肠道微生物在大豆抗原蛋白影响大菱鲆肠道健康中的作用机制

Limited marine resources dictate the increasing use of plant proteins in formulated diets for farmed fish. However, anti-nutritional factor (ANF) in plant sources reduced the utilization efficiency of plant protein by fish. The gut tract of fish consists of a very complex and dynamic microbial ecosystem that is very important from a nutritional, physiological and pathological point of view. Although the presence of gut microbiota in fish has been recognized, little is known about the bacterial communities and their establishment, diversity and most importantly their role in fish gut health. Previous studies of the author suggested that certain amount of soybean antigen protein in diets of turbot caused intestinal autochthonous microbiota profiles and alteration of intestinal microbiota. Based on these results, this study is aimed to screen the intestinal microbies which are significantly influenced by the dietary soybean antigen protein, using analysis of metagenomics and traditional microbiological techniques. Subsequently, the present study is to investigate the influences of re-adding of these screened bacteria in soybean antigen protein -supplemented/unsupplemented diets on the intestinal health of turbot, including the intestinal mucosal barriers, the intestinal mucosal immune system and gut microbiota variation. The primary-cultured intestinal epithelial cells isolated from turbot is also used as in vitro model to investigate the physiological effects of screened bacteria in the present study. This study could elucidate the interactions among dietary anti-nutritional factors, gut microbiota and gut health to a certain extent. This study is significant for elucidating the mechanisms involved in the effects of plant protein sources on gut health as well as provided the theoretical and technological support for the exploitation of probiotics with specific roles, i.e., alleviating the anti-nutritional activities of plant protein.

植物蛋白源中的抗营养因子能够损害鱼类健康，是影响植物蛋白替代鱼粉效果的主要原因之一。肠道健康是鱼类机体健康的重要保证。申请人前期研究发现饲料中添加一定剂量的大豆抗原蛋白能够改变肠道菌群结构并破坏大菱鲆肠道健康。本项目拟在前期研究基础上通过宏基因组学分析和微生物技术对大菱鲆肠道中与饲料大豆抗原蛋白（分别研究大豆球蛋白和β-伴大豆球蛋白）显著相关的微生物（可能益生菌和可能致病菌）进行筛选和分离培养，并通过在含有大豆抗原蛋白的饲料中添加筛选的可能益生菌、在不含大豆抗原蛋白饲料中添加筛选的可能致病菌对大菱鲆进行投喂养殖实验并结合体外肠道上皮细胞模型来研究大豆抗原蛋白引起的肠道微生物变化对大菱鲆肠道健康的影响，以探讨大豆抗原蛋白对大菱鲆肠道健康的影响中肠道微生物的介导作用机制。该研究不但对阐明植物蛋白影响鱼类健康的机制具有重要意义，还可为水产饲料产业中特定功能益生菌的开发和利用提供理论和技术支持。

针对植物蛋白源中的抗营养因子所引起的肉食性鱼类肠炎的问题，以肉食性海水养殖鱼类大菱鲆作为研究对象。从大菱鲆肠道微生物中分离、筛选、鉴定和培养与大豆抗原蛋白显著相关的肠道微生物，再通过在体内体外的实验中进行试验，探究肠道微生物对大豆抗原蛋白引起的大菱鲆肠炎的作用及机制。项目的主要成果包括：从大菱鲆肠道微生物分离、筛选、培养和鉴定与大豆抗原蛋白显著相关的细菌，获得6株芽孢杆菌全基因组分析；通过体内体外实验研究发现其中具有巨大益生潜能的4株菌T20、R32、E62和E4均能增强大菱鲆肠道黏膜屏障功能，均能从不同分类水平上改善大菱鲆肠道的菌群组成和结构，显著降低因摄入大豆抗原蛋白（β-伴大豆球蛋白，7S）而显著升高的软壁菌门以及支原体属的相对丰度，同时提高芽孢杆菌属的相对丰度。在项目的执行中，还获得了以下研究成果：建立大菱鲆肠道上皮细胞培养方法并构建7S引起的大菱鲆体外肠炎模型；测绘了大菱鲆幼鱼肠道微生物的核心组成结构；并经过筛选发现柠檬酸、谷氨酰胺和丁酸钠均能够通过增强大菱鲆的肠道黏膜屏障功能和改善肠道菌群结构促进肠道健康进而提高大菱鲆对植物蛋白的利用率；同时体外研究发现谷氨酰胺及丁酸钠对7S诱导的大菱鲆肠炎的防治作用效果及机制有所不同。本研究不但对阐明肠道微生物在植物蛋白源影响鱼类健康的机制具有重要意义，还为水产饲料产业中特定功能益生菌和功能性添加剂的开发和利用提供了理论和技术支持。本项目相关成果已发表学术论文16篇，其中SCI收录论文14篇，中文核心期刊2篇。