as glucan, lactic acid, and bacte-
riocin, and on the other hand, in order to allow lactic acid bacteria to produce more
functional natural components and improve their probiotic properties, more and
more lactic acid bacteria have been reported to have the ability to biotransform con-
jugated linoleic acid (CLA) over the past 20 years. Coakley et al. (2003) screened a
series of high-yield c9, t11-CLA Bifidobacteria from humans. Kishino et al. (2009)
evaluated the ability of several strains of Lactobacillus plantarum to biotransform
CLA, some of which convert more than 30% of linoleic acid to c9, t11-CLA, and
many lactic acid bacteria with biotransformation CLA capacity have also been
widely used in the development of a variety of fermented foods and functional
foods, such as probiotic yogurt, probiotic cheese, etc. (Florence et al. 2012; Alves
et al. 2013; Ye et al. 2013). In view of the numerous application values of lactic acid
bacteria, in order to obtain greater control and utilization of lactic acid bacteria and
obtain higher yields of target substances, bioengineering technology has been
applied to the research of lactic acid bacteria. Metabolomics technology has become
one of the most active and effective research methods in the field of bioengineering.
It is also widely used in the research to guide the optimization of lactic acid bacteria
fermentation process and increase the yield of target products.
6.6.2 Basic Metabolic Pathway of Lactic Acid Bacteria

6.6.2.1 Glucose Metabolism
For the metabolism of glucose, lactic acid bacteria use two different metabolic path-
ways: homolactic fermentation and heterolactic fermentation (Blandino et al. 2003).
Through the homolactic fermentation pathway, glucose is basically metabolized
into lactic acid. However, after the heterolactic fermentation pathway, in addition to
producing lactic acid, glucose also produces other fermentation products such as
ethanol, acetic acid, and carbon dioxide.
6.6.2.2 Fructose Metabolism
For the homolactic fermentation bacteria, fructose is phosphorylated by fructose

kinase to produce fructose 6-phosphate, fructose 6-phosphate produces pyruvate by
EMP pathway, and finally reduced to lactic acid; for heterolactic fermentation bac-
teria, a part of fructose is reduced as an electron acceptor to form the final product
mannitol after the action of mannitol dehydrogenase, but most of the fructose is
phosphorylated to form glucose 6-phosphate and produces end products such as
lactic acid, ethanol, acetic acid, and carbon dioxide in the HMP pathway.
6.6.2.3 Sucrose Metabolism
Sucrose is transported into cells through a permease or PTS system, and after glu-
cose and fructose are produced by sucrose hydrolase, it enters the main metabolic
pathway of the cell. It is then degraded to the final product via the EMP or HMP
pathway.
6.6.2.4 Malate Metabolism
Lactic acid bacteria can decarboxylate malic acid to produce lactic acid and carbon
dioxide by apple lactate. For some lactic acid bacteria, malic acid is metabolized to
produce pyruvic acid and carbon dioxide, and pyruvic acid is metabolized to pro-
duce metabolites such as lactic acid. In addition, malic acid can also be reacted to
form fumaric acid, which is then converted to succinic acid to form another meta-
bolic pathway.
6.6.2.5 Citric Acid Metabolism
The creamy flavor produced during the fermentation of dairy products is primarily
formed by the metabolism of citric acid. Citric acid metabolism may produce differ-
ent products depending on the genes and growth environment in which lactic acid
bacteria control citrate metabolism. After the citric acid enters the cell through the
transport carrier, it is decomposed into acetic acid and oxaloacetate by citrate lyase,
which generates pyruvate under the catalysis of oxaloacetate decarboxylase and
degrades into lactic acid, fumaric acid, acetic acid, and diacetyl or acetoin. Among
them, diacetyl and acetoin, although low in content, play an important role in flavor
regulation.
6.6.2.6 Amino Acid Metabolism
Among the amino acids metabolized by lactic acid bacteria, transaminase and
decarboxylase play an important role. In addition to these two enzymes, other
enzymes such as lyase and dehydrogenase play a role in the amino acid metabolism
of lactic acid bacteria. Under the action of decarboxylase, lactic acid bacteria can
convert glutamic acid into γ-aminobutyric acid. Under the action of dehydrogenase,
lactic acid bacteria can produce α-Ketoglutaric acid, an important metabolic inter-
mediate of glutamic acid, and α-Ketoglutaric acid produces glutamate under the
catalysis of a transaminase. Lactic acid bacteria can produce many flavor sub-
stances, such as ketones, aldehydes, and acids, due to incomplete metabolism of
such amino acids, which directly act to enhance the flavor of the fermented food or
as a substrate for the production of flavor substances.
6.6.3 pplication of Metabolomics in Lactic Acid Bacteria

A
Fermentation Engineering
The monitoring and optimization of the lactic acid bacteria fermentation process
requires the detection of a large number of parameters. The metabolomics research
tool can be used to investigate the material consumption and production during the
fermentation process of lactic acid bacteria, improve the detection flux, and help
reveal the biochemical network mechanism of the fermentation process that is ben-
eficial to optimize the process. On the one hand, researchers can use metabolomics
to find a more comprehensive metabolic profile of lactic acid bacteria in the fermen-
tation process, such as Hugenholtz et al. (2000), using nuclear magnetic resonance
technology to study the carbon metabolism and nitrogen metabolism pathways in

the fermentation process of lactic acid bacteria. Dynamic monitoring reveals a com-
prehensive physiology and genetics of lactic acid bacteria in the fermentation pro-
cess. Ramos et al. (2002) also used real-time dynamic monitoring of the synthesis
pathway of extracellular polysaccharides produced by lactic acid bacteria by NMR
technology and also quantitatively monitored the glycolysis pathway. In addition,
metabolomics techniques can be used to objectively study the changes in a meta-
bolic process of lactic acid bacteria and to guide the regulation and prediction of
component changes during fermentation. Some researchers have used LC-MS/MS
to monitor the metabolism of some key amino acids in lactic acid bacteria, so as to
monitor the whole fermentation process. It can be seen that the application of
metabolomics makes it possible to systematically analyze or control complex fer-
mentation processes.
6.7 pplication of Metabolomics in the Identification

A
6.7.1 Overview of Lactic Acid Bacteria Identification
Regarding the classification and identification of lactic acid bacteria, in addition to

morphological, physiological, and biochemical phenotypic characteristics as the
main indicators, genotype classification methods such as 16S rDNA sequencing,
DNA hybridization, and PCR fingerprinting have been developed, which is in rapid
progress with molecular biology closely related. However, the use of both genotype
and phenotypic methods for the classification of certain strains yields different
results. Therefore, combining these two methods has become a trend for different
classification purposes. Metabolomics has been increasingly used in the identifica-
tion of lactic acid bacteria as a classification method based on the metabolic pheno-
type of lactic acid bacteria.
6.7.2 tudy on Identification of Lactic Acid Bacteria Based

S
on Metabolomics Technology
At present, the identification of microorganisms in metabolomics is mostly concen-

trated on the use of NMR technology, GC-MS technology, and MALDI-TOF-MS
technology. As metabolomics is increasingly used in the identification of microor-
ganisms, some scholars have begun to try to identify lactic acid bacteria by metabo-
lomics. Del et al. (2009) established a method for determining the metabolic
fingerprint of microbial extracellular secretions in cheese by Fourier-transform
infrared spectroscopy (FTIR). This method can be used to distinguish different
strains and determine the fermentation characteristics of each strain. The molecular
biology approach is higher in resolution. Metabolic profiling methods not only have
higher resolution but also have the advantages of fast and high throughput, so they
are increasingly used in microbial classification.
6.7.3 pplication of MALDI-TOF-MS in the Identification

A
Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

(MALDI-TOF-MS) is a new type of soft ionization technology. The sample and the
substrate solution are added to the sample plate at a certain ratio, and the solvent is
volatilized to form a co-crystal of the sample and the matrix. The laser is used as an
energy source to radiate the co-crystal, and the matrix absorbs energy from the laser
to desorb the ionization of the sample. After flight time, the detector separates ions
of different mass-to-charge ratios (m/z), and the molecules to be measured are sepa-
rated by mass. By comparing with the database information, the corresponding
maps are screened and determined, and the identification results are obtained,
thereby distinguishing and identifying different bacterial genera, species, and sub-
species (Fenselau and Demirev 2001). The MALDI-TOF-MS technology is mainly
based on the analysis of various biomarkers of bacteria. By qualitative and quantita-
tive analysis of these biomarkers, the high-throughput strain information is finally
obtained, thereby completing the bacterial identification. The marker mainly
includes components such as proteins, nucleic acids, lipids, and the like.
This method has also been applied to the identification of lactic acid bacteria.
Nguyen et al. (2013) used MALDI-TOF-MS and molecular biology identification
methods to identify 881 strains of lactic acid bacteria isolated from fermented veg-
etables in Vietnam and successfully classified the strains according to characteristic
ions. Soro-Yao et al. (2014) also used MALDI-TOF-MS to classify lactic acid bac-
teria isolated from fermented grains, which showed higher throughput and resolu-
tion than molecular biology methods. Metabolomics-based MALDI-TOF-MS has
high-throughput and high-resolution characteristics for the identification of lactic
acid bacteria, and it has great reference value for the identification of high-resolution
lactic acid bacteria strains.
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Chapter 7
Functional Evaluation Model for Lactic
Acid Bacteria
Qixiao Zhai and Wei Chen
With the deepening exploration of the relation between the gut microbiota and the
host health, the health-promoting effect of lactic acid bacteria has been further ana-
lyzed and explored. Numerous studies have shown that the lactic acid bacteria not
only have the widely recognized functions like antagonizing pathogenic bacteria,
regulating gut microbiota, and improving immunity, but may also have a series of
special physiological functions such as antioxidant, anti-mutation, biological elimi-
nation of toxic and harmful substances, and reduction of cardiovascular diseases. At
present, many studies have attempted to develop a mature, accurate, and high-
throughput functional evaluation model for the characteristics mentioned above to
efficiently screen the strains, obtain probiotic strains with excellent physiological
characteristics, and then verify and evaluate its function by in vitro models, cell
models, as well as in vivo models. The establishment of these functional evaluation
models is beneficial to figure out the health-promoting benefits of lactic acid bacte-
ria and provides a good reference for the strain screening and physiological evalua-
tion of follow-up researchers.
7.1 istory and Development of Functional Evaluation

H
Model for Lactic Acid Bacteria
As the most commonly employed probiotics, lactic acid bacteria and their func-
tional evaluation are always the focus of relevant research. The most common func-
tional evaluations include the protection of strains on the gut health and the
regulating effect on the immune system. But with the deepening of the research,
functional evaluation is also starting to involve the prevention and treatment of
Q. Zhai · W. Chen (*)

https://doi.org/10.1007/978-981-13-7832-4_7
184 Q. Zhai and W. Chen
cardiovascular diseases, metabolic diseases, oral diseases, genital tract disease, skin
disease, mental disease, etc. Some novel features appeare, for example, Lactobacillus
reuteri relieves autism (Buffington et al. 2016), L. rhamnosus alleviates allergic
dermatitis (Hoang et al. 2010), L. kefir promotes wound healing (Huseini et al.
2012), L. acidophilus relieves intestinal pain (Rousseaux et al. 2007), etc. The dis-
covery and analysis of these new functions are inseparable from efficient and accu-
rate evaluation models.
The lactic acid bacteria functional evaluation model mainly involves in vitro
models, cell models, in vitro tissue models, in vivo models, human models, etc.
(Fig. 7.1). On account of the advantages of simple operation, short experimental
period, and low experimental cost, in vitro tests are often used as the first step in the
evaluation process to preliminarily determine the probiotic characteristics of the
strain. Then according to the need, the cell and in vitro tissue models or in vivo
models are selected for further function confirmation. At present, animal tests and
human clinical trials have been conducted by numerous researches to evaluate the
function of lactic acid bacteria in regulating gut microbiota. For example, animal
tests mainly focused on gut microbiota disorders caused by high-fat diets, high-
sugar diet, excessive use of antibiotics, pathogen infections, and so on. After giving
the animal lactic acid bacteria, the recovery of intestinal microecology could be
determined by the content of specific probiotics and pathogenic bacteria in the feces
or intestines. In the meantime, the composition of the intestinal microbiota could be
Fig. 7.1 Functional evaluation model of lactic acid bacteria

7 Functional Evaluation Model for Lactic Acid Bacteria 185
further evaluated by high-throughput sequencing methods such as metagenome.

Human experiments are mainly people who have a disorder of the gut microbiota
caused by various physiological abnormalities. After oral administration of the
strain for a period of time, changes in the gut microbiota could be determined,
which are similar to animal models. Furthermore, the antitumor function of lactic
acid bacteria can be evaluated by in vitro tests and animal and human experiments.
In vitro tests include inhibiting tumor cell growth, decomposing carcinogens, and
inhibiting telomerase in tumor cells. Animal experiments can mainly use spontane-
ous cancer animal model, induced cancer animal model, and tumor transplantable
cancer animal model. After oral administration of the strain for a period of time, the
shape, size, diffusivity, and immune activity of the tumor cells were examined to
determine the antitumor ability. There are also clinical studies that subjects are
given lactic acid bacteria to evaluate their inhibitory effect on cancer. (Serban 2014).
In 1899, Tisser isolated bifidobacteria from infant feces and found that it was
associated with infant nutrition and the frequency of diarrhea. The discovery and
exploration of the function of lactic acid bacteria began from this. Since Mechennikov
proposed the longevity hypothesis of yoghurt in 1907, the functional evaluation
model of lactic acid bacteria has been continuously developed and reformed. In
1915, Daviel Newman first used lactic acid bacteria to treat bladder infections. In
1922, Rettger and Cheplin reported on the clinical efficacy of Lactobacillus aci-
dophilus yoghurt on digestive function. From 1930 to 1935, Dr. Dai Tianyi isolated
a Lactobacillus casei strain named Shirota which could inhibit pathogenic bacteria
and could tolerate the gastric acid and bile salts and then reach the small intestine
smoothly. In 1957, Gordon et al. proposed an effective standard for lactobacillus
therapy in The Lancet. In 1966, Dr. Dai Tianyi found that taking Lactobacillus casei
strain Shirota orally can affect the gut microbiota of infants. From 1970 to 1980,
with the development of cell models and in vitro tissue models, plenty of researches
have demonstrated that the lactic acid bacteria and their fermentation products have
antitumor activity and could regulate immune response. In 2001, the World Health
Organization made a “general definition” of probiotics and systematically regulated
the functional evaluation and model application of lactic acid bacteria.
7.2 In Vitro Model
In lactic acid bacteria-related researches, the in vitro functional evaluation model is

one of the earliest and most widely used methods. This kind of model does not
involve living tissues and cells, so its evaluation is often convenient and has the
advantage of short experimental period, large experimental throughput, and low
experimental cost. At the same time, the in vitro model can simulate the internal
environment of the host, such as the common in vitro lactic acid bacteria gastroin-
testinal transit tolerance test. That is, simulating the environment including pH, bile
salt concentration, protease concentration, and other characteristics directly
investigates the survivability of lactic acid bacteria in the simulated environment

in vitro. In addition, in vitro models might be more preferable and intuitive when
studying the physiological characteristics of lactic acid bacteria compared to in vivo
models. However, the beneficial functions of lactic acid bacteria to the host are
generally more concerned in the cell models and in vivo models for the interaction
between the host and the strain. However, the physiological properties of the strain
are often hard to figure out extensively due to the interference of various compli-
cated factors. The in vitro model could intuitively eliminate the interference factors,
determine the single influencing component, and evaluate the health-promoting
effect of the lactic acid bacteria. For example, by functional evaluation of in vitro
adsorption models, in vitro antioxidant models, and so on, strains with excellent
physiological properties could be directly screened, which provides a basis for the
further evaluation in subsequent in vivo experiments.
7.2.1 In Vitro Anti-mutation Model
Carcinogens and mutagens have strong mutagenic and teratogenic effects on the
human body. The chemical pollution on foods and environment has become a major
factor in the development of cancer. Many researches have manifested that the lactic
acid bacteria and their fermented products could effectively inhibit tumorigenesis
and play anticancer and anti-mutation functions. The related mechanisms mainly
involve the adsorption and removal of mutagens (El-Nezami et al. 2000; Serrano-
Niño et al. 2015), inhibition of carcinogen-activating enzyme activity (Hugo et al.
2006; Wolf and Hammes 1988), regulation of host immune function (Viaud et al.
2013; Sivan et al. 2015), and so on. The following is a description of several com-
mon evaluation models for the in vitro anti-mutation function of lactic acid
bacteria.
7.2.1.1 4-Nitroquinoline-1-Oxide (4NQO) Experiment
4-Nitroquinoline-1-oxide (4NQO) is a nitroaromatic chemical. 4NQO in nature is

not uncommon due to the incomplete combustion of polycyclic aromatic hydrocar-
bons in the environment and the extensive use of nitrogen oxidation in the industry.
Numerous reports indicate that 4NQO is a strong chemical mutagen that could
induce mitochondrial membrane damage, directly shear the DNA chain, and cause
abnormal cell proliferation and canceration (Hawkins et al. 2010). Microgram
amount of 4NQO could induce breast cancer, oral cancer, and lymphoma in animal
models such as mice, rats, and rabbits. This chemical is also considered to be an
ideal model for cancer modeling by researchers (Dayan et al. 1997).
Numerous reports have shown that the lactic acid bacteria have the ability of
binding and removing mutagenic substances, thus could inhibit the absorption of
similar substances by the intestinal tract. In these studies, the 4NQO experiment is
a classic functional evaluation model. As early as 1970, Bond et al. studied the
inhibitory effect of 4NQO on the growth of Lactobacillus casei. Hosono (1986)
found that dairy products fermented by Lactobacillus bulgaricus and Streptococcus
lactis could effectively inhibit the mutagenicity of 4NQO. Borob’Eva et al. (1995)
found that lactic acid bacteria such as Lactobacillus delbrueckii and Bifidobacterium
significantly reduced the mutagenic effect of 4NQO on Salmonella typhimurium
with an inhibition rate of more than 50%. Lin and Chang (2000) found that
Bifidobacterium longum ATCC15708 and Lactobacillus acidophilus ATCC4356
could reduce the cytotoxicity of 4NQO on intestinal 407 cells CCRC60022 strain.
The inhibition rate reached 50% and 90%, respectively. At the same time, the inhibi-
tion rate of cell membrane lipid peroxidation reached 11–29%. Matar et al. (1997)
found that the fermented milk, whey, acetone extract, and protein of Lactobacillus
helveticus L89 could all inhibit the mutagenic effect of 4NQO. Analysis by high-
performance liquid chromatography showed that the fermented protein had the
strongest anti-mutation activity. Cenci et al. (2002) isolated 67 strains of lactic acid
bacteria from dairy products and evaluated their inhibition on 4NQO gene toxicity
by E. coli SOS chromogenic reaction. The results showed that 31 strains showed
strong inhibition rate (>75%), and the inhibition rate of some strains of Lactobacillus
casei, Lactobacillus plantarum, and Lactobacillus rhamnosus was even more than
90%. Fang et al. (2007) isolated a series of lactic acid bacteria strains from the feces
of the elderly in Changshou Village, Bama, and then evaluated their inhibition on
4NQO gene toxicity by E. coli SOS chromogenic reaction. The results showed that
the genotoxic clearance rates of Lactobacillus salivarius and Bifidobacterium were
both above 80%, while the clearance rates of the commercial Lactobacillus casei
strains and Lactobacillus rhamnosus strains were 73% and 9%, respectively. This
model could effectivity distinguish and screen lactic acid bacteria with different
anti-mutation characteristics. The authors also speculated that the relevant proper-
ties of lactic acid bacteria were directly or indirectly related to the metabolic activity
of the strain. Zhaoyong et al. (2011) further studied the conditions that the require-
ment for L. salivarius to degradate 4NQO. He found that when the concentration of
the strain cells reached 1011 cfu/ml, the obvious degradation was observed. And the
degradation product of 4NQO can be detected when the concentration of the cells
reaches 1013 cfu/ml.
In addition to classical and general test methods such as the mutagenicity detection
(Ames test), the SOS chromogenic reaction is a major method to evaluate the 4NQO
genotoxicity inhibition ability of lactic acid bacteria. The reaction principle is mainly
the exposure of E. coli PQ37 (genetically engineered bacteria) to the environment of
mutagenic substances. Under such circumstances, the RecA protease could decom-
pose the combined protein and activated the normal lac gene which was originally
deleted, finally resulting in the positive correlation between galactosidase expression
and mutagens. Then, the decomposition of o-nitrobenzene β-d-galactoside (ONPG)
by galactosidase could produce yellow soluble substances. Therefore, quantitative
determination can be performed (Quillardet and Hofnung 1985; Caldini et al. 2005).
This experiment is a commonly used method for analyzing the ability of lactic acid
bacteria to inhibit genotoxic effects of mutagenic chemicals due to its high s ensitivity
and high specificity. Moreover, quantitative analysis of 4NQO content can be mea-
sured by high-performance liquid chromatography (HPLC), gas chromatography
(GC), mass spectrometry (MS), or combined techniques. Therefore, the degradation
or adsorption ability of the strain could be concluded. For example, Verdenelli et al.
(2010) co-cultured Lactobacillus rhamnosus IMC501 with 4NQO, and then the
4NQO degradation was discovered by GC/MS. Zhaoyong et al. (2011) demonstrated
that intermediate product and final product of 4NQO degradation by lactic acid bac-
teria could be effectively detected by high-performance liquid chromatography. Its
liquid mobile phase consists of water [0.1% trifluoroacetic acid (TFA) added] and
methanol. The elution procedure was 0.01 min, 8% methanol; 3.0 min, 35% metha-
nol; 4.0 min, 35% methanol; 15.0 min, 77% methanol; 20.0 min, 88% methanol;
21.0 min, stopped. Wang et al. (2008) also demonstrated that high-performance liquid
chromatography can be an alternative to the SOS chromogenic reaction, with a cor-
relation coefficient (R2) between the two methods up to 0.991. At the same time, the
detection limit of liquid chromatography reaches 1.2 ng, which is more accurate and
sensitive than the SOS chromogenic method.
7.2.1.2 N-Methyl-N′-Nitro-N-Nitrosoguanidine (MNNG) Experiment
N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) is a nitrosamine chemical and a

class of alkylating agents with strong carcinogenic and mutagenic functions.
MNNG induces abnormal activation of the JNK/SAPK pathway in animals and
human cells, leading to DNA damage and chromosomal aberrations (Isabelle et al.
2012; Zhang et al. 2014a). Both epidemiology and animal experiments have shown
that MNNG can induce tumorigenesis in organs such as the digestive tract, liver,
and kidney (Jing et al. 2000; Manikandan et al. 2011; Matsumura et al. 2015).
Similar to 4NQO, there have also been many researches on the anti-mutation prop-
erties of lactic acid bacteria by MNNG experiments. Park and Rhee (2001) isolated
Lactobacillus plantarum KLAB21 from Korean kimchi. The whole cell, superna-
tant, and cell extract of this strain were found to have strong anti-mutation activity
against MNNG. Caldini et al. (2005) tested the anti-mutagenic properties of 65
lactic acid bacteria strains and found that more lactic acid bacteria could restrain the
genotoxicity caused by 4NQO, but only one strain of Lactobacillus acidophilus can
inhibit the mutagenic activity of MNNG. Ambalam et al. (2011) found that
Lactobacillus rhamnosus Lr231 isolated from human intestinal tract adsorbs
MNNG and reduces its mutagenic activity. The adsorption process is related to tei-
choic acid and sulfhydryl groups on the cell surface, as well as involves hydropho-
bic interaction. Jinggang and Hong (1998) used MNNG to induce the Escherichia
coli SOS reaction model and studied the anti-mutation characteristics of bifidobac-
teria. The results showed that Bifidobacterium whole bacteria, lipoteichoic acid,
cell wall peptidoglycan, and culture supernatant have a certain degree of anti-muta-
tion properties, in which lipoteichoic acid works best. They further found that
Bifidobacterium can effectively inhibit DNA damage induced by MNNG in mouse
intestinal mucosa by single-cell gel electrophoresis. The mechanism involved may
be that the MNNG-active group is masked by binding to MNNG, resulting in

decreased mutation activity (Jinggang et al. 1998).
Similar to the 4NQO experiment, the MNNG experiment was used to evaluate
the anti-mutation properties of lactic acid bacteria using common test methods such
as Ames method, SOS color reaction, liquid chromatography, mass spectrometry,
etc. (Caldini et al. 2005; Ambalam et al. 2011). In addition, single-cell gel electro-
phoresis, also known as comet assays, is often used to evaluate the results of MNNG
experiments. The mechanism of action is mainly that DNA damage leads to an
increase in fragmentation, looseness, and release of the DNA supercoiled structure,
and a comet-like fluorescent head and tail can be obtained by ethidium bromide
staining. Therefore, DNA damage and mutagenic effects of MNNG can be judged
based on the tail length of the comet and the DNA content (Wollowski et al. 1999).
7.2.2 The In Vitro Adsorption Model
According to the World Health Organization’s draft Global Food Safety Strategy,
food safety issues are primarily related to microbiological and chemical hazards.
The latter mainly includes natural toxic substances (such as microbial toxins) and
environmental pollutants (such as heavy metals), which have become important
safety hazard factors in food (Wei and Qixiao 2014). Lactic acid bacteria can effec-
tively absorb chemical substances such as heavy metal ions, biological toxins, and
pesticide residues due to their special surface structure. The adsorption mechanism
may involve group bonding, ion exchange, physical deposition, membrane perme-
ation, etc. on the surface of the cells and may also include active transport and
endocytosis of the cells, and these modes of action are interrelated to form a com-
plex adsorption mechanism. In order to look into the in vitro adsorption character-
istics of lactic acid bacteria, a variety of mature adsorption models have been
reported, which are described below.
7.2.2.1 Metal Ion Adsorption
Harmful metal pollution has become a serious food safety and health problem.
Take two common heavy metals such as lead and cadmium as examples. Lead is a
harmful metal that has pernicious effects on the body. It has significant toxic
effects on the liver, brain, kidney, and other organs. The ideal blood lead concen-
tration should be zero. Cadmium has serious toxic effects on the bones, liver, kid-
neys, reproductive system, and blood system of the body. It is listed as the seventh
toxic substance harmful to human health by the US Agency for Toxic Substances
and Disease Registry and the National Environmental Protection Agency. It is
classified as a class I carcinogen. Other toxic and harmful metals such as mercury,
arsenic, aluminum, manganese, etc. may also cause significant harm to the body.
At present, the problems of heavy metal pollution in foods are very common.
In recent years, surveys have shown that the content of heavy metals in rice, fruits,
and vegetables and aquatic products in developing countries including China is
seriously increasing (Zhang et al. 2014b; Zhai et al. 2015). However, there is cur-
rently no very efficient and safe means for removing harmful metals from food.
Lactic acid bacteria are considered to be food-safe microorganisms, and the spe-
cial adsorption characteristics of some strains on metal ions make them have the
potential to reduce the harmful metals in food. At present, many studies have used
different evaluation models to investigate the adsorption characteristics of lactic
acid bacteria and mainly consider the adsorption kinetics, thermodynamic proper-
ties, and adsorption stability of the strains.
Bhakta et al. (2012) isolated 255 strains of lactic acid bacteria from environmen-
tal sludge samples. After screening, 26 strains were resistant to heavy metals such
as lead and cadmium, and Lactobacillus reuteri Pb71-1 had the strongest removal
capacity (59%) to lead. Lactobacillus reuteri Cd70-13 has the strongest adsorption
capacity for cadmium (25%). In the first 24 h of adsorption, the adsorption capacity
of the intracellular substance is near the cell membrane. At two times, at 48 h, the
adsorption capacity of intracellular substances decreased, and the cell membrane
adsorption capacity was greatly increased. The test conditions were as follows: ini-
tial lead concentration 6 mg/L, initial bacterial concentration 3 g/L (wet weight),
culture temperature 37 °C, and culture time 2 h. Tian Fengwei et al. screened
Lactobacillus plantarum CCFM8661 with strong adsorption capacity for lead ions
and used HNO3 (15 mmol/L and 1.5 mmol/L) and EDTA (1.0 mmol/L and
0.1 mmol/L) solutions, as well as ultrapure water. Desorption experiments were car-
ried out on Lactobacillus plantarum CCFM8661 after the completion of adsorption
of lead ions. The results showed that the adsorption of Pb2+ by CCFM8661 was
stable and would not be easily desorbed. Through adsorption thermodynamic analy-
sis, a series of solutions with different initial lead concentrations were selected for
adsorption experiments, and equilibrium adsorption isotherms were used to balance
the adsorption amount and equilibrium solution concentration, and different ther-
modynamic models were used to desorb the fitting. The results show that CCFM8661
is suitable for Langmuir model (assuming that the adsorbent surface is uniform and
the adsorption energy is the same everywhere, and the adsorption process is mono-
layer adsorption. When the adsorbent surface is saturated with adsorbate, the
adsorption amount reaches the maximum value, corresponding to the physical
adsorption process). Both the Langmuir-Freundlich dual models have a good fit
(Yin et al. 2016).
A series of studies by Halttunen et al. (2007, 2010) measured the adsorption
capacity of ten strains of lactic acid bacteria including Lactobacillus rhamnosus,
Lactobacillus fermentum, Lactobacillus casei, Bifidobacterium longum, and
Bifidobacterium breve. Test conditions are as follows: initial cadmium concentra-
tion was 50 mg/L, initial pH was 6.0, initial bacterial concentration was 1 g/L (109
cells per ml), culture temperature was 22 °C, and culture time was 60 min. The
results showed that although each strain had a certain adsorption capacity, the dif-
ferences between the strains were significant and affected by the initial adsorption
conditions. To further analyze the adsorption thermodynamic properties of the
strain, the authors used different models to fit the existing adsorption isotherm data.
According to the Langmuir model, the authors demonstrated that the theoretical
monolayer maximum adsorption amount Qmax of Lactobacillus fermentum ME3
was 28.4 mg/g dry weight, Lactobacillus casei Qmax was 12.1 mg/g dry weight of
bacteria, and Lactobacillus rhamnosus (the Qmax of Lactobacillus rhamnosus GG,
LGG) was 13.2 mg/g dry weight of the cells. Qixiao (2015) screened a strain of
Lactobacillus plantarum CCFM8610 with strong adsorption capacity for cadmium.
Through adsorption thermodynamic analysis, it was found that the adsorption pro-
cess of CCFM8610 has the best goodness of fit for the Langmuir-Freundlich model
(R2 = 0.9928); the theoretical upper limit Qmax of the adsorption capacity of the
strain is significantly higher than the commercial probiotic strain such as
Lactobacillus rhamnosus LGG and Lactobacillus casei strains. Through adsorption
kinetic analysis, it was found that the adsorption of the strain was a fast and efficient
process, which accorded with the quasi-second-order kinetic equation, and reached
90% of the equilibrium adsorption amount at the time of 100 min. Further fitting
using the Weber-Morris model revealed that the adsorption process can be divided
into two phases, including the diffusion of cadmium ions from the solution onto the
surface of the cells and the binding of cadmium to the surface active sites of the cells
(Zhai et al. 2016). Ibrahim et al. (2006) found that the adsorption of cadmium by
L. rhamnosus LC-705 could better fit the Langmuir model, which Qmax was 13.2
mg/g dry weight of the cells, and was influenced by the initial cadmium concentra-
tion, the initial bacterial concentration, the adsorption time and the initial pH.
Through adsorption kinetic analysis, Teemu et al. (2008) found that the adsorption
of cadmium by Lactobacillus fermentum ME3 and Bifidobacterium longum 46 was
a rapid and efficient process and was associated with hydroxyl and phosphoryl
groups on the surface of the cells.
7.2.2.2 Microbial Toxin Adsorption
In general, microbial toxins refer to toxic chemicals produced by microorganisms

during their growth and reproduction or under specific environmental conditions.
The microbial toxins commonly found in people’s lives include aflatoxins, algal
toxins, patulin, botulinum toxin, and cholera toxin. These toxins are difficult to
remove once they contaminate food, posing a serious hazard to human food chain
safety. According to the characteristics of different microbial toxins, the main meth-
ods for toxin removal include physical methods, chemical methods, and biodegra-
dation methods. The biodegradation method does not use toxic and harmful
chemical agents and does not affect the taste of food and the loss of nutrients in
food. It is considered to be the best method of detoxification. The adsorption and
subtraction functions of lactic acid bacteria on microbial toxins have been widely
recognized. In different reports, there are some differences in the relevant adsorp-
tion models.
Halttunen et al. (2010) studied the competence of a series of lactic acid bacteria
to adsorb microcystins and aflatoxins. The test conditions were initial toxin
c oncentration of 100 μg/L (microcystins) and 4 mg/L (aflatoxin), initial pH 7.0, ini-
tial bacterial concentration of 2 g/L (microcystins) and 109cfu/ml (aflatoxin), cul-
ture temperature of 22 °C (microcystins) and 37 °C (aflatoxin), and culture time of
24 h (micro-cytotoxin) and 60 min (aflatoxin). The results showed that Lactobacillus
rhamnosus LC705 had the strongest adsorption capacity for microcystins, while
Bifidobacterium breve E8/99 had the strongest removal ability for aflatoxins. Wang
et al. (2010) examined the ability of three commercial lactic acid bacteria to reduce
microcystins. The initial conditions were 7.0, the initial bacterial concentration was
109 cfu/L, the culture temperature was 37 °C, and the culture time was 24 h.
Lactobacillus casei was found to have the best depletion ability, and the final
removal rate was up to 43%. At the same time, the initial cell activity, cell concen-
tration, and toxin concentration had an effect on the removal rate. After the addition
of exogenous carbon source glucose, the efficiency of the strain to reduce algal
toxin was significantly improved, reaching 92% after 24 h. Qian et al. (2012a)
screened a traditional fermented food to obtain a Lactobacillus casei BBE10-212
with high microcystin-reducing ability. The test conditions were as follows: initial
toxin concentration of 1000 μg/L, initial pH of 7.0, initial bacteria concentration of
109 cfu/ml, culture temperature of 37 °C, and culture time of 24 h. The addition of
5% glucose increased the algal toxin removal rate from 19% to 52%. El-Nezami
et al. (1998) studied the adsorption characteristics of aflatoxins B1 by five probiot-
ics in vitro. The test conditions were as follows: the initial toxin concentration was
5–50 mg/ml, the initial pH was 7.3, and the initial bacterial content was 109–1010 cfu/
ml. The culture temperature was 4~37 °C, and the culture time was 4~72 h. The
results showed that two strains of Lactobacillus rhamnosus (LGG and LC-705) had
rapid and efficient subtraction ability to aflatoxin B1, and the removal rate was 80%.
Fuchs et al. (2008) used high-performance liquid chromatography (HPLC) to
explore the degradation of patulin and ochratoxin by 30 strains of lactic acid bacte-
ria. It was found that the patulin-degrading rate of Bifidobacterium animalis VM12
is more than 80%; furthermore, the degradation rate of ochratoxin by Lactobacillus
acidophilus VM20 is above 95%. Subsequently, the cell experiments showed that
these strains can effectively alleviate the genotoxic damage caused by mycotoxin to
hepatocyte HepG2. Hatab et al. (2012) also detected the adsorption and removal of
patulin by a series of lactic acid strains by HPLC. Testing condition are as follows:
the initial conditions were 1 mg/L, the initial pH was 4.0, and the initial bacterial
concentration was 1010 cfu/ml, the culture temperature was 37 °C, and the culture
time was 24 h.
The results showed that the toxin-reducing efficiency of the strain was closely
related to the initial adsorption conditions and the activity of the strain.
Bifidobacterium bifidum 6071 and Lactobacillus rhamnosus 6149 had the highest
removal rate of patulin, which were 52.9% and 51.1%, respectively. El-Nezami
et al. (2002a) used gas chromatography-mass spectrometry to study the ability of
lactic acid bacteria to remove trichothecenes. Testing condition are as follows: the
initial conditions were 20 mg/L, the initial pH was 7.3, and the initial bacterial con-
centration was 1010 cfu/ml, the culture temperature was 37 °C, and the culture time
was 1 h. The results showed that the strain had specificity for the ability to remove
toxins. Lactobacillus rhamnosus LGG could remove four of the seven toxins
(removal rate varies from 18% to 93%), while Lactobacillus rhamnosus LC-705
could only remove two kinds (removal rate varies from 10% to 64%). El-Nezami’s
research team used high-performance liquid chromatography to further study the
removal mechanism of these food-grade lactic acid bacteria to corn ketene toxin.
The results showed that more than 55% of the toxins were quickly adsorbed after
contact with the strain, and the adsorption capacity and strain concentration were
observed. The authors speculate that strains remove zearalenone more by adsorp-
tion (rather than physiological metabolism) (El-Nezami et al. 2002b).
7.2.3 In Vitro Antioxidant Model
Oxidative stress and oxidative damage are important physiological and pathological
processes for the development of metabolic and systemic diseases in the body. They
have become the commonly used targets and research models in the development of
drugs and functional foods. Numerous studies have shown that lactic acid bacteria
may have the potential to alleviate oxidative stress caused by different factors,
which have been confirmed by many in vitro and in vivo tests. The antioxidant abili-
ties of lactic acid bacteria and its isolates in vitro, such as reducing activity, hydro-
gen peroxide tolerance, anti-lipid peroxidation, chelating properties of ferrous and
copper ions, DPPH free radicals, and hydroxyl radical scavenging ability, are often
used as an evaluation index to evaluate their antioxidant capacity. Next, we will
briefly introduce several commonly used models for evaluating the in vitro antioxi-
dant capacity of lactic acid bacteria.
7.2.3.1 DPPH Removal Model
DPPH is 1,1-diphenyl-2-trinitrophenylhydrazine, alias 1,1-diphenyl-2-picryl (free

radical), etc., which can be used for spectrophotometric determination of tocopherol
and aliphatic sulfur. Alcohols, reducing substances, are also commonly used as
polymerization inhibitors. Since 1958, the DPPH method has been widely used in
the quantitative determination of antioxidant capacity such as food samples and
biological samples. There is a single electron in the DPPH radical, which has a
strong absorption at 517 nm, and the alcohol solution is a purple solution. After
single electron pairing with the radical scavenger, its absorption gradually disap-
pears, and the degree of fading of the solution is quantitatively related to the number
of electrons received. Therefore, a rapid quantitative analysis of the scavenger
removal ability of the radical scavenger can be realized by a spectrophotometer.
A large number of experiments on the evaluation of in vitro antioxidant activity
of lactic acid bacteria based on DPPH clearance showed that lactic acid bacteria
have a strong ability to scavenge DPPH free radicals. Previous studies have
evaluated the ability of selected 30 strains of lactic acid bacteria to scavenge DPPH
free radicals and linoleic acid peroxidation and screened lactic acid bacteria L3 and
L4 strains with strong antioxidant activity (Jiang-wei and Yu-sheng 2005; Jiang-wei
et al. 2005). Gang et al. (2013) found that the DPPH clearance rate of extracellular
secretions of lactic acid bacteria changed widely, with a minimum of 0.031% and a
maximum of 98.153%. The clearest cell-free extract of lactic acid bacteria was La5
strain with a clearance rate of 98.153%. Wang Gang et al. (2013) determined the
DPPH free radical scavenging of cell-free extracts and intact cell suspensions of
different concentrations of lactic acid bacteria and found DPPH free radicals of
CCFM1106, CCFM8661, CCFM1566, and ATCC53103 strains when the cell con-
centration was 1010 cfu/ml. The clearance rate is over 60%. The highest scavenging
strain is CCFM1106, which is over 90%. Di et al. (2011) confirmed that cherry stem
extracts fermented with Lactobacillus acidophilus have higher DPPH free radical
scavenging ability. Zhan Jian-long et al. (2015) found that free radical scavenging
rate of SR6 (Lactobacillus LAB26) was higher than 60% when detected by DPPH
free radical scavenging experiments. They also studied the effects of fermentation
conditions on the antioxidant activity of lactic acid bacteria. It is believed that nitro-
gen source, carbon source, and growth factor are the main effects of DPPH free
radical scavenging rate. For SR6 strain, the degree of influence of the factors from
large to small is carbon source>nitrogen source>lactose.
7.2.3.2 Hydroxyl Radical Scavenging Model
Hydroxyl radical (·OH) is a free radical with great harm produced by human body
in the process of metabolism. It can cause oxidative damage to sugars, amino acids,
proteins, etc. in tissues, leading to cell necrosis and even cell mutation. The Fenton
reaction is a phenomenon in which a H2O2 solution is mixed with a Fe2+ solution
to produce a strong oxidation product. The mechanism is to generate a strong oxi-
dizing OH in the reaction system. The reaction equation is H2O2 + Fe2 + →·OH + H2O·
+Fe3+. At present, the Fenton reaction system has become a commonly used and
effective method to evaluate the antioxidant capacity of substances. In addition to
the Fenton reaction, there are many other methods that can be used to detect
hydroxyl radicals: non-Fenton-type reactions of H2O2 under the action of catalysts,
self-oxidation and reduction reactions of some thiol-containing organic compounds,
and ultraviolet radiation of H2O2.
Zhang Jiang-wei et al. (2005) found that the clearance rates of OH for L3 strains
and L4 strains were relatively high, reaching 83.5% and 45.7%, respectively. Hu
Xiao-li et al. (2009) established an in vitro model to simulate normal colon and iron-
stimulated colonic environment and obtained three lactic acid bacteria with hydroxyl
radical scavenging ability. The difference among strains was Lactobacillus rhamno-
sus> Lactobacillus paracasei> Lactobacillus, the highest clearance rate can reach
about 60%, and the hydroxyl radical scavenging rate of strains in different environ-
ments is different. Wang Gang et al. (2013) used Fenton method and found that,
when the cell concentration was 1010 cfu/ml, the clearance rate of ·OH of strains
such as CCFM8661, CCFM1566, and ATCC53103 was above 60%.
7.2.3.3 Anti-lipid Peroxidation Model
Oxygen free radical reaction and lipid peroxidation play a very important role in
human metabolism. Under normal conditions of coordination and dynamic balance,
the two are responsible for maintaining many physiological, biochemical, and
immune responses in the body. The lipid peroxidation products include malondial-
dehyde (MDA), 4-hydroxynonenal (HNE), and serum 8-isoprostane. The determi-
nation of MDA content can reflect the extent of lipid peroxidation damage to a
certain extent and is recognized as a good indicator of lipid peroxidation.
Lin and Yen (1999a) found that the inhibitory effects of cell extracts extracted
from Lactobacillus acidophilus and Bifidobacterium longum on linoleic acid per-
oxidation, osteoblast membrane lipid peroxidation, and fluorescent tissue pigment
accumulation, respectively, vary from 33% to 46%, 22% to 37%, and 20% to 39%.
At the same time, the cell-free extracts of the two strains have a certain ability to
scavenge the membrane lipid peroxidation product MDA. Lee et al. (2010) showed
that the complete cell suspension (IC) and cell-free extract (CFE) of L. casei KCTC
3260 inhibited the linoleic acid peroxidation ability, and the inhibition rate reached
46.2% and 72.9%. In the experiment of studying the resistance to linoleic acid per-
oxidation, Zhang Jiang-wei et al. (2005) found that 27 strains of lactic acid bacteria
inhibited the linoleic acid peroxidation reaction, the inhibition rate was at least
4.9%, and the highest was 75.2%. Liu Yang et al. (2012) found that the fermentation
supernatants and intracellular extracts of Lactobacillus fermentum, Lactobacillus
acidophilus, Lactococcus lactis, and Lactobacillus helveticus all showed certain
anti-lipid peroxidation ability; among them, the lactobacillus fermentation superna-
tant and intracellular extract have the highest anti-lipid peroxidation ability.
7.2.3.4 Reduction Activity Determination Model
There is a certain relationship between the reducing activity of organic compounds

and their oxidation resistance. The reduction ability of the substance can be evalu-
ated by the amount of Prussian blue (Fe4[Fe(CN)6]3) produced, and the magnitude
of the reducing ability of the substance to be detected is evaluated by the absorbance
at a wavelength of 700 nm. The larger the number of the A700nm, the stronger the
reducing ability of the substance will be detected.
Studies have shown that both the lactic acid bacteria L3 strain and the L4 strain
detected reductive substances in acellular extracts (Jiang-wei et al. 2005). The
experimental result of Xi et al. (2010) showed that the all of the selected 35 LAB
strains had reducing ability (A700nm = 0.001~3.169). Moreover, the extracellular
secretion had a better reducing capacity. The strain La29 had the strongest reducing
ability (A700nm = 3.168), while the La12 strain had the lowest reducing ability
(A700nm = 0.029). The fermentation supernatants of the four lactic acid bacteria
studied by Liu Yang et al. (2012) showed certain reducing ability, among which
Lactobacillus helveticus had the strongest reducing ability, while the intracellular
extracts of all strains did not detect any reducing ability.
7.2.3.5 Ferrous Ion Chelation Model
Many researches have shown that translation metals including copper and iron can
lead to the peroxidation of polyunsaturated fatty acids, which may cause further
damage to biological membranes and finally result in severe harm in genetic level.
Therefore, metal ion chelation ability is also an important indicator for the evalua-
tion of antioxidant capacity of probiotics. Studies showed that lactic acid bacteria
extracts have strong metal ion chelating ability.
A series of previous studies on the antioxidant effects of lactic acid bacteria on
intestinal mucosa found that Lactobacillus fermentum ME-3 increases antioxidant
level by chelating metal ions and reducing lipid peroxidation (Lee et al. 2010; Kai
et al. 2004). Studies have also shown that the antioxidant effect of Lactobacillus
casei KCTC 3260 is achieved by chelation of Fe2+ and Cu2+. The chelating abilities
were 10.6 mg/kg and 21.8 mg/kg, respectively. Lin and Yen’s (1999b, c) study
showed that cell-free extract of 1010 live lactic acid bacteria can chelate Fe2+ with
an amount of 2.5 × 10–6 ~ 72.7 × 10–6. Hu Xiao-li et al. (2009) established an
in vitro model that mimics the normal colon and iron-stimulated colonic environ-
ment. The ability of the three lactic acid bacteria to scavenge hydroxyl radicals in a
high-iron environment is consistent with their ability to inhibit proliferation of
enterococci and chelate ferrous ions. This indicates that in a high-iron environment,
the antibiotic ability of lactic acid bacteria was profoundly influenced by their fer-
rous ion chelating ability and inhibition for the proliferation of enterococci.
7.2.3.6 Hydrogen Peroxide Tolerance Model
Chemical formula of hydrogen peroxide is H2O2 with a strong oxidizing property.

Hydrogen peroxide can not only cause direct damage to cells or tissues but also
indirectly participate in the oxidation process as a precursor of hydroxyl radicals
(·OH). H2O2 stress is a widely used type of oxidative stress model. In general, lactic
acid bacteria are less tolerant to H2O2, but some lactic acid bacteria strains are resis-
tant to H2O2 and have potential antioxidant properties. Kullisaar et al. (2002) found
that Lactobacillus fermentum E-3 and E-8 strains with antioxidant activities can
survive 180 and 150 min when exposed to H2O2, while the survival time of
Lactobacillus fermentum E-338-1-1 without antioxidant activity was 90 min.
Lactobacillus have a variety of redox regulatory systems which are mainly com-
posed of glutathione system, thioredoxin system, and “NADH oxidase/NADH per-
oxidase” system. Researchers investigated Lactobacillus plantarum and
Lactobacillus brevis strains isolated from tomato and found that fermentation with
Lactobacillus plantarum POM1 and POM35 can significantly improve ascorbic
acid (ASC) and glutathione (GSH) content in tomato juice and also increase total
antioxidant capacity (TTA) levels (Cagno et al. 2009). Similarly, Kullisaar et al.
(2002) also concluded that most lactic acid bacteria could scavenge ·OH and H2O2
by producing superoxide dismutase and GSH. Wang Gang et al. (2013) used a
three-layer MRS plate containing H2O2 to screen H2O2-tolerant strains, and 125
strains were identified as lactic acid bacteria through Gram staining and contact
enzyme reaction detection.
7.2.4 In Vitro Simulated Gastrointestinal Tolerance Model
Probiotics exert their function mainly in the intestine. After oral ingestion, they need
to experience various adverse factors to survive in the gastrointestinal tract, such as
low pH caused by gastric acid, bile salts, and digestive enzymes. Tolerance of pro-
biotics to the gastrointestinal tract is an important indicator for screening and evalu-
ating beneficial bacteria. At present, commonly used digestive tract models include
artificial gastric fluid (mainly hydrochloric acid and buffer) model and artificial
intestinal fluid (mainly bile salts, digestive enzymes, and buffer) model. Besides,
many complex factors in the complex upper gastrointestinal should also be consid-
ered, such as the buffering effect of food on gastric acid, gastrointestinal motility,
the pH of the gastric juice as food intake changes, and the effects of digestive
enzymes. The following part lists some frequently used models adopted to evaluate
the tolerance of lactic acid bacteria to the gastrointestinal tract.
7.2.4.1 Mimical Gastric Fluid Model
Many reports have indicated that lactic acid bacteria are resistant to low pH, while
this ability varies among different lactic acid bacteria. According to Smith’s report
(Smith 2010), the rabbits were fed with a bacterial consortium by drinking water
with a concentration of 108 cfu/ml. After 24 h, the animals were slaughtered, and the
pH of the stomach contents was 1.2. Only Lactobacillus survived, which indicates
that Lactobacillus is more resistant to low pH environments than non-Lactobacillus
and survives in the digestive tract of animals. Conway et al. (1987) found that most
of Lactobacillus acidophilus survived after an incubation in pH 1.0 buffer for 0.5 h,
but when duration time continued to 1 h, no live bacteria could be detectable. There
were little effects on the survival of this strain when exposed to pH 3.0 and pH 5.0
buffers. Ruixia et al. (1996) studied the effects of bile salt and low pH environment
on the activity of lactic acid bacteria. It was found that lactic acid bacteria had cer-
tain tolerance to low pH environment, but the number of live bacteria decreased
with the decrease of pH. The results of Chen Lin et al. (2010) showed that several
strains were well-tolerating in simulated gastric fluid (pH = 3) environment and the
logarithmic decrease of live bacteria in 2–4 h was smaller than that of 0–2 h. Hao
Qinghong et al. (2011) reported that the viable count of JM-11 strain increased by
12.16% after incubation for 2.5 h in pH 2.0 environment and the viable count
increased by 32.97% after 3.5 h of culture. At pH 3.0, JM-11 was able to multiply,
and its viable count increased by 7.39%, 7.95%, and 16.48% after an incubation of
1.5 h, 2.5 h, and 3.5 h, respectively.
7.2.4.2 Mimical Intestinal Fluid Model
Many lactic acid bacteria have been reported to be tolerant to bile salts. Chen Lin
et al. (2010) found that many lactic acid bacteria strains could tolerate high concen-
trations of bile salts (0.3%) after a cultivation in a simulated intestinal fluid for 6 h.
The viable count of most strains decreased with the prolongation of culture time.
Hao Qinghong et al. (2011) studied the tolerance characteristics of chicken probi-
otic JM-11 and found that the survival rate of JM-11 strain reached 70.24% in 0.1%
bile salt environment. As the concentration of bile salt increased, the survival rate of
JM-11 strain decreased significantly, and 50% or even more bacteria could survive
in 0.3% bile salt environment. Kanno et al. (2012) studied the bile salt tolerance of
four Lactobacillus plantarum strains and one Leuconostoc mesogenes strain. These
strains were found to grow in lactic acid MRS medium containing 3 g/L of bile.
Zhang Dan-dan et al. (2014) showed that Lactobacillus helveticus has better toler-
ance to bile salts, and there are still 106 cfu/ml viable bacteria at 5 g/L bile salt.
Meng Xiangchen et al. (2015) studied the tolerance of bile salts of two Lactobacillus
plantarum strains. After an incubation in 0.3% bile salts for 8 h, no remarkable
variation was observed in the viable counts, which were maintained at 108 cfu/ml.
7.2.4.3 In Vitro Gastrointestinal Biomimetic Simulation System
Considering the gastrointestinal environment of the human body, the establishment

of an in vitro gastrointestinal biomimetic simulation system can more reliably eval-
uate the tolerance of the strain to the gastrointestinal environment. Plenty of studies
have been conducted to determine the digestive tract tolerance of probiotics using
some simple gastrointestinal simulation systems. Hernández-Ledesma et al. (2004,
2007) simulated gastrointestinal digestion as follows: firstly, prepare 1 L buffer con-
taining 500 mg/L peptide and 20 mg/g pepsin, and with pH 2.0; maintain this buffer
at 37 °C for 1.5 h using the water bath. Secondly, adjust pH to 7.5, add pancreatin
(40 mg/g), and continue the mixture to maintain at 37 °C for 150 min, and finally
inactivate at 95 °C for 5 min. In vitro simulated gastrointestinal digestive test used
by Zhang Yuanyuan et al. (2013) was as follows: 1.000 g of the sample is accurately
weighed, 20 ml of the prepared artificial gastric fluid is added and shaken evenly
and cultured at 37 °C for 1 h on a constant temperature shaking and then 80 ml
artificial intestinal juice is added, cultured at 37 °C, 120 r/min for 2 h. Pipette 1 ml
of the cultured suspension to be diluted in 9 ml of PBS buffer (dilution 10–3), and
perform serial dilution according to the approximate viable cell count of the sample.
Finally, the viable count was determined by the plate count method. On this basis,
many researchers have designed an in vitro gastrointestinal biomimetic simulator to
directly monitor the digestive process parameters and indicators through a computer
to achieve continuous or semicontinuous simulation of the digestion process.
Charteris et al. (2002) studied the digestive tolerance of Lactobacillus and
Bifidobacterium and found that Lactobacillus fermentum KLD was better tolerated
in the simulated upper digestive tract environment, while others had a mortality rate
greater than 90%. Mäkeläinen et al. (2009) studied the gastrointestinal tolerance of
lactic acid bacteria in cheese by treating the cheese with a simulated upper digestive
tract solution and then an artificial colon simulator (semicontinuous Enteromix®
human colon simulator). The analysis results of the fermentation broth showed that
the total number of Lactobacillus acidophilus, Lactobacillus rhamnosus, and
Lactobacillus showed similar growth in the upper digestive tract and colon and the
growth of other microorganisms was significantly influenced, indicating that the
lactic acid bacteria can tolerate the gastrointestinal environment and thus success-
fully adhere to epithelial cells and colonize the intestine to exert a beneficial effect.
Arroyo-LóPez et al. (2014) studied the in vitro dynamic gastrointestinal model and
found that Lactobacillus rhamnosus LGG, Lactobacillus pentosus TOMC-LAB2,
and Lactobacillus pentosus TOMC-LAB4 have strong gastrointestinal tolerance
when compared to E. coli.
7.3 Cell and Ex Vivo Tissue Model
Compared with animal and human experiments, cell and ex vivo tissue models are
high efficiency, less consumable, and suitable to mimic host physiology. Therefore,
they have become one of the most important and widely used models for studying
lactic acid bacteria. Cell and ex vivo tissue models can be used as pre- or supple-
mentary experiments for in vivo studies and with good stability and reproducibility.
Besides, these models can be also used in studying in-depth mechanism.
7.3.1 Cytotoxic Model
Cytotoxicity refers to the adverse effects of a physiological or functional disorder

caused by a drug or biologically active substance. These harmful effects act on a cell,
causing changes in its basic structure or physiological processes. And the following
methods are mainly employed to study cytotoxic characteristics of lactic acid bacteria.
7.3.1.1 MTT Model
3-(4,5-Dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric

method is generally used to detect cell survival and growth, originally proposed by
Mosmann, and later widely used in cytotoxicity evaluation. Succinate dehydroge-
nase in living cell mitochondria reduces yellow exogenous MTT to insoluble blue-
purple crystals (formazan) and deposits them in cells, whereas dead cells do not.
Dimethyl sulfoxide (DMSO) dissolves blue-violet crystals in cells, and the number
of viable cells can be detected by establishing a link between the amount of crystal-
lization produced and the number of viable cells. The absorbance at a wavelength of
490 nm was obtained by enzyme-linked immunosorbent assay, which indirectly
reflects the number of viable cells. The test process is simple, the degree of automa-
tion is high, the instrument is common and the structure is simple, and the relative
quantity and relative vitality of the cells can be determined. Based on these princi-
ples, MTT colorimetry is generally adopted to study the effect of lactic acid bacteria
on cell viability.
Thirabunyanon et al. (2009) obtained 54 lactic acid bacteria strains from fer-
mented milk. In the MTT experiment, colon cancer cells were co-cultured with
lactic acid bacteria and measured after 24 h of incubation. The results showed that
Lactobacillus fermentum RM28 significantly inhibited the proliferation of colon
cancer cells, indicating that this strain can be used as a potential functional food
probiotic or products to relieve colon cancer. Chen et al. (2015) studied the biotrans-
formation of aflatoxins in peanut meal by solid-state leavening agents, Streptococcus
thermophilus and Lactobacillus delbrueckii subsp. The aflatoxin and its derivatives
in the untreated peanut meal and fermented peanut meal were extracted with a
methanol-water mixed solution, and their cytotoxicity was analyzed by MTT assay
in L929 cell line. The results showed that untreated peanut meal exhibited strong
cell cytotoxicity while treated peanut meal had no obvious cytotoxicity, indicating
the derivatives of aflatoxin formed in fermentation had low toxicity. All of these
evidences suggest the feasibility of mycotoxin biotransformation by lactic acid bac-
teria. Zhang et al. (2015) studied the anticancer activity of malt extract fermented by
Lactobacillus plantarum dy-1 and verified the effects of unfermented malt extract
and fermented malt extract by Lactobacillus plantarum on cell proliferation. The
results showed that unfermented malt extract could not inhibit the proliferation of
HT-29 cell line, while fermented malt extract significantly inhibited the prolifera-
tion of HT-29 cell line, indicating that Lactobacillus plantarum fermented malt
extract has antitumor effect. Tsai et al. (2015) used MTT assay to determine the
inhibition effect of the antibacterial peptide m2163 and m2386 produced by
Lactobacillus casei ATCC 334 on the colon cancer cell line SW480. The results
showed that compared to PBS-treated SW480, the antibacterial peptides m2163 and
m2386 could inhibit its proliferation. They also compared the effects of the two
antibacterial peptides on both cancer cells (BFTC 905, Caco-2, CHOK1) and nor-
mal cells (H184B5F5/M10). The results showed that the antimicrobial peptides
m2163 and m2386 could selectively kill cancer cells but were not lethal to normal
cells. Similarly, Dilna et al. (2015) studied the toxicity of Lactobacillus plantarum
RJF4 on both cancer and normal cells. As a result, different doses of exopolysac-
charide in Lactobacillus plantarum showed toxicity on MiaPaCa2 human pancre-
atic cancer cell lines, while no toxicity was observed in normal fibroblasts,
suggesting a specific antiproliferative effect of extracellular polysaccharide of
Lactobacillus plantarum on tumor cells.
7.3.1.2 LDH Model
Lactate dehydrogenase (LDH) is an important enzyme that catalyzes the redox reac-
tion between lactic acid and pyruvic acid during glycolysis and gluconeogenesis.
The cytoplasm of living cells in almost all tissues has LDH. Under normal circum-
stances, LDH cannot penetrate the cell membrane, but the destruction of cell mem-
brane integrity caused by apoptosis or necrosis will cause LDH to be released
extracellularly. Under the action of LDH, NAD+ is reduced to form NADH, which
is then reduced to iodonitrotetrazolium (INT) by hydrogen donor phenazine
dimethyl sulfate (PMS), and INT accepts H+ and is reduced to fuchsia formazan.
The measurement was carried out by spectrophotometer, and the absorbance value
was determined at a wavelength of 490 nm. The value was positively linearly cor-
related with the lactate dehydrogenase activity, that is, the activity of lactate dehy-
drogenase can be quantitatively measured by colorimetry. The cytotoxicity of
endogenous and exogenous substances can be quantified by detecting the activity of
LDH released from the cells into the culture solution. LDH release is considered to
be an important indicator of cell membrane integrity and is commonly used in the
detection of cytotoxicity. Borruel et al. (2002) studied the effects of probiotics on
intestinal inflammation, co-cultured Lactobacillus casei DN-114001 and
Lactobacillus bulgaricus LB10 with intestinal lymphocytes, and detected cell via-
bility by LDH release method. The results showed that lactic acid bacteria had a
certain regulatory effect on local inflammation. Yang Jingqiu (2009) obtained lactic
acid bacteria with high antioxidant activity through experiments and studied its anti-
oxidant capacity. The cell model was established after subculture of CT-26 in intes-
tinal cancer cells. The antioxidant capacity of Lactobacillus acidophilus 874 was
analyzed by LDH release method. The results indicated that the LDH in the culture
supernatant of the intervention group was remarkably lower than that of the model
group, indicating that this strain can protect cells from H2O2 oxidative damage.
7.3.1.3 Mitochondrial Membrane Potential Model
Energy produced by respiration of mitochondria is stored in the inner membrane of

this organelle in the form of electrochemical potential energy and causes an asym-
metric distribution of proton and ions between inner and outer sides of the mito-
chondria membrane, which eventually forms a mitochondrial membrane potential
(ΔΨm, MMP). MMP in the normal range is a prerequisite for ensuring normal
physiological function of mitochondria, and its stability is conducive to maintaining
cell stability and normal function. The destruction of the normal membrane poten-
tial of mitochondria occurs before the appearance of apoptosis in the nucleus and is
considered to be one of the earliest events in the process of apoptosis cascade.
Therefore, mitochondrial membrane potential is an important parameter that reflects
the state of mitochondria in cells. Fluorescent probes commonly used in mitochon-
drial membrane potential models include JC-1, rhodamine 123 (Rh123), tetrameth-
ylrhodamine ethyl ester (TMRM), DioC6, etc. JC-1 is a novel cationic dye and a
sensitive marker of mitochondrial membrane potential, which is commonly used in

mitochondrial membrane potential detection. When the mitochondrial membrane
potential is high, JC-1 will concentrate in the matrix to form J-aggregates; when the
mitochondrial membrane potential is low, JC-1 cannot concentrate in the matrix,
maintaining the monomer state. If the JC-1 monomer is excited by 488 nm/514 nm
light, the monomer will emit green fluorescence with an absorption wavelength of
529 nm. If the J-aggregate is excited by 585 nm light, the aggregate will emit red
fluorescence with an absorption wavelength of 590 nm. Therefore, changes in mito-
chondrial membrane potential can be measured indirectly. Rhodamine 123 is a cat-
ionic fluorescent dye that stains mitochondria in living cells and enters the
mitochondrial matrix. The fluorescence intensity is reduced during the process.
When apoptosis occurs, the mitochondrial membrane structure is destroyed, the
mitochondrial membrane potential collapses, Rh123 in the membrane is released,
and fluorescence is generated, changes in mitochondrial membrane potential and
apoptosis can be detected by fluorescent signals. Rh123 is commonly used for the
measurement of mitochondrial membrane potential and does not have any toxicity
to cells. TMRM is a cationic fluorescent dye that penetrates the cell membrane and
can fluoresce at a wavelength of around 580 nm with a 543 nm laser. TMRM has
many advantages over other fluorescent probes. It only accumulates in the mito-
chondria because of changes in membrane potential, the toxicity is relatively small,
and the cell organ binding rate is low, and it is suitable for quantitative analysis of
mitochondrial membrane potential.
Wang et al. (2014) established a model of HT-29 cells to study the induction of
apoptosis by lactic acid bacteria. Using Rh123 as a fluorescent probe, the mitochon-
drial membrane potential of HT-29 cells was obtained by flow cytometry. The
results showed that X12, the M5, and K14 strains have the ability to induce apopto-
sis in HT-29 cells, which is associated with cell wall extracts of the three strains.
Ghoneum and Gimzewski (2014) studied the apoptotic effect of a new yoghurt
cereal product on human multidrug resistance (MDR) myeloid leukemia cells
in vitro and established a model for HL60/AR cells to explore apoptosis of the prod-
uct, and using TMRM as a fluorescent probe, the mitochondrial membrane potential
is detected by flow cytometry. The results indicate that the induction of apoptosis in
this product is related to the activation of caspase 3, the decrease of Bcl-2 expres-
sion level, and the decrease of mitochondrial membrane potential polarization, so
the yoghurt grain product can be used as a potential for MDR leukemia treatment
method.
7.3.2 Intestinal Cell Adhesion Models
Lactic acid bacteria can adhere to and colonize the intestine and form a biological
barrier of the intestinal mucosa, regulate the immune response, repair the damaged
gastrointestinal mucosa, and resist the interference of various harmful bacteria. The
ability of lactic acid bacteria to adhere to the intestinal tract of the host is one of the
important indicators for evaluating whether it is a probiotic. Intestinal cell adhesion

is rapid, sensitive, and intuitive. At present, cell lines such as Caco-2 and HT-29 are
mainly used as models. Tuomola and Salminen (1998) established an intestinal epi-
thelial cell model with Caco-2 cancer cells in the study to study the adhesion char-
acteristics of 12 different lactic acid bacteria. The study suggests that the probiotic
effect of lactic acid bacteria is related to its adhesion ability, but it cannot be used as
the only criterion for evaluation. Gopal et al. (2001) established a model of intesti-
nal cell adhesion using HT-29, Caco-2, and HT29-MTX cells to study the adhesion
characteristics and colonization characteristics of Lactobacillus rhamnosus DR20,
Lactobacillus acidophilus HN017, and Bifidobacterium DR10 strains. The results
showed that all the three strains had strong adhesion to intestinal cells. Tulini et al.
(2013) isolated a bacteriocin-producing lactic acid bacterium FT259 from cheese
and established a cell model with Caco-2 cells, which was found to have good intes-
tinal adhesion and inhibit the growth of Listeria monocytogenes. Li-dong et al.
(2014) isolated a dominant strain HUCM 201 from the starter kefir and found that
the strain has certain adhesion to Caco-2 cells. Chen Pei (2014) studied the adhesion
of 18 strains of lactic acid bacteria to Caco-2 cell line. The results showed that the
tested strains had certain adhesion to Caco-2 cell line, which was about 0.2–15.5
bacteria/cell. Among them, six strains of lactic acid bacteria have an adhesion
capacity greater than eight bacteria/cell.
7.3.3 Immunomodulatory Model
Lactic acid bacteria can regulate the body’s immune response, keep the body at a
normal immune level, and play an important role in the body’s various probiotic
functions. In recent years, the immunomodulatory function of lactic acid bacteria
has been broadly concerned and studied.
Lactic acid bacteria influence the secretion of cytokines. Zhu et al. (2011) studied
the influence of three lactic acid bacteria strains derived from centenarians on acti-
vated macrophages. In their study, mouse macrophage cell line RAW264.7 was
used, and then whole bacterial cells, bacterial cell walls, and cell-free extracts were
co-cultured with macrophages. Finally, the production and phagocytic activity of
NO and the production of IL-6 and TNF-α were tested. The results showed that these
three strains could increase the activity of macrophages by increasing phagocytic
activity and enhancing the expression of cytokines NO, IL-6, and TNF-α and could
regulate immune activity. Ren Dayong (2013) established a Caco-2 cell inflamma-
tory model to study the immunomodulatory effects of probiotic Lactobacillus.
Lactic acid bacteria were co-cultured with inflammatory cells, and the results
showed that Lactobacillus salivarius and Lactobacillus plantarum could downregu-
late the expression of cytokines such as IL-1α, IL-8, and NF-κB in Caco-2 cells,
suggesting their potentials in resisting pathogenic infection and alleviating inflam-
matory reaction. Chen Pei (2014) established an inflammatory model of HT-29 cells
to verify the regulation role of lactic acid bacteria on the mRNA expression and
secretion of cytokine. Chen Pei (2014) established an inflammatory model using

