Bioactive Peptide Functions

Biotechnol. J.
2007, 2, 435449
DOI 10.1002/biot.200700045
www.biotechnology-journal.com
Review
Putting microbes to work: Dairy fermentation, cell factories and 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, Ireland of Microbiology, University College, Cork, Ireland Pharmabiotic Centre, Cork, Ireland
2Department 3Alimentary
A variety of milk-derived biologically active peptides have been shown to exert both functional and physiological roles in vitro and in vivo, and because of this are of particular interest for food science and nutrition applications. Biological activities associated with such peptides include immunomodulatory, antibacterial, anti-hypertensive and opioid-like properties. Milk proteins are recognized as a primary source of bioactive peptides, which can be encrypted within the amino acid sequence of dairy proteins, requiring proteolysis for release and activation. Fermentation of milk proteins using the proteolytic systems of lactic acid bacteria is an attractive approach for generation of functional foods enriched in bioactive peptides given the low cost and positive nutritional image associated with fermented milk drinks and yoghurt. In Part II of this review, we focus on examples of milk-derived bioactive peptides and their associated health benefits, to illustrate the potential of this area for the design and improvement of future functional foods.
Received 12 December 2006 Revised 7 March 2007 Accepted 7 March 2007
Keywords: Casein Whey Proteolysis Lactobacilli Bioactive peptides
Introduction
Bioactive peptides are described as food-derived components (genuine or generated) that in addition to their nutritional value, exert a physiological effect in the body [1]. In 1950, Mellander [2] first described bioactive peptides when he reported that ingestion of casein-derived phosphorylated peptides led to enhanced vitamin D-independent calcification in rachitic infants. Since then, fundamental studies have opened a new field of research related to the generation of bioactive peptides from a variety of food proteins, with milk proteins currently being the primary source. Bioactive peptides can be latent or en-
crypted within the primary or parent proteins where proteolysis is required for their release and activation to exert a physiological response [3] on the various systems of the body. Some peptides also act as biocarriers by sequestering calcium and other minerals, thereby enhancing bioavailability [4]. In Part I of this review, we gave an overview on the release of encrypted bioactive peptides from a range of food protein sources, as well as the use of lactic acid bacteria (LAB) as cell factories for the de novo generation of bioactivities. Here we relate in more detail some examples of bioactive peptide functions.
2
Correspondence: Professor R. Paul Ross, Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland E-mail: Paul.Ross@teagasc.ie Fax: +353-2542229 Abbreviations: ACE, angiotensin-1-converting enzyme; BP, blood pressure; CPPs, caseinophosphopeptides; HHL, hippurly-histidyl-leucine; LAB, lactic acid bacteria; RAS, renin angiotensin system; SHR, spontaneously hypertensive rat; UHT, ultra high temperature
Bioactivities of released peptides
2.1 Role of angiotensin-converting enzyme inhibitors in the control of blood pressure
Hypertension is defined as a sustained increase in blood pressure (BP) and is a controllable risk factor in the development of a number of cardiovascular diseases such as stroke and coronary infarction. Even small decreases in
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Biotechnol. J. 2007, 2, 435449
BP result in a significantly reduced risk of cardiovascular disease, and a 5-mm Hg reduction in diastolic BP reduces the risk of heart disease by approximately 16% in hypertensive subjects [5]. Classically, the control of BP has been associated with the renin angiotensin system (RAS) (Fig. 1), while other regulators of BP include the neutral endopeptidase system, the endothelin-converting enzyme system and the kinin-nitric oxide system [5]. Central to RAS is an exopeptidase, angiotensin-converting enzyme (ACE), discovered by Skeggs et al. [6], which is responsible for the conversion of angiotensin I, a decapeptide generated by the action of rennin on the glycoprotein substrate known as angiotensinogen, to the vasoconstrictor octapeptide angiotensin II. There are two isoforms of human ACE, somatic (sACE) and germinal/testicular (gACE) forms [7], encoded by a single gene located on chromosome 17 at q23. This gene is 21 kb in length and contains 26 exons and 25 introns [8]. Human sACE is a type-I membranebound protein that consists of a 28-residue C-terminal cytosolic domain, a 22-residue hydrophobic transmembrane domain and a 1227-residue extracellular domain that is
heavily glycosylated and further divided into a 612residue N-terminal domain, linked by a 15-residue sequence to a 600-residue C-terminal domain [8]. The extracellular C-terminal domain and N-terminal domain contain a HEXXH sequence, which serves as the zincbinding ligands [8]. In sACE, the C-terminal domain is primarily involved in BP regulation, while the N-terminal domain is involved in the control of hematopoietic stem cell differentiation and proliferation [911]. Human gACE corresponds to the C-terminal domain of sACE [8, 12]. ACE inhibitors are thought to be competitive substrates for ACE. The C-terminal tripeptide sequence of the milk-derived ACE-inhibitor is the primary structural feature governing this inhibitory response as ACE appears to prefer substrates and inhibitors containing hydrophobic amino acid residues in the three C-terminal positions [13]. Generally, aliphatic, basic and aromatic residues are preferred in the penultimate positions, while aromatic, proline (Pro) and aliphatic residues are preferred in ultimate positions. The positive charge of Arg at the C terminus has also been shown to contribute to the ACEI-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 exopeptidase which cleaves from the C-terminal of various peptide substrates. In the renin angiotensin system (RAS), considered the major regulator of blood pressure (BP), ACE removes the C-terminal tripeptide His-Leu from angiotensin I resulting in the formation of angiotensin II, a potent octapeptide vasoconstrictor. 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, endothelium I, a potent vasoconstrictor is formed from Big Endothelium by the action of the enzyme ECE (EC 3.4.24.71). Endothelium I mediates vasoconstriction via 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 oxide 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 and macrophage stimulation. Milk protein-derived inhibitors of ACE may therefore prevent vasoconstriction and lower BP. In addition, inhibitors of ACE may be involved in the endocrine and immune systems.
