Study On The Protective Effect Of British Red Kidney Bean Antioxidant Peptides On Oxidatively Stressed Zebrafish

Under normal conditions,the production and scavenging of free radicals by the body is in a state of balance.Oxidative stress refers to the excessive production of reactive oxygen radicals(ROS)by the body under various stimuli,which exceeds the body’s own ability to scavenge them,resulting in an imbalance between oxidative and antioxidant states,causing tissue damage and various diseases.Antioxidant peptide is a natural antioxidant with simple structure,good stability under different conditions,no dangerous immune response,and widely studied not only for its strong antioxidant activity,but also for its nutritional and functional properties.In this paper,British red kidney bean protein was used as raw material and enzymatic digestion was carried out by alkaline protease,and the enzymatic digestion conditions of British red kidney bean protein were firstly optimized.The protective effect of the antioxidant peptide on2,2’-azodiisobutylamidine dihydrochloride(AAPH)induced oxidative stress was investigated.A preliminary study on the mechanism of in vivo antioxidant effect of British red kidney bean antioxidant peptide was conducted to provide theoretical basis for the development and application of the refined products of British red kidney bean antioxidant peptide.The main research contents and results are as follows:The optimum enzymatic conditions determined by single-factor test and orthogonal test were 10% substrate mass fraction,3h enzymatic time,50℃,p H 9.5.The enzymatic products were ultrafiltered,and it was found that the antioxidant activity of the fraction with relative molecular mass less than 1 k Da(BRKBPAPC)was the highest,and the antioxidant activity of the enzymatic conditions was significantly increased after the enzymatic digestion compared with the previous method(P<0.05),the DPPH radical scavenging rate increased by 28.62% and the reducing power increased by 5.30% compared with the previous method.The antioxidant activity of BRKBPAPC was significantly(P<0.05)increased after in vitro simulated digestion compared to the non-ultrafiltered peptide.Nutritional value analysis of British red kidney bean revealed that British red kidney bean is a good source of plant protein and BRKBPAPC a high quality plant-derived peptide.A model of oxidative stress zebrafish was established using 2,2’-azobis(isobutylamidine)dihydrochloride(AAPH)to investigate the effects of BRKBPAPC on the antioxidant system,lipid metabolism and behavior of oxidatively stressed zebrafish.Zebrafish were randomly divided into control group,model group,VC-positive control group,and BRKBPAPC low,medium and high dose groups.(1)Compared with the model group,BRKBPAPC significantly increased(P<0.05)catalase(CAT),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and total antioxidant capacity(T-AOC)and significantly decreased(P<0.05)malondialdehyde(MDA)content in zebrafish,with increasing treatment time and concentration the better.At 21 d of high-dose antioxidant peptide treatment,CAT,SOD,T-AOC,and GSH-Px increased to 28.29±3.29u/mgprot,126.59±6.39 u/mgprot,2.07±0.18 u/mgprot,and 84.75±7.77 u/mgprot of GSH-Px,respectively,and MDA content decreased to 3.35±0.29 nmol/mgprot.(2)Compared with the control group,the lipid metabolism of oxidatively stressed zebrafish was disrupted,with a significant increase in triglyceride(TG)(P<0.05)and a significant decrease in total cholesterol(TC),low-density lipoprotein(LDL-C)and high-density lipoprotein(HDL-C)content(P<0.05);while BRKBPAPC had the ability to regulate lipid metabolism in zebrafish and increased with the concentration of The concentration of BRKBPAPC in zebrafish gradually returned to the normal level.The TG decreased to 1.03 mmol/gprot;TC,LDL-C and HDL-C increased to 0.65 mmol/gprot,0.063 mmol/gprot and 0.444 mmol/gprot respectively by21 d of high dose BRKBPAPC treatment.(3)With the prolongation of treatment time,zebrafish in the model group were inactive,with reduced tactile wall reflex activity and shorter behavioral trajectories,indicating that AAPH caused oxidative stress in zebrafish and had a significant inhibitory effect on locomotor behavior.As the concentration of BRKBPAPC increased and the duration of action increased,the behavioral trajectories of zebrafish increased,and the number,time and distance of high-speed movements increased significantly(P<0.05).The zebrafish became lively and the trajectories increased,indicating that BRKBPAPC could effectively improve the behavioral weakening of zebrafish caused by oxidative stress.In conclusion,BRKBAPC has good in vivo antioxidant activity and has a protective effect on AAPH-induced oxidative stress zebrafish.This study provides a theoretical basis for the development of British red kidney bean products.