The effects of Clostridium butyricum HADIG-CB004 on intestinal microbial community in Kunming mouses

The aim of present study is to investigate the beneficial effect of Clostridium Butyricum HADIG-CB004 as a dietary supplement via modulating the intestinal microbiota in KM mouses. Twelve male KM mouses (average weight, 13 g, 2 weeks old) were purchased from Hunan Sileike Jingda Co. (Changsha, China). The KM mouses housed in plastic bottomed cages with reticulate stainless-steel covers under controlled light conditions (12 h light-dark cycle). The Kunming mouses had ad libitum access to food and water. The animals were divided into the following two treatment groups, each consisting of six mice: group 1 was normal control (NC) group, and received only drinking water and a standard mouse chow (basal diet) ad libitum for 12 days; group 2 was Clostridium Butyricum (CB) group, and was daily administered of basal diet supplemented with Clostridium Butyricum HADIG-CB004 at ten billion CFU per kilogram feed for 12 days. After 12 h of fasting, all mice were sacrificed by cervical dislocation on day 12 of feeding. Caecal contents were collected and sent to Chengdu Rhonin Biosciences Co.,Ltd. (Chengdu, China) for quantitative microbiological analysis. Total DNA was isolated from caecal contents using a Zymo Research BIOMICS DNA Microprep Kit (ZYMO RESEARCH, USA) following the manufacturer. The 8F and 1492R primer set was used to amplify the V4 region of bacterial 16S rRNA. Amplicons were purified with Zymoclean Gel Recovery Kit (ZYMO RESEARCH, USA). The final library was constructed using the Nanopore 16S Barcoding Kit, Ligation Sequencing Kit and Flow Cell Priming Kit, then sequenced on a Nanopore GridION (Oxford Nanopore Technologies Ltd., England).