Separation And Characterizationof Antioxidant Peptides From Bass Muscle And Their Protective Effects Against Oxidative Damage In HepG2 Cells

Bass?Lateolabrax japonicus?is a kind of food ingredient with high protein content?18.7%?and low fat content?2.1%?.Additionally,bass protein is rich in antioxidative amino acids including Tyr,Trp,Cys and Met,which makes it very appropriate for preparing antioxidant peptides by enzymatic hydrolysis.In this study,antioxidative bass muscle hydrolysates?BMHs?were prepared from bass muscle through controlled enzymatic hydrolysis.Gel chromatography was applied on the separation and purification of BMHs to obtain antioxidative fraction and mass-spectrometric technique was used to characterize the peptide sequence.Furthermore,the identified peptides were synthesized and their antioxidant activities were verified using in vitroantioxidant assays.The protective effects of these antioxidant peptides from bass muscle against oxidative damage in HepG2 cells were also studied.Firstly,BMHs were prepared by four proteases?Alcalase,Papain,Neutrase and Protamex?under appropriate conditions to screen the optimal protease.Results showed that BMH prepared by Papain showed the highest DH?25.61%?,protein recovery rate?78.71%?and small peptides content?MW<3000 Da accounting for 92.72%?as well as the highest Trolox equivalent?TE?values in ORAC and ABTS assays with 783.56 and 734.55?mol TE/g,respectively.Therefore,Papain was selected for the preparation of antioxidative BMH.Afterwards,BMH prepared by Papain was separated by gel filtration on Sephadex G-15and UPLC-ESI-Q-TOF-MS/MS was applied to identify the sequence of the active fraction.BMH was isolated to four fractions?F1,F2,F3 and F4?.The fraction F4 of small molecule showed the highest antioxidant activities.TE values of F4 in ORAC and ABTS assays reached to 4187.78 and 7070.58?mol TE/g,respectively.Furthermore,32 peptides were identified from F4.All these peptides were synthesized and 12 of them showed antioxidant activities in ORAC and ABTS assays.The sequence of these peptides were AW,GW,YE,YG,YP,GVW,NSW,VPVY,AAEGY,EYGTVVVFQ,GGGAGMLLK and HRDRLCVVQ.In the end,the synthetic antioxidant peptides identified from BMH were used to study their protective effects against H₂O₂-induced oxidative damage in HepG2 cells.Results showed that the cell viability of H₂O₂-treated HepG2 cells increased significantly?p<0.05?with pre-incubation of NSW,VPVY,AAEGY,HRDRLCVVQ and GGGAGMLLK for 24 h at the concentration of 10?M.Moreover,the cell viability of HepG2 cells treated with these peptides was higher than that treated with GSH at the same concentration?p<0.05?.Among these peptides,AAEGY demonstrated the strongest ability to resist H₂O₂-induced cell injuries at the concentration of 10?M with the cell viability reaching to 96.07%.In addition,all these antioxidant peptides at the concentration of 10?M could protect against oxidative damage in HepG2 cells by scavenging ROS,increasing the activitiy of antioxidase?i.e.,SOD,CAT and GSH-Px?and decreasing the MDA level.