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CHAPTER 2:

Biologically Active Peptides Derived from Milk Proteins

 

Anne Pihlanto-Leppälä*, Pirkko Antila^†, Ilari Paakkari^‡and Hannu Korhonen*

* Agricultural Research Centre of Finland, Food Research Institute, FIN-31600 Jokioinen, Finland

^† University of Helsinki, Department of Food Technology, Box 27, FIN-00014 Helsinki, Finland

^‡ University of Helsinki,  Department of Pharmacology and Toxicology, BOX 81, FIN-00014 Helsinki Finland

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Correspondence to:       Anne Pihlanto-Leppälä

Agricultural Research Centre of Finland,

Food Research Institute, FIN-31600 Jokioinen,

                                           Finland

                                           Tel.+ 358-3-4188276

                                           Fax + 358-3-4188444

e-mail: anne.pihlanto.leppala@mtt.fi

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1. Introduction

 

The major role of milk proteins is to supply amino acids and nitrogen to the young mammals and constitute an important part of dietary proteins for the adult. Intact milk proteins have also specific functions such as micelle formation. Furthermore, milk proteins have physiological importance, they facilitate uptake of several important nutrients such as trace elements and vitamins and contain a group of proteins which perform a protective function. This means that milk proteins are highly functional substances. During the last two decades it has become clear that milk proteins are a source of biologically active peptides. These peptides are inactive within the sequence of parent protein and can be released during gastrointestinal digestion or food processing. Once bioactive peptides are liberated, they may act as regulatory compounds with hormone-like activity. This aspect has been studied since 1979, and numerous peptides, which exhibit various activities such as opiate, antithrombotic or antihypertension activity, immunomodulation or mineral utilization properties, has been found.  Milk proteins are the most important source of bioactive peptides, though other animal as well as plant proteins also contain potential bioactive sequences. The  first biologically active peptide found in milk was opioid peptide followed by immunomodulating peptide.

 

The investigation techniques have included the following: the establishment of an assay system of biological activities, hydrolysis of proteins by digestive enzymes, isolation of peptides, determination of structures and synthesis of peptides. Investigation strategies have also comprised synthesis of peptides within milk proteins, based on sequence similarities of peptides having known biological activity. Most of the studies have so far been in vitro. The physiological functions of these peptides remain to be established in vivo.

 

In the present paper some aspects concerning biologically functional milk derived peptides, biochemical and  physiological properties as well as possible dietary application of peptides will be discussed.

 

2.  peptides with opioid activity

 

Opioid peptides are defined as peptides like enkephalins that have both affinity for opiate receptor and opiate-like effects which are inhibited by naloxone. The typical opioid peptides all originate from three precursor proteins- proopiomelanocortin (endorphins), proenkephalin (enkephalin) and prodynorphin (dynorphins) (Höllt, 1983). All of these typical opioid peptides have the same N-terminal sequence, Tyr-Gly-Gly-Phe. Opioid peptides exert their activity by binding to specific receptors of the target cell. Individual receptors are responsible for specific physiological effects, e.g., the m-receptor for emotional behavior and suppression of intestinal motility, the s-receptor for emotional behavior, and the k-receptor for sedation and food intake. The  opioid peptides derived from a variety of precursor proteins are called “atypical“ opioid peptides, since they carry various amino acid sequences at their N-terminal regions, only the  N-terminal tyrosine is conserved. The N-terminal  sequence of the atypical opioid peptides is  Tyr-X-Phe or Tyr-X₁-X₂-Phe. The tyrosine residue at the N-terminal  and the presence of another aromatic amino acid at third or fourth position is an important structural motif that fits into the binding site of the opioid receptors.

 

The first studied food derived opioid peptides were the b-casomorphins (Brantl et al., 1979). Opioid properties have been demonstrated for b-casein f60-70 as well as for fragments thereof. Morphiceptin, which is an amide derivative of b-casomorphin-4, is a highly specific opioid agonist for both m receptors in guinea pig ileum and morphine binding sites in rat brain.   Other b-casomorphins has also been characterized as m-type ligands (Chang et al., 1985). b-casomorphins were also found in analogous positions in sheep, water buffalo and human casein (Schlimme & Meisel 1995). Other milk opioid agonist peptides are a-casein derived exorphins, corresponding to bovine a_s1-casein f90-96. These a-casein exorphins  are -selective receptor ligands and can be separated from pepsin hydrolysate of a-casein (Loukas et al., 1983. Zioudrou et al., 1979). All bovine k-casein fragments having opioid activities - referred to as casoxins (k-casein f33-39, f25-34)  as well as lactoferrin fragments termed lactoferroxin - behave as opioid antagonists. The antagonistic activity of casoxin (k-casein f33-38) was lower than that of naloxone,  and  it can be regarded as a m- and k-selective receptor with low affinity (Yoshikawa et al., 1986; Chiba et al., 1989).

