Milk Derived Antimicrobial Bioactive Peptides: A Review

Abstract

In recent decades, bioactive peptides have attracted increasing interest as health promoting functional foods. A variety of naturally formed bioactive peptides have been found in fermented dairy products such as yogurt, sour milk, and cheese. Initially these peptides are inactive within the sequence of the parent protein molecule and can be generated by gastrointestinal digestion of milk, fermentation of milk with proteolytic starter cultures, and/or hydrolysis by proteolytic enzymes. Milk derived peptides exert a number of health beneficial activities, even upon oral administration. Bioactive peptides have a great impact on major body systems including the digestive, nervous, endocrine, cardiovascular, diabetes type II, obesity, and immune systems. Antimicrobial peptides are also an important ingredient of innate immunity, especially at mucosal surfaces such as lungs and small intestine that are constantly exposed to a range of potential pathogens. Therefore, it plays an important role in boosting natural immune protection by reducing the risk of chronic diseases. Bioactive peptides are considered as potent drugs with well-defined pharmacological residues and also used to formulate health-enhancing nutraceuticals.

Keywords:

• Milk protein
• Bioactive peptide
• Antimicrobial activity
• Purification

INTRODUCTION

Milk is an excellent source of bioactive peptides of high nutritive and pharmaceutical value. The interest on bioactive milk peptides is increasing because of several of their positive impacts on physiological and metabolic functions of human health.^[1] One of the systems present as a first line of defense in the innate immune system explains the activity of antimicrobial peptides (AMPs). These diverse multifunctional peptides were initially found from frogs and insects in the 1980s and a large number of additional AMPs have been isolated from insects, birds, fish, plants, and mammals, including humans.^[2–5] Since then, over 800 have been identified.^[6] Certain research articles described that initially these bioactive peptides are found in inactive form within the sequence of the precursor protein and can be released in different ways. Once they are released from precursor protein sequence, they ultimately influence body functions and human health.^[7,^8] The production of bioactive peptides in vivo following the digestion of milk proteins has been studied in the past present decade both in human and animal.^[9] AMPs are of 12 to 100 amino acids, relatively short and amphipathic having positive net charge of +2 to +9.^[2,^3] The expression of these AMPs can be constitutive by infectious stimuli including bacteria, bacterial molecules, or pro-inflammatory cytokines that induce innate immunity.^[10,^11] They neutralize the pathogenic effects of lipopolysaccharides (LPS), also able to enhance phagocytosis and accumulate various immune cells at inflammatory sites.^[12,^13] Due to the smaller size of AMPs, they facilitate the rapid diffusion and secretion of peptide outside the cells that promote the immediate defense response against pathogenic microbes.^[14,^15] Therefore, they display direct microbicidal activities toward bacteria, fungi, and some parasites by damaging their membrane or acting on other targets,^[14] whereas they also have an active role in adaptive immunity by enhancing the development of monocytes, dendritic cells, and T-cells.^[16–18] Host defense peptides (HDPs) are also known to be an innate defense regulators present in cattle milk and potentially protect against many parasitic challenges by stimulating immune system to infectious diseases including neonatal diarrheal disease.^[19–21] It has been proven that milk is a basic source of antimicrobial bioactive peptides that is not only a healthy nutrition but which also alleviates the consequences of lifestyle diseases. Researchers show a growing interest in developing several new functional food products commercially that contain bioactive peptides from milk proteins and exhibit antimicrobial activities on human health. BioPURE-GMP Davisco, USA can be taken as an example that carries κ-casein f(106-169) and show its inhibitory impact on bacterial pathogens.^[22]

AMPS FROM MILK PROTEIN

It has been recognized that bioactive AMPs are initially found in inactive form within the sequence of the precursor protein but when they are liberated, they may show their beneficial impact on human health. During the gastrointestinal digestion, digestive enzymes lead the possible release of AMPs in the intestine where they pass to the circulatory system and reach the target sites to inhibit pathogens.^[23] Milk derived AMPs have been released as a specific protein fragment from their precursor protein through one of the following ways.

