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Background: The effects of consuming hemp seed protein (HSP) as well as its hydrolysate-derived bioactive peptide (HSP+) on blood pressure (BP) has not, to our knowledge, been investigated in humans., Objectives: We aimed to investigate how consumption of HSP and its hydrolysate modulates 24-h systolic (SBP) and diastolic BP (DBP) and plasma biomarkers of BP compared with casein., Methods: In a double-blind, randomized, crossover design trial, 35 adults who had mild hypertension with SBP between 130 and 160 mmHg and DBP ≤110 mmHg were recruited. Participants were randomly assigned to varying sequences of 3 6-wk treatments, 50 g casein/d, 50 g HSP/d, or 45 g HSP plus 5 g HSP-derived bioactive peptides/d (HSP+), separated by a 2-wk washout period. Treatment effects were assessed with a linear mixed model with repeated measures., Results: Compared with casein, after HSP+ consumption, 24-h SBP and 24-h DBP decreased from 135.1 and 80.0 mmHg to 128.1 ± 1.6 (P < 0.0001) and 76.0 ± 1.4 mmHg (P < 0.0001), respectively, whereas these values were 133.5 ± 1.6 and 78.9 ± 1.4 mmHg after HSP consumption (P < 0.0001). There were no differences between the HSP and HSP+ consumption in plasma angiotensin-converting enzyme (ACE) activity, renin, or nitric oxide (NO) concentrations. However, these 2 treatments were able to lower both ACE and renin activities and raise NO concentration in plasma compared with casein., Conclusions: These results suggest that hemp protein consumption, as well as in combination with bioactive peptides, may have a role in the dietary management of hypertension. This trial was registered at clinicaltrials.gov as NCT03508895., Competing Interests: Conflict of interest The authors report no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pigeon pea protein was sequentially digested with pepsin followed by pancreatin and the hydrolysate separated into 18 fractions using reversed-phase high-performance liquid chromatography. Fractions were analyzed for in vitro antioxidant properties (radical scavenging, metal chelation, and ferric iron reducing ability) in addition to inhibition of renin and angiotensin-converting enzyme (ACE). The most active fractions were analyzed by mass spectroscopy followed by identification of 10 peptide sequences (7 pentapeptides and 3 hexapeptides). All the peptides showed a wide range of multifunctional activity by scavenging hydroxyl (31.9-66.8%) and superoxide (25.6-100.0%) radicals in addition to ACE inhibition (7.4-100%) with significant (p < .05) differences between the peptides. AGVTVS, TKDIG, TSRLG, GRIST, and SGEKI were the most active; however, AGVTVS had the highest hydrophobic residue and exhibited the strongest activity against ACE, renin as well as superoxide and hydroxyl radicals. PRACTICAL APPLICATIONS: There is an increasing attraction of researchers to food peptides especially from legume proteins. Enzymatic digestion as well as high performance liquid chromatography (HPLC) purification has become an important process used to separate peptides with significant biological activities and health-promoting effects. There is useful information regarding the bioactive and functional (in vitro antioxidant, antidiabetic, in vitro/in vivo antihypertensive) properties of hydrolyzed and ultra-filtered pigeon pea fractions but scant research output still exists for purified peptides from pigeon pea establishing their therapeutic potential. The present study aimed to separate peptide fractions from pigeon pea hydrolysate and identify available amino acid sequences from the parent protein. Therefore, peptide sequences generated from the most bioactive fractions showed prospects for the expanded industrial utilization of pigeon pea. Further promoting its application as functional ingredient or additive for alleviating angiotensin-converting enzyme-related diseases., (© 2022 Wiley Periodicals LLC.)

The nutritional-amino acid profile and mineral element of Moringa oleifera seed globulin (GLO) and its hydrolysates as well as the in vitro bioactive properties-antioxidant, alpha-amylase, and alpha-glucosidase inhibition of the GLO, hydrolysates, and membrane fractions were reported. The results showed that M. oleifera contained significant amounts of essential amino acids (EAA), which are more than the minimum required by the Food and Agricultural Organization for children, except for tryptophan, which was the limiting amino acid. However, hydrolysis mostly led to a reduction in the contents of the EAA. While the process of hydrolysis and the subsequent membrane fractionation produced peptides with improved activities in 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid radical scavenging ability and oxygen radical absorbance capacity, this process produced no activities in superoxide radical scavenging ability, α-amylase, and α-glucosidase inhibitory potentials of some of the hydrolysates and peptides fractions. In summary, M. oleifera seed peptide fraction (<3 kDa) from the alcalase-derived hydrolysate contains potent antioxidants but relatively low in vitro antidiabetic properties. PRACTICAL APPLICATIONS: Several studies have established the ability of proteins, including hydrolysate and peptide fractions to provide some bioactive properties such as antioxidant, antidiabetic, anti-inflammatory among others. However, because protein functionalities are influenced by several factors such as the source, type, processing method employed among others, research has continued to evaluate the bioactivities of proteins under different conditions. In this study, therefore, we reported the impact of processing methods (hydrolysis, enzyme type, and peptide size) on the nutritional, antioxidant, and in vitro antidiabetic properties of M. oleifera seed globulin, its hydrolysates, and membrane fractions. This information plays an important role in the further exploitation of M. oleifera seed proteins in the development of functional foods and nutraceuticals., (© 2022 Wiley Periodicals LLC.)

