Bioactive Milk Peptides, A Prospectus

INVITED REVIEW

Bioactive Milk Peptides: A Prospectus1

D. A. Clare* and H. E. Swaisgood†*Department of Food Science

†Departments of Food Science and BiochemistryNorth Carolina State University

Raleigh, NC 27695-7624

ABSTRACT

Bioactive peptides have been identified within theamino acid sequences of native milk proteins. Hydrolyticreactions, such as those catalyzed by digestive enzymes,result in their release. These peptides directly influencenumerous biological processes evoking behavioral, gas-trointestinal, hormonal, immunological, neurological,and nutritional responses. The specific bioreactions asso-ciated with each physiological class have been well char-acterized. Herein, we review the scientific literature andattempt to stimulate consideration of the continued useof bioactive peptides and their expanded development asa commercial product. Several applications have alreadyevolved. For example, phosphopeptides derived from ca-sein fractions are currently used as both dietary andpharmaceutical supplements. Potentially, the additionof bioactive peptides to food products could improve con-sumer safety as a result of their antimicrobial properties.Lastly, bioactive peptides may function as health careproducts, providing therapeutic value for either treat-ment of infection or prevention of disease.(Key words: bioactive peptide, milk proteins, func-tional foods)

Abbreviation key: ACE = angiotensin converting en-zyme, CPP = casein phosphopeptides.

INTRODUCTION

Milk contains components that provide critical nutri-tive elements, immunological protection, and biologicallyactive substances to both neonates and adults. In gen-eral, the major protein fractions in bovine milk includeα-LA, β-LG, caseins, immunoglobulins, lactoferrin, pro-teose-peptide fractions (heat-stable, acid soluble phos-phoglycoproteins), and minor whey proteins such astransferrin and serum albumin. From these, bioactivepeptides may be generated in vivo through gastrointesti-

Received October 15, 1999.Accepted December 26, 1999.Corresponding author: D. A. Clare; e-mail: [email protected] review was solicited by the Milk Proteins and Enzyme Com-

mittee of ADSA, R. Jimenez-Flores, Chairperson.

2000 J Dairy Sci 83:1187–1195 1187

nal processes. Often, this liberation serves to influencenumerous physiological responses as a result of theirhormone-like properties. These peptides, encoded withinthe sequences of native protein precursors, may also begenerated in vitro by enzymatic hydrolysis. In this case,peptides are purified from protein hydrolysates by vari-ous separation techniques and assayed for bioactivity.Lastly, physiologically active peptides have been chemi-cally synthesized to confirm the biological properties as-sociated with a specific amino acid sequence. There isconsiderable evidence that many bioactive peptidesserve in multifunctional capacities and often share com-mon structural features based on a defined, biospecificrole. Here, we review the primary classes of bioactivemilk peptides, based on their specific physiological func-tion, and provide a summary of general characteristicsassociated with each group.

MILK DEFENSE PEPTIDES

Antimicrobial Peptides

The total antibacterial effect in milk is greater thanthe sum of the individual contributions of immunoglobu-lin and nonimmunoglobulin defense proteins. This ismost likely due, at least in part, to their synergy. Anothercontributing factor may be the presence of naturally oc-curring bactericidal peptides, in addition to those gener-ated from inactive protein precursors.

Antimicrobial milk proteins, such as lactoferrin, weredescribed in early literature (9). During this time, reportsalso detailed the discovery of basic glycopeptides withbactericidal activity against various strains of Staphylo-coccus aureus and Streptococcus (40). In general, theirvalue for development as a commercial antimicrobialproduct was ignored. Recently, however, there has beena renewed interest in using bioactive peptides for appli-cation within the health care industry for these purposes.

Casecidin, obtained by chymosin digestion of caseinat neutral pH, was among the first defense peptidesactually purified and exhibited activity in vitro againstStaphylococcus, Sarcina, Bacillus subtilis, Diplococcuspneumoniae, and Streptococcus pyogenes (41; Table 1).Casocidin-I (bovine milk), a cationic αs2-CN derived pep-tide, inhibited growth of Escherichia coli and Staphylo-

CLARE AND SWAISGOOD1188

Table 1. Antimicrobial milk peptides.

