Food Protein-Derived Bioactive Peptides: Production, Processing, and Potential Health Benefits
Chibuike C. Udenigwe,
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: firstname.lastname@example.org).
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: email@example.com).
Abstract: Bioactive peptides (BAPs), derived through enzymatic hydrolysis of food proteins, have demonstrated potential for application as health-promoting agents against numerous human health and disease conditions, including cardiovascular disease, inflammation, and cancer. The feasibility of pharmacological application of these peptides depends on absorption and bioavailability in intact forms in target tissues, which in turn depends on structure of the peptides. Therefore, production and processing of peptides based on important structure-function parameters can lead to the production of potent peptides. This article reviews the literature on BAPs with emphasis on strategic production and processing methods as well as antihypertensive, anticancer, anticalmodulin, hypocholesterolemic, and multifunctional properties of the food protein-derived peptides. It is recommended that future research efforts on BAP should be directed toward elucidation of their in vivo molecular mechanisms of action, safety at various doses, and pharmacological activity in maintaining homeostasis during aberrant health conditions in human subjects.
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 definition, BAPs discussed in this review are food protein-derived peptides that possess beneficial 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 beneficial as a source of essential amino acids. This approach can provide the opportunity for diversification 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 benefits.
Production and Processing of Food Protein-Derived BAPs
Typical food sources
Numerous animal and plant food proteins have been exploited as sources of BAPs. Several studies on BAPs were conducted using animal proteins mostly milk proteins, casein and whey, egg and meat muscle proteins have also yielded BAPs. In addition, several BAPs have been produced from marine protein sources, including fish, salmon, oyster, macroalgae, squid, sea urchin, shrimp, snow crab, and seahorse (Table 1). Typical plant food proteins used for the production of BAPs include soy, pulses (lentil, chickpea, pea, and beans), oat, wheat, hemp seed, canola, and flaxseed. Based on the current literature, food proteins are selected as sources of BAPs based on 2 major criteria (1) a pursuit of value-added use of abundant underutilized proteins or protein-rich food industry by-products, and (2) utilization of proteins containing specific peptide sequences or amino acid residues of particular pharmacological interest. While these criteria are individually important, a combination of the 2 approaches can lead to the strategic selection of proteins that can produce high yields of defined potent peptide sequences. Recently, a QSAR-based in silico method was recently proposed for the prediction of food protein sources that can yield BAPs (Gu and others 2011). This approach could lead to the selection of excellent protein sources of BAPs only when details of the structure-function properties of active sequences are known. Moreover, detailed experimental work is needed to confirm actual production of the peptides, reproducibility, and substantiation of the feasibility of use of the in silico prediction method.
Table 1–. Sources and bioactive properties of marine protein-derived hydrolysates and peptides.
Peptide treatment enhanced mitogen-induced lymphocyte proliferation, natural killer (NK) cell activity, spleen CD4+ T helper cells, and secretion of cytokines (IL-2, IL-5, IL-6, IFN-γ); no effect observed on macrophage activity; peptides have potential for use as immune stimulant for disease prevention
Same as above but with irradiation-induced immune suppression in the ICR mice
Protected against gamma radiation induced immunosuppression by augmenting CD4+ T helper cells, enhancing spleen IL-12, reducing total NF-κB through I-κB induction, and inhibition of splenocyte apoptosis via increase in antiapoptotic Bcl-2 and decrease in proapoptotic Bax
Dose-dependent inhibition (up to 48%) of growth of sarcoma in mice; enhanced NK cells activity, spleen lymphocyte proliferation, and macrophage phagocytic rate; the observed immunostimulation could be responsible for the antitumor activity
Administered at 0, 5, or 10 mL/kg/d for 6 wk to D-galactose-induced aging ICR mice
SHP-1 down-regulated TPA-induced collagen release in cells via decresase in collagenases 1 and 3 expression; associated with blocking phosphorylation of NF-κB and p38 kinase cascade leading to decreased NO production, iNOS, and COX-2; beneficial effects for treatment of arthritis
Giant squid (Dosidicus gigas) gelatin
Seven proteases including Alcalase (Alc), Esperase (Esp), and Neutrase (Neu)
Crude protein hydrolysates containing a range of low- and high molecular size peptides
Alc- and Neu-prepared sample showed better ACE inhibition; Esp-prepared sample exhibited most potent cytotoxic effect against MCF-7 (human breast) and U87 (glioma) cancer cells (IC50 of 0.13 and 0.10 mg/mL, respectively); all samples also showed antioxidant properties (FRAP and metal chelation)
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 specific or nonspecific 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, enzyme–substrate 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 enzyme–protein 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 (Quirós 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 profiles 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 ultrafiltration and size-exclusion chromatography can be used to concentrate peptides of defined 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 structure-function 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 the molecular disease targets are inactivated by molecules with strong net positive or negative charges. In addition, a novel membrane technology known as electrodialysis-ultrafiltration (EDUF) can be used to separate cationic, anionic, and neutral peptides of defined molecular sizes (Firdaous and others 2009). This method has demonstrated high efficiency in selectively separating and concentrating low molecular size BAPs with net charges. Recently, the EDUF process was used to successfully fractionate net positively and negatively charged BAPs of low molecular sizes (300 to 700 Da) from snow crab by-product hydrolysates (Doyen and others 2011a, 2011b). Moreover, particular amino acids can be enriched in food peptide mixtures using adsorbent materials. For example, a peptide fraction rich in branched chain amino acids and low in aromatic amino acids can be obtained after protein hydrolysis by passing of the hydrolysates through a column packed with activated carbon or simply by mixing with activated carbon (Adachi and others 1993; Udenigwe and Aluko 2010). These fractionation processes often result in appreciable peptide yield depending on prevalence of the amino acid residues or peptides of interest within the hydrolysate product. Furthermore, extensive bioassay-guided purification steps can be carried out in order to produce pure peptides for further analysis especially for structure-function studies. The limitation of the latter process is the low yield of the resulting peptides, which may have to be synthesized for further studies. The low peptide yield decreases the feasibility of using food proteins as sources of BAPs. Therefore, for commercial production of functional food products containing BAPs, it will be worthwhile to develop applicable food-grade processing methods that will yield high amounts of highly active peptide mixtures. This approach requires an understanding of the structural requirements of the peptides for bioactivity, and exploiting the unique structural features in concentrating the particular peptides of interest during processing. In summary, the processes commonly used for the production and processing of BAPs are shown in Figure 1.
