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Annals of Biological Research, 2012, 3 (11):5313-5318

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ISSN 0976-1233
CODEN (USA): ABRNBW

Antimicrobial Peptides from Milk Proteins: A Prospectus

Sepideh Jabbari1*, Rahele Hasani2, Farshid Kafilzadeh3, Sahar Janfeshan4

1
Young Researcher Club, Astara Branch, Islamic Azad University, Astara, Iran.
2
Young Researcher Club, Birjand Branch, Islamic Azad University, Birjand, Iran.
3
Department of Biology, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
4
Department of Biology, Arsenjan Branch, Islamic Azad University, Arsenjan, Iran.
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ABSTRACT

Milk is a highest quality source of well-balanced nutrients and also displays a range of biological activities that
affects digestion, metabolic responses to absorbed nutrients, growth and development of specific organs, and
resistance to disease. Bioactive peptides have been identified within the amino acid sequences of native milk
proteins. Hydrolytic reactions, such as those catalyzed by digestive enzymes, result in their release. These peptides
directly influence numerous biological processes evoking behavioral, gastrointestinal, hormonal, immunological,
neurological, and nutritional responses.. The size of active sequences may vary from two to twenty amino acid
residues. The total antimicrobial effect in milk is greater than the sum of the individual contributions of
immunoglobulin and nonimmunoglobulin defense proteins such as lactoferrin, lactoferricin , and other peptides. A
variety of naturally formed bioactive peptides have been found in fermented dairy products, such as yoghurt, sour
milk and cheese. Bioactive peptides have the potential to be used in the formulation of health-enhancing
nutraceuticals, and as potent drugs with well defined pharmacological effects.

Keywords: milk proteins, bioactive peptides, antimicrobial peptides

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INTRODUCTION

Milk is a highest quality source of well-balanced nutrients and shows a various range of biological function.
Biological functions of milk are mainly due to milk peptides and proteins. Milk protein include of approximately
20% whey and 80% casein. Whey contains five major proteins, including -lactalbumin, glycomacropeptide, -
lactoglobulin, proteose peptone 3, immunoglobulins, and serum albumin, which together make up 85% of whey
protein. Casein contains s1-casein, s2- casein, -casein and -casein [1,2]

However, some of the biological function of milk protein ingredient is hidden, and is liberated upon proteolytic
action. Releases of biologically active milk peptides occur usually during digestion of milk proteins in the gut,
during enzymatic hydrolysis, chemical processes and fermentation. These bioactive peptides have important effect
on body function and promote body health. These peptides decrease risk factor of some disease and/or to prevent
disease development result of their hormone-like properties [3]. Upon oral administration, because of
gastrointestinal processes, bioactive peptides released and may influence the various body systems such as
cardiovascular, digestive, immune and nervous systems depending on their amino acid sequence. These health
promoting effects may be because of several known peptide sequences exhibiting antimicrobial, antioxidative,

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antithrombotic, antihypertensive and immunomodulatory activities [4]. Bioactive peptide functions depend on their
amino acid composition and sequence. The size of active peptides may vary from 2 to 20 amino acid residues [5].

Studies have been shown that several milk peptides exhibit multifunctional properties. The antimicrobial properties
of milk have been widely recognized for many years. The first time, in 1930, it was described that milk has active
inhibitors that decrease growth of streptococcal bacteria [6]. The antimicrobial activity of milk is mainly attributed
to immunoglobulins, and to non-immune proteins, such as lactoferrin (LF), lactoperoxidase and lysozyme [7]. In
addition, it has been demonstrated that breast-feeding of infants supplies protection from a wide range of respiratory
and entric infections. Antibacterial peptides are known as an important ingredient of innate immunity, especially at
mucosal surfaces such as the lungs and small intestine that are constantly exposed to a range of potential pathogens
[8, 9].

It has been demonstrated that a positive charge and amphiphilic of these peptides as major structural motifs
determining the interaction with bacterial membranes is their mechanism action. It has been recognized that some
antibacterial milk peptides can reach intracellular targets. More recently, it has been demonstrated several whey
proteins such as lactoferrin, lactoferricin, - Lactalbumin, - lactoglobulin have antimicrobial activity [10].
Antibacterial fragments also have been derived from s1-casein, s2 -casein, -casein and -casein. Studies have been
shown that these peptides have antimicrobial activity against a wide range of pathogenic organisms (e.g.
Escherichia, Helicobacter, Listeria, Salmonella and Staphylococcus), yeast and filamentous fungi [11].

