Overview on Milk Protein-derived Peptides

PII : S0958-6946(98)00059-4

Int. Dairy Journal 8 (1998) 363—373 ( 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$19.00#0.00

Overview on Milk Protein-derived Peptides Hans Meisel* Federal Dairy Research Centre, Institute for Chemistry and Physics, D-24121 Kiel, P.O. Box 6069, Germany ABSTRACT Milk protein-derived bioactive peptides include a variety of substances which are potential modulators of various regulatory processes in the body; many peptides reveal multifunctional bioactivities. Opioid peptides are opioid receptor ligands with agonistic or antagonistic activities. Angiotensin converting enzyme (ACE)-inhibitory peptides can exert an antihypertensive effect. Immunomodulating casein peptides stimulate proliferation of human lymphocytes and phagocytic activities of macrophages. Antimicrobial peptides kill sensitive microorganisms. Antithrombotic peptides inhibit fibrinogen binding to a specific receptor region on the platelet surface and also inhibit aggregation of platelets. Caseinophosphopeptides can form soluble organophosphate salts and may function as carriers for different minerals, especially calcium. In relation to their mode of action, bioactive peptides may reach target sites (e.g., receptors and enzymes) at the luminal side of the intestinal tract or, after absorption, in peripheral organs. Milk-derived peptides can be produced on an industrial scale and as a consequence these peptides have already been considered for application both as dietary supplements in ‘functional foods’ and as drugs. Hence, these peptides are claimed to be health enhancing nutraceuticals for food and pharmaceutical preparations. ( 1998 Elsevier Science Ltd. All rights reserved Keywords: bioactive peptides; milk proteins; functional foods; nutraceuticals

INTRODUCTION

BIOLOGICAL ACTIVITIES AND BIOCHEMICAL PROPERTIES

From the nutritional point of view, the protein fraction of milk contains many valuable components and biologically active substances. In the last two decades, a number of studies have been performed on bioactive peptides that are present in the amino acid sequence of milk proteins. Although other animal, as well as plant, proteins contain potential bioactive sequences, milk proteins are currently the main source of a range of biologically active peptides (Table 1). The structures of biologically active sequences were obtained from in vitro enzymatic and/or by in vivo gastrointestinal digests of the appropriate precursor proteins: Chemical synthesis has also been carried out to confirm the sequence of potential bioactive peptides. Milk protein-derived bioactive peptides are inactive within the sequence of the parent protein and can be released by enzymatic proteolysis, for example, during gastrointestinal digestion or during food processing. Once they are liberated in the body, bioactive peptides may act as regulatory compounds with hormone-like activity. Thus, these peptides represent potential health enhancing components for food and pharmaceutical applications. The following overview is based on recent reviews on the specific biochemical properties and possible dietary and pharmaceutical applications of peptides derived from milk proteins (Meisel, 1997a, b; Meisel and Schlimme, 1996).

Many milk-derived peptides reveal multifunctional properties: i.e., specific peptide sequences having two or more different biological activities have been reported (Table 2). For example, some regions in the primary structure of b-casein contain overlapping peptide sequences which exert different biological effects (Fig. 1). These regions have been considered as ‘strategic zones’ (Fiat and Jolle`s, 1989) which are partially protected from proteolytic breakdown. Opioid agonistic and antagonistic activities Opioid peptides, i.e., opioid receptor (k-, d- and i-type) ligands with agonistic activity, originate from different milk proteins and exert naloxone-inhibitable opioid activities in both receptor studies and during bioassays (Brantl et al., 1981). Milk-protein derived opioid peptides prolong gastrointestinal transit time, exert anti-diarrhoeal action (Daniel et al., 1990a, b), modulate intestinal transport of amino acids (Brandsch et al., 1994) and influence postprandial metabolism by stimulating secretion of insulin and somatostatin (Schusdziarra, 1983a, b). After intracerebral administration to experimental animals, opioid peptides modulate social behavior (Panksepp et al., 1984; Paroli, 1988) and produce analgesia (Chang et al., 1982; Matthies et al., 1984). The major exogenous opioid peptides, i.e. b-casomorphins, are fragments of the b-casein sequence 60—70 (Tyr—Pro—Phe—Pro—Gly—Pro—Ile—Pro—Asn—Ser—Leu) and have been characterized as k-type ligands (Teschemacher and Brantl, 1994). b-Casomorphin-5 and -7 were described as the first food-derived opioid peptides.

*Tel.: #431 609 2260; fax: #431 609 2390; e-mail: meisel@ bafm.de. 363

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Table 1. Bioactive Peptides Derived from Milk Proteins Bioactive peptide

Protein precursor

Casomorphins a-Lactorphin b-Lactorphin Lactoferroxins Casoxins Casokinins Immunopeptides

a-, b-Casein a-Lactalbumin b-Lactoglobulin Lactoferrin i-Casein a-, b-Casein a-, b-Casein

Lactoferricin Casoplatelins Phosphopeptides

Bioactivity

Opioid agonist Opioid agonist Opioid agonist Opioid antagonist Opioid antagonist ACE-inhibitory Immunomodulatory Lactoferrin Antimicrobial i-Casein, transferrin Antithrombotic a-, b-Casein Mineral binding