Milk peptidefragment Release protease Gram (+) activity Gram (−) activity Yeast and fungi* Reference

Casecidin Chymosin and chymotrypsin Staphylococcus 41αs1 and κ-CN Sarcina(MW ≅ 4000–6000) Bacillus subtilis

Diplococcus pneumoniaeStreptococcus pyogenes

Casocidin-I Synthetic peptide Staphylococcus carnosus Escherichia coli 103αs2-CN (f 165–203)

Isracidin Chymosin and chymotrypsin Staphylococcus aureus Candida albicans 41αs1-CN (f 1–23)

Lactoferricin B Pepsin Bacillus E. coli 0111 Candida albicans 4, 79, 89Lactoferrin (f 17–41) Listeria E. coli 0157H:7 Dermatophytes:

Streptococci Klebsiella *CryptococcusStaphylococci Proteus uniguttulatus

Pseudomonas *PenicillumSalmonella pinophilum

*Trichophytonmentagrophytes

coccus carnosus (103). Isracidin, an N-terminal segmentof αs1-CN B, protected mice against Staphylococcusaureus and Candida albicans. This peptide also safe-guarded sheep and cows against mastitis when injectedinto the udder at levels comparable to those observedwith standard antibiotic treatment (41).

Previous reports have focused on the antimicrobialpeptides generated by proteolytic digestion of bovine lac-toferrin. The hydrolysate was active against both Gram-positive (Bacillus, Listeria, and Streptococcus) andGram-negative (E. coli, Klebsiella, Salmonella, Proteus,and Pseudomonas) microorganisms in vitro. A patho-genic intestinal bacterium, E. coli 0111, was additionallyfound to be susceptible (89). A potent bactericidal peptidespecifically generated by pepsin degradation of lactofer-rin, so named lactoferricin B, also displayed antimicro-bial activity towards both Gram-positive and Gram-neg-ative microorganisms (32, 89). In recent literature, stud-ies indicated that lactoferricin B was active againstclinical isolates of enterohaemorrhagic E. coli 0157H:7at concentrations significantly less than either the lacto-ferrin hydrolysate or lactoferrin, itself (79). These prop-erties appear to be correlated with the net positivecharge of the peptide, which may kill susceptible micro-organisms by increasing cell membrane permeability.Present communications offer direct evidence for thegeneration of lactoferricin in human stomach after inges-tion of lactoferrin (39). There is conflicting data, however,regarding the “in vivo” antimicrobial properties of thepeptide, since the addition of 5% cow’s milk completelyameliorated much of the activity. The bactericidal prop-erties of lactoferricin were also reduced by adding in-

Journal of Dairy Science Vol. 83, No. 6, 2000

creasing concentrations of mucin to the assay mixture(32).

Milk also contains peptides that exhibit antifungalproperties (Table 1). Antifungal activity of lactoferrin orits peptides (ex. lactoferricin B), in combination withazole antifungal agents, has been demonstrated withCandida albicans (3, 90). Several filamentous fungi, in-cluding agents of skin disease (dermatophytes), werealso found to be susceptible to this mixture (4). The mostrecent data showed that lactoferrin-related compounds,combined with triazoles, inhibited growth of azole-resis-tant C. albicans hyphae (91).

PHYSIOLOGICALLY ACTIVE MILK PEPTIDES

In addition to providing immunodefense systems, milkalso contains other major peptide fractions that elicitbehavioral, neurological, physiological, and vasoregula-tory responses (Table 2). Often, the peptide displaysmultifunctional properties. Several articles reviewingthis topic have already been published (34, 53, 77, 87).Here, we categorize classifications of physiologically ac-tive peptides based on their primary biofunction.

