紫花苜蓿叶蛋白制备抗氧化肽酶解条件优化及氨基酸组成分析

Optimization of Enzymatic Hydrolysis Conditions for the Preparation of Antioxidant Peptides and Amino Acid Composition Analysis from Alfalfa Leaf Protein

为开发利用苜蓿叶蛋白资源,以紫花苜蓿叶蛋白为原料,利用碱性蛋白酶酶解制备抗氧化肽。在单因素试验基础上,以抗氧化活性为指标,选取酶解时间、碱性蛋白酶用量、酶解温度和pH值为考察因素。采用响应面试验优化,其最佳酶解条件为酶解时间240min、碱性蛋白酶用量4.80%、酶解温度60℃、pH11.60,在此条件下清除率达到64.25%。用制备色谱纯化并收集抗氧化性最强的抗氧化肽组分,表明纯化后的抗氧化肽具有更强的抗氧化活性。用高效液相色谱测定纯化后抗氧化肽的氨基酸组成,其组成为天冬氨酸(Asp)、甘氨酸(Gly)、组氨酸(His)、酪氨酸(Tyr)、缬氨酸(Val)、亮氨酸(Leu)、苯丙氨酸(Phe)、色氨酸(Trp)和赖氨酸(Lys)。

In order to develop and utilize alfalfa leaves as a good resource of protein,antioxidant peptides were preparedfrom alfalfa leaf protein by alkaline protease hydrolysis.Through one-factors-at-a-time experiments,hydrolysis time,enzyme concentration,hydrolysis temperature and pH were selected as independent variables for further optimization usingresponse surface methodology,and the response variable was antioxidant activity.The optimum conditions that providedthe maximum percentage scavenging of DPPH radical of64.25%were found to be hydrolysis time of240minutes,enzymeconcentration of4.80%,hydrolysis temperature of60℃and pH of11.60.Then the most potent antioxidant peptide waspurified and collected by preparative liquid chromatography,having stronger antioxidant activity than the crude hydrolysate.The amino acid composition of the antioxidant peptide was determined by high performance chromatography(HPLC)toconsist of Asp,Gly,His,Tyr,Val,Leu,Phe,Trp and Lys.