Afterwards, b-casomorphins were found in analogous positions in sheep, water buffalo and human b-casein (for review: Fiat and Jolle`s, 1989; Schlimme and Meisel, 1995). Three a-casein-derived exorphins corresponding to bovine a -casein peptide fragments 90—96 41 (Arg—Tyr—Leu—Gly—Tyr—Leu—Glu), 90—95 and 91—96 are d-selective receptor ligands (Loukas et al., 1983, 1990). Other milk protein-derived opioids such as a-lactorphin (Tyr—Gly—Leu—Phe ) NH ) and b-lactorphin (Tyr—Leu— 2 Leu—Phe ) NH ) correspond to peptide fragment 50—53 2 in both bovine and human a-lactalbumin and to fragment 102—105 in bovine b-lactoglobulin, respectively (Chiba and Yoshikawa, 1986). Another whey proteinderived opioid peptide, serorphin, was isolated from fragment 399—404 (Tyr—Gly—Phe—Asn—Ala) of serum albumin (Tani et al., 1994). The ‘atypical’ opioid peptides derived from milk proteins have N-terminal sequences different from that of the ‘typical’ endogenous opioid peptides, e.g., enkephalins, endorphins and dynorphins (Teschemacher and Brantl, 1994). The common structural feature among endogenous and exogenous opioid peptides is the presence of a tyrosine residue at the amino terminal end (except a-casein opioids) and the presence of another aromatic residue, e.g., phenylalanine or tyrosine, in the third or fourth position. This is an important structural motif that fits into the binding site of the opioid receptors. The negative potential, localized in the vicinity of the phenolic hydroxyl group of tyrosine, seems to be essential for opioid activity. Lack of the tyrosine residue results in a total absence of bioactivity (Chang et al., 1981). Opioid antagonists have been found in bovine and human i-casein (casoxins) and in a -casein (Chiba et al., 41 1989; Yoshikawa et al., 1994). Furthermore, the opioid antagonist lactoferroxin has been found in human lactoferrin (Yoshikawa et al., 1988). Various synthetic casoxins were isolated as C-terminally methoxylated peptides, e.g., the derivatives corresponding to the i-casein sequences 33—38 (Ser—Arg—Tyr—Pro—Ser—Tyr ) OCH ), 34—38 and 35—38 (Chiba and Yoshikawa, 1986). 3 The chemically modified casoxins were more active than the non-methoxylated fragments. The tryptic fragment corresponding to residues 25—34 (Tyr—Ile—Pro—Ile— Gln—Tyr—Val—Leu—Ser—Arg) of bovine i-casein, known as casoxin C, showed a relatively high opioid antagonistic activity in comparison to the esterified peptides (Chiba et al., 1989). Casoxins are opioid receptor ligands of the k-type with relatively low antagonistic potency as

compared with naloxone. They also bind to i-receptors where extension of the N-terminal amino acid sequence beyond the tyrosine residue seems to influence the binding to i-type receptors. Inhibition of the angiotensin-converting enzyme (ACE) ACE is a multifunctional enzyme that is located in different tissues and plays a key physiological role in the regulation of local levels of several endogenous bioactive peptides (Ondetti and Cushman, 1982; Bruneval et al., 1986). ACE has been classically associated with the renin-angiotensin system regulating peripheral blood pressure where ACE-inhibition results in an antihypertensive effect. Moreover, inhibition of ACE may influence different regulatory systems of the organism involved in modulating blood pressure, immune defense and nervous system activity (Meisel, 1993a). Several food protein sources contain ACE-inhibitory peptides (for review: Ariyoshi, 1993; Meisel, 1993a). The casein-derived ACE-inhibitors, or casokinins, represent different fragments of human and bovine casein. For example, highly active casokinins are the bovine a 41 casein sequence 23—27 (Phe—Phe—Val—Ala—Pro; IC " 50 6 kmol L~1) and the b-casein sequence 177—183 (Ala—Val—Pro—Tyr—Pro—Gln—Arg; IC "15 kmol L~1). 50 The opioid fragment b-casomorphin-7 reveals a low ACE-inhibitory activity (Meisel and Schlimme, 1994). Recently, the opioid sequences corresponding to b-lactorphin and related dipeptides have also been identified as ACE-inhibitory peptides with moderate activities (Mullally et al., 1996; Table 2; Fig. 2). A tryptic fragment of b-lactoglobulin 142—148 (Ala—Leu—Pro—Met—His— Ile—Arg) showed a higher activity (IC "42.6 kmol L~1) 50 and was found to be relatively resistant to further digestion (Mullally et al., 1997). Although the structure-activity relationship of ACEinhibitory peptides has not yet been established, these peptides show some common features. ACE is predominantly an ectoenzyme with two catalytic sites, one on each lobe on the extracellular portion (Johnston, 1992). Structure—activity correlations among different peptide inhibitors of ACE indicate that binding to ACE is strongly influenced by the C-terminal tripeptide sequence of the substrate. The C-terminal tripeptide residues can interact with three subsites of ACE (Ondetti and Cushman, 1982). ACE appears to prefer substrates or competitive inhibitors containing hydrophobic (aromatic or branched side chains) amino acid residues at each of the three C-terminal positions. Most casokinins have proline, lysine or arginine as the C-terminal residue. Structure-activity data suggest that the positive charge on the guanidino or the e-amino group of the C-terminal arginine and lysine side chain, respectively, contribute substantially to the inhibitory potency (Cheung et al., 1980; Meisel, 1993a, b; Ariyoshi, 1993). The replacement of the arginine residue at the C-terminal end can result in essentially inactive analogues (Fig. 2). There is evidence that the catalytic sites of ACE may differ in several properties and may have different conformational requirements so that ACE inhibitors may inhibit only one catalytic site. It is claimed that the mechanism of ACE inhibition also involves interaction to subsites not normally occupied by substrates or to an anionic inhibitor binding site that is different from the catalytic sites of the enzyme.

Table 2. Examples for Multifunctional Milk Protein-derived Biologically Active Peptides Peptide sequence!

Fragment

Name

Biological activity Opioid IC " 50

ACE-inhibitory IC $ 50

Immunomodulatory % of control%

!21/#26

b-CN (f60—70) b-CN (f60—66)

b-Casomorphin-11 b-Casomorphin-7

10 14

500

YPFPG

b-CN (f60—64)

b-Casomorphin-5

1.1

0

PGPIPN YQQPVLGPVR YIPIQYVLSR

b-CN (f63—68) b-CN (f193—202) i-CN (f25—34)

b-Casokinin-10 Casoxin C

TTMPLW

a -CN (f194—199) 41

50 (antagonist)

300 # (not quantified) 16

YLGYLE YLLF YL

a -CN (f91—96) 41 b-LG (f102—105) a -CN (f91—92) 41 b-LG (f102—103) b-LG (f104—105) a-LA (f50—53)

LF YGLF YG

a-LA (f50—51) a-LA (f18—19) i-CN (f38—39)

b-Lactorphin

45# 160

a-Lactorphin

300

#122/#139 !28/#14

#121/#162

172 122 349 733 '1000

#87/#101

Meisel (1986) Brantl et al. (1981) Meisel (1993a) Kayser and Meisel (1996) Brantl et al. (1981) Meisel (1993a) Migliore-Samour et al. (1989) Meisel and Schlimme (1994) Chiba et al. (1989) Yoshikawa et al. (1994) Maruyama et al. (1987) Migliore-Samour et al. (1989) Loukas et al. (1983) Chiba and Yoshikawa (1986) Mullally et al. (1996) Mullally et al. (1996) Chiba and Yoshikawa (1986) Mullally et al. (1996) Mullally et al. (1996) Kayser and Meisel (1996)

Overview on milk protein-derived peptides

YPFPGPIPNSL YPFPGPI

References

!The one-letter amino acid codes were used. "IC values (kmol L~1) are given for peptide concentrations inhibiting [3H]-naloxone binding by 50%. 50 #IC value (kmol L~1) is given for peptide concentration inhibiting [3H]-dihydromorphine binding, instead of [3H]-naloxone, by 50%. 50 $IC values (kmol L~1) are given for peptide concentrations inhibiting the activity of angiotensin-converting enzyme (ACE) by 50%. 50 % Figures indicate % stimulation (#) and inhibition (!), respectively, in relation to control ("100); the minimum/maximum value is given.