Food Protein-Derived BAPs and Human Health
As shown in Figure 2, food protein hydrolysates have exhibited potent biological activities such as antihypertensive, antioxidant, immunomodulatory, anticancer, antimicrobial, and lipid-lowering activities (Meisel 2004; Wang and Gonzalez De Mejia 2005; Korhonen and Pihlanto 2006; Pihlanto 2006; Aluko 2008a, 2008b), which are largely due to their constituent peptides. The specific bioactivity of food peptides against various molecular disease targets depends primarily on their structural properties such as chain length and physicochemical characteristics of the amino acid residues, for example, hydrophobicity, molecular charge, and side-chain bulkiness (Pripp and others 2005). Generally, the activity of these peptides against molecular disease targets are regarded as lower than synthetic peptidomimetics and drugs, but the use of dietary BAPs in intervention against human diseases offers many advantages, including safety of the natural product, low health cost, and the additional nutritional benefits of the peptides as source of beneficial and essential amino acids. The current literature contains a vast amount of information on food protein-derived BAPs with physiologically relevant bioactive properties. These peptides range in sizes from di-, tri-, and oligopeptides to high molecular weight polypeptides (Erdmann and others 2008; Hernández-Ledesma and others 2009a). Based on the bioactivities, a number of peptide-based food products have been developed and commercialized for use as human health-promoting agents; see review articles by Korhonen and Pihlanto (2006), Hartmann and Meisel (2007), and Fitzgerald and others (2011) for comprehensive lists of these food products and their health claims.
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 first and rate-limiting step of the RAS pathway. AT-I is then cleaved at the histidyl residue from the C-terminus by the activity of angiotensin I-converting enzyme (ACE) to produce AT-II. AT-II is a powerful vasoconstrictor that functions by binding to 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 is excessive level of AT-II, which causes severe blood vessel contractions and limited relaxation to produce high BP. Moreover, the kinin-NO system is involved in the production of bradykinin, which exerts its antihypertensive effects by eliciting reactions that increase intracellular Ca2+ concentration leading to activation of nitric oxide synthases (NOS) that produce NO, a powerful vasodilator. ACE degrades bradykinin, and increased concentration of ACE leads to dual effects such as the prevention of vasodilation and the activation of vasoconstriction. Based on the roles of ACE in the RAS pathway, inhibitors of this enzyme have been used as antihypertensive agents (Ibrahim 2006). Moreover, direct inhibition of renin can potentially provide better control of elevated BP than ACE inhibition since it prevents the synthesis of AT-I, which can be converted to AT-II in some tissues via an ACE-independent alternative chymase-catalyzed pathway (Segall and others 2007).
ACE-inhibiting food protein-derived peptides RAS-modulating food-derived peptides are primarily targeted against ACE activity. The pioneering work on naturally occurring ACE-inhibiting antihypertensive peptides from snake (Bothrops jararaca) venom (Ferreira and others 1970; Ondetti and others 1971) sprouted several investigations on the use of food protein-derived peptides as antihypertensive agents. Till date, several ACE-inhibiting peptides have been reported from an enormous list of plant and animal food proteins most especially milk, fish, egg, and soy proteins (see review articles by FitzGerald and others 2004; Hartman and Meisel 2007; Aluko 2008a, 2008b; Erdmann and others 2008). The technological aspect of production of antihypertensive food-derived peptides has also been reviewed (Aluko 2007). The amino acid sequences of a number of these peptides have been identified and related to their biological activities. The peptide inhibitory concentration that reduced ACE activity by 50% (IC50) were reported to be as low as 2, 5, and 9 μM for Val-Ala-Pro (αs1-casein f25–27), β-casein-derived Ile-Pro-Pro (f74–76), and Val-Pro-Pro (f84–86), respectively (Nakamura and others 1995; FitzGerald and others 2004).
