益生乳杆菌进入VBNC态过程中的抗逆机理研究

Lactic acid bacteria would inevitably encountered varieties of adverse conditions during the process of industrial production or entering the human gastrointestinal system, which caused some Lactic acid bacteria entered into the Viable but non-culturable (VBNC) state. However, the study on the mechanism of the formation of the VBNC state was not clear. In our study, Lb. casei Zhang was selected to observe, which had excellent probiotic characteristics. The strain was firstly cultivated in different stress conditions (e.g. low temperature, low pH value, and nutrient deficiencies), and the induction and detection of VBNC cells were examined. Differential expressions in key genes of Lb.casei Zhang during the induction period were analyzed by RNA-seq and quantitative PCR technologies. At the same time, the iTRAQ technology was used to compare the difference in protein composition between the normal cells and VBNC cells, and the differential expression proteins in key genes of Lb. casei Zhang during the induction period were analyzed ulteriorly, And the structure, properties and functions of the deferentially expressed genes were predicted. Fatty acid composition in cell membrane would be analyzed by GC. Finally, the key genes of lactic acid bacteria to maintain the activity would be obtained. On the above base, which will improve the fermentation activity of lactic acid bacteria and provide a scientific basis or heoretical for the development and utilization of lactic acid bacteria.

乳酸菌在生产应用过程中不可避免的要耐受各种环境压力，有些乳酸菌在不利自身生长条件下进入仍具有活力的“活的非可培养态”(Viable but Nonculturable, VBNC)，目前对其抗逆机制并不清楚。本研究以益生乳杆菌 Lb. casei Zhang为对象，通过在低温、低pH、营养缺乏等逆境条件下对其胁迫诱导，使其形成VBNC态，首先采用多种方法记录诱导过程中细胞活性和VBNC态细胞的变化；其次采用高通量RNA-seq转录组测序技术和RT-PCR技术分析Lb. casei Zhang进入低活力VBNC态过程中关键基因的表达变化；应用iTRAQ技术从蛋白组层面对比分析差异蛋白的变化；采用气相色谱检测诱导过程中细胞膜的脂肪酸变化，综合分析Lb. casei Zhang在进入VBNC态细胞维持低活性的抗逆机制。本研究将为益生乳杆菌的产品开发和提升乳酸菌活性奠定基础。

本项目将Lb. casei Zhang在生理盐水、低pH、低温、寡营养等多种胁迫条件下诱导VBNC态，通过多种非培养技术和平板计数方法结合检测其是否进入VBNC态，然后观察胁迫过程中的细胞形态变化、检测细胞膜脂肪酸变化；并通过转录组学、qPCR、蛋白组学及代谢组学等技术解析Lb. casei Zhang在逆境胁迫下的耐受机理。 . 基因表达方面，通过转录组学分析Lb. casei Zhang在低温低pH逆境胁迫下的基因表达差异及相关代谢途径；同时利用RT-PCR技术对差异表达基因进行分析，表达差异的12个基因主要涉及糖代谢、氨基酸代谢、细胞膜及脂肪酸以及应激蛋白合成等。 . 蛋白组学方面，Lb. casei Zhang VBNC态和正常态的差异蛋白显著富集在磷酸转移酶系统（PTS)、丙酮酸代谢、果糖和甘露糖代谢、丙氨酸、天冬氨酸和谷氨酸代谢等相关通路。. 代谢组学方面，利用气质联用仪分别分析 Lb. casei Zhang VBNC态与正常态、以及VBNC态在液体MRS培养基和脱脂乳中复苏的代谢物差异，均筛选出20多种主要差异代谢物，按类分析其作用机制。 . 细胞膜脂肪酸方面，建立了乳酸菌膜脂肪酸甲脂化提取方法，同时测定了VBNC态的脂肪酸组分和含量，并分析发现Lb. casei Zhang 通过增加不饱和脂肪脂肪酸含量增加细胞膜的流动性来抵抗逆境。 . 综上所述，Lb. casei Zhang进入VBNC态后通过磷酸转移酶系统（PTS）、丙酮酸代谢、ABC运输、以及氨基酸类、糖类、维生素类等变化来维持细胞的生存。这不仅为Lb. casei Zhang VBNC态的生存代谢提供一定的理论基础，也旨在提高益生乳酸菌在生产、贮藏及进入人体肠道系统过程中保持最高的活性提供新的思路。