Lastly, biologically active peptides have been chemically synthesized to prove the physiological properties
associated with a specific amino acid sequence. There is significant evidence that many bioactive peptides serve in
multifunctional roles and often share common structural features based on a defined, biospecific role [11].This
review will deal with the antimicrobial properties of milk protein derived peptides including peptides obtain from
enzymatic proteolysis and microbial fermentation. In vitro and in vivo studies of antimicrobial peptides will be
presented.

Whey proteins derived antibacterial peptides

Lactoferrin
Lactoferrin (Lf), a single-chain iron-binding glycoprotein, is the most important protein present in whey, due to the
numerous and diverse array of therapeutic properties it displays. Lactoferrin is able to influence on some strains of
Streptococcus mutans, Vibrio cholerae, Escherichia coli and Legionella pneumophila. Lf is a natural defense protein
present in most secretions commonly exposed to normal flora such as milk, colostrum, tears, pancreatic juice,
intestinal mucus, nasal secretions, saliva, bile, and genital secretions. Antimicrobial peptides such as lactoferrin
have are partly short (less than 100 amino acids), amphiphilic and positively charged. Cationicity, hydrophobicity,
and secondary structure have been implicated in the antimicrobial effect [12] .

Some studies have shown that human lactoferrin can directly destroys the outer membrane of gram-negative
bacteria, releases lipopolysaccharide (LPS) molecules from the membrane and enhances bacterial susceptibility to
hydrophobic antibiotics and human lysozyme. It has been suggested that the interrupt of the outer membrane
structure is most likely not the primary factor leading to cell death. The lactoferrin interact with and cross both the
outer and cytoplasmic membranes, and cause cell death by a multihit mechanism that involves action on more than
one ionic target [12].

Lactoferrin-derived peptide
Lactoferricin
Lactoferricin is a potent bactericidal peptide specifically generated by enzymatic degradation of lactoferrin, also
exhibit antimicrobial activity against both Gram-positive and Gram-negative microorganisms. Lactoferricin B
obtained from bovine lactoferrin and lactoferricin H obtained from human lactoferrin. The fragments were
characterised and named human (H) and bovine (B) lactoferricin [13]. Some studies have shown that lactoferricin B
has higher bacteriostatic and bactericidal activity than lactoferrin. Antimicrobial activity rather than lactoferrin.

It has been demonstrated that Lactoferricin starts electrostatic interaction with the negatively charged membranes of
bacteriaIn initial binding, lipopolysaccaride and teichoic acid as binding site in Gram-negative and Gram-positive
bacteria have been identified. It has been demonstrated the peptide approach the cytoplasm and suppress the
bacterial protein synthesis that exact mechanism is not clear [14]. The structureactivity relation of lactofericcin

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fragment has been studied during last years. Some studies have been shown that antimicrobial, antifungal,
antitumor, and antiviral properties of lactofericcin can be associated to tryptophan/Arginine-rich proportion of the
peptide. Also the anti inflammatory and immunomodulating properties are associated to a positively charged region
of the molecule [15].

Lactoferrampin
Lactoferrampin is an antimicrobial cationic domain in the N1-domain of lactoferrin. It includes amino acids 268-284
of bovine lactoferrin. It has been demonstrated that lactoferrampin display higher candidacidal activity than the
lactoferrin. Furthermore, lactoferrampin has antimicrobial activity against of Bucillus subtilis, Escherichia coli, and
Pseudomonas aeruginosa , but not against of the fermentingbacteria, Actinomyces naeslundii , Porphyromonas
gingivalis , Streptococcus mutans and Streptococcus sanguis. Lactoferrampin plays a critical role in membrane-
mediated activities of lactoferrin [16].

-lactoglobulin derived peptides

-Lactoglobulin exhibit around half of the whole protein in bovine whey, while human milk contains no -
lactoglobulin. Proteolytic digestion of bovine -lactoglobulin by trypsin bears four peptide segments with
bactericidal activity. These peptides corresponded to -lactoglobulin f(1520), f(2540), f(7883) and f(92100).
These peptides only effect on Gram-positive bacteria and inhibit them. The amino acid sequence of peptide fragment
f(92100) is alterd by replacing of Asp with Arg and the substitute of a Lys residue at the C-terminal end to enhance
the bactericidal activity to Gram-negative bacteria [17] .

-lactalbumin derived peptides

-Lactalbumin is one of the primary proteins determined in human and bovine milk. It includes approximately 20-25
percent of whey proteins and contains a wide variety of amino acids, such as essential and branched chain amino
acids. Some studies have been shown that -lactalbumin provided bactericidal peptides after digestion with trypsin
and chymotrypsin, but not with pepsin. It has bactericidal activity against of Gram-positive bacteria [18].