The mechanism of ACE inhibition by food protein-derived peptides has been studied and found to be via competitive inhibition (Sato and others 2002). This mode of enzyme inhibition is characterized by competition of the peptides with ACE substrate for the enzyme catalytic sites. Moreover, some peptides have also exhibited noncompetitive (for example, Leu-Trp and Ile-Tyr) and uncompetitive (for example, Ile-Trp and Phe-Tyr) modes of inhibition (Sato and others 2002) where the peptides bind other sites on the enzyme leading to changes in enzyme conformation and decreased activity. The above example shows that a single amino acid substitution, even with isomers, can greatly influence the nature of interactions between peptides and ACE. Thus, an understanding of the structural basis for potency has resulted in the discovery of more potent peptides. The molecular mechanism of ACE inhibition by peptides has been reviewed (Li and others 2004; Phelan and Kerins 2011) and hydrophobic amino acid residues of peptides are important structural feature for potency. QSAR studies by partial least squares projection of latent structure (PLS) indicated that C-terminal bulky hydrophobic amino acids (for example, Pro, Trp, Phe, and Tyr) and N-terminal aliphatic amino acids (for example, Leu, Ile, and Val) are necessary structural features of dipeptides and tripeptides for ACE inhibition (Wu and others 2006a) and that the last 4 C-terminal predominantly hydrophobic amino acid residues in 4 to 10 amino acid-containing peptides are important determinants for ACE inhibition (Wu and others 2006b). Moreover, amino acids with positive charge on the ɛ-amino group (for example, Arg and Lys) also contribute substantially to ACE inhibition if present at the C-terminal of peptides, possibly by interacting with anionic allosteric binding sites different from the active site of ACE (Vermeirssen and others 2004).
Renin-inhibiting food protein-derived peptides In addition to ACE inhibition, recent studies have demonstrated that food-derived peptides can also inhibit the activity of renin. This new approach to antihypertensive therapy by food-derived peptides can potentially provide better BP-lowering properties than inhibiting only ACE activity. The initial work reported that hydrolysis of flaxseed protein with different proteases followed by ultrafiltration yielded low molecular size (<1 kDa) fractions that exhibited moderate renin-inhibitory activities with IC50 of 1.22 to 2.81 mg/mL (Udenigwe and others 2009a). These peptide fractions inhibited renin activity in vitro through a mixed-type inhibition mode and also potently exhibited ACE inhibition at low concentrations. Thus, their dual roles as ACE and renin inhibitors can potentially enhance their antihypertensive effects. Similarly, other studies reported the presence of renin inhibitors in enzymatic hydrolysates of pea and hemp seed protein isolates. Girgih and others (2011) demonstrated that a simulated gastrointestinal digested hemp seed protein hydrolysate inhibited renin (IC50 of 0.81 mg/mL) and ACE (IC50 of 0.67 mg/mL) activities in vitro. The crude protein hydrolysate was found to be more potent than unhydrolyzed hemp seed protein and fractionated peptides of various molecular sizes as renin inhibitor. Moreover, Li and Aluko (2010) isolated 3 dipeptides (Ile-Arg, Lys-Phe, and Glu-Phe) from alcalase-prepared pea protein hydrolysates with the ability to moderately or weakly inhibit renin activity with IC50 of 9.2, 17.8, and 22.6 mM, respectively. The fact that these dipeptides exhibited lower activity than the crude peptide fraction suggests a possible synergistic activity of the peptides. Table 2 shows the sequence, renin- and ACE-inhibitory activities of the pea dipeptides. Based on these data, the structural requirements of dipeptides for renin inhibition was recently elucidated by PLS-based chemometrics and supported by experimental studies. Udenigwe and others (2011) recently reported that the presence of an N-terminal hydrophobic low molecular weight amino acid (for example, Ile, Leu, Ala, and Val) and a C-terminal bulky amino acid (for example, Trp, Phe, and Tyr) is required for potency against human renin. These features are similar to dipeptide structural requirements for ACE inhibition although there was no correlation between ACE- and renin-inhibitory activities of the dipeptides (Table 2). Based on these PLS models, previously reported antihypertensive dipeptide (Ile-Trp) was discovered as the most potent renin-inhibiting dipeptide and an effective ACE inhibitor (Table 2). These dual-potent activities in modulating RAS enzymes may have contributed to the pronounced BP-lowering activity of Ile-Trp compared to the other dipeptides (Sato and others 2002). These studies can lead to the discovery of safe natural highly potent antihypertensive agents and can also contribute to the elucidation of alternative mechanisms of action of previously reported food-derived BP-lowering peptides.
Table 2–. Predicted and observed renin-inhibitory activities (RI,%) of dipeptides and the corresponding ACE-inhibitory activities (ACEI); peptides with potency in inhibiting both RAS enzymes can be used as effective blood pressure-lowering agent during hypertension depending on bioavailability.
Predicted RI (%)a
Observed RI (%)a
n.d. = no data; n.a. = no activity.
