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Invited review: Physiological properties of bioactive peptides obtained from whey proteins

  • A.R. Madureira
    Affiliations
    Centro de Biotecnologia e Quımica Fina (CBQF)/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, R. Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal
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  • T. Tavares
    Affiliations
    Centro de Biotecnologia e Quımica Fina (CBQF)/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, R. Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal
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  • A.M.P. Gomes
    Affiliations
    Centro de Biotecnologia e Quımica Fina (CBQF)/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, R. Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal
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  • M.E. Pintado
    Affiliations
    Centro de Biotecnologia e Quımica Fina (CBQF)/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, R. Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal
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  • F.X. Malcata
    Correspondence
    Corresponding author.
    Affiliations
    Centro de Biotecnologia e Quımica Fina (CBQF)/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, R. Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal
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      Abstract

      Processing of whey proteins yields several bioactive peptides that can trigger physiological effects in the human body: on the nervous system via their opiate and ileum-contracting activities; on the cardiovascular system via their antithrombotic and antihypertensive activities; on the immune system via their antimicrobial and antiviral activities; and on the nutrition system via their digestibility and hypocholesterolemic effects. The specific physiological effects, as well the mechanisms by which they are achieved and the stabilities of the peptides obtained from various whey fractions during their gastrointestinal route, are specifically discussed in this review.

      Key words

      Introduction

      Whey proteins account for only about 20% (wt/wt) of the whole milk protein inventory, whereas caseins account for the most part. Whey proteins are globular molecules with substantial contents of α-helix motifs, in which acidic/basic and hydrophobic/hydrophilic AA are balanced throughout their sequences. The whey proteins include β-LG, α-LA, immunoglobulins, BSA, bovine lactoferrin (BLF), and lactoperoxidase, in addition to other minor proteinaceous components, such as glycomacropeptide (GMP), which is released from κ-casein in the first step of enzymatic cheesemaking. These proteins possess important nutritional and biological properties, particularly with regard to promotion of health and prevention of diseases and health conditions (
      • Madureira A.R.
      • Pereira C.I.
      • Gomes A.M.P.
      • Pintado M.E.
      • Malcata F.X.
      Bovine whey proteins—Overview on the main biological properties.
      ).
      Controlled hydrolysis of whey proteins releases bioactive peptides, most of which have not yet been characterized to the same degree as casein-derived peptides; see, for example, the comprehensive review by
      • Silva S.V.
      • Malcata F.X.
      Caseins as source of bioactive peptides.
      on this subject. However, whey peptides have the potential to play important roles in several areas of interest; namely as part of preventive and therapeutic health approaches, because of a favorable combination of various biochemical and physiological features (
      • Meisel H.
      Overview on milk protein-derived peptides.
      ). Experimental evidence exists that bioactive peptides can be released from α-LA, β-LG, BLF, and BSA; some of these bioactive peptides have received special designations: α- and β-lactorphin, β-lactotensin, serophin, albutensin A, lactoferricin B, and lactoferrampin (although many others exist).
      Besides being susceptible to inorganic (acid or alkaline) catalysis, whey proteins can be hydrolyzed via gastric, pancreatic, and microbial proteases, and thus generate peptides that may play physiological roles (Figure 1). These roles have been addressed by several researchers (
      • Shah N.P.
      Effects of milk-derived bioactives: An overview.
      ;
      • Smacchi E.
      • Gobbetti M.
      Bioactive peptides in dairy products, synthesis and interaction with proteolytic enzymes.
      ;
      • Baró L.
      • Jiménez J.
      • Martínez-Férez A.
      • Bouza J.
      Bioactive milk peptides and proteins.
      ;
      • Meisel H.
      • FitzGerald R.J.
      Biofunctional peptides from milk proteins, mineral binding and cytomodulatory effects.
      ;
      • Korhonen H.
      • Pihlanto A.
      Bioactive peptides and proteins.
      ) but with an emphasis on nutritional features. The physiological roles will be further discussed in this paper under a more global, yet integrated perspective. This review thus adds to other published work on the topic of whey protein characteristics and uses, and on the physiological roles of dairy peptides. However, it focuses specifically on biopeptides obtained from whey.
      Figure thumbnail gr1
      Figure 1Physiological framework of bioactivity of whey peptides.

      Production of Bioactive Peptides

      There are various modes of release of peptides (with biological activity) from precursor proteins or synthesis thereof from simpler molecules; the most frequent are described in Figure 2 and considered in detail below.
      Figure thumbnail gr2
      Figure 2Alternative modes of bioactive peptide generation. Peptide release and synthesis pathways.
      Starter and nonstarter bacteria are commonly used in the manufacture of dairy products and they take advantage of their proteolytic system, which contains at least 16 different peptidases. However, few bioactive peptides starting from whey proteins as precursors are typically obtained. This fact can be explained in several ways: more studies have been published pertaining to milk than whey, as more numerous dairy products are manufactured with whole milk than with whey, and some of them are released via rennet or other coagulants; and caseins are in higher proportion than whey proteins in milk, so they are more readily available for catalysis, although controversial discussions have been carried out on the resistance of whey proteins to breakdown by bacterial peptidases.
      The major whey proteins, α-LA and β-LG, are resistant to the endogenous enzymes present in milk, such as plasmin. Plasmin (EC 3.4.21.7) is a serine proteinase, similar to trypsin in its activity and characteristics; it hydrolyzes αS1-, αS2- and β-caseins, but has little or no activity on whey proteins (
      • Grufferty M.B.
      • Fox P.F.
      Milk alkaline proteinase.
      ;
      • Cassens P.W.J.R.
      • Visser S.
      • Gruppen H.
      • Voragen A.G.J.
      β-Lactoglobulin hydrolysis. 1. Peptide composition and functional properties of hydrolysates obtained by the action of plasmin, trypsin, and Staphylococcus aureus V8 protease.
      ). Plasmin cleaves proteins on the carboxyl side of K and R residues (
      • Kitchen B.J.
      Indigenous milk enzymes.
      ), with a preference for the former. Furthermore, unfolded β-LG can be a potent inhibitor of plasmin via thiol-disulfide binding (
      • Scollard P.G.
      • Beresford T.P.
      • Murphy P.M.
      • Kelly A.L.
      Barostability of milk plasmin activity.
      ).
      Despite the relative difficulty in obtaining peptides by microbial hydrolysis, enzymatic hydrolysis has been the most common route to produce bioactive peptides from whey proteins, and pancreatic enzymes (chiefly trypsin) have been associated with efforts toward production, as well as characterization and identification of many peptides (see Table 1). Trypsin cleaves at the C-terminal end of R and K residues, whereas chymotrypsin requires an aromatic or bulky nonpolar side chain (e.g., F, Y, W, L, or M) on the carboxyl side of the bond subject to cleavage.
      Table 1Peptides obtained from the main whey proteins, enzymes used to obtain those peptides, and resulting amino acid sequence and biological activity
      Source proteinEnzymePeptideAmino acid sequenceIdentityBioactivity
      ACE=angiotensin-I-converting enzyme.
      References
      α-LATrypsinf(1–5)EQLTKAntimicrobial against several gram-positive bacteria
      • Pellegrini A.
      • Thomas U.
      • Bramaz N.
      • Hunziker P.
      • von Fellenberg R.
      Isolation and identification of three bactericidal domains in the bovine α-lactalbumin molecule.
      f(17–31)S-Sf(109–114)
      Chymotrypsinf(61–68)S-Sf(75–80)
      Pepsinα-Lactorphinf(50–53)YGLFOpioid agonist; ACE-inhibitory; ileum contracting
      • Antila P.
      • Paakkari I.
      • Järvinen A.
      • Mattila M.J.
      • Laukkanen M.
      • Pihlanto-Leppälä A.
      • Mäntsälä P.
      • Hellman J.
      Opioid peptides derived from in vitro proteolysis of bovine whey proteins.
      • Mullally M.M.
      • Meisel H.
      • FitzGerald R.J.
      Synthetic peptides corresponding to α-LA and β-LG sequences with angiotensin-I-converting enzyme inhibitory activity.
      • Meisel H.
      • Schlimme E.
      Bioactive peptides derived from milk proteins: Ingredients for functional foods?.
      • Nurminen M.-L.
      • Sipola M.
      • Kaarto H.
      • Pihlanto-Leppälä A.
      • Piilola K.
      • Korpela R.
      • Tossavainen O.
      • Korhonen H.J.T.
      • Vapaatalo H.
      α-Lactorphin lowers blood pressure measured by radiotelemetry in normotensive and in spontaneously hypertensive rats.
      β-LGTrypsinf(15–20)VAGTWYACE-inhibitory; antimicrobial against several gram-positive bacteria
      • Ijäs H.
      • Collin M.
      • Finckenberg P.
      • Pihlanto-Leppälä A.
      • Korhonen H.
      • Korpela P.
      • Vapaatalo H.
      • Nurminem M.-L.
      Antihypertensive opioid-like milk peptide α-lactorphin: Lack of effect on behavioural tests in mice.
      • Pellegrini A.
      • Thomas U.
      • Bramaz N.
      • Hunziker P.
      • von Fellenberg R.
      Isolation and identification of three bactericidal domains in the bovine α-lactalbumin molecule.
      f(25–40)AASDISLLDAQSAPLRAntimicrobial against several gram-positive bacteria
      • Pellegrini A.
      • Dettling C.
      • Thomas U.
      • Hunziker P.
      Isolation and characterisation of four bactericidal domains in the bovine α-lactoglobulin.
      f(78–83)IPAVFK
      f(92–100)VLVLDTDYK
      Pepsin; trypsinß-Lactorphinf(102–105)YLLFOpioid agonist; ACE-inhibitory; ileum-contracting
      • Antila P.
      • Paakkari I.
      • Järvinen A.
      • Mattila M.J.
      • Laukkanen M.
      • Pihlanto-Leppälä A.
      • Mäntsälä P.
      • Hellman J.
      Opioid peptides derived from in vitro proteolysis of bovine whey proteins.
      • Mullally M.M.
      • Meisel H.
      • FitzGerald R.J.
      Synthetic peptides corresponding to α-LA and β-LG sequences with angiotensin-I-converting enzyme inhibitory activity.
      • Meisel H.
      • Schlimme E.
      Bioactive peptides derived from milk proteins: Ingredients for functional foods?.
      • Sipola M.
      • Finckenberg P.
      • Korpela R.
      • Vapaatalo H.
      • Nurminen M.-L.
      Effect of long-term intake of milk products on blood pressure in hypertensive rats.
      Porcine trypsinf(71–75)IIAEKHypocholesterolemic
      • Nagaoka S.
      • Futumura Y.
      • Miwa K.
      • Awano T.
      • Yamauchi K.
      • Kanamaru Y.
      • Tadashi K.
      • Kuwata T.
      Identification of novel hypocholesterolemic compound derived from bovine milk beta-lactoglobulin.
      Chymotrypsinß-Lactotensinf(146–149)HIRLACE-inhibitory; ileum contracting; antinoceptive activity; hypertensive activity
      • Pihlanto-Leppälä A.
      • Paakkari I.
      • Rinta-Koski M.
      • Antila P.
      Bioactive peptide derived from in vitro proteolysis of bovine β-lactoglobulin and its effect on smooth muscle.
      • Yamauchi R.
      • Usui H.
      • Yunden J.
      • Takenaka Y.
      • Tani F.
      • Yoshikawa M.
      Characterization of β-lactotensin a bioactive peptide derived from bovine β-lactoglobulin as a neurotensin agonist.
      Serum albuminTrypsinAlbutensin Af(208–216)AFKAWAVARIleum contracting
      • Yamauchi K.
      Biologically functional proteins of milk and peptides derived from milk proteins.
      Serophinf(399–404)YGFGNAOpioid activity
      • Tani F.
      • Shiota A.
      • Chiba H.
      • Yoshikawa M.
      Serophin an opioid peptide derived from bovine serum albumin.
      • Meisel H.
      • Schlimme E.
      Bioactive peptides derived from milk proteins: Ingredients for functional foods?.
      1 ACE = angiotensin-I-converting enzyme.
      In practice, enzymatic hydrolysis can be performed in 1 of 2 ways: in a batch or continuous manner (ultrafiltration for enzyme containment is often used in this case). Hydrolysis of whey proteins brought about by Alcalase (Novo Industry AS, Copenhagen, Denmark) using a membrane recycle reactor has been described (
      • Perea A.
      • Ugalde U.
      Continuous hydrolysis of whey proteins in a membrane recycle reactor.
      ), as has production of α-lactorphin via continuous hydrolysis of goat whey in an ultrafiltration reactor (
      • Bordenave S.
      • Sannier F.
      • Ricart G.
      • Piot J.M.
      Continuous hydrolysis of goat whey in an ultrafiltration reactor: Generation of alpha-lactorphin.
      ). Opioid peptides, such as α- and β-lactorphin, can also be obtained via selective ultrafiltration membranes (30 and 1 kDa cutoffs, respectively); this method was claimed (
      • Korhonen H.
      • Pihlanto A.
      Bioactive peptides and proteins.
      ) to produce final peptide mixtures with high angiotensin-I-converting enzyme (ACE)-inhibitory activity, owing to a selective concentration of low-molecular-weight peptides.
      Alternatively, one may resort to peptide synthesis, and the main typical approaches here are depicted in Figure 2; the most suitable method for synthesis depends mainly on the length and amount of the peptides sought. Chemical synthesis, starting from free AA, is normally used on the laboratory scale. Chemical synthesis exists in 2 variants, the liquid and the solid phase; the former is used for generation of short peptides, whereas the latter is more common for synthesis of peptides composed of 10 to 100 residues. Conversely, enzymatic synthesis is performed for shorter sequences, whereas DNA recombinant technology applies mainly to large peptides (
      • Iqbal G.
      • López-Fandiño R.
      • Jorba X.
      • Vulfson E.N.
      Biologically active peptides and enzymatic approaches to their production.
      ).

