Identification of microbial species active in starch digestion using Stable Isotope Probing.
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. A schematic presentation of RNA-SIP (Kovatcheva-Datchary et al, 2005).
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).
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