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minal Lys with a positive charge on the -amino group contributes substantially to the ACE-inhibitory potential [13, 14]. The consumption of fermented milk to maintain good health, including the regulation of BP, is a long tradition in several areas of the world (e.g., Japan, East Asia and France) [15]. ACE-inhibitory and anti-hypertensive (hypotensive) peptides from dairy origin have been known for several years and usually contain up to 10 amino acids [1618]. Strain selection is one of the main factors that influences the release of ACE inhibitors in dairy fermentations [18, 19]. Milk fermentations using LAB or their proteinases, have previously been described as a strategy to release ACE-inhibitory peptides from milk proteins, especially caseins, and ACE-inhibitory peptides have been isolated from many fermented milks and dairy commercial products (Table 1). The majority of milk protein-derived ACE inhibitors have moderate inhibitory potencies, usually within an IC50 range of 100500 mol/L; however, there are exceptions such as the casokinins s1-CN f (23-27) and -casein f (177-183) with IC50 values less than 20 mol/L [14]. Two potent ACE-inhibitory peptides Val-Pro-Pro [-casein f (84-86)] and Ile-Pro-Pro [-casein f (74-76)] derived from casein, were isolated from milk fermented with Lb. helveticus CP790 [20] and sour milk fermented with Lb. helveticus and Saccharomyces cerevisiae [21]. The latter two strains are used in the fermentation of Calpis sour milk (Calpis Co. Ltd., Tokyo, Japan), which is widely consumed in Japan for its anti-hypertensive properties. Lb. helveticus CHCC637 and Lb. helveticus CHCC641 were used to produce fermented milk rich in ACE inhibitors, which has
been shown clinically to cause a significant decrease in BP in spontaneously hypertensive rats (SHR) [22]. It has been reported that milk fermented with LAB species besides Lb. helveticus strains did not display any anti-hypertensive properties upon oral administration in SHR [1, 23]. Philanto-Leppl et al. [24] reported that casein and whey fermentates produced using LAB starters isolated from ropy milk, yoghurt and soured milk were poor sources of ACE-inhibitory peptides prior to digestion with pepsin and trypsin. Other studies have demonstrated ACE-inhibitory peptides following fermentation with different LAB species including Lb. delbrueckii subsp. bulgaricus SS1, Lc. lactis subsp. cremoris FT4, Lb. acidophilus, bifidobacteria, and Streptococcus thermophilus [2527]. Ultra-high temperature (UHT)-treated milk fermented with the probiotic Lb. rhamnosus GG and subsequently digested with the enzymes pepsin and trypsin was found to contain ACE-inhibitory activities, and the ACE-inhibitory peptides corresponding to Ala-Val-ProTyr-Pro-Gln-Arg and Tyr-Gln-Glu-Pro-Val-Leu-Gly-ProVal-Arg, which originated from -casein, were identified. Several opioid peptides were also identified and milk fermented by Lb. rhamnosus GG proved beneficial in several clinical studies when tested for the treatment of gastrointestinal infections such as diarrhea and rotavirus infection [28, 29]. The presence of ACE inhibitors has also been reported in several cheeses including Parmigiano-Reggiano [29], Gouda [25, 30], Cheddar [31] and ripened or cooked cheeses such as Comt [25, 32]. Anti-hypertensive peptides corresponding to s1-casein f (1-22) and s1-casein f (23-38) found in 6-month-old Parmigiano-Reggiano
Table 1. Summary of ACE-inhibitory activities of milk protein fermented with Lactic Acid Bacteria
Microorganism Lactobacillus helveticus Saccharomyces cerevisiae Lactobacillus helveticus Saccharomyces cerevisiae Lactobacillus helveticus LBK 16H Lactobacillus helveticus CPN 4 Lactobacillus helveticus CH CC637 Lactobacillus helveticus R389 Lactobacillus delbrueckii subsp. bulgaricus SS1 Lactococcus lactis ssp. cremoris FT4 Lactobacillus helveticus CP 790 proteinase Lactobacillus helveticus CM4 endopeptidase Lactobacillus helveticus JCM 1004 cell-free extract Lactobacillus helveticus R211 and R389 Lactobacillus delbrueckii ssp. bulgaricus Streptococcus salivarius ssp. thermophilus Lactococcus lactis biovar. diacetylactis Commercial dairy starters from yoghurt and ropy milk treated with enzymes Aspergillus oryzae protease
Substrate Milk Milk Whey protein Whey protein Milk Milk Milk Milk Milk Milk B-casein VPPL Skim milk Casein enriched milk Milk Milk Milk Whey, casein Skim Milk
Activity ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory ACE inhibitory
Reference [38] [38] [5, 16, 48] [5, 16, 48] [40] [56, 22] [56] [56] [56] [56] [24] [23] [22, 24] [22] [22] [22, 37] [37] [19] [41]
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cheese had disappeared following 15 months of ripening, suggesting that the appearance and subsequent disappearance of ACE-inhibitory peptides is influenced by the degree of proteolysis and ripening [33]. The presence of several ACE-inhibitory peptides in several ripened cheeses was reported, with the most potent inhibitory activities detected in short to medium-ripened cheeses such as Gouda [30]. The ACE-inhibitory peptides FFVAPFPEVFGK, corresponding to s1-casein f (23-34), and FFVAP corresponding to s1-casein f (23-27) previously shown to be ACE inhibitors by Maruyama et al. [34], were identified in Crescenza cheese [26]. Enzyme-modified cheese obtained by hydrolysis of pasteurized cheese homogenized with Neutrase and an enzyme preparation from Lb. casei, treated for 72 h was also found to contain ACE inhibitors with the amino acid sequences LTLTDVE corresponding to -casein f (125-131), YPQRDMPIQ corresponding to -casein f (180-197), PGPIP corresponding to -casein f (63-67) and PKHKEMPFPPKYPVEPFT corresponding to -casein f (104-120) [35]. Moreover, the antihypertensive peptides -casein f (193-202), -lactoglobulin f (147-148), lactoferrin f (288-289) and lactoferrin f (319320) were isolated from yoghurt, mature and vintage Cheddar and Feta cheese, respectively [36]. Additionally, the low-fat cheese Festivo, made using Lb. acidophilus and Bifidobacterium spp. with traditional starter cultures was also found to contain ACE inhibitors with potential anti-hypertensive effects after maturation for 13 weeks. However, ACE-inhibitory activity decreased when proteolysis exceeded a certain level during storage for 20 weeks [28]. To exert an anti-hypertensive effect, the bioavailability of ACE inhibitors is of paramount importance. ACEinhibitory activity of some peptides may be deactivated by their susceptibility to degradation by gastrointestinal, brush border, serum and blood proteinases and peptidases on route to the target organ(s). However, oligopeptide sequences containing encrypted bioactive peptides may also be activated and the potency of these oligopeptides increased, due to in vivo proteinase and peptidase activities. The presence of several previously identified ACE-inhibitors in two Spanish commercially available fermented milks following simulated gastrointestinal digestion has been reported [37]. Peptides corresponding to -casein f (202-209) and -casein f (203-209) with the amino acid sequences GPFPIIV and RGPFPIIV, respectively, are fragments of the ACE-inhibitory peptide LLYQQPVLGPVRGPFPIIV identified previously [23]. Using the SHR model, the anti-hypertensive effect of Calpis was demonstrated, which yielded a systolic BP decrease of 17.7 mm Hg following administration of 5 mL/kg body weight of Calpis sour milk drink over an 8-h period [38]. The anti-hypertensive effect of a fermented sour milk drink was demonstrated in human clinical trials, where a significant reduction in BP was obtained in mildly hypertensive patients, following oral consumption
