基于非酸非酶类物质及NirB调控的发酵乳杆菌RC4降解亚硝酸盐机理研究

亚硝酸盐含量超标已成为传统腌制食品的首要安全问题。乳酸菌能有效降低腌制食品的亚硝酸盐含量，但其降解方式和关键酶的调控机制尚不明确。前期研究筛选获得高效降解亚硝酸盐的发酵乳杆菌RC4，发现其除已知的酸降解、酶降解外，还存在一种新的方式，即非酸非酶类物质降解；且酶降解方式中的NADH亚硝酸盐还原酶（NirB）可能是关键酶。本课题拟通过代谢组学检测RC4代谢产物，并进行验证实验，以确定其非酸非酶类物质的组成、含量和作用方式。同时，构建NirB与绿色荧光蛋白的融合表达蛋白，研究NirB的亚细胞定位和运输途径；在RC4中过表达、敲除、回补nirB基因，测定菌体的亚硝酸盐降解率和生理特性的变化，以明确NirB的功能；进一步通过分析蛋白质组学和氮代谢途径的改变，揭示NirB调控亚硝酸盐降解机制。从非酸非酶类物质及NirB方面，系统解析乳酸菌降解亚硝酸盐机理，为生产低亚硝酸盐含量腌制食品提供理论依据。

Excessive nitrite content has become primary safety issue for traditional preserved foods. Lactic acid bacteria can effectively reduce the nitrite content of preserved foods, but the mechanisms of nitrite degradation are not clear. Lactobacillus fermentum RC4 with high nitrite degradation ability were screened in our previous work. A new non-acid non-enzymatic substances degradation mode was found besides the known acid degradation and enzymatic degradation. The metabolites of RC4 will be detected by metabolomics and validation experiments to determine the composition, content of non-acid non-enzymatic substances. The key enzyme for nitrite degradation, NADH nitrite reductase NirB, will be constructed and expressed by fusion with green fluorescent protein to detect the subcellular localization and transport pathway of NirB. Moreover, the overexpression, knock out and replenishment for the nirB gene in RC4 will be constructed and the nitrite degradation rate and physiological characteristics in combined or wild bacteria will be investigated to clarify the function of nirB. Proteomic changes and nitrogen metabolic transformation will be detected to analyze the regulation mechanism of NirB during nitrite degradation. The aim of this project is to systematically analyze the mechanism of degrading nitrite in RC4, which will provide theoretical guidance for the application of lactic acid bacteria in nitrite degradation for preserved food.