HT-29 cells, and compared to the control group, the expression levels of TNF-α,
IL-6, and IL-8 mRNA in L. casei CCFM0412 treatment group showed a significant
downregulating trend, while the expression levels of IL-4 and IL-10 mRNA in the L.
rhamnosus CCFM0528 and L. rhamnosus CCFM0528 treatment group showed a
significant upregulating trend, indicating their immunomodulatory effects. Jiang
et al. (2016) studied the effects of Lactobacillus plantarum NDC 75017 on the
expression of pro-inflammatory cytokines IL-1 and IL-6 and tumor necrosis factor
by Caco-2 cells. The results showed that Lactobacillus plantarum NDC 75017 can
regulate the immune processes associated with pro-inflammatory cytokines NF-κB
and p38 MAPK and has the potential to act as an immunomodulator, an important
type of functional foods. Rizzo et al. (2013) studied the effects of lactic acid bacteria
on the immune response of Streptococcus pyogenes-infected human laryngeal epi-
thelial cells Hep-2 and epithelial A549. The growth of S. pyogenes and the produc-
tion of IL-17 and IL-23 were determined. The results showed that Lactobacillus
plantarum inhibited the expression of TLR2/4 and the production of IL-17 and
IL-23 and had immunomodulatory effects. Zhai Qixiao (2015) studied the lactic
acid bacteria with the potential to reduce cadmium toxicity and found that lactic acid
bacteria can reverse the increase of cytokines such as TNF-α, IL-1β, IL-6, and
IL-8 in HT-29 cell line induced by cadmium exposure, indicating that probiotics can
regulate intestinal immunity.
7.3.4 Oxidative Stress Model
Oxidative stress is a series of adaptive responses caused by the imbalance between

the prooxidant component and the antioxidant component in the body’s cells.
Oxidative stress and its oxidative damage have become a basic pathological meta-
bolic process in the development and progression of diseases in different systems.
Cellular oxidative stress models are often used to evaluate the antioxidant capacity
of lactic acid bacteria.
Lin and Chang (2000) analyzed the antioxidant ability of both intact cells and
cell-free extracts of lactic acid bacteria, respectively. The results showed that
Lactobacillus acidophilus ATCC4356 and Bifidobacterium longum ATCC15708
strain can significantly reduce the cytotoxicity of 4-nitroquinoline-1-oxide (4NQO)
on intestinal cells and inhibit the occurrence of lipid peroxidation in cell membrane
differently. Yang Jingqiu (2009) studied the protection of oxidatively damaged CT-26
cell line by lactic acid bacteria and treated the CT-26 cell line with H2O2 to establish
an oxidative damage model. The experimental results show that the lactic acid bac-
teria can increase the antioxidant capacity of cells and reduce the content of malondi-
aldehyde, indicating that lactic acid bacteria may reduce the oxidative damage
caused by hydrogen peroxide by scavenging free radicals. Li Sheng-yu et al. (2013)
treated Caco-2 cell line with hydrogen peroxide for 12–48 h to establish oxidative
damage model, and then studied the antioxidant effect of Lactobacillus plantarum
C88. The results showed that Lactobacillus plantarum C88 showed strong antioxi-
dant activity by increasing the free radical scavenging ability and antioxidant enzyme
activity of Caco-2 cells. Achuthan et al. (2012) studied the antioxidant potential of
Indian intestinal probiotics and the ability to increase the host cell’s antioxidant
defense enzyme system under oxidative stress conditions. The results showed that
Lactobacillus could significantly upregulate the expression of catalase, superoxide
dismutase, and glutathione peroxidase in oxidative damage induced by H2O2 in
HT-29 cell line, so lactic acid bacteria can be used as a potential intervention for
diseases caused by oxidative stress. Lu Wei and Wei Ping (2013) established a model
of chicken embryo fibroblasts infected with avian infectious bronchitis virus to ana-
lyze the activity of lactic acid bacteria on intracellular superoxide dismutase and
glutathione peroxidase and analyzed the changes of malondialdehyde content. The
results showed that the six strains of lactic acid bacteria significantly increased the
total antioxidant capacity of the cells and decreased the content of malondialdehyde,
which can reduce the oxidative damage caused by free radicals. Wu et al. (2014)
established an oxidative stress cell model with Caco-2 cell line to study the antioxi-
dant capacity of lactic acid bacteria. The results showed that lactic acid bacteria can
improve the ability of cells to scavenge superoxide anion free radicals and total anti-
oxidant capacity. Zhai Qixiao (2015) studied the reduction of cadmium damage by
lactic acid bacteria and found that Lactobacillus plantarum CCFM8610 can signifi-
cantly reduce the content of reactive oxygen species (ROS) and malondialdehyde
(MDA) due to cadmium exposure, which proves that the strain has the effect of
relieving oxidative stress caused by cadmium exposure in cells.
7.4 Animal Model
Functional evaluation based on animal models is an approach which uses animal

model to investigate growth and metabolism and disease and homeostasis. Animal
models follow three principles: it should have a similar regulation process with the
human body, a similar behavioral appearance, as well as a similar response to drug
treatment. Therefore, functional evaluation of probiotics using animal models is one
of the most important links for the application of lactic acid bacteria. Even though
living animal models used in functional evaluation are economically expensive and
of long experimental period, relevant physiological and biochemical indicators
measured in these experiments can directly reflect the beneficial effect of lactic acid
bacteria on living animals. These studies can provide reference value for the
improvement of feeding formula for farmed animals and also guide the prediction
for the influence of lactic acid bacteria on the human physiology and pathology.
7.4.1 Rat Model
7.4.1.1 Variety, Strain, and Biological Characteristics
Rat (scientific name, Rattus norvegicus) is a mammalian, rodent, Muridae, Rattus

animal. Rat strains are mainly divided into inbred lines and closed groups. Different
varieties have different biological characteristics and medical applications. At pres-
ent, there are more than 100 inbred lines in rats, among which are mainly ACI,
F344/N, LEW, SHR, GH, BUF, and other lines. And common closed groups include
Wistar, SD, and Long-Evans.
Among them, ACI rats have a high incidence of spontaneous tumors such as
pituitary tumors, testicular tumors, adrenal tumors, and various congenital renal
abnormalities. F344 rats have a high incidence of breast tumors, pituitary tumors,
testicular stromal cell tumors, and leukemia and are acceptable to various trans-
planted tumors. LEW rats can be the host of tumors and can be transplanted into
many kinds of tumors; allergic encephalomyelitis, arthritis, and autoimmune com-
plex hemoglobinosis can be induced by experimental allergic adjuvants. SHR rats
have a high incidence of spontaneous hypertension and are often used in the screen-
ing of antihypertensive drugs. GH rats have hereditary hypertension combined with
cardiovascular disease and are often adopted to study the hypertension and related
cardiovascular diseases. Wistar rats are one of the most widely used rat breeds in the
laboratory because of their docile character, strong reproductive capacity, and low
tumor incidence. SD rats grow faster than Wistar rats and have strong disease resis-
tance, which are one of the most commonly used rat strains in toxicology and nutri-
tion research (Xinyou 1989; Lijun and Yunan 2012).
7.4.1.2 Applicable Functional Evaluation Model
Rats are one of the most widely used animals in pharmacology, tumor models, nutri-
tional metabolic disease models, hereditary disease models, neuroendocrine disease
models, and infectious disease models. Common pharmacological studies include
antihypertensive drugs, drugs for treating cardiovascular diseases, and drugs that
affect neurotransmitter release. At the same time, in the toxicological study of drugs,
rats are also often used to determine the maximum dose, metabolic changes, and
accumulation characteristics of the drug. Rats can replicate a variety of tumor mod-
els and are among the most commonly used animals in cancer research.
Rats are prone to liver cancer, and diethyl nitrosamine and diaminobiphenyl
(DAB) are commonly used to replicate rat liver cancer model; rat esophageal cancer
model is replicated by methylbenzyl nitrosamine. Because of their sensitivity to
nutrient deficiencies, rats are often used to establish a variety of battalion-deficient
disease models for the metabolism of vitamins, amino acids, calcium, and phospho-
rus. In recent years, rats have also been used in studies of alcoholism, atherosclero-
sis, malnutrition, and the like. In the introduction of rat breeds, it can be seen that
some strains of rats have spontaneous genetic diseases, such as GH rats and SHR
rats have a higher incidence of spontaneous hypertension. Such rats are often used
to establish genetic disease models such as epilepsy, obesity, hypertension, and
cataracts, making it easier to conduct in-depth studies of these diseases. Rats have a
developed pituitary-adrenal system, which has a strong stress response to external
stimuli. Experiments have been conducted to establish a rat model of stress gastric
ulcer. In addition, endocrine experiments such as the adrenal gland, pituitary, and
ovary can be performed by establishing a rat model of excised endocrine glands. It
is also possible to establish a rat model of paratyphoid fever and bronchial pneumo-
nia and then conduct an in-depth study on similar infectious diseases. At the same
time, rats are often used in infectious disease models such as pasteurellosis,
Staphylococcus infection (after hormone treatment), and Candida albicans.
Rats are an important experimental animal model for the functional evaluation of
lactic acid bacteria. Taking the diabetes model as an example, a rat model of type I
diabetes was successfully established by inoculating rats with streptozotocin (STZ),
and then the therapeutic effect of lactic acid bacteria on type I diabetes was explored
(Yeo 2010) A rat type II diabetes model was established by feeding a high-sucrose-
fat diet and injecting low-dose streptozotocin and explored the therapeutic benefits
of lactic acid bacteria-fermented camel milk on type II diabetes (Manaer et al.
2015). Specific methods are as follows: Male SPF Wistar rats weighing 160–200 g
were fed with a high-sugar and high-fat diet (67% basal diet, 2.5% cholesterol,
0.5% cholate, 10% lard, 15% carbohydrate). After 6 weeks of feeding, the rats in the
high-sugar and high-fat diet group were given STZ intraperitoneally at a dose of
30 mg/kg according to the body weight of the rats, while the normal diet group was
injected with the same amount of citrate buffer. The model of type II diabetes was
successfully established when the fasting blood glucose level of the mice was higher
than 11.1 mmol/L within 1 week after injection. Furthermore, the hypoglycemic
effect of lactic acid bacteria-fermented camel milk was investigated in groups. It
was found that high-density fermentation of camel milk (6.97 × 108 lactic acid bac-
teria +2.2 × 106 yeast) had significant hypoglycemic effect on type II diabetic rats.
He Qiuwen (2012) induced high-fat diet, injected low-dose STZ to obtain a rat
model of type II diabetes, and further assessed the effect of Lactobacillus casei
Zhang in preventing and treating type II diabetes in rats. The modeling method was
similar to the above report, but the STZ dose was changed to 40 mg/kg. The model
was successfully established when the fasting blood glucose was higher than
7 mmol/L after 1 week and the blood glucose was higher than 11 mmol/L 2 h after
meal. Through perfusion of the probiotics, it was found that the probiotic L. casei
Zhang had a good preventive and therapeutic effect on STZ-induced type II diabetes
in rats. The glucose content in rats was significantly regulated, and the blood glu-
cose tolerance was significantly improved.
Rats are also the most frequently used animal models in hyperlipidemia experi-
mental studies at home and abroad. In the modeling method, the high-fat feed
method is widely used because of its low difficulty in operation. Zhang Dong et al.
(2007) compared the differences in modeling results between different formulas by
changing the formula of high-fat diet. Specific methods are as follows: 60 Wistar
rats were randomly divided into 6 groups. In addition to the basic diet group, they
were divided into five groups, which were intragastrically administered with differ-
ent high-fat emulsions (formula 1 is lard, formula 2 is 20% lard + 10% cholesterol,
formula 3 is 20% lard + 10% cholesterol + 2% No. 3 bile salt, formula 4 is 20% lard
+ 2% cholesterol + 0.2% propyl thiouracil, and formula 5 is 15% lard + 6% choles-
terol + 2% biliary salt + 0.2% propyl thiouracil). These emulsions were adminis-
tered at a dose of 10 mg/kg every day, and blood was taken through the eyeball
every 10 days to measure the contents of total cholesterol, triglyceride, high-density
lipoprotein, and low-density lipoprotein in the blood. The results showed that bile
salt was combined with propylthiouracil, that is, formula 5 could get the ideal model
(10 days) faster, and the stability of each index was higher. Based on the rat high-fat
diet model, Banjoko et al. (2012) explored the probiotic effects of a mixture of vari-
ous lactic acid bacteria (Lactobacillus acidophilus DSM 20242, Bifidobacterium
DSM 20082, Lactobacillus helveticus CK 60) in regulating blood lipids. Usman and
Hosono (2001) explored the lipid-lowering effect of the high-fat Wistar rat model
by mixing lactic acid bacteria powder (Lactobacillus grisea SBT 0270) into high-fat
diet. The results showed that total cholesterol and total bile acid were significantly
decreased in the serum of rats in the probiotic powder treatment group. Lactic acid
bacteria can significantly reduce the reabsorption of cholate and increase the dis-
charge of sterols in feces.
For alcoholic liver injury, Du et al. (2003) fed 20% ethanol in Wistar rats daily.
After 8 weeks, the structure of rat hepatocyte membrane was destroyed, and liver
function was damaged, which behaves consistently with alcoholic liver function
damage in human. Thus, a rat model of chronic alcoholic liver injury was success-
fully established. Liu Keliang et al. (2015) administered SD rats with a dose of
50% alcohol at 12 ml/kg. Compared with the control group, the liver cells in the
model group were found to have severe diffuse steatosis, and more fat droplets
were observed in the cells. Thus, a model of acute alcoholic liver injury was suc-
cessfully created. Duan et al. (2002) gave lactic acid bacteria preparations before
feeding Wistar rats with ethanol. Finally, it was found that lactic acid bacteria can
effectively prevent severe macrovesicular lipid changes caused by ethanol, ensure
the histological structure of the liver is stable, reduce the absorption of ethanol in
the stomach, and reduce endotoxin translocation to prevent alcoholic liver damage.
Qing and Wang (2008) explored the probiotic effects of lactic acid bacteria by
establishing a rat model of steatohepatitis induced by ethanol. Specific methods are
as follows: 25 Wistar rats weighing 180–200 g were adopted and split into three
groups. The control group (five rats) were fed normally. The ethanol group (10) and
the antialcoholic group (10) were fed with ethanol at a dose of 10 g/kg daily. The
antialcoholic group was fed with a mixture of probiotics (containing Lactobacillus
acidophilus 4 × 1010 cfu/ml and Bifidobacterium longum 2.5 × 107 cfu/ml) at a dose
of 1.5 ml/100 g 30 min before feeding ethanol. The other groups were fed with the
same dose of normal saline. After continuous feeding for 5 days, the blood, liver,
and stomach were taken for sections, and the related enzyme activities (glutamic-
oxaloacetic aminotransferase, alanine aminotransferase, alkaline phosphatase,
etc.) were detected. The results showed that lactic acid bacteria can reduce the
concentration of ethanol in the blood by accelerating the initial decomposition of

ethanol in the stomach and liver. Compared with the ethanol group, lactic acid
bacteria can effectively protect the liver tissue structure and significantly reduce the
occurrence of alcoholic liver injury.
In addition, rats fed with a high-cholesterol diet can exhibit the symptom of high
cholesterol, the formation of which is similar to the human body, so it can be used
for high cholesterol-related regulation studies. Kim et al. (2010) explored the effect
of Lactobacillus-fermented soymilk on cholesterol content in rats by establishing a
high-cholesterol rat model. Methods are as follows: 50 male SD rats weighing 240–
260 g were randomly divided into five groups. One group was fed with standard diet
as control, and the other four groups were fed with high-fat diet containing 30%
shortening oil. In addition, the other four groups were fed with normal saline, inhib-
itor (positive control), 25% fermented soymilk, and 50% fermented soymilk 200 μl,
respectively. Daily intake and weight gain were recorded, and blood samples were
collected from the heart for analysis after one night fasting. The results showed that
fermented soybean milk could significantly reduce the LDL cholesterol content in
the blood. Pan and Zhang (2005) established a high-cholesterol model by feeding
rats with high-fat diet and evaluated the cholesterol-lowering and probiotic effects
of yoghurt produced by the cholesterol-lowering Lactococcus lactis subspecies
LQ-12. Forty-eight male SD rats weighing 180–220 g were divided into four groups
on average. They were fed with basic diet, high-fat diet (besides 86.5% basic diet,
12% lard, 1% cholesterol, and 0.5% bovine bile salt were added), high-fat diet +
sugary skimmed milk, and high-fat diet + yoghurt (made from the same amount of
sugary skimmed milk as the former group). Weekly intake and weight changes were
recorded, and the samples were tested at 14 days and 28 days, respectively. By mea-
suring the cholesterol content in the liver and feces, it was found that the cholesterol
content in the liver and feces of the yoghurt-feeding group decreased and the cho-
lesterol content in the feces increased, indicating that the Lactobacillus not only
promoted the excretion of excess cholesterol in vivo but also inhibited the choles-
terol synthesis to some extent.
7.4.2 Mouse Model
7.4.2.1 Variety Strains and Biological Characteristics
Mice are widely distributed all over the world. More than 1000 inbred lines and
independent outbreeding groups have been formed after long-term artificial feeding
and selective breeding. Mouse, as an experimental animal, has been the most fre-
quently used and thoroughly studied mammalian experimental animal in the world
since the seventeenth century. The strains of mice are mainly inbred lines, closed
populations, and mutant lines. Among them, there are about 250 inbred lines and
more than 350 mutant lines.
About 44% of inbred strain A mice aged 6 months showed lupus erythematosus
cells and antinuclear antibodies. It is deficient in complement C5 and is sensitive to
X-ray irradiation. Compared with other inbred mice, BALB/c mice had the highest
blood pressure and spontaneous hypertension and were susceptible to chronic pneu-
monia. CBA strain mice contain CBA/Br, CBA/Ca, CBA/J, CBA/St, CBA/H, and
other subspecies. They have high blood pressure and are highly sensitive to vitamin
K deficiency. AKR is deficient in complement C5, which is easy to induce immune
tolerance. It has low reproductive rate in common environment but good reproduc-
tion in aseptic and non-specific pathogen (SPF) environment and short growth
period. C57BL/6 mice are more sensitive to Mycobacterium tuberculosis and are
easy to induce immune tolerance. C3H mice include C3H/Bi, C3H/He, C3H/HeJ,
C3H/St, C3HeB/FeJ, C3H/DiSn, C3H/Sf, and other subfamilies. DBA/2 strain mice
were sensitive to pertussis histamine susceptibility factor and resistant to plasmo-
dium and typhus. The content of serum immunoglobulin in NZB strain mice was
remarkably larger than that in other strain mice. The specific manifestation was that
the content of IgM and IgG increased significantly, and the mice were susceptible to
autoimmune hemolytic anemia. Several classical models of the mutant line include
nude mice (hairless epidermis, T lymphocytes are damaged, and cellular immunity
is weak due to thymic dysplasia), dwarf mice (infertile, congenital hypoperfusion of
auxin and thyrotropin), diabetic mice (at 3–4 weeks of age, blood glucose increases
significantly, abnormal fat masses can be found in subcutaneous tissue of the subax-
illary and the groin), etc. The closed colony mice were KM mice, CFW mice, ICR
mice, CFW mice, NIH mice, etc.
Mice have been used in many functional evaluation models because of their diver-
sity and in-depth research. In immunological research, different types of immuno-
deficiency mice can be obtained by specific means, so as to better study the immune
mechanism and other immune-related aspects. At the same time, mice are often
used to produce monoclonal antibodies. In tumor models, mice are one of the most
widely used animal models. Many strains of mice have higher incidence of sponta-
neous tumors, which are similar to human tumors in terms of genesis, and are often
used for screening antineoplastic drugs and studying the related mechanisms. At the
same time, mice are often induced to carcinogenesis or directly transplanted tumor
cells for the study of tumor growth, metastasis, and treatment. Mice are often used
to establish infectious disease models to study the pathogenesis and treatment of
infectious diseases. Common infectious disease models include Salmonella model,
Schistosomiasis japonicum model, leptospirosis model, poliomyelitis model, mold
model, and so on. Mice are less resistant to many pathogens, so they are often used
in the study of various pathogens. Common pathogenic infection models include
influenza model, malaria model, encephalitis model, rabies model, and schistoso-
miasis infection model. Some strains of mice carry spontaneous genetic diseases, so
they are often used as ideal laboratory animals to explore the pathogenesis, genetic
characteristics, and treatment of related diseases. Common genetic disease models

include melanosis model, albinism model, familial obesity model, hereditary ane-
mia model, systemic lupus erythematosus model, and diabetes insipidus model
(Xinyou 1989; Lijun and Yunan 2012).
In the study of lactic acid bacteria, mice are commonly used animal models to
evaluate immune regulation. Li Li et al. (2011) established an animal model of dust
mite-sensitized and dust mite-stimulated female C57BL/6 mice and investigated
the effect of oral Lactobacillus on the immunoregulatory function of spleen cells in
mice with dust mite-induced allergic airway inflammation. The results showed that
oral Lactobacillus could induce the proliferation of CD4+ regulatory T lymphocyte
subsets in spleen cells of mice and reduce the content of Th1/Th2 cytokines by
releasing IL-10, thus inhibiting the allergic airway inflammation induced by dust
mites. Yuan et al. (2009) explored the relationship between the adhesion efficiency
of lactobacillus to Peyer’s Patch and its immunomodulatory effect, the results
showed that the adhesion of Lactobacillus increased the phagocytic activity of
abdominal macrophages in OVA-sensitized mice and increased the content of fecal
sIgA. Li et al. (2014) explored the immunomodulatory effect of Lactobacillus aci-
dophilus NCFM exopolysaccharide on BALB/C mice. Short-term expression of
interleukin-1 alpha (IL-1 alpha), chemokine C-C motif 2 (CCL2), tumor necrosis
factor alpha (TNF-alpha), and penetrating hormone 3 (PTX3) was detected in the
exopolysaccharide stimulation group. It has a certain inhibitory effect on colorectal
adenocarcinoma. In addition, the organ coefficient, intestinal emptying index, and
phagocyte index of mice stimulated by exopolysaccharide increased significantly.
Cross et al. (2002) established antigen-sensitized mice to investigate the induction
of T-cell factors Th1 and Th2 by Lactobacillus rhamnosus HN001. Specific steps
are as follows: 6-week-old female BALB/c mice were fed with basic diet at 22 °C
and 12-h light for 1 week. Thirty mice were randomly divided into two groups. The
bacterial group was fed with 109 CFU live bacteria daily, 50 μg ovalbumin (OVA)
with alum adjuvant was injected at 14 days and 21 days, and 20 μg OVA prepared
by physiological salts were injected at 26 days and 27 days. Samples were taken at
28 days.
In terms of antagonizing pathogens, Tsai et al. (2011) explored the effect of
mixed bacterial liquid and its additives on the ability of mice to resist Salmonella by
feeding mice with different kinds of mixed lactic acid bacteria and mixed bacteria
solution added skim milk. The results showed that different combinations of lactic
acid bacteria could affect the anti-Salmonella infection ability of mice. Specific
combinations often enhanced the anti-infection ability of mice liver and spleen.
Chen et al. (2013) explored the probiotic effect of heat-lethal Lactobacillus on anti-
Salmonella infection in mice by establishing a mouse model of Salmonella infec-
tion. The results showed that oral administration of the selected thermo-lethal
Lactobacillus mixture could effectively reduce the risk of infection by Salmonella
and infection-related inflammation in mice. The specific modeling steps were as
follows: 20 male inbred BALB/c mice weighing 24–28 g were randomly fed with
basic diet 1 week before the experiment, and then the mice were randomly divided
into four groups. The mice were fed with sterile saline, mixed live bacteria (the
density of each lactic acid bacteria was not less than 109 cfu/ml), mixed heat-killing
bacteria 1 (100 °C 30 min sterilization, the density of each lactic acid bacteria was
not less than 109 cfu/ml), and mixed thermo-lethal bacteria 2 (121 °C 15 min steril-
ization, the density of each lactic acid bacteria was not less than 109 cfu/ml). The
dose was 200 μl/day. After 7 days of feeding, the mice were inoculated with
3.0 × 107 cfu/ml of oral bacteria and 200 mu l of salmonella. The samples were
taken from the mice on the third and sixth day after infection.
Mouse models can also mimic many intestinal diseases, so as to study the regula-
tion of lactic acid bacteria on intestinal health. Taking inflammatory bowel disease
(IBD) as an example, according to different experimental needs, the establishment
of inflammatory bowel disease model in mice can be divided into four methods:
chemical-induced animal model (acetic acid, sodium glucan sulfate, trinitrobenzene
sulfonic acid, etc.), genetic engineering animal model (gene knockout or trans-
genic), cell transplantation animal model, and spontaneous animal model (Yuefan
and Nan 2011). Yao-Qing et al. (2011) established a mouse colitis model induced by
DSS. The inhibition effect of lactic acid bacteria on oxidative damage of colon
mucosa was studied. The results showed that the selected lactic acid bacteria was
able to inhibit the increase of free radical level in colon contents induced by DSS
and increase the antioxidant capacity of colon. Cha et al. (2014) established an
inflammatory bowel disease model in mice induced by trinitrobenzene sulfonic acid
(TNBS) and investigated the effect of lactic acid bacteria which inhibited the
decomposition of mucopolysaccharide on inflammatory bowel disease. It was found
that this kind of lactic acid bacteria could improve colon inflammation by inhibiting
intestinal bacteria to degrade mucopolysaccharide and inhibit the expression of
inflammatory cytokines. Cha et al. (2014) established an inflammatory bowel dis-
ease model in mice by injecting TNBS into the descending colon. The anti-
inflammatory effects of the four Lactobacillus mixed bacteria preparations were
investigated. It was found that the Lactobacillus preparations could effectively pro-
tect the colon from TNBS damage. Specific modeling steps are as follows: 6-week-
old female ICR mice were anesthetized and injected 2% TNBS (formulated by 50%
ethanol) 130 μl into the descending colon through the anus with a syringe. After
30 seconds of vertical posture, the mice were put back into the cage. Divided into
bacteria group and control group, the bacteria group was fed with different lactic
acid bacteria daily, and 2% TNBS was injected into colon 2 days later, and the con-
trol group was fed with normal saline, and 2% TNBS or 50% ethanol was injected
into colon 2 days later. Three days after colonic injection, correlation analysis was
performed (colon pathological section and related gene expression).
In regulating intestinal flora, Wu Jing et al. (2013) used antibiotic interference to
establish a mouse intestinal flora imbalance model and explored and proved the
probiotic effect of Lactobacillus and Bifidobacterium preparation on intestinal flora
imbalance mouse model. Yeom et al. (2015) established a mouse model of allergic
dermatitis induced by trimethylamine (TMA). By feeding the model mice with
Lactobacillus casei LCR35, it was observed that LCR35 could inhibit the develop-
ment of allergic dermatitis by balancing intestinal flora. Millette et al. (2008) estab-
lished a model of intestinal colonization against vancomycin enterococci. Through
screening and obtaining lactic acid bacteria producing Streptococcus lactis peptide
and fasciclin, the effect of reducing intestinal anti-vancomycin enterococci was
explored. Specific steps are as follows: female CF-1 mice weighing 25–30 g were
fed under SPF condition before modeling. The mice were subcutaneously injected
with clindamycin at a dose of 1.4 mg per day for 5 days. Three days after the injec-
tion, an enterococcal colonization model of vancomycin-resistant Enterococcus was
established by inoculating Enterococcus solution overnight cultured in brain heart
infusion (BHI) broth at a dose of 250 μl (containing about 108 cfu live bacteria)
through the stomach. Oral lactic acid bacteria test showed that oral lactic acid bac-
teria can effectively reduce the colonization of vancomycin-resistant Enterococcus
in the intestine.
7.4.3 Piglet/Miniature Pig Model
Currently, the breeds of pigs for biomedical research generally include common
pig, Homer pig, Nebraska pig, Khan Foster pig, Hommel pig, Pitman-Moore pig,
and Sinclair pig (because of the high cholesterol in the blood, scars in the arteries, a
typical atheromatous lesion in the artery), Von Wenli Bligh pig (with congenital
hemophilia for hemophilia study), Uktan pig (belonging to Mexico hairless pig, can
be used to study diabetes), German Gottingen pigs, and Japan OMINI mini pig
(cultured from small-scale black pigs in the northeast of China).
The digestive system, cardiovascular system, nutritional needs, bone develop-
ment, skin, and mineral metabolism of pigs are very similar to those of human
beings, so they are often chosen as animal models for functional evaluation. The
miniature pigs and piglets are more popular among researchers because of their low
cost, short feeding cycle, and convenient feeding. The average life-span of minia-
ture pigs is 16 years and the longest is 27 years. The weight of adult miniature pigs
is about 30 kg (6-month-old), and that of miniature pigs is about 15 kg. Saliva con-
tains more amylase. The stomach is a single chamber and can secrete a variety of
digestive enzymes. Gallbladder bile concentration capacity is low. Cecum contains
a large number of microorganisms and plays an important role in digestion. The
piglet stage is a critical period for the growth and development of pigs and the high-
est requirement for feed and feeding management. The health of piglets is very
important for the growth of pigs. At the same time, piglets have the characteristics
of rapid growth and short experimental cycle, and in the key period of the establish-
ment of intestinal flora and immune system, it is conducive to study the effects of
lactic acid bacteria on the establishment of intestinal flora and immune system. In
addition, the intestinal system and immune system of piglets are similar to that of
human beings. The model of piglets can simulate the effect of Lactobacillus on
infant health to some extent. This model can be used to study the probiotic effect of
lactic acid bacteria on piglets, such as the growth performance model, the intestinal
steady-state model, and so on, which can directly study the effects of lactic acid
bacteria on the health of piglets and provide the basis for developing probiotic feed
to promote healthy growth of pigs.
The functional evaluation of miniature pigs and piglets mainly includes cardiovas-
cular disease, immune function, nutritional and metabolic diseases, genetic dis-
eases, burns, cancer, and so on. It has the following characteristics: (1) The porcine
coronary circulation is similar to humans in anatomy and hemodynamics. Eating
high-cholesterol foods is as common as humans with typical lesions of atheroscle-
rosis. The atherosclerosis of the arteries and coronary arteries and blood vessels of
old pigs is similar to that of human, so pigs can be selected as the first choice for
atherosclerosis research. (2) Since piglets can only get maternal antibodies through
colostrum, there are very few γ-globulin and other immunoglobulins in the piglets
during the first few weeks of birth, and the immune response of the serum is very
low. There is no antibody in the body of aseptic pigs, and it can produce a good
immune response by contacting certain antigens. Therefore, these characteristics
can be used for immunological research. (3) The respiratory, urinary, and blood
systems of piglets and young pigs are similar to those of newborns and can be used
to study pediatric nutrition. (4) The tooth structure of miniature pig is similar to that
of human beings. Caries and cariogenic food can produce the same caries as human
beings. It is a good animal model for dental caries research. (5) Miniature pigs can
also be used to study congenital diseases such as red eye disease and small eye dis-
ease, congenital lymphedema, and porphyria. (6) The skin structure of pig is similar
to human, which can simulate the mechanism of humoral and metabolic changes of
burn skin. Specially made pig skin can be used as a cover for human burns, which
can shorten the healing time and reduce pain and infection. (7) American Sinclair
miniature pig has spontaneous skin melanoma before and after birth. This mela-
noma has the same disease and transmission pattern as human melanoma. Tumor
cell changes and clinical manifestations are very similar to those of human mela-
noma from benign to malignant. It is an ideal animal model for studying human
melanoma (Xinyou 1989; Lijun and Yunan 2012)
A large number of literatures reported that Lactobacillus feeding can effectively
improve the growth performance of piglets. The inadequate digestive system, poor
disease resistance, and unreasonable bacterial flora in the internal environment of
newborn piglets often lead to diarrhea, which not only hinders the growth of piglets
but also leads to the death of piglets and increases the economic losses in the pro-
cess of breeding. Establishment of growth performance model mainly studied the
effects of lactic acid bacteria on the health of piglets from the aspects of average
daily gain and feed conversion rate of piglets. Jin Lingmei (2000) reported that
microecological preparation at a dose of 0.2 g/one pig was administered to newborn
piglets 1–2 h before lactation for 3 consecutive days, once every 7 days, and to
35 days after weaning. The results showed that microecological preparation could
significantly improve the breeding rate and growth rate of piglets under the same
feeding conditions. Ross et al. (2010) found that feeding newborn piglets with
Lactobacillus and Enterococcus faecium could increase feed conversion rate and
daily gain of piglets. Estienne et al. (2005) also found that probiotics could increase
average daily gain and feed conversion rate in pigs. Guo Yong et al. (2013) fed seven
kinds of compound probiotics including Lactobacillus acidophilus, Lactobacillus
plantarum, Lactobacillus bulgaricus, and Lactobacillus rhamnosus to newborn pig-
lets and measured the body weight of newborn, 21-day-old, weaning age, and
70-day-old piglets. The results showed that the compound probiotics could effec-
tively reduce weaning stress and increase the survival rate and average daily gain of
piglets. Zhang Dongyan et al. (2011) used 64 weaned piglets (Duroc pig × Changbai
pig × Dabai pig ternary hybrid), the average body weight (16.57 + 0.23) kg, and
randomly divided into four groups, with four replicates in each group and four
heads per replicate. The control group was fed with basal diet, and the experimental
group was fed with basal diet supplemented with 0.25%, 0.50%, and 0.75% of
Lactobacillus reuteri, respectively, with a cycle of 30 days. The results showed that
the average daily gain of piglets with 0.75% lactobacillus was 20.07% (P < 0.05)
higher than that of the control group, and the feed weight was 14.90% (P < 0.05)
lower than that of the control group. Suo Cheng et al. (2012) used Lactobacillus
plantarum ZJ316 isolated from infant feces and added Lactobacillus plantarum
ZJ316 at a concentration of 1 × 109 cfu/ml, 5 × 109 cfu/ml, and 1010 cfu/ml in the
basal diet and added antibiotics for comparative experiments. The results showed
that feeding different concentrations of Lactobacillus plantarum ZJ316 can signifi-
cantly increase the daily weight gain and feed conversion rate of piglets and reduce
the ratio of meat to meat, diarrhea, and mortality.
The intestinal immune system of piglets is basically mature at the age of
4–7 weeks. Therefore, when piglets are early weaned, their autoimmunity is low
(immune antibody level and cellular immunity are low), and weaning interrupts
maternal immune antibody supply to piglets, which further reduces the imperfect
immune function of piglets. At the same time, weaning stress reduced the level of
circulating antibodies, inhibited the cellular immunity, and significantly inhibited
the immune function of piglets. Studies have reported that there are complex interac-
tions between lactic acid bacteria and host intestinal microecology (such as immune
cells, epithelial cells, and intrinsic microorganisms), which can produce immune
stimulation and regulate the immune system of piglets (Scharek et al. 2005).
Lähteinen et al. (2014) fed piglets weaned for 4–5 weeks and average body weight
(9.4 ± 1.7) kg with a concentration of 10 10 cfu/ml of Lactobacillus brevis ATCC8287
for 21 days. The results showed that TGF-β1 content in ileum decreased, while IL-6
content in cecum increased, indicating that Lactobacillus brevis ATCC8287 was
immune to intestinal tract regulation of disease. Liu et al. (2015) fed weaned piglets
with Lactobacillus brevis ZLB004. The initial average body weight of 144 weaned
piglets (Dullock pigs × Yorkshire pigs × Landrace pigs) was (15.60 + 0.13) kg. They
were randomly divided into 3 groups, 4 replicates in each group and 12 pigs per
replicate. The control group was fed with basic diet, and the experimental group was
supplemented with Lactobacillus brevis ZLB004 0.4 g/kg and 0.8 g/kg, respectively,
for 30 days. The results showed that feeding Lactobacillus brevis ZLB004 increased
the levels of interferon-γ and total protein in serum and decreased the levels of serum
bound globin and urea nitrogen, suggesting that Lactobacillus brevis ZLB004 could
improve the immune regulation function of weaned piglets (2015). Wang et al.
(2009) fed 108 cfu/ml Lactobacillus fermentum I5007 to piglets weaned for 24 days
and with an average body weight of (6.07 ± 0.63) kg at a dose of 20 ml/day. The
results showed that Lactobacillus fermentum I5007 could regulate the immune func-
tion of piglets by increasing the transformation of T lymphocytes and inducing the
expression of ileal cytokine. Parvizi et al. (2009) reported that Lactococcus lactis
could increase the secretion of IL-4 and SIGA in intestinal mucosa of weaned pig-
lets and enhance the intestinal mucosal immunity of piglets. Pollmann et al. (2005)
orally administered Lactobacillus acidophilus to aseptic piglets, resulting in
increased leukocyte count and serum total protein (mainly globulin) concentration.
Lactococcus lactis also has strong immune adjuvant properties, which can signifi-
cantly upregulate the expression of dendritic cells. Lactobacillus can affect the
immune response by influencing the level of cytokine production.
Influenced by weaning, diet, and environmental conditions, piglets are prone to
intestinal disorders of normal flora and rapid reduction of beneficial bacteria. The
harmful bacteria will multiply and produce harmful substances and stimulate the
damage of gastrointestinal mucosa, resulting in diarrhea, growth, and even death of
piglets (Inoue et al. 2005). Mathew et al. (1993) studied the changes of intestinal
flora in 28-day-old piglets before and after weaning. It was found that the number
of beneficial bacteria such as Lactobacillus in intestinal contents decreased after
weaning, while the number of harmful bacteria such as E. coli increased. Normal
intestinal flora of early weaned piglets has not been completely established, and the
amount of beneficial bacteria such as Lactobacillus in intestinal tract is low. Ohashi
et al. (2007) fed lactobacillus to weaned piglets and analyzed intestinal flora of
piglets by real-time fluorescence quantitative PCR (qPCR). It was found that after
feeding lactobacillus, the number of normal flora such as Lactobacillus in intestinal
tract of piglets increased and the number of harmful flora decreased, indicating that
Lactobacillus could maintain the level of intestinal flora of piglets and maintain the
intestinal health of piglets.
7.4.4 Poultry Model
Among poultry models with functional evaluation of lactic acid bacteria, chicken is
the most important strain. Domestic chicken is a species of chicks of Galliformes of
Aves of Chordata, which is a wide variety of chickens. They are widely used in the
experiment, such as “nine Jin yellow,” “Australian black chicken,” and “Avian
chicken.” In addition, with the development of reproductive technology and the
need of biotechnology experiments, SPF chicken is increasingly widely used.
Functional evaluation of chicken is as follows: (1) Coagulation test and immunol-

ogy test. Chicken has excellent coagulation mechanism, and in the experiment of
inflammatory phagocytic reaction, chicken red blood cells can be used as phago-
cytic foreign bodies of leukocytes in inflammatory exudates. (2) Sex hormone
research. After removal of male testes, the male characteristics are degraded, and
male hormones can be further studied. (3) Oncology research. Chickens can be used
not only for the study of viral leukemia but also for the study of the mechanism of
viral tumorigenesis and for the construction of animal models of tumor with chick-
ens.(4) Nutrition research. Chickens are suitable for the study of purine metabolism
and calcium and phosphorus metabolism, as well as for the study of vitamin D and
vitamin B 12 deficiency. (5) Research on infectious diseases. Arthritis and pneumo-
nia caused by streptococcal infection, bacterial endometritis, and mycoplasma
infection can be studied in chickens (Xinyou 1989; Lijun and Yunan 2012).
Poultry often suffer from overheating, overcooling, fright, feeding, infection, and
other stress factors in the process of breeding; these factors are easy to lead to the
imbalance of poultry digestive tract flora, resulting in decreased immunity, suscep-
tibility to disease, growth performance decline, or even death. People have been
using antibiotics to solve these problems, although the effect is remarkable, but
there are residues in the body, harmful to human health. Lactic acid bacteria can
regulate intestinal flora, improve growth performance, enhance immune function,
and hopefully become a substitute for antibiotics. Taking chicken as an example, the
effects of lactic acid bacteria on its health were studied by establishing growth per-
formance model, immune correlation model, and intestinal homeostasis model,
which are helpful for further exploring the probiotic effect of lactic acid bacteria and
providing certain help for subsequent scientific research workers.
The growth performance model was adopted to research the effects of lactic acid
bacteria on growth performance of chickens. The most common indexes to measure
growth performance were body weight as well as feed conversion ratio. Lactic acid
bacteria can promote the growth of chickens in the early growth stage, which can be
divided into 1–21-day growth stage and 22–42-day growth stage. Lan et al. (2003)
fed two lactic acid bacteria strains isolated from chicken intestine to white Leghorn
chickens and found that they gained 10.7% more weight than the control group. The
experimental conditions are as follows: 40 15-day-old white Leghorn chickens were
divided into two groups. The concentration of bacteria in the feed was 106 cfu/g, and
the experiment period was 40 days. Kalavathy et al. (2003) studied the effects of 12
kinds of Lactobacillus mixed bacteria powder on growth performance of broilers.
The weight gain and feed conversion rate of broilers fed with Lactobacillus at
1–21 days were not significantly different from those of control group. The weight
gain and feed conversion rate of broilers fed with Lactobacillus at 22–42 days and
1–42 days were significantly higher than those of control group (P < 0.05).
Compared with the control group, after feeding 42 days, the weight gain of the
experimental group was 4.7%, and the ratio of material to weight decreased by 0.18.
The experimental conditions are as follows: 136 1-day-old broiler hens were divided
into two groups, each group had 4 replicates, and each replicate was 17. The control
group was fed with two different basic diets on 1–21 days and 22–42 days. The
experiment group was fed with 0.1% Lactobacillus powder on the basis of the basic
diet, the temperature was 30 C, and the experiment period was 42 days.
The spleen, thymus, and bursa of Fabricius are important immune organs of
poultry. They are important indicators of the growth and development of the body’s
immune organs, representing the immune status of the body; spleen index, thymus
index, and bursa index, serum IgA and IgG, IL-2, SIgA in the duodenal chyme, T
lymphocyte transformation rate, etc. are also important immune indicators for
chickens. Therefore, the effects of lactic acid bacteria on chicken immunity can be
studied by analyzing the changes in these indicators in chickens after adding probi-
otics. Wang et al. (2008) fed broiler chickens with Lactobacillus plantarum P-8 and
found that feeding the bacteria increased the level of SIgA and IgA+ in the feces and
increased the number of CD3+, CD4+, and CD8+ T lymphocytes in the intestinal
tissues, thereby increasing immunity in broilers. Test conditions are as follows: 270
Cobb 500 broilers at 1-day-old age were divided into 3 groups, 6 replicates in each
group, 15 in each group. The control group was fed with basal feed, the model group
was fed with antibiotics-added feed, and the P-8 group was fed with basal feed
while the drinking water contained P-8 at a concentration of 2 × 106 cfu/ml. The
experiment period was 42 days. Hangzhou Bo-lin et al. (2008) added 1.5 × 108 cfu/
ml of Lactobacillus plantarum and Streptococcus faecalis to the feed, and the results
showed that both the mast bacteria and Streptococcus faecalis increased the phago-
cytic capacity of leukocytes, which increased by 16.1% and 15.47%, respectively,
compared with the control group.
Xie Quanxi et al. (2013) studied the effect of Lactobacillus plantarum on broiler
immunity. The study found that adding 4.0 × 105 cfu/g Lactobacillus plantarum to
basal feed can promote the development of spleen and bursa of broiler chickens,
increase serum IgA levels, and total the antioxidant capacity, and the level of SIgA
antibodies in the cecal contents enhance the body’s immunity. Chunyang et al.
(2002) showed that probiotics can significantly improve the growth and develop-
ment of immune organs in AA broilers and increase the spleen index and bursal
index at 28.d, which can reach 18.47% and 6.42%, respectively. Chen Jing et al.
(2012) studied the immune performance of Lactobacillus plantarum on broilers.
The spleen index and bursal index of Lactobacillus plantarum group were signifi-
cantly higher than those of the control group (P < 0.05), indicating that the addition
of appropriate amount of Lactobacillus plantarum increased the relative weight of
the immune organs, which promotes the maturation of the immune organs of broil-
ers. Wang Lifeng (2014) studied the effect of Lactobacillus plantarum P-8 on
broiler immunity. It was found that feeding the food containing the bacteria, the
thymus index, bursal index, and spleen index of broilers were significantly increased,
what’s more, the content of SIgA in serum IgG and intestinal juice increased signifi-
cantly. The cytokines Bu-1, IL-4, and IL-12 in intestinal tissues were significantly
higher than those in the control group (P < 0.05), indicating that the bacteria can
enhance the immune function of broilers.
In a study on the maintenance of intestinal homeostasis by lactic acid bacteria,

Pascual et al. (1999) found that Lactobacillus salivarius CTC2197 can prevent the
colonization of Salmonella C-114. After feeding the Leghorn chicken for 21 days,
it can be completely removed from the chicken intestine Salmonella; Jin et al.
(1998) showed that Lactobacillus significantly increased the number of Lactobacilli
in the cecal of broilers and reduced the number of E. coli; Li Shu-peng and Zhao
Xian-jun (2005) showed that after adding probiotics to the feed compared with the
control group, there was a significant increase in the number of lactic acid bacteria
in the intestines of chicks, and the number of E. coli was significantly decreased
(P < 0.05). Tang Feng et al. (2013) found that adding Lactobacillus acidophilus to
the feed can increase the number of Lactobacilli and Bifidobacteria in the chicks.
Reduce colonization and proliferation of E. coli and Salmonella in the intestine. Xu
Weisen (2008) isolated six strains of lactic acid bacteria from chicken intestinal
mucosa, studied its effect on chicken intestinal flora, found that lactic acid bacteria
can be stably colonized in the intestine, and inhibit the number of Salmonella
typhimurium and Staphylococcus aureus, especially chicken. The number of
Salmonella typhimurium was stable at 106 cfu/g, which was much lower than that of
the control group at 108 cfu/g, which was similar to the effect of feeding antibiotics.
Test conditions are as follows: 135 chickens were divided into 3 groups, 3 replicates
in each group, 15 replicates each. The control group was fed with basal diet, the
lactic acid bacteria group was fed with 3.0 × 109 cfu/g lactic acid bacteria, and the
antibiotic group was fed with 5 mg/kg oxytetracycline feed, fed for 55 days (differ-
ent basal feeds fed in different periods).
7.4.5 Fish Model
7.4.5.1 Variety Lines and Biological Characteristics
As an experimental research object in the fields of environmental protection and

biology, fish has obtained a large number of scientific research results. Fish breeds
quickly and has many kinds of types, which is an easily available experimental
material. Fish has unique advantages in the field of biomedicine, especially in the
toxicology and pharmacology of drugs. They are very sensitive to drugs and toxic
gases, and extremely small components can cause strong reactions. In the fish test,
the test indicators have habits in addition to death. Put the smaller fish into a certain
concentration of liquid medicine, which is suitable for studying Chinese herbal
medicines that have weak pharmacological effects and need to be taken for a long
time. Fish also has a good application in the field of oncology. All tissues of fresh-
water fish animals have lesions, similar to human tumors, so fish is an indispensable
material for carcinogenic exploration in water. The test method is simple, results are
easy to judge, and feeding management is simple; the fish is a good experimental
animal material. The fish immune system is the main mechanism for resisting the
invasion of foreign pathogenic microorganisms, including immune cells, immune
tissues, and humoral immune factors. The immune organs or tissues include the
kidney, spleen, thymus, and mucosal lymphoid tissues; immune cells include red
blood cells, white blood cells, etc.; immune factors include complement, serum
lysozyme, myeloperoxidase, and the like. Fish can maintain the normal function of
the body and the stability of its internal environment through non-specific and spe-
cific immune response mechanisms, but fish are more dependent on non-specific
immune responses.
There are many fish strains as animal models, of which zebrafish and tilapia are
the two main models. There are about 20 strains of zebrafish; they are small indi-
viduals, low cost of breeding, large-scale breeding, low water quality requirements,
in vitro fertilization, in vitro development, simultaneous embryo development and
fast speed, and many advantages in studying the molecular mechanism of embryo
development; and some even can be used as models for studying human diseases.
Tilapia has strong reproductive ability and heterosis and has good tolerance and
responsiveness to environmental stress, microbial infection, and disease damage.
Therefore, it is an excellent physiological and toxicological research model. In
addition, there are also swordtail fish (behavioral, neurological, chemical research),
green fin (genetic research), rainbow trout (growth performance, toxicology
research), and other strains suitable for functional evaluation models (Xinyou 1989;
Lijun and Yunan 2012).
The functional evaluation studies applicable to fish models mainly include three
types. The first one is developmental research: embryonic development mechanism
of fish animals is very similar to that of mammals, zebrafish can reach sexual matu-
rity in 90 days and lay eggs once in a few days under suitable conditions, and the
embryo of 1 day age have comparable levels of embryonic development with
humans of 3 months age. The embryo is transparent, and the embryonic develop-
ment such as embryonic cell movement, heartbeat, and brain region formation in the
intestinal phase can be observed. Combined with chimeric cloning, dye tracer, and
other analytical methods, the cell lineage analysis can be performed and applied to
the study of the origin of tissues and organs. The second is physiological toxicology
studies. Fish models can be used to assess aquaculture-related indicators such as
growth performance and feed utilization. Fish carp and kidney can be used for
membrane physiology research, and zebrafish are widely used in the research field
of circadian clock and motor nerve circuit. Tilapia and rainbow trout are important
models for studying water toxicology. The third is genetics research. There are
many kinds of fish, there are many specialized species and mutants, and the genetic
resources are very rich; the reproductive ability is strong, the cycle is short, the
genes related to quantitative traits are rich and easy to be intra-hybrid, and the sex
can be controlled manually, and genetic studies can be carried out by techniques
such as nuclear transfer and haplotypes.
In recent years, with the continuous expansion of high-density aquaculture in the

aquaculture industry, the breeding environment has been deteriorating, the stress
factors have been increasing, and the disease resistance of fish has decreased, result-
ing in frequent fish diseases, which has brought huge economic benefits to fish
farming loss. Therefore, research and development of additives that promote growth,
enhance immunity, and improve disease resistance have become hot topics in cur-
rent research. As a probiotic, lactic acid bacteria have been widely used to improve
the digestive function of human body, promote the growth of livestock, and improve
its disease resistance. The effects of probiotics on fish health were studied by evalu-
ating the effects of lactic acid bacteria on fish growth performance, immune-related
properties, and intestinal homeostasis. Suzer et al. (2008) showed that the addition
of lactic acid bacteria to the development of larvae of Sparus macrocephalus can
improve the growth performance of larvae. Son et al. (2009) studied the effects of
Lactobacillus plantarum on the growth performance of grouper, adding 106 cfu/kg,
108 cfu/kg, and 1010 cfu/kg of Lactobacillus plantarum to the grouper feed, and the
results showed that different concentrations of Lactobacillus plantarum the average
body weight, feed conversion rate, and survival rate of grouper were significantly
increased. Especially when the dosage was 1010 cfu/kg, the average body weight,
feed conversion rate, and survival rate were increased by 404.6%, 1.26 times, and
36.7%, respectively. Zeng Di-gang and Lei Ai-ying (2011) studied the effect of
Lactobacillus on the growth of tilapia by adding lyophilized powder containing
109cfu/g Lactobacillus to the feed with the addition amount of 0.1%, 0.2%, and
0.3%, the results showed that all three concentrations of lactic acid bacteria could
increase the weight gain rate of tilapia and reduce the feed coefficient. The most
significant increase was 0.3%, the weight gain rate increased by 11.3%, and the feed
coefficient decreased by 0.25. Zhao Qian et al. (2012b) studied the effects of lactic
acid bacteria on the growth performance of grass carp. The experimental conditions
were as follows: grass carp with an average body weight of (75 ± 5) g was raised in
an aquarium (0.9 m × 0.5 m × 0.5 m) with a water volume of 100 L, water tempera-
ture (24 ± 1) °C, 24-h continuous oxygenation, daily feeding amount of 1.5% of the
fish weight, adding lactic acid bacteria freeze-dried powder to the feed, so that the
feed lactic acid bacteria concentration is 0, 1 × 105 cfu/g, 1 × 106 cfu/g, 1 × 107 cfu/g,
1 × 108 cfu/g, and 1 × 109 cfu/g, fed for 80 days. The results showed that the addition
of different concentrations of lactic acid bacteria could promote the growth of grass
carp. The weight gain rate was the highest when the dosage was 106 cfu/g, which
was 26.47% higher than that of the blank group.
With the continuous understanding of the importance of the fish immune system,
in recent years, studies have been conducted to study the effects of lactic acid bac-
teria on fish immunity by adding a certain concentration of lactic acid bacteria to the
feed. The evaluation indexes mainly include enzyme activities such as serum lyso-
zyme, myeloperoxidase (MPO), alkaline phosphatase (AKP) and acid phosphatase
(ACP), complement C3, serum IgM content, phagocytic activity, and phagocytic
index. Panigrahi et al. (2004) fed rainbow trout with Lactobacillus rhamnosus, and
the results showed that Lactobacillus rhamnosus improved non-specific immunity
such as phagocytic activity and complement activity. Balcázar et al. (2007) fed rain-
bow trout with a mixture of Lactococcus lactis, Leuconostoc mesogenes, and
Lactobacillus sinensis for 2 weeks and found that the phagocytosis of leukocytes to
mildew in mucosal lymphoid tissues was enhanced. Gildberg et al. (1997) fed larvae
with lactic acid bacteria feed. After 3 weeks of feeding, the lactic acid bacteria
became one of the dominant bacteria in the intestinal larvae of the larvae, and the
disease resistance of the larvae was improved.
Liu Xiao-ling et al. (2013) studied the effects of Lactobacillus acidophilus on the
immunity of tilapia, including 0, 8 × 105 cfu/g, 4 × 106 cfu/g, 2 × 107 cfu/g, and
1.5 × 108 cfu/g. The feed of acidophilic lactic acid bacteria was fed to tilapia, and
the results showed that the addition of acidophilic lactic acid bacteria increased the
activity of superoxide dismutase (SOD), alkaline phosphatase (AKP), and acid
phosphatase (ACP) in tilapia, thereby improving the immunity of the fish. Liu Yu
et al. (2011) selected (22.35 ± 0.06) g of juvenile Jianye as the test object to study
the effects of Lactobacillus on its immunity and disease resistance. Add 109cfu/g
lyophilized powder of lactic acid bacteria to the feed; the addition amount is 0,
0.05%, 0.10%, 0.15%, 0.20%, and 0.25%; and the test period is 80 days, then chal-
lenge with Aeromonas hydrophila. The test period was 17 days. It was found that
the addition of Lactobacillus in the diet significantly affected the number of red
blood cells and white blood cells, survival rate after challenge, leukocyte phagocy-
tosis rate, IgM-like content, and complement C3 content and significantly increased
myeloperoxidase, serum lysozyme, and serum acid phosphatase activity, thereby
improving the immunity of juvenile Jianye.
The flora in the fish gut differs depending on the type of fish, the environment of
the water, and the composition of the feed. By adding lactic acid bacteria to the feed
to form a dominant flora in the fish gut, it is possible to regulate the balance of ben-
eficial bacteria and inhibit harmful bacteria, which can promote the health of fish
(Ringø and Gatesoupe 1998). Wang Fuqiang et al. (2005) fed gingivalis with
Lactobacillus rhamnosus P15 and Lactobacillus casei. The feed volume was
1.2 × 109 cfu/g and fed for 20 days. The results showed that the gingiva was found.
The number of aerobic heterotrophic bacteria and lactic acid bacteria in the channel
increased significantly, and the number of Vibrio decreased. Yin Jun-xia et al. (2007)
isolated a strain of Lactobacillus from the intestine of squid and studied its effect on
the intestinal flora of carp. It was found that the number of aerobic bacteria was
significantly reduced after feeding Lactobacillus preparation (P < 0.01). The total
number of oxygen bacteria, lactobacilli, and Bifidobacterium increased significantly
(P < 0.01), and the number of Clostridium perfringens decreased significantly
(P < 0.05), indicating that Lactobacillus is beneficial to improve intestinal flora.
Zhou Xiaobo et al. (2014) studied the effects of five kinds of lactic acid bacteria on
the intestinal flora of tilapia and found that the number of lactic acid bacteria and
Bifidobacterium in the intestine increased after adding different kinds of lactic acid
bacteria in the feed, but the total number of intestinal bacteria and the effect of E.
coli were not significant (P > 0.05), but both could reduce the number of E. coli to
varying degrees. Test conditions are as follows: Lactobacillus acidophilus,
Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus rhamnosus, and
Enterococcus faecalis, the concentration of bacteria was 108 cfu/g, the initial fish
weight was (3.49 g ± 0.01) g, the water temperature was 25–28 °C, the feeding
amount was 4%–6% of body weight, and the test period was 8 weeks.
7.4.6 Other Living Models