Inhibition of digestive enzymes such as α-amylase and pancreatic lipase (PL) is a promising therapeutic strategy for the treatment and management of chronic health conditions such as diabetes and obesity. Therefore, the aim of this work was to determine the enzyme inhibitory activity of polyphenol-rich aqueous extracts of Amaranthus viridis (AV), Solanum macrocarpon (SM) and Telfairia occidentalis (TO) leaves, which were harvested from plants produced using multiple urea fertilizer doses (0-80 kg N/ha). Fertilizer application was applied at two time points (at planting or 2 weeks after seedling emergence). Leaf extracts were obtained using aqueous extraction (1:20, leaves:water) for 4 h at 60°C followed by centrifugation and freeze-drying of the supernatant. Results showed that the extracts inhibited α-amylase, and pancreatic lipase dose-dependently with TO extracts having significantly ( p < 0.05) higher inhibitory activities for both enzymes. Fluorescence intensity and circular dichroism spectra in the presence and absence of leaf extracts indicate significant changes to the enzyme protein secondary and tertiary conformations. We conclude that the leaf extracts, especially from TO are potential agents for reducing calorie intake as a preventive or treatment tool against chronic diseases such as diabetes and obesity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Oluwagunwa, Alashi and Aluko.)

The demand for isolated seed proteins continues to increase but functionality in food systems can be greatly dependent on the extraction method. In this work, we report the physicochemical and functional properties of lentil seed proteins isolated using various protocols. Lentil flour was defatted followed by protein extraction using isoelectric pH precipitation (ISO) as well as NaOH (MEM&#95;NaOH) and NaCl (MEM&#95;NaCl) extractions coupled with membrane ultrafiltration. The MEM&#95;NaCl had significantly ( p < 0.05) higher protein content (90.28%) than the ISO (86.13%) and MEM&#95;NaOH (82.55%). At pH 3-5, the ISO was less soluble (2.26-11.84%) when compared to the MEM&#95;NaOH (25.74-27.22%) and MEM&#95;NaCl (27.78-40.98%). However, the ISO had higher yield and protein digestibility (48.45% and 89.82%) than MEM&#95;NaOH (35.05% and 77.87%) and MEM&#95;NaCl (13.35% and 77.61%), respectively. Near-UV circular dichroism spectra showed that the MEM&#95;NaOH had loose tertiary conformation at pH 3, 5, 7 and 9 while ISO and MEM&#95;NaCl had more compact structures at pH 7 and 9. The three protein isolates formed better emulsions (lower oil droplet sizes) at pH 7 and 9 when compared to pH 3 and 5. In contrast, foaming capacity was better at pH 5 than pH 3, 7, and 9.

Roasting of mustard seeds prior to oil extraction is a well-documented unit operation essential to produce canolol and other lipophilic sinapates. This study investigated the effectiveness of air frying as a seed roasting treatment operation for enhancing the recovery of lipophilic sinapates from various mustard samples and fractions/products. Air frying of seeds, powder, cake, bran, and flour from different mustard varieties was carried out at temperature-time combinations of 160, 170, and 180°C for 5, 10, 15, and 20 min, respectively. Oil was extracted using the Soxtec method. Lipophilic sinapates were extracted from the oil using equal volumes of hexane to methanol 70% (v/v) and quantified by high performance liquid chromatography-diode array detection (HPLC-DAD). The total phenolic content (TPC) and antioxidant activity of the oils were also evaluated. The results showed a time-temperature dependency for the recovery of major oil-soluble sinapates in all mustard samples and fractions. The optimum air frying condition 180°C for 15 min produced the maximum yield of canolol as well as other unidentified oil-soluble sinapates (retention time (RT)-7.7, RT-11.50, RT-14.95, and RT-16.24 min). The oil from lower grade yellow mustard seeds (LGYMS) roasted at 180°C for 20 mins specifically had the highest TPC (3402.22 ± 58.79 mg GAE/g oil), while LGYMS oils generally showed better antioxidant activities (2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric ion reducing antioxidant power (FRAP), and inhibition of linoleic acid oxidation) but were lower in metal ion chelating capacity. This information would be beneficial to the oil industry because air frying generated valuable canolol and other antioxidant lipophilic sinapates from mustard varieties and their fractions. PRACTICAL APPLICATION: A major limitation in the application of natural extracts in vegetable oils is the poor lipophilic nature of phenolic compounds. This study employed a new thermal treatment (air frying) in the recovery of canolol and other lipophilic antioxidants. Such treatments can enrich mustard-based ingredients with canolol and other lipophilic antioxidants for domestic and industrial applications., (© 2021 Institute of Food Technologists®.)

