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Milk Proteins: Bioactive Peptides☆
Milk Proteins: Bioactive Peptidesq Anne Pihlanto, Natural Resources Institute Finland (Luke), Jokioinen, Finland Ó 2016 Elsevier Inc. All rights reserved.
Introduction Structures and Function of Bioactive Peptides Possible Physiological Importance Production of Bioactive Peptides Conclusions Further Reading
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Introduction Bioactive peptides are compromised of two or more amino acid residues joined together by peptide bonds and are typically derived from enzymatic hydrolysis of proteins. The activity is based on their amino acid composition and sequence. Depending on proteolytic specificities, bioactive peptides can be released during processing and consumption of food proteins by gastric digestion, endogenous or exogenous proteolysis and by microbial enzyme action especially during fermentation. Some of these peptides share structural characteristics with endogenous peptides that act in the organisms as hormones, neurotransmitters, or regulatory peptides. Once they are released, bioactive peptides may exhibit wide range of biological functions, including antihypertensive, antioxidant, opioid, antimicrobial, activities and modulation of digestive enzymes, nutrient absorption and immune responses. Many milk-derived peptides have revealed multifunctional properties, i.e., specific peptide sequences may exert two or more different biological activities. Although, other animal as well as plant proteins contain potential bioactive sequences, milk proteins are currently the main source of biologically active peptides. Due the physiological and physico-chemical versatility, milk-borne peptides are regarded as highly prominent ingredients for health-promoting, functional foods or pharmaceutical preparations. The activity of antihypertensive peptides has been demonstrated in animal models and clinical trials, but for other peptides bioactivity has been mainly proved in cell cultures or in vitro assays. Moreover, there are many examples showing lack of correlation between in vitro and in vivo results, mainly due to the degradation of peptides during gastrointestinal digestion or the impossibility to reach target organ in a sufficient amount to exert the physiological effect. This article will discuss the current knowledge about milk-derived bioactive peptides and their potential application for promotion of human health.
Structures and Function of Bioactive Peptides Table 1 presents examples of structural properties of milk-derived bioactive peptides and Table 2 lists some examples of peptide sequences with different bioactivities measured by in vitro or in cell cultures. The exact function of peptides depends substantially on their structures, which in turn depend on the nature of their protein precursor, liberating protease specificity and production conditions. The classical approach for the discovery of bioactive peptides, including hydrolysis of proteins and fractionation steps, has been widely used to discover new bioactive peptides. The structures can be used as templates for designing more active peptides and peptidomimetics, and for structure-function relationship studies. The major drawbacks include limited sample scope, time consumption especially during purification steps, low yields of isolated peptides and the likelihood that potent peptides may not be discovered after extensive processing. The recent development and combination of computational tools and bioactive peptides databases have led to a growing importance of bioinformatic-driven approach or in silico analysis in the bioactive peptide discovery. Unlike the classical approach, the in silico approach provide a cost-effective strategy through the reduction of different steps of the traditional workflow. Among databases, BIOPEP emerges one of the most valued with more than 2500 entries classified according to specific biological activities and the opportunity of in silico proteolysis of more than 700 proteins. Food-derived peptides with opioid activity were first described in 1979 and were termed ‘exorphins’ on the basis of their structural similarity to endogenous ligands (endorphins and enkepahalins). Typical opioid peptides have the same N-terminal sequence, Tyr–GlyGly–Phe, and they exert activity by binding to specific receptors of the target cell e.g., the m-receptor for emotional behavior and suppression of intestinal motility, the sreceptor for emotional behavior, and the k-receptor for sedation and food intake. The food protein-derived opioid peptides are called ‘atypical’ opioid peptides, since they carry various amino acid sequences at their N-terminal region. Only the N-terminal tyrosine is conserved and the presence of another aromatic amino acid at the third or fourth position forms an important structural motif that fits into the binding site of the opioid receptors. The major opioid milk peptides, b-casomorphins, are fragments of the b-casein sequence 60–70, and are characterized as m-type
q Change History: March 2015. A. Pihlanto updated text, tables and references. Update of A. Pihlanto, Milk Protein Products: Bioactive Peptides. Encyclopedia of Dairy Sciences, 2nd Edition, 2011, Pages 879–886.
Reference Module in Food Sciences
http://dx.doi.org/10.1016/B978-0-08-100596-5.00932-X
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Milk Proteins: Bioactive Peptides
Table 1
Structural properties of selected bioactive peptides derived from milk proteins
Health
Activity
Cardiovascular diseases Blood pressure ACE inhibitory
Structural elements of peptides
Bioactive peptide
Precursor proteins
Pro or hydroxyl Pro, Tyr, Phe or Lys/Arg at C-terminus
Casokinins Lactokinins
as-, b- and k-Casein a-Lactalbumin b-Lactoglobulin as-, b- and k-Casein b-Lactoglobulin
Casoplatelins
k-Casein
Lactostatin Lactotensin
b-Lactoglobulin
Oxidative stress
Antioxidants
Amino acids His Tyr, Trp, Met, Lys, Cys in the sequence Peptides with Pro-His-His sequence Ile108, Lys112 and Asp115 residues, sugar content Low ratios of Met–Gly and Lys–Arg in protein, high amount of hydrophobic amino acids Ala or Pro at C-terminus Hydrophobic amino acid (preferably Trp) at N-terminus Branched chain amino acids, mainly Leu Peptide length Multiple Arg residues Highly positively charged sequence or certain amphiphilic character
Thrombosis
Antithrombotic
Blood lipid profile
Hypocholesterolemic and hypotriglyceridemic
Diabetes
DPP-IV inhibitors
Obesity
Satiety
Body defence, food quality
Antimicrobial
Nervous system
Opioid
Tyr at N-terminus, aromatic amino acid at 3rd or 4th position
b-casomorphin, a-casein exorphins Lactorphins
Bone and teeth health
Mineral absorption
Phosphorylated regions
Caseinophospho-peptides (CPP)
Whey proteins Caseins Whey proteins
Lactoferricin Casocidine, Isracidine
Lactoferrin a-Lactalbumin b-Lactoglobulin as-, b-Casein b-Casein a-Casein a-Lactalbumin b-Lactoglobulin as-, b-casein
ligands (Table 2). The a-casein exorphins were found to correspond to bovine as1-casein f(90–96), f(90–95) and f(91–96) and were s-selective receptor ligands. Whey proteins contain opioid-like sequences in their primary structure, namely a-and b-lactorphin. These peptides show low affinity for opioid receptors and are m-type receptor ligand. Opioid activity has, furthermore, been found in fragments from bovine serum albumin [serorphin, f(399–404)]. The milk-derived opioid-like peptides have shown multifunctional properties. In recent studies, b-casomorphin f(60–66), as1-casein exorphin f(90–94) and a-lactorphin showed the capability to induce mucin secretion in HT29-MTX cells. Concretely, a-lactorphin was able to up-regulate the expression of the major secreted gene encoded by these cells. Peptides from a peptic and tryptic digest of bovine and human k- and as1-caseins have been found to display opioid antagonist properties. Various C-terminally methoxylated casoxins have been identified, e.g., k-casein f(33–38) (casoxin-6), f(34–38) (casoxin-5) and f(35–38) (casoxin-4). Additionally, casoxin C [k-casein f(25–34)], casoxin A [k-casein f(35–41)] and casoxin B [k-casein f(61–64)] showed opioid antagonistic activity, but only casoxin C’s activity was comparable to the esterified casoxin-6 and 