of 95 mL Calpis over an 8-week period [39]. Lb. helveticus LBK-16H-fermented milk containing the bioactive peptides Val-Pro-Pro and Ile-Pro-Pro taken daily (150 mL LBK-16H-fermented milk/day) for 21 weeks, after a 2-week run-in period, also resulted in a BP-lowering effect in hypertensive subjects [40]. More recently, Mizuno et al. [41] demonstrated the anti-hypertensive properties of a casein hydrolysate containing the ACE inhibitors Val-ProPro and Ile-Pro-Pro, prepared using an Aspergillus oryzae protease, in a randomized, double-blind placebo-controlled study. The effect of intake of the casein hydrolysate was evaluated over a 12-week period on the BP of 144 subjects with either high-normal BP (n=104) or mild-hypertension (n=40) [41]. Additionally, the whey protein hydrolysate Biozate 1, containing a range of -lactoglobulin ACE-inhibitory peptides [42], resulted in a significant reduction in BP following oral ingestion of 20 g/day by 30 borderline hypertensive human male and female subjects over a 6-week period [43]. A summary of ACE-inhibitory peptides derived from fermentation using LAB or their proteases is provided (Table 1). One assay for measurement of ACE activity in vitro measures the rate of release of hippurate (product) from hippurly-histidyl-leucine (HHL) (substrate) following incubation of the ACE enzyme with substrate, in the presence of the potential ACE-inhibitory peptide [4447]. The anti-hypertensive effect of ACE-inhibitory peptides may be measured in vivo by direct measurement of BP via arterial cannulation or using tail cuffs in SHR following ingestion of the ACE-inhibitory peptides [48, 49].
2.2 Antimicrobial peptides
Milk represents a successful paradigm for preventing and limiting microbial infection, and understanding the molecules that participate in this protective process may potentially lead to novel antimicrobial therapies [3]. The collective antibacterial effect in milk due to the synergistic activity of naturally occurring peptides and defense proteins besides immunoglobulins, such as lactoferrin, lactoperoxidase and lysozyme is greater than any individual contribution [50]. These defense proteins can exert antimicrobial activities comparable to antibiotics, and therefore have potential as natural alternatives [51]. For example, bovine lactoferrin has shown strong antiviral activity against HIV and the human cytomegalovirus (HCMV), which is thought to act synergistically with HIV in patients with the acquired immunodeficiency syndrome [52]. In addition to these defense proteins, encrypted antimicrobial peptides within the parent protein, which may be released by enzymatic hydrolysis during digestion, may also contribute to the antimicrobial activity of milk [50]. The first antimicrobial peptide isolated from milk, termed lactenin, was identified by Simmes and Jones in 1930 [53], and resulted from the treatment of milk with
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rennet. This peptide exhibited antimicrobial activity against pathogenic strains of streptococci. Subsequently, a group of basic, glycosylated and high-molecular weight (5-kDa) milk-derived polypeptides called casecidins were identified following chymosin treatment of casein [54]. These exhibited antimicrobial activity against pathogenic Staphylococcus aureus and several lactobacilli [54, 55]. Subsequently, the antimicrobial peptide termed isracidin corresponding to s1-casein f (1-23) with a corresponding sequence 1RPKHPIKHQGLPQEVLNENLLRF23 was derived following treatment of s1-casein with chymosin [50, 54]. Isracidin inhibited the growth of S. aureus and Listeria monocytogenes, the principal agents of mastitis infections and protected against such infection in vivo at concentrations comparable with antibiotic use [54]. Most antimicrobial peptides reported to date have been released from the parent milk protein following heat and/or alkali treatment or enzymatic hydrolysis. Furthermore, a number of studies have reported the generation of antimicrobial peptides following dairy fermentations. Recently, an antimicrobial peptide corresponding to -CN f (184-210) with the amino acid sequence 184QELLLNPTHQYPVTQPLAPVHNPISV210, was produced by hydrolysis of human sodium caseinate with a partially purified proteinase of Lb. helveticus PR4 [56]. This peptide exhibited a broad spectrum of inhibition against potentially pathogenic bacteria such as S. aureus, Enterobacter faecium, Yersinia enterocolitica and Salmonella species [56]. Antibacterial activity was found in two water-soluble extracts of nine Italian cheese varieties that differed in the types of starter strains and biotechnological traits used [57]. Peptides with high levels of homology with N-terminal, C-terminal or whole fragments of known antimicrobial peptides were identified within these extracts. Water-soluble extracts derived from Caviocavallo cheese contained RPKHPIK corresponding to s1-casein f (1-7) and GLPQE corresponding to s1-casein f (10-14), which share homology with the intermediate fragments of isracidin [54, 57]. In another study, Dionysius et al. [54] reported on the isolation of the microbicidal peptide corresponding to s1-casein f (1-9) with the sequence RPKHPIKHQ in a yoghurt product. Other studies have identified the presence of whey protein-derived antimicrobial peptides, which were released following enzymatic cleavage with proteolytic enzymes and dairy fermentations with proteolytic LAB. Examples of antimicrobial peptides derived from whey include bovine and human lactoferricin, corresponding to bovine lactoferrin f (17-41) and human lactoferrin f (1-47), respectively, released following enzymatic digestion of bovine and human lactoferrin with trypsin and pepsin [52]. Both bovine and human lactoferricin display antimicrobial activity against a broadspectrum of gram-positive and gram-negative bacteria, including L. monocytogenes [50, 52]. In addition, two shorter peptide derivatives of lactoferricin B f (17-41), termed D-lactoferricin B and L-lacto-
ferricin B corresponding to f (17-31) showed a post-antibiotic effect (defined as an arrested bacterial growth following the removal of the active antibacterial agent) against E. coli [58]. Several peptides with bacteriocidal activity have also been obtained by enzymatic digestion of -lactalbumin and -lactoglobulin [26, 50, 52, 59]. Antibacterial peptides derived from milk proteins are thought to have membrane-lytic activity with specificities for prokaryotic cell membranes [54, 59, 60]. The generally accepted theory concerning the mechanism of action is the overwhelming disruption of microbial membranes, leading to ion and metabolite leakage, depolarization, disruption of membrane coupled respiration and ultimately cell death [61]. The mechanism of action involved in antibacterial peptide-mediated rupturing events remains unclear. However, three models of membrane rupturing have been proposed: the barrel-stave, carpet and toroidal models [62] as shown in Fig. 2. According to the barrel-stave model, antimicrobial peptides bind to the cell membrane, bound peptides recognize each other and oligomerize and subsequently this oligomer of peptides inserts into the hydrophobic core forming a transmembrane pore [62]. The carpet model suggests that antimicrobial peptides bind to and cover the surface of the target membrane, and electrostatic interactions between the peptide and the lipid head group causes membrane permeation [63]. In the toroidal model, peptides bind and interact with lipid head groups and impose a positive curvature force on the membranes, producing channels where the polar head group expands and forms toroidal pores [64]. Membrane-dependent processes such as translocation of cytotoxic peptides across the membrane to the cytoplasm, and transbilayer lipid diffusion are thought to be involved in the mechanism of action of some antimicrobial peptides [65]. Antimicrobial peptides have a broad spectrum of activity and additionally have the ability to distinguish between prokaryotic and eukaryotic cells based on the differences in the outer leaflet of mammalian and bacterial cell membranes. Mammalian cell membranes consist of phosphatidylcholine (PC), sphingomyelin and cholesterol, which are of neutral charge at physiological pH, whereas gram-positive bacterial cell membranes contain negatively charged phospholipids such as phosphatidylglycerol and cardiolipin [3, 56, 62, 66]. In addition to the cytoplasmic membrane, gram-negative bacteria contain an outer membrane that consists in its outer leaflet mostly of the polyanionic molecule lipopolysaccharide [62].
