Structural Stability and Viability of Microencapsulated Probiotic Bacteria: A Review

Structural Stability and Viability of
Microencapsulated Probiotic Bacteria: A Review

Rocı́o I. Corona-Hernandez, Emilio Álvarez-Parrilla, Jaime Lizardi-Mendoza, Alma R. Islas-Rubio, Laura. A. de la Rosa,
and Abraham Wall-Medrano
Abstract: Probiotics are live microorganisms that confer a number of health benefits when consumed in adequate
amounts, mostly due to improvement of intestinal microflora. Bacterial strains from the genera Lactobacillus, Bifidobacterium,
and Bacillus have been widely studied and are used to prepare ready-to-eat foods. However, the physicochemical stability
and bioavailability of these bacteria have represented a challenge for many years, particularly in nonrefrigerated foodstuffs.
Microencapsulation (ME) helps to improve the survival of these bacteria because it protects them from harsh conditions,
such as high temperature, pH, or salinity, during the preparation of a final food product and its gastrointestinal passage. The
most common coating materials used in the ME of probiotics are ionic polysaccharides, microbial exopolysaccharides,
and milk proteins, which exhibit different physicochemical features as well as mucoadhesion. Structurally, the survival
of improved bacteria depends on the quantity and strength of the functional groups located in the bacterial cell walls,
coating materials, and cross-linkers. However, studies addressing the role of these interacting groups and the resulting
metabolic impacts are still scarce. The fate of new probiotic-based products for the 21st century depends on the correct
selection of the bacterial strain, coating material, preparation technique, and food vehicle, which are all briefly reviewed
in this article.
Introduction fermented milks such as kefir, kurut, and yogurt (Cruz and others
Probiotics are defined by the World Health Organization as 2010; Fontana and others 2013), products which up to date are
“live microorganisms which, when administered in adequate considered as healthy foods (Cruz and others 2012). Yogurts
amounts, confer a health benefit on the host” (FAO/WHO are fortified milk products made with specific bacterial cultures
2001; Gawkowski and Chikindas 2013). Currently, there are many [mainly lactic acid bacteria (LAB)] and marketed as ready-to-eat
studies investigating probiotic bacteria and their benefits to hu- gels (Marafon and others 2011) or viscous beverages (Castro and
man health. Several public organizations, including the Interna- others 2013a,b) with or without modifications on their original
tional Dairy Federation (IDF), state that a food product must nutrient composition (Ibarra and others 2012) while kefir grains
contain a minimum of 107 colony-forming units per gram of are mixed cultures used for centuries for the production of the
food (CFU/g) to assure sufficient bioavailable bacteria to exert traditional fermented milk drink consisting of LAB (Lactobacillus
a functional effect within the body (IDF 1999; Mortazavian and brevis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus hel-
others 2007). veticus, Lactobacillus delbrueckii, and so on), yeasts (Kluyveromyces
Dairy products are the main carriers of probiotics and have sp., Candida sp., Torulopsis sp., and Saccharomyces sp.), strep-
led the market for many years. This relationship has existed for tococci (Streptococcus salivarius), lactococci (Lactobacillus lactis ssp.,
more than centuries, when people consumed large amounts of Lactobacillus thermophilus, Lactobacillus cremoris, Lactobacillus mesen-
teroides, and so on) and occasionally acetic acid bacteria (Simova
and others 2006). Other dairy foods, such as cheeses (Escobar and
others 2012; Lollo and others 2012; Karimi and others 2012a,b;
MS 20130333 Submitted 3/9/2013, Accepted 5/23/2013. Authors Corona- Minervini and others 2012; Alves and others 2013), milk protein
Hernandez, Álvarez-Parrilla, de la Rosa, and Wall-Medrano are with Departamento de beverages (Akalin and others 2012; Castro and others 2013a,b;
Ciencias Quı́mico-Biológicas, Instituto de Ciencias Biomédicas, Universidad Autónoma
de Ciudad Juárez. Anillo Envolvente del PRONAF y Estocolmo s/n, Ciudad Juárez Fontana and others 2013), and ice creams (Kebary and others
32310, Chihuahua, México. Author Lizardi-Mendoza is with Coordinación de Tec- 1998; Di Criscio and others 2010), have been used to incorpo-
nologı́a de Alimentos de Origen Animal, Centro de Investigación en Alimentación rate probiotics into our meal. Other nondairy foods with added
y Desarrollo, AC. Carretera a la Victoria km. 0.6, AP 1735, Hermosillo 83000, probiotics include baked goods (Altamirano-Fortoul and others
Sonora, México. Author Islas-Rubio is with Coordinación de Tecnologı́a de Alimentos
de Origen Vegetal, Centro de Investigación en Alimentación y Desarrollo, AC. Car-
2012; Trujillo-de Santiago and others 2012; Zanjani and oth-
retera a la Victoria km. 0.6, AP 1735, Hermosillo 83000, Sonora, México. Direct ers 2012), mayonnaise (Fahimdanesh and others 2012), beverages
inquiries to author Wall-Medrano (E-mail: [email protected]). (Gawkowski and Chikindas 2013), and soups made from legumes
and cereals (Barbosa and others 2012).

