Bioactive Peptide Functions

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Biotechnol. J. 2007, 2, 435–449 DOI 10.1002/biot.200700045 www.biotechnology-journal.com

1 Introduction

Bioactive peptides are described as ‘food-derived compo-nents (genuine or generated) that in addition to their nu-tritional value, exert a physiological effect in the body’ [1].In 1950, Mellander [2] first described bioactive peptideswhen he reported that ingestion of casein-derived phos-phorylated peptides led to enhanced vitamin D-inde-pendent calcification in rachitic infants. Since then, fun-damental studies have opened a new field of research re-lated to the generation of bioactive peptides from a vari-ety of food proteins, with milk proteins currently being theprimary source. Bioactive peptides can be latent or en-

crypted within the primary or parent proteins where pro-teolysis is required for their release and activation to ex-ert a physiological response [3] on the various systems ofthe body. Some peptides also act as biocarriers by se-questering calcium and other minerals, thereby enhan-cing bioavailability [4].

In Part I of this review, we gave an overview on the re-lease of encrypted bioactive peptides from a range of foodprotein sources, as well as the use of lactic acid bacteria(LAB) as cell factories for the de novo generation of bioac-tivities. Here we relate in more detail some examples ofbioactive peptide functions.

2 Bioactivities of released peptides

2.1 Role of angiotensin-converting enzyme inhibitors in thecontrol of blood pressure

Hypertension is defined as a sustained increase in bloodpressure (BP) and is a controllable risk factor in the devel-opment of a number of cardiovascular diseases such asstroke and coronary infarction. Even small decreases in

Review

Putting microbes to work: Dairy fermentation, cell factoriesand bioactive peptides. Part II: Bioactive peptide functions

Maria Hayes1, 2, Catherine Stanton1, 3, Gerald F. Fitzgerald2, 3 and R. Paul Ross1, 3

1Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland2Department of Microbiology, University College, Cork, Ireland3Alimentary Pharmabiotic Centre, Cork, Ireland

A variety of milk-derived biologically active peptides have been shown to exert both functional andphysiological roles in vitro and in vivo, and because of this are of particular interest for food scienceand nutrition applications. Biological activities associated with such peptides include im-munomodulatory, antibacterial, anti-hypertensive and opioid-like properties. Milk proteins are rec-ognized as a primary source of bioactive peptides, which can be encrypted within the amino acidsequence of dairy proteins, requiring proteolysis for release and activation. Fermentation of milkproteins using the proteolytic systems of lactic acid bacteria is an attractive approach for genera-tion of functional foods enriched in bioactive peptides given the low cost and positive nutritionalimage associated with fermented milk drinks and yoghurt. In Part II of this review, we focus on ex-amples of milk-derived bioactive peptides and their associated health benefits, to illustrate thepotential of this area for the design and improvement of future functional foods.

Keywords: Casein · Whey · Proteolysis · Lactobacilli · Bioactive peptides

Correspondence: Professor R. Paul Ross, Teagasc, Moorepark FoodResearch Centre, Fermoy, Co. Cork, IrelandE-mail: [email protected]: +353-2542229

Abbreviations: ACE, angiotensin-1-converting enzyme; BP, blood pressure;CPPs, caseinophosphopeptides; HHL, hippurly-histidyl-leucine; LAB, lacticacid bacteria; RAS, renin angiotensin system; SHR, spontaneously hyper-tensive rat; UHT, ultra high temperature

Received 12 December 2006Revised 7 March 2007Accepted 7 March 2007

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BiotechnologyJournal Biotechnol. J. 2007, 2, 435–449

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BP result in a significantly reduced risk of cardiovasculardisease, and a 5-mm Hg reduction in diastolic BP reducesthe risk of heart disease by approximately 16% in hyper-tensive subjects [5].

Classically, the control of BP has been associated withthe renin angiotensin system (RAS) (Fig. 1), while otherregulators of BP include the neutral endopeptidase sys-tem, the endothelin-converting enzyme system and thekinin-nitric oxide system [5]. Central to RAS is an ex-opeptidase, angiotensin-converting enzyme (ACE), dis-covered by Skeggs et al. [6], which is responsible for theconversion of angiotensin I, a decapeptide generated bythe action of rennin on the glycoprotein substrate knownas angiotensinogen, to the vasoconstrictor octapeptideangiotensin II. There are two isoforms of human ACE, so-matic (sACE) and germinal/testicular (gACE) forms [7],encoded by a single gene located on chromosome 17 atq23. This gene is 21 kb in length and contains 26 exonsand 25 introns [8]. Human sACE is a type-I membrane-bound protein that consists of a 28-residue C-terminal cy-tosolic domain, a 22-residue hydrophobic transmembranedomain and a 1227-residue extracellular domain that is

heavily glycosylated and further divided into a 612-residue N-terminal domain, linked by a 15-residue se-quence to a 600-residue C-terminal domain [8]. The ex-tracellular C-terminal domain and N-terminal domaincontain a HEXXH sequence, which serves as the zinc-binding ligands [8]. In sACE, the C-terminal domain is pri-marily involved in BP regulation, while the N-terminal do-main is involved in the control of hematopoietic stem celldifferentiation and proliferation [9–11]. Human gACE cor-responds to the C-terminal domain of sACE [8, 12].

ACE inhibitors are thought to be competitive sub-strates for ACE. The C-terminal tripeptide sequence ofthe milk-derived ACE-inhibitor is the primary structuralfeature governing this inhibitory response as ACE ap-pears to prefer substrates and inhibitors containing hy-drophobic amino acid residues in the three C-terminal po-sitions [13]. Generally, aliphatic, basic and aromaticresidues are preferred in the penultimate positions, whilearomatic, proline (Pro) and aliphatic residues are preferredin ultimate positions. The positive charge of Arg at the Cterminus has also been shown to contribute to the ACE-I-inhibitory potential of several peptides [1]. Also, a C-ter-

Figure 1. The rennin-angiotensin and kallikrein-kinin nitric oxide system. ACE (angiotensin-1-coverting enzyme EC 3.4.15.1) is a zinc metallopeptidase ex-opeptidase which cleaves from the C-terminal of various peptide substrates. In the renin angiotensin system (RAS), considered the major regulator ofblood pressure (BP), ACE removes the C-terminal tripeptide His-Leu from angiotensin I resulting in the formation of angiotensin II, a potent octapeptidevasoconstrictor. ACE also removes the C-terminal dipeptide from the vasodilator nonapeptide bradykinin resulting in the formation of inactive fragments.The catalyses of these two reactions by ACE allows for the regulation of peripheral BP. In addition, in response to stimulation by angiotensin II, endotheli-um I, a potent vasoconstrictor is formed from Big Endothelium by the action of the enzyme ECE (EC 3.4.24.71). Endothelium I mediates vasoconstrictionvia two receptors, which are both present in various tissues of the body. Endothelium I is also involved in sodium re-absorption in the nephron. Nitric ox-ide can inhibit the release of ECE. In addition, the kinin-nitric oxide system – a vasodilatory pathway-can be inhibited by the action of ACE on bradykinin.ACE may also affect the immune system by inactivation of bradykinin, which is known to be involved in lymphocyte migration, lymphokinin migration andmacrophage stimulation. Milk protein-derived inhibitors of ACE may therefore prevent vasoconstriction and lower BP. In addition, inhibitors of ACE may beinvolved in the endocrine and immune systems.



minal Lys with a positive charge on the ε-amino groupcontributes substantially to the ACE-inhibitory potential[13, 14].

