Antimicrobial

doi: 10.1111/j.1471-0307.2010.00584.
REVIEW
Antimicrobial peptides generated from milk proteins: a survey and prospects for application in the food industry. A review
NOREDDINE BENKERROUM*
Departement des Sciences Alimentaires et Nutritionnelles, Institut Agronomique et Veterinaire Hassan II, Instituts, 10101-Rabat, Morocco
Milk proteins constitute a natural reservoir of bioactive peptides with physiological and or antimicrobial properties, the release of which requires hydrolysis of the precursor molecules by digestive proteases or by fermentation with proteolytic micro-organisms. Depending on the digestive or microbial proteases used, an array of bioactive peptides would be released either from caseins or whey proteins, but only a small part of these peptides has so far been identied and characterised with respect to their antimicrobial activity. The antimicrobial peptides known thus far have proven to be potent inhibitors to the growth of a wide range of undesirable micro-organisms of health or spoilage signicance. Nevertheless, previous research work has largely been oriented towards their possible application in medicine, which has hindered their high potential as food-grade biopreservatives and or as supplements in functional foods. This review attempts to study the literature pertaining to antimicrobial peptides derived from major milk proteins (caseins, a-lactalbumin and b-lactoglobulin) upon hydrolysis either by digestive proteases or by fermentation with proteolytic lactic acid bacteria. Their possible application in the food industry and their mechanism of action will also be discussed. Reference antimicrobial peptides produced by living micro-organisms as innate immune defence components against microbial infections will occasionally be invoked for comparison purposes. Keywords Antimicrobial peptides, Milk proteins, Caseins, a-Lactalbumin, b-Lactoglobulin, Proteases, Lactic acid bacteria, Food industry.
INTRODUCTION Antimicrobial peptides produced by a wide variety of unicellular or multicellular living organisms as a rst-line defence against invading micro-organisms have attracted increased attention since the discovery of lysozyme (Fleming 1922). Many of these small molecules (< 10 kDa; 350 amino acid residues) have proven to be potent antimicrobial substances with promising applications in medicine or food preservation. Therefore, intensive research work has been carried out to detect, purify and characterise as many of these peptides as possible for application in industrial production. To date, a repertoire of more than 880 such peptides exist in international databases (Wang and Wang 2004; Rydengard et al. 2008). This number continues to grow, as sources other than living organisms, such as food proteins (Pellegrini 2003), can now be used to generate bioactive peptides. In particular, milk proteins have been recognised as important resources for peptides with biological activities. Once these peptides are released they exert, in addition to antimicrobial activities, other
*Author for corrrespondence. E-mail: n.benkerroum@gmail.com 2010 Society of Dairy Technology
interesting biological functions such as opioid, antithrombotic, immunomodulatory, antihypertensive or mineral carrying activities (Clare and Swaisgood 2000). Moreover, some antimicrobial peptides released from milk can also have other physiological functions (i.e. multifunctional peptides) (Meisel 1998; Clare and Swaisgood 2000; Tomita et al. 2002; Lopez-Exposito et al. 2007; Rydengard et al. 2008). Furthermore, the antimi crobial peptides present the advantage of being derived from a harmless and inexpensive source, and have therefore an undeniable potential for use in medicine or the food industry. In order to be active, these milk-derived peptides have to be rst released from their parent molecules. This can be achieved either by hydrolysis of the parent molecules (caseins and whey proteins) by digestive proteases (Clare and Swaisgood 2000) or by fermentation with selected proteolytic lactic acid bacteria (LAB) (Hayes et al. 2006); milk acidication followed by heat treatment has also been reported to generate active peptides (Zucht et al. 1995; Tomita et al. 2002). The present review will focus on antimicrobial peptides generated from
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Vol 63, No 3 August 2010 International Journal of Dairy Technology
Vol 63, No 3 August 2010
major milk proteins by digestive proteases or by proteolytic LAB. Their potential applications in the food industry and their antibacterial mechanisms of action are also discussed. Antimicrobial peptides generated from minor milk proteins (e.g. lactoferrin and lysozyme) are not considered in this review, as they have been extensively reviewed elsewhere (Tomita et al. 2002; Floris et al. 2003; Pellegrini 2003; Benkerroum 2008). ANTIMICROBIAL PEPTIDES G E N E R AT E D F R O M M I L K PROTEINS BY DIGESTIVE PROTEASES Major milk proteins [e.g. caseins, a-lactalbumin (a-La) and b-lactoglobulin (b-Lg)] constitute an important source for bioactive peptides reported to have a wide range of physiological properties (Meisel 1998; Clare and Swaisgood 2000; Pellegrini 2003). Among the array of known bioactive peptides, those exerting antimicrobial activities have received limited attention compared to their physiologically active counterparts (e.g. antihypertensive, antithrombotic and immunomodulatory peptides). This is due to the lack of interest in their commercial production within the healthcare industry as most of them are intended for medical use, mainly as antibiotics, and their production would be costly while being less potent than conventional antibiotics (Lahov and Regelson 1996). Now, however, there is a growing interest in the use of these antimicrobial peptides as foodgrade biopreservatives or as health-promoting food supplements. Such a trend is encouraged by the preference of consumers for lightly processed foods containing the least possible chemical additives, and the search for functional foods perceived as nutritious, healthy and prophylactic. This review deals with the main antimicrobial peptides released by action of digestive proteases from caseins and whey proteins, and how they would nd appropriate applications in the food industry. CASEIN-DERIVED ANTIMICROBIAL PEPTIDES
Antimicrobial peptides from a-caseins About three decades after the report of Jones and Simms (1930) on the presence of lactenin in milk treated with rennet and its antimicrobial activity against streptococci, several antibacterial peptides have been reported to be released from milk proteins upon hydrolysis with digestive proteases (Tables 1 and 2). These peptides are characterised to have a high diversity of structural and antimicrobial properties making it difcult to classify them in a specic group(s) of antimicrobial substances such as those proposed by Epand and Vogel (1999)
2010 Society of Dairy Technology
or by Reddy et al. (2004). Casecidins, obtained by chymosin digestion of as1-casein at neutral pH, were the rst reported milk-derived antimicrobial peptides (Lahov et al. 1971); and they constitute a family of basic glycopeptides with a relatively high molecular weight ($46 kDa). Upon discovery, casecidins were intended for therapeutic use to treat infectious diseases owing to their bactericidal activity against a wide range of Gram-positive bacteria of health signicance including staphylococci, Sarcina spp., Bacillus subtilis, Diplococcus pneumoniae and Streptococcus pyogenes (Lahov and Regelson 1996; Clare and Swaisgood 2000). However, their clinical performances were poor compared to commercially available antibiotics, and they required signicantly higher concentrations to inhibit, in vitro, sensitive bacteria (Lahov et al. 1971). Therefore, interest in casecidins has languished and their commercial development has been omitted. Nevertheless, such a discovery had the merit to revive interest of scientists in this area of research, and subsequent studies have characterised several novel milk-derived peptides with different antimicrobial properties and spectra of action (Tables 1 and 2). Isracidin is another antimicrobial peptide released by chymosin cleavage of bovine aS1-casein, which consists in a 23-aminoacid-residue fragment of aS1-casein at the N-terminal end [i.e., aS1-CN f(1-23)] (Hill et al. 1974). This cationic peptide has been reported to be active in vitro against a broad spectrum of Gram-positive and Gram-negative bacteria, but only at high concentrations ranging between 0.1 and 1 mg mL (Hill et al. 1974; Kolb 2001; Floris et al. 2003; Hayes et al. 2006). Conversely, in vivo, isracidin has proven competitive with antibiotics in therapeutic use and provided a strong protective effect in mice against Staphylococcus aureus, S. pyogenes and Listeria monocytogenes. An intramuscular injection of the peptide at levels similar to those used in standard antibiotherapy (10 lg per mouse) has prevented mice from lethal infections when administered before challenge with the above-mentioned pathogens (Kolb 2001). The peptide has also proven to be effective in protecting sheep and cows against mastitis when injected into the udder, and such protection has lasted beyond the period of treatment in a long-term immuneresponse manner (Lahov and Regelson 1996). Therefore, isracidin was considered to have both prophylactic and therapeutic effects; an observation that was corroborated by in vivo studies showing that intravenous injection of isracidin into mice stimulates their cellular and humoral immune responses, thereby protecting them from infection by Candida albicans (Lahov and Regelson 1996). The weak in vitro antimicrobial activity of isaracidin and its high efcacy in vivo represent a strong indication that it has an indirect mode of action as 321
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Cleaving protease Activity Gram positive bacteria Gram-positive and -negative bacteria Gram-positive and -negative bacteria, and yeasts Cationic peptide Cationic peptide Cationic peptide Cationic glycopeptides Reference Chymosin Chymosin R1PKHPIKHQGLPQEVLNENL-LRF23 NA Amino acid sequence (one letter code)a Main chemical properties (Lahov et al. 1971) (Lahov and Regelson 1996) (Hill et al. 1974)
