INVITED REVIEW Bioactive Milk Peptides: A Prospectus1

D. A. Clare* and H. E. Swaisgood

*Department of Food Science Departments of Food Science and Biochemistry North Carolina State University Raleigh, NC 27695-7624

ABSTRACT Bioactive peptides have been identied within the amino acid sequences of native milk proteins. Hydrolytic reactions, such as those catalyzed by digestive enzymes, result in their release. These peptides directly inuence numerous biological processes evoking behavioral, gastrointestinal, hormonal, immunological, neurological, and nutritional responses. The specic bioreactions associated with each physiological class have been well characterized. Herein, we review the scientic literature and attempt to stimulate consideration of the continued use of bioactive peptides and their expanded development as a commercial product. Several applications have already evolved. For example, phosphopeptides derived from casein fractions are currently used as both dietary and pharmaceutical supplements. Potentially, the addition of bioactive peptides to food products could improve consumer safety as a result of their antimicrobial properties. Lastly, bioactive peptides may function as health care products, providing therapeutic value for either treatment of infection or prevention of disease. (Key words: bioactive peptide, milk proteins, functional foods) Abbreviation key: ACE = angiotensin converting enzyme, CPP = casein phosphopeptides. INTRODUCTION Milk contains components that provide critical nutritive elements, immunological protection, and biologically active substances to both neonates and adults. In general, the major protein fractions in bovine milk include -LA, -LG, caseins, immunoglobulins, lactoferrin, proteose-peptide fractions (heat-stable, acid soluble phosphoglycoproteins), and minor whey proteins such as transferrin and serum albumin. From these, bioactive peptides may be generated in vivo through gastrointesti-

nal processes. Often, this liberation serves to inuence numerous physiological responses as a result of their hormone-like properties. These peptides, encoded within the sequences of native protein precursors, may also be generated in vitro by enzymatic hydrolysis. In this case, peptides are puried from protein hydrolysates by various separation techniques and assayed for bioactivity. Lastly, physiologically active peptides have been chemically synthesized to conrm the biological properties associated with a specic amino acid sequence. There is considerable evidence that many bioactive peptides serve in multifunctional capacities and often share common structural features based on a dened, biospecic role. Here, we review the primary classes of bioactive milk peptides, based on their specic physiological function, and provide a summary of general characteristics associated with each group. MILK DEFENSE PEPTIDES Antimicrobial Peptides The total antibacterial effect in milk is greater than the sum of the individual contributions of immunoglobulin and nonimmunoglobulin defense proteins. This is most likely due, at least in part, to their synergy. Another contributing factor may be the presence of naturally occurring bactericidal peptides, in addition to those generated from inactive protein precursors. Antimicrobial milk proteins, such as lactoferrin, were described in early literature (9). During this time, reports also detailed the discovery of basic glycopeptides with bactericidal activity against various strains of Staphylococcus aureus and Streptococcus (40). In general, their value for development as a commercial antimicrobial product was ignored. Recently, however, there has been a renewed interest in using bioactive peptides for application within the health care industry for these purposes. Casecidin, obtained by chymosin digestion of casein at neutral pH, was among the rst defense peptides actually puried and exhibited activity in vitro against Staphylococcus, Sarcina, Bacillus subtilis, Diplococcus pneumoniae, and Streptococcus pyogenes (41; Table 1). Casocidin-I (bovine milk), a cationic s2-CN derived peptide, inhibited growth of Escherichia coli and Staphylo-

Received October 15, 1999. Accepted December 26, 1999. Corresponding author: D. A. Clare; e-mail: debra_clare@ncsu.edu. 1 This review was solicited by the Milk Proteins and Enzyme Committee of ADSA, R. Jimenez-Flores, Chairperson. 2000 J Dairy Sci 83:11871195

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Table 1. Antimicrobial milk peptides. Milk peptide fragment Casecidin s1 and -CN (MW 40006000) Release protease Chymosin and chymotrypsin

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Gram (+) activity Staphylococcus Sarcina Bacillus subtilis Diplococcus pneumoniae Streptococcus pyogenes Staphylococcus carnosus

Gram () activity

Yeast and fungi*

Reference 41

Casocidin-I s2-CN (f 165203) Isracidin s1-CN (f 123) Lactoferricin B Lactoferrin (f 1741)