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Fig. 1. Schematic represention of the multifunctional activities in milk protein-derived peptides; strategic zones in the primary structure of bovine b-casein.

Fig. 2. Inhibition of ACE activity by endogenous and milk protein-derived peptides (at 100 kmol L~1). The data represent the percentage inhibition of activity compared with control (no peptide inhibitor present) (13). BK"bradykinin (RPPGFSPFR); dR9"desArg9-Bradykinin; NT"neurotensin (pyroELYENKPRPYIL); SP"substance P (RPKPQQFFGLM ) NH ); CK10"b-casokinin-10; CM7"b-casomorphin-7; 2 CM5"b-casomorphin-5; AL"a-lactorphin; BL"b-lactorphin.

Immunomodulatory effects The bioactivity of immunopeptides has been characterized by different in vitro and in vivo test systems. Casein-derived immunopeptides including fragments of a -casein (residues 194—199; Thr—Thr—Met—Pro— 41 Leu—Trp) and b-casein (residues 63—68; Pro—Gly—Pro— Ile—Pro—Asn and 191—193; Leu—Leu—Tyr) stimulate phagocytosis of sheep red blood cells by murine peritoneal macrophages and exert a protective effect against Klebsiella pneumoniae infection in mice after intravenous administration of peptides (Migliore-Samour et al., 1989). It is noteworthy, that the immunohexapeptide derived from b-casein represents the C-terminal part of bcasomorphin-11. The immunopeptide sequence in human b-casein corresponds to residues 54—59 (Parker et al., 1984). The C-terminal sequence 193—209 of b-casein (containing b-casokinin-10; Table 2), which was obtained from

a pepsin-chymosin digest of bovine casein, induced a significant proliferative response in rat lymphocytes (Coste et al., 1992). Recently, Kayser and Meisel (1996) reported that the immunoreactivity of human peripheral blood lymphocytes (PBL) was either stimulated or suppressed by various bioactive peptides derived from milk proteins. The peptides Tyr—Gly and Tyr—Gly—Gly corresponding to fragments of bovine a-lactalbumin (e.g., the N-terminal end of a-lactorphin) and i-casein, respectively, significantly enhanced the proliferation of PBL at concentrations ranging from 10~11 to 10~4 mol L~1. The peptide Tyr—Gly exhibited 93% of maximal stimulation at 10~9 mol L~1, while Tyr—Gly—Gly showed 74% of maximal stimulation at 10~12 mol L~1. Depending on peptide concentration, b-casokinin-10 and b-casomorphin-7 showed a suppression as well as a stimulation of lymphocyte proliferation (Table 2). b-Casomorphin-7 inhibits the proliferation of human colonic lamina propria lymphocytes (LPL) where the anti-proliferative effect was reversed by the opiate receptor antagonist naloxone (Elitsur and Luk, 1991). The mechanism by which milk protein derived peptides exert their immunomodulatory effects is not yet defined. However, the results obtained with LPL suggest that opioid peptides may affect the immunoreactivity of lymphocytes via the opiate receptor. There is indeed a remarkable relationship between the immune system and opioid peptides, because opioid k receptors for endorphins are present on T lymphocytes and human phagocytic leukocytes (Wybran et al. 1979; Lopker et al., 1980; Faith et al., 1984). Furthermore, it is known that lymphocytes and macrophages express receptors for many biologically active mediators. It has been suggested that an arginine residue at the N- or C-terminal region may be the dominating entity recognized by specific surface membrane receptors (Pagelow and Werner, 1986). The immunomodulatory ACE-inhibitor b-casokinin-10 (Table 2) as well as several other ACE-inhibitory peptides have arginine as the C-terminal residue. Antimicrobial activity Antimicrobial peptides have been derived from the whey protein lactoferrin (Tomita et al., 1991). Lactoferrin is an iron-binding glycoprotein present in most biological fluids of mammals including milk. It is widely

Overview on milk protein-derived peptides

considered to be an important component of the host defense against microbial infections. However, the antimicrobial mechanism of lactoferrin is more complex than simple binding of iron. The existence of an antimicrobial sequence near the N-terminus of lactoferrin in a region distinct from its iron-binding sites has been reported (Bellamy et al., 1992). The peptide fragment 17—41 having one intramolecular disulfide bond is generated in vitro upon enzymatic cleavage of lactoferrin with pepsin. This peptide, named lactoferricin, has bactericidal properties more potent than undigested lactoferrin suggesting its much smaller size may facilitate access to target sites on the microbial surface. The antimicrobial activity of lactoferricin and synthetic analogs seems to be correlated with the net positive charge of the peptides. A distinctive feature of lactoferricin is the relatively high proportion and asymmetric clustering of basic amino acid residues: eight of the 25 residues are basic amino acids, and among them 6 basic residues are clustered at the N-terminus with a-helical propensity (Kang et al., 1996). It is known that cationic amphipathic a-helical structures are related to antimicrobial activity by forming ion channels through membrane bilayers (Agawa et al., 1991). These cationic peptides kill sensitive microorganisms by increasing cell membrane permeability. Hence, it is assumed that lactoferricins exert their antibacterial effect by a similar mechanism (Bellamy et al., 1993). Recently, an a -casein fragment (position 165—203) 42 containing a high proportion (10 of 39) of basic amino acid residues was found to be an antibacterial agent (Zucht et al., 1995, 1996). This antibiotic peptide, named casocidin-I, can inhibit the growth of Esherichia coli and Staphylococcus carnosus.