Casein derived antibacterial peptides

S1-Casein derived peptides
Caseins contain antimicrobial peptides. The first time Jones and Simms revealed the antimicrobial properties of milk
[19]. They identified a substance capable of inhibiting the growth of Streptococci called lactenin. Subsequently,
some studies identified a group of basic high molecular weight polypeptides, called casecidins. Casecidin, obtained
by chymosin digestion of casein at neutral pH, and exhibited antimicrobial activity in vitro against some pathogenic
bacteria such as Staphylococcus, Sarcina, Bacillus subtilis, Diplococcus, pneumoniae, and Streptococcus pyogenes
[19].

Isracidin, is that N-terminal segment of s1-CN that inhibit the in vitro growth of Lactobacilli and other Gram-
positive bacteria. This peptide also protects sheep and cows against mastitis. Isracidin has a strong protective effect
against S. aureus, Streptococcus pyogenes and Listeria monocytogenes when administered at low dose as 10 g per
mouse prior to bacterial challenge [20].

S2-Casein derived peptides

Bovine S2-Casein was also shown to be a precursor of various peptide fragments with antibacterial activity. Zucht
et al. [21] describe the isolation and characterization of an antibacterial peptide, from bovine milk and called
casocidin-I. The peptide includes amino acids 165-203 of bovine caseinS2. Casocidin-I (bovine milk), is a cationic
s2-CN derived peptide that suppress growth of Gram-negative (E. coli) and Gram-positive (Staphylococcus
carnosus) bacteria. Recio and Visser [22] have recognized two antibacterial domains from bovine S2-casein, one of
this is [f(164279)] being included in the previously isolated fragment of S2-casein [f(150188)] and a new
antibacterial fragment at C terminus of the molecule [f(183207)] with more strong antibacterial activity.

Recently, researchers have recognized new antibacterial peptides from a chymosin digest of bovine sodium
caseinate, all of them originated from the C-terminal of bovine s2-casein f(181207), f(175207), f(164207). The
peptides have antimicrobial activity against a wide variety of Gram-positive and Gram-negative bacteria with low
concentration [23].Hydrolysis of bovine S2-casein also, induces four antibacterial peptides. These peptides
corresponded to amino acid residues 165170, 165181, 184208 and 203208 of bovine s2-casein. Fragments

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165181, 184208 were homologous to those previously identified in the bovine protein and f(165181) showed
potent antibacterial activity [23].

-Casein derived peptides

Kappacin
Liepke et al. [24] identified first antimicrobial peptide from -casein of human milk. This peptide was non-
glycosylated portion of human -casein f(63117) and it was obtained after acidification of human milk and
incubation with pepsin. Kappacin is an antimicrobial peptide that obtained from -casein [25]. Kappacin
corresponds to the non-glycosylated, phosphorylated form of caseinomacropeptide (CMP) which exhibited growth
inhibitory activity against Gram-positive (Streptococcus mutans) and Gram negative (Porphyromonas gingivals)
bacteria. It has been demonstrated that active component of this peptide that exhibit growth inhibitory activity
against Gram-positive (Streptococcus mutans) and Gram negative (Porphyromonas gingivals) bacteria is
phosphorylated and non-glycosylated. Also it has been demonstrated that non-phosphorylated and glycosylated
forms dont exhibit any activity against Streptococcus mutans [25].

The mechanism that by this kappacin limit gastrointestinal tract infection in the growing neonate, may be release of
kappacin in stomach that by which increase sensitivity of bacteria to gastric acid by collapsing important
transmembrane cation gradients. Molecular modeling suggests that the reason why the glycosylated forms of
kappacin dont have antimicrobial activity because of suguer moieties would block pore formation [26].

-Casecidin
-casecidin is a pentapeptide with antimicrobial activity that was isolated from trypisin digest of bovine -casecidin.
It can suppress growth of some pathogenic bacteria such as S. aureus, E.coli and S. typhimurium. Also this peptide
display cytotoxic activity against some mammalian cells such as human leukemic cell lines. Cytotoxic effect of this
peptide may be inducing apoptosis [27].

Caseinomacropeptide
Caseinomacropeptide (CMP) derive from -casecidin interacts with toxins, viruses and bacteria, thus it can promote
health. Glycosylated CMP suppresses the binding of cholera toxins to their oligosaccharide receptors on cell walls
and defends cells from infection induced by influenza virus. CMP also suppresses the adhesion of cariogenic
bacteria such as Streptococcus mutans, S.sanguis and S.sobrinus to the oral cavity and regulates the composition of
the dental plaque microbiota. This could help to influence acid formation in the dental plaque, in turn reducing
hydroxyapatite dissolution from tooth enamel and promoting remineralisation. For this, it has applied for oral care
products to prevent dental caries [28].

