antimicrobial peptides ijdt

REVIEWAntimicrobial peptides generated from milk proteins: asurvey 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

*Author forcorrrespondence. E-mail:[email protected]

� 2010 Society ofDairy Technology

Milk proteins constitute a natural reservoir of bioactive peptides with physiological and ⁄or antimicrobialproperties, the release of which requires hydrolysis of the precursor molecules by digestive proteases orby fermentation with proteolytic micro-organisms. Depending on the digestive or microbial proteasesused, 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 identified and characterised with respect to their antimicro-bial 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 significance. Nevertheless, previousresearch 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 byfermentation with proteolytic lactic acid bacteria. Their possible application in the food industry andtheir 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 beinvoked for comparison purposes.

Keywords Antimicrobial peptides, Milk proteins, Caseins, a-Lactalbumin, b-Lactoglobulin, Proteases,Lactic acid bacteria, Food industry.

INTRODUCT ION

Antimicrobial peptides produced by a wide varietyof unicellular or multicellular living organisms as afirst-line defence against invading micro-organismshave attracted increased attention since the discov-ery of lysozyme (Fleming 1922). Many of thesesmall molecules (< 10 kDa; 3–50 amino acidresidues) have proven to be potent antimicrobialsubstances with promising applications in medicineor food preservation. Therefore, intensive researchwork has been carried out to detect, purify andcharacterise as many of these peptides as possiblefor application in industrial production. To date, arepertoire of more than 880 such peptides exist ininternational databases (Wang and Wang 2004;Rydengard et al. 2008). This number continues togrow, as sources other than living organisms, suchas food proteins (Pellegrini 2003), can now be usedto generate bioactive peptides. In particular, milkproteins have been recognised as importantresources for peptides with biological activities.Once these peptides are released they exert, inaddition to antimicrobial activities, other

interesting biological functions such as opioid,antithrombotic, immunomodulatory, antihyperten-sive or mineral carrying activities (Clare andSwaisgood 2000). Moreover, some antimicrobialpeptides released from milk can also have otherphysiological functions (i.e. multifunctional pep-tides) (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 beingderived from a harmless and inexpensive source,and have therefore an undeniable potential for usein medicine or the food industry.In order to be active, these milk-derived peptides

have to be first released from their parent mole-cules. This can be achieved either by hydrolysis ofthe parent molecules (caseins and whey proteins)by digestive proteases (Clare and Swaisgood 2000)or by fermentation with selected proteolytic lacticacid bacteria (LAB) (Hayes et al. 2006); milk acid-ification followed by heat treatment has also beenreported to generate active peptides (Zucht et al.1995; Tomita et al. 2002). The present review willfocus on antimicrobial peptides generated from

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

doi: 10.1111/j.1471-0307.2010.00584.x

major milk proteins by digestive proteases or byproteolytic LAB. Their potential applications in thefood industry and their antibacterial mechanisms ofaction are also discussed. Antimicrobial peptidesgenerated from minor milk proteins (e.g. lactoferrinand lysozyme) are not considered in this review, asthey have been extensively reviewed elsewhere(Tomita et al. 2002; Floris et al. 2003; Pellegrini2003; Benkerroum 2008).

ANT IMICROBIAL PEPT IDESGENERATED FROM MILKPROTE INS BY DIGEST IVEPROTEASES

Major milk proteins [e.g. caseins, a-lactalbumin(a-La) and b-lactoglobulin (b-Lg)] constitute animportant source for bioactive peptides reported tohave a wide range of physiological properties(Meisel 1998; Clare and Swaisgood 2000; Pelleg-rini 2003). Among the array of known bioactivepeptides, those exerting antimicrobial activitieshave received limited attention compared to theirphysiologically active counterparts (e.g. antihyper-tensive, antithrombotic and immunomodulatorypeptides). This is due to the lack of interest in theircommercial production within the healthcareindustry as most of them are intended for medicaluse, mainly as antibiotics, and their productionwould be costly while being less potent thanconventional antibiotics (Lahov and Regelson1996). Now, however, there is a growing interest inthe use of these antimicrobial peptides as food-grade biopreservatives or as health-promoting foodsupplements. Such a trend is encouraged by thepreference of consumers for lightly processedfoods containing the least possible chemical addi-tives, and the search for functional foods perceivedas nutritious, healthy and prophylactic. This reviewdeals with the main antimicrobial peptides releasedby action of digestive proteases from caseins andwhey proteins, and how they would find appropri-ate applications in the food industry.

CASE IN -DER IVEDANT IMICROBIAL PEPT IDES

Antimicrobial peptides from a-caseinsAbout three decades after the report of Jones andSimms (1930) on the presence of lactenin in milktreated with rennet and its antimicrobial activityagainst streptococci, several antibacterial peptideshave been reported to be released from milkproteins upon hydrolysis with digestive proteases(Tables 1 and 2). These peptides are characterisedto have a high diversity of structural and antimicro-bial properties making it difficult to classify themin a specific group(s) of antimicrobial substancessuch as those proposed by Epand and Vogel (1999)

or by Reddy et al. (2004). Casecidins, obtained bychymosin digestion of as1-casein at neutral pH,were the first reported milk-derived antimicrobialpeptides (Lahov et al. 1971); and they constitute afamily of basic glycopeptides with a relatively highmolecular weight (�4–6 kDa). Upon discovery,casecidins were intended for therapeutic use totreat infectious diseases owing to their bactericidalactivity against a wide range of Gram-positivebacteria of health significance including staphylo-cocci, Sarcina spp., Bacillus subtilis, Diplococcuspneumoniae and Streptococcus pyogenes (Lahovand Regelson 1996; Clare and Swaisgood 2000).However, their clinical performances were poorcompared to commercially available antibiotics,and they required significantly higher concentra-tions to inhibit, in vitro, sensitive bacteria (Lahovet al. 1971). Therefore, interest in casecidins haslanguished and their commercial development hasbeen omitted. Nevertheless, such a discovery hadthe merit to revive interest of scientists in this areaof research, and subsequent studies have character-ised several novel milk-derived peptides with dif-ferent antimicrobial properties and spectra ofaction (Tables 1 and 2). Isracidin is another antimi-crobial peptide released by chymosin cleavage ofbovine aS1-casein, which consists in a 23-amino-acid-residue fragment of aS1-casein at the N-termi-nal end [i.e., aS1-CN f(1-23)] (Hill et al. 1974).This cationic peptide has been reported to be activein vitro against a broad spectrum of Gram-positiveand Gram-negative bacteria, but only at highconcentrations 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, isracidinhas proven competitive with antibiotics intherapeutic use and provided a strong protectiveeffect in mice against Staphylococcus aureus,S. pyogenes and Listeria monocytogenes. An intra-muscular injection of the peptide at levels similarto those used in standard antibiotherapy (10 lg permouse) has prevented mice from lethal infectionswhen administered before challenge with theabove-mentioned pathogens (Kolb 2001). The pep-tide has also proven to be effective in protectingsheep and cows against mastitis when injected intothe udder, and such protection has lasted beyondthe period of treatment in a long-term immune-response manner (Lahov and Regelson 1996).Therefore, isracidin was considered to have bothprophylactic and therapeutic effects; an observationthat was corroborated by in vivo studies showingthat intravenous injection of isracidin into micestimulates their cellular and humoral immuneresponses, thereby protecting them from infectionby Candida albicans (Lahov and Regelson 1996).The weak in vitro antimicrobial activity of isaraci-din and its high efficacy in vivo represent a strongindication that it has an indirect mode of action as

Vol 63, No 3 August 2010

� 2010 Society of Dairy Technology 321

Tab

le1Casein-derivedantimicrobialpeptides

released

bydigestiveprotease

action,and

theirmainproperties

Antimicrobial

peptide

Source

MW

(kDa)

Cleaving

protease

Aminoacid

sequence

(one

lettercode)a

Activity

Mainchem

ical

properties

Reference

Casecidins

bovine

a S1-casein

4-6

Chymosin

NA

Gram

positive

bacteria

Cationicglycopeptides

(Lahov

etal.1971)(Lahov

and

Regelson1996)

Isracidinf(1-23)

bovine

a S1-casein

2.7

Chymosin

R1PKHPIK

HQGLPQEVL-

NENL-LRF23

Gram-positiveand-negative

bacteria

Cationicandhydrophobic

peptide

Polyproline-helixlik

estructure

(Hilleta

l.1974)

(Malinetal.2001)

Casocidin-If(150-188)

bovine

a S2-casein

4.9

Trypsinb

K150TKLT

EEEKNRLNFLK-

KISQRYQKFA

LPQYLKT-

VYQHQ-K

188c

Gram-positiveand-negative

bacteria,and

yeasts

Cationicpeptide

(Zucht

etal.1995)(Forssmann

etal.2003)

f(181-207)

bovine

a S2-casein

3.3

Chymosin

KTVYQHQKAMKP-

WIQ

PKTK-V

IPYVRYL

Gram-positiveandgram

negative

bacteria

Cationicpeptide

(McC

annetal.2005)

f(180-207)

bovine

a S2-casein

3.4

Chymosin

LKTVYQHQKAMKP-

WIQ

PKT-KVIPYVRYL


negative

bacteria

Cationicpeptide

f(175-207)

bovine

a S2-casein

4.0

Chymosin

ALPQYLKTVYQHQ-

KAMKPW-IQPKTKVI-

PYVRYL


-negative

bacteria

Cationicpeptide

f(172-207)

bovine

a S2-casein

4.4

Chymosin

QKFA

LPQYLKTVYQHQ-

KAM-K

PWIQ

PKTKVI-

PYVRYL


-negative

bacteria

Cationicpeptide

f(164-207)

bovine

a S2-casein

5.4

Chymosin

LKKISQRYQKFA

LP-

QYLKTV-Y

QHQKAMKP-

WIQ

PKTKVIP-Y

VRYL

Gram-positiveand

Gram-negativebacteria

Cationicpeptide

f(165-170)

ovine

a S2-casein

0.7

Pepsin

LKKISQ

MainlyGram-positivebacteria

Cationicpeptide

(Lopez-Expositoetal.2006a)

f(165-181)

ovine

a S2-casein

2.2

Pepsin

LKKISQYYQKFA

WPQYL

Gram-positiveand


Cationicpeptide

f(184-208)

ovine

a S2-casein

3.0

Pepsin

VDQHQKAMKPWTQPKT-

NAIPYVR-Y

L


Cationicpeptide

f(203-208)

ovine

a S2-casein

0.8

Pepsin

PYVRYL


Cationicpeptide

KappacinAdf(106-169)

bovine

jcasein

6.8

chym

osin

A106IPPKKNQDKTEIPTIN

-

TIA

SGE-PTSTPTTEA-

VESTVATLED

RPEVIE

S-

PPEINTVQVTSTAV169e

Gram-positiveand


Anionicphosphopeptide

(Malkoskieta

l.2001)

f(42-49)

bovine

jcasein

1.0

Pepsin

YYQQKPVA

Gram-positiveand


Cationic

(Lopez-Expositoetal.2006b)

f(28-30)

bovine

jcasein

0.4

Pepsin

IQY

Mainlygram

-negativebacteria

Neutral

f(162-169)