2.3 Anti-thrombotic peptides
Thrombosis may be defined as the pathological condition in which improper activity of the hemostatic mechanism results in clot or thrombus formation in arteries, veins or the chambers of the heart. Both environmental factors and genetic variations in elements of the clotting cascade in-
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fluence thrombosis and consequently, atherosclerosis [67]. Three types of thrombi are recognized: the white thrombus, seen in arteries and which consists mainly of platelets; the red thrombus, which is composed mainly of fibrin and red cells; and the mixed thrombus, which consists of both white and red thrombi. During thrombus formation, circulating prothrombin is activated by platelets. In this process, other major steps take place such as the conversion of fibrinogen to fibrin, which then creates the fibrin matrix. All this takes place while platelets are being adhered and aggregated. In the mid-nineteenth century, Rudolf Ludwig Karl Virchow first described the phenomena he called embolism and thrombosis, and outlined three main factors that contribute to venous thrombosis, which are now known as the Virchow triad, i.e., abnormal vessel wall, abnormal blood flow and abnormal blood constituents. Thrombus composition is influenced by the velocity of blood flow at the site of formation. White platelet-rich thrombi are found in high flow systems, while red thrombi form in regions of stasis. The highshear rate in arteries prevents the accumulation of coagulation intermediates, therefore only platelets have the capacity to form thrombi to the area of damage via von Willebrand factor [68]. On the venous side of circulation, the thrombus consists of fibrin: thrombin can accumulate due to the slower flow rate and platelets play only a minor role. Arterial thrombosis is seen predominantly as myocardial infarction (heart attack, caused by thrombosis in a coronary artery), cerebrovascular arterial thrombosis (stroke, caused by thrombosis in a coronary artery), peripheral arterial thrombosis and ischemic stroke and is almost invariably associated with existing vessel wall disease, i.e., atherosclerosis. The most common forms of venous thrombosis are deep vein thrombosis of the leg and pulmonary embolism, with causes of venous thrombosis divided into those that are characterized by stasis and those reflecting abnormalities in blood plasma (hypercoagulability). Inhibitors of platelet aggregation are commonly used in the management of thrombosis to control further thrombus development. Peptides have been identified in blood and in dietary proteins that interfere with the formation of thrombi. For example, it was hypothesized that fibrinogen -chain and -casein may have evolved from a common ancestor during the past 450 million years [69]. In addition, milk protein-derived peptides have been described as possessing anti-thrombotic properties and their ability to prevent thrombus formation has been investigated. Indeed, Fiat et al. [70] proved that a homology exists between the mechanisms involved in milk clotting, defined by the interaction of -casein with chymosin and blood clotting, defined by the interaction of fibrinogen with thrombin. Anti-thrombotic peptides of food origin identified to date are mainly the result of enzymatic hydrolysis of -casein. Hydrolysis of bovine -casein by chymosin constitutes the first stage of milk clotting [71]. The
Phe105Met106 of -casein is rapidly hydrolyzed leading to release of the N-terminal fragment para--casein, and a soluble C-terminal fragment known as caseinomacropeptide from which tryptic anti-thrombotic peptides are derived [72]. The main anti-thrombotic peptide isolated from bovine -casein corresponding to f (106-116) with the amino acid sequence MAIPPKKNQDK and termed casoplatelin and fragments of this peptide, known as casoplateins, such as KNQDK f (112-116) and NDQK f (113-116), are structurally and functionally very similar to the C-terminal dodecapeptide of human fibrinogen -chain f (400-411) corresponding to the sequence HHLGGAKQAGDV. The amino acids, Ile108, Lys112 and Asp115 of -casein are in homologous positions as compared with -chain sequence of human fibrinogen. The three aforementioned residues of -casein are important for inhibition due to the competition between the antithrombotic -casein peptide and the -chain for the platelet receptors [72]. Additionally, similarities also exist between the fibrinogen -chain tetrapeptide with the amino acid sequence RGDX, and human lactoferrin with the amino acid sequence KRDS corresponding to lactoferrin f (39-42) [73]. KRDS inhibition of thrombin-induced platelet aggregation has been associated with an inhibition of the release of the dense granule protein serotonin, but RGDS has no effect on the release [74]. KRDS has been found to be anti-thrombotic in three different experimental thrombosis models in four different animal species [75]. However, RGDS has been found to induce detachment of endothelial cells in vitro and serious concerns exist relating to the toxicity of this sequence in vivo, although the sequence KRDS, found in human lactoferrin is not thought to have the potential detrimental effects of RGDX [73]. A tryptic hydrolysis of sheep -casein also produces antithrombotic peptides including KDQDK f (112-116), TAQVTSTEV f (163-171) and QVTSTEV f (165-171) [72]. It is thought that milk protein-derived anti-thrombotic peptides are absorbed into the bloodstream, as two peptides from human and bovine -caseinoglycopeptide respectively, have been identified in the plasma of 5-day-old newborns following ingestion of a cow milk-based formula [76]. Peptides exhibiting the sequences involved in anti-thrombotic activity have also been observed in fermented milk products such as yoghurt, where the antithrombotic peptide corresponding to -casein f (113-116) have been isolated [36]. The amino acid sequence PPK corresponding to -casein f (109-111) and sharing structure homology with the previously identified anti-thrombotic peptide MAIPPK [77] was isolated from the watersoluble extract of two commercially available Spanish fermented milk drinks [37]. Additionally, Gobbetti et al. (2004) suggested that -CN f (152-160) and f (155-160) isolated as ACE inhibitors may also possess anti-thrombotic activity [3].