Table 1 Casein-derived antimicrobial peptides released by digestive protease action, and their main properties
Antimicrobial peptide
Source
MW (kDa)
Casecidins
bovine aS1-casein
4-6
Isracidin f(1-23)
bovine aS1-casein
2.7
Casocidin-I f(150-188)
bovine aS2-casein
4.9
Trypsinb
Cationic and hydrophobic peptide Polyproline-helix like structure Cationic peptide
(Malin et al. 2001) (Zucht et al. 1995) (Forssmann et al. 2003) (McCann et al. 2005)
f(181-207) Chymosin Chymosin
bovine aS2-casein Chymosin
3.3
f(180-207)
bovine aS2-casein
3.4
f(175-207)
bovine aS2-casein
4.0
Gram-positive and gram negative bacteria Gram-positive and gram negative bacteria Gram-positive and gram-negative bacteria Gram-positive and gram-negative bacteria Gram-positive and Gram-negative bacteria Cationic peptide
f(172-207)
4.4
f(164-207)
5.4
Cationic peptide
f(165-170) f(165-181) Pepsin Pepsin chymosin
ovine aS2-casein ovine aS2-casein Pepsin Pepsin
0.7 2.2
K150TKLTEEEKNRLNFLKKISQRYQKFALPQYLKTVYQHQ-K188c KTVYQHQKAMKPWIQPKTK-VIPYVRYL LKTVYQHQKAMKPWIQPKT-KV IPYVRYL ALPQYLKTVYQHQKAMKPW-IQPKTKVIPYVRYL QKFALPQYLKTVYQHQKAM-KPWIQPKTKVIPYVRYL LKKISQRYQKFALPQYLKTV-YQHQKAMKPWIQPKTKVIP-YVRYL LKKISQ LKKISQYYQKFAWPQYL Cationic peptide Cationic peptide Cationic peptide Cationic peptide Anionic phosphopeptide Mainly Gram-positive bacteria Gram-positive and Gram-negative bacteria Mainly Gram-positive bacteria
(Lopez-Exposito et al. 2006a)
f(184-208)
ovine aS2-casein
3.0
f(203-208) Kappacin Ad f(106-169)
ovine aS2-casein bovine j casein
0.8 6.8
Mainly Gram-positive bacteria Gram-positive and Gram-negative bacteria
(Malkoski et al. 2001)
f(42-49) Pepsin Pepsin IQY VQVTSTAV
bovine j casein Pepsin
1.0
VDQHQKAMKPWTQPKTNAIPYVR-YL PYVRYL A106IPPKKNQDKTEIPTINTIASGE-PTSTPTTEAVESTVATLEDRPEVIESPPEI NTVQVTSTAV169e YYQQKPVA
Cationic Neutral Neutral
(Lopez-Exposito et al. 2006b)
f(28-30) f(162-169)
bovine j casein bovine j casein
0.4 0.8
Gram-positive and Gram-negative bacteria Mainly gram-negative bacteria Mainly Gram-positive bacteria
an immunostimulatory peptide to induce host-mediated nonspecic resistance to infectious microorganisms (Lahov and Regelson 1996; Kolb 2001). Nonetheless, like its predecessors casocidins, isracidin did not attract the anticipated commercial interest primarily due to the onerous and costly procedures for its purication and production on large scale batch fermentation for commercial use. Isomeric variation of the molecule during fermentation leading to inconsistencies in the purity of the nal product has been suggested as another technical limitation (Lahov and Regelson 1996; Ross et al. 2007). Its cationic nature may also account for the lack of commercial interest for therapeutic use. Cationic antimicrobial peptides are less effective in treating systemic diseases and tend to adsorb onto negatively charged surfaces, thereby reducing their effectiveness if they are administered remotely from the site of the infection to be treated (Dashper et al. 2007). Although no data, to our knowledge, are available regarding the possible application of isracidin in the food industry, its use appears promising and feasible. In a recent study, Hayes et al. (2006) demonstrated that isracidin is active against emerging pathogens of major concern to food safety, including Escherichia coli O157:H7, Enterobacter sakazakii and Staph. aureus. In addition, the antimicrobial activity of isracidin may be signicantly improved in food systems depending on the composition and intrinsic ecological parameters of the food matrix. Nisin, for example, the most widely used cationic peptide in the food industry is inactive or even partially degraded at pH values above 8.0 while under acid conditions it inhibits sensitive bacteria at nanogram concentrations (Hurst 1981; Benkerroum and Sandine 1988; Zasloff 2002; Benkerroum et al. 2003). Studies on the optimal parameters for antimicrobial activity of isracidin should, therefore, be carried out in the scope of its application in food preservation. To reduce the cost and labour of production, it may be possible to envisage the use of crude milk-based isracidin preparations instead of those highly puried as is required for pharmaceutical uses. Crude preparations from natural products, such as milk, do not raise food safety concerns and may thus be easily approved by health regulatory authorities for use in foods. MicrogardTM, lactoferrin and lactoferricins are examples of such crude preparations that have been legally approved for use in the food industry in many countries (Al-Zoreki et al. 1991; Tomita et al. 2002). Bovine as2-casein has also been reported to be a source of antimicrobial peptides among which casocidin-I was the rst to be well characterised (Zucht et al. 1995). It is a heat- and acid-stable peptide isolated from milk after hydrolysis of milk proteins by heat treatment under acidic conditions, 323
f(141-146) f(18-24) f(30-32) f(118-121) f(139-146)
f(64-75)
The number of the amino acid of the sequence (lower case) corresponds to the position of this amino acid in the parent mature protein. bOn the basis of the observation that C-and N-terminal ends of the peptide are potential tryptic digestion sites (Zucht et al. 1995). cUnderlined boldface sequence corresponds to a peptic digest with intrinsic growth-inhibitory activity (Recio and Visser 1999); it probably represents the active region of casocidin-I. dIn variant B, residues T136 and D148 are substituted for I136 and A148. eUnderlined boldface sequence represents the active region of kappacin A (Malkoski et al. 2001), R is a phosphorylated seryl residue.
a
Main chemical properties
Reference Neutral Cationic Neutral Anionic Anionic Pepsin Pepsin Pepsin Pepsin Pepsin 0.6 0.8 0.4 0.5 0.8 bovine j casein bovine j casein bovine j casein bovine j casein bovine j casein STVATL FSDKIAK YVL EIPT VESTVATL
Activity
Cleaving protease
Amino acid sequence (one letter code)a
MW (kDa)
Table 1 (Continued)
Antimicrobial peptide
Source
bovine j casein
1.3
Pepsin
PAAVRSPAQILQ
Weakly inhibitory Cationic peptide Mainly gram positive bacteria Mainly gram-negative bacteria Gram-positive and Gram-negative bacteria Gram-positive and Gram-negative bacteria
Cationic
Table 2 Antibacterial peptides derived from a-lactalbumin (a-La) or b-lactoglobulin (b-Lg) Sequence (one letter code) E1QLTK5 G17YGGVSLPEWVCTTF31 A109LCSEK114b C61KDDQNPH68 I75SCDKF80c V15AGTWY20 A25ASDISLLDAQSAPLR40 I78PAVFK83 V92LVLDTDYK100
a
Origin a-La a-La a-La b-Lg b-Lg b-Lg b- Lg
Amino acid fragment a f(1-5) f(17-31)S-S(109-114) (61-68)S-S(75-80) 1520 2540 7883 92100
Cleavage enzyme Trypsin Chymotrypsin Trypsin
References (Pellegrini et al. 1999)
(Pellegrini et al. 2001)
Numbers indicate the positions of the rst and last amino acids of the fragments as in the mature parent molecule. bTwo peptides held by disulde bond between cysteyl residues at positions 28 and 111. cTwo peptides held by a disulde bond between cysteyl residues at positions 61 and 77.
followed by different treatments to remove fat and most of the remaining high molecular-weight proteins. Basic peptides are then concentrated, chromatographically fractionated and eventually digested with proteases, e.g. pronase, endoproteinase Glu-c or trypsin (Zucht et al. 1995; Forssmann et al. 2001, 2003). Characterisation of the released peptides by Edman degradation reactions and mass spectroscopy has revealed casocidin-I as an as2casein-derived fragment consisting in 39 aminoacid residues [as2-CN f(150-188)] with both amino acid termini (i.e. C- and N-terminal) being tryptic cleavage sites (Zucht et al. 1995). In vitro antimicrobial activity tests showed that casocidin-I was inhibitory to two strains of E. coli and one strain of Staphylococcus carnosus in a dose-dependent manner. Forssmann et al. (2003) have further reported that casocidin-I inhibits the growth of several micro-organisms of hygienic and spoilage signicance including B. subtilis, Staph. carnosus, Staphylococcus epidermidis, Enterococcus faecium, E. coli and Rhodotorula rubra at concentrations ranging between 1 and 180 lg mL, B. subtilis was shown to be the most sensitive and Ent. faecium the most resistant. As regards the potential application of casocidin-I, Zucht et al. (1995) originally recommended using it in infant formulae to provide suckling infants with antibacterial peptides capable of inuencing the composition of the intestinal microora, especially those not present in human milk which is devoid of as2casein. The same authors later patented casocidinI, claiming its suitability to treat various dermatitis and mucosal infections of different origins (bacteria, fungi or parasites) as well as diarrheic gastroenteritis (Forssmann et al. 2003). They have also recommended using it in food preservation and in fermentation processes (Forssmann et al. 2001). However, most of these claims remain unconrmed; in particular the spectrum of action and clinical performances of the peptide. Application of casocidin-I as a food additive to enhance safety and keeping quality appears to be worthwhile considering. Yet, the onerous and costly procedure for the isolation and purication of the bioactive 324