7.4.6.1 Primate Model
Primates are also an important in vivo test model because of their similar physiolog-
ical and biochemical metabolism. Among them, the macaque model is the most
widely used, and the research is the most thorough. Rhesus monkeys can be used
physiologically in a variety of studies, such as brain function, blood circulation,
respiratory system, endocrine system, reproductive system, and gerontology.
In terms of disease models, macaques have made great contributions to the study
of infectious diseases. Human-specific infectious diseases such as polio and bacte-
rial dysentery are difficult to replicate in other experimental animals. They have
achieved good replication results in rhesus monkey experiments, and the clinical
manifestations are consistent with humans. The macaque is used as the sole experi-
mental animal for the manufacture and identification of polio vaccines. The suscep-
tibility of macaques to tubercle bacilli and dysentery bacilli makes their contribution
to the study of intestinal tuberculosis and bacillary disease particularly prominent.
Other infectious diseases such as viral diarrhea, malaria, influenza, viral hepatitis,
AIDS, measles, and other infectious diseases are also often used in animal experi-
ments. Rhesus monkeys also have significant advantages in pharmacology and toxi-
cology research. Parasitological studies have shown that macaques can be used for
human Plasmodium infection and can be an ideal screening drug model. In the
research of reproductive physiology, because of its physiological reproductive sys-
tem which is very similar to human beings, it plays an important role in the research
of new drugs such as birth control pills and analgesics, as well as in the study of
drug metabolism, and even becomes a necessary test before new drugs enter human
clinical trials. It can also be used for sexual behavior, vasectomy, and pregnancy
toxicity studies. There are differences in drug responses among different primates.
Studies have shown that the drug response of macaques and squirrels is most similar
to that of humans, especially central nervous drugs, and their effects in both qualita-
tive and quantitative aspects have shown good response to humans. For the study of
atherosclerosis, rhesus monkeys are similar to humans in terms of the nature of the
lesion, the location of the disease, the clinical symptoms, and the efficacy of the
drug. Rhesus macaques are suitable for the replication of chronic bronchitis models
and for the evaluation of phlegm and antitussive and antiasthmatic drugs because of
their large tracheal glands. Primates play an important role in radiology, blood typ-
ing, behavioral science, stomatology, oncology, vaccines, and other aspects. At the
same time, macaques are also an important model for organ transplantation (Xinyou
1989; Lijun and Yunan 2012).
In recent years, studies have been conducted to evaluate the immunomodulatory

function of lactic acid bacteria using a primate animal model. Klatt et al. (2013)
studied the effects of lactic acid bacteria (VSL combination preparation) and prebi-
otic (inulin) supplements on intestinal immunity of immunodeficiency virus (SIV)-
infected macaques. The results showed that the supplement increased the abundance
and enhanced function of antigen-presenting cells (APC) in the intestine, promoted
the remodeling of CD4+ T lymphocytes, and reduced the fibrosis of colon lymphoid
tissue. This study provides a good study for the study of intestinal immune regula-
tion in patients with HIV infection. Ortiz et al. (2016) also used the SIV-infected
macaque model to study the effects of probiotics and interleukin-21 (IL-21) on
Th17 cell expression, bacterial translocation, etc. and found that intestinal tract
homeostasis was maintained by probiotic intake. It can effectively inhibit pathologi-
cal damage caused by symptoms of immunodeficiency.
7.4.6.2 Nematode Model
As early as 1965, it has been suggested that nematodes can be used as model organ-
isms for studying animal development and nervous system, and Caenorhabditis
elegans is considered to be the most typical nematode model. In 1988, the cell
development process of C. elegans was clearly analyzed and became an important
model for studying the development of single-cell morphology. Later, Brenner and
Boyle (1996) clarified the cell division and differentiation process of C. elegans
from fertilized egg to adult and proposed the mechanism of programmed cell death
and thus won the Nobel Prize in Physiology or Medicine. Nematodes have simple
but precise anatomical structures, fast replication cycles, and translucent cell bod-
ies. Compared with large animals, the adults of C. elegans are small, have many
offspring, have short developmental cycles, are easy to culture, and have high
immune pathways. At the same time, sequencing of the complete genomic sequence
of the nematode has been achieved (40–50% of the sequence is homologous to
humans), making gene mutation experiments and PCR amplification possible. At
present, nematodes have been widely used in genetics, chemistry, immunology,
toxicology, and neurology (Xinyou 1989; Lijun and Yunan 2012).
In the study of lactic acid bacteria antagonizing pathogenic bacteria, some schol-
ars have used the nematode model to screen probiotics having the ability to suppress
Escherichia coli with enterotoxin-producing, and clarified the mechanism that
Lactobacillus zeae reduce the mortality of nematodes by inhibiting the expression
of enterotoxigenic genes in Escherichia coli (Zhou et al. 2014a, b). Ikeda et al.
(2007) used the C. elegans to study the protective effects of lactic acid bacteria on
nematode life-span and host defense under Salmonella stress. In the study of lactic
acid bacteria and the immune system, Kim and Mylonakis (2012) found eosinophils
and the NCFM can increase the immune response of nematodes, and Grompone
et al. (2012) also demonstrated that Lactobacillus rhamnosus has the effect of
relieving oxidative stress and inhibiting inflammatory response.
7.5 Human Model
7.5.1 Test Evaluation Model

7.5.1.1 Evaluation Criteria and Scope of Application
The crowd test is an important way to evaluate the function of health food. In the
“Technical Specifications for Health Food Inspection and Evaluation” promulgated
by the Ministry of Health (now the National Health and Family Planning
Commission), it is stipulated that 15 projects require functional evaluation of ani-
mal experiments and human trials, while the remaining 5 projects only need human
food test. In 2005, the “Administrative Measures for the Registration of Health
Foods” also used the functional test of human diet as a condition for approval. It
needs to be organized and implemented by an inspection agency, which can be a
medical institution or a scientific research unit. The test population is mostly healthy
and subhealthy (often avoiding disease patients), and the trial is not carried out in
stages (Ting et al. 2011). In principle, the test sample of the test must be a food that
has been toxicologically evaluated and confirmed to be safe. A detailed plan and
schedule should be developed prior to the trial, and an appropriate number of sub-
jects should be selected and approved by a qualified ethics committee. The subject’s
choice must strictly follow the voluntary principle and fully introduce the purpose
and content of the trial test to the subject in advance and eliminate their doubts.
Subjects need to have a reliable medical history and should read the informed con-
sent form carefully and accept the statement as determined on the consent form.
Before the start of the test, the reaction that may occur after use is estimated accord-
ing to the nature and function of the test sample, and the corresponding treatment
measures are prepared. In the test report, the number of effective cases in the test
group and the control group is generally not less than 50, and the test loss rate is
generally not more than 20%. The trial test period should not be less than 30 days in
principle and can be extended if necessary (Ting et al. 2011; Haifeng et al. 2012).
7.5.1.2 Typical Case Analysis
Lactic acid bacteria are generally considered to be recognized as safe food-grade

microorganisms. A large number of studies have been conducted to study the probi-
otic functions of lactic acid bacteria using a crowd-testing experiment.
In 2010, Bright Dairy Co., Ltd., conducted a human feeding test on the regula-
tion of blood lipids by Lactobacillus plantarum ST-III. The selection criteria for the
population is 18 to 65 years old, normal diets, no other disease except dyslipidemia
(the serum total cholesterol content was over 5.2 mmol/L). Exclusion criteria were
pregnant or lactating women, patients with severe cardiovascular disease, and short-
term use of blood lipid-regulating foods. 104 subjects were randomly divided into
the control group and the experimental group by random double-blind strategy. The
control group took normal skim milk, and the test group took skim milk containing
Lactobacillus plantarum ST-III once a day for 1 month. Subjects took blood from
the fasting vein before and after the start of the trial to determine serum total cho-
lesterol, triglyceride, high-density lipoprotein, total protein, alanine aminotransfer-
ase, aspartate aminotransferase, urea nitrogen, and the level of indicators such as
creatinine and the detection of routine indicators of urine and feces. The results
showed that the strain had a good function of regulating blood lipids and had no
adverse effects on the human body (Hong et al. 2010). In 2011, Yili Group con-
ducted a human-testing trial on the probiotic effects of “every benefit-added” com-
pound probiotic fermented milk beverage on immunity, digestion, and constipation.
The selection criteria for the population are subhealthy white-collar workers, peo-
ple with non-pathological mild constipation and occasional gastrointestinal symp-
toms, and people with insufficient intestinal motility. Exclusion criteria included
patients with chronic diseases, gastrointestinal diseases, diarrhea or chronic respira-
tory diseases, people with analgesic drugs such as aspirin, people who used laxa-
tives within 2 weeks before the start of the trial or who used probiotics in the first
10 days or who long-term used influenza-preventing drugs, pregnant women and
lactating women. In the end, 113 and 111 volunteers aged 25–45 were recruited in
Shanghai and Beijing, and their gender, age, height, weight, body mass index, and
blood pressure were recorded. Subjects were divided into a placebo group and a test
group using a double-blind randomized controlled strategy in which the placebo
group took the milk beverage and the test group took the milk beverage supple-
mented with the probiotic preparation. The time of administration is 10:00 every
morning for 12 weeks. The trial was followed up regularly to record the subject’s
symptoms, complaints, and adverse reactions. The results of the trial showed that
the incidence of upper respiratory tract infections and the cumulative number of
days of symptoms in subjects taking lactic acid bacteria beverages were signifi-
cantly lower than those in the placebo group. Compared with the placebo group, the
bloating of the volunteers in the experimental group was relieved, and the number
of bowel movements and defecation time were improved. This group of people’s
food test proved that “every benefit” compound probiotic-fermented milk drink can
improve human immunity, improve gastrointestinal function, and promote diges-
tion (Ying et al. 2012).
7.5.2 Clinical Application Model
7.5.2.1 Evaluation Criteria and Scope of Application
With the continuous digging of the function of lactic acid bacteria, its application
range gradually extends from food and health-care products to pharmaceutical or
pharmaceutical adjuvants. A large number of clinical trials have shown that lactic
acid bacteria can effectively prevent intestinal diseases such as constipation and
diarrhea (Table 7.1), and studies have shown that lactic acid bacteria have the
Table 7.1 Clinical application of probiotics for intestinal diseases

Targeting the
Probiotics crowd Clinical outcome
Lactobacillus reuteri 589 babies Defecation frequency increased by 30% after
1 month (Indrio et al. 2014)
Lactobacillus, 490 patients VSL probiotic preparations effectively
Propionibacterium faecalis with diarrhea reduce intestinal peristalsis and reduce the
mixed preparation incidence of diarrhea (Delia et al. 2007)
Bifidobacterium, lactic acid 75 Rotavirus- Probiotic intervention reduces the incidence
bacteria, Brady yeast mixed infected of childhood diarrhea and shortens the cycle
preparation children with of hospitalization for children (Teran et al.
diarrhea 2009)
Lactobacillus, 144 patients VSL probiotic preparations effectively
Propionibacterium faecalis with ulcerative reduce pathological scores and inhibit
mixed preparation colitis recurrence of blood in the stool and colitis
(Tursi et al. 2010)
Bifidobacterium longum 35 Crohn’s Reduce pathological scores, inhibit TNF-α
patients expression, and increase the number of
bifidobacteria in the gut (Steed et al. 2010)
Brady yeast/Lactobacillus C. difficile Inhibition of colonization and growth of C.
acidophilus/Lactobacillus infection difficile in the intestine, inhibition of the
rhamnosus/Bifidobacterium incidence, and recurrence of diarrhea caused
bifidum by C. difficile (Pozzoni et al. 2012; Pattani
et al. 2013)
potential to alleviate metabolic and immune diseases. When the strain is used as a
drug for clinical trials, it must be approved by the State Food and Drug Administration
and obtain clinical approvals. The qualified medical institutions shall strictly follow
the Drug Registration Management Measures and the Clinical Trial Management
Regulations. Before conducting clinical trials, it is necessary to carefully consider
the purpose of the trial, weigh the expected benefits and risks, and ensure that the
expected benefits exceed the potential hazards and that the test methods meet the
scientific and ethical requirements.
Clinical trials are more stringent in qualification requirements than trials.
Implementers need to have the appropriate professional and technical positions in
the medical institution and the expertise and experience required in clinical trials. In
addition to the routine trial design of double-blind randomized, positive, and pla-
cebo parallel crossovers, clinical trials also required stratified, multicenter, r epetitive
trial comparisons and screening of subjects according to the drug registration clas-
sification requirements. The subjects were mainly outpatients or hospitalized
patients. The trials were generally conducted in three phases or according to the
clinical requirements of the registration classification. Subjects have the right to
withdraw at any stage of the trial, and their medical treatment and rights are not
affected. If there is any health damage related to the trial, treatment and correspond-
ing compensation can be obtained (Ting et al. 2011; Faye et al. 2011).
7.5.2.2 Typical Case Analysis
The clinical application of lactic acid bacteria involves infectious diarrhea, consti-
pation, enteritis, tumors, allergies, genital diseases, etc. The most common ones are
the alleviation and prevention of various intestinal diseases.
In a multicenter, randomized, double-blind, controlled clinical trial published in
JAMA Pediatrics in 2014, Italian researchers recruited 589 neonates younger than
1 week of age, randomized to control and placebo, taking Roy’s Lactobacillus sp.
DSM17938 or placebo; the test lasted for 3 months, recording the number of nau-
sea, the duration of severe crying, the frequency of bowel movements, and the num-
ber of hospitalizations and treatments sought. The results showed that taking the
lactic acid bacteria reduced the baby’s crying time, reduced the number of nausea,
increased the number of bowel movements, and significantly reduced the financial
expenses of the parents ($118.71) and the government ($140.30) on the baby, proof
that Roy’s Latex DSM17938 can effectively alleviate intestinal dysfunction during
the first 3 months of life and reduce the financial costs of the family and the public
(Indrio et al. 2014). However, a large sample of clinical trials has found that lactic
acid bacteria have no significant effect in certain disease areas, indicating that strain
differences and individual and disease differences may be important factors for
clinical trials. Researchers from the Royal Australian Children’s Hospital and the
Canadian Children and Family Research Center conducted a phase III randomized
double-blind clinical trial from 2011 to 2012, recruiting 167 infants with colic in
3 months of age. Wessel’s criteria (freezing irritability for more than 3 h in 1 day or
more than 3 days in 1 week) were randomly divided into experimental group and
control group, respectively, given L. reuteri DSM17938 or placebo; the test lasted
1 month, the frequency of crying and fidgeting were recorded and the gut microbi-
ota of children were analyzed. The results showed that although no adverse reac-
tions occurred, the lactic acid bacteria did not play a role in relieving colic in infants
(Sung et al. 2014). A phase III randomized controlled trial published in the 2016
Lancet magazine also showed that Bifidobacterium breve BBG-001 did not play a
role in preventing necrotizing enterocolitis in preterm infants (Abrahamsson 2015).
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Chapter 8
Lactic Acid Bacteria and Gut Health
Haitao Li and Zhifeng Fang
8.1 Introduction
8.1.1 Gut Health
The gastrointestinal tract is a digestive organ system within any metazoan from
invertebrates to vertebrates. A growing large body of scientific evidence supports
the critical role of gut for human health (Flint et al. 2015). It takes in foods, digests
and absorbs nutrients and energy, and finally expels the remaining waste as feces.
More recently, its importance in immune system was also increasingly recognized.
However, it should be pointed out that a definition of gut health is still lacking
(Bischoff 2011). It is usually evaluated based on the following aspects: (1) digestion
and nutrition absorption, (2) immune response, (3) gastrointestinal disorders, and
(4) gut microbiome composition and functionality.
8.1.2 Gut Microbes
The gastrointestinal tract is an extremely complex dynamic ecosystem (Maccaferri

et al. 2012). It is widely accepted that the trillions of gut microbiota colonize human
intestinal tract. From a taxonomic viewpoint, gut microbiota mainly includes fungi,
bacteria, archaea, and viruses. All gut microbiota might generate a biomass of more
than 1.5 kg, and their combined genomes might be 100-fold of the human’s genome
(Gerard 2016). Of note, gut microbiota study has so far been focused on bacteria
which might be roughly divided into three categories: beneficial bacteria, neutral
bacteria, and harmful bacteria.
H. Li (*) · Z. Fang
https://doi.org/10.1007/978-981-13-7832-4_8
240 H. Li and Z. Fang
Emerging evidence indicated a pivotal role of gut microbes in host physiology.

Gut microbiota might naturally enhance food safety by suppressing food-borne ill-
ness, destroying naturally occurring toxins, and lowering allergic reactions (Hooper
et al. 2002; Bäckhed et al. 2005). Gut microbiota might low the risk of certain infec-
tious diseases by antagonizing pathogenic bacteria infection or inducing antibacte-
rial substances. Beyond these, they also involve in biosynthesis of short-chain fatty
acids (SCFAs) and certain vitamins. More recently, gut microbes have been identi-
fied as a “new organ” which might communicate with and/or complement our own
organs.
It is widely accepted that the gut microbiota composition is generally stable
within health adult individuals (Palmer et al. 2007). The gut microbial dysbiosis has
been recently implicated in various diseases, either inside or outside the gastrointes-
tinal tract. Various external factors such as foods, drugs, and even lifestyles were
reported to profoundly affect the gut microbiome as well as host health (Faith et al.
2013; Courtney et al. 2008; Dethlefsen and Gordon 2011; Scott et al. 2013). If gut
bacteria are making you ill, can swapping them makes you healthy? To answer the
question above, intestinal micro-ecology has become one of the hottest research
areas in biomedicine in the past 20 years (Quigley 2013).
8.1.3 Probiotic Lactic Acid Bacteria
Lactic acid bacteria are a group of gram-positive, catalase-negative, and nonsporu-

lating, aerotolerant bacteria which might ferment carbohydrates to lactic acid
(Hugenholtz and Smid 2002). They are widespread in nature ever in our gastrointes-
tinal tracts (Vlieg et al. 2011; Stolaki et al. 2012). Although lactic acid fermentation
is among the oldest forms of food preservation, accumulating evidence clearly indi-
cates that to extend food shelf-life is only the start of which lactic acid bacteria has
done to affect our life. For example, fermented foods have long had a reputation for
human health benefits. Multimillion-dollar industry runs by the concept that intro-
ducing lactic acid bacteria into gut might improve our health (Patrick et al. 2014).
Application of fermented foods has also been well-documented in folk medicines,
but it often relies on traditional beliefs rather than sciences.
Probiotics are defined as “live microorganisms which when administered in ade-
quate amounts confer a health benefit on the host.” So far, the literature on the health
benefits of probiotics has often focused on lactic acid bacteria. The original theory
of probiotics is generally attributed to the Nobel Laureate Elie Metchnikoff, who
hypothesized that the longevity of people in the Balkans might due to the bacteria
in yogurt in 1908. Unfortunately, Metchnikoff’s hypothesis remained dormant for
nearly a century. Over the last two decades, interests in probiotic lactic acid bacteria
have been rekindled for their potential benefits against various gastrointestinal dis-
eases such as bacterial infection, diarrhea, irritable bowel syndrome, inflammatory
bowel disease (IBD), and even tumorigenesis (Kitazawa et al. 2015). Although the
molecular underpinnings remain largely elusive, probiotic lactic acid bacteria might
8 Lactic Acid Bacteria and Gut Health 241
confer human health benefit, at least partially, by remodeling gut microbiota to be a

disease-free state (Table 8.1). Taken together, it may, therefore, be possible to pre-
vent and/or treat gastrointestinal disorders by probiotic LAB.
8.2 Irritable Bowel Syndrome
8.2.1 Epidemiology, Signs, and Symptoms
Irritable bowel syndrome (IBS), one common gastrointestinal disorder, might affect
10–15% of the general population worldwide (Didari et al. 2015). IBS not only
negatively affects the patients’ quality of life but also often incurs significant health-
care costs. Its primary symptoms are abdominal pain, diarrhea, constipation, and a
change in bowel habits (Soares 2014).
8.2.2 Histological and Molecular Pathogenesis
It should be pointed out that IBS is currently defined by symptom criteria. So far,
the etiology of IBS is still unclear as it occurs sometimes even without any obvious
histopathological abnormalities (Levesque et al. 2015). The associated risk factors
include genetic factors, stress, food sensitivity, small intestinal bacterial dysbiosis,
and gastroenteritis infection. For example, about 33% of IBS patients have family
history. More importantly, IBS patients from a same family even share a very simi-
lar signs and symptoms. Notably, stress and anxiety might trigger or aggravate
symptoms of IBS. In this regard, abnormal levels of several endocrine hormones
(e.g., 5-hydroxytryptamine, vasoactive intestinal peptide, somatostatin, glucagon,
and prostaglandin E2) have been observed. Although IBS is a non-communicable
disease, its risk might be significantly increased once the intestinal tract infection.
In addition, IBS has been associated with the genetic defects in innate immunity.
8.2.3 Gut Microbiota and Probiotic Intervention
More recently, several investigations suggest that gut microbiota might functionally
mediate IBS. Although gut microbial dysbiosis has been observed in IBS, it remains
to be determined whether such alternations are a cause or a consequence of IBS
(Collins 2014). Of note, certain probiotics (e.g., Bifidobacterium infantis 35624)
might greatly improve its clinical outcomes (Table 8.2). In this regard, probiotics
might act through diverse mechanisms such as directly enhancing the intestinal
mucosal barrier, reducing intestinal permeability, lowering bacterial translocation,
modulating the gut immunity, and even affecting the intestinal nervous system and
242
Table 8.1 Effects of lactic acid bacteria on gut bacteria

LAB strains Patients Clinical tails Dose and duration Main results
Lactobacillus rhamnosus GG Healthy individuals RDBPCc; 3 weeks; 1010 cfu No significant change
(Ma, Fb), Finland
New-born infants Open-label; 6 months; 109 cfu No significant change
USA
Lactobacillus paracasei Zhang Healthy individuals Open-label; 28 days; 1010 cfu An increase in Lactobacillus paracasei
China
Lactobacillus reuteri DSM17938 Cystic fibrosis patients RDBPC-COd; 6 months; 108 cfu A decrease in Gammaproteobacteria
Spain
Infants RDBPC; Italy 21 days; 108 cfu No significant change
Lactobacillus reuteri Hypercholesterolemia Random; UK 4 weeks; A significant increase in the
NCIMB30242 patients 3 × 109~1.8 × 1010 cfu Firmicutes/Bacteroidetes ratio
Lactobacillus paracasei DG Healthy individuals RDBPC-CO; 4 weeks; 2.4 × 1010 cfu An increase in Proteobacteria and
Italy Coprococcus, a decrease in Blautia
Bifidobacterium animalis subsp. Healthy individuals RDBPC; USA 4 weeks; 2.5 × 1010 cfu No significant change
lactis CNCM I-2494 Healthy individuals Open-label; 7 weeks; 2.5 × 1010 cfu No significant change
USA
Bifidobacterium breve M-16V + Pregnant women Open-label; Pregnant women: 4 weeks Pregnant women: a decrease in
Newborn infants Japan Proteobacteria
Infant: 6 months;
Bifidobacterium longum BB536 5 × 109 cfu Infant: an increase Bacteroides
VSL#3f IBS patients Open-label; HK 4 weeks; 1.8 × 1012 cfu A significant decrease in Bacteroides
Lactobacillus acidophilus NCFM Children with atopic RPCe; Denmark 8 weeks; 1010 cfu An increase in Clostridium and
Bifidobacterium lactis Bi-07 dermatitis Bifidobacterium
H. Li and Z. Fang
8
Bifidobacterium longum Bar33 Healthy individuals RDBPC; Italy 1 month; 109 cfu A significant decrease in Clostridium difficile
Lactobacillus helveticus Bar13
Lactobacillus rhamnosus GG IBS patients RDBPC; 5 months; 1.2 × 109 cfu No significant changes
Lactobacillus rhamnosusLc705 Finland
Propionibacterium freudenreichii
subsp. shermanii JS
Bifidobacterium animalis subsp.
lactis BB-12
Bifidobacterium animalis subsp. Healthy individuals Open-label; 7 weeks; 2.5 × 1010 cfu No significant changes
lactis CNCM I-2494 USA
IBS patients RDBPC; UK 4 weeks; 2.5 × 1010 cfu A decrease in Bilophila
Bifidobacterium animalis subsp. Healthy twins Open-label; 7 weeks; 2.5 × 1010 cfu An increase in polysaccharides degrading
Lactic Acid Bacteria and Gut Health
lactis CNCM I-2494 USA bacteria

Lactobacillus sp. HY7801 IBS patients RDBPC; Korea 8 weeks; 1.2 × 1010 cfu No significant changes
Bifidobacterium longum HY8004
Lactobacillus brevis HY7401
a
M male, bF female, cRDBPC randomized, double-blind, placebo-controlled clinical trial, dRDBPC-CO randomized, double-blind, placebo-controlled, cross-
clinical trial, eRPC randomized, placebo-controlled clinical trial (Derrien and van Hylckama Vlieg 2015)
243
Table 8.2 Effects of probiotic lactic acid bacteria on IBS

Strains and doses Duration Results References
Lactobacillus salivarius, 8 weeks No differences among O’Mahony et al. (2005)
1 × 1010 cfu treatment arms
Bifidobacterium infantis
35624, 1 × 1010 cfu
B. animalis DN173010, 4 weeks Ineffectiveness in Agrawal et al. (2009)
1.25 × 1010 cfu abdominal distension
Streptococcus thermophilus, and gastrointestinal
1.2 × 109 cfu transit
L. bulgaricus, 1.2 × 109 cfu
B. animalis DN173010, 6 weeks No significant Guyonnet et al. (2007)
1.25 × 1010 cfu improvement in quality
S. thermophilus, 1.2 × 109 cfu of life and symptoms
L. bulgaricus, 1.2 × 109 cfu
B. bifidum MIMBb75, 4 weeks Significant improvement Guglielmetti et al. (2011)
1 × 109 cfu after treatment
L. rhamnosus GG ATCC 20 weeks No differences among Kajander et al. (2008)
53103, 1 × 107 cfu treatment arms
L. rhamnosus Lc705
DSM7061, 1 × 107 cfu
Propionibacterium
freudenreichii, 1 × 107 cfu
B. animalis subsp. lactis,
1 × 107 cfu
BB-12 DSM 15954, 1 × 107 cfu
L. plantarum DSM 9843, 4 weeks Significant improvement Nobaek et al. (2000)
5 × 107 cfu in pain score
B. infantis 35624, 4 weeks Significant improvement Whorwell et al. (2006)
1 × 106–1 × 1010 cfu
S. thermophilus, 1 × 108 cfu 4 weeks No significant Zeng et al. (2008)
L. bulgaricus, 1 × 107 cfu improvement in mucosal
L. acidophilus, 1 × 107 cfu barrier function
B. longum, 1 × 107 cfu
L. acidophilus CUL-60 8 weeks Significant reduce in Williams et al. (2009)
NCIMB 30157 symptoms of IBS
CUL-21 NCIMB 30156
B. bifidum CUL-20
NCIMB 30153
B. lactis CUL-34 NCIMB
30172
Total 2.5 × 1010 cfu
L. paracasei subsp. paracasei 8 weeks Significant improvement Simren et al. (2010)
F19
L. acidophilus La5
B. animalis subsp. lactis BB12
5 × 107 cfu, each
brain signals (Andrade et al. 2015; Barberi et al. 2015; Canfora et al. 2015;
Chichlowski and Rudolph 2015; Kianifar et al. 2015; Martinez-Augustin et al.
2014; Mazurak et al. 2015; Meini et al. 2015; Moayyedi et al. 2010; Owaga et al.
2015; Stevenson et al. 2014).
8.3 Infectious Diarrhea
Infectious diarrhea (gastroenteritis) is a condition of having at least three loose or

liquid bowel movements each day. Infectious diarrhea might represent as a leading
cause of mortality among children under the age of 5, especially in those developing
countries (Dinleyici et al. 2012; Weichert et al. 2012). Worse, repeated infections
might lead to malnutrition, increase the risk of serious infections, and ultimately
negatively affect children growth and development (Vandenplas et al. 2011).
Depending on its duration, it has been classified into three main types: acute
(<14 days), persistent (14–29 days), or chronic (≥30 days). The primary symptoms
of infectious diarrhea include diarrhea, vomiting, and abdominal pain.
The primary causes of infectious diarrhea include viruses (rotavirus), bacteria (e.g.,
Escherichia coli, Campylobacter, Salmonella, Bacillus cereus, etc.), parasites (e.g.,
Giardia, Cryptosporidium, and Cyclospora), and fungi. Of note, antibiotic-
associated diarrhea is often related with Clostridium difficile. Compared with adult,
children are more predisposed to infectious diarrhea as they are less likely to prac-
tice good hygiene habits as well as normally under development of immunity.
Infectious diarrhea is normally an acute and self-limiting disease. It does not require
medication unless patient with dehydration or particularly severe symptoms (Gareau
et al. 2010). In nature, infectious diarrhea is a condition caused by gut microbial
dysbiosis (Sanders et al. 2013). Accordingly, probiotics have long been proposed
for infectious diarrhea management. Although probiotics (e.g., Saccharomyces bou-
lardii, Lactobacillus rhamnosus GG) show some promise in infectious diarrhea
(Canani et al. 2007; Islek et al. 2014; Saavedra et al. 1994; Saavedra 2000; Szajewska
et al. 2006; Szajewska and Kolodziej 2015), the overall results have been mixed
(Table 8.3). Moreover, such approaches might not for those critically ill hospitalized
patients.
Table 8.3 Effects of probiotic lactic acid bacteria on infectious diarrhea

L. casei 5 days Significant improvement in the Yazar et al. (2016)
L. plantarum duration of diarrhea in children
L. rhamnosus
Bifidobacterium
lactis
L. reuteri DSM 5 days Significantly shortens infectious Dinleyici et al.
17938, 1 × 108 cfu diarrhea in a pediatric outpatient (2015)
setting
L. rhamnosus R0011, 7 days Ineffectiveness in infectious Hegar et al. (2015)
1.9 × 109 cfu diarrhea in Indonesian children
L. acidophilus R0052,
0.1 × 109 cfu
B. animalis subsp. Hospitalization Ineffectiveness in preventing Hojsak et al.
lactis, 1 × 109 cfu period common infection in hospitalized (2015)
children
B. lactis B94, 5 days Significant improvement in Akin et al. (2014)
5 × 1010 cfu necrotizing enterocolitis
L. reuteri DSM 5 days Effective reduction in LOS in Dinleyici and
17938, 1 × 108 cfu hospitalized children Vandenplas (2014)
L. reuteri DSM 3 months Significant reduction in diarrhea in Gutierrez-
17938, 1 × 108 cfu preschool children Castrellon et al.
(2014)
L. acidophilus 5 days Significant improvement in the Dinleyici et al.
L. rhamnosus duration of infectious diarrhea and (2013)
B. bifidum length of hospital stay
B. longum
Enterococcus faecium
L. reuteri, 4 weeks Significant prevention of Cimperman et al.
1 × 108 cfu antibiotic-associated diarrhea in 2011
hospitalized adults
8.4 Inflammatory Bowel Disease
Inflammatory bowel disease (IBD), a group of chronic inflammatory disorders of

the intestinal tract, mainly includes Crohn’s disease (CD) and ulcerative colitis
(UC) (Kabeerdoss et al. 2015). They might affect the quality of life of 1.4 million
individuals in the United States. The most common symptoms of IBD are abdomi-
nal pain, diarrhea, bloody stools, and weight loss. Compared with healthy individu-
als, patients suffering long-term IBD might increase the risk of colorectal cancer.
The etiology of IBD is incompletely understood yet. IBD is widely recognized as a

complex disease which is trigged by the interaction between genetic and environ-
mental factors (Saad et al. 2013). Compared with others, Caucasian descent espe-
cially those in developed countries are more predisposed to IBD. The dysregulation
of innate immune mechanisms (e.g., TNFα, IL10, and ATG16L1 signaling path-
ways) has been implicated in the pathogenesis of IBD (Corthe et al. 2006).
IBD management is currently relied on nonspecific immunosuppressive agents

(such as steroids) and tumor necrosis factor (TNFα)-targeted therapy. However,
these treatments are not effective in all patients. Worse, side effects have dampened
enthusiasm for their long-term use.
Emerging evidence suggested a causal role of gut microbial dysbiosis in IBD
(Hardy et al. 2013). IBD patients are different from healthy individuals, either in the
community membership or abundance of gut microbiota. Probiotic use in IBD man-
agement has also been successful in multiple animal studies (Ahl et al. 2016).
Although highly expected by either mechanistic or animal studies (Sartor 2008;
Shanahan and Collins 2010), the clinical outcomes of probiotic use in IBD have
been mixed (Table 8.4). Most likely, upon rational utilization, certain probiotics
(e.g., Faecalibacterium prausnitzii, Bacteroides fragile, Bifidobacterium longum
BB536, E. coli Nissle 1917, and Clostridium species) might be helpful in IBD treat-
ment (Kato et al. 2004; Christensen 2006; Macfarlane et al. 2006; Saad et al. 2013;
Takeda 2009). One potential proposed approach is to induce rapid clinical remission
by corticosteroid and/or anti-TNFα therapy followed by probiotic interventions to
sustain remission. Of note, fecal microbiota transplant might be another useful ther-
apeutic strategy in IBD management. Mechanistically, probiotic might enhance
clinical outcome by modulating intestinal mucosal barrier, reducing intestinal per-
meability, and suppressing pathogenic bacteria translocation (Sartor 2004).
8.5 Necrotizing Enterocolitis
Necrotizing enterocolitis (NEC) is the death of tissue in the intestine. Despite all
modern advances in medical and surgical efforts, NEC still represents as a major
cause of neonatal morbidity and death, especially in premature and low-birth-weight
infants (Neu and Walker 2011; Zani and Pierro 2015). Currently, the mortality of
Table 8.4 Effects of probiotic lactic acid bacteria on IBD

Lactobacillus plantarum 21 days No significant improvement in Bengtsson et al.
299, 5 × 109 cfu ileal pouch function (2016)
Bifidobacterium infantis
Cure 21, 5 × 109 cfu
B. longum, 2 × 1011 cfu 1 month Significant improvement in UC Furrie (2005)
L. johnsonii LA1, 6 months No significant improvement in Marteau et al. (2006)
4 × 109 cfu CD recurrence
E. coli strain Nissle1917, 2 months Significant improvement in Harald et al. (2010)
1 × 108 cfu acute distal UC
VSL#3, 3.6 × 1011 cfu 2 months Significant improvement in Ng et al. (2010)
acute UC
B. longum, 2 × 1011 cfu 6 months Significant improvement in CD Steed et al. (2010)
VSL#3, 3.6 × 1011 cfu 2 months Significant improvement in UC Tursi et al. (2010)
B. animalis subsp. lactis 12 months No significant improvement in Wildt et al. (2011)
BB-12, UC
L. acidophilus La-5;
NEC is around 30%. Worse, even upon success treatment, the survival infants often
suffer malnutrition, growth retardation, and even neurologic abnormalities. As to
NEC’s symptoms, they mainly include feeding intolerance, vomiting, bloating,
diarrhea, and even bloody stools.
The exact etiology of NEC remains unclear, but growing evidence indicated that it
is a multifactorial disease. The potential risk factors for NEC include premature
birth, congenital heart disease, poor oxygen and blood supply, intestinal mucosal
immaturity, and bacterial infection (Akin et al. 2014; Bajwa et al. 2011; Cotten et al.
2009; Cummings 2015; Mai et al. 2011; Mercado-Lubo and McCormick 2010; Nabi
et al. 2006; Niemarkt et al. 2015). Compared with normal infants, the premature
and/or low-birth-weight infants are more predisposed to NEC as their gut are nor-
mally under development of immunity and thus prone to inflammation as well as
loss of epithelial integrity.
The management of NEC has currently relied on bowel rest therapy, orogastric tube,
intravenous fluids, and intravenous antibiotics. Accumulating evidence indicates an
association between NEC and gut microbiotal alternations. In this connection, a
decreased Firmicutes and increased Gammaproteobacteria were found in the gut

microbiota of infants with NEC. Most likely, such gut microbial dysbiosis might be
link with the antibiotic usage. Breastfeeding and probiotic uses have been strongly
recommended to lower the risk of NEC (Lin et al. 2008; Sharma and Shastri 2016;
Reali et al. 2015). Indeed, certain probiotics (e.g., Bifidobacterium, Lactobacillus,
and Saccharomyces) show promise in improving clinical outcomes for NEC
(Table 8.5). The gut of the newborn baby is generally accepted as sterile, and thus
the timing administration of probiotic might help them to establish rather normal
gut micro-ecology. Mechanistically, probiotic might reduce the incidence and mor-
tality of NEC by targeting pathogenic bacteria infection via their metabolic produc-
tions such as extracellular polysaccharide, lactic acid, short-chain fatty acids
(SCFA), and bacteriocin (Bird et al. 2010; Fleming et al. 2015; Gorelnikova and
Karpunina 2015; Hevia et al. 2015; Lim et al. 2015; Roy et al. 2006).
8.6 Colorectal Cancer
Colorectal cancer (CRC) is a malignant tumor that occurred in the colon or rectum.
It represents the third most common noncutaneous malignancy and affords the third
leading cause of cancer-related death (Brenner et al. 2014a, b). Despite the recent
improvements in preventive strategies, screening techniques, and development of
surgery and chemotherapy, the median survival period for metastatic colorectal can-
cer patients is only 24 months. The common symptoms of CRC might include
bloody stools, a change in bowel habits, body weight loss, and fatigue (e.g., extreme
tiredness or lack of energy).
During colorectal carcinogenesis, the transition from normal mucosa to adenoma

and final carcinoma is a protracted event as well as a multifactorial process (Baert-
Desurmont et al. 2016). The potential risk factors of CRC include family history,
obesity, lack of physical activity, high-fat diet, low dietary fiber intake, high red
meat intake, and smoking and alcohol use (Botteri et al. 2008; Crockett et al. 2011;
Declercq et al. 2015; Gallagher and LeRoith 2011; Germansky and Leffler 2011;
Fedirko et al. 2011; Han et al. 2015; Hannan et al. 2009; Odegaard et al. 2011;
Pajares and Perea 2015; Raufman et al. 2015; Wani et al. 2014). Molecular analyses
of colorectal carcinomas have led to a genetic model of colon carcinogenesis which
stemmed from the accumulation of a number of genetic alterations (e.g., APC, p53,
and K-Ras) and oncogenic protein overexpression (e.g., COX-2 and EGFR).
Table 8.5 Effects of probiotic lactic acid bacteria on NEC

B. breve BBG-001, 37 months No effect on premature Costeloe et al.
6.7 × 107~6.7 × 109 cfu infant NEC (2016)
B. infantis 6 weeks Effectively reduced the risk Bin-Nun et al.
S. thermophilus of NEC in very-low-birth- (2006)
B. bifidus weight neonates
Total 1 × 109 cfu
L. casei 30 days Significantly reduced the Braga et al. (2011)
B. breve morbidity of NEC in
Total very-low-birth-weight
3.5 × 107~3.5 × 109 cfu preterm infants
B. breve BBG-001, 36 weeks Invalid for NEC in very Costeloe et al.
1 × 109 cfu preterm infants (2015)
LGG, 6 × 109 cfu 7 days Ineffectiveness in NEC Dani et al. (2002)
prevention
Saccharomyces boulardii, During Ineffectiveness in reducing Demirel et al.
5 × 109 cfu hospitalization the morbidity of NEC, but (2013)
significant improvement in
feeding tolerance and sepsis
B. lactis, 5 × 109 cfu 8 weeks Effectiveness in NEC Dilli et al. (2015)
prevention
L. acidophilus, During Effectiveness in NEC Fernándezcarrocera
1 × 109 cfu/g hospitalization prevention in preterm (2013)
L. rhamnosus, newborns weighing less than
4.4 × 108 cfu 1500 g
L. casei, 1 × 109 cfu
L. plantarum,
1.76 × 108 cfu
B. infantis, 2.76 × 107 cfu
S. thermophilus,
6.6 × 105 cfu/
It is well-known that both high-fat diet and low dietary fiber intake might increase the
risk of CRC. Considering the fact that both of them greatly affect the composition of
the intestinal microbiota, gut microbial dysbiosis has long been suspected to func-
tionally mediate CRC development (Louis et al. 2014; Sears and Pardoll 2011).
Compared with healthy ones, CRC patients have a lower gut bacterial diversity but
more Fusobacterium nucleatum and Escherichia coli (Castellarin et al. 2012).
Further studies confirmed that they might potentiate intestinal tumorigenesis by
modulating the tumor-immune microenvironment. Certain probiotics show some
promise in CRC management (Table 8.6), but it still leaves much to be desired
(Ishikawa et al. 2005; Ma et al. 2010; Pearson et al. 2009; Pala et al. 2011; Rafter
Table 8.6 Effects of probiotic lactobacillus on CRC

L. casei BL23, 10 weeks Effectiveness in Lenoir (2016)
1 × 109 cfu chemoprevention of DMH-
induced CRC in C57BL/6 mice
VSL#3, 1.3 × 106 cfu 8 weeks Effectiveness in Chung et al.
chemoprevention of western- (2017)
style diet-induced CRC in
Balb/C mice
L. rhamnosus R0011 12 weeks Improvement in the quality of Lee et al. (2014)
L. acidophilus R0052 life in CRC survivors
Total 2 × 109 cfu
L. plantarum Pre-operation Significantly improve the Liu et al. (2011)
CGMCC 1258 6 days integrity of gut mucosal barrier
Post- operation and lower infectious
L. acidophilus LA-11
10 days complications
B. longum BL-88
B. longum BB 536, Pre-operation La1, but not BB 536, reduces Gianotti et al.
1 × 107 cfu 3 days the concentration of pathogens (2010)
Post- operation and modulates local immunity
L. johnsonii La1,
1 × 109 cfu 3 days
Lactobacillus Pre-radiation, Effectively reduced radiation- Ciorba et al.
rhamnosus GG, 3 days induced epithelial injury and (2012)
1 × 107 cfu improve crypt survival in mice
Bifidobacterium 7 and 14 days Commensal Bifidobacterium Sivan et al.
promotes antitumor immunity (2015)
and facilitates anti-PD-L1
efficacy
B. lactis, 1 × 1011 cfu 30 weeks The synbiotic combination of Leu et al. (2010)
RS and B. lactis significantly
protects against AOM-induced
CRC
B. lactis, 1 × 1011 cfu 4 weeks Induced unique changes in fecal Worthley et al.
microflora, but did not (2009)
significantly alter serum or
epithelial variables
L. acidophilus LA-5, Pre-operation Significantly reduced risk of Kotzampassi
1.75 × 109 cfu 15 days postoperative complications et al. (2015)
L. plantarum, Post-operation
0.5 × 109 cfu 15 days
B. lactis BB-12,
1.75 × 109 cfu
2002; Rafter et al. 2007; Rowland et al. 1998; Rowland 2009). It is worth noting that
probiotics might reduce side effects of radiation therapy (Ciorba et al. 2012) while
potently enhancing cancer immunotherapy (Chitapanarux et al. 2010; Moreno de
LeBlanc and Perdigón 2010; Vétizou et al. 2015).
8.7 robiotic Lactic Acid Bacteria: To the Future

P
and Beyond
8.7.1 Challenges to Probiotic Intervention
Accumulating data suggested the implication of gut microbial dysbiosis in multiple

gastrointestinal diseases. If gut bacteria are making you ill, can swapping them
make you healthy? Accordingly, gut microbiota is proposed as one promising
molecular target for gastrointestinal disorder management. People reasoned that
probiotic intervention might remodel a disease-prone microbiota pattern into a
disease-free state. Indeed, probiotics have shown some promise in several gastroin-
testinal disorders such as irritable bowel syndrome, infectious diarrhea, inflamma-
tory bowel disease, and even colorectal cancer, but the following bench-to-bedside
translation remains to be a big issue (Klein et al. 2010; Floch et al. 2011).
A success in clinical trial mainly depends on two factors: an effective and safe
drug and a selectively responsive subpopulation. In precision medicine, one trend is
rational drug design, which is largely based on a definite molecular target. The first
and most important question is how gut microbiota affect host physiology. Although
altered gut microbiota have been associated with various gastrointestinal diseases,
their causality remains to be further defined (Bäckhed et al. 2012). A better under-
standing of the etiology of gastrointestinal disorders as well as their cause-and-effect
relationship among gut microbiota, microbe-derived gut metabolites, and host is
thought to be an essential step forward.
The second question is how to design a clinical study on probiotics. Is it based on
epidemiological data and/or evidence-based medicine (Sanders and Levy 2011)?
Probiotic products are currently marketed as foods or dietary supplements, which
are normally not regulated as disease management. Then, how do we conduct stud-
ies to test the potential health benefit of probiotic in healthy ones? If performed
among a disease population, such kind of study should follow the standards of
drugs. Efficacy of probiotic intervention might be dependent on the probiotic strains
and/or dosage used. How the optimizing probiotic LAB strain, dose and even prod-
uct formulation are weight and judged? As outcomes must be clear and measurable
in a clinical study, what are the validated biomarkers used for targeted diseases? In
addition, has the optimal target population been clearly defined? To this end, nutri-
tion standards, dietary guidelines, and even genetic background of targeted popula-
tions must be considered.
Another issue is the safety of probiotics. Although probiotics are generally con-
sidered safe as they derived from traditional fermented foods or health gut, they
might cause adverse health consequences in certain cases (Guarner et al. 2011). If
gut bacteria are making you ill, can swapping them make you healthy? Please keep
in mind, probably, the reverse is also true. For example, some probiotics might
cause excessive immunity response, affect the metabolism of some drug, and even
carry and spread antibiotic resistance genes. Although probiotic might sometimes
rectify gut microbial dysbiosis, its administration in wrong way might further pro-
found microbial dysbiosis and thereby causes serious side effects, especially on
certain subgroup of populations. The suitability and safety of probiotic should be
studied by randomized, double-blind, placebo-controlled trials.
Taken together, before a large-scale clinical trial can be discussed, all those basic
information about the targeted populations as well as microbe used is required.
8.7.2 The Future of Probiotic Intervention
Recent studies clearly indicated that the gut microbiota play a critical role in host
physiology. However, an understanding of how they affect the host health is only
beginning to be elucidated. For example, beyond probiotic supplement, there are
many factors (e.g., as age, genetics, drug, diet, and even stress) influencing the com-
position of human microbiome. As a saying goes, you are what you eat. Diet
strongly affects human health, at least partially, by modulating gut microbiome.
And long-term dietary interventions may allow modulation of an individual’s
enterotype to improve health. To this end, the relationships among diet-microbe-
host should be more thoroughly studied and elucidated in the future.
Tomorrow’s probiotics might probably move beyond the microorganisms com-
monly used as probiotics today. For example, despite the presence of fungal and
viral members, studies in gut microbe so far have focused on the bacteria, and the
probiotics available to consumers also solely belong to lactic acid bacteria. Actually,
lactic acid bacteria itself is also a term which has no strict taxonomic significance.
Not surprisingly, such paradigm might be changed. Upon recent progression and
understanding of the gut microbiota, some specific strains (e.g., Akkermansia
muciniphila, Bacteroides fragilis, and Faecalibacterium prausnitzii) might be the
next-generation probiotics. Probiotic interventions outside the gastrointestinal tract
(e.g., diabetes, obesity, the metabolic syndrome, liver diseases, etc.) are also increas-
ingly recognized (Hojsak et al. 2010). Moreover, probiotics are normally nonpatho-
genic and noninvasive and non-colonizing bacterium, and thus recombinant
probiotics may represent an interesting direction in the future, especially to deliver
oral vaccine, improve natural immune responses, and restore antigen-specific toler-
ance (Takiishi et al. 2012). Advancements in this direction might largely lie in our
better understanding of their genetic-metabolic networks.
In summary, probiotics might have a huge potential in clinical application.
Although the overall efficacy of current probiotic intervention is still far to meet
the standard of medical care required for evidence-based medicine and some data
might be inconsistent or somewhat ever contradictory with each other, targeting
gut microbial dysbiosis by probiotics opens a new avenue to gastrointestinal dis-
eases management. Exciting times are definitely coming up for food microbiolo-
gist and gastroenterologists. Working together, we might create the next epoch in
this area.
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Chapter 9
Lactic Acid Bacteria and Host Immunity
Linlin Wang, Zhao He, Peijun Tian, and Gang Wang
9.1 Host Immunity
The immune system is the most effective barrier for the host to defend against the
invasion of external pathogens. The system consists of a series of immune organs,
immune cells, and immune active substances (immune molecules), which can detect
and eliminate non-autologous substances, such as foreign pathogens and foreign
bodies, and its own mutant cells. Among them, immune organs include bone mar-
row, spleen, lymph nodes, tonsils, small intestine collecting lymph nodes, appendix,
thymus, etc.; immune cells include lymphocytes, mononuclear phagocytic cells,
neutrophils, basophils, eosinophils, hypertrophy cells, platelets, etc.; immune mol-
ecules include the complement, immunoglobulin, interferon, interleukin, tumor
necrosis factor, etc. Different types of immune tissues, immune cells, and immune
molecules have different roles, and they coordinate the functions of various parts
through lymphocyte recycling and various immune molecules.
Higher animals have three immune defense lines. The first defense line is a phys-
ical barrier that blocks the invasion of external pathogens, including skin and
mucous membranes. Meanwhile, the secretions of skin and mucous membranes
have the function of immobilizing and killing microorganisms. The second defense
line includes non-specific immune cells and non-specific immune molecules in
body fluids that protect against the invasion of multiple pathogens. Both of the
above defense lines are inherent in higher animals and do not target a specific patho-
gen, so they are called non-specific immunity. The third defense line consists mainly
of immune organs such as thymus, lymph nodes and spleen, and lymphocytes,
L. Wang · Z. He · P. Tian · G. Wang (*)

e-mail: [email protected]; [email protected];
[email protected]
https://doi.org/10.1007/978-981-13-7832-4_9
262 L. Wang et al.
which includes “humoral immunity” mediated by B lymphocytes and “cellular

immunity” mediated by T lymphocytes. The third defense line is the defensive
mechanism that the body gradually establishes in the process of contact with the
external environment. It is the corresponding immune response that occurs during
the development process due to exposure to specific pathogens. This immune
response only works for a specific pathogens or foreign bodies, so they are called
specific immunity. Non-specific immunity and specific immunity work together to
ensure the health of the body.
9.1.1 Non-specific Immunity
Non-specific immunity is also known as innate immunity. Non-specific immunity

can quickly respond to a variety of invading pathogenic microorganisms and also
plays an important role in the establishment of specific immunity. Non-specific
immune systems can include tissue barriers (skin and mucosal systems, blood-brain
barrier, maternal-baby barriers, etc.), non-specific immune cells (macrophages,
neutrophils, eosinophils, dendritic cells, etc.), and non-specific immune molecules
(complement, interferon, lysozyme, etc.).
When an external pathogen invades, the non-specific immune system works
through an external physical barrier at first, including skin, mucous membranes, etc.
The skin and mucous membranes kill and eliminate exterior pathogens by their
mechanical barrier function, such as the scavenging effect of appendages (such as
cilia), and the bactericidal action of their secretions (such as lactic acid secreted by
the sweat glands and gastric acid secreted by the gastric mucosa). Once the patho-
gen enters the body through an external physical barrier, the second defense line
consisting of non-specific immune cells and non-specific immune molecules will
work. The phagocytosis of macrophages can effectively prevent the spread of patho-
gens in the body. Monocytes, neutrophils, eosinophils, etc. can also swallow, digest,
and eliminate the foreign pathogens. In addition, the lysozyme, complement, and
interferon in body fluids can also perform the functions of non-specific killing for-
eign pathogens.
Non-specific immunity has no specificity for the clearance of invading antigenic
substances and can clear it immediately once exposed to the pathogen. Therefore,
non-specific immunity has the characteristics of a wide range of effects, fast
response, stable action, and heritability. After the pathogen invades the body, non-
specific immunity is the first to act, and then specific immunity also takes part in the
pathogen clearing process. Non-specific immunity is the basis of specific immunity,
and the immune substance produced by specific immunity has the effect of enhanc-
ing non-specific immunity.
9 Lactic Acid Bacteria and Host Immunity 263
9.1.2 Specific Immunity
Specific immunity, also named as adaptive immunity or acquired immunity, is the

immune response that trigger by specific pathogen or antigen and can be obtained
by natural active immunization, artificial active immunization, natural passive
immunization, and artificial passive immunization. Specific immunity involves spe-
cific immunoglobulins and immune lymphocytes; therefore, it is divided into cel-
lular and humoral immunity. When the body is naturally infected with a certain
pathogen and healed during the growth and development process, the body will
have specific immunity to the pathogen. This process is called natural active immu-
nization. When the infected pathogen is obtained by artificial inoculation, it is called
artificial active immunization; if an immune effector such as immunoglobulin is
inoculated artificially, the body acquires the ability to target a certain pathogen spe-
cifically. This is called artificial passive immunization; Natural passive immuniza-
tion refers to the process by which a specific antibody enters a fetus or infant from
the mother through mother-to-child transmission (placenta or breast milk), thereby
giving the fetus or infant the ability to target a certain pathogen specifically. Specific
immunity is characterized by specificity, memory, positive and negative reactivity,
multiple cell involvement, individual characteristics, non-hereditary, and so on. The
formation process of specific immunity is generally divided into three stages: induc-
tion, response, and effect. When the antigen enters the body, the immune cells pro-
cess, present, and recognize the antigen, and this stage is called the antigen-sensing
stage. Subsequently, B lymphocytes and T lymphocytes proliferate, differentiate,
and form memory cells. This phase is called the reaction phase. The stage in which
the effector T lymphocytes, antibodies, and lymphokine exert immune effect is
called the effect phase.
9.1.2.1 Cellular Immunity
Cellular immunity is an immune response mediated by immune cells, which small

lymphocytes proliferate and differentiate and then attack the target cells or release
indirectly some lymphokine immune responses when the body is stimulated by anti-
gen. Among them, T lymphocyte is one of the main cells involved in cell immunity.
The formation process of cellular immunity is divided into three stages: induction,
response, and effect. T lymphocytes proliferate, differentiate, and transform into
effector T lymphocytes and exhibit a specific immune response after stimulated by
the antigen. When the effector T lymphocytes contact with antigen again, they clear
the antigen by its direct killing (cytotoxic T lymphocytes) and synergistic killing of
lymphokines (macrophage chemotactic factor, skin reactions factor, etc.). T lym-
phocytes can be divided into three categories according to their functions: cytotoxic
T cell (Tc), helper T cell (Th), and suppressor T cell (Ts). Cytotoxic T lymphocytes
are used to destroy foreign pathogens, which can recognize cells infected with
viruses (having MHC-antigen conjugates on the cell surface) or cancer cells, and
264 L. Wang et al.
secrete perforin to lyse target cells. Helper T lymphocytes have an auxiliary effect
on cytotoxic T lymphocytes, suppressor T lymphocytes, and B lymphocytes.
Macrophages phagocytize the invading pathogen, and then they send the molecule
complex of processed antigen molecule and the MHC class II molecule on the cell
surface and deliver the antigen to the helper T lymphocytes with the same MHC
class II molecule. In addition, the interleukin-1 secreted by macrophages can stimu-
late the secretion of interleukin-2 by the helper T lymphocytes and stimulate T
lymphocytes to differentiate into cytotoxic T lymphocytes as well as stimulating B
lymphocytes to differentiate into plasma cells and memory cells. Suppressor T lym-
phocytes can inhibit the activity of helper T lymphocytes, thereby inhibiting the
differentiation of B lymphocytes and the killing function of cytotoxic T lympho-
cytes, and negatively regulate specific immunity, which prevents the immune
response from being infinitely and continuously activated.
9.1.2.2 Humoral Immunity
Humoral immunity is the immune response which B lymphocytes proliferate and

differentiate into plasma cells, synthesize immunoglobulins (also known as antibod-
ies) that can specifically bind to the corresponding antigens, and release them into
the blood after the body stimulated by antigen. B lymphocytes play a major role in
humoral immunity. The generation of humoral immunity is also divided into three
stages: induction, response, and effect. The receptor molecule on the surface of B
lymphocytes (BCR) can combine with complementary antigen molecules. In addi-
tion, most of the antigen molecules can be finally presented to B lymphocytes via a
macrophage-helper T lymphocyte-B lymphocyte pathway. B lymphocytes are acti-
vated by antigenic stimulation and proliferate into a population of cells with the
same immunity. A part of the cell population develops into plasma cells, and each
plasma cell produces a large amount of antibody into the blood circulation; the anti-
body specifically binds to the corresponding antigen to clear the antigen. However,
the life span of the plasma cells is only a few days. The other part becomes memory
cells, which can keep the memory of the antigen for a long time after the antigen is
cleared in the body. When the same antigen reenters the body, the memory cells will
rapidly proliferate and differentiate into a large number of plasma cells to partici-
pate in the body’s secondary immune response, which produces a stronger specific
immune response and clears the antigen afterward. The antibodies produced by
plasma cells can be classified into five categories depending on different
structures.
1. IgG is the most abundant immunoglobulin in serum, accounting for 70% to 75%
of total immunoglobulin, and is the most persistent and important antibody in
primary immune response. It can promote phagocytosis of mononuclear macro-
phages, neutralize bacterial toxins, and combine with viral antigens to make
them noninfectious. It exists only in monomer form and can pass the maternal
and child barrier.
2. IgM is the first immunoglobulin produced by antigen-stimulated humoral