Antihypertensive Peptides (ACE Inhibitors)

Antihypertensive peptides inhibit the angiotensin con-verting enzyme (ACE) (61, 62, 92). ACE is a peptidyl-dipeptidase that cleaves dipeptides from the carboxy ter-minal end of the substrate. ACE converts angiotensin Ito angiotensin II, increasing blood pressure and alder-sterone, and inactivating the depressor action of bradyki-nin. ACE inhibitors derived from casein, or casokinins,

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Table 2. Examples of physiologically active milk peptides.

Samplenumber Peptide sequence1 Name AA2 segment Physiological classification Release protease Reference

1 FFVAP αs1-Casokinin-5 αs1-CN (f 23–27) ACE inhibitor Proline endopeptidase 462 AVPYPQR β-Casokinin-7 β-CN (f 177–183) ACE inhibitor Trypsin 463 YGLF α-Lactorphin α-LA (f 50–53) ACE inhibitor and opioid agonist Synthetic peptide 594 ALPMHIR β-Lactorphin β-LG (f 142–148) ACE inhibitor Trypsin 605 KVLPVPQ Antihypertensive β-CN (f 169–174) Antihypertensive peptide Lactobacillus 44

peptide CP790 protease& synthetic peptide

6 MAIPPKKNQDK Casoplatelin κ-CN (f 106–116) Antithrombotic Trypsin & 30synthetic peptide

7 KDQDK Thrombin inhibitory κ-CN glyco- Antithrombotic Trypsin 71peptide macropeptide

(f 112–116)8 KRDS Thrombin inhibitory Lactotransferrin Antithrombotic Pepsin 70

peptide (f 39–42)9 QMEAES*IS*S*S* Caseinophospho- αs1-CN Calcium binding and transport Trypsin 77

EEIVPNS*VEQK peptide (f 59–79)10 LLY β-CN Immunostimulatory (+) Synthetic 57

Immunopeptide (f 191–193)11 FKCRRWQWRMK Lactoferricin B Lactoferrin (f 17–41) Immunomodulatory (+) and Pepsin 2, 58

KLGAPSITCVRR antimicrobialAF

12 YQQPVLGPVR β-Casokinin-10 β-CN (f 193–202) Immunomodulatory (+/−) & Synthetic 55ACE Inhibitor

13 RYLGYLE α-Casein exorphin αs1-CN (f 90–96) Opioid agonist Pepsin 4314 YGFQNA Serorphin BSA (f 399–404) Opioid agonist Pepsin 8415 YLLF�NH2 β-Lactorphin β-LG (f 102–105) Opioid agonist = ACE Inhibitor Synthetic or Trypsin 59

(amide)16 YIPIQYVLSR Casoxin C κ-CN (f 25–34) Opioid antagonist Trypsin 1417 [YVPF PPF] Casoxin D αs1-CN (f 158–164) Opioid antagonist Pepsin-chymotrypsin 9618 YLGSGY-OCH3 Lactoferroxin A Lactoferrin (f 318– Opioid antagonist Pepsin 93

323)

1The one-letter amino acid codes were used; S* = Phosphoserine.2Amino acid.

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have been identified within the sequences of human β-and κ-CN (37, 38). They are also generated by trypticdigestion of bovine αs1- and β-CN (47, 55, 76). The C-terminal tripeptide sequence is the primary structuralfeature governing this inhibitory response (45), and re-ports indicated that the ACE binding pocket exhibiteda preference for hydrophobic amino acids at each of thesesites (52). A second characteristic of ACE inhibitory caso-kinins is the presence of a positively charged lysine orarginine at the carboxy terminal end (52). It was shownthat removal of this critical amino acid residue frombradykinin, an endogenous ACE inhibitor, resulted inproduction of an analogue that was essentially inac-tive (14).

ACE inhibitory peptides are also derived from bothαs1- and β-CN that are generated by the hydrolysis ofsour milk with the Lactobacillus helveticus CP790 extra-cellular protease. These peptides exhibited antihyper-tensive activity in spontaneously hypertensive rats asmonitored by systolic blood pressure (44, 62, 92, 93).A synthetic seven amino acid peptide, equivalent to asegment found in the β-CN hydrolysate, exhibited potentantihypertensive activity in these rats over an 8-h inter-val after oral administration (44). A third subclass, β-lactorphins, are sequestered within the primary aminoacid sequence of bovine β-LG and released by trypsin(60). Lastly, novel angiotensin-I converting enzyme(ACE) inhibition was detected in synthetic peptides thatcorresponded to sequences within both β-LG and α-LA(59).

