Soybean Protein Hydrolysates Research Articles

Bioactive peptide with antioxidant and anticancer activities from black soybean [Glycine max (L.) Merr.] byproduct: isolation, identification and molecular docking study

In this study, a novel peptide was isolated from the black soybean byproduct using bioactive guided isolation technique. Initially, the hydrolysate of black soybean protein was fractionated and separated sequentially by ultrafiltration, Sephadex G 25 chromatography and reverse-phase HPLC techniques. The peptide fractions that collected in each step were tested for their antioxidant capacity and anticancer activities against human liver (HepG2), lung (MCF-7) and cervical (Hela) cancer cell lines. The most active fraction was subjected to further purification. The final purified peptide fraction (F2-c) with the molecular weight of 455.0 Da showed highest DPPH free radical scavenging and hydroxyl radical scavenging activity with the IC50 values of 0.12 and 0.037 µM, respectively. Moreover, it showed high cytotoxic potential against HepG2, MCF-7 and Hela cells with the IC50 values of 0.22, 0.15 and 0.32 µM, respectively. The amino acid sequence of F2-c was identified as Leu/Ile-Val-Pro-Lys (L/I-VPK). Molecular docking studies revealed that the purified peptides effectively bound with four apoptosis related key proteins (XIAP, caspase-3, caspase-7, and Bcl-2) via hydrophobic effects and hydrogen bonds, amongst them, the peptide-caspase-3 binding showed the strongest binding energy. All the results suggested that the peptide fraction Leu/Ile-Val-Pro-Lys from black soybean byproduct with significant antioxidant and anticancer activities could be a good candidate for functional foods or related drugs.

Optimization of ACE inhibitory peptides from black soybean by microwave-assisted enzymatic method and study on its stability

To optimize the preparation of ACE inhibitory peptides from black soybean (Glycine max (L.) Merr.), three enzymatic methods were compared, with the technical conditions optimized by response surface analysis. The black soybean protein hydrolysate inhibited 70.38% of the ACE activity. The ACE inhibitory peptides were isolated from black soybean protein hydrolysates by macro-porous resin, ultrafiltration, and Sephadex G-15. Four fractions were obtained, with each fraction having some ACE inhibitory activity. Fraction III exhibited the highest activity of 90.78%, and a molecular weight range of 145–468 Da. The ACE inhibitory peptides were stable across a range of pH values (2–10), at temperatures <40 °C, and in the presence of metal ions (Ca2+, K+, Mg2+), but had little resistance to digestive enzymes. These results indicated that the ACE inhibitory peptides of black soybean are substrate-type inhibitory peptides.

Cationic peptides from enzymatic hydrolysates of soybean proteins exhibit LPS-neutralizing and angiogenic activities

In this study, we prepared fractions containing multifunctional cationic peptides by separating the commercial soybean protein hydrolysate Hinute-AM into 20 fractions. These fractions contained peptides with various isoelectric points (pI), as indicated by ampholyte-free isoelectric focusing (autofocusing). Thus, we purified and identified the cationic peptides from fractions 19 and 20, which had pH values greater than 10, using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among 19 identified cationic peptides, NKNAKPPSPR, PGKKNAIV, KSGPGMSPR, NVSKPPRVV, RKVGAGGRKPLG, and LPCVIGGVPKRV had high pI values and were included as chemically synthesized peptides in assays of various functions, including lipopolysaccharide (LPS)-neutralizing and angiogenic activities. Chromogenic LPS-neutralizing assays using Limulus amebocyte lysates showed that 50% effective concentrations of these six peptides were between 1.63 and 2.65μM, and were higher than that (0.12μM) of polymyxin B. Moreover, in tube-formation assays in human umbilical vein endothelial cells, all of the six cationic peptides except LPCVIGGVPKRV exhibited angiogenic activities similar to those of the positive control LL-37. In addition, the six identified cationic peptides had no hemolytic activity at concentrations up to 500μM in mammalian red blood cells. Our results demonstrate that five of the identified cationic peptides, excluding LPCVIGGVPKRV, have multiple functions and little or no hemolytic activity. These data indicate that fractions containing cationic peptides from Hinute-AM have the potential to be used as dietary supplements and functional ingredients in food products.