Synthetic peptide

Escherichia coli

103

Chymosin and chymotrypsin

Staphylococcus aureus

Candida albicans

41

Pepsin

Bacillus Listeria Streptococci Staphylococci

E. coli 0111 E. coli 0157H:7 Klebsiella Proteus Pseudomonas Salmonella

Candida albicans Dermatophytes: *Cryptococcus uniguttulatus *Penicillum pinophilum *Trichophyton mentagrophytes

4, 79, 89

coccus carnosus (103). Isracidin, an N-terminal segment of s1-CN B, protected mice against Staphylococcus aureus and Candida albicans. This peptide also safeguarded sheep and cows against mastitis when injected into the udder at levels comparable to those observed with standard antibiotic treatment (41). Previous reports have focused on the antimicrobial peptides generated by proteolytic digestion of bovine lactoferrin. The hydrolysate was active against both Grampositive (Bacillus, Listeria, and Streptococcus) and Gram-negative (E. coli, Klebsiella, Salmonella, Proteus, and Pseudomonas) microorganisms in vitro. A pathogenic intestinal bacterium, E. coli 0111, was additionally found to be susceptible (89). A potent bactericidal peptide specically generated by pepsin degradation of lactoferrin, so named lactoferricin B, also displayed antimicrobial activity towards both Gram-positive and Gram-negative microorganisms (32, 89). In recent literature, studies indicated that lactoferricin B was active against clinical isolates of enterohaemorrhagic E. coli 0157H:7 at concentrations signicantly less than either the lactoferrin hydrolysate or lactoferrin, itself (79). These properties appear to be correlated with the net positive charge of the peptide, which may kill susceptible microorganisms by increasing cell membrane permeability. Present communications offer direct evidence for the generation of lactoferricin in human stomach after ingestion of lactoferrin (39). There is conicting data, however, regarding the in vivo antimicrobial properties of the peptide, since the addition of 5% cows milk completely ameliorated much of the activity. The bactericidal properties of lactoferricin were also reduced by adding inJournal of Dairy Science Vol. 83, No. 6, 2000

creasing concentrations of mucin to the assay mixture (32). Milk also contains peptides that exhibit antifungal properties (Table 1). Antifungal activity of lactoferrin or its peptides (ex. lactoferricin B), in combination with azole antifungal agents, has been demonstrated with Candida albicans (3, 90). Several lamentous fungi, including agents of skin disease (dermatophytes), were also found to be susceptible to this mixture (4). The most recent data showed that lactoferrin-related compounds, combined with triazoles, inhibited growth of azole-resistant C. albicans hyphae (91). PHYSIOLOGICALLY ACTIVE MILK PEPTIDES In addition to providing immunodefense systems, milk also contains other major peptide fractions that elicit behavioral, neurological, physiological, and vasoregulatory responses (Table 2). Often, the peptide displays multifunctional properties. Several articles reviewing this topic have already been published (34, 53, 77, 87). Here, we categorize classications of physiologically active peptides based on their primary biofunction. Antihypertensive Peptides (ACE Inhibitors) Antihypertensive peptides inhibit the angiotensin converting enzyme (ACE) (61, 62, 92). ACE is a peptidyldipeptidase that cleaves dipeptides from the carboxy terminal end of the substrate. ACE converts angiotensin I to angiotensin II, increasing blood pressure and aldersterone, and inactivating the depressor action of bradykinin. ACE inhibitors derived from casein, or casokinins,

Table 2. Examples of physiologically active milk peptides. Sample number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1 2

Peptide sequence1 FFVAP AVPYPQR YGLF ALPMHIR KVLPVPQ MAIPPKKNQDK KDQDK KRDS QMEAES*IS*S*S* EEIVPNS*VEQK LLY FKCRRWQWRMK KLGAPSITCVRR AF YQQPVLGPVR RYLGYLE YGFQNA YLLF NH2 YIPIQYVLSR [YVPF PPF] YLGSGY-OCH3

Name s1-Casokinin-5 -Casokinin-7 -Lactorphin -Lactorphin Antihypertensive peptide Casoplatelin Thrombin inhibitory peptide Thrombin inhibitory peptide Caseinophosphopeptide Immunopeptide Lactoferricin B -Casokinin-10 -Casein exorphin Serorphin -Lactorphin (amide) Casoxin C Casoxin D Lactoferroxin A