Three main types of strategies can be followed to enhance the production of bioactive peptides from the milk proteins. The first one involves the proteolytic activity of lactic acid bacteria or food grade enzymes to release the peptides from their parent proteins. The second strategy exploits hydrolysis of the proteins by extracellular peptidases. The final strategy concerns the production of the bioactive peptides by microorganisms using recombinant DNA technology.^[24] It has been demonstrated that the combination of all the above methods is the most effective in production of AMPs.^[25,^26] There are several accumulating evidences suggesting that gastrointestinal digestion of different casein and whey proteins by different enzyme combinations of proteinases, namely pepsin, trypsin, alcalase, pancreatin, and chymotrypsin, etc., are able to generate AMPs.^[27–29] Many recent studies have been well-documented on the release of several AMPs by the activities of live proteolytic microorganisms or proteolytic enzyme in the process of fermentation.^[30,^31] In recent years, rapid progress has been made to describe proteolytic system of lactic acid bacteria namely Lactococcus lactis, Lactobacillus helveticus, and Lactobacillus delbrueckii var. bulgaricus. This system consists of a cell wall bound proteinase and several intracellular peptidases^[32] that provide the liberation of bioactive AMPs.

INHIBITORY ACTIVITY OF AMPS IN CORRESPONDS TO THEIR TYPES

Multifunctional properties of several antimicrobial milk peptides have been widely reported from the present study. Particularly, milk protein (80% casein and 20% whey) is a natural reservoir of bioactive peptides that exhibit an immune defense against several microbial infections and are used in formulation of potent drug with well-defined pharmaceutical effects.^[33] Whey constitutes five major proteins, such as α-lactalbumin, glycomacropeptide, β-lactoglobulin, protease peptone 3, immunoglobulins, and serum albumin, that together make up 85% of whey protein, whereas casein contains α_s1-casein, α_s2-casein, β-casein, and κ-casein.^[34,^35] Studies have been proven that these peptides are the potent inhibitors of pathogenic organisms like bacteria (e.g., Escherichia, Helicobacter, Listeria, Salmonella, and Staphylococcus), yeast, and filamentous fungi.^[36]

Casein Derived Antibacterial Peptides

During the last few years, it has become clear that dietary casein protein is a most abundant source of biological active peptides and widely categorized according to their corresponding proteins such as κ-casein, α_s1-casein, α_s2-casein, and β-casein.^[34,^35] κ-casein is a bovine protein which is hydrolyzed by the activity of chymosin enzyme to generate two polypeptides, such as (1) a hydrophobic N-terminal κ-casein f(1-105) and (2) a hydrophilic phosphorylated and glycosylated C-terminal κ-casein f(106-169), namely caseinomacropeptide (CMP).^[37] Glycosylated CMP and its derivatives, κ-casein–A f(138-158) may account for the inhibition of enter toxin and pathogenic adhesion to the cell wall and protects the cell from the infections mediated by Streptococcus mutans, S. sanguis, Porphyromonas gingivalis, and S. sobrinus.^[38,^39] Kappacin, the non-glycosylated, phosphorylated form of bovine CMP, can be obtained after incubation with pepsin and exhibits the growth inhibitory activity against Streptococcus mutans, S. sanguis, Porphyromonas gingivalis, and S. sobrinus.^[40] It may function as a surface active agent that forms pores in the cell membrane to reach the intracellular target to destroy the pathogens and in addition, it also limits the gastrointestinal tract infection by increasing sensitivity of bacteria to gastric juice that collapse the transmembrane cation gradients.^[29] κ-casecidin is obtained by trypsin digestion of κ-casein and displays its bactericidal activity against S. aureus, E. coli, and S. typhimurium.^[39] Apart from this, several other casein derived fragments including f(18-24), f(139-146), and f(30-32) have been proven the antimicrobial activity against the same pathogens.^[29] Research on casein-α_s1 born antimicrobial peptide have also revealed another two peptides known as caseicin A and caseicin B that provide inhibition of several pathogens (Staphylococcus, Sarcina, Bacillus subtilis, Diplococcus pneumoniae, and Streptococcus pyogenes) after digestion with chymosin enzyme.^[39] Casecidin, liberated by chymosin mediated digestion of casein-α_s1, has been reported to be active against a broad spectrum of pathogens.^[41] On the other hand, some cationic fragments including f(183–207), f(164–179), and f(165–203; casocidin-I) can be achieved by hydrolysis of α_s2-casein and provide a strong antimicrobial effect against the growth of gram-negative E. coli and gram-positive S. carnosus (Table 1).^[42] It has been demonstrated that N-terminal segment of α_s1-casein namely, isracidin is intended for therapeutic use to treat infections caused by S. aureus, Streptococcus pyogenes, and Listeria monocytogenes and also show a protective effect on sheep and cows against mastitis.^[33]