Solanum macrocarpon (eggplant) leaf protein isolate (ELI) was hydrolyzed using four different enzymes to produce hydrolysates from alcalase (AH), chymotrypsin (CH) pepsin (PH) and trypsin (TH). CH had an overall stronger antioxidant property and was separated using ultrafiltration membranes into <1, 1-3 and 3-5 kDa peptide fractions. Gel-permeation chromatography confirmed conversion of the ELI (average of 22 kDa) into protein hydrolysates that contained smaller peptides (<6 kDa). A total of 23 peptides consisting of tri and tetrapeptides were identified from the CH, which is a wider spectrum when compared to seven for AH and four each for TH and PH. CH exhibited stronger scavenging activities against DPPH and hydroxyl radicals. CH and TH exhibited the strongest inhibitions against angiotensin-converting enzyme. In contrast, AH was the strongest inhibitor of α-amylase while AH and PH had strong inhibitory activities against α-glucosidase when compared with other hydrolysates. Ultrafiltration fractionation produced peptides that were stronger ( p < 0.05) scavengers of DPPH, and hydroxyl radicals, in addition to better metal-chelating and enzyme inhibition agents. The study concluded that the eggplant protein hydrolysates and the UF fractions may find applications in tackling oxidative stress-related diseases and conditions involving excessive activities of the metabolic enzymes.

The aim of this work was to characterize the antioxidant properties of some of the peptides present in bromelain mung bean meal protein hydrolysate (MMPH). The MMPH was subjected to two rounds of bioassay-guided reversed-phase HPLC separation followed by peptide identification in the most potent fractions using tandem mass spectrometry. Twelve antioxidant peptides, namely, HC, CGN, LAN, CTN, LAF, CSGD, MMGW, QFAAD, ERF, EYW, FLQL, and QFAW were identified and assayed for antioxidant properties. CTN, HC, CGN, and CSGD were the most potent ( p < 0.05) DPPH radical scavengers with EC 50 values of 0.30, 0.29, 0.28, and 0.30 mg/mL, respectively, which are lower than the 0.03 mg/mL obtained for reduced glutathione (GSH). CTN, HC, CGN, and CSGD exhibited the most potent ( p < 0.05) scavenging activities against hydroxyl and superoxide radicals with EC 50 values that are similar to those of GSH. The cysteine-containing peptides also had stronger ferric reducing antioxidant power and metal chelation activity than peptides devoid of cysteine. In contrast, MMGW, ERF, and EYW had poor radical scavenging and metal chelation activities. We conclude that the availability of the sulfhydryl group may have enhanced antioxidant potency while the presence of bulky groups such phenylalanine and tryptophan had an opposite effect.

The aim of this study was to evaluate the nutritional value and antioxidant properties of aqueous extracts of some Bangladesh vegetables using fruits of ash gourd, bitter gourd, brinjal, okra, ridge gourd, snake gourd, and leaves of Indian spinach, kangkong, and stem amaranth. Proximate composition showed that the dried extracts were composed mainly of crude protein (14.6%-46.7%) and non-fibre carbohydrates (26.4%-53.5%). With the exception of stem amaranth, all the extracts had >40% DPPH radical scavenging ability at 0.5 mg/ml. In contrast metal chelation was lower, except in Indian spinach with ~46%. The ferric reducing antioxidant power (FRAP) was highest for the kangkong (10.9 mM Fe 3+ reduced), which is similar to the 9.9 mM for butylated hydroxytoluene (BHT). All the extracts suppressed linoleic acid oxidation better than BHT within the first 5 days of the incubation period. We conclude that the Indian spinach, kangkong, and okra could be considered as the most promising sources of antioxidant compounds. PRACTICAL APPLICATIONS: Vegetables are commonly consumed as part of a regular diet but the high water and fiber contents usually mean that large quantities are required to provide long-term health benefits. Therefore, in this work, aqueous extracts of nine Bangladesh vegetables were prepared to provide a more concentrated form of nutrients and bioactive compounds. The extracts had strong nutritional value based on the high contents of crude protein, potassium, iron, and non-fibre carbohydrates. The high content of polyphenolic compounds in the extracts can also provide health benefits, which was demonstrated through strong free radical scavenging, metal chelation, ferric iron reduction, and inhibition of linoleic acid oxidation. These vegetable extracts have the potential to be used as sources of bioactive compounds to prevent or treat non-communicable diseases that are associated with high oxidative stress., (© 2020 Wiley Periodicals LLC.)

Enzymatic hydrolysis can liberate bioactive peptides from protein materials, thus, pigeon pea was hydrolysed using thermoase. Crude hydrolysate (PPHT) was subjected to ultrafiltration using different molecular weight cutoffs to collect <1, 1-3, 3-5, 5-10, and >10 kDa peptide fractions. Fractions were analysed for in vitro antioxidative, antihypertensive, and antidiabetic properties. The peptide fractions had stronger DPPH • scavenging and renin inhibition when compared to PPHT. In contrast, ACE inhibition was stronger for the PPHT and <1 kDa peptide fraction while activity decreased as peptide size increased. The <1 kDa peptide also showed significantly stronger ferric reducing antioxidant power, OH • scavenging and inhibition of linoleic acid oxidation when compared to PPHT. α-amylase and α-glucosidase were inhibited by all the peptide fractions, though the 3-5 and >10 kDa had higher values. We conclude that the PPHT and peptide fractions could serve as potential ingredients to formulate antihypertensive and antidiabetic functional foods and nutraceuticals. PRACTICAL APPLICATIONS: Oxidative stress promotes the generation of free radicals, which have a significant impact in the pathogenesis of human chronic diseases such as cardiovascular impairment, cancer, and diabetes. Peptides generated from enzymatic hydrolysis of proteins have been identified to impart beneficial health effects. In this work, we showed that a thermoase digest of pigeon pea protein as well as the fractionated peptides had strong antioxidant properties in addition to exhibiting inhibitory activities against renin and angiotensin converting enzyme, the main therapeutic targets for antihypertensive agents. The peptide products also inhibited α-amylase and α-glucosidase activities, providing potential ingredients that can be used to formulate antidiabetic functional foods., (© 2020 Wiley Periodicals LLC.)

