酶法制备乳源钙螯合肽及其特性表征

Preparation and Characterization of Milk-derived Calcium Chelating Peptide by Enzymatic Method

以牛乳酪蛋白为原料,选用胰蛋白酶为水解酶,以钙螯合活性为指标,对底物浓度、加酶量、pH值、温度和酶解时间5个因素进行单因素试验和正交试验。采用高效凝胶过滤色谱法测定钙螯合肽的分子量分布,同时通过氨基酸自动分析法、紫外-可见光谱和红外光谱法来表征最优酶解条件下的钙螯合肽和钙肽螯合物的结构特性。试验结果表明:底物浓度7%,加酶量0.8%,pH8.7,温度53℃,时间3 h,为最优酶解条件,其钙螯合活性为46.78μg/mL。在此条件下获得的钙螯合肽中,分子量在3 kDa以下的肽组分占92.11%;氨基酸分析表明,谷氨酸、丝氨酸和天冬氨酸这几种具有较强螯合活性的氨基酸的含量明显增加;由紫外光谱和红外光谱的结果可得,钙螯合肽的结构在螯合前后发生了明显的变化,其主要结合位点是-NH2、-COOH。

Casein was used as the raw material,trypsin was selected as hydrolytic enzyme,and calcium chelating activity was taken as the index.Single-factor test and orthogonal test were carried out for five factors including substrate concentration,enzyme amount,pH,temperature and enzymatic hydrolysis time.The structure characteristics of calcium chelated peptides and calcium peptide chelates under optimal enzymatic hydrolysis were characterized by high performance gel filtration chromatography,amino acid automatic analysis,uv-visible spectrum and infrared spectroscopy.The results showed that:7%substrate concentration,0.8%enzyme dosage,pH 8.7,53℃hydrolysis temperature,and 3 h hydrolysis time were the optimal conditions with the activity of chelate calcium of 46.78μg/mL.The relative molecular weight of peptides below 3 kDa in the optimal enzymatic hydrolysis accounted for 92.11%.Amino acids analysis indicated that the content of glutamate,serine and aspartic acid with strong chelating activity had increased significantly after chelating reaction.According to the results of ultraviolet and infrared spectroscopy,the structure of calcium chelated peptide changed significantly before and after the chelation.And its main binding sites were-NH2,-COOH.