Analysis and direct quantification of Saccharomyces cerevisiae and Hanseniaspora guilliermondii populations during alcoholic fermentation by fluorescence in situ hybridization, flow cytometry and quantitative PCR.

Traditionally, it was assumed that non-Saccharomyces (NS) yeasts could only survive in the early stages of alcoholic fermentations. However, recent studies applying culture-independent methods have shown that NS populations persist throughout the fermentation process. The aim of the present work was to analyze and quantify Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) populations during alcoholic fermentations by plating and culture-independent methods, such as fluorescence in situ hybridization (FISH) and quantitative PCR (QPCR). Species-specific FISH probes labeled with fluorescein (FITC) were used to directly hybridize Sc and Hg cells from single and mixed cultures that were enumerated by epifluorescence microscopy and flow cytometry. Static and agitated fermentations were performed in synthetic grape juice and cell density as well as sugar consumption and ethanol production were determined throughout fermentations. Cell density values obtained by FISH and QPCR revealed the presence of high populations (107-108 cells/ml) of Sc and Hg throughout fermentations. Plate counts of both species did not show significant differences with culture-independent results in pure cultures. However, during mixed fermentations Hg lost its culturability after 4-6 days, while Sc remained culturable (about 108 cells/ml) throughout the entire fermentation (up to 10 days). The rRNA content of cells during mixed fermentations was also analyzed by flow cytometry in combination with FISH probes. The fluorescence intensity conferred by the species-specific FISH probes was considerably lower for Hg than for Sc. Moreover, the rRNA content of Hg cells, conversely to Sc cells, remained almost unchanged after boiling, which showed that rRNA stability is species-dependent.

Effect of oenological practices on microbial populations using culture-independent techniques.

Sulphur dioxide (SO(2)) addition and yeast inoculation are well-established practices in winemaking for restricting the growth of indigenous yeasts and bacterial populations. The effect of these oenological practices on wine microbial populations has been evaluated using culture-independent methods. These are quantitative PCR (qPCR) for the enumeration of yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB), and PCR-DGGE to determine the yeast and bacteria species diversity. The PCR-DGGE method detected a low yeast and bacteria species diversity. On the contrary, the specificity of the primers designed for the qPCR allowed that minor microbial groups such as Hanseniaspora were accurately quantified regardless of a large presence of other microbial groups such as Saccharomyces. From an oenological point of view, inoculation increased the proportion of Saccharomyces vs. non-Saccharomyces in a shorter time. Hanseniaspora increased during the first phase and decreased during the latter phases of the process, especially in the sulphited fermentations. Both yeast inoculation and SO(2) kept the LAB populations at very low level, while the AAB populations were hardly affected by these two practices.