Introduction
The human body is constantly subjected to physiological imbalances and exposure to extrinsic toxic substances that perturb normal functions leading to various health conditions. These aberrations can be controlled by physiological homeostasis, or through the use of health-promoting agents especially in acute and chronic conditions (Ames and others 1993). It is generally established that the nutritive and non-nutritive constituents of food can be used to modify the risk of developing or aggravating human disease conditions. In this regard, functional foods and nutraceuticals have emerged as adjuvant or alternative to chemotherapy especially in prevention and management of human diseases, and for maintaining optimum health state (Kris-Etherton and others 2002). This area of research has increasingly become the subject of various research programs as the health and well-being of consumers gradually became the primary focus of the food industry. There is a growing trend and interest in the use of food protein-derived peptides as intervention agents against chronic human diseases and for maintenance of general well-being. These peptides are produced by enzymatic hydrolysis of food proteins to release the peptide sequences, followed by posthydrolysis processing to isolate bioactive peptides (BAPs) from a complex mixture of other inactive molecules (Wang and Gonzalez De Mejia 2005; Korhonen and Pihlanto 2006; Hartmann and Meisel 2007; Aluko 2008a). These peptides are different from naturally occurring BAPs, such as endorphins, because they are generated by proteolysis of native
food proteins. By denition, BAPs discussed in this review are food protein-derived peptides that possess benecial pharmacological properties beyond normal and adequate nutrition (Hartmann and Meisel 2007). The food processing steps lead to concentration of the active peptides with the enhancement of the physiological activity of the products, which could also be nutritionally benecial as a source of essential amino acids. This approach can provide the opportunity for diversication of the use of agricultural crops and animal products beyond basic nutritional purposes, especially as a source of active ingredients for formulation of food products with health benets.
MS 20110594 Submitted 5/10/2011, Accepted 9/12/2011. Authors are with Dept. of Human Nutritional Sciences and author Aluko is also with the Richardson Centre for Functional Foods and Nutraceuticals, Univ. of Manitoba, 196 Innovation Drive, Winnipeg, MB R3T 2N2, Canada. Author Udenigwe is also with Dept. of Food Science, Univ. of Guelph, Guelph, ON N1G 2W1, Canada. Direct inquiries to author Aluko (E-mail: alukor@cc.umanitoba.ca).
Table 1Sources and bioactive properties of marine protein-derived hydrolysates and peptides. Protease Peptides (mostly 300 to 860 Da) produced after nanoltration, desalination, and cryoconcentration; fed 0 to 1.35 g/kg body weight to ICR mice for 4 wk Treatment/peptide property/sequence Outcome Reference Yang and others (2009)
Marine protein
Complex protease
Jellysh collagen
Protamex
A mixture of 6 proteases
Seven proteases including Alcalase (Alc), Esperase (Esp), and Neutrase (Neu)
Table 1Continued Protease Peptides rich in Gly, Pro, Ala, Glu Treatment/peptide property/sequence Outcome Reference Kim and others (2011)
Marine protein
Rocksh (Sebastes hubbsi) gelatin Dipeptides Ala-Pro and Val-Arg isolated after RP-HPLC
Alcalase, Flavourzyme
Alcalase, papain
Multifunctional property Moderate free radical (DPPH, superoxide, hydroxyl, alkyl) scavenging property; ACE inhibition with IC50 of 0.82 mg/mL ACE inhibition Ala-Pro and Val-Arg inhibited ACE activity with IC50 of 0.06 and 0.33 mg/mL, respectively 20- and 4-folds more potent than the crude hydrolysates Anticancer Peptides 1 and 2, from PA and PR, respectively, dose-dependently inhibited breast cancer (MCF-7) cells proliferation with IC50 of 8.1 and 8.8 M, respectively Anticancer Cationic fraction (KCl2, pH 6) showed most potent inhibitory activity against the viability of lung (A549), breast (BT549), colon (HCT15), and prostate (PC3) cancer cells at 1:10 and 1:100 dilutions
Protamex
Cryotin enzyme
Isolated a dodecapeptide Leu-Pro-His-ValLeu-Thr-Pro-Glu-Ala-Gly-Ala-Thr (1) and a hendecapeptide Pro-Thr-Ala-GluGly-Gly-Val-Tyr-Met-Val-Thr (2) after gel ltration and RP-HPLC Low molecular size net-charged peptide fractions (cationic = KCl2, anionic = KCl1) were generated after fractionation of the crude hydrolysates by electrodialysis-ultraltration at pH 3, 6, and 9 High molecular size (<10, 10 to 30, and >30 kDa) gastrointestinal resistant oligopeptide fractions Peptides were fractionated (<1, 1 to 3, 3 to 5, 5 to 10 kDa) by membrane ultraltration
Anticancer All samples showed time-dependent inhibition of proliferation of Caco-2 (colon) and HepG2 (liver) cancer cells (up to 60% inhibition by fractions <10 and 10 to 30 kDa) Antioxidant All samples exhibited antioxidant properties (DPPH scavenging and FRAP) but the <1 kDa fractions showed the best activities
Abbreviations: NF- B = nuclear factor- B; I -B = inhibitor of NF- B; DPPH = 2,2-diphenyl-1-picrylhydrazyl radical; FRAP = ferric-reducing antioxidant power; ACE = angiotensin I-converting enzyme; IL = interleukin; IFN = interferon; TPA = 12-O-tetradecanoylphorbol-13-acetate; detailed review about marine-derived bioactive peptides can be found in Kim and Wijesekara (2010), Harnedy and Fitzgerald (2011), Wilson and others (2011), and Fitzgerald and others (2011).