Amaranth leaf protein isolate (ALI) was hydrolyzed using four different proteases (alcalase, trypsin, pepsin, and chymotrypsin) followed by fractionation of the pepsin hydrolyzate (PH) into different sizes using ultrafiltration membrane. Gel permeation chromatography showed that all the hydrolyzates had smaller size peptides (<7 kDa) than the protein isolate (>32 kDa). The chymotrypsin hydrolyzate had higher contents of hydrophobic amino acid (44.95%) compared to alcalase (42.72%), pepsin (43.93%), and trypsin (40.95%) hydrolyzates. The PH had stronger DPPH, hydroxyl radical, and superoxide radical scavenging activities than the other protein hydrolyzates but weaker Ferric reducing antioxidant power and metal chelating activities when compared to the peptide fractions. The <1 kDa peptide fraction exhibited stronger DPPH, hydroxyl, and superoxide radicals scavenging activities than the higher molecular weight (>1 kDa) fractions. Fractionation of PH also resulted in enhanced inhibition of α-amylase and ACE activities but weaker α-glucosidase inhibition. PRACTICAL APPLICATIONS: ALI was hydrolyzed using four proteases to produce protein hydrolyzates. The most active of the hydrolyzate was then fractionated to produce fractions of different molecular sizes. The results of the analyses showed that the hydrolyzates and the fractions showed good antioxidant and enzyme inhibitory activities such as the inhibition of ACE, α-amylase, and glucosidase enzymes. The results suggest that the enzymatic hydrolyzates and peptide fractions could be used as ingredients in the nutraceutical and functional food industries to scavenge free radicals and inhibit angiotensin-converting enzyme activity., (© 2020 Wiley Periodicals LLC.)

An iron-binding mung bean protein hydrolysate (MBPH) was prepared using a continuous enzymatic membrane reactor followed by peptide separation on anion-exchange (AEC) and reverse-phase HPLC (RP-HPLC) columns. Amino acid sequences of peptides present in the RP-HPLC fraction with the strongest iron-binding capacity were identified using mass spectrometry, and ten peptides of 5-8 amino acids synthesized for antioxidant characterization. Five fractions (AF1- AF5) with higher iron-binding capacity (88.86 ± 6.43 to 153.59 ± 2.18 mg/g peptide) when compared to the MBPH (36.81 ± 0.93 mg/g peptide) were obtained from AEC. PAIDL had the significantly ( p < 0.05) highest iron-binding capacity, but LLLLG and LLGIL showed the strongest metal chelating activity. However, PAIDL (46.63%) and LLGIL (81.27%) had significantly ( p < 0.05) better DPPH radical scavenging activity than the other peptides. PAIDL and LLGIL were also the most effective ( p < 0.05) hydroxyl radical neutralizers with an effective concentration that scavenged 50% (EC 50 ) values of 0.09 and 0.37 mM, respectively. PAIDL and AIVIL showed the lowest EC 50 values of 0.07 mM each for superoxide radical scavenging activity. We conclude that short chain length in combination with leucine as the C -terminal amino acid residue contributed to the strong antioxidant properties of peptides in this study.