 

Opioid peptides from bovine caseins can be obtained by in vitro enzymatic hydrolysis (Pihlanto-Leppälä et al., 1994).  Precursors of b-casomorphins have also identified in Parmesan-cheese (Addeo et al., 1992). b-casomorphins have been detected in the duodenal chyme of minipigs ( Meisel, 1986) and in the human small intestine (Svedberg et al., 1985) as a consequence of in vivo digestion.

 

Whey proteins contain opioid-like sequences in their primary structure, namely a-lactalbumin (both bovine and human) f50-53 and b-lactoglobulin (bovine) f102-105. These peptides were termed a- and b-lactorphins (Yoshikawa et al., 1986).  Bovine blood serum albumin f399-404 - serorphins - have also opioid activity (Tani et al., 1994).    a-, and b-lactorphin can be  released from parent protein using in vitro proteolysis and various proteolytic enzymes (Antila et al., 1991). a-lactorphin exerts a weak but consistent opioid property in the smooth muscle and receptor binding, while b-lactorphin - in spite of the similar receptor binding affinity - exerts an apparently non-opioid stimulatory effect on the guinea pig ileum. a-, and b-lactorphins were m-type receptor ligands  (Paakkari et al., 1994).

 

3. peptides with Angiotensin I-converting enzyme inhibition activity

 

Angiotensin-I-converting enzyme (ACE) has been classically associated with the renin-angiotensin system regulating peripheral blood pressure. The enzyme can raise blood pressure by converting angiotensin I to a potent vasoconstrictor angiotensin II. Peptides which inhibit ACE have been isolated from many different food sources (Ariyoshi, 1993). ACE is a multifunctional enzyme, which also catalyses the degradation of vasodilator nonapeptide, bradykinin. At present specific inhibitors of ACE are used as antihypertensive drugs.

 

Maruyama et al. (1985, 1987) have shown that tryptic peptides from bovine a_s1-casein and b-casein are inhibitors of ACE. These peptides were termed casokinins and correspond   to fragments 23-34, 23-27 and 194-199 of a_s1-casein as well as fragment 177-183 of b-casein. Meisel and Schlimme (1994) have shown that the synthetic peptides from b-casein - b-casomorphin-7 (f60-66) as well as b-casokinin-10 (f193-202) - shows  ACE-inhibitory activity, within the range of known food derived ACE-inhibitors.  Synthetic b-casein peptide (f169-175) indicated a strong antyhypertensive activity in spontaneously hypertensive rats. The ACE-inhibitory activity of this peptide was quite low but increased after pancreatic digestion. a_s1-casein peptide (f104-109) had a strong ACE-inhibitory activity but no significant antihypertensive effect (Maeno et al., 1996).  Nakamura et al. (1995a) isolated two ACE-inhibitory peptides Val-Pro-Pro and Ile-Pro-Pro, from Calpis sour milk. ACE inhibitory activity was also found in  ripened cheese types and this activity increases during cheese maturation, but decreases when the proteolysis exceeds a certain level.  Inhibitory activity  in cheese may result from a complex mixture of small peptides (Meisel  et al., 1997).

 

 

Whey protein-derived opioid peptides (b-lactorphin and  a-lactorphin) have been shown to moderately inhibit ACE activity. The N-terminal dipeptide (Tyr-Leu) of b-lactorphin was  found to be the most potent inhibitor  (Mullally et al., 1996). Tryptic b-lactoglobulin peptide, corresponding to b-lactoglobulin f142-148, was found to be the most active ACE-inhibitory whey peptide so far reported (Mullally et al., 1997).

