为研发低致敏酱油,探究了低盐固态酱油酿造过程中大豆蛋白致敏原的变化规律。在建立实验室的模拟低盐固态酱油酿造的基础上,采集大豆未经处理、高压灭菌(121 ℃,8 min)、制曲阶段(44 h)、发酵阶段(30 d)、灭菌前和灭菌后等样品,利用聚丙烯酰胺凝胶电泳法(SDS-PAGE)测定酿造过程中蛋白的组成变化,用兔抗大豆多克隆抗体进行酶联免疫试验和免疫印迹实验分析发酵过程中大豆致敏原抗原性变化,用过敏病人血清进行酶联免疫试验测定大豆致敏原过敏原性变化。结果表明,经高压蒸煮、制曲、发酵和加热灭菌后,原料中的大豆蛋白条带减少或消失,其中制曲变化的最大。β-伴大豆球蛋白的α、α'亚基和大豆球蛋白的酸性亚基分别在制曲阶段开始降解,β-伴大豆球蛋白的β亚基及大豆球蛋白酸性亚基在制曲之后消失,大豆球蛋白的碱性亚基在整个酿造阶段变化不大。免疫印迹结果显示相同的结果,酿造过程中大豆过敏原的过敏性和抗原性逐渐降低,在发酵30 d后的生酱油中仍能在检测到大豆球蛋白,在灭菌后也没有完全降解,但是这些残留的大豆致敏原没有检测到IgE结合能力。酶联免疫试验结果表明,和原材料大豆相比,样品中的抗原性在经过4个酿造阶段高压蒸煮、制曲、发酵和加热灭菌之后分别下降了8.13%、39.00%、69.10%和87.06%,过敏原性分别下降了8.92%、71.66%、92.26%、98.45%。在酱油酿造过程中大豆致敏原逐步降解,制曲阶段对大豆致敏原的降解最大。酱油中仍残留有大豆球蛋白,但是没有检测残留蛋白的致敏性。
This study explored the degradation of soy allergen in the process of low-salt solid soy sauce brewing. Based on the establishment of a simulated low-salt solid soy sauce brewing laboratory,the samples of untreated soybean,autoclaved(121 ℃,8 min),koji-making stage(44 h),fermentation stage(30 d),pre-sterilization and sterile zation were taken. The composition of proteins in the brewing process was measured by polyacrylamide gel electrophoresis,and rabbit anti-soy polyclonal antibodies were used to perform enzyme-linked immunoassay and immunoblot experiments to analyze the changes in soybean allergen antigenicity during fermentation. SDS-PAGE results reflected that after autoclaved,koji,fermentation and heat sterilization,the soy protein bands in the raw materials decreased or disappeared and the variation of the koji was the largest in the four stage. The α and α' subunits of β-conglycinin and acidic subunits of glycinin begin to degrade during the koji making stage,the β subunit of β-conglycinin and glycinin acidic subunits disappeared after the koji,the basic subunits of glycinin did not change much throughout the brewing stage. The results of immunoblotting showed the similar results. The allergenicity and antigenicity of soybean allergens gradually decreased during the brewing process. After 30 days of fermentation,glycinin can still be detected in raw soy sauce,and was not completely degraded after sterilization,but these residual soybean allergens did not detect IgE binding capacity. The results of enzyme-linked immunosorbent assay showed that,compared with the raw material soybean,the antigenicity in the sample decreased by 8.13%,39.00%,69.10%,and 87.06% after high-pressure cooking,koji making,fermentation,and heat sterilization,respectively. The allergenicity decreased by 8.92%,71.66%,92.26%,and 98.45%,respectively. Soybean allergens gradually degraded during the soy sauce brewing process,the koji-forming stage had the greatest impact on the degradation of soybean allergens,Glycinin remained in the soy sauce,but the sensitization of the residual protein was not detected.