bovine

jcasein

0.8

Pepsin

VQVTSTAV


Neutral


322 � 2010 Society of Dairy Technology

an immunostimulatory peptide to induce host-med-iated nonspecific resistance to infectious micro-organisms (Lahov and Regelson 1996; Kolb2001). Nonetheless, like its predecessors casoci-dins, isracidin did not attract the anticipated com-mercial interest primarily due to the onerous andcostly procedures for its purification and produc-tion on large scale batch fermentation for commer-cial use. Isomeric variation of the molecule duringfermentation leading to inconsistencies in thepurity of the final product has been suggested asanother technical limitation (Lahov and Regelson1996; Ross et al. 2007). Its cationic nature mayalso account for the lack of commercial interest fortherapeutic use. Cationic antimicrobial peptides areless effective in treating systemic diseases and tendto adsorb onto negatively charged surfaces, therebyreducing their effectiveness if they are adminis-tered remotely from the site of the infection to betreated (Dashper et al. 2007). Although no data, toour knowledge, are available regarding the possibleapplication of isracidin in the food industry, its useappears promising and feasible. In a recent study,Hayes et al. (2006) demonstrated that isracidin isactive against emerging pathogens of majorconcern to food safety, including Escherichia coliO157:H7, Enterobacter sakazakii and Staph.aureus. In addition, the antimicrobial activity ofisracidin may be significantly improved in foodsystems depending on the composition and intrin-sic ecological parameters of the food matrix. Nisin,for example, the most widely used cationic peptidein the food industry is inactive or even partiallydegraded at pH values above 8.0 while under acidconditions it inhibits sensitive bacteria at nanogramconcentrations (Hurst 1981; Benkerroum andSandine 1988; Zasloff 2002; Benkerroum et al.2003). Studies on the optimal parameters forantimicrobial activity of isracidin should, therefore,be carried out in the scope of its application in foodpreservation. To reduce the cost and labour ofproduction, it may be possible to envisage the useof crude milk-based isracidin preparations insteadof those highly purified as is required for pharma-ceutical uses. Crude preparations from naturalproducts, such as milk, do not raise food safetyconcerns and may thus be easily approved byhealth regulatory authorities for use in foods. Mi-crogardTM, lactoferrin and lactoferricins are exam-ples of such crude preparations that have beenlegally approved for use in the food industry inmany countries (Al-Zoreki et al. 1991; Tomitaet al. 2002).Bovine as2-casein has also been reported to be a

source of antimicrobial peptides among whichcasocidin-I was the first to be well characterised(Zucht et al. 1995). It is a heat- and acid-stablepeptide isolated from milk after hydrolysis of milkproteins by heat treatment under acidic conditions,

Tab

le1(Continued)

Antimicrobial

peptide

Source

MW

(kDa)

Cleaving

protease

Aminoacid

sequence

(one

lettercode)a

Activity

Mainchem

ical

properties

Reference

f(141-146)

bovine

jcasein

0.6

Pepsin

STVATL

Weaklyinhibitory

Neutral

f(18-24)

bovine

jcasein

0.8

Pepsin

FSDKIA

KCationicpeptide

Cationic

f(30-32)

bovine

jcasein

0.4

Pepsin

YVL

Mainlygram

positive

bacteria

Neutral

f(118-121)

bovine

jcasein

0.5

Pepsin

EIPT

Mainlygram

-negativebacteria

Anionic

f(139-146)

bovine

jcasein

0.8

Pepsin

VESTVATL

Gram-positiveand


Anionic

f(64-75)

bovine

jcasein

1.3

Pepsin

PAAVRSPA

QILQ

Gram-positiveand


Cationic

a The

numberof

theam

inoacid

ofthesequence

(low

ercase)correspondstotheposition

ofthisam

inoacidin

theparentmatureprotein.

bOnthebasisof

theobservationthatC-and

N-terminalends

ofthepeptideare

potentialtrypticdigestionsites(Zuchtetal.1995).cUnderlined

boldface

sequence

correspondstoapepticdigestwithintrinsicgrow

th-inhibitoryactivity(Recio

andVisser1999);itprobablyrepresentstheactive

region

ofcasocidin-I.

dIn

variantB

,residuesT136andD148aresubstitutedforI136andA148.eUnderlinedboldface

sequence

representstheactive

region

ofkappacinA(M

alkoskieta

l.2001),

Risaphosphorylated

serylresidue.



followed by different treatments to remove fat andmost of the remaining high molecular-weightproteins. Basic peptides are then concentrated,chromatographically fractionated and eventuallydigested with proteases, e.g. pronase, endoprotein-ase Glu-c or trypsin (Zucht et al. 1995; Forssmannet al. 2001, 2003). Characterisation of the releasedpeptides by Edman degradation reactions and massspectroscopy has revealed casocidin-I as an as2-casein-derived fragment consisting in 39 amino-acid residues [as2-CN f(150-188)] with both aminoacid termini (i.e. C- and N-terminal) being trypticcleavage sites (Zucht et al. 1995). In vitro antimi-crobial activity tests showed that casocidin-I wasinhibitory to two strains of E. coli and one strain ofStaphylococcus carnosus in a dose-dependentmanner. Forssmann et al. (2003) have furtherreported that casocidin-I inhibits the growth ofseveral micro-organisms of hygienic and spoilagesignificance including B. subtilis, Staph. carnosus,Staphylococcus epidermidis, Enterococcus fae-cium, E. coli and Rhodotorula rubra at concentra-tions ranging between 1 and 180 lg ⁄mL,B. subtilis was shown to be the most sensitive andEnt. faecium the most resistant. As regards thepotential application of casocidin-I, Zucht et al.(1995) originally recommended using it in infantformulae to provide suckling infants with antibac-terial peptides capable of influencing the composi-tion of the intestinal microflora, especially thosenot present in human milk which is devoid of as2-casein. The same authors later patented casocidin-I, claiming its suitability to treat various dermatitisand mucosal infections of different origins (bacte-ria, fungi or parasites) as well as diarrheic gastroen-teritis (Forssmann et al. 2003). They have alsorecommended using it in food preservation and infermentation processes (Forssmann et al. 2001).However, most of these claims remain uncon-firmed; in particular the spectrum of action andclinical performances of the peptide. Applicationof casocidin-I as a food additive to enhance safetyand keeping quality appears to be worthwhileconsidering. Yet, the onerous and costly procedurefor the isolation and purification of the bioactive

peptide from milk may hinder its widespread appli-cation, as was pointed out by Ross et al. (2007).Crude milk-based preparations of casocidin-I maybe an appropriate alternative. A suggested proce-dure for the crude preparation of casocidin-I on thebasis of the purification technique described byZucht et al. (1995) is illustrated in Figure 1. Thiscrude preparation obtained according to Zuchtet al. (1995) may be further desalted and concen-trated by ultrafiltration and diafiltration, and thenfreeze- or spray-dried as is the case for industrialproduction of LactoferrinTM (Tomita et al. 2002).Although this procedure is not expected to yield ahigh purity product, the resulting crude preparationwould be more advantageous for food preservationthan a highly purified casocidin-I. Fractionation ofthe crude preparation of casocidin-I followed byantibacterial activity testing of the resulting frac-tions has revealed the presence of various minorantibacterial peptides having an additive antibacte-rial effect with each other and with casicidin-I(Zucht et al. 1995). Therefore, these authors haveanticipated that, beside casocidin-I, other antibacte-rial peptides with different masses would bereleased from the C-terminal region of bovine as2-casein beginning from residue 165 or 166. Indeed,subsequent studies have shown that the hydrolysisof bovine as2-casein with digestive proteasesgenerates various antibacterial peptides derivingfrom the C-terminal region starting from residue164 (Recio and Visser 1999; McCann et al. 2005;Lopez-Exposito et al. 2006a). Recio and Visser(1999) reported on the release of two antibacterialdomains by pepsin digestion of bovine as2-casein,f(164-179) and f(183-207), which were stronglyinhibitory to E. coli, Bacillus cereus and Strepto-coccus thermophilus. However, the originality ofthese peptides was controversial; the first fragmentwas considered only to be the active region ofcasocidin-I, as it is totally included in its sequence,and the second fragment has been assumed to be acomponent of the innate defence system of themammary gland due to structural similarities(Boman 1995; Kolb 2001). Furthermore, McCannet al. (2005) showed that chymosin digestion of

Table 2 Antibacterial peptides derived from a-lactalbumin (a-La) or b-lactoglobulin (b-Lg)

Sequence (one letter code) Origin Amino acid fragment a Cleavage enzyme References

E1QLTK5 a-La f(1-5) Trypsin (Pellegrini et al. 1999)

G17YGGVSLPEWVCTTF31 A109LCSEK114b a-La f(17-31)S-S(109-114)

C61KDDQNPH68 I75SCDKF80c a-La (61-68)S-S(75-80) Chymotrypsin

V15AGTWY20 b-Lg 15–20 Trypsin (Pellegrini et al. 2001)

A25ASDISLLDAQSAPLR40 b-Lg 25–40

I78PAVFK83 b-Lg 78–83

V92LVLDTDYK100 b- Lg 92–100

aNumbers indicate the positions of the first and last amino acids of the fragments as in the mature parent molecule. bTwo peptides held by disulfide bond

between cysteyl residues at positions 28 and 111. cTwo peptides held by a disulfide bond between cysteyl residues at positions 61 and 77.



bovine sodium caseinates releases five differentantibacterial peptides identified as fragments ofas2-casein C-terminal end (starting from residue164), and sharing the 27-amino acid-residuesequence f(181-207) of the casein C-terminalregion (Table 1). While four of these antimicrobialpeptides had not been reported earlier, the commonfragment, f(181-207), matched exactly one of thepeptides previously isolated from the pepsin hydro-lysate of bovine as2-casein and considered to bethe fragment containing the active domain f(183-207) (Recio and Visser 1999). The five antibacte-rial peptides characterised by McCann et al.(2005) inhibited a range of Gram-positive andGram-negative bacteria to different extents, thoughbeing generally more active against Gram-positivethan Gram-negative bacteria; the MICs againstL. monocytogenes, L. innocua and B. subtilis ran-ged between 4.8 and 21 lg ⁄mL, whereas the MICs

against S. Typhimurium, S. Enteridis and E. coliwere significantly higher and ranged between 21and > 171.2 lg ⁄mL. Of these peptides, f(181-207), f(175-207) and f(164-207) inhibited sensitiveGram-positive bacteria as effectively as nisin andlactoferricin B, suggesting that they have a highpotential to be used as food grade preservatives.Furthermore, the antibacterial domain f(164-179)reported by Recio and Visser (1999) was includedin only one of the five peptides reported byMcCann et al. (2005), indicating that it could notbe the active site of the antibacterial peptidesderived from the C-terminal region of bovine as2-casein, 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 peptidesdoes not correlate with the presence of a specificactive site nor does it with the size of the peptide;

Figure 1 Flow chart for crude preparation of casocidin-I (adapted from Zucht et al. 1995 and Tomita et al. 2002).



it was suggested to rather be due to the net electriccharge and hydrophobicity of the peptide.Similarly, antibacterial peptides derived from

ovine as2-casein were also demonstrated to beencrypted in the C-terminal region, and pepsindigestion of ovine as2-CN followed by a fraction-ation of the resulting hydrolysate by chromatogra-phy techniques yielded four major fractionsinhibitory against E. coli. Identification of thepeptides recovered from these fractions revealedthe presence of 10 peptides of different masses, allderiving from the C-terminal region starting fromresidue 165. However, antibacterial activity couldnot be attributed to specific peptides, as the frac-tions still contained more than one peptide at theultimate purification step (Lopez-Exposito et al.2006a). Therefore, selected fragments of the mostabundant in each fraction ⁄ sub-fraction (Table 1)were chemically synthesised and investigated fortheir antimicrobial activity against various Gram-positive and Gram-negative bacteria. On the struc-tural level, antibacterial peptides released by pepsindigestion of ovine as2-casein were generally smal-ler than those generated from bovine as2-casein (5–25 vs 27–44). Incidentally, two of the most activeof the ovine as2-casein peptides, f(165-181) andf(184-208), corresponded to the two antibacterialdomains of bovine as2-casein reported by Recioand Visser (1999), providing further evidence forthe originality of the latter peptides. However,differences in the antibacterial properties betweenthe two ovine fragments [i.e. f(165-181) and f(184-208)] and their bovine homologues [i.e. f(164-179)and f(183-207)] were observed when tested in thesame conditions and against the same indicatorstrains (Lopez-Exposito et al. 2006a). Ovine as2-CN f(165-181) was significantly more active thanits bovine homologue, while the bovine f(183-207)exhibited stronger antibacterial activity than theovine homologue (Lopez-Exposito et al. 2006a).

