doi: 10.1111/j.1471-0307.2011.00725.

REVIEW

Bioactive peptides in dairy products

JONGWOO CHOI, 1 LATHA SABIKHI, 2 ASHRAF HASSAN 1 and SANJEEV ANAND 1 *
1

Department of Dairy Science, South Dakota State University, Brookings, SD 57007, USA, and 2Department of Dairy Technology, National Dairy Research Institute, Karnal 132001, Haryana, India

Bioactive peptides are hydrolysates with specic amino acid sequences that exert a positive physiological inuence on the body. They are inert within the native protein, but once cleaved from the native protein by microbial or added enzymes and or gastrointestinal enzymes during the digestive process, they apply their benecial traits. Dairy products, particularly fermented products, are potential sources of bioactive peptides: several of them possess extra-nutritional physiological functions that qualify them to be classied under the Functional Foods label. Biological peptides in milk, the methods of their generation and their prevalence in dairy products are reviewed along with the reported health benets and safety aspects. Keywords Milk, Bioactive peptides, Dairy products, Functional food, Microbial fermentation.

INTRODUCTION Milk is not only a nutritious source of high-quality proteins, primarily due to its unique amino acid composition, but also a rich source of bioactive peptides. Many research papers report the recent work carried out on bioactive peptides derived from milk proteins (Yamamoto 1997; ScholzAhrens and Schrezenmeir 2000; Shah 2000; Bargeman et al. 2002; Gobbetti et al. 2002; Luhovyy et al. 2007; Haque and Chand 2008; Meisel 1997; Moller et al. 2008; Shahidi and Zhong 2008). Numerous peptides, whose sequences and locations are identied, have been shown to possess various activities such as opiate, antihypertensive, immunomodulator, antibacterial, anti-aggregating and antithrombotic effects (Fiat et al. 1993). These bioactive peptides benet the human physiological system through the cardiovascular, nervous, gastrointestinal and immune systems (Korhonen and Pihlanto 2006). As most of the bioactive peptides are inactive in the native protein sequence and are active only when released, milk proteins need to be hydrolysed into peptides prior to exerting these effects. They may be hydrolysed by the action of proteolytic enzymes during food-processing operations in vitro or by the gastric enzymes during the process of digestion in vivo. The naturally occurring enzymes in milk, coagulants and microbial enzymes can be

used to generate bioactive peptides from milk. Fermentation of milk proteins using the proteolytic systems of lactic acid bacteria (LAB) is a useful approach for the production of bioactive peptides. Of these, the tripeptides such as VPP and IPP obtained from milk fermented with Lactobacillus helveticus were identied as the most potent antihypertensive peptides (Nakamura et al. 1995a,b). It was conrmed that sour milk that contained these two peptides had antihypertensive effects (Hata et al. 1996). Recently, Butikofer et al. (2008) reported the presence of these peptides in different cheese varieties of Swiss origin. BIOACTIVE PEPTIDES IN MILK Milk contains a range of bioactive substances that impart extra-nutritional benets to the mammalian offspring. Some of these benets are evident in the native casein (CN), while others surface only after proteolysis of the proteins. The bioactivities from milk include modulators of digestive and gastrointestinal functions and those of hemodynamic regulators with potential effects on the gastrointestinal tract, hormones and growth factors. Whole CN is reported to inhibit the thrombin-induced platelet aggregation and thrombin-induced secretion of serotonin in vitro (Drouet et al. 1990). This kind of in vitro inhibition of platelet aggregation requires a very specic structural conformation. Human 1

*Author for correspondence. E-mail: sanjeev.anand@sdstate. edu 2011 Society of Dairy Technology

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lactoferrin injected as an intact molecule had a minimal effect of short duration on the formation of thrombi in an experimental animal (Sollier et al. 1990). These studies suggest that these proteins could be used to modify platelet function for prophylaxis. Important sites in these proteins were identied as 106 116 of j-CN and 3942 of human lactoferrin (Rutherfurd and Gill 2000). Several angiotensin I-converting enzyme (ACE) inhibitory peptides have also been isolated from the enzymatic hydrolysis of milk proteins (Hernandez-Ledesma et al. 2004). A cholesterol-lowering effect is another important property of milkderived biopeptides. The peptide with the IIAEK sequence that was isolated from b-lactoglobulin tryptic hydrolysate by Nagaoka et al. (2001) exhibited in rats a greater hypocholesterolaemic activity than the medicine b-sitosterol. In another study, McIntosh et al. (1995) demonstrated a protective role of dietary dairy proteins against tumour development in rats. The proteolytic activity for releasing the biopeptides varies with differentiation or functional state, or intramammary infection of the mammary gland (Schanbacher et al. 1997). Typical examples are the liberation of casomorphins in the mammary gland, and their transfer to the blood, nally to reach endogenous opiate receptors (Assargard et al. 1994; Koch et al. 1994). The latent bioactivities of milk proteins may also be activated by proteolytic action of enzymes from naturally occurring or added micro-organisms. For example, Hamel et al. (1985) rst identied b-casomorphin (b-CM) immunoreactive material in cow milk that had been incubated with various bacterial species, including LAB. PRODUCTION OF BIOACTIVE PEPTIDES The most prolic liberation of biopeptides during food processing occurs via proteolysis by microbial enzymes. Prominent among these are enzymes from LAB or secondary starters, naturally occurring enzymes in milk and coagulant or digestive enzymes used in cheesemaking. Industrial scale production of biologically active peptides can be achieved by the controlled hydrolysis of milk protein precursor, followed by membrane separation processes (Bargeman et al. 2002; Korhonen and Pihlanto 2003, 2006). This bulk production makes it possible for the bioactive peptides to be used as food ingredients for the production of functional foods.