The consumption of fermented milk to maintain goodhealth, including the regulation of BP, is a long tradition inseveral areas of the world (e.g., Japan, East Asia andFrance) [15]. ACE-inhibitory and anti-hypertensive (hy-potensive) peptides from dairy origin have been knownfor several years and usually contain up to 10 amino acids[16–18]. Strain selection is one of the main factors that in-fluences the release of ACE inhibitors in dairy fermenta-tions [18, 19]. Milk fermentations using LAB or their pro-teinases, have previously been described as a strategy torelease ACE-inhibitory peptides from milk proteins, espe-cially caseins, and ACE-inhibitory peptides have beenisolated from many fermented milks and dairy commer-cial products (Table 1).

The majority of milk protein-derived ACE inhibitorshave moderate inhibitory potencies, usually within anIC50 range of 100–500 µmol/L; however, there are excep-tions such as the casokinins αs1-CN f (23-27) and β-caseinf (177-183) with IC50 values less than 20 µmol/L [14]. Twopotent ACE-inhibitory peptides Val-Pro-Pro [β-casein f(84-86)] and Ile-Pro-Pro [β-casein f (74-76)] derived fromcasein, were isolated from milk fermented with Lb. hel-veticus CP790 [20] and sour milk fermented with Lb. hel-veticus and Saccharomyces cerevisiae [21]. The latter twostrains are used in the fermentation of Calpis® sour milk(Calpis Co. Ltd., Tokyo, Japan), which is widely consumedin Japan for its anti-hypertensive properties. Lb. helveti-cus CHCC637 and Lb. helveticus CHCC641 were used toproduce fermented milk rich in ACE inhibitors, which has

been shown clinically to cause a significant decrease inBP in spontaneously hypertensive rats (SHR) [22].

It has been reported that milk fermented with LABspecies besides Lb. helveticus strains did not display anyanti-hypertensive properties upon oral administration inSHR [1, 23]. Philanto-Leppälä et al. [24] reported that ca-sein and whey fermentates produced using LAB startersisolated from ‘ropy milk’, yoghurt and soured milk werepoor sources of ACE-inhibitory peptides prior to digestionwith pepsin and trypsin. Other studies have demonstrat-ed ACE-inhibitory peptides following fermentation withdifferent LAB species including Lb. delbrueckii subsp.bulgaricus SS1, Lc. lactis subsp. cremoris FT4, Lb. aci-dophilus, bifidobacteria, and Streptococcus thermophilus[25–27]. Ultra-high temperature (UHT)-treated milk fer-mented with the probiotic Lb. rhamnosus GG and subse-quently digested with the enzymes pepsin and trypsinwas found to contain ACE-inhibitory activities, and theACE-inhibitory peptides corresponding to Ala-Val-Pro-Tyr-Pro-Gln-Arg and Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Arg, which originated from β-casein, were identified.Several opioid peptides were also identified and milkfermented by Lb. rhamnosus GG proved beneficial inseveral clinical studies when tested for the treatment ofgastrointestinal infections such as diarrhea and rotavirusinfection [28, 29].

The presence of ACE inhibitors has also been report-ed in several cheeses including Parmigiano-Reggiano[29], Gouda [25, 30], Cheddar [31] and ripened or cookedcheeses such as Comté [25, 32]. Anti-hypertensive pep-tides corresponding to αs1-casein f (1-22) and αs1-casein f(23-38) found in 6-month-old Parmigiano-Reggiano

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Table 1. Summary of ACE-inhibitory activities of milk protein fermented with Lactic Acid Bacteria

Microorganism Substrate Activity Reference

Lactobacillus helveticus Milk ACE inhibitory [38]Saccharomyces cerevisiae Milk ACE inhibitory [38]Lactobacillus helveticus Whey protein ACE inhibitory [5, 16, 48]Saccharomyces cerevisiae Whey protein ACE inhibitory [5, 16, 48]Lactobacillus helveticus LBK 16H Milk ACE inhibitory [40]Lactobacillus helveticus CPN 4 Milk ACE inhibitory [56, 22]Lactobacillus helveticus CH CC637 Milk ACE inhibitory [56]Lactobacillus helveticus R389 Milk ACE inhibitory [56]Lactobacillus delbrueckii subsp. bulgaricus SS1 Milk ACE inhibitory [56]Lactococcus lactis ssp. cremoris FT4 Milk ACE inhibitory [56]Lactobacillus helveticus CP 790 proteinase B-casein ACE inhibitory [24]Lactobacillus helveticus CM4 endopeptidase VPPL ACE inhibitory [23]Lactobacillus helveticus JCM 1004 cell-free extract Skim milk ACE inhibitory [22, 24]Lactobacillus helveticus R211 and R389 Casein enriched milk ACE inhibitory [22]Lactobacillus delbrueckii ssp. bulgaricus Milk ACE inhibitory [22]Streptococcus salivarius ssp. thermophilus Milk ACE inhibitory [22, 37]Lactococcus lactis biovar. diacetylactis Milk ACE inhibitory [37]Commercial dairy starters from yoghurt and ‘ropy’ milk treated with enzymes Whey, casein ACE inhibitory [19]Aspergillus oryzae protease Skim Milk ACE inhibitory [41]




cheese had disappeared following 15 months of ripening,suggesting that the appearance and subsequent disap-pearance of ACE-inhibitory peptides is influenced by thedegree of proteolysis and ripening [33]. The presence ofseveral ACE-inhibitory peptides in several ripenedcheeses was reported, with the most potent inhibitory ac-tivities detected in short to medium-ripened cheesessuch as Gouda [30]. The ACE-inhibitory peptidesFFVAPFPEVFGK, corresponding to αs1-casein f (23-34),and FFVAP corresponding to αs1-casein f (23-27) previ-ously shown to be ACE inhibitors by Maruyama et al. [34],were identified in Crescenza cheese [26]. Enzyme-modi-fied cheese obtained by hydrolysis of pasteurized cheesehomogenized with Neutrase® and an enzyme preparationfrom Lb. casei, treated for 72 h was also found to containACE inhibitors with the amino acid sequences LTLTDVEcorresponding to β-casein f (125-131), YPQRDMPIQ cor-responding to β-casein f (180-197), PGPIP correspondingto β-casein f (63-67) and PKHKEMPFPPKYPVEPFT corre-sponding to β-casein f (104-120) [35]. Moreover, the anti-hypertensive peptides β-casein f (193-202), β-lactoglobu-lin f (147-148), lactoferrin f (288-289) and lactoferrin f (319-320) were isolated from yoghurt, mature and vintageCheddar and Feta cheese, respectively [36]. Additionally,the low-fat cheese “Festivo”, made using Lb. acidophilusand Bifidobacterium spp. with traditional starter cultureswas also found to contain ACE inhibitors with potentialanti-hypertensive effects after maturation for 13 weeks.However, ACE-inhibitory activity decreased when prote-olysis exceeded a certain level during storage for 20weeks [28].

