植物乳杆菌的肠道示踪及其对肠道菌群的调节机制

Lactic acid bacteria (LAB) is generally regarded as safe and plays important roles in ameliorating intestinal disorders, improving intestinal immunity and reducing the risk of cardiovascular diseases. Previous studies were focused on the changes of physiology, disease and gut microbiota of the host. However, very little is known about the fate of ingested LAB and about the systematic and mechanism knowledge on the distribution and quantities of ingested LAB and its regulatory mechanism on the gut microbiota. In this present project, Lactobacillus plantarum ST-III with clear genetic background and whole genome were selected as the objects of study. Green Fluorescence Protein (GFP) tracing and q-PCR techniques were combined to study the distribution and quantities of ST-III in the gut and determine the key factors by measuring the biochemical and immune parameters. Based on the characteristic of fructooligosaccharides (FOS) utilization, the mechanisms of ST-III on the composition of gut microbiota were performed through 16S rDNA metagenomics sequencing. The results will help us to further realize the physiological basis of LAB activities in the gut and provide the theoretic foundation for the functional evaluation of LAB and this project is very important in theory and practice.

乳酸菌是公认安全的微生物，对人体具有调节肠道紊乱、改善肠道免疫及降低心血管疾病风险等多种生理功能。既往的研究多关注宿主生理、疾病以及菌群等的变化，而乳酸菌进入宿主后的“命运”则研究很少，对乳酸菌在肠道内的分布、数量及调节肠道菌群的机制缺乏系统性和机理性的认识。本项目以遗传背景清晰、完成全基因组测序的植物乳杆菌ST-III为研究对象，采用绿色荧光蛋白GFP示踪结合q-PCR技术研究ST-III在肠道内的分布和数量规律，并通过测定生化和免疫指标解析关键影响因子；根据利用低聚果糖（FOS）的特性，采用16S rDNA宏基因组测序技术从FOS利用角度解析ST-III对肠道菌群的调节机制。研究结果有助于进一步认识乳酸菌在肠道内发挥作用的生理基础，并为乳酸菌的功能性评价与开发提供理论基础，具有重要的理论和实践意义。

植物乳杆菌具有多种生理功能，目前研究多关注它们对宿主的影响，对其进入宿主后的“命运”研究较少。本项目对植物乳杆菌进行绿色荧光蛋白（GFP）标记，采用GFP示踪技术分析植物乳杆菌在小鼠肠道内的分布和定殖情况，并结合16S rRNA宏基因组测序技术，从FOS利用角度探讨植物乳杆菌对小鼠粪便菌群的影响。.主要结果如下：.（1）对5株植物乳杆菌进行GFP标记，发现GFP标记不影响植物乳杆菌的耐酸、耐胆盐、耐低温、耐抗生素、FOS利用能力等生理特性。.（2）采用GFP示踪分析植物乳杆菌ST-III-GFP在小鼠肠道内分布，发现：灌胃30min后，ST-III-GFP的活菌检出率为47.6%，主要分布在小肠（99.8%）；灌胃1.5h后，检出率为29.1%，主要分布在小肠（37.0%）、盲肠（23.5%）和结肠（39.5%）；灌胃4h后，检出率为12.3%，主要分布在盲肠（34.6%）和结肠（61.2%）；灌胃8h后，检出率为10.6%，主要分布在盲肠（44.4%）和结肠（48.0%）。.（3）采用GFP示踪及活菌计数，发现给小鼠灌胃植物乳杆菌ST-III-GFP两周后，ST-III-GFP在小鼠肠道中的定殖水平稳定（5.1×109 CFU/g），回肠、盲肠和结肠分别定殖15.7%、18.8%和65.5%；FOS显著提高ST-III-GFP在小鼠肠道中的定殖速率，显著提高ST-III-GFP在回肠（24.1%）和盲肠（32.6%）的定殖数量。.（4）给小鼠灌胃植物乳杆菌ST-III四周后，16S rRNA宏基因组测序发现，小鼠粪便中厚壁菌门相对丰度显著降低，ST-III结合FOS同时干预后，小鼠粪便中乳杆菌属和双歧杆菌属的相对丰度显著增加，粪便中短链脂肪酸的含量也显著增加。.本项目研究揭示了植物乳杆菌进入宿主后的分布与定殖情况，为乳酸菌的功能性评价与开发提供理论基础。