发酵肉制品中基于氮代谢阻遏效应的组胺形成机制和控制研究

Histamine is one of the most toxic biogenic amines and has been considered as a food safety problem in fermented meat products. Histamine accumulation in fermented meat products is closely related to the selective absorption of histidine by microorganism during fermentation and histidine uptake is strictly regulated by nitrogen catabolite repression. The absorption of amino acids is regulated through permease regulation in nitrogen catabolite repression.Differences in gene expression between the Gap1p and Hip1p in Debaryomyces hansenii will be detected real-time quantitative PCR and Western blotting.Construction of Hip1p- and Gap1p- deficient strains will be constructed by using gene knockout technology to detect the functions of the Gap1p and Hip1p permease. The key regulatory transcription factors of Hip1p and Gap1p permease will be determined by using microarray technology to clarify the regulatory mechanism in transcription of Hip1p and Gap1p permease. The ubiquitination detectionvector to examine the ubiquitination process of Hip1p and Gap1p will be constructed based on the bimolecular fluorescence complementation technology. The histamine formation mechanism based on nitrogen catabolite repression in fermented meat products will be clarified according to the research in function, expression and post-translational modification of permease in Debaryomyces hansenii. On this basis, ubiquitination sites Hip1p permease mutant strain will be constructed according to Hip1p permease ubiquitination sites to eliminate nitrogen catabolite repression. This work will contribute to provide new ideas and strategies for effective prevention and control the formation of histamine in fermented meat products.

组胺的形成与组氨酸的吸收受氮代谢物阻遏效应调控密切相关。氮代谢物阻遏效应对氨基酸吸收调控的第一步是对氨基酸透性酶的调控。本课题以Debaryomyces hansenii为研究对象，研究组氨酸诱导下Gap1p和Hip1p基因与蛋白表达差异，确定负责转运组氨酸的透性酶；构建Hip1p-和Gap1p-缺陷菌株，研究Gap1p和Hip1p透性酶的功能；基因芯片技术确定Gap1p和Hip1p透性酶转录的关键调控因子，阐明透性酶转录水平的调控机制；双分子荧光互补技术构建泛素化检测载体，筛选与组氨酸代谢阻遏效应密切的泛素化修饰蛋白。从透性酶功能、表达和翻译后修饰的角度，揭示Debaryomyces hansenii对组氨酸的氮代谢物阻遏效应及调控机制，阐明发酵肉制品中基于氮代谢阻遏效应的组胺形成机制。构建透性酶泛素位点突变菌株，解除组氨酸氮代谢物阻遏效应，为从源头上消除发酵肉中组胺的产生提供新途径。

课题组紧密围绕发酵肉制品中基于氮代谢阻遏效应的组胺形成机制和控制开展研究工作。基于组胺合成调控的全局性和复杂性，课题组不局限于申报书中原定氨基酸转运蛋白，而是从碳代谢、氮代谢和能量代谢等途径全面解析氮代谢阻遏效应系统生物学解析和调控机制。通过四年研究工作，完成了申报项目-“发酵肉制品中基于氮代谢阻遏效应的组胺形成机制和控制研究”预定研究目标和研究内容，获得较理想研究成果：. （1）基于高通测序技术和传统微生物分离鉴定技术揭示发酵肉制品中作为功能菌的Debaryomyce hansenula具有产组胺的能力;（2）基于转录组学技术和蛋白组学技术从基因水平和蛋白组学水平，系统解析D. hansenula氮代谢阻遏效应对组胺合成的调控机制，明确氮代谢途径对组胺合成调控作用，证实我们提出的“组胺形成是由酵母对组氨酸的氮代谢物阻遏效应引起的科学假设”；（3）证实组胺合成过程中转运蛋白的重要性；（4）揭示D. hansenula的组氨酸脱羧酶体系属于磷酸吡哆醛依赖型；（5）揭示TORC1复合体控制组氨酸代谢，组胺合成代谢调控途径中，依赖泛素蛋白酶解途的氨基酸转运蛋白是重要关键调控位点；（6）基于基因敲除技术的D. hansenula工程菌的构建，验证依赖泛素蛋白酶解途的氨基酸转运蛋白是重要关键调控位点，提升发酵肉品食用安全性。. 通过项目实施，在国内外期刊上发表论文19篇，其中SCI 7篇，EI 3篇，中文核心期刊8篇；授权国家发明专利1项。项目成果-“微生物靶向调控生物胺合成技术”和“基于基因敲除技术构建D. hansenula工程菌”分别应用于发酵香肠和黑豆酱油酿造，显著提升发酵香肠和黑豆酱油品质和食用安全性，为四川高金实业集团股份有限公司、成都国酿食品有限责任公司、四川新希望味业集团等企业带来良好的经济效益，且两项成果分别荣获四川省科技进步奖二等奖和四川省食品工业科技进步奖一等奖。