AA2 segment s1-CN (f 2327) -CN (f 177183) -LA (f 5053) -LG (f 142148) -CN (f 169174) -CN (f 106116) -CN glycomacropeptide (f 112116) Lactotransferrin (f 3942) s1-CN (f 5979) -CN (f 191193) Lactoferrin (f 1741) -CN (f 193202) s1-CN (f 9096) BSA (f 399404) -LG (f 102105) -CN (f 2534) s1-CN (f 158164) Lactoferrin (f 318 323)

Physiological classication ACE inhibitor ACE inhibitor ACE inhibitor and opioid agonist ACE inhibitor Antihypertensive peptide Antithrombotic Antithrombotic Antithrombotic Calcium binding and transport Immunostimulatory (+) Immunomodulatory (+) and antimicrobial Immunomodulatory (+/) & ACE Inhibitor Opioid agonist Opioid agonist Opioid agonist = ACE Inhibitor Opioid antagonist Opioid antagonist Opioid antagonist

Release protease Proline endopeptidase Trypsin Synthetic peptide Trypsin Lactobacillus CP790 protease & synthetic peptide Trypsin & synthetic peptide Trypsin Pepsin Trypsin Synthetic Pepsin Synthetic Pepsin Pepsin Synthetic or Trypsin Trypsin Pepsin-chymotrypsin Pepsin

Reference 46 46 59 60 44

INVITED REVIEW BIOACTIVE PEPTIDES

30 71 70 77 57 2, 58 55 43 84 59 14 96 93

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The one-letter amino acid codes were used; S* = Phosphoserine. Amino acid.

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have been identied within the sequences of human and -CN (37, 38). They are also generated by tryptic digestion of bovine s1- and -CN (47, 55, 76). The Cterminal tripeptide sequence is the primary structural feature governing this inhibitory response (45), and reports indicated that the ACE binding pocket exhibited a preference for hydrophobic amino acids at each of these sites (52). A second characteristic of ACE inhibitory casokinins is the presence of a positively charged lysine or arginine at the carboxy terminal end (52). It was shown that removal of this critical amino acid residue from bradykinin, an endogenous ACE inhibitor, resulted in production of an analogue that was essentially inactive (14). ACE inhibitory peptides are also derived from both s1- and -CN that are generated by the hydrolysis of sour milk with the Lactobacillus helveticus CP790 extracellular protease. These peptides exhibited antihypertensive activity in spontaneously hypertensive rats as monitored by systolic blood pressure (44, 62, 92, 93). A synthetic seven amino acid peptide, equivalent to a segment found in the -CN hydrolysate, exhibited potent antihypertensive activity in these rats over an 8-h interval after oral administration (44). A third subclass, lactorphins, are sequestered within the primary amino acid sequence of bovine -LG and released by trypsin (60). Lastly, novel angiotensin-I converting enzyme (ACE) inhibition was detected in synthetic peptides that corresponded to sequences within both -LG and -LA (59). Antithrombotic Peptides Antithrombotic peptides are present in milk. Early on, it was learned that the mechanisms involved in milk clotting, dened by the interaction of -CN with chymosin and blood clotting processes, dened by the interaction of brinogen with thrombin, were comparable. In this regard, the C-terminal dodecapeptide of human brinogen -chain (residues 400 to 411) and the undecapeptide (residues 106 to 116) from bovine -CN are structurally and functionally quite similar. This casein-derived peptide sequence, termed casoplatelin, affected platelet function and inhibited both the aggregation of ADP-activated platelets and the binding of human brinogen -chain to its receptor region on the platelets surface (30). A smaller -CN fragment (residues 106 to 110), casopiastrin, was obtained from trypsin hydrolysates and exhibited antithrombotic activity by inhibiting brinogen binding (29, 30, 49). A second segment of the trypsin -CN fragment, residues 103 to 111, inhibited platelet aggregation but did not affect brinogen binding to the platelet receptor (19, 21, 30). Later, it was reported that biologically active peptides, isolated from both caJournal of Dairy Science Vol. 83, No. 6, 2000