apredicted and observed RI (%) for the dipeptides were analyzed at concentration of 3.2 mM, data were derived from Udenigwe and others (2011);
In vivo studies of food protein-derived antihypertensive peptides There has been some correlation between in vitro RAS enzyme inhibition and hypotensive activity of BAPs and vice versa. Recently, ACE- and renin-inhibitory hempseed protein hydrolysates induced a pronounced decrease in systolic BP (SBP) (−30 mmHg) in spontaneously hypertensive rats (SHR) after 8 h of oral gavage of 200 mg of sample/kg body weight (Girgih and others 2011). The popular milk-derived tripeptides, Ile-Pro-Pro and Val-Pro-Pro, are the active ingredients of hypotensive products Calpis AMEEL S and Evolus, and these tripeptides were reported to reduce SBP by −28.3 and −32.1 mmHg, respectively, in SHR (Nakamura and others 1995). In mildly hypertensive human subjects, Calpis reduced SBP and diastolic BP (DBP) by −14.1 and −6.9 mmHg, after consumption of 95 mL/d of the sour milk product for 8 wk (Hata and others 1996). Moreover, a randomized placebo controlled trial using 70 Caucasian subjects with stage 1 hypertension demonstrated that consumption of 7.5 mg of Ile-Pro-Pro per day for 4 wk reduced SBP and DBP by 3.8 and 2.3 mmHg, respectively (Boelsma and Kloek 2010). Therefore, the extent of activity of these peptides may be dependent on the nature of delivery system, dose, study duration, genetics, and stages of hypertension. In addition, a dairy whey protein-derived peptide product (BioZate) exhibited hypotensive activity in hypertensive human volunteers by decreasing SBP by −11 mmHg and DBP by −7 mmHg after 6 wk of consuming 20 g of the product per day (Pins and Keenan 2002). Contrary to these studies, a recent studies have demonstrated the lack of significant BP-lowering activity by putatively antihypertensive lactotripeptide-containing products in prehypertensive and hypertensive subjects following 24-h ambulatory BP monitoring (ABPM) (Van Mierlo and others 2009; Usinger and others 2010). These observations indicate that BP measurements such as ABPM can provide more precise data as opposed to single-point clinical/office measurements. Table 3 shows summaries of recent human clinical trials using lactotripeptides and the contradictory outcomes.
Table 3–. Human clinical studies with lactotripeptide (LTP)-based products on different stages of hypertension; contrasting results could be due to notable differences in study design, blood pressure measurement tool, study population, dose, and peptide delivery vehicle.
Dairy drink containing LTP (Ile-Pro-Pro and Val-Pro-Pro)
Multicentre crossover study with untreated hypertensive white subjects: study 1, 69 subjects received 200 g/d of dairy drink with lactotripeptides; study 2, 93 subjects received 100 g/d of same with 350 mg potassium for 4 wk; ABPM and OBPM
Contrary to previous reports, peptide products did not have any significant effect on mean 24-h ambulatory SBP and DBP compared to placebo in the 2 studies; office BP decreased but no difference was observed in treatments compared to placebo
LTP (Ile-Pro-Pro)-enriched milk protein hydrolysates (MPH)
Seventy prehypertensive and stage 1 hypertensive subjects consumed 15 mg of encapsulated MPH or placebo daily for 4 wk; OBPM
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 significant change in BP observed in prehypertensive subjects compared to placebo; MPH was well tolerated and safe to the subjects
Peptides from Lactobacillus helveticus fermented milk (FM) containing LTP (Ile-Pro-Pro and Val-Pro-Pro)
Ninety-four prehypertensive and borderline hypertensive subjects consumed 150-mL or 300-mL FM or placebo daily for 8 wk; ABPM and OBPM
FM showed no significant 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
Ninety-one previously treated and treatment-naive (newly diagnosed) stage 1 and stage 2 hypertensive subjects received a twice-daily 75-mg dose of peptide product for 6 wk; 24-h ABPM and OBPM
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
The inhibition of the physiological activities of ACE or renin by food protein-derived peptides ultimately lead to reduction in the amount of circulating AT-II, elevated level of bradykinin, decrease in ACE-induced contraction with concomitant decrease in elevated BP (Sipola and others 2001; Ruiz-Giménez and others 2010). For example, oral intake of a 100-mL drink containing 3 mg of Val-Tyr twice a day for 4 wk resulted in decreased plasma AT-II and aldosterone and increased AT-I with associated reduction in SBP by −9.3 mmHg in human subjects with mild hypertension (Kawasaki and others 2000). The observed pattern in the vasoactive peptides (AT) indicates in vivo ACE inhibition by Val-Tyr treatment. However, BP decrease was also observed in prehypertensive human subjects after administration of milk-derived Ile-Pro-Pro without significant change in plasma AT-I, AT-II, and renin activity (Boelsma and Kloek 2010). This suggests the possibility of existence of alternative routes for the activity of peptides. For example, a recent study showed that ACE-inhibiting trypsin-digested amaranth glutelins activated NO production through induction of endothelial NOS in cultured endothelial cells with concomitant induction of smooth muscle relaxation in isolated rat aortic segments (Barba de la Rosa and others 2010). The observed activity was attributed to the ability of the peptides to induce phosphorylation of endothelial NOS at Ser117 residue. Therefore, antihypertensive effects of food-derived peptides can be mediated through various other pathways other than modulation of RAS enzyme activity, though the observed effects in the study above could be as a result of increased bradykinin arising from ACE inhibition.
Meta-analyses A number of meta-analyses have been conducted on the hypotensive effects of food protein hydrolysates and peptides. Pripp (2008) reported a −5.3 and −2.4 mmHg decrease in SBP and DBP, respectively, in a meta-analysis of 17 clinical trials using peptides and protein hydrolysates from different sources (milk and fish). The heterogeneity of the samples constitutes a limitation for this study since it would be difficult to compare these data with studies that used homogeneous peptide samples. In another meta-analysis of 12 randomized controlled trials, Xu and others (2008) observed a similar effects on BP (−4.8 mmHg SBP, −2.2 mmHg DBP) with lactotripeptides (Val-Pro-Pro and Ile-Pro-Pro) in 623 prehypertensive and hypertensive subjects; more pronounced effect was observed with only hypertensive subjects. The homogeneity of the population studied may seem to have contributed to the uniformity of the results. A recent meta-analysis using 18 trials showed that the lactotripeptides decreased both SBP and DBP by slightly lower magnitude (−3.73 and −1.97 mmHg, respectively) and the effects were dependent on ethnic factors (Cicero and others 2011). Thus, the authors observed that the tripeptides-induced lowering of elevated BP was more pronounced in Asian subjects compared to Caucasian subjects, and was independent of age of subjects, length of study, dose of the lactotripeptides, and baseline BP. With the moderate peptide-induced decreases in BP, the consumption of hypotensive food-derived peptides can be combined with lifestyle changes in order to achieve substantial BP-lowering effects in severe hypertension.