Schlimme 1996; Mullally et al. 1997; Pihlanto-Leppala et al. 2000). Proteinase preparations sourced from bacteria, fungi, plants and animals have also been used (Abubakar et al. 1994; McDonagh and FitzGerald 1998). In addition, enzyme combinations including Alcalae, Chymosin, Pancreatin, Trypsin and Thermolysin can be used to release bioactive peptides from intact protein molecules (Phelan et al. 2009). ACE-inhibitory peptides having different IC50 values and corresponding to several a-lactalbumin and b-lactoglobulin fractions were produced by hydrolysis with combinations of pepsin, trypsin, chymotrypsin, pancreatin, elastase and carboxypeptidase (Mullally et al. 1997; McDonagh and FitzGerald 1998; Pihlanto-Leppala et al. 2000).

Enzymatic hydrolysis The most common method to produce bioactive peptides is through enzymatic hydrolysis of whole proteins. Most of the known biological peptides are released in vitro from their parent milk protein by hydrolysis with pancreatic proteinases, mostly trypsin. However, other enzyme activities and combinations of endoproteinases, including chymotrypsin, pepsin, thermolysin, pancreatin, elastase, carboxypeptidase and proline-specic endopeptidase, are also known to generate physiological peptides (Meisel and
2

Microbial fermentation Lactic acid bacteria utilise milk proteins as their prime source of essential and growth-stimulating amino acids (Juillard et al. 1995). As many dairy starter cultures are highly proteolytic, bioactive peptides can be generated by starter and nonstarter bacteria used in the manufacture of fermented dairy products (Ashar and Chand 2004). In addition to the use of live microorganisms, proteolytic enzymes isolated from LAB have also been successfully used to release bioactive peptides from milk proteins. Ueno et al. (2004) puried and characterised an endopeptidase from L. helveticus CM4 and demonstrated that this peptidase generated antihypertensive peptides using synthetic pro-peptides as substrates (Phelan et al. 2009). As LAB proteolytic systems are complex, there were several controversies regarding their ability to generate biopeptides. These peptides must be generated after the peptidase hydrolysis of long oligopeptides that are initially liberated through their proteinase activity. For example, it was suggested that during milk fermentation, probiotic strains (e.g. Lactobacillus GG) may produce several oligopeptides that generate biopeptides only after subsequent digestion by pepsin and trypsin (Rokka et al. 1997). As peptidase activity is intracellular in LAB, they probably contribute only after cell lysis, which is considered a rare event in fermented milks because of the short fermentation time (Meisel and Bockelmann 1999). At the same time, other studies have shown that LAB proteinases (Lactococcus sp. and Lactobacillus sp.) can hydrolyse more than 40% of the peptide bonds of as1-CN and b-CN, resulting in the formation of more than 100 different oligopeptides that are then actively degraded by the complex peptidase system (Juillard et al. 1995; Kunji et al. 1996; Mierau et al. 1997). Bulk production of bioactive peptides In the mass production of bioactive peptides used as ingredients for the manufacture of functional foods, it is necessary to have simple purication process. Starting materials or choice of enzymes can be an option for this purpose. Immobilised trypsin or glutamic acid-specic endopeptidase in uidised bed bioreactor was used to produce caseinophosphopeptides from as-CN and b-CN (Park and Allen 1998; Park et al. 1998). This
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process does not require the removal of enzymes from the reaction mixture. Ellegrd et al. (1999) combined dialtration and anion-exchange chromatography for the large-scale production of caseinophosphopeptides. They achieved yield efciency of 70% when acid caseinate was trypsinated. Angiotensin I-converting enzyme inhibitory peptides There are several reports on the preparation of ACE-inhibitory peptides. Protease N amino was employed to treat b-lactoglobulin, and the partial fractionated mixture obtained was found to have ACE-inhibitory effect whose main component was SAPLRVY with IC50 = 8 lM. This process does not need expensive isolation step (Ortiz-Chao et al. 2009). Ferreira et al. (2007) hydrolysed whey protein concentrates with trypsin and produced 1.109% of ALPMHIP as a percentage of freeze-dried products. It appears that production of ACE-inhibitory peptides is more efcient from skim milk as compared to whey protein or individual proteins, because both CN and whey proteins contain potential peptide sequences. In the light of this, fresh lowfat milk was fermented with LAB, followed by the hydrolysis of protease. This combined digestion produced 2.5 times higher IC50 than fermentation alone in reaction mixture (Chen et al. 2007b). A recycle ultraltration membrane reactor was used for the continuous production of antithrombotic peptides from tryptic digestion of caseinomacropeptides (CMP). Permeate was concentrated by nanoltration and freeze-dried. Bouhallab and Touze (1995) produced 2.65 g of small molecules (500 780 Da) from 34 g of CMP. In the production of antimicrobial peptides, as2-CN f (183207), Bargeman et al. (2002) used electro-membrane ltration that combines separation mechanism of membrane. Roughly prepared as2-CN from sodium caseinate was treated with porcine pepsin A, and hydrolysates were electro-membrane-ltered. The relative content of as2CN f (183207) was increased from 7.5% in the feed to 25% in the permeate product. BIOLOGICAL ACTIVITIES OF MILKDERIVED PEPTIDES Several biological activities have been reported in dairy-based peptides. These may be classied as antihypertensive or ACEinhibiting, antithrombotic, antimicrobial, opioid, immunomodulating and mineral binding properties, among several others. As these have been reviewed extensively earlier (Meisel 2005; Hartmann and Meisel 2007; Hayes et al. 2007a,b; Tidona et al. 2009; Madureira et al. 2010), they have only been summarised in Table 1. DAIRY PRODUCTS AS CARRIERS OF BIOACTIVE PEPTIDES Dairy products may contain bioactive peptides owing to the proteolytic action of naturally occurring enzymes in milk or by
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Table 1 Biological activities of milk-derived peptides Activity ACE-inhibitory Location a-, b-CN a-lactalbumin, b-lactoglobulin Lactoferrin aS1-CN J-CN J-CN aS1-CN a-, b-CN, a-lactalbumin, b-lactoglobulin a-, b-CN a-, b-CN a-lactalbumin b-lactoglobulin Lactoferrin J-CN J-CN a-, b-CN Lactoferrin Whey proteins Whey proteins (lactoferrin or bovine serum albumin) Casein Lactoferrin b-CN Nomenclature Casokinins Lactokinins Lactoferricin B Isracidin Kappacin Casoplatelins Casocidin Immunopeptides