peptide from milk may hinder its widespread application, as was pointed out by Ross et al. (2007). Crude milk-based preparations of casocidin-I may be an appropriate alternative. A suggested procedure for the crude preparation of casocidin-I on the basis of the purication technique described by Zucht et al. (1995) is illustrated in Figure 1. This crude preparation obtained according to Zucht et al. (1995) may be further desalted and concentrated by ultraltration and dialtration, and then freeze- or spray-dried as is the case for industrial production of LactoferrinTM (Tomita et al. 2002). Although this procedure is not expected to yield a high purity product, the resulting crude preparation would be more advantageous for food preservation than a highly puried casocidin-I. Fractionation of the crude preparation of casocidin-I followed by antibacterial activity testing of the resulting fractions has revealed the presence of various minor antibacterial peptides having an additive antibacterial effect with each other and with casicidin-I (Zucht et al. 1995). Therefore, these authors have anticipated that, beside casocidin-I, other antibacterial peptides with different masses would be released from the C-terminal region of bovine as2casein beginning from residue 165 or 166. Indeed, subsequent studies have shown that the hydrolysis of bovine as2-casein with digestive proteases generates various antibacterial peptides deriving from the C-terminal region starting from residue 164 (Recio and Visser 1999; McCann et al. 2005; Lopez-Exposito et al. 2006a). Recio and Visser (1999) reported on the release of two antibacterial domains by pepsin digestion of bovine as2-casein, f(164-179) and f(183-207), which were strongly inhibitory to E. coli, Bacillus cereus and Streptococcus thermophilus. However, the originality of these peptides was controversial; the rst fragment was considered only to be the active region of casocidin-I, as it is totally included in its sequence, and the second fragment has been assumed to be a component of the innate defence system of the mammary gland due to structural similarities (Boman 1995; Kolb 2001). Furthermore, McCann et al. (2005) showed that chymosin digestion of
Figure 1 Flow chart for crude preparation of casocidin-I (adapted from Zucht et al. 1995 and Tomita et al. 2002).
bovine sodium caseinates releases ve different antibacterial peptides identied as fragments of as2-casein C-terminal end (starting from residue 164), and sharing the 27-amino acid-residue sequence f(181-207) of the casein C-terminal region (Table 1). While four of these antimicrobial peptides had not been reported earlier, the common fragment, f(181-207), matched exactly one of the peptides previously isolated from the pepsin hydrolysate of bovine as2-casein and considered to be the fragment containing the active domain f(183207) (Recio and Visser 1999). The ve antibacterial peptides characterised by McCann et al. (2005) inhibited a range of Gram-positive and Gram-negative bacteria to different extents, though being generally more active against Gram-positive than Gram-negative bacteria; the MICs against L. monocytogenes, L. innocua and B. subtilis ranged between 4.8 and 21 lg mL, whereas the MICs
against S. Typhimurium, S. Enteridis and E. coli were signicantly higher and ranged between 21 and > 171.2 lg mL. Of these peptides, f(181207), f(175-207) and f(164-207) inhibited sensitive Gram-positive bacteria as effectively as nisin and lactoferricin B, suggesting that they have a high potential to be used as food grade preservatives. Furthermore, the antibacterial domain f(164-179) reported by Recio and Visser (1999) was included in only one of the ve peptides reported by McCann et al. (2005), indicating that it could not be the active site of the antibacterial peptides derived from the C-terminal region of bovine as2casein, contrary to what was suggested earlier (Kolb 2001). Indeed, according to McCann et al. (2005) and Lopez-Exposito et al. (2006a), the anti bacterial activity of the as2-CN-derived peptides does not correlate with the presence of a specic active site nor does it with the size of the peptide; 325
it was suggested to rather be due to the net electric charge and hydrophobicity of the peptide. Similarly, antibacterial peptides derived from ovine as2-casein were also demonstrated to be encrypted in the C-terminal region, and pepsin digestion of ovine as2-CN followed by a fractionation of the resulting hydrolysate by chromatography techniques yielded four major fractions inhibitory against E. coli. Identication of the peptides recovered from these fractions revealed the presence of 10 peptides of different masses, all deriving from the C-terminal region starting from residue 165. However, antibacterial activity could not be attributed to specic peptides, as the fractions still contained more than one peptide at the ultimate purication step (Lopez-Exposito et al. 2006a). Therefore, selected fragments of the most abundant in each fraction sub-fraction (Table 1) were chemically synthesised and investigated for their antimicrobial activity against various Grampositive and Gram-negative bacteria. On the structural level, antibacterial peptides released by pepsin digestion of ovine as2-casein were generally smaller than those generated from bovine as2-casein (5 25 vs 2744). Incidentally, two of the most active of the ovine as2-casein peptides, f(165-181) and f(184-208), corresponded to the two antibacterial domains of bovine as2-casein reported by Recio and Visser (1999), providing further evidence for the originality of the latter peptides. However, differences in the antibacterial properties between the two ovine fragments [i.e. f(165-181) and f(184208)] and their bovine homologues [i.e. f(164-179) and f(183-207)] were observed when tested in the same conditions and against the same indicator strains (Lopez-Exposito et al. 2006a). Ovine as2 CN f(165-181) was signicantly more active than its bovine homologue, while the bovine f(183-207) exhibited stronger antibacterial activity than the ovine homologue (Lopez-Exposito et al. 2006a).
Antimicrobial peptides from j-casein This class of milk proteins has also been demonstrated to be an important precursor of antibacterial peptides of potential use in food preservation and safety. The generation of such peptides from jcasein was rst demonstrated by Liepke et al. (2001) who puried an antibacterial peptide f(63117) from a hydrolysate obtained by acidication of human milk followed by pepsin hydrolysis, simulating the digestion process in infant stomachs. This peptide was shown to be inhibitory to Gram-positive and Gram-negative bacteria as well as yeasts, and was thus suggested for use in infant nutrition to stimulate the host defence system of the newborn. A subsequent study has demonstrated that a C-terminal chymosin-digest of bovine j-casein [j-CN f(106-169)], called caseinomacropeptide (CMP), exerts in vitro antibacterial activity against major
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oral pathogens (e.g. Streptococcus mutans, Porphyromonas gingivalis and Actinomyces naeslundii) and E. coli (Malkoski et al. 2001; Dashper et al. 2007). CMP is a highly heterogeneous polypeptide encompassing various glycosylated and phosphorylated forms (Saito and Itoh 1992; Talbo et al. 2001); and such heterogeneity is increased by the existing genetic variants of j-casein, with variants A and B being the most frequent (Creamer and Harris 1997). Malkoski et al. (2001) have demonstrated that the nonglycosylated, phosphorylated CMP is the only active form of the molecule, which they have designated kappacin. The antimicrobial activity of kappacin derived from variant B j-casein (j-CN-B) was, though, shown to be signicantly lower than that of variant-A kappacin; the activity of both peptides was lost or markedly reduced at neutral pH (Malkoski et al. 2001; Dashper et al. 2005, 2007). The difference in activities between kappacin fragments derived from variants A and B has mainly been attributed to the substitution of Asp148 in variant A for Ala148 in variant B (Dashper et al. 2005). Furthermore, digestion of kappacin-A with endoproteinase Glu-C generated various peptides among which only a phosphorylated one [Ser(P)149 j-CN-A f(138-158)] was active against S. mutans, indicating that the bactericidal domain of kappacin resides in this portion. Also, phosphorylation of the seryl residue at position 149 (Ser149) was shown to be crucial for antibacterial activity as was demonstrated by the susceptibility of S. mutans to chemically synthesised kappacin with phosphorylated Ser149 [Ser(P)149 k-CN-A(138-158)] and the resistance of the same strain to the corresponding synthetic fragment with nonphosphorylated seryl residue [Ser149 j-CN-A(138-158)]. Interestingly, the synthetic kappacin had higher potency than the puried peptide and its derivative f(138-158). Moreover, kappacin was shown to specically bind two zinc or calcium ions per mol, resulting in a signicant increase in its antimicrobial activity even at neutral pH. These divalent ions were suggested to help potentiate the activity of kappacin by inducing conformational changes in such a way to enhance the binding afnity of kappacin to the cell membrane (Dashper et al. 2005, see also the paragraph on the mechanism of action, below). The research work that has, so far, been conducted on kappacin has been largely oriented towards the use of the peptide as a pharmaceutical supplement for oral therapy (Malkoski et al. 2001; Dashper and Reynolds 2005; Dashper et al. 2005, 2007). In fact, kappacin has already been commercially available for dental care application by the Cooperative Research Centre for Oral Health Science (Australia) as KappacinTM or as KappaZinTM. The latter product consists in a combination of kappacin with zinc (1:1) to form an antimicrobial complex with enhanced antibacterial activity.