4. In general, the chemically modified peptides are more active than their non-methylated derivatives. Casoxins show a preference for m- and k-receptors with a relatively low potency compared to naloxone. Elevated blood pressure is one of the major independent risk factors for cardiovascular diseases. Angiotensin I-converting enzyme (ACE; dipeptidyl carboxypeptidase, EC 3.4.15.1) is one of the main regulators of blood pressure, thus, inhibition of this enzyme is considered one of the strategies for the treatment of hypertension. In recent years, a large number of ACE inhibitory peptides have been evaluated in vitro and in vivo from digests of various food proteins. Although the full mechanism of interaction between an inhibitor and the ACE is not known, it is highly possible that the peptide inhibitor interacts with the C-domain of ACE. Also some structural features, like amino acids with cyclic or aromatic rings (Tyr, Pro, Trp), are found at the C terminal of the peptides. The most commonly known peptides of milk origin are the ACE-inhibitory tripeptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), derived from b- and k-caseins. ACE-inhibitory activity has been detected in fragments 177–183 and 191–202 of b-casein and fragments 23–27 and 194–199 of as1-casein upon action by trypsin. Also, whey proteins contain ACE-inhibitory peptides; the highest activity has been found with peptides b-lg f(80–82), f(142–148) and a-la f(104–108). The IC50-value (inhibitor concentration leading to 50% inhibition) is used to estimate the effectiveness of different ACE-inhibitory peptides. The majority of milk-derived ACE-inhibitors have moderate inhibitory potencies, usually within an IC50 range of 100–500 mmol l1. However, in vitro activity is not always related to in vivo activity, since some peptides are susceptible to degradation or modification in the gut, vascular system and the liver. Moreover, some long-chain candidate peptides can be degraded and new active fragments are generated by gastrointestinal enzymes (Table 3). Another complication related to cardiovascular diseases is the inclination to develop thrombosis due to abnormalities in blood coagulation. There is a significant amount of molecular similarities involved in milk and blood clotting. Peptides that inhibit blood platelet aggregation and fibrinogen binding (g-chain) to platelet surface receptors are encrypted within the sequence of bovine
Table 2
Examples of milk protein-derived bioactive peptidesa Biological activity
Preparation
Substrate and enzymes applied Casein b-Casein as1-Casein k-Casein Casein
Jejunum (minipigs) Trypsin Trypsin Pepsin Pepsin Pepsin Trypsin Trypsin Trypsin Trypsin Trypsin
in vivo Synthesis a-Lactalbumin Pepsin Trypsin b-Lactoglobulin Pepsin þ trypsin Trypsin Lactoferrin Pepsin Glycomacropeptide Trypsin
Origin
Sequence b
Name
Opioid IC50 mM c
b-CN f(60–70)
YPFPGPIPNSL
b-Casomorphin-11
10
b-CN f(60–66) b-CN f(60–64) as1-CN f(90–96) as1-CN f(9095 k-CN f(33–38) k-CN f(25–34) as1-CN (23–34) as1-CN f(194– 199) b-CN f(1–25)4P as1-CN f(43–58) 2P as1-CN f66–74) b-CNf(193–202) a-LA f(50–53) a-LA f(104–108) b-LG f(102–105) b-LG f(142–148) LF f(17–41) k-CN f(106–116)
YPFPGPI YPFPG RYLGYLE RYLGYL SRYPSY YIPIQYVLSR FFVAPFPQVFGK TTMPLW
b-Casomorphin-7 b-Casomorphin-5 as1-Exorphin as1-Exorphin Casoxin-6 Casoxin-C Casokinin as1-Immunocasokinin
14 1.1 12 45 (Y) 250 (Y) 50
RELEELNVPGEIVES*LS*S*S*EESITR DIGS*ES*TEDQAMEDIM
Caseinophosphopeptide Caseinophosphopeptide
S*S*S*EEIVPN YEQPVLGPVR YGLF-NH2 WLAHK YLLF-NH2 ALPMHIR FKCRRWQWRMKKLGAPSITCVRRAF MAIPPKKNQDK
Caseinophosphopeptide b-Casokinin-10 aLactorphin Lactokinin bLactorphin Lactokinin Lactoferricin Casoplatelin
ACE-inhibitory IC50 mM d
Immunomodulatory e
500
21/þ26
77 16
þ162
Mucin secretion (% control)
282 234
[Ig-production
300 160
300 733 77 172 43
Ca2þ binding Kapp (l mol1)
629 328
28/þ14 201 453
Antimicrobial Antithrombotic
References used to generate this Table Antila, P., et al., 1991. Opioid peptides derived from in vitro proteolysis of bovine whey proteins. Int. Dairy J. 1, 215–229; Brantl, V., et al., 1981. Opioid activities of b-casomorphins. Life Sci. 28, 1903–1909; Chiba, H., et al., 1989. Opioid antagonist peptides derived from k-casein. J. Dairy Res. 56, 363–366; Chiba, H., Yoshikawa, M., 1986. Biologically functional peptides from food proteins: New opioid peptides from milk proteins. In: Feeney, R.E., Whitaker, J.R. (Eds.), Protein Tailoring for Food and Medical Uses. Marcel Dekker, New York, pp. 123–153; Fiat, A.-M., et al., 1993. Biologically active peptides from milk proteins with emphasis on two example concerning antithrombotic and immunomodulating activities. J. Dairy Sci. 76, 301–310; FitzGerald, R., 1998. Potential uses of caseinophosphopeptides. Int. Dairy J. 8, 451–457; Henschen, A., et al., 1979. Novel opioid peptides from casein (b-casomorphins) II. Structure of active components from bovine casein peptone. Hoppe-Seyler’s Z. Physiol. Chem. 360, 1217–1224; Loukas, S., et al., 1983. Opioid activities and structures of a-casein-derived exorphins. Biochemistry 22, 4567–4573; Maruyama, S., et al., 1987. Angiotensin I converting enzyme inhibitory activity of the C-terminal hexapeptide of as1-casein. Agric. Biol. Chem. 51, 2557–2561; Meisel, H., 1986. Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein. FEBS Lett. 196, 223–227; Meisel, H., Schlimme, E., 1994. Inhibitors of angiotensin-converting enzyme derived from bovine casein (Casokinins). In: Brantl, V., Teschemacher, H. (Eds.), b-casomorphins and Related Peptides Recent Developments. VCH Veinheim, pp. 66–72; Mullally, M.M., et al., 1996. Synthetic peptides corresponding to a-lactalbumin and b-lactoglobulin sequences with angiotensin-I-converting enzyme inhibitory activity. Biol. Chem. Hoppe-Seyler 377, 259–260; Mullally, M.M., et al., 1997. Identification of novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of b-lactoglobulin. FEBS Lett. 402, 99–101; Pihlanto-Leppälä, A., et al., 2000. Angiotensin I-converting enzyme inhibitory properties of whey protein digests: concentration and characterization of active peptides. J. Dairy Res. 67, 53–64; Tomita, M., et al., 1994. A review: the active peptide of lactoferrin. Acta Paediatr. Jpn. 36, 585–591; Martinez-Maqueda, D., et al., 2012. Food-derived peptides stimulate mucin secretion and gene expression in intestinal cells. J. Agric. Food Chem. 60, 8600–8605. b One letter amino acid codes used; Phosphoserin ¼ S*. c Opioid activities of synthetic peptides in an opiate receptor-binding assay IC50-values (mM) are given for peptide concentrations inhibiting [3H]-ligand binding by 50%; (Y) indicates antagonist activity. d Conditions for IC50 estimation are not identical; the concentration of peptide needed to inhibit 50% of the ACE activity. e Figures indicate the maximum % stimulation (þ) and/or inhibition (), respectively, in relation to control (¼100). a
Milk Proteins: Bioactive Peptides 3
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Milk Proteins: Bioactive Peptides
Table 3 Sequence
Antihypertensive peptides derived from milk proteins by enzymatic hydrolysis and fermentation Origin
Enzymatic treatments TTMPLW as1-CN f(194–199) FFVAPFPGVFGK as1-CN f(23–34) RYLGY as1-CN f(90–94) AYFYPEL as1-CN f(143–159) YGLF a-LA f(50–53) KVLPVPQ b-CN f(169–175) IPA b-LG f(80–82) LQKV b-LG f(58–61) LLF b-LG f(103–105) KVLPVP b-CN f(169–174) IAK k-CN f(22–24) YAKPVA k-CN f(61–66) YKVPQL as1-CN f(104–109) Fermentation VPP b-CN f(84–86) IPP b- or k-CN YP as1-, b- or k-CN LHLPLP b-CN f(133–138) LHLPLPL b-CN f(133–139) RPKHPIKHQ as1-CN f(1–9)
Preparation
IC50 (mM) a
Dose (mg kg1)
SBP mm Hg
Trypsin
16 77 0.71 6.58 733 1000 141 34.7 79.8 5 15.7 14.3 22
100 100 5 5 0.1 2 8 10 10 1 4 6 1
13.6 34.0 25.0 20.0 25 24 31 18.1 29.0 32.2 20.7 23.1 12.5
9 5 720 5.5 4.25 13.4
1.6 1 1 10 7 6.1–7.5
20 15.1 27 7.7 23.5 9.3
Pepsin
Proteinase K Thermolysin Digestive enzymes
Proteinase from L. helveticus CP790 L. helveticus & S. cerevisiae L. helveticus CPN4 E. faecalis Gouda cheese
a
The concentration of an ACE-inhibitor needed to inhibit 50% of ACE activity. Adapted from Hernandez-Ledesma, B., Garcia-Nebot, M., Fernandez-Tome, S., Amigo, L., Recio, I., 2014. Dairy protein hydrolyzates: peptides for health benefits. Int. Dairy J. 38, 82–100.