Antimicrobial

Antithrombotic Casoxin Immunomodulatory

Mineral binding Opioid agonist

Opioid antagonist Antigastric Osteoprotective

Caseinophosphopeptides (CPP) Casomorphins a-lactorphins b-lactorphins Lactoferroxins Casoxins CPP

Anticancer

Antioxidant Gut protection through stimulation of mucus release

b-casomorphin 7

Compiled from Meisel and Bockelmann 1999; Smacchi and Gobbetti 2000; Gobbetti et al. 2002; Hayes et al. 2007b; Moller et al. 2008; Tidona et al. 2009.

exogenous enzymes and by enzymes from microbial starters and are normally generated during dairy processing (Meisel 2005). The presence of these physiological peptides in dairy products enriches them and imparts to them some extra-nutritional functional attributes. While these peptides from dairy products are not as potent as the drugs commonly used in the treatment of the concerned ailments, milk products having moderate bioactivity constitute a naturally functional food group that is desirable in the daily diet. Most of the reports about biological activity of these peptides seem to revolve around controlling hypertension, probably owing to its vast prevalence the world over. Hypertension affects nearly 50 million people in the USA alone and close to one billion globally (Pachori et al. 2001; Cohen and Townsend 2002; Pins and Keenan 2002; National Heart, Lung and Blood Institute 2003). Some other physiological activities attributed to biopeptides found in dairy products are opioid, antimicrobial, 3

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Table 2 Biological activities of selected milk hydrolysates and fermented dairy products Product Ameal S (Calpis sour milk), Calpis Co. Japan Casein hydrolysate Casein DP containing the C12 peptide Kanebo Ltd., Japan Casein hydrolysate containing the C12 peptide DMV, Holland Cheddar cheese Comte cheese Crescenza, Gorgonzola cheeses Dahi Edam, Emmental, Festivo cheeses Evolus sour milk, Valio, Finland Feta, Swiss, Cheddar, Edam, Camembert cheeses Feta, Swiss, Cheddar, Edam, Camembert cheeses Feta, Swiss, Cheddar, Edam, Camembert cheeses Feta, Swiss, Cheddar, Edam, Camembert cheeses Gouda cheese, Havarti cheese LactiumTM -milk protein trypsin milk hydrolysate ING enriched with a-S casein, Cedex, France Mozzarella, Italico cheeses Parmesan, Reggiana cheeses Quarg Sour milk Sour milk Whey protein hydrolysate (BioPURE-a-lactalbumin), Davisco, USA Whey protein hydrolysate (BioPURE-GMP), Davisco, USA Physiological activity ACE-inhibitory activity Antihypertensive properties Antihypertensive properties Antihypertensive properties Phosphopeptides Phosphopeptides ACE-inhibitory activity ACE-inhibitory activity ACE-inhibitory activity ACE-inhibitory activity Antihypertensive properties ACE-inhibitory activity Immunomodulatory Antiamnesic Opioid activity ACE-inhibitory activity Anxiolytic-like activity ACE-inhibitory activity Opioid activity ACE-inhibitory activity Phosphopeptides Antihypertensive properties Opioid activity Antithrombotic activity Antimicrobial properties, Anticariogenic effects Antihypertensive properties ACE-inhibitory activity Immunomodulatory Antihypertensive properties Antiamnesic Microbiocidal Antithrombotic Reference Meisel and Bockelmann 1999 Meisel 2005 Silva and Malcata 2005 Meisel 2005 Meisel 2005 Gobbetti et al. 2004 Meisel 2005 Ryhanen et al. 2001 Meisel and Bockelmann 1999 FitzGerald et al. 2004 FitzGerald et al. 2004 FitzGerald et al. 2004 FitzGerald et al. 2004 Meisel and Bockelmann 1999 Meisel 2005 Meisel 2005 Meisel 2005 Meisel 2005 Silva and Malcata 2005 FitzGerald et al. 2004 Tidona et al. 2009 Hartmann and Meisel 2007