Antithrombotic Peptides

Antithrombotic peptides are present in milk. Early on,it was learned that the mechanisms involved in milkclotting, defined by the interaction of κ-CN with chy-mosin and blood clotting processes, defined by the inter-action of fibrinogen with thrombin, were comparable.In this regard, the C-terminal dodecapeptide of humanfibrinogen γ-chain (residues 400 to 411) and the undeca-peptide (residues 106 to 116) from bovine κ-CN are struc-turally and functionally quite similar. This casein-de-rived peptide sequence, termed casoplatelin, affectedplatelet function and inhibited both the aggregation ofADP-activated platelets and the binding of human fi-brinogen λ-chain to its receptor region on the platelets’surface (30). A smaller κ-CN fragment (residues 106 to110), casopiastrin, was obtained from trypsin hydroly-sates and exhibited antithrombotic activity by inhibitingfibrinogen binding (29, 30, 49). A second segment of thetrypsin κ-CN fragment, residues 103 to 111, inhibitedplatelet aggregation but did not affect fibrinogen bindingto the platelet receptor (19, 21, 30). Later, it was reportedthat biologically active peptides, isolated from both ca-


sein and lactotransferrin, inhibited platelet function(20, 50).

Antithrombotic peptides have also been derived fromκ-caseinoglycopeptides that were isolated from severalanimal species. Bovine κ-caseinoglycopeptide, the C-ter-minal end of κ-CN (residues 106 to 169), inhibited vonWillebrand factor-dependent platelet aggregation (12).Two antithrombotic peptides, derived from human andbovine κ-caseinoglycopeptides, have been identified inthe plasma of 5-d-old newborns after breast-feeding andingestion of cow’s milk based formula, respectively (10).The C-terminal residues (106 to 171) of sheep κ-casein,or κ-caseinoglycopeptide, decreased thrombin- and colla-gen-induced platelet aggregation in a dose dependentmanner (71).

Lastly, thrombin-induced platelet aggregation was in-hibited with pepsin digests of sheep and human lactofer-rin. A single peptide peak containing this activity wasobtained by reverse-phase chromatography of the hy-drolysate (70).

Caseinophosphopeptides

Casein phosphopeptides (CPP) have been identifiedafter trypsin release from αs1-, αs2-, and β-CN (34). Thephosphate residues, which are present as monoesters ofserine, occur mainly in clusters. Most CPP contain threeserine phosphate clusters followed by two glutamic acidresidues, form soluble organophosphate salts, and proba-bly function as carriers for different minerals, especiallycalcium (53, 75). These fractions exhibit different de-grees of phosphorylation, and a direct relationship be-tween the degree of phosphorylation and mineral chelat-ing ability has been described (34). In this event, αs2-CN > αs1-CN > β-CN > κ-CN; however, the distributionof their phosphoserine clusters is not uniform. It wasfurther demonstrated that the specific amino acid com-position associated with the phosphorylated binding sitealso influences the degree of calcium binding (1).

CPP are mostly resistant to enzymatic hydrolysis inthe gut and most often found in a complex with calciumphosphate (73). This complex formation results in anincreased solubility which, in turn, provides enhancedabsorption of calcium across the distal small intestinesof animals fed casein diets in comparison to control ani-mals fed soy-based diets (35, 99, 100). This passive trans-port system is the primary means of calcium absorptionunder physiological conditions and provides calcium re-quired for bone calcification (53). Caseinophosphopep-tides also inhibit caries lesions through recalcificationof the dental enamel. Hence, their application in thetreatment of dental diseases has been proposed (72).

