Antihypertensive effects of whey protein hydrolysate involve reshaping the gut microbiome in spontaneously hypertension rats

Novel angiotensin-converting enzyme(ACE)inhibitory peptides were identified from whey protein hydrolysates(WPH)in vitro in our previous study and the antihypertensive abilities of WPH in vivo were further investigated in the current study.Results indicated that WPH significantly inhibited the development of high blood pressure and tissue injuries caused by hypertension.WPH inhibited ACE activity(20.81%,P<0.01),and reduced renin concentration(P<0.05),thereby reducing systolic blood pressure(SBP)(12.63%,P<0.05)in spontaneously hypertensive rats.The increased Akkermansia,Bacteroides,and Lactobacillus abundance promoted high short chain fatty acid content in feces after WPH intervention.These changes jointly contributed to low blood pressure.The heart weight and cardiomyocyte injuries(hypertrophy and degeneration)were alleviated by WPH.The proteomic results revealed that 19 protein expressions in the heart mainly associated with the wingless/integrated(Wnt)signaling pathway and Apelin signaling pathway were altered after WPH supplementation.Notably,WPH alleviated serum oxidative stress,indicated by the decreased malondialdehyde content(P<0.01),enhanced total antioxidant capacity(P<0.01)and superoxide dismutase activity(P<0.01).The current study suggests that WPH exhibit promising antihypertensive abilities in vivo and could be a potential alternative for antihypertensive dietary supplements.

Food-derived protein hydrolysates and peptides:anxiolytic and antidepressant activities,characteristics,and mechanisms

Globally,the prevalence of anxiety and depression has reached epidemic proportions.Food-derived protein hydrolysates and peptides delivered through dietary supplementation can avoid the negative risks associated with traditional pharmaceuticals while delivering superior anxiolytic and antidepressant effects.This review summarizes current research on food-derived anxiolytic and antidepressant protein hydrolysates and peptides,and subsequently analyses their physicochemical characteristics and elaborates on their mechanisms.The aim of this work is to contribute to the in-depth study and provide a theoretical foundation for the development of related products to better serve patients with anxiety and depression.

Antioxidant and Anti-aging Activities of Silybum Marianum Protein Hydrolysate in Mice Treated with D-galactose

Objective In the present study, we investigated the antioxidant and anti‐aging effects of Silybum marianum protein hydrolysate（SMPH） in D‐galactose‐treated mice. Methods D‐galactose（500 mg/kg body weight） was intraperitoneally injected daily for 7 weeks to accelerate aging, and SMPH（400, 800, 1,200 mg/kg body weight, respectively） was simultaneously administered orally. The antioxidant and anti‐aging effects of SMPH in the liver and brain were measured by biochemical assays. Transmission electron microscopy（TEM） was performed to study the ultrastructure of liver mitochondria. Results SMPH decreased triglyceride and cholesterol levels in the D‐galactose‐treated mice. It significantly elevated the activities of superoxide dismutase（SOD） and glutathione peroxidase（GSH‐Px）, and total antioxidant capacity（T‐AOC）, which were suppressed by D‐galactose. Monoamine oxidase（MAO） and malondialdehyde（MDA） levels as well as the concentrations of caspase‐3 and 8‐OHd G in the liver and brain were significantly reduced by SMPH. Moreover, it increased Bcl‐2 levels in the liver and brain. Furthermore, SMPH significantly attenuated D‐galactose‐induced liver mitochondrial dysfunction by improving the activities of Na＋‐K＋‐ATPase and Ca2＋‐Mg2＋‐ATPase as well as mitochondrial membrane potential（ΔΨm） and fluidity. TEM showed that the degree of liver mitochondrial damage was significantly decreased by SMPH. Conclusion The results indicated that SMPH protects against D‐galactose‐induced accelerated aging in mice through its antioxidant and anti‐aging activities.