TABLE 1 Casein-derived AMPs released by digestive protease action, and their main properties^[33,42,^76–81]

Antimicrobial peptides	Source	Enzymatic activity	Activity against pathogens
Casecidins	bovine αs1-casein	Chymosin	Gram positive bacteria
Isracidin f(1-23)	bovine αs1-casein	Chymosin	Gram positive, negative bacteria
Casocidin-I f(150-188)	bovine αs2-casein	Trypsin	Gram positive, negative, yeasts
Casocidin-I f(181-207)	bovine αs2-casein	Chymosin	Gram positive, negative bacteria
Casocidin-I f(175-207)	bovine αs2-casein	Chymosin	Mainly Gram positive bacteria
Casocidin-I f(165-170)	bovine αs2-casein	Pepsin	Mainly Gram positive bacteria
Casocidin-I f(165-181)	bovine αs2-casein	Pepsin	Gram positive and negative bacteria
Kappacin f(106-169)	bovine κ casein	Pepsin	Mainly gram positive bacteria
Kappacin f(42-49)	bovine κ casein	Chymosin	Gram positive and negative bacteria

Whey Protein Derived Antibacterial Peptide

In recent years, whey derived AMPs have shown a measurable effect on health outcomes.^[43] Recent studies have shown that lactoferrin (Lf) is a non-haem iron binding multifunctional protein which is widely obtained in body secretions including colostrum, milk, tear, nasal secretion, saliva, bile, urine, and genital secretions. Its bacteriostatic effects have been well-documented both in vivo and in vitro against Bacillus stearothermophilus, B. subtilis, Clostridium spp., Haemophilus influenza,^[44] Streptococcus mutans, Vibrio cholerae, E. coli, and Legionella pneumophila.^[45] Bovine Lactoferricin, having 17–41 amino acid residues, is obtained from the basic N-terminal region of Lf.^[46] It is produced in gastrointestinal tract by gastric pepsin and exhibit antimicrobial activity against several gram-positive and gram-negative pathogens (such as E. coli, Listeria monocytogenes) including viruses and fungi.^[47,^48] Arginine and tryptophan residues are the major determinants of Lactoferricin that facilitates the interaction of positively charged residues of lactoferricin with negatively charges present in the inner core of LPS to disorganize the structure of outer membrane.^[47–49] Bovine lactoferrampin has been investigated most extensively and readily found in various species showing its antimicrobial activity against of B. subtilis, E. coli, and Pseudomonas aeruginosa where as several lines of evidences indicate that Lactoferrampin plays a critical role in membrane mediated activities of Lf.^[50] β-lactoglobulin derived peptides corresponded to β-lactoglobulin f(15–20), f(25–40), f(78–83), and f(92–100) obtained from β-lactoglobulin may function by inhibiting gram-positive bacteria.^[51] Studies showed that α-lactalbumin, yielded after digestion with trypsin and chymotrypsin, displays a wide array of mode of actions^[52] to execute its bactericidal activity against gram-positive bacteria with some examples as mentioned in Table 2. It has been observed that human milk contains a specific group of antimicrobial peptide, namely, beta-defensin-2 (hBD-2) that modulate the immune system^[53] by inhibiting overgrowth of potentially pathogenic enteric microorganisms, including Salmonella spp. and E. coli, as well as strains of S. marcescens, P. aeruginosa, and Acinetobacter baumanii. This peptide may help the child to avoid the development of inflammatory diseases and childhood infection.^[54] Antibacterial peptides from milk and whey proteins have been reported with clear inhibitory effects on various pathogenic cultures such as B. cereus, L. monocytogenes, S. aureus, S. typhi, S. dysentriae, and E. coli.^[55] It has been reported that gram-positive pathogens are more sensitive to bioactive peptides derived from whey as compare to gram-negative pathogens. Therefore, whey derived AMPs can be used as a major preventive against foodborne diseases and may also be utilized in the development of therapeutic foods to control the foodborne pathogens.^[55]