 

The structure-activity relationship of ACE-inhibitory peptides has not yet been established, because it has been observed that a large variety of peptides with different C-terminal amino acid sequence can serve as substrates. 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. It has been suggested that peptides which contain hydrophobic amino acids at these positions are potent inhibitors.  The side chains of  these amino acids are considered  to interact with the subsites at the active site of ACE (Ondetti & Cushman 1982). In studies on ACE inhibition by different structures, it was found that a C-terminal tryptophan, tyrosine, phenylalanine and proline residue was the most effective (Cheung et al., 1980). After the isolation of peptide inhibitors from snake venom it was realized that C-terminal proline can bind exceptionally well to ACE and can therefore provide  good substrates or inhibitors depending on other features of the sequence (Cheung et al., 1973).  Proline residues were also suggested as contributing to the potency of ACE-inhibitory peptides from food proteins (Nakamura et al., 1995). Furthermore, the positive charge -  as in the guanidine group of the C-terminal Arg - contributes to the ACE-inhibitory potency of several peptides, indicating that the binding site may be different from the catalytic site in ACE. Using molecular modeling it has been shown that inhibitory peptides possess a characteristic pattern different from that of inactive molecules: positive potential is located in nearly the same region at the C-terminus (Meisel, 1993).

 

4. Peptides with Immunomodulating activities

 

Immunomodulating peptides have been detected in human as well as in cow milk proteins (Migliore-Samour and Jolles, 1988). From human milk protein digests, two peptides, b-casein f54-59 and a-lactalbumin f51-53, enhance the phagocytic activity of macrophages both in mice and humans and enhance resistance against certain bacteria in mice (Migliore-Samour et al., 1989; Parker et al., 1984). Among the immunomodulating peptides isolated from bovine caseins, the b-casein f191-193, a_s1-casein C-terminal hexapeptide (f194-199) stimulated macrophages.  b-casein f63-68 stimulated in vitro phagocytosis, but this peptide as well as b-casein f191-193 failed to exert protection of mice in vivo (Fiat et al., 1993). The physiological mode of action is not known, but they may stimulate the proliferation and maturation of immune system cells. Synthetic peptides derived from milk proteins have been shown to enhance proliferation of human peripheral blood lymphocytes. These peptides were Tyr-Gly and Tyr-Gly-Gly and they correspond to fragments of bovine k-casein and a-lactalbumin. b-casomorphin-7 and b-casokinin-10 showed suppression and stimulation of lymphocyte proliferation depending on the peptide concentration (Kayser and Meisel, 1996).  Laffieneur et al. (1996) have shown that b-casein fermented by lactic acid bacteria have immunomodulatory activity  which might be related to interaction with monocyte-macrophage and T-helper cells, especially Th1-like cells. Sütas and coworkers (1996) showed that caseins hydrolyzed with Lactobacillus GG and digestive enzymes generate compounds with suppressive effects on lymphocyte proliferation. Several known immunostimulating peptides were identified from these hydrolysates (Rokka et al., 1997).

 

Another group of peptides which may be implicated in the stimulation of immunosystem are the ACE-inhibitors. Inhibition of ACE favors bradykinin formation and thus act as immunomodulators. Bradykinin, known as a mediator of the acute inflammatory process, is able to stimulate macrophages to enhance lymphocyte migration and increase secretion of lymphokinines (Pagelow and Werner, 1986). In this context it should be emphasised that peptides a_s1-casein f194-199, b-casein f60-66 and f193-202 have shown to have both immunostimulatory and ACE-inhibitory activities (Table 1).

 

The structure-activity relationship and the mechanism by which milk-derived peptides exert their immunomodulatory effects is not yet defined. It has been suggested that arginine in the N- or C-terminal region of peptide is important structural entity recognized by specific membrane bound receptors (Pagelow and Werner, 1986) . A common structural feature among some immunomodulatory peptides is the presence of arginine in the C-terminal.

 

5. peptides witn Mineral binding properties

 

Several phosphopeptides containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- have been identified from whole bovine casein. These sequences provide the peptides with the unique capacity to keep Ca, P and other mineral in a solution at intestinal pH.  Several phosphopeptides have been identified  from enzymatic digest of milk proteins, for example: a_s1-casein f43-58, f59-79, f43-79,  a_s2-casein f1-24 and  f46-70 and b-casein  f1-28, f2-28, f1-25, f33-48 (Gagnaire et al., 1996, Juilleart et al., 1989). The highly anionic character of these peptides renders them resistant to further proteolytic attack, allows them to form soluble complexes with calcium and prevents the formation of insoluble calcium phosphate  (Berrocal et al., 1986, Sato et al., 1986). The proportion of phosphopeptides interacting with colloidal calcium phosphate correlates with their relative  content of phosphoserine residues (Gagnaire et al., 1996).  Various phosphopeptide fractions revealed significant differences in their calcium-binding activities, which may be due to variant amino acid composition around the phosphorylated region (Meisel et al., 1991). 