Whey Protein Hydrolysate (Biozate 1), Davisco, USA Yoghurt Yoghurt Yoghurt Yoghurt Yoghurt Yoghurt

Meisel 2005 Meisel 2005 Meisel 2005 Fitzgerald and Murray 2006 Fitzgerald and Murray 2006 Fitzgerald and Murray 2006 Fitzgerald and Murray 2006

ACE, angiotensin I-converting enzyme. Compiled from Hartmann and Meisel 2007; Hayes et al. 2007a,b; Tidona et al. 2009.

antithrombotic, antioxidative, immunomodulating and cytomodulating properties. Table 2 lists the biological activities of some milk hydrolysates and fermented dairy products.

Fermented dairy products

Angiotensin I-converting enzyme inhibitory peptides Calpis, a Japanese soft drink made from skim milk fermented by L. helveticus CP790 and Saccharomyces cerevisiae, reportedly contains ACE-inhibitory peptides (Nakamura et al. 1995a). When fed to spontaneously hypertensive rats (SHR) at a dosage of 5 mL kg of body weight, these peptides decreased the systolic blood pressure (BP) after 68 h 4

of administration (Nakamura et al. 1995b). These peptides (-Val-Pro-Pro- and -Ile-Pro-Pro-) were detected in a heat-treated solubilised fraction from the abdominal aorta of the rats fed on the sour milk, but not in rats given unfermented milk (Masuda et al. 1996). These workers also suggested these peptides were not decomposed by digestive enzymes and were absorbed directly. Meisel et al. (1997) reported that the BP of hypertensive patients decreased after 4 and 8 weeks of daily ingestion of 95 mL of sour milk containing 1.21.6 mg of VPP and IPP. These peptides originating from both aS1- and b-CN were puried from the fermented milk and were also present in milk fermented with L. helveticus (Yamamoto et al. 1994b) and CN hydrolysates produced by extracellular proteinases of
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L. helveticus CP790 (Yamamoto et al. 1994a). Evolus, a Finnish milk product produced by L. helveticus LBK-16H strain as a starter, contained the tripeptide IPP and exerted similar antihypertensive effect (Seppo et al. 2002). In a placebo-controlled randomised study, 39 hypertensive patients were given 150 mL per day of either a control or a L. helveticus LBK-16H-fermented milk for 21 weeks after a 2 week run-in period. After the experiment, there was a mean difference of 6.7 3 and 3.6 1.9 mm Hg in systolic and diastolic BPs, respectively, between the test and control groups (Seppo et al. 2003). L. helveticus CHCC637 and L. helveticus CHCC641 were used to produce fermented milk rich in ACE-inhibitors (Fuglsang et al. 2002) and has been shown clinically to cause a decrease in BP in SHR. Interestingly, milk fermented with LAB species besides L. helveticus strains did not display antihypertensive properties upon oral administration in these animals (Vermeirssen et al. 2004). Hernandez-Ledesma et al. (2004) reported the presence of several previously identied ACE-inhibitors in two Spanish commercially available fermented milks following simulated gastrointestinal digestion. The antihypertensive peptide b-CN fraction (b-CN f) 193202 was isolated from yoghurt (Dionysius et al. 2000). Furushiro et al. (1990) reported an antihypertensive effect of a cell extract from L. casei. Compounds puried from the cell lysate were polysaccharideglycopeptide complexes and had a molecular weight of 180 000 (Furushiro et al. 1993). However, the enhanced biosynthesis of prostaglandin I2 and the resulting decrease in peripheral vascular resistance and not ACE-inhibitory activity was behind the antihypertensive action of these peptides, as suggested by Sawada et al. (1990). Inhibition of ACE-converting enzyme increased in whey separated from low-fat milk fermented by ve mixed LAB (L. casei, L. acidophilus, Streptococcus thermophilus, L. bulgaricus and Bidobacterium) followed by enzymic action by a protease Prozyme 6 (Chen et al. 2007a), as proved by a 22.6- and 21.5-mm Hg decrease, respectively, in the systolic and diastolic BP in SHR. The rats were subjected to 8 weeks of oral administration