Wine-related non-Saccharomyces yeasts are becoming more widely used in oenological practice for their ability to confer wine a more complex satisfying aroma, but their metabolism remains unknown. Our study explored the nitrogen utilisation profile of three popular non-Saccharomyces species, Torulaspora delbrueckii, Metschnikowia pulcherrima and Metschnikowia fructicola. The nitrogen source preferences to support growth and fermentation as well as the uptake order of different nitrogen sources during wine fermentation were investigated. While T. delbrueckii and S. cerevisiae strains shared the same nitrogen source preferences, Metschnikowia sp. displayed a lower capacity to efficiently use the preferred nitrogen compounds, but were able to assimilate a wider range of amino acids. During alcoholic fermentation, the non-Saccharomyces strains consumed different nitrogen sources in a similar order as S. cerevisiae, but not as quickly. Furthermore, when all the nitrogen sources were supplied in the same amount, their assimilation order was similarly affected for both S. cerevisiae and non-Saccharomyces strains. Under this condition, the rate of nitrogen source consumption of non-Saccharomyces strains and S. cerevisiae was comparable. Overall, this study expands our understanding about the preferences and consumption rates of individual nitrogen sources by the investigated non-Saccharomyces yeasts in a wine environment. This knowledge provides useful information for a more efficient exploitation of non-Saccharomyces strains that improves the management of the wine fermentation.

During alcoholic fermentation, many parameters, including the nitrogen composition of the must, can affect aroma production. The aim of this study was to examine the impact of several types of nitrogen sources added at different times during fermentation. Nitrogen was added as ammonium or a mixture of amino acids at the beginning of fermentation or at the start of the stationary phase. These conditions were tested with two Saccharomyces cerevisiae strains that have different nitrogen requirements. The additions systematically reduced the fermentation duration. The aroma production was impacted by both the timing of the addition and the composition of the nitrogen source. Propanol appeared to be a metabolic marker of the presence of assimilable nitrogen in the must. The production of ethyl esters was slightly higher after the addition of any type of nitrogen; the production of higher alcohols other than propanol was unchanged, and acetate esters were overproduced due to the overexpression of the genes ATF1 and ATF2. Finally the parameter affecting the most the synthesis of beneficial aromas was the addition timing: The supply of organic nitrogen at the beginning of the stationary phase was more favorable for the synthesis of beneficial aromas.

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.