TABLE 2 Antibacterial peptides derived from whey protein^[51,⁵²,^82–84]

Antimicrobial peptides	Source	Enzymaticactivity	Activity againstpathogens
β-lactoglobulin f(15–20)	β-lactoglobulin	Trypsin	Gram positive
β-lactoglobulin f(25–40)	β-lactoglobulin	Trypsin	Gram positive
Lactoferricin f(17-41)	Lactoferrin	Pepsin	Escherichia coli, Listeria monocytogenes,viruses, and fungi
β-lactoglobulin f(78–83)	β-lactoglobulin	Trypsin	Gram positive
β-lactoglobulin f(92–100)	β-lactoglobulin	Trypsin	Gram positive

MECHANISMS BEHIND INHIBITORY ACTIVITY OF AMPS

Of the several mechanisms describing inhibitory action of AMPs, cell membrane permeabilization by forming transmembrane pore is the sole mode of action of AMPs. A thick rigid protective covering of prokaryotic cell contains negatively charged components, such as lipopolysaccharides (LPS; gram-negative bacteria) and teichoic acid (gram-positive bacteria). The primary interaction of AMP to its target cell is the initial electrostatic attraction between cationic AMPs and anionic components of bacterial cell wall.^[56] In gram-negative bacteria, AMP binds with surface LPS to partially neutralize it by displacing divalent polyanionic cations that leads to the disruption of cell envelope and reach into cytoplasmic membrane in a process named as self-promoted uptake.^[57] This process describes different mechanisms acting behind it and can be presented as several models including barrel stave model, carpet, toroidal, or aggregate channel model.^[56,⁵⁸,^59] After initial electrostatic binding to the outer membrane in bacteria, some AMPs get positioned on target surface membrane to become stave in a barrel-like cluster in such a way that, its side chains direct the hydrophobic lipid core of the membrane and the hydrophilic surfaces of peptides point facilitate pores. Leakage of cellular components through these transmembrane pores causes cell death.^[60,61] However, in other cases AMPs align parallel to the cell surface of target as a local carpet. When the threshold concentration reached, AMPs cause membrane permeation, eventually causing cell death.^[62] Early studies suggested that some AMPs insert themselves into bacterial cell membranes in a perpendicular orientation to form toroidal-shaped transmembrane pores with micellar formation to adopt irreversible membrane disruption, leading to abrupt lysis.^[63] In contrast, it has been also proposed that several other AMPs exert the inhibitory mechanism by forming un-structural aggregates on bacterial cell membrane which provides channels for ion leakage.^[64] Membrane damage mechanisms of AMPs can be categorized as energy dependent and energy independent uptake. After disruption of outer envelope, AMPs arrive at the cytoplasmic membrane where they pass to interfacial region of target cell to inhibit essential cellular processes.^[65] Bioinformatics approaches are also incorporated in bioactive peptide research for the prediction and selection of active properties.^[66]