To exert an anti-hypertensive effect, the bioavailabili-ty of ACE inhibitors is of paramount importance. ACE-inhibitory activity of some peptides may be deactivat-ed by their susceptibility to degradation by gastrointesti-nal, brush border, serum and blood proteinases and pep-tidases on route to the target organ(s). However,oligopeptide sequences containing encrypted bioactivepeptides may also be activated and the potency of theseoligopeptides increased, due to in vivo proteinase andpeptidase activities. The presence of several previouslyidentified ACE-inhibitors in two Spanish commerciallyavailable fermented milks following simulated gastroin-testinal digestion has been reported [37]. Peptides corres-ponding to β-casein f (202-209) and β-casein f (203-209)with the amino acid sequences GPFPIIV and RGPFPIIV,respectively, are fragments of the ACE-inhibitory peptideLLYQQPVLGPVRGPFPIIV identified previously [23].

Using the SHR model, the anti-hypertensive effect ofCalpis® was demonstrated, which yielded a systolic BPdecrease of 17.7 mm Hg following administration of5 mL/kg body weight of Calpis© sour milk drink over an8-h period [38]. The anti-hypertensive effect of a ferment-ed sour milk drink was demonstrated in human clinicaltrials, where a significant reduction in BP was obtained inmildly hypertensive patients, following oral consumption

of 95 mL Calpis® over an 8-week period [39]. Lb. helveti-cus LBK-16H-fermented milk containing the bioactivepeptides Val-Pro-Pro and Ile-Pro-Pro taken daily (150 mLLBK-16H-fermented milk/day) for 21 weeks, after a2-week run-in period, also resulted in a BP-lowering effectin hypertensive subjects [40]. More recently, Mizuno et al.[41] demonstrated the anti-hypertensive properties of acasein hydrolysate containing the ACE inhibitors Val-Pro-Pro and Ile-Pro-Pro, prepared using an Aspergillus oryzaeprotease, in a randomized, double-blind placebo-con-trolled study. The effect of intake of the casein hydrolysatewas evaluated over a 12-week period on the BP of 144 sub-jects with either high-normal BP (n=104) or mild-hyper-tension (n=40) [41]. Additionally, the whey protein hy-drolysate Biozate® 1, containing a range of β-lactoglobu-lin ACE-inhibitory peptides [42], resulted in a significantreduction in BP following oral ingestion of 20 g/day by30 borderline hypertensive human male and female sub-jects over a 6-week period [43]. A summary of ACE-in-hibitory peptides derived from fermentation using LAB ortheir proteases is provided (Table 1).

One assay for measurement of ACE activity in vitromeasures the rate of release of hippurate (product) fromhippurly-histidyl-leucine (HHL) (substrate) following in-cubation of the ACE enzyme with substrate, in the pres-ence of the potential ACE-inhibitory peptide [44–47]. Theanti-hypertensive effect of ACE-inhibitory peptides maybe measured in vivo by direct measurement of BP via ar-terial cannulation or using tail cuffs in SHR following in-gestion of the ACE-inhibitory peptides [48, 49].

2.2 Antimicrobial peptides

Milk represents a successful paradigm for preventing andlimiting microbial infection, and understanding the mole-cules that participate in this protective process may po-tentially lead to novel antimicrobial therapies [3]. The col-lective antibacterial effect in milk due to the synergisticactivity of naturally occurring peptides and defense pro-teins besides immunoglobulins, such as lactoferrin, lac-toperoxidase and lysozyme is greater than any individualcontribution [50]. These defense proteins can exert an-timicrobial activities comparable to antibiotics, andtherefore have potential as natural alternatives [51]. Forexample, bovine lactoferrin has shown strong antiviral ac-tivity against HIV and the human cytomegalovirus(HCMV), which is thought to act synergistically with HIVin patients with the acquired immunodeficiency syn-drome [52]. In addition to these defense proteins, en-crypted antimicrobial peptides within the parent protein,which may be released by enzymatic hydrolysis duringdigestion, may also contribute to the antimicrobial activ-ity of milk [50].

The first antimicrobial peptide isolated from milk,termed lactenin, was identified by Simmes and Jones in1930 [53], and resulted from the treatment of milk with



rennet. This peptide exhibited antimicrobial activityagainst pathogenic strains of streptococci. Subsequently,a group of basic, glycosylated and high-molecular weight(5-kDa) milk-derived polypeptides called casecidins wereidentified following chymosin treatment of casein [54].These exhibited antimicrobial activity against pathogen-ic Staphylococcus aureus and several lactobacilli [54, 55].Subsequently, the antimicrobial peptide termed isracidincorresponding to αs1-casein f (1-23) with a correspondingsequence 1RPKHPIKHQGLPQEVLNENLLRF23 was de-rived following treatment of αs1-casein with chymosin[50, 54]. Isracidin inhibited the growth of S. aureus andListeria monocytogenes, the principal agents of mastitisinfections and protected against such infection in vivo atconcentrations comparable with antibiotic use [54].

Most antimicrobial peptides reported to date havebeen released from the parent milk protein followingheat and/or alkali treatment or enzymatic hydrolysis.Furthermore, a number of studies have reported the gen-eration of antimicrobial peptides following dairy fermen-tations. Recently, an antimicrobial peptide correspondingto β-CN f (184-210) with the amino acid sequence

184QELLLNPTHQYPVTQPLAPVHNPISV210, was producedby hydrolysis of human sodium caseinate with a partiallypurified proteinase of Lb. helveticus PR4 [56]. This pep-tide exhibited a broad spectrum of inhibition against po-tentially pathogenic bacteria such as S. aureus, Enter-obacter faecium, Yersinia enterocolitica and Salmonellaspecies [56]. Antibacterial activity was found in two wa-ter-soluble extracts of nine Italian cheese varieties thatdiffered in the types of starter strains and biotechnologi-cal traits used [57]. Peptides with high levels of homologywith N-terminal, C-terminal or whole fragments of knownantimicrobial peptides were identified within these ex-tracts. Water-soluble extracts derived from Caviocavallocheese contained RPKHPIK corresponding to αs1-caseinf (1-7) and GLPQE corresponding to αs1-casein f (10-14),which share homology with the intermediate fragmentsof isracidin [54, 57]. In another study, Dionysius et al. [54]reported on the isolation of the microbicidal peptide cor-responding to αs1-casein f (1-9) with the sequence RPKH-PIKHQ in a yoghurt product. Other studies have identifiedthe presence of whey protein-derived antimicrobial pep-tides, which were released following enzymatic cleavagewith proteolytic enzymes and dairy fermentations withproteolytic LAB. Examples of antimicrobial peptides de-rived from whey include bovine and human lactoferricin,corresponding to bovine lactoferrin f (17-41) and humanlactoferrin f (1-47), respectively, released following enzy-matic digestion of bovine and human lactoferrin withtrypsin and pepsin [52]. Both bovine and human lacto-ferricin display antimicrobial activity against a broad-spectrum of gram-positive and gram-negative bacteria,including L. monocytogenes [50, 52].