C 2013 Institute of Food Technologists®
614 Comprehensive Reviews in Food Science and Food Safety r Vol. 12, 2013 doi: 10.1111/1541-4337.12030
Encapsulation of probiotic bacteria . . .
However, in spite of the growing popularity of probiotics, which others 2010). To overcome this problem, several technological al-
account for a global market of more than 19 billion dollars in 2011 ternatives have been proposed that include the addition of chem-
(BCC Research 2011), the viability of the bacterial strains involved ical compounds (such as nitrogen), enzymatic treatments (Cruz
and the quality of the products that contain them are questionable. and others 2012), and the use of adequate plastic packaging sys-
A simple exploration of the global market shows that the currently tems (Cruz and others 2013), although these technologies could
employed probiotic strains exhibit little or no survival in final increase the production costs of these dairy foods and so, a care-
goods, and the food vehicles that carries them are still scarce (Ross ful selection of strains nonsusceptible to redox changes is prefer-
and others 2005). Also, from a marketing perspective, the scientific able. For example, L. acidophilus (LA) and Bifidobacterium spp. are
support for permissible health statements in several countries (van both capable of mounting a cellular response against oxygen, and
Loveren and others 2012; Shimizu 2012) has limited the type of NADH oxidase and peroxidase appear to play an important role
bacterial strains that are generally regarded as safe (GRAS; Allen in the oxygen-scavenging mechanism. Finally, it has been well
and others 2012) or can be employed as foods for specified health documented that probiotic viability rapidly decreases within dairy
uses (FOSHU; Duran and Valenzuela 2010; Shimizu 2012). foods above refrigeration temperatures and therefore a rigorous
Due to the adverse physicochemical conditions that probiotic cold chain is mandatory to ensure their quality and functionality,
bacteria are exposed to during the preparation and gastrointestinal entailing high costs and a complicated logistic for their distribution
(GI) passage of a food product, their survival is at risk (Prakash (Trujillo-de Santiago and others 2012).
and others 2011). Therefore, in the characterization of a pro- Cheeses provide a valuable alternative compared to fermented
biotic food, both the viability (within foods) and bioavailability dairy as vehicles for probiotic delivery (Karimi and others
(within the host) of the microorganism involved must be ex- 2012a,b). They have been shown to be a favorable environment for
amined, among other parameters (Figueroa-Gonzalez and others probiotic bacteria during both storage and GI transit, since their
2011). Fortunately, there are several technologies that help to solid matrix and fat content protects probiotics from harsh condi-
improve the survival of these bacteria during the manufactur- tions (Escobar and others 2012). However, not all cheeses behave
ing process. Microencapsulation (ME) by means of electrostatic, on the same way as probiotic carriers because both compaction
extrusion/coacervation, or emulsification (freeze-, fluidized-, or level and pH has a strong influence on the viability, release, and
spray-drying) treatments is one method used to improve survival metabolic impact of certain probiotics (Escobar and others 2012;
(Vidhyalakshmi and others 2009). However, the addition of mi- Lollo and others 2012). Also, in case of nonrefrigerated food-
croencapsulated live bacteria to prepared foods is a relatively new stuffs such as baked goods (Altamirano-Fortoul and others 2012;
alternative, although it presents huge potential within the probi- Trujillo-de Santiago and others 2012; Zanjani and others 2012),
otic market (Heidebach and others 2012). The bacterial survival and beverages (Gawkowski and Chikindas 2013), the thermoresis-
rate also depends on the bacterial strain involved and the chem- tance of probiotic strains has severely limited their incorporation
ical nature of the artificial or natural matrix in which it will be within these foods.
contained (Del Piano and others 2006; Gawkowski and Chikindas In conclusion, due to the several physicochemical changes oc-
2013; Gebara and others 2013). Lastly, whatever technology is curring during the preparation and storage of probiotic foods and
used to increase the viability of probiotic strains in prepared foods, due to the complex metabolism implicit to each strain, several food
the resulted products must be subjected to several resistance tests technologies should be employed to guarantee the survival of pro-
(such as against temperature, pH, salinity, and bile salts) to demon- biotics within dairy (Gomes and others 2011; Ibarra and others
strate their potential bioavailability (Ding and Shah 2007; Riaz 2012) and nondairy formulations (Granato and others 2010a,b).
and Masud 2013). Among them, immobilized cell technologies (ICT), such as ME,
In this article, we discuss the positive influence of ME on resis- may reduce the contact between these bacteria and their physico-
tance to these physicochemical factors for the most common pro- chemical stressors (Selmer-Olsen and others 1999; Heidebach and
biotic strains reported in the scientific literature and found in the others 2012).
global probiotic market. Well-studied materials for ME as well as
some physicochemical and structural aspects related to interacting Methods for Bacterial Immobilization
functional groups located in bacterial cell walls, covering materi- The loss of probiotic viability within food products (especially
als and cross-linkers, are also discussed. Although there are several fermented ones) and in the acidic-bile conditions of the GI tract
comprehensive reviews on ME of probiotics, to our knowledge, has encouraged researchers to find new efficient methods for im-
there are still very few articles that address the structural relation- proving bacterial viability (Mortazavian and others 2007). The
ships established during their preparation, the influence of several chemical nature and physicochemical properties of a particular
physicochemical factors in these relationships, and the potential coating material intrinsically dictate the most convenient entrap-
impact at physiological level. ment method. The selection of an entrapment method depends on
many factors, including the potential for large-scale production,
Survival of Probiotic Bacteria in Processed Foods cost, particle shape and resistance and, most importantly, the re-
During food processing and storage, probiotic bacteria are ex- sulting viable bacterial count. A comprehensive decision pathway
posed to several challenges that compromise their survival. Oxida- for selecting a convenient immobilization technique is depicted
tive stress, temperature, acid-base changes, and molecular entrap- in Figure 1. Freeze- and spray-drying methods applied to direct
ments are just few examples. Information on the influence of these probiotic cultures maintain their viability within foodstuffs and
and many other factors has been largely studied in dairy foods. pharmaceutical formulations and, in some cases, improve the re-
Fermented dairy formulations are highly susceptible to spoilage sistance of the microorganisms during their passage through the GI
because of their high water activity (Aw) and nutrient availability tract (Dolly and others 2011; Figueroa-Gonzalez and others 2011).
(Trujillo-de Santiago and others 2012). Oxidative stress caused by The low temperatures and water sublimation that occur during
oxygen diffusion of fluid yogurts during storage, since the anaer- freeze-drying are less harmful to microorganisms than the higher
obic nature of several bacterial strains is compromised (Cruz and temperatures used in spray-drying, and the bacterial viability