k-casein, referred as casoplatelins. Several casoplatelins have been reported to have an antithrombotic effect in vitro and in guinea pigs after parenteral administration. k-Casein f(106–116) inhibited ADP-induced platelet aggregation and combined with the fibrinogen receptor of blood platelets, consequently preventing fibrinogen binding with blood platelets. The two smaller tryptic peptides (k-casein f(106–112) and f(113–116)) exerted an effect on platelet aggregation but did not inhibit fibrinogen binding. Oxidative stress is also one possible factor for initiation and evolution of cardiovascular diseases. Natural antioxidants provide additional benefits to the endogenous defence strategies in the battle against oxidative stress. Antioxidant properties to prevent enzymatic (lipoxygenase) and non-enzymatic peroxidation of essential fatty acids have been found in peptides derived from milk proteins. The identified peptides are encrypted in the sequences of as1-, b- and k-caseins and b-lactoglobulin. The antioxidative properties of the peptides are related to their structure and hydrophobicity. Hypocholesterolemic effects have been reported for whey-derived peptides. The mechanism involved in these effects remains to be clarified, but preliminary results suggests that the amino acid composition may play a key influence Two b-lactoglobulin derived peptides Ile-Ile-Ala-Glu-Lys (f71–75, lactostatin) and His-Ile-Arg-Leu (f146–149, lactotensin), are suggested to have hypocholesterolomic properties. Dipeptidyl peptidase IV (DPP-IV) inhibitors are among the newest medication introduced to the type 2 diabetes. These agents can enhance the endogenous concentrations of the active incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Bioinformatic approach has been used to predict the tryptic release of several synthetic Trpcontaining DPP-IV inhibiting peptides from dairy proteins, but it resulted in little success possibly due to narrow enzyme specificity. Some potent DPP-IV inhibitory peptides Phe-Leu-Gln-Pro [b-cn f(87–91)], Ile-Pro-Ile [k-casein f(26–28)] and Trp–Pro have been identified after gastric digestion. Studies on the effects of peptides on immunomodulation have been conducted in vitro with cells of specific and unspecific immune system. Immunomodulatory peptides can enhance immune cell functions, measured as lymphocyte proliferation, natural killer cell activity, antibody synthesis and cytokine regulation. The casein-derived peptides, including as1-casein f(194–199), b-casein f(63–68) and f(191–193), stimulate human and murine macrophages. Synthetic peptides (Tyr–Gly and Tyr-Gly-Gly) have been shown to enhance the proliferation of human peripheral blood lymphocytes. b-Casomorphin-7 and b-casokinin-10 suppress lymphocyte proliferation at low concentrations but are stimulatory at higher concentrations. b-Casein f(193–199) induces a significant proliferative response in rat lymphocytes. Bioactive peptides in yoghurt preparations decrease cell proliferation with Caco-2-cells or IEC-6-cells. as1-Casein f(59–79) and b-casein f(1–25), having a phosphoserine-rich region, have been found to display mitogenic activity and enhance immunoglobulin production in mouse spleen cells. Lactoferrin-derived peptides have been reported to influence in several cell type e.g., human monocytic and polymorphonuclear leucocytes cell lines, experiments which are involved in immune and inflammatory actions of the body. The antimicrobial activity of milk is associated mainly with lactoferrin. Moreover, peptides released from lactoferrin by digestion with pepsin or by heat treatment at an acidic pH show higher antimicrobial potency than undigested lactoferrin. The identified
Milk Proteins: Bioactive Peptides
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antimicrobial peptides, named lactoferricin, originate from the N-terminal region of the molecule and have antimicrobial activity against various Gram-positive and Gram-negative bacteria, yeast and filamentous fungi. For example, lactoferricin B killed four clinical isolates of enterohaemorrhagic Escherichia coli O157:H7 within 3 h at concentrations above 10 mg ml1. Additionally, as2-casein f(165–203) (Casocidin-I) purified from bovine milk hydrolyzed by a serine protease inhibits the growth of E. coli and Staphylococcus carnosus. The antimicrobial activity of lactoferricins and casocidin-I seems to be correlated with the net positive charge on the peptides. A distinctive feature is the high proportion and asymmetric clustering of basic amino acid residues. It is generally known that cationic amphipathic a-helical structures are related to antimicrobial activity by forming ion channels through membrane bilayers. Lactoferricins disrupt the normal membrane permeability of the cell, which is at least partly responsible for the antibacterial mechanism of lactoferrin-derived peptides. Peptides generated by enzymatic cleavage from a-lactalbumin and b-lactoglobulin are negatively charged and exhibit antimicrobial activities mostly against Gram-positive bacteria. Tryptic digestion of casein proteins yields casinophosphopeptides (CPPs) from the polar N-terminus region, which contains clusters of phosphorylated seryl residues. For example: as1-casein f(43–58), f(59–79), f(43–79), as2-casein f(1–24) and f(46–70) and b-casein f(1–28), f(2–28), f(1–25), f(33–48) have been isolated from the tryptic hydrolyzate of whole casein. These phosphorylated clusters have been hypothesized to be responsible for the interaction between caseins and calcium phosphate that leads to the formation of casein micelles. CPPs retain the ability of whole casein to stabilize calcium and phosphate ions through the formation of complexes, thus enhancing their general bioavailability.
Possible Physiological Importance The potential physiological role of bioactive peptides derived from milk proteins and the in situ formation of these peptides in the gastrointestinal tract is still largely unknown. One of the greatest challenges in developing peptides as food ingredients has been proving their in vivo efficacy. It mainly depends on the capacity of peptides, after being orally ingested, to reach the target organs in an intact and active form in substantial concentrations. The problem of absorption of bioactive peptides is still an open question due to differences occurring between the forms and transport of the individual peptides. Caco-2 cell models are found to be a helpful tool to predict their oral bioavailability. In the last years, studies demonstrated the bioavailability of potential antihypertensive tripeptides (VPP and IPP). The tripeptides have been detected in aortal tissues using animal model and ACE activity in aorta was lower than in control group. Paracellular transport was found to be the main form of transport of VPP. The bioavailability of IPP has been assessed in human studies, and maximum peptide concentration in plasma after ingestion of peptide enriched beverage was 897 157 p.m. Studies have shown no intact transepithelial passage of b-casomorphins; therefore, it is generally concluded that the physiological influences are limited to the gastrointestinal tract. The situation is different in neonates where passive transport across intestinal mucosal membranes occurs. This may lead to physiological responses such as an analgesic effect resulting in calmness and sleep in infants. Small CPPs were detected in the stomach, duodenal and ileostomy fluids of humans after milk ingestion. Most bioactive peptides are not absorbed in the intestinal tract. Hence peptides may either act directly in the intestinal tract or via receptors and cell signaling in the gut. Numerous studies in spontaneously hypertensive rats (SHR) as well as in hypertensive human volunteers have been performed to determine the antihypertensive effect of milk-derived peptides. These studies demonstrated that several ACE-inhibitory peptides significantly reduce blood pressure, either after intravenous or oral administration. Most of the human intervention studies have been focused on the consumption of products containing the tripeptides, IPP and VPP. Maximum blood pressure reductions approximate 13 mmHg (systolic blood pressure, SBP) and 8 mmHg (diastolic blood pressure, DBP) after active treatment compared to placebo, and are likely to reach after 8–12 weeks of treatment. Effective dosages of tripeptides range from 3.07 to 52.5 mg day1. Meta-analyses performed with published data of clinical trials have been reported. Nineteen randomized clinical intervention trials with small daily doses (2.0–10.2 mg) of milk casein-derived tripeptides showed an overall lowering of 4.0 mmHg of SBP in mildly hypertensive subjects. A meta-analysis carried out with 28 clinical trials reported a mean reduction of SBP of 1.66 mmHg. Another, meta-analysis with 18 clinical trials, reported that the tripeptides reduce SBP in Asian subjects while this effect was lower in Caucasian individual. Moreover, some clinical trials did not find an effect on human blood pressure in Dutch and Danish subjects after consumption fermented milk with tripeptides. The findings suggest that genetics and/or dietary patterns might exert an important influence on the antihypertensive effects of peptides IPP and VPP. Moreover, the European Food Safety Authority (EFSA) Panel on Dietetic Products, Nutrition and Allergies (NDA) has concluded that the evidence presented to date on the antihypertensive effects of peptides VPP and IPP is insufficient to establish the relationship between the consumption of these peptides and the maintenance of normal blood pressure. Other milk protein-derived hydrolyzates and peptides have been tested in clinical trials. Consumption of whey protein hydrolyzate for 6 weeks resulted in reduction of SBP and DBP, of hypertensive subjects. However, peptides responsible for the observed effect have not been identified. More recently, yoghurt enriched with other milk-derived peptides, Arg-Tyr-Leu-Gly-Tyr and Ala-Tyr-Phe-Tyr-Pro-Glu-Leu, reduced blood pressure in hypertensive humans. The consumption of dairy proteins, particularly whey protein, has been shown in a number of intervention studies to have beneficial effects on glucose metabolism. The antidiabetic effect of whey protein have been suggested to be attributable to its content of bioactive peptides which, following their release during gastrointestinal digestion, could stimulate the secretion of gut-derived hormones and/or inhibit enzymes involved in glycemia homeostasis. Inhibition of DPP-IV may be one of the potential mechanisms contributing to the reported mechanisms of whey proteins. So far few studies have reported the in vitro DPP-IV inhibitory
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Milk Proteins: Bioactive Peptides