In addition, two shorter peptide derivatives of lacto-ferricin B f (17-41), termed D-lactoferricin B and L-lacto-

ferricin B corresponding to f (17-31) showed a post-an-tibiotic effect (defined as an arrested bacterial growth fol-lowing the removal of the active antibacterial agent)against E. coli [58]. Several peptides with bacteriocidalactivity have also been obtained by enzymatic digestionof α-lactalbumin and β-lactoglobulin [26, 50, 52, 59]. An-tibacterial peptides derived from milk proteins arethought to have membrane-lytic activity with specifici-ties for prokaryotic cell membranes [54, 59, 60]. The gen-erally accepted theory concerning the mechanism of ac-tion is the overwhelming disruption of microbial mem-branes, leading to ion and metabolite leakage, depolariza-tion, disruption of membrane coupled respiration andultimately cell death [61]. The mechanism of action in-volved in antibacterial peptide-mediated rupturingevents remains unclear. However, three models of mem-brane rupturing have been proposed: the barrel-stave,carpet and toroidal models [62] as shown in Fig. 2. Ac-cording to the barrel-stave model, antimicrobial peptidesbind to the cell membrane, bound peptides recognizeeach other and oligomerize and subsequently thisoligomer of peptides inserts into the hydrophobic coreforming a transmembrane pore [62]. The carpet modelsuggests that antimicrobial peptides bind to and coverthe surface of the target membrane, and electrostatic in-teractions between the peptide and the lipid head groupcauses membrane permeation [63]. In the toroidal model,peptides bind and interact with lipid head groups and im-pose a positive curvature force on the membranes, pro-ducing channels where the polar head group expands andforms “toroidal” pores [64]. Membrane-dependentprocesses such as translocation of cytotoxic peptidesacross the membrane to the cytoplasm, and transbilayerlipid diffusion are thought to be involved in the mecha-nism of action of some antimicrobial peptides [65]. An-timicrobial peptides have a broad spectrum of activityand additionally have the ability to distinguish betweenprokaryotic and eukaryotic cells based on the differencesin the outer leaflet of mammalian and bacterial cell mem-branes. Mammalian cell membranes consist of phos-phatidylcholine (PC), sphingomyelin and cholesterol,which are of neutral charge at physiological pH, whereasgram-positive bacterial cell membranes contain nega-tively charged phospholipids such as phosphatidylgly-cerol and cardiolipin [3, 56, 62, 66]. In addition to thecytoplasmic membrane, gram-negative bacteria containan outer membrane that consists in its outer leaflet mostlyof the polyanionic molecule lipopolysaccharide [62].

2.3 Anti-thrombotic peptides

Thrombosis may be defined as the pathological conditionin which improper activity of the hemostatic mechanismresults in clot or thrombus formation in arteries, veins orthe chambers of the heart. Both environmental factors andgenetic variations in elements of the clotting cascade in-





fluence thrombosis and consequently, atherosclerosis[67]. Three types of thrombi are recognized: the whitethrombus, seen in arteries and which consists mainly ofplatelets; the red thrombus, which is composed mainly offibrin and red cells; and the mixed thrombus, which con-sists of both white and red thrombi. During thrombus for-mation, circulating prothrombin is activated by platelets.In this process, other major steps take place such as theconversion of fibrinogen to fibrin, which then creates thefibrin matrix. All this takes place while platelets are beingadhered and aggregated. In the mid-nineteenth century,Rudolf Ludwig Karl Virchow first described the phenom-ena he called “embolism” and “thrombosis”, and outlinedthree main factors that contribute to venous thrombosis,which are now known as the Virchow triad, i.e., ‘abnormalvessel wall’, ‘abnormal blood flow’ and ‘abnormal bloodconstituents’. Thrombus composition is influenced by thevelocity of blood flow at the site of formation. Whiteplatelet-rich thrombi are found in high flow systems,while red thrombi form in regions of stasis. The high-shear rate in arteries prevents the accumulation of coag-ulation intermediates, therefore only platelets have thecapacity to form thrombi to the area of damage via vonWillebrand factor [68]. On the venous side of circulation,the thrombus consists of fibrin: thrombin can accumulatedue to the slower flow rate and platelets play only a minorrole. Arterial thrombosis is seen predominantly as my-ocardial infarction (heart attack, caused by thrombosis ina coronary artery), cerebrovascular arterial thrombosis(stroke, caused by thrombosis in a coronary artery), pe-ripheral arterial thrombosis and ischemic stroke and is al-most invariably associated with existing vessel wall dis-ease, i.e., atherosclerosis. The most common forms of ve-nous thrombosis are deep vein thrombosis of the leg andpulmonary embolism, with causes of venous thrombosisdivided into those that are characterized by stasis andthose reflecting abnormalities in blood plasma (hyperco-agulability). Inhibitors of platelet aggregation are com-monly used in the management of thrombosis to controlfurther thrombus development.

Peptides have been identified in blood and in dietaryproteins that interfere with the formation of thrombi. Forexample, it was hypothesized that fibrinogen γ-chain andκ-casein may have evolved from a common ancestorduring the past 450 million years [69]. In addition, milkprotein-derived peptides have been described as pos-sessing anti-thrombotic properties and their ability toprevent thrombus formation has been investigated. In-deed, Fiat et al. [70] proved that a homology exists be-tween the mechanisms involved in milk clotting, definedby the interaction of κ-casein with chymosin and bloodclotting, defined by the interaction of fibrinogen withthrombin. Anti-thrombotic peptides of food origin identi-fied to date are mainly the result of enzymatic hydrolysisof κ-casein. Hydrolysis of bovine κ-casein by chymosinconstitutes the first stage of milk clotting [71]. The

