IsoLife はライフサイエンスのための安定同位体標識植物製品を供給します。 stabile isotopes

Applications  |  Nutrition: Starch Metabolism and Prebiotic Effects (RNA-SIP)


Identification of microbial species active in starch digestion using Stable Isotope Probing.

Introduction

Potential beneficial or deleterious effects of starch in the diet will depend on its digestion and fermentation characteristics (Topping et al., 2003). Part of the starch, the resistant starch (RS), escapes from digestion and is fermented in the large intestine. RS may have pre-biotic effects in the colon by stimulating the growth of specific probiotic bacteria like Lactobacillus acidophilus and Bifidobacterium spec. However, it is still not known which members of the colon microflora are involved in the starch metabolism in situ.

Stable Isotope Solution: 13C-labelled biomarkers

13C-labelled prebiotics like RS or other components from different plant species, purified or in the natural food matrix, can be used to feed humans or animals to quantify digestion and fermentation characteristics. The high 13C enrichment levels enable the analyses of the microbial population, either in vivo or in vitro systems like TIM (TNO gastro-Intestinal Models; www.tno.nl) during a long period. The active microbial species involved in the fermentation process can be identified by extracting and analyzing 13C-DNA from faeces. This method, called stable isotope probing (SIP), has been used with success in ecology (Boschker et al., 1998; Radajewski et al., 2000). modified for 13C-RNA analysis (Manefield et al., 2002), and has been used succesfully in RNA-SIP with IsoLife's uniformly labelled potato starch in TIM (Figure 1).

Figure 1
Figure 1. A schematic presentation of RNA-SIP (Kovatcheva-Datchary et al, 2005).

Results

Stimulation of functional probiotic microbial species that are metabolizing starch and are thus linked to prebiotic effects of starches can be measured by RNA-SIP and subsequent analysis of 13C-RNA.

To identify the active starch consumers differences in 16S rRNA were investigated using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) fingerprinting with different restriction enzymes. The fingerprints showed an increase in several T-RF's that probably represented Bifidobacteria as apparent starch consumers (Kovatcheva-Datchary, 2005; Award winning publication).

References

Boschker HTS, SC Nold, P Wellsbury, D Bos, W de Graaf, R Pel, RJ Parkes, TE Cappenberg. 1998.
Direct linking of microbial populations to specific biogeochemical processes by 13C-labelling of biomarkers.
Nature 392: 801-805

Kovatcheva-Datchary P, M Egbert, A Maathuis, H Smidt, WM de Vos, K Venema. 2005.
Which bacteria metabolise starch in the human colon?
Poster presentation Biomedicine in the Post-Genomic Era, Dec 1-3, Mexico City.

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Manefield M, AS Whiteley, N Ostle, P Ineson, MJ Bailey. 2002.
Technical considerations for RNA-based stable isotope probing.
Rapid Communications in Mass Spectrometry 16: 2179-2183.

Radajewski S, P Ineson, NR Parekh, JC Murrell. 2000.
Stable-isotope probing as a tool in microbial ecology.
Nature 403: 646-649.

Schell M. 2002.
In: Friendly tenants in the human gut: The genome of B. longum. Ed. B. Reinert.
www.genomenewsnetwork.org/articles/10_02/bifido.shtml

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. doi:10.11/j.11462-2920.2008.01815.x

 

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