Antimicrobial peptides from j-caseinThis class of milk proteins has also been demon-strated to be an important precursor of antibacterialpeptides of potential use in food preservation andsafety. The generation of such peptides from j-casein was first demonstrated by Liepke et al.(2001) who purified an antibacterial peptide f(63-117) from a hydrolysate obtained by acidification ofhuman milk followed by pepsin hydrolysis, simu-lating the digestion process in infant stomachs. Thispeptide was shown to be inhibitory to Gram-posi-tive and Gram-negative bacteria as well as yeasts,and was thus suggested for use in infant nutrition tostimulate the host defence system of the newborn.A subsequent study has demonstrated that a C-ter-minal chymosin-digest of bovine j-casein [j-CNf(106-169)], called caseinomacropeptide (CMP),exerts in vitro antibacterial activity against major

oral pathogens (e.g. Streptococcus mutans,Porphyromonas gingivalis and Actinomyces naes-lundii) and E. coli (Malkoski et al. 2001; Dashperet al. 2007). CMP is a highly heterogeneous poly-peptide encompassing various glycosylated andphosphorylated forms (Saito and Itoh 1992; Talboet al. 2001); and such heterogeneity is increased bythe existing genetic variants of j-casein, with vari-ants A and B being the most frequent (Creamer andHarris 1997). Malkoski et al. (2001) have demon-strated that the nonglycosylated, phosphorylatedCMP is the only active form of the molecule, whichthey have designated kappacin. The antimicrobialactivity of kappacin derived from variant B j-casein(j-CN-B) was, though, shown to be significantlylower than that of variant-A kappacin; the activityof both peptides was lost or markedly reduced atneutral pH (Malkoski et al. 2001; Dashper et al.2005, 2007). The difference in activities betweenkappacin fragments derived from variants A and Bhas mainly been attributed to the substitution ofAsp148 in variant A for Ala148 in variant B (Dashperet al. 2005). Furthermore, digestion of kappacin-Awith endoproteinase Glu-C generated various pep-tides among which only a phosphorylated one[Ser(P)149 j-CN-A f(138-158)] was active againstS. mutans, indicating that the bactericidal domain ofkappacin resides in this portion. Also, phosphoryla-tion of the seryl residue at position 149 (Ser149) wasshown to be crucial for antibacterial activity as wasdemonstrated by the susceptibility of S. mutans tochemically synthesised kappacin with phosphory-lated Ser149 [Ser(P)149 k-CN-A(138-158)] and theresistance of the same strain to the correspondingsynthetic fragment with nonphosphorylated serylresidue [Ser149 j-CN-A(138-158)]. Interestingly,the synthetic kappacin had higher potency than thepurified peptide and its derivative f(138-158).Moreover, kappacin was shown to specifically bindtwo zinc or calcium ions per mol, resulting in a sig-nificant increase in its antimicrobial activity even atneutral pH. These divalent ions were suggested tohelp potentiate the activity of kappacin by inducingconformational changes in such a way to enhancethe binding affinity of kappacin to the cell mem-brane (Dashper et al. 2005, see also the paragraphon the mechanism of action, below).The research work that has, so far, been

conducted on kappacin has been largely orientedtowards the use of the peptide as a pharmaceuticalsupplement for oral therapy (Malkoski et al. 2001;Dashper and Reynolds 2005; Dashper et al. 2005,2007). In fact, kappacin has already been commer-cially available for dental care application by theCooperative Research Centre for Oral Health Sci-ence (Australia) as KappacinTM or as KappaZinTM.The latter product consists in a combination ofkappacin with zinc (1:1) to form an antimicrobialcomplex with enhanced antibacterial activity.



Apart from this application, kappacin may alsobe a suitable and safe food-grade biopreservativewith high potential for application in the foodindustry due to its ability to inhibit Gram-positiveand Gram-negative bacteria and to resist proteo-lytic enzymes, in addition to its presumed safetyas it is derived from j-casein that has a long his-tory of safe consumption in dairy products. As amatter of fact, the vast majority of known cheesevarieties already contain some amount of kappa-cin in the form of CMP resulting from milkcoagulation with rennet or other clotting enzymesof animal or plant origin that hydrolyse j-caseinat the same cleavage site (see Abd-El Salam andBenkerroum 2006). The antimicrobial effect ofkappacin would be particularly enhanced in dairyproducts which are naturally rich in calcium ionsshown to potentiate the antibacterial activity ofthe peptide (Dashper et al. 2005). Furthermore,in most cheeses, an excess of calcium is addedduring manufacture to accelerate milk clotting,thereby increasing the amount of calcium avail-able for binding kappacin. Further studies areneeded to substantiate such observations, and todetermine the optimal conditions as well the tar-get food products on a sound basis. Health riskassessment studies, especially concerning thepotential allergenicity of the peptide have also tobe carried out prior to any practical use.In another study on the generation of antibacte-

rial peptides from milk caseins, Lopez-Expositoet al. (2006b) have identified 21 fragments in sixdifferent active HPLC fractions recovered from apepsin hydrolysate of commercial j-casein. How-ever, a thorough purification and identification ofthese digests revealed that only 12 (recovered from5 active fractions) were actually released from j-casein, while the others were derived from the a orb caseins contaminating the commercial j-caseinused in the study, as revealed by capillary electro-phoresis. Of the 12 j-casein-derived peptides, nineof the most abundant in the corresponding activeHPLC fractions (Table 1) were chemicallysynthesised and tested for their antimicrobial activ-ity against Gram-positive (L. innocua and Staph.carnosus) and Gram-negative (E. coli, S. marces-cens) bacteria. These peptides inhibited indicatorstrains to different extents, with fragments f(18-24), f(30-32) and f(139-146) being the most inhibi-tory, inducing a reduction of the initial counts ofthe most sensitive bacteria by more than six loga-rithmic units after 2 h of incubation. Nonetheless,S. marcescens was only moderately inhibited by allthe peptides, and its initial count was reduced by0.20–0.59 logarithmic units depending on thepeptide, with the exception of peptide f(30-32)which reduced the initial count of the bacterium bythree logarithmic units after 2 h of incubation(Lopez-Exposito et al. 2006b).

Antimicrobial peptides from whey proteinsWhey proteins (a-La, b-Lg, serum albumin, immu-noglobulins, lactoferrin, lysozyme, etc.) are knownto be valuable sources for bioactive peptides withhealth promoting or antimicrobial properties (Floriset al. 2003). Among these, lactoferrin and lyso-zyme possess inherent antimicrobial activities, andalso release antimicrobial fragments by proteolyticdigestion (Tomita et al. 2002; Touch et al. 2004).The latter two whey-protein components and theantimicrobial peptides derived therefrom are thebest studied, and have extensively been reviewedelsewhere (Salton 1957; Pellegrini et al. 1992;Tomita et al. 1994, 2002; Floris et al. 2003;Pellegrini 2003; Benkerroum 2008). On the con-trary, antimicrobial peptides derived from a-La andb-Lg have received much less attention despite thescientific evidence for their existence and thepotential of a-La and b-Lg as bioactive peptideprecursors (Fiat et al. 1993; Meisel 1998; Shaha2000; Pellegrini 2003). In a preliminary study, Pi-hlanto-Leppala et al. (1999) showed that proteo-lytic digestion of bovine a-LA and b-Lg withseven different proteases yielded various hydroly-sates exerting bacteriostatic activities against E.coli, while the parent molecules failed to inhibit thebacterium under the same experimental conditions.The most active hydrolysates were obtained by acombined action of pepsine and trypsin, and havea molecular mass less than 1 kDa; however, theinhibitory peptides were not further characterisedor sequenced. In two other studies, a-lactalbulinand b-Lg were digested with pepsine, trypsin orchymotrypsin, and the hydrolysates were fraction-ated chromatographically, and the growth-inhibi-tory activity of each fraction was monitored; theactive peptides were then purified and character-ised (Pellegrini et al. 1999, 2001). While no anti-bacterial activity was associated with the pepticdigests of a-LA, hydrolysis of the protein withtrypsin or chymotrypsin yielded three antibacterialfragments; two tryptic and one chymotryptic(Table 2). The chymotryptic fragment and one ofthe two tryptic fragments have a dimeric structure,each of which is composed of two subunits heldtogether by a disulfide bridge, and they wereshown to be significantly more inhibitory than themonomeric pentapeptide released from a-LA, withthe tryptic dimer being the most active (Pellegriniet al. 1999). However, it has not been demon-strated whether or not the disulfide bridges of thedimeric peptides account for such increased antimi-crobial activity, as is the case for other antimicro-bial peptides where the formation of intra- orintermolecular disulfide bridges, via specificassembly or post-translational rearrangementmechanisms, is crucial to their activity (Romeoet al. 1988; Hill et al. 1991; Simmaco et al. 1998;



Reddy et al. 2004; Jenssen et al. 2006). The disul-fide bridges of the a-LA-derived dimeric peptidesdo not appear to result from a specific assemblymechanism, as they are naturally present in the par-ent molecule (Ikeguchi et al. 1998). They mayonly remain intact, holding the two subunitstogether, after the hydrolysis of the protein asdepicted in Figure 2.As for the potential of b-Lg to generate antimi-

crobial peptides, Pellegrini et al. (2001) haveshown that the tryptic digestion of the proteinyielded four antibacterial peptides with differentlengths (6–16 residues) and different amino acidsequences, which were inhibitory to Gram-positivebacteria only (Table 2). However, modification ofthe nine amino-acid peptide VLVLDTDYK bysubstituting the Asp98 residue for Arg and adding alysyl residue at the C-terminus has made E. coli sus-ceptible to the resulting peptide (VLVLDTRYKK),thereby extending the spectrum of action to Gram-negative bacteria (Pellegrini et al. 2001). Thesestudies have lead the authors to recommend themajor whey proteins (i.e. a-La and b-Lg), after par-tial digestion by proteases, for a possible antimicro-bial function in therapy or in the food industry(Pellegrini et al. 1999, 2001). Yet, none of the sevenpeptides generated from a-LA and b-Lg has foundan industrial application; they would suffer fromthree main limitations: (i) the narrow spectrum ofaction limited to Gram-positive bacteria, (ii) themoderate antimicrobial potency and (iii) the poten-tial allergenicity of the peptides, particularly thosederived from b-lactoglobulin which is known to bea major bovine milk allergen with numerousepitopes (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 thatthe allergenicity of peptides derived from milk

proteins is significantly reduced compared to theprecursor molecules (Fritsche et al. 2005; Asselinet al. 2006), others have demonstrated thatb-Lg-derived peptides containing epitopes retainedbetween 40% and 97% allergenicity of the parentmolecule (Selo et al. 1999). Therefore, whey-derived antimicrobial peptides should be scrutinisedon a case-by-case basis for allergenicity at differentconcentrations for their anticipated use in the foodindustry.

