青春双歧杆菌ATCC15703酵解莲子低聚糖异构单体的多级动态响应增殖机理研究

The gradually declined number of bifidobacteria in the human body with age increased and the nutritional competition with other intestinal microflora result in the low utilization of carbon source. However, bifidobacteria and other probiotics could make use of the glycolysis of functional oligosaccharides by repression effects of metabolites to form the dominant flora under the competition. Our previous study showed that lotus seed oligosaccharides (LOS) could make the targeted regulation to Bifidobacterium adolescentis, which may be related to the highly similar structure of several isomeric monomers in oligosaccharides. The proposed project will investigate Bifidobacterium adolescentis ATCC 15703 induced glycolysis of the key structure differences of the intracellular metabolic sites between isomeric monomers in LOS by the metagenomics, proteomics, bioinformatics and computer simulation techniques. The different metabolic pathways for the monomers of lotus seeds oligosaccharides would be studied by comparative genomics techniques. The signal mapping pathway between cell glucosidase responses and extracellular oligosaccharide will be established by dynamic zymogram. The catalyzed hydrolysis mechanism of glycoside hydrolases to sugar monomers could be studied by molecular simulation technology. Additionally, the bacterial evoked rejection and induction inhibitory effect induced by conformation of LOS will be explained, and the responses of interactive mechanisms would be established among the monomers of LOS-glycoside hydrolases-Bifidobacterium adolescentis. The proposed project expects to provide a theoretical basis for artificial modification of prebiotic foods.

人体内双歧杆菌的数量随年龄的增长逐渐下降，且与其他肠道菌群存在营养竞争而导致其对碳源利用率低。但双歧杆菌等益生菌可通过代谢物阻遏效应酵解功能性低聚糖，从而在肠道竞争中胜出形成优势菌群。本课题组前期研究发现，莲子低聚糖可靶向性调控青春双歧杆菌数量，这可能与几种糖单体的结构紧密相关。本项目运用基因组学、蛋白质组学、生物信息学和计算机模拟技术探讨青春双歧杆菌ATCC 15703酵解莲子低聚糖异构单体的关键结构差异性代谢位点，通过比较基因组学技术研究菌体对莲子低聚糖各单体的不同代谢通路；采用动态酶谱技术构建菌体各时刻感应胞外低聚糖与应答糖苷酶的信号映射通路；并联合分子模拟技术研究糖苷水解酶对糖单体的水解催化机理；进而阐明莲子低聚糖构象对菌体产生的诱发排斥和感应抑制效应，建立莲子低聚糖单体-糖苷水解酶-青春双歧杆菌间的相互作用响应机制，为益生元类食品的人工改造提供理论依据。

为深入系统地研究低聚糖与双歧杆菌的增殖机制和构效关系，本项目选取莲子低聚糖（LOS）与其各单体和青春双歧杆菌ATCC 15703作为研究对象，构建菌体对不同低聚糖单体的代谢通路模型；运用蛋白组学、分子模拟技术阐明莲子低聚糖单体与酶的作用机制，主要研究结论如下：.1 莲子低聚糖单体高效提取分离工艺及其益生元活性研究：当提取时间为325.00 s，液料比10.00 mL/g，超声波功率300.46 W，微波功率250.00 W的超声波-微波辅助提取工艺时，莲子低聚糖得率较高，30mL/min的制备色谱流速为莲子低聚糖各单体的较优分离流速，所分离的低聚糖单体纯度高。将莲子低聚糖灌胃便秘的模型小鼠后发现，低聚糖促进了小鼠肠道内容物蠕动并促进排便，进而缓解便秘。不同莲子低聚糖单体对小鼠粪便微生物组成的影响有所差异并具有一定的构效关系。.2 青春双歧杆菌ATCC 15703 酵解莲子低聚糖的代谢路径研究：运用基因转录技术对菌体增殖效果相对较好的LOS4低聚糖进行菌体增殖研究后发现，LOS4相比LOS3-1增殖效果较好的效果主要在于，低聚糖经菌体内化后通过专用的细胞内酶将LOS4通过磷酸化或转糖基化进行水解成单糖，随后通过异构酶和酮糖激酶的作用，经糖酵解和戊糖转化最终转化为磷酸烯醇丙酮酸，并进一步参与了TCA循环被菌体代谢。.3 莲子低聚糖调控菌体蛋白质生物信息学分析：研究表明青春双歧杆菌ATCC15703利用莲子低聚糖各单体碳源时主要以分子转运、小分子代谢等过程，主要以跨膜转运形式为主，代谢路径与双歧杆菌偏好莲子低聚糖中葡萄糖与果糖有关，酶表达与转录过程基本一致。.4 莲子低聚糖与菌体糖苷酶相互作用分析：甘露糖苷酶与LOS3-2底物、葡萄糖苷酶与LOS4底物、果糖苷酶与LOS4底物对接后结合效果较佳，不同莲子低聚糖单体作为底物与糖苷酶对接主要的分子相互作用方式为氢键、范德华作用力和静电作用力，范德华力是莲子低聚糖与酶发生作用的主要作用力。甘露糖相对于葡萄糖和果糖对于青春双歧杆菌的利用能力较差，糖苷键连接类型和组成是对菌体利用糖的能力影响较大。