      Peptides with Antihypertensive and Antithrombotic Activities

      One of the major risk factors for cardiovascular disease is elevated blood pressure. Angiotensin I-converting enzyme plays a crucial role in the regulation of blood pressure, because it promotes the conversion of angiotensin I to the potent vasoconstrictor angiotensin II and inactivates the vasodilator bradykinin (Figure 3). By inhibiting these processes, synthetic ACE inhibitors have long been used as antihypertensive agents. Milk proteins were identified as sources of ACE inhibitory peptides and are currently the best-known class of bioactive peptides.
      Figure thumbnail gr3
      Figure 3Schematic representation of the renin-angiotensin system, demonstrating the balance between angiotensin and bradykinin. Angiotensin-I-converting enzyme (ACE) plays a central role in converting angiotensin I to angiotensin II and in deactivating bradykinin.

      Peptides from α-LA and β-LG

      The ACE-inhibiting peptides derived from casein are termed casokinins, whereas those derived from whey (α-LA and β-LG) are called lactokinins (
      • FitzGerald R.J.
      • Meisel H.
      Lactokinins: Whey protein-derived ACE inhibitory peptides.
      ). Characterization of hydrolysates of whey proteins, including the amino acid sequences of peptides therein that exhibit in vitro ACE-inhibiting activity or in vivo antihypertensive effects, is provided in Table 2. Inhibition of ACE is classically measured as the concentration of compound needed to inhibit 50% of the original ACE activity (IC50;
      • Gerdes S.K.
      • Harper J.W.
      • Miller G.
      Applications Monograph: Cardiovascular Health Bioactive Components of Whey and Cardiovascular Health.
      ). The heptapeptide ALPMHIR from β-LG is the most potent ACE-inhibitor (IC50 = 43 μM) isolated from whey to date. As shown in Table 2, trypsin has been the most widely used enzyme to produce hydrolysates with reasonable ACE-inhibiting activity.
      Table 2Angiotensin I-converting enzyme (ACE)-inhibiting peptides obtained from the main whey proteins, as well as enzymes used to obtain them, resulting amino acid sequence and in vitro ACE inhibition
      Source proteinEnzymeAA sequenceIdentityIn vitro ACE inhibition
      Given as inhibitory concentration (mg/mL) that produces 50% of maximum effect (IC50).
      Reference
      α-LATrypsinf(50–51)YG1,522
      • Mullally M.M.
      • Meisel H.
      • FitzGerald R.J.
      Synthetic peptides corresponding to α-LA and β-LG sequences with angiotensin-I-converting enzyme inhibitory activity.
      Pepsin + trypsin + chymotrypsinf(50–52)YGL409
      • Pihlanto-Leppälä A.
      • Koskinen P.
      • Piilola K.
      • Tupasela T.
      • Korhonen H.
      Angiotensin I-converting enzyme inhibitory properties of whey protein digests: Concentration and characterization of active peptides.
      Pepsinf(50–53)YGLF733
      Blood pressure reduction effect.
      • Mullally M.M.
      • Meisel H.
      • FitzGerald R.J.
      Synthetic peptides corresponding to α-LA and β-LG sequences with angiotensin-I-converting enzyme inhibitory activity.
      Trypsinf(99–108)VGINYWLAHK327
      • Pihlanto-Leppälä A.
      • Koskinen P.
      • Piilola K.
      • Tupasela T.
      • Korhonen H.
      Angiotensin I-converting enzyme inhibitory properties of whey protein digests: Concentration and characterization of active peptides.
      f(104–108)WLAHK77
      β-LGTrypsinf(22–25)LAMA1,062
      • Pihlanto-Leppälä A.
      • Koskinen P.
      • Piilola K.
      • Tupasela T.
      • Korhonen H.
      Angiotensin I-converting enzyme inhibitory properties of whey protein digests: Concentration and characterization of active peptides.
      f(32–40)LDAQASPLR635
      f(81–83)VKF1,029
      • Mullally M.M.
      • Meisel H.
      • FitzGerald R.J.
      Angiotensin-I-converting enzyme inhibitory activities of gastric and pancreatic proteinase digests of whey proteins.
      f(142–148)ALPMHIR43
      Syntheticf(102–103)YL122
      • Mullally M.M.
      • Meisel H.
      • FitzGerald R.J.
      Synthetic peptides corresponding to α-LA and β-LG sequences with angiotensin-I-converting enzyme inhibitory activity.
      f(104–105)LF349
      Pepsin + trypsin + chymotrypsinf(94–100)VLDTDYK946
      • Pihlanto-Leppälä A.
      • Koskinen P.
      • Piilola K.
      • Tupasela T.
      • Korhonen H.
      Angiotensin I-converting enzyme inhibitory properties of whey protein digests: Concentration and characterization of active peptides.
      f(102–105)YLLF172
      • Chiba H.
      • Yoshikawa M.
      Bioactive peptides derived from food proteins.
      f(106–111)CMENSA788
      • Pihlanto-Leppälä A.
      • Koskinen P.
      • Piilola K.
      • Tupasela T.
      • Korhonen H.
      Angiotensin I-converting enzyme inhibitory properties of whey protein digests: Concentration and characterization of active peptides.
      f(142–146)ALPMH521
      WheyFermentation + trypsin + chymotrypsinα-LA f(105–110)LAHKAL621
      • Pihlanto-Leppälä A.
      • Rokka T.
      • Korhonen H.
      Angiotensin I-converting enzyme inhibitory peptides from bovine milk proteins.
      β-LA f(9–14)GLDIQK580
      • Pihlanto-Leppälä A.
      • Rokka T.
      • Korhonen H.
      Angiotensin I-converting enzyme inhibitory peptides from bovine milk proteins.
      β-LA f(15–20)VAGTWY1,682
      • Pihlanto-Leppälä A.
      • Rokka T.
      • Korhonen H.
      Angiotensin I-converting enzyme inhibitory peptides from bovine milk proteins.
      Proteinase Kf(78–80)IPA141
      • Abubakar A.
      • Saito T.
      • Kitazawa H.
      • Kawai Y.
      • Itoh T.
      Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion.
      Fermentation by cheese microfloraα-LA f(104–108)WLAHK77
      • Didelot S.
      • Bordenave-Juchereau S.
      • Rosenfeld E.
      • Piot J.-M.
      • Sannier F.
      Peptides released from acid goat whey by a yeast-lactobacillus association isolated from cheese microflora.
      1 Given as inhibitory concentration (mg/mL) that produces 50% of maximum effect (IC50).
      2 Blood pressure reduction effect.
      At present, the main challenge in the production of bioactive peptides by enzymatic hydrolysis in vitro is finding the suitable enzyme and hydrolysis conditions that enhance bioactivity and yield in their production. Digestion of α-LA and β-LG by enzymes (e.g., pepsin, α-chymotrypsin, pancreatin, elastase, or carboxypeptidase A and B) indicates that trypsin is normally required to produce high ACE-inhibitory activity from these whey proteins (
      • Pihlanto-Leppälä A.
      • Koskinen P.
      • Piilola K.
      • Tupasela T.
      • Korhonen H.
      Angiotensin I-converting enzyme inhibitory properties of whey protein digests: Concentration and characterization of active peptides.
      ); for example, the peptides f(104–108) and f(142–148), released from α-LA and β-LG via trypsin, possess ACE-inhibiting activities of 77 and 43 μM, respectively. On the other hand, the gastrointestinal protease elastase is associated with a poor yield of ACE-inhibitory peptides from α-LA and β-LG (
      • Mullally M.M.
      • Meisel H.
      • FitzGerald R.J.
      Angiotensin-I-converting enzyme inhibitory activities of gastric and pancreatic proteinase digests of whey proteins.
      ;
      • Pihlanto-Leppälä A.
      • Koskinen P.
      • Piilola K.
      • Tupasela T.
      • Korhonen H.
      Angiotensin I-converting enzyme inhibitory properties of whey protein digests: Concentration and characterization of active peptides.
      ). A new food-grade proteolytic preparation was tested for the production of novel β-LG–derived ACE inhibitory peptides (
      • Ortiz-Chao P.
      • Gomez-Ruiz J.A.
      • Rastall R.A.
      • Mills D.
      • Cramer R.
      • Pihlanto A.
      • Korhonen H.
      • Jauregi P.
      Production of novel ACE inhibitory peptides from β-lactoglobulin using Protease N Amano.
      ). Protease N Amano (EC 3.4.24.28) is a commercial proteolytic mixture produced by Bacillus subtilis fermentation, which was found to produce very complex peptide mixtures; the partially fractionated hydrolysates already had very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterized. It corresponded to β-LG f(36–42) and had an IC50 value of 8°μM, which is considerably lower than that of the most potent ACE inhibitory peptides derived from bovine β-LG.
      The importance of hydrophobic amino acid residues in the peptide sequence toward ACE inhibition has been discussed at some length (
      • Cheung H.S.
      • Wang F.L.
      • Ondetti M.A.
      • Sabo E.F.
      • Cushman D.W.
      Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOH-terminal dipeptide sequence.
      ): such aromatic amino (W, Y, and F) or imino (P) C-terminal residues contribute to expression of ACE-inhibitory activity; those C-terminal residues can indeed interact with many subsites of ACE (
      • Ondetti M.A.
      • Cushman D.W.
      Enzymes of the renin-angiotensin system and their inhibitors.
      ), whereas a positive charge, such as that in the guanidine group of R, is important for ACE inhibition (
      • Meisel H.
      Overview on milk protein-derived peptides.
      ): ALPM is composed of hydrophobic amino acids, and HIR has a hydrophobic (I) amino acid. It seems that the dipeptide YG is the major component responsible for such an activity in α-lactorphin, whereas the same holds for lactoferrin regarding β-lactorphin. As shown in Figure 4, breakdown of peptides may either strengthen their ACE-inhibiting capacity (e.g., VGINYWLAHK → WLAHK, YLLF → YL, and HIRL → IR) or weaken it (e.g., ALPMHIR → ALPMH → ALPM, YLLF → LF, and HIRL → RL); however, no apparent general trend emerges from this observation.
      Figure thumbnail gr4