ANT IMICROBIAL PEPT IDESRELEASED FROM MILK PROTE INSBY FERMENTAT ION WITH LACT ICAC ID BACTER IA

Degradation of milk proteins by LAB as a meansto fulfil their nutritional requirements for essentialamino acids is well documented (Abraham et al.1993; Fira et al. 2001; Matar et al. 2003). There-fore, the use of LAB or their proteases to generatebioactive peptides from milk proteins is attractingincreased attention; and such bioactive peptides areexpected to be fundamentally different from thosereleased by digestive proteinases which differ frommicrobial proteinases in specificity and mode ofaction (Atlan et al. 1990; Laloi et al. 1991; Sasakiet al. 1995; Matar et al. 2003).Few studies, however, have been carried out to

identify the peptides generated from milk proteinsby LAB with respect to their biological activities;and most of these studies have focused on thehealth-promoting properties (Clare and Swaisgood2000; Gobbetti et al. 2000; Quiros et al. 2007). Ina survey on bioactive peptides released from milk-caseinates from different domestic mammals upondigestion with a partially purified proteinase fromLactobacillus 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 unspecific cleavage regardless of the

protease. Fragments held by a disulfide 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.



reported that among the numerous resulting bioac-tive peptides, only one peptide deriving fromhuman b-casein-hydrolysate, [b-CN f(184-210)],exhibited antibacterial activity. This peptideshowed a very large spectrum of action againstGram-positive and Gram-negative bacteria, includ-ing Ent. faecium, Bacillus megaterium, E. coli, L.innocua, Salmonella spp., Yersinia enterocolitica,and Staph. aureus, and MIC for a sensitive E. colistrain was �50 lg ⁄mL. On the other hand, frac-tionation of water-soluble extracts from nine differ-ent Italian ripened-cheese varieties has revealed thepresence of several antimicrobial peptides with apotent inhibitory activity against a wide range ofbacteria of health and spoilage significance (Rizz-ello et al. 2005). Yet, the release of these antibacte-rial peptides could not be specifically ascribed toLAB, as chymosin, ripening moulds or nonstarterlactic acid bacteria also could be responsible forsuch biopeptides release.More recently, Hayes et al. (2006) provided

further evidence for the generation of antimicrobialpeptides by fermentation of milk-caseins withLAB. In this study, sodium caseinate from bovineas2-casein was fermented with a proteolytic strainof Lactobacillus acidophilus, and the fermentatewas passed through a size exclusion cartridge-filterto discard peptides having a molecular weightgreater than 10 kDa. The filtrate was fractionated,and the peptides in fractions showing antagonisticactivity against E. coli were purified by RP-HPLCand sequenced, revealing the presence of threeshort antibacterial peptides which have been desig-nated caseicins A, B and C (Table 3). Caseicins Aand B exerted a potent inhibitory activity againstGram-positive and Gram-negative bacteria includ-ing food borne pathogens of health concern, suchas Listeria innocua, E. coli O157:H7, Ent. sak-azakii and S. mutans (Hayes et al. 2006). TheMICs 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 ofthese peptides as food-grade biopreservatives or asfunctional-food supplements. In view of thepotency of caseicins A and B against Ent. sak-azakii, Hayes et al. (2006) have recommendedusing the peptides to control the pathogen in milk-based infant formulae. Indeed, these products are

known to be the major reservoir for Ent. sakazakiiwhich is responsible for a distinctive syndrome ofmeningitis with a mortality rate as high as 80% inneonates (Nazarowec-White and Farber 1997).Such an application is even more appropriate thatthe pathogen is difficult to control by conventionalmeans for quality assurance (Nazarowec-Whiteand Farber 1997; Hayes et al. 2006; Ross et al.2007). As matter of fact, these peptides would finda larger application in food preservation, either intheir purified state or as components of casein-fermentate of Lb. acidophilus, owing to their largespectrum of action covering bacteria of health andspoilage significance in addition to their anticipatedsafety and consumer acceptability. The LAB usedas biological catalysts have a long history of safeuse in food fermentation and already benefit fromthe ‘generally recognised as safe’ status, and themilk proteins used as substrates are reputed fortheir safety and health benefits.The use of such ‘biological kits’ comprising

proteolytic LAB as catalysts and milk or milk pro-teins as substrates to release different antimicrobialpeptides represents a novel and promising strategyfor food preservation. It offers a great versatility totarget specific undesirable bacteria and foods byusing different combinations of proteolytic LABand substrates (e.g. a, b or j-caseins or wheyproteins) to design tailored antimicrobial peptidesfor ‘in-built’ preservation technology. In somedairy products, for example, selected proteolyticLAB may be used as part of the starter or adjunctstarter cultures to produce, in situ, the desired anti-microbial peptides during processing. Furthermore,proteolytic LAB would produce a greater diversityof antimicrobial peptides from milk proteins com-pared to digestive proteases, due to the complexityand multiplicity of microbial proteolytic systems(Reid and Coolbear 1998; Deutsch et al. 2000).Strains of LAB have been shown to cleave peptidebonds 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 overlappingpeptide sequences with various biological activi-ties, but which are protected from breakdown bydigestive 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 ofLactobacillus acidophilus (Hayes et al. 2006)

Antibacterial peptide

Sequence (number of

amino acid residues) Spectrum of action

Caseicin A as1-CN f(21-29) IKHQGLPQE (9) E. coli, Ent. sakazakii, Lb. bulgaricus, L. innocua, S. mutans

Caseicin B as1-CN f(30-37) VLNENLLR (8) As above

Caseicin C as1-CN f(195-208) SDIPNPIGSENSEK (14) L. innocuaa

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



et al. (2006), the generation of caseicins A and Bfrom bovine as1-casein has been shown to requirea combined action of three endopeptidases and pro-teinases. Nonetheless, the complexity and multi-plicity of proteolytic systems in LAB may also bea disadvantage if not adequately managed. Anexcessive proteolytic activity results in further deg-radation of the antibacterial peptides, once releasedfrom the precursor molecules, into smaller inactivefragments (Rizzello et al. 2005). Therefore, whensuch antimicrobial substances are to be generatedfrom milk proteins by fermentation with proteo-lytic LAB, the proteolysis should be managed sothat the ratio of soluble nitrogen to total nitrogen(Nsoluble ⁄Ntotal) remains between 12% and 24%(Rizzello et al. 2005). Water-soluble extracts fromcheese types with long ripening or intense proteol-ysis (Nsoluble ⁄Ntotal ‡ 30%) have been shown to bedevoid of antibacterial peptides, contrary to theircounterparts from cheese samples with a limitedproteolysis (Addeo et al. 1992; Meisel et al. 1997;Rizzello et al. 2005). Similarly, Lopez-Expositoet al. (2007) showed that the antimicrobial activityproduced by hydrolysis of ovine as2-casein andbovine j-casein with pepsin digestion was partiallyor completely lost when the caseins were digestedfor a longer period than 30 min and 2 h respec-tively. In addition, sequential digestion of caseinswith pepsin and a mixture of trypsin and chymo-trypsin has yielded inactive digests, while the case-ins had generated antimicrobial peptides whendigested separately with the same enzymes(Lopez-Exposito et al. 2006b).Another potential advantage of the preservation

strategy using proteolytic LAB is the possibility touse specific bacteriocin-producing strains of LABwhich are also able to generate antimicrobial pep-tides from food proteins. In this regard, a strain ofLactobacillus curvatus has been shown to producetwo distinct bacteriocins and to generate an antimi-crobial peptide from the tryptic digest of b-caseinpresent in de Man, Rogosa and Sharp (MRS) broth(Ghalfi et al. 2010), and the effectiveness of thisLactobacillus strain in food preservation has beendemonstrated (Ghalfi et al. 2006a,b, 2007). Thismight extend the spectrum of action and increasethe overall efficacy of the peptides in a synergisticmanner as in ‘preservative systems’, as was dem-onstrated in vitro for nisin and the bovine milk-derived peptides aS2-casein f(183-207) against L.monocytogenes (Lopez-Exposito et al. 2008b).

MECHANISM OF ACT ION OFANT IMICROBIAL MILK -DER IVEDPEPT IDES

General considerationsFew and sparse studies have been done to elucidatethe mechanism of action of antimicrobial peptides

derived from milk proteins. The variability in theiramino acid composition, structure and physico-chemical characteristics (Table 4) suggests that theyact in different ways on sensitive bacteria. Yet, thereis a general agreement that, regardless of the precisemechanism of action, all antimicrobial peptides pri-marily act on the plasma membrane through theestablishment of electrostatic bonding between thepeptide and plasma membrane components, at leastduring early steps of a peptide-mediated killing pro-cess (Zasloff 2002; Reddy et al. 2004). Such aninteraction results in one of two major effects: (i) theformation of transient transmembrane channels thatalter the membrane permeability and ⁄or energygeneration while preserving the cell integrity or (ii)the disruption of the plasma membrane with a con-sequent disintegration of the whole cell (Epand andVogel 1999; Jenssen et al. 2006; Park et al. 2008).It is generally admitted that the electrostatic bondingis only possible if the peptide has a shape in whichclusters of hydrophobic and cationic amino acidsare spatially organised in an amphipathic form, withthe hydrophobic sectors on one side and the cationic(i.e. hydrophilic) sectors on the opposite side. Insuch a conformation, the hydrophobic sectors inter-act with the lipids of the membrane, while the cat-ionic sectors interact with its negatively chargedphosphate groups (Brogden 2005). Furthermore,the potency of amphipathic antimicrobial peptideshas 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 plasmamembrane as a first step in a killing process wherethe subsequent steps involve one of five differentmechanisms described as the ‘barrel-stave’, ‘car-pet’, ‘detergent’, ‘toroidal pore’ or ‘aggregates’models (for further reading, see Zasloff 2002; Red-dy et al. 2004; Brogden 2005; Jenssen et al. 2006).Nonetheless, the ultimate killing target is not alwaysthe plasma membrane itself, as the peptide maytranslocate into the cytoplasm and act on intracellu-lar components including nucleic acids, proteins orlipids thereby interfering with DNA replication andexpression, enzymatic reactions or macromolecularsynthesis (Carlsson et al. 1998; Epand and Vogel1999; Minervini et al. 2003a; Ulvatne et al. 2004;Jenssen et al. 2006), or else cause the cytoplasm toflocculate (Brogden 2005).