of the diluted whey (12.5 mg whey powder mL). The quantity of the bioactive peptides in the enzyme-facilitated lactic fermentation was more than that from the lactic fermentation alone, 32.8 vs 5.8 mg g whey powder (Tsai et al. 2008). ACE-inhibitory peptides were isolated from whey fractions from standardised milks fermented by L. delbrueckii ssp. bulgaricus alone, as well as in combination with S. salivarius ssp. thermophilus and Lactococcus lactis biovar. diacetylactis (Ashar and Chand 2004). Both the peptides (Ser-Lys-Val-Tyr-Pro-Phe-Pro-GlyPro-lle, f5766, IC50 1.7 mg mL, quantity 0.4 mg mL, from L. delbrueckii ssp. bulgaricus alone; Ser-Lys-Val-Tyr-Pro, f5761, IC50 1.4 mg mL, quantity 0.2 mg mL, from L. delbrueckii ssp. bulgaricus, S. salivarius ssp. thermophilus and Lac. lactis biovar. diacetylactis 1:0.25:0.25) were from b-CN and were markedly stable to digestive enzymes, acidic and alkaline pH, as well as during storage at 5 and 10C for 4 days. Two fermented milks made with strains of L. delbrueckii ssp.
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bulgaricus SS1 and Lac. lactis subsp. cremoris FT4 contained ACE-inhibitory peptides that contained higher proportions of hydrophobic residues (Gobbetti et al. 2000). Fractions that had the most inhibitory activity were from milk fermented by L. delbrueckii subsp. bulgaricus SS1 and contained the sequences of b-CN f614, f714, f7382, f7482 and f7582. Those from milk fermented by Lac. lactis subsp. cremoris FT4 contained the sequences of b-CN f714, f4752 and f169175 and j-CN f155160 and f152160. Some of these peptides were also synthesised. The 50% inhibitory concentrations (IC50s) of the crude puried fractions containing the peptide mixture were low (8.011.2 mg L). The ACE-inhibitory activity was conrmed in the synthesised peptides when used individually, but IC50s were considerably higher. This may be due to the stronger inhibitory effect of the peptide mixtures with respect to the activity of individual peptides. As reported by others, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin. Antithrombotic peptides Matar and Goulet (1996) used a mutant strain of L. helveticus that lacked X-prolyl-dipeptidyl aminopeptidase activity in the production of fermented milk containing the opioid b-CM 14 (f6063). This peptidase has the ability to alter the amino acid sequence in the peptide that is responsible for the physiological activity. An antithrombotic peptide corresponding to j-CN f113116 has been isolated from yoghurt (Dionysius et al. 2000). An antithrombotic peptide PPK corresponding to j-CN f109111 and having the same structure homology with an earlier-identied antithrombotic peptide MAIPPK (Mazoyer et al. 1992) was isolated from the water-soluble extract of two commercially available Spanish fermented milk drinks (HernandezLedesma et al. 2004). Gobbetti et al. (2004) also reported that j-CN f152160 and f155160 isolated as ACE-inhibitors may also possess antithrombotic activity. The probiotic action of several LAB strains includes the synthesis of specic bioactive peptides. Milk fermented with a human lactobacillus strain was found benecial when tested in several clinical studies for the treatment of gastrointestinal infections such as diarrhoea and rotavirus infection (Kaila et al. 1992). The production of fragments of aS1- and b-CN, and of a-lactalbumin, with different degrees of immunostimulating, opioid and ACE-inhibitory activities in UHT milk fermented by the same probiotic Lactobacillus GG strain and subsequently digested by the enzymes pepsin and trypsin may explain some of the probiotic action of the strain (Rokka et al. 1997). Casein phosphopeptides Casein phosphopeptides (CPPs) are phosphorylated CNderived peptides produced by proteolytic digestion of as1-, as2and -CN and have been found useful in the treatment of dental caries (Cross et al. 2004, 2005). As suggested by Walker et al. (2006), liquid milk has the majority of the CN and calcium and 5