Apart from this application, kappacin may also be a suitable and safe food-grade biopreservative with high potential for application in the food industry due to its ability to inhibit Gram-positive and Gram-negative bacteria and to resist proteolytic enzymes, in addition to its presumed safety as it is derived from j-casein that has a long history of safe consumption in dairy products. As a matter of fact, the vast majority of known cheese varieties already contain some amount of kappacin in the form of CMP resulting from milk coagulation with rennet or other clotting enzymes of animal or plant origin that hydrolyse j-casein at the same cleavage site (see Abd-El Salam and Benkerroum 2006). The antimicrobial effect of kappacin would be particularly enhanced in dairy products which are naturally rich in calcium ions shown to potentiate the antibacterial activity of the peptide (Dashper et al. 2005). Furthermore, in most cheeses, an excess of calcium is added during manufacture to accelerate milk clotting, thereby increasing the amount of calcium available for binding kappacin. Further studies are needed to substantiate such observations, and to determine the optimal conditions as well the target food products on a sound basis. Health risk assessment studies, especially concerning the potential allergenicity of the peptide have also to be carried out prior to any practical use. In another study on the generation of antibacterial peptides from milk caseins, Lopez-Exposito et al. (2006b) have identied 21 fragments in six different active HPLC fractions recovered from a pepsin hydrolysate of commercial j-casein. However, a thorough purication and identication of these digests revealed that only 12 (recovered from 5 active fractions) were actually released from jcasein, while the others were derived from the a or b caseins contaminating the commercial j-casein used in the study, as revealed by capillary electrophoresis. Of the 12 j-casein-derived peptides, nine of the most abundant in the corresponding active HPLC fractions (Table 1) were chemically synthesised and tested for their antimicrobial activity against Gram-positive (L. innocua and Staph. carnosus) and Gram-negative (E. coli, S. marcescens) bacteria. These peptides inhibited indicator strains to different extents, with fragments f(1824), f(30-32) and f(139-146) being the most inhibitory, inducing a reduction of the initial counts of the most sensitive bacteria by more than six logarithmic units after 2 h of incubation. Nonetheless, S. marcescens was only moderately inhibited by all the peptides, and its initial count was reduced by 0.200.59 logarithmic units depending on the peptide, with the exception of peptide f(30-32) which reduced the initial count of the bacterium by three logarithmic units after 2 h of incubation (Lopez-Exposito et al. 2006b).
Antimicrobial peptides from whey proteins Whey proteins (a-La, b-Lg, serum albumin, immunoglobulins, lactoferrin, lysozyme, etc.) are known to be valuable sources for bioactive peptides with health promoting or antimicrobial properties (Floris et al. 2003). Among these, lactoferrin and lysozyme possess inherent antimicrobial activities, and also release antimicrobial fragments by proteolytic digestion (Tomita et al. 2002; Touch et al. 2004). The latter two whey-protein components and the antimicrobial peptides derived therefrom are the best studied, and have extensively been reviewed elsewhere (Salton 1957; Pellegrini et al. 1992; Tomita et al. 1994, 2002; Floris et al. 2003; Pellegrini 2003; Benkerroum 2008). On the contrary, antimicrobial peptides derived from a-La and b-Lg have received much less attention despite the scientic evidence for their existence and the potential of a-La and b-Lg as bioactive peptide precursors (Fiat et al. 1993; Meisel 1998; Shaha 2000; Pellegrini 2003). In a preliminary study, Pihlanto-Leppala et al. (1999) showed that proteolytic digestion of bovine a-LA and b-Lg with seven different proteases yielded various hydrolysates exerting bacteriostatic activities against E. coli, while the parent molecules failed to inhibit the bacterium under the same experimental conditions. The most active hydrolysates were obtained by a combined action of pepsine and trypsin, and have a molecular mass less than 1 kDa; however, the inhibitory peptides were not further characterised or sequenced. In two other studies, a-lactalbulin and b-Lg were digested with pepsine, trypsin or chymotrypsin, and the hydrolysates were fractionated chromatographically, and the growth-inhibitory activity of each fraction was monitored; the active peptides were then puried and characterised (Pellegrini et al. 1999, 2001). While no antibacterial activity was associated with the peptic digests of a-LA, hydrolysis of the protein with trypsin or chymotrypsin yielded three antibacterial fragments; two tryptic and one chymotryptic (Table 2). The chymotryptic fragment and one of the two tryptic fragments have a dimeric structure, each of which is composed of two subunits held together by a disulde bridge, and they were shown to be signicantly more inhibitory than the monomeric pentapeptide released from a-LA, with the tryptic dimer being the most active (Pellegrini et al. 1999). However, it has not been demonstrated whether or not the disulde bridges of the dimeric peptides account for such increased antimicrobial activity, as is the case for other antimicrobial peptides where the formation of intra- or intermolecular disulde bridges, via specic assembly or post-translational rearrangement mechanisms, is crucial to their activity (Romeo et al. 1988; Hill et al. 1991; Simmaco et al. 1998;
327
Reddy et al. 2004; Jenssen et al. 2006). The disulde bridges of the a-LA-derived dimeric peptides do not appear to result from a specic assembly mechanism, as they are naturally present in the parent molecule (Ikeguchi et al. 1998). They may only remain intact, holding the two subunits together, after the hydrolysis of the protein as depicted in Figure 2. As for the potential of b-Lg to generate antimicrobial peptides, Pellegrini et al. (2001) have shown that the tryptic digestion of the protein yielded four antibacterial peptides with different lengths (616 residues) and different amino acid sequences, which were inhibitory to Gram-positive bacteria only (Table 2). However, modication of the nine amino-acid peptide VLVLDTDYK by substituting the Asp98 residue for Arg and adding a lysyl residue at the C-terminus has made E. coli susceptible to the resulting peptide (VLVLDTRYKK), thereby extending the spectrum of action to Gramnegative bacteria (Pellegrini et al. 2001). These studies have lead the authors to recommend the major whey proteins (i.e. a-La and b-Lg), after partial digestion by proteases, for a possible antimicrobial function in therapy or in the food industry (Pellegrini et al. 1999, 2001). Yet, none of the seven peptides generated from a-LA and b-Lg has found an industrial application; they would suffer from three main limitations: (i) the narrow spectrum of action limited to Gram-positive bacteria, (ii) the moderate antimicrobial potency and (iii) the potential allergenicity of the peptides, particularly those derived from b-lactoglobulin which is known to be a major bovine milk allergen with numerous epitopes (regions of the molecule able to bind IgE) scattered all along the molecule (Natale et al. 1989; Selo et al. 1999). Although authors have shown that the allergenicity of peptides derived from milk
proteins is signicantly reduced compared to the precursor molecules (Fritsche et al. 2005; Asselin et al. 2006), others have demonstrated that b-Lg-derived peptides containing epitopes retained between 40% and 97% allergenicity of the parent molecule (Selo et al. 1999). Therefore, whey derived antimicrobial peptides should be scrutinised on a case-by-case basis for allergenicity at different concentrations for their anticipated use in the food industry. ANTIMICROBIAL PEPTIDES RELEASED FROM MILK PROTEINS B Y F E R M E N TAT I O N W I T H L A C T I C ACID BACTERIA Degradation of milk proteins by LAB as a means to full their nutritional requirements for essential amino acids is well documented (Abraham et al. 1993; Fira et al. 2001; Matar et al. 2003). Therefore, the use of LAB or their proteases to generate bioactive peptides from milk proteins is attracting increased attention; and such bioactive peptides are expected to be fundamentally different from those released by digestive proteinases which differ from microbial proteinases in specicity and mode of action (Atlan et al. 1990; Laloi et al. 1991; Sasaki et al. 1995; Matar et al. 2003). Few studies, however, have been carried out to identify the peptides generated from milk proteins by LAB with respect to their biological activities; and most of these studies have focused on the health-promoting properties (Clare and Swaisgood 2000; Gobbetti et al. 2000; Quiros et al. 2007). In a survey on bioactive peptides released from milkcaseinates from different domestic mammals upon digestion with a partially puried proteinase from Lactobacillus helveticus, Minervini et al. (2003b)
Figure 2 Schematic representation of bovine a-lactalbumin amino acid sequence (one-letter code amino acids), showing the breakage sites of trypsin (hollow letters font) and chymotrypsin (grey letters) that releases the antimicrobial peptides (underlined sequences) described by Pellegrini et al. (1999) as depicted by black (breakage with trypsin) and grey (breakage with chymotrysin) arrows. Dashed arrows (black or grey) indicate an unspecic cleavage regardless of the protease. Fragments held by a disulde bridge are underlined in the same manner (double or single underline); the sequence underlined by a single dashed line is a monomeric antimicrobial pentapeptide. NB: ... represents omitted residues of bovine a-lactalbumin, which are not relevant to this study.
328
reported that among the numerous resulting bioactive peptides, only one peptide deriving from human b-casein-hydrolysate, [b-CN f(184-210)], exhibited antibacterial activity. This peptide showed a very large spectrum of action against Gram-positive and Gram-negative bacteria, including Ent. faecium, Bacillus megaterium, E. coli, L. innocua, Salmonella spp., Yersinia enterocolitica, and Staph. aureus, and MIC for a sensitive E. coli strain was $50 lg mL. On the other hand, fractionation of water-soluble extracts from nine different Italian ripened-cheese varieties has revealed the presence of several antimicrobial peptides with a potent inhibitory activity against a wide range of bacteria of health and spoilage signicance (Rizzello et al. 2005). Yet, the release of these antibacterial peptides could not be specically ascribed to LAB, as chymosin, ripening moulds or nonstarter lactic acid bacteria also could be responsible for such biopeptides release. More recently, Hayes et al. (2006) provided further evidence for the generation of antimicrobial peptides by fermentation of milk-caseins with LAB. In this study, sodium caseinate from bovine as2-casein was fermented with a proteolytic strain of Lactobacillus acidophilus, and the fermentate was passed through a size exclusion cartridge-lter to discard peptides having a molecular weight greater than 10 kDa. The ltrate was fractionated, and the peptides in fractions showing antagonistic activity against E. coli were puried by RP-HPLC and sequenced, revealing the presence of three short antibacterial peptides which have been designated caseicins A, B and C (Table 3). Caseicins A and B exerted a potent inhibitory activity against Gram-positive and Gram-negative bacteria including food borne pathogens of health concern, such as Listeria innocua, E. coli O157:H7, Ent. sakazakii and S. mutans (Hayes et al. 2006). The MICs of caseicin A and caseicin B against an E. coli strain were: 0.05 (0.05 mM) and 0.2 (0.22 mM) mg mL respectively, suggesting potential use of these peptides as food-grade biopreservatives or as functional-food supplements. In view of the potency of caseicins A and B against Ent. sakazakii, Hayes et al. (2006) have recommended using the peptides to control the pathogen in milkbased infant formulae. Indeed, these products are