Case of milk protein-derived antimicrobialpeptidesGiven the diversity of the milk protein-derivedantimicrobial peptides in their net electric charges,secondary structures or hydrophobicity ⁄hydrophi-licity properties (Table 4), it is difficult to antici-pate a priori how or if they would exertantimicrobial activities. Preliminary studies havebeen conducted in this regard, and are discussedherein. 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


(amino-acid sequence) Name

Net

charge

(pH 7)a,bIsoelectric

point a

Molecular

mass

(kDa)aAverage

hydrophilicitya

Hydrophilic

residues ⁄ totalresidues (%)a

Predicted

secondary

structurec

IKHQGLPQE Bovine

as1-CN f(21-29)

Caseicin A 0.1 7.8 1.05 0.3 44 C

VLNENLLR Bovine as1-CNf(30-37)

Caseicin B 0b 7.0 0.97 )0.1 50 C

SDIPNPIGSENSEK Bovine

as1-CN f(195-208)

Caseicin C )2 3.8 1.49 0.7 64 C

RPKHPIKHQGLPQEVL

NENLLRF Bovine as1-CNf(1-23)

Isracidin 2.2 10.6 2.8 0.2 43 H ⁄C

KTKLTEEEKNRLNFLKKI

SQRYQKFALP-QYLKT

VYQHQK Bovine aS2-CNf(150-188)

Casocidin-I 7.1 10.5 4.87 0.4 54 H ⁄C

LKKISQRYQKFALPQY

Bovine aS2-CN f(164-179)

Unnamed

(casocidin-I fragment)

4.0 10.5 2.01 0 50 H ⁄C

YQHQKAMKPWIQPKTK

VIPYV-RYL Bovine

aS2-CN f(183-207)

Unnamedd 5.1 10.5 3.02 )0.2 33 C ⁄E

KTVYQHQKAMKP-

WIQPKTKV-

IPYVRYL Bovine

as2-CN f(181-207)

Cr1 6.1 10.6 3.3 )0.2 33 C ⁄H ⁄E

LKTVYQHQKAMKP

WIQPKTKV-

IPYVRYL Bovine

as2-CN f(180-207)

Cr3 6.1 10.6 3.5 )0.2 32 C ⁄H ⁄E

ALPQYLKTVYQHQKA-

MKPWIQP-KTKVIPYV-

RYL Bovine as2-CNf(175-207)

Cr4 6.1 10.4 4.0 )0.3 30 C ⁄H ⁄E

QKFALPQYLKTVYQHQ-

KAMK-PWIQPKTKVIPYV-

RYL Bovine as2-CNf(172-207)

Cr7 7.1 10.5 4.4 )0.3 33 C ⁄H ⁄E

LKKISQRYQKFALP-

QYLKTVYQ-HQKAM-

KPWIQPKTKVIPYVRYL

Bovine as2-CN f(164-207)

Cr5 ⁄Cr6 10.1 10.7 5.5 )0.1 35 C ⁄H ⁄E

LKKISQ Ovine as2-CNf(165-170)

Unnamed 2 10.6 0.7 0.5 67 C

LKKISQYYQKFAWPQYL

Ovine as2-CN f(165-181)

Unnamed 3 10 2.2 )0.5 41 C ⁄E

VDQHQKAMKPWTQPKT-

NAIPY-VRYLOvineas2-CNf(184-208)

Unnamed 3.1 10.2 3.0 )0.1 36 C ⁄E

PYVRYL Ovine as2-CNf(203-208)

Unnamed 1.0 9.6 0.8 )0.8 17 C

AIPPKKNQDKTEIPTINTI-

ASGEP-TSTPTTEAVEST-

VATLEDSPEVI-ES-

PPEINTVQ-VTSTAV

Bovine j-CN f(106-169)

Kappacine )7 3.8 6.67 0.2 37 C ⁄H

AVESTVATLEDSPEVIES-

PPE Bovine j-CN f(136-156)

Kappacin fragment )6 2.8 2.20 0.4 38 H ⁄C ⁄E

YYQQKPVA Bovine

j-CN f(42-49)

Unnamed 1.0 9.5 1.0 )0.4 38 C ⁄E



been carried out on kappacin by using liposome-swelling assays (Dashper et al. 2005). In this study,the authors have demonstrated that a concentrationranging between 30 and 50 lM of kappacin-Aincreased the permeability of liposomes in aconcentration-dependant manner, indicating thatthe peptide permeabilises the plasma membrane ofsensitive bacteria. According to the authors, kappa-cin would disrupt the plasma membrane in a simi-lar manner as the cationic a-helical peptides, inspite of the fact that kappacin is strongly anionic(seven negative charges) and has a random-coiledsecondary structure in an aqueous environment[Table 4, (Dashper et al. 2005)]. It has beensuggested, however, that once in contact with thesurface of bacterial cell-membrane, kappacinwould shift to an amphipathic a-helical structure.This assumption was supported by the ability of

kappacin to form an a-helix in presence of triflu-oroethanol and excess divalent metal ions (e.g.Ca++ or Zn++); an environment that mimics the sur-face of the plasma membrane. In fact, it is believed,although debated, that all of the antimicrobialpeptides, regardless of their original conformation,would adopt an amphipathic a-helical conforma-tion once in the vicinity of the plasma membranein response to the prevailing physico-chemicalparameters such as the ionic strength, pH, EH,temperature, metal ions, membrane lipids (Gennaroand Zanetti 2000; Park et al. 2000, 2008). Never-theless, the study of Malkoski et al. (2001) did notprovide further evidence for the ultimate killingtarget of the peptide, nor did it give an explana-tion regarding the permeabilisation of the outermembrane to kappacin which has been, other-wise, shown to potently inhibit the growth of

Table 4 (Continued)


(amino-acid sequence) Name

Net

charge

(pH 7)a,bIsoelectric

point a

Molecular

mass

(kDa)aAverage

hydrophilicitya

Hydrophilic

residues ⁄ totalresidues (%)a

Predicted

secondary

structurec

IQY Bovine j-CN f(28-30) Unnamed 0.0 5.9 0.4 )1.3 33 C

VQVTSTAV Bovine j-CNf(162-169)

Unnamed 0.0 6.0 0.8 )0.7 25 C

STVATL Bovine j-CNf(141-146)

Unnamed 0.0 6.0 0.6 )0.7 17 C ⁄E

FSDKIAK Bovine j-CNf(18-24)

Unnamed 1.0 9.9 0.8 0.6 57 C ⁄H

YVL Bovine j-CN f(30-32) Unnamed 0.0 5.9 0.4 )1.9 0 C

EIPT Bovine j-CN f(118-

121)

Unnamed )1.0 3.3 0.5 0.2 25 C

VESTVATL Bovine j-CNf(139-146)

Unnamed )1.0 3.3 0.8 )0.3 25 C ⁄E

PAAVRSPAQILQ Bovine j-CN f(64-75)

Unnamed 1.0 11 1.3 )0.2 33 C ⁄E ⁄H

EQLTK Bovine a-La f(1-5) Unnamed 0 6.9 0.62 0.8 60 C

GYGGVSLPEWVCTTFAL-

CSEK Bovine a-La f(17-

31)S-S(109-114)

Unnamed )1.1 4.3 2.25 )0.4 24 NPf

CKDDQNPH ISCDKF

Bovine a-La f(61-68)S-S(75-

80)

Unnamed )1 5.2 1.65 0.6 57 NP

VAGTWY Bovine b- Lg

f(15–20)

Unnamed 0 5.9 0.70 )1.3 0 C

AASDISLLDAQSAPLR

Bovine b- Gl f(25–40)Unnamed )1 3.9 1.63 0.1 44 C ⁄C ⁄H

IPAVFK Bovine b- Gl f(78–83)

Unnamed 1 10.1 0.67 )0.5 17 C

VLVLDTDYK Bovine b- Glf(92–100)

Unnamed )1 3.9 1.07 0 37 E ⁄C

aData obtained by calculations using ‘Peptide Property Calculator’ facility; available at: http://www.innovagen.se/custom-peptide-synthesis/peptide-

property-calculator/peptide-property-calculator.asp. bThe overall electric charge was calculated by an algorithm using the formula:

Z ¼P

iNi

10pKai10pHþ10pKai �

P

jNj

10pH

10pHþ10pKaj where Ni is the number, and pKai the pKa values of the N-terminus and the side chains of Arginine, Lysine and

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, exten-

ded. dC-terminal fragment of bovine aS2-casein.eForms an a-helix in presence of trifluoroethanol (TFE) and excess divalent metal ions (e.g. Ca++ or

Zn++); Dashper et al. (2005). fNot provided.



Gram-negative bacteria. The permeabilisation ofthe plasma membrane as revealed by liposome-swelling assay is not an unequivocal evidence, byitself, for the membranolytic activity of the peptide;it does not always correlate with the antimicrobialactivity but could only be a route for antimicrobialpeptides to reach alternative intracellular targets(Wu et al. 1999; Rydengard et al. 2008). Further-more, the validity of model membranes (e.g. lipo-some) in mechanistic studies has been criticiseddue to the high complexity of the plasma mem-brane compared with liposomes (Park et al. 2008).Although model membranes have been extensivelyused to study the mechanism of action of antibacte-rial substances and have proven useful, they haveoften been associated to other analyses and testssuch as electron microscopy, X-ray crystallo-graphy, NMR spectroscopy, Fournier transforminfrared, use of dyes, conductivity measurements,circular dichroism or neutron diffusion for soundand convincing conclusions (Brogden 2005). Asfor the passage of kappacin across the outer mem-brane of sensitive Gram-negative bacteria, it shouldbe noted that it is unlikely to be self-promoted orpassive through porins due to the anionic hydro-phobic nature of the peptide in addition to its rela-tively high molecular weight (6670 Da). Anionicpeptides are especially restricted from crossing theOM mainly because of the electric repulsionbetween the negative charges of the peptides andthose of phosphate groups of the lipopolysaccha-ride (LPS). In addition, the OM porins are imper-meable to large (> 900 Da) or hydrophobicpeptides due to the small size of the porin-channelswhich are, in addition, filled with water (Engstromet al. 1984; Nikaido et al. 1991; Carlsson et al.1998). Further studies are thus needed to provideclearer insights into the molecular events leading tocell-death of either Gram-positive or Gram-nega-tive bacteria upon exposure to kappacin.Recently, Lopez-Exposito et al. (2008a) have