Production and processing methods BAPs are encrypted in the primary structure of plant and animal proteins as inactive amino acid sequences but they can be released by fermentation, food processing, and enzyme-catalyzed proteolysis in vitro or in the digestive tract after human consumption (Hartmann and Meisel 2007; Aluko 2008b). In most cases, these protein hydrolysates and peptides have demonstrated better bioactivity compared to their parent proteins, and this shows that hydrolysis of peptide bonds is important in liberating the potent peptides. Several factors affect the bioactive properties of the peptides including the enzymes used for hydrolysis, processing conditions, and the size of the resulting peptides, which greatly affects their absorption across the enterocytes and bioavailability in target tissues. Most reported BAPs are produced by in vitro enzymatic hydrolysis or fermentation. After selecting an appropriate food protein, enzymatic hydrolysis is performed using single or multiple specic or nonspecic proteases to release peptides of interest. Simulated gastrointestinal enzymatic process has also been used to mimic normal human digestion of proteins to evaluate the possibility of releasing potent BAPs after normal consumption of food proteins. The latter strategy could be cost-effective since extensive processing of the peptide product will not be needed. Some factors to consider in producing BAPs include hydrolysis time, degree of hydrolysis of the proteins, enzymesubstrate ratios, and pretreatment of the protein prior to hydrolysis. For example, thermal treatment of proteins can enhance enzymatic hydrolysis (Inouye and others 2009) possibly by increasing enzymeprotein interactions due to thermal-induced unfolding of the proteins. In addition, sonication and hydrostatic pressure treatments of food proteins have separately resulted in enhanced hydrolysis and release of potent BAPs (Quir os and others 2007; Wu and Majumder 2009). Furthermore, it is feasible to scale-up production of peptides from laboratory scale to pilot and industrial plant scales with conserved peptide proles and bioactivity of the resulting products (Wang and others 2010). A challenge often faced in food protein-derived peptide research is to obtain high-yield peptide products with potent bioactivity. This limitation results in carrying out further processing of the enzymatic food protein hydrolysates. Therefore, after protein hydrolysis, the resulting peptide product is further processed based on physicochemical and structural properties of the constituent peptides in a bid to enhance bioactivity. The peptide properties that are often focused on include size, net charge, and hydrophobicity, depending on the targeted pharmacological uses. Membrane ultraltration and size-exclusion chromatography can be used to concentrate peptides of dened molecular weight ranges, especially for obtaining fractions containing low molecular weight peptides that can withstand further in vivo proteolytic digestion. In addition, reverse-phase HPLC on a hydrophobic column matrix can be used to fractionate peptides based on their hydrophobic properties (Pownall and others 2010), especially when studying the structurefunction properties of peptides. Peptide fractions of particular net charges can be obtained by chromatography using selective ion-exchange columns (Li and Aluko 2005; Pownall and others 2011). This processing approach is very useful especially when
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Antihypertensive peptides Physiological regulation of blood pressure (BP). BP is physiologically controlled by the renin-angiotensin system (RAS) and the kinin-nitric oxide (NO) system (Figure 3). The RAS involves activation of angiotensinogen by the proteolytic activity of renin which converts it to angiotensin (AT)-I. This reaction is the rst and rate-limiting step of the RAS pathway. AT-I is then cleaved at the histidyl residue from the C-terminus by the activAntioxidant ity of angiotensin I-converting enzyme (ACE) to produce AT-II. Activities Immunomodulatory Antihypertensive AT-II is a powerful vasoconstrictor that functions by binding to Properties Activity receptors, located in tissues all over the body, to elicit physiological reaction cascades that lead to blood vessel contractions that maintain normal BP. However, in pathological conditions, there Food ProteinLipid-lowering Anticancer is excessive level of AT-II, which causes severe blood vessel conProperties Derived Peptides Properties tractions and limited relaxation to produce high BP. Moreover, the kinin-NO system is involved in the production of bradykinin, Liver Disease Treatment which exerts its antihypertensive effects by eliciting reactions that Antimicrobial (High Fischer ratio 2+ Anti-inflammatory Activity increase intracellular Ca concentration leading to activation of peptides) Properties nitric oxide synthases (NOS) that produce NO, a powerful vaMultifunctional sodilator. ACE degrades bradykinin, and increased concentration Properties of ACE leads to dual effects such as the prevention of vasodilation and the activation of vasoconstriction. Based on the roles of ACE Figure 2Bioactive properties of food protein-derived peptides relevant to in the RAS pathway, inhibitors of this enzyme have been used as the promotion of human health and disease prevention.