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phosphate ions bound in micelles and, therefore, may not be available to promote enamel remineralisation. This is because the bound calcium in CN micelles in milk is unable to diffuse across a relatively intact enamel surface layer into a subsurface lesion. Yoghurt is high in calcium and phosphorus as fermentation does not affect the mineral content of milk. As the pH of yoghurt is lower compared with that of milk, calcium is present in yoghurt, mostly in ionic form (Adolfsson et al. 2004). The CPP in yoghurt is higher than that in milk because of proteolytic activity of micro-organisms contained in the yoghurt (Rasic and Kurmann 1983). Petti et al. (2008) reported that in vitro yoghurt containing S. thermophilus and L. bulgaricus had bactericidal activity against S. mutans and suggested that the decrease in the oral level of these streptococci in vivo as a result of daily yoghurt intake was partly due to direct and selective bactericidal activity of yoghurt against S. mutans. Survival rates after 15 min incubation with fat-free plain yoghurt were 8% (S. mutans 6519T), 12% (S. mutans 31738), 35% (S. oralis 25671) and >50% (all other species tested), and after 30 min, 0.01% (S. mutans) and >10% (all other species tested). Cantile (2009) studied the effectiveness of synthetic and natural (from yoghurt) CPPs in vitro and in vivo in prevention of dental caries. The capability of the CPPs to prevent demineralisation and promote remineralisation of early enamel lesions was tested by preserving one group in distilled water, treating the next group with a demineralising solution for producing articial caries and giving the samples in the third group the same treatment, but with the addition of synthetic or natural CPPs. The effects of these procedures were evaluated by quantitative (change in weight and calcium titration) and qualitative [scanning electron microscopy (SEM)] analyses. Acid dissolution of human enamel was reduced by over 50% in vitro with synthetic CPPs, while there was signicant difference in the weight changes between the groups with and without natural CPPs in vitro (Ferrazzano et al. 2008). The SEM observation also showed the protective effects of natural CPPs. In the in vivo study, human subjects were fed on 125 g of commercial plain fruit yoghurt twice a day for 2 weeks, and salivary S. mutans and lactobacilli were enumerated weekly. S. mutans count decreased in the test group compared to the control group, while there was no effect on the salivary lactobacilli. Cytomodulatory peptides These have been isolated from a variety of fermented dairy products. Dialysate and anion-exchange fraction of yoghurt showed signicant inhibitory action against tumours in a mouse assay on cultured mammalian intestinal Caco-2 and IEC-6 cells (Ayebo et al. 1982). Cytomodulatory peptides that inuenced colon Caco-2 kinetics in vitro were identied by MacDonald et al. (1994) from CN hydrolysates using the yoghurt culture. Yoghurt contains peptides in the range of 50010 000 Da, which in vitro reduces the risk of colon cancer by limiting cancer cell proliferation, as demonstrated by a 6

cell culture model system (Ganjam et al. 1997). The opioid peptides b-CM and as1-exorphins have also exhibited antiproliferative effects by inhibiting human prostrate cancer cell lines LNCaP, PC 3 and DU145 through partial interaction with opioid receptors (Kampa et al. 1997). A j-CN-derived peptide with antioxidant activity was isolated from milk fermented with L. delbrueckii subsp. bulgaricus (Kudoh et al. 2003). Another peptide (SKVLPVPQ) was identied from two commercial Spanish fermented milk beverages with the aid of L. helveticus and Sac. cerevisiae, which exhibited antioxidant activities (Hernandez-Ledesma et al. 2004).