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 of antioxidant peptides include pea, soy, fish, quinoa, flaxseed, milk casein, whey, and egg (Aluko and Monu 2003; Pihlanto 2006; Humiski and Aluko 2007; Erdmann and others 2008; Udenigwe and others 2009b). The antioxidant properties of these peptides include scavenging or quenching of reactive oxygen species (ROS)/free radicals and inhibition of ROS-induced oxidation of biological macromolecules such as lipids, proteins, and DNA. The radical-quenching activities of food antioxidants are due to the ability of the antioxidants to participate in single electron transfer reaction (Huang and others 2005); thus, the abundance of peptidic amino acid residues that can transfer electrons to the free radicals at physiological pH can contribute to enhanced antioxidative property. Other mechanisms of antioxidant activity of peptides include transition metal chelating activity and ferric reducing power.
Some factors that may affect the antioxidant activity of food protein hydrolysates include specificity of proteases used for hydrolysis, degree of hydrolysis, and the structural properties of the resulting peptides, including molecular size, hydrophobicity, and amino acid composition (Pihlanto 2006). The amount of histidine, cysteine, proline, methionine, and aromatic amino acids have been reported to contribute to the antioxidant activity of food peptides. Structure-function studies using a number of synthetic peptides revealed that histidine residue of peptides can chelate metal ion, quench active oxygen, and scavenge .OH (Chen and others 1996; Chen and others 1998) and these properties were attributed to its imidazole group, which can participate in hydrogen atom transfer and single electron transfer reactions (Chan and Decker 1994). Similar potent antioxidant activity has also been reported for a histidine-containing dipeptide, carnosine (β-Ala-His), derived from muscle cells (Chan and others 1994). Moreover, the addition of hydrophobic amino acids, proline and leucine, to the N-terminus of a dipeptide His-His resulted in enhanced antioxidative property of the peptides, and these new peptides also displayed synergistic effects when combined with nonpeptide antioxidants (Chen and others 1996). Hydrophobic amino acids are important for enhancement of the antioxidant properties of peptides since they can increase the accessibility of the antioxidant peptides to hydrophobic cellular targets such as the polyunsaturated chain of fatty acids of biological membranes (Chen and others 1998). Moreover, the electron-dense aromatic rings of phenylalanine, tyrosine, and tryptophan residues of peptides can contribute to the chelating of pro-oxidant metal ions whereas phenylalanine can also scavenge ˙OH radicals to form more stable para-, meta-, or ortho-substituted hydroxylated derivatives (Sun and others 1993). Therefore, the specific contribution of individual amino acid residues to the antioxidant activity of a peptide depends largely on the nature of the ROS/free radical and the reaction medium. However, it is not clear how these “antioxidant” amino acid residues contribute to the antioxidant activity of a peptide mixture typical of food protein hydrolysates, or the possible positive or negative contributions of other amino acid residues present in the hydrolysates. It is important to delineate these possible amino acid contributions in order to strategically process the hydrolysates to yield peptide mixtures containing amino acid residues of interest.
In addition to the direct antioxidant activity due to the sulfhydryl functional group, cysteine residues of peptides can also serve as precursor for the synthesis of glutathione (γ-L-glutamyl-L-cysteinylglycine), a ubiquitous cellular antioxidant tripeptide, thereby contributing toward regeneration of the physiological antioxidant defense system (Meisel 2005). Moreover, food-derived peptides can also display antioxidant property by induction of gene expression of proteins that protect cellular components from oxidative stress-induced deterioration. In endothelial cells, a dipeptide Met-Tyr derived from sardine muscle protein stimulated the expression of heme oxygenase-1 and ferritin leading to a sustained cellular protection from oxidative stress (Erdmann and others 2006). In addition, a recent study observed that casein hydrolysates generated with different proteases exhibited varying antioxidant activities, independent of degree of hydrolysis, in human Jurkat T cells by increasing cellular catalase activity and amount of reduced glutathione but without any effect on superoxide dismutase (SOD) activity (Phelan and others 2009a; Lahart and other 2011). Although the casein hydrolysates showed dose-dependent decrease in viability and growth of the human Jurkat T cells, lower doses retained the beneficial antioxidant properties without any effect on membrane integrity. In D-galactose-induced aging ICR mice, oral intake of jellyfish collagen hydrolysates prepared with Protamex induced increase in SOD and glutathione peroxidase with concomitant decrease in serum and hepatic malondialdehyde, an oxidative stress marker (Ding and others 2011). Although these effects are promising, it will be worthwhile for researchers to focus more on the effects of these peptides in human subjects in order to evaluate mechanisms of action and possible application of these peptides in formulating health-promoting food products.