INVITED REVIEW BIOACTIVE PEPTIDES 1191

Immunomodulatory Peptides

Immunomodulatory milk peptides affect both the im-mune system and cell proliferation responses. As dis-cussed previously, β-casokinins inhibit ACE enzymesthat are responsible for inactivating bradykinin, a hor-mone with immune enhancing effects. Thus, this chainof events indirectly produces an overall immunostimula-tory response. Peptides derived from casein hydrolysateswere shown to increase phagocytotic activity of humanmacrophages against aging red blood cells and augmentphagocytosis of sheep red blood cells by murine perito-neal macrophages in vitro (21, 31, 57). Immunostimula-tory activity against Klebsiella pneumoniae was demon-strated in vivo using rats treated intravenously with ahexapeptide obtained by hydrolysis of human β-CN (57,65). Most recently, lactoferricin B, obtained by hydrolysisof lactoferrin with pepsin, was found to promote phago-cytic activity of human neutrophils via dual mechanismsthat may involve direct binding to the neutrophil andopsonin-like activity (58).

Small peptides, corresponding to the N-terminal endof bovine α-LA (dipeptide) and κ-CN (tripeptide), sig-nificantly increased proliferation of human peripheralblood lymphocytes (33), while the C-terminal sequenceof bovine β-CN (193 to 209), obtained by hydrolysis withpepsin-chymosin, induced a similar response in rats (16).Bioactive peptides in yogurt preparations actually de-creased cell proliferation with IEC-6 or Caco-2 cells. Thisreport may explain, in part, why consumption of yogurthas been associated with a reduced incidence of coloncancer (23). Kayser and Meisel (33) have described bothstimulatory and suppressive immune responses of hu-man lymphocytes to milk derived peptides.

In general, the mechanisms by which these milk-de-rived peptides exert either their immunopotentiating ef-fects or influence proliferative responses are not cur-rently known; however, one example suggests that theopioid milk peptide, β-casomorphin (Table 3), may exertan inhibitory effect on the proliferation of human laminapropria lymphocytes in vitro via the opiate receptor (18).This antiproliferative response was reversed by the opi-ate receptor antagonist, naloxone.

Opioid Milk Peptides

The major opioid peptides are fragments of β-CN,called β-casomorphins, due to their exogenous origin andmorphine-like properties (8, 26, 97); however, they havealso been obtained from pepsin hydrolysis of bovine αs1-CN fractions (43, 54, 66, 102). Similar peptides havebeen reported from human β-CN fractions (24, 98), andthe Y-P-F sequence, which is common to bovine β-caso-morphin, was also found to be present in the primarystructure of human β-CN. Various synthetic derivatives


have been made and among these, Y-P-F-V-NH2 (valmu-ceptin) and Y-P-F(D)-V-NH2 (D-valmuceptin) show highaffinity for their receptor (97). Brantl and co-workersreported opioid activity from synthetic tetra- and penta-peptide fragments of human β-casein (7). Opioid peptideshave been generated in vitro by enzymatic digestion ofβ-caseins from cows, water buffalo, and sheep (68 and77, 67, 74, respectively). In general, the α- and β-CNfragments produce agonist responses, while those de-rived from κ-CN elicit antagonist effects. Opioid peptidesmay be further subdivided into classifications accordingto the specific milk protein from which they were derived,and these categories are summarized in Table 3. It isnoteworthy that bioactive peptides are generated frommost of the major proteins in both bovine and humanmilk.

a. Structure and function. “Typical” opioid pep-tides, or endorphins, are derived from proenkephalin,propiomelanocortin, and prodynorphin and exhibit adefinite N-terminal sequence Y-G-G-F (15, 86). Milk-derived peptides, generated by hydrolysis of various pre-cursor proteins such as α- and β-CN, α-LA, and β-LG,are called “atypical,” exomorphic, agonist peptides andexhibit morphine-like activity (102). Their primarystructure (i.e., Y-X1-F or Y-X1-X2-F or Y) differs from theamino terminal sequence of the “typical” endogenousopioid peptide defined above. With the exception of αs1-CN, most share a common sequence feature, defined bya N-terminal tyrosine residue, that is absolutely essen-tial for activity (13, 27). Typically, a second aromaticamino acid residue, such as phenylalanine or tyrosine,is also present in the third or fourth position. This struc-tural motif fits well into the binding pocket of the opioidreceptor. One of the most potent milk-derived opioidpeptides, β-casomorphin-4-amide (or morphiceptin), alsocontains a proline that is crucial for its function. Thisresidue reportedly maintains the proper orientation ofthe tyrosine and phenylalanine side chains (56).

