Efficient and direct identification of fructose fermenting and non-fermenting bacteria from calf gut microbiota using stable isotope probing and modified T-RFLP.

The aim of this study was the development of an efficient method to identify the prebiotics-assimilating-bacteria in gut microbiota using DNA-stable isotope probing (DNA-SIP) technology. For efficient probing of microbiota with stable isotopes, a small-scale repeated batch culture using a low-carbon-source-containing medium was developed. Fecal samples from cattle were inoculated and [U-(13)C]-fructose was applied to the culture after 24 h stabilization. Organic acid production, pH value of the period and the total diversity of microorganisms of the culture were successfully maintained during the chasing period. DNA samples were extracted from the culture and were subjected to isopycnic centrifugation and fractionation in order to separate fructose fermenters from non-fermenters. T-RFLP (Terminal Restriction Fragment Length Polymorphism) and the modified T-RFLP of each fraction suggested that Streptococcus bovis was the most dominant fructose fermenter in this culture. In addition, we improved the modified T-RFLP method and successfully identified Lactobacillus vitulinus and Megasphaella eldenii as minor fructose-fermenters and several species of Clostridium cluster IV as non-fermenters. From these results we concluded that the methods shown here provide a means for assessing the importance of individual prebiotics on gut microbiota.