Soybean-Derived Glycine–Arginine Dipeptide Administration Promotes Neurotrophic Factor Expression in the Mouse Brain

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays an important role in cognitive abilities, including memory and learning. We demonstrated that soybean protein hydrolysate (SPH) diet suppresses age-related cognitive decline via the upregulation of BDNF in a mouse model of senescence. Our purpose was to identify novel bioactive peptides in SPH, which enhance BDNF expression. We treated mouse primary astrocytes with SPH as well as with its positively charged chromatographic fraction. Significant increases in the expression of BDNF were observed in the treatment with positively charged fraction of SPH. Among the synthesized peptides, the dipeptide glycine-arginine (GR) increased BDNF expression in vitro, andLC-TOF-MS analysis showed the presence of GR in the SPH. Furthermore, its administration in vivo increased the expression of BDNF in the cerebral cortex and the number of neurons in hippocampus and cerebral cortex. These data indicate that GR might promote neurogenesis by upregulating BDNF levels.

Purification and a molecular docking study of α-glucosidase-inhibitory peptides from a soybean protein hydrolysate with ultrasonic pretreatment

Soybean protein was hydrolyzed using trypsin with ultrasound pretreatment to obtain a soybean protein hydrolysate. The latter was separated sequentially by ultrafiltration, ion exchange chromatography, gel filtration chromatography, and reversed-phase high-performance liquid chromatography. A highly active component with molecular weight less than 5 kDa, obtained by ultrafiltration, significantly reduced the levels of fasting blood glucose in mice. α-Glucosidase-inhibitory activity of fraction C-III-2a separated by reversed-phase high-performance liquid chromatography was the highest, and the half-inhibitory concentration (IC50) was 0.049 mg/mL. Amino acid sequences of two tripeptides from fraction C-III-2a were identified as Gly-Ser-Arg and Glu-Ala-Lys by ion trap tandem mass spectrometry. Molecular docking analyses revealed that the α-glucosidase inhibition by the peptides could be mainly attributed to the formation of five strong hydrogen bonds between Glu-Ala-Lys and His-674, Asp-518, Arg-600, Asp-616, and Asp-282 in α-glucosidase, and to four hydrogen bonds between Gly-Ser-Arg and residues Asp-282, Asp-518, and Asp-616. The results indicated that an α-glucosidase-inhibitory peptide may have hypoglycemic efficacy when used as an ingredient in novel functional foods.

Flavor Compounds in Pixian Broad-Bean Paste: Non-Volatile Organic Acids and Amino Acids.

Non-volatile organic acids and amino acids are important flavor compounds in Pixian broad-bean paste, which is a traditional Chinese seasoning product. In this study, non-volatile organic acids, formed in the broad-bean paste due to the metabolism of large molecular compounds, are qualitatively and quantitatively determined by high-performance liquid chromatography (HPLC). Amino acids, mainly produced by hydrolysis of soybean proteins, were determined by the amino acid automatic analyzer. Results indicated that seven common organic acids and eighteen common amino acids were found in six Pixian broad-bean paste samples. The content of citric acid was found to be the highest in each sample, between 4.1 mg/g to 6.3 mg/g, and malic acid were between 2.1 mg/g to 3.6 mg/g ranked as the second. Moreover, fumaric acid was first detected in fermented bean pastes albeit with a low content. For amino acids, savory with lower sour taste including glutamine (Gln), glutamic acid (Glu), aspartic acid (Asp) and asparagines (Asn) were the most abundant, noted to be 6.5 mg/g, 4.0 mg/g, 6.4 mg/g, 4.9 mg/g, 6.2 mg/g and 10.2 mg/g, and bitter taste amino acids followed. More importantly, as important flavor materials in Pixian broad-bean paste, these two groups of substances are expected to be used to evaluate and represent the flavor quality of Pixian broad-bean paste. Moreover, the results revealed that citric acid, glutamic acid, methionine and proline were the most important flavor compounds. These findings are agreat contribution for evaluating the quality and further assessment of Pixian broad-bean paste.

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 kDa), SPH-III (5–10 kDa) 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 H2O2-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/mL) and reducing capacity while SPH-III had the best metal ion-chelating activity (IC50 = 0.29 mg/mL). 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.