Exorphins have been isolated from peptic hydrolysatesof α-casein fractions as well. In general, their structuresdiffer considerably from those of β-caseinomorphins. Ac-tive fractions were shown to be a mixture of two separatepeptides derived from α-casein fragments #90–95 and#90–96. The sequences were determined as listed, [R90-Y-L-G-Y-L95-(E96)], in which case the latter peptideproved to be more effective. The N-terminal arginineresidue was also reported to be essential for activity (43).

b. Opioid agonists. β-Casein peptides were amongthe first reported opioid peptides (8). β-Casomorphinsare fragments corresponding to the 60 to 70th aminoacid residues of bovine β-CN, considered the “strategiczone,” and are classified as µ-type receptor ligands (36).Three exorphins, derived from bovine αs1-CN, wereshown to be selective for δ-receptors (43). Certain proteo-


Table 3. Opioid milk peptides.

Protein substrate Peptide name Amino acid segment References

Bovine αs1-CN αs1-Casein exorphins f 90–95, f 90–96, and f 91–96 43Human αs1-CN Casoxin D f 158–164 96Human β-CN β-Casomorphin (4,5) f 51–54 and f 51–55 7Bovine and human κ-CN Casoxin A, B, and C f 25–34, f 35–41, and f 57–60 97, 95, 14Bovine and human α-LA α-Lactorphin f 50–53 15, 19Bovine β-LG β-Lactorphin f 102–105 21, 97Lactotransferrin Lactoferroxins A, B, and C f 318–323, F 536–540, and f 673–679 83BSA Serophin f 399–404 84

lytic bacteria, such as Pseudomonas aeruginosa and Ba-cillus cereus, also produce high levels of β-casomorphinswhen inoculated and grown in milk (25).β-Caseinomorphins are resistant to enzymes of the

gastrointestinal tract and have been detected in vivo inthe intestinal chyme of minipigs (51) and human smallintestines (81). Because their absorption in the gut hasnot been observed in adults, it is generally concludedthat the physiological influences are limited to the gas-trointestinal tract with important effects on intestinaltransit time, amino acid uptake, and water balance (78,86). Once they enter the bloodstream, they are rapidlydegraded (53). In contrast, passive transport of β-casein-omorphins across intestinal mucosal membranes doesoccur in neonates, which may experience physiologicalresponses such as an analgesic effect on the nervoussystem resulting in calmness and sleep in infants (80).A precursor ofβ-casomorphin was reported in the plasmaof newborn calves and infants after ingestion of bovinemilk (85). In pregnant or lactating women, β-casomor-phins originate in the milk (5, 94), pass through themammary tissue, and possibly influence the release ofprolactin and oxytocin (86). More recently, Chabance etal. (11) showed that many peptides derived from αs1-,β-, or κ-CN, and κ-caseino-glycomacropeptide can be de-tected in the stomach of adults after consumption of milkor yogurt.

Casomorphins, as opioid ligands, modulate social be-havior (64, 66), increase analgesic behavior (48, 66), pro-long gastrointestinal transient time by inhibiting intesti-nal peristalsis and motility (88), exert antisecretory(antidiarrheal) action (17), modulate amino acid trans-port (6), and stimulate endocrine responses such as thesecretion of insulin and somatostatin (54). Opioid-likemilk peptides also play a regulatory role regarding appe-tite by modifying endocrine activity of the pancreas, re-sulting in an increase of insulin output (63). Presently,data suggest that intracerebroventricular β-casomor-phin1-7 stimulates uptake of a high fat diet in rats fastedovernight. Enterostatin inhibited this effect, as did nal-oxone, a general opioid antagonist. Ligand binding stud-ies indicated that at high dosages, β-casomorphin1-7 and


enterostatin may interact with the same low affinityreceptor to modulate intake of dietary fat (42).

c. Opioid antagonists. Opioid antagonists suppressthe agonist activity of enkephalin. Yoshikawa et al. (95)reported that a chloroform and methanol extract froma peptic digest of bovine κ-CN bound to opioid receptorsof rat brain. The peptide was methylated at the C-termi-nal end and exhibited antagonist effects selective for theµ- and κ-type of opioid receptor. The peptide was thusnamed casoxin.

