高静压与温度协同诱导芽孢萌发的细胞与分子机制

Sterilization is one of the main food processing units, which can effectively control the formation of microbial toxins and chemical noxious substances formed during food heating processing. Spore killing is the most difficult issue in the low-acid food sterilization. Usually, the high-temperature sterilization (121℃) is used to kill the spores in food industry. However, the food quality and nutrition are destroyed, and some toxic substances may generate and accumulate. High hydrostatic pressure (HHP) processing is a commercial non-thermal sterilization technology, since it can better maintain the food flavor, taste and nutrition. However, the inactivation of bacterial spores by pressure alone is not possible. The commercial sterilization can be attained under HHP combined with high temperature (>110℃). Therefore, it is necessary to explore the novel technology to inactive bacterial spore. The previous studies indicated that germination of dormant spores is the first crucial step in the return of spores to vegetative growth. Hence, we proposed a new idea "inducing spore germination homogenously before killing them" in our preliminary studies, and we found a new method of killing spores (8-log killing effect) under lower pressure (200MPa) combined with lower temperature (80℃) and the addition of muropeptide from bacterial cell wall. In this proposal, we hypothesize that HHP and heat ("double shocks") have significant effects on the inner membrane microenvironment of spore and receptor activation. The cellular and molecular mechanism of "double shock" on collaborative spore germination will be systematically studied. A "double shock and double induce" theory of spore germination will be established. This study will provide a theoretical basis for finding a new killing spore technology with lower pressure and lower temperature, while controlling the formation of microbial toxins and chemical noxious substances during sterilization processing.

杀菌是食品加工过程中有效控制生物毒素和因高温引起化学有害物形成的核心单元。杀灭芽孢是低酸性食品杀菌技术最关键的难点和热点。传统的高温灭菌法（121℃）能够彻底杀灭芽孢，但会引起食品品质下降和有害物的形成。高静压技术是一种能产业化的非热杀菌技术，目前这种技术杀灭芽孢同样需要结合高温（>110℃）。申请人团队基于芽孢萌发途径假说，利用芽孢萌发的生物力，提出“先同步快速萌发，后全部杀灭”的新思路，发现高静压协同温度（“双激”）与细菌胞壁肽对芽孢具有约8个对数的杀灭效果，实现温度与压力大幅降低。本项目拟系统研究“双激”协同诱导芽孢萌发的细胞与分子机制，揭示“双激”对芽孢萌发信使、芽孢内膜受体微环境的形成及与受体激活的关系，构建“双激双诱”协同诱导芽孢萌发的理论体系，确立芽孢全部萌发的新靶点。为创建降温降压杀菌技术的革命性突破，实现加工过程中有害物的减少和生物毒素的有效控制奠定科学基础。

芽孢是一类细菌在营养缺乏条件下形成的休眠体，因具有极强的抗性而难以被杀灭，对食品安全产生重大威胁，因此成为食品工业中一个亟待解决的难题。萌发是芽孢复苏的起始阶段，该过程伴随着DPA的释放、芽孢核水化、皮层降解等生理过程，同时芽孢抗性消失更易被杀灭。本项目利用高静压和温度条件，结合细菌胞壁肽，实现8个对数芽孢的“先萌发，后全杀灭”效果。并进一步研究其萌发机制，发现温压处理可以促进PrkC蛋白的活性改变，增加胞壁肽进入芽孢内部的数量，提高芽孢萌发效率，实现温压结合胞壁肽促进芽孢萌发的协同作用。明确了PrkC激酶诱导芽孢萌发途径的互作蛋白，初步揭示其在RNA降解、群体效应、氧化磷酸化等生物学过程中起催化作用。阐明了磷酸酶YwlE蛋白的精氨酸磷酸化/去磷酸化修饰在萌发过程中的调控作用，发现了Tig蛋白磷酸化会破坏其与核糖体的结合从而影响芽孢萌发过程中的蛋白合成，SigA蛋白磷酸化则会影响芽孢萌发中的基因转录过程，而YwlE对Tig和SigA蛋白进行的精氨酸去磷酸化作用可以保证芽孢萌发中的转录和翻译正常进行。进一步确定了磷酸酶YwlE蛋白的互作蛋白，并进行功能研究，发现芽孢形成过程中RocG调控的谷氨酸代谢决定休眠芽孢中ATP的含量，从而对芽孢萌发能力起到调控作用。以上的研究结果阐明了温压结合胞壁肽诱导芽孢萌发的作用机制以及精氨酸去磷酸化在芽孢萌发途径中的分子机制。这完善了芽孢萌发机制的理论体系，为寻找芽孢萌发的新靶点，创新超高压杀灭芽孢技术提供理论基础和数据参考。