In Vivo Assessment of Resistant Starch Degradation by the Caecal Microbiota of Mice Using RNA-Based Stable Isotope Probing-A Proof-of-Principle Study

Resistant starch (RS) is the digestion resistant fraction of complex polysaccharide starch. By reaching the large bowel, RS can function as a prebiotic carbohydrate, i.e., it can shape the structure and activity of bowel bacterial communities towards a profile that confers health benefits. However, knowledge about the fate of RS in complex intestinal communities and the microbial members involved in its degradation is limited. In this study, 16S ribosomal RNA (rRNA)-based stable isotope probing (RNA-SIP) was used to identify mouse bowel bacteria involved in the assimilation of RS or its derivatives directly in their natural gut habitat. Stable-isotope [U¹³C]-labeled native potato starch was administrated to mice, and caecal contents were collected before 0 h and 2 h and 4 h after administration. 'Heavy', isotope-labeled [¹³C]RNA species, presumably derived from bacteria that have metabolized the labeled starch, were separated from 'light', unlabeled [¹²C]RNA species by fractionation of isolated total RNA in isopycnic-density gradients. Inspection of different density gradients showed a continuous increase in 'heavy' 16S rRNA in caecal samples over the course of the experiment. Sequencing analyses of unlabeled and labeled 16S amplicons particularly suggested a group of unclassified Clostridiales, Dorea, and a few other taxa (Bacteroides, Turicibacter) to be most actively involved in starch assimilation in vivo. In addition, metabolic product analyses revealed that the predominant ¹³C-labeled short chain fatty acid (SCFA) in caecal contents produced from the [U¹³C] starch was butyrate. For the first time, this study provides insights into the metabolic transformation of RS by intestinal bacterial communities directly within a gut ecosystem, which will finally help to better understand its prebiotic potential and possible applications in human health.