Enzymatic hydrolysis of proteins produces bioactive peptides that have the potential to provide health benefits. This study examined the inflammatory- and immune-modulating properties of a flavourzyme-derived sunflower protein hydrolysate (SPH) and peptides. The SPH was fractionated into <1, 1-3, 3-5, and >5 kDa peptides by membrane ultrafiltration. The SPH blunted IL-1β stimulated NFκB activation and boosted IL-4/GM-CSF induced expression of surface markers CD14 and CD86, indicating maturation into a dendritic cell (DC) phenotype. Testing of SPH membrane ultrafiltration and HPLC fractions indicated that smaller and non-polar peptides were the most potent, respectively. Four novel peptides (YFVP, SGRDP, MVWGP and TGSYTEGWS) were identified and all of them blunted IL-1β stimulated NFκB activation. The peptides also boosted IL-4/GM-CSF induction of CD14, while only MVWGP and TGSYTEGWS boosted the expression of CD86. MVWGP was the most potent immune-modulatory peptide across all cellular assays, which was attributed to the presence of a methionine residue., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The aim of the study was to determine the in vitro enzyme inhibition activities of aqueous polyphenolic extracts of nine popular Bangladeshi vegetables, namely ash gourd, bitter gourd, brinjal, Indian spinach, kangkong, okra, ridge gourd, snake gourd, and stem amaranth. Polyphenolic glycosides were the major compounds present in the extracts. Inhibition of α-amylase (up to 100% at 1 mg/mL) was stronger than α-glucosidase inhibition (up to 70.78% at 10 mg/mL). The Indian spinach extract was the strongest inhibitor of pancreatic lipase activity (IC 50 = 276.77 µg/mL), which was significantly better than that of orlistat (381.16 µg/mL), a drug. Ash gourd (76.51%), brinjal (72.48%), and snake gourd (66.82%) extracts were the most effective inhibitors of angiotensin-converting enzyme (ACE), an enzyme whose excessive activities have been associated with hypertension. Brinjal also had a significantly higher renin-inhibitory activity than the other vegetable extracts. We conclude that the vegetable extracts may have the ability to reduce enzyme activities that have been associated with hyperglycemia, hyperlipidemia, and hypertension., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Being averse to bitter taste is a common phenomenon for humans and other animals, which requires the pharmaceutical and food industries to source compounds that can block bitterness intensity and increase consumer acceptability. In this work, beef protein alcalase hydrolysates (BPAH) and chymotrypsin hydrolysates (BPCH) were reacted with glucose to initiate Maillard reactions that led to the formation of glycated or advanced glycation end products (AGEs), BPAH-AGEs and BPCH-AGEs, respectively. The degree of glycation was higher for the BPAH-AGEs (47-55%) than the BPCH-AGEs (30-38%). Analysis by an electronic tongue instrument showed that BPAH-AGEs and BPCH-AGEs had bitterness scores that were significantly ( p < 0.05) less than quinine. The addition of BPAH-AGEs or BPCH-AGEs to quinine led to significant ( p < 0.05) reductions (up to 38%) in bitterness intensity of quinine. The use of 3% hydrolysate to react with glucose yielded glycated peptides with a stronger ability to reduce quinine bitterness than when 1% was used. Calcium release from HEK293T cells stably expressing the T2R4 human bitter taste receptor was significantly ( p < 0.05) attenuated by BPAH-AGEs (up to 96%) and BPCH-AGEs (up to 92%) when compared to the BPAH (62%) and BPCH (3%) or quinine (0%). We concluded that BPAH-AGEs and BPCH-AGEs may be used as bitter taste blockers to formulate better tasting foods.

The aim of this work was to determine the antioxidant properties of aqueous extracts of vegetable leaf-fortified bread as well as estimate the contents of polyphenolic compounds. Enriched bread was produced from wheat flour fortified at 1, 2, and 3% (w/w) with dried leafy vegetable powders from Amaranthus viridis, Solanum macrocarpon, and Telfairia occidentalis. Gallic acid was the most abundant soluble polyphenol in the control bread and the content in the control bread was significantly higher (p < 0.05) than in all the fortified bread samples. Fortification of bread especially at 3% level resulted in significantly (p < 0.05) higher concentrations of other polyphenols (myricetin, catechin, quercetin, and rutin) compared to the control bread. The fortified bread extracts had significantly (p < 0.05) more effective antioxidants than the control for DPPH radical scavenging activity, ferric iron reducing antioxidant power, metal chelation, and inhibition of linoleic acid peroxidation. PRACTICAL APPLICATIONS: Bread is one of the consumed foods and could be used as a suitable carrier of bioactive compounds. Leafy vegetables contain high levels of polyphenols that could provide beneficial effects by contributing to improved health status of consumers. Therefore, incorporation of leafy vegetables into leavened bread could provide a means of enhancing polyphenol consumption. In this work, we showed that soluble polyphenols were enriched in vegetable-fortified bread. The polyphenol-rich extracts of the fortified bread demonstrated better free radical scavenging and inhibition of unsaturated fatty acid oxidation activities than the regular bread. Therefore, regular consumption of vegetable leaf-fortified bread could lead to reduced oxidative stress and associated chronic diseases in human beings. The vegetable leaf fortification could also serve as a suitable means of enhancing the shelf life of wheat bread., (© 2018 Wiley Periodicals, Inc.)

The aim of this work was to determine bioavailability and in vivo calmodulin-dependent-phosphodiesterase (CaMPDE)-inhibitory activity of six flaxseed-protein-derived peptides (AGA, AKLMS, QIAK, RWIQ, QQAKQ, and KQLSTGC) after oral administration to Wistar rats. Initial experiments tested the cytotoxicity and cellular-transport potentials of the peptides using Caco-2 cells. The cytotoxicity assay indicated that none of the six peptides had an adverse effect on the proliferation and viability of the Caco-2 cells, whereas the transport assay confirmed peptide translocation across the cell membrane. However, only two of the peptides (AGA and RWIQ) were detected in the rat serum up to 90 min postgavage, with traces of RWIQ persisting in serum 1 week after oral gavage. The six peptides inhibited plasma activity of CaMPDE with AGA (34.63%), QIAK (36.66%), and KQLSTGC (34.21%) being the most effective 30 min after gavage. In contrast, only AGA maintained significant plasma-CaMPDE-activity inhibition (44.35%) after 60 min.