A new method for determination of antithrombotic activity of egg white protein hydrolysate by microplate reader

A new method for the determination of antithrombotic activity of egg white protein hydrolysate （EWPH） was developed using a microplate reader. Reaction was carried out at 37℃and pH 7.2 with fibrinogen concentration 0.1%. Microplate reading was conducted at 405 nm. Inhibition rate of EWPH on thrombin activity showed linearity （R2 = 0.9971）, when the inhibition rate was in the range of 10-90%. The lower limit of detection （LLD, at 99.7% probability） and the biological limit of detection （BLD, at 99.7% probability） of the method were 10.643 and 40 mg/mL, respectively. The repeatability standard deviation （R.S.D.） was 1.08%. The standard deviation of the method was ±0.027 AT-U.

Calcium-binding ability of soy protein hydrolysates

This present study investigated the ability of various soy protein hydrolysates (SPHs) in binding calcium. It was demonstrated that the amount of Ca-bound depended greatly on the SPHs obtained using different proteases, which included: neutrase, flavourzyme, protease M and pepsin. The maximum level of Ca-bound (66.9 mg/g) occurred when protease M was used to hydrolyze soy protein. Peptide fragments exhibiting high Ca-binding capacity had molecular weights of either 14.4 or 8–9 kDa. The level of Ca-bound increased linearly with the increment of carboxyl content in SPHs, and further deamidation on SPHs from protease M improved Ca-binding of the hydrolysate.

Effects of brewers' spent grain protein hydrolysates on gas production, ruminal fermentation characteristics, microbial protein synthesis and microbial community in an artificial rumen fed a high grain diet

Background: Brewers' spent grain(BSG) typically contains 20% – 29% crude protein(CP) with high concentrations of glutamine, proline and hydrophobic and non-polar amino acid, making it an ideal material for producing valueadded products like bioactive peptides which have antioxidant properties. For this study, protein was extracted from BSG, hydrolyzed with 1% alcalase and flavourzyme, with the generated protein hydrolysates(AlcH and FlaH)showing antioxidant activities. This study evaluated the effects of AlcH and FlaH on gas production, ruminal fermentation characteristics, nutrient disappearance, microbial protein synthesis and microbial community using an artificial rumen system(RUSITEC) fed a high-grain diet.Results: As compared to the control of grain only, supplementation of FlaH decreased(P < 0.01) disappearances of dry matter(DM), organic matter(OM), CP and starch, without affecting fibre disappearances;while AlcH had no effect on nutrient disappearance. Neither AlcH nor FlaH affected gas production or VFA profiles, however they increased(P < 0.01) NH_3-N and decreased(P < 0.01) H_2 production. Supplementation of FlaH decreased(P < 0.01)the percentage of CH_4 in total gas and dissolved-CH_4(dCH_4) in dissolved gas. Addition of monensin reduced(P < 0.01) disappearance of nutrients, improved fermentation efficiency and reduced CH_4 and H_2 emissions.Total microbial nitrogen production was decreased(P < 0.05) but the proportion of feed particle associated(FPA) bacteria was increased with FlaH and monensin supplementation. Numbers of OTUs and Shannon diversity indices of FPA microbial community were unaffected by AlcH and FlaH;whereas both indices were reduced(P < 0.05) by monensin. Taxonomic analysis revealed no effect of AlcH and FlaH on the relative abundance(RA) of bacteria at phylum level, whereas monensin reduced(P < 0.05) the RA of Firmicutes and Bacteroidetes and enhanced Proteobacteria. Supplementation of FlaH enhanced(P < 0.05) the RA of genus Prevotella, reduced Selenomonas, Shuttleworthia, Bifidobacterium and Dialister as compared to control;monensin reduced(P < 0.05) RA of genus Prevotella but enhaced Succinivibrio.Conclusions: The supplementation of FlaH in high-grain diets may potentially protect CP and starch from ruminal degradation, without adversely affecting fibre degradation and VFA profiles. It also showed promising effects on reducing CH_4 production by suppressing H_2 production. Protein enzymatic hydrolysates from BSG using flavourzyme showed potential application to high value-added bio-products.