immune response. It is a macroglobulin, which has the largest molecular mass
unable to pass the maternal and child barrier. Its bactericidal, lytic, warming, and
agglutinating effects are higher than IgG. IgM plays a foremost role in the early
defense of the body.
3. IgA is divided into serotype and secretory type. Secretory IgA is a major compo-
nent of the mucosal defense system, which attaches pathogens to the mucosal
surface and prevent their multiplying and spreading. Serotype IgA mediates
antibody-dependent cell-mediated cytotoxicity (ADCC).
4. IgE is the latest immunoglobulin that can initiate both an immediate allergic
reaction and a delayed allergic reaction.
5. The immune function of IgD is still unclear. It mainly exists on the surface of
mature B lymphocytes, which may be related to the recognition and differentia-
tion of B lymphocytes.
9.2 Host Immunity Regulated by Lactic Acid Bacteria
Lactic acid bacteria can stimulate intestinal mucosal to change composition and
affect the mucosal immune system for regulating immune function, including regu-
lating the balance of Th1 and Th2 cellular immune responses, enhancing the activity
of NK cells and macrophages, and affecting the secretion of cytokines and the pro-
duction of antibodies. Small intestinal epithelial cells play an important role in regu-
lating innate and adaptive immunity, which can recognize different antigens and
response to different bacteria or antigenic substances. Some studies report that dif-
ferent lactic acid bacteria can stimulate intestinal epithelial cells to release different
pro-inflammatory or anti-inflammatory factors. Bacterial DNA derived from VSL#3
can reduce IL-8 expression in intestinal epithelial cells by inhibiting the activity of
epithelial nuclear receptor NF-κB to inhibit the development of enteritis (Jijon et al.
2004). L. plantarum regulates the body’s immune tolerance by changing NF-κB
signaling pathway of intestinal mucosal cells (Ko et al. 2007). These findings sug-
gest that lactic acid bacteria can affect mucosal and systemic immune function by
regulating the secretion or expression of cytokines and chemokines.
Some human experiments have found that probiotics can significantly enhance
the immune cell receptor activity and regulate the production of immune-related
cytokines to regulate the host’s immune function. The study found that the levels
of IFN-γ, IFN-α, IL-10, and IL-2 were significantly higher in the subjects who
received yogurt and/or probiotics than the control group (Medina et al. 2007). The
level of IFN-γ and TGF-β in cord blood of the pregnant women who took the B.
lactis HN019 and L. rhamnosus HN001 orally increased (Prescott et al. 2008). In
a 6-month infant test, the level of IL-10, total IgA, total IgE, and C-reactive pro-
tein (CRP) in infants who were administrated with B. breve Bb99 or
Propionibacterium freudenreichii subsp. shermanii significantly increased when
266 L. Wang et al.
compared to the control group (Marschan et al. 2008). In a double-blind and ran-
domized trial, the serum TNF-α and IL-10 levels were negatively correlated with
the intestinal B. longum and B. animalis abundance in the subjects who treated with
B. longum 2C and B. longum 46. But the TGF-β level was positively correlated with
B. breve abundance (Ouwehand et al. 2008). The immunities of the elderly people
who took L. rhamnosus GG and oligofructose were improved by increasing the
production of IL-lβ, IL-6, and IL-8 in serum (Amati et al. 2010). However, some
researchers have found that probiotics have little or no regulation to the levels of
immune cytokines. For example, the production of IFN-γ was not regulated in
healthy people (Nagao et al. 2000a; Olivares et al. 2006a; Schultz et al. 2003;
Takeda and Okumura 2007); L. acidophilus LAVRI-A1 had no effect on the innate
immune response in the infants younger than 6 months during the activation and
development of innate immune system (Taylor et al. 2006b). These different
results may be caused by different immune status in each group, different types of
probiotics, and different doses. In fact, existing studies have demonstrated that
different probiotic strains have different abilities to induce immune cells to secrete
cytokines (Niers et al. 2005).
9.2.1 Lactic Acid Bacteria Regulate Host Immunity
The intestinal mucosa is an important barrier for the host to protect against influence
from harmful substances or the environment. The intestine contains a large number
of complex bacteria, which play an important role in regulating the function of the
host’s normal intestinal mucosal immune system. The difference in the composition
and quantity of the strain directly or indirectly affects the host’s immune system
functions. Lactic acid bacterium is one of the main dominant bacteria in intestinal
microbes, which can be widely found on the surface of mucous membranes (mainly
gastrointestinal mucosa and genital tract mucosa). After entering the intestine, the
lactic acid bacteria can adhere to the epithelial cells and stimulate the intestinal
lymphoid tissues to regulate the immune response. The interaction between lactic
acid bacteria and mucous membranes affects the intestinal immune response. The
regulation of non-specific immunity by lactic acid bacteria mainly includes improv-
ing the barrier function of mucosa, enhancing the phagocytic ability of phagocytic
cells, stimulating immune cells to produce various cytokines (such as pro-
inflammatory factors IL-12, IFN-γ, and TNF-α, anti-inflammatory factor TGF-β,
and IL-10) for controlling the intensity of inflammatory response, and regulating the
activity of NK cells, dendritic cells, macrophages, production of antibody etc.
(Fig. 9.1).
Fig. 9.1 Lactic acid

bacteria regulate non-
specific immunity
9.2.2 Intestine Mucosa
The intestinal mucosa is the inner surface layer of the intestine, existing in the out-
side of intestinal epithelial cells. Intestine mucosa is the first barrier to contact with
exogenous immunogens. A stable intestinal mucosal layer can prevent the cross of
some abnormal food components or microbial antigens and damage caused by
immune activity. Destruction of the intestinal mucosa would induce food allergies
and intestinal diseases. A large number of animal experiments have shown that lac-
tic acid bacteria can regulate the secretion of intestinal mucus. Lactic acid bacteria
can enhance the stability of the mucosa through promoting the expression of mucin
(MUC1, MUC2, and MUC3) in intestinal epithelial cells. The study found that the
expression of intestinal mucin MUC2 and MUC3 was significantly increased in
piglets after feeding with L. fermentum I5007 (Yu et al. 2008). I5007 can promote
protein expression which was associated with lipid metabolism, cell structure, and
viability in jejunal mucosal proteins (Wang et al. 2012). Studies have also found that
probiotics can effectively prevent and repair mucosal damage caused by food aller-
gies or drug (Rosenfeldt et al. 2003). Lactic acid bacteria interfered with the genera-
tion of pro-inflammatory responses by stimulating the production of secretory IgA
and other defense molecules such as defensins (Malin et al. 1996; Schlee et al.
2008). Lactic acid bacteria protected the integrity of tight junctions in epithelial
cells by inducing the expression of cytoprotective proteins such as mucin (Mack
et al. 2003). In addition, some lactic acid bacteria can promote the repairment of
268 L. Wang et al.
body tissues by secreting certain factors and inhibiting the adhesion of pathogens,
thereby maintaining normal immune function and protecting the body’s health
(Sherman et al. 2005; Yamaguchi and Ma 2003).
Intervention of neonatal intestinal microbes can induce host immune function to
a more stable and anti-invasive direction. L. fermentum I5007 can remarkably
increase the amount of IgA in serum of pig; thus it can improve the body’s humoral
immunity (Liu et al. 2014). L. sobrius DSM16698T significantly restricted the
secretion of IL-8 but promoting IL-10 expression, which was induced by enterotoxin-
producing Escherichia coli (Roselli et al. 2007). L. plantarum ATCC8014 can
restrain the decrease of IL-8 which was secreted by Caco-2 cells through mediating
of TNF-α and maintain the epithelial barrier function. In addition, it can also restrain
inflammatory responses by altering the ERK signaling pathway (Ko et al. 2007).
Some studies showed that probiotics can affect innate immune defense mechanisms,
particularly to regulate the activity of phagocytic and NK cell. L. fermentum I5007
can also significantly reduce the expression of IL-1β in the ileum of pigs and pro-
mote the formation of immune homeostasis in piglets by regulating innate immu-
nity and alleviate diarrhea caused by rotavirus (Vlasova et al. 2013).
9.2.3 Cell Phagocytosis
The regulation of lactic acid bacteria on immune phagocytic cells mainly reflected
in two aspects: interfering with the ability to phagocytosis and the expression and
secretion of cytokines (Fig. 9.2). Lactic acid bacteria regulate host immunity mainly
by affecting immune cells, such as monocytes, macrophages, neutrophils, NK cells,
and dendritic cells. In healthy people with different levels of immune ability, probi-
otics can enhance the phagocytic activity of leukocytes (monocytes/macrophages
and polymorphonuclear cells) in peripheral blood, especially in patients with low
immunity. Some studies have shown that the intake of probiotics can restore the
decline in function of swallowed cells caused by senescence to some extent. In a
study that focused on the elderly group, the subjects treated by yogurt with L. rham-
nosus HN001 or B. lactis HN019 for 3–6 weeks showed an enhanced leukocyte
swallowing activity compared to the control (Arunachalam et al. 2000; Gill et al.
2001a; Gill and Rutherfurd 2001a). Administration of probiotic cheese which added
with L. rhamnosus HN001 and L. acidophilus NCFM (109 CFU/day, for 4 weeks)
significantly enhanced the phagocytic activity of granulocytes and monocytes in the
elderly (average age 86 years old) (Ibrahim et al. 2010). More importantly, probiot-
ics improve immune cell phagocytosis more significantly in people with weak
immunity (Gill et al. 2001c). In addition, the increase in phagocytosis is also associ-
ated with age, and the improvement in immune function is more obvious in the
elderly over 70 years old compared with people under 70 years old. It is worth not-
ing that some studies have found that taking probiotics has no effect on immune
function (Spanhaak et al. 1998). This may be related to the poor ability of the strain,
non-optimal dose, and the insufficient viable count of probiotics. Studies have
Fig. 9.2 Lactic acid

bacteria regulate immune
cell activity or
phagocytosis
shown that different types and doses of lactic acid bacteria have different capacities
to regulate immunity (Donnet-Hughes et al. 1999).
Monocytes are a kind of immune cells with phagocytic ability in the blood, and
they are important components of non-specific immunity in the body. Many lactic
acid bacteria can activate monocytes and induce them to produce various cytokines.
The consumption of L. rhamnosus and L. acidophilus significantly enhance the
phagocytic capacity of mononuclear cells in peripheral blood (Sheih et al. 2001).
Co-culture of Lactobacillus, intestinal epithelial cells and CD14 monocytes. CD14
monocytes can be transformed into CD141ow and CD161ow monocytes when co-
cultured with Lactobacillus, accompanied with the reduction of CD80, CD86, and
CD58 on the cell surface. Meanwhile, the anti-inflammatory factors IL-10 was
secreted to reduce the immune response (Evrard et al. 2011). In the clinical experi-
ment, researchers found that taking probiotics L. johnsonii Lal, L. acidophilus 74–2,
B. lactis 420, B. animalis subsp. lactis Bb12, L. rhamnosus HNOO1, or B. lactis
HN019 can make the phagocytic ability of leukocytes in peripheral blood (PMN
and monocytes) of healthy subjects have a significant increase (Gill and Rutherfurd
2001a; Gill et al. 2001a, b, c; Klein et al. 2008; Schiffiin et al. 1995, 1997). Other
clinical trials have also shown that taking probiotics such as L. paracasei LTH2579,
B. animalis subsp. lactis DGCC420, L. acidophilus, L. paracasei Lpc-37, L. aci-
dophilus 74–2, and B. lactis 420 can significantly enhanced the activity of mono-
cytes and granulocytes in the immune system of healthy individuals (Klein et al.
2008; Roessler et al. 2008). Taking B. lactis Bi-07 not only remarkably improved
the phagocytic ability of normal human monocytes but also enhanced the phago-
cytic ability of human granulocytes (Maneerat et al. 2013). Intake of L. salivarius
270 L. Wang et al.
CECT5713 can significantly increase the proportion of monocytes and NK cells in

all leukocytes (Sierra et al. 2010).
Macrophages are immune cells which exist in tissues and have the phagocytic
activity; they are generally differentiated from monocytes. Research has indicated
that lactic acid bacteria have a great influence on macrophages. Oral administration
of L. rhamnosus and L. acidophilus significantly enhanced the phagocytic ability of
macrophages in the peritoneal cavity of mice (Kaushal and Kansal 2014; Pena and
Versalovic 2003). The macrophages in respiratory tract of mice were activated, and
their phagocytic capacity was promoted after intranasal instillation of L. fermentum
(Cangemi et al. 2000). Except for changing the phagocytosis capability, lactobacilli
can also alter cytokines which was produced by macrophages. Some studies have
shown that L. bulgaricus can stimulate macrophages that generate TNFα and IL-6,
thus affecting host immunity (Marin et al. 1998). L. casei DN-114001 can increase
the production of CXCL1, CXCL2, and CCL20 and induce more macrophage
aggregated (Tien et al. 2006). In a study of healthy elderly people, administration of
L. johnsonii Lal, Streptococcus thermophilus, B. animalis subsp. lactis Bb12, and B.
lactis HN019 can enhance the phagocytic activity of granulocytes and monocytes
(Fukushima et al. 2007; Schiffrin et al. 1995, 1997). L. casei CRL 431 inhibited the
infection of Salmonella by macrophage phagocytosis (Castillo et al. 2013).
Polymorphonuclear (PMN) is generally referred to as neutrophils. When patho-
gens enter the body, neutrophils immediately reach the invading site to exert phago-
cytosis, and neutrophils show more phagocytic ability than monocytes. Edible
probiotics can enhance the oxidative and bactericidal ability of neutrophils, and the
increase of phagocytic activity is related to the dose of probiotics. The enhancement
could maintained for several weeks even after stopping taking the probiotics
(Donnet-Hughes et al. 1999; Parra et al. 2004; Schiffrin et al. 1995). LGG strains
can activate the neutrophils and promote their phagocytosis by stimulating the
expression of phagocytic receptors CR1, CR3, FcyRI, and FcaR in healthy people.
However, in the people who have allergy to the milk, administration of LGG showed
the opponent effect and worsen the allergic reactions (Pelto et al. 1998). A study of
healthy elderly indicated that taking probiotic B. lactis (HN019, DR10TM strains)
can significantly enhance the phagocytic ability of polymorphonuclear cells, which
can enhance the bactericidal capacity mediated by phagocytosis (Arunachalam
et al. 2000).
NK cells are important components of the non-specific immune system and play
an important role in the production of anti-tumor, IFN-γ, and other anti-infective
cytokines. Several studies have shown that probiotics can affect the activity of NK
cells. Administration of L. casei DN114001 can enhance the activity of NK cells in
lactating women at 6 months postpartum, but it didn’t have significant effect on T
lymphocytes and B lymphocytes (Ortiz et al. 2008). Using probiotics can signifi-
cantly enhance the activity of NK cell in healthy middle-aged subjects (30–45 years
old) and smokers (20–60 years old) (Takeda and Okumura 2007). Numerous studies
have also shown that drinking yogurt or fermented beverages containing probiotics
can obviously improve the activity of NK cells in peripheral blood of healthy people
and the proportion of NK cells (Chiang et al. 2000; Meyer et al. 2006; Olivares et al.
2006b; Sheih et al. 2001). L. casei Shirota can increase the number of NK cells in
the spleen, promote the cytotoxicity to cancer cells, enhance the cell activity of NK,
and induce the expression of cytokines such as IL-6 and IL-10 (Dong et al. 2010).
NK cells (CD3-/CD16 + CD15+) can be activated by stimulating L. johnsonii, pro-
ducing activated antigen CD69 and secreting IFN-γ and IL-12, but this activation
requires the presence of autologous monocytes (Haller et al. 2000, 2002). L. casei
subsp. casei enhanced the activity of NK cell in healthy individuals and induced
IL-12 production in peripheral blood (Ogawa et al. 2006). Yogurt with L. gasseri
CECT 5714 and L. coryniformis CECT 5711 induced a significant increase in the
number of NK cells and raised the concentration of IgA simultaneously (Olivares
et al. 2006a). In healthy middle-aged populations, long-term intake of B. lactis
HN019 can not only increase phagocytosis of monocytes but also enhance the activ-
ity of NK cells (Arunachalam et al. 2000; Chiang et al. 2000; Gill et al. 2001a, b, c).
Animal experiments have also shown that the L. casei subsp. casei can significantly
enhance the activity of NK cells in mice (Ogawa et al. 2006).
Dendritic cells (DCs) exist in a variety of tissues, especially in the mucosa of the
lumen that communicates with the outside world. The main function of DCs is to
present antigens and produce cytokines. Different lactobacilli can induce DCs to
produce different kinds of cytokines, and the production of cytokines is related to
the dose of Lactobacillus (Verbeek et al. 2010). L. rhamnosus can downregulate the
expression of CD14 to mediate immune responses (Evrard et al. 2011; Prescott
et al. 2008). The probiotic product VSL#3 significantly inhibited LPS and induced
DCs to produce the anti-inflammatory factor IL-10 (Hart et al. 2004; Mariman et al.
2014). L. reuteri and L. casei also induced DCs to produce IL-10 (Smits et al. 2005).
L. gasseri OLL2809 and L13-Ia significantly facilitated maturation of DCs in mice
(Luongo et al. 2013). B. breve activated intestinal DCs to produce IL-10 and IL-27
to regulate intestinal immune responses (Jeon et al. 2012). L. acidophilus NCFM
and VSL#3 strongly induced DCs to produce IL-12p70, while L. salivarius Ls-33
and B. infantis 35624 were more likely to induce DCs to produce IL-10 (Gad et al.
2011). B. breve BbC50SN promoted the expression of CD83,CD86, and HLA-DR
by activating the Toll-like receptor 2 (TLR2) signaling pathway, thereby facilitating
DCs to mature; once DCs become mature, the secretion of IL-10 increase. VSL #3
can also significantly affect the maturation of DCs (Hart et al. 2004; Hoarau et al.
2006). S. thermophilus THS, B. breve Bb99, and L. lactis subsp. cremoris ARH74
all can induce the maturation of DCs, but S. thermophilus mainly induced the
expression of pro-inflammatory factors TNF-a, IL-12, IL-6, CCL20, and IFN-γ,
while B. breve and L. lactis tended to induce the production of the anti-inflammatory
factor IL-10 (Latvala et al. 2008).
It is well known that stress can lead to a decline in the body’s immunity. A study
focusing on the effects of probiotics on the immunity of students during the exam
(L. casei DN-114001, 100 ml/day, for 6 weeks) found that probiotic intervention
significantly increased absolute quantity of lymphocytes and CD56 cells in students
(Marcos et al. 2004). These results clearly showed that probiotics can significantly
improve the immunity of healthy individuals. However, a recent study on infants
and children found that probiotics L. acidophilus LAVRI-A1 (Lafti L10) had no
272 L. Wang et al.
effect on innate immunity in infants aged 0–6 months, and there is no difference in
the ability of Pansorbin or LPS to stimulate monocyte producing cytokines as well
as the ability of antigen presentation in immunocompetent cells compared to con-
trols (Taylor et al. 2006a). It indicated that the probiotic did not affect the innate
immune response of the infant. The reasons about this phenomenon are not clear,
possibly related to factors such as the immune regulation ability of the strain, the
measurement of immune parameters, and the sampling time.
In addition to regulating the phagocytic activity of immune cells, some studies
have also found that lactic acid bacteria can promote the secretion of pro-
inflammatory factors IL-6 and IL-8 by intestinal endothelial cells, thereby inducing
inflammation of intestinal epithelial cells (Ruiz et al. 2005; Vinderola et al. 2005).
9.2.4 Lactic Acid Bacteria Modulate Specific Immunity
Specific immunity requires stimulation of specific antigens. Some immune cells

will preserve the memory of corresponding antigens, once the immune cells are
activated to produce an immune response. Specific immunity can be divided into
humoral immunity and cellular immunity. Probiotics can affect specific immunity,
especially those immune responses aimed at virus and bacterial vaccine.
9.2.4.1 Cellular Immunity
Lactic acid bacteria can affect cellular immunity by modulating the proliferation
and differentiation of lymphocytes and producing cytokines. Studies have suggested
that L. casei Shirota can promote the expression of IL-12 in spleen T lymphocytes
(Dong et al. 2013). Lactic acid bacteria can also increase the proliferation of T lym-
phocytes against mitogens and increase the number of T lymphocytes. In recent
years, there have been some reports about the effects of Lactobacillus on delayed-
type hypersensitivity (DTH). L. casei Shirota live bacteria can improve the activity
and/or proliferative capacity of memory Thl cells and enhance the antigen-specific
DTH effect in mice, and this effect is related to the time and dose of L. casei Shirota
into the body. Taking L. rhamnosus significantly inhibited the proliferation of T
lymphocytes and the production of IL-2, IL-4, and IL-10 in healthy people (Braat
et al. 2004). L. reuteri and L. casei significantly promote the differentiation of
human T lymphocytes (Smits et al. 2005) (Fig. 9.3).
Studies have suggested that probiotics can mediate the differentiation of effector
T lymphocytes or regulatory T lymphocytes by regulating TLR signaling pathways;
lactobacilli and bifidobacteria can also induce DC to produce various cytokines
(e.g., the secretion of IL-10) (Baumgart and Carding 2007; O’Mahony et al. 2006;
Forchielli and Walker 2005). Healthy young people significantly increase the num-
ber of T lymphocytes (CD4 + CD8+) and NK cell (Kim et al. 2006) after taking
Bacillus polyfermenticus. Healthy young women eating yogurt containing large
Fig. 9.3 Lactic acid bacteria regulates cellular immunity
amounts of lactic acid bacteria can significantly increase the cytotoxicity of T lym-
phocytes (CD8+), but it has no significant effect on activated T lymphocytes (Meyer
et al. 2006). In double-blind population trials of different ages, long-term use of L.
gasseri PA 16/8, B. longum SP 07/3, and B. bifidum MF20/5 significantly increase
the quantity of cytotoxic T lymphocytes (CD8+) and helper T lymphocytes (CD4+),
and short-term use of these bacteria can significantly increase the number of T lym-
phocytes (CD45 + CD3+), Th cells (CD45 + CD3 + CD4+), and cytotoxic T lym-
phocytes (CD45 + CD3 + CD8+) (Winkler et al. 2005). L. rhamnosus GG can
significantly increase the activity of CD4+ T lymphocyte (Schultz et al. 2003). L.
reuteri and L. casei are able to induce Treg cells producing IL-10 by modulating
DC, and B. infantis 35624 can repress the infection of Salmonella typhimurium by
decreasing NF-Kb activity (O’Mahony et al. 2008; Smits et al. 2005). However,
other human experiments have found that L. gasseri CECT 5714 and L. corynifor-
mis CECT5711 binding S. thermophilus did not affect the percentage of Th cells
(CD4+), cytotoxic T lymphocytes (CD8+), and inhibitory T lymphocytes (Olivares
et al. 2006a). In addition to increasing the activity of NK cells, L. casei strain Shirota
did not affect the differentiation of T lymphocytes and the proportion of various T
lymphocytes [total T lymphocytes (CD3+), Th (CD4+), and T inhibitory/cytotoxic
cells (CD8+)](Nagao et al. 2000b; Spanhaak et al. 1998). Taking L. acidophilus or
B. animalis subsp. B12 did not affect the proportion of total T lymphocytes (CD3+),
activated T lymphocytes (CD3 + HLA-DR+), B lymphocytes (CD19+), Th cells
274 L. Wang et al.
(CD3 + CD4+), cytotoxic T lymphocytes (CD3 + CD8+), and NK cell

(CD3-CD16 + CD56+) in blood (Schiffrin et al. 1995, 1997).
Several different lactic acid bacteria, such as B. bifidum, B. longum, and B. pseu-
docatenulatum, can induce the secretion of IL-10 and the expression of CD83 in the
blood of the spinal cord, so that the immune response develops toward the Th2
direction (Young et al. 2004). The VSL#3 (a mixture of several lactic acid bacteria)
can affect the DC maturation by promoting the production of IL-10 (Hart et al.
2004), and alleviate the pro-inflammatory effect by reducing lipopolysaccharide
(LPS)-induced IL-12 production. However, different strains have different effects.
For example, some strains of B. longum can induce Th2 to produce IL-4 and IL-10,
while others can promote the differentiation of IFN-γ and TNF-α producing Th1
(Menard et al. 2008). Studies have also found that L. reuteri 5289 significantly
inhibits the secretion of IL-12 induced by pathogens or other lactic acid bacteria
(Amar et al. 2015). B. longum can strongly stimulate the production of IL-10 and
pro-inflammatory factor TNF-a; especially B. longum W11 can induce the produc-
tion of Th1 cytokines, but B. longum NCIMB 8809 and BIF 53 can induce low
levels of Th1 cytokines and high levels of IL-10 (Medina et al. 2007; Menard et al.
2008). L. reuteri and L. casei are capable of inducing DC to differentiate into IL-10-
secretingTreg cells by interaction with cell adhesion molecules (Smits et al. 2005).
L. salivarius or L. rhamnosus Lr32, but not L. acidophilus NCFM, is capable of
inducing IL-10-secreting Treg cells (CD4 + CD25+) (Foligne et al. 2007). L. rham-
nosus HN001 can induce the production of Th1 and Th2 cytokines simultaneously
(Cross et al. 2002). L. reuteri and L. brevis strongly induce the expression of Th1
pro-inflammatory cytokines (Maassen et al. 2000).
9.2.4.2 Humoral Immunity
Lactic acid bacteria can affect humoral immunity and increase the body’s antibodies
against foreign antigens (Fig. 9.4). However some lactobacilli in allergic people can
mitigate or inhabit anaphylaxis by inhabiting the production of antibodies against
antigens. Lactobacillus can also enhance the proliferative capacity of B lympho-
cytes against mitogens. For example, L. rhamnosus and L. acidophilus can signifi-
cantly increase the production of serum antibodies in normal humans (Mastrandrea
et al. 2004; Piirainen et al. 2008; Vissers et al. 2011), but in allergic patients and
allergic animal models, some lactobacilli such as L. casei Shirota, L. plantarum, and
L. acidophilus make the differentiation of Th cells shifts to Th1, which reduces the
Th1 type cytokine IL-12, and thus greatly reduces the production and secretion of
IgGl and IgE (Saliganti et al. 2015; Shida et al. 2002). Lactic acid bacteria can also
significantly affect the proportion of IgG1 and IgG2a after antigenic stimulation
(Maassen et al. 2003).
Many probiotics have been shown to enhance human mucosal immune response,
with studies covering different ages from infant to old age. The mucosal immune
system function is immature for a certain period of time after birth, for example, the
number of cells that produce IgA in newborns is relatively small. Sig A is an impor-
Fig. 9.4 Lactic acid bacteria regulate humoral immunity
tant antibody for mucosal immunity, because it protects and maintains mucosal
homeostasis and plays a key role in local anti-infective processes. L. plantarum and
L. lactis can increase the number of plasma cells that produce SIgA (Daniel et al.
2006). Studies have shown that adding bifidobacteria to infants aged 15–31 months,
and total IgA in infant feces during intervention, is more severe than before inter-
vention (Fukushima et al. 1998). Similar studies have also found that the levels of
IgA in healthy or allergic children increased significantly after taking L. corynifor-
mis and L. gasseri (Lara et al. 2007; Martinez et al. 2009). In contrast, another study
found that the addition of B. animalis subsp. lactis to infant formula had no
significant effect on secretory IgA in infant feces (Bakker et al. 2006; Phuapradit
et al. 1999). In another study, B. lactis HN019 and L. rhamnosus HN001 interven-
tions were given to pregnant women for 2 to 5 weeks before delivery, and their IgA
levels were significantly increased during lactation (Prescott et al. 2008).
Probiotics not only improve the immunity of infants and young children but also
improve the immune level of the elderly. A recent randomized, double-blind,
placebo-controlled dietary intervention trial found that the intake of heat-inactivated
276 L. Wang et al.
probiotic L. pentosus b240 in elderly subjects was effective in improving the aver-
age secretion rate and concentration of salivary SIgA (Shimizu et al. 2014; Kotani
et al. 2010). In addition, older subjects treated with L. acidophilus NCFM and lac-
titol had similar improvements (Ouwehand et al. 2009). However, other studies
using different strains (L. paracasei subsp. paracasei CRL431, L. johnsonii La1, B.
animalis subsp. lactis Bb12, L. gasseri CECT 5714, and L. coryniformis CECT
5711) found that taking probiotic supplements have no effect on healthy adult’s
mucosal SIgA levels (Christensen et al. 2006; Marteau et al. 1997; Olivares et al.
2006b). In conclusion, probiotics have significantly different mucosal immunity
effects in children, healthy adults, and the elderly, suggesting that the physiological
functions of probiotics are very complex.
During viral vaccination, probiotic can significantly enhance the vaccinator’s
acquired immunity ability and allow them obtain a better immune response. In a
randomized, double-blind, placebo-controlled study, subjects were first vaccinated
with live attenuated poliovirus. Compared with patients taking placebo (chemically
acidic milk), virus-neutralizing antibody response, polio-specific serum IgG and
IgA levels were both significantly increased in volunteers who consumed yogurt
(Fukushima et al. 1998). Inoculation of polio vaccine in healthy young people found
that probiotics L. rhamnosus GG or L. acidophilus CRL431 can increase the con-
centration of specific neutralizing antibodies induced by viral vaccines, as well as
the formation of specific IgA and IgG. However, the effect of these two lactic acid
bacteria on the titers of poliovirus antibodies 1, 2, and 3 are different, indicating that
they are specific to the strains (de Vrese et al. 2005b). Newborns taking B. breve
C50 and S. thermophilus can significantly increase the concentration of poliovirus-
specific antibody IgA after vaccination (Mullie et al. 2004). However, in the study
of healthy full-term infants, it was found that taking L. paracasei subsp. paracasei
strain F19, DTaP, and Hib while vaccinating did not induce polio antibody response
caused by poliovirus (West et al. 2008). One study compared the antibody response
of infants to hepatitis B vaccine and DTPA-hepatitis B vaccine after administration
of probiotics or placebo and found that the antibody response to hepatitis B surface
antigen was significantly improved in those subjects taking probiotics (Soh et al.
2010). In a randomized, double-blind human trial, L. casei DN-114001 was found
to significantly enhance the specific immune response of the elderly to H1N1,
H3N2, and B mixed influenza viruses. After vaccinating a newborn with a rotavirus
vaccine, infants taking L. casei strain GG got a more production of rotavirus-specific
antibodies IgM; at the same time, the level of antibodies IgA also increased (Isolauri
et al. 1995; Kaila et al. 1995). However, in another experiment about influenza vac-
cines, the administration of probiotic L. fermentum CECT5716 promoted the
production of Th1 cytokines, increased the ratio of Th and Tc cells, and improved
the content of total IgM, but did not increase the content of anti-influenza virus-
specific IgM (Olivares et al. 2007). These results indicate that the mechanism and
effect of probiotics on virus immunity are not the same.
Many studies have shown that probiotics can affect the effectiveness of bacterial
vaccines. Studies have shown that taking B. animalis subsp. lactis Bb12 and L. aci-
dophilus La1 can significantly increase the production of Salmonella-specific sero-
type IgA antibodies as well as the immune response of IgA-secreting cells in healthy
subjects. Similar studies have also found that the level of anti-Salmonella IgA
increased in healthy adult volunteers taking LGG and Salmonella vaccines (Fang
et al. 2000; Perdigon et al. 1995; Link et al. 1994). B. lactis B1–04 and L. acidophi-
lus La-14 have an auxiliary activity against the immune response produced by
Vibrio cholerae vaccine, which can increase the concentration of human serum
immunoglobulin and accelerate the process of immune response (Paineau et al.
2008). A randomized, double-blind trial of neonates found that infants taking
L. acidophilus LAVRI-A1 produced lower levels of IL-10 in the antigenic response
induced by the tetanus toxin vaccine (Taylor et al. 2006a). However, other studies
have shown that high-risk children who are naturally exposed to the microbial envi-
ronment vaccinated DTP/Hib vaccine or anti-pneumococcal vaccine combined with
yogurt fermented with S. thermophilus and L. casei probiotics as an adjuvant, and
no improvement in antibody response was observed in all study groups. In a ran-
domized, double-blind trial, newborn infants were vaccinated with DPT (DTwP,
diphtheria, pertussis, tetanus, and Haemophilus toxin), and administration of probi-
otics significantly increased the concentration of Haemophilus IgG, but have no
obvious effect in several others (Kukkonen et al. 2006). A weak self-immunity
resulted from the exposure to microorganisms in a poor environment at the early age
may be the reason why probiotic supplements show no additional immune stimulat-
ing effects (Perez et al. 2010).
These studies have shown that the immune response of virus antigens is gener-
ally consistent in all age groups (infants, children, adults, and the elderly) without
considering probiotic strains as immunoadjuvants. For polio vaccines, taking probi-
otics alone, rather than in combined with other vaccines, seems to have a more
pronounced effect; in addition, differences in probiotic strains and the degree of
early exposure to other microorganisms affect the immune effect of bacterial anti-
gens (e.g., Salmonella, arcs) in infants and young children. Why can probiotics
regulate specific immune responses? One possibility is that probiotics affect the
immunological recognition of immune cells and the function of effector cells
through extracellular antigens. Studies have found that probiotics can upregulate the
expression of pattern recognition receptors (such as TLR, MHC-I) in immune-
activated cells and promote the secretion of some cytokines (Miettinen et al. 2008;
Villena et al. 2014). However, the exact molecular mechanism of probiotics as an
adjuvant to regulate specific immune responses is still not clear.
9.3 I mmunomodulatory Effects of Lactic Acid Bacteria Cell

Components
Lactic acid bacteria act as an immunoadjuvant to exert immunomodulatory effects,

but the specific molecular mechanism for its action remains unclear. Live bacteria
and its cell wall components, metabolites, decomposition products (polypeptides),
278 L. Wang et al.
and other molecules have the function of stimulating immune response. For exam-
ple, lymphocytes and macrophages have the function of recognizing peptidoglycan
molecules, and teichuronic acid stimulates monocytes to produce cytokines such as
TNF-α and IL-6 in vitro. Studies have found that lactic acid bacteria can mediate
immune regulation through pattern recognition receptors of host cell (PRRs), such
as TLRs and NOD-like receptors (NLRs). Compared with the antigen receptors in
the acquired immune system, these pattern recognition receptors have a broader
spectrum of specificity and play an important role in the innate immune system and
mainly identify the components of lactic acid bacteria cell wall such as lipopolysac-
charide (LPS), peptides glycan (PG), lipoteichoic acid (LTA), and cell wall lipopro-
tein (Medzhitov 2007).
9.3.1 I mmunomodulatory Effects of Intact Cells

and Metabolites
Lactic acid bacteria can inhibit the growth and reproduction of pathogenic bacteria in
the host by competing for nutrients. At the same time, the colonization of lactobacil-
lus to the intestine can competitively reject the adhesion and colonization to intesti-
nal epithelial cells by endogenous and exogenous potential pathogenic bacteria in the
intestine. L. johnsonii and L. acidophilus can chelate iron hydroxide on the surface
of their cells, thereby limiting the use of iron by pathogenic bacteria in the intestine
(Elli et al. 2000). A large number of studies have found that colonization and adhe-
sion in the intestine are prerequisite for lactobacillus to play its role. The lactic acid
bacteria can specifically adhere to host cells through teichoic acid, polysaccharides,
and S-layer proteins on the cell surface (Deepika and Charalampopoulos 2010).
Studies have shown that the cytokine induced by Gram-positive bacteria is IL-12,
which is more biased toward the immune response of Th1, while the cytokine
induced by Gram-negative bacteria is IL-10. Most of the lactic acid bacteria are
Gram-positive bacteria, which can stimulate the immune response of intestinal-
related lymphoid tissues after entering the intestine. And the effect is affected by the
degree of contacting with the lymphoid tissue when the cells are colonized (Perdigon
et al. 2002). Teichoic acid is a component of the bacterial cell wall and is only found
in Gram-positive bacteria, which activates the TLR signaling pathway to regulate
the host’s immune response. Studies have found that lipoteichoic acid molecules of
L. casei and L. fermentum can promote the development of inflammation in the
body because it activates the TLR2 signaling pathway and induces the production of
TNF-α in murine macrophages and spleen cells (Matsuguchi et al. 2003).The
administration of L. rhamnosus RW-9595 M can induce the production of TNF-α,
IL-6, and IL-12. However, studies have also shown that the cell wall polysaccharide
of L. casei Shirota can stimulate macrophages and inhibit the production of cyto-
kines (Bleau et al. 2010; Yasuda et al. 2008). Traditionally, lactic acid bacteria must
be colonized and multiplied in the intestines to play their role, but studies also have
shown that lactic acid bacteria colonized transiently can also stimulate the intestinal
immune system, especially for patients taking antibiotics or diarrheic. Eating lactic
acid bacteria can regulate the balance of the immune system and help the restore of
intestinal flora (Naidu et al. 1999).
Probiotic DNA from VSL#3 can inhibit NF-κB activation by stabilizing IκB levels
and inhibiting ubiquitin-dependent protein degradation, which also inhibits IL-8 secre-
tion by inhibiting the inflammatory signaling pathway p38 (Jijon et al. 2004). B. breve
C50 and the soluble substances it secreted also can activate NF-kB and p38 signaling
pathways, inhibiting the secretion of CXCL8 in epidermal cells (Heuvelin et al. 2009).
The secretion of L. reuteri ATCC PTA6475 inhibits the production of lymphocyte
TNF-α by activating C-Jun and AP-1 signaling pathways (Iyer et al. 2008).
LAB also has a regulatory effect on the intestinal barrier by producing some
proteins without direct contact with the cells. The study has found that by activating
Erkl/2 and PKC signaling pathways, p40 and p75 proteins encoded by L. rhamno-
sus GG can protect the tight junctions and barrier functions of intestinal epithelial
cells. P40 protein has a beneficial effect on gut immunity by activating the epider-
mal growth factor receptor EGFR signaling pathway (Seth et al. 2008; Yan et al.
2011). In addition, organic acid (mainly lactic acid), the main metabolite of LAB, is
also a significant antibacterial substance. Organic acid can inhibit the growth and
reproduction of harmful bacteria (such as Salmonella, pathogenic Escherichia coli,
etc.) by chelating metal ions, changing the permeability of cell membranes and
reducing intestinal pH. As a matter of fact, many bacteriostatic effects of LAB are
realized by producing lactate. The hydrogen peroxide synthesized by LAB could
effectively kill the Salmonella and disintegrate the cell membrane of the
Staphylococcus aureus as well, so that the cellular content is exuded to protect the
intestinal barrier.
9.3.2 Immunomodulatory Effects of Exopolysaccharides
Among the metabolic components of LAB, the most studied immunomodulatory

function is extracellular polysaccharide. Study has shown that EPS produced by L.
kefiranofaciens ATCC 43761 can induce the response of mucosal immune system
(Vinderola et al. 2006). The polysaccharide-peptidoglycan (PSPG) is usually pro-
duced after N-acetyl muramidase digesting heat-inactivated LAB, which molecular
mass is generally greater than 30 kDa and polysaccharide portion is mainly com-
posed of rhamnose, glucose, galactose, glucosamine, and galactosamine. Previous
research has found that taking PSPG could improve the survival rate of mice infected
with Listeria monocytogenes or Pseudomonas aeruginosa, and the number of
infected bacteria in the abdomen and liver of PSPG treatment group is significantly
reduced. The anti-infective ability of PSPG is mainly related to the activation of
macrophage activity (Lee et al. 2004). PSPG can relieve chronic inflammation by
inhibiting IL-6 and IFN-γ while promoting IL-12 and TNF-ɑ (Matsumoto et al.
2005; Shida et al. 2006). Peptidoglycan isolated from L. delbrueckii subsp.
280 L. Wang et al.
bulgaricus has the ability to convert murine pre-T lymphocytes into positive Theta
cells and is able to activate complement (Prokop’Ev et al. 1987).
EPS secreted by LAB to the environment during cells growth and metabolism is
a kind of carbohydrate. According to its distribution, it can be divided into two
types: one is closely attached to the surface of bacteria called capsular polysaccha-
ride (CPS); another is loosely distributed on the surface of bacteria called slime
polysaccharide (SPS). The CPS can affect the adhesion of bacteria. In one study, a
L. rhamnosus GC mutant showed a relatively weaker ability to produce CPS with its
adhesion, and biofilm-forming capacity decreased and became sensitive to immune
factors such as innate antibacterial peptides in the host, which indicates high level
of the CPS protecting L. rhamnosus GC against intestinal innate immune factors
(Lebeer et al. 2011). The EPS of L. casei Shirota inhibits macrophage from produc-
ing pro-inflammatory cytokine IL-6, thereby suppressing the host immune response
(Yasuda et al. 2008). The kefiran secreted by the L. kefiranofaciens can increase the
level of IgA in animals and regulate the secretion of immune cytokines associated
with T lymphocytes (Vinderola et al. 2006). The EPS isolated from the L. del-
brueckii subsp. bulgaricus OLL1073R-1 and NCFB2483 can effectively promote
the mitosis of B lymphocytes and inhibit the secretion of IFN-γ, thereby regulating
the immune function of the host (Kano et al. 2002; Kitazawa et al. 1998).
9.3.3 Immunomodulatory Effects of Cell Wall Components
The cell wall of LAB is mainly composed of peptidoglycan, teichoic acid, surface
proteins, and EPS, which can recognize specific PRRs on the intestinal mucosa of
the host. PRR plays a significant role in infection and innate immunity, which Toll-
like receptors and NOD-like receptors act as extracellular and intracellular pattern
recognition receptors. The surface component of LAB is important for its function.
Through the interaction between related molecular patterns and PRR, a series of
immune signals are generated to regulate intestinal barrier function. Many researches
have revealed that dead LABs also have the ability to stimulate immune response:
Co-culture of heat-inactivated Bifidobacterium with macrophages significantly
increased the production of TNF-ɑ, IL-6, and NO (Hur et al. 2004). In a study, mice
were intragastric administered of fermented milk containing activated and inacti-
vated L. rhamnosus HN001 for 14 days, with a supplement of cholera toxin. The
result showed that both activated and inactivated L. rhamnosus HN001 could
improve the phagocytosis of leukocytes in the blood, while activated LAB signifi-
cantly increased the antibody response to cholera toxin on mice intestinal mucosa
(Gill and Rutherfurd 2001b). In addition, animal experiments have shown that
Deodan (an lysozyme degradation) isolated from the cell wall of L. delbrueckii
subsp. bulgaricus can reduce the production of endogenous TNF-ɑ in control group
(Guencheva et al. 1992; Popova et al. 1993).
Peptidoglycan and its derived cell wall peptide are active compounds in which
LABs exert a probiotic function. Peptidoglycan is a major component forming the
cell wall of LAB, which regulates innate immunity by activating the TLR2 signal-
ing pathway. The diaminopimelic acid-type (Dap-type) fragment in peptidoglycan
plays a major recognition function, followed by L-lysine type (Lys type) fragment
(Bron et al. 2012). However, the downstream signaling pathways of microbial pep-
tidoglycan and other non-acylated cell wall components of diverse species may be
different. Some studies have shown that peptidoglycan activates NLR for signal
transduction, rather than TLR to regulate host immune response (Macho et al. 2011;
Travassos et al. 2004; Volz et al. 2010). A recent study found that lipoproteins of
Bifidobacterium and other LABs interact with TLR2, lipopolysaccharide, and
NOD2 to exert immunoregulatory functions (Zeuthen et al. 2008).
Teichoic acids are found within the cell walls of many LABs. According to their
different anchored ways on the cell surface, they are usually divided into two types:
wall teichoic acid (WTA) and lipoteichoic acid (LTA). WTA does not penetrate into
the plasma membrane, it is covalently linked to the N-acetylmuramic acid residue
of peptidoglycan by phosphodiester bond, and the terminal end of the LTA is cova-
lently linked to the oligosaccharide of glycolipid in the plasma membrane. LTA can
not only adhere to intestinal epithelial cells but also act as a ligand to activate the
TLR2 signaling pathway in host immune cells, thereby regulating the production of
TNF-ɑ. D-Alanylation of the LTA backbone of L. plantarum WCFS1 and L. planta-
rum L-137 are required for recognition of TLR2 receptors, whereas L. plantarum
KCTC10887BP and L. rhamnosus GG71 don’t require such precise modification
for TLR2 signal activation (Bron et al. 2012). Other evidence suggests that L. plan-
tarum NCIMB8826 (L. plantarum WCFS1 mutant) can induce less TLR2-dependent
pro-inflammatory cytokines and increase IL-10 secretion (Grangette et al. 2005). In
fact, although L. rhamnosus GG mutant reduces the TLR2 expression and the secre-
tion of downstream pro-inflammatory cytokines, its inflammation inhibitory effect
is not significant, and the adhesion to cells is affected (Claes et al. 2010; Lebeer
et al. 2012). These results indicate that D-Ala cylation of the LTA backbone can
enhance the anti-inflammatory function. A recent study is consistent with the fact
that the WTA and the LTA in L. casei Shirota and L. plantarum ATCC 14917 act
synergistically in TLR2 signaling pathway-mediated IL-10 production, but the
mechanism needs further study (Kaji et al. 2010). In conclusion, we find that the
effects of WTA and LTA on the activation of TLR2 and NOD2 signaling pathways
are not consistent and more research on their molecular mechanism is needed.
Surface protein is the single-stranded protein in the outermost cell wall of LAB,
as a part of the complex structure of the cell wall. It regulates LAB’s ability of
adhering to epithelial cells and inhibits the adhesion of pathogens (Bron et al. 2012).
The SLP protein of L. acidophilus NCFM can be recognized by the dendritic cell-
specific ICAM-3 grabbing nonintegrin (CD209DC-SIGN) and inhibits host inflam-
matory response. The SlpA protein-mutant strain will induce the host to produce
more pro-inflammatory cytokines such as IL-12, TNF-ɑ, and IL-1β (Konstantinov
et al. 2008). The sortase-dependent protein (SDP) on the cell wall is a kind of sur-
face protein containing a conserved LPXTG motif, which plays an important role in
282 L. Wang et al.
the adhesion of LAB to host cells. The SrtA of L. plantarum WCFS1 can significantly
affect the function of host dendritic cells, indicating SDP may play a part of func-
tion in host immunological regulation (Remus et al. 2013).
Studies relevant to LAB and immune system are summarized as follows accord-
ing to cell tests, animal experiments, and human experiments, as shown in Tables
9.1, 9.2, and 9.3 (↑ indicates the number, expression increases; ↓ indicates the oppo-
site condition).
Table 9.1 In vitro studies

Subject Strains Results Reference
IPEC-J2 cells L. reuteri DSM17938, ATCC Intestinal inflammation↓ Liu et al.
PTA4659, ATCC PTA 5289, (2010)
and ATCC PTA6475
HT-29 cell VSL3 Inflammation↓ Jijon et al.
(2004)
Caco-2 L. plantarum IL-8↓ Ko et al.
(2007)
Peripheral blood B. longum NCIMB 8809 and IFN-γ and TNF-α↑ Medina et al.
Mononuclear cells BIF53 (2007)
Dendritic cells Streptococcus pyogenes and Semi-mature DCs Veckman
L. rhamnosus et al. (2003)
Human Streptococcus pyogenes, Th1 chemokine↑ Veckman
macrophages L. rhamnosus GG et al. (2004)
Peripheral blood L. rhamnosus strain GG IL-10, IL-4↑; TNF-α, IL-6, Schultz et al.
cells IFN-γ↓ (2003)
PBMCs Lactobacilli and bifidobacteria IL-10↑, IL-12p70, IL-5, Niers et al.
IL-13↓ (2005)
Caco-2 cells VSL#3 β-Defensin 2↑ Schlee et al.
(2008)
Mouse L. casei Shirota TNF-α, IL-12, IL-10, and Yasuda et al.
macrophage cell IL-6↑ (2008)
lines or to mouse
spleen cells
Dendritic cells L. rhamnosus Lcr35 TNF-α, IL-1β, IL-12p70, Evrard et al.
IL-12p40, and IL-23↑ (2011)
Mice macrophage L. rhamnosus GG TNF-α↑ Pena and
Versalovic
(2003)
RAW 264.7 and L. bulgaricus, B. adolescentis, IL-2, IL-5, TNF-α↑ Marin et al.
EL4.IL-2 and B. bifidum (1998)
Caco-2 and L. casei Shigella Anti-inflammatory effect Tien et al.
HEK293T (2006)
PBMCs L. casei Shirota NK cell activity↑ Dong et al.
(2010)
NK cell Staphylococcus aureus, NK cell activity↑ Haller et al.
L. johnsonii La (2000)
(continued)

PBMCs L. johnsonii, L. sakei IFN-γ, IL-12↑ Haller et al.
(2002)
Human dendritic L. salivarius, L. paracasei Early cytokines response Verbeek et al.
cells subsp. paracasei, (2010)
L. coryniformis, B. animalis
subsp. lactis
Human dendritic VSL#3 CXCL9, CXCL10, CCL2, Mariman
cells CCL7, and CCL8↓ et al. (2014)
Human dendritic VSL#3 IL-10↑, IL-12, TNF-α↓ Hart et al.
cells (2004)
Human dendritic L. reuteri and L. casei IL-10↑ Smits et al.
cells (2005)
Human dendritic L. gasseri IL-12, TNF-α, IL-10↑ Luongo et al.
cells (2013)
Human dendritic L. acidophilus NCFM, IL-10, IL-12p70 ↑ Gad et al.
cells VSL#3, L. salivarius Ls-33, (2011)
B. infantis 35,624,
Escherichia coli Nissle 1917
Dendritic cells B. breve DC maturation, survival, Hoarau et al.
IL-10↑ (2006)
Human dendritic S. thermophilus THS, B. breve DC maturation, IL-10↑ Latvala et al.
cells Bb99, L. lactis subsp. (2008)
cremoris ARH74
Murine intestinal L. casei CRL431, IL-6↑ Vinderola
epithelial cells L. helveticus R389 et al. (2005)
Rat intestinal B. animalis subsp. lactis Trigger innate signal Ruiz et al.
epithelial cells BB12 transduction and pro- (2005)
inflammatory gene
expression
Human dendritic L. rhamnosus IL-2, IL-4, IL-10↓T cell Braat et al.
cells hyporesponsiveness↑ (2004)
Human dendritic B. bifidum, B. longum, and IL-10↑ Young et al.
cells B. pseudocatenulatum, (2004)
B. infantis
Human dendritic L. reuteri 5289, L. acidophilus IL-12↓ Amar et al.
cells NCFM IL-12↑ (2015)
PBMC L. acidophilus, L. plantarum, IL-13↓ Vissers et al.
and L. fermentum (2011)
Human L. rhamnosus GG TLR2 expression↑ Miettinen
macrophages et al. (2008)
Porcine intestinal L. rhamnosus CRL1505 IFNs, IFN-γ, and regulatory Villena et al.
epithelial and IL-10 ↑ (2014)
antigen presenting
cells
RAW264.7 L. casei, L. fermentum TNF-α↑ Matsuguchi
et al. (2003)
Mice macrophages L. rhamnosus RW-9595 M IL-10↑ Bleau et al.
(2010)
284 L. Wang et al.
Table 9.2 Clinical studies

Mice L. rhamnosus HN001 Immunity↑ Gill et al. (2001a)
Mice L. acidophilus and B. bifidum Phagocytic activities↑ Kaushal and Kansal
(2014)
Mice L. Fermentum Macrophage↑ Cangemi de
Gutierrez et al.
(2000)
Mice L. casei CRL 431, L. delbrueckii subsp. Anti-infection, Castillo et al.
bulgaricus CRL 423, L. acidophilus inflammatory (2013)
CRL 730 response↓
Mice L. casei subsp. casei NK cell activities ↑ Ogawa et al. (2006)
Mice B. breve IL-10↑ Jeon et al. (2012)
Mice L. acidophilus Mouse lymphocyte↑ Kirjavainen et al.
L. casei, L. gasseri and L. rhamnosus Mouse lymphocyte↓ (1999)
Mice B. infantis 35624 Treg cells↑ O’Mahony et al.
(2008)
Mice B. longum NCC2705 TH1 and TH2 Menard et al.
cytokines↑ (2008)
Mice L. rhamnosus HN001 Th1 and Th2 Cross et al. (2002)
cytokines↑
Mice L. reuteri and L. brevis TNF-α, IL-2, and/or Maassen et al.
IL-1β↑ (2000)
Mice L. casei Shirota IgE and IgG1 Shida et al. (2002)
responses↓
Mice L. rhamnosus (MTCC 5897) IgA+ cells, IFN- γ↑; Saliganti et al.
IL-4↓ (2015)
Mice L. murines and L. casei IgG1/IgG2a ratios↑ Maassen et al.
(2003)
Mice L. plantarum and L. lactis IgA↑ Daniel et al. (2006)
Mice L. casei, L. delbrueckii subsp. IL-10 and IL-4↑ Perdigon et al.
bulgaricus, and Streptococcus IL-2 and IL-12↓ (2002)
thermophiles
L. acidophilus
9.4 Conclusion
Accumulated clinical and animal studies have shown that lactic acid bacteria have a
good effect on improving intestinal barrier and regulating immunity. Lactic acid
bacteria can regulate both body-specific and non-specific immunity, including regu-
lation of Th1, Th3, and Th2 immune reaction balance, regulation of NK cell and
macrophage activity, regulation of dendritic cell maturation, regulation of
inflammation-associated cytokines secretion, as well as vaccines that induce the
production of antibodies, and so on; At the same time, lactic acid bacteria is impor-
tant in intestinal flora structure, intestinal villus structure, epithelial cell permeabil-
ity, tight junction and secretion of cell mucus; lactic acid bacteria play an important
Table 9.3 Clinical studies

40 children, L. reuteri ATCC 55730 IL-10↑, IL-1β, TNFα, Oliva et al.
ulcerative colitis IL-8↓ (2012)
474 pregnant women L. rhamnosus HN001, IFN-γ↑ Prescott et al.
B. lactis HN019 (2008)
98 infancy(1-6 m) L. rhamnosus GG, Allergy↓ Marschan
L. rhamnosus LC705, et al. (2008)
B. breve Bb99,
Propionibacterium
freudenreichii subsp.
shermanii JS
209 elderly B. longum strains 2C and 46, TGF-β1↑, IL-10↓ Ouwehand
B. animalis subsp. lactis et al. (2008)
Bb-12
10 elderly L. rhamnosus GG IL-1β, IL-6, and IL-8↓ Amati et al.
(2010)
9 healthy and 10 L. casei Shirota NK activity↑ Takeda and
elderly Okumura
(2007)
231 pregnant women L. acidophilus LAVRI-A1 No effect on infancy Taylor et al.
allergy (2006a, b)
20 1–13-year-old L. rhamnosus 19070–2 and No effect on IL-2, IL-4, Rosenfeldt
children L. reuteri DSM 122460 IL-10, or IFN-γ et al. (2003)
Positive effect on atopic
dermatitis, IgE↑
14 children with L. casei strain GG IgA↑ Malin et al.
Crohn’s disease (1996)
25 elderly B. lactis HN019 IFN-γ↑ Arunachalam
et al. (2000)
L. rhamnosus HN001, Natural killer cell activity Gill et al.
B. lactis HN019 ↑ (2001a, b, c)
31 elderly L. rhamnosus HN001 and Innate immunity↑ Ibrahim et al.
L. acidophilus NCFM (2010)
20 elderly L. casei strain Shirota No effect Spanhaak
et al. (1998)
L. johnsonii La1 No effect Donnet-
Hughes et al.
(1999)
52 healthy middle- L. rhamnosus HN001 NK cell activity, Sheih et al.
aged and elderly polymorphonuclear (2001)
volunteers activity↑
30 healthy elderly B. lactis HN019 Helper (CD4+), activated Gill et al.
(CD25+) T lymphocytes (2001a, b, c)
and natural killer cells↑
Healthy L. acidophilus La1 or No effect Schiffrin et al.
B. animalis subsp. lactis (1995)
Bb12
(continued)
286 L. Wang et al.

26 healthy L. acidophilus 74–2, Phagocytic activity↑ Klein et al.
B. animalis subsp. lactis (2008)
DGCC420
15 healthy adults and L. paracasei Lpc-37, Phagocytic activity↑ Roessler et al.
15 patients with AD L. acidophilus 74–2, and (2008)
B. animalis subsp. lactis
DGCC 420
37 healthy B. lactis Bi-07 Phagocytic activity↑ Maneerat et al.
(2013)
40 healthy adults L. salivarius CECT5713 NK cells, IL-10↑ Sierra et al.
(2010)
24 elderly over age L. johnsonii La1 (NCC533) TNF-α↓ Fukushima
70 et al. (2007)
45 healthy L. casei DN114001 Oxidative burst capacity Donnet-
of monocyte↓ Hughes et al.
(1999)
8 milk-hypersensitive L. GG (ATCC 53103) CR1, CR3, FcγRIII, and Pelto et al.
and healthy adults FcαR in neutrophils↑ (1998)
104 lactating mothers L. casei DN11400 TNF-α modestly↓ Arunachalam
et al. (2000)
50 healthy B. lactis HN019 PMN cell phagocytosis, Chiang et al.
NK cell activity↑ (2000)
33 young healthy Conventional yogurt Cytotoxic T Meyer et al.
women lymphocytes↑ (2006)
30 healthy L. gasseri CECT 5714, Natural killer (NK) cells, Olivares et al.
L. coryniformis CECT 5711 IgA↑ (2006a, b)
63 students L. casei DN-114001 Lymphocytes↑ Marcos et al.
(2004)
30 healthy old L. casei Shirota NK cell activity, IL-10↑ Dong et al.
(2013)
35 adults Probiotic Bacillus IgG, CD4+/CD8+ T cells Kim et al.
polyfermenticus, Bispan and NK cells↑ (2006)
strain
479 adults L. gasseri PA 16/8, Cold episodes↓CD8+, de Vrese et al.
B. longum SP 07/3, CD4+ T cells↑ (2005a)
B. bifidum MF20/5
9 healthy and 10 L. casei Shirota NK activity↑ Nagao et al.
elderly (2000b)
14 consecutive L. acidophilus, Circulating CD34+ cell↓ Mastrandrea
subjects L. delbrueckii, and et al. (2004)
Streptococcus thermophilus
19 patients L. rhamnosus GG IgA↑ Piirainen et al.
(2008)
7 healthy children Bifidobacterium IgA↑ Fukushima
et al. (1998)
(continued)

44 allergic children L. gasseri CECT5714 and IgA↑, IgE↓ Martinez et al.
L. coryniformis CECT5711 (2009)
30 children L. coryniformis CECT5711 Aggressions and Lara et al.
and L. gasseri CECT5714 infections↓ (2007)
175 children B. animalis subsp. lactis Rotavirus infection↓ Phuapradit
Bb12 et al. (1999)
19 infants B. animalis SIgA ↑ Bakker et al.
(2006)
80 healthy elderly L. pentosus b240 SIgA ↑ Kotani et al.
(2010)
98 low physical L. pentosus b240 SIgA ↑ Shimizu et al.
fitness elderly (2014)
51 healthy elderly L. acidophilus NCFM IgA↑ Ouwehand
et al. (2009)
12 healthy L. johnsonii strain La1 IgA↑ Marteau et al.
(1997)
71 young healthy B. animalis ssp. lactis No immune function Christensen
adults (BB-12), L. paracasei subsp. et al. (2006)
paracasei
64 healthy adults L. rhamnosus GG or Neutralizing antibody de Vrese et al.
L. acidophilus CRL431 titers ↑ (2005b)
30 infants Fermented infant formula IgA↑ Mullie et al.
(B. longum/B. infantis and (2004)
B. breve)
89 infants L. paracasei subsp. Immune responses↑ West et al.
paracasei F19 (2008)
29 infants B. longum BL 999, Specific antibody Soh et al.
L. rhamnosus LPR responses↑ (2010)
222 elderly L. casei DN-114001 Antibody responses to Boge et al.
influenza vaccination↑ (2009)
50 adults L. fermentum CECT5716 Th1 response, virus- Olivares et al.
neutralizing antibodies↑ (2007)
Infants L. casei GG Immune responses ↑ Isolauri et al.
(1995)
41 children L. casei GG IgA↑ Kaila et al.
(1995)
30 healthy adults Lactobacillus GG, L. lactis IgA, immune responses↑ Fang et al.
(2000)
30 healthy adults L. acidophilus La1 and IgA↑ Link et al.
bifidobacteria (1994)
83 healthy B. lactis Bl-04, Immune responses↑ Perdigon et al.
L. acidophilus La-14 (1995)
231 pregnant women L. acidophilus LAVRI-A1 No effect on allergen- Taylor et al.
with allergic disease specific responses (2006b)
and positive allergen
skin prick test
(continued)
288 L. Wang et al.