Phe105–Met106 of κ-casein is rapidly hydrolyzed leading torelease of the N-terminal fragment para-κ-casein, and asoluble C-terminal fragment known as caseinomacropep-tide from which tryptic anti-thrombotic peptides are de-rived [72]. The main anti-thrombotic peptide isolated frombovine κ-casein corresponding to f (106-116) with theamino acid sequence MAIPPKKNQDK and termed caso-platelin and fragments of this peptide, known as caso-plateins, such as KNQDK f (112-116) and NDQKf (113-116), are structurally and functionally very similar tothe C-terminal dodecapeptide of human fibrinogenγ-chain f (400-411) corresponding to the sequenceHHLGGAKQAGDV. The amino acids, Ile108, Lys112 andAsp115 of κ-casein are in homologous positions as com-pared with γ-chain sequence of human fibrinogen. Thethree aforementioned residues of κ-casein are importantfor inhibition due to the competition between the anti-thrombotic κ-casein peptide and the γ-chain for theplatelet receptors [72]. Additionally, similarities also existbetween the fibrinogen α-chain tetrapeptide with theamino acid sequence RGDX, and human lactoferrin withthe amino acid sequence KRDS corresponding to lacto-ferrin f (39-42) [73]. KRDS inhibition of thrombin-inducedplatelet aggregation has been associated with an inhibi-tion of the release of the dense granule protein serotonin,but RGDS has no effect on the release [74]. KRDS has beenfound to be anti-thrombotic in three different experimen-tal thrombosis models in four different animal species [75].However, RGDS has been found to induce detachment ofendothelial cells in vitro and serious concerns exist relat-ing to the toxicity of this sequence in vivo, although thesequence KRDS, found in human lactoferrin is not thoughtto have the potential detrimental effects of RGDX [73]. Atryptic hydrolysis of sheep κ-casein also produces anti-thrombotic peptides including KDQDK f (112-116),TAQVTSTEV f (163-171) and QVTSTEV f (165-171) [72]. Itis thought that milk protein-derived anti-thrombotic pep-tides are absorbed into the bloodstream, as two peptidesfrom human and bovine κ-caseinoglycopeptide respec-tively, have been identified in the plasma of 5-day-oldnewborns following ingestion of a cow milk-based formu-la [76]. Peptides exhibiting the sequences involved inanti-thrombotic activity have also been observed in fer-mented milk products such as yoghurt, where the anti-thrombotic peptide corresponding to κ-casein f (113-116)have been isolated [36]. The amino acid sequence PPKcorresponding to κ-casein f (109-111) and sharing struc-ture homology with the previously identified anti-throm-botic peptide MAIPPK [77] was isolated from the water-soluble extract of two commercially available Spanish fer-mented milk drinks [37]. Additionally, Gobbetti et al.(2004) suggested that κ-CN f (152-160) and f (155-160) iso-lated as ACE inhibitors may also possess anti-thromboticactivity [3].



2.4 Opioid peptides derived from milk protein fermentationusing LAB

The active ingredient in opium, named morphine after theGreek god of dreams, was isolated by Serturner in 1803

[78]. Several chemical classes exhibit morphine-like phar-macological effects, such as the phenylpiperidines, ben-zomorphans and octahydroisoquinolines, and these aretermed opioid drugs and defined as ‘agents that bind toor otherwise influence opioid receptors’ [78]. Opioid re-


Figure 2. Models for antimicrobial peptide and peptide-lipid interactions. The toroidal-pore and the barrel-stave models are similar and based on the for-mation of aqueous pores. According to the barrel-stave model (A) peptides attach to the membrane via electrostatic interactions. On the membrane sur-face, peptides adopt α-helical conformation and assemble into bundles. These bundles then insert into the membrane and form pores by interacting theirhydrophobic part with the hydrophobic core of the membrane. The toroidal-pore or wormhole model (B) is similar to the barrel-stave model as the pep-tides attach to the membrane via electrostatic interactions. The peptides adopt α-helical conformations but no bundle is formed. The α-helical peptideskeep their hydrophilic part in contact with the hydrophilic head groups of lipid membrane and bend the membrane to form a pore. In this model, peptidesinteract with lipid head groups during the whole pore formation process. In the carpet model (C), α-helical conformation is not initially required and thepeptides bind preferentially to the lipid head groups. On the membrane surface, the peptides undergo reorientation and realignment with their hydrophilicsurface facing lipid head groups, while the hydrophobic surface faces the hydrophobic core of the membrane. Once a critical local concentration is reached,transient holes form, leading to the eventual collapse of the membrane. The small cylinders represent the antimicrobial peptide. The shaded area repre-sents the head group region of the lipid bilayer.




ceptors are integral membrane proteins of the centralnervous system thought to be responsible for mediatingeffects such as the analgesic effect, feelings of euphoria,myosis, constipation and changes in the endocrine im-mune system produced by ingestion of opioid drugs inman. At least three types of opioid receptors are known todate; these are termed µ- (morphine), δ- (enkephalin) andκ- (dynorphin) receptors. A further receptor named theε-opioid receptor has also been reported [79]. Additional-ly, a further member of the opioid receptor family (ORL1)was identified by Mollereau et al. [80] that mediates anti-opioid effects. These receptors are members of the G pro-tein-coupled receptor family and upon activation, the ac-tive state of the receptor can then couple to a G proteinvia interactions with the intracellular loops (and the C-ter-minal in some receptors), which then initiates the subse-quent intracellular signaling cascade which regulates ef-fector systems such as adenylyl cyclase, phosphatidyl-in-ositol-3 kinase, the MAP kinase pathway, Ca2+ channels,and K+ channels.

Opioid peptides, i.e., opioid receptor ligands derivedfrom milk proteins that exhibit naloxone inhibitable opi-oid activities, have been divided into two groups desig-nated as ‘typical’ and ‘atypical’. As opioid receptor lig-ands, these peptides can be expected to behave like oth-er opioids acting as agonists or antagonists, binding to re-ceptors and eliciting effects in cells and tissue whereopioids are known to be active. ‘Typical’ endogenous opi-oid peptides include the enkephalins, endorphins anddynorphins derived from proenkephalin, propiome-lanocortin and prodynorphin and these peptides exhibitthe definite N-terminal sequence Tyr-Gly-Gly-Phe [81,82]. “Atypical” opioid peptides are characterized by an N-terminal sequence Tyr-X-Phe or Tyr-X1-X2-Phe. The pres-ence of Tyr and an aromatic amino acid form a structuralmotif important in ligand-receptor binding [81]. Specificligand-receptor interaction results in specific physiologi-cal functions. The µ-receptor is thought to be responsiblefor emotional behavior and peristalsis or intestinal motili-ty affecting intestinal transport of electrolytes, the κ- re-ceptor is also thought to be responsible for emotional be-havior, while the δ- receptor is thought responsible for se-dation, analgesia and food intake [83].

The caseins (αs1-, αs2-, β-, κ-) and whey proteins arepotential sources of opioid peptides. β-Casomorphins, thefirst identified opioid peptides initially described in thebovine β-casein sequence, correspond to fragments of theβ-casein sequence 60-70 corresponding to the amino acidsequence f (Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser-Leu)and are the most extensively studied peptides to date [59,81]. They are also found in analogous positions in sheep,water buffalo, and human β-caseins. The well-preservedprimary structure of these peptides suggests that β-caso-morphins are important biologically active molecules, andraises the question of the significance of these opioid pep-tides in diet. β-Casomorphins are resistant to the action

of gastrointestinal proteolytic enzymes and could elicitphysiological effects in the intestine [84], such as opioid,anti-hypertensive, immunomodulatory and antidepres-sant activities as well as anti-secretory or anti-diarrhealactivities [16]. They show a natural affinity for the µ-re-ceptor and have been shown to cause analgesia, apneaand changes in the sleeping patterns of neonatal rats [83].

