豆奶在乳酸菌发酵期间的致敏性变化及其机制研究

Fermentation is one of the effective methods to reduce the allergenicity of food, which plays an important role in the control of food allergens. In this proposal, soybean was used as raw material, and the fermented soymilk was prepared by lactic acid bacteria. During the course of the fermented soymilk production, the allergic serum IgE binding is regarded as an indicator for the allergenicity, and the choice of suitable strain and the optimization of fermentation conditions will be done to reduce significantly the allergenicity of soymilk. Based on the above work, the ELISA, Western-blotting, liquid chromatography tandem mass spectrometry, sensitization and challenge of mice model will be used to evaluate the changes in total allergenicity, main allergic protein allergenicity and sensitization ability of soymilk during the fermentation. Simultaneously, the changes in composition and chemical structure of the main allergenic protein in soymilk will be characterized by electrophoresis, circular dichroism, UV spectroscopy, fluorescence spectroscopy, and mass spectrometry. In this proposal, the relationship between the allergenicity of fermented soybean milk and the structure of main allergen during the course of fermentation will be illustrated, and it will reveal the molecular mechanism of degrading milk allergy caused by lactic acid bacteria. Furthermore, it may provide theoretical basis for the development of soybean desensitization technology and production of hypoallergenic soybean products.

发酵法是降低食物过敏原致敏性的有效方法之一，在食物过敏原的加工控制中具有重要作用。本项目拟以豆奶为研究对象，利用乳酸菌发酵制备酸豆奶，以过敏患者血清IgE结合能力为致敏性评价指标，优选降低豆奶致敏性显著的菌种，并优化发酵工艺条件；在此基础上，采用ELISA、免疫印迹、液质联用和小鼠致敏激发模型等方法探讨豆奶发酵期间总蛋白致敏性、主要过敏原致敏性和致敏激发能力的变化规律；同时，借助电泳、圆二色光谱、紫外光谱、荧光光谱、质谱等技术解析和表征发酵期间大豆过敏原的结构变化，明确主要过敏原关键表位降解规律。通过本项目的实施，将阐明发酵豆奶致敏性变化与过敏原结构改变之间的关联性，最终揭示乳酸菌发酵降低豆奶致敏性的分子机制，为大豆蛋白脱敏技术研究和低致敏豆制品的开发提供理论依据。

豆奶营养丰富，然而，大豆致敏性一定程度上限制了豆奶的利用。微生物发酵被认为是降低致敏性的最佳方法；且发酵能降解豆奶中抗营养因子，发酵豆奶所含的益生菌还具有调节肠道菌群平衡等功效。菌种选取和工艺优化是生物发酵中两个关键环节。因此，开展菌种优选和发酵工艺优化以期降低豆奶致敏性，并建立发酵期间豆奶致敏性变化与过敏原及其关键表位降解的关联分析具有研究价值。. 本研究以豆奶为对象，利用乳酸菌发酵豆奶，以发酵后豆奶中蛋白降解为指标进行菌种优选和发酵工艺优化；然后采用ELISA、细胞致敏模型和小鼠致敏激发模型系统探讨豆奶发酵前后过敏原致敏性的变化规律；同时，借助圆二色谱、紫外光谱、荧光光谱、免疫印迹和质谱等技术表征发酵后大豆过敏蛋白的结构变化，明确主要过敏原关键表位降解规律。结果表明：选取短乳杆菌与乳杆菌属进行液态发酵，短乳杆菌和乳杆菌属的接种比例为3:2，总接种量为4%，发酵温度为33℃，发酵时间为24 h，大豆蛋白的降解程度最大；与豆奶中蛋白相比，发酵豆奶中蛋白与过敏血清IgE结合能力显著降低；发酵豆奶中蛋白的胃肠消化产物激发KU812细胞产生的氨基己糖激酶、组胺和IL-4显著降低；动物实验结果显示，发酵豆奶组小鼠过敏症状减弱，空肠绒毛萎缩、脱落减轻，血清中特异性抗体、Th2型细胞因子和过敏效应细胞脱颗粒水平均显著低于豆奶组小鼠；表明发酵后豆奶致敏性显著降低。豆奶经过发酵之后，主要过敏蛋白发生了水解，内部相关基团暴露，结构变得更加松散；β-伴大豆球蛋白的α、ά 和β亚基条带几乎完全消失，且大豆球蛋白的酸性亚基也基本消失；大豆蛋白中五个人类IgE结合表位被降解，表明豆奶在发酵后主要过敏蛋白一些线性表位被降解破坏，推测这可能是导致其致敏性降低的主要原因。本课题工作将对开发低致敏豆奶提供科学依据和技术支撑。