Protein hydrolysates in animal nutrition：Industrial production, bioactive peptides,and functional significance

Recent years have witnessed growing interest in the role of peptides in animal nutrition. Chemical, enzymatic, or microbial hydrolysis of proteins in animal by-products or plant-source feedstuffs before feeding is an attractive means of generating high-quality small or large peptides that have both nutritional and physiological or regulatory functions in livestock, poultry and fish. These peptides may also be formed from ingested proteins in the gastrointestinal tract, but the types of resultant peptides can vary greatly with the physiological conditions of the animals and the composition of the diets. In the small intestine, large peptides are hydrolyzed to small peptides,which are absorbed into enterocytes faster than free amino acids（AAs） to provide a more balanced pattern of AAs in the blood circulation. Some peptides of plant or animal sources also have antimicrobial, antioxidant,antihypertensive, and immunomodulatory activities. Those peptides which confer biological functions beyond their nutritional value are called bioactive peptides. They are usually 2–20 AA residues in length but may consist of 〉20AA residues. Inclusion of some（e.g. 2–8%） animal-protein hydrolysates（e.g., porcine intestine, porcine mucosa,salmon viscera, or poultry tissue hydrolysates） or soybean protein hydrolysates in practical corn-and soybean mealbased diets can ensure desirable rates of growth performance and feed efficiency in weanling pigs, young calves,post-hatching poultry, and fish. Thus, protein hydrolysates hold promise in optimizing the nutrition of domestic and companion animals, as well as their health（particularly gut health） and well-being.

Lima Bean(Phaseolus lunatus)Protein Hydrolysates with ACE-I Inhibitory Activity

Several protein sources can be used to produce bioactive peptides with angiotensin I-converting enzyme (ACE) inhibittory activity. Protein concentrates from ungerminated and germinated lima bean Phaseolus lunatus seed flours were hydrolyzed with Alcalase 2.4 L or pepsin-pancreatin sequential hydrolysis, and ACE inhibitory activity measured in the different hydrolysis treatments. Protein hydrolysate production was analyzed with a 23 factorial design with four replicates of the central treatment. Evaluated factors were protein concentrate source (ungerminated seeds, PC1;germinated seeds, PC2), enzyme/substrate ratio E/S (1/50 or 1/10) and hydrolysis time (0.5 or 2.0 h for Alcalase;1 or 3 h for pepsin-pancreatin). Degree of hydrolysis (DH) was high for the Alcalase hydrolysates (24.12% 58.94%), but the pepsin-pancreatin hydrolysates exhibited the highest ACE inhibitory activity (IC50 = 0.250 0.692 mg/mL). Under the tested conditions, the hydrolysates with the highest ACE inhibitory activity were produced with sequential pepsin-pancreatin using either PC1 at 1 h hydrolysis time and a 1/10 E/S ratio or PC2 at 1 h hydrolysis time and a 1/50 E/S ratio. Lima bean protein hydrolysates prepared with Alcalase or pepsin-pancreatin are a potential ingredient in the production of physiologically functional foods with antihypertensive activity.

Effect of Different Treatments on Antioxidative Stability of the Scallop Protein Hydrolysates

Effects of different treatments on the antioxidant activity of scallop protein hydrolysates (SPH) were evaluated using DPPH radical scavenging activity and reducing power. Results showed that the antioxidant activity of SPH had good heating-resistance from 25°C to 65°C. The antioxidant activity of SPH could retain under acidic environment, but rapidly reduced under alkaline conditions. Addition of D-galactose, D-xylose, and D-fructose at 65°C could increase the antioxidant activity of SPH, but no such effect was not observed at this temperature. With the increase of storage time, the antioxidant activity of SPH gradually decreased. Moreover, pepsin digestion treatment slightly reduced the antioxidant activity of SPH, and further trypsin and mixed enzyme (trypsin + chymotrypsin) digestion significantly reduced this activity (p < 0.05). In conclusion, SPH may be used as food ingredients or food supplements in different food fields.

Improvement of Vitis amurensis Rupr.grape quality by using fish protein hydrolysates as fertilizer