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exert an influence on absorption of calcium or other minerals and trace elements in the intestine. Furthermore, it has been shown that calcium-binding phosphopeptides can have an anticariogenic effect in that they inhibit caries lesions through recalcification of the dental enamel (Reynolds, 1987). About 30% of the phosphorous of milk is bound via monoester linkages to seryl residues of casein. Accordingly, several mineral binding caseinophosphopeptides, corresponding to different phosphorylated regions of a -, 41 a - and b-casein, have been isolated from enzymatic 42 casein digests (Berrocal et al., 1989; Meisel et al., 1991a). Most casein phosphopeptides contain a serine phosphate cluster and glutamyl residues in the sequence of three phosphoseryl groups followed by two glutamic acid residues. The negatively charged side chains, in particular the phosphate groups, of these amino acids represent the binding sites for minerals. However, different phosphopeptide fractions of casein have significant differences in their calcium binding activity (Fig. 2). The significant differences in their calcium binding activity can be attributed to the influence of further amino acids around the phosphorylated binding sites. PHYSIOLOGICAL SIGNIFICANCE Milk-protein derived bioactive peptides may function as exogenous regulatory substances. Although a number of studies indicate the regulatory potential of these peptides, their role as food hormones or ‘formones’ needs further clarification. To exert physiological effects in vivo, bioactive peptides must be released by proteolysis and then reach their target sites at the luminal side of the intestinal tract or, after absorption, in peripheral organs.

Antithrombotic activity Release of active peptides from milk proteins Casoplatelins are peptides derived from the C-terminal part (caseinoglycomacropeptide) of bovine i-casein. The casoplatelins are inhibitors of the aggregation of ADPactivated platelets as well as binding of human fibrinogen c-chain to a specific receptor site on the platelet surface (Jolle`s et al., 1986). The main antithrombotic peptides of i-casein are the sequence 106—116 (Met—Ala—Ile—Pro— Pro—Lys—Lys—Asn—Gln—Asp—Lys) and the smaller fragments 106—112, 112—116, 113—116 (Bouhallab et al., 1992). On a molecular level, the clotting of blood and milk shows a large number of similarities (Jolle`s and Caen, 1991). The interacting region of the fibrinogen c-chain in platelet aggregation is the C-terminal dodecapeptide sequence which itself possesses similar inhibitory effects as the tryptic i-casein fragments. Three amino acid residues (Ile108, Lys112, Asp115) of the aforementioned unodecapeptide of i-casein are, indeed, in homologous positions as compared with the c-chain sequence of human fibrinogen (Fiat et al., 1989). These residues seem to be important for the inhibitory effect which is due to the competition between antithrombotic peptides and the c-chain for the platelet receptors. Mineral binding properties Phosphopeptides can form soluble organophosphate salts and may function as carriers for different minerals, especially calcium (Sato et al., 1986). Hence they might

In addition to the possible liberation of bioactive peptides during intestinal proteolysis, such peptides may already be generated during manufacture of several milk products and thus be ingested as food components. For example, partially hydrolyzed milk proteins for hypoallergenic infant formulae and for clinical applications in enteral nutrition consist exclusively of peptides. Cheese contains phosphopeptides as natural constituents and secondary proteolysis during cheese ripening leads to formation of various ACE-inhibitory peptides (Meisel et al., 1997). A number of caseolytic bacterial species used in production of some types of cheese and other milk products can produce casomorphins (Hamel et al., 1985) as well as casokinins (Yamamoto et al., 1994). It is notable that structural and chemical changes occur during the processing of food proteins and may affect the profile of bioactive peptides subsequently produced during protein digestion. For example, heatinduced dephosphorylation of casein in milk can lead to both a structural change and a reduced level of phosphopeptides with the consequence that their potential regulatory effect as carriers for minerals will be impaired (Meisel et al., 1991b; Meisel and Schlimme, 1995). Furthermore, digestive enzymes react with structurally altered proteins as if they were different substrates. In particular, heat and/or alkali treatment can generate additional hydrolysis-resistant inter- and intramolecular covalent bonds (Liardon and Ledermann,

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H. Meisel Table 3. Maximum Yields of Bioactive Peptides Derived from Caseins and Whey Proteins

Bioactive peptides

Opioid agonists b-Casomorphin-5 a -Casein-Exorphin 41 a-Lactorphin b-Lactorphin! Serorphin Opioid antagonists Casoxin A Casoxin B Casoxin C ACE-inhibitory activities a -Casokinin-5 41 a - Casokinin-7 41 a - Casokinin-6" 41 b-Casokinin-7 b-Casokinin-10" Immunomodulatory activities a -Casokinin-6 41 b-Casokinin-10 b-Casein-fragment a Lactalbumin-fragment Antithrombic activities i-Casein-fragment i-Casein-fragment Mineral binding activities a -Caseinophosphate 1 a -Caseinophosphate 1 b-Caseinophosphate

1g precursor protein (fragment)

mg bioactive peptide

b-Casein a -Casein 41 a-Lactalbumin b-Lactoglobulin Bov. serum albumin

(f60—64) (f90—96) (f50—53) (f102—105) (f399—404)

27.2 43.3 39.0 33.3 11.9

i-Casein i-Casein i-Casein

(f35—42) (f58—61) (f25—34)

58.0 34.7 74.4

a -Casein 41 a -Casein 41 a -Casein 41 b-Casein b-Casein

(f23—27) (f28—34) (f194—199) (f177—183) (f193—202)

27.6 39.4 35.6 39.1 55.0

b-Casein a-Lactalbumin

(f191—193) (f18—19)

18.5 17.8

i-Casein i-Casein

(f103—111) (f113—116)

61.3 29.3

a -Casein 41 a -Casein 41 b-Casein

(f43—58) (f59—79) (f1—25)

32.4 45.8 48.1

!Acts also as ACE-inhibitor. "Shows also immunomodulatory activities.

