Rotimi E. Aluko

Food protein-derived bioactive peptides: production, processing, and potential health benefits.

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.

Amino Acid Composition and Antioxidant Properties of Pea Seed (Pisum sativum L.) Enzymatic Protein Hydrolysate Fractions

The amino acid composition and antioxidant activities of peptide fractions obtained from HPLC separation of a pea protein hydrolysate (PPH) were studied. Thermolysin hydrolysis of pea protein isolate and ultrafiltration (3 kDa molecular weight cutoff membrane) yielded a PPH that was separated into five fractions (F1-F5) on a C(18) reverse phase HPLC column. The fractions that eluted later from the column (F3-F5) contained higher contents hydrophobic and aromatic amino acids when compared to fractions that eluted early or the original PPH. Fractions F3-F5 also exhibited the strongest radical scavenging and metal chelating activities; however, hydrophobic character did not seem to contribute to reducing power of the peptides. In comparison to glutathione, the peptide fractions had significantly higher (p < 0.05) ability to inhibit linoleic acid oxidation and chelate metals. In contrast, glutathione had significantly higher (p < 0.05) free radical scavenging properties than the peptide fractions.

Potential of resveratrol in anticancer and anti-inflammatory therapy

Phytochemicals present in food have shown significant prospects in the treatment and management of a vast array of human diseases. Resveratrol is a stilbene-type aromatic phytoalexin predominantly found in grapes, peanuts, berries, turmeric, and other food products. Resveratrol has been reported to exhibit several physiological activities including anticancer and anti-inflammatory activities in vitro and in experimental animal models, as well as in humans. Anticancer activity of this compound is mainly due to induction of apoptosis via several pathways, as well as alteration of gene expressions, all leading to a decrease in tumor initiation, promotion, and progression. Resveratrol exhibits anti-inflammatory activity through modulation of enzymes and pathways that produce mediators of inflammation and also induction of programmed cell death in activated immune cells. Resveratrol has been shown to produce no adverse effects, even when consumed at high concentrations. Hence, resveratrol possesses good potential to be used as an adjunctive or alternative therapy for cancer and inflammatory diseases.

Identification and inhibitory properties of multifunctional peptides from pea protein hydrolysate.

Pea protein isolate was hydrolyzed with alcalase, and the hydrolysate passed through a 1 kDa cutoff ultrafiltration membrane. The permeate was freeze-dried and fractionated on a cationic solid-phase extraction (SPE) column. All fractions were tested for their inhibitory activities against angiotensin-converting enzyme (ACE), renin, and calmodulin-dependent phosphodiesterase 1 (CaMPDE). With the exception of the first eluted fraction, inhibitory properties of the SPE fractions against CaMPDE (but not ACE and renin) were directly related to cationic character (residence time on the column). However, the fraction that eluted with 1% ammonium hydroxide (SPE 1%) had the highest peptide yield and was subsequently fractionated using two consecutive rounds of reversed-phase high-performance liquid chromatography to obtain three peaks with major peptides identified as IR, KF, and EF by ultra performance liquid chromatography-tandem mass spectrometry. The three dipeptides showed weak inhibitory properties toward CaMPDE but strong inhibitions (IC50 values <25 mM) of ACE and renin. In general, the peptides had higher potency against ACE than against renin. It is indicated from our results that these peptides may be used as potential ingredients to formulate multifunctional food products and nutraceuticals.

Effect of peptide size on antioxidant properties of African yam bean seed (Sphenostylis stenocarpa) protein hydrolysate fractions.

Enzymatic hydrolysate of African yam bean seed protein isolate was prepared by treatment with alcalase. The hydrolysate was further fractionated into peptide sizes of <1, 1–3, 3–5 and 5–10 kDa using membrane ultrafiltration. The protein hydrolysate (APH) and its membrane ultrafiltration fractions were assayed for in vitro antioxidant activities. The <1 kDa peptides exhibited significantly better (p < 0.05) ferric reducing power, diphenyl-1-picryhydradzyl (DPPH) and hydroxyl radical scavenging activities when compared to peptide fractions of higher molecular weights. The high activity of <1 kDa peptides in these antioxidant assay systems may be related to the high levels of total hydrophobic and aromatic amino acids. In comparison to glutathione (GSH), the APH and its membrane fractions had significantly higher (p < 0.05) ability to chelate metal ions. In contrast, GSH had significantly greater (p < 0.05) ferric reducing power and free radical scavenging activities than APH and its membrane fractions. The APH and its membrane fractions effectively inhibited lipid peroxidation, results that were concentration dependent. The activity of APH and its membrane fractions against linoleic acid oxidation was higher when compared to that of GSH but lower than that of butylated hydroxyl toluene (BHT). The results show potential use of APH and its membrane fractions as antioxidants in the management of oxidative stress-related metabolic disorders and in the prevention of lipid oxidation in food products.

Improved method for direct high-performance liquid chromatography assay of angiotensin-converting enzyme-catalyzed reactions.