C 2013 Institute of Food Technologists® Vol. 12, 2013 r Comprehensive Reviews in Food Science and Food Safety 615
Figure 1–Cell immobilization techniques.
is improved when drying with coating materials (Oliveira and nozzle, and in-situ polymerization methods. Excellent reviews ad-
other 2007). However, not all strains can be subjected to freeze- dressing these methods have been published (Vidhyalakshmi and
and spray-drying methods without dying in the process (Leja and others 2009; Cook and others 2012).
others 2009; Ghandi and others 2013).
Other alternatives for increasing probiotic viability are ME and
other immobilization techniques. ME is the process of forming a Selection of Probiotic Bacteria
continuous coating around an inner matrix that is wholly con- The design of a probiotic food begins with the function-specific
tained within a capsule wall (Figure 2), while immobilization selection of the strain (Islam and others 2010). Several health
refers to the trapping of materials throughout a particular ma- protection mechanisms of probiotic microorganisms are exerted
trix (Vidhyalakshmi and others 2009). ME is commonly used to within the gut and internal organs (Prakash and others 2011):
pack solids, liquids, or gases in micrometric beads consisting of var- (a) production of pathogen-inhibitory substances, (b) competition
ious materials, usually via extrusive/coacervation methods when with pathogenic bacteria for epithelial adhesion sites, (c) nutri-
immobilizing microorganisms. For example, alginate beads can be ent competition and production, (d) degradation of toxins and
formed by both extrusion and emulsion methods (Krasaekoopt toxin receptors, and (e) modulating immune and nonspecific host
and others 2003). These beads can release their content at a con- responses. From a clinical standpoint, the expected functionality
trolled rate and enable a gradual interaction of the inner materials is generally associated with the prevention and/or treatment of
with the environment under certain physicochemical conditions, GI and immunological disorders associated with a great number
such as for bacteria within the GI milieu (Parra 2010). Recently, of organic disturbances, ranging from a simple decrease in in-
the viability (within foods) and bioavailability (within the host) of testinal motility (Itsaranuwat and others 2003) to more complex
probiotic bacteria were shown to be 5 times higher when using immunological difficulties (Gill and Prassad 2008). In particular,
ME (Del Piano and others 2010), leading to an extended shelf-life the benefits of probiotics related to allergic disorders, such as atopic
of food products. Moreover, multistage coating further increases eczema (Kalliomaki and others 2001) or acute rhinitis (Giovannini
bacterial survival (Mokarram and others 2009). However, there are and others 2007), and intestinal disorders, such as constipation and
still significant hurdles with respect to currently available methods diarrhea (Allen and others 2012), irritable bowel syndrome (Hun
for probiotic ME . This is mainly due to the fact that important 2009), and Crohn´s disease (Shen and others 2009), have been sup-
physicochemical characteristics of microcapsules (such as particle ported by numerous studies, with species/strain-specific patterns
size and type of covering material) appear to be in conflict with the being observed (Prakash and others 2011).
requirements arising from their application in certain food prod- The genera Lactobacillus, Bifidobacterium, and Bacillus have been
ucts (Heidebach and others 2012). Lastly, other immobilization widely used to prepare ready-to-eat foods and tested in controlled
techniques include ultrasonic spray-drying (Semyonov and others trials (Gawkowski and Chikindas 2013). However, the expected
2011), fluidized bed coating (Goderska and Czarnecki 2008), elec- functional effect of the bacteria is determined by the probiotic
trostatic (Anal and Singh 2007; Ding and Shah 2009), pan- and strain and other factors, such as the timing of the intervention,
air suspension-coating (Semyonov and others 2012), vibrational the stage of disease progression (Kalliomaki and others 2001), and
616 Comprehensive Reviews in Food Science and Food Safety r Vol. 12, 2013
Figure 2–Microencapsulation of probiotic bacteria. Dotted circles represent bacteria–bacteria and bacteria-coating material interactions.
the host’s predominant microflora (Prakash and others 2011) and life. As mentioned in a previous section, oxygen toxicity is widely
metabolic condition (Thomas and Greer 2010). considered to be responsible for bacterial death (Talwalkar and
In a systematic PubMed search performed in March 2013, Lac- Kailasapathy 2004): as the oxygen concentration rises, the levels of
tobacillus and Bifidobacterium were found to be the genera that lactic acid produced by LA and the lactate:acetate ratio produced
were most commonly reported as “probiotic” (MESH; J02.500. by bifidobacteria is reduced.
456.500; 10506 entries), returning 4500 and 1881 entries, respec- Another group of bacteria with confirmed probiotic effects in
tively. Both genera consist of Gram-positive lactic acid producers vitro and in vivo is the endospore-forming, microaerophilic Gram-
that colonize the human gut at birth and during breastfeeding positive bacteria that belong to the Bacillus, Sporolactobacillus, and
(Moore and others 2011; Prakash and others 2011), but nearly dis- Brevibacillus genera (Jurenka 2012). When sporulated, these bacte-
appear in adulthood (Gavini and others 2001). Lactobacillus is the ria are metabolically inactive but are extremely resistant to harsh
main genus found in marketed foodstuffs (BCC Research 2011), treatments such as extreme heat, drying, freezing, chemical treat-
and several species have been patented. Several species and strains ments, and radiation (Furukawa and others 2005), although they
belonging to this genus have been shown to prevent antibiotic-, are unstable in food systems with high Aw such as fruit purees
rotavirus-, and Clostridium difficile-associated diarrhea (Guandalini (Cerruti and others 2000). Due to their dormancy and resistance,
2011), in addition to conferring other health benefits. Among these spores can survive for years in the absence of nutrients, but
Lactobacillus species, LA (894 PubMed entries) is one of the main when a proper stimulus is detected by germinant receptors (GRs),
probiotic species colonizing animal and human guts (Moore and they can rapidly germinate and grow (Ramirez-Peralta and others
others 2011) and when consumed in a sufficient amount, they are 2012).
capable of creating a balance between beneficial and potentially Bacillus coagulans (BC, formerly Bacillus sporogenes), a thermotol-
harmful microflora (Šušković and others 2001). The metabolic ac- erant facultative anaerobe lactic acid bacterium, is the most com-
tivity of LA in fermented foods results in the production of char- mon species of the genus Bacillus in manufactured goods. BC (37
acteristic organoleptic properties and inhibits food spoilage (Jafarei PubMed entries) has not been studied as much as Bacillus subtilis
and Tajabadi 2011). Comparative genomic analysis between Lac- or Bacillus cereus, although it has resulted in the greatest number of
tobacillus species has identified unique gene targets in LA that are pharmaceutical and prepared food developments (including bread
responsible for functional properties such as adhesion to mucin and and low-calorie sweeteners) and it is not considered harmful (Bora
intestinal epithelial cells, acid and bile tolerance, and quorum sens- and others 2009; Jurenka 2012). The complete genome sequence
ing (Klaenhammer and others 2008). Lactobacillus rhamnosus (880 of BC strain 36D1 was recently published by Rhee and others
PubMed entries) and L. casei (635 PubMed entries), 2 other strains (2011), and its predicted proteome shows a dual phylogeny with
commonly found in marketed foods, differ in their genomic con- Bacillus spp. and Lactobacillus spp. It is noteworthy that the devel-
tent, metabolic and mucus-binding capacities, and host-signaling opment of novel food products containing BC coincide with the
capabilities (Douillard and others 2013). publication of controlled trials supporting its probiotic potential
Bifidobacterium lactis (403 PubMed entries) and Bifidobacterium (Baron 2009; Drago and De Vecchi 2009). In particular, Gane-
longum (354 PubMed entries) are the most frequently mentioned den BC30 TM (patent name for BC GBI-30, 6086; 9 PubMed
probiotics in PubMed from the genus Bifidobacterium, which in- entries) is commonly found in the international pharmaceutical
cludes 32 species of Gram-positive anaerobic, ramified bacteria market and has recently been commercialized as a food ingredient.
with no motility. Studies addressing the distribution of bifidobac- From a technological perspective, GanedenBC30 withstands high-
teria in feces from infants and adults have shown a typical life- temperature processes such as baking and boiling, low-temperature
cycle adaptation (Gavini and others 2001). As in the case of Lacto- processes such as freezing and refrigeration, and high-pressure ap-
bacillus spp., Bifidobacterium spp. also inhibit intestinal colonization plications like extrusion and roll forming. From a clinical perspec-
by harmful bacteria, display antidiarrheal properties, and produce tive, this microorganism is nonpathogenic, nontoxigenic, and is
bioactive compounds, such as conjugated linoleic acid (CLA) iso- recommended for the treatment of some immunological and in-
mers (Russell and others 2011). Lactobacillus spp. and Bifidobac- flammatory disorders (Jensen and others 2010; Kimmel and others
terium spp. are usually found together within commercial foodstuffs 2010) due to its capacity to support the maturation of mononuclear
(mainly in yogurt-like and fermented commodities) with GRAS phagocytes toward both macrophage and dendritic cell phenotypes
and/or FOSHU recognition (Allen and others 2012). However, and to reduce the numbers of CD14+ CD16+ pro-inflammatory
the efficacy of these probiotic foods is limited by their short shelf- cells (Benson and others 2012). Ganedenbc30TM is currently on