Selection of food protein source Protein isolation Enzymatic hydrolysis
Figure 1Schematic diagram showing steps toward the production and processing of food protein-derived bioactive peptides.
Inactivation of enzyme(s)
Ultrafiltration
Post-hydrolysis processing
Electrodialysisultrafiltration
Chromatography
Sizeexclusion
RP-HPLC
Anionexchange
HPLC purification
Pure peptides
Angiotensinogen Renin Angiotensin-I Chymase Angiotensin-II Angiotensin Iconverting enzyme (ACE) Bioactive peptides
Bradykinin
AT receptor-mediated vasoconstriction
Figure 3The blood pressure regulating renin-angiotensin system (RAS) pathway showing potential molecular targets (renin and angiotensinconverting enzyme, ACE) for bioactive peptides. Inhibition of renin reduces the possibility of producing angiotensin-II via an ACE-independent chymase-catalyzed reaction.
Peptides from Lactobacillus helveticus fermented milk (FM) containing LTP (Ile-Pro-Pro and Val-Pro-Pro) LTP product (AmealPeptide)
MPH decreased SBP and DBP by 3.8 and 2.3 mmHg, respectively; no difference was found in plasma renin activity, AT-I or AT-II; no signicant change in BP observed in prehypertensive subjects compared to placebo; MPH was well tolerated and safe to the subjects FM showed no signicant effect on SBP and DBP compared to placebo using both ABPM and OBPM; no effects on plasma lipids; observed effect was not superior to effects of lifestyle intervention for lowering BP ABPM showed peptide-induced decreased daytime SBP (3.6 mmHg) and mean 24-h SBP (2 mmHg); OBPM was not reliable for BP measurement due to detected placebo effect which was minimal using ABPM; effect on daytime SBP was more pronounced in treatment-naive subjects compared to placebo
Abbreviations: ABPM = 24-h ambulatory BP; OBPM = ofce BP measurements; AT = angiotensin; SBP = systolic blood pressure; DBP = diastolic blood pressure.
Food protein-derived antioxidant peptides Dietary consumption of antioxidants can supplement the endogenous enzymatic and nonenzymatic antioxidant systems against oxidative stress (Fang and others 2002). Although synthetic food antioxidants have been widely applied in the food industry for food preservation, the use of food-derived peptides has generated interest as both food preservative and health products. There is abundant literature information on several food protein hydrolysates and peptides with antioxidant properties in various oxidative reaction systems. Plant and animal food protein sources
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Food protein-derived calmodulin (CaM)-binding peptides CaM is a ubiquitous negatively charged 148-amino acid (16.6 kDa) Ca2+ -binding protein that is involved in the activation of several important proteins in response to increased intracellular Ca2+ concentration (Ikura and others 1992; Hooks and Means 2001). Some clinically important enzymes that require Ca2+ /CaM activation include endothelial and neuronal NOS, cyclic nucleotide phosphodiesterase 1 (CaMPDE), adenosine triphosphatase, phospholipase A2 , adenylate cyclase, and protein kinase II (Itano and others 1980). Thus, CaM plays important roles in several cellular processes including cell growth, cell proliferation, neurotransmission, vasodilation, and smooth muscle contraction (Cho and others 1998). Therefore, CaMbinding natural compounds can be used for the prevention and amelioration of diseases induced or exacerbated by increased activity of CaM-dependent enzyme (Mart nez-Luis and others 2007). Considering the roles of CaM in human health conditions, CaM-binding agents can be used as multifunctional agents for ameliorating disease conditions. The amino acid sequences of many natural CaM-binding proteins and peptides revealed the presence of repeated positively charged (cationic) and hydrophobic amino acid residues at the CaM-binding sites (ONeil and DeGrado 1990). These structural features are thought to be more important than the specic amino acid sequence in determining afnity of peptides for CaM (Kizawa and others 1995). The afnity of the cationic residues for the net negatively charged CaM led to a rationale to use cationic peptides as CaM-binding agents (Itano and others 1980; Barnett and others 1983). A number of food protein-derived cationic peptides have been reported to bind CaM leading to the inhibition of CaM-dependent enzymes. Earlier works by Kizawa and others (1995) and Kizawa (1997) reported the isolation of CaM-binding peptides from casein, specically s2 -casein (f164179, f183206, f183207, and f90109), which inhibited CaMPDE activation with IC50 values of 38, 6.9, 1.1 and 1.0 M, respectively, without any effects on the basal PDE activity. These activities are lower than the inhibition of CaM-induced PDE activity by an anti-CaM drug (calmidazolium) with IC50 of 0.12 M and a microbial metabolite