-Casein derived peptides

Studies have been shown that antimicrobial peptide derived from -Casein suppress a large spectrum of Gram-
positive and Gram-negative bacteria including potentially pathogenic bacteria of clinical interests such as
Enterococcus faecium, Bacillus megaterium, E.coli K-12, Staphylococcus aureus, Yersinia enterocolitica. This
peptide have 26 amino acids, contain a higher content of nonpolar hydrophobic residues, lower positive charge and
some pro residues at C-terminal which makes it resistant to further digestion by trypsin and chymotrypsin [29].
Fractions of human -casein have also a protective effect against Klebsiella pneumoniae in mice. The
immunomodulatory peptide derived from bovine -casein , viz. f193-209 , was shown to enhance the antimicrobial
activity of mouse macrophages [30].

Lysozyme-derived peptides
It has been shown that milk lysozyme has antibacterial activity against both Gram-positive and some Gram-negative
bacteria that are completely tolerant to egg white lysozyme. Amino acid residues 87-114 of chicken lysozyme and
87-115 of human- milk lysozyme were potently bactericidal strongly Gram-positive and Gram-negative bacteria.
Both lysozyme fragments can be used for the design of novel antimicrobial peptides [26].

In vivo studies on milk-derived antibacterial peptides

There are several documents about antimicrobial activity of bioactive milk peptides but very limited studies have
done in vivo using animal model or in human clinical trials. It has been demonstrated that that bioactive milk
peptides may modulate the intestinal microflora when they formed during milk digestion in vivo and protect host
against pathogen microorganisms [31].

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Some researchers have shown in vivo activity of isracidin that is derived from S1-casein f(123) [20]. Isracidin is
required in much smaller quantities in vivo to employ a protective effect against bacterial than in vitro. In mice it
exerts a protective function against Listeria monocytogenes, Streptococcus pyogens and Staphylococcus aureus. In
cows with mastitis, isracidin was successful in treatment of chronic streptococcal infection.

Future perspectives for antimicrobial peptides from milk proteins

Antibiotic resistant bacteria is a great concerns that treat both animal and human health. Thus the necessarily of
developing compatible antibiotic alternatives for feed and medical industries has been arisen. Many of researches
have been paid attention to antimicrobial peptides derived from animals and plants because of their effective
antimicrobial activity against pathogen microorganism compared with current antibiotics [32]. Antimicrobial
peptides obtained from food proteins such as milk have more advantage to those derived from harmless substances,
therefore they could have used with safety in medicine and food industry. Antibacterial domains of such proteins
may have a potential use drug therapy, functional foods, or infant formulas [33].

Application of applicable protocols such as membrane processing and chromatographic isolation may also be an
area of future interest in the extraction of potent biofunctional peptides from fermented dairy products and their
subsequent utilization as functional food ingredients [11]. Developments in nano-encapsulation and nanoemulsion
technology, as well as research into delivery systems, may finally lead to the generation of specific formulations
containing bioactive peptides with anti-microbial activity. Antimicrobial peptides from food protein merit attention
due to their mechanism of activity, which makes microbial resistance doubtful, and for increasing the functional
values of foods. Bioactive peptides have the potencial to be used in the formulation of health-enhancing
nutraceuticals, and as potent drugs with well defined pharmacological effects [4,34].

CONCLUSION

In summary, milk contains various peptide sequences that influence vital physiological functions and regulate many
biochemical processes. The interest on bioactive milk peptides is increasing because milk proteins are available in
great amounts with high degree of purity at low price which, under a technological aspect, make them attractive in
the search of bioactive peptides. The potential physiological effects of these peptides have been examined to supply
a better understanding of their effects.

Further work is also required to synthesize modified peptide sequences in order to find pharmacologically active
peptides with higher potency and longer duration of action. Furthermore, there is a need to develop technologies by
means of which active peptide fractions can be produced and enriched.

The future applications of bioactive peptides look promising in the field of food industry as food preservatives and
nutraceuticals and also as natural drug in pharmaceutical industry.

Milk proteins are not only essential to supply nutrition and specific immunological protection to the neonate but that
they also provide bioactive peptides which defened the organism against infectious agents. These antibacterial
peptides havebeen derived from lactoferrin, S 1,S 2,, and -casein, -Lactalbumin, -lactoglobulin,
proteosepeptone-3 and lysozyme. Antibacterial domains of milk proteins may have a potential use in therapy,
functional foods, or infant formulas.

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