sein and lactotransferrin, inhibited platelet function (20, 50). Antithrombotic peptides have also been derived from -caseinoglycopeptides that were isolated from several animal species. Bovine -caseinoglycopeptide, the C-terminal end of -CN (residues 106 to 169), inhibited von Willebrand factor-dependent platelet aggregation (12). Two antithrombotic peptides, derived from human and bovine -caseinoglycopeptides, have been identied in the plasma of 5-d-old newborns after breast-feeding and ingestion of cows milk based formula, respectively (10). The C-terminal residues (106 to 171) of sheep -casein, or -caseinoglycopeptide, decreased thrombin- and collagen-induced platelet aggregation in a dose dependent manner (71). Lastly, thrombin-induced platelet aggregation was inhibited with pepsin digests of sheep and human lactoferrin. A single peptide peak containing this activity was obtained by reverse-phase chromatography of the hydrolysate (70). Caseinophosphopeptides Casein phosphopeptides (CPP) have been identied after trypsin release from s1-, s2-, and -CN (34). The phosphate residues, which are present as monoesters of serine, occur mainly in clusters. Most CPP contain three serine phosphate clusters followed by two glutamic acid residues, form soluble organophosphate salts, and probably function as carriers for different minerals, especially calcium (53, 75). These fractions exhibit different degrees of phosphorylation, and a direct relationship between the degree of phosphorylation and mineral chelating ability has been described (34). In this event, s2CN > s1-CN > -CN > -CN; however, the distribution of their phosphoserine clusters is not uniform. It was further demonstrated that the specic amino acid composition associated with the phosphorylated binding site also inuences the degree of calcium binding (1). CPP are mostly resistant to enzymatic hydrolysis in the gut and most often found in a complex with calcium phosphate (73). This complex formation results in an increased solubility which, in turn, provides enhanced absorption of calcium across the distal small intestines of animals fed casein diets in comparison to control animals fed soy-based diets (35, 99, 100). This passive transport system is the primary means of calcium absorption under physiological conditions and provides calcium required for bone calcication (53). Caseinophosphopeptides also inhibit caries lesions through recalcication of the dental enamel. Hence, their application in the treatment of dental diseases has been proposed (72).

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Immunomodulatory Peptides Immunomodulatory milk peptides affect both the immune system and cell proliferation responses. As discussed previously, -casokinins inhibit ACE enzymes that are responsible for inactivating bradykinin, a hormone with immune enhancing effects. Thus, this chain of events indirectly produces an overall immunostimulatory response. Peptides derived from casein hydrolysates were shown to increase phagocytotic activity of human macrophages against aging red blood cells and augment phagocytosis of sheep red blood cells by murine peritoneal macrophages in vitro (21, 31, 57). Immunostimulatory activity against Klebsiella pneumoniae was demonstrated in vivo using rats treated intravenously with a hexapeptide obtained by hydrolysis of human -CN (57, 65). Most recently, lactoferricin B, obtained by hydrolysis of lactoferrin with pepsin, was found to promote phagocytic activity of human neutrophils via dual mechanisms that may involve direct binding to the neutrophil and opsonin-like activity (58). Small peptides, corresponding to the N-terminal end of bovine -LA (dipeptide) and -CN (tripeptide), signicantly increased proliferation of human peripheral blood lymphocytes (33), while the C-terminal sequence of bovine -CN (193 to 209), obtained by hydrolysis with pepsin-chymosin, induced a similar response in rats (16). Bioactive peptides in yogurt preparations actually decreased cell proliferation with IEC-6 or Caco-2 cells. This report may explain, in part, why consumption of yogurt has been associated with a reduced incidence of colon cancer (23). Kayser and Meisel (33) have described both stimulatory and suppressive immune responses of human lymphocytes to milk derived peptides. In general, the mechanisms by which these milk-derived peptides exert either their immunopotentiating effects or inuence proliferative responses are not currently known; however, one example suggests that the opioid milk peptide, -casomorphin (Table 3), may exert an inhibitory effect on the proliferation of human lamina propria lymphocytes in vitro via the opiate receptor (18). This antiproliferative response was reversed by the opiate receptor antagonist, naloxone. Opioid Milk Peptides The major opioid peptides are fragments of -CN, called -casomorphins, due to their exogenous origin and morphine-like properties (8, 26, 97); however, they have also been obtained from pepsin hydrolysis of bovine s1CN fractions (43, 54, 66, 102). Similar peptides have been reported from human -CN fractions (24, 98), and the Y-P-F sequence, which is common to bovine -casomorphin, was also found to be present in the primary structure of human -CN. Various synthetic derivatives