track for getting the GRAS approval by the U.S. Food and Drug
Administration (FDA) for use in food and beverages (Basiotis and
others 2011).
Selection of Coating Materials

Another aspect to consider when microencapsulating probiotics
is the chemical nature of the coating material. It has been exten-
sively shown that ME techniques increase the viability of probiotics
both within foods and during their passage through the GI tract
(Chandramouli and others 2004). However, coating materials be-
have in structurally different ways and, therefore, their capacity to
protect living microorganisms and/or deliver bioactive substances
also varies (Riaz and Masud 2013). Also, the effectiveness of any
material depends not upon its capsule-forming capability, strength,
and enhancing viability but also on its cheapness, availability, and
biocompatibility. Many materials have been used to immobilize
bacteria, such as gelling polysaccharides (such as starch, cellulose,
alginate, pectin, carrageenan, and chitosan), proteins (soy, whey,
Figure 3–Alginate: α-L-guluronic acid (G), β-D-mannuronic acid (M)
casein, gelatin, and β-lactoglobulin), and lipids (waxes), all of them monomers and block types.
extensively studied (Reid and others 2007; Imran and others 2010;
Rokka and Rantamäki 2010). The most commonly used mate-
rials in the ME of Lactobacillus spp. and Bifidobacterium spp. are alginate increases with its concentration, showing pseudoplastic
ionic polysaccharides (specially alginate and chitosan), microbial behavior (Kesavan and others 2010). However, phosphate, citrate,
exopolysaccharides (for example gellan and xanthan gums), and and chelating agents compete for calcium ions, thus weakening
milk proteins (Truelstrup-Hansen and others 2002; Islam and oth- alginate gels.
ers 2010), while several different lipid emulsions have been used Chitosan, a linear cationic/basic homoglycan [β-(1-4)-linked d-
for ME of Bacillus sp. This strain-specific material selection is a glucosamine and N-acetyl-d-glucosamine], mainly obtained from
result of several factors, most notably the accumulated evidence crustacean exoskeletons, modifies the gut microbiota (with the
regarding the physicochemical properties of new materials or the exception of Bifidobacterium strains; Koppová and others 2012)
molecular interactions between these materials, the bacteria, and through its antibacterial activity (Goy and others 2009). The first
the host’s GI epithelial tissue (Alli and others 2011; Prakash and reports addressing the use of chitosan to immobilize lactic bacte-
others 2011). ria originated in the beginning of the 20th century (Le-Tien and
In addition to the protection capacity of coating materials pre- others 2004). Improved stability under simulated GI conditions
venting bacterial degradation along the GI tract, selection of has been observed for L. casei (Koo and others 2001), Bifidobac-
materials with convenient biochemical features is highly desir- terium breve (Cook and others 2011), L. gasseri and Bifidobacterium
able. For example, excellent muco-adhesive properties are typical bifidum (Chávarri and others 2010) following ME using alginate
of hydrophilic polymers such as alginate and chitosan, and this and chitosan. Structural studies have shown highly stable polyelec-
property is useful for enhancing the in situ delivery of bacteria trolyte complexes (Figure 5) between alginate (anion) and chitosan
along the GI tract (Gombotz and Wee 2012; Chen and others (cation), either in the form of multilayer films (Lawrie and oth-
2013). Hydrophilic polymers possess charged and/or nonionic ers 2007) or rough microspheres (Lucinda-Silva and Evangelista
functional groups capable of forming hydrogen bonds with mu- 2005), which explains the observed resistance to harsh GI condi-
cosal surfaces (Dhawan and others 2004; Khutoryanskiy 2011). tions and colon delivery of L. rhamnosus and LA (Sohail and others
ME with alginate, an anionic/acidic diheteroglycan [(1-4)-linked- 2011). However, the amount of chitosan and the method used to
β-d-mannuronate (M) and its C-5 epimer α-l-guluronate (G); produce microcapsules modify the gut muco-adhesion proper-
Figure 3] extracted from the cell wall of brown algae, increases ties of chitosan (Dhawan and others 2004) and alginate-chitosan
the odds of survival for several species from the Bifidobacterium and particles (Wittaya-areekul and others 2006). Additionally, ME of
Lactobacillus genera by up to 80% to 95% (Krasaekoopt and others LA through the layer-by-layer self-assembly of chitosan and car-
2004; Prakash and others 2011). Strains microencapsulated in al- boxymethyl cellulose (3 nanolayers) not only enhances its survival
ginate have been included in food formulations such as ice cream, rates in simulated gastric (SGF) and intestinal fluids (SIF) but also
frozen yogurt, and mayonnaise (Kebary and others 1998; Khalil serves to reduce viability losses during freezing and freeze-drying
and Mansour 1998; Krasaekoopt and others 2003). From a molec- (Priya and others 2011).
ular stand point, alginate in the presence of Ca2+ produces a par- Microbial polymers, proteins, and peptides have also been
ticularly strong molecular framework. As a result, cold-prepared, used as coating materials for colon-targeted delivery of probi-
thermoirreversible, and freeze-thaw-stable microcapsules can be otics (Jimenez-Pranteda and others 2012a). Alginate combined
obtained. However, to form a gel with calcium, alginates need with gellan gum, a linear acidic triheteroglycan [d-Glc(β1→4)
to contain sufficient amounts of G-monomers, a certain propor- d-GlcA(β1→4) d-Glc(β1→4)l-Rha(α1→3)] exopolysaccharide
tion of which must occur in G-rich blocks. Ca2+ fits into G obtained from Sphingomonas paucimobilis (Fialho and others 2008),
blocks such as “eggs in an egg box” (Onsøyen 2001), binding improves the thermotolerance of B. bifidum (Chen and others
alginate polymers together by forming junction zones (Figure 4). 2007). When alginate is combined with xanthan gum, an im-
The alginate-to-calcium concentration and the ionic strength of proved survival of LA (LA14) and B. lactis (BI-07) in acidic condi-
the medium influence the viscoelastic properties of alginate beads tions was observed (Albertini and others 2010), while gellan com-
(Ouwerx and others 1998), while the muco-adhesion force of bined with xanthan gum in a 0.75:1 ratio improved the survival
Figure 4–Alginate requires G-G blocks to form a gel, which is known as the “egg box” model.
complex (Oliveira and others 2007). It is noteworthy that, among