High quality wine grape is the first factor to satisfy the wine-maker to produce superior quality wines.In this work,a fish protein hydrolysates derived from deep-sea salmon or cod,was used as fertilizer to improve Shuanghong(Vitis amurensis Rupr.)grape quality.Application of powder fish protein hydrolysates fertilizer by spraying directly to foliar(4.5 kg/ha),to root(4.5 kg/ha)or to the both foliar(4.5 kg/ha)and root(4.5 kg/ha)were carried out respectively,during the period from 2–3 weeks before flowering until 2–3 weeks before harvest.Grape sampling was made at both veraison and harvest.Different classes of phenolic compounds,including 10 phenolic acids and 11 proanthocyanidins in grape seeds and 10 anthocyanins in grape skins were determined by UHPLC or HPLC;total anthocyanins and total polyphenols in grape skins were determined by spectrophotometric method.Antioxidant activities of the phenolic compounds were evaluated by ABTS and FRAP methods.The results showed that the application of fish protein hydrolysates fertilizer to both root and foliar led to a significant improvement of berry quality at harvest/maturity,i.e.,increasing 12.13%total polyphenols(TP),8.3%total anthocyanins 11.03%ABTS and 45.81%FRAP compared with control.Besides,both root and foliar application resulted in the highest individual anthocyanins and proanthocyanidins in grapes.These results suggest that the both root and foliar application could be of great interest for the viticulturists to produce grapes with higher polyphenolic content and antioxidant activity.Although this work was experimented with Vitis amurensis Rupr.grapes,the proposed methods would also be applicable to other grape varieties for the purposes of increasing anthocyanin or polyphenol contents.

The effect of selected hemp seed protein hydrolysates in modulating vascular function

Reduced bioavailability of nitric oxide along with an increase in inflammatory cytokines,reactive oxygen species,cell adhesion molecules,and platelet hyperactivity are underlying causes of endothelial dysfunction.Bioactive peptides derived from various plants have been shown to attenuate biomarkers of endothelial dysfunction.The objective of this study was to determine the effects of hemp seed protein hydrolysates(HSPH)in modulating biomarkers of endothelial dysfunction using an in vitro cell culture model and an ex vivo platelet activity assay.HUVEC cells were treated with HSPH from commercial variant X and ECO-commercial variant Y,followed by hydrogen peroxide to simulate oxidative stress and inflammation.Biomarkers of endothelial dysfunction were then measured to determine the protective properties of HSPH.Blood samples collected from healthy volunteers were used to determine the effect of the HSPH on ADP and hydrogen peroxide-induced platelet activity,using flow cytometry.The HSPH reduced the production of reactive oxygen species,inflammatory cytokines IL-8,IL-12p70 and IL-1β,and adhesion molecule vascular cell adhesion molecule 1.Additionally,treatment with HSPH also reduced platelet activation marker CD62P ex vivo.The results obtained from this study suggest HSPH alleviates biomarkers of endothelial dysfunction by reducing oxidative stress,inflammation and platelet activation.

Investigation of hydrolysis conditions and properties on protein hydrolysates from flatfish skin

Response surface method （RSM）, based on Box-Behnken design, was used to optimize the enzymatic hydrolysis conditions of flatfish skin protein hydrolysates （FSPH）. Among the tested proteases, the combination of nutrase and trypsin was selected. The optimal hydrolysis conditions were as follows： pH 7.3, temperature 51.8℃, and the enzyme/substrate （E/S） ratio 2.5; under these conditions, the maximum peptide yield （PY） was 69.41 =1：0.43%. The physiochemical analysis showed that the amino acids （His, Asp and Glu） of FSPH accounted for 18.15%, and FSPH was a mixture of polypeptides mostly distributed among 900-2000 Da. FSPH could exhibit a 93% chelating effect on ferrous ion at a concentration of 400 gg/mL, and also a notable reducing power. This study showed bioprocess for the production of FSPH for the first time, which had a good potential for valuable ingredients in the food, cosmetic and medicine industries.

Optimization of hydrolysis conditions for the production of protein hydrolysates from fish wastes using response surface methodology

The recycling of waste from food processing is an important industrial procedure for obtaining protein sources with high added value.In this study,trout(Onchorynchus mykiss),anchovy(Engraulis encrasicolus),and whiting(Merlangius merlangus)wastes were hydrolyzed.The hydrolyzation procedure was investigated under the optimum predicted conditions defined by time,temperature,and enzyme ratio(E/S)using alkaline protease(pH 8),Protamex(pH 7),and Flavourzyme(pH 7).The hydrolysis conditions that showed the best degree of hydrolysis(DH%)were optimized using response surface methodology(RSM)with the central composite design(CCD)and Box-Behnken design(BBD)models.The effects of three independent variables,temperature(40-60℃),time(1-8 h),and enzyme concentrations(1-2%),were examined for model optimization.It was determined that the DH%varies between 50.92%and 74.30% according to the type of fish waste and enzyme.The highest degree of hydrolysis was observed from trout waste(74.30%)and the lowest degree from whiting waste(50.92%)with Flavourenzyme.From this study,it has been shown that different degrees of hydrolysis and protein recovery can be obtained,depending on fish species,waste composition,enzyme type,and hydrolysis method.