known to be the major reservoir for Ent. sakazakii which is responsible for a distinctive syndrome of meningitis with a mortality rate as high as 80% in neonates (Nazarowec-White and Farber 1997). Such an application is even more appropriate that the pathogen is difcult to control by conventional means for quality assurance (Nazarowec-White and Farber 1997; Hayes et al. 2006; Ross et al. 2007). As matter of fact, these peptides would nd a larger application in food preservation, either in their puried state or as components of caseinfermentate of Lb. acidophilus, owing to their large spectrum of action covering bacteria of health and spoilage signicance in addition to their anticipated safety and consumer acceptability. The LAB used as biological catalysts have a long history of safe use in food fermentation and already benet from the generally recognised as safe status, and the milk proteins used as substrates are reputed for their safety and health benets. The use of such biological kits comprising proteolytic LAB as catalysts and milk or milk proteins as substrates to release different antimicrobial peptides represents a novel and promising strategy for food preservation. It offers a great versatility to target specic undesirable bacteria and foods by using different combinations of proteolytic LAB and substrates (e.g. a, b or j-caseins or whey proteins) to design tailored antimicrobial peptides for in-built preservation technology. In some dairy products, for example, selected proteolytic LAB may be used as part of the starter or adjunct starter cultures to produce, in situ, the desired antimicrobial peptides during processing. Furthermore, proteolytic LAB would produce a greater diversity of antimicrobial peptides from milk proteins compared to digestive proteases, due to the complexity and multiplicity of microbial proteolytic systems (Reid and Coolbear 1998; Deutsch et al. 2000). Strains of LAB have been shown to cleave peptide bonds not normally cleaved by digestive proteases, such as those involving the imino group of proline (Atlan et al. 1990), and to hydrolyse the so-called strategic regions of casein containing overlapping peptide sequences with various biological activities, but which are protected from breakdown by digestive proteases (Atlan et al. 1990; Fiat et al. 1993; Rizzello et al. 2005). According to Hayes
Table 3 Antibacterial peptides (caseicins) produced from bovine as1-casein by fermentation with a proteolytic strain of Lactobacillus acidophilus (Hayes et al. 2006) Sequence (number of amino acid residues) IKHQGLPQE (9) VLNENLLR (8) SDIPNPIGSENSEK (14)
Antibacterial peptide Caseicin A as1-CN f(21-29) Caseicin B as1-CN f(30-37) Caseicin C as1-CN f(195-208)
a
Spectrum of action E. coli, Ent. sakazakii, Lb. bulgaricus, L. innocua, S. mutans As above L. innocuaa
A weak inhibitory activity compared to that of caseicins A and B.
329
et al. (2006), the generation of caseicins A and B from bovine as1-casein has been shown to require a combined action of three endopeptidases and proteinases. Nonetheless, the complexity and multiplicity of proteolytic systems in LAB may also be a disadvantage if not adequately managed. An excessive proteolytic activity results in further degradation of the antibacterial peptides, once released from the precursor molecules, into smaller inactive fragments (Rizzello et al. 2005). Therefore, when such antimicrobial substances are to be generated from milk proteins by fermentation with proteolytic LAB, the proteolysis should be managed so that the ratio of soluble nitrogen to total nitrogen (Nsoluble Ntotal) remains between 12% and 24% (Rizzello et al. 2005). Water-soluble extracts from cheese types with long ripening or intense proteolysis (Nsoluble Ntotal 30%) have been shown to be devoid of antibacterial peptides, contrary to their counterparts from cheese samples with a limited proteolysis (Addeo et al. 1992; Meisel et al. 1997; Rizzello et al. 2005). Similarly, Lopez-Exposito et al. (2007) showed that the antimicrobial activity produced by hydrolysis of ovine as2-casein and bovine j-casein with pepsin digestion was partially or completely lost when the caseins were digested for a longer period than 30 min and 2 h respectively. In addition, sequential digestion of caseins with pepsin and a mixture of trypsin and chymotrypsin has yielded inactive digests, while the caseins had generated antimicrobial peptides when digested separately with the same enzymes (Lopez-Exposito et al. 2006b). Another potential advantage of the preservation strategy using proteolytic LAB is the possibility to use specic bacteriocin-producing strains of LAB which are also able to generate antimicrobial peptides from food proteins. In this regard, a strain of Lactobacillus curvatus has been shown to produce two distinct bacteriocins and to generate an antimicrobial peptide from the tryptic digest of b-casein present in de Man, Rogosa and Sharp (MRS) broth (Ghal et al. 2010), and the effectiveness of this Lactobacillus strain in food preservation has been demonstrated (Ghal et al. 2006a,b, 2007). This might extend the spectrum of action and increase the overall efcacy of the peptides in a synergistic manner as in preservative systems, as was demonstrated in vitro for nisin and the bovine milkderived peptides aS2-casein f(183-207) against L. monocytogenes (Lopez-Exposito et al. 2008b). MECHANISM OF ACTION OF ANTIMICROBIAL MILK-DERIVED PEPTIDES
derived from milk proteins. The variability in their amino acid composition, structure and physicochemical characteristics (Table 4) suggests that they act in different ways on sensitive bacteria. Yet, there is a general agreement that, regardless of the precise mechanism of action, all antimicrobial peptides primarily act on the plasma membrane through the establishment of electrostatic bonding between the peptide and plasma membrane components, at least during early steps of a peptide-mediated killing process (Zasloff 2002; Reddy et al. 2004). Such an interaction results in one of two major effects: (i) the formation of transient transmembrane channels that alter the membrane permeability and or energy generation while preserving the cell integrity or (ii) the disruption of the plasma membrane with a consequent disintegration of the whole cell (Epand and Vogel 1999; Jenssen et al. 2006; Park et al. 2008). It is generally admitted that the electrostatic bonding is only possible if the peptide has a shape in which clusters of hydrophobic and cationic amino acids are spatially organised in an amphipathic form, with the hydrophobic sectors on one side and the cationic (i.e. hydrophilic) sectors on the opposite side. In such a conformation, the hydrophobic sectors interact with the lipids of the membrane, while the cationic sectors interact with its negatively charged phosphate groups (Brogden 2005). Furthermore, the potency of amphipathic antimicrobial peptides has been correlated with their a-helical content (Uteng et al. 2003; Lee et al. 2006; Park et al. 2008).The peptide may thus attach to the plasma membrane as a rst step in a killing process where the subsequent steps involve one of ve different mechanisms described as the barrel-stave, carpet, detergent, toroidal pore or aggregates models (for further reading, see Zasloff 2002; Reddy et al. 2004; Brogden 2005; Jenssen et al. 2006). Nonetheless, the ultimate killing target is not always the plasma membrane itself, as the peptide may translocate into the cytoplasm and act on intracellular components including nucleic acids, proteins or lipids thereby interfering with DNA replication and expression, enzymatic reactions or macromolecular synthesis (Carlsson et al. 1998; Epand and Vogel 1999; Minervini et al. 2003a; Ulvatne et al. 2004; Jenssen et al. 2006), or else cause the cytoplasm to occulate (Brogden 2005).
General considerations Few and sparse studies have been done to elucidate the mechanism of action of antimicrobial peptides
330
Case of milk protein-derived antimicrobial peptides Given the diversity of the milk protein-derived antimicrobial peptides in their net electric charges, secondary structures or hydrophobicity hydrophilicity properties (Table 4), it is difcult to anticipate a priori how or if they would exert antimicrobial activities. Preliminary studies have been conducted in this regard, and are discussed herein. One of the most advanced such studies has
Table 4 Main characteristics of known protein-derived antibacterial peptides expected to impact their mode of action Net charge (pH 7)a,b 0.1 0b )2 2.2 Molecular mass (kDa)a 1.05 0.97 1.49 2.8 Hydrophilic residues total residues (%)a 44 50 64 43 Predicted secondary structurec C C C HC
Antibacterial peptide (amino-acid sequence) IKHQGLPQE Bovine as1-CN f(21-29) VLNENLLR Bovine as1-CN f(30-37) SDIPNPIGSENSEK Bovine as1-CN f(195-208) RPKHPIKHQGLPQEVL NENLLRF Bovine as1-CN f(1-23) KTKLTEEEKNRLNFLKKI SQRYQKFALP-QYLKT VYQHQK Bovine aS2-CN f(150-188) LKKISQRYQKFALPQY Bovine aS2-CN f(164-179) YQHQKAMKPWIQPKTK VIPYV-RYL Bovine aS2-CN f(183-207) KTVYQHQKAMKPWIQPKTKVIPYVRYL Bovine as2-CN f(181-207) LKTVYQHQKAMKP WIQPKTKVIPYVRYL Bovine as2-CN f(180-207) ALPQYLKTVYQHQKAMKPWIQP-KTKVIPYVRYL Bovine as2-CN f(175-207) QKFALPQYLKTVYQHQKAMK-PWIQPKTKVIPYVRYL Bovine as2-CN f(172-207) LKKISQRYQKFALPQYLKTVYQ-HQKAMKPWIQPKTKVIPYVRYL Bovine as2-CN f(164-207) LKKISQ Ovine as2-CN f(165-170) LKKISQYYQKFAWPQYL Ovine as2-CN f(165-181) VDQHQKAMKPWTQPKTNAIPY-VRYL Ovine as2-CN f(184-208) PYVRYL Ovine as2-CN f(203-208) AIPPKKNQDKTEIPTINTIASGEP-TSTPTTEAVESTVATLEDSPEVI-ESPPEINTVQ-VTSTAV Bovine j-CN f(106-169) AVESTVATLEDSPEVIESPPE Bovine j-CN f(136-156) YYQQKPVA Bovine j-CN f(42-49)