the basic amino acids, only histidine possesses the ability to form
polyelectrolyte complexes with alginate near a physiological pH
(pKR = 6.0). However, only 2 histidines are present in the 178
amino acids (19.9 Da) of β-lactoglobulin (β-LG), which is the
major whey protein found in the milk of ruminants. Additionally,
polyelectrolyte complexes with poly-l-lysine (PLL) have been ob-
tained, which confer mechanical stability, selective permeability,
and bacterial immunoprotection (Prakash and others 2011).
Lastly, prebiotic ingredients have also been used as encapsulating
agents. Their advantages over other covering materials rely on
the fact that, besides being nondigestible carbohydrates, they also
have beneficial effects for the host, by selectively stimulating the
growth and/or activity of probiotic bacteria (prebiosis) within the
colon (Fritzen-Freire and others 2012). Formulations with pre
and probiotics are known as Synbiotics which have been shown to
Figure 5–Alginate G-monomer (A)-chitosan glucosamine residue enhance the host`s immune response upon regular consumption
(B) interactions. (Gourbeyre and others 2011; Jirillo and others 2012). Synbiotics
have been successfully included in cheese (Alves and others 2013),
of Lactobacillus plantarum and L. rhamnosus against simulated bile yogurts (Malpeli and others 2012), and ice creams (Han and others
(Jimenez-Pranteda and others 2012b). Alginate combined with 2012). However, although functional drinks, and other probiotic
whey protein in a stable proportion (40%/60%) produces a highly functional foods, are available in a relatively high number, the
cross-linked matrix (Figure 6) that protects microparticles from market of prebiotics (including Synbiotic formulations) is still small
acid digestion (pH 1.2) but dissolves at pH 7.5, which is a phe- and fragmented (Bigliardi and Galati 2013).
nomenon that is highly desirable for the delivery of drugs (Hebrard Inulin [highly polymerized fructose (PF)], oligofructose (low
and others 2013) and bifidobacteria (Picot and Lacroix 2004; He- PF), and resistant starch are three of the most commonly used
brard and others 2010). This phenomenon is also observed for B. prebiotics for ME (Escobar and others 2012; Fritzen-Freire and
lactis (Bi 01) and LA (LAC 4) when they are ME in a casein/pectin others 2012). All 3 have been scarcely used alone but there are

