核桃粕源抗氧化活性肽的酶解制备及活性分析

Enzymatic hydrolysis preparation and activity analysis of antioxidant peptides derived from walnut dregs

目的:深度利用核桃粕蛋白。方法:采用分级分离法从核桃粕中提取蛋白质后,分别用胰蛋白酶、木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和风味蛋白酶5种蛋白酶对其进行水解,并比较分析胰蛋白酶酶解物(trypsin hydrolysate,TH)、木瓜蛋白酶酶解物(papain hydrolysate,PH)、中性蛋白酶酶解物(dispase hydrolysate,DH)、碱性蛋白酶酶解物(alcalase hydrolysate,AH)和风味蛋白酶酶解物(flavourzyme hydrolysate,FPH)的抗氧化活性;以DPPH自由基清除率、ABTS自由基清除率和羟自由基清除率为检测指标分析不同浓度AH的体外抗氧化性;采用LC-MS/MS技术鉴定AH中所有肽的序列,并利用PeptideRanker、BIOPEP-UWM数据库和分子对接工具AutoDock1.5.6筛选潜在的核桃抗氧化活性肽;将上述两条肽分别与Keap1蛋白进行计算机模拟分子对接,并利用斑马鱼胚胎氧化损伤模型,对人工合成的两条抗氧化活性肽的抗氧化活性进行分析。结果:5种酶解物中抗氧化活性较好的AH对DPPH自由基、ABTS自由基和羟自由基的最佳清除率分别为(71.56±0.75)%,(60.63±0.45)%,(98.63±0.06)%。LC-MS/MS鉴定并通过计算机分析预测得到两条活性最优的肽,即:ALWPF和PLRWPF;两条抗氧化活性肽均能与Keap1蛋白的活性部位结合,其结合能分别为-9.2,-9.6 kJ/mol。ALWPF和PLRWPF肽处理斑马鱼胚胎的安全质量浓度范围分别为0~50,0~30μg/mL。结论:从核桃粕中鉴定出两条抗氧化肽,其对斑马鱼胚胎具有保护作用。

Objective:This study focused on the identification and activity analysis of antioxidant active peptides in walnut dregs protein hydrolysates.Methods:Protein was extracted from walnut meal by fractionation method,and five kinds of proteolytic enzymes,including trypsin,papain,neutral protease,alkaline protease and flavor protease,were used to hydrolyze the proteins.The antioxidant activity of trypsin hydrolysate(TH),papain hydrolysate(PH),dispase hydrolysate(DH),alcalase hydrolysate(AH)and flavourzyme hydrolysate(FPH)were evaluated and compared.The in vitro antioxidant activity of different concentrations of AH was determined by the detection indexes of DPPH free radical scavenging rate,ABTS free radical scavenging rate and hydroxyl free radical scavenging rate.The sequences of all peptides in AH were identified by LC-MS/MS technology,and the potential antioxidant active peptides of walnut were screened by PeptideRanker,BIOPEP-UWM database and molecular docking tool AutoDock1.5.6,and the two antioxidant active peptides were obtained.The above two peptides were simulated with Keap1 protein,and the antioxidant activity of the two antioxidant peptides was analyzed by using the zebrafish embryo oxidative damage model.Results:The optimal scavenging rates of AH with better antioxidant activity among the five enzymatic hydrolysates were(71.56±0.75)%,(60.63±0.45)% and(98.63±0.06)%,respectively.LC-MS/MS was identified by computer analysis to obtain two active ALWPF and PLRWPF peptides,both of which could bind to the active sites of Keap1 protein,with binding energies of-9.2 and-9.6 kJ/mol,respectively.The safe concentrations of zebrafish embryos treated with ALWPF and PLRWPF peptides ranged from 0~50μg/mL and 0~30μg/mL,respectively.Conclusion:Two antioxidant peptides were obtained from walnut meal,and the antioxidant effects of them were verified by zebrafish embryo model.The results of this study can provide a basis for the in-depth utilization of walnut protein.