保加利亚乳杆菌CH9调控CEP生物合成影响发酵乳凝胶性质的关键因子和途径分析

During yogurt fermentation, cell wall proteinase (CEP) of Lactobacillus delbrueckii subsp. bulgaricus was necessary for the growth of Streptococcus thermophilus and the rate of acid production. In our previous work, it was indicated that the action regularity of the hydrolysis ability and purified CEP of Lactobacillus delbrueckii subsp. bulgaricus were different for gel properties of fermented milk. In the present study, PrtB gene and LPKKT gene knockout mutant strains of Lactobacillus delbrueckii subsp. bulgaricus CH9 will be constructed by double-crossover homologous recombination for three existing form models of CEP, which were envelope-associated, released, and purified, respectively. The role of different existing form of CEP playing in effect of Lactobacillus delbrueckii subsp. bulgaricus CH9 on the fermentation process will be analyzed by comparing difference between the wild and mutant strains. Mechanisms of regulation of CEP biosynthesis in Lactobacillus delbrueckii subsp. bulgaricus CH9 improving gel properties of fermented milk will be explained systematically through investigating growth metabolism, protein metabolism and the expression level of proteolytic system by using biological chemistry and molecular biology technologies. This project will provide a theoretical basis for the mechanisms controlling the regulation of PrtB biosynthesis and the key factors of the regulation at the transcriptional level, and lay a theoretical foundation for the research and application of CEP for quality improvement of fermented dairy products.

酸奶发酵过程中，保加利亚乳杆菌细胞壁蛋白酶（CEP）直接影响嗜热链球菌的生长和发酵产酸速度。前期研究发现，保加利亚乳杆菌蛋白水解能力和其纯化CEP活力对发酵乳凝胶性质影响的作用规律不同。在此基础上，本项目拟采用同源序列双交换构建PrtB-突变株和LPKKT基因阻断株，构建保加利亚乳杆菌CH9菌株CEP绑定于细胞壁、游离、提取纯化三种状态模型，分析突变株发酵乳的凝胶性质，揭示CEP影响发酵过程的作用规律；同时基于生化和分子水平，分析CEP生物合成对保加利亚乳杆菌生长代谢、蛋白质代谢和蛋白水解系统转录表达的影响，明确在转录水平调控CEP生物合成影响发酵乳凝胶性质的确切途径。本项目将为研究PrtB生物合成的调控机制及在转录水平调控的关键因子提供理论依据，为研究CEP影响发酵乳品质的作用机制及CEP的应用奠定理论基础。

保加利亚乳杆菌细胞壁蛋白酶(CEP)直接影响酸奶发酵过程中嗜热链球菌的生长和发酵产酸速度，其蛋白水解能力和其纯化CEP活力影响发酵乳凝胶性质。为研究保加利亚乳杆菌CEP影响乳发酵的作用机制，在本研究中，通过PCR获得了保加利亚乳杆菌CH9的细胞壁蛋白酶基因序列，并进行了生物信息学分析，该基因序列所对应的蛋白酶是亲水且稳定的胞外蛋白；利用5 mol/L LiCl提取获得了保加利亚乳杆菌CEP，大小为190 kD，通过质谱鉴定验证所提取的酶来自保加利亚乳杆菌细胞壁；利用提取的CEP水解脱脂乳中的酪蛋白效果明显；以葡萄糖酸-δ-内酯（GDL）酸化乳为模型研究CEP对酸化乳凝胶性质的影响，发现CEP水解可以改善凝胶质构，降低脱水收缩系数，促进凝胶过程。通过peptide-rich和peptide-deficient条件下保加利亚乳杆菌Labelfree 定量蛋白组和基于UHPLC-Q-TOF MS技术的代谢组分析，认为双组份系统SecY、Opp转移系统、群体感应LuxS、Peptidase S8（AT236_01166)、mtHSP70 proteaseDNA下调，参与保加利亚乳杆菌蛋白质代谢调控。