potencies of whey hydrolyzates and peptides. A b-lg derived peptide, Val-Ala-Gly-Thr-Trp-Tyr, presented hypoglycaemic effects in the oral glucose tolerance test in mice. This in vivo effect has been also demonstrated for the peptide Leu-Pro-Gln-Asn-Ile-ProPro-Leu, a casein derived fragment described as DPP-IV inhibitor. Diet plays a key role in plasma lipids profile, and consequently it is used as a strategy to prevent or decrease the incidence of cardiovascular diseases. It was observed that tryptic whey protein hydrolyzate displayed a hypocholesterolemic effect in rats and the effect was related to a peptide called lactostatin, Ile-Ile-Ala-Glu-Lys (b-lg f 71–75). Another b-lg-derived peptide b-lactotensin (His-Ile-Arg-Leu) decreased total cholesterol and low density lipoproteins-cholesterol content in mice fed a cholesterol-enriched diet. The systems involved in the defence mechanism of the body are both varied and complex and the main focus are on two peptide groups, namely immunomodulatory and antimicrobial peptides. Only a very limited amount of information is available on the in vivo effects. The Tyr–Gly and Tyr–GlyGly peptides modulate the lymphokine production in vitro. Moreover, these peptides were used successfully for immunotherapy of human immunodefiency virus infection. Furthermore, a study showed that oral administration of a commercial caseinophosphopeptide preparation [consisted of as2-casein f(1–32) and b-casein f(1–28)] enhanced intestinal IgA levels in piglets. A hexapeptide [human b-casein f(54–59)] and a tripeptide Gly-Leu-Phe, protected mice against Klebsiella pneumoniae infection after intravenous infection. An antimicrobial peptide, as1-casein f(1–23), isracidin, has been shown to exhibit in vivo activity against Staphylococcus aureus and Candida albicans at concentrations that are comparable with known antibiotics. Moreover, bactericidal peptides may assist in protecting against microbial challenge, especially in the neonatal intestinal tract, and thus support the non-immune defence of the gut. The immunostimulating activity may also have a direct effect on their resistance to bacterial and viral infections of adult humans. There are also indications that milk-derived peptides, especially lactoferricin B could be used to prevent different steps of cancer, including initiation, promotion and progression. Mineral deficiencies are the most important nutritional problems worldwide, with the iron deficiency being the most common. Mineral fortification is one of the best and most common strategies to prevent this deficiency. It has been proposed that the CPPs could increase the mineral solubility at intestinal pH, modulating its bioavailability. Published data on the effect of CPPs in animal and human studies are contradictory. A rat model system indicates that CPPs increase passive calcium transport in the distal small intestine. However, no evidence has been supplied on the effectiveness of CPPs in increasing passive calcium absorption in humans. In a recent study, it was concluded that CPPs cannot enhance calcium absorption in the gut. Concerning the iron bioavailability, a positive influence of CPPs on this parameter has been reported. An increase in the iron liver storage was found when iron deficient rats were supplemented with iron and hydrolyzed b-casein or b-casein f(1–25)4P. In humans, the effect of CPPs on iron bioavailability is controversial. The results indicate that the effects of CPPs can be influenced by food matrix (solid or liquid) as well as phytate content. Besides, CPPs can have anticariogenic properties, based on their ability to stabilize and localize calcium and phosphate ions at the tooth surface, promote remineralization of enamel sub-surface lesions. It is generally accepted that protein is the most satiating macronutrient. Several studies speculate that peptides released from dietary proteins during digestion can initiate several satiety signals from the gut and thus prevent further food intake. Because these peptides act on the intestinal site they do not need to be absorbed into the systemic circulation. It has been suggested that caseinderived peptides induce satiety by independent activation of both opioid and cholecystokinin receptors in rats. Moreover, caseinand whey protein-derived peptides appear to stimulate GLP-1 release. It remains to be determined which peptides are responsible for the above mentioned effects.
Production of Bioactive Peptides The ability of lactic acid bacteria or their purified peptidases to release bioactive peptides have been evaluated in many studies. Many of these works concern the formation of ACE-inhibitory peptides, certainly because of the ease to detect their presence through the use of a simple enzymatic assay. The release of peptides has been shown to depend on the proteolytic activity of the microbes. The most prominent strains include Lactobacillus helveticus, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Enterococcus faecalis. Especially, L. helveticus strains are capable of releasing the well-characterized tripeptides, VPP and IPP. It should be noted that hydrolysis of whey protein concentrate with crude proteinase from L. helveticus led to the isolation of ACE-inhibitory peptide derived from b-lactoglobulin (f148–153). Fermentation with L. helveticus LH-2 yielded immune peptides [b-casein f(143–154); f(145–160), f(148–154) and a-lactalbumin f(115–122)]. Another common way to produce bioactive peptides is enzymatic hydrolysis by a range of different gastric enzymes, such as pepsin, trypsin and pancreatin as well as microbial enzymes such as thermolysin and alcalase. After hydrolysis, the peptides in hydrolyzates have been fractionated and enriched using different methods, such as precipitation with salts or solvent, ultrafiltration and chromatography. Application of an ultrafiltration membrane reactor, for the continuous extraction of permeates enriched with bioactive fragments has been described for the production of antithrombotic peptides. Membranes consisting of negatively charged materials have been used to desalt whey protein hydrolyzates and to enrich cationic peptides with antibacterial properties. This technique provides new possibilities for enriching peptides with a low molecular mass and is easily up-scaled. Cheese constitutes one of the major sources of bioactive peptides among commercial dairy products due to the proteolytic activities during cheese ripening by natural milk proteases and peptidases, rennet and microbial flora. The occurrence of bioactive peptides in cheese varieties has been reported in several studies, and the activity depends on the maturation of cheese. CPPs are produced during cheese ripening due to plasmin and proteolytic enzymes derived from lactic acid bacteria.
Milk Proteins: Bioactive Peptides
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Several ACE-inhibitory peptides, including the tripeptides VPP and IPP has been found in several commercially available cheeses at physiological relevant concentrations. Opioid peptides, b-casomorphin-7, have been quantified in different cheese varieties, concentrations of 0.15 mg kg1 in Brie cheese. Fermented milks, yoghurts and kefir are another important group of commercial dairy products that can include bioactive peptides in their composition due to milk protein proteolysis by microbial fermentation. At least, ACE-inhibitory, antioxidant, immunomodulatory and opioid peptides have been detected in commercial products.
Conclusions The research area of bioactive peptides is at its beginning, most of the studies have mostly focused in vitro and animal studies, with the lack of strong clinical studies to substantiate the efficacy and support future health claims. Since metabolic disorders are significant global public health problem bioactive peptides may be of vital interest in maintaining a healthy population. However, it appears that translation of the milk-derived peptides in health promotion has met challenges that can potentially impact efficacy, consumer acceptability, commercialization and approval of peptide products. To meet the challenges, emphasis should be placed on enhancing taste, gastric stability and bioavailability of peptides, providing clinical evidence to support the health effects as well as optimizing their inclusion into food products to limit undesirable reaction. In these studies there is a need to identify and validate biomarkers that conclusively are related to a certain health benefits. Future clinical trials could benefit from data on genotype, metabolomic profiles and proteomics or transcriptomics data of the volunteers. In addition, sources to produce bioactive peptides should be sustainable, to reduce the heavy reliance on primary human food. Accordingly, research emphasis is shifting towards valorisation of protein-rich industrial by-products for inclusion into human food systems in hydrolyzed forms containing bioactive peptides. Moreover, there is need to develop modern, sustainable, techniques to enrich active peptides from food protein and to facilitate the production in huge amounts for the market. Given the facts that milk-derived bioactive peptides are safe, they are good candidates for inclusion in healthy lifestyle changes to prevent or reduce diseases such as cardiovascular diseases and obesity. In summary, more emphasis should be placed on translating the promising results into commercially available peptide products with defined mechanisms of physiological functions.
Further Reading Boelsma, E., Kloek, J., 2008. Lactotripeptides and antihypertensive effects: a critical review. Br. J. Nutr. http://dx.doi.org/10.1017/S0007114508137722. Published online by Cambridge University Press, December 05, 2008. Erdmann, K., Cheung, B.W.Y., Schröder, H., 2008. The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. J. Nutr. Biochem. 19, 643–654. Gauthier, S.F., Pouliot, Y., Saint-Sauver, D., 2006. Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins. Int. Dairy J. 16, 1315–1323. Korhonen, H., Pihlanto, A., 2006. Bioactive peptides: production and functionality. Int. Dairy J. 16, 945–960. Korhonen, H., Pihlanto, A., 2007. Technological options for the production of health-promoting proteins and peptides derived from milk and colostrum. Curr. Pharm. Des. 13, 829–843. Möller, N.P., Scholz-Ahrens, K.E., Roos, N., Schrezenmeir, J., 2008. Bioactive peptides and proteins from foods: indication for health effects. Eur. J. Nutr. 47, 171–182. Murray, B.A., FitzGerald, R.J., 2007. Angiotensin converting enzyme inhibitory peptides derived from food proteins: biochemistry, bioactivity and production. Curr. Pharm. Des. 13, 773–791. Pihlanto, A., 2006. Antioxidative peptides derived from milk proteins. Int. Dairy J. 16, 1306–1314. Hafeez, Z., Cakir-Kiefer, C., Roux, E., Perrin, C., Miclo, L., Dary-Mourot, A., 2014. Strategies of producing bioactive peptides from milk proteins to functionalize fermented milk products. Food Res. Int. 63, 71–80. Hernandez-Ledesma, B., Garcia-Nebot, M., Fernandez-Tome, S., Amigo, L., Recio, I., 2014. Dairy protein hydrolysates: peptides for health benefits. Int. Dairy J. 38, 82–100. Udenigwe, C., 2014. Bioinformatics approaches, prospects and challenges of food bioactive peptide research. Trends Food Sci. Technol. 36, 137–143.
