Impact of CO2 overpressure on yeast mitochondrial associated proteome during the "prise de mousse" of sparkling wine production.

The "prise de mousse" stage during sparkling wine elaboration by the traditional method (Champenoise) involves a second fermentation in a sealed bottle followed by a prolonged aging period, known to contribute significantly to the unique organoleptic properties of these wines. During this stage, CO2 overpressure, nutrient starvation and high ethanol concentrations are stress factors that affect yeast cells viability and metabolism. Since mitochondria are responsible for energy generation and are required for cell aging and response to numerous stresses, we hypothesized that these organelles may play an essential role during the prise de mousse. The objective of this study is to characterize the mitochondrial response of a Saccharomyces cerevisiae strain traditionally used in sparkling wine production along the "prise de mousse" and study the effect of CO2 overpressure through a proteomic analysis. We observed that pressure negatively affects the content of mitochondrion-related proteome, especially to those proteins involved in tricarboxylic acid cycle. However, proteins required for the branched-amino acid synthesis, implied in wine aromas, and respiratory chain, also previously reported by transcriptomic analyses, were found over-represented in the sealed bottles. Multivariate analysis of proteins required for tricarboxylic cycle, respiratory chain and amino acid metabolism revealed differences in concentrations, allowing the wine samples to group depending on the time and CO2 overpressure parameters. Ethanol content along the second fermentation could be the main reason for this changing behavior observed at proteomic level. Further research including genetic studies, determination of ROS, characterization of mitochondrial activity and targeted metabolomics analyses is required. The list of mitochondrial proteins provided in this work will lead to a better understanding of the yeast behavior under these conditions of special interest in the wine industry.

A Differential Proteomic Approach to Characterize the Cell Wall Adaptive Response to CO2 Overpressure during Sparkling Wine-Making Process.

In this study, a first proteomic approach was carried out to characterize the adaptive response of cell wall-related proteins to endogenous CO2 overpressure, which is typical of second fermentation conditions, in two wine Saccharomyces cerevisiae strains (P29, a conventional second fermentation strain, and G1, a flor yeast strain implicated in sherry wine making). The results showed a high number of cell wall proteins in flor yeast G1 under pressure, highlighting content at the first month of aging. The cell wall proteomic response to pressure in flor yeast G1 was characterized by an increase in both the number and content of cell wall proteins involved in glucan remodeling and mannoproteins. On the other hand, cell wall proteins responsible for glucan assembly, cell adhesion, and lipid metabolism stood out in P29. Over-represented proteins under pressure were involved in cell wall integrity (Ecm33p and Pst1p), protein folding (Ssa1p and Ssa2p), and glucan remodeling (Exg2p and Scw4p). Flocculation-related proteins were not identified under pressure conditions. The use of flor yeasts for sparkling wine elaboration and improvement is proposed. Further research based on the genetic engineering of wine yeast using those genes from protein biomarkers under pressure alongside the second fermentation in bottle is required to achieve improvements.

Autophagic Proteome in Two Saccharomyces cerevisiae Strains During Second Fermentation for Sparkling Wine Elaboration.

A correlation between autophagy and autolysis has been proposed in order to acceleratethe acquisition of wine organoleptic properties during sparkling wine elaboration. In this context, aproteomic analysis was carried out in two industrial Saccharomyces cerevisiae strains (P29,conventional sparkling wine strain and G1, implicated in sherry wine elaboration) with the aim ofstudying the autophagy-related proteome and comparing the effect of CO2 overpressure duringsparkling wine elaboration. In general, a detrimental effect of pressure and second fermentationdevelopment on autophagy-related proteome was observed in both strains, although it was morepronounced in flor yeast strain G1. Proteins mainly involved in autophagy regulation andautophagosome formation in flor yeast G1, and those required for vesicle nucleation and expansionin P29 strain, highlighted in sealed bottle. Proteins Sec2 and Sec18 were detected 3-fold underpressure conditions in P29 and G1 strains, respectively. Moreover, 'fingerprinting' obtained frommultivariate data analysis established differences in autophagy-related proteome between strainsand conditions. Further research is needed to achieve more solid conclusions and design strategiesto promote autophagy for an accelerated autolysis, thus reducing cost and time production, as wellas acquisition of good organoleptic properties.

First Proteomic Approach to Identify Cell Death Biomarkers in Wine Yeasts during Sparkling Wine Production.

Apoptosis and later autolysis are biological processes which take place in Saccharomyces cerevisiae during industrial fermentation processes, which involve costly and time-consuming aging periods. Therefore, the identification of potential cell death biomarkers can contribute to the creation of a long-term strategy in order to improve and accelerate the winemaking process. Here, we performed a proteomic analysis based on the detection of possible apoptosis and autolysis protein biomarkers in two industrial yeast strains commonly used in post-fermentative processes (sparkling wine secondary fermentation and biological aging) under typical sparkling wine elaboration conditions. Pressure had a negatively effect on viability for flor yeast, whereas the sparkling wine strain seems to be more adapted to these conditions. Flor yeast strain experienced an increase in content of apoptosis-related proteins, glucanases and vacuolar proteases at the first month of aging. Significant correlations between viability and apoptosis proteins were established in both yeast strains. Multivariate analysis based on the proteome of each process allowed to distinguish among samples and strains. The proteomic profile obtained in this study could provide useful information on the selection of wine strains and yeast behavior during sparkling wine elaboration. Additionally, the use of flor yeasts for sparkling wine improvement and elaboration is proposed.