 

The formation of caseinphosphopeptide has been observed during in vitro digestion of bovine caseins and specific caseinphosphopeptide  residues have been identified in the intestinal content of minipigs after ingestion of a diet containing casein (Meisel and Frister, 1989). Caseinphosphopeptide can be formed also during cheese ripening due to plasmin and microbial protease activity during ripening ( Roudot-Algaron et al., 1994, Singh et al., 1997)

 

6. peptides with Antithrombotic activity

 

Functional similarities between milk and blood coagulation as well as sequence homologies exist in the fibrinogen g-chain  and k-casein (Jolles and Caen, 1991). Jolles et al. (1986) showed that bovine k-casein f106-116 inhibited platelet aggregation and combined with the receptor site, consequently preventing fibrinogen binding with blood platelets. This inhibition was dependent on peptide concentration. The two smaller tryptic peptides (k-casein f106-112 and f113-116) exerted a much more minimal effect on platelet aggregation and did not inhibit fibrinogen binding. These peptides are referred to as casoplatelins. The behavior of k-casein f106-116 is similar to that of the C-terminal peptide of the human fibrinogen g-chain (Fiat et al., 1989).

 

7. Peptides with Antimicrobial activity

 

The antimicrobial activity of milk is mainly associated with minor whey proteins, namely lactoferrin. This protein has bacteriostatic and bactericidial properties attributed to its ability to chelate iron  or to bind to bacterial surfaces. Tomita et al. (1991) found out that pepsin digestion of bovine lactoferrin produces potent bactericidial peptide, and that the antimicrobial potency of hydrolysate was higher than that of undigested lactoferrin. Dionysius and Milne (1997) have identified two peptides from the N-terminal of lactoferrin which displayed antimicrobial activity toward a number of pathogenic and food spoilage micro-organisms. These results indicated that the bactericidial mechanism is independent of iron because the identified peptides are distinct from the iron-binding site of the molecule. It is possible that the active peptides have an affinity for the bactericidal cell surface and act by disrupting the essential membrane functions. No effect has been detected against  Bifidobacterium, therefore  lactoferrin derived peptides may positively affect the intestinal flora. a_s1-casein f1-23 obtained from chymosin hydrolysis, has been shown to have antibacterial activity against Satphylococcus aureus and Candida albicans (Lahov and Regelson, 1996).

 

8. Other activities

 

Yamauchi (1992) has reported that two peptides derived from serum albumin and b-lactoglobulin induced contraction of guinea pig ileum longitudinal muscle when the test was completed without electric stimulation in the absence of agonist. The peptides were referred as to “peptides acting on smooth muscle,“ and they contained serum albumin f208-216 (albutensin A) and b-lactoglobulin f146-149 (b-lactotensin). b-lactotensin was released during hydrolysis with chymotrypsin and it had a non-opioid contracting effect on guinea pig smooth muscle (Pihlanto-Leppälä et al., 1997).

 

Some of the peptides derived from milk proteins have more than one functional significance, peptides from the sequence 60-70 of b-casein show  immunostimulatory, opioid and ACE-inhibitory activities. Such sequence is defined as strategic zone (Migliore-Samour and Jolles, 1988). This sequence is protected from proteolysis because of its high hydrophobicity and the presence of proline residues. In addition to the strategic zone, some other multifunctional peptides can be liberated from milk proteins, for example a_s1-casein  f194-199 and b-casein f177-183 have immunomodulatory and ACE-inhibitory activity. Also from whey proteins multifunctional peptides can be liberated, e.g. b-lactoglobulin f102-105 (b-lactorphin) shows both ACE-inhibitory and opioid activity.

