胆盐水解酶提高乳酸菌肠道存活力的作用机制研究

To improve the survival of lactic acid bacteria in gastrointestinal (GI) tract is a pressing issue that needs to be addressed in the research and application of lactic acid bacteria. In this study, the survival of the lactic acid bacteria will be studied by cloning and expressing bsh gene into lactic acid bacteria. As our preliminary work showed that BSH was capable of enhancing the survival of lactic acid bacteria in GI tract. Further study revealed that there was no improvement on the bile tolerance of BSH recombinant strains, however, the adherence of BSH recombinant strains to Caco-2 cells was enhanced significantly, which suggested that adherence played an important role in the above-mentioned function of BSH. This was a new finding. Based on our previous results, an in-depth investigation will be carried out on BSH recombinant strains: their survival in the GI tract of the mice will be studied through murine experiments; their adhesion properties will be characterized using Caco-2 cell model; the difference on cell wall protein expression will be studied via comparative proteomic analysis. This study, if carried out smoothly, will enable us to identify the adhesin, which is associated with BSH expression, and its pattern of variation; furthermore, it will reveal the molecular mechanisms of BSH gene’s function to enhance adherence of lactic acid bacteria to intestinal cells. This will lay a good foundation for systematically elaborating the mechanism of BSH’s capability to improve the GI survival of lactic acid bacteria. It will also provide a theoretical basis for the development and utilization of lactic acid bacteria in food industry.

提高乳酸菌肠道存活力是乳酸菌研究及应用中迫切需要解决的问题。本项目将bsh基因克隆重组至乳酸菌中进行研究，前期预实验初步证明BSH具有提高乳酸菌肠道存活力的作用。进一步研究显示BSH重组菌的胆盐耐受力没有提高，但是与Caco-2细胞黏附性明显增强，推测后者是上述作用的重要机制，这是一个新的发现。在此基础上本项目将以BSH重组乳酸菌为对象进行深入研究：通过动物实验研究其在小鼠肠道内存活情况；通过Caco-2细胞模型研究其黏附特性；通过比较蛋白质组学研究其细胞壁蛋白表达差异性。本项目的顺利进行将探明与BSH表达相关的乳酸菌黏附素及其变化规律，揭示BSH增强乳酸菌与肠道细胞黏附性的分子机理，为系统阐述BSH提高乳酸菌肠道存活力的作用机制奠定基础，也为生产上乳酸菌的开发利用提供理论依据。

提高乳酸菌肠道存活力是乳酸菌研究及应用中迫切需要解决的问题。本项目将小鼠肠道来源的bsh基因克隆重组至乳酸菌中进行研究，动物实验结果显示BSH重组菌在肠道存活时间高于空载对照组，说明了BSH具有提高乳酸菌肠道存活力的作用。进一步研究显示BSH重组菌与Caco-2细胞黏附性明显增强，推测后者是上述作用的重要机制。随后利用iTRAQ蛋白质组测序方法研究了BSH重组乳酸菌由BSH表达影响的差异蛋白表达情况，本项目采用iTRAQ蛋白质组测序研究BSH重组乳酸菌蛋白表达情况，共鉴定到 1712 个蛋白，定量结果中发现的存在显著差异的蛋白数为 214（B_L:400_L）和251（B_H:400_H）。其中，BSH表达差异蛋白在 “Metabolic pathways”、“Microbial metabolism in diverse environments”、 “ABC transporters”、 “Ribosome” 和“Two-component system”等功能蛋白方面存在差异。其中， “Metabolic pathways” 功能蛋白的差异最大，上调和下调的蛋白分别占61.9% 和 77.14% 。更重要的是，对差异蛋白结果分析发现由BSH表达影响的23个蛋白差异显著，其中多个粘附蛋白上调。本项目按照计划进行，进展顺利，研究结果有效揭示了BSH增强乳酸菌与肠道细胞黏附性的分子机理，为系统阐述BSH提高乳酸菌肠道存活力的作用机制奠定基础，也为生产上乳酸菌的开发利用提供理论依据。