It has been found that the extracellular PI-type pro-teinase of Lc. lactis hydrolyses β-casein, and results in thegeneration of several oligopeptides including the β-ca-sein sequence corresponding to f (60-68), which formspart of β-casomorphin-11 [85]. The production of highconcentrations of β-casomorphin in milk inoculated withproteolytic bacteria such as Pseudomonas aeruginosaand Bacillus cereus has also been reported [86]. Hy-drophobic peptides were isolated from milk fermentedwith a wild-type strain and a mutant strain of Lb. helveti-cus L89 deficient in X-prolyl-dipeptidyl aminopeptidase(X-PDAP. E. C. 3. 4. 14. 5), an enzyme specific for peptidebonds on the carboxyl side of proline residues. β-Caso-morphin-4 f (60-63), was detected in a peptide extract de-rived from milk fermented with the X-PDAP-deficient mu-tant but not in the milk fermentation using the wild typeLb. helveticus L89 strain [87]. The proteolytic breakdownof β-casein during the ripening of Edam cheese (with orwithout bifidobacteria) resulted in the formation of β-ca-somorphin 3, which was present in all the Edam cheesesamples during ripening, enhancing the beneficial phys-iological effects [88]. Additionally, the peptide fragmentYPFP corresponding to bovine β-CN f (60-63) was isolatedfrom Australian vintage cheddar, and is thought to be anopioid agonist peptide as it shares sequence homologieswith the opioid peptides β-casomorphin-4 and β-caso-morphin-7 corresponding to bovine β-casein f (60-64) andf (60-67), respectively [36]. Furthermore, opioid peptidesreleased by enzymatic proteolysis of Lb. rhamnosus GGfermented UHT milk were identified [28], and these cor-respond to peptides from bovine αs1-casein and peptidesfrom the documented strategic zone of bovine β-casein,known to contain peptides with a variety of bioactivities[14]. Orally administered milk-protein-derived opioid pep-tides exert anti-diarrheal action [89, 90], modulate intes-tinal transport of amino acids and influence postprandialmetabolism by stimulating secretion of insulin and so-matostatin [14]. Indeed, Isolauri et al. (1991) found that Lb.rhamnosus GG-fermented milk or freeze-dried powderwas effective in shortening the course of acute diarrhea inchildren via augmentation of the local immune systemresponse [91]. However, the formation of casomorphins infermented milk products due to LAB proteolytic activityseems unlikely due to the presence of PepX in all LAB [92].The common structural feature among endogenous andexogenous opioid peptides is the presence of a tyrosineresidue at the amino terminal end (with the exception ofα-casein opioids) and the presence of another aromaticresidue in the third or fourth position. The negative po-



tential, localized around the phenolic hydroxyl group of ty-rosine is essential for opioid activity. Lack of the tyrosineresidue results in a diminished opioid effect and the Proresidue, located in position two, is crucial for the bioac-tivity of casomorphins, as it is thought to keep the tyro-sine and phenylalanine residues in the correct orientation[83, 92]. Therefore, casomorphins provide an ideal sub-strate for PepX activity due to the alternating X-Pro se-quence [17, 92, 93].

Opioid peptides are also found encrypted within theprimary sequence of whey proteins such as lactoferrin,β-lactoglobulin (β-lg) and bovine serum albumin (BSA).β-lactorphin corresponding to sequence YLLF fromβ-lg f (102-105) was identified in whey fermented withKluyveromyces marxianus var. marxianus [94]. This pep-tide, identified previously as a µ-opioid receptor agonistwith low potency [95], also exhibited anti-hypertensiveproperties when injected subcutaneously in young SHR(1–100 µg/kg body weight) [48]. In addition, opioid pep-tides were derived from α-lactalbumin followingpepsin/trypsin treatment of Lb. rhamnosus GG-ferment-ed UHT milk [28]. Opioid peptides are biologically very po-tent, and even micromolar amounts of released peptidesmay be sufficient to exert physiological effects [30, 83].Opioid peptides have been found in the small intestinalcontents of adults following ingestion of cows’ milk [14,96], but have not been detected in the plasma of adultmammals, suggesting that the opioid receptors of the in-testinal brush border membrane is the main target site forphysiological effects of milk-derived opioid peptides [95].

2.5 Immunomodulatory peptides

The functions of the immune system include recognitionof pathogens or foreign materials and mounting a re-sponse to eliminate it. Initially, exposure to a foreignpathogen affects the innate, nonspecific immune re-sponse. In this process cytokines may be released. Cy-tokines (pro- and anti-inflammatory) are regulators of hostresponses to infection, inflammation and trauma. Cy-tokines inhibit the synthesis of interleukin-1 (IL-1), tumornecrosis factor (TNF) and other major proinflammatory cy-tokines. After ingestion, orally administered antigenscome into contact with the gut-associated lymphoid tis-sue (GALT), a well-organized immune network that pro-tects the host from infection and also from hyperstimula-tion [97]. The immune response can be divided into twocategories; innate immunity and the adaptive immune re-sponse. Both involve multiple cellular interactions. Thecell types involved in mediating immunity include lym-phocytes and accessory cells such as macrophages, anti-gen-presenting cells and epithelial cells. Oral tolerance isthe active act of suppression (non-response) to antigensdelivered via the oral route and the host develops certainoral tolerances as neonates. A phenomenon linked to oraltolerance and suppression of the immune system is con-

trolled or physiological inflammation in the gut [98]. Reg-ulation of this non-response is controlled by breakdown ofproteins in the gut and activation of suppressor T cells[98]. Cell types involved include CD8+ T cells located insplenocytes and the cytokine-producing CD4+ T (Th)helper cells present in Peyer’s patches, which promoteoral tolerance via the secretion of TGF-β and to a lesser ex-tent IL-10 and IL-4 [98]. TGF-β promotes an isotypeswitch in B cells from IgM to IgA, which results in selec-tive production of IgA, suppression of IgG and IgM secre-tion in the GI tract, resulting in oral tolerance [98]. Oral tol-erance can be abrogated and an immune response in-duced. This immune response is mainly humorally medi-ated through immunoglobulin A (IgA)-producing cellsand secretory IgA, which neutralizes and prevents entryof pathogenic antigens, reducing the risk of diseasecaused by microorganisms entering through the oralroute.

A number of reports exist concerning the up- anddown-regulation of the immune system by milk-derivedimmunomodulatory peptides and fermented milk prod-ucts. Milk-derived peptides are known to affect cells ofthe immune system and consequently to affect down-stream immunological responses and cellular functions[99]. An immune induction response associated with milkproteins was observed after trypsin-hydrolyzed humanmilk was found to contain immunostimulating activity[100]. Subsequently, a hexapeptide corresponding to hu-man β-casein f (54-59), with the amino acid sequence Val-Glu-Pro-Ile-Pro-Tyr was found to induce the phagocytosisof sheep red blood cells and to enhance the resistance ofmice to Klebsiella pneumoniae infection when adminis-tered intravenously [101, 102]. Lb. helveticus-fermentedmilk has demonstrated immunomodulating effects onlymphocyte proliferation in vitro [103] and the abilityto stimulate the phagocytic activity of pulmonarymacrophages. This strain is known to have high prote-olytic activity, causing the release of oligopeptides fromdigestion of milk proteins. Matar et al. [87] identified im-munostimulatory peptides generated through fermenta-tion of milk proteins using LAB, specifically Lb. helveticuscommonly used in the manufacture of Swiss-type cheeseand other fermented milk products. The immunostimula-tory and antitumor properties of peptidic fractions issuedfrom milk fermented with Lb. helveticus R389 were alsoexamined [104]. The humoral immune response was as-sessed by measuring the number of IgA-secreting cellsand the antitumor response was monitored by studyingthe regression of subcutaneously implanted fibrosarco-mas following administration of three fractions from theLb. helveticus R389 milk fermentation, to mice [104]. TheIgA-producing cell count was found to increase signifi-cantly and a decrease in fibrosarcoma size was also ob-served [104]. Lb. paracasei was shown to induce an oraltolerance by proteolytic generation of immunomodulato-ry peptides from β-lactoglobulin (β-lg) [105]. This study re-