Name Caseicin A Caseicin B Caseicin C Isracidin
Isoelectric point a 7.8 7.0 3.8 10.6
Average hydrophilicitya 0.3 )0.1 0.7 0.2
Casocidin-I
7.1
10.5
4.87
0.4
54
HC
Unnamed (casocidin-I fragment) Unnamedd
4.0 5.1
10.5 10.5
2.01 3.02
0 )0.2
50 33
HC CE
Cr1
6.1
10.6
3.3
)0.2
33
CHE
Cr3
6.1
10.6
3.5
)0.2
32
CHE
Cr4
6.1
10.4
4.0
)0.3
30
CHE
Cr7
7.1
10.5
4.4
)0.3
33
CHE
Cr5 Cr6
10.1
10.7
5.5
)0.1
35
CHE
Unnamed Unnamed Unnamed
2 3 3.1
10.6 10 10.2
0.7 2.2 3.0
0.5 )0.5 )0.1
67 41 36
C CE CE
Unnamed Kappacine
1.0 )7
9.6 3.8
0.8 6.67
)0.8 0.2
17 37
C CH
Kappacin fragment Unnamed
)6 1.0
2.8 9.5
2.20 1.0
0.4 )0.4
38 38
HCE CE
331
Table 4 (Continued) Net charge (pH 7)a,b 0.0 0.0 0.0 1.0 0.0 )1.0 )1.0 1.0 0 )1.1 Molecular mass (kDa)a 0.4 0.8 0.6 0.8 0.4 0.5 0.8 1.3 0.62 2.25 Hydrophilic residues total residues (%)a 33 25 17 57 0 25 25 33 60 24 Predicted secondary structurec C C CE CH C C CE CEH C NPf
Antibacterial peptide (amino-acid sequence) IQY Bovine j-CN f(28-30) VQVTSTAV Bovine j-CN f(162-169) STVATL Bovine j-CN f(141-146) FSDKIAK Bovine j-CN f(18-24) YVL Bovine j-CN f(30-32) EIPT Bovine j-CN f(118121) VESTVATL Bovine j-CN f(139-146) PAAVRSPAQILQ Bovine jCN f(64-75) EQLTK Bovine a-La f(1-5) GYGGVSLPEWVCTTF ALCSEK Bovine a-La f(1731)S-S(109-114) CKDDQNPH ISCDKF Bovine a-La f(61-68)S-S(7580) VAGTWY Bovine b- Lg f(1520) AASDISLLDAQSAPLR Bovine b- Gl f(2540) IPAVFK Bovine b- Gl f(78 83) VLVLDTDYK Bovine b- Gl f(92100)
a
Name Unnamed Unnamed Unnamed Unnamed Unnamed Unnamed Unnamed Unnamed Unnamed Unnamed
Isoelectric point a 5.9 6.0 6.0 9.9 5.9 3.3 3.3 11 6.9 4.3
Average hydrophilicitya )1.3 )0.7 )0.7 0.6 )1.9 0.2 )0.3 )0.2 0.8 )0.4
Unnamed
)1
5.2
1.65
0.6
57
NP
Unnamed Unnamed Unnamed Unnamed
0 )1 1 )1
5.9 3.9 10.1 3.9
0.70 1.63 0.67 1.07
)1.3 0.1 )0.5 0
0 44 17 37
C CCH C EC
Data obtained by calculations using Peptide Property Calculator facility; available at: http://www.innovagen.se/custom-peptide-synthesis/peptideproperty-calculator/peptide-property-calculator.asp. bThe overall electric charge was calculated by an algorithm using the formula: P P pH 10pKa Z Ni 10pH 10ipKai Nj 10pH10 pKaj where Ni is the number, and pKai the pKa values of the N-terminus and the side chains of Arginine, Lysine and 10 j i Histidine. The j-index pertain to the C-terminus and the aspartic acid, glutamic acid, cysteine, tyrosine amino acids. cUsing Scratch protein predictor program, SSpro in ExPASy web server; available at: http://www.igb.uci.edu/tools/scratch/ (Cheng et al. 2005); C, random coil; H: a-helix; E, extended. dC-terminal fragment of bovine aS2-casein. eForms an a-helix in presence of triuoroethanol (TFE) and excess divalent metal ions (e.g. Ca++ or Zn++); Dashper et al. (2005). fNot provided.
been carried out on kappacin by using liposomeswelling assays (Dashper et al. 2005). In this study, the authors have demonstrated that a concentration ranging between 30 and 50 lM of kappacin-A increased the permeability of liposomes in a concentration-dependant manner, indicating that the peptide permeabilises the plasma membrane of sensitive bacteria. According to the authors, kappacin would disrupt the plasma membrane in a similar manner as the cationic a-helical peptides, in spite of the fact that kappacin is strongly anionic (seven negative charges) and has a random-coiled secondary structure in an aqueous environment [Table 4, (Dashper et al. 2005)]. It has been suggested, however, that once in contact with the surface of bacterial cell-membrane, kappacin would shift to an amphipathic a-helical structure. This assumption was supported by the ability of 332
kappacin to form an a-helix in presence of triuoroethanol and excess divalent metal ions (e.g. Ca++ or Zn++); an environment that mimics the surface of the plasma membrane. In fact, it is believed, although debated, that all of the antimicrobial peptides, regardless of their original conformation, would adopt an amphipathic a-helical conformation once in the vicinity of the plasma membrane in response to the prevailing physico-chemical parameters such as the ionic strength, pH, EH, temperature, metal ions, membrane lipids (Gennaro and Zanetti 2000; Park et al. 2000, 2008). Nevertheless, the study of Malkoski et al. (2001) did not provide further evidence for the ultimate killing target of the peptide, nor did it give an explanation regarding the permeabilisation of the outer membrane to kappacin which has been, otherwise, shown to potently inhibit the growth of
Gram-negative bacteria. The permeabilisation of the plasma membrane as revealed by liposomeswelling assay is not an unequivocal evidence, by itself, for the membranolytic activity of the peptide; it does not always correlate with the antimicrobial activity but could only be a route for antimicrobial peptides to reach alternative intracellular targets (Wu et al. 1999; Rydengard et al. 2008). Further more, the validity of model membranes (e.g. liposome) in mechanistic studies has been criticised due to the high complexity of the plasma membrane compared with liposomes (Park et al. 2008). Although model membranes have been extensively used to study the mechanism of action of antibacterial substances and have proven useful, they have often been associated to other analyses and tests such as electron microscopy, X-ray crystallography, NMR spectroscopy, Fournier transform infrared, use of dyes, conductivity measurements, circular dichroism or neutron diffusion for sound and convincing conclusions (Brogden 2005). As for the passage of kappacin across the outer membrane of sensitive Gram-negative bacteria, it should be noted that it is unlikely to be self-promoted or passive through porins due to the anionic hydrophobic nature of the peptide in addition to its relatively high molecular weight (6670 Da). Anionic peptides are especially restricted from crossing the OM mainly because of the electric repulsion between the negative charges of the peptides and those of phosphate groups of the lipopolysaccharide (LPS). In addition, the OM porins are impermeable to large (> 900 Da) or hydrophobic peptides due to the small size of the porin-channels which are, in addition, lled with water (Engstrom et al. 1984; Nikaido et al. 1991; Carlsson et al. 1998). Further studies are thus needed to provide clearer insights into the molecular events leading to cell-death of either Gram-positive or Gram-negative bacteria upon exposure to kappacin. Recently, Lopez-Exposito et al. (2008a) have investigated the permeabilisation of the cell envelopes of E. coli and Staph. carnosus by the cationic bovine milk-derived aS2-casein f(183-207) and the subsequent morphological changes leading to the death of sensitive bacteria. The results of the study revealed that the peptide initially binds to the LPS of the OM in the Gram-negative bacterium and to the lipoteichoic acid (LTA) of the cell wall in the Gram-positive bacterium. The identication of these binding sites was achieved by competition studies showing that the required concentration of as2-casein f(183-207) to inhibit the growth of sensitive bacteria has increased linearly with the increase in the amounts of soluble LPS or LTA added to a mixture of the peptide and each of E. coli or Staph. carnosus respectively (Lopez-Exposito et al. 2008a). Cationic antibacte rial peptides have been reported to attach to the
LPS in Gram-negative bacteria (Vaara 1992; Piers et al. 1994) and teichoic or LTA in Gram-positive bacteria (Vorland et al. 1999; Ulvatne et al. 2004), primarily by electrostatic interactions. However, attachment of these peptides through specic protein receptors present on the OM or by means of polylysyl residues at the C-terminal end of the peptides has also been demonstrated (Vaara 1992; Carlsson et al. 1998; Brogden 2005). Although the rational used by Lopez-Exposito et al. (2008a) in these competition studies provides a convincing evidence for the validity of the attachment of the as2-casein f(183-207) to the cell envelopes of sensitive bacteria, the exact mechanisms of such an attachment remain to be elucidated. As for the permeabilisation of the cell envelopes to the bovine as2-casein f(183-207), chromogenic dye tests performed in the same study have demonstrated that the peptide induces concurrent permeabilisation of the inner and outer membranes in E. coli (Lopez Exposito et al. 2008a). This has been evidenced by recording the translocation of nitrocen (normally prevented from crossing the OM due to its large size) into the OM of E. coli in presence of the peptide, as traced by the development of a red colour upon cleavage of nitrocen molecules by the b-lactamase normally located in the periplasmic space. Similarly, the permeabilisation of the inner membrane was demonstrated by the alteration of the selectivity of a galactoside permease-decient mutant of E. coli (ML-35p) to Ortho-nitrophenylb-galactoside (ONPG). The passive passage of the ONPG across the cytoplasmic membrane, reporting on the alteration of the selectivity of the membrane, was demonstrated by monitoring the hydrolysis of ONPG within the cytoplasm by a constitutively produced b-galactosidase by the mutant strain of E. coli. Hydrolysis of the reporter molecule (ONPG) upon incubation with E. coli in presence of aS2-casein f(183-207) was revealed by the appearance of a yellow colour due to the liberation of ONP. Furthermore, transmission electron microscopy (TEM) studies have shown that the translocation of the antimicrobial peptide across the OM of E. coli and the peptidoglycan layer of Staph. aureus occurs via the formation of pores in both envelopes (Lopez-Exposito et al. 2008a). Destabilisation of the OM of Gram-negative bacteria by cationic peptides with subsequent pore formation as a result of displacement of the divalent cations (e.g. Ca++ and Mg++) that cement adjacent LPS molecules, has been reported earlier (Vaara 1992; Brogden 2005). In the Gram-positive bacterium, hydrophobic interactions between the tryptophan residues from the antimicrobial peptide and the lipid-A from the peptidoglycan layer have been suggested as a possible mechanism for the pore formation and permeabilisation of the cell wall (Farnaud et al. 2004). As for the ultimate killing 333