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, CaM-binding natural compounds can be used for the prevention and amelioration of diseases induced or exacerbated by increased activity of CaM-dependent enzyme (Martínez-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 (O’Neil and DeGrado 1990). These structural features are thought to be more important than the specific amino acid sequence in determining affinity of peptides for CaM (Kizawa and others 1995). The affinity 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, specifically αs2-casein (f164–179, f183–206, f183–207, and f90–109), which inhibited CaMPDE activation with IC50 values of 38, 6.9, 1.1 and 1.0 μM, 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 μM and a microbial metabolite (KS-505a) with IC50 of 0.065 μM (Martínez-Luis and others 2007) although αs2-casein f183–207 and f90–109 show potential for further consideration. Based on the work with αs2-casein peptides, our laboratory has explored other cationic amino acid-rich food protein sources for the production of CaM-binding peptides. Li and Aluko (2005) reported that pea protein-derived cationic peptide fraction inhibited CaM-dependent protein kinase II activity via the competitive mode of inhibition. In other similar studies, 2 cationic peptide mixtures fractionated from Alcalase-prepared flaxseed protein hydrolysates bound CaM with concomitant inhibition of the activities of endothelial and neuronal NOS (Omoni and Aluko 2006a, 2006b) via the mixed-type and noncompetitive modes of inhibition, respectively. The authors reported that these activities were due to decreased α-helix/unfolding of CaM and increased rigidity of the Ca2+/CaM complex due to binding of the cationic peptides. Moreover, a recent study have also shown that cationic peptide fractions from egg white lysozyme can simultaneously inhibit CaMPDE and also act as antioxidants (You and others 2010), which makes these peptides good candidates for use against multiple disease conditions. These cationic peptides can be easily purified from inactive enzymatic food protein hydrolysates using ion-exchange columns or electrodialysis, due to their unique physicochemical characteristics (Kizawa and others 1995). Although these peptides have shown unique interaction with CaM leading to potent inhibition of CaM-dependent enzymes, there is currently a dearth of information on absorption, bioavailability, and pharmacological activity of food protein-derived CaM-binding peptides in ameliorating specific human health and disease conditions.
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 fish 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 (β-conglycinin). The α+α′ 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 identified from the α′ subunit and sequenced (Lovati and others 2000). This 24-amino acid peptide that corresponds to position 127 to 150 of the α′ 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 identified 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 purified isoflavone-free soy 7S β-conglycinin using bacterial proteases. The resulting 7S-peptides showed hypotriglyceridemic properties by altering gene expressions related to triacylglycerol synthesis and also decreased Apo B-100 accumulation in Hep G2 cells partly due to increase in LDL receptor mRNA expression (Mochizuki and others 2009). Apo B-100 is a functional component of very low-density lipoprotein (VLDL) and its degradation reduces VLDL synthesis. These observations supported a previous study that showed that soy β-conglycinin possesses beneficial effects on plasma triacylglycerol in humans (Kohno and others 2006).
In addition to alterations of gene expressions, soy protein hydrolysates and constituent peptides also exhibited hypocholesterolemic activity by binding bile acids and neutral sterols in the intestine leading to increased fecal removal (Cho and others 2007; Yang and others 2007). The ability of the soy protein hydrolysates to bind bile acids may depend in part on their insoluble high molecular weight peptide fraction rich in hydrophobic amino acids (Higaki and others 2006), as earlier observed for high molecular weight fraction of the tryptic digest of buckwheat protein (Kayashita and others 1997). This shows that even though large BAPs may not be able to cross the intestinal epithelium into blood circulation to exert their beneficial lipid-lowering effects in the hepatocytes and other cellular locations, they might be useful in cholesterol homeostasis by enhancing fecal removal of bile acids and exogenous cholesterol from the intestine depending on their hydrophobic properties. Two soybeans-derived peptide products based on lunasin (Lunasin XP® and LunaSoy™) have been commercialized as cholesterol-lowering food ingredients (SoyLabs 2011). Lunasin is a 43 amino acid-containing polypeptide (molecular weight 5.4 kDa) found in soybeans, barley, rye, and wheat (Wang and others 2008; Hernández-Ledesma and others 2009a). Lunasin exerts its hypocholesterolemic activity by blocking acetylation of histone H3 Lys14 residue thereby reducing the production of HMG-CoA reductase with concomitant decrease in cholesterol biosynthesis; lunasin also increases cellular production of LDL receptors leading to removal of plasma LDL cholesterol (SoyLabs 2011). Structure-function studies are needed to understand the structural requirements for the lipid-lowering properties of peptides. Further larger human studies are required to confirm the beneficial effects of these products in different hypercholesterolemic populations, and to evaluate the overall contribution toward management of cardiovascular disease.