1986; Friedman, 1992). Such processing conditions also promote the racemic conversion of L amino acids into D isomers and, consequently, lead to undigestible peptide bonds. The possible formation of undigestible peptide sequences during food processing merits special attention, since this may promote both formation and absorption of bioactive peptides which do not occur naturally in the precursor protein. After ingestion of 1 g casein and whey protein, respectively, a maximum luminal formation of relatively high amounts of bioactive peptides is possible (Table 3). Bioactive peptides that have been produced by limited proteolysis during processing and/or intestinal digestion of milk proteins could be further digested by intestinal proteinases or brush-border peptidases. However, evidence for the liberation of b-casomorphins (Svedberg et al., 1985; Meisel and Frister, 1989) and casein phosphopeptides (Meisel and Frister, 1988; Kasai et al., 1992) from casein into the intestinal lumen of mammals after intake of milk or a casein-containing diet, i.e., under in vivo conditions, has already been obtained. The opioid b-CN(f60—70) and the phosphorylated a -CN(f66—74)3P 41 were isolated and chemically characterized from the soluble part of intestinal chyme in Go¨ttingen minipigs (Meisel, 1986; Meisel and Frister, 1988). A similar phosphopeptide a -CN(f59—79)5P obtained from 41 tryptic casein hydrolysate also showed relatively high in vivo stability (Brommage et al., 1991). This fragment was more slowly dephosphorylated than other fragments

and was the most abundant phosphopeptide in the ileum of rats. Intestinal and peripheral target sites of milk-protein derived peptides Orally ingested peptides and intestinal proteolytic products may produce local effects in the gastrointestinal tract or they may enter peripheral blood intact and exert systemic effects (for review: Gardner, 1984). Di- and tripeptides, such as the immunopotentiatory Tyr—Gly and Tyr—Gly—Gly as well as several ACE-inhibitors, can pass across the intestine in quantitatively significant amounts to reach peripheral target sites. Inhibition of ACE which is located in different tissues (e.g. plasma, lung, kidney, heart, skeletal muscle, pancreas, brain, mammary arteries, testes, uterus, intestine) may influence different regulatory systems of the organism (Bruneval et al., 1986; Johnston, 1992; Meisel, 1993a). When ACE-inhibitory peptides were orally given to rats, blood pressure was reduced in a dose-dependent manner (Suetsuna and Osajima, 1989). The possible interaction of casokinins with ACE located as a brush border membrane-bound enzyme (Ondetti et al., 1982) on the luminal surface of human jejunum may affect electrolyte and water transport as a result of the decreased activity of angiotensin II (Levens,1986; Stevens, 1988). Opioid receptors are located in the nervous, endocrine and immune systems as well as in the intestinal tract of

Overview on milk protein-derived peptides

the mammalian organism and can interact with their endogenous ligands as well as with exogenous opioids and opioid antagonists (Teschemacher and Brantl, 1994). Orally given opioid peptides derived from milk proteins are able to modulate absorption processes in the gut. The enhancement of net water and electrolyte absorption by b-casomorphins in the small and large intestine is a major component of their antidiarrhoeal action which could be mediated via subepithelial opioid receptors or specific luminal binding sites at the brush border membrane (Tome´ et al., 1987, Brandsch et al., 1994). b-Casomorphins are claimed to be rapidly degraded once they enter the blood stream. However, the presence of bcasomorphin-7 immunoreactive material has been demonstrated in the plasma of newborn calves after their first milk intake (Umbach et al., 1985). This material revealed a greater molar mass than b-casomorphin-7 and thus has been considered as a b-casomorphin precursor. Such pre-casomorphins could reach any potential site of action in the organism to elicit effects after liberation of the protected active sequence from the precursor molecule. Opioid casein fragments have not been detected in the plasma of adult mammals (Umbach et al., 1985; Teschemacher et al., 1986). Thus, only the neonatal intestine appears to be permeable to (pre-) casomorphins. It has been shown that calcium binding phosphopeptides can have an anticariogenic effect in that they inhibit caries lesions through recalcification of the dental enamel (Reynolds, 1987). The formation of soluble complexes with calcium and the proteolytic resistance of phosphopeptides in the intestinal lumen is a prerequisite for their function as mineral carriers, because—under normal physiological conditions—the paracellular transport of calcium plays a major role, independent of vitamin D, in calcium absorption from the distal small intestine (for review: Kitts and Yuan, 1992). Hence they might exert an influence on the absorption of calcium or other minerals and trace elements in the intestine. There is considerable controversy as to the physiological significance of the enhancement of calcium absorption by casein phosphopeptides. This discrepancy is obviously due to differences in the composition of phosphopeptide preparations (mostly chemically not well defined mixtures) which can differ significantly in mineral binding activity (Meisel, 1997a). Endogenous release of the active substance Irrespective of the potential function of milk-derived peptides as exogenous regulators, it is also possible that certain peptides from milk may influence directly the maternal organism. For example, casomorphins can be liberated in the mammary gland, transferred to the blood and then reach endogenous opiate receptors (Assarga> rd et al., 1994; Koch et al., 1994). In this way, casomorphins may participate in the endocrine regulation of pregnancy, e.g., by stimulation of prolactin release (Yen et al., 1985). Another target of casomorphins in pregnant or lactating mammals may be the cardiovascular system. These peptides can exert a positive inotropic and antiarrhythmic effect and thus may have a cardioprotective function (Mentz et al., 1990). It is not yet clear how such effects are of physiological importance.

