植物乳杆菌JCM 1149与嗜酸乳杆菌JCM 1132饲喂斑马鱼益生机制的比较研究

The purpose of the present project is to compare the probiotic mechanisms of Lactobacillus plantarum subsp. plantarum JCM 1149 (adhesive) and L. acidophilus (non-adhesive) on zebrafish (Danio rerio)―the important vertebrate model organism and to provide the research basis for development of novel and highly efficient probiotic products in aquaculture. Their respective key genes required for intestinal adhesion or/and quorum sensing (QS) control were identified by means of gene knock-out and activity-recovery techniques through feeding trials of zebrafish. Parameters of probiotic effectiveness were assessed, including the count of adherent cells, the morphology of intestinal mucosa, the activities of intestinal alkaline phosphatease (LAP), nuclear factor κ-light chain-enhancer of activated B cells (NF-κB), immunoglobulin M (IgM) and the relative percentage of survival (RPS) upon an immersion challenge with bacterial pathogen in vivo. The prerequisite that "a probiotic bacterial strain must be adhesive to host intestinal mucus/mucosa" was questioned based on the evidences that dietary non-adhesive strain JCM 1132 had immune protective effects on zebrafish. This recognized criterion for selecting probiotic strains will be revised once the genes required for intestinal adhesion and their contributions to probiotic effectiveness of strain JCM 1149 were validated. In addition, based on the observations that the immune protective effects of strain JCM1132 were strictly controlled by its density, we assumed that its probiotic effectiveness is regulated by QS. After identification of the QS control genes and their functions in strain JCM 1132, the importance to use specific QS signal molecular as a tool to manipulate the probiotic effectiveness of non-adesive Lactobacillus sp. will be explored.

本项目旨在比较植物乳杆菌JCM 1149(斑马鱼肠道定植)与嗜酸乳杆菌JCM 1132(非定植)对斑马鱼-一种重要模式脊椎动物的益生机制，拟为研发新型、高效水产用微生态制剂奠定基础。通过构建突变株与活性回复株,结合斑马鱼饲喂试验以判定其定植决定基因或(和)群体感应(QS)调控决定基因，益生效应评价手段包括肠道定植计数、黏膜形态观察、碱性磷酸酶(LAP)、核转录因子κB(NF-κB)、免疫球蛋白M(IgM)活性分析及浸浴攻毒免疫保护率(RPS)测定等：以非定植JCM 1132存在免疫保护效应，质疑"宿主消化道可定植"这一水产益生菌的筛选前提，通过判定JCM 1149斑马鱼肠道定植决定基因及益生贡献，拟修订这一公认标准；以JCM 1132免疫保护效应受菌群密度调控，推测其益生效应受QS机制调控，通过判定其QS调控决定基因、类型及益生贡献，拟了解QS信号分子对于非定植乳酸杆菌益生效应的调控价值。

本研究在原计划的基础上，用鼠李糖乳杆菌LGG菌毛蛋白SpaC敲除突变株ΔSpaC-LGG mutant替代植物乳杆菌JCM 1149开展粘附效应机制研究，用Lactobacillus plantarum WCFS1群体感应相关基因luxS的敲除突变株ΔluxS-WCFS1 mutant替代嗜酸乳杆菌JCM 1132群体感应敲除突变株开展乳杆菌益生效应群体感应调控机制研究。为排除宿主消化道背景菌群对研究结果的干扰，直接依托无菌斑马鱼模型开展不同菌株宿主免疫保护效应比较研究。宿主消化道mucin诱导了乳杆菌肠道主要粘附基因的上调表达，缺失菌毛蛋白，并未降低LGG肠道粘附数量，却改变ΔSpaC LGG肠道粘附空间类型，将LGG从黏膜粘附类型改变成黏液黏膜均匀分布类型，并显著提升LGG宿主免疫保护力；无菌斑马鱼模型表明嗜酸乳杆菌JCM 1132宿主免疫保护表现群体感应调控效应，其群体感应信分子AIP可增强宿主免疫保护。上述成果为研制基于微生物群体感应调控的新型、高效、廉价的水产用微生态制剂奠定了基础。以第一作者或通讯作者发表SCI收录论文19篇，中文核心期刊2篇。获授权中国发明专利14项。毕业硕士研究生6名，博士研究生1名，出站博士后2名。执行项目提出“鱼类消化道菌群益生效应元件应用优势策略”，4项授权发明专利实施转让，转让收益525.6万元。主持获得大北农科技成果奖一等奖（大北农集团）、中国水产行业科技创新奖一等奖。

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