Mung bean seed is a well-known plant protein consumed in Asian countries but the protein is usually retrieved as a waste product during starch production. This study investigated the anti-allergic property of mung bean protein hydrolysates (MBPH) produced by enzymatic hydrolysis using non-gastrointestinal (non-GI), GI and a combination of non-GI+GI enzymes. The hydrolysates were investigated for any anti-allergic property by detecting the amount of β-hexosaminidase released in RBL-2H3 cells, and complemented with the MTT assay to show cell viability. It was found that MBPH hydrolyzed by a combination of flavourzyme (non-GI enzyme) and pancreatin (GI enzyme) exhibited the highest anti-allergic activity (135.61%), followed by those produced with alcalase, a non-GI enzyme (121.74%) and 80.32% for pancreatin (GI enzyme). Minimal toxicity (<30%) of all hydrolysates on RBL-2H3 cells line was observed. The results suggest that MBPH can potentially serve as a hypoallergenic food ingredient or supplement. PRACTICAL APPLICATIONS: Mung bean (Vigna radiata L. (Wilczek)) is also known as "green gram" and it is an excellent source of protein. The major mung bean storage proteins are the globulin, albumin and legumin, which are also referred to as legume allergens. Our study showed that mung bean peptides obtained after enzymatic hydrolysis influenced β-hexosaminidase inhibition without any toxic effect on RBL-2H3 cells. This indicates that mung bean allergenicity can be reduced after enzymatic hydrolysis and the protein hydrolysates could be as a hypoallergic food, ingredient, supplement and/or protein substitute in the formulation of food products., (© 2018 Wiley Periodicals, Inc.)

Moringa oleifera seed globulin was hydrolyzed with trypsin and fractionated to produce <1, 1-3, and 3-5 kDa peptide sizes. These were evaluated for antioxidant properties: DPPH, hydroxyl radical scavenging assays, FRAP, and metal chelation tests; and in vitro antihypertensive properties: ACE and renin inhibition. Membrane fractionation led to improved antioxidative properties of 29.13% (<1 kDa), 180% (<1 kDa), and 30.58% (1-3 kDa) for DPPH, FRAP, and metal iron chelation, respectively. There was however 48.77% reduction (1-3 kDa) in hydroxyl radical scavenging activity. There was also improvement in ACE inhibitory potentials of the peptides with the 1-3 kDa peptide showing significantly highest ACE inhibition (72.48%)but very low (17.64%, 1-3 kDa) inhibition against the renin. It was concluded that hydrolysis of M oleifera seed globulin with trypsin produced peptides and peptide fractions with potential antioxidant and antihypertensive properties.

The aim of this work was to compare the antioxidant and angiotensin converting enzyme (ACE) inhibitory properties of Moringa oleifera seed protein isolate (ISO) and its enzymatic protein hydrolysates. ISO was subjected to enzymatic (alcalase, pepsin and trypsin) hydrolysis to obtain alcalase isolate, pepsin isolate and trypsin isolate hydrolysates (AIH, PIH, TIH). Amino acid composition was similar for the samples except that TIH had lower Sulphur-containing amino acids while PIH was lower in tryptophan. All the samples were tested for antioxidant properties through free radical scavenging abilities such as 2,2 diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical scavenging assays as well as ferric reducing antioxidant power (FRAP) and metal ion chelation assays. The maximum percentage inhibition obtained for the samples from the different assays are: DPPH, 36% (PIH); FRAP, 0.04% (PIH); hydroxyl radical scavenging activity, 42.98% (ISO); and inhibition of metal ion chelation, 29.46% (AIH). AIH (79.3%) had the highest ACE-inhibitory activity followed by TIH (75.1%) while PIH (43.0%) had the least. Generally, the hydrolysis process produced hydrolysates with improved antioxidant and ACE-inhibitory properties when compared to the isolate. We conclude that enzymatic hydrolysis with alcalase, pepsin and trypsin may be used to produce M. oleifera seed protein hydrolysates with potential to be used as ingredients for the formulation of functional foods and nutraceuticals.

Legumes are rich sources of protein in human diet and their consumption has been associated with the prevention of chronic diseases attributable to their bioactive components. Pigeon pea ( Cajanus cajan ) is an underutilized legume with relatively high protein content (~24%). Protein hydrolysates were prepared from pea isolate by enzymatic hydrolysis using pepsin and pancreatin. Hydrolysates were evaluated for their amino acid composition, antioxidant properties, in vitro and in vivo antihypertensive properties. The hydrolysates had high hydrophobic amino acids, especially isoleucine, phenylalanine, and leucine. Pepsin-pancreatin-hydrolyzed pea protein (PPHPp) showed significantly higher ability to scavenge DPPH˙ while pancreatin-hydrolyzed pea protein (PPHPa) had higher ˙OH, ABTS˙ + scavenging, Fe 3+ reducing and linoleic acid peroxidation inhibition. PPHPp exhibited superior angiotensin-converting enzyme inhibition (61.82%) while PPHPa showed higher renin inhibition (14.28%). PPHPp exhibited strong antihypertensive effect, showing an instantaneous systolic blood pressure lowering effect (-26.12 mmHg) within 2-h post-oral administration. Pigeon pea protein hydrolysate (especially from pancreatin digest) could therefore, be a promising source of bioactive peptides and potential ingredient for formulation of functional foods against oxidative stress and hypertension.