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POTENTIAL USES IN FUNCTIONAL FOODS AND PHARMACEUTICALS Even if some bioactive peptides are not released under physiological conditions in vivo, they could be produced commercially and used as nutraceuticals. A nutraceutical is any substance that is a food or a part of a food that provides medical or health benefits, including prevention and treatment of disease (DeFelice, 1995). However, it is advisable to make a clear distinction between health enhancing nutraceuticals for prevention and those for treatment of disease where pharmacologically active compounds (drugs) are needed. Nutraceuticals for disease prevention are suitable as ingredients in functional foods. A physiologically functional food has been defined as a food derived from naturally occurring substances that can and should be consumed as part of the daily diet, and which serves to regulate or otherwise affect a particular bodily process when ingested (Schmidl, 1993). Functional foods should not be confused with medical food products that are designed to supply missing nutrients or to treat patients suffering from dietrelated diseases. Casein-derived peptides, which can be manufactured on industrial scale, have already been considered for interesting applications both as dietary supplements and as pharmaceutical preparations. Preparations of casein phosphopeptides were obtained from enzymatic hydrolysates by ion exchange chromatography (Kunst, 1992) or by aggregation of hydrolysate peptides with bivalent cations in combination with ultrafiltration (Brule et al., 1982). Phosphopeptide mixtures are commercially available as spray-dried peptide powders. Regarding the possible application of casein phosphopeptides as mineral carriers, they have been proposed for use in dietary products such as bread, cake, flour, beverages, chewing gum and in pharmaceutical preparations such as tablets, toothpaste and dental filling material (Reynolds, 1987). The pharmaceutical products are intended for use in the treatment of dental disease and for rarefying bone disease. b-Casomorphins have also been produced by genetic engineering techniques followed by enzymatic or chemical cleavage of the microbial fusion protein to liberate the required peptide (Carnie et al., 1989). These recombinant b-casomorphins are intended for oral administration in order to increase animal performance, e.g., weight gain or milk yield. Several attempts were made to synthesize modified b-casomorphin sequences in order to find pharmacologically active peptides with higher analgesic potency, altered side effects and longer duration of action. A considerable increase in analgesic or antidiarrhoeal activity was obtained by substitution of L- with D-amino acids, e.g. Pro2 and Pro4, and by C-terminal amidation (Matthies et al., 1984; Daniel et al., 1990b; Mansfeld et al., 1990; Erll et al., 1994). Examples of chemically modified potent opioid peptides include morphiceptin (b-casomorphin-4-amide) and casokefamide (D-Ala2,4, Tyr5-b- casomorphin-5-amide). Modifications to the natural casomorphins do not only influence the affinity of the resulting analogs to opiate receptors, but also alter their pharmacokinetics, particularly their inactivation by proteolytic/peptideolytic enzymes. Tryptic hydrolysates of casein, containing ACE-inhibitory peptides, were suggested to be useful ingredients in functional foods to prevent hypertension. This finding is

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based on studies with normotensive and mildly hypertensive volunteers who had ingested 10 g of a tryptic casein hydrolysate twice a day for 4 weeks (Sekiya et al., 1992). Recently, a placebo-controlled study showed that the daily intake of 95 ml of Calpis sour milk results in a significant decrease of blood pressure in hypertensive subjects (Hata et al., 1996). The sour milk drink contains two ACE-inhibitory tripeptides, Val—Pro—Pro and Ile—Pro—Pro, which were not present in the placebo drink. The di- and tripeptides corresponding to the immunomodulating sequences (Table 2) derived from milk proteins were also found to be the active components in a dialysed leukocyte extract from normal donors which was recently used in a large multicenter trial to inhibit the development of infections in patients with pre-AIDS (Hadden, 1991). Encouraging results after a bi-weekly treatment of 93 patients with an AIDS-related syndrome showed a significantly reduced tendency to progress to a clinically relevant endpoint or to AIDS. A 11-residue fragment of lactoferricin had a lower hemolytic activity while keeping its antimicrobial activity (Kang et al., 1996). This peptide seems to be beneficial for the development of peptide antibiotics as therapeutic agents with low toxicity. Application of an ultrafiltration membrane reactor for continuous extraction of permeate enriched with small bioactive fragments has been described for production of antithrombotic peptides (Bouhallab et al., 1992). Data on the pharmacological significance of peptides with antithrombotic activity are not yet available. It is tempting to speculate that such peptides or analogs will find pharmaceutical applications in the future. The examples mentioned above clearly show that food protein-derived bioactive peptides fall within the food/drug interface. Whether a product of the food/drug interface should be considered as a food, food additive, dietary supplement, medical food product or a drug depends on the bioactive ingredient itself, what safety risks are associated with its use, what population benefits the most from its use and government regulations. In the near future, it is claimed that nutraceuticals containing bioactive peptides will dramatically penetrate the food and pharmaceutical markets. Food companies, especially in the dairy sector, are now beginning to realize that nutraceuticals have great commercial significance. However, the nutraceutical market is still confused mainly because of the ‘grey zone’ between an allowed nutrient content claim and a prohibited health claim (Fig. 3). A health claim implies a relationship exists between a food or food ingredient and a disease or health-related

condition. In Japan, new regulations have been developed granting functional food products individual health claims for specific health needs based on approved and sufficient research data (Schmidl, 1993). CONCLUSIONS It seems necessary to identify foods containing health enhancing ingredients as ‘more beneficial’ rather than ‘less harmful’. Based on this concept, food researchers are presently considering different bioactive peptides as health enhancing nutraceuticals for use in functional foods. Besides supplementation of food, production of desirable bioactive peptides during food processing, e.g., by use of specific enzymes or genetically transformed microorganisms, would be interesting for future research work. Efforts should also be focused on building screening libraries containing structure—activity data of foodderived bioactive peptides to facilitate identification of new biologically active sequences. Several bioactive peptides may be used as highly active drugs having a well-defined pharmacological effect, for example, in the treatment of diarrhoea (casomorphins), hypertension (casokinins), thrombosis (casoplatelins), dental diseases as well as mineral malabsorption (casein phosphopeptides) and immunodeficiency (immunopeptides). Drugs reveal well-defined pharmacological effects and should not be recommended for application until they have been proven clinically effective in humans. A nutraceutical represents a bioactive substance having a non-nutrient character, and thus has to be considered as an aid in maintaining good health. It is important to gain sufficient data based on human studies as well as human cell culture models to demonstrate the health enhancing effect of a peptide preparation. The beneficial effect of a potential nutraceutical which has moderate bioactivity may be provable only after a long (lifelong?) ingestion period. The question of what kinds of bioactive peptides are beneficial and desirable as food constituents or as drugs should be always carefully examined. However, the possibilities for the design of new dietary products and drugs to help to reduce or control diet-related chronic diseases looks promising. According to the present state of knowledge caseinophosphopeptides and ACE-inhibitory peptides are the most favourite bioactive peptides for application to foodstuffs formulated to provide specific health benefits. ACKNOWLEDGEMENTS Prof. E. Schlimme is gratefully acknowledged for the generous and stimulating support of the work on bioactive peptides and Dr. Richard J. FitzGerald (Limerick, Ireland) for helpful discussions. REFERENCES

Fig. 3. Schematic representation of the link between ‘allowed’ and ‘prohibited’ claims. Nutrient claim implies a relationship exists between a food or a nutrient or a nutraceutical contained in a food and a health-related condition or disease (according to Coussement, 1995).

