碱性蛋白酶水解乳清分离蛋白工艺优化

Process optimization of alkaline protease hydrolyzing whey protein isolate

中文关键词:  乳清分离蛋白  碱性蛋白酶  致敏性  酶解  响应面法  十二烷基硫酸钠-聚丙烯酰胺凝胶电泳  高效液相色谱法  酶联免疫吸附法

英文关键词:residues whey protein isolate  alkaline protease  allergenicity  enzymatic hydrolysis  response surface methodology  sodium dodecyl sulfate-polyacrylamide gel electrophoresis  high performance liquid chromatography  enzyme linked immunosorbent assay

中文摘要:

      目的 降低乳清分离蛋白中的致敏蛋白含量, 制备低致敏性乳制品。方法 利用碱性蛋白酶水解乳清分离蛋白, 研究酶添加量、初始pH、酶解时间以及温度对乳清分离蛋白水解度的影响。在单因素的实验基础上, 采用Box-Behnken实验设计方法进行四因素三水平的响应面优化实验。结果 在P<0.05的水平下, 4个因素对乳清分离蛋白的水解度都有显著影响。最优的水解工艺为: 酶添加量6.4%、初始pH 11、酶解时间4 h、温度60 ℃。乳清分离蛋白在此条件下水解后, 水解度达到21.11%。酶解液的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)分析显示, 经过这一优化工艺水解, 10 kDa以上的蛋白基本全部被降解。高效液相色谱法(high performance liquid chromatography, HPLC)分析酶解产物的多肽及蛋白质分子量分布, 结果显示酶解产物的分子量大都分布在3.5 kDa及以下。采用间接竞争酶联免疫吸附法原理测定2种标志性致敏蛋白(β-乳球蛋白和α-乳白蛋白)的残余抗原性, 发现2种致敏蛋白的残余抗原性也有不同程度的降低。结论 通过碱性蛋白酶水解后, 乳清分离蛋白中具有致敏性的大分子蛋白转变为小分子的肽类, 从而降低了致敏性。

英文摘要:

      Objective To reduce the content of sensitized protein in whey protein isolate (WPI) and prepare hypersensitive dairy products. Methods WPI was hydrolyzed by alkaline protease and the effects of enzyme addition, initial pH, hydrolysis time and temperature on the degree of hydrolysis of WPI were studied. On the basis of single-factor experiments, the response surface optimization experiment of 4 factors and 3 levels was carried out by using Box-Behnken experimental design method. Results At the level of P<0.05, 4 factors had significant effects on the degree of hydrolysis of WPI. The optimal hydrolysis process was as follows: enzyme addition 6.4%, initial pH 11, enzymatic hydrolysis time 4 h and 60 ℃. After hydrolyzed under this process, the degree of hydrolysis of WPI reached 21.11%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the hydrolysate showed that all the proteins above 10 kDa were degraded by this optimized process. The results of high performance liquid chromatography (HPLC) analysis of the molecular weight distribution of peptides and proteins showed that most of the molecular weights of the hydrolysates were below 3.5 kDa. The residual antigenicity of 2 landmark sensitizing proteins (β-lactoglobulin and α-lactalbumin) was determined by indirect competitive enzyme linked immunosorbent assay principle. It was found that the residual antigenicity of 2 allergenic proteins also decreased to varying degrees. Conclusion After alkaline protease hydrolysis, the allergenic macromolecular proteins in WPI are transformed into small molecular peptides, thus reducing it’s allergenicity.