桑叶清蛋白调节肠道菌群与改善机体免疫的相关性及机制

With the development of multi-omics, the relationship between diet, gut microbiota, and host immunity is attracting more and more attention. Studies showed that intestinal microorganism could mediate the interaction between protein metabolism and host immune response. However, studies on the regulation of immunity by dietary protein based on intestinal microbiota are rarely reported. And the key bacteria that mediate protein metabolism and host immunity are still unclear. Due to the nutritious, mulberry leaves are widely used as functional food. Our previous studies showed that albumin MP extracted from mulberry leaf could promote the secretion of immune factors, increase the diversity of gut microbiota, and promote the colonization of Lactobacilli. However, the key intestinal microbiota regulated by MP and its role in improving immunity need to be further studied. This project intends to analyze the effects of MP on gut microbiota and immune system through animal experiments, and identify key bacteria that can mediate MP metabolism and regulate immunity by combining microbial genomics, in vitro fermentation and selective culture experiments. The molecular mechanism of immune regulation of key metabolites will be analyzed by metabonomics, cell biology and other techniques. And then, the mechanism of intestinal microbiota mediating MP to improve immunity will be illuminated. This research is conducive to further revealing the intrinsic relationship between protein, immunity, and gut microbiota, which is meaningful for improving host health by regulating intestinal microbiota with dietary proteins.

多组学技术的发展使饮食-肠道菌群-宿主免疫的相关性研究备受关注。研究表明肠道微生物能够介导蛋白质代谢与宿主免疫应答之间的相互作用。然而基于肠道菌群探究膳食蛋白调节机体免疫的研究有限，介导蛋白质代谢与宿主免疫的关键细菌尚不明确。桑叶因营养丰富已被用作功能食品。申请人前期研究表明，从桑叶中分离的清蛋白MP可促进免疫因子分泌，显著增加菌群多样性、促进乳酸菌等定殖。但MP调节的关键肠道菌群及其在改善机体免疫中的作用有待进一步研究。本项目拟通过动物实验分析MP对小鼠肠道菌群及免疫体系的影响，并结合微生物基因组学、体外发酵及选择性培养实验，分离鉴定出可介导MP调节免疫的关键细菌；通过代谢组学、细胞生物学等技术分析关键细菌代谢产物的免疫调节机制，进而揭示肠道菌群介导MP改善机体免疫的机制。本研究有利于进一步揭示蛋白质、肠道菌群、免疫之间的内在关系，对利用膳食蛋白调节肠道菌群改善宿主健康具有重要意义。

多组学技术的发展使饮食-肠道菌群-宿主免疫的相关性研究备受关注。研究表明肠道微生物能够介导蛋白质代谢与宿主免疫应答之间的相互作用。然而基于肠道菌群探究膳食蛋白调节机体免疫的研究有限，介导蛋白质代谢与宿主免疫的关键细菌尚不明确。桑叶因营养丰富已被用作功能食品。申请人前期研究表明，从桑叶中分离的清蛋白可促进免疫因子分泌，显著增加菌群多样性、促进乳酸菌等定殖。但其调节的关键肠道菌群及其在改善机体免疫中的作用有待进一步研究。本项目首先对桑叶清蛋白进行分离纯化、结构鉴定，得到高纯度的桑叶蛋白GUMP，通过动物实验分析GUMP对小鼠肠道菌群及免疫体系的影响，结合微生物基因组学、体外发酵及选择性培养实验，分离鉴定出可介导GUMP调节免疫的关键细菌；通过代谢组学、细胞生物学等技术分析关键细菌代谢产物的免疫调节机制，进而揭示肠道菌群介导GUMP改善机体免疫的机制。结果表明：（1）GUMP是一种蛋白含量89%、糖含量9%的糖蛋白；（2）不同剂量的GUMP在体内外均具有免疫活性和肠道菌群调节能力，中剂量组的综合活性最优；（3）筛选出6个关键菌属，即乳球菌属、乳杆菌属、双歧杆菌属、脱硫弧菌属、Odoribacter、拟杆菌属。通过选择性培养及体外发酵二次验证，获得4个关键菌种，即粪肠球菌、格式乳球菌、台湾乳杆菌、假长双歧杆菌；（4）根据特征菌属与代谢产物的相关性分析，筛选出6种关键代谢产物：L-甘氨酸、半胱胺，乙醇胺、棕榈酸、丁酸、异戊酸；（5）测定6种代谢产物的细胞毒性及免疫活性。结果表明，相同浓度范围内丁酸、异戊酸、L-甘氨酸、半胱胺的细胞存活率高于棕榈酸及乙醇胺；同时丁酸、L-甘氨酸的NO产生能力较高。目前正进行多个细胞因子的测定，以期选出活性较高的2种产物进行免疫机制研究。本研究有利于进一步揭示蛋白质、肠道菌群、免疫之间的内在关系，对利用膳食蛋白调节肠道菌群改善宿主健康具有重要的现实意义。