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Koch, G., Lange, E., Link, G., Bo¨deker, R.-H. and Teschemacher, H. (1994) Analysis of clinical data and plasma levels of b-casomorphin-8 immunoreactive material in pregnant and puerperal women. In b-Casomorphins and Related Peptides: Recent Developments, eds V. Brantl and H. Teschemacher. VCH, Weinheim, pp. 227—239. Kunst, A. (1992) Process to isolate phosphopeptides. European Patent Application 0 467 199 A. Levens, N. R. (1986) Response of isolated rat jejunum to angiotensin peptides. American Journal of Physiology 251, G559—G566. Liardon, R. and Ledermann, S. (1986) Racemization kinetics of free and protein-bound amino acids under moderate alkaline treatment. Journal of Agricultural and Food Chemistry 34, 557—565. Lopker, A., Abood, L. G., Hoss, W. and Lionetti, F. J. (1980) Stereoselective muscarinic acetylcholine and opiate receptors in human phagocytic leukocytes. Biochemical Pharmacology 29, 1361—1365. Loukas, S., Varoucha, D., Zioudrou, C., Streaty, R. A. and Klee, W. A. (1983) Opioid activities and structures of a-caseinderived exorphins. Biochemistry 22, 4567—4573. Loukas, S., Panetsos, F., Donga, E. and Zioudrou, C. (1990) Selective d-antagonist peptides, analogs of a-casein exorphin, as probes for the opioid receptor. In b-Casomorphins and Related Peptides, eds F. Nyberg and V. Brantl. FyrisTryck AB, Uppsala, pp. 143—149. Mansfeld, R., Kautni, J., Grunert, E., Brantl, V. and Jo¨chle, W. (1990) Clinical application of bovine b-casomorphins for treatment of calf diarrhea. In b-Casomorphins and Related Peptides, eds F. Nyberg and V. Brantl. Fyris-Tryck AB, Uppsala, pp. 105—108. Maruyama, S., Mitachi, H., Awaya, J., Kurono, M., Tomizuka, N. and Suzuki, H. (1987) Angiotensin I-converting enzyme inhibitory activity of the C-terminal hexapeptide of a 41 casein. Agricultural and Biological Chemistry 54, 2557—2561. Matthies, H., Stark, H. and Hartrodt, B. (1984) Derivatives of b-casomorphins with high analgesic potency. Peptides 5, 463—470. Meisel, H. (1986) Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein. FEBS ¸etters 196, 223—227. Meisel, H. (1993a) Casokinins as inhibitors of AngiotensinConverting-Enzyme. In New Perspectives in Infant Nutrition, eds G. Sawatzki and B. Renner. Thieme, Stuttgart, New York, pp. 153—159. Meisel, H. (1993b) Casokinins as bioactive peptides in the primary structure of casein. In Food Proteins—Structure Functionality, eds K. D. Schwenke and R. Mothes. VCH, Weinheim, New York, pp. 67—75. Meisel, H. (1997a) Biochemical properties of bioactive peptides derived from milk proteins: potential nutraceuticals for food and pharmacological applications. ¸ivestock and Production Science (in press). Meisel, H. (1997b) Biochemical properties of regulatory peptides derived from milk proteins. Biopolymers (Peptide Science) 43, 119—128. Meisel, H. and Frister, H. (1988) Chemical characterization of a caseinophosphopeptide isolated from in vivo digests of a casein diet. Biological Chemistry Hoppe-Seyler 369, 1275—1279. Meisel, H. and Frister, H. (1989) Chemical characterization of bioactive peptides from in vivo digests of casein. Journal of Dairy Research 56, 343—349. Meisel, H. and Schlimme, E. (1990) Milk proteins: precursors of bioactive peptides. ¹rends in Food Science and ¹echnology 1, 41—43. Meisel, H. and Schlimme, E. (1994) Inhibitors of AngiotensinConverting-Enzyme Derived from Bovine Casein (Casokinins). In b-Casomorphins and Related Peptides: Recent Developments, eds V. Brantl and H. Teschemacher. VCH, Weinheim, pp. 27—33.

Meisel, H. and Schlimme, E. (1995) Casein-gebundener Phosphor und der Gehalt an freien Aminosa¨uren in unterschiedlich wa¨rmebehandelter Milch. Kieler Milchwirtschaftliche Forschungsberichte 47, 289—295. Meisel, H. and Schlimme, E. (1996) Bioactive peptides derived from milk proteins: ingredients for functional foods? Kieler Milchwirtschaftliche Forschungsberichte 48, 343—357. Meisel, H., Behrens, S. and Schlimme, E. (1991a) CalciumBindungsstudie an Phosphopeptidfraktionen aus der in vitro—Proteolyse von Casein. Kieler Milchwirtschaftliche Forschungsberichte 43, 199—212. Meisel, H., Andersson, H., Buhl, K., Erbersdobler, H. and Schlimme, E. (1991b) Heat-induced changes in caseinderived phosphopeptides. Zeitschrift fu¨ r Erna¨ hrungswissenschaft 29, 227—232. Meisel, H., Goepfert, A. and Gu¨nther, S. (1997) Occurence of ACE inhibitory peptides in milk products. Milchwissenschaft 52, 307—311. Mentz, P., Neubert, K., Liebmann, C., Hoffmann, S., Schrader, U. and Barth, A. (1990) In b-Casomorphins: possible physiological significance. In Proceedings from the 1st International Symposium on b-Casomorphins and Related Peptides, eds F. Nyberg and V. Brantl. Fyris-Tryck AB, Uppsala, pp. 143—149. Migliore-Samour. D., Floc`h, F. and Jolle`s, P. (1989) Biologically active casein peptides implicated in immunomodulation. Journal of Dairy Research 56, 357—362. Mullally, M. M., Meisel, H. and FitzGerald, R. J. (1996) Synthetic peptides corresponding to a-lactalbumin and b-lactoglobulin sequences with angiotensin-I-converting enzyme inhibitory activity. Biological Chemistry Hoppe-Seyler 377, 259—260. Mullally, M. M., Meisel, H. and FitzGerald, R. J. (1997) Identification of novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of b-lactoglobulin. FEBS ¸etters 402, 99—101. Ondetti, M. A. and Cushman, D. W. (1982) Enzymes of the renin-angiotensin system and their inhibitors. Annual Reviews of Biochemistry 51, 283—308. Pagelow, I. and Werner, H. (1986) Immunomodulation by some oligopeptides. Methods and Findings of Experimental Clinical Pharmacology 8, 91—95. Panksepp, J., Normansell, L., Siviy, S., Rossi, J. and Zolovick, A. J. (1984) Casomorphins reduce separation distress in chicks. Peptides 5, 829—831. Parker, F., Migliore-Samour, D., Floc`h, F., Zerial, A., Werner, G. H., Jolle`s, J., Casaretto, M., Zahn, H. and Jolle`s, P. (1984) Immunostimulating hexapeptide from human casein: amino acid sequence, synthesis and biological properties. European Journal of Biochemistry 145, 677—682. Paroli, E. (1988) Opioid peptides from food (the exorphins). ¼orld Reviews of Nutrition and Dietetics 55, 58—97. Reynolds, E. (1987) Phosphopeptides. PCT International Patent Application WO 87/07615 A1. Sato, R., Naguchi, T. and Naito, H. (1986) Casein phosphopeptide (CPP) enhance calcium absorption from the ligated segment of rat small intestine. Journal of Nutrition Science and »itaminology 32, 67—76. Schlimme, E. and Meisel, H. (1995) Bioactive peptides derived from milk proteins. Structural, physiological and analytical aspects. Nahrung-Food 39, 1—20. Schmidl, M. K. (1993) Food products for medical purposes. ¹rends in Food Science and ¹echnology 4, 163—168. Schusdziarra, V., Schick, R., de la Fuente, A., Holland, A., Brantl, V. and Pfeiffer, E.F. (1983a) Effect of b-casomorphins on somatostatin release in dogs. Endocrinology 112, 1948—1951. Schusdziarra, V., Schick, R., de la Fuente, A., Specht, J., Klier, M., Brantl, V. and Pfeiffer, E. F. (1983b) Effect of b-casomorphins and analogs on insulin release in dogs. Endocrinology 112, 885—889.