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2.4 Opioid peptides derived from milk protein fermentation using LAB
The active ingredient in opium, named morphine after the Greek god of dreams, was isolated by Serturner in 1803
[78]. Several chemical classes exhibit morphine-like pharmacological effects, such as the phenylpiperidines, benzomorphans and octahydroisoquinolines, and these are termed opioid drugs and defined as agents that bind to or 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 formation of aqueous pores. According to the barrel-stave model (A) peptides attach to the membrane via electrostatic interactions. On the membrane surface, peptides adopt -helical conformation and assemble into bundles. These bundles then insert into the membrane and form pores by interacting their hydrophobic part with the hydrophobic core of the membrane. The toroidal-pore or wormhole model (B) is similar to the barrel-stave model as the peptides attach to the membrane via electrostatic interactions. The peptides adopt -helical conformations but no bundle is formed. The -helical peptides keep their hydrophilic part in contact with the hydrophilic head groups of lipid membrane and bend the membrane to form a pore. In this model, peptides interact with lipid head groups during the whole pore formation process. In the carpet model (C), -helical conformation is not initially required and the peptides bind preferentially to the lipid head groups. On the membrane surface, the peptides undergo reorientation and realignment with their hydrophilic surface 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 represents the head group region of the lipid bilayer.
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ceptors are integral membrane proteins of the central nervous system thought to be responsible for mediating effects such as the analgesic effect, feelings of euphoria, myosis, constipation and changes in the endocrine immune system produced by ingestion of opioid drugs in man. At least three types of opioid receptors are known to date; these are termed - (morphine), - (enkephalin) and - (dynorphin) receptors. A further receptor named the -opioid receptor has also been reported [79]. Additionally, a further member of the opioid receptor family (ORL1) was identified by Mollereau et al. [80] that mediates antiopioid effects. These receptors are members of the G protein-coupled receptor family and upon activation, the active state of the receptor can then couple to a G protein via interactions with the intracellular loops (and the C-terminal in some receptors), which then initiates the subsequent intracellular signaling cascade which regulates effector systems such as adenylyl cyclase, phosphatidyl-inositol-3 kinase, the MAP kinase pathway, Ca2+ channels, and K+ channels. Opioid peptides, i.e., opioid receptor ligands derived from milk proteins that exhibit naloxone inhibitable opioid activities, have been divided into two groups designated as typical and atypical. As opioid receptor ligands, these peptides can be expected to behave like other opioids acting as agonists or antagonists, binding to receptors and eliciting effects in cells and tissue where opioids are known to be active. Typical endogenous opioid peptides include the enkephalins, endorphins and dynorphins derived from proenkephalin, propiomelanocortin and prodynorphin and these peptides exhibit the definite N-terminal sequence Tyr-Gly-Gly-Phe [81, 82]. Atypical opioid peptides are characterized by an Nterminal sequence Tyr-X-Phe or Tyr-X1-X2-Phe. The presence of Tyr and an aromatic amino acid form a structural motif important in ligand-receptor binding [81]. Specific ligand-receptor interaction results in specific physiological functions. The -receptor is thought to be responsible for emotional behavior and peristalsis or intestinal motility affecting intestinal transport of electrolytes, the - receptor is also thought to be responsible for emotional behavior, while the - receptor is thought responsible for sedation, analgesia and food intake [83]. The caseins (s1-, s2-, -, -) and whey proteins are potential sources of opioid peptides. -Casomorphins, the first identified opioid peptides initially described in the bovine -casein sequence, correspond to fragments of the -casein sequence 60-70 corresponding to the amino acid sequence 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 -caseins. The well-preserved primary structure of these peptides suggests that -casomorphins are important biologically active molecules, and raises the question of the significance of these opioid peptides in diet. -Casomorphins are resistant to the action
of gastrointestinal proteolytic enzymes and could elicit physiological effects in the intestine [84], such as opioid, anti-hypertensive, immunomodulatory and antidepressant activities as well as anti-secretory or anti-diarrheal activities [16]. They show a natural affinity for the -receptor and have been shown to cause analgesia, apnea and changes in the sleeping patterns of neonatal rats [83]. It has been found that the extracellular PI-type proteinase of Lc. lactis hydrolyses -casein, and results in the generation of several oligopeptides including the -casein sequence corresponding to f (60-68), which forms part of -casomorphin-11 [85]. The production of high concentrations of -casomorphin in milk inoculated with proteolytic bacteria such as Pseudomonas aeruginosa and Bacillus cereus has also been reported [86]. Hydrophobic peptides were isolated from milk fermented with a wild-type strain and a mutant strain of Lb. helveticus L89 deficient in X-prolyl-dipeptidyl aminopeptidase (X-PDAP. E. C. 3. 4. 14. 5), an enzyme specific for peptide bonds on the carboxyl side of proline residues. -Casomorphin-4 f (60-63), was detected in a peptide extract derived from milk fermented with the X-PDAP-deficient mutant but not in the milk fermentation using the wild type Lb. helveticus L89 strain [87]. The proteolytic breakdown of -casein during the ripening of Edam cheese (with or without bifidobacteria) resulted in the formation of -casomorphin 3, which was present in all the Edam cheese samples during ripening, enhancing the beneficial physiological effects [88]. Additionally, the peptide fragment YPFP corresponding to bovine -CN f (60-63) was isolated from Australian vintage cheddar, and is thought to be an opioid agonist peptide as it shares sequence homologies with the opioid peptides -casomorphin-4 and -casomorphin-7 corresponding to bovine -casein f (60-64) and f (60-67), respectively [36]. Furthermore, opioid peptides released by enzymatic proteolysis of Lb. rhamnosus GG fermented UHT milk were identified [28], and these correspond to peptides from bovine s1-casein and peptides from the documented strategic zone of bovine -casein, known to contain peptides with a variety of bioactivities [14]. Orally administered milk-protein-derived opioid peptides exert anti-diarrheal action [89, 90], modulate intestinal transport of amino acids and influence postprandial metabolism by stimulating secretion of insulin and somatostatin [14]. Indeed, Isolauri et al. (1991) found that Lb. rhamnosus GG-fermented milk or freeze-dried powder was effective in shortening the course of acute diarrhea in children via augmentation of the local immune system response [91]. However, the formation of casomorphins in fermented milk products due to LAB proteolytic activity seems unlikely due to the presence of PepX in all LAB [92]. The common structural feature among endogenous and exogenous opioid peptides is the presence of a tyrosine residue at the amino terminal end (with the exception of -casein opioids) and the presence of another aromatic residue in the third or fourth position. The negative po-
