两步酶解法制备低致敏性乳清蛋白及其致敏性评价

Preparation of hypoallergenic whey protein isolate hydrolysates by two-step enzymatic hydrolysis and its allergenicity evaluation

目的探究两步复合酶解工艺处理对牛乳清蛋白(whey protein isolate,WPI)结构和致敏性的影响。方法实验采用邻苯二甲醛法、体积排阻色谱法分析WPI水解物在不同酶解时间下的水解程度,同时采用酶联免疫吸附法(enzyme-linked immunoassay,ELISA)、细胞模型和小鼠模型对水解物进行体外和体内的致敏性评估,并利用超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)分析水解物中的残留过敏原表位。结果WPI经碱性蛋白酶和木瓜蛋白酶顺序酶解3.0 h后水解度达到14.19%,在水解2.0~3.0 h期间,WPI水解物主要由小肽(<5.0 kDa)组成。水解3.0 h后,水解产物与特异性抗体的结合能力降低了97.83%,细胞模型和小鼠模型结果证实,酶解产物的体外、体内致敏性显著降低,与空白对照组无显著性差异。UPLC-MS/MS结果表明WPI水解3.0 h产物中70.59%的肽段不含过敏原表位。结论复合酶解工艺有效降低了WPI致敏性,本研究构建的致敏性综合评价体系可为低敏乳制品水解工艺的开发提供指导。

Objective To investigate the effects of two-step compound enzymatic hydrolysis on the structure and allergenicity of whey protein isolate(WPI).Methods The degree of hydrolysis of WPI hydrolysates at different hydrolysis time was analyzed by the o-phthalaldehyde method and size exclusion chromatography.At the same time,enzyme-linked immunoassay(ELISA),cell models and mouse models were used to evaluate the allergenicity of the hydrolysates in vitro and in vivo,and ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)was used to analyze the residual allergen epitopes in hydrolysates.Results After sequential hydrolysis of WPI by alcalase and papain for 3.0 hours,the degree of hydrolysis of the sample reached 14.19%.During the hydrolysis of 2.0-3.0 hours,the WPI hydrolysates were primarily made up of tiny peptides(<5.0 kDa).After hydrolysis for 3.0 hours,the binding ability of the hydrolysates to the specific antibody decreased by 97.83%.The cell model and mouse model results confirmed that the in vitro and in vivo allergenicity of the hydrolysate were significantly reduced,and there was no significant difference compared with the control group.The results of UPLC-MS/MS showed that 70.59%of the hydrolyzed peptides failed to find allergen epitopes after hydrolyzing WPI for 3.0 hours.Conclusion The compound enzymatic hydrolysis process effectively reduces the allergenicity of WPI,and the comprehensive evaluation system of allergenicity established in this study provides insightful guidance for the development of hypoallergenic dairy products.