Peptides with anticancer properties have also been reported from foods. A number of studies on anticancer peptides have been focused on lunasin (Wang and others 2008; Hernández-Ledesma and others 2009a). The anticancer property of lunasin is predominantly against chemical and viral oncogene-induced cancers, and based on the modulation of histone (H) acetylation and deacetylation pathways specifically by inhibiting histone acetyl transferase (HAT). This leads to inhibition of acetylation of H3 and H4, repression of cell cycle progression (arrest at G1/S phase), and apoptosis in cancer cells (Hernández-Ledesma and others 2009a). Although lunasin showed excellent potential as anticancer agent in cell cultures, its large molecular size raises questions as to its absorption and use as an orally bioavailable health-promoting agent. Dia and others (2009a) reported that 4.5% of lunasin was absorbed in human subjects that consumed lunasin-containing soy protein. They also observed other lunasin-derived peptide sequences in the plasma, which could be attributed to degradation by gastrointestinal proteases and plasma peptidases. Another study reported efficient absorption of lunasin from rye consumption into the liver, kidney, and blood, and the tissue-derived extracts retained the anticancer HAT-inhibitory property of the parent molecule (Jeong and others 2009). It has been suggested that the activity of protease inhibitors present in lunasin-containing whole food contributed to the resistance of lunasin against gastrointestinal digestion as opposed to its synthetic form (Hernández-Ledesma and others 2009a). Additional studies are needed to understand the mechanism of lunasin activity, absorption kinetics into the blood circulation and cancer cell targets, and application as effective food-derived anticancer nutraceutical.
In addition to lunasin, other soy protein-derived peptides have also shown promising activities for anticancer therapy. Wang and others (2008) reported that enzymatic hydrolysates from different soy varieties inhibited the viability of cultured leukemia cells (L1210) with IC50 values of 3.5 to 6.2 mg/mL, which were significantly lower than the activity of lunasin (IC50 of 0.078 mg/mL). Moreover, a lunasin-containing glutelin fraction of Amaranthus hypochondriacus, when digested with trypsin, induced programmed cell death (apoptosis) in cervical cancer (HeLa) cells by 30% and 38% at 1 and 5 μg/mL, respectively (Silva-Sánchez and others 2008). It was not reported whether the anticancer peptides were derived from lunasin primary sequence or from other protein precursors present within the fraction. A similar study also demonstrated that a soy protein-derived hydrophobic peptide fraction exhibited cytotoxicity with IC50 of 0.16 mg/mL against macrophage-like murine tumor cell line (P388D1) by arresting cell cycle progression at the G2/M phases (Kim and others 2000). Recently, Hsu and others (2011) isolated 2 large peptides (Leu-Pro-His-Val-Leu-Thr-Pro-Glu-Ala-Gly-Ala-Thr and Pro-Thr-Ala-Glu-Gly-Gly-Val-Tyr-Met-Val-Thr) from tuna dark muscle by-product hydrolyzed with papain and protease XXII. These peptides exhibited dose-dependent antiproliferative activities against cultured breast cancer (MCF-7) cells with IC50 of 8.1 and 8.8 μM, respectively. Thus, enzymatic hydrolysis of food proteins can release BAPs with anticancer properties. This was clearly demonstrated in a study where the antiproliferative activity of A. mantegazzianus protein hydrolysates was twice the activity of the parent protein (Barrio and Añon 2010). Recently, low molecular size peptides from Pacific oyster hydrolysates were reported to induce dose-dependent inhibition of growth of transplanted murine sarcoma in BALB/c mice possibly via increased immunostimulation (Wang and others 2010). Other marine food-derived anticancer peptides are presented in Table 1. Based on these studies, detailed animal studies and clinical human trials are highly needed to evaluate the physiological anticancer activities of these peptides.
Immunomodulatory and anti-inflammatory peptides
Immunomodulation involves suppression or stimulation of human immune functions. Immunomodulatory food peptides act by enhancing the functions of immune system including regulation of cytokine expression, antibody production, and ROS-induced immune functions (Hartmann and Meisel 2007; Yang and others 2009). For example, a tryptic digest of rice protein improved immune function by promoting phagocytosis and increasing superoxide anion production in human polymorphonuclear leukocytes (Takahashi and others 1994). In addition, egg-derived peptides also showed immunostimulating activities and were used to increase immune functions during cancer immunotherapy (Mine and Kovacs-Nolan 2006). Moreover, a recent work showed that oral administration of a pea protein hydrolysate to mice led to reduced NO production by activated macrophages as well as reduced secretion of the proinflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-6, by up to 35% and 80%, respectively (Ndiaye and others 2011). In human volunteers, consumption of 3 g/d of wheat protein hydrolysate for 6 d increased activity of natural killer cells (Horiguchi and others 2005). Furthermore, another study demonstrated that whey protein-derived peptides can activate cellular immune functions (Gauthier and others 2006).
The anti-inflammatory properties of food-derived peptides have been reported mostly in modulating endotoxin-induced production of proinflammatory responses in macrophages. For example, soy lunasin and lunasin-like peptides exhibited anti-inflammatory properties by decreasing ROS production, TNF-α, IL-6, IL-1β, nuclear factor-κB (NF-κB) levels, and down-regulation NO/PGE2 synthesis and inducible NOS/COX-2 expressions in activated macrophages (Dia and others 2009b; Gonzalez de Mejia and Dia 2009; Hernández-Ledesma and others 2009b). The activity of lunasin was due to the suppression of nuclear translocation of p65/p50 subunits of NF-κB in RAW264.7 macrophage, which reduces binding of NF-κB to target genes with concomitant inhibition of proinflammatory markers gene activation and the gene products, for example, IL-6, inducible NOS, COX-2 (Gonzalez de Mejia and Dia 2009). In addition, casein hydrolysates were reported to increase concanavalin A (ConA)-stimulated T helper (Th)-1 produced IL-2 level but not Th-2 produced IL-10 in human Jurkat T cells; this shows that the peptides acted via T cell-mediated immune response and not humoral or antibody-mediated immune response (Phelan and others 2009a). Furthermore, food-derived peptides have also protected against radiation-induced immunosuppression. A recent study reported that a peptide fraction from Chum salmon collagen hydrolysates protected against gamma radiation-induced immunosuppression in mice by augmenting CD4+ Th cells, enhancing spleen production of IL-12, inducing I-κB thereby reducing NF-κB, and inhibiting splenocyte apoptosis (Yang and others 2010). As with anticancer and CaM-binding peptides, future clinical intervention studies using these peptides are needed to evaluate their efficacy, pharmacokinetics, and possible use for the formulation of functional food products.
