大豆抗原蛋白对大菱鲆肠道微生态及机体免疫力的影响

Aquaculture is the fastest growing food production sector of the world. To sustain such high rates of increase in aquaculture production, a matching increase in the levels of production of fish feeds is required. Limited marine resources dictate the increased use of plant proteins in formulated diets for farmed fish species. However, plant anti-nutritional factor (ANF) is one of the main reasons that reduced growth and damaged the health of 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 nutrition and health.It has been demonstrated that the growth and health of animals was largely influenced by the gut microbiota of fish. Because conventional characterization of microorganisms has depended on cultivation-based techniques, our understanding of the gut microbiota is restricted to those that can be cultured. Most of recent studies have used molecular technology for the analysis of microbiota present in the intestine of animals, but there still remain doubts about the complete microbial composition. Metagenomics is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms, which reveals the previously hidden diversity of microscopic life, and offers a powerful lens for viewing the microbial world. High throughput sequencing of 16S rDNA amplicons is a more efficient method to analyze the composition, distribution, and abundance of intestinal microbiota, and thus a more efficient method to investigate the profiling of gut microbiota. The present study is to investigate the effects of one of the main anti-nutritional factors in plant protein source, soybean antigen protein, on the profiling of gut microbiota,intestinal immunity and immunity of turbot, one of the most important marine culture species in China, using high throughput sequencing analysis of 16S rDNA. Furthermore, to expound the relationship among gut microbiota variation, intestinal immunity and health of fish. Thus, the mechanisms of effects of plant ANF on fish growth and health could be elucidated based on effects of different dietary soybean antigen protein (glycinin and β-conglycinin) levels on gut microbiota composition, amount and distribution of turbot, as well as effects of different dietary soybean antigen protein levels on histology and expression of immune related genes of turbot intestine. This study is significant for elucidating the mechanisms of effects of plant protein sources on fish growth and health.

植物蛋白源中的抗营养因子能够抑制鱼类正常生长并损害鱼类健康，是影响植物蛋白替代鱼粉的主要原因之一。动物肠道微生态平衡是机体正常生长和健康的重要保证。16S rDNA宏基因组高通量测序技术能更全面真实地反映肠道微生物群体的物种组成、分布及丰度信息，从而更高效准确地探究动物肠道菌群结构。前期研究中发现饲料中添加大豆抗原蛋白投喂大菱鲆能引起鱼体肠道组织形态出现病理性症状。本项目以大菱鲆为研究对象，以大豆抗原蛋白作为抗营养因子代表，基于16S rDNA宏基因组高通量测序分析技术，研究饲料中不同添加量的大豆抗原蛋白（大豆球蛋白、β-伴大豆球蛋白）对大菱鲆肠道菌群种类组成、分布和丰度等的影响，并结合肠道免疫相关基因表达、肠道组织形态学研究以及鱼体非特异性免疫力的检测，从肠道微生态的角度探讨植物蛋白源中抗营养因子对鱼体生长和健康的影响，对阐明植物蛋白源影响鱼类生长和健康的机制具有重要意义。

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。动物肠道微生态平衡是机体正常生长和健康的重要保证。而16S rDNA高通量测序技术能更全面真实地反映肠道微生物群体的物种组成、分布及丰度信息，从而更高效准确地探究动物肠道菌群结构。本项目基于以上研究背景选择主要抗营养因子大豆抗原蛋白（包括β-伴大豆球蛋白和大豆球蛋白），以重要海水养殖鱼类大菱鲆为研究对象，通过分别在饲料中添加不同剂量的β-伴大豆球蛋白和大豆球蛋白，研究两种抗原蛋白对大菱鲆肠道微生态（包括肠道组织结构和肠道菌群结构）、生长性能和机体免疫的影响。主要研究结果如下：饲料中添加低水平的β-伴大豆球蛋白（2%）对大菱鲆的存活、生长性能、消化率以及肠道组织形态（形态观察和结构完整性相关基因表达）以及肠道菌群结构（门水平和属水平）均无显著影响，而且还具有一定的免疫增强剂效果。但是饲料中添加高水平β-伴大豆球蛋白（4%和8%）能够显著降低大菱鲆的生长性能、破坏肠道组织结构、引起肠道炎症反应并改变肠道菌群结构，在8%处理组变化尤为明显。但4%和8%处理组的大菱鲆摄食率反而显著升高，可见β-伴大豆球蛋白主要是通过对肠道微生态平衡的破坏而降低饲料利用率进而抑制生长。与β-伴大豆球蛋白相比，大菱鲆对大豆球蛋白的敏感性要弱一些。只有在饲料中添加12%的大豆球蛋白显著破坏肠道组织结构、引起肠道炎症反应并改变肠道菌群结构。综上，在应用豆粕替代鱼粉时抗原蛋白尤其是β-伴大豆球蛋白的含量必须严格控制，并且这种营养抗性作用主要是通过破坏肠道微生态进而抑制生长和破坏机体免疫。本项目首次比较系统的研究纯化的抗原蛋白对海水养殖肉食性鱼类的生长、机体免疫和肠道微生态进行研究，尤其是从肠道微生态的角度探讨植物蛋白源中抗营养因子对鱼体生长和健康的影响，发现肠道微生态在豆粕替代鱼粉中的重要作用，这对阐明植物蛋白源影响鱼类生长和健康的机制具有重要意义。此外，本项目为筛选和培养可能益生菌提供了研究基础，这为阐明植物蛋白在养殖鱼类中的营养抗性作用机理，为鱼类饲料中提高植物蛋白利用效率的相关营养策略的开发（如具相关特定功能的益生菌的开发）提供理论依据。