基于非水溶性膳食纤维基质的双歧杆菌生物膜成膜规律及调控机制

The formation of microbial biofilm is a mechanism of improving stress resistance and environmental adaptation. Current study mainly focused on how to prevent and to inactivate the biofilm formation of pathogenic and spoilage bacteria. To understand this mechanism and thereby to improve the environmental stress tolerance should improve the controllable and uni-directional biofilm formation of Bifidobacterium and other probiotics, should have strong impact on both theories and industry practice for the large scale and high efficient production of active probiotics. However, the mechanism of the biofilm formation of Bifidobacterium remains unclear. The aim of this project is to analyze the rules of biofilm formation and regulation mechanism of Bifidobacterium based on insoluble dietary fiber matrix. First of all, to establish a biofilm formation model based on insoluble dietary fiber, and thereby reveals the differences of spatial conformation, structural constituent and growth dynamics of Bifidobacterium biofilm based on various media by using scanning electronic microscopy, confocal laser scanning microscopy and single-cell force spectroscopy; Then, to further investigate the mechanism of restriction impact of the surface characteristics of biofilm-forming medium and the conditions on the Bifidobacterium biofilm formation based on the fermentation of insoluble dietary fiber; Furthermore, to systematically study the genetic basis and metabolic pathways of Bifidobacterium biofilm formation via comparative genomics and transcriptomics analysis; And finally, to elucidate the biofilm regulation mechanism of Bifidobacterium by using the combined techniques of metabolomics and transcriptomics, and targeted gene knockout. The purpose of this project is to provide a theoretical basis for the establishment of uni-directional and controllable strategy of Bifidobacterium biofilm formation.

生物膜形成是微生物提高抗逆性的重要环境适应机制，现有研究主要集中于防止致病菌和腐败菌生物膜的形成和成膜后的杀灭。如何利用生物膜的这一特性和机制，实现双歧杆菌等有益微生物生物膜的高效形成和定向调控，对活性益生菌的规模化及高效制备具有革新性的理论意义和现实的应用价值。然而双歧杆菌生物膜的成膜规律及调控机制还不清楚。本项目通过建立双歧杆菌基于非水溶性膳食纤维基质的生物膜成膜模型，结合扫描电子显微镜、激光共聚焦显微技术及单细胞力谱，揭示双歧杆菌生物膜基于不同基质的空间构象、结构组分和生长动力学差异性；同时明确基质表面特性和培养条件对生物膜发酵成膜的限制机制；进一步基于多组学联用技术，系统认识双歧杆菌成膜的基因基础和调控网络；结合基因靶向敲除验证，最终阐明双歧杆菌生物膜的成膜调控机制。本项目的实施可以为双歧杆菌生物膜的定向及可控形成提供理论基础，并为实际应用提供理论指导。

双歧杆菌是人们最常食用的益生菌，但在生产加工过程极易死亡。生物膜是细菌提高环境抗性的一种自我保护机制，具有重要的工业应用价值。目前，生物膜发酵体系以孔板静态发酵为主，然而由于成膜面积小且成膜量低，缺乏工业生产潜能。尽管对生物膜已有大量研究，但生物膜形成机制尚未在许多细菌物种中得到充分阐明，对于双歧杆菌尤其如此。本研究以双歧杆菌为研究对象，探究了双歧杆菌生物膜的成膜差异性，并对成膜功能基因进行了挖掘。基于此，本研究构建了双歧杆菌生物膜发酵体系，并研究了双歧杆菌成膜机制，最后使用不同发酵条件进行了生物膜发酵调控。主要结果如下：1）实验涉及114株双歧杆菌的生物膜具有种间和株间差异性，空间结构主要表现为“扁平状”和“蘑菇状”。2）双歧杆菌表面性质与其成膜能力存在相关性，成膜菌株表面疏水性及路易斯酸特性较强。疏水性菌株更易在疏水性材料表面粘附，亲水性菌株更易在亲水性材料表面粘附，表面带正电或电中性的材料更利于菌株的粘附。3）构建了基于非水溶性膳食纤维的双歧杆菌生物膜发酵体系，且发酵条件显著地影响成膜。双歧杆菌在表面粗糙且多孔的葡萄籽粉上成膜能力最强。0.05%胆盐促进多数双歧杆菌的生物膜形成，0.2%胆盐几乎抑制所有双歧杆菌生物膜形成。4）两歧双歧杆菌、假小链双歧杆菌和长双歧杆菌的成膜机制分别主要以QseC/QseB调控、c-di-GMP调控和AI-2/QS调控为主。5）1.0%葡萄糖和pH 6.0可促进双歧杆菌生物膜形成，2 mM苏氨酸可促进两歧双歧杆菌生物膜形成。双歧杆菌生物膜发酵与肽聚糖，氨基酸和有机酸代谢有关。本课题构建了基于非水溶性膳食纤维的双歧杆菌体外动态生物膜发酵体系，有助于系统认识双歧杆菌的成膜机制和调控网络。本课题既可以为双歧杆菌生物膜的定向及可控形成提供理论基础，也以为益生菌生物膜机制研究提供思路进而推动益生菌生物膜产业的发展。