Partial 26S rDNA restriction analysis as a tool to characterise non-Saccharomyces yeasts present during red wine...

Restriction patterns of amplified regions of ribosomal large subunit RNA encoding genes (26S rDNA) were evaluated as a routine methodology to examine yeast species diversity during red wine fermentation. The results were confirmed by sequencing of D1/D2 region of 26S rDNA. Red wine production was carried out using a yeast starter culture together with different commercial products, namely enzymes, fermentation activators and tannins and their influence on the non-Saccharomyces yeast population was studied. Yeast strains were isolated using lysine agar as a selective medium for non-Saccharomyces yeasts, after morphological characterisation of colonies. Amplification of 26S rDNA followed by digestion with three restriction enzymes applied to the 121 isolates, generated 19 profiles and a very high correlation with sequencing results was achieved. Although a starter yeast culture was added, results showed that several yeast species were present during all stages of fermentation, independent of the conditions tested, emphasizing the diversity of microorganisms associated with winemaking. On the other hand commercial additives did not significantly influence the diversity of yeast population during the fermentation process. For non-Saccharomyces strains, restriction patterns of a PCR amplified 26S rDNA region proved to be an adequate tool for clustering strains at species level and enabled the monitoring of yeast population dynamics during red wine fermentation.