Antioxidant potential and physicochemical properties of protein hydrolysates from body parts of North Atlantic sea cucumber(Cucumaria frondosa)

Protein hydrolysates were prepared from North Atlantic sea cucumber(Cucumaria frondosa)body wall(BW),and processing by-product flower(FL)and internal organs(IN).Sea cucumber proteins from these three tissues were hydrolysed with selected endopeptidases and exopeptidases.The enzymes used were Alcalase(A),and Corolase(C)as endopeptidases and Flavourzyme(F)with both endo-and exopeptidase functions.These were employed individually or in combination under controlled conditions.The hydrolysates so prepared were subsequently analysed for their antioxidant potential and functionalities in food systems for the first time.Hydrolysates treated with the combination of A and F exhibited the highest radical scavenging activity against DPPH and ABTS radicals.The highest metal chelation activity was observed for samples hydrolysed with the combination of enzymes(C+F and A+F).All treatments inhibited beta-carotene bleaching in an oil-in-water emulsion and TBARS production in a meat model system.In addition,sea cucumber protein hydrolysates were more than 75%soluble over a pH range of 2–12.Hydrolysed proteins were also effective in enhancing water holding capacity in a meat model system compared to their untreated counterparts.The amino acids of sea cucumber protein hydrolysates had desirable profiles with glutamic acid as the predominant component in samples analysed.These findings demonstrate the desirable functionalities of hydrolysates from North Atlantic sea cucumber and their potential for use as functional food ingredients.

Whole wheat cracker fortified with biocalcium and protein hydrolysate powders from salmon frame: characteristics and nutritional value

Objectives:This study aimed to develop whole wheat crackers fortified with biocalcium(BC)and protein hydrolysate(PH)powders from salmon frame at varying substitution levels.Materials and Methods:Whole wheat cracker fortified with BC and protein hydrolysate PH powders from salmon frame was produced.BC and PH powders or their mixtures at different ratios(3:1,1:1,1:3)were used to fortify the dough mix at a substitution level of 16.67%based on the whole wheat flour content.The physical,textural,sensory,and nutritional profiles of the crackers produced were examined and compared with crackers without fortification.Results:The weight,colour,textural properties,and thickness of the crackers varied with the addition of different ratios of BC and PH powders.The incorporation of BC/PH at ratio(3:1)showed no negative effect on sensory properties.The crackers thus produced possessed higher protein,fat,calcium,phosphorus,sodium,and cholesterol but lower carbohydrate,sugar,fibre,and energy value than the control crackers without fortification.The crackers contained 0.026-0.045 mg/100 g polyunsaturated fatty acid.Developed crackers had a denser structure and were less porous than the control crackers as shown in scanning electron microscopic images.In scanning electron microscopy-energy dispersive X-ray spectroscopic(SEM-EDX)analysis,the crackers fortified with BC/PH at ratio(3:1)had higher calcium and phosphorus contents and intensity than the control.Conclusions:This study demonstrated that the addition of BC and PH powders obtained from salmon frame represent a promising means of increasing the nutritive values of crackers.

Changes in Antioxidant Activity of Alcalase-Hydrolyzed Soybean Hydrolysate under Simulated Gastrointestinal Digestion and Transepithelial Transport

Peptides from Alcalase-hydrolyzed soybean protein hydrolysate(SPH)may hold the potential as natural antioxidants.In addition,the effect of human gastrointestinal(GI)tract on peptide bioavailability needs to be explored.In this study,the impact of simulated GI digestion and transepithelial transport on various antioxidant properties of SPH were investigated.SPH displayed DPPH radical scavenging(IC50=4.22 mg/m L),ABTS·+radical scavenging(IC50=2.93 mg/m L),reducing power and metal ion-chelating activities(IC50=0.67 mg/m L).Furthermore,SPH significantly(P<0.05)inhibited the generation of intracellular reactive oxygen species(ROS)in Caco-2 cells.After simulated GI digestion,the antioxidant properties of SPH were enhanced,except for a decrease in ABTS·+radical scavenging activity.After transepithelial transport,the permeates maintained partial antioxidant activity and the LC-MS/MS data further identified the absorbed soybean peptides.These results suggest that SPH contains the antioxidant peptides that are potentially bioavailable and can be regarded as a promising source of functional food ingredients.