Cheese Several cheeses have been reported to contain biological peptides, the type and quantity depending on the starter cultures and the ripening conditions employed. Cell-bound proteinases of LAB release many oligopeptides that are further hydrolysed by peptidases into shorter fragments and amino acids, which contribute directly, or as precursors, to avour in cheeses (Fox et al. 1984; Stepaniak and Fox 1995). Several of these peptides are also bioactive. Meisel et al. (1997) reported the presence of low molecular weight ACE-inhibitory peptides in several ripened cheeses. However, the effect of these peptides lasts only to a limited extent, as they are further degraded to inactive fragments as ripening progressed. The ACE-inhibitory activity in mediumaged Gouda was about double that of the long-ripened Gouda. An aS1-CN-derived antihypertensive peptide, isolated from 6-month-ripened Parmesan cheese, was not present on ripening for 15 months (Addeo et al.1992). ACE-inhibiting peptides have been isolated from several Italian cheeses characterised by short (Crescenza and Italico) and medium (Gorgonzola) ripening period (Smacchi and Gobbetti 1998). Crescenza (Smacchi and Gobbetti 1998) and Cheddar cheeses (Stepaniak et al. 1995) contained antihypertensive fractions of b-CN (f5872) that included the b-CM-7 sequence. While b-CMs in cheeses were further hydrolysed by the proteolytic enzymes of Lac. lactis subsp. cremoris, the degradation was slow and dependent on the pH and salt concentration of the cheese (Muehlenkamp and Warthesen 1996). Low ACE-inhibition index was found in Quarg, an unripened cheese with limited proteolysis (Meisel et al. 1997). Gomez-Ruiz et al. (2002) monitored the ACE-inhibitory activity of water-soluble extracts during ripening, from Manchego cheeses manufactured with different starter cultures. Although they identied 22 peptide fragments in nine fractions, the complexity of the collected fractions after three chromatographic steps prevented the assignment of a single peptide responsible for the ACE-inhibitory activity. However, f199 204 from ovine b-CN, which was included in one of the most active fractions, was chemically synthesised and had an IC50 value of 592 mm. The same group (Gomez-Ruiz et al. 2006) also studied Protected Denomination of Origin (PDO) cheeses for the presence of biopeptides. They identied 41
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ACE-inhibitory peptides, mainly derived from b- and as1-CN ` in six Spanish cheeses (Cabrales, Idiazabal, Roncal, Manchego, ` n and a goats milk cheese ve of them with PDO). Maho Cabrales cheese that had the highest proteolysis index also showed the highest and also had the highest inhibitory activity ` while Mahon showed the lowest. Ryhanen et al. (2001) analysed Festivo cheese containing a mixture of 12 different strains of Lactococcus sp., Leuconostoc sp., Propionibacterium sp., Lactobacillus sp. and Bidobacterium sp. for its ACE-inhibitory activity. They found that inhibitory effect increased during ripening and remained active for a limited period till a certain level of proteolysis was exceeded. The contents of the two antihypertensive peptides VPP and IPP were determined in 101 samples of 10 different Swiss cheese varieties. While the average total concentration varied widely, the lowest average concentration was 19.1 mg kg in LEtivaz cheese and the greatest at 182.2 mg kg was found in Appenzeller cheese (Butikofer et al. 2008). They concluded that these traditional cheese varieties contain similar concentrations of the two antihypertensive peptides to the commercial fermented milk products. Peptide fractions prepared from Emmental, Blue, Edam, Gouda and Harvati cheeses exhibited antihypertensive action in SHR (Saito et al. 2000). ACE-inhibitory peptides were also detected in Gamalost, Castello, Brie, Pultost, Noevegia, Port Salut and Kesam cheeses when Pripp et al. (2006) investigated the relationship between proteolysis and ACE-inhibition. Antihypertensive peptides b-lactoglobulin f147148, lactoferrin f288289 and lactoferrin f319320 were isolated from mature and vintage Cheddar and feta cheese, respectively (Dionysius et al. 2000). Haileselassie et al. (1999) puried and identied several bioactive peptides from enzyme-modied cheese (EMC) prepared using a combination of Neutrase, L. casei enzymes and Debitrase. The Neutrase digest contained a b-CM 7 (b-CN f6066). Sabikhi and Mathur (2001) reported the presence of b-CM 3 in probiotic Edam cheese, although longer casomorphins were not detectable. The presence of bioactive peptides, which are naturally formed in cheese, depends probably on the equilibrium between their formation and the enzymatic degradation during the ripening process. The peptide fragment YPFP corresponding to bovine b-CN f6063 was isolated from Australian vintage Cheddar and was classied as an opioid agonist peptide as it shares sequence homologies with the opioid peptides b-CM 4 and b-CM 7 corresponding to bovine b-CN f6064 and b-CN f6067, respectively (Dionysius et al. 2000). Caseinophosphopeptides are produced during ripening of cooked curd cheeses such as Comte (Roudot-Algaron et al. 1994) or Grana Padano (Pellegrino et al. 1997). Anticarcinogenic peptides were present in a cheese slurry made using Lac. lactis subsp. lactis as starter culture (Kim et al. 1995). Rizzello et al. (2005) found antibacterial activity in two watersoluble extracts of nine Italian cheese varieties that varied in starter strains and biotechnological parameters employed.
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These extracts had peptides with high levels of homology with N-terminal, C-terminal or whole fragments of known antimicrobial peptides. Water-soluble extracts derived from Caviocavallo cheese contained RPKHPIK corresponding to as1-CN f17 and GLPQE corresponding to as1-CN f1014. These peptides are homologous with the intermediate fragments of isracidin (Rizzello et al. 2005; Lahov and Regelson 1996).