ported that unsequenced acidic immunomodulatory pep-tides of less than 1000 kDa in size stimulated IL-10 pro-duction and repressed lymphocyte proliferation [105]. Ad-ditionally, the immunomodulatory effect on human bloodlymphocytes and the down-regulation of cytokine pro-duction following fermentation of milk total casein, αs1-,β- and κ-caseins with a protease of Lb. rhamnosus GG,followed by enzymatic treatment with pepsin and trypsinwas reported [106]. The suppression of T cell activation byLb. rhamnosus GG degraded bovine casein was investi-gated, and it was observed that the digests reduced IL-2expression and inhibited kinase C translocation, bothmarkers for suppression of T cell activation [104, 107]. In-duction of a humoral immune response was observed byfeeding mice with Lb. helveticus-fermented milk follow-ing infection of the mice with E. coli O157:H7 [104, 108].An increase in the number of IgA+ B cells in the small in-testine was reported. The discovery that a protease defi-cient derivative of Lb. helveticus did not produce thesame increase in IgA+ B cells supports the concept thatthe proteolytic machinery of Lb. helveticus produces im-munomodulatory peptides from milk proteins [18, 104].This is also supported by Laffineur et al. [103], whoshowed that β-casein fermented by LAB displayed im-munomodulatory activities not related to interaction withmonocyte-macrophage and Th cells.

ACE inhibitors could also be viewed as bradykinin po-tentiating peptides and therefore act as immunomodula-tors [83]. A number of fermented milk products such asyoghurt are associated with an increased immune re-sponse due to components independent of bacteria suchas immunomodulatory peptides (Table 2). Perdigon et al.[109] reported that the supernatant of fermented milk cul-tured with Lb. casei and Lb. acidophilus strains increasedthe immune response independent of the presence of lac-tobacilli. In addition, filtered yoghurt devoid of microor-

ganisms was found to increase interferon-γ (IFN-γ) pro-duction and natural killer (NK) cell activity of human pe-ripheral blood lymphocytes [110, 111]. Dionysius et al.(2000) isolated the immunomodulatory peptidesβ-CN f (193-209) and β-CN f (192-209) from yoghurt andfermented milks as well as several types of cheese includ-ing Feta and Camembert [36]. The immunomodulatorypeptide β-CN f (193-209) with the corresponding se-quence YQQPVLGPVRGPFPIIV was shown previously toenhance proliferation of rat lymphocytes [112]. In addi-tion, this peptide was isolated from two commercial milkdrinks and a fragment corresponding to the amino acidsequence GPVRGPFPII displayed ACE-inhibitory activi-ty, further supporting the concept that ACE-inhibitorsmay also act as immunomodulators by acting asbradykinin-potentiating peptides [37].

2.6 Cytomodulatory peptides

Cytomodulatory peptides inhibit cancer cell growth andstimulate the activity of immunocompetent cells andneonatal intestinal cells, respectively. The cytomodulato-ry effects of peptidic fractions from milk fermented withLb. helveticus were studied in mice [104]. Additionally,cytomodulatory effects of milk fermented by five bacteri-al species, i.e., Bifidobacterium infantis, Bifidobacteriumbifidum, Bifidobacterium animalis, Lb. acidophilus, andLb. paracasei in a human breast cancer cell line were re-ported, which were found to be due to the presence ofnonbacterial peptides and compounds generated by thebacteria from milk [113]. MacDonald et al. [114] developeda cell culture based assay to identify cytomodulatory pep-tides generated by hydrolysis of casein with the yoghurtculture starter strains, Lb. delbrueckii ssp. bulgaricus andStreptococcus thermophilus. Cytomodulatory peptidesthat influenced colon Caco-2 kinetics in vitro were identi-

Table 2. Immunomodulatory peptides derived from milk proteins

Protein type Peptide sequence Amino acid sequence Reference

αs1-casein (bovine) isracidin (αs1-CN f(1-23) RPKHPIKHQGLPQEVLNENLLRF [54]αs1-casein (bovine) αs1-CN f (194-199) TTMPLW [37]αs2-casein (bovine) αs2-CN f (1-32) KNTMEHVSSSEESIISQETYKQEKNMAINPSK [36, 112]β-casein (bovine) β-CN f (1-28) RELEELNVPGEIVESLSSSEESITRINK [36, 112]β-casein (bovine) β-CN f (63-68) PGPIPN [36, 112]β-casein (bovine) β-CN f (191-193) LLY [36, 112]β-casein (bovine) β-CN f (191-209) LLYQEPVLGPVRGPFPIIV [102, 112]β-casein (human) β-CN f (54-59) VEPIPY [102, 112]α-lactalbumin hydrolysed α-lactalbumin [26, 59]β-lactoglobulin hydrolysed β-lactoglobulin [26, 50]Lactoferrin Residues 17-41 termed FKCRRWQWRMKKLGAPSITCVRRAF [58, 52]

lactoferricin BProline-rich polypeptide nonapeptide fragment VESYVPLFP [112](ovine colostral whey)Tuftsin tetrapeptide IgG Fc region f (289-292) YKPR [112](human Fc region of IgG)



fied. In addition, bovine skimmed milk hydrolyzed withcell-free extract of Saccharomyces cerevisiae reportedlyexhibited anti-proliferative activity against human HL-60leukemia cells [115]. In a mouse model, De Moreno deLeBlanc et al. [116] demonstrated that 7 days of cyclicalfeeding with milk fermented with Lb. helveticus R389 re-sulted in a delay of tumor development, which was relat-ed principally to a decrease in the cytokine IL-6, normallyimplicated in the synthesis of estrogen in both normal andtumor-invaded breasts in mice, and an increase in the cy-tokine IL-10. The increase in IL-10 was not observed in amurine model for milk fermented with Lb. helveticus L89,a proteolytic-deficient variant of Lb. helveticus R389. Theability of milk fermented with probiotic bacteria to en-hance immune responses as reflected in macrophage cy-tokine production was also assessed, and it was reportedthat substances independent of the presence of live bac-terial cells could enhance immune responses [117].

Cytomodulatory peptides have been isolated from avariety of fermented dairy products. Dialysate and anionexchange fraction of yoghurt showed significant inhibito-ry action against tumors in a mouse assay on culturedmammalian intestinal Caco-2 and IEC-6 cells [118]. Anti-proliferative peptides have also been isolated from Goudacheese [119], and lyophilized extracts of Gouda were re-ported to inhibit leukemia cells at concentrations as lowas 1 pmol/L [119]. The vast majority of tumor promotersare potent inhibitors of apoptosis and, therefore, apopto-sis-inducing peptides can be classified as potential anti-carcinogens [14]. It has been reported that cytomodulato-ry and immunomodulatory peptides operate as specificsignals that can trigger the viability of cancer cells exert-ing protective effects in cancer development [14]. Theopioid peptides β-casomorphin and αs1-exorphins havealso exhibited anti-proliferative effects by inhibiting hu-man prostrate cancer cell lines LNCaP, PC 3 and DU145through partial interaction with opioid receptors [120].