Casoxins A and B have been chemically synthesizedand correspond to amino acid sequences within bothbovine and human κ-CN (14, 15). Casoxin C is an opioidantagonist, obtained from tryptic digests of bovine κ-CN(14), that also functions as an agonist for C3a receptors(82). Lastly, casoxin D, purified from humanαs1-CN frac-tions, elicits an opioid antagonist response (96). In gen-eral, the chemically modified casoxins are more activethat their nonmethylated derivatives (14, 15).

Lactoferroxins are antagonists generated from humanlactoferrin (83). Initially, a chloroform and methanol ex-tract from a peptic digest of lactoferrin was assayed foractivity, and the results indicated that the opioid proper-ties were similar to those of naloxone, a known antago-nist ligand. Peptides derived from pepsin digestion,alone, were minimally effective, while those purifiedfrom a methyl-esterified fraction were significantly morepotent. HPLC analyses resulted in purification of threeseparate active fractions designated lactoferroxin A, B,and C, respectively. It was determined that the α-car-bonyl group of each was methyl esterified based on com-parison of bioactivity measurements and HPLC reten-tion times to those of corresponding synthetic peptides.Like casoxins, the chemically modified peptides may notactually exist in vivo. Lactoferroxin A, residues 318 to323, showed a preference for µ-receptors. On the otherhand, lactoferroxin B and C, derived from residues 536to 540 and 673 to 679, respectively, exhibited a higherpropensity for κ-receptors (83).

Miscellaneous Peptides

Physiologically active peptides that directly affect gas-trointestinal functions have also been documented. Ca-


somorphins slow gastric motility and emptying in nonru-minants (22), while caseinomacropeptide, a 64-aminoacid glycopeptide released from κ-CN by gastric prote-ases, exerts its effects on digestive function by inhibitinggastric acid secretions (101).

Several other milk-derived peptides have been de-scribed in the literature. Atrial natriuretic factor, or atri-opeptin, is a peptide found naturally occurring in humanmilk (28). This peptide functions as a strong diuretic,natriuretic, and vasorelaxant, and plays an importantrole in circulatory adaptation to extrauterine life. Morerecently, a peptide, obtained by in vitro proteolysis ofbovine β-LG, was found to exert its effect on smoothmuscle (69).

CONCLUSIONS

In summary, milk contains numerous peptide se-quences that affect crucial physiological functions andmodulate many regulatory processes. These include, butare not limited to hormone secretion (casomorphins),immune defense (casokinins, casomorphins, and immu-nopeptides), nutrient uptake (phosphopeptides and caso-morphins), and neurological information transmission(casokinins). Industrial-scale processes are already es-tablished for the recovery of nondenatured, biologicallyactive, whey proteins that are produced as a direct by-product during cheese manufacturing. Caseins are man-ufactured on an equally large scale for food productssuch as cottage cheese. Thus, it should be economicallypossible to salvage these protein fractions and generateactive peptides from them for use as dietary supple-ments, safe and effective natural preservatives, andmilk-based nutraceuticals. This field of research prom-ises to contribute novel biologically active peptides de-rived from milk proteins that significantly impact boththe food and health care industries.

ACKNOWLEDGMENTS

This work was partially supported by the SoutheastDairy Foods Research Center, Dept. of Food Science,North Carolina State University, Raleigh, NC, project#5-44408. The authors would like to gratefully acknowl-edge Hans Meisel for his significant scientific contribu-tions to this field of research and the careful reviewingof this manuscript.

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