Multifunctional peptides have been discovered from some food proteins and have been reported to possess more than one significant physiologically relevant bioactive property. Studies on milk proteins demonstrated that a hexapeptide (TTMPLW) derived from αS1-casien (f194–199) by trypsin-catalyzed digestion exhibited both ACE-inhibitory and immunomodulatory activities (Meisel 2004) while a β-lactoglobulin-derived β-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 α-lactorphin (YGLF), α-immunocasokinin (TTMPLW), β-casomorphin-7 (YPFPGPI), and β-casokinin (AVPYPQR) are regarded as multifunctional, some possessing in vivo bioactive properties (Meisel 2004). Moreover, crude chymotryptic α-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 2008). Other partially purified food peptides from quinoa/pea proteins and hen egg white lysozyme have also displayed multifunctionality as ACE-inhibiting antioxidants (Aluko and Monu 2003; Humiski and Aluko 2007) and CaMPDE-inhibiting antioxidants (You and others 2010), respectively. The multifunctional bioactive properties of protein hydrolysates and peptides derived from marine foods are shown in Table 1. The multiple bioactivities displayed by these peptides can increase their impact toward the amelioration of more than one disease target or multiple symptoms of a disease, such as cardiovascular disease, since many human diseases are interrelated in terms of etiology and progression. Therefore, the conditions for generating and processing bioactive food protein hydrolyastes can be carefully designed to yield multifunctional peptides with diverse applications in maintaining optimum human health.
Delivery and Bioavailability of BAPs
BAPs can be administered using different vehicles such as beverages and bakery products (reviewed by Fitzgerald and others 2011; Hernández-Ledesma and others 2011). In vitro bioactivity of peptides does not generally translate into in vivo pharmacological effects due to concerns about absorption, bioavailability, and susceptibility of the peptides to degradation into inactive fragments by physiological enzymes (Vermeirssen and others 2004; Hernández-Ledesma and others 2011). For example, peptides derived from milk proteins MAP1 and MAP2 showed in vitro ACE-inhibitory activity (MPH2 was more potent than MPH1) but only MAP1 reduced BP of hypertensive subjects (Boelsma and Klooek 2010). Thus, in order to use food-derived peptides as enterally potent health-promoting agents, they must show stability against gastrointestinal proteases and be absorbed through the enterocytes to the serum without degradation by brush border and serum peptidases (FitzGerald and others 2004). This ensures that the original peptide sequences that displayed in vitro bioactivity are conserved and delivered to the cellular sites of action. Microencapsulation has been explored for delivery of BAPs to enhance their stability and absorption (Rocha and others 2009; Hwang and others 2010). Moreover, bioavailability of BAPs depends on physicochemical properties of the peptides such as charge, molecular size, lipophilicity, and solubility; smaller peptides are transported across the enterocytes through intestinal-expressed peptide transporters whereas oligopeptides may be absorbed by passive transport through hydrophobic regions of membrane epithelia or tight junctions (Darewicz and others 2011). In this regard, it would be desirable to investigate the bioactivity of small peptides (dipeptides, tri-peptides, and small oligopeptides); peptides of small sizes have demonstrated in vivo bioactivity, resistance to peptidolysis and can be absorbed intact into blood circulation (Matsui and others 2002; Foltz and others 2007). Lactotripeptides (Ile-Pro-Pro, Val-Pro-Pro) were detected in nanomolar amounts when given with yogurt as delivery medium in fasted and fed states in normotensive subjects (Foltz and others 2007). Furthermore, peptides that act in the gastrointestinal tract (for example, cholesterol-binding and anoretic peptides) do not have to be absorbed to exert their biological properties (Wang and Gonzalez De Mejia 2005).
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 production of peptides. Numerous studies have shown lack of toxicity of these peptides in cell cultures. The safety aspects of milk-derived peptides have been reviewed (Phelan and others 2009b). Moreover, a recent study reported that both single dose (2000 mg/kg) and repeated daily dose (1000 mg/kg for 4 wk) of casein hydrolysates containing antihypertensive peptides (αs1-casein f90–94 and f143–149) resulted in no adverse effects on clinical (blood biochemical, hematology, organ weight ratios, histopathological) parameters or mortality in rats (Anadón and others 2010). The sample doses represent peptide amounts that are well above those needed to observe pharmacological activities. Furthermore, in prehypertensive and hypertensive human subjects, lactotripeptide (Ile-Pro-Pro)-rich milk protein hydrolysates were found to be well tolerated and without any significant adverse effects on serum chemistry and urine parameters when compared with subjects that received placebo treatment (Boelsma and Klooek 2010; Germino and others 2010). Therefore, food protein-derived BAPs are generally safe but care should be taken to avoid processing techniques that would negatively affect peptide quality and safety.
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.
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.