饲用益生芽孢杆菌孢子体内萌发机制研究

The germination of spores from feed probiotic Bacillus in vivo is one of the problems associated with the mechanism of probiotics from the source of Bacillus, which plays core roles in the selection of Bacillus isolates and its efficacy in animal production. Early reports based on occasional results of parts of experimental animals showed that the spores could germinate in vivo, but no systematic and explicit conclusions were drawn till now. In this project, the factors including Bacillus characterization and intestinal environments are concerned to study the interaction between the spore and animal gastrointesinte. Bacillus isolates are screened based on the growth characteristics and two isolates with significant difference in growth rate are used in mice experiments. The differences in the germination and metabolism activity of the two strains are prominently compared. The differences of antimicrobial activity of potential Bacillus are further explored when the spore is mixed with germinates or spore inhibitors. Based on the analyses methods of real-time and specific detection and fluorescence labeling and imaging technology, the germination mechanism of probiotic Bacillus in protecting intestinal health in vivo will be elucidated and benefit the theoretical basis for Bacillus application in animal production.

芽孢杆菌的孢子在动物体内萌发是关系到芽孢类益生菌发挥作用机制的核心问题，对于菌株的选育及其应用稳定性方面具有重要意义。前期研究初步证实芽孢能在体内萌发转变成营养体细胞，但只是个别实验动物的偶然现象，缺乏系统性结论。本项目综合考虑到芽孢杆菌的细胞特性及动物体内的环境因素，系统探讨菌和动物胃肠互作效应，即以菌株的生长速率作为首要选育条件，通过筛选获得具有生长速率迥异的两种芽孢杆菌菌株，比较这两种菌株在体内的萌发及代谢活性差异；以及通过诱导和抑制芽孢的萌发来比较益生芽孢杆菌体内代谢活性差异，研究中综合利用实时特异性监测、生物发光与成像等定量检测方法，系统阐明外源芽孢在动物肠道中萌发调控机制，为益生芽孢杆菌的进一步应用提供理论基础。

芽孢杆菌的孢子在动物体内萌发是芽孢类益生菌发挥益生活性的关键，芽孢杆菌的体内萌发特性一方面与菌株的个体差异相关，即菌株是否存在与萌发相关的特异性萌发受体，另一方面与环境中的营养性萌发剂的激活相关。本项目建立DPA-EuCl3-CYDTA的荧光反应体系实现了DPA物质的精确测定，不仅实现了芽孢的快速定量测定（其检测限达到了103CFU/ml）；而且用于芽孢的特异性萌发响应的测定，基于此筛选分离得到了两株萌发响应显著差异的菌株（即萌发高响应菌株B. subtilis S-2和萌发不响应菌株B. subtilis 312）。为了特异性测定芽孢杆菌在胃肠道代谢对肠毒性大肠杆菌的拮抗活性，构建了一种红色荧光蛋白mCherry标记的肠毒性大肠杆菌菌株，采用了CRISPR/Cas9的基因编辑技术实现了mCherry在肠毒性大肠杆菌K99基因组上的定点插入与表达；荧光标记后的菌株采用荧光分光光度计检测时其菌株检测限为104CFU/ml；该菌株作为病原菌攻毒菌株可以显著导致肠道炎性因子的上调。基于芽孢DPA的检测进一步研究外源芽孢在胃肠道内的分布、增殖情况，发现在大鼠的胃部均没有检测到DPA，在小肠内检出的DPA浓度较低，大肠中DPA的浓度较高，且萌发能力强的菌株B. subtilis S-2其在肠道中分布的DPA的数量比萌发能力弱的菌株B. subtilis 312显著增加。L-Ala介导的B. subtilis S-2芽孢灌服大鼠后其日增重和采食量显著高于阳性试验组（即攻毒组），并能显著增加部分肠段对E. coli K99-mCherry荧光菌株拮抗活性。该结果表明L-Ala作为营养性萌发剂促进芽孢在体内的萌发和增殖并进一步增强其体内的代谢活性，提高其对肠道屏障的保护效果。