Cytomodulatory activity is usually measured in vitroby measuring the growth rate of cancer cell lines such asCaco-2 cells (colon and breast cancer), LNCaP, PC 3 andDU145 (prostate cancer) cells and HL-60 (human leukemiacancer) cells in the presence of inhibitory peptides [114].Cytomodulatory activity of milk protein-derived peptidescan be measured using animals challenged with tumorcells. The rate of tumor development and the presence ofcytokines in serum, mammary gland tissue and tumor-isolated cells is monitored post administration. Thismethod has been used to study the effects of Lb. helveti-cus R389-fermented milk on a murine breast cancer mod-el [116].

2.7 Mineral binding properties of caseinophosphopeptidesand implications for bone health

Bone consists of a collagen matrix into which calcium-rich hydroxyapatite is deposited and the accumulation

and maintenance of bone is the result of a continuousprocess of formation mediated by osteoblasts (cells thatarise from fibroblasts and which, as they mature, are as-sociated with the production of bone) and resorption fa-cilitated by osteoclasts (large multinuclear cells associat-ed with the absorption and removal of bone). Osteoporo-sis is a systemic skeletal disease characterized by lowbone mass and deterioration of bone tissue with in-creased bone fragility and vulnerability to fracture [121].An accumulation of scientific evidence suggests that ad-equate dietary calcium has a significant role in protectionagainst osteoporosis in later life [122]. Milk is a rich die-tary source of calcium and its absorption may be en-hanced when present in association with caseinophos-phopeptides (CPPs). CPPs refer to casein-derived phos-phorylated peptides, which contain single and multiplephosphoryl residues, and these phosphopeptides are re-leased by enzymatic hydrolysis of αs1-, αs2-, β- and κ-ca-seins both in vitro and in vivo [59]. Due to the high con-tent of negative charges, these peptides efficiently binddivalent cations and, therefore, may act as biocarriers fortrace elements such as Fe, Mn, Cu and Se . CPPs gener-ally refer to peptides generated after enzymatic treatmentwith trypsin [14, 123], and which enhance absorption ofcalcium across the distal small intestine [18, 30, 123].CPPs are used in the food industry as ingredients or forti-fiers in some low mineral-containing foods and drinks. Forexample, breakfast cereal sprayed with CPPs and tooth-paste containing CPPs are available [30]. Several phos-phopeptides have been identified in the water-solublefraction of Cheddar cheese derived from αs1-, αs2-, and β-casein [124], while phosphopeptides including β-caseinf (15-28) have been identified in the pH 4.6 soluble extractof Parmigiano-Reggiano cheese. Furthermore, 13 low mo-lecular mass phosphopeptides were isolated from the wa-ter-soluble extract of Comté cheese [32]. It is thought thatacid phosphatase activity in cheese originates from milkand starter bacteria, which contributes to the dephos-phorylation of phosphopeptides in cheese leading to fla-vor development [125, 126]. While some controversy sur-rounds CPPs in terms of ability to enhance Ca2+ absorp-tion in vivo, a number of reports involving animal studieshave revealed positive effect of CPPs on Ca2+ absorption[14, 18]. The addition of CPP to calcium-fortified milk in-creased calcium absorption in young male rats but no ef-fect was observed when the animals were given unforti-fied milk [127]. Lb. helveticus-fermented milk whey hasbeen reported to contain bioactive components that in-crease osteoblastic bone formation in vitro [128]. An-giotensin II has been shown to affect bone by decreasingosteoblast differentiation and stimulating osteoclasticbone resorption [128]. Bradykinin receptors are present inhuman osteoblast cell lines and, although it has no re-ported effect on osteoblast proliferation, it stimulatesprostaglandin synthesis, which has been shown to stim-ulate osteoblastic differentiation and bone formation





[129]. It has been suggested that Lb. helveticus-ferment-ed milk whey affects bone formation through prost-aglandins such as prostaglandin E2 (PGE2) [130], althoughthe mechanisms of action of such peptides on bone healthare not fully elucidated.

2.8 Antioxidant activity of fermented milks generated usingLAB

The imbalance between free radical formation (chemicalcompound that contains one or more unpaired electrons)and the mechanisms involved in their elimination resultsin oxidative stress, which lies at the baseline of many dis-eases, such as degenerative diseases associated with ag-ing [37, 131].

It has been reported that milk proteins and fermentedmilks are among the dietary sources of natural antioxi-dants, in the form of antioxidant peptides. For example,αs1-casein f (144-149) hexapeptide corresponding to theamino acid sequence YFYPEL was found to possess a po-tent superoxide anion radical scavenging activity. Otherpeptides derived from tryptic cleavage of β-casein exhib-ited potent inhibition of lipooxygenase activity. Quench-ing of free radicals by oxidation of amino acid residues incasein is thought to be involved in the mechanisms ofaction of bovine casein derived antioxidant peptides. Aκ-casein derived peptide with 2, 2-diphenyl-1-picryl-hidrazyl activity was recently isolated from milk ferment-ed with Lb. delbrueckii subsp. bulgaricus [132]. Anotherpeptide corresponding to the amino acid sequenceSKVLPVPQ was identified from two commercial Spanishfermented milk drinks manufactured with Lb. helveticusand Saccharomyces cerevisiae, which, based on struc-ture exhibited antioxidant activities [37]. A fragment ofSQSKVLPVPQ, with the amino acid sequence VLPVPQKwas identified previously as an antioxidant peptide [133].The antioxidant activity of whey protein derived peptideshave been reported along with the antioxidant activity ofwhey itself, which is likely due to the presence of cys-teine-rich proteins that aid in the synthesis of glutathione,a potent intracellular antioxidant [14, 59]. For example, theantioxidant activity of the peptide YYSLAMAASDI de-rived from a corolase® PP β-lactoglobulin A hydrolysatewas reported [134]. Neither the structure-activity rela-tionship of antioxidant peptides nor their mechanisms ofaction are yet fully understood. However, following thescreening of 40 peptides structurally related to the an-tioxidant peptide LLPHH isolated from soy protein, thetripeptide PHH was identified as the active center [135].

3 Conclusion

Research is demonstrating that milk contains a plethoraof bioactive peptides that can positively impact on humanhealth. Such sequences can be released and made

bioavailable through proteolysis by digestive enzymes orthrough the action of (gut) bacteria. These released pep-tides have been shown to exhibit a range of propertiesincluding antimicrobial, anti-hypertensive and immunos-timulation. Systematic mining of milk for these “latent”health components has enormous potential to form thebasis of new ranges of Functional Foods.

This review describes the main groups of bioactivepeptides generated through microbial fermentation ofmilk protein using LAB, and discusses the bioprotective-ness, physiological importance and influence that thesehave on the body.

Maria Hayes is in receipt of a Teagasc Walsh Fellowship.This work was funded by the Irish Government under theNational Development Plan, 2000-2006, the European Re-search and Development Fund and Science FoundationIreland (SFI).

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