have been made and among these, Y-P-F-V-NH2 (valmuceptin) and Y-P-F(D)-V-NH2 (D-valmuceptin) show high afnity for their receptor (97). Brantl and co-workers reported opioid activity from synthetic tetra- and pentapeptide fragments of human -casein (7). Opioid peptides have been generated in vitro by enzymatic digestion of -caseins from cows, water buffalo, and sheep (68 and 77, 67, 74, respectively). In general, the - and -CN fragments produce agonist responses, while those derived from -CN elicit antagonist effects. Opioid peptides may be further subdivided into classications according to the specic milk protein from which they were derived, and these categories are summarized in Table 3. It is noteworthy that bioactive peptides are generated from most of the major proteins in both bovine and human milk. a. Structure and function. Typical opioid peptides, or endorphins, are derived from proenkephalin, propiomelanocortin, and prodynorphin and exhibit a denite N-terminal sequence Y-G-G-F (15, 86). Milkderived peptides, generated by hydrolysis of various precursor proteins such as - and -CN, -LA, and -LG, are called atypical, exomorphic, agonist peptides and exhibit morphine-like activity (102). Their primary structure (i.e., Y-X1-F or Y-X1-X2-F or Y) differs from the amino terminal sequence of the typical endogenous opioid peptide dened above. With the exception of s1CN, most share a common sequence feature, dened by a N-terminal tyrosine residue, that is absolutely essential for activity (13, 27). Typically, a second aromatic amino acid residue, such as phenylalanine or tyrosine, is also present in the third or fourth position. This structural motif ts well into the binding pocket of the opioid receptor. One of the most potent milk-derived opioid peptides, -casomorphin-4-amide (or morphiceptin), also contains a proline that is crucial for its function. This residue reportedly maintains the proper orientation of the tyrosine and phenylalanine side chains (56). Exorphins have been isolated from peptic hydrolysates of -casein fractions as well. In general, their structures differ considerably from those of -caseinomorphins. Active fractions were shown to be a mixture of two separate peptides derived from -casein fragments #9095 and #9096. The sequences were determined as listed, [R90Y-L-G-Y-L95-(E96)], in which case the latter peptide proved to be more effective. The N-terminal arginine residue was also reported to be essential for activity (43). b. Opioid agonists. -Casein peptides were among the rst reported opioid peptides (8). -Casomorphins are fragments corresponding to the 60 to 70th amino acid residues of bovine -CN, considered the strategic zone, and are classied as -type receptor ligands (36). Three exorphins, derived from bovine s1-CN, were shown to be selective for -receptors (43). Certain proteoJournal of Dairy Science Vol. 83, No. 6, 2000

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Table 3. Opioid milk peptides. Protein substrate Bovine s1-CN Human s1-CN Human -CN Bovine and human -CN Bovine and human -LA Bovine -LG Lactotransferrin BSA Peptide name

CLARE AND SWAISGOOD

Amino acid segment f f f f f f f f 9095, f 9096, and f 9196 158164 5154 and f 5155 2534, f 3541, and f 5760 5053 102105 318323, F 536540, and f 673679 399404

References 43 96 7 97, 95, 14 15, 19 21, 97 83 84

s1-Casein exorphins Casoxin D -Casomorphin (4,5) Casoxin A, B, and C -Lactorphin -Lactorphin Lactoferroxins A, B, and C Serophin

