酵母菌与乳酸菌协同发酵降低面筋蛋白致敏性的机制研究

In recent years, the incidence of wheat allergy showed an increasing trend. Mantou, as one of the major traditional wheat flour fermentation products in China, occupies a very important position in the food consumption of residents. In previous studies, it was found that in the process of making steamed buns with the sourdough, the allergenicity of the dough first increased and then decreased, and the dough sensitized intensity of the dough fermented by “old buns” in different regions was different. In order to reveal its mechanism, this project intends to screen the dominant yeasts and lactic acid bacteria that affect allergenicity from the hypoallergenic sourdough, and to carry out relevant research on gluten protein. Metagenomics and metabolomics methods were used to investigate the interactions of yeast and lactic acid bacteria in the micro-ecological fermentation environment and their effects on dough properties. Column chromatography would be used to separate and purify the highly sensitizing proteins in gluten proteins. Proteomics and immunology would be used to investigate the changes of the composition and structure of highly allergenic proteins during fermentation, and the structure-activity relationship between them and allergenicity was established. Clarify the mechanism of synergistic fermentation of yeast and lactobacillus to reduce gluten allergenicity. Finally, the allergenicity, quality and nutritional properties of the processed steamed bread products were evaluated, which laid a theoretical foundation for the further development of safe and effective hypoallergenic wheat products.

近年来，小麦引起的过敏发病率呈逐年上升的趋势。馒头作为我国主要的传统小麦粉发酵制品之一，在居民膳食消费中占有十分重要的地位。前期研究发现采用“老酵头”制作馒头过程中，面团的致敏性出现先升高后降低的现象，且不同地区“老酵头”发酵的面团致敏强度不同。为了揭示其机理，本课题拟从低致敏性老酵头中筛选影响致敏性的优势酵母菌与乳酸菌，以面筋蛋白为对象开展相关研究。采用宏基因组学及代谢组学的方法，探讨微生态发酵环境中酵母菌与乳酸菌的互作代谢规律及其对面团特性的影响规律。采用柱层析法分离纯化面筋蛋白中的高致敏性蛋白，利用蛋白组学、免疫学等方法，研究发酵过程中高致敏蛋白组成与结构的变化规律，建立其与致敏性之间的构效关系，阐明酵母菌与乳酸菌协同发酵降低面筋蛋白致敏性的作用机制。最后通过对加工制成的馒头制品进行致敏性、品质特性及营养特性等方面的评价，为进一步开发安全有效的低致敏小麦制品奠定理论基础。

我国是小麦种植和进口大国，馒头作为我国主要的小麦粉发酵制品之一，在居民膳食消费中占有十分重要的地位。研究表明小麦是引发中国人严重过敏反应的最常见诱因，青少年（10-17岁）和成人（≥18岁）是小麦过敏的高发人群。目前，治疗食物过敏最有效的办法就是避免接触致敏性食物，然而，完全避免小麦制品的摄入非常困难，且小麦中富含多种人体生长所必需的微量元素，饮食规避容易导致患者营养不良。因此，通过合适的加工手段改性食物致敏原，生产低致敏或无致敏性的产品对于保障小麦食品的安全性，改善过敏患者的膳食结构具有重要的现实意义。本研究首先利用高通量测序对我国具有地域代表性的酸面团菌群组成进行分析，然后通过传统的分离方法对酸面团中主要微生物进行分离、鉴定和筛选。筛出具有代表性的菌株，再将其接种至面团中，利用转录组学和蛋白质组学相关技术，从微生物互作和蛋白结构变化两个角度探究发酵影响小麦蛋白致敏性的机制。最后利用体外胃肠消化模型和致敏小鼠模型进一步研究发酵产物的消化抗性和致敏性，并对前期的致敏性体外评价结果进行验证。主要结果如下：.（1）不同地区的酸面团样品中细菌的多样性不同，但优势菌属的类别没有显著性差异；筛选出了一株具有较高蛋白水解活性的乳酸菌XZ31；以发酵力为指标筛选出了两株具有较强发酵力的酵母菌JM1和JM4；.（2）发酵过程中致敏原蛋白的水解主要依赖于XZ31的代谢活动,酵母菌可以加速XZ31对蛋白的代谢活动；.（3）发酵过程中小麦可溶蛋白肽的水解程度大于难溶蛋白肽，致敏原蛋白Tri a 28 和Tri a 19含量显著降低；XZ31通过增强蛋白二硫键的交换作用以及破坏蛋白的非共价聚集程度降低麦谷蛋白聚合度，在麦谷蛋白解聚的同时，肽段延展并发生了显著的水解；.（4）相比于XZ31单独发酵，其与酵母菌复配发酵产物能更显著地减轻致敏小鼠的临床症状和肠道屏障的损伤。