Hypolipidemic and hypocholesterolemic peptides Protease-aided hydrolysis of food proteins can also release peptide sequences that possess cholesterol and lipid-lowering activities. Food protein sources of hypocholesterolemic and hypolipidemic peptides include soy protein (Nagaoka and others 1999; Aoyama and others 2000; Cho and others 2007), milk protein (Kirana and others 2005), buckwheat protein (Kayashita and others 1997), egg white protein (Manso and others 2008), and sh protein (Wergedahl and others 2004). However, enzymatic hydrolysis can also lead to reduced lipid-lowering activity of food proteins (Kayashita and others 1997). Most literatures on lipid-lowering peptides were focused on soy protein hydrolysates and peptides. The hypocholesterolemic and hypolipidemic properties of soy protein hydrolysates reported in animals (Aoyama and others 2000) and in humans (Hori and others 2001) have been partly attributed to the soy 7S globulin ( -conglycinin). The + subunit of this protein strongly upregulated the expression of low-density lipoprotein (LDL) receptor in cultured hepatocytes leading to an increase in LDL uptake and degradation (Lovati and others 1998). The peptide region responsible for the activity has been identied from the subunit and sequenced (Lovati and others 2000). This 24-amino acid peptide that corresponds to position 127 to 150 of the subunit displayed potential in modulating cholesterol homeostasis by increasing LDL receptor-mediated LDL uptake in Hep G2 cells (Lovati and others 2000). Moreover, Cho and others (2008) also identied an octapeptide (FVVNATSN) from the enzymatic digest of soy protein as the most active stimulator of LDL receptor transcription in Hep T9A4 human hepatic cells. Thus, proteolytic digestion of the soy protein was important for releasing more active small peptides with improved cardioprotective property. This has also been demonstrated in a study by Mochizuki and others (2009) that produced BAPs from puried isoavone-free
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Multifunctional peptides Multifunctional peptides have been discovered from some food proteins and have been reported to possess more than one signicant physiologically relevant bioactive property. Studies on milk proteins demonstrated that a hexapeptide (TTMPLW) derived from S1 -casien (f194199) by trypsin-catalyzed digestion exhibited both ACE-inhibitory and immunomodulatory activities (Meisel 2004) while a -lactoglobulin-derived -lactorphin (YLLF) inhibited ACE activity and also possessed opioid-like activity (Antila and others 1991; Mullally and others 1997). In addition, several other milk-derived peptides such as -lactorphin (YGLF), -immunocasokinin (TTMPLW), casomorphin-7 (YPFPGPI), and -casokinin (AVPYPQR) are regarded as multifunctional, some possessing in vivo bioactive properties (Meisel 2004). Moreover, crude chymotryptic -casein hydrolysates displayed several in vitro bioactivities such as ACE and propyl endopeptidase inhibition, antioxidant, Zn2+ -binding, and antibacterial activities (Srinivas and Prakash 2010). Four peptides (GFHI, DFHING, FHG, and GLSDGEWQ) present in beef sarcoplasmic protein hydrolysate were reported to possess anticancer, antimicrobial, and ACE-inhibitory properties (Jang and others
Safety of BAPs
Till date, there has been little concern about safety of food protein-derived BAPs since the body would normally hydrolyze food proteins into peptides (Wang and Gonzalez De Mejia 2005) and food-grade enzymes and processes are utilized for industrial
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Conclusions
The current literature has shown that peptides derived from enzymatic food protein hydrolysates possess remarkable multifunctional activities relevant to the sustenance of human health. This research area is continuously growing with the discovery of new molecular disease targets. While a lot of information exists on the various bioactivities of food protein-derived peptides, future research efforts should be directed toward evaluation of in vivo health-promoting effects, bioavailability, and pharmacokinetics in human subjects, elucidation of the molecular mechanisms of action and overall possible use as health-promoting agents in food systems. Moreover, the safety of these peptide-based products should also be evaluated prior to commercialization especially after extensive food processing that may affect the natural integrity and quality of the constituent peptides.
Acknowledgments
The research program of REA is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). CCU acknowledges the support from NSERC through an Alexander Graham Bell Canada Graduate Scholarship for his doctoral studies.
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