Introduction Structures and Function of Bioactive Peptides Possible Physiological Importance Production of Bioactive Peptides Conclusions Further Reading
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Introduction Bioactive peptides are compromised of two or more amino acid residues joined together by peptide bonds and are typically derived from enzymatic hydrolysis of proteins. The activity is based on their amino acid composition and sequence. Depending on proteolytic specificities, bioactive peptides can be released during processing and consumption of food proteins by gastric digestion, endogenous or exogenous proteolysis and by microbial enzyme action especially during fermentation. Some of these peptides share structural characteristics with endogenous peptides that act in the organisms as hormones, neurotransmitters, or regulatory peptides. Once they are released, bioactive peptides may exhibit wide range of biological functions, including antihypertensive, antioxidant, opioid, antimicrobial, activities and modulation of digestive enzymes, nutrient absorption and immune responses. Many milk-derived peptides have revealed multifunctional properties, i.e., specific peptide sequences may exert two or more different biological activities. Although, other animal as well as plant proteins contain potential bioactive sequences, milk proteins are currently the main source of biologically active peptides. Due the physiological and physico-chemical versatility, milk-borne peptides are regarded as highly prominent ingredients for health-promoting, functional foods or pharmaceutical preparations. The activity of antihypertensive peptides has been demonstrated in animal models and clinical trials, but for other peptides bioactivity has been mainly proved in cell cultures or in vitro assays. Moreover, there are many examples showing lack of correlation between in vitro and in vivo results, mainly due to the degradation of peptides during gastrointestinal digestion or the impossibility to reach target organ in a sufficient amount to exert the physiological effect. This article will discuss the current knowledge about milk-derived bioactive peptides and their potential application for promotion of human health.
Structures and Function of Bioactive Peptides Table 1 presents examples of structural properties of milk-derived bioactive peptides and Table 2 lists some examples of peptide sequences with different bioactivities measured by in vitro or in cell cultures. The exact function of peptides depends substantially on their structures, which in turn depend on the nature of their protein precursor, liberating protease specificity and production conditions. The classical approach for the discovery of bioactive peptides, including hydrolysis of proteins and fractionation steps, has been widely used to discover new bioactive peptides. The structures can be used as templates for designing more active peptides and peptidomimetics, and for structure-function relationship studies. The major drawbacks include limited sample scope, time consumption especially during purification steps, low yields of isolated peptides and the likelihood that potent peptides may not be discovered after extensive processing. The recent development and combination of computational tools and bioactive peptides databases have led to a growing importance of bioinformatic-driven approach or in silico analysis in the bioactive peptide discovery. Unlike the classical approach, the in silico approach provide a cost-effective strategy through the reduction of different steps of the traditional workflow. Among databases, BIOPEP emerges one of the most valued with more than 2500 entries classified according to specific biological activities and the opportunity of in silico proteolysis of more than 700 proteins. Food-derived peptides with opioid activity were first described in 1979 and were termed ‘exorphins’ on the basis of their structural similarity to endogenous ligands (endorphins and enkepahalins). Typical opioid peptides have the same N-terminal sequence, Tyr–GlyGly–Phe, and they exert activity by binding to specific receptors of the target cell e.g., the m-receptor for emotional behavior and suppression of intestinal motility, the sreceptor for emotional behavior, and the k-receptor for sedation and food intake. The food protein-derived opioid peptides are called ‘atypical’ opioid peptides, since they carry various amino acid sequences at their N-terminal region. Only the N-terminal tyrosine is conserved and the presence of another aromatic amino acid at the third or fourth position forms an important structural motif that fits into the binding site of the opioid receptors. The major opioid milk peptides, b-casomorphins, are fragments of the b-casein sequence 60–70, and are characterized as m-type
q Change History: March 2015. A. Pihlanto updated text, tables and references. Update of A. Pihlanto, Milk Protein Products: Bioactive Peptides. Encyclopedia of Dairy Sciences, 2nd Edition, 2011, Pages 879–886.
Reference Module in Food Sciences
http://dx.doi.org/10.1016/B978-0-08-100596-5.00932-X
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Milk Proteins: Bioactive Peptides
Table 1
Structural properties of selected bioactive peptides derived from milk proteins
Health
Activity
Cardiovascular diseases Blood pressure ACE inhibitory
Structural elements of peptides
Bioactive peptide
Precursor proteins
Pro or hydroxyl Pro, Tyr, Phe or Lys/Arg at C-terminus
Casokinins Lactokinins
as-, b- and k-Casein a-Lactalbumin b-Lactoglobulin as-, b- and k-Casein b-Lactoglobulin
Casoplatelins
k-Casein
Lactostatin Lactotensin
b-Lactoglobulin
Oxidative stress
Antioxidants
Amino acids His Tyr, Trp, Met, Lys, Cys in the sequence Peptides with Pro-His-His sequence Ile108, Lys112 and Asp115 residues, sugar content Low ratios of Met–Gly and Lys–Arg in protein, high amount of hydrophobic amino acids Ala or Pro at C-terminus Hydrophobic amino acid (preferably Trp) at N-terminus Branched chain amino acids, mainly Leu Peptide length Multiple Arg residues Highly positively charged sequence or certain amphiphilic character
Thrombosis
Antithrombotic
Blood lipid profile
Hypocholesterolemic and hypotriglyceridemic
Diabetes
DPP-IV inhibitors
Obesity
Satiety
Body defence, food quality
Antimicrobial
Nervous system
Opioid
Tyr at N-terminus, aromatic amino acid at 3rd or 4th position
b-casomorphin, a-casein exorphins Lactorphins
Bone and teeth health
Mineral absorption
Phosphorylated regions
Caseinophospho-peptides (CPP)
Whey proteins Caseins Whey proteins
Lactoferricin Casocidine, Isracidine
Lactoferrin a-Lactalbumin b-Lactoglobulin as-, b-Casein b-Casein a-Casein a-Lactalbumin b-Lactoglobulin as-, b-casein
ligands (Table 2). The a-casein exorphins were found to correspond to bovine as1-casein f(90–96), f(90–95) and f(91–96) and were s-selective receptor ligands. Whey proteins contain opioid-like sequences in their primary structure, namely a-and b-lactorphin. These peptides show low affinity for opioid receptors and are m-type receptor ligand. Opioid activity has, furthermore, been found in fragments from bovine serum albumin [serorphin, f(399–404)]. The milk-derived opioid-like peptides have shown multifunctional properties. In recent studies, b-casomorphin f(60–66), as1-casein exorphin f(90–94) and a-lactorphin showed the capability to induce mucin secretion in HT29-MTX cells. Concretely, a-lactorphin was able to up-regulate the expression of the major secreted gene encoded by these cells. Peptides from a peptic and tryptic digest of bovine and human k- and as1-caseins have been found to display opioid antagonist properties. Various C-terminally methoxylated casoxins have been identified, e.g., k-casein f(33–38) (casoxin-6), f(34–38) (casoxin-5) and f(35–38) (casoxin-4). Additionally, casoxin C [k-casein f(25–34)], casoxin A [k-casein f(35–41)] and casoxin B [k-casein f(61–64)] showed opioid antagonistic activity, but only casoxin C’s activity was comparable to the esterified casoxin-6 and 4. In general, the chemically modified peptides are more active than their non-methylated derivatives. Casoxins show a