Abstract
The use of controlled mixed inocula of Saccharomyces cerevisiae and non-Saccharomyces yeasts is a common practice in winemaking, with Torulaspora delbrueckii, Lachancea thermotolerans and Metschnikowia pulcherrima being the most commonly used non-Saccharomyces species. Although S. cerevisiae is usually the dominant yeast at the end of mixed fermentations, some non-Saccharomyces species are also able to reach the late stages; such species may not grow in culture media, which is a status known as viable but non-culturable (VBNC). Thus, an accurate methodology to properly monitor viable yeast population dynamics during alcoholic fermentation is required to understand microbial interactions and the contribution of each species to the final product. Quantitative PCR (qPCR) has been found to be a good and sensitive method for determining the identity of the cell population, but it cannot distinguish the DNA from living and dead cells, which can overestimate the final population results. To address this shortcoming, viability dyes can be used to avoid the amplification and, therefore, the quantification of DNA from non-viable cells. In this study, we validated the use of PMAxx dye (an optimized version of propidium monoazide (PMA) dye) coupled with qPCR (PMAxx-qPCR), as a tool to monitor the viable population dynamics of the most common yeast species used in wine mixed fermentations (S. cerevisiae, T. delbrueckii, L. thermotolerans and M. pulcherrima), comparing the results with non-dyed qPCR and colony counting on differential medium. Our results showed that the PMAxx-qPCR assay used in this study is a reliable, specific and fast method for quantifying these four yeast species during the alcoholic fermentation process, being able to distinguish between living and dead yeast populations. Moreover, the entry into VBNC status was observed for the first time in L. thermotolerans and S. cerevisiae during alcoholic fermentation. Further studies are needed to unravel which compounds trigger this VBNC state during alcoholic fermentation in these species, which would help to better understand yeast interactions.
Highlights
Alcoholic fermentation of grape must is mainly driven by Saccharomyces cerevisiae, which quickly dominates the fermentation of grape must in wine production
Four yeast species were used in this study: S. cerevisiae QA23 (Lallemand Inc., Montreal, QC, Canada) (Sc), T. delbrueckii Biodiva (Lallemand Inc., Canada) (Td), L. thermotolerans 1 (Lt) and M. pulcherrima CECT 13131 (Mp) isolated from grape must from the Priorat region (URV collection) [25]
As PMAxx can only enter cells with compromised or damaged membranes, but not intact cells, predominantly, DNA from living cells will be detected by Quantitative PCR (qPCR)
Summary
Alcoholic fermentation of grape must is mainly driven by Saccharomyces cerevisiae, which quickly dominates the fermentation of grape must in wine production. In the last years, there has been an increasing interest in studying other fermentative yeasts, which are generally referred to as non-Saccharomyces yeasts, due to the ability of some of them to improve the complexity of wines by increasing the concentration of aromatic molecules, such as terpenoids, esters, higher. Foods 2020, 9, 1373 alcohols or other molecules of interest, such as glycerol [1,2,3,4,5,6] Another advantage is the potential of some species to reduce the alcohol content of wines, a feature increasingly sought after in this industry [7,8,9]. L. thermotolerans increases wine natural acidity due to increased lactic acid production [13,19,20] and T. delbrueckii has a positive influence on the overall impression of the obtained wines, improving the aroma quality and the varietal character [19,21,22]
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