Figure 6–Hypothetical interactions between basic amino acids (P) and alginate (G-monomer). Exposed R-groups from the tetrapeptide Arg-R-His-Lys
(outside gray circle).
several studies that report their combination with other dietary and luminal carbohydrates and participate in biofilm formation
carbohydrates (Martin and others 2013; Okuro and others 2013) (Ambalam and others 2012). The major structural component of
or proteins (Bedani and others 2013; Fritzen-Freire and others the bacterial cell wall is peptidoglycan (a.k.a. murein; Figure 7c,
2013) with which they form stable molecular complexes, lead- gray circle), a complex polymer made up of glycan strands of re-
ing to different ME products with specific physicochemical and peating disaccharide residues cross-linked via short peptides and
physiological features. For instance, Raftilose P95 when added at organized in a “helical cabling mesh,” which is responsible for
1.5% (w/v) to yogurt improves the viability of Lactobacillus spp. producing the closed-bag configuration of the bacterial cell wall
and Bifidobacterium spp. by 1.42 log during 4 wk of storage at 4 ◦ C and its viability (Hayhurst and others 2008; Jafarei and Tajabadi
(Capela and others 2006), spray-dried microcapsules made with 2011). The cell walls of Lactobacillus spp., Bifidobacterium spp. and
different combinations of reconstituted skim milk and inulin or Bacillus spp. contain 30 to 40 layers of murein associated with
oligofructose showed higher protection for Bifidobacterium BB-12 covalently bound polymers derived from teichoic acid (TA, an
under simulated GI conditions and under extreme heat conditions anionic phosphate-rich polymer, accounting for 50% to 60% of
(Fritzen-Freire and others 2013), and ME in calcium alginate plus the cell wall weight), lipoteichoic acid (LTA, a macroamphiphile
resistant starch increased the survival rate of L. acidophilus La5 in polyglycerol-phosphate polymer derived from TA), teichuronic
Iranian white brined cheese after 6 mo of storage (Mirzaei and acid (a polysaccharide consisting of d-glucose and N-acetyl-d-
others 2012). mannosaminuronic acid), and other “non classical” polysaccha-
rides (Jafarei and Tajabadi 2011; Potekhina and others 2011). Two
Cell Adhesion and Functional Group Interactions important features of TA that contribute to cell adhesion, biofilm
The nature of all of the possible physicochemical interactions production, and immunoreactivity are the branching of TA from
between the bacterial cell wall, the host’s intestinal mucosa, and the cell wall and beyond and the presence of d-alanine residues;
the coating material is another important consideration. While ad- both contribute to the cell wall polarization, TA binding capac-
hesion of the cell walls of probiotics (Van Tassell and Miller 2011, ity, electromechanical properties, and the resistance to antimi-
Figure 7c) and complex carbohydrates (Khutoryanskiy 2011; Fig- crobial cationic peptides (Neuhaus and Braddiley 2003). Murein,
ure 7d) to the host’s intestinal epithelial mucosa (Figure 7b) has TA, and LTA form a polyanionic matrix that exhibits functions
been well documented in many scientific reviews, this triad as a related to the elasticity, porosity, tensile strength, and electro-
whole (Figure 7e) has not been studied in depth. From a struc- static steering of Gram-positive cell walls (Neuhaus and Braddiley
tural standpoint, improved bacterial survival and delivery within 2003).
the colon depend on the type, number, and bonding strength of However, TA is quite diverse in its structure and abundance,
all functional groups located in the bacterial cell wall, the coating depending on the strain, rate of growth, pH of the medium,
material, and the cross-linkers. However, physicochemical stud- carbon source, and availability of phosphate ions (Jafarei and
ies addressing the role of these interacting groups are still scarce, Tajabadi 2011). For example, in BC (AHU 1638) the main
although this aspect is of particular importance for the efficient linkage oligomer is Glc(β1→4)GlcNAc, and TA is conjugated
delivery of probiotic bacteria at different stages during their GI to poly(galactosyl(1→2)glycerol phosphate) (Potekhina and oth-
passage (Anal and Singh 2007). ers 2011), while other polyglycerol-phosphate derivates of LTA
The cell wall of Gram-positive bacteria is a dynamic structure. are more prominent in B. bifidum and L. rhamnosus ATCC
Several enzymes coordinately assemble, transport, polymerize, and 7469. In L. lactis, the distribution of d-alanyl esters correlates
cross-link all of the cell wall precursors (Morata de Ambrosi and with the random distribution of α-d-galactopyranosyl residues
others 1998). Protein and carbohydrate moieties embedded in of LTA (Neuhaus and Braddiley 2003). It is noteworthy that
the bacterial cell wall promote the adherence to the intestinal bile salts upregulate genes from the dlt operon in L. rhamnosus
mucosa (van Tassell and Miller 2011; Yasuda and others 2011) GG (Koskenniemi and others 2011) and L. plantarum (Bron and
Figure 7–Hypothetical interactions between the bacterial cell wall, a coating material (chitosan) and intestinal mucin. (A) Gray circles represent
intestinal epithelial cells, and white circles represent goblet cells. (B) Mucin-containing proteins (MUC) are characterized by tandem repeats of Thr,
Pro, and Ser residues with O-linked-(N-GalNAc) oligosaccharides (broken lines) and cysteine-rich regions (Cis-Cis blocks), which promote
intermolecular disulfide bonding. (C) Bacterial adhesion and biofilm formation over the intestinal mucosa depends on many interactions between the
ionic and hydrophobic species present in mucin and complementary adhesion groups located in bacterial cell wall murein (gray circle), including
teichoic acid (TA), lipoteichoic acid (LTA), mucin-binding proteins (MBD), and cell wall lectins (CWL). Some of these interactions are cross-linked with
calcium ions. (D) Chitosan (D1, cation) binds to glycosylated mucin residues such as sialic acid (D2, anion) through noncovalent interactions
(electrostatic and hydrogen bonds). (E) Ionizable groups located in each of the 3 components of the triad (bacteria-coating material-mucin) interact
with each other in a competitive manner.
others 2006), including those involved in d-alanylation of LTA; Based on the above information, the existence of physicochem-
this transcriptional regulation increases the net charge and hy- ical interactions between bacterial cell walls and ME coating ma-
drophobic protein content of the bacterial cell walls, but reduces terials is not surprising (Figure 7e). In fact, these interactions are
the hydrophobicity of LTA, thus modifying the bacterial capac- the basis for the immobilization of bacteria for soymilk fermen-
ity to produce biofilms (Ambalam and others 2012) and to in- tation (Lye and others 2012), functional sweetener production
teract with the intestinal epithelium (Figure 7a) and its mucin (Xu and others 2012), and bacterial protease release (Mrudula
layer (Figure 7b), which are hydrophobic (Ambalam and others and Shyam 2012). For instance, interactions with cationic ma-
2012). terials such as chitosan are mediated by electrostatic forces with
Intestinal mucin-associated glycoproteins polymerize, form- negatively charged moieties (specifically TA) present in the cell
ing a framework involving extensive disulfide bonding between wall, presumably due to competing with available calcium ions
cysteine-rich regions of protein cores (Figure 7b). It creates the (Figure 7c). Because this interaction results in cell wall distortion-
characteristic viscoelastic mucus (van Tassell and Miller 2011). disruption and exposes bacteria to osmotic shock, such cationic
Lactobacilli spp., Bifidobacterium spp., and Bacillus spp. can adhere polysaccharides are considered potent (<200 ppm) antimicrobials
to this mucus by cell wall murein polysaccharides (Morata de against Gram-positive bacteria, such as Staphylococcus aureus and
Ambrosini and others 1998), mucin-binding proteins (MBP, van Listeria monocytogenes, and, at higher concentrations, against Lacto-
Tassell and Miller 2011), and lectins (CWL, Yasuda and others bacilli (Goy and others 2009), while oleoyl-chitosan nanoparticles
2011). While TA and its d-alanyl residues (among other functional (a more lipophilic particle) inhibit Escherichia coli and S. aureus by
groups) drive the adhesive electrostatic force, LTA contributes to damaging the cell membrane and other intracellular targets (Xing
the hydrophobic interactions between bacterial cell walls and mu- and others 2009). Triantafilou and others (2012) demonstrated that
cus. Additionally, as mentioned earlier, mucoadhesion of complex blood proteins such as apolipoproteins, LDL, and transferrin inac-
polysaccharides is also necessary for targeted GI delivery of en- tivate LTA, reducing its proinflammatory potential. This finding
trapped bioactive substances (Thongborisute and others 2006). As may indicate that dietary proteins can also bind to LTA, reducing
a consequence, the mucoadhesion of bacterial cell walls and lu- or facilitating bacterial release within the GI tract. However, the
minal polysaccharides results from concerted passive, electrostatic, number of studies investigating the role of other coating materials
hydrophobic, and steric interactions (Dianawati and Shah 2011). is insufficient to draw further conclusions regarding the nature

