Study of the capability of rRNA to be a marker of viable wine yeast cells

2017 edition

Mercè Sunyer Figueres

Cells in Viable But Not Culturable (VBNC) cell state do not have the capacity of growing, but can be metabolic active. Some wine yeasts in VBNC state have an important role during alcoholic fermentation, affecting the quality of final wine, thus it is important to detect and quantify them as a part of viable cells. VBNC state difficulties the discernment between viable and dead cells: culture-dependent methods underestimate the viable population, because they cannot detect cells in VBNC state; the majority of culture-independent methods overestimate the viable population, because they use markers present in dead cells. Nowadays, the challenge of studies that tries to obtain the total number of viable cells is to find a marker able to detect all the live cells, without detecting the dead ones. The aim of this study is to determine the capacity of rRNA to be a viable cell marker, through the analysis of its stability in lysed cells. Firstly, we tested the effect of different lysis treatments in wine yeasts (one strain of Saccharomyces cerevisiae and three strains of non-Saccharomyces). Treatments with high temperature and antimicrobial DMDC (dimethyl dicarbonate) lysed the yeast completely. After that, we quantified the rRNA during 48h after lysing the cells with the two treatments. In order to do the quantification, RNA was extracted and through RT-PCR it was transformed into cDNA, which was quantified with qPCR. The results confirm that rRNA is stable during 48h after cellular lysis with these treatments. To sum up, it seems that rRNA is not a good cellular viability marker in the tested wine yeast. Therefore, it is necessary to keep looking for a marker to quantify viable cells.