      Figure 4Evolution in degree of angiotensin-I-converting enzyme (ACE) inhibition, upon breakdown of selected whey peptides.
      It should be emphasized that a high ACE-inhibiting activity in vitro does not necessarily imply a high antihypertensive activity in vivo; unfortunately, only a few in vivo studies encompassing whey protein hydrolysates are available to date (e.g.,
      • Nakamura Y.
      • Yamamoto N.
      • Sakai K.
      • Takano T.
      Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme.
      ;
      • Abubakar A.
      • Saito T.
      • Kitazawa H.
      • Kawai Y.
      • Itoh T.
      Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion.
      ;
      • Yamamoto N.
      • Maeno M.
      • Takano T.
      Purification and characterization of an antihypertensive peptide from a yogurt-like product fermented by Lactobacillus helveticus CPN4.
      ), which accordingly provide only limited validation of that statement. In particular, the peptides f(50–53) (α-lactorphin) (
      • Nurminen M.-L.
      • Sipola M.
      • Kaarto H.
      • Pihlanto-Leppälä A.
      • Piilola K.
      • Korpela R.
      • Tossavainen O.
      • Korhonen H.J.T.
      • Vapaatalo H.
      α-Lactorphin lowers blood pressure measured by radiotelemetry in normotensive and in spontaneously hypertensive rats.
      ) and f(78–80) (lactosin A) from α-LA (
      • Abubakar A.
      • Saito T.
      • Kitazawa H.
      • Kawai Y.
      • Itoh T.
      Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion.
      ), as well as f(142–145) (lactosin B) from β-LG (
      • Murakami M.
      • Tonouchi H.
      • Takahashi R.
      • Kitazawa H.
      • Kawai Y.
      • Negishi H.
      • Saito T.
      Structural analysis of a new anti-hypertensive peptide (β-lactosin B) isolated from a commercial whey product.
      ) were shown to reduce blood pressure in vivo. Existence of a (negative) correlation between in vitro ACE inhibition and in vivo blood pressure decrease has been illustrated (
      • Yamamoto N.
      • Maeno M.
      • Takano T.
      Purification and characterization of an antihypertensive peptide from a yogurt-like product fermented by Lactobacillus helveticus CPN4.
      ): hydrolysates brought about by trypsin and actinase (a fungal proteinase from Actinomyces spp.) possess a relatively high in vitro ACE-inhibiting activity (141 μM), but yield a relatively large blood pressure decrease in spontaneously hypertensive rats, whereas chemically synthesized lactosin B (APLM) showed a high IC50 value (928 μM), thus indicating a weak ACE-inhibitory activity (
      • Walsh D.J.
      • Bernard H.
      • Murray B.A.
      • MacDonald J.
      • Pentzien A.-K.
      • Wright G.A.
      • Wal J.-M.
      • Struthers A.D.
      • Meisel H.
      • FitzGerald R.J.
      In vitro generation and stability of the lactokinin β-lactoglobulin fragment (142–148).
      ).
      In addition to inhibition of ACE, the exact molecular mechanisms by which the active peptides act to exert their antihypertensive effect are not fully explained, which demands future research in this area. Some of the mechanisms proposed are described in Table 3 and all proceed through increase of vascular relaxation.
      Table 3Proposed mechanisms for some of the biological activities of peptides derived from whey proteins hydrolysis
      Peptide name/sequenceBiological functionMechanism proposedReference
      f(142–148)AntihypertensiveInhibits release of vasoconstrictor endothelin-1 (ET-1)
      • Maes W.
      • van Camp J.
      • Vermeirssen V.
      • Hemeryck M.
      • Ketelslegers J.M.
      • Chrezenmeir J.
      • van Oosteveldt P.
      • Huyghebaert A.
      Influence of the lactokinin Ala-Leu-Pro- Met-His-Ile-Arg (ALPMHIR) on the release of endothelin-1 by endothelial cells.
      α-Lactorphin f(50–53)AntihypertensiveNitric oxide-sensitive mechanism (in vivo)
      • Sipola M.
      • Finckenberg P.
      • Korpela R.
      • Vapaatalo H.
      • Nurminen M.-L.
      Effect of long-term intake of milk products on blood pressure in hypertensive rats.
      β-Lactorphin f(10–105)Antihypertensive
      β-Lactotensin f(146–149)AntihypertensiveMediated by neurotensin receptor NT2
      • Yamauchi R.
      • Usui H.
      • Yunden J.
      • Takenaka Y.
      • Tani F.
      • Yoshikawa M.
      Characterization of β-lactotensin a bioactive peptide derived from bovine β-lactoglobulin as a neurotensin agonist.
      α-Lactorphin; ß-Lactorphin; serophinOpioidDecrease of blood pressure by blocking opioid receptor antagonist naloxone
      • Nurminen M.-L.
      • Sipola M.
      • Kaarto H.
      • Pihlanto-Leppälä A.
      • Piilola K.
      • Korpela R.
      • Tossavainen O.
      • Korhonen H.J.T.
      • Vapaatalo H.
      α-Lactorphin lowers blood pressure measured by radiotelemetry in normotensive and in spontaneously hypertensive rats.
      ;
      • Ijäs H.
      • Collin M.
      • Finckenberg P.
      • Pihlanto-Leppälä A.
      • Korhonen H.
      • Korpela P.
      • Vapaatalo H.
      • Nurminem M.-L.
      Antihypertensive opioid-like milk peptide α-lactorphin: Lack of effect on behavioural tests in mice.
      AlbutensinOpioidVia complement of C3a and C5a receptors
      • Takahashi M.
      • Moriguchi S.M.
      • Suganuma T.
      • Shiota H.
      • Takenaka A.
      • Tani Y.
      • Ryuzo F.S.
      • Yoshikawa M.
      Albutensin A an ileum-contracting peptide derived from serum albumin acts through both receptors for complements C3a and C5a.
      AntinociceptiveMediated by neurotensin and dopamine antagonist receptors NT2 and D1
      • Yamauchi R.
      • Usui H.
      • Yunden J.
      • Takenaka Y.
      • Tani F.
      • Yoshikawa M.
      Characterization of β-lactotensin a bioactive peptide derived from bovine β-lactoglobulin as a neurotensin agonist.
      Food intake regulationMediated by complementing C3a receptor
      • Ohinata K.
      • Inui A.
      • Asakawa A.
      • Wada K.
      • Wada E.
      • Yoshikawa M.
      Albutensin A and complement C3a decrease food intake in mice.
      GlycomacropeptideFood intake regulationRelease of cholecystokinin
      • Beucher M.
      • Levenez F.
      • Yvon Y.
      • Corring T.
      Effect of caseinomacropeptide (CMP) of cholecystokinin (CCK) release in rats.
      At present, fragments of β-LG resulting from hydrolysis of whey protein isolate are marketed as BioZate (Danisco Foods International, Le Sueur, MN), which is claimed to reduce blood pressure. A placebo-controlled study has been conducted with the BioZate 1 product in 30 borderline hypertensive subjects for 6 wk, in which the placebo was unhydrolyzed whey protein isolate. A reduction in blood pressure of 8°mmHg was obtained compared with the placebo group (
      • Pins J.J.
      • Keenan J.M.
      Effects of whey peptides on cardiovascular disease risk factors.
      ).

      Peptides with Opioid and Ileum-Contracting Activities

      Whey proteins can release opioid peptides, which are atypical because of their structure (Y-X1-X2-F, where X1 and X2 denote generic residues) that enables binding to cell receptors and makes them responsible for specific physiological effects (Figure 5). α-Lactorphin, β-lactorphin, and β-lactotensin are opioid peptides (
      • Antila P.
      • Paakkari I.
      • Järvinen A.
      • Mattila M.J.
      • Laukkanen M.
      • Pihlanto-Leppälä A.
      • Mäntsälä P.
      • Hellman J.
      Opioid peptides derived from in vitro proteolysis of bovine whey proteins.
      ;
      • Pihlanto-Leppälä A.
      • Paakkari I.
      • Rinta-Koski M.
      • Antila P.
      Bioactive peptide derived from in vitro proteolysis of bovine β-lactoglobulin and its effect on smooth muscle.
      ) that also have antihypertensive functions. The proposed mechanism by which the opioid effect is achieved by such peptides is depicted in Table 3. Receptors involved include neurotensin receptor (NT) 1, which is a bioactive 13-AA neuropeptide that acts in the bovine hypothalamus and is involved in hypotension, food intake suppression, and analgesia, in addition to ileum contraction (
      • Tyler-McMahon B.M.
      • Bolles M.
      • Richelson E.
      Neurotensin: Peptide for the next millennium.
      ); NT2; and dopamine antagonist 1 (D1) (
      • Yamauchi R.
      • Usui H.
      • Yunden J.
      • Takenaka Y.
      • Tani F.
      • Yoshikawa M.
      Characterization of β-lactotensin a bioactive peptide derived from bovine β-lactoglobulin as a neurotensin agonist.
      ). α-Lactorphin was found to lower blood pressure via interaction with opioid receptors, but not via ACE inhibition. In fact, subcutaneous administration of the synthetic form of α-lactorphin in conscious, spontaneously hypertensive rats and in normotensive Wistar Kyoto rats led to lower blood pressures without affecting their heart rate (monitored by continuous radiotelemetry).
      Figure thumbnail gr5
      Figure 5Action of atypical opioid peptides onto specific receptors (σ, μ, κ) and physiological effects thereof. X1 and X2 represent generic endo amino acid residues.
      The opioid peptide produced from β-LG is β-lactotensin, which induces contraction in guinea pig ileum longitudinal muscle without electric stimulation in the absence of agonist up to a concentration of 10−6 M (
      • Yamauchi K.
      Biologically functional proteins of milk and peptides derived from milk proteins.
      ). This researcher discovered, in addition, that the smooth muscle-contracting effect of β-lactotensin is not mediated by an opioid-like mechanism. Furthermore, albutensin A, which is derived from BSA via tryptic digestion, exhibits an ileum-contracting activity (
      • Tani F.
      • Shiota A.
      • Chiba H.
      • Yoshikawa M.
      Serophin an opioid peptide derived from bovine serum albumin.
      ).