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tential, localized around the phenolic hydroxyl group of tyrosine is essential for opioid activity. Lack of the tyrosine residue results in a diminished opioid effect and the Pro residue, located in position two, is crucial for the bioactivity of casomorphins, as it is thought to keep the tyrosine and phenylalanine residues in the correct orientation [83, 92]. Therefore, casomorphins provide an ideal substrate for PepX activity due to the alternating X-Pro sequence [17, 92, 93]. Opioid peptides are also found encrypted within the primary sequence of whey proteins such as lactoferrin, -lactoglobulin (-lg) and bovine serum albumin (BSA). -lactorphin corresponding to sequence YLLF from -lg f (102-105) was identified in whey fermented with Kluyveromyces marxianus var. marxianus [94]. This peptide, identified previously as a -opioid receptor agonist with low potency [95], also exhibited anti-hypertensive properties when injected subcutaneously in young SHR (1100 g/kg body weight) [48]. In addition, opioid peptides were derived from -lactalbumin following pepsin/trypsin treatment of Lb. rhamnosus GG-fermented UHT milk [28]. Opioid peptides are biologically very potent, and even micromolar amounts of released peptides may be sufficient to exert physiological effects [30, 83]. Opioid peptides have been found in the small intestinal contents of adults following ingestion of cows milk [14, 96], but have not been detected in the plasma of adult mammals, suggesting that the opioid receptors of the intestinal brush border membrane is the main target site for physiological effects of milk-derived opioid peptides [95].
2.5 Immunomodulatory peptides
The functions of the immune system include recognition of pathogens or foreign materials and mounting a response to eliminate it. Initially, exposure to a foreign pathogen affects the innate, nonspecific immune response. In this process cytokines may be released. Cytokines (pro- and anti-inflammatory) are regulators of host responses to infection, inflammation and trauma. Cytokines inhibit the synthesis of interleukin-1 (IL-1), tumor necrosis factor (TNF) and other major proinflammatory cytokines. After ingestion, orally administered antigens come into contact with the gut-associated lymphoid tissue (GALT), a well-organized immune network that protects the host from infection and also from hyperstimulation [97]. The immune response can be divided into two categories; innate immunity and the adaptive immune response. Both involve multiple cellular interactions. The cell types involved in mediating immunity include lymphocytes and accessory cells such as macrophages, antigen-presenting cells and epithelial cells. Oral tolerance is the active act of suppression (non-response) to antigens delivered via the oral route and the host develops certain oral tolerances as neonates. A phenomenon linked to oral tolerance and suppression of the immune system is con-
trolled or physiological inflammation in the gut [98]. Regulation of this non-response is controlled by breakdown of proteins in the gut and activation of suppressor T cells [98]. Cell types involved include CD8+ T cells located in splenocytes and the cytokine-producing CD4+ T (Th) helper cells present in Peyers patches, which promote oral tolerance via the secretion of TGF- and to a lesser extent IL-10 and IL-4 [98]. TGF- promotes an isotype switch in B cells from IgM to IgA, which results in selective production of IgA, suppression of IgG and IgM secretion in the GI tract, resulting in oral tolerance [98]. Oral tolerance can be abrogated and an immune response induced. This immune response is mainly humorally mediated through immunoglobulin A (IgA)-producing cells and secretory IgA, which neutralizes and prevents entry of pathogenic antigens, reducing the risk of disease caused by microorganisms entering through the oral route. A number of reports exist concerning the up- and down-regulation of the immune system by milk-derived immunomodulatory peptides and fermented milk products. Milk-derived peptides are known to affect cells of the immune system and consequently to affect downstream immunological responses and cellular functions [99]. An immune induction response associated with milk proteins was observed after trypsin-hydrolyzed human milk was found to contain immunostimulating activity [100]. Subsequently, a hexapeptide corresponding to human -casein f (54-59), with the amino acid sequence ValGlu-Pro-Ile-Pro-Tyr was found to induce the phagocytosis of sheep red blood cells and to enhance the resistance of mice to Klebsiella pneumoniae infection when administered intravenously [101, 102]. Lb. helveticus-fermented milk has demonstrated immunomodulating effects on lymphocyte proliferation in vitro [103] and the ability to stimulate the phagocytic activity of pulmonary macrophages. This strain is known to have high proteolytic activity, causing the release of oligopeptides from digestion of milk proteins. Matar et al. [87] identified immunostimulatory peptides generated through fermentation of milk proteins using LAB, specifically Lb. helveticus commonly used in the manufacture of Swiss-type cheese and other fermented milk products. The immunostimulatory and antitumor properties of peptidic fractions issued from milk fermented with Lb. helveticus R389 were also examined [104]. The humoral immune response was assessed by measuring the number of IgA-secreting cells and the antitumor response was monitored by studying the regression of subcutaneously implanted fibrosarcomas following administration of three fractions from the Lb. helveticus R389 milk fermentation, to mice [104]. The IgA-producing cell count was found to increase significantly and a decrease in fibrosarcoma size was also observed [104]. Lb. paracasei was shown to induce an oral tolerance by proteolytic generation of immunomodulatory peptides from -lactoglobulin (-lg) [105]. This study re-