preference for m- and k-receptors with a relatively low potency compared to naloxone. Elevated blood pressure is one of the major independent risk factors for cardiovascular diseases. Angiotensin I-converting enzyme (ACE; dipeptidyl carboxypeptidase, EC 3.4.15.1) is one of the main regulators of blood pressure, thus, inhibition of this enzyme is considered one of the strategies for the treatment of hypertension. In recent years, a large number of ACE inhibitory peptides have been evaluated in vitro and in vivo from digests of various food proteins. Although the full mechanism of interaction between an inhibitor and the ACE is not known, it is highly possible that the peptide inhibitor interacts with the C-domain of ACE. Also some structural features, like amino acids with cyclic or aromatic rings (Tyr, Pro, Trp), are found at the C terminal of the peptides. The most commonly known peptides of milk origin are the ACE-inhibitory tripeptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), derived from b- and k-caseins. ACE-inhibitory activity has been detected in fragments 177–183 and 191–202 of b-casein and fragments 23–27 and 194–199 of as1-casein upon action by trypsin. Also, whey proteins contain ACE-inhibitory peptides; the highest activity has been found with peptides b-lg f(80–82), f(142–148) and a-la f(104–108). The IC50-value (inhibitor concentration leading to 50% inhibition) is used to estimate the effectiveness of different ACE-inhibitory peptides. The majority of milk-derived ACE-inhibitors have moderate inhibitory potencies, usually within an IC50 range of 100–500 mmol l1. However, in vitro activity is not always related to in vivo activity, since some peptides are susceptible to degradation or modification in the gut, vascular system and the liver. Moreover, some long-chain candidate peptides can be degraded and new active fragments are generated by gastrointestinal enzymes (Table 3). Another complication related to cardiovascular diseases is the inclination to develop thrombosis due to abnormalities in blood coagulation. There is a significant amount of molecular similarities involved in milk and blood clotting. Peptides that inhibit blood platelet aggregation and fibrinogen binding (g-chain) to platelet surface receptors are encrypted within the sequence of bovine
Table 2
Examples of milk protein-derived bioactive peptidesa Biological activity
Preparation
Substrate and enzymes applied Casein b-Casein as1-Casein k-Casein Casein
Jejunum (minipigs) Trypsin Trypsin Pepsin Pepsin Pepsin Trypsin Trypsin Trypsin Trypsin Trypsin
in vivo Synthesis a-Lactalbumin Pepsin Trypsin b-Lactoglobulin Pepsin þ trypsin Trypsin Lactoferrin Pepsin Glycomacropeptide Trypsin
Origin
Sequence b
Name
Opioid IC50 mM c
b-CN f(60–70)
YPFPGPIPNSL
b-Casomorphin-11
10
b-CN f(60–66) b-CN f(60–64) as1-CN f(90–96) as1-CN f(9095 k-CN f(33–38) k-CN f(25–34) as1-CN (23–34) as1-CN f(194– 199) b-CN f(1–25)4P as1-CN f(43–58) 2P as1-CN f66–74) b-CNf(193–202) a-LA f(50–53) a-LA f(104–108) b-LG f(102–105) b-LG f(142–148) LF f(17–41) k-CN f(106–116)
YPFPGPI YPFPG RYLGYLE RYLGYL SRYPSY YIPIQYVLSR FFVAPFPQVFGK TTMPLW
b-Casomorphin-7 b-Casomorphin-5 as1-Exorphin as1-Exorphin Casoxin-6 Casoxin-C Casokinin as1-Immunocasokinin
14 1.1 12 45 (Y) 250 (Y) 50
RELEELNVPGEIVES*LS*S*S*EESITR DIGS*ES*TEDQAMEDIM
Caseinophosphopeptide Caseinophosphopeptide
S*S*S*EEIVPN YEQPVLGPVR YGLF-NH2 WLAHK YLLF-NH2 ALPMHIR FKCRRWQWRMKKLGAPSITCVRRAF MAIPPKKNQDK
Caseinophosphopeptide b-Casokinin-10 aLactorphin Lactokinin bLactorphin Lactokinin Lactoferricin Casoplatelin
ACE-inhibitory IC50 mM d
Immunomodulatory e
500
21/þ26
77 16
þ162
Mucin secretion (% control)
282 234
[Ig-production
300 160
300 733 77 172 43
Ca2þ binding Kapp (l mol1)
629 328
28/þ14 201 453
Antimicrobial Antithrombotic
References used to generate this Table Antila, P., et al., 1991. Opioid peptides derived from in vitro proteolysis of bovine whey proteins. Int. Dairy J. 1, 215–229; Brantl, V., et al., 1981. Opioid activities of b-casomorphins. Life Sci. 28, 1903–1909; Chiba, H., et al., 1989. Opioid antagonist peptides derived from k-casein. J. Dairy Res. 56, 363–366; Chiba, H., Yoshikawa, M., 1986. Biologically functional peptides from food proteins: New opioid peptides from milk proteins. In: Feeney, R.E., Whitaker, J.R. (Eds.), Protein Tailoring for Food and Medical Uses. Marcel Dekker, New York, pp. 123–153; Fiat, A.-M., et al., 1993. Biologically active peptides from milk proteins with emphasis on two example concerning antithrombotic and immunomodulating activities. J. Dairy Sci. 76, 301–310; FitzGerald, R., 1998. Potential uses of caseinophosphopeptides. Int. Dairy J. 8, 451–457; Henschen, A., et al., 1979. Novel opioid peptides from casein (b-casomorphins) II. Structure of active components from bovine casein peptone. Hoppe-Seyler’s Z. Physiol. Chem. 360, 1217–1224; Loukas, S., et al., 1983. Opioid activities and structures of a-casein-derived exorphins. Biochemistry 22, 4567–4573; Maruyama, S., et al., 1987. Angiotensin I converting enzyme inhibitory activity of the C-terminal hexapeptide of as1-casein. Agric. Biol. Chem. 51, 2557–2561; Meisel, H., 1986. Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein. FEBS Lett. 196, 223–227; Meisel, H., Schlimme, E., 1994. Inhibitors of angiotensin-converting enzyme derived from bovine casein (Casokinins). In: Brantl, V., Teschemacher, H. (Eds.), b-casomorphins and Related Peptides Recent Developments. VCH Veinheim, pp. 66–72; Mullally, M.M., et al., 1996. Synthetic peptides corresponding to a-lactalbumin and b-lactoglobulin sequences with angiotensin-I-converting enzyme inhibitory activity. Biol. Chem. Hoppe-Seyler 377, 259–260; Mullally, M.M., et al., 1997. Identification of novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of b-lactoglobulin. FEBS Lett. 402, 99–101; Pihlanto-Leppälä, A., et al., 2000. Angiotensin I-converting enzyme inhibitory properties of whey protein digests: concentration and characterization of active peptides. J. Dairy Res. 67, 53–64; Tomita, M., et al., 1994. A review: the active peptide of lactoferrin. Acta Paediatr. Jpn. 36, 585–591; Martinez-Maqueda, D., et al., 2012. Food-derived peptides stimulate mucin secretion and gene expression in intestinal cells. J. Agric. Food Chem. 60, 8600–8605. b One letter amino acid codes used; Phosphoserin ¼ S*. c Opioid activities of synthetic peptides in an opiate receptor-binding assay IC50-values (mM) are given for peptide concentrations inhibiting [3H]-ligand binding by 50%; (Y) indicates antagonist activity. d Conditions for IC50 estimation are not identical; the concentration of peptide needed to inhibit 50% of the ACE activity. e Figures indicate the maximum % stimulation (þ) and/or inhibition (), respectively, in relation to control (¼100). a
Milk Proteins: Bioactive Peptides 3
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Milk Proteins: Bioactive Peptides
Table 3 Sequence
Antihypertensive peptides derived from milk proteins by enzymatic hydrolysis and fermentation Origin
Enzymatic treatments TTMPLW as1-CN f(194–199) FFVAPFPGVFGK as1-CN f(23–34) RYLGY as1-CN f(90–94) AYFYPEL as1-CN f(143–159) YGLF a-LA f(50–53) KVLPVPQ b-CN f(169–175) IPA b-LG f(80–82) LQKV b-LG f(58–61) LLF b-LG f(103–105) KVLPVP b-CN f(169–174) IAK k-CN f(22–24) YAKPVA k-CN f(61–66) YKVPQL as1-CN f(104–109) Fermentation VPP b-CN f(84–86) IPP b- or k-CN YP as1-, b- or k-CN LHLPLP b-CN f(133–138) LHLPLPL b-CN f(133–139) RPKHPIKHQ as1-CN f(1–9)
Preparation
IC50 (mM) a
Dose (mg kg1)
SBP mm Hg
Trypsin
16 77 0.71 6.58 733 1000 141 34.7 79.8 5 15.7 14.3 22
100 100 5 5 0.1 2 8 10 10 1 4 6 1
13.6 34.0 25.0 20.0 25 24 31 18.1 29.0 32.2 20.7 23.1 12.5
9 5 720 5.5 4.25 13.4
1.6 1 1 10 7 6.1–7.5
20 15.1 27 7.7 23.5 9.3
Pepsin
Proteinase K Thermolysin Digestive enzymes
Proteinase from L. helveticus CP790 L. helveticus & S. cerevisiae L. helveticus CPN4 E. faecalis Gouda cheese
a
The concentration of an ACE-inhibitor needed to inhibit 50% of ACE activity. Adapted from Hernandez-Ledesma, B., Garcia-Nebot, M., Fernandez-Tome, S., Amigo, L., Recio, I., 2014. Dairy protein hydrolyzates: peptides for health benefits. Int. Dairy J. 38, 82–100.