Plastein reaction enhanced bile-acid binding capacity of soybean protein hydrolysates and whey protein hydrolysates

Plastein reaction is a modification reaction that can improve the functional properties of protein hydrolysate. The product of the reaction is a thixotropic aggregation of peptides. This study investigated the formation condition of soybean-whey plastein and bile acid binding capacity of plastein. Soy protein and whey protein were hydrolyzed by pepsin. The mixture (1:1, w/w) of two hydrolysates was modified by pepsin again. After the reaction, the decrease in free amino groups and the turbidity of the modified hydrolysate were measured to obtain appropriate reaction condition. Results showed that the concentration of hydrolysates 40% (w/v), enzyme ratio of 2.0KU/g protein, pH 5.0, 37°C, reaction time of 3.0h respectively, were showed maximum changes in protein hydrolysates. Tricine SDS-PAGE analysis under denaturing conditions revealed that whey protein was more sensitive to pepsin and yielded different polypeptides (PPs) of molecular weight ranged from 3.5-17 kDa. However, a high molecular weight PP was completely hydrolyzed while PPs of 14.2-26 kDa were partially digested after pepsin treatment. Native page analysis further revealed the presence of a high-molecular weight PP in crude and purified plastein product. The bile acid binding capacity was improved by the plastein reaction. The amount of binding sodium deoxycholate, sodium taurocholate, and sodium cholate were 0.75, 2.0 and 1.87μmol/100mg respectively.

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/mL), ABTS+ radical scavenging (IC50 = 2.93 mg/mL), reducing power and metal ion-chelating activities (IC50 = 0.67 mg/mL). Furthermore, SPH significantly (P < .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.

Hydrolysis of Soybean Proteins with Kamchatka Crab Hepatopancreas Enzyme Complex

To perform hydrolysis with the enzyme complex from the hepatopancreas of the Kamchatka crab, a protein mixture was isolated from soybean meal by extraction at alkaline pH values. Extractable low-molecular impurities were removed by ultrafiltration and precipitation of proteins with alcohol. The amino acid composition of the obtained protein extract turned out to be similar to the composition of the fish meal traditionally used in the production of fish feeds. Analysis of the products of fermentolysis by DDS-electrophoresis, HPLC, and mass spectrometry showed a high degree of hydrolysis of soybean proteins. Depending on the time of fermentolysis, the hydrolysates contained up to 60% (18 h of hydrolysis) of free amino acids (the fraction of the weight of the hydrolyzed protein mixture) and short peptides (2–20 amino acid residues).

Peptide supplementation to nutrient‐adequate diets enhanced internal egg quality during storage in hens at peak production

There is paucity of information on the use of dietary peptides in laying hens and its effects on egg production and quality. In the current study, peptide from enzymatic hydrolysis of soybean protein was incorporated into laying hens' diets to investigate its effect on egg production and internal egg quality. There were no treatment effects on egg production (average hen day production was 96%) during the experiment. Final body weight of the hens increased quadratically (P < 0.05) in response to peptide supplementation. There were no significant effects of peptide supplementation on internal egg quality of the fresh eggs. Peptide supplementation tended to increase yolk colour (P < 0.10) in eggs collected at 4 weeks of the study and stored at room temperature for 14 days. For the eggs collected at 8 weeks of the experiment and stored at room temperature for 14 days, peptide supplementation linearly increased (P < 0.05) albumen height, Haugh unit and yolk index but linearly decreased (P < 0.01) yolk width. Peptide supplementation to laying hens at peak production, receiving diets meeting their nutrient requirement, did not improve hen production but positively helped to maintain hens' body weight and egg quality during storage. © 2017 Society of Chemical Industry.

Interaction mode of calcium-binding peptides and Caco-2 cell membrane

In our previous studies, soluble soybean protein hydrolysate (SPH)–calcium complexes were shown to promote the calcium uptake of Caco-2 cells. However, the calcium transport mode involved remains unknown. In this article, several experiments were carried out via cytological analysis to investigate the calcium transport mode of peptides with low calcium binding capacities (F1), peptides with high calcium-binding capacities (F2), and their separate calcium complexes (F1–Ca and F2–Ca) when interacting with cell membranes. The interaction between one of them and a cell membrane is the first step in intracellular transport, as indicated by fluorescence blue shift experiments and acrylamide quenching experiments. The results of zeta potential experiments showed that only the “charge neutralization” phenomenon occurs when the F1 peptide or F1–Ca complex interacts with cell membranes and thus cannot be transported into cells. On the contrary, in an F2 at high concentrations or F2–Ca complex, a “charge recovery” phenomenon occurs apart from “charge neutralization” and can thus be transported into cells through endocytosis.