investigated the permeabilisation of the cell enve-lopes of E. coli and Staph. carnosus by the cationicbovine milk-derived aS2-casein f(183-207) and thesubsequent morphological changes leading to thedeath of sensitive bacteria. The results of the studyrevealed that the peptide initially binds to the LPSof the OM in the Gram-negative bacterium and tothe lipoteichoic acid (LTA) of the cell wall in theGram-positive bacterium. The identification ofthese binding sites was achieved by competitionstudies showing that the required concentration ofas2-casein f(183-207) to inhibit the growth ofsensitive bacteria has increased linearly with theincrease in the amounts of soluble LPS or LTAadded to a mixture of the peptide and each ofE. 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; Pierset al. 1994) and teichoic or LTA in Gram-positivebacteria (Vorland et al. 1999; Ulvatne et al. 2004),primarily by electrostatic interactions. However,attachment of these peptides through specific pro-tein receptors present on the OM or by means ofpolylysyl residues at the C-terminal end of the pep-tides has also been demonstrated (Vaara 1992;Carlsson et al. 1998; Brogden 2005). Although therational used by Lopez-Exposito et al. (2008a) inthese competition studies provides a convincingevidence for the validity of the attachment of theas2-casein f(183-207) to the cell envelopes of sen-sitive bacteria, the exact mechanisms of such anattachment remain to be elucidated. As for the per-meabilisation of the cell envelopes to the bovineas2-casein f(183-207), chromogenic dye tests per-formed in the same study have demonstrated thatthe peptide induces concurrent permeabilisation ofthe inner and outer membranes in E. coli (Lopez-Exposito et al. 2008a). This has been evidenced byrecording the translocation of nitrocefin (normallyprevented from crossing the OM due to its largesize) into the OM of E. coli in presence of thepeptide, as traced by the development of a red col-our upon cleavage of nitrocefin molecules by theb-lactamase normally located in the periplasmicspace. Similarly, the permeabilisation of the innermembrane was demonstrated by the alteration ofthe selectivity of a galactoside permease-deficientmutant of E. coli (ML-35p) to Ortho-nitrophenyl-b-galactoside (ONPG). The passive passage of theONPG across the cytoplasmic membrane, report-ing on the alteration of the selectivity of the mem-brane, was demonstrated by monitoring thehydrolysis of ONPG within the cytoplasm by aconstitutively produced b-galactosidase by themutant strain of E. coli. Hydrolysis of the reportermolecule (ONPG) upon incubation with E. coli inpresence of aS2-casein f(183-207) was revealed bythe appearance of a yellow colour due to the libera-tion of ONP. Furthermore, transmission electronmicroscopy (TEM) studies have shown that thetranslocation of the antimicrobial peptide acrossthe OM of E. coli and the peptidoglycan layer ofStaph. aureus occurs via the formation of pores inboth envelopes (Lopez-Exposito et al. 2008a).Destabilisation of the OM of Gram-negative bacte-ria by cationic peptides with subsequent pore for-mation as a result of displacement of the divalentcations (e.g. Ca++ and Mg++) that cement adjacentLPS molecules, has been reported earlier (Vaara1992; Brogden 2005). In the Gram-positive bacte-rium, hydrophobic interactions between the trypto-phan residues from the antimicrobial peptide andthe lipid-A from the peptidoglycan layer have beensuggested as a possible mechanism for the poreformation and permeabilisation of the cell wall(Farnaud et al. 2004). As for the ultimate killing



step of sensitive bacteria by the bovine aS2-caseinf(183-207), TEM studies showed that the peptideessentially acts by promoting the condensation ofthe cytoplasm in the Gram-negative bacterium (E.coli ML-35p) (Lopez-Exposito et al. 2008a), ashas been reported for anionic peptides (Brogden2005), while in the Gram-positive bacterium(Staph. carnosus CECT 4491T), it was suggestedto act by a different mechanism involving the dis-ruption of the plasma membrane with a consequentleakage 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 mainlyconsisted in amino-acid substitution experimentsand related observations to determine the role ofspecific amino acids in the overall antimicrobialactivity. Therefore, they did not provide a clearunderstanding of the precise mechanism of actionof the studied peptides. In this regard, several stud-ies have investigated the effect of hydrophilic-ity ⁄hydrophobicity on the antimicrobial activity ofmilk-derived peptides. For example, Pellegriniet al. (1999) have shown that substitution of Leu23

for the structurally similar but more hydrophobicIle in the sequence of the dimeric tryptic-digest ofa-LA [a-La f(17-31)S-S(109-114)] (Table 2) hasdrastically reduced the antimicrobial activity of thepeptide. Conversely, the substitution of theuncharged Asp98 residue for the positively chargedArg and addition of a lysyl residue at the C-termi-nus of the antimicrobial peptide VLVLDTDYKreleased by tryptic digestion of b-Lg (Table 2), in away to increase the overall hydrophilicity, hasextended the antimicrobial activity of the resultingpeptide (VLVLDTNYKK) to Gram-negativebacteria (Pellegrini et al. 2001). Also, kappacin Bthat differs from kappacin A by the substitution oftwo hydrophilic amino acid residues at positions136 and 148 (residues Thr136 and Asp148 inkappacin A) for hydrophobic residues (Ile136 andAla148 in kappacin B) has been shown to exertsignificantly reduced or no antibacterial activitycompared to kappacin A (Malkoski et al. 2001).Such studies concur to suggest that the antimicro-bial activity of the peptides is adversely affected byincreased hydrophobicity, and is stimulated by theincrease in hydrophilicity. However, such conclu-sion is not valid for all milk-derived antimicrobialpeptides, and, on the contrary, increased hydropho-bicity has been shown to enhance the antibacterialeffectiveness of some antimicrobial peptides(Lopez-Exposito et al. 2006a,b). In addition, themost hydrophilic caseicin C and the pentapeptide(EQLTK) released from aS2-casein and a-LArespectively (Table 4) were shown to have theweakest activity compared with the less hydro-philic or the hydrophobic antimicrobial peptidesreleased in the same conditions (Pellegrini et al.

1999; Hayes et al. 2006). Similar observation hasbeen made in the case of an antimicrobial peptideof bacterial origin by Lee et al. (2006) whoshowed that the activity of a native antimicrobialpeptide produced by Helicobacter pylori [i.e. f(2-20) derived from the N-terminus of ribosomal pro-tein L1] against bacteria and yeasts has been signif-icantly enhanced by increased hydrophobicity,while an opposite effect has resulted by increasingthe hydrophilicity. In fact, it has been consideredthat the net electric charge of antimicrobial pep-tides, especially the cationic ones, is the main fea-ture that determines the antimicrobial activity(Pellegrini et al. 1992; Bradshaw 2003). Therefore,the increase in the antimicrobial activity of the pep-tide VLVLDTNYKK obtained by substituting theuncharged Asp98 residue for the positively chargedArg and addition of a lysyl residue at the C-termi-nus of the b-Lg f(92-100) i.e., V92LVLDTDYK100,could be attributed to the increase in the net posi-tive charge rather than hydrophilicity. Also, Lopez-Exposito et al. (2006a) have explained theincreased antibacterial activity of bovine as2-caseinf(183-207) compared to its ovine homologuef(184-208) by the higher positive electric charge ofthe former (i.e. 5.1 vs 1), as these peptides differ infour amino acid residues (Table 4). The sameexplanation has been provided for the decrease inantimicrobial activity of the b-casein-derived pep-tides RNKKI and RINKK upon loss of their N-ter-minal arginyl and C-terminal lysyl residuesrespectively, with a consequent reduction in the netpositive charge (Lopez-Exposito et al. 2006b). Theimportance of these particular positively chargedresidues (i.e. lysine and arginine) to the antimicro-bial effect of cationic peptides has also been dem-onstrated in antibiotics (Hancock et al. 1995;Hancock and Lehrer 1998).Despite such conflicting considerations regard-

ing the means by which protein-derived peptidesexert their antimicrobial activity, there is a growingbody of evidence that the antimicrobial activity ofa peptide is related to an optimal balance betweenthe hydrophobic and hydrophilic residues so as toobtain an a-helix with an appropriate amphipathic-ity in the presence of target bacteria, rather than tothe average hydrophobicity or net electric charge.This is consistent with the studies that have demon-strated that the positioning of charged and hydro-phobic residues is crucial for peptides to exhibitantimicrobial activity, as they would influence thetertiary structure (Wieprecht et al. 1997; Dathe andWieprecht 1999). Furthermore, minor modifica-tions or substitutions in ⁄of amino acids in thesequence of a bioactive peptide that do not affectthe amphipathicity or the net electric charge, suchas the phosphorylation of specific amino acid orchemical modification of the C or N-termini, havebeen shown to affect dramatically the peptide’s



secondary and tertiary structures, and consequentlyits antimicrobial activity (Perez-Paya et al. 1994;Uteng et al. 2003; Dashper et al. 2007). Therefore,any changes leading to the formation of stableamphipathic a-helix appear to increase the activityof antibacterial peptides (Lee et al. 2006; Parket al. 2008), indicating that antimicrobial activityis primarily dependent on electrostatic interactionwith the cell-membrane as suggested earlier (Jens-sen et al. 2006). This is in agreement with theobservation that some antimicrobial peptideswould show more affinity to the polarised bacterialmembranes than to the zwitterionic membranes ofeukaryotic cells such as those of yeast and moulds(Zasloff 2002). It also explains why most of themilk-derived antimicrobial peptides are essentiallyactive against bacteria. However, other authorssuggest that antimicrobial peptides would also rec-ognise components of cell-envelopes of prokary-otic or eukaryotic organisms with which theyspecifically interact to exert their growth inhibitoryeffect (for review, see Epand and Vogel 1999). Forexample, the potency of the antimicrobial peptideproduced by H. pylori against bacteria and fungi(yeast and moulds) has been associated with thepeptide’s binding affinity to cell-wall components(Park et al. 2008). These authors have demon-strated that MICs of the peptide and modifiedderivatives towards sensitive micro-organisms cor-related well with their respective in vitro bindingaffinities to glycopeptides, chitin or LPS. Further-more, the peptide shown to be active against chi-tin-expressing Penicillium strains, failed to inhibitPhytophthora strains lacking chitin in their cellwall (Park et al. 2008). The role of the cell wall inthe antimicrobial activity of peptides is not wellunderstood and should be investigated in moredepth in the case of milk-derived and other antimi-crobial peptides, as it could profoundly change thecurrent understanding of the mechanism of actionof bioactive peptides in relation to micro-organismsas well as to host cells. It is worth mentioning thatsmall antimicrobial peptides with three to fiveamino acid residues have received the least atten-tion as regards the study of their mechanism ofaction, and would act by different mechanism(s) asyet to be elucidated (Lopez-Exposito et al. 2006b).

CONCLUS IONS

The high genetic variability in milk proteins amongspecies or within the same species suggests a greatpossibility to release bioactive peptides with vari-ous structures and functions. Although, only fewmilk-derived antimicrobial peptides are currentlyknown and characterised, this number is expectedto grow rapidly in the future due to renewed inter-est in their application in the food industry and inmedicine. Among the prominent advantages of

these peptides are their low likelihood to developresistant microbial strains in addition to their widespectrum of action, which in some cases includesbacteria (Gram-positive and Gram-negative),viruses, fungi and protozoans (Forssmann et al.2003). Milk proteins provide, therefore, an oppor-tunity to tailor specific peptides by using differentproteases or LAB, or their combinations; and thesepeptides can be used as food-grade additives in apurified form, or as milk-based hydrolysates orfermentates. The possibility for milk-derivedpeptides to have other functional properties besidethe antimicrobial activity is another advantage ofparamount importance to food industry as suchpeptides could be used to promote food safety andstability while insuring the functionality of foods,thereby increasing their added value in conformitywith the current consumers demand.It should be emphasised, however, that the

understanding of the exact mechanisms by whichmilk protein-derived antimicrobial peptides act onsensitive bacteria is important as it would allow tai-loring or designing new potent molecules withextended spectrum of action or enhanced antimi-crobial activities of known peptides. This aspecthas been poorly studied so far and warrants dueconsideration.

A C KNOWL EDG EMEN T S

A deep appreciation is expressed to Dr William E.Sandine, Emeritus Distinguished Professor of Micro-biology at Oregon State University for reviewing thepre-publication manuscript.

R E F E R E N C E S

Abd-El Salam M and Benkerroum N (2006) North African

cheeses. In Brined Cheeses, pp 139–187. Tamime A Y,

ed. London: Willey Blackwell Publishing.