A rapid and sensitive assay was developed for determination of the activity of angiotensin-converting enzyme (ACE) in the presence of inhibitory peptides present in soybean protein hydrolysates. The method utilizes reversed-phase high-performance liquid chromatography (HPLC) to separate and quantify hippuryl-histidyl-leucine (HHL) and hippuric acid (HA). HHL and HA were separated on a Symmetry C18 column by gradient elution that used mixtures of trifluoroacetic acid TFA)-acetonitrile and TFA-water as solvents. Analytical time and baseline separation of HA from HHL were improved over previous HPLC methods. In comparison to the standard spectrophotometric method, the new HPLC method obviates the need for ethyl acetate extraction of HA but requires direct injection of the ACE reaction mixture onto the HPLC column.

Chemometric Analysis of the Amino Acid Requirements of Antioxidant Food Protein Hydrolysates

The contributions of individual amino acid residues or groups of amino acids to antioxidant activities of some food protein hydrolysates were investigated using partial least squares (PLS) regression method. PLS models were computed with amino acid composition and 3-z scale descriptors in the X-matrix and antioxidant activities of the samples in the Y-matrix; models were validated by cross-validation and permutation tests. Based on coefficients of the resulting models, it was observed that sulfur-containing (SCAA), acidic and hydrophobic amino acids had strong positive effects on scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and H2O2 radicals in addition to ferric reducing antioxidant power. For superoxide radicals, only lysine and leucine showed strong positive contributions while SCAA had strong negative contributions to scavenging by the protein hydrolysates. In contrast, positively-charged amino acids strongly contributed negatively to ferric reducing antioxidant power and scavenging of DPPH and H2O2 radicals. Therefore, food protein hydrolysates containing appropriate amounts of amino acids with strong contribution properties could be potential candidates for use as potent antioxidant agents. We conclude that information presented in this work could support the development of low cost methods that will efficiently generate potent antioxidant peptide mixtures from food proteins without the need for costly peptide purification.

Polypeptide profile and functional properties of defatted meals and protein isolates of canola seeds

Laboratory-defatted meals from four types of canola seeds were analysed for protein profile by reducing and non-reducing forms of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In the absence of a reducing agent (2-mercaptoethanol), four major polypeptide bands (16, 18, 30 and 53 kDa) were prominent in similar ratios in all varieties. In the presence of mercaptoethanol, significant reductions in intensity of the major bands occurred, suggesting that the major polypeptides contained smaller units which were held together by disulphide bonds. Meals from Brassica napus seeds had higher protein solubilities than meals from Brassica rapa seeds. Meals with higher protein solubility values also had higher foaming capacity (FC) values. Generally, the acid-precipitated (pH 4.0) protein isolates (APPIs) had higher FC values than the calcium-precipitated isolates (CPPIs). On the other hand, the CPPIs formed emulsions with higher values of emulsifying activity index (EAI) when compared to the APPIs. The results indicate that variations in functional properties of protein isolates and meals between the four seed types were probably due to differences in protein conformation in aqueous solutions rather than differences in polypeptide composition. © 2001 Society of Chemical Industry

Antioxidant and angiotensin converting enzyme-inhibitory properties of a flaxseed protein-derived high Fischer ratio peptide mixture.

Hydrolysis of flaxseed proteins using thermolysin and pronase followed by mixing with activated carbon, centrifugation and filtration yielded a filtrate (peptide mixture) with a Fischer ratio (branched-chain amino acids/aromatic amino acids) of 23.65 and a phenylalanine + tyrosine content of 1.11%. Gel permeation chromatography showed that the flaxseed peptide sample contained mainly low molecular weight peptides (<4 kDa). The high Fischer ratio peptide sample exhibited antioxidant property by scavenging 2,2-diphenyl-1-picrylhydrazyl radical, superoxide radical, hydroxyl radical, and also by protecting linoleic acid from oxidation. In addition, the peptide mixture showed potential antihypertensive properties by inhibiting angiotensin I-converting enzyme in a mixed-type inhibition pattern. Protein hydrolysates with Fischer ratio higher than 20 and phenylalanine + tyrosine content lower than 2% have been used to treat patients with hepatic encephalopathy; thus, this multifunctional flaxseed peptide mixture could be used to formulate food products with multiple human health benefits during liver diseases, oxidative stress and hypertension.

Antihypertensive Peptides from Food Proteins

Bioactive peptides are encrypted within the primary structure of food proteins where they remain inactive until released by enzymatic hydrolysis. Once released from the parent protein, certain peptides have the ability to modulate the renin-angiotensin system (RAS) because they decrease activities of renin or angiotensin-converting enzyme (ACE), the two main enzymes that regulate mammalian blood pressure. These antihypertensive peptides can also enhance the endothelial nitric oxide synthase (eNOS) pathway to increase nitric oxide (NO) levels within vascular walls and promote vasodilation. The peptides can block the interactions between angiotensin II (vasoconstrictor) and angiotensin receptors, which can contribute to reduced blood pressure. This review focuses on the methods that are involved in antihypertensive peptide production from food sources, including fractionation protocols that are used to enrich bioactive peptide content and enhance peptide activity. It also discusses mechanisms that are believed to be involved in the antihypertensive activity of these peptides.