人肠道内Faecalibacterium prausnitzii的分离、鉴定及优良菌株筛选研究

Isolation, Identification and Excellent Strain Screening of Faecalibacterium prausnitzii in Human Intestinal Tract

本研究通过特异性引物PCR、ERIC-PCR指纹图谱,从一健康人体肠道内筛选到20种ERIC类型的分离物。选取不同ERIC类型的代表菌株进行16S r RNA全长基因测序并在NCBI中进行BLAST,发现15种代表菌株的16S r RNA基因序列与库中的普拉梭菌（Faecalibacterium prausnitzii）相似性均达到97%以上,初步将这些代表菌株鉴定为普拉梭菌。为了筛选出与人体健康具有密切相关性且生物学活性强的优良菌株,通过综合分析15种ERIC类型的分离物的分离丰度及遗传距离,从鉴定出的15株代表菌株中挑选了7株候选优良菌株,进行胆汁酸盐耐受、丁酸盐产生和对肠屏障功能的障影响等评价。研究表明：胆汁酸盐浓度高于0.1%时,F18、F22、F109和F139菌株仍然可以生长,表现出较强的耐胆汁酸能力;菌株间产丁酸盐能力差异显著,F31的丁酸盐产量最高,其次为F18、F20、F33和F139,且这4株菌株间产丁酸盐能力无显著差异;菌株F31使肠屏障功能受损,而其他菌株无明显影响。综合实验数据,筛选出F18和F139为优良菌株。本研究成功实现了普拉梭菌的分离鉴定及优良菌株筛选,为后续的体内实验研究提供了重要研究基础。

In this research, 20 isolates of ERIC-types were screened from a healthy human intestinal tract by specific primers PCR and ERIC-PCR fingerprinting technology. Representative strains of different ERiC-types were selected, and the 16S rRNA full-length gene was sequenced as well as BLAST was conducted in NCBI. It was found that 15 representative isolates were preliminary identified to be Faecalibacterium prausnitzii as they share 〉97% sequence identity with 16S rRNA gene sequences ofFaecalibacterium prausnitzii from NCBI. In order to screen excellent strains of strong biological activity closely related to human health, the isolation abundance and genetic distance of ! 5 ERiC-type isolates were comprehensively analyzed. Then 7 excellent strain candidates were chosen from 15 representative strains to evaluate the bile salt tolerance, butyrate-production and effects on intestinal barrier function. The results showed that F18, F22, FI09 and Fl39 strains survived when the bile salts concentration was higher than 0.1%, which meant they had strong tolerance to bile acid. The butyrate production capacity showed significantly different among strains, F31 was the strongest butyrate-producer, followed by F18, F20, F33 and F139. There was no significant difference among the four strains in butyrate producing. The intestinal barrier function was damaged by F31, while the other strains had no obvious effect. According to the experimental data, F18 and F139 were chosen to be target excellent strains. This research successfully isolated, identified and screened excellent strains of Faecalibacterium prausnitzii, which would lay important foundation for further researches in vivo.