To date, there are no specific guidelines for the use of bioacidifying yeasts in winemaking. In this work, we aimed to characterize an oenological strain of Lachancea thermotolerans (Lt), a non-Saccharomyces lactic acid-producing yeast, and to test different sequential inoculation conditions with Saccharomyces cerevisiae (Sc). The results of bench scale vinifications showed that both the strategy of inoculating Sc 12 h after Lt and the mixing of Lt and Sc during fermentation delivered an acceptable increase in lactic acid (2 g/L) and a decrease in pH (about 0.15 units). Therefore, both strategies were implemented in winery experiments. Our results at the cellar scale showed no increase in acidity, which was likely due to the presence of indigenous yeasts. Overall, our experience shows the difficulty of translating laboratory protocols into cellar experiments and calls for further research into new strategies for implementing acidifying yeasts.

The phenomenon of global warming has been demonstrated to induce the premature ripening of grapes, resulting in a concomitant increase in sugar and a decrease in acidity. This can result in the production of wines that are characterised by an imbalance in terms of alcohol content and acidity. The utilisation of lactic acid-producing Lachancea thermotolerans yeast in fermentation represents a promising strategy for reducing the pH of must without the need for chemical acidification. However, it is important to note that the fermentation rate and lactic acid production can be influenced by several factors present in the must. The present study focused on the effect of copper, used in organic vineyards to limit mildew, on biomass and lactic acid production by two L. thermotolerans oenological strains in laboratory-scale fermentations. The methodology used is based on an innovative integrative approach combining conventional microbiological, spectroscopic, cytometric and transcriptomic analyses. The results obtained demonstrated that the addition of copper increased lactic acid production in comparison with the control condition and this effect was strain-dependent, reaching up to 16.15 g/L for strain Lt1 under conditions of high copper concentration (20 mg/L). The monitoring of microbial populations demonstrated discrepancies in the fermentation kinetics in conditions exhibiting varying concentrations of copper and showed that an increased duration of cell generation is evident under conditions exhibiting elevated copper concentrations, particularly in the context of the Lt1 strain. Furthermore, the analysis of the samples using flow cytometry revealed the oxidative status of the two strains throughout the fermentation process. Transcriptomic data indicated that metabolic pathways involved in cell communication, ribosomal and cell wall activity of L. thermotolerans were modulated by the presence of copper. The findings of this study demonstrate strain-specific effects of copper on L. thermotolerans, which may be of interest to winemakers, particularly those engaged in organic viticulture who intend to use acidifying yeasts during warm vintages.

We applied flow cytometry (FCM) to monitor microbial dynamics during grape must fermentation at the winery scale. Experiments were performed on Pinot Noir grapes using three distinct winemaking protocols: inoculation with active dried yeast, Pied-de-Cuve, and spontaneous fermentation. FCM enabled the assessment of yeast viability and metabolic activity, as well as the detection and monitoring of viable bacterial populations during alcoholic fermentation. Amplicon-based DNA sequencing was performed to characterize the associated microbial communities and evaluate protocol-specific effects. Trends identified by amplicon sequencing were partially mirrored by patterns observed in unsupervised FCM analysis. Overall, our results indicate that FCM is a practical tool for monitoring microbial dynamics during fermentation, providing near–real-time information that can support monitoring strategies and risk management in winemaking.