      Peptides with Antimicrobial and Immunomodulatory Activities

      Peptides from α-LA and β-LG

      Peptides were produced (
      • Pellegrini A.
      • Thomas U.
      • Bramaz N.
      • Hunziker P.
      • von Fellenberg R.
      Isolation and identification of three bactericidal domains in the bovine α-lactalbumin molecule.
      ,
      • Pellegrini A.
      • Dettling C.
      • Thomas U.
      • Hunziker P.
      Isolation and characterisation of four bactericidal domains in the bovine α-lactoglobulin.
      ) via proteolytic digestion of α-LA and β-LG by endopeptidases and were found to possess bactericidal properties, mainly against gram-positive bacteria (see Table 4).
      Table 4Peptides obtained from lactoferrin, enzymes used to obtain them, and resulting amino acid sequence and antimicrobial activity
      EnzymePeptideAmino acid sequenceIdentityAntimicrobial activityReferences
      Pepsin or chymosinLactoferricin Bf(17–41/42)FKCRRWQWRMKKLGAPSICVRRAF/AGram-positive and gram-negative bacteria
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Identification of the bactericidal domain of lactoferrin.
      ,
      • Bellamy W.
      • Takase M.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin.
      • Hoek K.S.
      • Milne J.M.
      • Grieve P.A.
      • Dionysius D.A.
      • Smith R.
      Antibacterial activity of bovine lactoferrin-derived peptides.
      • Recio I.
      • Visser S.
      Two ion-exchange chromatographic methods for the isolation of antibacterial peptides from lactoferrin: In situ enzymatic hydrolysis on an ion-exchange membrane.
      Chymosinf(1–16)S-S(17–48)APRKNVRWCTISQPEWFKCRRWQWRMKKLGAPSITCVRRAFALECIRAEscherichia coli
      • Hoek K.S.
      • Milne J.M.
      • Grieve P.A.
      • Dionysius D.A.
      • Smith R.
      Antibacterial activity of bovine lactoferrin-derived peptides.
      Pepsinf(1–16)S-S(45–48)APRKNVRWCTISQPEWCIRAMicrococcus flavus
      • Recio I.
      • Visser S.
      Two ion-exchange chromatographic methods for the isolation of antibacterial peptides from lactoferrin: In situ enzymatic hydrolysis on an ion-exchange membrane.
      Pepsinf(1–11)S-S(17–47)APRKNVRWCTIFKCRRWQWRMKKLGAPSITCVRRAFALECIRAM. flavus
      • Recio I.
      • Visser S.
      Two ion-exchange chromatographic methods for the isolation of antibacterial peptides from lactoferrin: In situ enzymatic hydrolysis on an ion-exchange membrane.
      Syntheticf(17–30)FKCRRWQWRMKKLGOral pathogenic bacteria
      • Groenink J.
      • Walgreen-Weterings E.
      • van’t Hof W.
      • Veerman E.C.
      • Amerongen A.V.N.
      Cationic amphipathic peptides, derived from bovine and human lactoferrins, with antimicrobial activity against oral pathogens.
      Syntheticf(19–37)CRRWQWRMKKLGAPSICVOral pathogenic bacteria
      PepsinLactoferricin CGoat f(14–42)PEWSKCYQWQRRMRKLGAPSITCVRRTSARRWQWRMKKLGAPSICVALRAM. flavus
      • Recio I.
      • Visser S.
      Two ion-exchange chromatographic methods for the isolation of antibacterial peptides from lactoferrin: In situ enzymatic hydrolysis on an ion-exchange membrane.
      SyntheticGoat f(17–41)SKCYQWQRRMRKLGAPSITCVRRTSE. coli
      • Vorland L.H.
      • Ulvatne H.
      • Rekdal O.
      • Svendsen J.S.
      Initial binding sites of antimicrobial peptides in Staphylococcus aureus and Escherichia coli.
      SyntheticLactoferricin MMurine f(17–41)EKCLRWQNEMRKVGGPPLSCVStaphylococcus aureus, E. coli
      SyntheticLfpepHuman f(118–140)TKCFQWQRNMRKVRGPPVSCIKRCandida albicans
      • Hammer J.
      • Haheim H.
      • Gutterberg T.J.
      Bovine lactoferrin is more efficient than bovine lactoferricin in inhibiting HSV-I/II replication in vitro.
      SyntheticKaliocin-1Human f(153–183)FFSASCVPGADKGQFPNLCRLCAGTGENKCAC. albicans
      SyntheticHuman lysozyme f(87–115)DNIADAVACAKRVVRDPQGIRAWVAWRNRGram-positive and gram-negative bacteria
      • Ibrahim E.H.
      • Sherman G.
      • Ward S.
      • Fraser V.J.
      • Kollef M.H.
      The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting.
      SyntheticLactoferrampinf(268–284)WKLLSKAQEKFGKNKRSC. albicans, E. coli, Bacillus subtilis and Pseudomonas aeruginosa
      • van der Kraan M.I.A.
      • Groenink J.
      • Nazmi K.
      • Veerman E.C.I.
      • Bolscher J.G.J.
      • Amerongen A.V.N.
      Lactoferrampin: A novel antimicrobial peptide in the N1-domain of bovine lactoferrin.
      f(265–284)DLIKLLSKAQEKFGKNKRS
      • van der Kraan M.I.A.
      • Nazmi K.
      • Teeken A.
      • Groenink J.
      • van’t Hoff W.
      • Veerman E.C.I.
      Lactoferrampin an antimicrobial peptide of bovine lactoferrin exhibits its candidacidal activity by a cluster of positively charged residues at the C-terminus in combination with a helix facilitating N-terminal part.
      Trypsin-mediated hydrolyses of bovine α-LA yielded interesting polypeptide fragments, which are depicted in Table 1; similar digestion of β-LG yielded 4 peptide fragments, all of which possess bactericidal activity. When synthesized in vitro, those 4 peptides were thought (
      • Pellegrini A.
      • Thomas U.
      • Bramaz N.
      • Hunziker P.
      • von Fellenberg R.
      Isolation and identification of three bactericidal domains in the bovine α-lactalbumin molecule.
      ) to exert bactericidal effects against gram-positive bacteria only, and Bacillus subtilis was the most susceptible among the species tested. In the case of peptide f(92–100), its amino acid sequence was then deliberately modified to ascertain the structural requirements for antibacterial activity. Replacement of D98 by R, and addition of K at the C-terminus yielded a peptide with even stronger bactericidal activity against the gram-negative bacteria Escherichia coli and Bordetella bronchiseptica, but a significantly reduced antibacterial capacity resulted toward B. subtilis. After a database search centered on this sequence, a high degree of homology was found with opsin, f(55–64), a human blue-sensitive peptide that is responsible for color discrimination. A peptide with this sequence was synthesized in vitro and assayed for bactericidal activity; it was strongly active against several bacterial strains (
      • Pellegrini A.
      • Dettling C.
      • Thomas U.
      • Hunziker P.
      Isolation and characterisation of four bactericidal domains in the bovine α-lactoglobulin.
      ).
      Hydrolysis of whey proteins has been proven to alter the biological activity of the proteins with respect to their ability to change immune system (immunomodulation). Two synthetic peptides, YG and YGG, corresponding to f(50–51) and f(18–20) of α-LA, respectively, enhanced both in vitro proliferation and protein synthesis of concanavalin A–stimulated human peripheral blood lymphocytes (
      • Kayser H.
      • Meisel H.
      Stimulation of human peripheral blood lymphocytes by bioactive peptides derived from bovine milk proteins.
      ). In vitro proliferation of murine spleen lymphocytes was stimulated by microfiltered whey protein isolates, and production of IgG was enhanced by purified β-LG, but such effects were reduced following hydrolysis by a trypsin-chymotrypsin mixture (
      • Wong K.F.
      • Middleton N.
      • Montgomery M.
      • Dey M.
      • Carr R.I.
      Immunostimulation of murine spleen cells by materials associated with bovine milk protein fractions.
      ;
      • Mercier A.
      • Gauthier S.F.
      • Fliss I.
      Immunomodulating effects of whey proteins and their enzymatic digests.
      ). Recently, whey peptide fractions derived from trypsin-chymotrypsin digestion of whey protein isolates were shown to modulate components of the immune response of a noninfected and E. coli–infected murine model, inducing increases in IgA in the absence of infection (
      • Saint-Sauveur D.
      • Gauthier S.F.
      • Boutin Y.
      • Montoni A.
      • Fliss I.
      Effect of feeding whey peptide fractions on the immune response in healthy and Escherichia coli infected mice.
      ).
      Whey proteins also induce oral tolerance: β-LG (mainly) and peptides obtained from enzymatic hydrolysis of whey proteins (e.g., tryptic peptides of bovine β-LG) are able to induce oral tolerance in mice (
      • Pecquet S.
      • Bovetto L.
      • Maynard F.
      • Fritsché R.
      Peptides obtained by tryptic hydrolysis of bovine β-lactoglobulin induce specific oral tolerance in mice.
      ). Oral tolerance is the mucosal and systemic antigen-specific immunological unresponsiveness caused by oral administration of a dietary antigen, which will prevent the digestive IgE–mediated hypersensitivity reactions to food antigens. Acidic peptides were further demonstrated, upon separation by isoelectric focusing of a tryptic-chymotryptic hydrolysate of β-LG, to stimulate splenocyte proliferation and IFN-γ production in vitro (
      • Prioult G.
      • Pecquet S.
      • Fliss I.
      Stimulation of interleukin-10 production by acidic beta-lactoglobulin-derived peptides hydrolyzed with Lactobacillus paracasei NCC2461 peptidases.
      ). However, hydrolysis of this peptide fraction brought about by Lactobacillus paracasei peptidases repressed lymphocyte stimulation, upregulated IL-10 production, and downregulated IFN-γ and IL-4 secretion. The authors thus concluded that L. paracasei apparently induces in vivo oral tolerance to β-LG by degrading acidic peptides and releasing immunomodulatory peptides that stimulate regulatory T cells, which function as major immunosuppressive agents, via secretion of IL-10 (
      • Prioult G.
      • Pecquet S.
      • Fliss I.
      Stimulation of interleukin-10 production by acidic beta-lactoglobulin-derived peptides hydrolyzed with Lactobacillus paracasei NCC2461 peptidases.
      ).
      The synthetic peptide GLF, corresponding to f(51–53) of α-LA, significantly increased phagocytosis of sheep red blood cells by murine peritoneal macrophages and protected mice against lethal Klebsiella pneumonia infections (
      • Berthou J.
      • Migliore-Samour D.
      • Lifchitz A.
      • Delettre J.
      • Floch F.
      • Jolles P.
      Immunostimulating properties and three-dimensional structure of two tripeptides from human and cow caseins.
      ). This peptide also stimulated, in a dose-dependent manner, binding of human senescent red blood cells to human monocytic-macrophage cells, as well as phagocytosis by the latter (
      • Gattegno L.
      • Migliore-Samour D.
      • Saffar L.
      • Jolles P.
      Enhancement of phagocytic activity of human monocytic–macrophagic cells by immunostimulating peptides from human casein.
      ). This activity correlates well with the presence of specific binding sites on human blood phagocytic cells (
      • Jaziri M.
      • Migliore-Samour D.
      • Casabianca-Pignède M.-R.
      • Keddad K.
      • Morgat J.-L.
      • Jollès P.
      Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr immunostimulating peptides from human milk proteins.
      ).
      The antimicrobial activity and immunostimulatory activity of hydrolysates of α-LA and β-LG have been shown to act jointly (
      • Biziulevicius G.A.
      • Kislukhina O.V.
      • Kazlauskaite J.
      • Zukaite V.
      Food-protein enzymatic hydrolysates possess both antimicrobial and immunostimulatory activities: A “cause and effect” theory of bifunctionality.
      ). These hydrolysates not only stimulated the autolytic system of naturally autolysing and some naturally nonautolysing microbial strains, but also increased the phagocytic ability of peritoneal macrophages in mice after oral administration, thus suggesting a relationship between both activities.

