乳酸菌对β-伴大豆球蛋白降解规律及对过敏原性的影响机制

Soy allergy has focused numerous researchers’ and consumers’ attention. β-conglycinin is an important soy allergen and contains very stable epitopes, which are difficult to be degraded by conventional technologies. Lactic acid bacteria are able to reduce the allergenicity of β-conglycinin and demonstrated selectivity in β-conglycinin degradation. However, little has been understand about the mechanism of allergenicity alternations by lactic acid bacteria. Few investigations were conducted to understand whether epitopes of β-conglycinin can be completely degraded by this process, and the degradation sites and the composition of the degradation products are unknown. Therefore, this project is proposed to firstly conduct β-conglycinin proteolytic degradation using the proteases separated from Lactobacillus plantarum B1-6, a strain previously separated by our team exerted potent degradation of legume 7S globulin, aimed at reduction of its allergenicity. Then, investigations of changes of β-conglycinin during degradation will be carried out by differential proteomic profiling techniques. A peptidomic protocol will be used to study the degradation sites as well as the primary structure of the peptides. After that, immunoinformatics tools will be applied to predict allergenicity of the degradation products as well as their higher structures. The results will be further confirmed by immunoblotting. The goal of this project is to reveal the degradation profile of β-conglycinin by lactic acid bacteria and to understand the mechanism of lactic acid bacteria to alternate the allergenicity of β-conglycinin. This project will be further considered to provide solid theoretical base for the future applications of desensitization techniques using lactic acid bacteria.

大豆过敏是研究者和消费者关注的焦点问题。β-伴大豆球蛋白是重要的大豆过敏原，具有稳定的过敏原表位，难以被常规技术降解。乳酸菌发酵可降低β-伴大豆球蛋白的过敏原性，并已证实，乳酸菌对β-伴大豆球蛋白存在选择性降解作用，但降解位点及降解产物的组成尚不清楚，能否完全降解过敏原表位尚不明确，导致乳酸菌脱敏的内在机制难以阐释。本项目针对此进行研究，首先以降低过敏原性为目标，采用本项目组前期分离的对豆类7S球蛋白具有强降解作用的植物乳杆菌B1-6酶解β-伴大豆球蛋白，然后利用差异蛋白质组学研究降解前后β-伴大豆球蛋白的变化规律，利用肽组学明确降解位点及降解产物的一级结构，接着，采用免疫信息学技术，预测降解产物的高级结构及过敏原性，最后，运用免迹印迹的方法对过敏原性结果进行验证。目的是从蛋白质组学与肽组学角度，揭示乳酸菌对β-伴大豆球蛋白降解规律及对过敏原性的影响机制，为乳酸菌发酵脱敏技术提供理论指导。

大豆过敏发生率不断上升成为研究者和消费者关注的焦点问题。β-伴大豆球蛋白、大豆球蛋白等主要大豆过敏原蛋白具有非常稳定的过敏原表位，难以被常规技术降解。乳酸菌发酵可降低大豆蛋白的过敏原性，因此成为极具潜力的脱敏方式。本项目首先从9株具有潜在大豆蛋白脱敏能力的乳酸菌中筛选得到一株植物乳杆菌B1-6可显著降低大豆蛋白IgE免疫反应性，通过条件优化，得到最优的发酵时间为48小时、碳氮比为2：5、大豆蛋白浓度为5%（m/v）时大豆蛋白IgE免疫反应性最低。对植物乳杆菌B1-6脱敏大豆蛋白机理研究发现，植物乳杆菌B1-6主要通过蛋白降解及对蛋白高级结构改变的共同作用对致敏性产生影响。发酵前后蛋白质组学实验表明在1024种鉴定蛋白中，表达量显著差异蛋白数目45种，其中上调蛋白14种，下调蛋白31种。发酵后大豆蛋白游离巯基含量降低，二级结构发生显著变化、疏水作用力增加，而氢键作用力降低，微观结构趋于致密，质地坚实。进一步采用模拟胃肠道消化对发酵前后大豆蛋白致敏性进行分析，结果表明，发酵后大豆蛋白致敏性在胃肠道消化后显著低于未发酵大豆蛋白，这可能是由于发酵导致大豆蛋白聚集体的生成引发了大豆蛋白消化行为的改变。该系列研究结果揭示了植物乳杆菌B1-6对大豆蛋白脱敏机制，为乳酸菌发酵脱敏技术提供有效的理论指导，对于脱敏大豆蛋白深层次的开发具有重要的理论和实践意义。