 

 

9. Possible physiological importance

 

Bioactive peptides are widely distributed among milk proteins. This fact suggests the physiological importance of these peptides. Although the potency of these milk derived peptides is smaller than those of endogenous peptides or peptide-based drugs, they may well have physiological effects, because milk proteins are usually ingested in fairly large amounts.  To exert physiological effects in vivo, bioactive peptides must be released during intestinal digestion and then reach their target sites at the luminal side of the intestinal tract or after reabsorption, in the peripheral organs.  Many studies have shown in vitro formation of bioactive peptides and in some studies in vivo formation has also been found. In addition to liberation during in vitro or in vivo digestion,  bioactive peptides may also be liberated during the manufacture of milk products.  For example,  hydrolyzed milk proteins used for hypoallergenic infant formulae,  for clinical application and as  food ingredients, consist exclusively of peptides.  Proteolysis during milk fermentation and cheese ripening  lead  to the formation of various peptides. Indeed, casomorphins, ACE-inhibitory peptides and phosphopeptides have been found from fermented milk products. 

 

There are different possible intestinal and peripheral target sites of the active substance.  As opioid receptor ligands, these peptides can be expected to behave like other opioids i.e. to act as agonist or antagonist, to bind to receptors and elicit effects at all cells or tissues where opioids are known to do this, mediated via signal transduction pathways as known for opioids already.  Indirect evidence suggest the presence of b-casomorphins in the intestinal of humans after milk ingestion (Svedberg et al., 1985), whereas milk derived opioid peptides do not seem to permeate into the cardiovascular compartment, in more than neglible amounts, in adult mammals. Enzymatic degradation of peptides in the intestinal wall and in the blood appear to prevent  (Teschemacher et al., 1986).  It seems likely that casomorphins   participate in the control of gastrointestinal function in adults  (Tome et al., 1987). Casomorphins have been found to prolong gastrointestinal transit time and exert antidiarrhoeal action (Daniel et al.,1991). The physiological significance of opioid peptides may be different in pregnant, puerperal women and in neonates. b-casomorphin immunoreactive materials, obviously representing b-casein cleavage products larger than b-casomorphins, i.e., potential b-casomorphin precursors,  have been found in plasma and in the cardiovascular compartment in women during pregnancy and lactation and new-born animals (Teschemacher et al., 1997).  These  findings indicate the pharmacological activity of b-casomorphins not for physiological significance. 

 

Inhibition of ACE, which is located in different tissues (e.g. plasma, lung, kidney, heart, skeletal muscle, pancreas brain) may influence different regulatory systems (Ondetti and Cushman 1982). When ACE-inhibitory peptides (Val-Pro-Pro and Ile-Pro-Pro) were given to spontaneously hypertensive rats, the blood pressure was reduced dose dependently. The peptide mixture, or the fermented milk containing the peptides did not change the blood pressure (Nakamura et al., 1995b).  Masuda et al. (1996) detected two ACE-inhibitory tripeptides, present in fermented milk product, in aorta after oral administration of fermented milk in spontaneously hypertensive rats. Also the ACE activity in fractions from aorta was lower in rats given fermented milk than in control group. The results indicate that these tripeptides are absorbed directly without being decomposed by digestive enzymes, reach the abdominal aorta, inhibit the ACE and show antihypertensive activity.

 

Caseinphosphopeptides have  been shown to have anticariogenic properties, based on their ability to localize amorphous phosphate in dental plaque (Reynold, 1994). Caseinphosphopeptide residues have been isolated in the intestinal contents of pigs and rats fed casein, indicating the in vivo formation. Most minerals are dissociated from the food,  as a result of the low pH in the stomach, subsequently transferring to the duodenum. These ions may gradually become insoluble as pH increases. When phosphopeptides are present, metal ions may be rebound in soluble complexes instead of precipitated with other compounds and thereby rendering them more absorbable (Sato et al., 1986) There is, however considerable controversy as to the physiological significance of the enhancement of intestinal calcium paracellular absorption by caseinphosphopeptide. Disagreement centers on the conclusions drawn from the various experimental methods used to evaluate calcium bioavailability in vivo, which may involve different endpoint measurements. The disagreements are partly due to different compositions of phosphopeptide preparations, which have been in the studies  and might lead to different calcium-binding activities as shown by Meisel et al. (1991).