k-casein, referred as casoplatelins. Several casoplatelins have been reported to have an antithrombotic effect in vitro and in guinea pigs after parenteral administration. k-Casein f(106–116) inhibited ADP-induced platelet aggregation and combined with the fibrinogen receptor of blood platelets, consequently preventing fibrinogen binding with blood platelets. The two smaller tryptic peptides (k-casein f(106–112) and f(113–116)) exerted an effect on platelet aggregation but did not inhibit fibrinogen binding. Oxidative stress is also one possible factor for initiation and evolution of cardiovascular diseases. Natural antioxidants provide additional benefits to the endogenous defence strategies in the battle against oxidative stress. Antioxidant properties to prevent enzymatic (lipoxygenase) and non-enzymatic peroxidation of essential fatty acids have been found in peptides derived from milk proteins. The identified peptides are encrypted in the sequences of as1-, b- and k-caseins and b-lactoglobulin. The antioxidative properties of the peptides are related to their structure and hydrophobicity. Hypocholesterolemic effects have been reported for whey-derived peptides. The mechanism involved in these effects remains to be clarified, but preliminary results suggests that the amino acid composition may play a key influence Two b-lactoglobulin derived peptides Ile-Ile-Ala-Glu-Lys (f71–75, lactostatin) and His-Ile-Arg-Leu (f146–149, lactotensin), are suggested to have hypocholesterolomic properties. Dipeptidyl peptidase IV (DPP-IV) inhibitors are among the newest medication introduced to the type 2 diabetes. These agents can enhance the endogenous concentrations of the active incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Bioinformatic approach has been used to predict the tryptic release of several synthetic Trpcontaining DPP-IV inhibiting peptides from dairy proteins, but it resulted in little success possibly due to narrow enzyme specificity. Some potent DPP-IV inhibitory peptides Phe-Leu-Gln-Pro [b-cn f(87–91)], Ile-Pro-Ile [k-casein f(26–28)] and Trp–Pro have been identified after gastric digestion. Studies on the effects of peptides on immunomodulation have been conducted in vitro with cells of specific and unspecific immune system. Immunomodulatory peptides can enhance immune cell functions, measured as lymphocyte proliferation, natural killer cell activity, antibody synthesis and cytokine regulation. The casein-derived peptides, including as1-casein f(194–199), b-casein f(63–68) and f(191–193), stimulate human and murine macrophages. Synthetic peptides (Tyr–Gly and Tyr-Gly-Gly) have been shown to enhance the proliferation of human peripheral blood lymphocytes. b-Casomorphin-7 and b-casokinin-10 suppress lymphocyte proliferation at low concentrations but are stimulatory at higher concentrations. b-Casein f(193–199) induces a significant proliferative response in rat lymphocytes. Bioactive peptides in yoghurt preparations decrease cell proliferation with Caco-2-cells or IEC-6-cells. as1-Casein f(59–79) and b-casein f(1–25), having a phosphoserine-rich region, have been found to display mitogenic activity and enhance immunoglobulin production in mouse spleen cells. Lactoferrin-derived peptides have been reported to influence in several cell type e.g., human monocytic and polymorphonuclear leucocytes cell lines, experiments which are involved in immune and inflammatory actions of the body. The antimicrobial activity of milk is associated mainly with lactoferrin. Moreover, peptides released from lactoferrin by digestion with pepsin or by heat treatment at an acidic pH show higher antimicrobial potency than undigested lactoferrin. The identified
Milk Proteins: Bioactive Peptides
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antimicrobial peptides, named lactoferricin, originate from the N-terminal region of the molecule and have antimicrobial activity against various Gram-positive and Gram-negative bacteria, yeast and filamentous fungi. For example, lactoferricin B killed four clinical isolates of enterohaemorrhagic Escherichia coli O157:H7 within 3 h at concentrations above 10 mg ml1. Additionally, as2-casein f(165–203) (Casocidin-I) purified from bovine milk hydrolyzed by a serine protease inhibits the growth of E. coli and Staphylococcus carnosus. The antimicrobial activity of lactoferricins and casocidin-I seems to be correlated with the net positive charge on the peptides. A distinctive feature is the high proportion and asymmetric clustering of basic amino acid residues. It is generally known that cationic amphipathic a-helical structures are related to antimicrobial activity by forming ion channels through membrane bilayers. Lactoferricins disrupt the normal membrane permeability of the cell, which is at least partly responsible for the antibacterial mechanism of lactoferrin-derived peptides. Peptides generated by enzymatic cleavage from a-lactalbumin and b-lactoglobulin are negatively charged and exhibit antimicrobial activities mostly against Gram-positive bacteria. Tryptic digestion of casein proteins yields casinophosphopeptides (CPPs) from the polar N-terminus region, which contains clusters of phosphorylated seryl residues. For example: as1-casein f(43–58), f(59–79), f(43–79), as2-casein f(1–24) and f(46–70) and b-casein f(1–28), f(2–28), f(1–25), f(33–48) have been isolated from the tryptic hydrolyzate of whole casein. These phosphorylated clusters have been hypothesized to be responsible for the interaction between caseins and calcium phosphate that leads to the formation of casein micelles. CPPs retain the ability of whole casein to stabilize calcium and phosphate ions through the formation of complexes, thus enhancing their general bioavailability.
Possible Physiological Importance The potential physiological role of bioactive peptides derived from milk proteins and the in situ formation of these peptides in the gastrointestinal tract is still largely unknown. One of the greatest challenges in developing peptides as food ingredients has been proving their in vivo efficacy. It mainly depends on the capacity of peptides, after being orally ingested, to reach the target organs in an intact and active form in substantial concentrations. The problem of absorption of bioactive peptides is still an open question due to differences occurring between the forms and transport of the individual peptides. Caco-2 cell models are found to be a helpful tool to predict their oral bioavailability. In the last years, studies demonstrated the bioavailability of potential antihypertensive tripeptides (VPP and IPP). The tripeptides have been detected in aortal tissues using animal model and ACE activity in aorta was lower than in control group. Paracellular transport was found to be the main form of transport of VPP. The bioavailability of IPP has been assessed in human studies, and maximum peptide concentration in plasma after ingestion of peptide enriched beverage was 897 157 p.m. Studies have shown no intact transepithelial passage of b-casomorphins; therefore, it is generally concluded that the physiological influences are limited to the gastrointestinal tract. The situation is different in neonates where passive transport across intestinal mucosal membranes occurs. This may lead to physiological responses such as an analgesic effect resulting in calmness and sleep in infants. Small CPPs were detected in the stomach, duodenal and ileostomy fluids of humans after milk ingestion. Most bioactive peptides are not absorbed in the intestinal tract. Hence peptides may either act directly in the intestinal tract or via receptors and cell signaling in the gut. Numerous studies in spontaneously hypertensive rats (SHR) as well as in hypertensive human volunteers have been performed to determine the antihypertensive effect of milk-derived peptides. These studies demonstrated that several ACE-inhibitory peptides significantly reduce blood pressure, either after intravenous or oral administration. Most of the human intervention studies have been focused on the consumption of products containing the tripeptides, IPP and VPP. Maximum blood pressure reductions approximate 13 mmHg (systolic blood pressure, SBP) and 8 mmHg (diastolic blood pressure, DBP) after active treatment compared to placebo, and are likely to reach after 8–12 weeks of treatment. Effective dosages of tripeptides range from 3.07 to 52.5 mg day1. Meta-analyses performed with published data of clinical trials have been reported. Nineteen randomized clinical intervention trials with small daily doses (2.0–10.2 mg) of milk casein-derived tripeptides showed an overall lowering of 4.0 mmHg of SBP in mildly hypertensive subjects. A meta-analysis carried out with 28 clinical trials reported a mean reduction of SBP of 1.66 mmHg. Another, meta-analysis with 18 clinical trials, reported that the tripeptides reduce SBP in Asian subjects while this effect was lower in Caucasian individual. Moreover, some clinical trials did not find an effect on human blood pressure in Dutch and Danish subjects after consumption fermented milk with tripeptides. The findings suggest that genetics and/or dietary patterns might exert an important influence on the antihypertensive effects of peptides IPP and VPP. Moreover, the European Food Safety Authority (EFSA) Panel on Dietetic Products, Nutrition and Allergies (NDA) has concluded that the evidence presented to date on the antihypertensive effects of peptides VPP and IPP is insufficient to establish the relationship between the consumption of these peptides and the maintenance of normal blood pressure. Other milk protein-derived hydrolyzates and peptides have been tested in clinical trials. Consumption of whey protein hydrolyzate for 6 weeks resulted in reduction of SBP and DBP, of hypertensive subjects. However, peptides responsible for the observed effect have not been identified. More recently, yoghurt enriched with other milk-derived peptides, Arg-Tyr-Leu-Gly-Tyr and Ala-Tyr-Phe-Tyr-Pro-Glu-Leu, reduced blood pressure in hypertensive humans. The consumption of dairy proteins, particularly whey protein, has been shown in a number of intervention studies to have beneficial effects on glucose metabolism. The antidiabetic effect of whey protein have been suggested to be attributable to its content of bioactive peptides which, following their release during gastrointestinal digestion, could stimulate the secretion of gut-derived hormones and/or inhibit enzymes involved in glycemia homeostasis. Inhibition of DPP-IV may be one of the potential mechanisms contributing to the reported mechanisms of whey proteins. So far few studies have reported the in vitro DPP-IV inhibitory
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Milk Proteins: Bioactive Peptides