Antioxidant Activity and Protective Effects of Alcalase-Hydrolyzed Soybean Hydrolysate in Human Intestinal Epithelial Caco-2 Cells

Soybeans are known as a promising source of bioactive peptides.However,knowledge on the antioxidant behaviors of soybean protein hydrolysate(SPH)in the human intestinal epithelium is limited.In this study,SPH was prepared with Alcalase and subsequently ultrafiltered into four peptide fractions as SPH-I(<3 kDa),SPH-II(3~5 k Da),SPH-III(5~10 k Da)and SPH-IV(>10 kDa).The antioxidant properties of SPH and membrane fractions were investigated using different chemical assays and their protective effects against oxidative stress were evaluated using H2 O2-stressed human intestinal Caco-2 cells.Results showed that SPH-I exhibited the strongest 2,2-diphenyl-1-picrylhydrazyl(DPPH)radical scavenging activity(IC50=2.56 mg/m L)and reducing capacity while SPH-III had the best metal ion-chelating activity(IC50=0.29 mg/m L).Both SPH and the peptide fractions dose-dependently suppressed intracellular reactive oxygen species(ROS)accumulation induced by H2O2 in Caco-2 cells,but the strongest inhibitory effect was observed for SPH-I.Amino acid(AA)results revealed that SPH-I was rich in hydrophobic and antioxidant AAs,which could contribute to its stronger antioxidant properties.Additionally,SPH-I protected Caco-2 cells from H2O2-induced oxidative stress via inhibiting lipid peroxidation and stimulating antioxidant enzyme activities.These results suggest that SPH-I and constitutive peptides can be beneficial ingredients with antioxidant properties and protective effects against ROS-mediated intestinal injury.

Systematic functional analysis and potential application of a serine protease from cold-adapted Planococcus bacterium

In this study, a gene encoding serine protease(PmSpr288)from cold-adapted bacterium, namely Planococcus maritimus XJ11, was cloned and overexpressed in Escherichia coli BL21(DE3). Bioinformatics analysis revealed that PmSpr288 belongs to serine protease S8 superfamily with a classical catalytic triad comprised by the Asp49, His86 and Ser251. Moreover, PmSpr288 was found to be active over broad alkaline pH and low-moderate temperature, and exhibited wide range of protein substrate specificity. In addition, PmSpr288 was able to hydrolyze the meat proteins actin and myosin, and molecular docking results suggested that the crucial interaction between PmSpr288 and actin/myosin complexes was mainly occupied by hydrogen bonds. The muscle protein hydrolysates of silver carp prepared by PmSpr288 was shown to have antioxidant activity via DPPH radical scavenging assay, which presented an IC_(50) valve of 1.309 mg/mL. In conclusion, these characteristics imply that PmSpr288 has potential biotechnological application prospect for the production of bioactive peptides.

Recovery of Residual Brewer’s Yeast by Electroactivation

Yeasts resulting from the brewing process (RBY) are a valuable by-product, with an important content of minerals, vitamins and, especially, proteins. The purpose of the research was the electroactivation of RBY and the simultaneous obtaining of two products—protein concentrates and hydrolyzed protein from residual brewer’s yeast. Electroactivation is a non-residual process, without the use of chemical reagents and relatively inexpensive. The variation of the electroactivation conditions allowed the separation of 90% - 94% of the proteins in the form of protein concentrates. During the process, it is attested to increase the pH value and decrease the redox potential, which characterizes the multiple redox processes that take place in the cathode cell, including sedimentation at the isoelectric point. The presence of albumin in the protein fractions of RBY allows the formation of protein complexes with calcium, attributing a higher biological value to the obtained products.