微生态制剂调节便秘、腹泻人群肠道菌群结构与产短链脂肪酸关键菌属的相关性

为探究微生态制剂对健康、便秘和腹泻人群肠道菌群结构的调节能力。本研究以健康、便秘和腹泻人群为对象,令其定时、定量摄入水苏糖(stachyose tetrahydrate,Sta)、益生菌纯粉(probiotics power,PP)、益生菌剂(probotic preparations,PPrs)3种微生态制剂,共6周,采集新鲜粪便样本并提取DNA,利用Ion torrent PGM二代测序技术进行16S r RNA V3区扩增子测序,并用气相色谱检测粪便中的短链脂肪酸(short-chain fatty acids,SCFAs)表达水平,最后联合多变量统计学分析方法对测序数据进行多样性分析。结果表明:绝大多数序列属于硬壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes),约占总序列数的94.37%。随着微生态制剂的摄入,受试人群肠道菌群的群落结构多样性明显增加,毛螺菌科(Lachnospiraceae)中的Blautia、Lachnospira以及瘤胃菌科(Ruminococcaceae)中的Faecalibacterium、Oscillospir等与产SCFAs相关的菌属都明显地增长,其中Blautia和Faecalibacterium与SCFAs含量呈显著正相关。SCFAs的含量以及肠道中相应菌群的增长与减少都与微生态制剂的成分相关,在3组受试人群中,服用Sta组,丙酸含量显著增加,乙酸与丁酸含量也在2周左右有所增加,并伴随产SCFAs的菌属快速且大量增长;PP组肠道中只有乙酸含量有所增加,丙酸和丁酸含量呈现降低的趋势,而产SCFAs的菌属增长较明显;服用PPrs组,乙酸和丁酸含量明显增加,且便秘和腹泻人群在停止服用后,其SCFAs的含量接近于健康人群,常见的Bifidobacterium、Lactobacillus、Parabacteroides等外源性益生菌均明显增长,可能性致病菌相对丰度降低,表明服用PPrs对肠道菌群结构的调节作用以及影响更大。此外,根据肠型的分析,Bacteroides和Prevotella在饮食的共同驱动下会调整并改变肠型,而仅通过所选择的微生态制剂的作用,在驱动肠型改变方面不显著。综上所述,肠道疾病状态的人群服用微生态制剂后,肠道菌群结构向正常人群的状态调整,菌群多样性和SCFAs表达水平提高,表现出持续抑制肠道有害细菌生长,促进有益菌的增殖,以此来维持肠道菌群结构的稳态。经扩增子测序分析获得初步结论为:微生态制剂有改变腹泻、便秘人群肠道菌群整体结构的功效,并且PPrs要比单一的Sta或PP调整肠道菌群的能力更突出。

Relationship between Microecologics and the Expression of Short Chain Fatty Acids Synthesis Genes in Key Bacterial Genera in the Regulation of Intestinal Flora Structure in Populations with Constipation and Diarrhea

This study was devised to explore the ability of probiotics to regulate the gut microbiota in healthy, constipated and diarrheal populations. These populations were asked to ingest three different microecologics: storyose tetrahydrate (Sta), probiotics power (PP), and probiotics preparations (PPrs) at a fixed dose at fixed times daily for 6 weeks. Fresh fecal samples were collected for DNA extraction. The V3 region of the 16S rRNA gene was sequenced using Ion Torrent PGM and short- chain fatty acids (SCFAs) in feces was quantified by gas chromatography. The sequencing data was used to make diversity analysis by multivariate statistical analysis. The results showed that the most identified sequences were from Firmicutes and Bacteroidetes, accounting for 94.37% of the total number of sequences. Ingestion of probiotics significantly increased the structural diversity of intestinal flora in the tested populations. Significant growth rates were observed for the intestinal bacteria associated with SCFAs, such as Blautia and Lachnospira in the Lachnospiraceae family and Faecalibacterium and Oscillospir in the Ruminococcaceae family. Furthermore, Blautia and Faecalibacterium were positively correlated with SCFAs. In addition, changes in both the contents of SCFAs and the corresponding intestinal microbial communities were related to the composition of probiotics. Propionic acid content significantly was increased by Sta ingestion; moreover, the contents of acetic acid and butyric acid were likewise increased at about 2 weeks along with rapid and highly efficient growth of the SCFAs-producing strains for all three populations. PP caused an increase in acetic acid but led to a decreasing trend of propionic and butyric acid, accompanied by significant growth of the SCFAs-producing bacteria. After taking PPrs, the contents of acetic acid and butyric acid were significantly increased in the subjects, and the fecal content of SCFAs in constipated and diarrheal populations was close to that in healthy people. Concomitantly, the exogenous probiotics such as Bifidobacterium, Lactobacillus and Parabacteroides showed a significant increase, and the relative abundance of possible pathogenic bacteria decreased, indicating that PPrs exert greater regulation on the gut microbiota structure. In addition, enterotype analysis showed that Bacteroides and Prevotella could be adjusted driven by the diet, thus changing the enterotype, but no significant change was achieved by simply using the microecologics. In summary, the gut microbiota structure of populations with gut diseases can be adjusted toward the normal after ingestion of microecologics through increasing the bacterial community diversity and the expression of fecal SCFAs. Microecologics can sustainably inhibit harmful bacteria and promotes beneficial bacteria in the gut, thereby maintaining the stability of intestinal flora structure. The results of 16S rDNA PCR amplification and sequencing show that probiotics can change the overall structure of the intestinal flora in patients with constipation and diarrhea, and the composite PPrs are more effective than single Sta and PP.