      Peptides from κ-Casein

      Glycomacropeptide is found in sweet (but not acid) whey and is released when chymosin (the main enzyme of rennet) acts on κ-casein in the preliminary cheesemaking step that eventually leads to αS- and β-casein precipitation. Such a peptide corresponds to the (hydrophilic) C-terminal portion of its substrate molecule and contains oligosaccharides that are O-linked to T and S residues. Glycomacropeptide is composed of 64 AA residues, with an overall molecular weight of 6.7 kDa. A unique AA sequence is found within GMP: aromatic AA are absent, but the sequence is rich in branched-chain AA. The degree of glycosylation of GMP is variable and influenced by the stage of lactation of the producing female and the genetic phenotype of κ-casein (
      • Dziuba J.
      • Minkiewicz P.
      Influence of glycosylation on content of both micelle-stabilizing ability and biological properties of the C-terminal fragments of cow's κ-casein.
      ); its molecular properties have been reviewed to some length (
      • Dziuba J.
      • Minkiewicz P.
      Influence of glycosylation on content of both micelle-stabilizing ability and biological properties of the C-terminal fragments of cow's κ-casein.
      ;
      • el-Salam A.M.H.
      • el-Shibiny S.
      • Buchheim W.
      Characteristics and potential uses of the casein macropeptide.
      ).
      The metabolic activity of GMP is thought to depend on the content and structure of its sugar moieties, which participate in stabilization of the whole κ-casein complex. The 2 most important carbohydrate components are N-acetylneuraminic acid and N-acetylgalactosamine (
      • Brody E.P.
      Biological activities of bovine glycomacropeptide.
      ). The cleavage sites of various enzymes on the C-terminal part of bovine GMP were studied by
      • Dziuba J.
      • Minkiewicz P.
      Influence of glycosylation on content of both micelle-stabilizing ability and biological properties of the C-terminal fragments of cow's κ-casein.
      . The pathway followed by the carbohydrate moieties, which eventually determine the biological function of GMP, is still not well understood; in fact, smaller sugar-free peptides released by trypsin and chymotrypsin cannot preserve the biological function of the original GMP.
      In vitro approaches have indicated that GMP prevents adhesion of cariogenic bacteria to tooth surfaces, thus suggesting that such a whey peptide is capable of inhibiting dental plaque and caries buildup (
      • Schupbach P.
      • Neeser J.R.
      • Golliard M.
      • Rouvet M.
      • Guggenheim B.
      Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutant streptococci.
      ). It has also been shown (
      • Neeser J.-R.
      • Chambaz A.
      • del Vedovo S.
      • Prigent M.-J.
      • Guggenheim B.
      Specific and nonspecific inhibition of adhesion of oral actinomyces and streptococci to erythrocytes and polystyrene by caseinoglycopeptide derivatives.
      ) that GMP decreases the extent of bacterial adhesion of actinomycetes and streptococci, the binding of cholera toxin to its receptor (
      • Kawasaki Y.
      • Isoda H.
      • Tanimoto M.
      • Dosako S.
      • Idota T.
      • Ahiko K.
      Inhibition by lactoferrin and casein glycomacropeptide of binding of cholera toxin to its receptor.
      ), and the binding of the heat-labile enterotoxins LT-I and LT-II of E. coli. This glycoprotein exhibits antiviral activity against hemagglutinin from the influenza virus (
      • Kawasaki Y.
      • Kawakami H.
      • Tanimoto M.
      • Dosako S.
      • Tomizawa A.
      • Kotake M.
      • Nakajima I.
      pH-Dependent molecular weight changes of κ-casein glycomacropeptide and its preparation by ultrafiltration.
      ). Most studies of the biological activity of GMP were done in vitro, so they will eventually require in vivo validation.
      Proliferation and phagocytic activities (via incorporation of fluorescent beads) of human macrophage-like cells (U937) were significantly enhanced in the presence of GMP (
      • Li E.W.
      • Mine Y.
      Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophage-like cells U937.
      ). Furthermore, digestion of GMP with pepsin led to higher proliferation and phagocytic activities, indicating that the enhanced immunostimulatory effect of GMP is due mainly to pepsin-digested fragments of it;
      • Li E.W.
      • Mine Y.
      Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophage-like cells U937.
      also showed that both the carbohydrate and the polypeptide chain compositions of GMP are essential for such stimulating effects to occur.