Overview on milk protein-derived peptides Sekiya, S., Kobayashi, Y., Kita, E., Imamura, Y. and Toyama, S. (1992) Antihypertensive effects of tryptic hydrolysates of casein on normotensive and hypertensive volunteers (in Japan). Journal of Japanese Society of Nutrition and Food Science 45, 513—517. Stevens, B. R., Fernandez, A., Kneer, C., Cerda. J. J., Phillips, M. I. and Woodward, E. R. (1988) Human intestinal brush border angiotensin-converting enzyme activity and its inhibition by antihypertensive ramipril. Gastroenterology 94, 942—94. Suetsuna, K. and Osajima, K. (1989) Blood pressure reduction and vasodilatory effects in vivo of peptides originating from sardine muscle. Journal of Japanese Society of Nutrition and Food Science (in Japan) 42, 47—54. Svedberg, J., de Haas, J., Leimenstoll, G., Paul, F. and Teschemacher, H. (1985) Demonstration of b-casomorphin immunoreactive materials in in vitro digests of bovine milk and in small intestine contents after bovine milk ingestion in adult humans. Peptides 6, 825—830. Tani, F., Shiota, A., Chiba, H. and Yoshikawa, M. (1994) Serorphin, an opioid peptide derived from bovine serum albumin. In b-Casomorphins and Related Peptides: Recent Developments. eds V. Brantl and H. Teschemacher. VCH, Weinheim, pp. 49—53. Teschemacher, H. and Brantl, V. (1994) Milk protein derived atypical opioid peptides and related compounds with opioid antagonist activity. In b-Casomorphins and Related Peptides: Recent Developments, eds V. Brantl and H. Teschemacher. VCH, Weinheim, pp. 3—17. Teschemacher, H., Umbach, M., Hamel, U., Praetorius, K., Ahnert-Hilger, G., Brantl, V., Lottspeich, F. and Henschen, A. (1986) No evidence for the presence of b-casomorphins in human plasma after ingestion of cows’ milk or milk products. Journal of Dairy Research 53, 135—138. Tome´, D., Dumontier, A. M., Hautefeuille, M. and Desjeux, J. F. (1987) Opiate activity and transepithelial passage of intact b-casomorphins in rabbit ileum. American Journal of Physiology 253, G737—G744. Tomita, M., Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., and Kawase, K. (1991) Potent antibac-

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terial peptides generated by pepsin digestion of bovine lactoferrin. Journal of Dairy Science 74, 4137—4142. Umbach, M., Teschemacher, H., Praetorius, K., Hirschha¨user, R. and Bostedt, H. (1985) Demonstration of a b-casomorphin immunoreactive material in the plasma of newborn calves after milk intake. Regulatory Peptides 12, 223—230. Wybran, J., Appelboom, T., Famacy, J. P. and Govaerts, A. (1979) Suggestive evidence for receptors for morphine and methionine-enkephalin on normal human blood T lymphocytes. Journal of Immunology 123, 1068—1070. Yamamoto, N., Akino, A. and Takano, T. (1994) Antihypertensive effect of the peptides derived from casein by an extracellular proteinase from ¸actobacillus helveticus CP790. Journal of Dairy Science 77, 917—922. Yen, S. S. C., Quigley, M. E., Reid, R. L., Ropert, J. F. and Cetel, N. S. (1985) Neuroendocrinology of opioid peptides and their role in the control of gonadotropin and prolactin secretion. American Journal of Obstetics and Gynecology 152, 485—493. Yoshikawa, M, Tani, F. and Chiba, H. (1988) Structure-activity relationship of opioid antagonist peptides derived from milk proteins. In Peptide Chemistry, ed. T. Shiba. Protein Research Foundation, Osaka, pp. 473—476. Yoshikawa, M, Tani, F., Shiota, H., Usui, H., Kurahashi, K. and Chiba, H. D. (1994) Casoxin D, an opioid antagonist/ileum-contracting/vasorelaxing peptide derived from human a -casein. In b-Casomorphins and Related Peptides: 41 Recent Developments, eds V. Brantl and H. Teschemacher. VCH, Weinheim, pp. 43—48. Zucht, H. D., Raida, M., Andermann, K., Ma¨gert, H.-J. and Forssman, W. G. (1995) Casocidin-I: a casein-a derived 42 peptide exhibits antibacterial activity. FEBS ¸etters 372, 185—188. Zucht, H. D., Forssmann, W.-G., Raida, M. and Adermann, K. (1996) Verfahren zur Gewinnung eines antibiotisch wirksamen Pra¨parates aus der bovinen Milch und zu deren synthetischen Darstellung. Patent Offenlegungsschrift DE 44 44 753 A 1, pp. 1—9.