芡实多肽的分步酶解法制备及抗氧化活性研究

Two-step Enzymatic Preparation and Antioxidant Activity in vitro of Peptides from Euryale ferox

为探索制备芡实生物活性肽的最佳工艺条件,以水解度为评价指标,芡实蛋白为原料,利用双酶分步次序酶解的方式结合单因素和正交试验设计优化芡实多肽的制备工艺,综合ABTS自由基、羟基自由基(OH·)清除活性及铁氰化钾还原效果评价了芡实多肽的抗氧化性,并对其相对分子质量分布与氨基酸构成进行了分析。结果表明:先用中性蛋白酶于pH8.0、温度45℃、底物浓度5%、加酶量3000 U/g的条件下水解3 h,再用碱性蛋白酶于pH9.5、温度50℃、底物浓度3%、加酶量4000 U/g的条件下水解3 h,此时水解度可达46.8%,为芡实多肽的最佳制备工艺条件。芡实多肽对ABTS自由基、羟基自由基和还原力的半数抑制浓度(IC50)分别为1.42、0.98、6.29 mg/mL,抗氧化能力较强。芡实多肽中相对分子质量低于1000 u的肽比例为91.26%,主要为小分子低聚寡肽,且富含酸性氨基酸和疏水氨基酸。双酶分步酶解制备芡实多肽的工艺可有效改善酶解效果,成功制备出具有良好抗氧化能力的小分子肽。

This study was designed to optimize the enzymatic preparation of bioactive peptides from Euryale ferox protein.The hydrolysis conditions were optimized based on degree of hydrolysis(DH)with one-factor-at-a-time method and orthogonal array design.Three different methods including ABTS radical scavenging,hydroxyl radical(OH·)scavenging and reducing power were used for evaluating the antioxidant activities of Euryale ferox peptides.Moreover,the molecular weight distribution and amino acid composition of the peptide were analyzed.The optimal technological parameters for preparing Euryale ferox peptide were determined as follows.Euryale ferox protein were hydrolyzed firstly with 3000 U/g neutrase at an initial pH of 8.0 and 45℃with a substrate concentration of 5%for 3 h,and then with 4000 U/g alcalase for 3 h at 50℃with a substrate concentration of 3%after pH adjustment to 9.5.Under these conditions,the DH was up to 46.8%.With the increase of the concentration of Euryale ferox peptides,the scavenging rate of ABTS and hydroxyl radical increased significantly in a dose-dependent manner.The half inhibitory concentration(IC50)for ABTS,hydroxyl radical scavenging rate and reducing power were 1.42,0.98,6.29 mg/mL,respectively,demonstrating that Euryale ferox peptides had strong antioxidant activity.Moreover,the molecular weight of peptides prepared below 1000 u accounted for 91.26%.The majority of them were small-molecule oligopeptides,which are rich in acidic amino acids and hydrophobic amino acids.It was concluded that neutrase-alcalase hydrolysis would provide an effcient way to prepare Euryale ferox peptides with excellent antioxidant ability.