The aim of this work was to determine the T2R4 bitter taste receptor-blocking ability of enzymatic beef protein hydrolysates and identified peptide sequences. Beef protein was hydrolyzed with each of six commercial enzymes (alcalase, chymotrypsin, trypsin, pepsin, flavourzyme, and thermoase). Electronic tongue measurements showed that the hydrolysates had significantly ( p < 0.05) lower bitter scores than quinine. Addition of the hydrolysates to quinine led to reduced bitterness intensity of quinine with trypsin and pepsin hydrolysates being the most effective. Addition of the hydrolysates to HEK293T cells that heterologously express one of the bitter taste receptors (T2R4) showed alcalase, thermoase, pepsin, and trypsin hydrolysates as the most effective in reducing calcium mobilization. Eight peptides that were identified from the alcalase and chymotrypsin hydrolysates also suppressed quinine-dependent calcium release from T2R4 with AGDDAPRAVF and ETSARHL being the most effective. We conclude that short peptide lengths or the presence of multiple serine residues may not be desirable structural requirements for blocking quinine-dependent T2R4 activation.

Proteins from tilapia frame and skin can potentially be precursors of antihypertensive peptides according to the result of BIOPEP analyses. The aim was to generate peptides with inhibitory effects against angiotensin-converting enzyme (ACE) and renin from tilapia frame and skin protein isolates (FPI and SPI). The most active hydrolysate was then tested for blood pressure-lowering ability in spontaneously hypertensive rats (SHRs). Tilapia frame and skin protein hydrolysates (FPHs and SPHs) were respectively produced from FPI and SPI hydrolysis using pepsin, papain, or bromelain. The ACE-inhibitory activities of tilapia protein hydrolysates with varying degree of hydrolysis (DH) were evaluated. In order to enhance the activity, the hydrolysate was fractionated into four fractions (<1 kDa, 1-3 kDa, 3-5 kDa, and 5-10 kDa) and the one with the greatest ability to inhibit in vitro ACE and renin activities was subjected to oral administration (100 mg/kg body weight) to SHRs. Systolic and diastolic blood pressure (SBP and DBP), mean arterial pressure (MAP), and heart rates (HR) were subsequently measured within 24 h. The pepsin-hydrolyzed FPH (FPHPe) with the highest DH (23%) possessed the strongest ACE-inhibitory activity (IC 50 : 0.57 mg/mL). Its <1 kDa ultrafiltration fraction (FPHPe1) suppressed both ACE (IC 50 : 0.41 mg/mL) and renin activities more effectively than larger peptides. In addition, FPHPe1 significantly ( p  < 0.05) reduced SBP (maximum -33 mmHg), DBP (maximum -24 mmHg), MAP (maximum -28 mmHg), and HR (maximum -58 beats) in SHRs. FPHPe1 showed both in vitro and in vivo antihypertensive effects, which suggest tilapia processing coproducts may be valuable protein raw materials for producing antihypertensive peptides., Competing Interests: No potential conflict of interest was reported by the authors.

In this work, the inhibitory effects of potato patatin-derived peptides Trp-Gly (WG) and Pro-Arg-Tyr (PRY) on angiotensin I-converting enzyme (ACE) and renin activities were investigated using kinetics, intrinsic fluorescence and molecular docking. The results indicated that PRY was a more potent ACE- and renin-inhibitory peptide than WG. Enzyme inhibition kinetics showed that WG and PRY inhibited ACE activity through mixed-type and competitive modes, respectively. The inhibitory mechanism of WG and PRY towards renin was determined to be mixed-type. PRY exhibited stronger affinity towards ACE and renin molecules, when compared to WG as determined by intrinsic fluorescence intensity. Molecular docking data confirmed that the higher inhibitory potency of PRY might be attributed to formation of more hydrogen bonds with the enzyme's active site or non-active sites that distorted the configuration necessary for catalysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: An increased rate of high blood pressure has led to critical human hypertensive conditions in most nations. In the present study, bambara protein hydrolysates (BPHs) obtained using three different proteases (alcalase, trypsin and pepsin) and their peptide fractions (molecular weight: 10, 5, 3 and 1 kDa) were investigated for antihypertensive and antioxidant activities., Results: Alcalase hydrolysate contained the highest amount of low molecular weight (LMW) peptides compared to pepsin and trypsin hydrolysates. LMW peptides fractions (<1 kDa) exhibited the highest inhibitory activity against angiotensin-converting enzyme (ACE) for all the enzymes hydrolysates. For renin inhibition, alcalase hydrolysate showed the highest inhibition at 59% compared to other hydrolysates and their corresponding membrane fractions. The antioxidant power of bambara protein hydrolysates and peptide fractions was evaluated through the inhibition of linoleic acid peroxidation and ABTS scavenging activity. Among the hydrolysates, alcalase exhibited the highest inhibition of linoleic acid oxidation. Furthermore, all BPHs were able to scavenge ABTS •+ to a three-fold greater extent compared to the isolate., Conclusion: BPH and LMW peptide fractions could potentially serve as useful ingredients in the formulation of functional foods and nutraceuticals against high blood pressure and oxidative stress. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