of their interaction with probiotic cell walls and their metabolic tolerance can be improved by using a double layer of prebiotics as
impact within the host. a coating material. In this regard, Chen and others (2007) eval-
uated the survival of B. bifidum treated with heat (75 ◦ C for one
Thermotolerance of ME Live Bacteria min), gastric juices, and bile salts after 2 mo of storage using pre-
Once the critical requirements are established and immobi- biotics containing different concentrations of the bacterium and
lized probiotic bacteria have been obtained, the shelf-life (viabil- 2% sodium alginate mixed with 1% gellan gum as coating mate-
ity within foods) and resistance to GI conditions (bioavailability rials. Following these treatments, the probiotic count remained at
within the host) must be analyzed. Temperature is a critical factor 105 to 106 CFU/g for the microcapsules. However, in the studies
for the viability of probiotic bacteria in refrigerated and nonrefrig- mentioned above, the temperatures did not exceed 75 ◦ C, and the
erated foodstuffs (Altamirano-Fortoul and others 2012). At tem- exposure time tended to decrease as the temperature increased.
peratures ≤4 ◦ C, the survival of LA and some bifidobacteria im- Spore-forming Bacillus bacteria, like BC, are highly resistant
proves for several weeks (Sakai and others 1997), but freeze-drying to physical threats such as heat, drying, radiation, and chemical
at lower temperatures results in a lower survival rate (Dianawati agents such as hydrogen peroxide (Furukawa and others 2005).
and Shah 2011). Mosilhey (2003) found that microencapsulated Under controlled culture conditions, sporulation can be induced
LA was stable up to 15 wk at 5 ◦ C, which makes it ideal for use in in solution or solid cultures. Sporulation confers protection against
refrigerated foods, such as dairy products (Shah 2000). However, sterilization at high temperatures in an aqueous environment, en-
the viability of LA is largely dependent on the type of coating ma- ables the formation of biofilms in a solid environment (Kolari and
terial employed: a greater loss of viability occurs under refrigerated others 2001), and creates true bridges of communication between
storage conditions when using acacia gum (AG) or gelatin (GE) as the vegetative and growing states (Shank and others 2011). Fu-
a coating material compared to AG combined with protein (SP) rukawa and others (2005) described how sporulation during heat
or soymilk (SM). treatment (85 ◦ C for 30 min) increased the heat resistance and
In contrast, the number of reports addressing the effects of ME survival of BC, B. cereus, and Bacillus licheniformis. The relative
on bacterial tolerance at high temperatures is limited. In theory, hydrophobicity of the spore surface was shown to be increased
the viability of encapsulated bacteria increases in direct propor- during the treatment, contributing to the formation of spore clus-
tion to the amount of coating material (Chen and others 2007). ters. Rosemberg and others (2005) observed that incubation at 70
Mosilhey (2003) determined the effect of different temperatures ◦ C for 25 min eliminated all vegetative forms of BC. In spite of
on LA microencapsulated with different combinations of materi- the apparent resistance of BC to temperature and pH, the Aw in
als. Non-ME LA showed a decrease in viability from 1010 to 102 finished goods should be taken into account to ensure BC avail-
CFU/g between 45 and 65 ◦ C for 30 min. Using 65 ◦ C as a refer- ability, as its vegetative state (spore) is lost at Aw > 0.9 (Cerrutti
ence temperature, the loss of viability in LA microencapsulated in and others 2000). Based on these findings, heat treatment of spores
AG, SP, SM, gelatin (GE), and whey protein (WP) was evaluated prior to ME may increase their thermal resistance (Bora and others
using LA suspensions of 109 to 1010 CFU/g. Coating LA with 2009) and water permeability. However, in a systematic PubMed
GE or AG was found to increase its resistance to temperature- search conducted in March 2013, no studies were found addressing
inactivation resulting in a viability of 103 to 104 CFU/g. Similar the ME of BC (MESH, B03.510.100.100.218).
results were observed by Kim and others (2008) for LA (ATCC Finally, a few investigators have attempted to incorporate probi-
43121) microencapsulated in alginate and by Mandal and others otics into food under high temperature (including pasteurized
(2006) for L. casei (NCDC-298) microencapsulated in alginate, or baked products). Most probiotic foods on the market are
though with slight variations in the temperature range. The most either refrigerated (yogurts and milk-derived products) or are
important contribution reported by Mosilhey (2003) was the ob- stored/consumed at room temperature (mayonnaise), and even
servation that the formation of protein–carbohydrate crystallizable novel foods consisting of fermented cereal substrates (Rathore and
complexes (AG + SM, SP, and WM) improved LA viability in others 2012), such as emmer beverages (Coda and others 2011),
thermal treatments conducted at 60 ◦ C (104 to 106 CFU/g) and are produced and stored at low temperatures. ME of LA in calcium
63 ◦ C (104 to 105 CFU/g) for 30 min; AG + SM was the only alginate has been found to improve heat tolerance under temper-
combination that resisted a temperature of 65 ◦ C (102 CFU/g). atures of 55 to 65 ◦ C for 20 min (Selmer-Olsen and others 1999).
During the process of AG (anion) coacervation with WP or SP Soares and others (2004) evaluated the thermal breakdown of al-
(cations), the number of electrostatic interactions (Figure 6) is di- ginic acid (HAlg) and sodium alginate (NaAlg) in the presence
rectly related to the viscoelastic properties of the resulting AG + of N2 and air (90 mL/min air pressure) using differential scan-
WP colloidal complex (Salazar-Montoya and others 2012), in- ning calorimetry (SCD) and observed 2 and 3 breakdown phases,
creasing its thermotolerance. respectively. The first phase (endothermic) observed in the acid
Recoating of “primary” microcapsules with additional materials corresponded to a dehydration reaction occurring at 80 ◦ C (N2 )
can help to prevent the exposure of probiotics to oxygen during or between 75 and 96 ◦ C (air), whereas the first phase occurred
storage and improve their stability at a low pH and high tem- at higher temperatures for NaAlg. Despite the thermotolerance of
perature. Some of the materials employed for recoating include alginate, its use has some disadvantages, as capsules composed of
chitosan, poly-l-lysine, alginate, and several starches, gums, and this material are more sensitive to acidity (Mortazavian and oth-
gelatins (Krasaekoopt and others 2004; Mortazavian and others ers 2008). Krasaekoopt and others (2004) demonstrated that LA
2007, 2008). However, no significant differences were found in in alginate microcapsules coated with chitosan shows increased
the viability of microencapsulated and nonencapsulated LA 43121 survival in bile salts as compared to uncoated bacteria.
in prebiotics such as fructooligosaccharides, lactulose, and raffinose The probiotic resistance to temperatures between 65 and
when polyvinyl acetate phthalate was used as a coating material 100 ◦ C observed when LA is microencapsulated in alginate, with
(second layer) in an aquose system (Eun and others 2007). Nev- or without copolymerization with other materials (for example
ertheless, double-ME increased bacterial tolerance at 55 ◦ C for proteins), may represent an area of opportunity for the possible in-
180 and 240 min as compared to single-ME, indicating that heat corporation of this microorganism into baked and/or pasteurized
products. Altamirano-Fortoul and others (2012) recently assessed chitosan-alginate microparticles. Therefore, the diffusion of bile
the viability of LA microencapsulated in a complex matrix of WP, salts into the matrix core can be limited, protecting the probiotic