STRATEGIES FOLLOWED FOR PURIFICATION

For the detection and identification of novel AMPs, a strategy of purification has been developed by means of ion exchange chromatography (IEC), high-performance liquid chromatography (HPLC), reverse-phase liquid chromatography (RP-HPLC), Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).^[67,^68] Cell free supernatant of culture containing peptide of interest is concentrated by using ammonium sulfate through the application of salting out, in which the salt solution is added to the sample until desired saturation of salt is reached. The range of saturation always varies with respect to the samples. Therefore, the percentage of salt concentration involved in the precipitation of the peptide of interest must be assayed before going through the above procedure.^[69,^70] The suspension is washed twice by centrifugation with a phosphate buffer and can be desalted by using benzoylated membrane with the same buffer.^[71] Several scientific researches demonstrate that AMPs exist as cationic or anionic form depending upon their isoelectric points and readily adsorbed to cationic exchange resin or anionic exchange resin used in ion exchange column chromatography. Furthermore, the column is equilibrated with phosphate buffer and desired peptides can be eluted with the starting buffer containing an increasing salt (NaCl) concentration.^[72] The methodology of ammonium sulfate precipitation, ultra-filtration, and IEC could be used to produce a partially purified sample. The complete fractionation of resulting AMPs are only achieved by HPLC, RP-HPLC using RP-C18 column and trifluoroacetic acid (TFA) in acetonitrile solvent. The whole process of HPLC can be carried out by following four steps (1) 0.1% TFA in acetonitrile is used to prepare two same solvents (solvent A, solvent B) in the first step with a flow rate of 2 mL/min, gradient of 0–60% B in 45 min, (2) follows the same condition as used in first step with a gradient of 20–50%, (3) 0.1% TFA in methanol is used in the preparation of solvent B with a gradient of 40–70% B in 45 min, and (4) finally, the ion exchange column is used with two solvents (solvent A: 10 mM phosphate buffer, solvent B:10 mM phosphate buffer with 1M NaCl) where the flow rate 0.75 mL/min and the gradient is measured as 0–60% B within 60 min.^[67] In addition to HPLC, another useful technique, such as RP-HPLC is also widely used in this purpose. RP-HPLC is an analytical and preparatory HPLC which has shown to be a valuable for analysis consisting of an automated gradient controller pump module, an automatic sampling device and a photodiode array detector, whereas all chromatographic data are plotted and analyzed by using millennium software.^[68] In the protocol of this technique, acetonitrile and HPLC-grade water containing 0.1% TFA are most frequently used as a mobile phase where several varieties of silica-based column (C₄-C₁₈) are recommended according to the peptide characterization. Peptides with microbial inhibitory activity are supposed to be found in fractions collected in between retention time.^[73–75] SDS-PAGE is employed as a useful analytical purification technique using Coomassie blue stain or Silver stain.^[71] Peptide sequence identification in correspond to their molecular mass are employed by using Liquid chromatography–mass spectrometry (LC-MS) and matrix assisted laser desorption ionization - time of flight (MALDI-TOF).^[68]

CONCLUSION

Development of antibiotics against antibiotic-resistant microorganisms is a growing concern of recent scientific research. Milk is part of our daily food intake as an array of AMPs which has been the focus of intense research in formulation of nutraceutical foods and drugs. Although limited milk derived AMPs are discovered currently, it is supposed to be obtained several such unique peptides having great potential in clinical and food aspects. Further researches are needed to decode the beneficial pathogen inhibitory effects of dietary antimicrobial peptide that can be possible by development of economically feasible industrial and scientific methods. Industrial scale processes, including advance in peptidomic analysis, nano-encapsulation, and microencapsulation, are widely employed to increase the stability of peptides during enzymatic digestion. Sound knowledge of exact inhibitory mechanisms followed by different peptides against pathogens would give an emphasis in formulation of potent drugs. This field of study promises to devolve wide varieties of pharmaceutical food products and drugs having a great impact on human health and physiology.