不同碳水化合物对正常人肠道菌群体外模拟效果的比较

Effects of different carbohydrates on the simulation of human intestinal bacterial flora with in vitro culture

目的 比较不同碳水化合物对正常人肠道菌群体外模拟效果的影响,找出适合肠道菌群体外模拟的条件,研究肠道微生物体外制备方法.方法 采集3名正常志愿者的粪便样品,利用以淀粉为主要碳源的培养基VI和以淀粉＋多糖为主要碳源的培养基XP,在体外模拟发酵模型中对其菌群结构进行模拟培养.通过提取发酵液细菌基因组DNA,运用PCR-变性梯度凝胶电泳（PCR-DGGE）和16S rRNA基因高通量测序来评价模拟系统的稳定性和模拟效果,通过测定发酵液短链脂肪酸含量来推断细菌代谢情况.结果 体外发酵模型连续运行8d后,3名志愿者的发酵液细菌PCR-DGGE条带一致,不同培养基存在特异的细菌条带.细菌16S rRNA基因高通量测序结果表明,3名志愿者的粪便样品主要由4个细菌门组成,分别是拟杆菌门、厚壁菌门、变形菌门和放线菌门.在考虑细菌百分含量的情况下,VI培养基对3名志愿者肠道菌群的模拟相似性是0.847、0.825、0.968,XP培养基为0.927、0.926、0.836.当不考虑细菌百分含量,只考虑细菌种类时,VI培养基对3名志愿者肠道菌群的模拟相似性是0.553、0.580、0.623,XP培养基为0.617、0.520、0.574.VI和XP培养基模拟效果存在个体差异,VI培养基对志愿者3菌群的模拟效果更好,XP培养基对志愿者1、2菌群的模拟效果更好.VI和XP培养基均能较好地模拟拟杆菌和毛螺旋菌属细菌的生长,但VI和XP培养基中也存在各自特异生长的细菌.在发酵罐内检测到短链脂肪酸的含量是15～35 mmol/L,产生最多的是丁酸和丙酸.结论 发酵系统可用于肠道细菌的模拟培养,不同碳水化合物对肠道细菌的体外模拟效果有不同影响,能体外模拟培养出相同的细菌,也有在各培养基中特异性生长的细菌,但对3名志愿者肠道细菌模拟相似性均在80％以上.

Objective To investigate the optimal growth condition of human fecal bacterial flora in vitro by comparing the effect of different carbohydrates as cultural media.Methods Three fecal samples （1, 2, 3） were collected and inoculated into a single-stage chemostat system, in which starch medium （VI） and starch polysaccharide medium（XP） were used.Samples were collected for bacterial genomic DNA extraction and polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） analysis.Bacterial composition and short chain fatty acid （SCFA） were then analyzed.Results The single stage chemostat system reached steady after operating 8 days, when evaluated by the PCR-DGGE.Bacterial 16s rRNA high-throughput sequencing showed that the intestinal bacteria of these three volunteers was mainly composed of four bacterial phyla, namely, Bacteroidetes, Firmicutes, Proteobacteria and Actinobncteria.When the influence of bacterial abundance was considered, the similarity of bacterial composition between the original fecal samples to the harvested flora after culture was 0.847, 0.825, 0.968 in VI medium and 0.927, 0.926, 0.836 in XP medium, respectively.The similarity was decreased to 0.553, 0.580, 0.623 with VI medium and 0.617, 0.520, 0.574 with XP medium, when the number of bacterial species was considered.The variation of host individual also influenced the simulation.VI medium favored fecal sample 3, while XP medium more benefited sample 1 and 2.Bacteroides and Lachnospiraceae_incertae_sedis grew in both VI and XP medium.However, some species were only detected in VI medium and some were specifically found in the XP medium.The SCFA concentration in fermenters was 15-35 mmol/L, mainly propionate and butyrate.Conclusions The chemostat system works for stimulating human gut bacterial flora in vitro.The bacterial composition is affected by different carbohydrate in the culture medium yet with close simulation higher than 80％.