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ported that unsequenced acidic immunomodulatory peptides of less than 1000 kDa in size stimulated IL-10 production and repressed lymphocyte proliferation [105]. Additionally, the immunomodulatory effect on human blood lymphocytes and the down-regulation of cytokine production following fermentation of milk total casein, s1-, - and -caseins with a protease of Lb. rhamnosus GG, followed by enzymatic treatment with pepsin and trypsin was reported [106]. The suppression of T cell activation by Lb. rhamnosus GG degraded bovine casein was investigated, and it was observed that the digests reduced IL-2 expression and inhibited kinase C translocation, both markers for suppression of T cell activation [104, 107]. Induction of a humoral immune response was observed by feeding mice with Lb. helveticus-fermented milk following infection of the mice with E. coli O157:H7 [104, 108]. An increase in the number of IgA+ B cells in the small intestine was reported. The discovery that a protease deficient derivative of Lb. helveticus did not produce the same increase in IgA+ B cells supports the concept that the proteolytic machinery of Lb. helveticus produces immunomodulatory peptides from milk proteins [18, 104]. This is also supported by Laffineur et al. [103], who showed that -casein fermented by LAB displayed immunomodulatory activities not related to interaction with monocyte-macrophage and Th cells. ACE inhibitors could also be viewed as bradykinin potentiating peptides and therefore act as immunomodulators [83]. A number of fermented milk products such as yoghurt are associated with an increased immune response due to components independent of bacteria such as immunomodulatory peptides (Table 2). Perdigon et al. [109] reported that the supernatant of fermented milk cultured with Lb. casei and Lb. acidophilus strains increased the immune response independent of the presence of lactobacilli. In addition, filtered yoghurt devoid of microorTable 2. Immunomodulatory peptides derived from milk proteins
ganisms was found to increase interferon- (IFN-) production and natural killer (NK) cell activity of human peripheral blood lymphocytes [110, 111]. Dionysius et al. (2000) isolated the immunomodulatory peptides -CN f (193-209) and -CN f (192-209) from yoghurt and fermented milks as well as several types of cheese including Feta and Camembert [36]. The immunomodulatory peptide -CN f (193-209) with the corresponding sequence YQQPVLGPVRGPFPIIV was shown previously to enhance proliferation of rat lymphocytes [112]. In addition, this peptide was isolated from two commercial milk drinks and a fragment corresponding to the amino acid sequence GPVRGPFPII displayed ACE-inhibitory activity, further supporting the concept that ACE-inhibitors may also act as immunomodulators by acting as bradykinin-potentiating peptides [37].
2.6 Cytomodulatory peptides
Cytomodulatory peptides inhibit cancer cell growth and stimulate the activity of immunocompetent cells and neonatal intestinal cells, respectively. The cytomodulatory effects of peptidic fractions from milk fermented with Lb. helveticus were studied in mice [104]. Additionally, cytomodulatory effects of milk fermented by five bacterial species, i.e., Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium animalis, Lb. acidophilus, and Lb. paracasei in a human breast cancer cell line were reported, which were found to be due to the presence of nonbacterial peptides and compounds generated by the bacteria from milk [113]. MacDonald et al. [114] developed a cell culture based assay to identify cytomodulatory peptides generated by hydrolysis of casein with the yoghurt culture starter strains, Lb. delbrueckii ssp. bulgaricus and Streptococcus thermophilus. Cytomodulatory peptides that influenced colon Caco-2 kinetics in vitro were identi-
Protein type s1-casein (bovine) s1-casein (bovine) s2-casein (bovine) -casein (bovine) -casein (bovine) -casein (bovine) -casein (bovine) -casein (human) -lactalbumin -lactoglobulin Lactoferrin Proline-rich polypeptide (ovine colostral whey) Tuftsin tetrapeptide (human Fc region of IgG)
Peptide sequence isracidin (s1-CN f(1-23) s1-CN f (194-199) s2-CN f (1-32) -CN f (1-28) -CN f (63-68) -CN f (191-193) -CN f (191-209) -CN f (54-59) hydrolysed -lactalbumin hydrolysed -lactoglobulin Residues 17-41 termed lactoferricin B nonapeptide fragment IgG Fc region f (289-292)
Amino acid sequence RPKHPIKHQGLPQEVLNENLLRF TTMPLW KNTMEHVSSSEESIISQETYKQEKNMAINPSK RELEELNVPGEIVESLSSSEESITRINK PGPIPN LLY LLYQEPVLGPVRGPFPIIV VEPIPY
Reference [54] [37] [36, 112] [36, 112] [36, 112] [36, 112] [102, 112] [102, 112] [26, 59] [26, 50] [58, 52] [112] [112]
FKCRRWQWRMKKLGAPSITCVRRAF VESYVPLFP YKPR
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fied. In addition, bovine skimmed milk hydrolyzed with cell-free extract of Saccharomyces cerevisiae reportedly exhibited anti-proliferative activity against human HL-60 leukemia cells [115]. In a mouse model, De Moreno de LeBlanc et al. [116] demonstrated that 7 days of cyclical feeding with milk fermented with Lb. helveticus R389 resulted in a delay of tumor development, which was related principally to a decrease in the cytokine IL-6, normally implicated in the synthesis of estrogen in both normal and tumor-invaded breasts in mice, and an increase in the cytokine IL-10. The increase in IL-10 was not observed in a murine model for milk fermented with Lb. helveticus L89, a proteolytic-deficient variant of Lb. helveticus R389. The ability of milk fermented with probiotic bacteria to enhance immune responses as reflected in macrophage cytokine production was also assessed, and it was reported that substances independent of the presence of live bacterial cells could enhance immune responses [117]. Cytomodulatory peptides have been isolated from a variety of fermented dairy products. Dialysate and anion exchange fraction of yoghurt showed significant inhibitory action against tumors in a mouse assay on cultured mammalian intestinal Caco-2 and IEC-6 cells [118]. Antiproliferative peptides have also been isolated from Gouda cheese [119], and lyophilized extracts of Gouda were reported to inhibit leukemia cells at concentrations as low as 1 pmol/L [119]. The vast majority of tumor promoters are potent inhibitors of apoptosis and, therefore, apoptosis-inducing peptides can be classified as potential anticarcinogens [14]. It has been reported that cytomodulatory and immunomodulatory peptides operate as specific signals that can trigger the viability of cancer cells exerting protective effects in cancer development [14]. The opioid peptides -casomorphin and s1-exorphins have also exhibited anti-proliferative effects by inhibiting human prostrate cancer cell lines LNCaP, PC 3 and DU145 through partial interaction with opioid receptors [120]. Cytomodulatory activity is usually measured in vitro by measuring the growth rate of cancer cell lines such as Caco-2 cells (colon and breast cancer), LNCaP, PC 3 and DU145 (prostate cancer) cells and HL-60 (human leukemia cancer) cells in the presence of inhibitory peptides [114]. Cytomodulatory activity of milk protein-derived peptides can be measured using animals challenged with tumor cells. The rate of tumor development and the presence of cytokines in serum, mammary gland tissue and tumorisolated cells is monitored post administration. This method has been used to study the effects of Lb. helveticus R389-fermented milk on a murine breast cancer model [116].