Infants L. rhamnosus GG(ATCC No effect on vaccine- Kukkonen
53103), L. rhamnosus specific responses et al., (2006)
LC705, B. breve Bbi99,
P. freudenreichii subsp.
shermanii
162 children S. thermophilus and L. casei, No effect Perez et al.
with L. acidophilus (2010)
role in many functions such as body nutrition, biological barrier, and anti-tumor.
With the deeper understanding of lactic acid bacteria and immune regulation mech-
anism, the immune regulation function of lactic acid bacteria will attract more and
more attention. It should be noted that the immunomodulatory effects of different
lactic acid bacteria strains are distinctly different, so it is of great practical signifi-
cance to screen lactic acid bacteria with great specific functions.
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Chapter 10
Commercial Strains of Lactic Acid
Bacteria with Health Benefits
Xin Tang and Jichun Zhao
10.1 Lactobacillus casei Shirota
10.1.1 he Discovery and Development of Lactobacillus casei

T
Strain Shirota
10.1.1.1 The Discovery of Lactobacillus casei Shirota
In the 1930s, Lactobacillus casei strain Shirota (LcS) is isolated and intensively
cultured by Japanese scientist Dr. Minoru Shirota. Dr. Minoru Shirota initially
wanted to find a biological pathway to antagonize pathogenic bacteria. Inspired by
Elie Metchnikoff’s theory of longevity in Bulgarian yogurt, Dr. Minoru Shirota
worked mainly on the Bulgarian yogurt strain (Lactobacillus delbrueckii subsp. bul-
garicus) during his studies. However, the results were not satisfactory. Dr. Minoru
Shirota found that the Bulgarian yogurt strain could not be stored in the form of live
bacteria. This strain was incapable of tolerating the environment of high acid (stom-
ach) and high bile salt (small intestine) in the digestive tract. Since then, Dr. Minoru
Shirota had selected high-activity lactic acid bacteria that can pass through the
digestive tract from many strains of lactic acid bacteria based on acid- and bile-
tolerant salts. Later, this strain of lactic acid bacteria was named Lactobacillus casei
strain Shirota (Sako et al. 2011).
Dr. Minoru Shirota was not only discovered and intensively cultivated the LcS
strain, but more importantly, after 5 years of the discovery of the LcS strain, he
developed a new fermented milk drink with this strain, named Yakult.
X. Tang (*)
J. Zhao
Southwest University, Chongqing, China
https://doi.org/10.1007/978-981-13-7832-4_10
298 X. Tang and J. Zhao
10.1.1.2 Early Studies of LcS Strain in Japan
From the 1960s to 1970s, Dr. Minoru Shirota and other Japanese scientists con-
ducted a series of clinical experiments around Yakult and LcS strain. One study on
the effect of Yakult on the incidence of shigellosis in soldiers demonstrated that
Yakult products containing LcS have a significant role in preventing specific patho-
gen infections (Kotani et al. 1961). In addition, Japanese scientists had also explored
the effects of Yakult on intestinal microbes in infants. Dr. Minoru Shirota studied
the probiotic effects of Yakult on fecal microbes in children aged 2–6 years. The
results showed that drinking Yakult for 4 weeks significantly increased the number
of Lactobacillus and Bifidobacterium strain, reduced the number of Gram-negative
Bacillus and Enterococcus, and decreased the pH of the feces (Shirota et al. 1966).
10.1.1.3 Development of LcS and Yakult in the World
In 1935, Yakult probiotic beverages were produced and sold in Japan. In 1964,
Yakult’s first overseas branch was established in Taiwan, China. Since then, it has
sold Yakult in more than 32 countries and regions around the world, including
Brazil, Thailand, South Korea, Mexico, Australia, the United Kingdom, Germany,
and Mainland China. At the same time, in addition to the Tokyo Central Research
Institute, in 2005, the Yakult Society Institute of Europe was established in
Belgium.
10.1.2 Safety Studies of LcS
Traditional fermented foods, such as fermented dairy products, are derived from
human production and life practices and have been widely accepted for their long-
term consumption. The Food and Drug Administration (FDA) states that foods with
a history of more than 30 years can be considered safe and harmless. Since its birth
in Japan in 1935, Yakult (containing LcS strain) has been consumed for more than
80 years with no safety problems. In addition, in January 2013, the LcS strain was
obtained the FDA’s generally recognized safety certification for food safety (GRAS
Notice No. 429). Therefore, there is almost no safety problem with the LcS strain
and its products.
Nevertheless, Yakult has conducted a series of safety evaluation experiments,
including acute and chronic toxicity tests, genotoxicity experiments, and terato-
genic experiments. In the acute toxicity test of a 2 g/kg dose of LcS in rats, no
abnormalities such as lethality, diarrhea, and weight loss were observed. There were
no abnormalities in the chronic toxicity tests based on body weight, organ weight,
food intake, water intake, urine index, biochemical indicators, and pathological tis-
sue sections. The acute toxicity test in rats also showed that the LcS strain was safe
10 Commercial Strains of Lactic Acid Bacteria with Health Benefits 299
and harmless. The Ames experiment also showed that the strain was not mutagenic
(Miyazaki and Matsuzaki 2008). In addition, a large number of clinical trials have
shown that LcS strains have proven to be safe and harmless, both for adults and
children. In another preliminary study of patients with severe illness, no evidence of
placeholder or bacteremia associated with the LcS strain was found, suggesting that
the L. casei Shirota is not only very healthy for the healthy population but also safe
for the patient population (Srinivasan et al. 2006).
10.1.3 Functional Study of LcS
10.1.3.1 Modulation of Gut Microbiota
In the early study, Dr. Minoru Shirota (Shirota et al. 1966) found that consumption
of Yakult induced an increase in the number of Lactobacillus and Bifidobacterium
and a decrease in the number of Enterococcus and Enterobacter in the feces of chil-
dren and adults. The pH of the feces also decreased. It is considered to be an impor-
tant cause of the reduction of harmful bacteria. In another study, 20 healthy men
were administrated with LcS strain (109 cfu/ml) fermented milk and placebo (unfer-
mented milk) three times, respectively (Spanhaak et al. 1998). Compared with pla-
cebo group, the content of Lactobacillus was increased in the fermented milk group,
especially LcS strain with a concentration of 107 cfu/g wet feces. At the same time,
the number of Bifidobacterium has also increased significantly. In conclusion, the
beneficial effects of LcS on the composition of the intestine microbiota are mainly
caused by an increase in the number of beneficial bacteria such as Lactobacillus and
Bifidobacterium, and reduction of conditional pathogens such as Enterobacter and
Enterococcus or toxin-producing strains.
In addition, Shimizu and Shibamoto (1964) first reported on the modulation of
intestinal metabolic activity by LcS strains – drinking Yakult can reduce the produc-
tion of gas in the intestine. Spanhaak et al. (1998) found that intake of LcS fer-
mented milk can inhibit bacterial β-glucuronidase and β-glucosidase activities in
the human intestinal tract. These enzymes are thought to metabolize to produce
carcinogens. A random crossover human trial conducted by De Preter et al. (2008)
also confirmed that LcS can inhibit β-glucuronidase activity.
10.1.3.2 Prevention of Infectious Diseases
The research aim of Dr. Minoru Shirota was to find a biological approach to inhibit
the infection of pathogenic bacteria. A human trial conducted by Kotani et al. (1961)
showed that LcS strains have a preventive effect on patient infected with Shigella
flexneri. Later, scientists have studied the effects of LcS strains on bacterial patho-
gens and viral pathogens such as Helicobacter pylori and Listeria monocytogenes.
Bacteria
Sgouras et al. (2004) revealed that the LcS treatment can significantly reduce the
colonization of Helicobacter pylori in the gastric cavity and gastric mucosa in vitro
inhibition assay and in mouse infected by Helicobacter pylori, accompanied with a
reduce in related chronic and active gastric mucosal inflammation. de Waard et al.
(2002) investigated the effect of LcS strains on a rat model of Listeria monocyto-
genes infection. The results showed that administration of LcS strains significantly
reduced the number of Listeria monocytogenes in organs such as the stomach,
cecum, faeces, spleen, and liver. The level of specific enzyme alanine transferase
indicating liver function was also significantly reduced. This enhanced resistance to
L. monocytogenes is supposed to be related to the mediated immunity of LcS strains.
Lin et al. (2015) reported that LcS can inhibit the growth of Streptococcus mutans
and the formation of biofilm. A double-blind trial by Yadav et al. (2014) which dem-
onstrated that the level of S. mutans in the saliva of children was as high as 105 cfu/
ml was reduced by 34% after LcS drinking for 20 days. In another study, 7–12-year-
old children who received LcS ice cream or beverages were found to have signifi-
cantly reduced levels of Streptococcus mutans in the saliva. After 90 days of elution,
the level of Streptococcus mutans in the saliva of LcS group was also lower than that
of the control group (Mahantesha et al. 2015). These studies indicate that LcS
strains may be beneficial to prevent dental caries. Fayol-Messaoudi et al. (2005)
showed that LcS antagonized the activity of Salmonella typhimurium by non-lactic
acid molecules in the cell-free supernatant of lactic acid bacteria, and complete
inhibition of the bacteria is the result of pH lowering.
Virus
Hori et al. (2001) found that after being treated with LcS for 3 days, the titers of
influenza virus in the upper respiratory tract were reduced to 1/10 of the model
group, whose mice were infected with influenza virus by intranasal infection. The
survival rate of the LcS group was also significantly higher than that of the model
group. The intranasal ingestion of LcS strains may enhance the cellular immunity of
the upper respiratory tract, thereby effectively inhibiting influenza virus infection.
Similar results were obtained when the LcS strain was administered to mice by oral
ingestion. In the LcS group, the activity of the natural killer cell and the level of
IL-12 levels in the lung tissue were significantly increased, indicating that the oral
administration of LcS strains initiated the immature immune system of neonatal and
young rats and enhanced its ability to fight influenza virus infection. It suggests that
oral administration of LcS may accelerate the innate immune response in the upper
respiratory tract of infants and children, thereby protecting children against influ-
enza virus infection (Yasui et al. 2004).
Relieve Diarrhea
A randomized, double-blind, placebo-based clinical trial was aimed to evaluate the

effects of live Yakult on antibiotic-associated diarrhea and duration of diarrhea.
After a 2-month clinical trial, no significant difference was observed in the inci-
dence of diarrhea between the experimental and placebo groups, with durations of
4d and 5d, respectively. Yakult administration tended to shorten the duration of diar-
rhea, but it is not significantly different from the placebo group (Wright et al. 2015).
Wong et al. (2014) examined the nutritional intervention of LcS on the antibiotic-
associated diarrhea in patient with spinal cord injury. During antibiotic treatment,
164 patients with spinal cord injury were randomized to receive 6.5 × 109 cfu of LcS
or without probiotics. The study demonstrated that probiotics significantly reduced
the incidence of antibiotic-associated diarrhea (17.1% in the probiotic group and
54% in the control group).
Improve Constipation
Early studies in Japan have shown that in a double-blind clinical trial, Yakult drink-
ing can significantly increase the frequency of bowel movements and improve stool
stiffness (Ogawa et al. 1974). In addition, a double-blind, randomized, placebo trial
conducted by Mazlyn et al. (2013) administered LcS strain to 90 adults with func-
tional constipation (no other disease in the body). The main indicators of the experi-
ment were the severity of constipation and the frequency of bowel movements. The
secondary indicators were fecal consistency and fecal count. As a result, it was
found that the intake of LcS for 4 weeks had no effect on the improvement of con-
stipation severity, frequency of defecation, and stool consistency and amount.
However, considering that the subject (LcS) is not a drug, with long-term intake
characteristics, as it is re-estimated at 10%, in the fourth week, the severity of con-
stipation is significantly improved.
Constipation is also a common secondary symptom in patients with Parkinson’s
disease. Cassani et al. (2011) evaluated the improvement of chronic constipation in
patients with Parkinson’s disease by combining LcS with traditional dietary therapy.
Compared to the control group, after the intake of fermented milk (containing
6.5 × 109 cfu of LcS) for 5 weeks, the number of days of normal stool consistency
increased significantly, while the number of days in which patients felt bloating,
experienced abdominal pain, and incomplete bowel movements decreased
significantly.
In addition to regular and secondary constipation, some special physiological
periods are accompanied by constipation symptoms, such as women’s postpartum
stage. One recent study randomized 40 women who gave birth naturally, taking a
bottle of fermented milk or placebo containing 6.5 × 109 cfu of LcS per day for
6 weeks. During the study period, subjects completed their bowel habits (including
the number of bowel movements, stool consistency, or presence of hemorrhoids)
and completed the constipation-related symptoms (PAC-SYM) and quality of life
(PAC-QOL) questionnaires. The probiotic group scored a better score on the total
PAC-SYM and PAC-SYM minor abdominal symptoms, rectal symptoms, and PAC-
QOL sub-satisfaction (compared to the control group). In the probiotic group, there
were two to four cases of acne in the first 3 weeks, and it decreased in the fourth
week. The acne cases were zero in most of the fifth and sixth weeks. In contrast, the
placebo group did not change significantly from the beginning of four acne cases
until the sixth week. The study showed that continuous intake of fermented milk
containing LcS strains can alleviate constipation-related symptoms and make bowel
movements smoother, and postpartum women can eliminate acne symptoms more
quickly (Sakai et al. 2015).
Modulation of Immune Activity
Natural killer cells are important components of the innate immune system of the
human body and play an important role in targeting tumor cells and virus-infected
cells. Nine healthy volunteers drank fermented milk containing 4 × 1010 cfu of LcS
for live bacteria for 3 weeks to determine the natural killer cell activity and other
immune functions of the subjects. It was found that natural killer cell activity
increased significantly after 3 weeks of ingestion of fermented milk and maintained
an upward trend for the next 3 weeks. This effect is particularly pronounced in indi-
viduals with low natural killer cell activity (Nagao et al. 2000). In human trials in
healthy populations, natural killer cell activity is inversely related to the number of
cigarettes smoked, while drinking Yakult with LcS strains can restore reduced natu-
ral killer cell activity (Morimoto et al. 2005). In addition, Dong et al. (2013) found
that consumption of LcS significantly increases natural killer cell activity and
decreases the expression of CD25 on resting T lymphocytes. In addition, the ratio of
interleukin-10 (IL-10) to IL-12 increased after intake of probiotics compared with
uningested probiotics, indicating an improvement in inflammatory conditions.
LcS may also have the effect of reducing inflammation and supporting immune
function in immunocompromised populations, such as HIV-infected patients. Thirty
HIV male patients receiving antiretroviral therapy continued to drink two bottles of
Yakult fermented milk containing LcS strains per day. After 4 weeks, the T lympho-
cytes of probiotics were increased, and CD56+ cells increased significantly. The
mRNA levels of factor-β (TGF-β), IL-10, and IL-12 and the expression of
interleukin-1β (IL-1β) were significantly reduced. At the same time, caspase C is
decreased, showing a decrease in the level of inflammation and a reduced risk of
cardiovascular disease. This suggests that LcS may be an economical and practical
strategy to support the immune function of HIV-infected patients in the future
(Falasca et al. 2015).
The immunomodulatory effects of LcS strains have also been reported in a large
number of animal experiments. Shida et al. (2002) evaluated the immunoglobulin E
(IgE) response and allergy of LcS strains to a food allergy model, ovalbumin-
specific T-lymphocyte receptor genetically modified mice (OVA-TCR-Tg). The
effect of the reaction was found to be that the intake of probiotics inhibited
immunoglobulin E response, immunoglobulin G1 (IgG1) response, and systemic
allergic response in the food allergy model. At the same time, Matsuzaki et al.
(1998) found that oral administration of heat-killed LcS was effective in inhibiting
IgE production in serum in mice model.
Antitumor Effect
Yokokura et al. (1981) firstly reported the effect of LcS strains on cancer. Aso and
Akazan (1992) conducted the first human experiment on the development of super-
ficial bladder cancer by LcS. During the 430-day experiment, patients who had
undergone urethrotomy were given or not given LcS while taking the drug. After the
experiment, the cumulative tumor recurrence rate of the probiotic group was signifi-
cantly lower than that of the control group. Studies by Aso et al. (1995) and Ohashi
et al. (2002) have shown that LcS has a positive effect on preventing the recurrence
of bladder cancer and reducing the risk of its development.
Kaga et al. (2013) examined the effects of complex of LcS strain with soymilk
on 2-amino-1-methyl-6-phenylimidazolium-induced breast cancer rat model, con-
firming that LcS strains can inhibit the growth of tumor.
10.1.4 Representative Products of LcS
Five years after the LcS strain was discovered by Dr. Minoru Shirota, it was used in
fermented milk drink, and it was sold in the market. In order to let people around the
world better understand this product, Dr. Minoru Shirota named it “Yakult” in
Esperanto. “Yakult” means “yogurt” (yogurt), but Yakult is not a yogurt, but a fer-
mented skim milk drink. Up to today, as its founders expected, Yakult has been sold
at least in 32 countries.
Yakult contains 1 × 106 cfu/mL LcS strain and other main ingredients such as
water, white sugar, skim milk powder, glucose, and food flavor. Compared with the
classic red packaging, the blue packaging of Yakult has lower sugar content and
healthier while ensuring the same amount of active probiotics. At the present, the
average daily consumption of Yakult is about 30 million bottles per day.
10.2 Lactobacillus rhamnosus GG
10.2.1 he Discovery and Development of Lactobacillus

T
rhamnosus GG
Lactobacillus rhamnosus GG (LGG) is a probiotic strain selected from human feces

by Gorbach and Goldin, professors at North Carolina State University in the United
States. Named by the discoverer, LGG is now one of the world’s most known pro-
biotics. LGG is also the most probiotic bacteria reported in the world. There are
more than 1000 scientific publications on LGG and more than 300 clinical studies
(up to April 2017, the data obtained from Hansen website). Since 1990, LGG has
been using additives in food and dietary supplements worldwide.
10.2.2 Safety Studies of LGG
The Food and Drug Administration (FDA) has recognized the safety of LGG in
infant food. Based on the qualified presumption of safety (QPS) concept, the
European Food Safety Authority (EFSA) has recognized LGG’s QPS status and
permitted it for food processing. In addition, LGG has been used in fresh milk prod-
ucts since 1990 and has a long history of safe use in dairy products.
As the country where LGG was first sold, a study evaluated the possible effects
of LGG consumption of probiotics in Finland on increased Lactobacillus-associated
bacteremia from 1990 to 2000. The isolation rate of Lactobacillus in all blood sam-
ples is 0.02%, and the isolation rate of all bacteremia blood samples is 0.2%; the
trend of increased lactis-associated bacteremia was not observed. Among them, in
1995–2000, the annual incidence of bacteremia in Finland was 0.3 per 100,000.
Although 11 isolates of 66 species of bacteremia at the species level were identical
to LGG, no increase in LGG consumption was associated with an increase in asso-
ciated bacteremia (Salminen et al. 2002). The risks of lactobacilli or bifidobacteria
are similar to the risk of commensal infections. The risks of LGG products are
negligible for consumers, even for people with impaired immune function (Borriello
et al. 2003).
10.2.3 Functional Study of LGG

10.2.3.1 Prevention of Diarrhea
Vanderhoof et al. (1999) examined the protective effects of LGG on diarrhea caused
by acute oral infection in children. The study enrolled 202 children between
6 months and 10 years of age who were given double-blind random doses of 1 × 1010
to 2 × 1010 cfu LGG per day while receiving antibiotic therapy. The guardian answers
questions every 3 days. Questions include the presence or absence of gastrointesti-
nal symptoms, frequency of bowel movements, and stool consistency. The results
showed that 25 of the placebo-treated volunteers developed diarrhea, compared
with only 7 in the LGG-treated group. LGG significantly reduced the frequency of
defecation and increased the stool consistency compared with the placebo group.
Basu et al. (2007b) examined the effects of LGG powder (6 × 107 cfu) on chil-
dren with acute watery diarrhea in India. The results showed that there was no sig-
nificant difference between the intervention group and the control group regardless
of the daily diarrhea frequency or the duration of diarrhea, or the vomiting status
and hospital stay, indicating that the supplement of LGG at the current dose has no
protective effect on acute diarrhea in children. In the follow-up study, for patients
with acute watery diarrhea for more than 1 year, the doses of LGG were increased
to 1010 cfu and 1012 cfu, respectively. The frequency, duration, and length of hospital
stay were significantly lower than the control group (Basu et al. 2009). Another
similar study examined the effects of LGG on children with persistent diarrhea. The
results showed that the duration of diarrhea in the intervention group was signifi-
cantly lower than that in the control group (5.3d and 9.2d, respectively), and the
length of hospital stay was significantly reduced. The vomiting response was also
controlled (Basu et al. 2007a). LGG has different effects on different types of diar-
rhea, and the amount of LGG is also a key factor.
Salminen et al. (2004) explored the role of LGG in the improvement of gastroin-
testinal symptoms during noninfectious diarrhea in HIV patients receiving retrovi-
ral therapy. In this placebo-controlled, crossover study, HIV-infected individuals
with diarrhea were randomized to receive different doses of LGG (1 × 1010 to
5 × 1010 cfu) twice a day for 2 weeks. The main gastrointestinal symptoms include
the number of bowel movements per day, the level of stool hardness (water, hemi-
hydrate, loose, hard, or foamy), and gastrointestinal symptoms (flatulence, stomach
pain, bloating disorders, and overall good). The results showed no significant differ-
ences in noninfectious diarrhea and gastrointestinal symptoms between the LGG-
treated group and the placebo group. However, the number of HIV RNA copies and
CD4 cells in the blood remained stable during the study period, indicating that HIV
patients are well tolerated by LGG treatment itself.
Rotavirus is an important cause of infectious diarrhea in infants and young chil-
dren. Oral-specific immunoglobulin can be passively immunized to the human body
and has rapid effects on diarrhea caused by rotavirus infection. Pant et al. (2007)
evaluated different probiotics with therapeutic potential and their role in anti-
rotavirus antibody complexes in a mouse rotavirus diarrhea model. The results show
that LGG has the strongest ability to reduce the prevalence, duration, and severity
of diarrhea. LGG combined with antibody can significantly reduce diarrhea mea-
surement indicators, prevent histopathological changes, and reduce the viral load of
the small intestine.
For induced diarrhea in newborn mice infected with human rotavirus, a low dose
(2 × 105 cfu), a medium dose (2 × 107 cfu), and a high dose (2 × 109 cfu) of LGG
were administrated before and after infection with rotavirus. The duration of
d iarrhea was significantly shortened. The vacuolization of jejunal epithelial cells

was significantly reduced, and the high-dose LGG protection before infection was
better, indicating that the protective effect of LGG on rotavirus-induced diarrhea
was highly correlated with the time and quantity of LGG administration (Zhang
et al. 2013). However, the molecular mechanism by which LGG mediates defense
against rotavirus infection remains unclear. Wu et al. (2013) used a pig model to
study the protective effect of LGG feeding on viral enteritis. It was found that LGG
feeding did not cause autophagy in pig small intestine, but viral infection could
induce self-phagocytosis. Viral infection increased the protein levels of the autoph-
agy markers ATG16L1 and Beclin-1, as well as the autophagy regulator mTOR. LGG
treatment resulted in normal expression of autophagy markers between viral gastro-
enteritis, induced apoptosis, and prevented the virus.
Zhang et al. (2010) investigated the effect of LGG on the incidence and severity
of diarrhea in weaned piglets induced by Escherichia coli (E. coli) K88. Compared
with the control group, LGG significantly reduced the incidence of diarrhea and had
higher concentrations of SIgA in the jejunum and ileum, as well as higher concen-
trations of TNF-α and interleukin-1 (IL-1). In contrast, in the model set, the number
of fecal E. coli increased significantly, while the number of lactobacilli and bifido-
bacteria decreased. Therefore, the mechanism by which LGG improves diarrhea in
piglets after weaning may be achieved by regulating intestinal microbes, enhancing
intestinal defense capacity, and regulating systemic inflammatory factors.
10.2.3.2 Immunology and Allergic Reactions
Studies have shown that oral administration of LGG for 5 days to 4 weeks can alle-
viate gastrointestinal inflammation and atopic dermatitis. Pessi et al. (2000) added
LGG to a diet of nine children with atopic dermatitis at a daily dose of 2 × 1010 cfu
for 4 weeks. The results showed that serum IL-10 levels were significantly different
after LGG intake for a period of time before LGG intake and LGG intake. Oral LGG
increased serum IL-10 levels. An increase in IL-10 content confirms the anti-
inflammatory properties of LGG. Piirainen et al. (2008) examined the effects of
LGG on oral immune responses in adolescents and adults with an allergic effect on
birch pollen. Subjects received LGG or placebo for 5.5 months and started
2.5 months before the birch flowering season. After 5.5 months, rbet v1- and rMal
d1-specific IgA levels were increased from baseline in the LGG group compared
with placebo, but the levels of serum rBet v1-specific IgE were similar between
groups. In the LGG group, rBet v1-specific IgE levels were positively correlated
with total IgA and IgG produced by saliva stimulation. In the placebo group, rBet
v1-specific IgE levels were inversely correlated with stimulated levels of rBet v1
and rMal d1 IgA. Increased salivary allergen-specific IgA levels indicate that LGG
is immunostimulatory to the oral mucosa.
10.2.4 Representative Products of LGG
LGG’s first commercial probiotic product, part of the Gefilus brand, was launched
in Finland in 1990. Today, LGG has been used in a variety of products, including
fermented milk, single-day mini bottles, pasteurized milk, cheese, and some milk-
free products (juice drinks and dietary supplements).
10.2.4.1 LGG®
LGG® is a registered commercial brand and the patent is owned by the Valio com-
pany in Finland. The functional characteristics and safety of LGG® are the same as
in the “LGG” section above.
Culturelle is a probiotic supplement brand under the US healthcare company
i-Health. Its products are divided into three series: digestive health, immune health,
and child health. The main ingredients of Culturelle Digestive Health Probiotics are
ten billion active probiotics LGG and prebiotic inulin. The stated functions of prod-
uct include helping the digestive system to operate, reducing indigestion, alleviating
sporadic diarrhea, reducing gas and abdominal pain, and supporting the immune
system. Digestive health products are available in capsules and chewable tablets to
meet the needs of different people. And immune health is divided into two catego-
ries: daily support and enhanced immunity. The Health and Happiness Immune
Support product contains 15 billion active LGG. The product claims to support the
body’s natural defenses, support the immune system, maintain health, and restore
the natural balance of beneficial bacteria in the digestive tract. Another vegetarian
health and happiness immune product contains no gluten and no milk and is 100%
LGG powder. The children’s health probiotic products are available in small packets
and chewable tablets. The main function is to help children’s digestive tract work
well and support healthy immune system.
10.2.4.2 LGG®Extra
Overview
LGG®Extra is a multi-strain probiotic combining LGG with three other strains,

including Lactobacillus rhamnosus LC705 (DSM 7061) and Propionibacterium sp.
(Propionibacterium freudenreichii subsp. shermanii PJS) (PJS, DSM 7065) and
Bifidobacterium animalis subsp. lactis BB-12 (DSM 15954). The content of each
strain was 107 cfu/mL. According to previous studies, LC705 and PJS have the
effect of inhibiting the growth of yeast and mold. Bifidobacterium naturally coexists
in the healthy intestine and are generally considered to be related to human intesti-
nal health. Because the purpose of the probiotic combination is to work on the small
intestine and colon, BB-12 is included in the combination.
Security Issues
All strains of the LGG®Extra probiotic mix are among the microorganisms approved
for use by the European Food Safety Authority’s “Safety Qualification.” LGG and
BB-12 are also approved by the US Food and Drug Administration (FDA) and can
be added to infant food. All probiotic strains in this combination have a long history
of safe use: PJS has been used in cheese production in Finland since 1979 as an
adjunct starter; LC705 has been used in cheese production since 1995; LGG has
been used in fresh dairy products since 1990; and BB-12 has been in use since the
1980s. To date, no reports have been published on the safety issues of each strain.
Therefore, LGG®Extra is safe and harmless.
Clinical Function Research
The adhesion of probiotics to intestinal mucosa and epithelial cells is an important

indicator for evaluating probiotics, especially in terms of inhibiting adhesion and
immune effects of pathogenic bacteria. Each strain of the LGG®Extra probiotic
combination differed in adhesion. LGG has good adhesion to intestinal mucosa and
epithelial cells, while LC705 strain has poor adhesion. Gene sequencing revealed
that the possible cause was that the specific cilia structure on the surface of LGG
cells had human mucin junction synapses, whereas LC705 did not (Kankainen et al.
2009). In addition, BB-12 has a good adhesion ability, and the adhesion ability of
PJS is different in different studies. The adhesion ability of probiotics is closely
related to their retention in the human gastrointestinal tract. However, if the probi-
otic combination is embedded in a capsule or food system, each strain in the com-
bination can pass well through the stomach and small intestine.
The LGG®Extra Intestinal Probiotic Combination has no significant effect on the
intestinal microbial composition of healthy volunteers. Generally, healthy volun-
teers have higher Bifidobacterium and Lactobacillus. At lower baseline levels, the
probiotic combination can increase the level of lactobacilli.
In addition, there have been reports of studies on the combination of probiotics
for immune regulation and clearance of dietary toxins.
10.3 Bifidobacterium animalis subsp. lactis BB-12
10.3.1 The Discovery and Development of BB-12
Bifidobacterium animalis subsp. lactis BB-12 (BB-12) is a strain selected by Chr.

Hansen from its fermented dairy products. Formerly named Bifidobacterium lactis
BB-12, BB-12 has excellent acid- and bile-tolerant properties, so it can reach the
large intestine through the upper digestive tract in the form of live bacteria.
Since 1985, BB-12 has been used in food and dietary ingredients. As of July
2015, there are more than 360 scientific papers on BB-12, including more than 165
clinical studies. BB-12 is the most reported bifidobacteria in the world (Note: The
data obtained from the official website of Ke Hansen, http://www.chr-hansen. com/
zh).
Currently, BB-12 has been used in baby food formulas, dietary supplements,
fermented milk, and other products. In addition, because BB-12 has no negative
impact on the appearance and flavor of the product, it can also survive in the product
for a long time, so it is favored by people.
10.3.2 Safety of BB-12
At present, millions of people around the world have consumed products containing
BB-12, and no negative reports have been reported. BB-12 has been certified by the
US Food and Drug Administration (FDA) as a “generally considered safe” strain
(GRAS Notice No. GRN 00 0049) and can be used in formulas for infants of
4 months or older.
Merenstein et al. (2015) used BB-12 as a drug and conducted a Phase 1 safety
trial in accordance with FDA requirements. Forty patients with respiratory infec-
tions who received antibiotics were randomized to receive 4 ounces of BB-12 yogurt
or no added control yogurt for ten consecutive days. Safety and tolerance were
assessed by the number of negative effect events reported. A total of 165 nonserious
negative events were reported, but there was no difference between the control
group and the BB-12 group, explaining that healthy adults taking BB-12 while tak-
ing antibiotics are safe and have good tolerance.
10.3.3 Functional Study of BB-12
10.3.3.1 Prevention of Infection
Related studies have shown that BB-12 can increase mucosal resistance to infection
by increasing the level of native IgA in healthy infants (Fukushima et al. 1998). In
a double-blind, placebo trial conducted in Finland from 2004 to 2007, 109 1-month-
old babies were randomized to receive BB-12-containing tablets or placebo tablets
via a new slow-release pacifier. The baby is given twice daily (BB-12 daily dose is
1.0 × 1010 cfu), which lasts from 1 month of age to 8 months of age. It was found
that the characteristics of the placebo group and BB-12 group at the starting point
were similar. In the group receiving BB-12 tablets, BB-12 was detected in the feces
of 62% of the infants. There was no correlation between the nipple sucking time and
the incidence of acute otitis media. There were no significant differences between
the two groups in terms of reported gastrointestinal symptoms, otitis media, and
antibiotic use. However, infants receiving BB-12 experienced fewer respiratory

infections than the placebo group. This indicates that intake of BB-12 in infants and
young children may reduce respiratory infections (Taipale et al. 2011).
Because of multiple stresses, lack of sleep, and concentrated accommodation,
college students are more susceptible to upper respiratory tract infections than nor-
mal adults. Smith et al. (2013) launched a double-blind, randomized trial to exam-
ine the effects of BB-12 and LGG powder (daily dose of at least 109 cfu) on the
quality of life of 231 healthy college students with upper respiratory tract infections
for 12 weeks. The mean duration of upper respiratory tract infection was signifi-
cantly shorter (2 days), and the mean severity score was significantly lower (34%
lower) than the placebo group, indicating that probiotics increased health quality of
life during upper respiratory tract infections. There was no difference between the
days of absenteeism, but the days of absenteeism in the probiotic intake group were
significantly lower than the number of days in school. This suggests that LGG and
BB-12 may have a mitigating effect on the decline in the quality of life of college
students with upper respiratory tract infections.
An anaerobic fermentation experiment in vitro also showed that synbiotics
including BB-12 have an inhibitory effect on Escherichia coli and Campylobacter
jejuni. In this experiment, the anaerobic fermentation system contained human
feces, two probiotics (Lactobacillus plantarum 0407 and BB-12), and a mixture of
oligofructose and xylooligosaccharide (50:50, w/w). In the batch fermentation
experiment, although the pH of the culture system was neutral, both pathogenic
bacteria were inhibited by the synbiotics. In the continuous fermentation experi-
ment, only Campylobacter jejuni was inhibited. The mechanism by which the syn-
biotics antagonize pathogenic bacteria is not clear, but it has been found that acetic
acid and lactic acid directly play an antagonistic role, rather than the antagonism
caused by a decrease in pH of the system (Fooks and Gibson 2003).
10.3.3.2 Immune Effect
One of the mechanisms of the anti-infective effect of probiotics may be non-specific

initiation of immunity. In a Japanese study, NAN BF (BB-12 content of about
109 cfu/200 mL) containing BB-12 was given to seven healthy Japanese children
(aged 15–30 months) for 21 days. It was found that BB-12 was detected in stool
samples of five children (71%) during the intake of formula, and there was a slight
increase in bifidobacteria in all feces. Total IgA and anti-poliovirus IgA levels in
feces were significantly higher than pre-intake levels during the intake of probiotic-
containing formulas. It indicates that the increase of local IgA levels caused by
ingestion of probiotic formula may be beneficial to enhance the resistance of gastro-
intestinal mucosa to infection.
A randomized, double-blind, and placebo-controlled experiment examined the
regulation of BB-12 and Lactobacillus paracasei sub-combination L. casei 431® on
the immune system of influenza vaccine models in healthy populations (Rizzardini
et al. 2012). Subjects were given at least 109 cfu of BB-12® (capsule) or L. casei
431® (milk drink) or the corresponding placebo (placebo or placebo acidized milk
drink) once daily for 6 weeks. After 2 weeks, the seasonal flu vaccine was given. In
the BB-12 and L. casei 431 ingestion groups, the change in vaccine-specific plasma
IgG antibodies IgG1 and IgG3 was significantly greater than the initial placebo
group. In terms of the number of subjects with a significant increase in specific IgG
(defined as ≥ two times the initial level), the number of probiotic groups in the two
groups was also significantly greater than the number in the corresponding placebo
group. The mean increase in vaccine-specific secretory IgA in the saliva of the pro-
biotic group was also significantly higher than in the placebo group. Similar results
were observed for total antibody concentrations. However, there was no difference
between plasma cytokines or innate immune parameters. Studies have shown that
BB-12® or L. casei 431® supplementation may be an effective means of increasing
immune function by increasing systemic and mucosal immune responses.
10.3.3.3 Prevention and Treatment of Diarrhea
Diarrhea is induced by many causes, and rotavirus infection is one of the main
causes of diarrhea in children. Studies have shown that BB-12 intake is associated
with fecal rotavirus excretion during diarrhea. A study in Thailand explored the
effects of formula supplements (mainly milk) with the addition of probiotics on the
development of diarrheal diseases. The study selected 175 children, aged
6–36 months, according to the type of formula, divided into three groups: milk-
based formula (control group), formula added with BB-12, and formula added with
BB-12 and Streptococcus thermophilus. At the end of the 8-month experiment,
30.4% of the children in the control group had a more than fourfold increase in
rotavirus-specific antibody titers in saliva samples, indicating subclinical rotavirus
infection. The antibody titers of most children in the other two groups did not
change significantly from the initial period of the experiment compared with the end
of the experiment. It is indicated that children’s intake of formula containing bifido-
bacteria has a protective effect on rotavirus infection (Phuapradit et al. 1999).
There are also reports that mixtures of BB-12 and other probiotic have a signifi-
cant impact on human bowel movements. Studies have shown that BB-12 can
reduce the incidence of acute diarrhea in infants and children after 12 months of
administration with Streptococcus thermophilus. Larsen et al. (2006) explored the
combination of BB-12 and Lactobacillus paracasei subsp. paracasei (CRL-431)
(108 cfu, 109 cfu, 1010 cfu, and 1011 cfu) on human blood lipids, fecal recovery, and
bowel habits in a randomized, double-blind, placebo-controlled study. The results
showed that the stool BB-12 detection rate increased significantly with the increase
of the dose of the mixed probiotics. In the 1011 cfu/d BB-12 group, 13 of the 15
volunteers detected BB-12 in the feces. CRL-431 was not detected in all groups of
stool samples. Supplementation of probiotics did not alter intestinal microbial com-
position. However, the stool consistency increased linearly with increasing doses of
probiotics administered.
In addition, some animal models are also used to explain the alleviation of diar-
rhea by probiotics. Studies have shown that probiotics have the ability to alleviate
diarrhea in children and enhance systemic-specific mucosal antibody responses, but
T-lymphocyte responses have not been confirmed. A study by Chattha et al. (2013)
explored T lymphocytes and cytokines against human rotavirus-attenuated vaccines
(AttHRV) and human rotavirus virulence vaccine (HRV) response in gnotobiotic
pigs colonized with LGG and BB-12. Probiotic colonized and unvaccinated pigs
(probiotics) had a lower rate of diarrhea than unproportioned probiotics and unvac-
cinated pigs (control group). The higher protective effect of the probiotic group is
consistent with higher ileal regulatory T lymphocytes before and after vaccination,
as well as higher serum TGF-β and lower serum and biliary proinflammatory factors
after access to the virus. Compared to vaccinated pigs (vaccine groups) that are not
colonized with probiotics, probiotic colonization enhances serum congenital IFN-α,
spleen and circulatory system IFN-γ, T lymphocytes, and serum Th1 cytokines but
decreases serum Th2 cytokines. Therefore, LGG + BB-12 induced a systemic Th1
immune response (for oral AttHRV), which was consistent with a reduction in the
severity of diarrhea in the vaccine + probiotic group and a reduction in virus shed-
ding after access. Therefore, the probiotic LGG + BB-12 exerts different immuno-
modulatory effects, and the oral AttHRV vaccine enhances the Th1 cell response
and virulence HRV and induces regulatory T-lymphocyte response.
10.3.3.4 Alleviation of Constipation
Eskesen et al. (2015) explored the effects of BB-12 on the frequency of bowel
movements and gastrointestinal comfort (defined as total abdominal discomfort) in
healthy adults with low bowel movements and abdominal discomfort. In this 4-week
randomized, double-blind, placebo-controlled trial, the frequency of defecation in
the probiotic treatment group was significantly higher than in the placebo group.
The effects of different doses of probiotics (1.0 × 109 cfu or 1.0 × 1010 cfu) on the
frequency of defecation were similar, indicating that the effect of 1.0 × 109 cfu/d
probiotics has reached the upper limit. Overall, 4 weeks of BB-12 intake has clinical
benefit associated with bowel movements, indicating that BB-12® improves gastro-
intestinal health for those whose symptoms are not yet severe enough to consult a
physician.
10.3.3.5 Prevention of Dental Caries
Studys also explored the preventive effects of BB-12 on dental caries. In a double-
blind, placebo-controlled clinical study, infants aged 1–2 months were randomized
to receive probiotic BB-12 (1010 cfu/d), xylitol, or sorbitol (sugar alcohol dose 200–
600 mg/d) until 2 years old. The colonization rate of Streptococcus mutans in
2-year-old children was quite low, 6% for BB-12 group, 31% for xylitol group, and
10% for sorbitol group. The levels of lactic acid bacteria and yeast did not differ
between groups. As for the oral samples of children in the three treatment groups,
the number of BB-12 barely exceeded the detection limit at 8 months of age, but
BB-12 was no longer detected in the samples at 2 years of age. The results suggest
that early intake of BB-12 in children does not cause long-term colonization of this
probiotic in the oral cavity, nor does it significantly affect the streptococci coloniza-
tion in children (Taipale et al. 2012). Two years after the end of the study, the clini-
cal examination and questionnaire data of the children (4 years old) were re-collected,
and the international dental caries examination and evaluation system was used to
evaluate the incidence of dental caries and to detect oral hygiene status and
Streptococcus mutans. The results showed that BB-12 intake during infancy did not
increase or decrease the incidence of dental caries in 4-year-old low-cavity popula-
tion (Taipale et al. 2013). Another randomized, controlled, double-blind trial evalu-
ated the effects of oral LGG and BB-12 on the number of Streptococcus mutans,
plaque, gingival inflammation, and oral microbes in healthy adults. After 4 weeks of
volunteer intake of two probiotic combinations or placebo, the plaque index and
gingival index were reduced, whereas probiotics had no effect on oral microbes
(Toiviainen et al. 2015).
10.3.4 Representative Products of BB-12
10.3.4.1 BB-12®
BB-12® is a registered trademark of Chr. Hansen A/S, Denmark. Currently, BB-12®

has been developed into a variety of probiotic products, including single strain
forms and products in combination with other strains. In addition, BB-12® is also
commonly used in fermented dairy products such as yogurt and lactic acid bacteria
beverages.
At present, the probiotic products containing BB-12 circulating in the market
mainly include the following.
Nemans Probiotics
The Nemans probiotic series are divided into children’s probiotic powder and adult
and female special type of probiotic soft capsule according to the applicable subjects.
The probiotic powder for children consists of two probiotics, BB-12 and LGG. It is
suitable for children over 0 years old and has the functions of assisting nutrient absorp-
tion and improving immunity. The probiotics are microencapsulated and have a shelf
life of 730 days. Each strip contains at least 4.5 billion live bacteria and can be brewed
at 37 °C warm water. The adult type of probiotic soft capsule contains Lactobacillus
acidophilus, Bifidobacterium, Streptococcus thermophilus, and Lactobacillus del-
brueckii subsp. bulgaricus. It is suitable for ordinary adults and pregnant women, and
each capsule contains at least 4.5 billion live bacteria. The product has various physi-
ological functions such as relieving constipation, balancing intestinal flora, clearing
Helicobacter pylori, and enhancing the colonization of bifidobacteria.
Nitta Probiotic Powder
Nitta probiotics are produced by the Danish company Hansen and the main ingredi-
ent is BB-12. Each pack contains six billion live bacteria. Nitta probiotics have the
effect of enhancing immunity, effectively preventing diarrhea in children and adults,
and alleviating allergic eczema.
Puractive® Probiotics
The strain contained in Puractive® probiotics is BB-12. Puractive, Pure and Active,
means “the purest source of vitality.” Puractive® probiotics are available in both
family and baby packs. Puractive® family products are mainly composed of skim
milk powder, glucose, inulin, white sugar, maltodextrin, silica, and vanillin, in addi-
tion to the probiotics themselves. The main ingredients of Puractive® baby products
are maltodextrin and BB-12.
10.3.4.2 LGG®Extra
See Sect. 10.2 LGG®Extra for an introduction.
10.4 Lactobacillus gasseri LG21
10.4.1 The Discovery and Development of LG21
Probiotics are generally considered to have physiological functions of inhibiting

pathogenic bacteria, are safe and inexpensive, and are promising to play an impor-
tant role in the treatment of pathogenic bacteria infection. Helicobacter pylori is a
common pathogen that inhabits the pyloric part of the human stomach, causing
chronic inflammation, stomach ulcers, and even stomach cancer (Calvino-Fernandez
and Parra-Cid 2010). Helicobacter pylori has high urease activity and can catalyze
the production of ammonia and carbon dioxide in the stomach, so Helicobacter
pylori can survive in a strong acid environment. In order to obtain probiotics with
excellent ability to antagonize Helicobacter pylori, researchers in Meiji Dairy of
Japan conducted a series of screening work through in vitro and animal
experiments.
First, in vitro tests were used to evaluate the tolerance of lactic acid bacteria to
gastric acid, the ability to proliferate in an acidic environment, the adhesion to gas-
tric epithelial cells, the ability to inhibit Helicobacter pylori during co-culture, and
the physiological characteristics associated with antagonism of Helicobacter pylori.
Three strains of Lactobacillus gasseri LOL2716 (LG21) and No.6 and Lactobacillus
salivarius WB1004 (WB1004) were screened from 203 strains of Lactobacillus.

Subsequently, the protective effects of the three Lactobacillus strains on a mouse
model of Helicobacter pylori infection were examined. It was found that oral
administration of LG21 and WB1004 effectively eradicated Helicobacter pylori in
mice, and LG21-treated mice had the lowest anti-Helicobacter pylori IgG levels.
Therefore, LG21 was screened as the most suitable lactic acid bacteria for antago-
nizing H. pylori (Kimura 2004). In addition, studies have also found that LG21 also
has a certain inhibitory effect on Helicobacter pylori-tolerant clarithromycin strains
(Charteris et al. 1998).
In recent years, the effects of LG21 on Helicobacter pylori infection have been
verified not only in animal experiments (Uchida and Kurakazu 2004; Uchida et al.
2010) but also are discussed in a large number of human experiments in adults,
children, and asymptomatic infections (Deguchi et al. 2012; Fujimura et al. 2006;
Gotteland and Cruchet 2003; Igarashi et al. 2014; Kawai et al. 2006; Ojetti et al.
2012; Sakamoto et al. 2001; Shimizu et al. 2002).
10.4.2 Safety Study of LG21
Relevant researches currently carried out, including animal experiments and human
experiments, have not involved adverse reactions related to LG21.
10.4.3 Functional Study of LG21
Antagonizing Helicobacter pylori infection is the most important function of LG21.

Therefore, published research, whether animal or human, mainly focuses on the
prevention and treatment of Helicobacter pylori-related diseases. However, a small
number of studies have explored the potential probiotic effects of LG21 on other
upper digestive tract diseases and colon cancer.
10.4.3.1 Antagonizing Helicobacter pylori Infection
In Vitro Test
Through co-culturing LG21 with Helicobacter pylori, Fujimura et al. (2012) found
that DL-lactic acid produced by LG21 transformed Helicobacter pylori into a glob-
ular shape, inhibiting the reproduction of Helicobacter pylori, which is one of the
important mechanisms of LG21 antagonizing Helicobacter pylori. In addition,
LG21 has strong acid resistance compared to other lactobacilli. Another co-culture
study demonstrated that Helicobacter pylori-induced IL-8 was inhibited when
LG21 was less than 1% of Helicobacter pylori. IL-8 induces the accumulation of
neutrophils in the gastric mucosa. The results of IL-8 in gastric biopsies from
patients with Helicobacter pylori infection also confirmed that LG21 can inhibit
IL-8 production in the intestinal mucosa (Tamura et al. 2006).
Human Experiment
Sakamoto et al. (2001) examined the effects of LG21 on the colonization of

Helicobacter pylori in adults and the associated inflammation. Thirty-one
Helicobacter pylori-infected yogurts were consumed daily for 8 weeks. [13C] urea
breath test and serum pepsinogen assay showed that ingestion of LG21 significantly
improved the Helicobacter pylori infection status and effectively inhibited
Helicobacter pylori on the one hand and reduced gastric mucosal inflammation on
the other hand. However, children infected with Helicobacter pylori consumed
LG21 yogurt for 8 weeks, and the results were not satisfactory. Twelve children
with Helicobacter pylori infection, who had an average age of 12.1 years, con-
sumed 120 g of yogurt containing LG21 twice a day (i.e., 240 g/d). The serum
pepsinogen I/II value at 4 weeks after the intake of LG21 yogurt was significantly
higher than that before, suggesting inhibition of Helicobacter pylori-associated
gastric mucosal inflammation. However, at 2 weeks after the end of LG21 yogurt
intake, [13C] urea respiration test results, fecal Helicobacter pylori antigen levels
and serum pepsinogen I/II values were not significantly different from those before
LG21 intake, indicating that LG21 intake did not reduce gastric mucosal
Helicobacter pylori after the end of the experiment. Density did not alleviate the
associated inflammation. Compared with adult subjects, children consumed more
LG21 yogurt (180 g/d for adults and 240 g/d for children), but the results were
contrary. One of the reasons may be that probiotics are limited to Helicobacter
pylori during consumption, and Helicobacter pylori regains vitality after probiotic
intake ceases (Shimizu et al. 2002).
At present, the treatment of Helicobacter pylori-related diseases mainly uses a
triple method, that is, a proton pump inhibitor combined with two antibiotic treat-
ments. The disadvantages of this method include strong adverse reactions, easy
drug resistance, and high recurrence rate. Studies have shown that eradication of
Helicobacter pylori can reduce the recurrence rate of gastric ulcer, but the emergence
of resistant strains affects its eradication effect. Probiotics-assisted antibiotics in the
treatment of Helicobacter pylori-related diseases can improve efficacy and reduce
adverse reactions. In a randomized, controlled clinical trial, 229 patients were ran-
domized to receive 1-week triple therapy including rabeprazole (10 mg twice daily),
amoxicillin (750 mg twice daily), and clarithromycin (200 mg twice daily) or triple
therapy plus yogurt with LG21. In the yogurt plus triple therapy group, LG21
(112 g) yogurt was administered twice daily for 4 weeks (pretreatment for 3 weeks
and antibiotic treatment for 1 week). The results showed that 188 of the 229 samples
detected Helicobacter pylori susceptible to clarithromycin. The infection rate of
Helicobacter pylori resistant to clarithromycin was 27.1%. Overall clearance (intent
treatment/treatment) was 69.3%/74.5% in the triple therapy group and 82.6%/85.6%
in the yogurt plus triple therapy group. The yogurt plus triple therapy group had a
higher clearance of major clarithromycin-resistant strains than the triple therapy

group (38.5% vs. 28.0%). This study confirmed that the addition of LG21 yogurt for
4 weeks improved the efficacy of triple therapy for Helicobacter pylori infection
(Deguchi et al. 2012).
The therapeutic mechanism of LG21 for Helicobacter pylori infection is closely
related to the physiological function of LG21. LG21 is highly resistant to gastric
acid and can grow in an acidic environment. The study examined LG21 in the stom-
ach of LG21, revealing that one of the mechanisms by which LG21 inhibits
Helicobacter pylori colonization in vivo may be LG21 in gastric mucus. The study
examined LG21 in the stomach of LG21, revealing that one of the mechanisms by
which LG21 inhibits Helicobacter pylori colonization in vivo may be the competi-
tive occupancy of LG21 in gastric mucus layer (Fujimura et al. 2006). In addition,
LG21 can produce a large amount of lactic acid, which can also inhibit the growth
of Helicobacter pylori. Compared with hydrochloric acid of the same pH, lactic
acid has a stronger inhibitory effect on the growth of Helicobacter pylori.
10.4.3.2 Prevention of Upper Gastrointestinal Tract Disease
Studies on the probiotic effects of probiotics on gastrointestinal function have

focused on improving defecation but less on preventing ulcers and promoting ulcer
healing. LG21 can effectively inhibit Helicobacter pylori, and it also has the effect
of improving inflammation of the gastric mucosa. Therefore, its potential in the
prevention and treatment of ulcers has gradually gained the attention of
researchers.
Animal Experiment
Uchida and Kurakazu (2004) examined whether LG21 yogurt has protective effects
on acute gastric mucosal lesions or gastric antrum ulcers in rats and analyzed the
mechanism of gastric protective effects. Results showed that LG21 yogurt signifi-
cantly inhibited the formation of acute gastric injury, and this protective effect was
dose-dependent. LG21 yogurt treatment also significantly increased the production
of prostaglandin E2 in the gastric mucosa, and also significantly reduced the length
of gastric mucosal fold staining, significantly inhibiting the formation of gastric
antrum ulcer. It can be observed that the administration of LG21 yogurt is beneficial
to prevent gastric ulcer. Endogenous prostaglandins are also one of the mechanisms
of gastric protection in LG21 yogurt.
LG21 yogurt or gamma-irradiated LG21 yogurt was orally administered to rats
twice a day for a total of 10 days with a dose of 5 mL per kg. LG21 yogurt remark-
ably accelerated ulcer recovery, while gamma-irradiated LG21 yogurt did not work.
However, both yogurts significantly inhibited HCl-induced gastric erosion damage
and increased the production of gastric mucosal prostaglandin E2. This suggests
that living cells are necessary to accelerate the healing of gastric ulcers and not to
inhibit gastric damage (Uchida et al. 2010).
Human Experiment
Some studies have shown that LG21 may have potential for therapy. Symptoms of
upper gastrointestinal functional diseases, such as gastroesophageal reflux disease
and functional dyspepsia, often occur without any organ or systemic disease that
can explain the symptoms. To this end, Igarashi et al. (2014) studied the relationship
between gastric-related biomarker levels and symptoms, finding that the frequency
of symptoms of esophageal reflux disease decreased, while the value of pepsinogen
I increased after treatment with LG21. Multiple regression analysis showed a strong
correlation between the frequency score of symptoms of esophageal reflux disease
and pepsinogen I. Therefore, LG21 and the like are substances that can increase the
level of serum pepsinogen I and may be used to treat upper gastrointestinal func-
tional diseases.
Akama et al. (2011) examined whether probiotic LG21 has a protective effect on
the destruction of gastric mucosal integrity caused by aspirin. In the first study,
urine sugar excretion experiments were performed in 29 volunteers who received
high doses of aspirin before and after LG21 intake for 4 weeks. And in the second
study, 37 patients received low-dose aspirin therapy after LG21 treatment for
16 weeks, and urine sugar excretion experiments were also conducted. The results
showed that in the previous study, urinary sucrose excretion in LG21 treatment
group decreased significantly after aspirin intake. In the latter study, urinary sucrose
excretion decreased significantly during LG21 treatment, whereas during no LG21
treatment, the difference was not significant. During the LG21 treatment, the num-
ber of occult blood positives decreased. Regular intake of LG21 may protect the
integrity of gastric mucosal permeability under aspirin.
10.4.3.3 Prevention of Colon Cancer
Ohara et al. (2010) explored the possibility of probiotic LG21 treatment to prevent
colorectal cancer. The study selected 10 patients with colorectal cancer and 20
healthy individuals. The results showed that the detection rate of Lactobacillus in
the healthy control group was significantly higher than that in the colon cancer
group, and the total Clostridium perfringens was lower than that in the colorectal
cancer group. The pH of the stool in the colorectal cancer group showed alkali tox-
icity, and the total short-chain fatty acids in the feces was often lower than in the
healthy group. After the intake of probiotics, the detection rate of lactobacilli
increased, the total Clostridium perfringens decreased, the synthesis of fecal spoil-
age products was inhibited, and the short-chain fatty acid isobutyric acid increased.
The activity of IL-1β and NK cells in the blood was significantly higher from the
fourth week than the value before the probiotic intake. Compared with healthy con-
trols, the intestinal environment of patients with colorectal cancer deteriorates, and
the intake of probiotics can improve the intestinal environment, suggesting that pro-
biotics have the potential to prevent colorectal cancer.
10.4.4 Representative Products of LG21
Meiji Dairy’s LG21 series of yogurt, due to the addition of lactic acid bacteria
LG21, which can antagonize Helicobacter pylori, can reduce Helicobacter pylori
and protect the gastric mucosa. Therefore, it is also considered to reduce the inci-
dence of gastric cancer.
10.5 Lactobacillus casei CRL 431
10.5.1 The Discovery and Development of CRL 431
Lactobacillus paracasei subsp. paracasei CRL 431 (ATCC 55544) is a probiotic

isolated from the feces of healthy children by the Universidad Nacional de Tucumán.
In the vast majority of published research papers, its name is Lactobacillus casei
CRL 431 (Lactobacillus casei CRL 431). Lactobacillus casei 431 is its other name.
For ease of discussion and avoidance of confusion, the name Lactobacillus casei
CRL 431 is used in this section.
The commercial strain of Lactobacillus casei CRL 431 is L. casei 431®, which
has been used as an additive in food and food ingredients since 1995. In fermented
milk, CRL 431 is very viable and can also be used as a dietary ingredient in the form
of a lyophilized product.
10.5.2 Safety of CRL 431
Lactobacillus casei CRL 431 has been used in the food industry for more than
20 years as a microorganism that constitutes a normal intestinal flora. No cases of
CRL 431-related diseases or injuries have been reported in the consumer popula-
tion. A series of clinical trials from adult to infant did not reveal serious adverse
events.
A study to evaluate the safety of Bifidobacterium lactis subsp. lactis BB-12 and
Lactobacillus casei CRL 431 mixed vaccine for neonates. A total of 126 neonates
were enrolled, randomized to receive BB-12 and CRL431 formula or probiotic-free
formula within 3 months of birth. The results showed that all infants had normal
growth and development, and there was no significant difference in body weight,
height, and brain circumference. Experimental records showed significant differ-
ences between the experimental and control groups in terms of crying and sleep
time, number of infections perceived by parents, use of antibiotics, number of visits
to doctors, and negative events. Infants with probiotics have softer feces and more
frequent bowel movements. Studies have shown that CRL431 and BB-12 are safe
for young infants as a formula supplement and have no negative impact on infant
growth and behavior (Vlieger et al. 2009).
The US Food and Drug Administration (FDA) has identified L. casei 431® as
“generally recognized as safe” (GRAS). In addition, the FDA has also granted the
status of “new dietary ingredient” (NDI) to Lactobacillus paracasei. In 2007, the
European Food Safety Authority (EFSA) also awarded a qualified presumption of
safety (QPS) for Lactobacillus paracasei at the species level. Therefore, we can
think that CRL 431 is safe and harmless.
10.5.3 The Functional Study of CRL 431
Clinical experimental data show that Lactobacillus casei CRL 431 has the follow-
ing beneficial functions: regulating immune function, reducing diarrhea and abdom-
inal pain caused by pathogenic bacteria, and shortening the duration of common
cold and influenza symptoms.
10.5.3.1 Inhibition of Pathogenic Bacteria Infection
Prevention of pathogenic infections is one of the most studied aspects of the func-
tion of Lactobacillus casei CRL 431. Villena et al. (2009) examined the effects of
CRL 431 live and dead bacteria on the resistance of mice with malnutrition to pneu-
mococcal resistance. In malnourished mice, lung (S. pneumoniae) colonization and
lung injury are more severe, and leukocyte supplementation damage is more severe,
and antibodies and cytokines are more reduced. Ingestion of Lactobacillus casei
CRL 431 increased the resistance of malnourished mice to infection. Balanced
Dietary Addition CRL 431 live bacteria and balanced diets added to the dead group
prevented the spread of pathogenic bacteria into the bloodstream, inducing lung
clearance in mice. A balanced diet supplemented with live or dead bacteria increased
TNF-α production and respiratory phagocytic activity, whereas the balanced dietary
control did not. In addition, IL-4 and IL-10 were significantly increased in the live
and dead groups and were associated with increased specific IgA in the respiratory
tract. Nasal probiotic treatment initiated systemic and respiratory-specific IgG pro-
duction. A comparative study of live bacteria treatment and dead bacteria treatment
found that live bacteria play an important role in maximizing protection.
Diarrhea caused by Salmonella infection is one of the leading causes of morbid-
ity and mortality in children in developing countries. Salmonella can cause a variety
of diseases, including gastroenteritis and enteric fever. Probiotics are considered to
be one of the possible ways to combat Salmonella infection. de LeBlanc et al.
(2010) used a mouse model to investigate the preventive and therapeutic effects of
Lactobacillus casei CRL 431 on Salmonella enteritidis typhoid serotypes. The
results showed that the administration of CRL 431 to mice for 7 days before infec-
tion of pathogenic bacteria reduced the severity of Salmonella enteritidis typhoid
serotype infection, confirming the best effect of continuous administration.