lytic bacteria, such as Pseudomonas aeruginosa and Bacillus cereus, also produce high levels of -casomorphins when inoculated and grown in milk (25). -Caseinomorphins are resistant to enzymes of the gastrointestinal tract and have been detected in vivo in the intestinal chyme of minipigs (51) and human small intestines (81). Because their absorption in the gut has not been observed in adults, it is generally concluded that the physiological inuences are limited to the gastrointestinal tract with important effects on intestinal transit time, amino acid uptake, and water balance (78, 86). Once they enter the bloodstream, they are rapidly degraded (53). In contrast, passive transport of -caseinomorphins across intestinal mucosal membranes does occur in neonates, which may experience physiological responses such as an analgesic effect on the nervous system resulting in calmness and sleep in infants (80). A precursor of -casomorphin was reported in the plasma of newborn calves and infants after ingestion of bovine milk (85). In pregnant or lactating women, -casomorphins originate in the milk (5, 94), pass through the mammary tissue, and possibly inuence the release of prolactin and oxytocin (86). More recently, Chabance et al. (11) showed that many peptides derived from s1-, -, or -CN, and -caseino-glycomacropeptide can be detected in the stomach of adults after consumption of milk or yogurt. Casomorphins, as opioid ligands, modulate social behavior (64, 66), increase analgesic behavior (48, 66), prolong gastrointestinal transient time by inhibiting intestinal peristalsis and motility (88), exert antisecretory (antidiarrheal) action (17), modulate amino acid transport (6), and stimulate endocrine responses such as the secretion of insulin and somatostatin (54). Opioid-like milk peptides also play a regulatory role regarding appetite by modifying endocrine activity of the pancreas, resulting in an increase of insulin output (63). Presently, data suggest that intracerebroventricular -casomorphin1-7 stimulates uptake of a high fat diet in rats fasted overnight. Enterostatin inhibited this effect, as did naloxone, a general opioid antagonist. Ligand binding studies indicated that at high dosages, -casomorphin1-7 and
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enterostatin may interact with the same low afnity receptor to modulate intake of dietary fat (42). c. Opioid antagonists. Opioid antagonists suppress the agonist activity of enkephalin. Yoshikawa et al. (95) reported that a chloroform and methanol extract from a peptic digest of bovine -CN bound to opioid receptors of rat brain. The peptide was methylated at the C-terminal end and exhibited antagonist effects selective for the - and -type of opioid receptor. The peptide was thus named casoxin. Casoxins A and B have been chemically synthesized and correspond to amino acid sequences within both bovine and human -CN (14, 15). Casoxin C is an opioid antagonist, obtained from tryptic digests of bovine -CN (14), that also functions as an agonist for C3a receptors (82). Lastly, casoxin D, puried from human s1-CN fractions, elicits an opioid antagonist response (96). In general, the chemically modied casoxins are more active that their nonmethylated derivatives (14, 15). Lactoferroxins are antagonists generated from human lactoferrin (83). Initially, a chloroform and methanol extract from a peptic digest of lactoferrin was assayed for activity, and the results indicated that the opioid properties were similar to those of naloxone, a known antagonist ligand. Peptides derived from pepsin digestion, alone, were minimally effective, while those puried from a methyl-esteried fraction were signicantly more potent. HPLC analyses resulted in purication of three separate active fractions designated lactoferroxin A, B, and C, respectively. It was determined that the -carbonyl group of each was methyl esteried based on comparison of bioactivity measurements and HPLC retention times to those of corresponding synthetic peptides. Like casoxins, the chemically modied peptides may not actually exist in vivo. Lactoferroxin A, residues 318 to 323, showed a preference for -receptors. On the other hand, lactoferroxin B and C, derived from residues 536 to 540 and 673 to 679, respectively, exhibited a higher propensity for -receptors (83). Miscellaneous Peptides Physiologically active peptides that directly affect gastrointestinal functions have also been documented. Ca-

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somorphins slow gastric motility and emptying in nonruminants (22), while caseinomacropeptide, a 64-amino acid glycopeptide released from -CN by gastric proteases, exerts its effects on digestive function by inhibiting gastric acid secretions (101). Several other milk-derived peptides have been described in the literature. Atrial natriuretic factor, or atriopeptin, is a peptide found naturally occurring in human milk (28). This peptide functions as a strong diuretic, natriuretic, and vasorelaxant, and plays an important role in circulatory adaptation to extrauterine life. More recently, a peptide, obtained by in vitro proteolysis of bovine -LG, was found to exert its effect on smooth muscle (69). CONCLUSIONS In summary, milk contains numerous peptide sequences that affect crucial physiological functions and modulate many regulatory processes. These include, but are not limited to hormone secretion (casomorphins), immune defense (casokinins, casomorphins, and immunopeptides), nutrient uptake (phosphopeptides and casomorphins), and neurological information transmission (casokinins). Industrial-scale processes are already established for the recovery of nondenatured, biologically active, whey proteins that are produced as a direct byproduct during cheese manufacturing. Caseins are manufactured on an equally large scale for food products such as cottage cheese. Thus, it should be economically possible to salvage these protein fractions and generate active peptides from them for use as dietary supplements, safe and effective natural preservatives, and milk-based nutraceuticals. This eld of research promises to contribute novel biologically active peptides derived from milk proteins that signicantly impact both the food and health care industries. ACKNOWLEDGMENTS This work was partially supported by the Southeast Dairy Foods Research Center, Dept. of Food Science, North Carolina State University, Raleigh, NC, project #5-44408. The authors would like to gratefully acknowledge Hans Meisel for his signicant scientic contributions to this eld of research and the careful reviewing of this manuscript. REFERENCES
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