Chibuike C. Udenigwe

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.

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.

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.

Blood Pressure Lowering Effect of a Pea Protein Hydrolysate in Hypertensive Rats and Humans

The blood pressure lowering effect of a pea protein hydrolysate (PPH) that contained <3 kDa peptides, isolated by membrane ultrafiltration from the thermolysin digest of pea protein isolate (PPI), was examined using different rat models of hypertension as well as hypertensive human subjects. The PPH showed weak in vitro activities against renin and angiotensin converting enzyme (ACE) with inhibitory activities of 17 and 19%, respectively, at 1 mg/mL test concentration. Oral administration of the PPH to spontaneously hypertensive rats (SHR) at doses of 100 and 200 mg/kg body weight led to a lowering of hourly systolic blood pressure (SBP), with a maximum reduction of 19 mmHg at 4 h. In contrast, orally administered unhydrolyzed PPI had no blood pressure reducing effect in SHR, suggesting that thermolysin hydrolysis may have been responsible for releasing bioactive peptides from the native protein. Oral administration of the PPH to the Han:SPRD-cy rat (a model of chronic kidney disease) over an 8-week period led to 29 and 25 mmHg reductions in SBP and diastolic blood pressure, respectively. The PPH-fed rats had lower plasma levels of angiotensin II, the major vasopressor involved in development of hypertension, but there was no effect on plasma activity or renal mRNA levels of ACE. However, renal expression of renin mRNA levels was reduced by approximately 50% in the PPH-fed rats, suggesting that reduced renin may be responsible for the reduced levels of angiotensin II. In a 3-week randomized double blind placebo-controlled crossover human intervention trial (7 volunteers), significant (p<0.05) reductions (over placebo) in SBP of 5 and 6 mmHg were obtained in the second and third weeks, respectively, for the PPH group. Therefore, thermolysin derived bioactive peptides from PPH reduced blood pressure in hypertensive rats and human subjects, likely via effects on the renal angiotensin system.

Anti-diabetic and antihypertensive activities of two flaxseed protein hydrolysate fractions revealed following their simultaneous separation by electrodialysis with ultrafiltration membranes

Flaxseed protein hydrolysate has been fractionated by electrodialysis with two ultrafiltration membranes (20 and 50 kDa) stacked in the system for the recovery of two specific cationic peptide fractions (KCl-F1 and KCl-F2). After 360 min of treatment, peptide migration increased as a function of time in KCl compartments. Moreover, the use of two different ultrafiltration membrane allowed concentration of the 300-400 and 400-500 Da molecular weight range peptides in the KCl-F1 and KCl-F2 fractions, respectively, compared to the initial hydrolysate. After mass spectrometry analysis, higher amounts of low molecular weight peptides were recovered in the KCl-F2 compartment while relatively higher molecular weight peptides were more detected in the KCl-F1 compartment. Amino acid analysis showed that His, Lys and Arg were especially concentrated in the KCl compartments. Finally, glucose-transport assay demonstrated that the KCl-F2 fraction increased glucose uptake while oral administration of KCl-F1 and final FPH decreased systolic blood pressure.

Low molecular weight flaxseed protein-derived arginine-containing peptides reduced blood pressure of spontaneously hypertensive rats faster than amino acid form of arginine and native flaxseed protein

Flaxseed protein isolate (FPI) contains high amount of arginine, which plays important physiological roles especially as nitric oxide precursor in the vascular endothelium. Arginine-rich peptides can be generated from FPI and used as a source of nitric oxide, which can produce in vivo vasodilatory effects during hypertension. Enzymatic hydrolysis of FPI with trypsin and pronase resulted in a hydrolysate that was fractionated using electrodialysis-ultrafiltration (EDUF). EDUF experiment resulted in migration of peptides to the anionic and cationic recovery compartments. Compared to FPI with 11% arginine, about one-third of the cationic fraction was composed of arginine. Thirteen potential peptide sequences were identified to be present in the cationic compartment of which 12 contained at least one arginine residue. None of the peptides identified from the anionic compartment contained arginine. Oral administration of the cationic peptides (200mg/kgbodywt.) to spontaneously hypertensive rats resulted in a more rapid decrease in systolic blood pressure when compared to similar amounts of FPI or the amino acid form of arginine. It was concluded that the rapid effect of the arginine-rich peptide product suggests faster rate of peptide absorption than amino acids and this may be exploited to provide fast relief from hypertension.

Evaluation of the in vitro antioxidant properties of a cod (Gadus morhua) protein hydrolysate and peptide fractions

Mechanically-deboned cod muscle proteins were sequentially hydrolysed using pepsin and a trypsin+chymotrypsin combination, which was followed by passing the digest through a 1 kDa equipped tangential flow filtration system; the permeate (<1 kDa peptides) was collected as the cod protein hydrolysate (CPH). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to separate the CPH into four peptide fractions (CF1-CF4) and their in vitro antioxidant properties investigated. Results showed that most of the peptide fractions (CF2-CF4) displayed significantly higher (p<0.05) oxygen radical absorbance capacity values (698-942 μM Trolox equivalents, TE/g) and 2,2-diphenyl-1-picrylhydrazyl scavenging activities (17-32%) than those of CPH (613 μM TE/g and 19%, respectively). However, the unfractionated CPH displayed improved capability to scavenge superoxide and hydroxyl radicals as well as significantly higher (p<0.05) ferric iron reduction and chelation of iron than the RP-HPLC peptides. The CPH and peptide fractions displayed a dose-dependent inhibition of linoleic acid oxidation.

Encapsulation of food protein hydrolysates and peptides: a review

Food protein hydrolysates and peptides are considered a category of promising functional food ingredients. However, commercial application of protein hydrolysates and their constituent peptides can be impeded by their low bioavailability, bitter taste, hygroscopicity and likelihood of interacting with the food matrix. Encapsulation as a delivery mechanism can be used to overcome these challenges for improving the bioavailability and organoleptic properties of the peptides. Proteins, polysaccharides and lipids are the three carrier systems that have been utilized in food peptide encapsulation. The protein and polysaccharide systems mainly aim at masking the bitter taste and reducing the hygroscopicity of protein hydrolysates, whereas the lipid-based systems are intended for use in enhancing the bioavailability and biostability of encapsulated peptides. A spray drying technique is largely used to achieve microencapsulation in both protein and polysaccharide systems while, generally, liposomes are prepared by a film hydration technique. However, it is seen that the encapsulation efficiency (EE) of peptides using the liposome model is relatively lower since the entropy-driven liposome formation is uncontrolled and spontaneous. Achieving adequate EE through cost effective techniques is indispensable for encapsulation to be applicable to bioactive peptide-based product commercialization. Furthermore, the design of high quality functional foods requires detailed understanding of the release mechanism and kinetics, gastrointestinal stability, bioavailability and physiological bioactivity of the encapsulated peptide products.