Continuous administration of probiotics reduces the number of pathogens in the
intestine and beyond the intestine. The mechanism of action of CRL 431 may
involve both innate and acquired immune responses. Intake of CRL 431 reduces
neutrophil infiltration, thereby reducing intestinal inflammation; triggering phago-
cytic activity of multiple macrophages such as the Peyer’s patches, spleen, and peri-
toneum; and increasing the amount of IgA+ cells in the lamina propria of the small
intestine (with increased S-IgA release in pathogens against intestinal pathogens).
To further understand how CRL 431 works, subsequent studies examined the effects
of probiotics on the expression and secretion of pro-inflammatory and anti-
inflammatory factors in intestinal immune responses and analyzed difference in
Toll-like receptor expression in healthy and infected mice. It was found that the
intake of Lactobacillus casei CRL 431 increased the expression of TLR1, TLR4,
and TLR9 in healthy mice and increased the secretion of TNF-α, IFN-γ, and IL-10 in
Peyer’s patches. Probiotics are continuously administered before and after
Salmonella infection, which is achieved by regulating the inflammation of the lam-
ina propria of the small intestine, reducing TNF-α, and increasing the production of
IL-6 and IL-10. Oral ingestion of Lactobacillus casei CRL 431 induced changes in
cytokines. These changes suggest that probiotics that protect against Salmonella
infection may be involved in the regulation of immune function.
10.5.3.2 Prevention and Treatment of Diarrhea
Lactobacillus casei CRL 431 and Lactobacillus acidophilus fermented milk are
used to treat infantile diarrhea and can restore fecal flora imbalance caused by diar-
rhea. Levels of Lactobacillus more than 106 cfu/g feces are beneficial to keep chil-
dren healthy, indicating two Lactobacillus mixtures have potential in the treatment
of gastrointestinal disorders (Gonzalez et al. 1995).
Lactobacillus casei CRL 431 is also effective in treating intestinal hyperplasia-
associated diarrhea. Patients taking oral CRL 431 and Lactobacillus acidophilus
CERLA for three consecutive weeks significantly reduced the average number of
bowel movements per day until 15 days after stopping the intake. In addition, mixed
probiotic treatment significantly reduced the hydrogen concentration in the breath-
ing gas, indicating that mixed probiotics are effective in treating chronic diarrhea
associated with bacterial overgrowth (Gaon et al. 2002).
10.5.3.3 Immune Regulation
Studies have shown that Lactobacillus casei CRL 431 has functions such as regulat-
ing phagocytosis and stimulating the production of cytokines and antibodies. CRL
431 is administered orally to mice, stimulating an increase in the anti-inflammatory
factors IL-10 and IL-14 in intestinal immune cells, enhancing the IgG1 response,
and favoring Th2 balance (Perdigon et al. 2002). CRL 431 also induced an increase
in IgA+ cells and IL-6-secreting cells. At the same time, phagocytic and dendritic
cell surface receptors CD-206 and TLR-2 increased, indicating that the main mech-
anism of probiotic CRL 431 on the immune system is the clonal expansion of innate
immune IgA B lymphocytes (Galdeano and Perdigon 2006).
10.5.3.4 Affecting Obesity
Obesity is a chronic disease associated with inflammatory responses in which cyto-

kines play an important role. Novotny Nunez et al. (2015) evaluated the effect of
Lactobacillus casei CRL 431 on cytokine response in high-fat diet model mice. The
results showed that probiotic CRL 431 intake showed an anti-inflammatory response
to high-fat diet-fed mice: reduced levels of pro-inflammatory factors such as IL-6,
IL-17, and TFN-α. The intake of probiotics is associated with a decrease in immune-
infiltrating cells in the liver of mice fed with a high-fat diet, which reduces the secre-
tion of MCP-1 in adipocytes. It is also associated with a decrease in macrophage
accumulation in adipose tissue, indicating that Lactobacillus casei CRL 431 exhib-
its an anti-inflammatory effect when administered to a high-fat diet model mouse.
Intake of CRL431 increases the number of IgA+ cells and macrophages in the small
intestine, suggesting a regulatory effect on the innate immune system (Novotny
Nunez et al. 2014).
Obesity is closely related to intestinal microbial changes (Bajzer and Seeley
2006). Lactobacillus casei CRL 431 also acts on intestinal microbes in obese mice
and increases the number of Bacteroides and Bifidobacterium (Novotny Nunez
et al. 2014).
10.5.3.5 Inhibiting Tumors
Bonet et al. (2005) studied the inhibitory mechanisms of oral Lactobacillus casei
CRL 431 on fibrosarcoma (non-intestinal tumor, NIT) and cancer (intestinal tumor,
IT). Lactobacillus casei (1.2 × 109 cfu/d per mouse) was administered for two con-
secutive days before induction of tumors in BALB/c mice. After IT induction, the
frequency of oral administration of Lactobacillus casei was once every 5 days for
five consecutive months. The percentage of tumor inhibition in NIT mice was deter-
mined by tumor morphology studies and serum and spleen TNF-α levels, whereas
for IT mice, macroscopic and histopathological studies and intestinal IgA (+) and
TNF-α (+). The number of cells is determined. The results showed that Lactobacillus
casei CRL 431 inhibited the growth of both tumors. In NIT animals given with
Lactobacillus casei, high concentrations of TNF-α in the serum play an important
role in inhibiting tumor growth because TNF-α has lytic activity against tumor cells.
In IT mice, increased IgA(+) and TNF-α(+) cells may be involved in inhibition. This
suggests that screening for probiotics with antitumor activity cannot be limited to
the determination of antitumor-related cytokines, as different cancers have a multi-
factorial etiology.
Aragon et al. (2014) explored the effect of Lactobacillus casei CRL 431 fer-
mented milk on a mouse model of breast cancer. Studies have found that intake of
probiotics delays or blocks tumor progression, which is associated with the regula-
tion of tumor-induced immune responses. In mice given with fermented milk, the
area of blood vessels in the tumor decreased, the tumor decreased, and the adverse
reactions decreased. After the tumor is detected, the intake of probiotics is beneficial
for delaying tumor growth. Lactobacillus casei CRL 431 fermented milk can acti-
vate the immune system, respond to breast tumors, and prevent or delay tumor
growth. Aragon et al. (2015) then investigated whether administration of CRL 431
fermented milk to mice can affect not only tumor growth but also tumor cell over-
flow and lung metastasis if the tumor can be detected. The results showed that fer-
mented milk was administered to mice, which reduced or inhibited tumor growth,
tumor vascularization, tumor cell overflow, and lung metastasis. These effects are
associated with modulation of immune responses such as reduction of tumor and
pulmonary macrophage infiltration. Ingestion of fermented milk maintains an
increase in antitumor response-related CD8(+) lymphocytes and may be involved in
an increase in the immune response CD4(+).
10.5.4 Representative Products of CRL 431
L. casei 431® probiotic strains have been used worldwide for food and food ingre-
dients since 1995.
NU-TRISH® Probiotics starter products include other varieties of NU-TRISH
starters such as Probio-Tec®, Yo-Fast, and L. casei 431®. The strain of L. casei 431®
starter is Lactobacillus paracasei subsp. paracasei CRL 431, a medium-temperature
lactic acid bacteria species. This strain is mainly used to produce probiotic milk
products and can also be mixed with other dairy strains.
10.6 Lactobacillus acidophilus NCFM
10.6.1 iscovery and Development of Lactobacillus

D
acidophilus NCFM
10.6.1.1 Discovery of Lactobacillus acidophilus NCFM
Lactobacillus acidophilus NCFM was isolated from human feces by the US North
Carolina Food Microbiology (NCFM) in the early 1970s; thus, it was named NCFM
(Sanders and Klaenhammer 2001). Lactobacillus acidophilus NCFM mainly exists
in the small intestine and antagonizes pathogenic microorganisms by releasing lac-
tic acid, acetic acid, and antibiotics.
10.6.1.2 Development of Lactobacillus acidophilus NCFM
Lactobacillus acidophilus NCFM is now marketed by Danisco and is the first

Lactobacillus acidophilus strain whose genome was sequenced (Altermann et al.
2005). Genome research has identified a number of genes which are important for
probiotics and support their functions in maintaining and restoring gastrointestinal
health. These identified genes are associated with bacteriocin, sugar and probiotic
metabolism, adhesion to human cells, lactose metabolism, and physiological stress
such as acid and bile tolerance (Azcarate-Peril et al. 2004; Han et al. 2011; Man
et al. 2014). Metabolic reconstruction based on genome also verified the adaptabil-
ity of Lactobacillus acidophilus NCFM to gastrointestinal environment. In addition,
analysis of the NCFM genome sequence of Lactobacillus acidophilus confirmed
that it had transferable genetic factors associated with antibiotic resistance
(Altermann et al. 2005). Lactobacillus acidophilus NCFM has been shown to be
able to exert antimicrobial activity. The production of antimicrobial substances by
NCFM has been confirmed from the genomic sequence of the strain. Twelve hypo-
thetical genes have been identified to be involved in the production and action of
antimicrobial substances (Altermann et al. 2005; Dobson et al. 2007).
10.6.2 Safety Study of Lactobacillus acidophilus NCFM
Lactobacillus acidophilus NCFM has a long history of research. It has been widely
used in yogurt and other products in the past few decades. Its excellent safety record
and ease of commercial use further enhance its application value. The safety of
Lactobacillus acidophilus NCFM is based on its long-term history of safe use and
systematic testing in immunodeficient mice. Neither neonatal nor adult mice died
after eating high levels of Lactobacillus acidophilus NCFM. The history of the safe
use of Lactobacillus acidophilus NCFM can be traced back to the beginning of its
production and marketing in the United States and other countries around the world
as a food and dietary supplement. NCFM is also a scientifically recorded strain for
probiotic commercial use.
China’s Ministry of Health announced in 2011 that Lactobacillus acidophilus is
a kind of strain that can be used in infant food. As the most widely studied strain of
Lactobacillus acidophilus, NCFM has been widely used in fermented dairy prod-
ucts, pharmaceuticals, infant food, and probiotics. Lactobacillus acidophilus NCFM
as a supplement and fermented and non-fermented food has been commercially
produced and has been proved to have good stability for fermented products to pro-
vide a good flavor (Laličić-Petronijević et al. 2015). In addition, Lactobacillus aci-
dophilus NCFM can utilize fructooligosaccharides and other probiotic substances,
providing an opportunity for the development of synbiotic functional foods
(Barrangou et al. 2003; Endo et al. 2012).
10.6.3 Functional Study of Lactobacillus acidophilus NCFM
10.6.3.1 Inhibition of Harmful Bacteria and Pathogens
Antimicrobial activity is an important indicator for the competitive elimination or

inhibition of harmful or pathogenic intestinal microbial activities by probiotics.
Antimicrobial compounds produced by probiotics include organic acids (such as
lactic acid and acetic acid), hydrogen peroxide, diacetyl, beta-
hydroxypropionaldehyde, bacteriostatic peptides and proteins (De Vuyst and
Vandamme 1994; Stoyanova et al. 2012). Laboratory tests and co-culture experi-
ments have repeatedly demonstrated the ability of many lactic acid bacteria to
inhibit a range of microorganisms (pathogens, spoilage bacteria and other lactic
acid bacteria). In vitro studies have shown that most Lactobacillus usually produce
bacteriocins (a class of proteins that exhibit bactericidal or antimicrobial activity),
killing related species. Bacteriocins produced by Lactobacillus acidophilus NCFM
have been found to inhibit another Lactobacillus species, Lactobacillus deltoides.
However, the bacteriocins function and activity in vivo is not very clear (Lee and
Salminen 2009). Lactobacillus acidophilus NCFM can antagonize many food-
borne pathogens, such as Staphylococcus aureus, Salmonella, pathogenic
Escherichia coli, Clostridium perfringens. The results show that NCFM can inhibit
80–90% of these pathogens, and the inhibitors may be organic acids, hydrogen per-
oxide or other antibacterial components (Bengmark 2013; Hougaard et al. 2016).
Lactobacillus acidophilus NCFM was found to produce a specific bacteriocin,
lettuce B, whose biochemical and genetic properties have been extensively studied.
In vitro tests showed that the antibacterial activity of lettuce B was only against
other Lactobacillus and Enterococcus faecalis, but not against pathogenic bacteria
(Barefoot and Klaenhammer 1983). The molecular weight of lettuce B is 6500,
which is sensitive to protease K and Streptomyces proteinase. This proves that let-
tuce B has the characteristics of protein. The bacteriocin is stable to heat (121 °C, 3
min, pH 5), cold (−20 °C) and chemical ionizer (beta-mercaptoethanol, 8 M urea,
1% SDS) (Barefoot et al. 1994).
10.6.3.2 Intestinal Survival and Adhesion
In vitro studies have shown that Lactobacillus acidophilus NCFM has the necessary
preconditions for probiotics to survive digestion in the gastrointestinal tract. The
strain is resistant to bile acids, low pH and digestive enzymes (Daniel et al. 2006;
Ruiz et al. 2013). In addition, Lactobacillus acidophilus NCFM has been shown to
adhere to human epithelial cell lines and human intestinal mucus (Collado et al.
2007; 满朝新 et al. 2014). This adhesion can be further improved by adding Ca2+.
Through adhesion and copolymerization of intestinal mucus, Lactobacillus aci-
dophilus NCFM can also prevent the adhesion of certain pathogens (Collado et al.
2007, 2008). Genomic analysis of Lactobacillus acidophilus NCFM showed that
there were some potential important genes related to adhesion process, including
mucin-binding protein, fibronectin and so on. Lactobacillus acidophilus NCFM has
encoded gene clusters producing extracellular polysaccharides, which may also
affect its adhesion ability (Altermann et al. 2005). In addition, oral adhesion and
survival of Lactobacillus acidophilus NCFM have also been studied (Haukioja et al.
2006).
Oral administration of 1010 cfu Lactobacillus acidophilus NCFM in healthy adult
volunteers increased the number of Lactobacillus in feces, especially Lactobacillus
acidophilus, which had not been detected before the intervention but became the
dominant strain after the intervention. There was no significant difference in the
levels of bifidobacteria and sulfide-producing bacteria (Sui et al. 2002). Recently,
the ability of Lactobacillus acidophilus NCFM to survive in the human gastrointes-
tinal tract has been confirmed by clinical trials, which also found that the strain,
together with lactosol, can increase the amount of bifidobacteria in feces (Ouwehand
et al. 2009).
10.6.3.3 Hypolipidemic Effects
Reducing total cholesterol or LDL levels in human plasma is thought to reduce the
risk of cardiovascular disease, and taking probiotic products is recommended as a
dietary intervention to reduce blood lipids (Ebel et al. 2014). The laboratory results
of Lactobacillus acidophilus NCFM showed that it had the ability to remove cho-
lesterol from the culture medium (Gilliland et al. 1985; Santos et al. 2013).
Lactobacillus acidophilus NCFM has been reported to remove cholesterol both in
the presence of bile in the gut and in the absence of oxygen. Other Lactobacillus
acidophilus studies showed that the ability of the strain to remove cholesterol was
dependent on the strain. However, these in vitro studies do not prove that
Lactobacillus acidophilus NCFM can also reduce blood lipids in humans, and clini-
cal comparative experimental analysis of different strains of Lactobacillus acidoph-
ilus NCFM that can remove cholesterol and can not remove cholesterol can confirm
the role of Lactobacillus acidophilus NCFM in reducing blood lipids. To study the
effects of different dairy products on plasma cholesterol, Thompson et al. conducted
a nine-week human trial on 12 healthy volunteers who were fed Sweet Acidophilus
milk, a low-fat milk product registered as a trademark at the North Carolina Dairy
Agency, and 2 × 106 cfu Lactobacillus acidophilus NCFM per milliliter was added
(Thompson et al. 1982). The results showed that different dairy products, including
Sweet Acidophilus milk, did not affect serum cholesterol levels. Although no NCFM
intake was reported in this study, the amount of Lactobacillus acidophilus NCFM
obtained from Sweet Acidophilus milk intake was 2 × 108 cfu per day, suggesting
that the body may need to consume more NCFM to achieve lipid-lowering effects.
10.6.3.4 Remission Lactose Intolerance
Lactose intolerance is a common condition in most non northwestern European

ancestry. Milk is rich in protein and calcium, which can provide abundant nutrition
for human beings, but milk also contains more lactose, and lactose cannot be
absorbed by the intestine, it must be decomposed into monosaccharide to be
absorbed by the intestine and into the blood. Lactose is not well broken down into
monosaccharides if the body lacks it, and lactose enters the colon to produce acid
and gas, causing a series of symptoms of lactose intolerance (Misselwitz et al.
2013). Lactose intolerance can block the absorption of calcium and minerals in the
body, leading to osteoporosis, and studies have shown that lactose intolerance can
also lead to loss of appetite (Mattar et al. 2012; Usai-Satta et al. 2012). A phenom-
enon of lactose intolerance is the production of hydrogen after consumption of lac-
tose. Although Lactobacillus acidophilus NCFM does not always reduce hydrogen
production, it has been shown to reduce the symptoms of lactose intolerance in
children (Montes et al. 1995; Sohail et al. 2013). Lactose can cause intestinal dis-
comfort in certain people because it is fermented in the colon after intestinal diges-
tion. Fermented dairy products have been shown several times to improve the host’s
lactose tolerance compared with non-fermented and lactose-based foods, partly
because Lactobacillus can be a source of lactase in the small intestine, thereby help-
ing lactose-deficient people digest lactose (Mesrine et al. 2013; Patil and Ghetia
2014). Studies have shown that Streptococcus thermophilus and Lactobacillus bul-
garicus (non-enterogenous strains) in yogurt starters generally reduce hydrogen
production and lactose dyspepsia symptoms better than enterogenous Lactobacillus,
and some studies have found that probiotic Lactobacillus can improve symptoms of
sugar digestion and lactose intolerance in lactase-deficient people (Lin et al. 1998;
Yang et al. 2013). The ability of a strain to digest lactose in humans is not always
the amount of total lactase in the cell. This may be due to the difference in the ability
of different strains to utilize lactase in the small intestine (stability of lactase, trans-
port capacity of lactose, release capacity of lactose) or to differences in cell lysis
and activity analysis (Brown-Esters et al. 2012; Mustapha et al. 1997).
10.6.3.5 Anticancer
Many animal and human experiments have examined the effects of intestinal flora
on the host. The activity of some enzymes (such as azo reductase, beta-glucuronidase,
nitroreductase) in intestinal microorganisms is closely related to carcinogenesis
(Kumar et al. 2015). Intervention experiments in humans showed that azo reductase,
beta-glucuronidase and nitroreductase activities were decreased when the subjects
ate milk daily containing 109–1010 cfu Lactobacillus acidophilus NCFM (Goldin
et al. 1980). Lactobacillus acidophilus NCFM and antibiotics can reduce the inci-
dence of colon cancer in rats (Goldin and Gorbach 1984b). Goldin et al. studied the
effect of Lactobacillus acidophilus NCFM on free amine levels in feces of meat-fed
rats and found that the level of free amine in rats fed NCFM was significantly
reduced (Goldin and Gorbach, 1984a). A blind study (placebo-controlled) of the
effects of Lactobacillus acidophilus NCFM on colon abnormalities in mutagenic
rats showed that the inhibition rates of 2% and 4% Lactobacillus acidophilus NCFM
on colon cancer in rats were 29% and 39%, respectively. In conclusion, these results
support the positive role of Lactobacillus acidophilus NCFM in reducing intestinal
carcinogenesis.
10.6.3.6 Other Physiological Functions
Lactobacillus acidophilus NCFM significantly reduced the incidence of systemic

candidiasis and prolonged survival in mice with immunocompromised Candida
albicans infection (Matsubara et al. 2012; Wagner et al. 1998). In addition, prophy-
lactic feeding of Lactobacillus acidophilus NCFM in mice may help protect the
colon from proliferating due to Citrobacter (Varcoe et al. 2003). In addition, for
intestinal bacterial overgrowth, Lactobacillus acidophilus NCFM has been shown
to be beneficial in altering intestinal flora metabolism by reducing levels of dimeth-
ylamine and nitrodimethylamine (Dunn et al. 1998).
Lactobacillus acidophilus NCFM maintains the stability of human intestinal
flora in antibiotic therapy and binds to soluble fibers and glutamine, and has been
shown to improve antiretroviral therapy-related diarrhea (Engelbrektson et al.
2006). One of the latest discoveries of Lactobacillus acidophilus NCFM is that it
induces opioid and cannabinoid receptors in the gut of laboratory animals, thereby
alleviating bowel pain (Rousseaux et al. 2007). Alleviation of bowel pain may have
significant effects in some areas, such as irritable bowel syndrome and infantile
colic. Lactobacillus acidophilus NCFM has been shown to be effective in irritable
bowel syndrome animal models, but this requires further confirmation by human
experiments (Clarke et al. 2012).
In a recent large randomized controlled trial, taking a single strain of Lactobacillus
acidophilus NCFM or combining it with Bifidobacterium Bi-07 can reduce com-
mon respiratory infections, such as fever, cough and runny nose, in children in child
care centers (Ouwehand et al. 2008). The duration of these symptoms, absence from
work due to illness, and antibiotic use also decreased significantly within six months
of taking probiotics.
Lactobacillus acidophilus NCFM can regulate immune response. Combined
with lactosol, Lactobacillus acidophilus NCFM increased the levels of PGE2 and
spermidine in feces of elderly healthy volunteers, suggesting that it reduced intesti-
nal inflammation and enhanced cell protection (Ouwehand et al. 2009). Lactobacillus
acidophilus NCFM regulates antibody response to Candida albicans has been con-
firmed in mouse models. (Wagner et al. 2000). Recently, immunomodulatory effects
of Lactobacillus acidophilus NCFM on serum immunoglobulin have been found in
oral vaccines for healthy adults (Paineau et al. 2008). The enhanced immune
response in healthy volunteers may partly explain the preventive effect of
Lactobacillus acidophilus NCFM on common respiratory tract infections
(Ouwehand et al. 2008).
10.6.4 epresentative Products of Lactobacillus

R
acidophilus NCFM
10.6.4.1 Howaru Premium Probiotics
Howaru premium probiotics comes from DuPont Group, the main ingredient con-
tains Lactobacillus acidophilus NCFM. It has different formulations for different
groups of people. It adds super-strong bifidofactor fructooligosaccharides to infants
aged 0–6, which can effectively increase the intestinal bifidobacteria of infants and
provide multiple protection for Chinese professionals. Irregular and unhealthy eat-
ing habits add three distinct health ingredients: functional patented probiotics,
superbifidus, and effective ingredients patented to reduce fatty liver. In addition to
ensuring probiotics reach the gut to the greatest extent, they also protect the liver,
delay aging and other health benefits. For those with a long-term low number of
probiotics in the body, adding functional patented probiotics to the formula can
further increase the value of probiotics in the body to play a multiplier and stronger
health effect. For women with a need to maintain their figure and slow down aging,
add super bifidofactor fructooligosaccharides to help bifidobacteria. The rapid
growth of bacteria in a short time, as well as collagen, double play the role of skin
repair at the same time add natural betaine, not only can protect the liver, while
eliminating the body’s free radicals to maintain health, slow down aging.
10.6.4.2 Metagenics Super Lactobacillus Capsules
Metagenics super Lactobacillus capsules comes from a company Metagenics

founded in 1983. This capsule product can provide 1 billion and 500 million live
bacteria per capsules during the shelf life. The main ingredients of the product are
Lactobacillus acidophilus NCFM and Bifidobacterium lactis, which maintain intes-
tinal health. Immunoglobulin is added to promote intestinal environmental health.
10.7 Lactobacillus acidophilus LA-5

D
acidophilus LA-5
10.7.1.1 Discovery of Lactobacillus acidophilus LA-5
Lactobacillus acidophilus LA-5 was originally used in probiotic dairy products by

Chr. Hansen A/S company. It has been used in dietary supplements and fermented
dairy products all over the world with excellent clinical records. Lactobacillus aci-
dophilus LA-5 has the functions of regulating intestinal flora balance and treating
diarrhea caused by pathogenic bacteria and antibiotics. It can also improve human
immune regulation.
10.7.1.2 Development of Lactobacillus acidophilus LA-5
Lactobacillus acidophilus LA-5 has no adverse effects on the taste, appearance and
taste of the product. In addition, it survives in products for a long time, can be pre-
served until consumer consumption has been active, and has many probiotic func-
tions. Lactobacillus acidophilus LA-5 can survive in the stomach and small
intestine, depending on its strong tolerance to gastric acid, bile acid and digestive
enzymes (Ranadheera et al. 2012). Lactobacillus acidophilus LA-5 can adhere to
intestinal mucosa. It was successfully validated in fecal examination after oral
administration of Lactobacillus acidophilus LA-5 (Saarela et al. 2007). Lactobacillus
acidophilus can improve the intestinal flora and inhibit the growth of pathogenic
bacteria, thus improving the digestive and immune system of the human body and
enhancing its resistance. Diarrhea can expel probiotics from the intestine and
increase the risk of infection. However, Lactobacillus acidophilus can prevent intes-
tinal infection and diarrhea caused by eating unclean food. Studies have shown that
increasing the number of Lactobacillus acidophilus in the intestine can alleviate
intestinal allergies, prevent vaginal infections caused by Candida proliferation, and
reduce cholesterol. Lactobacillus acidophilus can also alleviate a variety of herpes
infections (van Baarlen et al. 2013).
10.7.2 Safety Study of Lactobacillus acidophilus LA-5
Using microbes as human probiotics is obviously a big problem. As part of the

intestinal flora of normal people, Lactobacillus is generally considered safe, and
millions of people have eaten Lactobacillus acidophilus LA-5 without any negative
reports.
10.7.3 Function Study of Lactobacillus acidophilus LA-5
10.7.3.1 Maintaining Intestinal Flora Balance
Lactic acid bacteria can produce organic acid, hydrogen peroxide, bacteriocin and
other antagonistic substances in the process of growth and metabolism, which can
effectively inhibit the growth and reproduction of harmful bacteria in human body,
alleviate gastrointestinal infection and diarrhea caused by antibiotics (Cizeikiene
et al. 2013). Lactic acid bacteria adhere to human intestinal tract and colonize to
form a biological barrier, which can inhibit the adhesion of pathogenic bacteria on
intestinal mucosa, inhibit their growth and reproduction by competing with patho-
genic bacteria for nutrients, and prevent the absorption of harmful substances.
Lactic acid bacteria exist naturally in the intestinal tract. The main end products of
glucose fermentation are lactic acid, acetic acid and hydrogen peroxide. The envi-
ronment in which these metabolites form is unfavorable to the growth of potential
pathogenic microorganisms. Lactic acid bacteria control the intestinal pH through
acid production, thus inhibiting the growth of many potential pathogens and spoil-
age bacteria. In vitro, Lactobacillus acidophilus LA-5 increased the amount of ace-
tic acid and propionic acid produced by colonic bacteria before a stable flora was
established, suggesting that Lactobacillus acidophilus LA-5 supplementation could
alter the fermentation process in the colon (Jiang and Savaiano 1995).
Lactobacillus acidophilus LA-5 can also produce bacteriocin CH5. CH5 not
only has a wide range of antimicrobial activities, but also inhibits some yeasts and
fungi (Plockova et al. 1997). The inhibition rate of Lactobacillus acidophilus LA-5
on Salmonella typhimurium reached 60%. The growth of Campylobacter jejuni
slowed down with the increase of the concentration of Lactobacillus acidophilus
LA-5 acellular fermentation extract (Ding et al. 2005; Salminen et al. 1996; Zeinhom
et al. 2012). In some intestinal inflammatory diseases, the disorder of intestinal flora
has great influence on it. After feeding Lactobacillus acidophilus LA-5 and
Bifidobacterium animal BB-12, the number of Bifidobacterium and Lactobacillus in
the intestine of patients undergoing ileal pouch-anal anastomosis increased, while
the number of stools per day decreased, and some gastrointestinal diseases also
improved (Laake et al. 2003). After feeding Lactobacillus acidophilus LA-5 and
Bifidobacterium animals BB-12, the number of liquid fecal days per week decreased
from 6 days to 1 day in patients with collagen colitis and diarrhea, and increased
from 4.5 days to 6 days in the placebo group (Wildt et al. 2006).
10.7.3.2 Treating Diarrhea
Diarrhea is the most common health problem when tourists go to areas with poor
sanitation. Acute diarrhea is mainly caused by eating water or food infected with
Escherichia coli, Salmonella, Shigella or Campylobacter. Some studies on the use
of probiotics to prevent diarrhea among travelers have emerged (Guarino et al.
2015). A study of tourists traveling to Egypt found that taking Lactobacillus aci-
dophilus LA-5 together with Bifidobacterium animalis BB-12, Streptococcus ther-
mophilus STY-31 and Lactobacillus bulgaricus LBY-27 significantly reduced the
incidence of diarrhea in the experimental group, and the diarrhea rate (43%) was
much lower than that in the placebo group (71%) (Black et al. 1989).
Antibiotic therapy can also lead to diarrhea, and many patients will develop diar-
rhea after anti infection treatment. Probiotics can play a role in a variety of gastro-
intestinal diseases, and have been proved to be effective in preventing and treating
diarrhea. During oral antibiotics, intestinal flora can be disrupted and diarrhea may
occur, thereby expanding the harmful effects of potential pathogenic bacteria in the
intestine. Helicobacter pylori triple therapy is a high dose of antibiotics, which
causes great damage to human body and many adverse effects. When antibiotic
therapy was supplemented with yogurt containing Lactobacillus acidophilus LA-5
and Bifidobacterium animalis BB-12, patients with Helicobacter pylori infection
experienced fewer vomiting, constipation and diarrhea (compared with the placebo
group of blank yogurt) (Sheu et al. 2002, 2006). In another study, six weeks after
taking yogurt containing Lactobacillus acidophilus LA-5 and Bifidobacterium ani-
malis BB-12, Helicobacter pylori levels in antrum and gastric inflammation were
reduced and improved (Wang et al. 2004). Other studies have shown that ampicillin
or clindamycin combined with Lactobacillus acidophilus LA-5 and Bifidobacterium
animals BB-12 can normalize intestinal flora faster than placebo (Black et al. 1991;
Nord et al. 1997).
10.7.3.3 Improving Immune Regulation
Lactobacillus acidophilus plays an important role in improving immune response.

Evidence shows that Lactobacillus acidophilus plays an important role in intestinal
immune regulation in humans and animals. The gastrointestinal system has specific
substances that react to antigens from diet, thus causing immune response. These
substances constitute the intestinal immune system. They are lymphoid tissues
(B-cells, T-cells, macrophages and appendages) that contain the immune cells nec-
essary for induction of immune responses. Intestinal-associated lymphoid tissue is
the largest organ in the immune system. The health function of many probiotics may
be related to the regulation of immune function. Lactobacillus acidophilus LA-5 is
associated with non-specific induction of cytokine production and phagocytic activ-
ity, as well as with specific stimulation such as antibody production.
The acellular extract of Lactobacillus acidophilus LA-5 was added to cultured
mouse macrophage-like cells in vitro. It was found that the acellular extract of
Lactobacillus acidophilus LA-5 enhanced the phagocytosis of inert particles or
Salmonella (Hatcher and Lambrecht 1993). Lactobacillus acidophilus LA-5 can
also induce immune response and cytokine signaling compounds. The ability of 10
strains of Lactobacillus to induce the release of three cytokines was compared
in vitro. The three cytokines were tumor necrosis factor-alpha (TNF-alpha), inter-
leukin-6 (IL-6) and interleukin-10 (IL-10). The results showed that Lactobacillus
acidophilus LA-5 was one of the four strains with the best effect of inducing TNF-
alpha, and its inducing release of TNF-alpha. The volume was significantly higher
than that of the blank group (Miettinen et al. 1996). In one study, mice were fed
regular yogurt, supplemented with Lactobacillus acidophilus LA-5 and
Bifidobacterium animals BB-12 yogurt, or non-fermented milk, and then orally
inoculated with cholera toxin to mimic intestinal infection. The results showed that
the specific antibody concentration of cholera toxin in feces and serum of mice fed
with Lactobacillus acidophilus LA-5 and Bifidobacterium bifidum BB-12 was
higher than that of mice fed with conventional yogurt and non-fermented milk
(Tejada-Simon et al. 1999).
10.7.3.4 Other Physiological Functions
Constipation is a common disease, which usually occurs in all ages, especially in

the elderly. One of the main causes of constipation is the lack of intestinal probiot-
ics. In this case, the putrefaction bacteria in the intestine will produce a large num-
ber of enterotoxins and harmful substances, causing serious damage and pollution
to the intestine, slowing down intestinal peristalsis, and increasing intestinal pH,
leading to intestinal dysfunction. Probiotics can inhibit the growth of harmful bac-
teria, but also decompose harmful substances in the intestine. In addition, they can
also produce organic acids to reduce intestinal pH and promote intestinal peristalsis,
thereby improving constipation. Constipation is common in developed countries,
while Lactobacillus supplementation may improve natural defecation time. Elderly
patients with chronic constipation were randomly given unfermented milk or milk
supplemented with probiotic Lactobacillus acidophilus LA-5 and Bifidobacterium
animals BB-12. It was found that the frequency of intestinal motility was signifi-
cantly improved in patients with probiotic group, and no adverse effects were
reported.(Alm et al. 1993).
Some probiotics have been proven to improve lactose digestion by releasing beta
galactosidase (Gobinath and Prapulla 2014). Although studies have shown that lac-
tose intolerance symptoms such as abdominal pain, abdominal distension, diarrhea
and constipation in Lactobacillus acidophilus LA-5, Bifidobacterium animalis
BB-12, Streptococcus thermophilus STY-31 and Lactobacillus bulgaricus LBY-27
have not been significantly improved (Hove et al. 1994). However, the addition of
Lactobacillus acidophilus LA-5 to milk significantly reduced the expiratory hydro-
gen value in lactose intolerant subjects, suggesting that Lactobacillus acidophilus
LA-5 can improve the degradation of lactose (Lin et al. 1991). In vitro studies also
found that Lactobacillus acidophilus LA-5 added to the medium decreased lactose
concentration and increased the activity of beta-galactosidase, which also showed
that Lactobacillus acidophilus LA-5 could improve lactose fermentation (Jiang and
Savaiano 1997).
The effects of Lactobacillus acidophilus LA-5 on human health include inhibit-
ing the growth of breast cancer cells (Biffi et al. 1997). In addition, feeding
Lactobacillus acidophilus LA-5 and Bifidobacterium animalis BB-12 during or
after chemotherapy significantly reduced the frequency of fever symptoms in

patients with acute leukemia, with an average of 8 days in the placebo group and
12 days in the probiotics group (Ellegaard et al. 1992). In addition, Lactobacillus
acidophilus LA-5 has been found to reduce cholesterol in humans, rodents and
in vitro, thereby improving lipid metabolism (El-Gawad et al. 2005; Remagni et al.
2013).
10.7.4 epresentative Products of Lactobacillus

R
acidophilus LA-5
10.7.4.1 Enjone Original Power Probiotics
The main ingredients of Enjone original power probiotics are Lactobacillus aci-
dophilus LA-5 and Bifidobacterium animal BB-12. At the same time, sufficient
fructooligosaccharides and dietary fibers are added to facilitate the colonization of
probiotics in the human intestine. Junle Force is a compound probiotic product. The
probiotics in this product can not only promote the digestion and absorption of food,
but also promote the synthesis of vitamins, amino acids and other active substances
in human body. Lactobacillus acidophilus LA-5 and Bifidobacterium animalis
BB-12, the probiotic bacteria contained in this product, can participate in and pro-
mote the synthesis of nutrients in human body when they lack some nutrients in
human body, so as to achieve a balanced state of nutrition. The product can also
inhibit and eliminate toxins in the body. Lactobacillus acidophilus LA-5 contained
in this product can compete with harmful bacteria for space and resources and
inhibit the growth of harmful bacteria.
10.7.4.2 Hemei Qingyi Suit
Hemei qingyi suit is an international standard “probiotic product” made from

Danish CHR-HANSEN company and Dutch SENSUS company. It is known as
“microecological agent” abroad. Its main components are Bifidobacterium animalis
BB-12, Lactobacillus acidophilus LA-5 and konjac powder. The product can regu-
late the balance of human intestinal tract, promote human body to synthesize a
variety of nutrient active substances, thereby improving human immunity. It can
also alleviate acute and chronic diarrhea, habitual constipation, colitis and indiges-
tion caused by intestinal flora disorders.
10.8 Bifidobacterium lactis HN019
10.8.1 iscovery and Development of Bifidobacterium lactis

D
HN019
10.8.1.1 Discovery of Bifidobacterium lactis HN019
Bifidobacterium lactis HN019 was isolated by the New Zealand Dairy Research
Institute Strain Preservation Center in view of its immunoregulatory and acid and
bile tolerance characteristics (Prasad et al. 1998). This strain is marketed by Danisco
as HOWARU bifidobacteria and New Zealand Fonterra as Bifidobacterium lactis
DR10 (Gopal et al. 2005).
10.8.1.2 Development of Bifidobacterium lactis HN019
In vitro studies have shown that Bifidobacterium lactis HN019 has the necessary
prerequisites for survival in the gastrointestinal tract-bile resistance, low pH and
digestive enzymes (Chen et al. 2012; Prasad et al. 1998). In addition, the strain has
been shown to adhere to human epithelial cells (Gopal et al. 2001a). Bifidobacterium
lactis HN019 has been found to have good stability in dietary supplements, dairy
products, non-dairy products, and to provide a good flavor for fermented products
(Ahmed et al. 2007). Moreover, the strain is capable of utilizing galactooligosac-
charide, which provides a choice for the development of synbiotic functional foods
(Gopal et al. 2001b). From the study, it was found that Bifidobacterium lactis
HN019 has a wide range of effects in immune health (Sanders 2006). In the Article
13 program of the European Union, the immune health and natural defense efficacy
of this strain has been submitted.
10.8.2 Safety Study of Bifidobacterium lactis HN019
The safety study of Bifidobacterium lactis HN019 has been matured, and the clini-
cal trial did not find that the strain contains antibiotic resistance gene (Zhou et al.
2005). This strain, like other probiotics such as Lactobacillus rhamnosus, is essen-
tially vancomycin-resistant and sensitive to some clinically effective antibiotics
(Delgado et al. 2005). And the toxicity test did not find any negative effects. The
LD50 of this strain was over 50 g/kg/day in mice, while the acceptable daily intake
for 70 kg was 35 g of dried bacteria (Zhou et al. 2000b). This strain does not degrade
intestinal mucus and therefore does not affect the intestinal mucosal barrier (Shu
et al. 1999). The safety of HN019 strain has also been shown by other toxicity tests
(Zhou et al. 2000a). In addition, no negative effects were found in the human studies
of Bifidobacterium lactis HN019, and the history of commercial use also indicates
the safety of the strain.
10.8.3 Functional Study of Bifidobacterium lactis HN019
10.8.3.1 Regulate Intestinal Flora
There are various microorganisms in the human intestine, which we call the intesti-
nal flora. Under normal circumstances, the intestinal flora forms an ecological bal-
ance in each species and quantity, but when the intestinal environment changes in
the human body, such as long-term use of antibiotics, this will lead to inhibition of
some antibiotic-sensitive strains. To increase the number of other antibiotic-resistant
microorganisms, causing intestinal flora imbalance. Bifidobacterium lactis HN019
has been shown to increase the levels of bifidobacteria and lactobacilli in human
feces, which is beneficial to intestinal health, while taking this activity does not alter
other flora and metabolic activities (Ahmed et al. 2007; Gopal et al. 2003).
10.8.3.2 Regulate the Immune System
Bifidobacterium lactis HN019 has the function of regulating the immune system,
and a large number of animal and human experiments have demonstrated this func-
tion. Studies have focused on immunodeficient middle-aged and elderly people for
human models, in which non-specific (natural) immune responses have been
improved. This immune response does not need to be induced and is always “pre-
pared for action,” providing the first line of defense against infection. Bifidobacterium
lactis HN019 has been shown to enhance the cytotoxic activity of natural killer cells
and the phagocytic activity of peripheral blood mononuclear cells. This improve-
ment is particularly clear in subjects with poor initial immune response. However,
although Bifidobacterium lactis HN019 can enhance the phagocytic activity of
phagocytic cells after 6 weeks of cessation of administration, like the usual probi-
otic strains, the improvement of the non-specific immune response disappears after
stopping the administration of the probiotics. Although Bifidobacterium lactis
HN019 increases the non-specific immune response, this does not occur arbitrarily.
Animal studies have shown that Bifidobacterium lactis HN019 does not have any
risk of an inflammatory response.
Bifidobacterium lactis HN019 maintains intestinal balance by activating immune
responses in animal and human intestinal epithelial cells. Studies have shown that
Bifidobacterium lactis HN019 can contact the intestinal epithelial cells INT-407 and
enter the interior of the cells after 6 h of action. Transcriptional analysis indicated
that the intestinal epithelial cells INT-407 were able to express the cytokines IL-1β,
IL-8, TNF-α, and TGF-β1. As a mediator of inflammatory response, IL-1β, IL-8,
and TNF-α are pro-inflammatory factors. IL-1β can induce the expression of
immune-related genes, activate pro-inflammatory cytokines of lymphocytes, pro-

mote the expression of leukocyte adhesion molecules, and induce inflammatory
cells to enter the intestinal lesions, causing intestinal tissue damage. Since the tran-
scription level of IL-1β cytokines is positively correlated with the inflammatory
degree of inflammatory bowel disease, it is often used clinically as an indicator for
judging the severity and therapeutic effect of inflammation (Gan et al. 2002). IL-8
is often produced in leukocytes, macrophages, and T lymphocytes, which promote
acute cellular inflammatory responses and produce certain signals that activate the
cellular mucosal immune response early in microbial invasion. To date, we believe
that IL-1β-, IL-6-, and TNF-α-induced inflammations are mainly caused by the
induction of IL-8 cytokines, and its transcription level can be used as an important
indicator for judging the degree of inflammatory bowel disease (Gan et al. 2002).
TNF-α is a cytokine in innate immunity, involved in both natural and acquired
immunity, and is capable of inducing an inflammatory response. The study found
that compared with the control group, the live bacteria of Bifidobacterium lactis
HN019 did not induce IL-lβ and IL-8, but only a short-term upregulation of TNF-α,
heat-activated Bifidobacterium lactis HN019. The expression level of IL-8 was only
slightly upregulated, but the expression levels of IL-1β, IL-8, and TNF-α were sig-
nificantly regulated by Salmonella typhimurium, and the stimulating effect of
Salmonella typhimurium on the expression level of TNF-α was strong. Live bacteria
in Bifidobacterium lactis HN019. The results showed that the live and dead bacteria
of Bifidobacterium lactis HN019 had no obvious stimulating effect on intestinal
epithelial cells and did not cause inflammatory reaction. Therefore, it is concluded
that intestinal cells may recognize and tolerate milk double by some means.
Bifidobacterium HN019, so Bifidobacterium lactis HN019 can be used in people
with chronic inflammatory diseases (Liu 2009).
When Bifidobacterium lactis HN019 and Salmonella typhimurium acted together
on INT-407, a small upregulation of IL-1β showed a significant upregulation of
TNF-α expression, but the amplitude was still much less than the individual stimu-
lation of Salmonella typhimurium, indicating Bifidobacterium lactis HN019 is able
to alleviate the pre-inflammatory response induced by Salmonella typhimurium.
When the two strains work together, both strains can continue to grow in the culture
supernatant, but the growth rate of Salmonella typhimurium is significantly higher
than that of Bifidobacterium lactis HN019, which also leads to the bacterial count
of Salmonella typhimurium. The time was gradually higher than that of
Bifidobacterium lactis HN019, and its effect was stronger than that of Bifidobacterium
lactis HN019, indicating that Bifidobacterium lactis HN019 could not completely
eliminate the inflammatory reaction caused by Salmonella typhimurium. In the
interaction group, Bifidobacterium lactis HN019 also significantly reduced the
expression of pro-inflammatory cytokines induced by Salmonella typhimurium, and
the response of cells to Salmonella typhimurium was also reduced after pretreat-
ment with Bifidobacterium lactis HN019. No longer induce or induce only low
levels of proinflammatory cytokine expression (Liu 2009). These findings indicate
that Bifidobacterium lactis HN019 can indirectly regulate the immune response of
intestinal epithelial cells to Salmonella typhimurium and reduce its inflammatory
response, thereby maintaining intestinal balance. At the protein level, both live and
dead bacteria of Bifidobacterium lactis HN019 can reduce IL-8 production. Both
the lipopolysaccharide (LPS) group and the Bifidobacterium lactis HN019 + lipo-
polysaccharide (BLPS) group increased the secretion of IL-8, and the interaction
between LPS and Bifidobacterium lactis HN019 significantly reduced IL-8 produc-
tion. These protein level analysis results were basically consistent with the tran-
script level results, which also confirmed the anti-inflammatory activity possessed
by Bifidobacterium lactis HN019 from the protein level. IL-8 is closely related to
the occurrence of inflammation. Intestinal pathogens are associated with the patho-
genesis of inflammatory bowel disease (IBD), and HN019 can reduce the produc-
tion of IL-8, which is HN019 in IBD and other inflammatory diseases. The
application provides a theoretical basis. However, it is unclear what mechanism of
Bifidobacterium lactis HN019 downregulates inflammatory factors. However, stud-
ies have found that lipoteichoic acid (LTA) of Lactobacillus can inhibit the secretion
of IL-8 by LPS, and studies have found that LTA of Lactobacillus plantarum is
related to immunity and anti-inflammatory ability (Grangette et al. 2005). However,
it is not clear which mechanism exerts anti-inflammatory effects on bifidobacteria,
and its mechanism needs further study.
10.8.3.3 Relieve Disease Risk
Many harmful bacteria that cause inflammation are colonized in the intestine, but
there are also many beneficial bacteria with anti-inflammatory activity in the intes-
tinal tract. These beneficial bacteria can inhibit inflammation and protect the intes-
tinal tract from inflammation. Even supplementation of probiotics with
anti-inflammatory activity in the intestine where inflammatory lesions have occurred
can alleviate inflammation. In the protection or treatment of diseases by probiotics,
since humans are not exposed to pathogens, the efficacy of probiotics is usually
studied using animals. This method is also applied to the study of Bifidobacterium
lactis HN019. One group of mice was fed with Bifidobacterium lactis HN019 for
1 week, while the control group did not, and these mice were subsequently infected
with Salmonella typhimurium. After 3 weeks, only 7% of the control group sur-
vived, while 80% of the mice fed with Bifidobacterium lactis HN019 were still alive
(Shu et al. 2000). Other studies have confirmed that Bifidobacterium lactis HN019
has activity in relieving gastrointestinal diseases (Shu et al. 2001). A double-blind,
placebo-controlled study involving more than 600 children between the ages of 1
and 3 showed that the use of Bifidobacterium lactis HN019 reduced the incidence
and prevalence of dysentery compared with galactooligosaccharides. In addition,
taking the Bifidobacterium lactis HN019 group also reduced the number of days of
severe symptoms or fever and the incidence of middle ear infections. And the level
and growth of iron are also significantly improved compared with the control group
(Sazawal et al. 2004).
Toll-like receptors (TLR)2 and 4 play a major role in bacterial infection and are
often selected as subjects of the study. TLR2 mainly recognizes peptidoglycans of
Gram-positive bacteria and lipoproteins of mycobacteria. TLR4 mainly recognizes

LPS of Gram-negative bacteria. Previous reports indicate that intestinal epithelial
cells can only express low levels of TLR4 and cannot express TLR4. Synergistic
molecular myeloid differentiation protein-2 (MD-2) (Abreu et al. 2001). Studies
have found that small intestinal epithelial cells INT-407 cannot detect TLR expres-
sion at the transcriptional level without bacterial stimulation, but can express TLR4
and MD-2. Both Bifidobacterium lactis HN019 and Salmonella typhimurium upreg-
ulated the expression of TLR2 gene, but the upregulation of Bifidobacterium lactis
HN019 was much smaller than that of Salmonella typhimurium. Although the inter-
action group of the two strains was able to transiently upregulate TLR2, the magni-
tude was smaller than that of the single stimulation group. Salmonella typhimurium
contains four components that activate TLRs: LPS, bacterial lipoprotein, flagellin,
and CpG DNA, which activate TLR4, TLR2, TLR5, and TLR9, respectively (Gerold
et al. 2007). After the mouse was infected with Salmonella typhimurium, the TLR2
molecule of the mouse spleen cells was upregulated, while in the intestinal epithe-
lial cells, TLR2 was also upregulated by LPS (Arce et al. 2010; Tötemeyer et al.
2003). At the protein level, inactivated Salmonella typhimurium and LPS-stimulated
cells do not detect TLR2 expression, which may be due to a negative feedback inhi-
bition of the cells themselves or may be due to Salmonella typhimurium live and
dead bacteria and LPS. There is a difference in function, and no change is seen at
the protein level. At the transcriptional level, the live and dead bacteria of
Bifidobacterium lactis HN019 can upregulate the expression of TLR4, while at the
protein level, in addition to the live and dead bacteria of Bifidobacterium lactis
HN019, LPS group, BLPS group, and LPS. The pretreatment group also upregu-
lated the expression of TLR4, and LPS, BLPS, and LPS pretreatment groups all had
LPS involvement. Therefore, LPS can induce TLR4 receptor expression, while
Bifidobacterium lactis HN019 acts as a Gram-positive bacterium, which can also
upregulate the expression of TLR4, and Bifidobacterium lactis HN019 can increase
INT-407 cells to Gram by upregulating TLR4. Negative bacteria sensitivity (Liu
2009).
10.8.4 epresentative Products of Bifidobacterium lactis

R
HN019
10.8.4.1 Changmin Probiotic Powder
The ratio of Bifidobacterium lactis HN019 to Lactobacillus rhamnosus HN001 in

Changmin probiotic powder is 2:1. Changmin probiotic powder is antiallergic and
can improve eczema and allergic rhinitis. Especially for infants and young children,
taking Changmin probiotic powder can prevent allergies for the baby. The powder
can mainly reduce the stimulation of allergens on infants and young children and
can improve the tolerance of infants and young children to allergens.
10.8.4.2 Douglas iFlora Multiple Probiotics
iFlora’s various probiotics contain 16 potent strains, including Bifidobacterium lac-

tis HN019, which has an improved effect on the gastrointestinal tract and possesses
immune function. It also contains prebiotics that work synergistically with probiot-
ics to help the human body get better from many diseases and maintain the health of
the gut flora. Among them, Bifidobacterium lactis HN019 is a characteristic strain
of various probiotics of iFlora. Clinical studies have shown that this characteristic
strain can enhance the immune system of adults and the elderly, and pregnant
women can also provide immune support for the fetus.
10.9 Lactobacillus plantarum ST-III

D
plantarum ST-III
10.9.1.1 Discovery of Lactobacillus plantarum ST-III
Lactobacillus plantarum ST-III is a probiotic strain obtained from the traditional

Chinese kimchi screening of cholesterol-lowering strains by the Food Biotechnology
Center of Jiangnan University in 2000. The kimchi raw materials used are from the
farmer’s pickled clams in Tianpingshan, Suzhou, and Jiangsu (Zhang et al. 2002).
Lactobacillus plantarum ST-III is a kind of Lactobacillus, which is a Gram-positive
bacterium, anaerobic or facultative anaerobic, and the strain has a straight or curved
rod shape, and in addition to the beneficial effects of common Lactobacillus, it can
be used to regulate blood lipids.
10.9.1.2 Development of Lactobacillus plantarum ST-III
Lactobacillus plantarum has a very close relationship with human life, and it can
survive in a variety of stressful environments. Lactobacillus plantarum is generally
found in the late stages of the fermentation process, mainly due to the high acid
resistance of this strain. Among fermented foods such as yogurt, fermented sausage,
bread, and kimchi, Lactobacillus plantarum is often used to improve the classifica-
tion of foods, improve the nutritional status of foods, and extend the shelf life of
foods. Lactobacillus plantarum is also widely used in aquaculture, and is the most
common silage-fermented lactic acid bacteria, which is used as an additive for
microecological feed. In addition, Lactobacillus plantarum is also used as a bio-
preservative in the food industry. Nowadays, Lactobacillus plantarum ST-III has
been put into commercial production by Bright Dairy Co., Ltd. The extensive
research of Lactobacillus plantarum ST-III also provides theoretical basis and tech-
nical support for its industrial applications. The results showed that Lactobacillus
plantarum ST-II has the effect of inhibiting the growth of pathogenic bacteria and
regulating the intestinal flora. In addition, ST-III can form a protective barrier in the
intestine to prevent the intestinal tract from being attacked by harmful bacteria and
harmful substances. At the same time, the strain can also improve the body’s immu-
nity and reduce the body’s cholesterol levels. Lactobacillus plantarum ST-III has
obtained a number of invention patents, and Lactobacillus plantarum ST-III is more
tolerant to the gastrointestinal environment and can be colonized in the body, which
provides a guarantee for its probiotic effect in the intestine. In 2008, the plant
Lactobacillus ST-III space breeding experiment has been carried out by the shen-
zhou VII, the strains will also carry the goddess of the moon in 2017 No. 5 in
380000 km away from the earth, the use of space microgravity environment again
for breeding, the bacterial strains of multiple space breeding for Chinese dairy
industry is pioneering work, which will promote the vigorous development of the
industry.
10.9.2 Study on the Safety of Lactobacillus plantarum ST-III
Lactobacillus plantarum ST-III is isolated from healthy foods (traditional kimchi),

and the Lactobacillus plantarum is also included in the “List of Species for Foods”
published by the Ministry of Health in 2010, and this strain is industrialized in the
production and sales process of Bright Dairy. There were no adverse reactions
reported, which indicated that Lactobacillus plantarum ST-III possessed food-grade
safety.
10.9.3 Functional Study of Lactobacillus plantarum ST-III

10.9.3.1 Cholesterol Lowering
Cardiovascular disease is a type of disease associated with the circulatory system

(heart, blood vessels, etc.), which can be classified into acute cardiovascular disease
and chronic cardiovascular disease, and is usually closely related to arteriosclerosis.
In recent decades, the prevalence of cardiovascular disease and its mortality rate
have also increased year by year, and it has become one of the most important dis-
eases of human death. Hyperlipidemia refers to a cholesterol content or a triglycer-
ide content in the blood exceeding a limited value, which is a risk factor for
cardiovascular disease. But if only the blood cholesterol level exceeds the standard,
we will call the condition hypercholesterolemia. So far, there are many drugs for
cholesterol lowering on the market, but humans still hope to achieve cholesterol-
lowering effects through non-pharmacological treatment. It has been reported that
some probiotics can lower the body’s cholesterol content and have no side effects on
the human body, and Lactobacillus plantarum ST-III is a probiotic that has a
cholesterol-lowering function verified by in vitro tests and in vivo experiments. In

vitro studies have found that Lactobacillus plantarum reduces cholesterol levels by
synergistic action of assimilation and precipitation, and the assimilation plays a
dominant role, which can reduce cholesterol content by 76.38 μg/mL (25.5%), and
precipitation is auxiliary, which can reduce 44.62 μg/mL (14.85%) of cholesterol.
The study also found that the effect of precipitation was closely related to the pH
value. When the pH was 6.0, the amount of cholesterol was reduced by 29 μg/mL,
and when the pH was not controlled, the cholesterol was reduced by 41 μg/
mL. These results indicated that under low pH conditions, the precipitation effect of
Lactobacillus plantarum ST-III strain is more significant (Juan Liu et al. 2008).
With hypercholesterolemia rats for animal models in research on plant Lactobacillus
ST-III three different doses’ (21 mL/kg bw, 42 mL/kg bw and 125 mL/kg bw) active
influence on blood lipid levels of rats, the results found that three doses of plant
Lactobacillus ST-III activity milk can significantly reduce the cholesterol content in
serum of rats, but have no significant effect on lower triglycerides (Sun et al. 2005).
Therefore, ST-III does have the function of lowering blood lipid, especially for
hypercholesterolemia.
The addition of Lactobacillus plantarum to yogurt significantly reduced the level
of cholesterol in yogurt, which was about onefold lower than that of yogurt fer-
mented only by S. thermophilus and Lactobacillus bulgaricus. Coronary heart dis-
ease and atherosclerosis are directly related to serum total cholesterol, while
Lactobacillus plantarum has a cholesterol-lowering effect and therefore has an
adjuvant therapeutic effect on these diseases. Liu Wei et al. found that the applica-
tion of the selected Lactobacillus plantarum ST-III fermented yogurt decreased its
cholesterol content to a building, compared to 0.3 mg/g in raw milk and 0.2 mg/g in
traditional yogurt. Lactobacillus yogurt has a cholesterol content of only 0.122 mg/g
(Yanyan Liu et al. 2011).
10.9.3.2 Inhibition of Intestinal Pathogens
Lactic acid bacteria have inhibitory effects on spoilage bacteria and pathogenic
microorganisms, and their bacteriostatic action may depend on metabolites such as
organic acids, hydrogen peroxide, diacetyl, and bacteriocin, some of which can only
inhibit one of the same genera. Bacteria, while some bacteriocins can inhibit Gram-
positive bacteria extensively (Cizeikiene et al. 2013; Gálvez et al. 2014; Schillinger
2014).The results showed that the fermentation products of Lactobacillus planta-
rum ST-III could inhibit Staphylococcus aureus and Salmonella enteritidis, and the
strain had higher antibacterial activity against Staphylococcus aureus than S. enter-
itidis. It was found that the antibacterial efficiency of the strain increased with the
prolongation of the action time. The fermentation broth was treated with catalase,
trypsin, and proteinase K, and the bacteriostatic activity was analyzed in compari-
son with the blank MRS medium. The results showed that the bacteriostasis of
hydrogen peroxide in the fermentation product was eliminated. The bacteriostatic
component contains a protein product. The pH value of the blank MRS was also
adjusted to the same as that of the fermentation product by organic acids such as
lactic acid, and it was found that there was no significant difference in the antibacte-
rial activity, thereby eliminating the antibacterial effect of the organic acid on the
two indicator bacteria and confirming the production of the strain bacteriocin (Ji
et al. 2013). The bacteriocin produced by Lactobacillus plantarum ST-III is stable
in acidity and weak acidity and can maintain high antibacterial activity, but as the
pH value increases, its antibacterial efficiency will have a downward trend, and
when the pH rises by 7, the bacteriocin activity was almost zero. In addition to the
pH requirements, the bacteriostatic effect of the bacteriocin on the two strains is
also affected by the concentration. When the concentration drops to 1/4, the bacte-
riostatic effect is still significant, but when it drops to 1/8 concentration, the antibac-
terial effect is no longer obvious. Staphylococcus aureus is a representative strain of
Gram-positive bacteria, and Salmonella enteritidis is a representative strain of
Gram-negative bacteria, and Lactobacillus plantarum ST-III has significant inhibi-
tory effects on both representative strains. Therefore, it can be inferred that the
bacteriocin produced by Lactobacillus plantarum ST-III should be a class of bacte-
riocins capable of inhibiting the growth of a wide range of strains. The study on the
fermentation of supernatant of Lactobacillus plantarum ST-III showed that it also
had a certain inhibitory effect on Staphylococcus aureus and Salmonella enteritidis.
Nowadays, Lactobacillus plantarum ST-III has been approved for use in foods. If it
can utilize the antibacterial ability of the bacteria, it can also improve the shelf life
of the food to which the bacteria are added, improve the method of sterilization of
the food, and reduce food nutrition. Although the bacteriostatic action of bacteriocin
produced by Lactobacillus plantarum ST-III has been confirmed, if there is a need
to apply its characteristics to the production and processing of food, there are still
many studies that need to be continued. First, heat treatment is a sterilization method
commonly used in food processing, and therefore the thermal stability of the
Lactobacillus plantarum ST-III metabolite needs further study. In addition, for the
bacteriocin produced by Lactobacillus plantarum ST-III, its physicochemical prop-
erties and molecular structure still need to be further isolated and purified to study
its antibacterial mechanism, thereby improving its application prospect in food.
10.9.3.3 Regulate Intestinal Flora
A wide variety of microorganisms form a more complex ecosystem in the gut, and
the system is closely related to the growth and metabolism of the host. When the
host is healthy, the intestinal flora has usually a stable and balanced system.
However, when the physiological environment of the intestinal flora is subjected to
some stress, it will cause disorder of the host intestinal microflora, resulting in a
large number of pathogenic bacteria. Breeding, thus endangering the health of the
host. Studies have found that the addition of probiotics can regulate the intestinal
flora and promote the health of the human body. This is because the metabolites
produced by the probiotics have an antagonistic effect on the pathogenic bacteria,
and the competitive rejection caused by the probiotics colonizing the intestinal
mucosa can also form a protective barrier for the intestine (Blaut 2002). Animal
experiments showed that Lactobacillus plantarum ST-III has a certain regulatory
effect on the intestinal flora of mice, which can selectively increase the number of
probiotics such as lactobacilli in the intestine and inhibit pathogenic bacteria such
as gas production. The growth of Clostridium perfringens regulates floral balance.
The study found that the effective dose of Lactobacillus plantarum ST-III to regu-
late the intestinal flora of mice is 6 × 08 cfu/mL-1. If the concentration of the bacte-
ria is continuously increased, the bacterial population in the stool samples of mice
is not significant. The mice were tested for the efficacy of Lactobacillus plantarum
ST-III gavage. The results showed that the control effect of the bacteria still existed
after stopping the gavage for 3–5 days, but 7 days after stopping the gavage, various
intestines of the mice. The number of indicator bacteria returned to the level before
the gavage. Lactobacillus plantarum ST-III can inhibit the growth and reproduction
of pathogenic bacteria in the intestine of the tested mice, increase the number of
probiotics, and improve the intestinal bacteria growth by continuously inhaling
6 × 108 cfu/mL live bacteria for 15 days. The efficacy of the group can continue for
3–5 days after stopping the gavage (Xu et al. 2006). Therefore, in view of the inher-
ent cholesterol-lowering function of Lactobacillus plantarum ST-III, combined
with its regulation of the intestinal flora, the application of this product to foods,
especially dairy products with special functions, has a bright future.
10.9.3.4 Lose Weight
With the improvement of people’s living standards and the improvement of living
conditions, overweight and obesity are increasing, and the age of onset is getting
smaller and smaller, often accompanied by metabolic diseases such as hypertension
and diabetes, which seriously threaten human health. Simple obesity is mainly
caused by genetics or overnutrition. Such patients have no symptoms of metabolic
disorders and often have a genetic history of family obesity. The survey report
shows that the proportion of simple obesity among adolescents in China is very
high. A physical examination at a school in Beijing found that 27.8% of the youth
in the school were overweight. The mental and psychological stress of adolescents
with obesity is even greater than the disease itself, which causes them to feel infe-
rior and affect their personal development and interpersonal relationships. From the
research report, it is found that many weight loss methods used by adolescents, such
as diet pills, dieting, etc., have a serious impact on their growth and development,
which is not conducive to their healthy growth.
Eating diet pills and dieting have seriously affected the growth of adolescents’
body and are not conducive to the development of their various tissues and organs.
In addition, severe weight changes in a short period of time can cause metabolic
disorders in the human body. A large number of experimental reports indicate that
adolescent obesity is a chronic disease caused by hereditary or bad habits, and
weight control has become an important means of preventing obesity, hypertension,
and diabetes, and it has received more and more attention. On the one hand, many
young people have become popular for weight loss in their perfect body, but the
common methods of weight control and weight loss often use some drugs, and their
side effects are very large. As a probiotic, Lactobacillus plantarum ST-III has a
weight loss effect and has no side effects on the human body. Animal experiments
found that rats taking ST-III had significantly reduced body weight compared with
the blank group, which had the function of assisting weight loss.
10.9.4 epresentative Products of Lactobacillus plantarum