Abraham A O, de Antoni G L and Anon M C (1993) Proteo-

lytic activity of Lactobacillus bulgaricus grown in milk.

Journal of Dairy Science 76 1498–1505.

Addeo F, Chianese L, Salzano A, Sacchi R, Cappuccio U,

Ferranti P and Malorni A (1992) Characterization of the

12% trichloro-acetic acid-insoluble oligopeptides of

Parmigiano-Reggiano cheese. Journal of Dairy Research

59 401–411.

Al-Zoreki N, Ayres J W and Sandine W E (1991) Antimicro-

bial activity of MicrogardTM against food spoilage and

pathogenic microorganisms. Journal of Dairy Science 74

758–763.

Asselin J, Hebert J and Amiot J (2006) Effects of in vitro

proteolysis on the allergenicity of major whey proteins.

Journal of Food Science 54 1037–1039.

Atlan D, Laloi P and Portalier R (1990) X-prolyl-dipeptidyl

aminopeptidase of Lactobacillus delbrueckii subsp.

bulgaricus: characterization of the enzyme and isolation

of deficient mutants. Applied and Environmental Micro-

biology 56 2174–2179.



Benkerroum N (2008) Antimicrobial activity of lysozyme

with special relevance to milk. African Journal of Biotech-

nology 7 4856–4867.

Benkerroum N and Sandine W E (1988) Inhibitory action of

nisin on Listeria monocytogenes. Journal of Dairy

Science 71 3237–3245.

Benkerroum N, Daoudi A and Kamal M (2003) Behavior of

Listeria monocytogenes in a raw sausage (merguez) in

presence of a bacteriocin-producing strain as a protective

culture.Meat Science 63 53–58.

Boman H G (1995) Peptide antibiotics and their role in innate

immunity. Annual Reviews of Immunology 13 61–92.

Bradshaw J (2003) Cationic antimicrobial peptides: issues for

potential clinical use. BioDrugs 17 233–240.

Brogden K A (2005) Antimicrobial peptides: pore formers or

metabolic inhibitors in bacteria? Nature Reviews in Micro-

biology 3 238–250.

Carlsson A, Nystrom T, de Cock H and Bennich H (1998)

Attacin – an insect immune protein – binds LPS and trig-

gers the specific inhibition of bacterial outer-membrane

protein synthesis.Microbiology 144 (Pt 8) 2179–2188.

Cheng J, Randall A, Sweredoski M and Baldi P (2005)

SCRATCH: a protein structure and structural feature

prediction server. Nucleic Acid Research 33 w72–w76.

Clare D A and Swaisgood H E (2000) Bioactive milk peptides:

a prospectus. Journal of Dairy Science 83 1187–1195.

Creamer L K and Harris D P (1997) Relationship between

milk protein polymorphism and physico-chemical proper-

ties: Milk Protein Polymorphism. Special Issue. Interna-

tional Dairy Federation 9702 110–123.

Dashper S G and Reynolds E G (2005) Combating dental

decay.Microbiology Australia 26 107–109.

Dashper S G, O’Brien-Simpson N M, Cross K J, Paolini R A,

Hoffmann B, Catmull D V, Malkoski M and Reynolds E C

(2005) Divalent metal cations increase the activity of the

antimicrobial peptide Kappacin. Antimicrobial Agents and

Chemotherapy 49 2322–2328.

Dashper S G, Liu S Wand Reynolds E C (2007) Antibacterial

peptides and their potential as oral therapeutic agents.

International Journal of Peptide Research and Therapeu-

tics 13 505–516.

Dathe M and Wieprecht T (1999) Structural features of helical

antimicrobial peptides: their potential to modulate activity

on model membranes and biological cells. Biochimica et

Biophysica Acta (BBA) – Biomembranes 1462 71–87.

Deutsch S-M, Molle D, Gagnaire V, Piot M, Atlan D and

Lortal S (2000) Hydrolysis of sequenced b-casein pep-

tides provides new insight into peptidase activity from

thermophilic lactic acid bacteria and highlights intrinsic

resistance of phosphopeptides. Applied and Environmen-

tal Microbiology 66 5360–5367.

Engstrom P, Carlsson A, Engstrom A, Tao Z J and Bennich H

(1984) The antibacterial effect of attacins from the silk

moth Hyalophora cecropia is directed against the outer

membrane of Escherichia coli. EMBO Journal 3

3347–3351.

Epand R M and Vogel H J (1999) Diversity of antimicrobial

peptides and their mechanisms of action. A review.

Biochimica et Biophysica Acta 1462 11–28.

Farnaud S, Spiller C, Moriarty L C, Patel A, Gant V, Odell E

W and Evans R W (2004) Interactions of lactoferricin-

derived peptides with LPS and antimicrobial activity.

FEMS (Federation of European Microbiological Socie-

ties) Microbiology Letters 233 193–199.

Fiat A M, Migliore-Samour D, Jolles P, Drouet L, Sollier C B

and Caen J (1993) Biologically active peptides from milk

with emphasis on two examples concerning antithrombot-

ic and immunomodulating activities. Journal of Dairy

Science 76 301–310.

Fira D, Kojic M, Banina A, Spasojevic I, Strahinic I and

Topisirovic L (2001) Characterization of cell envelope-

associated proteinases of thermophilic lactobacilli.

Journal of Applied Microbiology 90 123–130.

Fleming A (1922) On a remarkable bacteriolytic element

found in tissues and secretions. Proceedings of the Royal

Society of London. Series B 93 306–317.

Floris R, Recio I, Berkhout B and Visser S (2003)

Antibacterial and antiviral effects of milk proteins and

derivatives thereof. Current Pharmaceutical Design 9

1257–1275.

Forssmann W-G, Zucht H-D, Raida M, Adermann K and

Magert H-J (2001) Antibiotic peptides from bovine milk.

European Patent EP19960910013. 20 June 2001.

Forssmann W-G, Zucht H-D, Raida M, Adermann K and

Magert H-J (2003) Antibiotic peptides from bovine milk.

United States Patent 657984917. 17 June 2003.

Fritsche R, Adel-Patient K, Bernard H, Martin-Paschoud C,

Schwarz C, Ah-Leung S and Wal J-M (2005) IgE-

mediated rat mast cell triggering with tryptic and syn-

thetic peptides of bovine b-Lactoglobulin. InternationalArchives of Allergy and Immunology 138 291–297.

Gennaro R and Zanetti M (2000) Structural features and bio-

logical activities of the cathelicidin-derived antimicrobial

peptides. Biopolymers 55 31–49.

Ghalfi H, Allaoui A, Destain J, Benkerroum N and Thonart P

(2006a) Bacteriocin activity by Lactobacillus curvatus

CWBI-B28 to inactivate Listeria monocytogenes in Cold-

Smoked salmon during 4�C storage. Journal of Food

Protection 69 1066–1071.

Ghalfi H, Kouakou P, Duroy M, Daoudi A, Benkerroum N

and Thonart P (2006b) Application of an antilisterial

bacteriocin-producing strain of Lactobacillus curvatus

CWBI-B28 as a protective culture in bacon meat and

effect of fat and sodium nitrites on bacteriocin production

and activity. Food Science and Technology International

12 325–333.

Ghalfi H, Benkerroum N, Doguiet D D K, Bensaid M and

Thonart P (2007) Effectiveness of cell-adsorbed bacterio-

cin produced by Lactobacillus curvatus CWBI-B28 and

selected essential oils to control Listeria monocytogenes

in pork meat during cold storage. Letters in Applied

Microbiology 44 268–273.

Ghalfi H, Benkerroum N, Ongena M, Bensaid M and Thonart P

(2010) Production of three anti-listerial peptides by Lacto-

bacillus curvatus in MRS broth. Food Research Inter-

national 43 33–39.

Gobbetti M, Ferranti P, Smacchi E, Goffredi F and Addeo F

(2000) Production of angiotensin-I-converting-enzyme-

inhibitory peptides in fermented milks started by Lactoba-

cillus delbrueckii subsp. bulgaricus SS1 and Lactococcus

lactis subsp. cremoris FT4. Applied and Environmental

Microbiology 66 3898–3904.

Hancock R E and Lehrer R (1998) Cationic peptides: a new

source of antibiotics. Trends in Biotechnology 16 82–88.



Hancock R E W, Falla T and Brown M (1995) Cationic bacte-

ricidal peptides. Advances in Microbial Physiology 37

135–175.

Hayes M, Ross R P, Fitzgerald G F, Hill C and Stanton C

(2006) Casein-derived antimicrobial peptides generated

by Lactobacillus acidophilus DPC6026. Applied and

Environmental Microbiology 72 2260–2264.

Hill R D, Lahov E and Givol D (1974) A rennin-sensitive

bond in alpha-s1 B-casein. Journal of Dairy Research 41

147–153.

Hill C P, Yee J, Selsted M E and Eisenberg D (1991) Crystal

structure of defensin HNP-3, an amphipathic dimer:

mechanisms of membrane permeabilization. Science Asia

251 1482–1485.

Hurst A (1981) Nisin: a review. Advances in Applied Micro-

biololgy 27 85–119.

Ikeguchi M, Fujino M, Kato M, Kuwajima K and Sugai S

(1998) Transition state in the folding of {alpha}-lactalbu-

min probed by the 6-120 disulfide bond. Protein science 7

1564–1574.

Jenssen H, Hamill P and Hancock R E W (2006) Peptide

antimicrobial agents. Clinical Microbiology Reviews 19

491–511.

Jones F S and Simms H S (1930) The bacterial growth inhibi-

tor (lactenin) of milk. The Journal of Experimental Medi-

cine 51 327–339.

Kolb A F (2001) The prospects of modifying the antimicrobial

properties of milk. Biotechnology advances 19 299–316.

Lahov E and Regelson W (1996) Antibacterial and immuno-

stimulating casein-derived substances from milk: caseci-

din, isracidin peptides. Food and Chemical Toxicology 34

131–145.

Lahov E, Edelsten D, Sode-Mogensen M T and Sofer E

(1971) Properties of basic glycopeptides from cow milk

protein by heat.Milchwissenschaft 26 489–495.

Laloi P, Atlan D, Blanc B, Gilbert C and Portalier R (1991)

Cell-wall-associated proteinase of Lactobacillus del-

brueckii subsp. bulgaricus CNRZ 397: differential extrac-

tion, purification and properties the enzyme. Applied

Microbiology and Biotechnology 36 196–204.

Lee K H, Lee D G, Park Y, Kang D I, Shin S Y, Hahm K S

and Kim Y (2006) Interactions between the antimicrobial

peptide, HP (2-20) and its analogues derived from Heli-

cobacter pylori, and membrane. Biochemical Journal 394

105–114.

Liepke C, Zucht H D, Forssmann W G and Standker L (2001)

Purification of novel peptide antibiotics from human milk.

Journal of Chromatography B 752 369–377.

Lopez-Exposito I, Gomez-Ruiz J A, Amigo L and Recio I

(2006a) Identification of antibacterial peptides from ovine

as2-casein. International Dairy Journal 16 1072–1080.Lopez-Exposito I, Minervini F, Amigo L and Recio I (2006b)

Identification of antibacterial peptides from bovine kappa-

casein. Journal of Food Protection 69 2992–2997.

