乳酸菌来源胞外囊泡对断奶仔猪道菌群的调控及分子机制

The intestinal tract is not only the main place for animal digestion and absorption, but also the largest immune organ and "bacterial library" in the body. Changes in intestinal microbiota are associated with a variety of intestinal diseases. Lactobacillus is the dominant intestinal microbiota, has a strong colonization resistance, can effectively balance or improve the composition of intestinal microbiota. Up to now, extracellular vesicles (EVs) were found in bacterial secretions. EVs contains small RNAs, mRNAs, proteins, etc., which may be a new information material transmitted by bacteria to the outside world. In this study, we hypothesize that lactobacillus EVs be able to regulate the intestinal microbiota balance. Firstly, the lactobacillus-derived EVs were isolated and identified, and the small RNAs in EVs were analyzed by high-throughput sequencing. Then EVs is used to feed the animals, and 16S rDNA sequencing was performed to analyze the changes of faecal microbiota. Finally, bioinformatics analysis and experiments were used to verify the regulatory effect of EVs small RNAs on intestinal microbiota and determine influence of EVs on intestinal microbiota and the mechanism of action. The expected results of this project can clarify the physiological function of lactobacillus-derived EVs on intestinal microbiota, and provide novel idea to improve piglet intestinal health.

肠道既是动物消化吸收的主要场所，也是体内最大的免疫器官和“细菌库”。肠道菌群的变化与多种肠道疾病有关。乳酸菌是肠道优势菌群，具有较强的定植抵抗力，可有效平衡或改善肠道菌群的组成。近来发现细菌分泌物中除已知成分外还含有胞外囊泡（Extracellular vesicles, EVs）。EVs中含有small RNAs、mRNAs、蛋白质等，可能是细菌向外界传递的一种新的信息物质。本项目提出乳酸菌EVs调控仔猪肠道菌群平衡的假设。首先对乳酸菌来源的EVs进行分离和鉴定，应用测序对EVs中small RNAs进行解析；然后通过对饲喂EVs动物粪便进行16S rDNA测序，解析菌群变化；最后使用生物信息学分析及试验验证EVs中small RNAs对猪肠菌群的调节作用，确定EVs对肠道菌群的影响及其作用机制。该项目的预期结果可以明确乳酸菌来源EVs对肠道菌群的生理功能，为改善仔猪肠道健康提供新思路。

本项目主要研究屎肠球菌来源的细胞外囊泡(Enterococcus faecium-Extracellular Vesicles, Efm-EVs)对动物肠道菌群的调节作用及机制。项目首先对Efm-EVs进行了分离鉴定，通过转录组、蛋白质组、代谢组方法对其活性成分进行了解析；其次通过小鼠灌胃示踪试验确定了Efm-EVs在动物体内的代谢和分布情况；最后分别通过小鼠活体试验及仔猪粪便培养试验研究了Efm-EVs对动物肠道微生物区系的影响。具体结果如下：.[1]通过超速离心的方法可以成功分离Efm-EVs；通过TEM及NTA对Efm-EVs进行鉴定发现Efm-EVs具有典型的圆形或杯形外观，Efm-EVs平均大小为94.34 nm， 粒径主峰为188.5 nm， 20-200nm占比为50.2%。.[2]通过转录组学、蛋白质组学、代谢组学对Efm-EVs进行组成成分解析；分别鉴定得到了2122种RNAs分子、711种蛋白质分子和519种代谢产物。.[3]通过标记示踪，灌胃后Efm-EVs主要分布于小鼠的消化道内。.[4]小鼠活体试验结果显示，灌胃Efm-EVs不影响动物的生长性能，显著提高了小鼠肠道疣微菌科UCG009（Ruminococcaceae_UCG_009）的丰度。.[5]仔猪粪便培养结果显示，添加Efm-EVs不影响粪便pH，而显著提高了粪便菌群的数量；添加Efm-EVs显著提高了丁酸和异戊酸的水平，显著降低了戊酸的水平；添加Efm-EVs显著提高了g_[eubacterium] eligens_group丰度，降低了g_Escherichia_Shigella丰度。.综上所述，Efm-EVs中富含RNA、蛋白质、代谢产物等生物活性成分，通过口服给药后Efm-EVs主要在动物的消化道中分布，Efm-EVs主要通过靶向肠道疣微菌科和肠杆菌科调节肠道菌群结构，并最终调节了挥发性脂肪酸的产生。