R
ST-III
10.9.4.1 Bright “Changyou” Series of Fermented Milk
The most important component of the bright “Changyou” series of fermented milk
is Lactobacillus plantarum ST-III. With this excellent strain, Bright Dairy is also
praised as the “first brand of unobstructed yogurt.” After 2012, the formula of
“Changyou” was upgraded to “Lactobacillus plantarum ST-III.” In 2013, Changyou
launched “zero fat.” Zero fat and low calories are better for our health. The impor-
tant probiotic Lactobacillus plantarum ST-III regulates the flora, lowers cholesterol,
and loses weight in the Changyou series. Compared with other lactic acid bacteria
products, this product can be used as a weight loss auxiliary product without side
effects.
10.9.4.2 right “Plant Vitality” Series of Active Lactic Acid Bacteria

B
Milk Drinks
Bright “Plant Vitality” series of active lactic acid bacteria milk beverages are lactic
acid bacteria beverages that are dependent on Lactobacillus plantarum ST-III. The
applicable population is young workers or students. Due to the pressure of their
work and school, plus day and night, irregular diet lifestyle habits are often plagued
by intestinal health problems. This product features zero fat without burden and can
be used as an auxiliary product for weight loss.
10.10 Lactobacillus casei Zhang
10.10.1 The Discovery and Development of L. casei Zhang
Lactobacillus casei Zhang (L. casei Zhang) is a Lactobacillus strain isolated from
Zhenglan Flag, Xilin Gol League, Inner Mongolia by Professor Zhang Heping’s
research team from Laboratory of Dairy Biotechnology and Engineering, Inner
Mongolia Agricultural University (YueYing et al. 2006). 16S rDNA homology anal-
ysis showed that the strain had 100% homology with the standard strain Lactobacillus
casei ATCC334 in GenBank. L. casei Zhang has good acid resistance, bile salt resis-
tance, and intestinal colonization ability. At the same time, the research showed that
the strain has many functions such as anti-oxidation, antagonizing pathogenic bac-
teria infection, regulating immunity, lowering blood sugar, and lowering blood fat
(Guo et al. 2009). Compared with the commercial strains L. acidophilus NCFM,
LGG, L. casei Shirota, and Bifidobacterium animalis subsp. lactis BB-12, L. casei
Zhang fermented milk has higher acid production capacity and stronger proteolytic
activity under refrigeration conditions. After 28 days of refrigeration, it has similar
viable counts (1.0 × 109 cfu/ml) with other commercial strains, indicating that L.
casei Zhang has broad application potential in functional foods and fermented foods
(Guo et al. 2009).
10.10.2 Safety Study of L. casei Zhang
Up to now, no adverse reactions related to L. casei Zhang have been reported in

animal experiments and production applications. In addition, in a human experi-
ment evaluating the effects of L. casei on intestinal microbes, 24 adults succes-
sively consumed L. casei Zhang for 28 days without adverse reactions. Through
analyzing intestinal microbial changes by 454 pyrosequencing, it is found that L.
casei Zhang changed the composition of intestinal microbes. At the genus level, L.
casei Zhang is positively associated with Plasmodium, Lactobacillus, Clostridium
leptum, Propionibacterium, Bifidobacterium, and unidentified genus Bacteroides
and Trichophyton, whereas it is negatively correlated with Clostridium,
Phascolarctobacterium, Serratia, Enterococcus, Shigella, and Shewanella (Zhang
et al. 2014). Another human experiment conducted by three different ages (youth,
middle-aged, and elderly) of Chinese people also yielded similar results. L. casei
Zhang can regulate intestinal microbes by increasing the content of short-chain
unsaturated fatty acids and lowering the total bile acid content, which has a benefi-
cial effect on the human body. At the same time, L. casei Zhang also has an inhibi-
tory effect on potential pathogenic bacteria such as Pseudomonas and Acinetobacter.
In the subjects of different ages, the intestinal bacteria Bifidobacterium and
Bacteroides fragilis are slightly different. None of the above human experiments
showed side effects due to the intake of L. casei Zhang. However, rigorous and
standardized safety evaluation experiments are still very necessary.
10.10.3 Functional Study of L. casei Zhang
10.10.3.1 Antioxidant and Protective Ability Against Liver Damage
In the animal experiment, the SOD activity and serum GSH-Px activity of the rat
liver homogenate were significantly higher than that of the control group after the
normal Wister rats were intragastrically administered with 4 × 109 cfu dose of L.
casei Zhang. Liver malondialdehyde (MDA) content was significantly lower than
that of the control group, indicating that L. casei Zhang has good antioxidant capac-
ity. In addition, the antioxidant capacity of L. casei Zhang is significantly higher
than that of another L. casei MG1–4 in the same study, indicating that the antioxi-
dant capacity of Lactobacillus is strain specific (Wang et al. 2009). In another study,
a rat model of liver injury was induced using a combination of 50 g/kg lipopolysac-
charide (LPS) and 300 mg/kg D-galactose (D-GalN), and 109 cfu was administered
daily 30 days before drug exposure. L. casei Zhang not only significantly reduced
the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT),
decreased liver MDA content, and increased SOD activity but also decreased LPS/
D-GalN-induced liver NO production and activated inducible expression of nitric
oxide synthase (iNOS) and TNF-α. In addition, the intake of L. casei Zhang signifi-
cantly downregulated the mRNA and protein expression levels of liver TLR4, I-κB
phosphorylation level, and nuclear transcription factor-κB (NF-κB) levels. This
suggests that the protective effect of L. casei Zhang on LPS/D-GalN-induced liver
injury is due to its good antioxidant capacity and, on the other hand, its immuno-
modulatory capacity (Wang et al. 2013a). Since then, L. casei Zhang has further
explored the protective mechanism of LPS/D-GalN-induced liver injury (Gong
et al. 2013). Compared with the model group (LPS/D-GalN), the hepatocyte necro-
sis of L. casei Zhang group was significantly reduced. The results of immunohisto-
chemistry showed that the levels of TLR4 and p-ERK in the probiotic group also
had the same trend but were decreased. The expression of PPARγ was also restored,
suggesting that the protective mechanism of L. casei Zhang may be related to the
downregulation of TLR and ERK expression and promotion of PPARγ expression.
10.10.3.2 Antagonizing Pathogenic Bacteria Infection
L. casei Zhang protects pathogenic Escherichia coli O157 and K88 exposure stud-
ies, 15 days before the exposure of pathogenic bacteria, L. paracasei Zhang, a dose
of 10 ml/kg BW (probiotic concentration 2 × 108 cfu/ml). After 5 days of exposure
to E. coli, the mortality rate was 66.67%, while the mortality of the probiotic preven-
tion group and the treatment group were only 16.67% and 5.56%, respectively. E.
coli K88 itself is less pathogenic, so the probiotics are not much improved. After
4 days of exposure to pathogenic Escherichia coli, the intestinal microbes of the

model group and the probiotic group were analyzed and found. A large number of L.
casei Zhang were detected in the probiotic group, and the total number of E. coli was
significantly decreased, E. coli O157 and E. coli K88. The protective effect of L.
casei Zhang on mice exposed to pathogenic bacteria may be related to colonization
and inhibition of pathogenic bacteria activity in the intestine (HePing et al. 2007).
10.10.3.3 Immunomodulatory Effects
At present, a large number of studies have shown that probiotics have immuno-
modulatory functions. L. casei Zhang is a Lactobacillus strain with multiple probi-
otic functions, and its potential immunomodulatory effects have also received the
attention of researchers. Wang et al. (2013b) evaluated the immunomodulatory
effects of L. casei Zhang on the expression of cytokines and Toll-like receptors in
RAW264.7 macrophages. The probiotics were found to promote nitric oxide, TNF-
α, and IL-6 and produce IFN-β. At the same time, the transcription of inducible
nitric oxide synthase (iNOS) can be enhanced. The immunostimulatory effect of the
heat-killing L. casei Zhang was significantly reduced compared to live bacteria.
Another difference is that live bacteria promote TLR2 mRNA transcription, while
thermophilic probiotics enhance TLR2, TLR3, TLR4, and TLR9 transcription. In
addition, live bacteria significantly inhibited the levels of poly-inosinic acid-
stimulated NO, iNOS, and TNF-α while enhancing the expression of IFN-β. These
results indicate that L. casei Zhang improves the function of the innate immune
system by enhancing the transcription of Toll-like receptors and enhancing the pro-
duction of proinflammatory mediators and type I interferons in macrophages.
At the same time, the in vivo test also confirmed the immunomodulatory effects
of L. casei Zhang. In one study, the live and dead bacteria of L. casei Zhang were
administered to normal mice for several days. Its results found that only live bacte-
ria can cause a wide range of immune responses, in which IFN-γ content is increased,
while the TNF-α content is decreased, and this effect is dose-dependent. In addition,
the transcription of interleukin-2 (IL-2) and IL-2 receptor genes was significantly
increased, while the proportion of T-lymphocyte subsets was not affected. L. casei
Zhang can induce intestinal mucosal responses by enhancing the production of
secretory immunoglobulin A (SIgA) and by affecting systemic immunity through
cytokines released into the blood. This suggests that L. casei Zhang can affect the
immune response in a dose-dependent manner (Ya et al. 2008). In the study of L.
casei Zhang intervention in LPS/D-GalN-induced liver injury, the intake of probiot-
ics improved the significant increase in endotoxin, TNF-α, IL-1β, and nitric oxide
production caused by drug challenge, and significantly LPS/GalN-triggered phos-
phorylation of ERK, JNK, and p-38 MAPK was inhibited, while expression of
TLR2, TLR9, and PPAR-γ was enhanced. In addition, the study also found that the
intake of L. casei Zhang prevented intestinal damage and believed that protection
was achieved by increasing the level of intestinal ecology and fecal bacillus and
bifidobacteria (Wang et al. 2016).
The study also examined the effects of L. casei Zhang on intestinal immune
function in mice. Live L. casei Zhang was intragastrically administered to mice at
high (1 × 109 cfu), medium (0.5 × 109 cfu), and low (0.25 × 109 cfu) doses for
11 days. After 11 days of intragastric administration, the number of Peyer’s patches
in the high-dose group was significantly higher than that in the control group
(10.4 ± 1.6 vs. 8.0 ± 1.6, P < 0.5). In the same high-dose group, even the middle-
dose group was scored. The value was significantly higher than the control group
(9.0 ± 2.0, 8.9 ± 1.5 vs. 6.8 ± 1.6, P < 0.05). The average number and average high
density values of CD3+- and IgA+-positive cells in different parts of mice in the
high-dose group were significantly higher than those in the control group, indicat-
ing that L. casei Zhang has a regulatory effect on intestinal mucosal immunity in
mice (Rui-ting et al. 2009).
10.10.3.4 Relieving Hyperlipidemia
In vitro, L. casei Zhang has a good tolerance and adsorption capacity for choles-
terol. The results showed that serum TC and TG levels in the high-fat diet group
were significantly higher than those in the normal diet group after 14 days of high-
fat diet, indicating that the hyperlipidemia model was successfully established. At
this time, compared with the high-fat diet group, serum TC was significantly
decreased (P < 0.01), while HDL-C content was significantly increased (P < 0.05).
At 28 days, heat-killed probiotics significantly reduced TC content (high-fat diet
group, 2.09 ± 0.26 vs. heat-killed bacterial group, 1.82 ± 0.26, P < 0.05), whereas
for TG, LDL-C, and HDL-C level, the effect is not obvious or has no effect.
Comparing the bile acid content in the feces, the bile acid excretion of the heat-
killed cells group was significantly higher than that of the high-fat diet group
(P < 0.05), while the excretion of the live bacteria group was not significantly dif-
ferent from the model group. This indicates that the cholesterol-lowering effect of
L. casei Zhang is achieved by adsorbtion and assimilation of cholesterol and inhibi-
tion of the reabsorption of bile acids into the intestinal liver (YueYing et al. 2006).
It is worth noting that there is a big difference in the ability to kill cholesterol and
lower blood fat in living cells and heat-killing cells, but the cause is still unknown.
In addition, the effects of different doses of probiotics may also differ. Zhang et al.
(2012) and Zhong et al. (2012) studied the effect of high (2.0 × 1010 cfu/d), medium
(2.0 × 109 cfu/d) and low (2.0 × 108 cfu/d) doses and probiotic fermented beverage
[2.0 × 108 cfu/(ml·d)] on liver lipids in hypercholesterolemic rats. The high-fat diet
was fed for 2 weeks. After confirming the successful modeling, the probiotics inter-
vention group began to administer the probiotics while continuing to maintain the
high-fat diet (the control group was given with a normal feed) for 4 weeks. Compared
with the model group, the liver TC levels of the probiotics dose group and the fer-
mented beverage group were significantly lower (P < 0.01), but the difference
between the three different dose groups was not significant. For liver TG, all probi-
otics dose groups significantly reduced their content (P < 0.05), and the three dose
groups decreased dose TG capacity in a dose-effect relationship. Each probiotic
group had no significant effect on fecal TG, but compared to the model group and
the fermented beverage group, the three dose groups of L. casei Zhang significantly
increased the level of fecal total bile acid. In addition, probiotics in the three dose
groups significantly increased serum apolipoprotein ApoAI levels and decreased
ApoB levels, which also ameliorated the pathological damage of liver tissue. L.
casei Zhang at 2.0 × 108 cfu/d and above can improve liver lipid metabolism in rats.
The possible mechanism is to increase total bile acid levels, reduce their reabsorp-
tion, and regulate serum apolipoprotein levels
In another study, the dose of L. casei Zhang to alleviate hyperlipidemia was
explored. After administration of L. casei Zhang in high-cholesterol model rats,
serum TG and LDL-C levels were reduced by 31.5% and 30.8%, respectively. DNA
microarray results showed that 755 genes involved in bioregulation and cell and
metabolic processes were diversely expressed in the liver of probiotic-treated rats,
in which among them, 324 genes were upregulated and 431 genes were downregu-
lated. In the upregulated group, genes such as Acsl1, Hadh, Acaa2, Acads, and
gcdH, which are involved in the oxidation of fatty acids and encode enzymes that
catalyze key steps in the synthesis and dehydrogenation of CoA, are characterized.
Lactobacillus may accelerate fatty acid catabolism by enhancing the hydrazine oxi-
dation step, thereby reducing levels of free fatty acids and TG in the serum and liver.
In addition, L. casei Zhang can regulate the body’s lipid metabolism balance to
convert TG, which is the main storage form of fatty acids, into free fatty acids and
reduce TG concentrations in the serum and liver (Zhong et al. 2012).
10.10.4 Application Study of Lactobacillus casei Zhang
In order to achieve a large-scale production of L. casei Zhang, the research team

carried out a series of biological characteristics and processing application research
on the probiotics (He 2010), including medium optimization, high-density fermen-
tation, freeze-drying, and mixed fermentation of strains and application in fer-
mented dairy products. According to the growth characteristics and nutritional
needs of L. casei Zhang, the composition and proportion of the medium carbon
source, nitrogen source, and other salt components were optimized for L. casei
Zhang. Finally, the optimal medium (1000 ml medium) was obtained: 20.9 g of
glucose, 10.45 g of yeast powder, 10.45 g of soy peptone, 3.51 g of dipotassium
hydrogen phosphate, 14.64 g of sodium acetate, 2.34 g of sodium citrate, 1 g of
magnesium sulfate heptahydrate, 0.054 g of sulfuric acid pentahydrate, 0.01 g of
copper sulfate pentahydrate, 1g of Tween-80. L. casei Zhang was cultured in an
optimal medium at 37 °C for 18 h, and the cells of the 1 ml fermentation broth
reached 4.78 × 109 cfu, which was about ten times that of the MRS medium under
the same culture conditions (PengFei et al. 2008). In order to achieve high-density
culture, the later studies compared the two fermentation modes of batch culture and
fed-batch culture and further optimized the factors affecting the growth of the cells.
The results showed that the batch culture method was more suitable for the culture
of L. casei Zhang. The high-density culture condition adjusted the amount of glu-
cose to 80–100 g (per 1000 ml), and the pH was maintained at about 5.9 by adding
ammonia water, and the anaerobic state was maintained by intermittent nitrogen
gas. The batch culture was carried out at 37 °C for 10–12 h, and the number of cells
per unit volume was increased by more than seven times compared with the previ-
ously optimized medium culture (Yan et al. 2009). The optimization of high-density
medium and culture conditions laid a solid foundation for the industrial production
of L. casei Zhang.
Freeze-drying is an effective means to maintain the activity of bacteria and has a
wide range of applications in the production of starter. In order to improve the bac-
terial activity of L. casei Zhang after freeze-drying, the researchers analyzed and
compared the protective effects of various lyoprotectants and found that using glu-
tathione as a protective agent can increase the survival rate by 86.6%. Further stud-
ies have shown that glutathione can maintain a higher ratio of unsaturated fatty
acids and shorter saturated fatty acid chains in cell membranes (Zhang et al. 2016).
At the same time, a lot of research has been carried out on the application of L. casei
Zhang in fermented foods, especially fermented dairy products. The combination of
L. casei Zhang and Bifidobacterium lactis V9 in lactic acid bacteria beverages
showed that the co-fermentation of two probiotics had a synergistic effect.
Bifidobacterium promoted the proliferation of L. casei Zhang and improved the
taste of fermentation of Lactobacillus casei (Chen et al. 2015). In addition, L. casei
Zhang is also used for soymilk fermentation and cheese production (Dantas et al.
2016; Dong et al. 2008; Li et al. 2012).
10.11 Lactobacillus plantarum CCFM8610

D
plantarum CCFM8610
Lactobacillus plantarum CCFM8610 is a probiotic Lactobacillus isolated from

Chinese traditional fermented pickles by the probiotic theory and technology
research team of Jiangnan University in 2008. The 16S rDNA of Lactobacillus
plantarum CCFM8610 was identified by molecular biology. The 16S rDNA
sequence of Lactobacillus plantarum CCFM8610 was obtained by direct sequenc-
ing of the PCR products. By comparison and analysis with several Lactobacillus
plantarum model strains by NCBI BLAST, 99% homology was found. Therefore,
the strain was identified as Lactobacillus plantarum. The probiotic function of
Lactobacillus plantarum CCFM8610 was systematically evaluated by in vitro
screening and animal model, and related products such as yogurt were developed
according to the special probiotic characteristics of the strain.
10.11.2 Safety Study of Lactobacillus plantarum CCFM8610
At present, there are no reports of toxic and side effects of Lactobacillus plantarum
CCFM8610 in related studies, including animal and human experiments. Animal
experiments showed that Lactobacillus plantarum CCFM8610 alone did not signifi-
cantly affect the body weight of mice and the contents of Ca, Fe, Zn, Mg, and other
essential elements in liver and kidney tissues. At the same time, there was no signifi-
cant difference in liver and kidney oxidation index and histopathological index
between mice and control group (Zhai et al. 2014). Human experiments also showed
that no obvious side effects of Lactobacillus plantarum CCFM8610 on the human
body were observed. Therefore, we can believe that CCFM8610 is safe and
harmless.
10.11.3 asic Physiological Characteristics of Lactobacillus

B
plantarum CCFM8610
10.11.3.1 Tolerance to Simulate Gastric Juice and Intestinal Fluid
Lactobacillus plantarum CCFM8610 has excellent ability to tolerate gastric acid

and intestinal juice in the digestive system. The survival rates of simulated gastric
juice and intestinal juice in vitro are 88.72% and 92.98%, respectively. It shows that
the strain can successfully pass the two barriers of gastric juice and intestinal juice
and maintain high activity in the intestinal tract of the host (Zhai et al. 2015b).
10.11.3.2 Scavenging Free Radicals and Antioxidant Capacity
Lactobacillus plantarum CCFM8610 has good DPPH-free radical and hydroxyl-

free radical scavenging capacity, as well as good reductive capacity and anti-lipid
peroxidation ability. Among the 11 tested strains, the antioxidant property of
Lactobacillus plantarum CCFM8610 was the most similar to that of the control
strain LGG (Zhai et al. 2015b). LGG is a probiotic strain widely used in food indus-
try and widely studied in clinical experiments. It has excellent antioxidant activity
both in vitro and in vivo (Forsyth et al. 2009; Goyal et al. 2013).
10.11.3.3 Culture Characteristics and Freeze-Drying Process
Lactobacillus plantarum CCFM8610 could grow well in food-grade multiplication

medium. After 8–10 h of culture, the concentration of live bacteria exceeded
4 × 109 cfu/mL. The strain can be prepared by freeze-drying. With protective agents
including skim milk, trehalose, and sucrose, the survival rate of freeze-drying is
over 90%, and the concentration of viable bacteria is over 3 × 1011 cfu/g (Cui et al.
2016a, b).
10.11.4 robiotic Functional Properties of Lactobacillus

P
plantarum CCFM8610
10.11.4.1 Excellent Cadmium Adsorption and Tolerance
The cadmium tolerance and cadmium adsorption capacity of 33 lactic acid bacteria
including Lactobacillus plantarum CCFM8610 were investigated. The results
showed that Lactobacillus plantarum CCFM8610 had the strongest cadmium
adsorption capacity under different initial cadmium concentrations. The strain has a
strong tolerance to cadmium ions, and its MIC to cadmium exceeds 1000 mg/L,
which is more than 20 times that of other strains tested (Zhai et al. 2015b). Previous
studies have reported that when the MIC of cadmium exceeds 100 mg/L or
112.4 mg/L (Akinbowale et al. 2007; Matyar et al. 2008), it can be considered that
the strain is tolerant to cadmium. According to this definition, Lactobacillus planta-
rum CCFM8610 is a cadmium-tolerant strain.
10.11.4.2 Specific Cadmium Adsorption Mechanism
The cadmium ion adsorption mechanism of Lactobacillus plantarum CCFM8610

was analyzed in vitro (Zhai et al. 2016). The results showed that most cadmium was
adsorbed on the cell wall and cell membrane, only about 10% of cadmium ions
entered the protoplast, and the amino and carboxyl groups on the surface of the
bacteria played an important role in adsorbing cadmium. Thermodynamic analysis
showed that the adsorption process of CCFM8610 had the best goodness of fit for
Langmuir-Freundlich model (R2 = 0.9928). The theoretical upper limit of Qmax was
significantly higher than that of commercial probiotic strains such as Lactobacillus
rhamnosus LGG and Lactobacillus casei substitutes (Halttunen et al. 2007).
Therefore, Lactobacillus plantarum CCFM8610 has its unique mechanism of cad-
mium ion adsorption, and its theoretical adsorption capacity is higher than that of
commercial strains reported in the past.
10.11.4.3 Specific Cadmium Tolerance Mechanism
Using Lactobacillus plantarum CCFM8610 with strong resistance to cadmium and

Lactobacillus plantarum CCFM191 sensitive to cadmium as research objects, the
mechanism of cadmium tolerance of Lactobacillus plantarum was studied by iTRAQ
comparative proteomics method (Zhai et al. 2017). The results showed that CCFM8610
had a specific protein molecular mechanism related to cadmium tolerance, involving
the tolerance characteristic of proteins such as prophage P2b protein 18 and energy
metabolism pathways related to cadmium tolerance. At the same time, Lactobacillus
plantarum CCFM8610 has inherent cadmium tolerance properties in its natural state
and specific response mode after cadmium stimulation, which together determine the
molecular mechanism of its strong cadmium tolerance.
10.11.4.4 Remission of Cadmium Toxicity In Vivo
Reducing Cadmium Content in Tissues
In acute cadmium-exposed mice model, Lactobacillus plantarum CCFM8610 could

effectively increase the cadmium content in feces of cadmium-exposed mice, while
the cadmium content in the liver and kidney of mice decreased significantly (Zhai
et al. 2013, 2014). As Lactobacillus plantarum CCFM8610 has excellent cadmium
adsorption ability, it may absorb cadmium ion before intestinal epithelial cells
absorb cadmium ion and discharge cadmium ion through feces, so as to reduce
cadmium content in tissues after entering the intestinal tract of mice. It is worth not-
ing that the effect of living bacteria is significantly better than that of dead bacteria,
which may be due to the fact that live Lactobacillus plantarum CCFM8610 can
promote intestinal peristalsis, thus promoting cadmium excretion with feces.
In the chronic mice model of cadmium exposure, intragastric administration of
Lactobacillus plantarum CCFM8610 could effectively increase the cadmium con-
tent in feces at each time point within 8 weeks. Correspondingly, the cadmium con-
tent in the liver and kidney of mice also decreased significantly with CCFM8610.
Alleviating Oxidative Stress
Cadmium exposure leads to inactivation of antioxidant enzymes and production of

reactive oxygen species (ROS), leading to oxidative damage, tissue lesions, and
physiological damage, which are the main mechanisms of cadmium toxicity dam-
age to the body (Liu et al. 2009). Lactobacillus plantarum CCFM8610 has excellent
antioxidant properties. It can effectively reduce MDA concentration in host tissues;
restore GSH levels and SOD, GPx, and CAT activities; and alleviate oxidative dam-
age caused by acute and chronic cadmium exposure. Further studies have shown
that the mitigation of oxidative damage is relatively independent and is not a follow-
up result of the reduction of cadmium content in tissues (Zhai et al. 2013, 2014).
Regulating Intestinal Barrier and Intestinal Flora
Tight junction proteins play an important role in intestinal barrier (Qin and Gao
2005). Cadmium exposure to the destruction of tight junction proteins will lead to
cadmium ions more easily absorbed by the intestine and then cause toxic effects on
the body. Feeding Lactobacillus plantarum CCFM8610 significantly increased the
expression of tight junction proteins (occludin, claudin-1, ZO-1, and ZO-2),
decreased blood endotoxin levels, and restored the increase of intestinal cell perme-
ability caused by cadmium exposure. These results suggest that Lactobacillus plan-
tarum CCFM8610 may alleviate the damage of cadmium toxicity to tight junction
proteins and protect the integrity of intestinal barrier, thereby reducing the absorp-
tion of cadmium in the intestine.
Recent studies have shown that there is an interaction between cadmium and
intestinal flora. Cadmium has toxic effects on many intestinal microbes and can
cause disorders of intestinal flora (Fazeli et al. 2011; Liu et al. 2014). Conversely,
intestinal flora can also modulate cadmium toxicity and bioavailability of cadmium
(Breton et al. 2013). At the same time, a large number of studies have shown that
probiotics have the function of regulating intestinal flora. Considering the above
two points, Lactobacillus plantarum CCFM8610 as a probiotic also has the poten-
tial to alleviate cadmium toxicity by regulating intestinal flora. At present, the
mechanism needs further study.
10.11.5 pplication Experiment of Lactobacillus plantarum

A
CCFM8610
10.11.5.1 Trial of Mixed Bacteria Powder for Food Experiment
Compound bacterial powder containing seven probiotics such as Lactobacillus

plantarum CCFM8610 can effectively alleviate constipation, dyspepsia, and other
symptoms, alleviate immune diseases such as eczema and dermatitis, and improve
intestinal symptoms such as abdominal pain and diarrhea in autistic children.
10.11.5.2 Experiment of Fermented Soymilk
Lactobacillus plantarum CCFM8610 was used to ferment yogurt and compared

with the control strain CCFM8004 (commercial starter) (Zhai et al. 2015a). The
results showed that the antioxidant properties of CCFM8610 fermented soybean
milk were significantly better than that of CCFM8004 fermented soybean milk, and
the content of aglycones was also significantly higher than that of CCFM8004 fer-
mented soybean milk. CCFM8610 fermented yogurt obtained higher scores after
sensory evaluation by professional evaluation team, even surpassed commercial
starter CCFM8004 fermented yogurt.
Animal experiments also showed that the protective effect of unfermented soy-
bean milk on mice exposed to chronic cadmium was very limited, but Lactobacillus
plantarum CCFM8610 fermented soybean milk could effectively alleviate the toxic
damage caused by chronic cadmium exposure in mice, which was manifested in
promoting excretion of cadmium through feces, reducing cadmium content in tis-
sues, and restoring the oxidation caused by cadmium exposure. The protective
effect of stress and physiological damage-related indicators was significantly better
than that of the control strain CCFM8004.
10.11.5.3 Experiment on Fermented Fruit and Vegetable Juices
Nine kinds of fruit and vegetable juices (apple, tomato, and cucumber juices from
Jiangsu, Hunan, and Jiangxi provinces) were collected, and their cadmium content
was determined to study the ability of probiotics to reduce heavy metals in fruit and
vegetable juices (Zhai et al. 2016). After 2 h of cultivation and centrifugation,
Lactobacillus plantarum CCFM8610 can effectively remove 67–82% of cadmium
from fruit and vegetable juices. Long-term fermentation of fruit and vegetable juices
(36 h) with the strain could also significantly reduce cadmium content (56–81%).
The results showed that the strain could be used as a food safety microorganism to
control cadmium content in fruit and vegetable juice products. The method was
simple, safe, and effective and could avoid the negative effects of chemical treat-
ment on fruit and vegetable juices.
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Chapter 11
Safety Evaluation of Lactic Acid Bacteria
Wei Chen, Leilei Yu, and Ying Shi
Fermented food containing active lactic acid bacteria (LAB) has been consumed
worldwide for nearly a thousand years. The initial reason for adding lactic acid
bacteria to the food system is to reduce the overall pH as its characteristics of pro-
ducing lactic acid in the natural fermentation process, thereby inhibiting the patho-
genic microorganisms and food spoilage, as well as prolonging the food shelf life.
With the development of functional food, LAB with different functional character-
istics and physiological activities have been used in food fermentation, which not
only prolongs the shelf life of food but also improves the functional quality of food.
The main purpose of developing fermented food with LAB is to improve the health
of consumers, so the concept of “probiotics” is introduced into fermented food, and
safety evaluation is indispensable in such functional products.
Lactobacillus and Bifidobacterium are the most common LAB used in food at
present, which do not have any pathogenic characteristics. In general, they are seldom
involved in common infections (except for Enterococcus). However, some rare cases
of infections, mainly preclinical assessments of new or mixed strains, have been
reported. Infections usually occur in people with serious underlying diseases, such as
immunocompromised people or patients with hepatitis (Besselink et al. 2008a). As a
result, such infections are extremely rare with little data to support risk factors for the
general population. Many LAB strains, such as Lactobacillus and Bifidobacterium,
have a good safety record in daily life. These strains are also important components
of gut microbiota in healthy people and play an important role in many metabolic
processes. In view of the particularity of LAB in functional foods and their important
role in human health, the International Dairy Foods Association (IDFA) has formu-
lated a document on the safety assessment of microorganisms in foods. The safety of
LAB commonly used in food, including Lactobacillus and Bifidobacterium, has also
W. Chen (*) · L. Yu · Y. Shi

e-mail: [email protected]; [email protected]; [email protected]
https://doi.org/10.1007/978-981-13-7832-4_11
372 W. Chen et al.
been stipulated in the Qualified Presumption of Safety (QPS) document established

by the European Food Safety Agency (EFSA). Most of LAB keep a safety record so
far, and even many products still maintain good monitoring data after they are on the
market (Salminen et al. 2002).
In view of the importance of this issue, the safety of LAB, especially for the new
and mixed LAB strains, without safety assessment and history of safe use, must be
strictly and systematically evaluated. Strict prevention of food safety problems
caused by microorganisms is important for the development of safe and healthy
functional probiotic food containing novel active LAB, and it is also a challenge in
current and future food development.
11.1 evelopment and Present Situation of Lactic Acid

D
Bacteria Safety Evaluation
11.1.1 he History of Safety Evaluation of Lactic Acid

T
Bacteria
11.1.1.1 Potential Pathogenicity and Toxicity of Lactic Acid Bacteria
Translocation and Bacteremia of Lactic Acid Bacteria
The migration ability of LAB is related to its pathogenicity, and the direct method
to measure the migration translocation ability of LAB is to give probiotics orally
and count the number of bacteria invading various organs and tissues, including the
blood, liver, spleen, kidneys, and mesenteric lymph nodes. These studies have dem-
onstrated the safety of probiotics. Kabeir et al. confirmed that B. pseudocatenula-
tum did not migrate into the liver and blood of mice after oral feeding strains (Kabeir
et al. 2008). Moreover, all organs and tissues, including the blood, liver, kidneys,
spleen, and mesenteric lymph nodes, were analyzed after 7 days of administration
with B. longum BB536; the results showed that no bacteria (including
Bifidobacterium) was found (Abe et al. 2010). For Lactobacillus, Paturi et al.
pointed out that Lactobacillus acidophilus and Lactobacillus paracasei did not
translocate to the spleen, liver, and blood of mice (Paturi et al. 2008). However,
early trials were conducted in disease-free normal mice, a model for healthy people,
and most probiotic-induced sepsis or bacteremia occurred in patients with disease,
not in healthy people. Therefore, using immunodeficient animals as susceptible
populations (including infants or immunodeficiency patients), such as models of
leukemia, premature infants, severe AIDS, and postoperative patients, are more
appropriate for detecting translocation and sepsis.
Subsequently, several studies established mouse model of immunodeficiency for
observing bacterial translocation and subsequent septicemia (Kawahara et al. 2015;
Matsumoto et al. 2008). Cyclophosphamide is an immunosuppressive agent which
is used in cancer patients sometimes and can induce immune damage in mice. The
11 Safety Evaluation of Lactic Acid Bacteria 373
results indicated that these mice have low white blood cell counts and become prone
to intestinal cell migration. In this model, Pseudomonas aeruginosa was given, and
these bacteria were observed in the blood and liver of mice, and all mice died within
a few weeks. Bifidobacterium longum BB536 was administered orally before and
after administration of P. aeruginosa in the immunocompromised mouse. No B.
longum BB536 was detected in the liver and blood of mice, and the mortality caused
by P. aeruginosa was inhibited after administration of B. longum BB536. These
findings suggest that probiotic testing is relatively safe even in patients with low
immune function and bacterial migration. Therefore, immunodeficient animals can
simulate the status of immunocompromised people such as premature infants, the
elderly, and critically ill patients. It is important to study whether probiotics can be
used in healthy patients.
All probiotics may be susceptible to bacterial migration in severely damaged
bowel walls, such as severe gastrointestinal injury, major diseases, and extensive use
of antibiotics. However, bacterial translocation is significantly distinct from septice-
mia or endocarditis. If translocated bacteria do not cause sepsis, endocarditis, infec-
tion, or any other disease, it will not threaten the health and can be removed from the
body (Yazawa et al. 2000). Conversely, if translocated probiotic can cause sepsis or
infection, they should not be used (even if their translocation ability is low).
Previous studies have reported that when the germfree mice were given oral
administration of pathogenic Escherichia coli O-111, most mice died within a few
weeks as Escherichia coli O-111 migration and the production of enterotoxin in the
organs (Turroni et al. 2014; Yamazaki et al. 1985). However, when the germfree
mice were treated with Bifidobacterium longum BB536, the strain was observed in
organs such as the liver, kidney, and mesenteric lymph nodes in the first 4 weeks, but
no mice died, but after 4–8 weeks of production of specific IgG and IgA, the strain
was not detected in all organs (Turroni et al. 2014; Yamazaki et al. 1985). The results
indicated that B. longum BB536 strain did not have infection side effects even
though it migrated and it is still safe for patients at risk of bacterial migration
(Yamasaki et al. 2012). After oral gavage B. longum BB536 to germfree mice, B.
longum BB536 was found to be transferred to the liver, mesenteric lymph nodes,
and kidneys by organ counting. It was detected in every organ during the first
2 weeks, but was not detected after 4 weeks; no disease or death was found in all
mice. In addition, the strain has been used in immunocompromised patients or in
conjunction with anticancer drugs, including cyclophosphamide, to confirm the effi-
cacy of probiotics against monilial infection. Monilia proliferation in patients
resulting from taking antileukemic drugs can be reduced by Bifidobacterium inter-
vention, and probiotics do not have any side effect.
Lactic Acid Bacteria and the Production of Harmful Metabolites
Biogenic amines (BAs) are a group of small nitrogen compounds with biological
activity formed by the decarboxylation of amino acids or the amination of alde-
hydes and ketones (Maijala et al. 1995). It is ubiquitous in fermented foods, such as
374 W. Chen et al.
fermented meat products, fermented seafood, fermented dairy products, and fer-
mented vegetables (Shukla et al. 2015; Cvetkovic et al. 2015; An et al. 2015;
Piersanti et al. 2014; De Mey et al. 2014; Kuley et al. 2011; Bunkova et al. 2010).
The BA types and contents in different products or different samples of congeneric
products are different, and they are influenced by environmental factors and fermen-
tation process (Cvetkovic et al. 2015; Piersanti et al. 2014; Li et al. 2014). BA in
food is mainly from the decarboxylation of free amino acids by amino acid decar-
boxylase generated by microorganisms.
BAs are bioactive organic alkaloids with important physiological functions, such
as brain activity, gastric acid secretion, immunity, cell growth, and differentiation
(Jagu et al. 2015). BAs are maintained at normal concentrations in tissues and cells
through constant biosynthesis and metabolic decomposition (Linsalata and Russo
2008). However, excessive intake of BAs can break this balance and cause cardio-
vascular and neurological damage, leading to elevated blood pressure, rapid heart-
beat, increased blood sugar levels, excessive adrenaline secretion, and headaches,
and sometimes mainly gastrointestinal disorders, such as vomiting, diarrhea, and
abdominal spasm (Kovacova-Hanuskova et al. 2015). It is also pointed out that the
content of BAs synthesized by the organism on its own can meet the basic physio-
logical function requirements without external supplements as long as the amino
acid intake is adequate (Kalac 2014). Because the toxicity of BAs varies, and the
detoxification ability of different populations against BAs is also very different, so
the requirements of BAs level in food vary from country to country.
It is reported that the probiotics with the ability of BA production in foods include
Enterococcus and Lactococcus, tyramine-producing bacteria isolated from cheese
(Ladero et al. 2010). Lorencov et al. isolated 81 LAB from dairy products and beer
to test their ability to produce BAs (Lorencová et al. 2012). The results showed that
50 strains had decarboxylase activity; 70% of the strains isolated from beer had an
excellent ability to produce tyramine. Most of the LAB had decarboxylase activity;
these LAB can increase the content of BAs in food, thereby threatening food safety
and consumer health. It is noteworthy that some probiotics, such as Bifidobacterium
and Lactobacillus rhamnosus, can also increase the levels of BAs in foods. Yüceer
et al. also found that 68% of enterococci isolated from Turkish sausages can pro-
duce tyramine, which may have an adverse impact on consumer health (Yüceer and
Banu 2015).
The Mucin Degradation Activity of Lactic Acid Bacteria
Increased mucin degrading activity of bacteria leads to more permeability of intes-

tinal wall and further bacterial translocation (Gork et al. 1999). On the one hand,
some researchers have reported that probiotics have or do not have mucin-degrading
activity. They have demonstrated that L. rhamnosus, L. acidophilus, and
Bifidobacterium lactis do not decompose mucin in vitro. Abe et al. (2010) also stud-
ied the mucin degradation activity of different strains of Bifidobacterium (including
B. longum, B. breve, and B. longum subsp. infantis). It was initially observed that
Bifidobacterium grew slower in the medium with mucin as the sole carbon source
than in the glucose medium. Subsequently, they used sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) and stool samples as a positive
control; the results confirmed that the Bifidobacterium did not decompose mucin.
On the other hand, although most of B. longum and all tested B. pseudocatenulatum
do not have these properties, some bifidobacteria, such as B. bifidum, B. breve, and
B. longum, have mucin-degrading activity and contain gene encoding for mucin-
degrading enzyme (Egan et al. 2014; Ruas-Madiedo et al. 2008). Since mucin plays
an important role in preventing bacteria from invading the intestine, it is important
to determine whether probiotics used in food have mucin degradation activity, i.e.,
the ability to damage the mucin layer. These findings suggest that mucin decompo-
sition activity should be investigated at the strain level.
Vesterlund et al. studied the extracellular matrix proteins, mucus, blood cell
adhesion properties, as well as the avoidance of the ability of inducing respiratory
infection and the resistance characteristics of human serum in polymorphonuclear
leukocyte (PMN), of 44 strains isolated from feces, 5 strains isolated from blood,
and 15 strains isolated from probiotic products (including 3 strains from dairy prod-
ucts) (Vesterlund et al. 2007). Among the tested strains, the strains adhering to col-
lagen, fibrinogen, and mucus were specific, while the strains isolated from feces,
blood, and probiotics showed no significant statistical difference. However, the
probability of respiratory infection induced by strains from probiotics in PMN was
lower than by those from blood (P < 0.05). In addition, there was a positive correla-
tion between adhesion collagen and respiratory infection in strains from feces
(P < 0.05). In the determination of serum resistance, probiotics showed a tendency
to be less sensitive to human serum-mediated death than strains from feces
(P = 0.07). Compared with fecal or probiotic strains, no detectable virulence factors
were found in blood strains, suggesting that these factors do not pose a risk when
considering the safety of probiotics. However, the importance of probiotic adhesion
to mucus, low induction of respiratory infections in PMN, and low resistance to
human serum-mediated killing may require further assessment by animal models
and epidemiological data.
The “Toxicity” of Lactic Acid Bacteria
In the long history of thousands of years, food safety issues related to LAB were
rare, and the incidence of related human diseases has been extremely low (exclud-
ing the Enterococcus and Streptococcus strains mentioned above). Safety assess-
ment of LAB is also closely related to human scientific progress and technological
development, so there are some hesitations when referring to terms such as “toxic-
ity,” “virulence factor,” and “pathogenicity” related to LAB. The terms “toxicity”
and “virulence factor” have been temporarily used in the following discussion
before the emergence of a better explanation of the disease terminology caused by
LAB. Even for Enterococcus, the concept of pathogenic factors is less precise than
376 W. Chen et al.
those commonly recognized as Gram-positive pathogens such as Botulinum,

Staphylococcus aureus, Listeria, and Bacillus cereus in foods. However, consider-
able progress has been made in the safety assessment of enterococci in recent years,
such as the well-described factors associated with specific stages of enterococci
infection (Franz and Holzapfel 2004). Johnson studied pathogenic strains at the
specific stage that could cause infection and invade host tissues, resist the host’s
specific and non-specific defense system, cause pathological changes in the host,
directly produce toxins, and indirectly cause inflammation (Johnson 1994). The
virulence factors of enterococci involve all four stages mentioned above (Table 11.1).
Franz et al. reviewed the mechanisms of these virulence factors in infection (Franz
and Holzapfel 2004; Franz et al. 2003). It is noteworthy that Eaton et al. reported
that enterococci isolated from food have one or more virulence factors, but the inci-
dence of probiotic enterococcal virulence factors is significantly lower than that of
food strains (Eaton and Gasson 2001; Franz et al. 2001). This suggests that
Enterococcus carries virulent factors in food and poses a safety risk, especially for
people with underlying diseases. To sum up, the European VRE (vancomycin-
resistant Enterococcus) is likely to be transferred through the food chain, so for
enterococcal risk strains with antibiotic resistance or carrying virulent factors, we
should focus on the supervision of food transport routes.
Table 11.1 The virulence factors and toxic effects of enterococci (Franz et al. 2003)
Virulence factor Related toxic phases
Polymer Adhesion: adherence to eukaryotic cells or promotion of
colonization
Invasion: invasion of eukaryotic cells
Promoting migration: adhesion to extracellular matrix
proteins
Evading host immune response: enhancing immune cell
survival
Cell lysin Release of eukaryotic toxin
Evasion of host immune response: lysis of immune cells
Gelatinase Displacement: hydrolysis of various biological peptides,
such as collagen and fibrin.
Evasion of host innate immune response: hydrolysis of
antimicrobial peptides
Enterococci surface protein Evading host immune response: presenting bacterial
surface recognition and adhesion matrix molecular
characteristics
Enterococcus faecium or Promoting migration: adhesion of extracellular matrix
Enterococcus faecium collagen proteins
adhesin Evading host immune response: presenting bacterial
surface recognition and adhesion matrix molecular
characteristics
Hyaluronidase Displacement: degrading hyaluronic acid (a major
extracellular matrix)
Because non-enterococcal LAB have a long history of food safety, its possibility
of toxicity is very low, and it is difficult to find “virulence factors” in them. Although
it has been reported that some strains, such as Weissella confusa, have alpha-
hemolytic phenotypes and some L. lactis in dairy products have been confirmed to
have a hemolysin III gene (Olano et al. 2001; Bolotin et al. 1999), the effects of
these hemolytic characteristics are negligible, because no infection induced by
these hemolytic characteristics was reported so far. Recently, many reports were
about further sequencing of LAB genome; the potential virulence exists in their
genome informations (Bolotin et al. 1999; Kwon et al. 2015; Kwak et al. 2015; Yu
et al. 2012; Pridmore et al. 2004; Kleerebezem et al. 2003). The genome of probiotic
L. yoelii NCC 533 showed a protein with 50% amino acid sequence similarity to the
IgA protease of pathogenic Streptococcus, which may be related to the hydrolysis
of extracellular proteins or adhesion to mucosal surfaces (Pridmore et al. 2004). IgA
protease can prevent bacteria trapped in mucous layer and is thought to be immune
from host immune defense. However, the IgA protease can only be regarded as an
attribute of the probiotic strain without obvious virulence factors. Therefore, the
role of IgA protease may be related to the colonization of probiotics in the gastroin-
testinal tract, rather than toxicity.
Vesterlund et al. studied the presumed risk factors in feces, blood, and
Lactobacillus strains (Vesterlund et al. 2007). The adhesion of extracellular proteins
and mucus, hemolysis, the ability of monocytes in peripheral blood to avoid respira-
tory burst, and the tolerance to human serum were studied. For the test strains, adhe-
sion to collagen, fibrinogen, and mucus was strain-specific, and no significant
difference was observed between blood, feces, and beneficial biomass. This study
did not find any clear virulence factors in Lactobacillus.
11.1.1.2 Safety Events and Current Opinions of Lactic Acid Bacteria
For special cases with relatively low immunity or congenital infections, ingestion of
specific lactic acid bacteria in a certain period of time would induce immunoreac-
tion, including hypersensitivity reactions, so special individuals should be cautious
when selecting lactic acid bacteria products. In the 1990s, Schwab et al. reported
that the ingestion of lactic acid bacteria through non-oral routes would cause
immune responses and cause joint pain and complications (Schwab 1993). Studies
have shown that the symptoms of these over-immune reactions are mediated by
cytokine secreted by lactic acid bacteria.
Cases of probiotic translocation have been reported in the past decade. Kunz
et al. observed two cases of LGG bacteremia during the treatment with lactobacilli
(Kunz et al. 2004). In 2005, another case of LGG bacteremia was associated with
the use of probiotics. In addition, Land et al. demonstrated that lactobacilli sepsis
induced by ingestion of probiotics in two pediatric patients is not easy to find out
(Land et al. 2005). Similarly, LeDoux et al. reported L. acidophilus bacteremia hap-
pened after ingesting probiotics (LeDoux et al. 2006). These cases have sounded the
alarm to us. Although all probiotics that caused bacteremia were reported in patients,
378 W. Chen et al.
such as short-term bowel syndrome, and AIDS or premature babies, it is important

to consider the risk of some probiotics causing bacterial translocations, sepsis, and
bacteremia. In a systematic review of the safety of probiotics in patients with nutri-
tional support, Whelan et al. reported that 32 patients in 20 adverse events were
infected with LGG or Saccharomyces boulardii (Whelan 2010). They concluded
that certain probiotic products (single or combination) may increase the risk of
complications in a particular patient population.
A 5-year-old patient developed D-lactic acidosis after bowel resection. His con-
dition improved after the treatment, but acidosis was followed by intake of mixed
LAB supplement. It may be due to over-colonization of the genus producing lactic
acid in the intestinal tract (Ku 2006). It has been reported that the intake of LAB
caused methemoglobinemia in infants (Nitrate 1995). This is because LAB in the
mouth have strong nitroreductase activity, which will cause the nitrate in the food to
form nitrite, and the lesion occurs in the acidic stomach environment. At the same
time, as a precursor of nitrosamines, the presence of nitrite may also have a hidden
danger of cancer. The lactic acid bacteria in the intestine have strong amino decar-
boxylase activity, which can act on free amino acids to convert them into biogenic
amines, leading to food poisoning disorders such as nausea and vomiting.
In addition, Besselink et al. reported that probiotic ingestion has increased the
mortality rate of severe acute pancreatitis, and probiotics are not administered in
this category of patients (Besselink et al. 2008a). In recent years, some consumers
have developed bacteremia after consuming large amounts of dairy products con-
taining Lactobacillus casei. These cases demonstrate that the safety of probiotics is
not just an accidental isolated event, but is always present in clinical treatment. The
bacterial translocation caused by these probiotic strains and subsequent cases of
sepsis or endocarditis are a warning to us and emphasize the need to investigate the
translocation capacity of probiotics and take it as an aspect of safety evaluation.
Combined with these events, probiotics cannot be considered to be harmless
adjuncts to enteral nutrition, especially in critically ill patients or patients at risk for
nonocclusive mesenteric ischemia. The probiotic interventions are not recom-
mended in specific patient populations, such as those with severe acute
pancreatitis.
11.1.1.3 Development of Lactic Acid Bacteria Safety Evaluation
To date, clinical cases of infection caused by lactic acid bacteria have rarely been found
in healthy people. In most cases of clinical infection caused by lactic acid bacteria, the
lactic acid bacteria that cause infection are almost exclusively derived from the free
bacterial flora in patients. In 1938, the first case of endocarditis caused by Lactobacillus
infection occurred. In the following 55 years, 53 cases of endocardial inflammation
caused by Lactobacillus were found, indicating the incidence was very low.
Lactobacillus is widely distributed in humans as a common commensal flora. Compared
with another type of commensal flora such as Streptococcus or Staphylococcus, the
ratio of lactobacilli to endocardial inflammation is very low (0.05%).
In September 1993, the International Association of Microbiology Societies

(IUMS) believed that probiotics including lactic acid bacteria had pathogenic risks,
such as invasive or toxic arguments lacking food or clinical case evidence support.
However, probiotic productions include not only bifidobacteria but also enterococci,
propionic acid bacteria, or brewer’s yeast; therefore it is necessary to confirm the
safety of the relevant edible supplement strains.
The status of probiotic LAB (including Lactobacillus and Bifidobacteria) is gen-
erally recognized as safe (GRAS) in four aspects: human trials, animal models,
in vitro tests, and market research on probiotic products. The human experiment
was mainly based on 143 human clinical trials conducted from 1961 to 1998. It was
confirmed that oral probiotic lactic acid bacteria had no adverse side effects, and
7526 subjects all responded well, thus confirming its safety. In animal experiments,
oral or injection probiotics did not cause suppurative infection, acute toxicity, bac-
teremia, etc. in mice, rats, and guinea pig models. Not only that, probiotics can
significantly extend the life-span in animal experiments. At the same time, lactic
acid bacteria were not invasive to human lymphoma cells in vitro.
Although safety incidents rarely occur, with the increase of bacterial resistance
and the continuous discovery of pathogenic factors in enterococci, people are pay-
ing more attention to the safety of probiotics. In the European Union seminar in
early 2000, the safety of LAB was discussed, and the risk of infection by lactic acid
bacteria was low except for Enterococcus. However, considering that the probiotics
currently used are separated from different infectious parts, the monitoring needs
for commercial lactic acid bacteria such as L. rhamnosus are continued, and the
safety of probiotics should always be kept in the industrial application.
Internationally, there are different ways and various categories of regulations that
limit the use of lactic acid bacteria in food. There are differences between countries,
which are not necessarily related to strain’s function. According to the Federal
Food, Drug, and Cosmetic Act in 1958, the cultured microorganisms used in the
United States are defined as “food additives,” and new products are subject to safety
standards before they are launched. However, according to Section 210 of the act,
certain substances are expressly excluded by the US Food and Drug Administration
(FDA), and the substances of the generally recognized as safe (GRAS) are specifi-
cally mentioned. Therefore, the FDA also lists specific familiar microorganisms that
are considered safe. Although this type is not GRAS, they are microorganisms tra-
ditionally used in the food industry.
11.1.2 afety and Risk Status of Different Uses of Lactic Acid

S
Bacteria
The current reports on the harmful effects of probiotic strains are less, while the
demand for new strains is increasing, and there is a huge market and industrial pro-
duction potential. Therefore, we cannot assume that the new strains have the same
safety as the traditional strains that have been tested. Domestic research on the
380 W. Chen et al.
safety of lactic acid bacteria focuses on two aspects: one is the probiotic mechanism
and functional research related to lactic acid bacteria; the other is the potential abil-
ity of lactic acid bacteria as a safety strain to become a genetic engineering carrier.
Although research on intestinal microbes and other lactic acid bacteria has become
a hot topic in recent years, there are few studies on the safety of lactic acid bacteria,
and it showed that domestic safety awareness and attention on lactic acid bacteria
are low, which still stays in the “very safe” evaluation stage, indicating that there is
insufficient understanding of the potential risks and hazards of lactic acid bacteria
from consumers to researchers to food safety departments and lack of crisis aware-
ness. At some international seminars about lactic acid bacteria, the safety issues of
related probiotics have also been mentioned or fully valued. Researchers, especially
enterprises and R&D institutions, have invested more energy into the functional
research of lactic acid bacteria, and the safety problems are greatly neglected.
11.1.2.1 Safety Status of Lactic Acid Bacteria in Food
Status of Antibiotic Resistance Genes in Lactic Acid Bacteria
Recently, it has been reported that many intestinal bacteria have antibiotic resistance
genes. Masco et al. found that some bifidobacteria isolated from human, animal,
and probiotic products have tetracycline resistance activity, and the tetW gene is
responsible for drug resistance expression in all strains (Masco et al. 2006). Aires
et al. reported that bifidobacteria isolated from humans contain 26% and 7% of the
tetW and tetM genes, indicating that many bacteria in our gut acquired antibiotic
resistance, such as tetW, and having the risk that the antibiotic resistance gene has
been transferred to harmful bacteria (Aires et al. 2007). Liu and Pop created a data-
base of antibiotic resistance genes (Liu and Pop 2009), and we continue to expand
the resistance gene library of probiotics including bifidobacteria and lactobacilli in
this book. Some antibiotics such as tetW, tetC, tetL, and cata-1 are expressed in
some strains of Bifidobacterium and Lactobacillus (Table 11.2). The tetW gene is
Table 11.2 List of antibiotic resistance genes carried by bifidobacteria in the antibiotic resistance
gene databasea
Drug
resistance Bifidobacterium listed in the Lactobacillus listed in
Antibiotic type gene database the database
Tetracycline tetW B. longum subsp. infantis, B. L. reuteri, L. johnsonii
bifidum, B. animalis subsp. lactis
Tetracycline tetC B. thermophilum, Bifidobacterium L. reuteri, L. paracasei
spp., B. bifidum
Tetracycline tetL B. thermophilum L. sakei
Chloramphenicol cata-1 B. adolescentis
Chloramphenicol ermX B. thermophilum
The database of antibiotic resistance genes: http://ardb.cbcb.umd.edu/index.html
a
present in various bifidobacteria such as Bifidobacterium longum, Bifidobacterium

animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium longum subsp.
infantis, and Bifidobacterium thermophilus. Therefore, various studies have shown
that many bifidobacteria and lactobacilli in the human gut have already carried anti-
biotic resistance genes and that this antibiotic resistance may be present in patho-
genic bacteria, posing a potential threat.
Some reports have revealed the source of antibiotic resistance and how these
genes are transmitted to bifidobacteria and other bacteria and studied the antibiotic
susceptibility in probiotic products containing bifidobacteria in the Japanese mar-
ket. In the report, Bifidobacterium spp. are isolated from various probiotic products,
including Bifidobacterium longum subsp., Bifidobacterium breve, and
Bifidobacterium animalis subsp. lactis, in which all Bifidobacterium animalis subsp.
lactis, have tetracycline resistance and carry the tetW gene. Kastner et al. also found
that B. lactis isolated from probiotic products contained the tetW gene (Duran et al.
2012; Kastner et al. 2006). In addition, Gueimonde et al. reported that all B. anima-
lis subsp. lactis strains isolated from products including yogurt, fermented food, or
probiotic supplements carry tetracycline resistance gene tetW (Gueimonde et al.
2010). These findings indicate that there are many commercial products of bifido-
bacteria probiotics containing the tetW gene, and the existence of the gene tetW is
the minimum requirement of safety assessment on the selection of probiotic
Bifidobacterium strains.
Safety Status of Lactic Acid Bacteria Products Infected with Human Body
Because the usage amount of lactic acid bacteria is very large, the safety evaluation
of lactic acid bacteria is very important. The safety of some probiotics with a long
history is clear. For a long time in the past, Lactobacillus, Leuconostoc, Pediococcus,
and Streptococcus thermophilus have been used in food processing. There was a
long history that people consumed foods containing lactic acid bacteria or their
metabolites (Ishibashi and Yamazaki 2001; Yan et al. 2015; Doron and Snydman
2015). Until now, the safety of these bacteria has not been questioned, and reports
of the harmful effects of these bacteria are extremely rare (except for pathogenic
enterococci and streptococci). The infection cases caused by Lactobacillus and
Bifidobacterium are very rare, and the probability of causing endocarditis or bacte-
remia is only 0.05–0.40%. But if early diagnosis and appropriate treatment are not
performed, the overall mortality rate is high, close to 25% (Cannon et al. 2005). It
has been reported that Leuconostoc causes less than 0.01% of bacteremia cases, and
lactic acid bacteria are also associated with epidemic diseases in specific hospitals
in Spain or India (Bou et al. 2008; Taneja et al. 2005). Enterococcus is a major
exception in lactic acid bacteria (without pathogenic streptococci), and its probabil-
ity of causing bacteremia is 5–15% (Aguirre and Collins 1993; Wang and Wang
2014; Gasser 1994; Salminen et al. 1998; Saxelin et al. 1996).
Most of the lactic acid bacteria that cause infection in the population belong to
Enterococcus faecalis and Enterococcus faecium (Johnson 1994; Franz et al. 2003;
382 W. Chen et al.
Oguntoyinbo and Okueso 2013; Valenzuela et al. 2009; Murray 1990), but other
lactic acid bacteria, including L. rhamnosus, L. acidophilus, L. paracasei, L. casei,
L. curvatus, L. sphaericus, L. salivarius, Leuconostoc lactis, Leuconostoc citreum,
Leuconostoc pseudomesenteroides, Pediococcus acidilactici, and have also been
reported in human infections. Cannon et al. (2005) reported that 241 cases of endo-
carditis and bacteremia were caused by Lactobacillus between 1950 and 2003.
Lactobacillus casei (35.7%) and Lactobacillus rhamnosus (22.9%) are the most
common strains causing infective lactobacilli and endocarditis. In general, the vast
majority of patients infected with lactic acid bacteria have a potential disease, which
make them more susceptible to infection. Age and pregnancy are not risk factors for
lactic acid bacteria infection. For enterococci, infective endocarditis is prone to
occur in patients with underlying heart disease. Risk factors of endocarditis include
genitourinary diseases, abortion, or urinary tract infections. Risk factors of bactere-
mia caused by Enterococcus include underlying diseases, urethral or vascular
bypass, surgery, severe burns, multiple trauma, or antibiotic treatment (Franz et al.
2003; Morandi et al. 2013; Fortina et al. 2008). For Pediococcus infections, there is
a risk of the following underlying disease such as diabetes, tuberculosis, vascular
complications, burns, thyroid hyperactivity, abdominal surgery, stomach tube feed-
ing (whether pregnant or not).
The individuals with low immunity are generally more susceptible to be infected.
However, consumption of probiotics (lactic acid bacteria) does not increase the
infection chance in these populations (Borriello et al. 2003). In contrast, probiotics
are widely used in the clinic for the treatment of diseases. Probiotic lactobacillus is
reported to treat children with severe underlying diseases and diarrhea (Land et al.
2005). The probiotic strain L. rhamnosus is also used to treat diarrhea caused by
invasive diseases. Besselink et al. (2008a, b) and van Baal et al. (2011) found that
oral administration probiotics containing four lactic acid bacteria and two
Bifidobacterium longum can improve therapy efficacy in severe acute pancreatitis
(van Baal et al. 2011; Besselink et al. 2008b). Nevertheless, probiotic prevention
does not reduce the risk of infection complications and is even associated with an
increased risk of mortality.
11.1.2.2 Safety Status of Clinical Medicinal Lactic Acid Bacteria
With the development of microecology research on lactic acid bacteria, there is grow-
ing concern on the health of gut microbes, and related microecological preparations
are also emerging. However, there are still security concerns on using lactobacillus
and other bacterial formulations in health care and clinical care. Common medical
probiotic preparations generally include a single live bacterial preparation, such as
bifidobacterium live bacteria capsule, lactasinum biofermin, etc.; multi-bacterial
combined preparations, such as bifidobacteria triple live bacteria capsules; and dead
bacteria preparations, such as oral lactobacillus dispersion. Besides, functional explo-
ration of probiotics is not limited to the intestines; other organs, such as the skin,
vagina, oral cavity, etc., are also well studied and have been clinically applied.
At present, probiotic lactic acid bacteria applied to clinical or health-care drugs

are continuously supplemented and being developed; the microecological drugs that
have obtained the drug approval number in China are mainly active bacteria probi-
otic preparations. These probiotics are mainly lactic acid bacteria and some Bacillus
and physiological fungi. In general, the normal gut microbes consist of three types
of bacteria: resident bacteria, symbiotic bacteria, and passing bacteria. Among
them, the resident bacteria are relatively stable, indispensable, and low in immuno-
genicity and mostly are anaerobic bacteria; the symbiotic bacteria rely on the origi-
nal bacteria to exert physiological functions, which cover most lactic acid bacteria
in the gut, while passing bacteria, including nonpathogenic bacteria and conditional
pathogens, can colonize in the host intestinal and cause disease when the immune
function is impaired or the resident bacteria are disordered. It can be seen that the
drug lactic acid bacteria as gut microecological preparation can reach the target
organ through the host digestive tract barrier, colonize the digestive tract in a certain
period of time, and exert a physiological action and can be metabolized naturally
from the host. Although there is great potential of lactobacillus in domestic medi-
cine, the related research and development is still in start-up stage, and it is urgent
to introduce and implement relevant medical safety regulations. How to rationally
use lactobacillus in clinical and other medical medicines and what safety problems
exist in probiotic products have not been solved.
Lactic acid bacteria are not the main drug in clinical medicinal. High school medi-
cal practice in a small Canada town showed that only 31% of resident had clinical
knowledge of probiotics, while 24% believed that probiotics do not work in medical
practice (Edmunds 2001). However, 76% of physicians believe that probiotics are very
useful in their clinical practice; the potential market and the lack of relevant experience
in this area are worrisome. Many health-care professionals, such as hospital practitio-
ners, physiotherapists, massage therapists, and herbalists, routinely use products con-
taining lactobacilli, bifidobacteria, or other probiotics. However, due to the limitations
of relevant training, doctors may not be able to access relevant evaluations and cannot
discuss the advantages and disadvantages of the so-called nontraditional, complemen-
tary, alternative medicines, which contain probiotics. The government plans to con-
duct policy to manage such drugs separately from other categories; it is difficult for
small businesses to find suitable probiotic-related regulations and drug licenses. At the
same time, when doctors use or recommend these drugs, they may not able to provide
relevant tests to show these drugs have no clinical side effects and are reliable, since it
is difficult to ensure that the large number of strains used in clinical practice has been
sufficiently tested under current research fund conditions.
There are many factors that have prompted doctors to test and evaluate probiotic
strains and other drug substitutes, including fluctuations in the multidrug resistance
genes carried by pathogens, increased demand for natural medicines, and some pro-
biotic effectiveness evidenced by scientific and clinical experiments. Governments
and research institutions should promote relevant regulations and guidance as soon
as possible to ensure the reliability of clinical application strains. Without such a
product identification model, doctors will know very little about the probiotics they
provide to patients, which is very unfavorable for clinical use. However, the current
384 W. Chen et al.
situation is still severe; the analysis of probiotic strains shows that the strains cur-
rently available for drugs, food, or dietary supplements are difficult to meet clinical
medicinal standards.
11.1.2.3 Safety Status of Lactic Acid Bacteria in Feed
Feed enters the human food chain through animal products, so its safety is directly
related to food safety and human health. In the 1980s, the successive occurrence of
major feed safety incidents caused widespread international concern, and the feed
safety issues caused the international wide attention, and it was considered as important
as the food safety issues. Microorganisms were added to feed as an additive and were
generally considered to be a new green additive that is naturally safe and not harmful to
health. With the development of economy and technology, people have higher require-
ments for health and safety, and relevant regulations and requirements in the process of
applying microorganisms to feed are needed to ensure their development.
There are few reports on the safety of lactic acid bacteria supplemented in the feed
in China, which cannot fully explain the safety of lactic acid bacteria. Existing safety
problems cannot be ignored though we started late, and relevant research has not
been carried out by the government. Current scientific research are mostly based on
foreign research and analysis, and research experiment indicators on the feed pro-
duction mainly focused on daily gain, feed conversion rate, mortality, and morbidity,
but there are few indicators around safety. Research on microbial strains can be based
on several questions in key steps in the production process: screening the source of
the strain, the strain variation during the breeding process, the mutual exclusion
between the strains, the toxicology of the finished feed, and epidemiological analy-
sis. In addition, the amount of microorganism supplemented in feed should be analo-
gous to that of common chemical drugs and antibiotics, and it can have a beneficial
effect only when the corresponding amount of additives were attained, that is, the
number of viable bacteria reaches a certain cfu/ml and then the formulated product
meets the corresponding requirements. However, there is almost no regulation on the
content of lactic acid bacteria in the products for feed addition at present, so further
research and improvement of relevant specifications are needed in these aspects.
11.2 valuation Methods and Principles of Lactic Acid