Peptides produced by milk-clotting enzymes Milk-clotting and digestive enzymes have also been studied for their capacity to produce bioactive peptides. Some of these, produced in vitro by chymosin, trypsin, chymotrypsin and cellbound proteinases from Lac. lactis subsp. cremoris, are b-CN f193209, f194209, f6997, f141163 and f6984 (Monnet et al. 1989; Reid et al. 1991; Visser 1993; Fox et al. 1995; Stepaniak et al. 1995, 1996). These fragments were either bitter or contained sequences of bitter peptides (Visser 1993; Fox et al. 1995). Of these, b-CN f193209 and f194209 had antihypertensive properties (Yamamoto et al. 1994a). Raw and sterilised ovine and caprine cheeses manufactured with enzymes from Cinara cardunculus as the coagulant agent contained peptides bearing ACE-inhibitory and antioxidant activity (Verissimo et al. 1995). Two aspartic proteases, cardonsis A and B, which resemble chymosin and pepsin, respectively, in activity and specicity, were responsible for the milk-clotting activity. Glycomacropeptide (GMP), the large peptide that forms a part of the hydrophilic portion of j-CN after rennet cleavage, also has several reported biological activities. GMP regulates digestion without being absorbed, the active components being hydrolysis of the macropeptide by pepsin (Madureira et al. 2010). GMP can be included in diets aimed at controlling liver ailments as well as phenylketuronia (El-Salam et al. 1996; Smithers et al. 1996). This macropeptide also binds cholera and Escherichia coli enterotoxins, inhibits bacterial and viral adhesion, promotes bidobacterial growth and modulates immune system responses (Brody 2000). GMP prevented cariogenic bacterial adhesion in an in vitro model (Schupbach et al. 1996) by altering the microbial population dominated by S. mutans and S. sangius to a less cariogenic population predominated by Actinomycetes viscous. It has been patented as an antimicrobial agent effective against S. mutans (Neeser 1991a,b). Other products Selected infant formulae (adapted, follow-up, probiotic followup, toddler and probiotic toddler) were analysed by an in vitro enzymatic process simulating physiological digestion (Miquel et al. 2005) for the presence of CN phosphopeptides. The peptide aS2-CN (f119) 4P was present in all the samples analysed. The probiotic formulae had phosphopeptides that were not present in the other formulae, probably due to the fermentative action of bidobacteria. The study suggested that gastric digestion of these infant formulae promote the formation of bioactive peptides in the gastrointestinal tract.
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MANUFACTURE OF FUNCTIONAL FOODS ENRICHED WITH BIOACTIVE PEPTIDES The vast evidence found in literature suggests there is potential for application of bioactive peptides in functional foods and pharmaceutical products. Calpis is certied as a food for specialized health use (FOSHU) in Japan with a health claim on the label (Shimizu 2002). Over 400 foods have been placed under the FOSHU label since its establishment in 1993 (Fitzgerald and Murray 2006). Yakoult made by fermentation using L. casei subsp. shirota is another FOSHU-labelled product popular globally (Shah 2007). CN-derived peptides have been considered for use as dietary supplements, and pharmaceutical preparations (Gobbetti et al. 2002). Caseinophosphopeptides and GMP have been patented for use in common personal hygiene products to prevent dental caries. GMP was patented as an antimicrobial agent effective against S. mutans (Neeser 1991a,b). Some product manufacturers have incorporated GMP into patent-protected commercial products. Some examples are hydrolysed gelatin as a stabiliser, combination of GMP and uoride and use of xylitol as synergistic agent (Zhang and Gaffar 1998a,b, 2001). A patent was led that uses CPP in chewing gum compositions by the Warner-Lambert Company (Aimutis 2004). Caseinophosphopeptides also has possible applications as a mineral carrier in bread, cake, our and beverage, and as pharmaceutical preparations such as tablets, and dental lling materials (Reynolds 1987). Several bioactive peptides may be used as highly active drugs having specic pharmacological effects in the treatment of ailments. Some examples are casomorphins for diarrhoea, casokinins for hypertension, casoplatelins for thrombosis and immunopeptides for immunodeciency (Meisel 2005). Studies on the synthesis of modied peptide sequences aimed at higher potency, altered side effects and longer duration of action (Matthies et al. 1984; Daniel et al. 1990) involved morphiceptin (b-CM 4 amide) and casokefamide (b-CM 5 amide). SAFETY ASPECTS OF BIOACTIVE PEPTIDES For food and pharmaceutical applications of biological active peptides, it is important to consider their safety and toxicity. Maeno et al. (2005) reported that CN hydrolysates and VPP originating from powdered fermented milk exerted no specic target organ toxicity. There was also no evidence to support establishment of either the lowest-observed-effect level (LOEL) or maximally tolerated dose (MTD), both being >2 g per kg per day for up to 28 consecutive days in rats. They concluded that, under these conditions, the MTD and no-observed-effect level (NOEL) for powered CN hydrolysates administered once daily for 13 weeks were >1 g per kg per day or >6 mg of VPP plus IPP per kg per day. Doorten et al. (2009) reported that Tensugard, a functional food ingredient containing large amounts of IPP, had an overall intake of 2 g Tensugard per kg per day as the no-observed-adverse-effect level (NOAEL), 8

which is equivalent to 40 mg of IPP. Owing to their lower molecular weight, peptides are more reactive than the native proteins. For this reason, it is important to ascertain their safety, which would include absence of toxicity, cytotoxicity and allergenicity (Korhonen and Pihlanto 2006). Strict and unambiguous legislation is an absolute necessity to ensure the protection of consumers from potentially harmful or misleading products. CONCLUSIONS As milk proteins are precursors of numerous bioactive peptides, dairy products, particularly fermented products and fermentates, may be regarded as functional foods and targeted for imparting several biologically benecial attributes. Bioactive peptides that are present in abundance in milk, fermented milks and cheeses can be physiologically activated at target sites. CN-derived peptides can be manufactured on an industrial scale and used as dietary supplements and as pharmaceutical preparations. However, there is a need to make a clear distinction between health-promoting nutraceuticals for prevention and pharmacological applications (drugs) for the treatment of diseases and to label the product accordingly. Food industry is now exploiting these bioactive peptides, and several studies are being directed to their bioavailability within the food matrix during extended storage periods. Thus, it is evident that bioactive peptides could offer an excellent basis for the novel concept of personalised nutrition. It is also important to stress that no compromise should be permitted in the regulations that ascertain the safety of newly developed products. REFERENCES
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2011 Society of Dairy Technology