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Applications  |  Nutrition: Inulin Metabolism in rats (RNA-SIP)


Identification of microbial species active in the bowel of rats using Stable Isotope Probing.

Introduction

Polymeric plant components may pass undigested to the large bowel where they become carbon substrates for bacteria. The large bowel accommodates numerous species occupying a wide range of ecological niches where they practice their often specific functions. Identification of the bowel bacteria involved in fermentation processes will lead to a better insight of the functioning of the bowel in relation to effects of prebiotics or functional foods. In this study, decomposition of inulin and fructo-oligosaccharides was studied in the bowel of rats.

Stable Isotope Solution: 13C-labelled biomarkers

13C-labelled prebiotics like fructo-oligosaccharides derived from inulin can be used to clarify digestion characteristics in the bowel. High 13C-enrichment levels also enable the analyses of the microbial population during a longer period, either in in vitro systems like TIM (TNO gastro-Intestinal Model) or in vivo. The active microbial species involved in the digestion process can be identified by extracting and analyzing 13C-RNA from faeces. This method, called RNA-stable isotope probing (RNA-SIP), has been successfully used in vitro with 13C-labelled potato starch (Kovatcheva et al. 2009) and here its effectiveness to identify active bacterial groups using inulin and fructo-oligosaccharides as a carbon source is demonstrated in rats. During the study, the rats were fed with a diet containing IsoLife's uniformly labelled 13C-Inulin. Several hours after administration, cecal samples were taken, RNA extracted and subsequently centrifuged over a density gradient to separate 12C-RNA from 13C-enriched RNA. After converting the RNA to cDNA, 16S rRNA sequences in cDNA were amplified by PCR prior to sequencing.

Results

Most of the RNA obtained after 6 to 24 hours was present in the denser fractions: little 12C -RNA was present in the samples, indicating an active population of 13C-Inulin consumers. It was shown that Bacteroides uniformis was able to directly use inulin as a carbon source (trophic level 1), whereas Blautia glucerasea, Clostridium indolis, and Bifidobacterium animalis fermented the fructo-oligosaccharides derived from inulin degradation (trophic level 2) (Figure 1).

13C Inulin in bacteria

Figure 1. Abundances of 16S 501 rRNA gene sequences from bacterial groups (Tannock et al, 2014).

The authors conclude that RNA-SIP is useful to identify bacteria at different trophical levels and provides valuable information about the dynamics of inulin in a rats bowel

References

Tannock GW, B Lawley, K Munro, IM. Sims, J Lee, CA. Butts, N Roy. 2014.
RNA-stable isotope probing (RNA-SIP) shows carbon utilization from inulin by 2 specific bacterial populations in the large bowel of rats.
Applied Environmental Microbiology doi:10.1128/AEM.03799-13.

Kovatcheva-Datchary P, M Egbert, A Maathuis, M Rajilic-Stojanovic, AA de Graaf, H Smidt, WM de Vos, K Venema. 2009.
Linking phylogenetic identities of bacteria to starch fermentation in an in vitro model of the large intestine by RNA-based stable isotope probing.
Environmental Microbiology 11: 914-926.

 

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