step of sensitive bacteria by the bovine aS2-casein f(183-207), TEM studies showed that the peptide essentially acts by promoting the condensation of the cytoplasm in the Gram-negative bacterium (E. coli ML-35p) (Lopez-Exposito et al. 2008a), as has been reported for anionic peptides (Brogden 2005), while in the Gram-positive bacterium (Staph. carnosus CECT 4491T), it was suggested to act by a different mechanism involving the disruption of the plasma membrane with a consequent leakage of the cytoplasmic content out of the cell (Lopez-Exposito et al. 2008a). The other studies on the mechanism of action of milk-derived antimicrobial peptides have mainly consisted in amino-acid substitution experiments and related observations to determine the role of specic amino acids in the overall antimicrobial activity. Therefore, they did not provide a clear understanding of the precise mechanism of action of the studied peptides. In this regard, several studies have investigated the effect of hydrophilicity hydrophobicity on the antimicrobial activity of milk-derived peptides. For example, Pellegrini et al. (1999) have shown that substitution of Leu23 for the structurally similar but more hydrophobic Ile in the sequence of the dimeric tryptic-digest of a-LA [a-La f(17-31)S-S(109-114)] (Table 2) has drastically reduced the antimicrobial activity of the peptide. Conversely, the substitution of the uncharged Asp98 residue for the positively charged Arg and addition of a lysyl residue at the C-terminus of the antimicrobial peptide VLVLDTDYK released by tryptic digestion of b-Lg (Table 2), in a way to increase the overall hydrophilicity, has extended the antimicrobial activity of the resulting peptide (VLVLDTNYKK) to Gram-negative bacteria (Pellegrini et al. 2001). Also, kappacin B that differs from kappacin A by the substitution of two hydrophilic amino acid residues at positions 136 and 148 (residues Thr136 and Asp148 in kappacin A) for hydrophobic residues (Ile136 and Ala148 in kappacin B) has been shown to exert signicantly reduced or no antibacterial activity compared to kappacin A (Malkoski et al. 2001). Such studies concur to suggest that the antimicrobial activity of the peptides is adversely affected by increased hydrophobicity, and is stimulated by the increase in hydrophilicity. However, such conclusion is not valid for all milk-derived antimicrobial peptides, and, on the contrary, increased hydrophobicity has been shown to enhance the antibacterial effectiveness of some antimicrobial peptides (Lopez-Exposito et al. 2006a,b). In addition, the most hydrophilic caseicin C and the pentapeptide (EQLTK) released from aS2-casein and a-LA respectively (Table 4) were shown to have the weakest activity compared with the less hydrophilic or the hydrophobic antimicrobial peptides released in the same conditions (Pellegrini et al. 334
1999; Hayes et al. 2006). Similar observation has been made in the case of an antimicrobial peptide of bacterial origin by Lee et al. (2006) who showed that the activity of a native antimicrobial peptide produced by Helicobacter pylori [i.e. f(220) derived from the N-terminus of ribosomal protein L1] against bacteria and yeasts has been significantly enhanced by increased hydrophobicity, while an opposite effect has resulted by increasing the hydrophilicity. In fact, it has been considered that the net electric charge of antimicrobial peptides, especially the cationic ones, is the main feature that determines the antimicrobial activity (Pellegrini et al. 1992; Bradshaw 2003). Therefore, the increase in the antimicrobial activity of the peptide VLVLDTNYKK obtained by substituting the uncharged Asp98 residue for the positively charged Arg and addition of a lysyl residue at the C-terminus of the b-Lg f(92-100) i.e., V92LVLDTDYK100, could be attributed to the increase in the net positive charge rather than hydrophilicity. Also, Lopez Exposito et al. (2006a) have explained the increased antibacterial activity of bovine as2-casein f(183-207) compared to its ovine homologue f(184-208) by the higher positive electric charge of the former (i.e. 5.1 vs 1), as these peptides differ in four amino acid residues (Table 4). The same explanation has been provided for the decrease in antimicrobial activity of the b-casein-derived peptides RNKKI and RINKK upon loss of their N-terminal arginyl and C-terminal lysyl residues respectively, with a consequent reduction in the net positive charge (Lopez-Exposito et al. 2006b). The importance of these particular positively charged residues (i.e. lysine and arginine) to the antimicrobial effect of cationic peptides has also been demonstrated in antibiotics (Hancock et al. 1995; Hancock and Lehrer 1998). Despite such conicting considerations regarding the means by which protein-derived peptides exert their antimicrobial activity, there is a growing body of evidence that the antimicrobial activity of a peptide is related to an optimal balance between the hydrophobic and hydrophilic residues so as to obtain an a-helix with an appropriate amphipathicity in the presence of target bacteria, rather than to the average hydrophobicity or net electric charge. This is consistent with the studies that have demonstrated that the positioning of charged and hydrophobic residues is crucial for peptides to exhibit antimicrobial activity, as they would inuence the tertiary structure (Wieprecht et al. 1997; Dathe and Wieprecht 1999). Furthermore, minor modications or substitutions in of amino acids in the sequence of a bioactive peptide that do not affect the amphipathicity or the net electric charge, such as the phosphorylation of specic amino acid or chemical modication of the C or N-termini, have been shown to affect dramatically the peptides
secondary and tertiary structures, and consequently its antimicrobial activity (Perez-Paya et al. 1994; Uteng et al. 2003; Dashper et al. 2007). Therefore, any changes leading to the formation of stable amphipathic a-helix appear to increase the activity of antibacterial peptides (Lee et al. 2006; Park et al. 2008), indicating that antimicrobial activity is primarily dependent on electrostatic interaction with the cell-membrane as suggested earlier (Jenssen et al. 2006). This is in agreement with the observation that some antimicrobial peptides would show more afnity to the polarised bacterial membranes than to the zwitterionic membranes of eukaryotic cells such as those of yeast and moulds (Zasloff 2002). It also explains why most of the milk-derived antimicrobial peptides are essentially active against bacteria. However, other authors suggest that antimicrobial peptides would also recognise components of cell-envelopes of prokaryotic or eukaryotic organisms with which they specically interact to exert their growth inhibitory effect (for review, see Epand and Vogel 1999). For example, the potency of the antimicrobial peptide produced by H. pylori against bacteria and fungi (yeast and moulds) has been associated with the peptides binding afnity to cell-wall components (Park et al. 2008). These authors have demonstrated that MICs of the peptide and modied derivatives towards sensitive micro-organisms correlated well with their respective in vitro binding afnities to glycopeptides, chitin or LPS. Furthermore, the peptide shown to be active against chitin-expressing Penicillium strains, failed to inhibit Phytophthora strains lacking chitin in their cell wall (Park et al. 2008). The role of the cell wall in the antimicrobial activity of peptides is not well understood and should be investigated in more depth in the case of milk-derived and other antimicrobial peptides, as it could profoundly change the current understanding of the mechanism of action of bioactive peptides in relation to micro-organisms as well as to host cells. It is worth mentioning that small antimicrobial peptides with three to ve amino acid residues have received the least attention as regards the study of their mechanism of action, and would act by different mechanism(s) as yet to be elucidated (Lopez-Exposito et al. 2006b). CONCLUSIONS The high genetic variability in milk proteins among species or within the same species suggests a great possibility to release bioactive peptides with various structures and functions. Although, only few milk-derived antimicrobial peptides are currently known and characterised, this number is expected to grow rapidly in the future due to renewed interest in their application in the food industry and in medicine. Among the prominent advantages of
these peptides are their low likelihood to develop resistant microbial strains in addition to their wide spectrum of action, which in some cases includes bacteria (Gram-positive and Gram-negative), viruses, fungi and protozoans (Forssmann et al. 2003). Milk proteins provide, therefore, an opportunity to tailor specic peptides by using different proteases or LAB, or their combinations; and these peptides can be used as food-grade additives in a puried form, or as milk-based hydrolysates or fermentates. The possibility for milk-derived peptides to have other functional properties beside the antimicrobial activity is another advantage of paramount importance to food industry as such peptides could be used to promote food safety and stability while insuring the functionality of foods, thereby increasing their added value in conformity with the current consumers demand. It should be emphasised, however, that the understanding of the exact mechanisms by which milk protein-derived antimicrobial peptides act on sensitive bacteria is important as it would allow tailoring or designing new potent molecules with extended spectrum of action or enhanced antimicrobial activities of known peptides. This aspect has been poorly studied so far and warrants due consideration. ACKNOWLEDGEMENTS
A deep appreciation is expressed to Dr William E. Sandine, Emeritus Distinguished Professor of Microbiology at Oregon State University for reviewing the pre-publication manuscript.
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doi: 10.1111/j.1471-0307.2010.00584.