Characterization of soy protein hydrolysates produced by varying subcritical water processing temperature

Abstract The objective of this study was to investigate the effect of subcritical water processing (SWP) temperature on the hydrolysis pattern and quality characteristics of soybean protein hydrolysates. Two common cultivars of soybean were subjected to SWP at temperature ranging from 150 to 250 °C and pressure of 22 MPa. The highest free amino group content and yield were obtained at a processing temperature of 190 °C for both the cultivars ( p

Industrial Applications of Selected JFS Articles

Industrial Applications of Selected <i>JFS</i> Articles

Sport Nutrition Drinks Based on Octopus Protein Hydrolysate

&lt;p&gt;Abstract&lt;br /&gt;Sport nutrition drinks are well-known in escalating athlete’s performance and endurance. These product developed from whey protein hydrolysates and soybean protein hydrolysates have already been recognized, however expansion from marine product is comparatively rare. Octopus (Octopus cyanea) widely acknowledged containing taurine and rich in amino acids is potential to be developed as ingredient for sport nutrition drink. The aims of this study were to create and characterize sport nutrition drinks based on marine peptides through Octopus protein hydrolyzate. Octopus protein hydrolysate has 77.78±2.69% degree of hydrolysis and 751.02±10.63 mg / 100g taurine. Sports nutrition drinks with the addition of 4% Octopus protein hydrolyzate was acceptable sensory panelists, and the serving size of 600 ml contained taurine 726.06±0.82 mg and detected 17 types of amino acids.&lt;/p&gt;

Sport Nutrition Drinks Based on Octopus Protein Hydrolysate

Sport nutrition drinks are well-known in escalating athlete’s performance and endurance. These product developed from whey protein hydrolysates and soybean protein hydrolysates have already been recognized, however expansion from marine product is comparatively rare. Octopus (Octopus cyanea) widely acknowledged containing taurine and rich in amino acids is potential to be developed as ingredient for sport nutrition drink. The aims of this study were to create and characterize sport nutrition drinks based on marine peptides through Octopus protein hydrolyzate. Octopus protein hydrolysate has 77.78 ± 2.69% degree of hydrolysis and 751.02 ± 10.63 mg / 100g taurine. Sports nutrition drinks with the addition of 4% Octopus protein hydrolyzate was acceptable sensory panelists, and the serving size of 600 ml contained taurine 726.06 ± 0.82 mg and detected 17 types of amino acids.

In vitro antioxidant activities of the novel pentapeptides Ser-His-Glu-Cys-Asn and Leu-Pro-Phe-Ala-Met and the relationship between activity and peptide secondary structure.

Using high-performance liquid chromatography/tandem mass spectrometry, two novel antioxidant pentapeptides [Ser-His-Glu-Cys-Asn (SHECN) and Leu-Pro-Phe-Ala-Met (LPFAM)] were identified from 1-3-kDa soybean protein hydrolysates (SPH). The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to evaluate cytotoxicity in HepG2 cells. Antioxidant activity was measured using in vitro assays, including the cellular antioxidant activity assay (CAA), 2,2-diphenyl-1-picrylhydrazyl or 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) inhibition, and oxygen radical absorbance capacity (ORAC) assays. Finally, the secondary structure was determined using circular dichroism (CD). The results revealed that two novel peptides were nontoxic and possessed antioxidant activity. SHECN had significantly higher antioxidant activity than LPFAM (P < 0.05). The CAA value of SHECN was 776.22 µmol QE 100 g-1 . SHECN also showed significant DPPH inhibition (70.18 ± 4.06%) and ABTS inhibition (88.16 ± 0.76%). It had normalized ORAC values of 0.3000 ± 0.0070 µmol GE mg-1 and 0.0900 ± 0.0020 µmol TE mg-1 , respectively. The results of the CD analysis demonstrated that, compared to LPFAM, which had much lower antioxidant activity, SHECN had a high β-sheet content and reduced α-helix content. The results indicated that SHECN possessed high antioxidant activity. A higher β-sheet content and lower content levels of α-helix appear to be correlated with antioxidant activity. © 2016 Society of Chemical Industry.