      Peptides from Lactoferrin

      Lactoferrin (LF) derivatives deserve special mention among whey peptides with antimicrobial activity. To date, the 2 most-studied lactoferricins (LFcin) are those derived from bovine and human LF (BLFcin and HLFcin, respectively). The 25-residue bovine peptide (f17–41) of LF is a more potent antimicrobial compound than its 47-residue counterpart derived from human LF (
      • Facon M.J.
      • Sakura B.J.
      Antibacterial activity of lactoferricin, lysozyme and EDTA against Salmonella enteritidis.
      ).
      Several biological properties have been claimed for BLFcin (
      • Wakabayashi H.
      • Takase M.
      • Tomita M.
      Lactoferricin derived from milk protein lactoferrin.
      ). It possesses antimicrobial activity against gram-negative and gram-positive bacteria and yeasts, as described in Tables 4 and 5(
      • Shin K.
      • Yamauchi K.
      • Teraguchi S.
      • Hayasawa H.
      • Tomita M.
      • Otsuka Y.
      • Yamazaki S.
      Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157 7.
      ;
      • Lupetti A.
      • Paulusma-Annema A.
      • Welling M.M.
      • Senesi S.
      • van Dissel J.T.
      • Nibbering P.H.
      Candidacidal activities of human lactoferrin peptides derived from the N terminus.
      ); however, some strains of Streptococcus lactis and Lactobacillus casei proved resistant to BLFcin (
      • Korhonen H.
      Antibacterial and antiviral activities of whey proteins, the importance of whey and whey components in food and nutrition.
      ). It has several putative modes of action: 1) cell surface binding (e.g., to E. coli and B. subtilis) (
      • Bellamy W.
      • Wakabayashi H.
      • Takase M.
      • Kawase K.
      • Shimamura S.
      • Tomita M.
      Role of cell binding in the antibacterial mechanism of lactoferricin B.
      ); 2) damage in cell bacteria via membrane disruption, and in fungi via changes in ultrastructural features (
      • Yamauchi K.
      • Tomita M.
      • Giehl T.J.
      • Ellison R.T.
      Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment.
      ); 3) release of LPS, and consequent disruption of the outer membrane (
      • Kang J.H.
      • Lee M.K.
      • Kim K.L.
      • Hahm K.-S.
      Structure-biological activity relationships of 11-residue highly basic peptide segment of bovine lactoferrin.
      ); 4) interaction (of a BLFcin 11-residue peptide) with bacterial phospholipid membranes (
      • Jack R.W.
      • Bierbaum G.
      • Sahl H.-G.
      Lantibiotics and Related Peptides.
      ); 5) disruption of essential cell-membrane functions via formation of ion channels in artificial membranes (
      • Samuelsen Ø.
      • Haukland H.H.
      • Ulvatne H.
      • Vorland L.H.
      Anti-complement effects of lactoferrin-derived peptides.
      ); and 6) effects in the cytoplasm contents and consequent action upon the cell surface (
      • Shin K.
      • Yamauchi K.
      • Teraguchi S.
      • Hayasawa H.
      • Tomita M.
      • Otsuka Y.
      • Yamazaki S.
      Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157 7.
      ). Bovine LFcin lacks the iron-binding region of its source protein, BLF, so it follows a different antimicrobial mechanism.
      Bovine LFcin, like LF, possesses immunomodulating and anti-inflammatory properties; it apparently inhibits the classical complement pathway and reduces the inhibitory properties of serum against E. coli in a concentration-dependent manner (
      • Samuelsen Ø.
      • Haukland H.H.
      • Ulvatne H.
      • Vorland L.H.
      Anti-complement effects of lactoferrin-derived peptides.
      ). However, no inhibitory effect was observed on the alternative complement pathway by other authors (
      • Mattsby-Balzer I.
      • Roseanu A.
      • Motas C.
      • Elverfors J.
      • Engberg I.
      • Hanson L.A.
      Lactoferrin or a fragment thereof inhibits the endotoxin-induced interleukin-6 response in human monocytic cells.
      ;
      • Vorland L.H.
      • Ulvatne H.
      • Rekdal O.
      • Svendsen J.S.
      Initial binding sites of antimicrobial peptides in Staphylococcus aureus and Escherichia coli.
      ); this was explained by the capacity of BLFcin to inhibit LPS-induced cytokine response in human monocytic cells.
      Lactoferricin exhibits a synergistic capacity with antifungal compounds such as azole agents (
      • Wakabayashi H.
      • Abe S.
      • Okutomi T.
      • Tansho S.
      • Kawase K.
      • Yamaguchi H.
      Cooperative anti-Candida effects of lactoferrin or its peptides in combination with azole antifungal agents.
      ). Lactoferricin also exerts antiviral activity (Table 5) against human cytomegalovirus and is able to inhibit actual invasion thereby (
      • Andersen J.H.
      • Osbakk S.A.
      • Vorland L.H.
      • Travik T.
      • Gutterberg T.J.
      Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomelogavirus into human fibroblasts.
      ). Bovine LF is more effective than BLFcinB against herpes simplex virus 1 and 2 (
      • Andersen J.H.
      • Jenssen H.
      • Gutterberg T.J.
      Lactoferrin and lactoferricin inhibit Herpes simplex 1 and 2 infection and exhibit synergy when combined with acyclovir.
      ), suggesting that the native protein possesses other regions that contribute to the aforementioned phenomenon (
      • Siciliano R.
      • Rega B.
      • Marchetti M.
      • Seganti L.
      • Antonini G.
      • Valenti P.
      Bovine lactoferrin peptidic fragments involved in inhibition of herpes simplex virus type 1 infection.
      ;
      • Hammer J.
      • Haheim H.
      • Gutterberg T.J.
      Bovine lactoferrin is more efficient than bovine lactoferricin in inhibiting HSV-I/II replication in vitro.
      ); this higher activity was also observed toward hepatitis C virus (
      • Ikeda M.
      • Nozaki A.
      • Sugiyama K.
      • Tanaka T.
      • Naganuma A.
      • Tanaka K.
      • Sekihara H.
      • Shimotohno K.
      • Saito M.
      • Kato N.
      Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells.
      ). Furthermore, BLFcin was demonstrated (
      • Shimazaki K.
      • Tazume T.
      • Uji K.
      • Tanaka M.
      • Kumura H.
      • Mikawa K.
      • Shimo-Oka T.
      Properties of a heparin-binding peptide derived from bovine lactoferrin.
      ) to bind glycosaminoglycans, heparin in particular.
      Table 5Specific targets of antimicrobial features of lactoferricin and lactoferrampin both derived from lactoferrin hydrolysis
      Peptide and antimicrobial activity targetReference
      Lactoferricin
       Gram-negative
      Escherichia coli O157:H7
      • Shin K.
      • Yamauchi K.
      • Teraguchi S.
      • Hayasawa H.
      • Tomita M.
      • Otsuka Y.
      • Yamazaki S.
      Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157 7.
      ;
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Identification of the bactericidal domain of lactoferrin.
      ,
      • Bellamy W.
      • Takase M.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin.
      ;
      • Tomita M.
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin.
      Salmonella spp.
      • Dionysius D.A.
      • Milne J.M.
      Antibacterial peptides of bovine lactoferrin 494: Purification and characterization.
      Klebsiella pneumoniae
      • Shin K.
      • Yamauchi K.
      • Teraguchi S.
      • Hayasawa H.
      • Tomita M.
      • Otsuka Y.
      • Yamazaki S.
      Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157 7.
      ;
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Identification of the bactericidal domain of lactoferrin.
      Yersinia enterocolitica
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Identification of the bactericidal domain of lactoferrin.
      Pseudomonas aeruginosa
      • Yamauchi K.
      • Tomita M.
      • Giehl T.J.
      • Ellison R.T.
      Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment.
      ;
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Identification of the bactericidal domain of lactoferrin.
      ,
      • Bellamy W.
      • Takase M.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin.
       Gram-positive
      Listeria monocytogenes
      • Bellamy W.
      • Takase M.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin.
      Bacillus spp.
      • Shin K.
      • Yamauchi K.
      • Teraguchi S.
      • Hayasawa H.
      • Tomita M.
      • Otsuka Y.
      • Yamazaki S.
      Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157 7.
      ;
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Identification of the bactericidal domain of lactoferrin.
      Clostridium spp.
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Identification of the bactericidal domain of lactoferrin.
      Corynebacterium spp.
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Identification of the bactericidal domain of lactoferrin.
      Enterococcus faecalis
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Identification of the bactericidal domain of lactoferrin.
      Streptococcus spp.
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Identification of the bactericidal domain of lactoferrin.
       Yeasts
      Candida albicans
      • Viejo-Díaz M.
      • Andrés M.T.
      • Fierro J.F.
      Different anti-Candida activities of two lactoferrin-derived peptides Lfpep and kaliocin-1.
      ;
      • Bellamy W.
      • Takase M.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin.
      Trichosporum cutaneum
      • Bellamy W.
      • Yamauchi K.
      • Wakabayashi H.
      • Takase M.
      • Takakura N.
      • Shimamura S.
      Antifungal properties of lactoferricin B, a peptide derived from the N-terminal region of bovine lactoferrin.
       Dermatophytes
      Trychophyton spp.
      • Bellamy W.
      • Yamauchi K.
      • Wakabayashi H.
      • Takase M.
      • Takakura N.
      • Shimamura S.
      Antifungal properties of lactoferricin B, a peptide derived from the N-terminal region of bovine lactoferrin.
      Nannizzia spp.
      • Bellamy W.
      • Yamauchi K.
      • Wakabayashi H.
      • Takase M.
      • Takakura N.
      • Shimamura S.
      Antifungal properties of lactoferricin B, a peptide derived from the N-terminal region of bovine lactoferrin.
       Other filamentous fungi
      Aspergillus spp.
      • Bellamy W.
      • Yamauchi K.
      • Wakabayashi H.
      • Takase M.
      • Takakura N.
      • Shimamura S.
      Antifungal properties of lactoferricin B, a peptide derived from the N-terminal region of bovine lactoferrin.
      Penicillium spp.
       Parasites
      Toxoplasma gondi
      • Bellamy W.
      • Yamauchi K.
      • Wakabayashi H.
      • Takase M.
      • Takakura N.
      • Shimamura S.
      Antifungal properties of lactoferricin B, a peptide derived from the N-terminal region of bovine lactoferrin.
       Viruses
        Human cytomelogalovirus
      • Andersen J.H.
      • Osbakk S.A.
      • Vorland L.H.
      • Travik T.
      • Gutterberg T.J.
      Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomelogavirus into human fibroblasts.
        Hepatitis C virus
      • Ikeda M.
      • Nozaki A.
      • Sugiyama K.
      • Tanaka T.
      • Naganuma A.
      • Tanaka K.
      • Sekihara H.
      • Shimotohno K.
      • Saito M.
      • Kato N.
      Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells.
        Herpes simplex virus 1 and 2
      • Andersen J.H.
      • Osbakk S.A.
      • Vorland L.H.
      • Travik T.
      • Gutterberg T.J.
      Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomelogavirus into human fibroblasts.
        Feline calcivirus
      • McCann K.B.
      • Lee A.
      • Wan J.
      • Roginski H.
      • Coventry M.J.
      The effect of bovine lactoferrin and lactoferricin B on the ability of feline calicivirus (a norovirus surrogate) and poliovirus to infect cell cultures.
        Adenovirus
      • di Biase A.M.
      • Pietrantoni A.
      • Tinari A.
      Heparin-interacting sites of bovine lactoferrin are involved in anti-adenovirus activity.
        Echovirus
      • Pietrantoni A.
      • Ammendolia M.G.
      • Tinari A.
      • Siciliano R.
      • Valenti P.
      • Superti F.
      Bovine lactoferrin peptidic fragments involved in inhibition of echovirus 6 in vitro infection.
       Lactoferrampin
      Escherichia coli
      • van der Kraan M.I.A.
      • Groenink J.
      • Nazmi K.
      • Veerman E.C.I.
      • Bolscher J.G.J.
      • Amerongen A.V.N.
      Lactoferrampin: A novel antimicrobial peptide in the N1-domain of bovine lactoferrin.
      Pseudomonas aeruginosa
      Bacillus subtilis
      Candida albicans
      The N-acylated, d-enantiomer peptide derivatives of BLFcin are believed (
      • Wakabayashi H.
      • Matsumoto H.
      • Hashimoto K.
      • Teraguchi S.
      • Takase M.
      • Hayasawa H.
      N-Acylated and d-enantiomer derivatives of a nonamer core peptide of lactoferricin B showing improved antimicrobial activity.
      ) to possess antimicrobial activities greater than those of the native peptide against both bacteria and fungi; the most potent peptide, conjugated with an 11-carbon chain-acyl group, showed 2 to 8 times lower MIC than BLFcin.
      Several other LF-derived peptides exist (Table 5), indicating that LF hydrolysates besides LFcins may yield a number of antimicrobial peptides against various bacteria.
      Two synthetic peptides from human LF were described (
      • Viejo-Díaz M.
      • Andrés M.T.
      • Fierro J.F.
      Different anti-Candida activities of two lactoferrin-derived peptides Lfpep and kaliocin-1.
      ): Lfpep, a cationic peptide with bactericidal and giardicidal effects, and kaliocin-1, a novel bactericidal peptide that shares the highly homologous sequence with the transferrin family of proteins (Table 4). Both proteins possess fungicidal activity against Candida spp. The killing activity of LFpep on Candida albicans is mediated by its permeabilizing activity, whereas kaliocin-1 is unable to disrupt the cytoplasmatic membrane – as indicated by its inability to allow permeation of propidium iodide, and by the small amount of K+ released.
      Lactoferrampin (BLFampin) was recently identified in the N1-domain of BLF, and a primary structure close to that of BLFcinB was reported (
      • Vogel H.J.
      • Shibli D.J.
      • Jing W.
      • Lohmeiher-Vogel E.M.
      • Epand R.F.
      • Epand R.M.
      Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides.
      ; Table 5). Besides BLFampin, several shorter peptides have been studied (
      • van der Kraan M.I.A.
      • Groenink J.
      • Nazmi K.
      • Veerman E.C.I.
      • Bolscher J.G.J.
      • Amerongen A.V.N.
      Lactoferrampin: A novel antimicrobial peptide in the N1-domain of bovine lactoferrin.
      ). The peptide f(265–284) of BLFampin possesses a higher activity than the original BLFampin against yeasts, probably because of its higher propensity to adopt an α-helical conformation (
      • Schmidt R.H.
      • Packardm V.S.
      • Morris H.A.
      Effects of processing on whey protein functionality.
      ). Lactoferrampin peptide f(265–284) and BLFcinB f(17–30) can be translocated across the membranes of C. albicans and E. coli to exhibit large effects upon their plasma membrane integrity, such as induction of distinct vesicle-like structures in the membrane by BLFampin, and detachment of the outer membrane and emergence of surface protrusions in the latter by BLFcin B; this peptide also showed a particularly strong effect against pathogenicity of E. coli.

      Peptides with Nutrition System Activities

      Hydrolysis of proteins in general, and of whey proteins in particular, has been shown to improve digestibility (and to better regulate the digestive process), as well as specifically decrease cholesterol levels. Improvement of digestibility is beneficial for patients who suffer from digestion disorders such as cystic fibrosis, short bowel syndrome, or pancreatitis, and that improvement may easily be achieved via (nonspecific) hydrolysis of whey proteins (
      • Yvon M.
      • Beucher S.
      • Guilloteau P.
      • le Huerou-Luron I.
      • Corring T.
      Effects of caseinomacropeptide (CMP) on digestion regulation.
      ).