 

 

10. Possible applications of bioactive peptides

 

Peptides with biological activity could be produced in several ways. The most common methods are: processing of foods using heat, alkali or acid conditions that hydrolyze proteins, enzymatic hydrolysis of food proteins  and microbial activity of fermented foods. Although bioactive peptides do exist in a number of processed and fermented foods, their true physiological functions in humans are unknown. In healthy individual, eating a varied diet, the presence of bioactive peptides may help keep the nervous, immune and digestive systems in a well-maintained state. The future potential value of bioactive peptides in the diet may be their ability to affect certain pathological conditions, although this has yet to be proven.  Casein derived peptides have already found interesting applications as dietary supplements (phosphopeptides) and as pharmaceutical preparations (phosphopeptides, b-casomorphins)  (Brule et al., 1982, Reynolds, 1987).  The efficacy and safe conditions of use of these peptides in animals and in humans remain to be proven. At present, ACE-inhibitory peptides and  phosphopeptides are an important area in which bioactive peptides may be found to be useful  ingredients for dietary applications.

 

11. Conclusions

 

Emzymatic digestion of milk proteins represents an important supply or numerous peptides that may have biological activity. The physiological role of these peptides is not yet fully understood.  Peptides have been shown to exert beneficial physiological effects. These findings introduces new perspectives in the nutritional and technological evaluation of milk and milk products. These milk peptides may be considered as food additive and perhaps as starting components for some drug developments. Casein derived peptides have already found interesting applications as dietary supplements and pharmaceutical preparations. Today we know that some of the biologically active peptides have  can be released during the in vivo digestion, however we don’t know if these peptides have physiological function. More recearch is needed to fully understand the functional significance of these substances.

 

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Table 1. Examples of biolocically active  peptides derived from bovine casein proteins.

 

Precursor protein	Fragment	Peptide sequence	Name	Function	Ref.
b-casein	60-70	Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser-Leu	b-casomorphin-11	opioid agonist	Meisel, 1986
	60-66	Tyr-Pro-Phe-Pro-Gly-Pro-Ile	b-casomorphin-7	opioid agonist, ACE-inhibition, immunomodulation	Brantl et al., 1979, Meisel and Schlimme, 1994, Kayser et al., 1996
	60-64	Tyr-Pro-Phe-Pro-Gly	b-casomorphin-5	opioid agonist	Henschen et al., 1979
	177-183	Ala-Val-Pro-Tyr-Pro-Gln-Arg	b-casokinin-7	ACE-inhibition	Maruyama et al.,1985
	193-202	Tyr-Gln-Gln-Pro-Val-Leu-Gly-Pro-Val-Arg	b-casokinin-10	ACE-inhibition,  immunomodulation	Meisel and Schlimme 1994, Kayser et al., 1996
	169-175	Lys-Val-Leu-Pro-Val-Pro-Gln		ACE-inhibition	Maeno et al., 1996
	63-68	Pro-Gly-Pro-Ile-Pro-Asn		immunomodulation	Migliore-Samour and Jolles, 1988
	191-193	Leu-Leu-Tyr		immunomodulation	Berthou et al., 1987
	1-25	Arg-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu-Ser(P)-Leu-Ser(P)Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Thr-Arg	casein phosphopeptide	stimulation of mineral absorption	Gagnaire et al., 1996
as1-casein	90-96	Arg-Tyr-Leu-Gly-Tyr-Leu-Glu	a-casein  exorphin	opioid agonist	Loukas et al., 1983
	90-95	Arg-Tyr-Leu-Gly-Tyr-Leu	a-casein  exorphin	opioid agonist	Loukas et al., 1983
	91-96	Tyr-Leu-Gly-Tyr-Leu-Glu	a-casein  exorphin	opioid agonist	Loukas et al., 1983
	23-34	Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys		ACE-inhibition	Maruyama  and Suzuki, 1982
	23-27	Phe-Phe-Val-Ala-Pro	a-casokinin-5	ACE-inhibition	Maruyama et al.,1987b
	104-109	Tyr-Lys-Val-Pro-Gln-Leu		ACE-inhibition	Maeno et al., 1996
	194-199	Thr-Thr-Met-Pro-Leu-Trp	a-casokinin-6	ACE-inhibition, immunomodulation	Maruyama et al.,1987, Fiat et al., 1993
k-casein	33-39	Ser-Arg-Tyr-Pro-Ser-Tyr-OH	casoxin	opioid antagonist	Chiba et al., 1989
	25-34	Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-Ser-Arg	casoxin C	opioid antagonist	Chiba et al., 1989
	106-116	Met-Ala-Ile-Pro-Pro-Lys-Lys-Asn-Gln-Asp-Lys	Casoplatelin	antithrombotic	Jolles et al., 1986