*Author for corrrespondence. E-mail: n.benkerroum@gmail.com 2010 Society of Dairy Technology

Vol 63, No 3 August 2010 International Journal of Dairy Technology

Vol 63, No 3 August 2010

Cationic and hydrophobic peptide Polyproline-helix like structure Cationic peptide

f(181-207) Chymosin Chymosin

bovine aS2-casein Chymosin

2010 Society of Dairy Technology

bovine aS2-casein Chymosin

bovine aS2-casein Chymosin

f(165-170) f(165-181) Pepsin Pepsin chymosin

ovine aS2-casein ovine aS2-casein Pepsin Pepsin

(Lopez-Exposito et al. 2006a)

f(203-208) Kappacin Ad f(106-169)

ovine aS2-casein bovine j casein

Mainly Gram-positive bacteria Gram-positive and Gram-negative bacteria

(Malkoski et al. 2001)

f(42-49) Pepsin Pepsin IQY VQVTSTAV

bovine j casein Pepsin

VDQHQKAMKPWTQPKTNAIPYVR-YL PYVRYL A106IPPKKNQDKTEIPTINTIASGE-PTSTPTTEAVESTVATLEDRPEVIESPPEI NTVQVTSTAV169e YYQQKPVA

Cationic Neutral Neutral

(Lopez-Exposito et al. 2006b)

bovine j casein bovine j casein

Vol 63, No 3 August 2010

f(141-146) f(18-24) f(30-32) f(118-121) f(139-146)

2010 Society of Dairy Technology

Main chemical properties

Amino acid sequence (one letter code)a

Vol 63, No 3 August 2010

Origin a-La a-La a-La b-Lg b-Lg b-Lg b- Lg

Cleavage enzyme Trypsin Chymotrypsin Trypsin

References (Pellegrini et al. 1999)

(Pellegrini et al. 2001)

Vol 63, No 3 August 2010

Vol 63, No 3 August 2010

Vol 63, No 3 August 2010

Vol 63, No 3 August 2010

2010 Society of Dairy Technology

Vol 63, No 3 August 2010

A weak inhibitory activity compared to that of caseicins A and B.

2010 Society of Dairy Technology

Vol 63, No 3 August 2010

Vol 63, No 3 August 2010

Name Caseicin A Caseicin B Caseicin C Isracidin

Isoelectric point a 7.8 7.0 3.8 10.6

Average hydrophilicitya 0.3 )0.1 0.7 0.2

Unnamed (casocidin-I fragment) Unnamedd

Unnamed Unnamed Unnamed

0.7 2.2 3.0

0.5 )0.5 )0.1

Kappacin fragment Unnamed

2010 Society of Dairy Technology

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Unnamed Unnamed Unnamed Unnamed

5.9 3.9 10.1 3.9

0.70 1.63 0.67 1.07

)1.3 0.1 )0.5 0

Vol 63, No 3 August 2010

Vol 63, No 3 August 2010

Vol 63, No 3 August 2010

Vol 63, No 3 August 2010

2010 Society of Dairy Technology

Vol 63, No 3 August 2010

2010 Society of Dairy Technology