CcpA调控抗冻干胁迫保加利亚乳杆菌应激蛋白合成机制的研究

乳酸菌冻干过程中受多重胁迫作用导致菌株活力丧失、死亡仍是制约我国乳酸菌发酵剂生产的瓶颈。项目前期工作已明确经氯化钠刺激后的保加利亚乳杆菌抗冻干胁迫能力显著提高，提高糖代谢途径所涉及的关键酶(PFK,PYK，LDH)表达水平是其抗冻胁迫能力提升的重要因素，并发现CcpA是糖代谢酶及相关代谢重要调控因子。本项目采用同源双交换法构建的CcpA缺失株，通过HPLC法测定代谢组分，通过i-TRAQ技术和qRT-PCR分析糖代谢关键酶、盐应激蛋白的变化及相关基因表达水平，结合相关蛋白活性和基因转录水平数据，明确代谢物差异；同时比对研究控氧和盐刺激协同作用下以上代谢变化对乳杆菌抗冻干能力的影响，利用生物信息学和相应数据库对蛋白和转录结果进行分析，结合CcpA-cre位点相互作用研究，共同揭示出基于CcpA调控糖代谢关键酶表达来提高乳杆菌抗冻干胁迫能力的机制，为进一步明确乳酸菌抗胁迫的机制提供理论支持。

Lactic acid bacteria strain will result in loss of vitality and even cell death under freeze-dried process with osmotic pressure stress ,that is still the bottleneck for Chinese DVS industry. The preliminary project has clearly gived that anti-freezing dry stress capacity of Lactobacillus bulgaricus was significantly increased after stimulated by sodium chloride ,and at the same time , which key enzymes involved in glucose metabolic pathways of lactobacillus have a direct adjustment to anti-freeze-drying capcity,and the CcpA (catabolite control protein) is an important regulatory factor for sugar metabolic enzymes to enhance the anti-stress ability of Lactobacillus under freeze-drying.In this project, we will construct CcpA mutant strains by homologous pairs of exchange method, determine the metabolic components by HPLC method, analyze the key enzymes and the expression level of fluorescence two-dimensional difference gel electrophoresis and changes of enzyme by qRT-PCR technology. Combining with metabolic changes, the relevant protein activity, and gene transcription level results, clear intermediate and final metabolites. Research that anti-freeze-dry ability of Lactobacillus bulgricus was also preformed under oxygen control and salt stimulation. Bioinformatics tools and the corresponding database is used to analyze the result of the proteins and transcription, revealing the expression of key enzymes of glucose metabolism regulation mechanism based on CcpA to improve the anti freeze tolerance of Lactobacillus,in order to further clarify the anti-stress mechanism of lactic acid bacteria to provide theoretical support.