CMC, pectin, inulin, and fresh agave syrup via spray-drying, ex- bacteria from interacting with the bile salts (Koo and others 2001;
posed at 65 ◦ C, 2 bar of pressure, for 135 min. Subsequently, the Yu and others 2001). Lucinda-Silva and Evangelista (2005) pre-
microcapsules were resuspended in various starch solutions (1% to pared alginate-chitosan microparticles and observed a reduction in
2%, w/v), sprayed evenly (118 cm2 ) over a cold dough (0.1 to 0.2 their electrical conductivity, indicating a high level of coordination
g/bread), and the bread was baked for 16 min at 180 ◦ C. Before between these materials at a ratio of 1:1.5 to 1:1.7. Thus, a bal-
baking, the bread crust contained 108 CFU/g of LA, which was anced ratio of coating materials is essential to reduce the numbers
reduced to 107 and 106 CFU/g after baking and 24 h of storage, re- of unconjugated functional groups that are free to bind bacterial
spectively. From the data presented above, it is clear that additional cell walls.
layers and materials with a different chemical nature can provide
improved thermotolerance to microencapsulated probiotics. Sensory aspects of ME in probiotic foods
It is well known that the addition of probiotics to dairy and
Resistance of ME Live Bacteria to GI Conditions nondairy foods, modify their sensory attributes. Their addition to
The resistance of various probiotic bacteria, with or without cheese in a suitable culture composition, does not considerably
ME, to simultaneous changes in pH and in the presence of gas- change the flavor and/or other sensory characteristics of the final
tric juices has also been examined. Mokarram and others (2009) product, when compared to control cheeses (Karimi and others
studied the influence of the number of layers of alginate on the 2012). However, the impact of probiotic bacteria on cheese flavor
survival of LA (PTCC1643) and L. rhamnosus (PTCC1637) in is dependent on the bacterial strain used and its metabolic activity
simulated GI conditions. In this study, ME with a double layer of during cheese production and storage. In particular, excessive pro-
alginate (about 100 μm) was found to provide the best protection teolysis and over acidification during cheese ripening decreases its
against a loss of viability for both bacteria in gastric (pH 1.5, 2 acceptability by consumers (Michaelidou and others 2003; Milesi
h) and intestinal (pH 7.25, 2 h) juices. Chandramouli and others and others 2009; Karimi and others 2011). The selection of differ-
(2004) and Kim and others (2008) also showed that ME with algi- ent probiotic strain has profound effects on the sensory attributes:
nate increased the survival of LA CSCC 2400 and ATCC 43121, Escobar and others (2012) studied the effect of probiotics and fava
respectively, in GI conditions. Lyer and Kailasapathy (2005) re- bean starch added to panela cheese (a soft cheese), observing that
ported that LA microencapsulated in 1 or 2 layers of chitosan starchless cheese supplemented with L. rhamnosus GG (1 × 108
and resistant starch showed a loss of viability of 1 × 102 or 3 CFU/g) showed greater consumer acceptance in terms of cheese
× 103 CFU/g, respectively, in simulated GI conditions, while in compactness, hardness, moisture, and softness, whereas that sup-
non-microencapsulated LA the observed loss of viability was 1 × plemented with B. breve scored better in creamy and milky flavor
105 CFU/g. attributes. In probiotic ice creams, it has been observed that the
Mandal and others (2006) found that the viability of L. casei original milk fat level, pH, probiotic strain used, overrun (incor-
(NCDC-298) improved as the concentration of alginate increased poration of air) level, and the use of stabilizing hydrocolloids are
at pH 1.5 for 3 h, and Li and others (2010) confirmed this result critical quality parameters that modify consumer’s sensory percep-
in L. casei (ATCC 393) microencapsulated in alginate-chitosan- tion and preference (Soukolis and others 2010; Cruz and others
carboxymethyl chitosan. Similar results were reported by Sun and 2012; Ferraz and others 2012). Nondairy foods (like probiotic
Griffiths (2000) for Bacillus infantis ATCC 15697 microencapsu- fruit beverages) also change their sensory attributes upon addition
lated in gellan gum (0.75%) and xanthan (1%) compared to the and/or fermentation with probiotic bacteria (de Souza and others
non-microencapsulated bacteria at pH 1.5 to 2.5. Sun and Grif- 2012; Hassan and others 2012). Although the generation of new
fiths (2000) also found that microencapsulated B. infantis added to sensory characteristics in probiotic-supplemented foods might be
pasteurized yogurt survived 5 wk under refrigeration. However, desirable in some cases, food habits of some consumers may find
Trindade and Grosso (2000) reported that ME of B. lactis and LA these characteristics unpleasant.
with calcium alginate was ineffective in protecting cells exposed ME of probiotic strains can avoid many of the problems associ-
to bile salts at concentrations of 2% and 4%, respectively. ated with their addition in free form. Undesirable characteristics,
Studies describing GI resistance when several bacteria are ex- such as those mentioned above, in both dairy (Özer and oth-
amined simultaneously have highlighted a species-specific trend. ers 2008, 2009) and nondairy formulations can be avoided by
Ding and Shah (2009) evaluated the effect of acid (pH 2.0) on the a convenient selection of a probiotic strain and an entrapment
viability (1010 CFU/g) of LA, L. rhamnosus, B. longum, L. salivar- method. As mentioned in previous sections, the confinement of
ius, L. plantarum, L. paracasei, and B. lactis (type BI-O4 and Bi-07) probiotic bacteria within semipermeable polymeric microspheres
microencapsulated in guar and xanthan gums, carob, alginate, and (1 to 1000 μM) enables the physical isolation of bacteria from
carrageenan. LA was one of the microorganisms that was most the external environment while maintaining a hospitable internal
tolerant (105 to 108 CFU/g) to acidity (pH 2.0, 2 h) and to bile microenvironment (Rathore and others 2013). The addition of
salts (taurocholic acid), especially when it was microencapsulated microencapsulated L. casei has no significant effect on the sensory
in alginate, xanthan gum, and carrageenan. properties of nonfermented ice cream (Homayouni and others
Resistance to bile salts also depends on the type of coating 2008) and cream-filled cakes (Zanjani and others 2012) and dry
material applied. Murata and others (1999) coated chitosan with sausage batter containing either nonencapsulated or microencap-
alginate and observed that chitosan has the ability to bind bile sulated (in alginate) Lactobacillus reuteri show the same sensory pro-
salts. This finding could indicate that the effectiveness of algi- file (Muthukumarasamy and Holley 2006); interestingly, the ME of
nate in protecting a probiotic within its matrix may be decreased L. casei and B. bifidum improved the sensory characteristics of may-
due to the incorrect selection of a second material, as in the onnaises (Fahimdanesh and others 2012). However, it is mandatory
case of chitosan. However, the formation of an insoluble com- to consider the shape and size of final microspheres do to their
plex between chitosan and bile salts takes place on the surface of possibility to affect the palatability of products containing them.

The smoothness of yogurt supplemented with microencapsulated

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Structural Stability and Viability of Microencapsulated Probiotic Bacteria: A Review

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Structural Stability and Viability of

Microencapsulated Probiotic Bacteria: A Review

Figure 1–Cell immobilization techniques.

Selection of Coating Materials

complex (Oliveira and others 2007). It is noteworthy that, among

The smoothness of yogurt supplemented with microencapsulated

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