Abstract: [Objective] To investigate the effects of calcium ions on the gut microbiota of mice fed a high-fat diet. [Methods] Thirty 6- to 8-week-old male C57BL/6J mice were randomly divided into three groups: a standard diet control group （C group）, a high-fat diet group （M group）, and a high-fat diet supplemented with calcium chloride treatment group （D group）, with 10 mice in each group. The C group was fed a standard diet and given regular drinking water, the M group was fed a high-fat diet and given regular drinking water, and the D group was fed a high-fat diet and given drinking water supplemented with 1.5 g/100 mL calcium chloride. After 11 weeks of continuous feeding, fecal samples were collected from each group. The gut microbiota was sequenced using 16S rRNA high-throughput sequencing technology. Species richness, evenness, and diversity were calculated using Perl and R programming languages, and shared and unique OTUs were compared among samples. Alpha diversity （Shannon index） and richness （ACE and Chao1 indices） were analyzed using QIIME software. Differences in microbiota between groups were analyzed using the Wilcox test and LDA histogram from the R STATS package. The PICRUSt software was used to analyze the species composition information obtained from the 16S rRNA sequencing data, and in combination with the Kyoto Encyclopedia of Genes and Genomes （KEGG） database, the functional genes of the microbial communities in each group were classified and analyzed for enrichment differences. [Results] ①A total of 953 922 valid sequences were obtained from the sequencing of nine samples across the three groups. Analysis of the ACE index, Chao1 index, and Shannon index showed no significant differences in species richness and diversity among the groups （P>0.05）. ②Analysis of species relative abundance at the genus and species levels revealed that the dominant genera in the C, M, and D groups were Dubosiella, Lachnospiraceae NK4A136 group, and Ileibacterium, with relative abundances of 14.34%, 14.49%, and 21.69%, respectively. The dominant species in the C, M, and D groups were Clostridiales bacterium CIEAF 020, Ileibacterium valens, and Ileibacterium valens, with relative abundances of 1.36%, 7.24%, and 21.69%, respectively. ③ Analysis of microbiota differences between groups showed that the D group microbiota differed from the C and M groups, primarily characterized by Ileibacterium, Ileibacterium valens, Proteobacteria, Bacilli, Erysipelotrichales, and Erysipelotrichaceae. ④ PICRUSt prediction of functional gene composition revealed significant differences （P<0.05） between the D and M groups in functional gene pathways such as carbon fixation, pyruvate metabolism, and starch and sucrose metabolism, suggesting that calcium chloride treatment may influence gene expression in these metabolic pathways. [Conclusion] Adding calcium ions to a high-fat diet can change the structure of the intestinal microbiota of mice, increase the number of specific microorganisms, and may affect the metabolic pathways related to energy intake and fat accumulation, providing a new perspective for the application of calcium ions in the prevention of obesity.

Key words: calcium ions, obesity, gut microbiota, 16S rRNA high-throughput sequencing, mice