Study On The Preparation Of Peptide From Walnut Protein By Enzymatic Hydrolysis And Its Antioxidant Activity

Walnut protein is a high quality dietary protein with rich essential amino acids.Walnut oil residue contains a large number of proteins.Protein extracting from defatted walnut meal and natural active peptides preparied from defatted walnut protein hydrolysate can not only develop walnut protein resources,but also increase the additional value of walnut.It has great significance and broad prospects for the research of walnut protein properties.Two proteases including trypsin and alkaline protease were used to hydrolyze degreased walnut meal step by step.The antioxidant activity of the hydrolysate was measured.The antioxidant peptides with high antioxidant activity were obtained by isolation and purification.The antioxidant mechanism of the antioxidant peptides was also studied.The main results were summarized as follows:1.Screening the best enzyme to prepare walnut antioxidant peptides:Walnut protein were prepared by trypsin and alkaline protease enzymatic hydrolysis under thier optimal conditions and DPPH radical scavenging rate were used as evaluation indexes.Under the conditions of substrate concentration 30 mg/mL,37℃ for 1.5 h,3%addition of enzyme,pH 8.0 of trypsin and then substrate concentration 30 mg/mL,50℃ for 1.5 h,3%addition of enzyme,pH 8.0 of alkaline protease.The maximum degree of hydrolysis of the enzymatic hydrolysate of defatted walnut protein was 9.27±0.03%.2.Isolation,purification and structure identification of defatted walnut protein antioxidant peptides:defatted walnut protein hydrolysate(DWPH)was ultrafiltered,of which U-3 had the strongest antioxidant capacity.U-3 was washed out of 4 components by Sephadex G-25 gel chromatography,and F4 had the highest ability to scavenging DPPH radical.Nine peaks were purified from F4 by RP-HPLC.Among them,peak P6 scavenging DPPH radical scavenging capacity was the highest.LC-MS/MS was used to detect peak P6,and 23 potential peptides with potential antioxidant activity were identified.Three potential antioxidant peptides,EELEQMEDEE,LQQPPFLE and YVPHWNLN,were selected to synthesize and determine their antioxidant activities.The results showed that EELEQMEDEE could effectively chelate Fe2+ and YVPHWNLN peptide had high ORAC capability.3.Investigating the cellular and molecular mechanisms of polypeptide-induced oxidative damage in HepG2 cells by H2O2.Cells were cultured with appropriate concentrations of polypeptides and the cell viability was detected by MTT assay.The content of oxygen free radicals(ROS)was detected by DCFH-DA probe.The expression level of protein molecules was determined by western blotting.The results showed that LQQPPFLE(60,80 and 100 μg/mL)and YVPHWNLN(60,80 and 100 μg/mL)could protect HepG2 cells from oxidative damage induced by hydrogen peroxide,effectively reduce ROS content in damaged cells,improve ROS scavenging ability and antioxidant ability of cells.LQQPPFLE(60,80 and 100 μg/mL)and YVPHWNLN(60,80 and 100μg/mL)regulated the expression of antioxidant enzymes and phase II detoxifying enzymes through Nrf2/HO-1 signaling pathway.