Lopez-Exposito I, Quiros A, Amigo L and Recio I (2007)

Casein hydrolysates as a source of antimicrobial, antioxi-

dant and antihypertensive peptides. Le Lait 87 241–249.

Lopez-Exposito I, Amigo L and Recio I (2008a) Identification

of the initial binding sites of alpha S2-casein f(183-207)

and effect on bacterial membranes and cell morphology.

Biochimica et Biophysica Acta (BBA)-Biomembranes

1778 2444–2449.

Lopez-Exposito I, Pellegrini A, Amigo L and Recio I (2008b)

Synergistic effect between different milk-derived peptides

and proteins. Journal of Dairy Science 91 2184–2189.

Malin E L, Alaimo M H, Brown E M, Aramini J M, Germann

M W, Farrell H M Jr, McSweeney P L and Fox P F

(2001) Solution structures of casein peptides: NMR,

FTIR, CD, and molecular modeling studies of alphas1-

casein, 1-23. Journal of Protein Chemistry 20 391–404.

Malkoski M, Dashper S G, O’brien-Simpson N M, Talbo G

H, Macris M, Cross K J and Reynolds E C (2001) Kappa-

cin, a novel antibacterial peptide from bovine milk. Anti-

microbial Agents and Chemotherapy 45 2309–2315.

Matar C, LeBlanc J G, Martin L and Perdigon G (2003)

Biologically active peptides released in fermented milk:

role and functions. In Handbook of Fermented Functional

Foods, pp 177–199. Farnworth E R, ed. Boca Raton:

CRC Press Inc.

McCann K B, Shiell B J, Michalski W P, Lee A, Wan J,

Roginski H and Coventry M J (2005) Isolation and char-

acterisation of antibacterial peptides derived from the

f(164-207) region of bovine as2-casein. International

Dairy Journal 15 133–143.

Meisel H (1998) Overview on milk protein-derived peptides.

International Dairy Journal 8 363–373.

Meisel H, Goepfert A and Gunther S (1997) ACE-inhibitory

activities in milk products.Milchwissenschaft 52 307–311.

Minervini F, Algaron F, Rizzello C G, Fox P F, Monnet Vand

Gobbetti M (2003a) Angiotensin I-converting-enzyme-

inhibitory and antibacterial peptides from Lactobacillus

helveticus PR4 proteinase-hydrolyzed caseins of milk

from six species. Applied and Environmental Microbio-

logy 69 5297–5305.

Minervini F, Algaron F, Rizzello C G, Ox P F F, Monnet V

and Gobbetti M (2003b) Angiotensin I-converting-

enzyme-inhibitory and antibacterial peptides from Lacto-

bacillus helveticus PR4 proteinase-hydrolyzed caseins of

milk from six species. Applied Environmental Microbiol-

ogy 6 5297–5305.

Natale M, Bisson C, Monti G, Peltran A, Garoffo L P, Valen-

tini S, Fabris C, Bertino E, Coscia A and Conti A (1989)

Cow’s milk allergens identification by two-dimensional

immunoblotting and mass spectrometry. Molecular Nutri-

tion and Food Research 48 363–369.

Nazarowec-White M and Farber J M (1997) Enterobacter

sakazakii: a review. International Journal of Food Micro-

biology 34 103–113.

Nikaido H, Nikaido K and Harayama S (1991) Identification

and characterization of porins in Pseudomonas aerugin-

osa. Journal of Biological Chemistry 266 770–779.

Park C B, Yi K S, Matsuzaki K, KimM S and Kim S C (2000)

Structure-activity analysis of buforin II, a histone H2A-

derived antimicrobial peptide: the proline hinge is responsi-

ble for the cell-penetrating ability of buforin II.Proceedings

of the National Academy of Sciences, USA 97 8245–8250.

Park S-C, Kim M-H, Hossain M A, Shin S Y, Kim Y, Stella

L, Wade J D, Park Y K and Hahm K-S (2008) Amphi-

pathic a-helical peptide, HP (2-20), and its analogues

derived from Helicobacter pylori: pore formation mecha-

nism in various lipid compositions. Biochemica et Biophy-

sica Acta 1778 229–241.

Pellegrini A (2003) Antimicrobial peptides from food

proteins. Current Pharmaceutical Design 9 1225–1238.



Pellegrini A, Thomas U, von Fellenberg R and Wild P (1992)

Bactericidal activities of lysozyme and aprotinin against

gram-negative and gram-positive bacteria related to their

basic character. Journal of Applied Bacteriology 72

180–187.

Pellegrini A, Thomas U, Bramaz N, Hunziker P and von

Fellenberg R (1999) Isolation and identification of three

bactericidal domains in the bovine a-lactalbumin mole-

cule. Biochimie et Biophisica Acta (BBA) 1426 439–448.

Pellegrini A, Dettling C, Thomas U and Hunziker P (2001)

Isolation and characterization of four bactericidal domains

in the bovine b-lactoglobulin. Biochimica et Biophysica

Acta 1526 131–140.

Perez-Paya E, Houghten R A and Blondelle S E (1994) Deter-

mination of the secondary structure of selected melittin

analogues with different haemolytic activities. Biochemi-

cal Journal 299 (Pt 2) 587–591.

Piers K L, Brown M H and Hancock R E (1994) Improve-

ment of outer membrane-permeabilizing and lipopolysac-

charide-binding activities of an antimicrobial cationic

peptide by C-terminal modification. Antimicrobial Agents

Chemotherapy 38 2311–2316.

Pihlanto-Leppala A, Marnila P, Hubert L, Rokka T, Korhonen

H J T and Karp M (1999) The effect of a-lactalbumin and

b-lactoglobulin hydrolysates on the metabolic activity of

Escherichia coli JM103. Journal of Applied Microbiology

87 540–545.

Quiros A, Ramos M, Muguerza B, Delgado M A, Miguel M,

Aleixandre A and Recio I (2007) Identification of novel

antihypertensive peptides in milk fermented with Entero-

coccus faecalis. International Dairy Journal 17 33–41.

Recio I and Visser S (1999) Identification of two distinct anti-

bacterial domains within the sequence of bovine alpha s2-

casein. Biochimica et Biophysica Acta 1428 314–326.

Reddy K V R, Yedery R D and Aranha C (2004) Antimicro-

bial peptides: premises and promises. International Jour-

nal of Antimicrobial Agents 24 536–547.

Reid J and Coolbear T (1998) Altered specificity of lactococcal

proteinase PI (Lactocepin I) in humectant systems reflect-

ing the water activity and salt content of cheddar cheese.

Applied Environmental Microbiology 64 588–593.

Rizzello C G, Losito I, Gobbetti M, Carbonara T, De Bari M

D and Zambonin P G (2005) Antibacterial activities of

peptides from the water-soluble extracts of Italian cheese

varieties. Journal of Dairy Science 88 2348–2360.

Romeo D, Skerlavaj B, Bolognesi M and Gennaro R (1988)

Structure and bactericidal activity of an antibiotic dodeca-

peptide purified from bovine neutrophils. Journal of Bio-

logical Chemistry 263 9573–9575.

Ross P, Stanton C, Hill C and Fitzgerald G (2007) Casein-

derived antimicrobial peptides and Lactobacillus strains

that produce them. Patent PCT ⁄ IE2006 ⁄ 000130. 24 May

2007.

Rydengard V, Shannon O, Lundqvist K, Kacprzyk L, Chal-

upka A, Morgelin M, Jahnen-Dechent W, Malmsten M,

Schmidtchen A and Olsson A-K (2008) Histidine-rich

glycoprotein protects from systemic Candida infection.

PLoS Pathogens 4 e1000116 doi:1000110.1001371/jour-

nal.ppat.1000116.

Saito T and Itoh T (1992) Variations and distributions of

O-glycosidically linked sugar chains in bovine-casein.

Journal of Dairy Science 75 1768–1774.

Salton M R J (1957) The properties of lysozyme and its action

on microorganisms. Bacteriological Reviews 21 82–100.

Sasaki M, Bosman B W and Tan P S T (1995) Comparison of

proteolytic activities in various lactobacilli. Journal of

Dairy Research 62 601–610.

Selo I, Clement G, Bernard H, Chatel J, Creminon C, Peltre G

and Wal J (1999) Allergy to bovine b-lactoglobulin: speci-ficity of human IgE to tryptic peptides. Clinical Experi-

mental Allergy 29 1055–1063.

Shaha N P (2000) Effects of milk-derived bioactives: an over-

view. British Journal of Nutrition 84 3–10.

Simmaco M, Mignogna G and Barra D (1998) Antimicrobial

peptides from amphibian skin: what do they tell us?

Biopolymers 47 435–450.

Talbo G H, Suckau D, Malkoski M and Reynolds E C (2001)

MALDI-PSD MS analysis of the phosphorylation sites of

caseinomacropeptide. Peptides 22 1093–1098.

Tomita M, Takase M, Bellamy W and Shimamura S (1994) A

review: the active peptide of lactoferrin. Acta paediatrica

Japonica 36 585–591.

Tomita M, Wakabayashi H, Yamauchi K, Teraguchi S and Ha-

yasawa H (2002) Bovine lactoferrin and lactoferricin

derived from milk: production and applications. Biochem-

istry and Cell Biology 80 109–112.

Touch V, Hayakawa S and Saitoh K (2004) Relationships

between conformational changes and antimicrobial activ-

ity of lysozyme upon reduction of its disulfide bonds.

Food Chemistry 84 421–428.

Ulvatne H, Samuelsen O, Haukland H H, Kramer M and

Vorland L H (2004) Lactoferricin B inhibits bacterial mac-

romolecular synthesis in Escherichia coli and Bacillus

subtilis. FEMS Microbiology Letters 237 377–384.

Uteng M, Hauge H H, Markwick P R, Fimland G, Mantzilas

D, Nissen-Meyer J and Muhle-Goll C (2003) Three-

dimensional structure in lipid micelles of the pediocin-like

antimicrobial peptide sakacin P and a sakacin P variant

that is structurally stabilized by an inserted C-terminal

disulfide bridge. Biochemistry 42 11417–114126.

Vaara M (1992) Agents that increase the permeability of the

outer membrane.Microbiological Reviews 56 395–411.

Vorland L H, Ulvatne H, Rekdal O and Svendsen J S (1999)

Initial binding sites of antimicrobial peptides in Staphylo-

coccus aureus and Escherichia coli. Scandinavian journal

of infectious diseases 31 467–473.

Wang Z and Wang G (2004) APD: the antimicrobial database.

Nucleic Acids Research 32 D590–D592.

Wieprecht T, Dathe M, Epand R M, Beyermann M, Krause E,

Maloy W L, MacDonald D L and Bienert M (1997) Influ-

ence of the angle subtended by the positively charged

helix face on the membrane activity of amphipathic, anti-

bacterial peptides. Biochemistry 36 12869–12880.

Wu T, Samaranayake L P, Leung W K and Sullivan P A

(1999) Inhibition of growth and secreted aspartyl protein-

ase production in Candida albicans by lysozyme. Journal

of Medical Microbiology 48 721–730.

Zasloff M (2002) Antimicrobial peptides of multicellular

organisms. Nature 415 389–395.

Zucht H D, Raida M, Adermann K, Magert H J and Forss-

mann W G (1995) Casocidin-I: A casein-alpha s2 derived

peptide exhibits antibacterial activity. FEBS Letters 372

185–188.



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