      Peptides from GMP

      The protein source has been evaluated for its effect on satiety and food intake in humans. Glycomacropeptide can, in particular, act as a regulator of the digestive function without being absorbed (
      • Yvon M.
      • Beucher S.
      • Guilloteau P.
      • le Huerou-Luron I.
      • Corring T.
      Effects of caseinomacropeptide (CMP) on digestion regulation.
      ). This conclusion was reached upon injection of GMP, which led to suppression of gastric secretions (
      • Chernikov M.P.
      • Nikolskaya G.V.
      • Stan E.Y.
      • Shlygun G.K.
      • Vasilevskaya L.S.
      Biological role of κ-casein glycomacropeptide.
      ); the active components were peptides formed via breakdown of GMP effected by pepsin (at the low pH typically prevailing in the stomach). Glycomacropeptide suppresses gastric digestion and promotes satiety by induction of cholecystokinin, which is a hormone that regulates energy and food intake by intestinal cells; it also stimulates gall bladder contraction and bowel motility, regulates gastric emptying, and stimulates release of enzymes by the pancreas (
      • Beucher M.
      • Levenez F.
      • Yvon Y.
      • Corring T.
      Effect of caseinomacropeptide (CMP) of cholecystokinin (CCK) release in rats.
      ). The proposed regulation of food intake mechanisms is depicted in Table 3.
      Glycomacropeptide can be included in diets aimed at controlling various liver diseases, in which branched-chain AA residues are used as a carbon source (
      • el-Salam A.M.H.
      • el-Shibiny S.
      • Buchheim W.
      Characteristics and potential uses of the casein macropeptide.
      ). The short-term effect of mixtures of whey protein and GMP versus a carbohydrate control on satiety in healthy adult humans has been assessed (
      • Lam S.M.
      • Moughan P.J.
      • Awati A.
      • Morton H.R.
      The influence of whey protein and glycomacropeptide on satiety in adult humans.
      ). There is some evidence that whey proteins and their components enhance satiety over a short-term period compared with carbohydrate, but there was no consistent effect of whey protein either alone or coupled with GMP.
      The most important nutritional role that has been associated with GMP derives from its use as an ingredient in diets designed for people suffering from phenylketonuria (
      • Smithers G.W.
      • Ballard J.B.
      • Copeland A.D.
      • Kirthi J.A.
      • Dionysius D.A.
      • Francis G.L.
      • Goddard C.
      • Grieve P.A.
      • Mcintosh G.H.
      • Mitchell I.R.
      • Pearce R.J.
      • Regester G.O.
      Symposium: Advances in dairy foods processing and engineering new opportunities from the isolation and utilization of whey proteins.
      ); these patients are unable to metabolize F and therefore must ingest diets free of Phe (GMP lacks F). In particular, a peptide-fortified fruit gel low in F, and targeted at these individuals, was successfully developed (
      • Marshall S.C.
      Casein macropeptide from whey—A new product opportunity.
      ).

      Peptides from Other Sources

      Several dietary proteins have been shown to influence serum cholesterol levels (
      • Zhang X.
      • Beynen A.C.
      Influence of dietary fish proteins on plasma and liver cholesterol concentrations in rats.
      ;
      • Potter S.
      Overview of proposed mechanisms for the hypocholesterolemic effect of soy.
      ); however, few data concern the effect of derivatives of whey proteins on cholesterol metabolism. A hypocholesterolemic peptide (IIAQK) derived from β-LG can act effectively on serum cholesterol levels to an extent above that of β-sitosterol (
      • Nagaoka S.
      • Futumura Y.
      • Miwa K.
      • Awano T.
      • Yamauchi K.
      • Kanamaru Y.
      • Tadashi K.
      • Kuwata T.
      Identification of novel hypocholesterolemic compound derived from bovine milk beta-lactoglobulin.
      ). β-Lactotensin showed hypocholesterolemic activity after administration to mice for 2 d at a dose of 30 mg/kg i.p. or 100 mg/kg p.o. (
      • Yamauchi R.
      • Ohinata K.
      • Yoshikawa M.
      β-Lactotensin and neurotensin rapidly reduce serum cholesterol via NT2 receptor.
      ).
      The effect of albutensin A on food intake in mice was also studied (Table 3); this peptide delays gastric emptying and elevates blood glucose levels (
      • Ohinata K.
      • Inui A.
      • Asakawa A.
      • Wada K.
      • Wada E.
      • Yoshikawa M.
      Albutensin A and complement C3a decrease food intake in mice.
      ), so it mayeventually be used in human diets to promote weight loss and prevent obesity.
      Finally, hydrolysis can be applied to destroy protein epitopes responsible for allergic reactions in sensitive individuals; for example, infants suffering from cow's milk protein allergy (
      • Boza J.J.
      • Martínez-Augustin O.
      • Gil A.
      Nutritional and antigenic characterization of an enzymatic whey protein hydrolysate.
      ;
      • Martin-Esteban M.
      • Garcia-Ara M.C.
      • Banque-Molas M.
      • Boyano-Martinez M.T.
      • Martin-Munoz F.
      • Diaz-Pena J.M.
      Evaluation of an extensively hydrolyzed casein-whey protein formula in immediate cow's milk protein hypersensitivity.
      ;
      • Halken S.
      • Hansen K.S.
      • Jacobsen H.P.
      • Estmann A.
      • Faelling A.E.
      • Hansen L.G.
      • Kier S.R.
      • Lassen K.
      • Lintrup M.
      • Mortensen M.
      • Ibsen K.K.
      • Osterballe O.
      • Host A.
      Comparison of a partially hydrolyzed infant formula with two extensively hydrolyzed formulas for allergy prevention: A prospective randomized study.
      ); this general feature may also be taken advantage of in the case of whey peptides.

      Other Peptides with Bioactivities

      In contrast to the basic whey protein fractions, little is known on the possible benefits of the acidic whey protein fractions. The acidic (i.e., low isoelectric point) protein component of whey is known to contain phosphorylated proteins and peptides (
      • Sorensen E.S.
      • Petersen T.
      Purification and characterization of three proteins from the proteose peptone fraction of bovine milk.
      ;

      I. R., J. Cornish, N. W. Haggarty, and K. P. Palmano. 2004. Bone health compositions derived from milk. US patent US2004052860. New Zealand Dairy Board, assignee.

      ), some of which may play a role in calcium absorption. Additionally, the whey acidic protein complement contains osteopontin and likely fragments of it (e.g., free potassium; K. P. Palmano, LactoPharma, Fonterra Research Center, Palmerston North, New Zealand; unpublished data), which are essential to bone mineralization (
      • Bayless K.J.
      • David G.E.
      • Meininger G.A.
      Isolation and biological properties of osteopontin from bovine milk.
      ;
      • Denhardt D.T.
      • Noda M.
      Osteopontin expression and function: Role in bone remodelling.
      ). An acidic protein fraction isolated from mineral acid whey protein concentrate was recently shown to have antiresorptive effects in vitro (

      I. R., J. Cornish, N. W. Haggarty, and K. P. Palmano. 2004. Bone health compositions derived from milk. US patent US2004052860. New Zealand Dairy Board, assignee.

      ); such a fraction also exhibited bone bioactivity in vivo in ovariectomized rat (
      • Kruger M.C.
      • Plimmer G.G.
      • Schollum L.M.
      • Haggarty N.
      • Ram S.
      • Palmano K.
      The effect of whey acidic protein fractions on bone loss in the ovariectomised rat.
      ).
      The proteose-peptone component 3 (PP3) is a phosphorylated glycoprotein isolated from bovine milk, but usually released only in whey (
      • Sorensen E.S.
      • Petersen T.
      Purification and characterization of three proteins from the proteose peptone fraction of bovine milk.
      ); it is also known as lactophoricin. It comprises a polypeptide backbone of 135 AA residues (including 5 phosphorylated S residues, 2 T-linked O-glycosylations, and 1 N-glycosylation site; it has been cloned, and its cDNA sequence has been determined (
      • Johnsen L.B.
      • Sùrensen E.S.
      • Petersen T.E.
      • Berglund L.
      Characterization of a bovine mammary gland PP3 cDNA reveals homology with mouse and rat adhesion molecule GlyCAM-1.
      ). The exact function of PP3 in vivo is still unresolved; however, several studies (
      • Girardet J.M.
      • Linden G.
      • Loye S.
      • Courthaudon J.L.
      • Lorient D.
      Study of a mechanism of lipolysis inhibition by bovine milk proteose peptone component-3.
      ) have shown that PP3 has the ability to inhibit lipoprotein lipase activity, thus suggesting a potential role as inhibitor of spontaneous lipolysis in milk. Immunological studies found PP3 residues in the milk fat globule membrane and have shown that PP3 forms multimeric aggregates in bovine whey (
      • Sorensen E.S.
      • Rasmussen L.K.
      • Moller L.
      • Petersen T.E.
      The localization and multimeric nature of component PP3 in bovine milk: Purification and characterization of PP3 from caprine and ovine milks.
      ).
      Considering the pore-forming ability of the f(113–135) C-terminal peptide of bovine PP3, it is conceivable that this peptide interacts with natural lipidic bilayers, such as bacterial membranes. Antimicrobial and hemolytic assays were therefore carried out, using the corresponding synthetic peptide; it presented inhibitory growth activity against both gram-negative and gram-positive bacteria, but no hemolytic activity in the concentration range tested (<200 μM;
      • Campagna S.
      • Mathot A.-G.
      • Fleury Y.
      • Girardet J.-M.
      • Gaillard J.-L.
      Antibacterial activity of lactophoricin, a synthetic 23-residue peptide derived from the sequence of bovine milk component-3 of proteose peptone.
      ).