E
Bacteria Safety
11.2.1 eneral Safety Evaluation Methods for Lactic Acid

G
Bacteria
Recently, with the development and prevalence of lactic acid bacteria and related
products, some serious side effects including the sepsis caused by bacterial translo-
cation and the horizontal transfer of antibiotic resistance genes can happen, and the
safety of probiotics, especially a large proportion of lactic acid bacteria, has been
attracting more and more attention. In response to these safety concerns, manufac-
turers must demonstrate the safety of probiotics on species levels, as experimental
studies do not demonstrate that all probiotic strains have the same properties. On
one hand, probiotics with antibiotic resistance genes should not be used in industrial
production in order to prevent the outbreak of pathogenic bacteria with antibiotic
resistance, since current studies cannot prove whether the possible gene of horizon-
tal transfer is located on chromosome DNA. On the other hand, there is a need to
improve hygiene standards in the production process to prevent probiotics from
being contaminated by pathogenic bacteria or allergens to ensure the safety of the
probiotics.
The following discussion will focus on the most common safety issues of lactic
acid bacteria involving the drug resistance, translocation, and production of harmful
metabolites of the bacteria. Courvalin reviewed the safety of the probiotics on anti-
biotic resistance and proposed an algorithm based on the antibiotic resistance for
antibiotic selection of probiotics (Courvalin 2006). Based on this method, we can
determine whether a strain is suitable for probiotics based on the following few
steps. Firstly, we need to know the phenotype of antibiotic resistance, and only the
strain with no resistance to specific antibiotics for probiotic and the strain with resis-
tance to specific antibiotics can be confirmed safe when the antibiotic resistance
genes are conserved. Secondly, if the transferability of the bacteria cannot be
assessed, we need to determine whether the antibiotic gene is available in consider-
ation of the gene transferred to other bacteria, and the strain can be safe only when
the genes are proved to be conserved in chromosomes. For example, if the DNA
fragment contains homologous cognate of family genes, the strain is credible. This
rule is not difficult to understand and can make the whole study extremely
meaningful.
In recent years, with the wide spread of many antibiotic resistance genes, con-
cerns have been growing that probiotics carry pathogenic genes and cause patho-
genic bacteria to have antibiotic resistance genes. Probiotics carrying antibiotic
resistance genes cannot be used in food chains and feeding areas as recommended
by FAO/WHO or EFSA. To prevent the spread of antibiotic resistance genes in the
environment (including our gut), all probiotic product manufacturers need to review
the product from the perspective of antibiotic resistance and select the appropriate
probiotics according to the rules set by Courvalin (Courvalin 2006).
11.2.1.1 Evaluation of Drug Resistance of Lactic Acid Bacteria
Overview of Drug Resistance and Drug Resistance Genes of Lactic Acid

Bacteria
Lactic acid bacteria strains isolated from feces of human infected by diseases, par-
ticularly Enterococcus, are known to be resistant to various antibiotics (Klare et al.
1995a). Enterococcus may be born with an intrinsic resistance gene, and the
386 W. Chen et al.
resistance gene is located on the chromosome or acquired, and its resistance gene is
located in the plasmid or transposon (Murray 1990; Klare et al. 1995a; Clewell
1990). Intrinsic antibiotic resistance includes antibiotics of cephalosporin, low con-
centration of β-lactam, sulfonamide, low concentration of clindamycin and amino-
glycoside antibiotics, and acquired resistance includes antibiotics of
chloramphenicol, erythromycin, and high horizontal clindamycin, aminoglycoside
antibiotics, tetracycline, high concentrations of beta-lactams, quinolones, and gly-
copeptides such as vancomycin (Murray 1990; Klare et al. 1995a; Leclercq 1997).
The resistance to vancomycin is especially a concern for it is considered to be the
last treatment for infectious diseases caused by multidrug-resistant enterococci. In
the mid-1990s, anti-vancomycin enterococci (VRE) in Europe proved to be derived
from farm animals and probably due to the extensive use of the glycopeptide anti-
biotic vancomycin (Bjorkeng et al. 2011; Klare et al. 1995b). VRE have also been
shown to be isolated from various farm animals, which are the main cause of VRE
(Klare et al. 1995b; McDonald et al. 1997). Therefore, in 1997, the EU prohibited
the use of vancomycin in animal husbandry. In 1999, the United States approved
the use of streptavidin B/A-quinapoddine-dafopridine (Synercid®, King’s
Pharmaceuticals, Bristol, TN) for the treatment of VRE. Jensen et al. reported that
anti-streptavidin strains were transferred from farm animals to farmers, indicating
that enterococci-resistant strains are increasing (Jensen et al. 2000). Recently, new
antibiotics such as linezolid and daptomycin have been successfully used in the
treatment of VRE; however, the emergence of drug-resistant strains seems to be
only a matter of time, and multidrug-resistant enterococci strains with these antibi-
otics are constantly emerging.
Non-enterococci can be obtained from medicine and food, some of which are
inherently resistant and the others are transferable. Leuconostoc, Pediococcus, and
some Lactobacillus such as L. rhamnosus, L. paracasei, L. plantarum, and L. reuteri
are inherently resistant to vancomycin, while most Lactococcus and L. acidophilus
are susceptible to it (Klare et al. 2007; Ammor et al. 2007; Delgado et al. 2005;
Danielsen and Wind 2003). Klein et al. proposed that the resistance of Leuconostoc,
Pediococcus, and Lactobacillus to vancomycin was due to the D-alanine-D-lactic
acid structure in its peptidoglycan (Klein et al. 2000).
Most of the Lactobacillus, Lactococcus, Streptococcus, and Pediococcus are
resistant to metronidazole because they lack the hydrogenase activity (Ammor et al.
2007). On the one hand, most Lactobacillus, Pediococcus, and Leuconostoc can
tolerate high concentrations of cephalosporins. On the other hand, Lactobacillus
and Lactococcus are sensitive to penicillin and β-lactam antibiotics but resistant to
cephalosporin. And the resistance of Streptococcus thermophilus to penicillin G and
ampicillin is highly variable (Ammor et al. 2007; Temmerman et al. 2003; Katla
et al. 2001). Penicillin G and imipenem, a β-lactam antibiotic, are generally active
against Pediococcus; however, some reports suggested that some Pediococcus are
resistant to β-lactam (Ammor et al. 2007).
Lactobacillus, Leuconostoc, and Lactococcus are generally intolerant to antibi-
otics that inhibit protein synthesis, such as chloramphenicol, erythromycin,
clindamycin, and tetracycline antibiotics, but are resistant to aminoglycoside antibi-
otics such as neomycin, kanamycin, streptomycin, and gentamicin tolerance

(Ammor et al. 2007; Delgado et al. 2005; Danielsen and Wind 2003; Temmerman
et al. 2003; Katla et al. 2001; Hummel et al. 2007; Gevers et al. 2003). Some
Lactobacillus, Lactococcus, and Pediococcus strains have been shown to be highly
resistant to chloramphenicol, clindamycin, streptomycin, erythromycin, and tetra-
cycline (Klare et al. 2007; Ammor et al. 2007; Temmerman et al. 2003). In many
cases, antibiotic resistance is attributed to the presence of drug-resistant genes, for
instance, Lactobacillus plantarum (Ahn et al. 1992) and Lactobacillus reuteri (Lin
et al. 1996) have toxin acetyltransferase (CAT) gene. Moreover, erm(B) and erm(T)
genes encoding erythromycin are present in Lactobacillus, Lactococcus, and
Pediococcus (Ammor et al. 2007; Florez et al. 2006; Stroman et al. 2003). The resis-
tance of macrolide antibiotics is related to the mutation of 23S RNA gene locus
from A to G, and the resistance of lactic acid bacteria to tetracycline is related to
tetracycline resistance genes such as tet (K, M, O, Q, S, W) (Ammor et al. 2007;
Gevers et al. 2003; Huys et al. 2004). Rojo-Bezares et al. found the tetracycline
resistance gene tet (L) in Pediococcus parvulus from wine (Rojo-Bezares et al.
2006). At the same time, it has been reported that aminoglycoside antibiotic resis-
tance genes such as aac(6′)le-aph(2″) la, aac(6′)-aph(2″), ant(6) and aph(3′)-llla
exist in some Lactobacillus and Lactococcus. Studies also showed that multiple
drug transporters (Mdt, LmrP) are associated with resistance of lactobacilli to anti-
biotics such as streptavidin, tetracycline, and macrolides (Putman et al. 2001;
Perreten et al. 2001). Lactobacilli are generally resistant to most nucleic acid syn-
thesis inhibitors such as enrofloxacin, pefloxacin, norfloxacin, nalidixic acid, sul-
fonamide, trimethoprim, and metronidazole, and their drug resistance is intrinsic
resistance rather than acquired resistance (Ammor et al. 2007; Han et al. 2015;
Charteris et al. 1998).
In conclusion, it is recommended to use β-lactam (penicillin or ampicillin) and
aminoglycosides (usually gentamicin) for synergistic treatment in the case of
Lactobacillus infection, and erythromycin or clindamycin is available as an optional
adjuvant treatment (Cannon et al. 2005; Danielsen et al. 2007).
Risk Assessment of Lactic Acid Bacteria Carrying Antibiotic Resistance Genes
Teuber et al. warned the presence of antibiotic resistance genes in the human gut
microbiota and food chain (Besselink et al. 2008a). Unfortunately, despite these
warnings, many studies have shown that antibiotic resistance genes remain increased
in the past 10 years, and Enterobacteriaceae carrying the antibiotic resistance gene
NDM-1 has also aroused great concern. Since the transfer of antibiotic resistance
genes to pathogenic bacteria may raise serious problems, careful investigation and
ongoing monitoring of the potential drug-resistant gene transfer in the environment
must be conducted.
Probiotic products contain yogurt, infant formula, cheese, and dietary supple-
ments, which may contain a wide variety of bacterial species (Phillips et al. 2006;
Champagne et al. 2005; Vinderola et al. 2000). A large number of consumers con-
388 W. Chen et al.
sume a significant amount of probiotics each year, and these probiotic strains can
interact very closely with many high-density gut microbiota. Therefore, antibiotic
resistance genes can be transferred to pathogens or gut microbiota by probiotic
bacteria, which can increase the risk of widespread spread of these genes or bacteria
in our gut or environment.
Some studies indicate that the sequence of tetracycline resistance gene tetW in
the tetracycline-resistant Clostridium difficile isolated from humans was found to
have 99% homologous similarity to that of B. longum F8 strain gene sequence
(Roberts et al. 2015; Spigaglia et al. 2008). This finding suggests that the tetW gene
may be derived from a gene exchange between the two strains (which is very
unlikely) or derived from the microbial source sequence gene in the same gut.
Study also suggests that the tetW gene may spread through horizontal gene trans-
fer. In fact, Jacobsen et al. have demonstrated horizontal gene transfer from
Lactobacillus plantarum to Enterococcus faecalis in the gut of sterile mice (Roberts
et al. 2015; Spigaglia et al. 2008). These experiments showed that the human intes-
tinal tract may provide a good environment for the horizontal transmission of gene
levels (including antibiotic resistance genes). For these reasons, whether antibiotic
resistance genes presented in probiotic strains need to be explored and resolved.
The Measurement of Lactic Acid Bacteria Resistance
Since there is no unified method and standard for establishing drug resistance tests
for lactic acid bacteria, it is difficult to evaluate the drug resistance genes of lactic
acid bacteria. Lactic acid bacteria are different from pathogenic bacteria and are not
the main cause of disease in the body. In addition, many lactic acid bacteria have
high nutritional requirements, so they are only suitable for living in nutritious envi-
ronment. This has led to routine drug resistance test media, such as Mueller-Hinton
or Iso-Sensitest media, which are often not suitable for drug resistance testing of
Lactobacillus, Leuconostoc, or Pediococcus (Klare et al. 2007). Herra et al. pointed
out that the growth of Lactobacillus was inhibited when Wilkens-Chalgren agar
medium recommended by the National Committee for Clinical Laboratory
Standards (NCCLS) was used to determine anaerobic resistance (Herra et al. 1995).
However, if 5% horse blood is contained in the Wilkens-Chalgren agar medium, the
Lactobacillus will grow well. Besides, the choice of medium in the resistance test
can greatly affect the minimum inhibitory concentration (MIC). For the selection of
the medium, the nutrition in the medium is best suited to test bacterial growth. Then,
the medium has a suitable gel structure to facilitate the diffusion of antibiotics and
repeated experiments. Finally, known or unknown components of the medium do
not interact with the antibiotics or have a little interaction (Huys et al. 2002). It is
well known that the results of the test will vary with changes in the concentration of
cations and essential nutrients such as thymine and folic acid in the medium.
Therefore, in addition to the composition of the medium, the number of inoculation,
the temperature of the culture, the composition of the gas, and the culture period
may affect the results of the strain resistance test.
In order to solve the problem of test medium standardization, Klare et al. modi-
fied a medium to test the susceptibility of lactic acid bacteria (Klare et al. 2007).
It is a mixture of 90% Iso-Sensitest medium and 10% MRS medium. It can be
added or not added with L-cysteine when testing the susceptibility of lactic acid
bacteria. This medium can provide nutrient-rich lactic acid bacteria (not including
Enterococcus) to grow, and the MIC of different lactic acid bacteria can be well
detected. The results of Klare et al. showed that the MIC value was similar to the
Mueller-Hinton medium of which cation concentration was adjusted and was
supplemented with horse blood (Klare et al. 2005). Mueller-Hinton is a medium
recommended by the Clinical and Laboratory Standards Institute (CLSI) in 2007
for testing the complex nutritional microbial resistance. Tosi et al. developed a S.
thermophilus test medium (SSM) formulation for the specific detection of resis-
tance to S. thermophilus (Tosi et al. 2007). This medium can well test the atypical
tetracycline resistance of the strain. The SSM medium was formulated with 90%
Iso-Sensitest medium and 10% M17 medium supplemented with 0.5% (w/v)
lactose.
Lactobacillaceae family contains a rich variety of genus contains plenty of spe-
cies, it is therefore difficult to choose a MIC threshold to separate resistant and
nonresistant strains. In brief, a resistance threshold can be set on this genus when
most strains in a genus have similar resistance to an antibiotic. However, for most
lactic acid bacteria, this setting is too simple, because different species have great
differences in resistance to different antibiotics. The European Commission’s
Committee on Animal Nutrition provides a list of drug resistance thresholds for
lactic acid bacteria (not including Enterococcus), which is an immature list because
it only includes thresholds for Pediococcus- and Lactobacillus-related genera and
does not consider the difference between species. Danielsen and Wind believed that
there are still many shortcomings in using this method to study the drug resistance
of lactic acid bacteria, because many strains have strong drug resistance, and their
threshold will be much higher compared to those drug sensitivity strains (Danielsen
and Wind 2003). The Scientific Commission for Animal Nutrition (SCAN) gives a
MIC threshold concentration of 1 μg/ml for gentamicin; however, Danielsen con-
sidered that this value is too low, and it is recommended to use the threshold of
128 μg/ml for the resistance of L. paracasei, L. plantarum, L. sinensis, L. pentosus,
L. brevis, and L. rhamnosus and use a threshold of 256 μg/ml for testing for L. aci-
dophilus resistance. At the same time, Danielsen also believes that the streptomycin
threshold of 16 μg/ml set by SCAN is too low and should be adjusted to >256 μg/
ml for all lactic acid bacteria. In addition, they believed that the 4 μg/ml erythromy-
cin threshold recommended by SCAN is too high, leading to the undetectable resis-
tance of certain lactobacilli. For the erythromycin threshold, the recommended
amount is 1 μg/ml for L. acidophilus, L. sphaericus, and L. curvus, 2 μg/ml for L.
paracasei and L. rhamnosus, and 4 μg/ml for L. plantarum and L. pentosus. In 2005,
the Animal Feed Additives and Products Group (FEEDA) gave a threshold table for
13 antibiotics for lactic acid bacteria that can distinguish between homolactic fer-
mentation and heterolactic fermentation (Lactobacillus plantarum, Enterococcus,
Pediococcus, Leuconostoc, Lactobacillus lactis, and Streptococcus thermophilus).
390 W. Chen et al.
Klare et al. studied the resistance of 16 antibiotics to different lactic acid bacteria
strains, including Lactobacillus plantarum, Pediococcus, and Lactococcus (n = 473)
(Klare et al. 2007). They proposed the experimental epidemiological cutoff (ECOFF)
to describe the innate or acquired drug resistance of the strain. Klare et al. deter-
mined the ECOFF values of 12 lactic acid bacteria against different antibiotics,
respectively. The strains are including Lactobacillus pentosus, Lactobacillus rham-
nosus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus fermen-
tum, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus,
Lactobacillus johnsonii, Lactobacillus delbrueckii, and Lactobacillus reuteri.
Therefore, the MIC threshold has a clear understanding. As predicted by the safety
eligibility presumption, the MIC threshold can help us to determine the drug resis-
tance of the strain and filter the strain with high resistance, to study its drug resis-
tance gene and the transformability of this gene.
11.2.1.2 Evaluation of Lactic Acid Bacteria Translocation and Migration
Safety Testing of Lactic Acid Bacteria Migration Ability
In conjunction with the safety studies of the previous sections, in order to confirm
the safety of probiotics associated with bacterial migration or sepsis, the following
studies must be performed:
1. Viscolytic activity
2. Bacterial translocation ability in traditional animal models
3. Intestinal surface structure in animal models
4. Bacterial translocation ability in immunocompromised animal models
5. Infection activity after bacterial translocation
6. Clinical study of healthy people
7. Clinical study of susceptible people
Probiotics are usually consumed as food, and all people have the opportunity to
consume probiotic products, including susceptible people. And most people get the
expected health benefits after taking probiotics. However, it is necessary to confirm
the validity of the probiotic consumption and provide adequate safety data, includ-
ing acute and chronic toxicity, bacterial translocation, and sensitivity to vulnerable
populations (Tompkins et al. 2008). In addition, some probiotic strains have poten-
tial risk of translocation and sepsis, which can pose serious health problems.
Although there are a large number of potential probiotics, not all strains are identi-
cal. Since probiotics have many positive implications for human health, probiotic
producers must choose the right strain and confirm that the source is safe, to avoid
losing consumer trust or triggering safety incidents.
The Possibility of Horizontal Transfer of Antibiotic Resistance from Lactic

Acid Bacteria
The researchers questioned the transfer of tetW from B. animalis subsp. lactis to
other bacteria, including pathogens. Kazimierczak et al. have shown that the prob-
ability of gene-level metastasis in B. longum and B. adolescentis is quite low
(Kazimierczak et al. 2006). At present, no studies have reported the transfer of tetW
from B. animalis subsp. lactis to other bacteria.
In general, horizontal gene transfer occurs by transduction, transduction, trans-
poson, or natural transformation (Levy and Marshall 2004). Transduction is medi-
ated by phage, but few studies have been conducted on phage or bifidobacteria
transduction. Transfection is often occurring in our intestines, but in many cases, the
plasmid needs to transmit genes to the recipient cells. Since most tetW genes are
located on ribosomal DNA rather than in plasmids, it is unlikely that tetW will be
transferred by trans-binding (Ammor et al. 2008a; Ammor et al. 2008b).
It has been reported that transposons such as Tn916 and Tn5276 are mediated by
the horizontal transfer of genes in lactic acid bacteria (Nawaz et al. 2011; Clementi
and Aquilanti 2011; Mathur and Singh 2005). Although a recent research suggested
that the transposable gene tetW of the B. animalis subsp. lactis is upregulated and
the co-transcription of the tetW gene is identical to the transposase gene, there is no
evidence that tetW participates in transposon expression (Gueimonde et al. 2010). In
addition, since the recipient cells can accept naked strands of DNA, natural transfor-
mation allows all chromosomal sequences to be transferred to the recipient cells in
a condition acceptable to the recipient cells. Although natural transformation can
only change a few restricted species, studies have reported surprising results that
Vibrio cholerae has never displayed the natural transformation of antibiotic resis-
tance genes (Yamamoto et al. 2010; Meibom et al. 2005). In their report, antibiotic
resistance genes can be transferred to Vibrio cholerae by natural transformation
only when the growth conditions are very similar to the natural environment, such
as on the surface of the crab shell; antibiotic resistance genes can be transferred to
Vibrio cholerae by natural transformation. However, even the same strain did not
undergo natural transformation in the same environment in vitro. It can be specu-
lated that natural growth conditions include the presence of some key substances (in
this case chitin), high cell density, auxotrophy, or stress conditions, in which case
competent cells are produced and natural transformation is activated.
Considering that the cell density in our intestinal environment is much higher
than that in laboratory using in vitro liquid culture and the large strands of DNA
released in dead bacterial cells are present around the intestinal bacterial cells, nutri-
ent limitation in the intestine may be a normal state. Because the most readily avail-
able nutrients have been absorbed by humans, only the remaining nutrients are
being contested by a considerable number of gut microbes.
In addition, it has been reported that bile salts normally present in the intestine
can induce tetracycline-resistant phenotypic expression in bifidobacteria (Noriega
et al. 2005). Therefore, the intestinal environment may be beneficial for natural
392 W. Chen et al.
transformation. In the decades of research on streptococci, we have known that in

the laboratory environment, only some streptococci have the ability to naturally
transform (Tomasz and Hotchkiss 1964). However, a recent discovery of a novel
mechanism for regulating natural transformation in Streptococcus suggests that this
property may spread more widely in Streptococcus than in previous views
(Havarstein 2010; Johnsborg and Havarstein 2009). Moreover, Chen and Dubnau
pointed out that various bacteria are competent and have their own DNA extraction
system, which indicates that a variety of bacteria in the human gut can occupy
naked-stranded DNA (including tetW and flanking sequences), although further dis-
cussion and investigation can prove that the B. animalis subsp. lactis may be the
origin of the widely spread tetW gene in the food chain, but this possibility cannot
be denied (Chen and Dubnau 2004).
Antibiotic Resistance Gene in Lactic Acid Bacteria
To the best of our knowledge, lactic acid bacteria such as L. crispatus, L. planta-
rum, L. johnsonii, L. reuteri, and E. faecalis are antibiotic resistant, containing
antibiotic resistance genes ermB, tetM, and tetW (Klare et al. 2007; Mathur and
Singh 2005).
Since most of these lactic acid bacteria are used as starters for meat and dairy
products or as probiotic products, they may present a potential risk of transferring
these genes to intestinal bacteria and pathogenic bacteria. In particular, some
Lactobacillus and Enterococcus contain plasmids, and in vivo studies have shown
that plasmid-carrying antibiotic resistance genes in Lactobacillus can be transferred
to other bacteria (Jacobsen et al. 2007). Since many bacteria contained in the human
intestinal flora can obtain exogenous DNA through conjugation, regular horizontal
gene transfer among bacteria can occur in our intestines.
In view of the above problems and based on the fact that bacteria isolated from
dairy products and meat products carry antibiotic resistance genes, bacterial strains
carrying these resistance genes should not be used as feed additives, unless it can be
proved to be caused by chromosomal mutations. That is recommended by the
European Food Safety Authority (EFSA) technical manual for animal feed additives
and products in the FEEDAP section.
Evaluation of Drug Resistance
With the increasing use of antibiotics and the application of some new antimicrobial
compounds to treatment, drug resistance appears in the treatment, and with the
spread of bacteria, the food chain is also one of the ways in which drug resistance
between humans and animals spreads. Concisely, cured meat products and fer-
mented dairy products are a major mediator of bacterial resistance (Versporten et al.
2016; Ponce de Leon-Rosales et al. 2015; Coccolini et al. 2015). Therefore, it is
important to conduct anti-antibiotic gene detection and its potential transmission
mechanism for lactic acid bacteria used in foods, which can effectively reduce the
risk of lactic acid strains carrying drug-resistant genes infecting hosts (Ramakrishnan
and Sriram 2015; Koga et al. 2015).
Zhang et al. performed a comprehensive safety assessment based on genome-
wide sequence and corresponding phenotype of Lactobacillus plantarum JDM1, a
commercial probiotic strain widely used in China (Zhang et al. 2012). At the same
time, the minimum inhibitory concentration (MIC) of JDM1 against 16 antibiotics
and the production of biogenic amines by traditional oral toxicity test were deter-
mined. Overall, JDM1 contains 51 antibiotic resistance-related genes, 126 virulence-
related genes, and 23 adverse metabolic-related genes. However, this strain does not
contain a gene encoded by toxin or hemolysin, and the safety-related gene can
hardly be transferred. This approach can be extended to provide a deep safety sur-
vey of new probiotic strains and greatly reveal its underlying risk factors and
molecular mechanisms. However, not all genes are effective depending on environ-
mental conditions, so this analysis only reveals the theoretical maximum level of
risk.
1. Antibiotic Sensitivity Test
In the safety evaluation of lactic acid bacteria, the evaluation of drug resistance
is very important. Twenty-four antibiotic resistance tests were carried out on 22
strains of Lactobacillus and 9 strains of Bifidobacteria, which are commonly used
in Chinese probiotics and health-care products. The results showed that Lactobacillus
was higher than the Bifidobacteria in the resistance to the antibiotics selected for the
experiment. And all strains are more resistant to nalidixic acid, vancomycin, and
fosfomycin.
2. Antibiotic Resistance Gene Detection
Numerous studies have confirmed that almost all antibiotic resistance in lactic
acid bacteria is nonmetastatic, but drug resistance factors associated with binding
plasmids and transposons may cause drug resistance to shift among intestinal flora
(Fruchart et al. 1997).
11.2.1.3 Evaluation of Harmful Metabolites of Lactic Acid Bacteria
The detection of D-lactic acid is classified into L-lactic acid and D-lactic acid
according to the difference in optical activity. Since the human body only contains
enzymes that metabolize L-lactic acid, excessive D-lactic acid may cause metabolic
disorders and even acidosis. The normal body does not cause obvious disease
response to a small amount of D-lactic acid, but it can cause D-lactic acidosis, intes-
tinal discomfort, and even coma for patients with short bowel syndrome. It is unsuit-
able for infants and other susceptible people to use D-lactic acid-producing lactic
acid bacteria (Fukushima et al. 2014).
The nitroreductase activity was tested to demonstrate that the lactic acid bacteria
have strong nitrate reductase activity. When the human body ingests foods contain-
394 W. Chen et al.
ing more nitrate components, it will be reduced to nitrite by the bacteria in the body
and then formed carcinogen nitrosamines or other intestinal endotoxins. Marino
et al. tested their amino decarboxylase activity against lactic acid bacteria isolated
from Italian cheese according to the method of Bover-Cid and Holzapfel (Marino
et al. 2008).
In addition, due to the presence of amino acid decarboxylase activity in some
lactobacilli, decarboxylation occurs in foods to reduce the amino acids to biogenic
amines (Bover-Cid and Holzapfel 1999; De Llano and Cuesta 1998). The substances
gradually accumulate in the human body, and excessive amounts lead to poisoning.
At the same time, the decarboxylation reaction also leads to the production of nitro-
samines. Therefore, before the products enter the market, the activity of amino
decarboxylase should be tested on related lactic acid bacteria products. The method
can refer to Marino et al. detecting the amino decarboxylase activity of lactic acid
bacteria in Italian cheese (Marino et al. 2008).
11.2.2 afety Evaluation Specifications for Different

S
Functional Lactic Acid Bacteria Products
11.2.2.1 Safety Evaluation Specifications for Lactic Acid Bacteria in Food
Safety evaluation is the most basic requirement for functional lactic acid bacteria.
Although most of the functional lactic acid bacteria are currently isolated from tra-
ditional fermented foods or human intestinal microbial systems, we cannot fully
guarantee their safety as their particularity as food. In previous reports, it was also
found that some lactic acid bacteria were isolated in the diseased or infected tissues.
In some cases, specific lactic acid bacteria also turned into conditional pathogenic
bacteria. It can be seen that the safety evaluation of lactic acid bacteria in functional
probiotic foods is particularly important.
The Food and Agriculture Organization (FAO) and the World Health Organization
(WHO) have proposed guidelines for the safety evaluation of probiotic strains in
foods. The necessary evaluation steps include drug resistance evaluation, human
intervention evaluation, metabolic activity evaluation, epidemiological evaluation
in circulation, animal-targeted infection evaluation, and special activity evaluation,
such as hemolysis test evaluation, toxigenic test evaluation, and other basic aspects.
Classifying from the experimental categories, the safety evaluation of lactic acid
bacteria is generally divided into pathogenicity, toxicity detection, metabolic activ-
ity detection, and strain intrinsic property detection. Classifying from the subject, it
is generally divided into in vitro experiments, animal targeting experiments, and
human clinical intervention experiment.
Many studies report the health benefits of probiotics, such as improving the
intestinal environment, enhancing immunity, reducing the risk of allergies, reducing
diarrhea, improving constipation, and reducing the risk of cancer (Xie et al. 2014;
Farid et al. 2011; Preter et al. 2007; Fujii et al. 2006; Xiao et al. 2006; Ogata et al.
1999; Colombel et al. 1987). Consumers purchase probiotic products based on these
expectations. However, some of the concerns about the safety of probiotics, includ-
ing the infection (such as sepsis) and antibiotic resistance genes, have been reported
(Whelan 2010; Courvalin 2006; Snydman 2008). Since most probiotic consumption
is food rather than drug, any potential risk factors for human health within the pro-
biotic product must be excluded.
Another unique feature is that probiotics can be ingested by a variety of people
at any time, including susceptible people, such as infants, children, the elderly, and
patients. Therefore, not only the effectiveness to healthy people needs to be consid-
ered but also the safety of risk groups (Liong 2008). In 2008, an important issue
concerning the safety of probiotics was reported. Clinical studies of severe acute
pancreatitis in the Netherlands have shown an increased risk of mortality by probi-
otics (Besselink et al. 2008a). This finding warns of social and scientific research
that the safety of probiotic must be taken seriously and considered.
Probiotic products have been widely used in foods in the world, such as yogurt,
baby formula, and dietary supplements. Some risk factors such as drug resistance
are present in probiotics and can be spread globally. In addition, many antibiotic
resistance genes are present in intestinal bacteria has been reported. We need to
continue to pay attention to these concerns. At the same time, the quality of probi-
otic products is another important part of the safety of probiotics. Probiotics have
been added to many infant or child formulas in recent years (Abe et al. 2009). Since
pathogenic bacteria such as Salmonella contamination is one of the biggest prob-
lems with formula, the production of probiotic powder in formula requires high
hygienic specifications (Cahill et al. 2008). Besides, allergen contamination can
cause serious problems; in order to make consumers consume probiotic products
safely, these potential problems should be solved. .
In recent years, people have gradually begun to understand the benefits of LAB
to the human body. Toxic diarrhea, antibiotic-induced diarrhea (AAD), allergies,
inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and constipa-
tion can be improved by probiotics. The different strains have different degrees of
beneficial effects on acid and bile salt tolerance, intestinal adhesion, clinical-specific
effects, and host health. LAB are one of the most widely used probiotics. It is gener-
ally accepted that the use of LAB to ferment products is feasible and safe. However,
a few studies have shown that LAB can be isolated from patients with endocarditis,
sepsis, liver abscess, and urinary tract infection, so the safety of LAB has attracted
people’s attention. Lara-Villoslada et al. (2007a, b) evaluated the oral toxicity of the
isolated probiotic L. coryniformis CECT5711 and Lactobacillus gasseri CECT
5714 (Lara-Villoslada et al. 2007a). In an oral toxicity analysis of 20 Balb/c mice
after oral administration of CECT5711 or CECT5714 (1010 cfu/d) for 30 days, the
results indicated that CECT5711 and CECT5714 do not have harmful enzyme
activity and inherent antibiotic resistance characteristics. After ingestion of the two
strains, the mice had no side effects on their body weight or diet, no bacteremia in
the liver or spleen, and no treatment-related bacterial translocations in these organs.
The liver glutathione content and plasma lipid peroxidation concentration of mice
in the probiotic group were not statistically different from the control group.
396 W. Chen et al.
Probiotic treatment did not cause changes in biochemical and blood parameters, so
it can be inferred that CECT5711 and CECT5714 are not pathogenic and may be
safe for human health. It is necessary to evaluate the safety of newly selected LAB
that can be used as probiotics, especially for food.
At present, the safety evaluation of LAB in domestic and abroad is mainly aimed
at the toxicological evaluation of foods with microorganisms as raw materials or
starter. It is necessary to conduct toxicity tests, including three teratogenic mutation
experiments and 30d feeding experiments.
Yakabe et al. (2009) reported that the probiotic Lactobacillus sp. KB290, iso-
lated from plant, has functions to improve intestinal health and stimulate immunity
(Yakabe et al. 2009). The genotype; acute, subacute, and subchronic toxicity; and
the potential for bacterial migration of KB290 were studied deeply. The result of
bacterial back mutation experiments, using preincubation method, showed that
KB290 was not mutagenizable. In a single oral toxicity test, the maximum nontoxic
amount of KB290 was 109 cfu/ml (20 ml/kg). In a subacute trial, after daily
administration of 108 cfu/kg, 109 cfu/kg, or 1010 cfu/kg of KB290 to rats for 2 weeks,
we did not observe a significant therapeutic-related effect or the evidence of bacte-
rial translocation in the gastrointestinal tract. When the rats were orally adminis-
tered with KB290 for 13 weeks (subchronic test), there was still no significant
treatment-related effect and no significant toxicological effects. Based on these
results, we believed that the highest dose tested (1010 cfu/kg/d) is the level of no
visible harmful effects (NOAEL). These results indicated that KB290 is safe for
human.
1. Salmonella Reverse Mutation Assay
The Salmonella Reversal Mutation Test (Ames Test) was established and devel-
oped by Ames and his team at the University of California for more than 10 years.
This method has been widely used for the detection of mutations and antimutagenic
ability and the screening of carcinogens because of its advantages of speediness,
simplicity, sensitivity, economy, and suitability for testing mixtures. Ebringer et al.
(1995) tested the mutation and anti-mutation ability of Lactobacillus spp. CCM4179
and CCM4180 by Ames test (Ebringer et al. 1995). The results showed that there
was no obvious mutagenic effect.
2. Bone Marrow Micronucleus Test
This method is suitable for determining the mutagenic effects of a substance. In
the late stage of cell mitosis, when chromosomes enter a daughter cell to form a
nucleus, the chromosome or chromatid fragment remaining in the cytoplasm is
called a micronucleus. After the terminal phase, a subnucleus that is much smaller
than the nucleus is formed. Domestic researchers performed bone marrow micro-
nucleus experiments on Yiliduo brand active LAB milk drink using SPF-class NIH
mice. The micronucleus test results were negative at doses of 5.00–40.00 g/(kg·BW),
indicating that Yiliduo had no mutagenic effects at the somatic level.
3. Mice Sperm Abnormality Test

Many chromosomal genes directly or indirectly determine the morphology of
sperm, and sperm abnormalities are the result of genetic mutations, indicating
changes in related genes and their protein products. The method is suitable for eval-
uating the genotoxicity of health foods to male germ cells.
The detection of total glutathione in the liver. The reduced glutathione (GSH)
exerts the ability to resist oxidation and scavenge free radicals mainly through the
active sulfhydryl group on the side chain of cysteine and clinically acts as an impor-
tant pharmaceutical ingredient for protecting liver. Lara-Villoslada et al. (2009)
studied the GSH level in liver tissue after continuously giving L. fermentum
CECT5716 to Balb/c mice for 2 days (Lara-Villoslada et al. 2009). The GSH levels
before or after gavage were basically equal, indicating that the CECT5716 had no
negative effects on liver function. In addition, it has been reported that L. rhamnosus
CCFM1107 can improve alcoholic liver injury (Tian et al. 2015).
The detection of serum malondialdehyde (MDA) concentration. Peroxidation
reaction occurs with the free radicals in the organism acting on the lipids. The final
product is malondialdehyde, which causes cytotoxicity as the polymerization of
molecules, such as proteins and nucleic acids. Due to the low concentration and
instability of free radicals in the body, the MDA level is generally used for reflecting
the metabolism of free radicals in the body. Therefore, it can indirectly reflect the
damage degree of cells attacked by free radicals. Lara-Villoslada et al. (2009) used
HPLC to determine the MDA level in the blood after continuous gavage of L. fer-
mentum CB5716 for 28 days in Balb/c mice (Lara-Villoslada et al. 2009). The
results showed that there was no significant difference between the mice in CB5716
group and control group.
The detection of serum amyloid A (SAA) protein. SAA, an acute phase-reactive
protein, is associated with high-density lipoprotein (HDL) and can regulate HDL
metabolism during inflammation. SAA concentration is a sensitive indicator of
inflammation in the early stages of infectious diseases and a plasma indicator for the
analysis of sepsis. Lara-Villoslada et al. (2007a, b) intraperitoneally injected L. sali-
varius CECT5713 in Balb/c mice; SAA level in blood was detected by ELISA
(Lara-Villoslada et al. 2007b). The results showed that SAA concentration increased
in the short term after injection but can return to normal levels soon.
The detection of bile salt hydroxylase activity. Some LAB have the ability to
degrade bile salts (Ren et al. 2011). If bile salt dissociates in the small intestine, it
will affect the digestion and absorption of fat. If it does not dissociate in the large
intestine, it also will affect the enterohepatic circulation and then affect the diges-
tion and absorption of fat. Bacterial metabolism affects the content of secondary
bile salts in bile acids, which can directly affect the activity of bile salt hydroxylase
and indirectly affect the solubility of bile salts by affecting the acidity and alkalinity
of the colon. Therefore, LAB do not have bile salt hydroxylase activity in vivo.
Some studies examined the concentration of bile salt hydroxylase in LAB by MRS
culture in vitro and isotope tracing methods in vivo (Wang et al. 2015; Abouakil
et al. 1989).
398 W. Chen et al.
11.2.2.2 LAB Evaluation Criteria in Clinical or Health Care
The Joint Working Group of FAO and WHO drafted new guidelines for the probi-
otic evaluation in food in May 2002. FAO and WHO and the countries they repre-
sent have established evaluation criteria based on the definition of probiotics and the
minimum requirements for accurate health verification. Although the FAO and
WHO reports were primarily in food field, a number of recommendations including
the definition of probiotics were adopted at the International Science Conference in
May 2002. Based on these guidelines, several important specifications and stan-
dards must be introduced to ensure that physicians know the quality and reliability
of the products they prescribed or recommend, the primarily points as shown in the
following.
Strain Identification
The first consideration is using internationally recognized methods to confirm and

represent the genus and species of strain, such as DND-DNA hybridization and 16S
rRNA-encoded DNA sequencing (Wang et al. 2002). The most important methods
of strains differentiation are pulsed electric field gel electrophoresis and random
amplified polymorphic DNA technology. Strain differentiation and characterization
can also be performed by determining the genetic elements other than chromo-
somes, such as plasmids. Once the strain has been identified, it must be named
according to bacterial name approval list and update the list.
The second consideration is to define a strain name according to the probiotic
function that is consistent with its characteristics, such as L. rhamnosus GG (LGG).
This will enable doctors and consumers to track publications related to the strain
and verify the strain do exhibit the probiotic properties. An important example of a
strain name causing confusion is that a so-called probiotic yogurt mentioned in a
publication has side effects on patients, but in fact, 12 of the 16 strains mentioned
have no probiotic characteristics (Sipsas et al. 2002). Similarly, the benefits of
strains claimed on a website or label, which are not in the product or never been
proven (Reid et al. 2001). This is not to say that such products are defective or unre-
liable, but manufacturers need to improve the probiotic characteristics of the
products.
In Vitro and In Vivo Tests
In the early 1980s, some research teams discovered probiotic strains of Lactobacillus
and provided a useful screening system in vitro. Therefore, characteristics such as
cell adhesion, acid production, bacteriocin, hydroperoxide, and ability to inhibit
adhesion of pathogens are considered to be the most important factors for conferring
functional properties of probiotics (Gopal et al. 2001; Reid and Bruce 2001). These
methods can also be used for strain differentiation, but a single method is not
sufficient. In short, before these factors are used for predicting the effects of probiot-
ics on the human body, they need to be expressed in vivo and verify the main mecha-
nism. In vitro tests such as bile salt tolerance can be associated with gastric survival
tests in vivo; spermicide resistance can help probiotics survive in the vagina.
Many other trials that need to be validated in vivo still have advantages in assess-
ing and characterizing the body and studying the underlying mechanisms. For
example, Lactobacillus can adhere to intestinal cells and signal mucus products,
thereby preventing the adhesion of pathogens, which was very valuable and pro-
posed a new function of probiotics in the intestine. In addition, the study also
revealed the mechanism by which Lactobacillus interferes with the pathogenesis of
E. coli, including analysis of the inflammatory effects of lipopolysaccharide antago-
nized by competition of lipoic acid with soluble CD14 (lipopolysaccharide binding
protein) (Vidal et al. 2002). The important concept shows that probiotics can directly
benefit the host.
The Safety
Probiotics are living organisms, so they can infect the host. Historical data indicate
that LAB and bifidobacteria in the form of capsules are safe for humans. This con-
clusion is supported by their normal symbiosis with bacteria in mammals and their
safe use in different food and supplement products. Despite this, it has been reported
that they have side effects, including rare systemic infections. Some susceptible
populations, such as immunocompromised individuals and those with symptoms of
intestinal bleeding, should be cautious when taking live bacteria (Marteau 2002).
Patients with susceptibility to arthritis or other complications should also be careful
to ensure that they do not induce excessive immune stimuli.
In order to establish safety regulations for probiotics, FAO and WHO recom-
mend the east tested groups of probiotic strains for experimental evaluation, includ-
ing antibiotic resistance evaluation, metabolite activity, toxin production, hemolytic
activity, infection in immunocompromised animal models, and incidence of side
effects in the human and in consumers. But the side effects of Lactobacillus strains
are very few. A large number of studies confirm their functional effects, and few
studies research side effects.
Phase II of Clinical Study
The second phase of the clinical study primarily assessed the effectiveness of a
product relative to placebo. For an individual, clinical outcomes should have statis-
tically and biologically significant improvements in symptoms, life quality, reduc-
tion in disease risk or duration, and rapid recovery from disease. More credible
clinical evidence is needed in a wider range of clinical examples. These studies need
to provide the doctor with the strain name, form of production, and the effectiveness
of these strains in some respects. L. rhamnosus GG and L. reuteri SD2222 have a lot
400 W. Chen et al.
of good clinical data, but it is not possible to simply apply the strains to the clinic.
The LGG strain is a liquid form product from Finland, while the US product is put
into a capsule. The study of SD2222 is even less clear with fewer articles in the
relevant reports, and it is not a product that can be sold in labels in the United States.
Phase III of Clinical Study
The third phase of the clinical study is to evaluate the effectiveness of the product
compared to the standard treatment of a particular disease. In general, on the one
hand, it is necessary to provide the results of randomized and double-blind experi-
ments, determine the sample amount and use it reasonably, and obtain truthful
results. Such experiments need to consider life quality assessments and risk-benefit
ratios. For example, 3-hydroxy-3-methylglutaryl coenzyme as a reductase inhibitor
product may reduce HDL cholesterol levels by 45% but may also cause rhabdomy-
olysis, kidney damage, or death. On the other hand, the results of animal experi-
ments showed that taking 104 cfu/d of L. reuteri CRL1098 for 7 days can prevent
hypercholesterolemia and have a 17% probability of causing HDL to become LDL
(Taranto et al. 2000). It may be reappeared in the human body and then will provide
a significant improvement in clinical treatment to no side effects. In other words,
careful planning and extensive evaluation for results are required before deciding
whether probiotics should be applied to phase III clinical trials.
Health Claim and Product Launch
In more cases, probiotics are allowed in foods only in general health conditions. In
terms of drug use, it is demonstrated that probiotics were superior to placebo in
certain situations. A three-stage clinical study is required for a specific condition at
the time of drug review. Clear and specific statements and labels should be marked
on the market to let patients know the true benefits of this probiotic drug. For exam-
ple, statements such as the information of “reducing the incidence and severity of
infant rotavirus diarrhea” are more accurate than “improving gut health.” In addi-
tion, the disclaimers stated on some company websites are enforced unless they can
be proven, which will greatly increase consumer trust for product (Reid et al. 2001).
11.2.2.3 Criterion of LAB for Feed and Crops
The safety of microorganisms as feed additives needs to be standardized and

improved. In addition to the existing additives and veterinary drug management
regulations, some factors, such as animal safety, consumer safety, and environmen-
tal safety, must be considered. In the application process, it should be proved that it
is safe and harmless through rigorous experiments before it can be widely applied.
The Identification of LAB
Since LAB are generally recognized as safe (GRAS), the strains added to the feed
must belong to the certification safety catalog, and strain identification is the basis
of safety regulations. Bacterial identification can be performed by 16S rDNA
sequence analysis, PCR product analysis, and so on. Berger’s Manual of Systematic
Archaea and Bacteriology (2015) can be used for reference if necessary, including
strain isolation and purification, morphological observation, sugar fermentation
experiments, and physiological and biochemical property analysis.
Operating Environment and Personnel Safety
In the process of feed production and addition, it is necessary to ensure the safety of
animals and breeders and keep qualified environment. Therefore, it is significant to
pass the drug resistance and toxicological tests of LAB, including general drug
resistance test- and toxicology test-related experiments, such as carcinogenicity
test, acute toxicity testing, and so on. At the same time, considering the relative bal-
ance of the types and quantities of microorganisms in the environment and the self-
regulation within a certain range, large-scale feeding of single or multiple LAB
strains will impact the number of bacteria in the surrounding environment and will
endanger the microbial ecosystem once the formation of dominant flora. This haz-
ard will not have a major impact in the short term, but it will also significantly
change the composition of environmental microbes in the long-term, posing a
potential hazard on maintenance of ecological balance.
Safety for Animals
Although the breeding cycle is relatively short from the birth of the animal to the
slaughter, the long-term LAB feeding experiments are still necessary, which can most
directly prove the harmlessness of the product. Generally, long-term feeding experi-
ments need to be fed for 5–7 years, which proves that it has no adverse effects on
animal growth and development. In addition, the effects of feeding LAB feed on ani-
mal reproduction must be further verified. When its safety on heredity has been veri-
fied, the product’s safety can be determined, and thus it can be widely launched.
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Lactic Acid Bacteria

ISBN 978-981-13-7831-7 ISBN 978-981-13-7832-4 (eBook)

© Springer Nature Singapore Pte Ltd. and Science Press 2019

1.1  ection I Overview and Definition of Lactic Acid

1.1.1 Overview of Lactic Acid Bacteria

© Springer Nature Singapore Pte Ltd. and Science Press 2019 1

1.1.2 The Definition and Membership of Lactic Acid Bacteria

Lactic acid bacteria are an important class of prokaryotic microorganisms. However,

descriptions of lactobacillus from the aspects of cell morphology characteristics and

1.2  ection II Development History and Applications

1.2.1  tage Division of Development Course and Applications

1.2.2  evelopment Course of the Practical Application

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

1.2.2.1  nconscious Use of Lactic Acid Bacteria Derived from Empirical

 ifferent Types of Lactic Acid Bacteria-Fermented Dairy Products Are

 ermentation of Fruits and Vegetables and Fermented Grains by Lactic Acid

 ong-Term Practical Application Makes People Realize that Lactic Acid

1.2.2.2 Practical Application of Lactic Acid Bacteria Based on Science

With the development of microbiology represented by the research of Pasteur and

 ommercial Production of Yogurt and Other Lactic Acid Bacteria-Fermented

Application of Lactic Acid Bacteria Fermentation Products

Drugs and Medical Applications of Lactic Acid Bacteria

1.2.3  he Development of Scientific Research on Lactic Acid

1.2.3.1 Pasteur’s Contribution to Lactic Acid Bacteria

development of related science. For lactic acid bacteria, Pasteur’s contribution is

1.2.3.2 Discovery of Some Important Lactic Acid Bacteria

As early as 1847, C. Blondeau studied the fermentation process of lactic acid,

1.2.3.3  stablishment of the Basis and Application System of Lactic Acid

In addition to his contribution to basic biology, bacterial taxonomy, Orla-Jensen’s

1.2.3.4  nalysis of Basic Physiological Metabolism of Lactic Acid

The physiological metabolism of lactic acid bacteria mainly involves nutrition

1.2.3.5 Molecular Biology Period of Lactic Acid Bacteria Research

1.2.3.6  xploration and Application of the Function of Lactic Acid

posed by several scientists represented by Metchnikoff, which believes that lactic

function of the intestine to systemic metabolism, neurodevelopment, mental cogni-

1.3  ection III Application of Lactic Acid Bacteria in Human

1.3.1 Lactic Acid Bacteria and Food Manufacturing Industry

1.3.2  actic Acid Bacteria and Farming and Aquaculture

1.3.3 Lactic Acid Bacteria and Medical Health Industry

1.3.4  actic Acid Bacteria and Industrial Manufacturing

saccharide, γ-aminobutyric acid, food flavor substances, short-chain organic acids,

1.4  ection IV Lactic Acid Bacteria Science and Technology

1.4.1  omposition of Lactic Acid Bacteria Science

1.4.2  evelopment Frontier in Research and Application

1.4.2.1 Research on Basic Microbiology of Lactic Acid Bacteria

Under the guidance of macrogenomic information of environmental microorganisms,

1.4.2.2  igh-Efficiency Biomanufacturing Research of Lactic Acid

bacteria for specific applications such as bio-manufacturing, food, and medicine.

1.4.2.3  tudy on Environmental Physiology and Cell Response of Lactic

1.4.2.5  he Development of Lactic Acid Bacteria for Food and Animal

grain food, natural or recombinant lactobacillus strains that can significantly

1.5 Section V Organization Structure of the Book

Lactobacillus science and technology is a rapidly growing field. The author’s

Fig. 1.3 The organization and chapters of the book

Grigoroff S (1905) Etude sur le laitfermenté comestible: le Kissélo-mléko de Bulgarie. Revue

Lactic acid bacteria (LAB), a class of commonly existing microorganisms in nature,

1.1 ection I Overview and Definition of Lactic Acid

1.1.1 Overview of Lactic Acid Bacteria

1.1.2 The Definition and Membership of Lactic Acid Bacteria

1.2 ection II Development History and Applications

1.2.1 tage Division of Development Course and Applications

1.2.2 evelopment Course of the Practical Application

1.2.2.1 nconscious Use of Lactic Acid Bacteria Derived from Empirical

ifferent Types of Lactic Acid Bacteria-Fermented Dairy Products Are

ermentation of Fruits and Vegetables and Fermented Grains by Lactic Acid

ong-Term Practical Application Makes People Realize that Lactic Acid

1.2.2.2 Practical Application of Lactic Acid Bacteria Based on Science

ommercial Production of Yogurt and Other Lactic Acid Bacteria-Fermented

Application of Lactic Acid Bacteria Fermentation Products

Drugs and Medical Applications of Lactic Acid Bacteria

1.2.3 he Development of Scientific Research on Lactic Acid

1.2.3.1 Pasteur’s Contribution to Lactic Acid Bacteria

1.2.3.2 Discovery of Some Important Lactic Acid Bacteria

1.2.3.3 stablishment of the Basis and Application System of Lactic Acid

1.2.3.4 nalysis of Basic Physiological Metabolism of Lactic Acid

1.2.3.5 Molecular Biology Period of Lactic Acid Bacteria Research

1.2.3.6 xploration and Application of the Function of Lactic Acid

1.3 ection III Application of Lactic Acid Bacteria in Human

1.3.1 Lactic Acid Bacteria and Food Manufacturing Industry

1.3.2 actic Acid Bacteria and Farming and Aquaculture

1.3.3 Lactic Acid Bacteria and Medical Health Industry

1.3.4 actic Acid Bacteria and Industrial Manufacturing

1.4 ection IV Lactic Acid Bacteria Science and Technology

1.4.1 omposition of Lactic Acid Bacteria Science

1.4.2 evelopment Frontier in Research and Application

1.4.2.1 Research on Basic Microbiology of Lactic Acid Bacteria

1.4.2.2 igh-Efficiency Biomanufacturing Research of Lactic Acid

1.4.2.3 tudy on Environmental Physiology and Cell Response of Lactic

1.4.2.5 he Development of Lactic Acid Bacteria for Food and Animal

1.5 Section V Organization Structure of the Book

2.1 LAB-Associated Gene Expression Systems

2.1.1 Host Strains in Expression Systems for LAB

2.1.2 Vectors in LAB-Based Gene Expression System

2.1.2.1 Plasmids in LAB

2.1.2.2 Cloning Vectors for LAB

2.1.2.3 Expression Vectors for LAB

2.1.3 Intracellular Expression Systems for LAB

2.1.4 Secreted Expression Systems for LAB

2.1.4.1 Features of Secretion Vectors in LAB

2.1.4.2 Secretion Systems Based on Usp45 Signal Peptide

2.1.4.3 Secretion Systems Based on the Signal Peptide of S-Layer Protein

2.1.4.4 Secretion Systems Based on Other Types of Signal Peptides

2.1.5 he Surface Expression System of Lactic Acid Bacteria

2.1.5.2 Cell Wall Anchoring Expression System

2.1.6 Applications of LAB-Based Gene Expression System

2.1.6.1 LAB as Vaccine Delivery Vehicles

2.1.6.2 Recombinant LAB in Enzyme Preparations

2.1.6.3 Recombinant LAB in Metabolic Regulation

2.2 Food-Grade Expression System for Lactic Acid Bacteria

2.2.1 Basic Requirements of Food-Grade Expression System

2.2.2 Selective Marker of Food-Grade

2.2.2.1 Saccharide Utilization Selective Markers

2.2.2.2 Auxotrophic Complementary Selection Markers

2.2.2.3 Bacteriocin Resistance Markers

2.2.2.4 Selection Markers for Heavy Metal Resistance

2.2.3 Food-Grade Inducer

2.2.4 ood-Grade Expression System of LAB and Its

2.3 Gene Knockout System in Lactic Acid Bacteria

2.3.1 he Mechanisms and Characteristics of Gene Knockout

2.3.1.1 Gene Knockout Based on Homologous Recombination

2.3.2 ene Knockout Based on Improved Homologous

2.3.2.2 omologous Recombination with a Temperature-Sensitive Type

2.3.3 ene Knockout System Based on Site-Specific

2.3.3.1 cre/loxp Recombination System

2.3.3.2 TP901-1/att Recombination System

2.3.3.3 Linear Transformation Knockout System

2.3.4 New Gene Knockout Method

2.3.5 The Common Vectors and Their Application