桑椹低聚糖EMOS促鼠李糖乳杆菌增殖作用构效关系及其分子机制研究

Lactobacillus rhamnosus (LGG), which has strong colonization ability, is one of the core probiotic bacteria that maintain the structure and function of intestinal flora. It is an important research field of probiotic functional food to explore the regulation factor of LGG proliferation promoting and its mechanism of action from natural plants. Previous studies have found that mulberry oligosaccharide prepared by enzymatic method (EMOS) had significant LGG proliferative activity in vitro, and the proliferative activity was better than that of mulberry oligosaccharides prepared by physical or chemical methods and commercial prebiotics: isomaltooligosaccharide (IMO), galactooligosaccharides (GOS). However the mechanism and structure-activity relationship of EMOS promotive LGG proliferative activity in vitro is unknown. Accordingly, in this project, UPLC/Q-TOF-MS, GC-MS and NMR were used to characterize the physicochemical properties of EMOS oligosaccharide structure and sugar chain structure, and the structure-activity relationship of EMOS-induced LGG proliferation activity was investigated. Based on the RNA-Seq technique, the differentially expressed genes of LGG cultured from EMOS were ascertained from the mRNA transcription level, and qRT-PCR was used to further verify the molecular signaling pathways involved in the regulation of LGG proliferation. UPLC/Q-TOF-MS analyzed the changes of LGG metabolites by EMOS, and the molecular mechanism of EMOS promoting LGG proliferation was elucidated from the levels of gene transcription and metabolic pathway. The research results will provide a theoretical basis for the research and development of mulberry probiotic food.

肠道定植能力强的鼠李糖乳杆菌（LGG）是维持肠道菌群结构及其功能的核心益生菌之一，从天然植物中挖掘促LGG增殖功能因子并探明其作用机制是益生元食品研究的重要内容。项目组前期研究发现，酶法制备的桑椹低聚糖（EMOS）具有显著的体外促LGG增殖活性，且效果优于理化法制备的桑椹低聚糖及IMO、GOS等商品化益生元，但其作用机制及构效关系不明。据此，本项目拟采用UPLC/Q-TOF-MS、GC-MS和NMR表征EMOS寡糖构成及糖链结构等理化特性，明确EMOS促LGG增殖活性构效关系；基于RNA-Seq技术由mRNA转录水平探明EMOS培养LGG差异表达基因，结合qRT-PCR进一步验证促LGG增殖调控相关的分子信号通路；UPLC/Q-TOF-MS解析EMOS培养LGG代谢产物变化规律，结合基因转录和代谢通路两个层面阐释EMOS促LGG增殖调控分子机制。研究结果将为桑椹益生元食品研发提供理论依据。

桑椹富含多糖、花青素和多酚等生物活性物质，具有改善便秘、调节肠道菌群、降血糖、降血脂等多种保健功能。利用酶法制备桑椹低聚糖，通过色谱柱法对低聚糖进行分离纯化得出一种名为 EMOS-1a 的水溶性低聚糖。通过紫外光谱、傅里叶变换红外光谱和气相色谱-质谱法研究纯化馏分的化学结构，表明半乳糖是 EMOS-1a 的主要成分。化学分析表明，EMOS-1a 的糖醛酸和硫酸盐含量分别为 5.6% 和 8.35%，而凝胶渗透色谱显示 EMOS-1a 的平均分子量为 987 Da。抗氧化活性表明EMOS-1a表现出对1,1-二苯基-2-苦基肼自由基清除活性、Trolox 等效抗氧化能力和铁还原抗氧化能力的浓度依赖性。添加4% (w/v) EMOS-1a时，鼠李糖乳杆菌增殖水平达到1420±16%。这些结果表明低聚糖 EMOS-1a 可用作益生元制剂中的天然抗氧化剂。.在MRS培养基添加有桑椹低聚半乳糖（MGO）下，对鼠李糖乳杆菌GG（LGG）进行转录水平上的相关基因表达研究。在稳定生长期，在MRS培养基中添加MPS和MGO的差异基因比添加GOS的差异基因多约63%和132%，上调基因数MPS和MGO比GOS多约18%和66%。在促益生菌增殖方面，MGO对LGG的促增殖率也是高于GOS。为了进一步了解这些基因的生物学功能，通过与KEGG库比对，找出显著性富集的 Pathway 进行分析。在MRS培养基中添加MGO后培养LGG情况下，随生长速率上升都上调的120个基因主要富集于膜运输28个、氨基酸代谢21 个和碳水化合物代谢10 个等通路上。在MRS培养基中添加MGO后培养LGG情况下，随生长速率上升都下调的153个基因主要富集于碳水化合物代谢45 个、膜运输15个、氨基酸代谢11 个和核苷酸代谢11个。对差异基因和差异代谢物做合并分析，筛选出差异最多的KEGG 途径进行分析，在MRS培养基中加入MGO后，差异基因或差异代谢产物数量最多的5个途径是ABC转运蛋白、果糖和甘露糖代谢、PTS、氨基糖和核苷酸糖代谢、嘌呤代谢。MRS培养基添加MGO后LGG的半乳糖代谢通路中发生上调的基因是gatB，发生下调的基因是lacC、galK、galU和galE。MRS培养基添加MGO后LGG的糖酵解/糖异生通路中发生上调的基因是bglA，发生下调的基因是fbaA、pdhA、pdhB、aceF和lpd。