      Stability of Bioactive Peptides

      Following oral ingestion, milk components are digested and eventually absorbed in the gastrointestinal tract (Figure 1). Digestion of proteins is initiated in the stomach and is normally brought about by pepsin under the strongly acidic conditions prevailing in that organ. After this step, the digestion products are further hydrolyzed by pancreatic enzymes such as trypsin and chymotrypsin, as well as by membrane peptidases (
      • Tomé D.
      • Ledoux N.
      Nutritional and physiological role of milk protein components.
      ). Gastrointestinal digestion depends upon availability of ACE-inhibitory peptides; proteases present in the gastrointestinal tract may hydrolyze such peptides and thus alter their activity. Research experiments have been conducted that simulate gastrointestinal digestion, from batch dialysis bags (
      • Gauthier S.F.
      • Vachon C.
      • Savoie L.
      Enzymatic conditions of an in vitro method to study protein digestion.
      ;
      • Pihlanto-Leppälä A.
      • Rokka T.
      • Korhonen H.
      Angiotensin I-converting enzyme inhibitory peptides from bovine milk proteins.
      ;
      • Oomen A.G.
      • Hack A.
      • Minekus M.
      • Zeijdner E.
      • Cornelis C.
      • Schoeters G.
      • Verstraete W.
      • van de Wiele T.
      • Wragg J.
      • Rompelberg C.J.M.
      • Sips A.
      • van Wijnen J.H.
      Comparison of five in vitro digestion models to study the bioaccessibility of soil contaminants.
      ) to more complex computer-controlled models, including artificial stomachs (
      • Yvon M.
      • Beucher S.
      • Scanff P.
      • Thirouin S.
      • Pelissier J.P.
      In vitro simulation of gastric digestion of milk proteins: Comparison between in vitro and in vivo data.
      ), the TIM gastrointestinal tract model (
      • Minekus M.
      • Marteau P.
      • Havenaar R.
      • Huis in’t Veld J.H.J.
      A multi compartmental dynamic computer-controlled model simulating the stomach and small intestine.
      ), the SHIME model (simulator of the human intestinal microbial ecosystem;
      • Molly K.
      • van de Woestyne M.
      • Verstraete W.
      Development of a 5-step multi-chamber reactor as a simulation of the human intestinal microbial ecosystem.
      ), and models coupled to cell culture (
      • Glahn R.P.
      • Wortley G.M.
      • South P.K.
      • Miller D.D.
      Inhibition of iron uptake by phytic acid tannic acid and ZnCl2: Studies using an in vitro digestion/Caco-2 cell model.
      ). Some of them use pancreatin as a small intestine enzyme source, whereas others resort to relatively pure trypsin and chymotrypsin.
      To exert their physiological effects in vivo, bioactive peptides have to reach their target sites at the luminal side of the intestinal tract or at specific peripheral organs following absorption (
      • Meisel H.
      • Schlimme E.
      Bioactive peptides derived from milk proteins: Ingredients for functional foods?.
      ;
      • Langley-Danysz P.
      Des hydrolysats protéiques pour développer des aliments santé.
      ;
      • Meisel H.
      Overview on milk protein-derived peptides.
      ). There is considerable evidence suggesting that intact proteins and peptide fragments can enter the blood circulation to reach physiologically important levels (
      • Mills E.N.C.
      • Alcocer M.J.C.
      • Morgan M.R.A.
      Biochemical interactions of food-derived peptides.
      ). However, some peptides exhibit bioactivity in vitro but are ineffective in vivo, which suggests gastrointestinal degradation. Some researchers have identified potent in vitro ACE-inhibitory activity for some milk-derived peptides that cannot exert antihypertensive effects in vivo (
      • Maruyama S.
      • Mitachi H.
      • Awaya J.
      • Kurono M.
      • Tomizuka N.
      • Suzuki H.
      Angiotensin I-converting enzyme inhibitory activity of the C-terminal hexapeptide of αS1-casein.
      ;
      • Maeno M.
      • Yamamoto N.
      • Takano T.
      Identification of an antihypertensive peptide from casein hydrolysate produced by a proteinase from Lactobacillus helveticus CP790.
      ;
      • Abubakar A.
      • Saito T.
      • Kitazawa H.
      • Kawai Y.
      • Itoh T.
      Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion.
      ;
      • Saito T.
      • Nakamura T.
      • Kitazawa H.
      • Kawai Y.
      • Itoh T.
      Isolation and structural analysis of antihypertensive peptides that exist naturally in Gouda cheese.
      ). Another example is β-LG f(142–148); despite reports that it can be transported intact across Caco-2b cell monolayers (
      • Vermeirssen V.
      • Deplacke B.
      • Tappenden K.A.
      • van Camp J.
      • Gaskins H.R.
      • Verstraete W.
      Intestinal transport of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg through a Caco-2 Bbe monolayer.
      ), recent studies (
      • Walsh D.J.
      • Bernard H.
      • Murray B.A.
      • MacDonald J.
      • Pentzien A.-K.
      • Wright G.A.
      • Wal J.-M.
      • Struthers A.D.
      • Meisel H.
      • FitzGerald R.J.
      In vitro generation and stability of the lactokinin β-lactoglobulin fragment (142–148).
      ;
      • Roufik S.
      • Gauthier S.F.
      • Turgeon S.L.
      In vitro digestibility of bioactive peptides derived from bovine β-lactoglobulin.
      ) have shown that this peptide is degraded during (simulated) gastrointestinal digestion. Its stability was further tested (
      • Walsh D.J.
      • Bernard H.
      • Murray B.A.
      • MacDonald J.
      • Pentzien A.-K.
      • Wright G.A.
      • Wal J.-M.
      • Struthers A.D.
      • Meisel H.
      • FitzGerald R.J.
      In vitro generation and stability of the lactokinin β-lactoglobulin fragment (142–148).
      ), so we can conclude that it is not sufficiently stable in vivo to gastrointestinal and serum proteinases and peptidases (e.g., pepsin and colorases), and that it is unable to exhibit ACE-inhibiting activity in vivo. Afterwards, peptides β-LG f(15–20), f(102–105), and f(142–148), known for their biological activities (Table 1), were tested for stability during digestion (
      • Roufik S.
      • Gauthier S.F.
      • Turgeon S.L.
      In vitro digestibility of bioactive peptides derived from bovine β-lactoglobulin.
      ). When incubated with pepsin, f(142–148) remained intact, whereas β-LG f(15–20) and f(102–105) were hydrolyzed to some extent (less than 31%); conversely, chymotrypsin hydrolyzed β-LG f(142–148) up to 99.8%. Furthermore, peptides obtained by fermentation sometimes exert higher ACE-inhibitory activity following in vitro digestion; for example, whey protein fermented by Saccharomyces cerevisiae (
      • Sorensen E.S.
      • Petersen T.
      Purification and characterization of three proteins from the proteose peptone fraction of bovine milk.
      ). These results suggest that lactokinin β-LG f(142–148) and other bioactive peptides may need protection against gastric or intestinal enzymatic degradation in order for their physiological effects be fully displayed in vivo.

      Protein–Peptide Interactions

      The interactions of peptides with proteins have been shown to induce changes in the conformation of the target protein, which enhances its resistance to some aggressive processes. There are 2 types of interactions: 1) specific interactions, which include peptide interaction with a protein membrane receptor for propagation of information through a signaling system, inhibition of an enzyme with a peptide, and formation of molecular complexes—this process may play an important role in a wide range of biological processes; and 2) nonspecific protein–peptide interactions, which are observed in food systems. Such interactions apply mainly to protein hydrolysates that consist of mixtures of intact protein and peptides.
      Hydrolysates of a whey protein isolate brought about by a seryl protease from Bacillus licheniformis could aggregate nonhydrolyzed whey proteins; these peptides have apparent molecular weights ranging from 1,400 to 7,500 Da (under reducing conditions). Such protein–peptide interactions depend on a balance between hydrophobic attraction and electrostatic repulsion (
      • Creusot N.
      • Gruppen H.
      Enzyme-induced aggregation and gelation of proteins.
      ).
      β-Lactoglobulin was shown to interact with whey peptide fractions, thus increasing their resistance to thermodenaturation (
      • Barbeau J.
      • Gauthier S.F.
      • Pouliot Y.
      Thermal stabilization of β-lactoblobulin by whey peptide factions.
      ). Whole protein (in a zymogen-like vector) was suggested as a carrier to protect the bioactive peptides from gastric digestion (
      • Gauthier S.F.
      • Pouliot Y.
      Functional and biological properties of peptides obtained by enzymatic hydrolysis of whey proteins.
      ). Study of peptide–peptide and peptide–protein interactions during fractionation of hydrolysates by nanofiltration, aiming at recovery of specific peptide fractions, led these authors to conclude that the opioid peptide f(102–105) binds to the inner cavity of β-LG. Furthermore, interaction between β-LG and its daughter peptide f(142–148) was hypothesized (
      • Roufik S.
      • Gauthier S.F.
      • Turgeon S.L.
      In vitro digestibility of bioactive peptides derived from bovine β-lactoglobulin.
      ,
      • Roufik S.
      • Gauthier S.F.
      • Sylvie L.T.
      Physicochemical characterization and in vitro digestibility of β-lactoglobulin/β-Lg f142–148 complexes.
      ); β-LG f(142–148) was able to bind inside the hydrophobic calyx of the protein or near the interface of the dimer. In vitro chymotryptic hydrolysis of β-LG A to peptide complexes suggested that hydrolysis during gastrointestinal digestion can be delayed, hence allowing delivery of intact lactokinin β-LG f(142–148) closer to the sites of intestinal absorption (
      • Roufik S.
      • Gauthier S.F.
      • Sylvie L.T.
      Physicochemical characterization and in vitro digestibility of β-lactoglobulin/β-Lg f142–148 complexes.
      ).
      α-Lactalbumin is known to interact with peptides containing clusters of basic amino acid residues in close proximity with hydrophobic amino acid ones (
      • Gurgel P.V.
      • Carbonell R.G.
      • Swaisgood H.E.
      Identification of peptide ligands generated by combinatorial chemistry that bind α-lactalbumin.
      ) such as melittin, a 26-residue cytolytic peptide from bee venom. The binding of α-lactalbumin to the synthetic peptide WHWRKR was used to develop a purification strategy for that protein.

      Final Remarks

      Use of selective membranes to isolate and eventually purify whey proteins has substantially increased the number and the depth of studies encompassing those molecules and their hydrolysates. Most whey peptides bearing biological activity are released by enzymatic hydrolysis, but microbial fermentation can also be used for this purpose. Increasing availability of whey protein concentrates in the market and generalization of fermentation technology has helped promote interest in production of bioactive peptides by microbial fermentation as an alternative to enzymatic routes.
      Many new products with bioactive peptides have been launched in the market. There is a growing body of evidence that whey peptides exhibit physiological activities on specific components of the immune response system. In addition, ACE-inhibitory whey peptides can play roles in blood pressure regulation and hypertension; unfortunately, a high ACE-inhibiting activity in vitro does not necessarily correlate with a high antihypertensive activity in vivo. Release of peptides with high ACE-inhibiting activity is not limited by the protein and enzyme sources, but the highest activities are typically found in peptides released by trypsin.
      Differences in methodologies, in the nature of raw materials, and in model systems have led to several disparate results. Hence, conclusions drawn from in vitro models need to be consistently validated with physiological data obtained in vivo, so that the potential of whey peptides in immunomodulation can be fully demonstrated.
      For a candidate peptide to be labeled as bioactive, its resistance to gastrointestinal conditions must be determined in advance. The exact mechanisms by which whey peptides exert their bioactivities upon reaching the intestine need further elucidation; for example, whether their effect is mediated directly in the gut lumen or through receptors on the intestinal cell wall. Therefore, in vivo studies are essential not only to validate the physiological effects of tentative bioactive peptides, but also to confirm whether they will require protection from gastrointestinal enzymes when orally administered.
      Future research should focus on novel hydrolysis pathways for breakdown of whey proteins and peptides, brought about by unusual proteases aimed at releasing unique amino acid sequences; these might include enzymes from the native microbiota of dairy products or from plant rennets. Furthermore, molecular studies concerning the mechanisms by which bioactive peptides exert their activities are to be undertaken.

      Acknowledgments

      Partial funding for this research work was provided via project PROBIOSORO , coordinated by F. X. Malcata and administered by Agência de Inovação – POCTI: Programa Operacional de Ciência, Tecnologia e Inovação. Funding for A. R. Madureira and T. Tavares was via PhD fellowships supervised by F. X. Malcata and administered by Fundação para a Ciência e a Tecnologia (refs. SFRH/BD/18500/2004 and SFRH/BD/31604/2006).

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