Detection of butyrate from a Clostridium cluster I (Clostridium sensu stricto) strain, Clostridium tertium, under anaerobic conditions

Lactic acid bacteria and bifidobacteria are widely used as probiotics. However, butyrate-producing bacteria remain less explored despite the critical role of butyrate in gut health. Our metagenomic analysis of the gut microbiota of obese mice given fructooligosaccharides as a prebiotic showed potential butyrate producers in Clostridium sensu stricto. In particular, the butyrate-producing ability of Clostridium tertium, which belongs to the genus Clostridium sensu stricto, had not been clarified. In this study, we examined how effective C. tertium is as a butyrate producer to evaluate its potential as a novel probiotic bacterium. Protein BLAST (BLASTP) searches for butyrate kinase (BUK) proteins in the genomes of C. tertium identified two proteins, and polymerase chain reaction (PCR) confirmed the presence of two buk genes and phosphate butyryltransferase (ptb), a buk gene adjacent gene. Ion-exclusion HPLC analysis detected lactate, formate, acetate, and butyrate in cultures of C. tertium, with a butyrate concentration of 6.5 ± 1.6 mM, comparable to that produced by Clostridium butyricum under the same conditions. These results demonstrate that C. tertium is a butyrate-producing bacterium and suggests its potential as a novel probiotic species.