A reverse transcription-quantitative polymerase chain reaction system for evaluating intestinal butyrate production by fecal bacteria.

Intestinal butyrate is key in maintaining host health. As an indicator of butyrate production, the fecal butyrate concentration is used for assessing health status; however, it may not be a suitable marker as it only accounts for residual fecal butyrate following intestinal absorption. Therefore, we aimed to develop a novel quantitative system for evaluating intestinal butyrate production. We designed a primer set targeting the gene encoding butyryl-coenzyme A (CoA):acetate CoA-transferase (but), a key enzyme involved in butyrate synthesis. The new primer set specifically reacted with but-harboring bacteria, including Faecalibacterium, suggesting its suitability for the comprehensive and accurate detection of intestinal butyrate-producing bacteria. We established a system for measuring but mRNA levels using the novel primer set and found that the but expression levels were more closely correlated with butyrate production than but DNA copy number in an in vitro culture model. Finally, we verified the effects of synbiotics by fecal analysis based on this primer set and found that synbiotics significantly increased but expression levels, but not DNA copy number. In conclusion, our but expression quantification system based on a novel primer set is a promising tool for evaluating butyrate production and verifying the efficacy of probiotics and prebiotics.IMPORTANCEIntestinal butyrate has diverse functions, including intestinal barrier function enhancement and anti-inflammatory effects, and has therapeutic potential for metabolic and neurological health. Butyrate affects human health, and intestinal butyrate production must be accurately measured for understanding health status. However, as over 95% of the butyrate produced by intestinal bacteria is absorbed in the intestinal tract, direct fecal butyrate concentration measurement may not accurately reflect the amount produced by intestinal bacteria. Herein, we developed a system for measuring the expression levels of butyrate-producing genes in intestinal bacteria and demonstrated its suitability for estimating butyrate production capacity. This system can be used in the development of probiotics, prebiotics, and synbiotics. As butyrate-based preventive and therapeutic methods for various diseases are developed in the future, this system will aid in verifying their effectiveness and elucidating the mechanisms of action.