Hemp seed protein and its hydrolysate vs. casein protein consumption in adults with hypertension: a double-blind crossover study.

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. OBJECTIVE: We aimed to investigate how consumption of HSP and its hydrolysate modulates 24-hr systolic and diastolic BP (24-hrSBP and 24-hrDBP) and plasma biomarkers of BP compared to casein. DESIGN: In a double-blind, randomized, cross-over design trial, 35 adults who had mild hypertension with SBP between 130 and 160 millimeters of mercury (mmHg), and DBP ≤110 mmHg were recruited. Participants were randomly assigned to varying sequences of three 6-week treatments, 50 g/d of casein, HSP, and HSP, 45 g/d plus HSP derived bioactive peptides (HSP+), 5 g/d separated by 2-week washout period. Treatment effects were assessed by linear mixed model with repeated measures. RESULTS: Compared with casein, after HSP+ consumption, 24hrSBP and 24hrDBP decreased from 135.1 and 80.0 mmHg to 128.1 ± 1.6 (P<.0001) and 76.0 ± 1.4 mmHg (P<.0001) respectively whereas these values were 133.5 ± 1.6 and 78.9 ± 1.4 after HSP consumption (P<.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 two 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.

Pigeon pea penta- and hexapeptides with antioxidant properties also inhibit renin and angiotensin-I-converting enzyme activities.

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.

Inhibition of the in vitro Activities of α-Amylase and Pancreatic Lipase by Aqueous Extracts of Amaranthus viridis, Solanum macrocarpon and Telfairia occidentalis Leaves.

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.

Comparative Study of the Structural and Functional Properties of Membrane-Isolated and Isoelectric pH Precipitated Green Lentil Seed Protein Isolates.

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_NaOH) and NaCl (MEM_NaCl) extractions coupled with membrane ultrafiltration. The MEM_NaCl had significantly (p < 0.05) higher protein content (90.28%) than the ISO (86.13%) and MEM_NaOH (82.55%). At pH 3-5, the ISO was less soluble (2.26-11.84%) when compared to the MEM_NaOH (25.74-27.22%) and MEM_NaCl (27.78-40.98%). However, the ISO had higher yield and protein digestibility (48.45% and 89.82%) than MEM_NaOH (35.05% and 77.87%) and MEM_NaCl (13.35% and 77.61%), respectively. Near-UV circular dichroism spectra showed that the MEM_NaOH had loose tertiary conformation at pH 3, 5, 7 and 9 while ISO and MEM_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.

Air frying pretreatment and the recovery of lipophilic sinapates from the oil fraction of mustard samples.

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.

Effect of Protease Type and Peptide Size on the In Vitro Antioxidant, Antihypertensive and Anti-Diabetic Activities of Eggplant Leaf Protein Hydrolysates.

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.

Functional Characterization of Mung Bean Meal Protein-Derived Antioxidant Peptides.

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 EC50 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 EC50 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.

Thermoase-hydrolysed pigeon pea protein and its membrane fractions possess in vitro bioactive properties (antioxidative, antihypertensive, and antidiabetic).

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.

Chemical composition and in vitro antioxidant properties of water-soluble extracts obtained from Bangladesh vegetables.

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 Fe3+ 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.

Antioxidant and enzymes inhibitory properties of Amaranth leaf protein hydrolyzates and ultrafiltration peptide fractions.

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.

Structure and Function of Mung Bean Protein-Derived Iron-Binding Antioxidant Peptides.

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% (EC50) values of 0.09 and 0.37 mM, respectively. PAIDL and AIVIL showed the lowest EC50 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.

Inhibitory Activities of Polyphenolic Extracts of Bangladeshi Vegetables against α-Amylase, α-Glucosidase, Pancreatic Lipase, Renin, and Angiotensin-Converting Enzyme.

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 (IC50 = 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.

Enzymatically derived sunflower protein hydrolysate and peptides inhibit NFκB and promote monocyte differentiation to a dendritic cell phenotype.

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-1ß 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-1ß 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.

Glycated Beef Protein Hydrolysates as Sources of Bitter Taste Modifiers.

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.

Anti-allergic activity of mung bean (Vigna radiata (L.) Wilczek) protein hydrolysates produced by enzymatic hydrolysis using non-gastrointestinal and gastrointestinal enzymes.

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 ß-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 ß-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.

Polyphenol composition and antioxidant properties of vegetable leaf-fortified bread.

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.

Transport, Bioavailability, Safety, and Calmodulin-Dependent-Phosphodiesterase-Inhibitory Properties of Flaxseed-Derived Bioactive Peptides.

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.

Antioxidant properties, ACE/renin inhibitory activities of pigeon pea hydrolysates and effects on systolic blood pressure of spontaneously hypertensive rats.

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, Fe3+ 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.

Beef Protein-Derived Peptides as Bitter Taste Receptor T2R4 Blockers.

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.

Antihypertensive properties of tilapia (Oreochromis spp.) frame and skin enzymatic protein hydrolysates.

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 (IC50: 0.57 mg/mL). Its <1 kDa ultrafiltration fraction (FPHPe1) suppressed both ACE (IC50: 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.