蔬菜发酵乳酸菌降解亚硝酸盐相关基因分析及增强降解机制

When involved in the vegetable fermentation, lactic acid bacteria (LAB) initiate nitrite depleting due to the impact of vegetable matrix and fermentation conditions. Up to date, molecular mechanism of nitrite depletion with lactic acid bacteria in vegetable fermention is rarely reported, reuslting in its being the one of the hot and difficult point in studying safty of traditional fermented food. To ascertain the molecular modeling of special metabolic behavior of lactic acid bacteria in vegetable fermentation, we plan to improve the promoter trap technology to screen out the inducing expression gene of LAB in fermentation vegetable, overcoming the shortcomings of the interference of the constitutive promoter and loss of down-regulated genes. Moreover, we screen out genes and promoters related to nitrite depleting metabolism, enhance gene expression of nitrite reductase , and disclose the theoritical basis for enhancing nitrite depletion and optimizating the vegetable fermentation processing, strain breeding and raw material controlling, according to the effect of nitrite depletion on gene regulation expression. Since the biotechnology level of Chinese traditional vegetable fermentation is backward, this project will provide foundation for fermention vegetable further processing. It will also provide research models and new ideas for the study of the interaction between microbiology and traditional fermented food matrix.

受蔬菜基质和发酵条件等因素的影响，乳酸菌参与蔬菜发酵降解亚硝酸盐时，代谢途径受到多重基因调控，产生不同的降解效果。乳酸菌中降解亚硝酸盐代谢的分子机理研究目前鲜见报道，成为提升蔬菜发酵食品安全性研究的难点和关键问题之一。为确定蔬菜发酵中乳酸菌降解亚硝酸盐代谢行为的分子作用背景，本项目拟改进启动子陷阱技术，克服组成型启动子干扰和不能筛选下调表达基因的缺点，筛选乳酸菌在蔬菜发酵降解亚硝酸盐过程中被诱导表达的基因，进而从中筛选与蔬菜发酵乳酸菌降解亚硝酸盐代谢相关的基因，捕获相关启动子，增强表达亚硝酸盐还原酶基因，研究影响这些基因调控表达的理化因素，阐明增强乳酸菌降解亚硝酸盐降解机制，为蔬菜发酵工艺优化、菌种选育及原料控制等提供理论依据。本项目将为蔬菜发酵深加工提供技术基础，也为微生物与食品基质(属传统发酵食品)的相互作用研究提供研究模式和新的思路。

研究如何降低或消除发酵蔬菜中已有亚硝酸盐，提高食用安全性，促进发酵蔬菜产业的健康、持续发展，具有重要的理论意义和应用价值。.项目筛选鉴定出蔬菜发酵过程中降解亚硝酸盐的优势乳酸菌株，以条件致死基因和抗性基因作为双报告基因，改进现有启动子陷阱技术，筛选降解亚硝酸盐相关基因。为揭示亚硝酸盐在乳酸菌中吸收转运、NIR还原降解过程的多基因调控机理，增强蔬菜发酵降解亚硝酸盐工艺提供理论依据。.课题为1年期资助课题，课题组在资金、时间紧的条件下，完成了以下研究：.1、环境因素诱导乳酸菌降解亚硝酸盐含量动态分析.研究了不同贮存条件对蔬菜中亚硝酸盐含量的影响、不同种类蔬菜含亚硝酸盐量，阐述了蔬菜自身以及贮存因素对亚硝酸盐含量的影响。对发酵中菌群变化进行分析以总菌落数、大肠杆菌菌落数、乳酸菌菌落数为指标，分析自然发酵中菌群变化与泡菜亚硝酸盐含量的关系。进行了分离乳酸菌的鉴定，得到需要的优势降解菌株，为后续诱变育种提供基础材料。.2、降解亚硝酸盐菌株的60Co 射线诱变选育.课题组在中国工业微生物中心购买了三株菌：Lactobacillus plantarum CICC20765、Leuconostoc mesenteroides CICC21859及Lactobacillus brevis CICC6239。设计诱变方案分别对以上三株菌进行60Co 射线诱变选育，根据致死率和正突变率选取最佳诱变剂量反复筛选得到降解亚硝酸盐优势菌株，对诱变前后生长量、降解亚硝酸盐特性、亚硝酸盐还原酶的酶活进行对比研究。为分离乳酸菌降解亚硝酸盐相关基因提供出发菌株。.3、利用启动子陷阱技术构建亚硝酸盐降解过程中相关基因表达的启动子陷阱文库.以肠膜明串珠菌为受体细胞，提取肠膜明串珠菌基因组DNA，并酶切成0.5 - 1.5的片段，插入启动子陷阱载体的erm GT和URA3基因的上游，重组质粒PHY300plk，构建启动子陷阱载体转化大肠杆菌扩增重组质粒，提取重组质粒电转化肠膜明串珠菌，挑取阳性克隆测序分析，即得到肠膜明串珠菌的启动子陷阱文库。利用显色反应筛选出文库中含有诱导表达降解亚硝酸盐相关基因（片段）的克隆子。.4、课题发表研究论文1篇。