Non-Saccharomyces yeasts are currently widely used in winemaking to enhance aroma profile diversity among wines. The use of Metschnikowia pulcherrima in sequential inoculation with S. cerevisiae was compared to the inoculation of a pure culture of S. cerevisiae. Moreover, various concentrations of sugar, nitrogen and lipids were tested in synthetic must to assess their impact on fermentation and its outcomes using a Box-Behnken design. Due to its phenotypic specificities, early inoculation with M. pulcherrima led to important modifications, first altering the fermentation kinetics. This may relate, at least in part, to the depletion of some nitrogen sources by M. pulcherrima during the first part of fermentation. Beyond these negative interactions on fermentation performance, comparisons between pure cultures and sequentially inoculated cultures revealed changes in the distribution of carbon fluxes during fermentation in presence of M. pulcherrima, resulting in a positive impact on the production of central carbon metabolites and aromas. Furthermore, the expression of varietal thiols was strongly increased as a consequence of positive interactions between the two species. The mechanism of this release still needs to be investigated. Significant differences in the final concentrations of fermentative and varietal aromas depending on the initial must composition were obtained under both inoculation strategies. Interestingly, the response to changes in nutrient availability varied according to the inoculation modality. In particular, a greater incidence of lipids on the production of fatty acids and their ethyl esters derivatives was found during sequential fermentation compared with pure culture, to be viewed in combination with the metabolic characteristics of M. pulcherrima regarding the production of volatile compounds from acetyl-CoA. Overall, the importance of managing nutrient availability under M. pulcherrima/S. cerevisiae sequential inoculation in order to derive the maximum benefit from the potentialities of the non-Saccharomyces species while carrying out fermentation to dryness was highlighted.

Summary: Nitrogen and lipids are key nutrients of grape must that influence the production of fermentative aromas by wine yeast, and we have previously shown that a strong interaction exists between these two nutrients. However, more than 90% of the acids and higher alcohols (and their acetate ester derivatives) were derived from intermediates produced by the carbon central metabolism ( CCM ). The objective of this study was to determine how variations in nitrogen and lipid resources can modulate the contribution of nitrogen and carbon metabolisms for the production of fermentative aromas. A quantitative analysis of metabolism using ¹³ C‐labelled leucine and valine showed that nitrogen availability affected the part of the catabolism of N‐containing compounds, the formation of α‐ketoacids from CCM and the redistribution of fluxes around these precursors, explaining the optimum production of higher alcohols occurring at an intermediate nitrogen content. Moreover, nitrogen content modulated the total production of acids and higher alcohols differently, through variations in the redox state of cells. We also demonstrated that the phytosterol content, modifying the intracellular availability of acetyl‐CoA, can influence the flux distribution, especially the formation of higher alcohols and the conversion of α‐ketoisovalerate to α‐ketoisocaproate.

Wine is a particularly complex beverage resulting from the combination of several factors, with yeasts being highlighted due to their fundamental role in its development. For many years, non-Saccharomyces yeasts were believed to be sources of spoilage and contamination, but this idea was challenged, and many of these yeasts are starting to be explored for their beneficial input to wine character. Among this group, Torulaspora delbrueckii is gaining relevance within the wine industry, owing to its low volatile acidity production, increased release of aromatic compounds and enhanced color intensity. In addition, this yeast was also attracting interest in other biotechnological areas, such as bread and beer fermentation. In this work, a set of 40 T. delbrueckii strains, of varied geographical and technological origins, was gathered in order to characterize the phenotypic behavior of this species, focusing on different parameters of biotechnological interest. The fermentative performance of the strains was also evaluated through individual fermentations in synthetic grape must with the isolates’ metabolic profile being assessed by HPLC. Data analysis revealed that T. delbrueckii growth is significantly affected by high temperature (37 °C) and ethanol concentrations (up to 18%), alongside 1.5 mM SO₂, showing variable fermentative power and yields. Our computation models suggest that the technological origin of the strains seems to prevail over the geographical origin as regards the influence on yeast properties. The inter-strain variability and profile of the products through the fermentative processes reinforce the potential of T. delbrueckii from a biotechnological point of view.

Beyond the production of positive aromas during alcoholic fermentation, Saccharomyces cerevisiae metabolism also results in the formation of volatile compounds detrimental to wine quality, including a wide range of volatile sulfur compounds (VSCs). The formation of these VSCs during wine fermentation is strongly variable and depends on biological and environmental factors. First, the comparison of the VSCs profile of 22 S. cerevisiae strains provided a comprehensive overview of the intra-species diversity in VSCs production: according to their genetic background, strains synthetized from 1 to 6 different sulfur molecules, in a 1- to 30-fold concentration range. The impact of fermentation parameters on VSCs production was then investigated. We identified yeast assimilable nitrogen, cysteine, methionine and pantothenic acid contents – but not SO₂ content – as the main factors modulating VSCs production. In particular, ethylthioacetate and all the VSCs deriving from methionine catabolism displayed a maximal production at yeast assimilable nitrogen concentrations around 250 mg/L; pantothenic acid had a positive impact on compounds deriving from methionine catabolism through the Ehrlich pathway but a negative one on the production of thioesters. Overall, these results highlight those factors to be taken into account to modulate the formation of negative VSCs and limit their content in wines.