Molecular analysis of the interactions between starter cultures during wine fermentation stars

Abstract

In recent years, the wine industry has been facing major challenges such as climate change, one of the consequences of which is the increase in the concentration of sugars in grapes at harvest time and, consequently, in the alcohol content of wines. On the other hand, the widespread use of Saccharomyces cerevisiae strains as fermentation starters has led to a perception of uniformity or standardization of the aromatic profile of wines. In both cases, part of the proposals for alcohol reduction and product diversification involve the use of starter cultures of alternative species to S. cerevisiae, commonly referred to as non-Saccharomyces. These new starter cultures are usually combined with some strain of S. cerevisiae in order to avoid stuck fermentation. The use of more than one species as starter culture implies that there is more than one population with a similar number of individuals performing the fermentation process simultaneously. In this way, the interactions that are established between them acquire a relevance that until now had not been valued. Today, it is widely accepted that the outcome of a fermentation process involving two or more inoculated yeast species will be different from the weighted average of the corresponding individual cultures. Interspecific interactions between wine yeasts take place at various levels, including interference competition, exploitation competition, and exchange of metabolic intermediates, among others. Some interactions could be a simple consequence of the metabolism of each yeast in a context where metabolic intermediates and end products of other yeasts are present. However, there are clear indications, in some cases, of specific recognition between interacting yeasts. In general terms, this doctoral thesis aims to deepen the knowledge of how microbial communities behave in wine, analyzing such behavior mainly from a transcriptomic and proteomic point of view, trying to anticipate their biological responses in this complex matrix. In this sense, the first chapter of the thesis focuses on the practical impact of using oenological yeasts that present the prion [GAR+] element in the fermentation of natural grape must. Parameters such as real alcohol reduction, volatile acidity produced, fermentation kinetics and fermentation time under both aerobic and anaerobic conditions are evaluated. In addition, the isolation frequency, heritability and penetrance values of this type of prion in six industrial strains of S. cerevisiae with different genetic backgrounds are analyzed. Among the main results, the low phenotypic penetrance of this epigenetic element and the absence of reduction in alcoholic yield are highlighted. The second chapter of the thesis focuses on the study of short-term yeast-yeast interactions to prevent changes in nutrient composition from affecting yeast metabolic activity. The transcriptional response of S. cerevisiae to co-culture, in only three hours, with the yeast Metschnikowia pulcherrima has been evaluated. In addition, the responses obtained in this chapter have been compared with those obtained in a previous work carried out by this research group in which the short-term response of S. cerevisiae was also analyzed, but against co-culture with yeasts of the species Torulaspora delbrueckii, Candida sake and Hanseniaspora uvarum. The results show that S. cerevisiae responds to M. pulcherrima in a more intense manner than to other yeast species, involving a repression of respiratory activity, in addition to the induction of glycolysis, which is common to all the responses analyzed. In turn, the third and fourth chapters of this PhD thesis focus on the study of EVs as complex structures with potential participation in the communication processes between yeast cells under oenological fermentation conditions. Specifically, the third chapter not only evaluates whether oenological yeasts are capable of producing EVs in an oenological context, but also aims to analyze which proteins are mostly present in EV-enriched fractions by means of a proteomic analysis. This study has shown, as described in the literature, an enrichment in proteins related to the cell wall and glycolysis. In addition to the hypothesis that EVs act as signal carriers in the interactions between different wine yeast species, the fourth chapter of this doctoral thesis evaluates whether the protein content of EVs is, in turn, affected by these interactions. The results show a clear impact of co-cultures on protein composition in all cases. In addition, although no firm conclusions can be drawn, there are indications that some yeasts shown common factors in their response to different competitors, while others induce in other yeasts a response that shows some commmon factor among several of them. Taken together, the results shown in these last two chapters point to a potential role of EVs in communication between different oenological yeast species. In general terms, this doctoral thesis contributes not only to the fundamental knowledge of long-term ecological interactions, but also provides a glimpse of their potential industrial applicability. In this sense, a precise knowledge of the interaction mechanisms would allow the design of new mixed starter cultures based on Saccharomyces and non-Saccharomyces yeasts, as well as, the optimal conditions for their application.