The aromatic profile of wine distillates from Ugni blanc grape musts is influenced by the nitrogen nutrition (organic vs. inorganic) of Saccharomyces cerevisiae.

Although the impact of nitrogen nutrition on the production of fermentative aromas in oenological fermentation is well known today, one may wonder whether the effects studied are the same when winemaking takes place at high turbidities, specifically for the production of wines intended for cognac distillation. To that effect, a fermentation robot was used to analyze 30 different fermentation conditions at two turbidity levels with several factors tested: (i) initial addition of nitrogen either organic (with a mixture of amino acids - MixAA) or inorganic with di-ammonium phosphate (DAP) at different concentrations, (ii) variation of the ratio of inorganic/organic nitrogen (MixAA and DAP) and (iii) addition of different single amino acids (alanine, arginine, aspartic acid and glutamic acid). A metabolomic analysis was carried out on all resulting wines to have a global vision of the impact of nitrogen on more than sixty aromatic molecules of various families. Then, at the end of the alcoholic fermentation, the wines were micro-distilled. A first interesting observation was that the aroma profiles of both wines and distillates were close, indicating that the concentration factor is rather similar for the different aromas studied. Secondly, the fermentation kinetics and aroma results have shown that the nitrogen concentration effect prevailed over the nature of nitrogen. Although the lipid concentration was in excess, an interaction between the assimilable nitrogen and lipid contents was still observed in wines or in micro-distillates. Alanine is involved in the synthesis of acetaldehyde, isobutanol, isoamyl alcohol and isoamyl acetate. Finally, it was demonstrated that modifying the ratio of assimilable nitrogen in musts is not an interesting technological response to improve the aromatic profile of wines and brandies. Indeed, unbalance the physiological ratio of the must by adding a single source of assimilable nitrogen (organic or inorganic) has been shown to deregulate the synthesis of most of the fermentation aromas produced by the yeast. Wine metabolomic analysis confirmed the results that had been observed in micro-distillates but also in the other aromatic families, especially on terpenes. The contribution of solid particles, but also yeast biosynthesis (via sterol management in must) to wine terpenes is discussed. Indeed, the synthesis of terpenes in this oenological context seems to be favored, especially since the concentration of assimilable nitrogen (in addition to the lipid content) favor their accumulation in the medium. A non-negligible vintage effect on the terpene profile was also demonstrated with variations in their distribution depending on the years. Thus, the present study focuses on the metabolism of wine yeasts under different environmental conditions (nitrogen and lipid content) and on the impact of distillation on the fate of flavor compounds. The results highlight once again the complexity of metabolic fluxes and of the impact of nitrogen source (nature and amount) and of lipids. Furthermore, this study demonstrates that beyond the varietal origin of terpenes, the part resulting from the de novo synthesis by the yeast during the fermentation cannot be neglected in the context of cognac winemaking with high levels of turbidity.

Influence of ergosterol and phytosterols on wine alcoholic fermentation with Saccharomyces cerevisiae strains.

Sterols are a fraction of the eukaryotic lipidome that is essential for the maintenance of cell membrane integrity and its good functionality. During alcoholic fermentation, they enhance yeast growth, metabolism and viability, as well as resistance to high sugar content and ethanol stress. Grape musts clarified in excess lead to the loss of solid particles rich in sterols, resulting in sluggish and stuck fermentations. Two sterol sources can help Saccharomyces cerevisiae yeasts to adapt to fermentation stress conditions: ergosterol (synthesized by yeast under aerobic conditions) and phytosterols (plant sterols imported by yeast cells from grape musts under anaerobiosis). Little is known about the physiological impact of phytosterols assimilation in comparison with ergosterol and the influence of sterol type on fermentation kinetics parameters. Moreover, studies to date have analyzed a limited number of yeast strains. Thus, the aim of this work was to compare the performances of a set of Saccharomyces cerevisiae wine strains that represent the diversity of industrial wine yeast, fermenting with phytosterols or ergosterol under two conditions: sterol limitation (sterol starvation) and high sugar content (the most common stress during fermentation). Results indicated that yeast cell viability was negatively impacted by both stressful conditions, resulting in sluggish and stuck fermentations. This study revealed the huge phenotype diversity of the S. cerevisiae strains tested, in particular in terms of cell viability. Indeed, strains with better viability maintenance completed fermentation earlier. Interestingly, we showed for the first time that sterol type differently affects a wide variety of phenotype, such as viability, biomass, fermentation kinetics parameters and biosynthesis of carbon central metabolism (CCM) metabolites. Ergosterol allowed preserving more viable cells at the end of fermentation and, as a consequence, a better completion of fermentation in both conditions tested, even if phytosterols also enabled the completion of alcoholic fermentation for almost all strains. These results highlighted the essential role of sterols during wine alcoholic fermentation to ensure yeast growth and avoid sluggish or stuck fermentations. Finally, this study emphasizes the importance of taking into account sterol types available during wine fermentation.

Impact of high lipid contents on the production of fermentative aromas during white wine fermentation.

In Cognac, the musts are rich in grape solids and fermentations are usually run with turbidities ranging between 500 and 1500 NTU (nephelometric turbidity unit). These conditions, considered favourable for generating the desired organoleptic profiles of the final Eaux-de-vies, are unusual in winemaking, and, consequently, their impact on yeast metabolism is poorly understood. This study aims to better describe and understand the synthesis of fermentative aromas in such lipid-excess conditions, while integrating the effect of two other very important parameters: the initial concentration of assimilable nitrogen and the temperature of fermentation. To reach this objective, a Box-Behnken design was implemented to describe and model the simple effects of these factors as well as their interactions. Although the lipid concentration was very high, impacts on the production of fermentative aromas were observed. Indeed, high lipid levels promoted the synthesis of higher alcohols. Observing this effect was surprising because there is no metabolic connection between the anabolic pathways of production of these alcohols and the lipid pathway. This effect may be partly explained by impairment in the activity of alcohol acetyl transferases in the presence of lipids, which catalyse the conversion of higher alcohols into the corresponding esters. Therefore, in this study, the negative impact of turbidity was very significant on acetate esters related to the production of acetyl-CoA, which was the main molecule disturbed by the strong presence of lipids. Finally, and more surprisingly, lipid intake did not impact the synthesis of ethyl esters, which depended on the concentration of exogenous lipids. KEY POINTS: • Innovative work on the fermentation of white wine musts with very high lipid contents. • Precise fermentation management and monitoring in Cognac-making conditions. • Experimental design to study the impact of lipids, assimilable nitrogen and temperature on fermentative aroma synthesis.