potencies of whey hydrolyzates and peptides. A b-lg derived peptide, Val-Ala-Gly-Thr-Trp-Tyr, presented hypoglycaemic effects in the oral glucose tolerance test in mice. This in vivo effect has been also demonstrated for the peptide Leu-Pro-Gln-Asn-Ile-ProPro-Leu, a casein derived fragment described as DPP-IV inhibitor. Diet plays a key role in plasma lipids profile, and consequently it is used as a strategy to prevent or decrease the incidence of cardiovascular diseases. It was observed that tryptic whey protein hydrolyzate displayed a hypocholesterolemic effect in rats and the effect was related to a peptide called lactostatin, Ile-Ile-Ala-Glu-Lys (b-lg f 71–75). Another b-lg-derived peptide b-lactotensin (His-Ile-Arg-Leu) decreased total cholesterol and low density lipoproteins-cholesterol content in mice fed a cholesterol-enriched diet. The systems involved in the defence mechanism of the body are both varied and complex and the main focus are on two peptide groups, namely immunomodulatory and antimicrobial peptides. Only a very limited amount of information is available on the in vivo effects. The Tyr–Gly and Tyr–GlyGly peptides modulate the lymphokine production in vitro. Moreover, these peptides were used successfully for immunotherapy of human immunodefiency virus infection. Furthermore, a study showed that oral administration of a commercial caseinophosphopeptide preparation [consisted of as2-casein f(1–32) and b-casein f(1–28)] enhanced intestinal IgA levels in piglets. A hexapeptide [human b-casein f(54–59)] and a tripeptide Gly-Leu-Phe, protected mice against Klebsiella pneumoniae infection after intravenous infection. An antimicrobial peptide, as1-casein f(1–23), isracidin, has been shown to exhibit in vivo activity against Staphylococcus aureus and Candida albicans at concentrations that are comparable with known antibiotics. Moreover, bactericidal peptides may assist in protecting against microbial challenge, especially in the neonatal intestinal tract, and thus support the non-immune defence of the gut. The immunostimulating activity may also have a direct effect on their resistance to bacterial and viral infections of adult humans. There are also indications that milk-derived peptides, especially lactoferricin B could be used to prevent different steps of cancer, including initiation, promotion and progression. Mineral deficiencies are the most important nutritional problems worldwide, with the iron deficiency being the most common. Mineral fortification is one of the best and most common strategies to prevent this deficiency. It has been proposed that the CPPs could increase the mineral solubility at intestinal pH, modulating its bioavailability. Published data on the effect of CPPs in animal and human studies are contradictory. A rat model system indicates that CPPs increase passive calcium transport in the distal small intestine. However, no evidence has been supplied on the effectiveness of CPPs in increasing passive calcium absorption in humans. In a recent study, it was concluded that CPPs cannot enhance calcium absorption in the gut. Concerning the iron bioavailability, a positive influence of CPPs on this parameter has been reported. An increase in the iron liver storage was found when iron deficient rats were supplemented with iron and hydrolyzed b-casein or b-casein f(1–25)4P. In humans, the effect of CPPs on iron bioavailability is controversial. The results indicate that the effects of CPPs can be influenced by food matrix (solid or liquid) as well as phytate content. Besides, CPPs can have anticariogenic properties, based on their ability to stabilize and localize calcium and phosphate ions at the tooth surface, promote remineralization of enamel sub-surface lesions. It is generally accepted that protein is the most satiating macronutrient. Several studies speculate that peptides released from dietary proteins during digestion can initiate several satiety signals from the gut and thus prevent further food intake. Because these peptides act on the intestinal site they do not need to be absorbed into the systemic circulation. It has been suggested that caseinderived peptides induce satiety by independent activation of both opioid and cholecystokinin receptors in rats. Moreover, caseinand whey protein-derived peptides appear to stimulate GLP-1 release. It remains to be determined which peptides are responsible for the above mentioned effects.
Production of Bioactive Peptides The ability of lactic acid bacteria or their purified peptidases to release bioactive peptides have been evaluated in many studies. Many of these works concern the formation of ACE-inhibitory peptides, certainly because of the ease to detect their presence through the use of a simple enzymatic assay. The release of peptides has been shown to depend on the proteolytic activity of the microbes. The most prominent strains include Lactobacillus helveticus, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Enterococcus faecalis. Especially, L. helveticus strains are capable of releasing the well-characterized tripeptides, VPP and IPP. It should be noted that hydrolysis of whey protein concentrate with crude proteinase from L. helveticus led to the isolation of ACE-inhibitory peptide derived from b-lactoglobulin (f148–153). Fermentation with L. helveticus LH-2 yielded immune peptides [b-casein f(143–154); f(145–160), f(148–154) and a-lactalbumin f(115–122)]. Another common way to produce bioactive peptides is enzymatic hydrolysis by a range of different gastric enzymes, such as pepsin, trypsin and pancreatin as well as microbial enzymes such as thermolysin and alcalase. After hydrolysis, the peptides in hydrolyzates have been fractionated and enriched using different methods, such as precipitation with salts or solvent, ultrafiltration and chromatography. Application of an ultrafiltration membrane reactor, for the continuous extraction of permeates enriched with bioactive fragments has been described for the production of antithrombotic peptides. Membranes consisting of negatively charged materials have been used to desalt whey protein hydrolyzates and to enrich cationic peptides with antibacterial properties. This technique provides new possibilities for enriching peptides with a low molecular mass and is easily up-scaled. Cheese constitutes one of the major sources of bioactive peptides among commercial dairy products due to the proteolytic activities during cheese ripening by natural milk proteases and peptidases, rennet and microbial flora. The occurrence of bioactive peptides in cheese varieties has been reported in several studies, and the activity depends on the maturation of cheese. CPPs are produced during cheese ripening due to plasmin and proteolytic enzymes derived from lactic acid bacteria.
Milk Proteins: Bioactive Peptides
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Several ACE-inhibitory peptides, including the tripeptides VPP and IPP has been found in several commercially available cheeses at physiological relevant concentrations. Opioid peptides, b-casomorphin-7, have been quantified in different cheese varieties, concentrations of 0.15 mg kg1 in Brie cheese. Fermented milks, yoghurts and kefir are another important group of commercial dairy products that can include bioactive peptides in their composition due to milk protein proteolysis by microbial fermentation. At least, ACE-inhibitory, antioxidant, immunomodulatory and opioid peptides have been detected in commercial products.
Conclusions The research area of bioactive peptides is at its beginning, most of the studies have mostly focused in vitro and animal studies, with the lack of strong clinical studies to substantiate the efficacy and support future health claims. Since metabolic disorders are significant global public health problem bioactive peptides may be of vital interest in maintaining a healthy population. However, it appears that translation of the milk-derived peptides in health promotion has met challenges that can potentially impact efficacy, consumer acceptability, commercialization and approval of peptide products. To meet the challenges, emphasis should be placed on enhancing taste, gastric stability and bioavailability of peptides, providing clinical evidence to support the health effects as well as optimizing their inclusion into food products to limit undesirable reaction. In these studies there is a need to identify and validate biomarkers that conclusively are related to a certain health benefits. Future clinical trials could benefit from data on genotype, metabolomic profiles and proteomics or transcriptomics data of the volunteers. In addition, sources to produce bioactive peptides should be sustainable, to reduce the heavy reliance on primary human food. Accordingly, research emphasis is shifting towards valorisation of protein-rich industrial by-products for inclusion into human food systems in hydrolyzed forms containing bioactive peptides. Moreover, there is need to develop modern, sustainable, techniques to enrich active peptides from food protein and to facilitate the production in huge amounts for the market. Given the facts that milk-derived bioactive peptides are safe, they are good candidates for inclusion in healthy lifestyle changes to prevent or reduce diseases such as cardiovascular diseases and obesity. In summary, more emphasis should be placed on translating the promising results into commercially available peptide products with defined mechanisms of physiological functions.
Further Reading Boelsma, E., Kloek, J., 2008. Lactotripeptides and antihypertensive effects: a critical review. Br. J. Nutr. http://dx.doi.org/10.1017/S0007114508137722. Published online by Cambridge University Press, December 05, 2008. Erdmann, K., Cheung, B.W.Y., Schröder, H., 2008. The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. J. Nutr. Biochem. 19, 643–654. Gauthier, S.F., Pouliot, Y., Saint-Sauver, D., 2006. Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins. Int. Dairy J. 16, 1315–1323. Korhonen, H., Pihlanto, A., 2006. Bioactive peptides: production and functionality. Int. Dairy J. 16, 945–960. Korhonen, H., Pihlanto, A., 2007. Technological options for the production of health-promoting proteins and peptides derived from milk and colostrum. Curr. Pharm. Des. 13, 829–843. Möller, N.P., Scholz-Ahrens, K.E., Roos, N., Schrezenmeir, J., 2008. Bioactive peptides and proteins from foods: indication for health effects. Eur. J. Nutr. 47, 171–182. Murray, B.A., FitzGerald, R.J., 2007. Angiotensin converting enzyme inhibitory peptides derived from food proteins: biochemistry, bioactivity and production. Curr. Pharm. Des. 13, 773–791. Pihlanto, A., 2006. Antioxidative peptides derived from milk proteins. Int. Dairy J. 16, 1306–1314. Hafeez, Z., Cakir-Kiefer, C., Roux, E., Perrin, C., Miclo, L., Dary-Mourot, A., 2014. Strategies of producing bioactive peptides from milk proteins to functionalize fermented milk products. Food Res. Int. 63, 71–80. Hernandez-Ledesma, B., Garcia-Nebot, M., Fernandez-Tome, S., Amigo, L., Recio, I., 2014. Dairy protein hydrolysates: peptides for health benefits. Int. Dairy J. 38, 82–100. Udenigwe, C., 2014. Bioinformatics approaches, prospects and challenges of food bioactive peptide research. Trends Food Sci. Technol. 36, 137–143.