Evaluation of the Antioxidant and Antiproliferative Effects of Three Peptide Fractions of Germinated Soybeans on Breast and Cervical Cancer Cell Lines.

Soybeans are an important source of bioactive molecules, such as peptides, which generation can improve through germination. In this study, the antioxidant and antiproliferative activities of three peptide fractions (>10kDa, 5-10kDa and <5kDa) that were obtained by ultrafiltration of soybean protein hydrolysate after sixdays of germination were evaluated. The antioxidant activities of the peptide fractions were assessed by reducing power, Cu+2 and Fe+2 chelation and OH· scavenging assays, whereas their antiproliferative effects against cervical (HeLa, SiHa, CasKi) and breast (MCF7 and MDA-MB-231) cancer cell lines were evaluated by the MTT assay. Apoptosis was determined by Hoechst-PI staining. The most active peptide fraction (MAPF) was the >10kDa fraction, which showed the greatest antioxidant and antiproliferative activity. The most sensitive cancer cell lines were the HeLa, CasKi and MDA-MB-231 cells, which had IC50 values of 16.2, 14.3 and 15.2mg/mL, respectively, and apoptotic indices above 50% after 6 or 8h of exposure. The effect of MAPF on normal cells (HaCaT) was minimal. The amino acid composition of MAPF was characterized by high proline, phenylalanine and tyrosine content, and MALDI-TOF/TOF analysis showed six signals with molecular weights of 12 to 42kDa.

Influence of microcrystalline cellulose on the microrheological property and freeze-thaw stability of soybean protein hydrolysate stabilized curcumin emulsion

The impact of microcrystalline cellulose (MCC) on the physical stability, microrheological property and freeze-thaw stability of papain hydrolysate of soybean isolate protein (SPIH) stabilized curcumin emulsion was studied. Curcumin emulsions were prepared by Microfluidizer containing SPIH and various concentrations of MCC. The curcumin emulsions were assessed by zeta potential, average particle size through dynamic light scattering technique (DLS), stability index by Turbiscan and microrheological behavior through diffusive wave spectroscopy (DWS) technique. With addition of MCC, the negative charge of the droplets was increased, which indicated that the negatively charged fraction from MCC interacted with the protein at the interface. An increase in droplet size and decrease in TSI were noted with increasing MCC concentration. It demonstrated that enough MCC changed microrheological property of curcumin emulsions from purely viscous to viscoelastic over the range of decorrelation times, indicating that the droplets were not free to move due to the droplets network interaction. Freeze-thaw stability analysis demonstrated that MCC brought about a remarkable improvement, which can be attributed to the enhancement of repulsive steric forces between the curcumin droplets. It demonstrated that combined enzymatic hydrolysate of protein and addition of polysaccharide MCC could be an effective method for protecting bioactive compound in emulsions.

Study on Optimal Conditions of Alcalase Enzymatic Hydrolysis of Soybean Protein Isolate

Soybean protein isolate was hydrolyzed to obtain soybean polypeptide solution using Alcalase as hydrolase. Degree of hydrolysis and the recovery rate of protein were used to characterize the soybean protein hydrolysis reaction result. Influence factors of soybean protein hydrolysis reaction including the substrate concentration, temperature, pH, enzyme concentration characterized by E/S (ratio of Enzyme and Substrate) and hydrolysis time were systematically studied with single factor and multi-level experimental. The optimal reaction conditions of soybean protein isolate hydrolyzed by Alcalase were that the substrate concentration was 8%; E/S was 3.6 AU/100 g substrate; pH was 8.0; temperature was 60°C and hydrolysis time was 2 h. The study also showed that the hydrolytic activity of the Alcalase to soybean protein isolate was stronger in the front 1 h; the hydrolytic activity of Alcalase weakened gradually with time when reaction time was longer than 2 h.