Study On The Preparation And Antioxidant Activity Of Proteasome Peptides From Hydrilla Verticillata

At present,antioxidant peptides derived from natural protein enzymatic hydrolysis are favored because of their relatively simple structure,strong activity,easy absorption,safety and non-toxic side effects.Hydrilla is a kind of Hydrilla of hydrocharidae with high protein content,which is a good raw material for preparing antioxidant peptides.However,the black algal protein has not been fully utilized,and the high value resource is low.At present,there are few reports on the preparation of antioxidant peptides from hydrillin.Therefore,in this study,hydrillin was used as raw material to hydrolyze hydrillin and prepare antioxidant peptides.The antioxidant peptides were isolated,purified and sequenced,and their antioxidant activities in vitro and in cells were studied,so as to provide a basis for the development and high value resource utilization of hydrillin.The specific research contents are as follows:1)Firstly,the degree of hydrolysis and antioxidant activity of pepsin,trypsin,alkaline protease and papain hydrolysates were determined to select the best protease for the enzymatic hydrolysis of hydrillin,and then the response surface method was used to optimize the conditions of hydrillin on the basis of single factor experiment.The results showed that trypsin was the best protease to hydrolyze the black algal protein,and the optimal conditions for trypsin to hydrolyze the black algal protein were as follows:the ratio of enzyme to substrate was 8.6%,the time was 5.9 h,and the p H value was 7.3.The scavenging rates of ABTS,DPPH,superoxide anion and hydroxyl radical were 65.9%,40.1%,50.1%and 25.3%,respectively.2)The trypsin hydrolysates of black algae were separated by ultrafiltration,Sephadex G-25 gel column and semi prepared reverse HPLC,and the D-3 fractions（1000μg/m L）the scavenging rates of ABTS,DPPH,superoxide anion and hydroxyl radical were 96.03%,57.32%,58.25%and 40.38%,respectively.According to the amino acid sequence of D-3 component,two peptides TCLGER and TCLGPK with strong antioxidant activity were selected and synthesized.The antioxidant activities of TCLGER and TCLGPK were higher.3)The oxidative stress model of HepG2 cells was established.The experimental results showed that 0.8 mmol/l H₂O₂ was used.After the intervention of different concentrations of TCLGER and TCLGPK,the survival rate of oxidized Hep G2 cells increased significantly,the apoptosis rate was significantly reduced,ROS content was significantly reduced,MDA content decreased,SOD enzyme activity,cat enzyme activity and GSH-Px enzyme activity increased significantly.The survival rate of damaged cells treated with 1 mg/ml TCLGER and TCLGPK was 93.51%and 95.68%,the apoptosis rate was 2.33%and 2.03%,MDA content was 2.9 and 2.4 u/mg prot,and the relative fluorescence intensity of ROS in cells was 40156 a.u.and 46455 a.u.SOD enzyme activity was 95.97 u/mg prot and 98.31 u/mg prot respectively;Cat enzyme activity was divided into 5.87 u/mg prot and 6.24 u/mg prot;GSH-Px enzyme activity was divided into 1.23 u/mg prot and 1.01 u/mg prot.