2.7 Mineral binding properties of caseinophosphopeptides and implications for bone health
Bone consists of a collagen matrix into which calciumrich hydroxyapatite is deposited and the accumulation
and maintenance of bone is the result of a continuous process of formation mediated by osteoblasts (cells that arise from fibroblasts and which, as they mature, are associated with the production of bone) and resorption facilitated by osteoclasts (large multinuclear cells associated with the absorption and removal of bone). Osteoporosis is a systemic skeletal disease characterized by low bone mass and deterioration of bone tissue with increased bone fragility and vulnerability to fracture [121]. An accumulation of scientific evidence suggests that adequate dietary calcium has a significant role in protection against osteoporosis in later life [122]. Milk is a rich dietary source of calcium and its absorption may be enhanced when present in association with caseinophosphopeptides (CPPs). CPPs refer to casein-derived phosphorylated peptides, which contain single and multiple phosphoryl residues, and these phosphopeptides are released by enzymatic hydrolysis of s1-, s2-, - and -caseins both in vitro and in vivo [59]. Due to the high content of negative charges, these peptides efficiently bind divalent cations and, therefore, may act as biocarriers for trace elements such as Fe, Mn, Cu and Se . CPPs generally refer to peptides generated after enzymatic treatment with trypsin [14, 123], and which enhance absorption of calcium across the distal small intestine [18, 30, 123]. CPPs are used in the food industry as ingredients or fortifiers in some low mineral-containing foods and drinks. For example, breakfast cereal sprayed with CPPs and toothpaste containing CPPs are available [30]. Several phosphopeptides have been identified in the water-soluble fraction of Cheddar cheese derived from s1-, s2-, and casein [124], while phosphopeptides including -casein f (15-28) have been identified in the pH 4.6 soluble extract of Parmigiano-Reggiano cheese. Furthermore, 13 low molecular mass phosphopeptides were isolated from the water-soluble extract of Comt cheese [32]. It is thought that acid phosphatase activity in cheese originates from milk and starter bacteria, which contributes to the dephosphorylation of phosphopeptides in cheese leading to flavor development [125, 126]. While some controversy surrounds CPPs in terms of ability to enhance Ca2+ absorption in vivo, a number of reports involving animal studies have revealed positive effect of CPPs on Ca2+ absorption [14, 18]. The addition of CPP to calcium-fortified milk increased calcium absorption in young male rats but no effect was observed when the animals were given unfortified milk [127]. Lb. helveticus-fermented milk whey has been reported to contain bioactive components that increase osteoblastic bone formation in vitro [128]. Angiotensin II has been shown to affect bone by decreasing osteoblast differentiation and stimulating osteoclastic bone resorption [128]. Bradykinin receptors are present in human osteoblast cell lines and, although it has no reported effect on osteoblast proliferation, it stimulates prostaglandin synthesis, which has been shown to stimulate osteoblastic differentiation and bone formation
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[129]. It has been suggested that Lb. helveticus-fermented milk whey affects bone formation through prostaglandins such as prostaglandin E2 (PGE2) [130], although the mechanisms of action of such peptides on bone health are not fully elucidated.
2.8 Antioxidant activity of fermented milks generated using LAB
The imbalance between free radical formation (chemical compound that contains one or more unpaired electrons) and the mechanisms involved in their elimination results in oxidative stress, which lies at the baseline of many diseases, such as degenerative diseases associated with aging [37, 131]. It has been reported that milk proteins and fermented milks are among the dietary sources of natural antioxidants, in the form of antioxidant peptides. For example, s1-casein f (144-149) hexapeptide corresponding to the amino acid sequence YFYPEL was found to possess a potent superoxide anion radical scavenging activity. Other peptides derived from tryptic cleavage of -casein exhibited potent inhibition of lipooxygenase activity. Quenching of free radicals by oxidation of amino acid residues in casein is thought to be involved in the mechanisms of action of bovine casein derived antioxidant peptides. A -casein derived peptide with 2, 2-diphenyl-1-picrylhidrazyl activity was recently isolated from milk fermented with Lb. delbrueckii subsp. bulgaricus [132]. Another peptide corresponding to the amino acid sequence SKVLPVPQ was identified from two commercial Spanish fermented milk drinks manufactured with Lb. helveticus and Saccharomyces cerevisiae, which, based on structure exhibited antioxidant activities [37]. A fragment of SQSKVLPVPQ, with the amino acid sequence VLPVPQK was identified previously as an antioxidant peptide [133]. The antioxidant activity of whey protein derived peptides have been reported along with the antioxidant activity of whey itself, which is likely due to the presence of cysteine-rich proteins that aid in the synthesis of glutathione, a potent intracellular antioxidant [14, 59]. For example, the antioxidant activity of the peptide YYSLAMAASDI derived from a corolase PP -lactoglobulin A hydrolysate was reported [134]. Neither the structure-activity relationship of antioxidant peptides nor their mechanisms of action are yet fully understood. However, following the screening of 40 peptides structurally related to the antioxidant peptide LLPHH isolated from soy protein, the tripeptide PHH was identified as the active center [135].
bioavailable through proteolysis by digestive enzymes or through the action of (gut) bacteria. These released peptides have been shown to exhibit a range of properties including antimicrobial, anti-hypertensive and immunostimulation. Systematic mining of milk for these latent health components has enormous potential to form the basis of new ranges of Functional Foods. This review describes the main groups of bioactive peptides generated through microbial fermentation of milk protein using LAB, and discusses the bioprotectiveness, physiological importance and influence that these have on the body.
Maria Hayes is in receipt of a Teagasc Walsh Fellowship. This work was funded by the Irish Government under the National Development Plan, 2000-2006, the European Research and Development Fund and Science Foundation Ireland (SFI).
References
Conclusion
Research is demonstrating that milk contains a plethora of bioactive peptides that can positively impact on human health. Such sequences can be released and made
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Bioactive Peptide Functions

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Biotechnol. J.

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

Keywords: Casein Whey Proteolysis Lactobacilli Bioactive peptides

Bioactivities of released peptides

2.1 Role of angiotensin-converting enzyme inhibitors in the control of blood pressure

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

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2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

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2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2007, 2, 435449

2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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