In this study, the bambara protein isolate (BPI) was digested with three proteases (alcalase, trypsin and pepsin), to produce bambara protein hydrolysates (BPHs). These hydrolysates were passed through ultrafiltration membranes to obtain peptide fractions of different sizes (<1, 1-3, 3-5 and 5-10 kDa). The hydrolysates and their peptide fractions were investigated for antioxidant activities. The membrane fractions showed that peptides with sizes <3 kDa had significantly (p < 0.05) reduced surface hydrophobicity when compared with peptides >3 kDa. This is in agreement with the result obtained for the ferric reducing power, metal chelating and hydroxyl radical scavenging activities where higher molecular weight peptides exhibited better activity (p < 0.05) when compared to low molecular weight peptide fractions. However, for all the hydrolysates, the low molecular weight peptides were more effective diphenyl-1-picrylhydrazyl (DPPH) radical scavengers but not superoxide radicals when compared to the bigger peptides. In comparison with glutathione (GSH), BPHs and their membrane fractions had better (p < 0.05) reducing power and ability to chelate metal ions except for the pepsin hydrolysate and its membrane fractions that did not show any metal chelating activity. However, the 5-10 kDa pepsin hydrolysate peptide fractions had greater (88%) hydroxyl scavenging activity than GSH, alcalase and trypsin hydrolysates (82%). These findings show the potential use of BPHs and their peptide fraction as antioxidants in reducing food spoilage or management of oxidative stress-related metabolic disorders.

This study assessed the ability of canola protein isolate (CPI) and enzymatic hydrolysates (CPHs) to inhibit adipogenic differentiation of C3H10T1/2 murine mesenchymal stem cells in vitro. Cell viability was maintained at concentrations of 60 μg/ml of sample. Cells treated with Alcalase hydrolysate demonstrated a higher reduction in anti-adipogenic differentiation through quantitation by oil-red O staining. qPCR analysis showed that CPI and CPH-treated cells significantly inhibited PPARγ expression, a key transcription factor involved in adipocyte differentiation, as evident in an ∼ 60-80% fold reduction of PPARγ mRNA. Immunofluorescence staining for PPARγ protein also showed a reduced expression in some treated cells when compared to differentiated untreated cells. The 50% inhibition concentration (IC50) of CPI, CPHs and their membrane ultrafiltration fractions on pancreatic lipase (PL) activity ranged between 0.75 and 2.5 mg/ml, (p < 0.05) for the hydrolysed and unhydrolysed samples. These findings demonstrate that CPI and CPHs contain bioactive components which can modulate in vitro adipocyte differentiation., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

This report shows the antioxidant effects of a hemp seed meal protein hydrolysate (HMH) in spontaneously hypertensive rats (SHR). Defatted hemp seed meal was hydrolyzed consecutively with pepsin and pancreatin to yield HMH, which was incorporated into rat feed as a source of antioxidant peptides. Young (8-week old) SHRs were divided into three groups (8 rats/group) and fed diets that contained 0.0%, 0.5% or 1.0% (w/w) HMH for eight weeks; half of the rats were sacrificed for blood collection. After a 4-week washout period, the remaining 20-week old SHRs were fed for an additional four weeks and sacrificed for blood collection. Plasma total antioxidant capacity (TAC) and superoxide dismutase (SOD), catalase (CAT) and total peroxides (TPx) levels were determined. Results showed that plasma TAC, CAT and SOD levels decreased in the older 20-week old SHRs when compared to the young SHRs. The presence of HMH in the diets led to significant (p < 0.05) increases in plasma SOD and CAT levels in both young and adult SHR groups; these increases were accompanied by decreases in TPx levels. The results suggest that HMH contained antioxidant peptides that reduced the rate of lipid peroxidation in SHRs with enhanced antioxidant enzyme levels and total antioxidant capacity.

Antioxidant activities of canola protein hydrolysates (CPHs) and peptide fractions prepared using five proteases and ultrafiltration membranes (1, 3, 5, and 10kDa) were investigated. CPHs had similar and adequate quantities of essential amino acids. The effective concentration that scavenged 50% (EC50) of the ABTS(+) was greatest for the <1kDa pancreatin fraction at 10.1μg/ml. CPHs and peptide fractions scavenged DPPH(+) with most of the EC50 values being <1.0mg/ml. Scavenging of superoxide radical was generally weak, except for the <1kDa pepsin peptide fraction that had a value of 51%. All CPHs inhibited linoleic acid oxidation with greater efficiency observed for pepsin hydrolysates. The oxygen radical absorbance capacity of Alcalase, chymotrypsin and pepsin hydrolysates was found to be better than that of glutathione (GSH) (p<0.05). These results show that CPHs have the potential to be used as bioactive ingredients in the formulation of functional foods against oxidative stress., (Copyright © 2013 Elsevier Ltd. All rights reserved.)