Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. In the present study, we have supplemented nitrogen-deficient fermentations with a mixture of ammonium and amino acids at various stages throughout the alcoholic fermentation. The timing of the nitrogen additions influenced the biomass yield, the fermentation performance, the patterns of ammonium and amino acid consumption, and the production of secondary metabolites. These nitrogen additions induced a nitrogen-repressed situation in the cells, and this situation determined which nitrogen sources were selected. Glutamine and tryptophan were the main amino acids consumed in all the fermentations. Ammonium is the preferred nitrogen source for biomass production but was hardly consumed when it was added in the final stages of the fermentation. The higher ammonium consumption in some fermentations correlated with a greater synthesis of glycerol, acetate, and acetaldehyde but with a lower synthesis of higher alcohols.

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We carried out fermentations with several nitrogen sources in different concentrations and studied nitrogen regulation by following the transcriptional profile of the general amino-acid permease (GAP1) and the ammonium permeases (MEP1, MEP2, MEP3). In wine fermentations the cells evolve from a nitrogen-repressed situation at the beginning of the process to a nitrogen-derepressed situation as the nitrogen is consumed. These nitrogen-repressed/derepressed conditions determined the different patterns of ammonium and amino-acid consumption. Arginine and alanine were hardly used under the repressed conditions, while the uptake of branched-chain and aromatic amino acids increased.

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Wine produced at low temperature is often considered to have improved sensory qualities. To investigate the effects of temperature on winemaking, the expression patterns during the industrial fermentation process carried out at 13 degrees C and 25 degrees C were compared, and correlated with physiological and biochemical data, including viability, fermentation byproducts and lipid content of the cells. From a total of 535 ORFs that were significantly differentially expressed between the 13 degrees C and 25 degrees C fermentations, two significant transcription programmes were identified. A cold-stress response was expressed at the initial stage of the fermentation, and this was followed by a transcription pattern of upregulated genes concerned with the cell cycle, growth control and maintenance in the middle and late stages of the process at 13 degrees C with respect to 25 degrees C. These expression patterns were correlated with higher cell viability at low temperature. The other relevant transcriptomic difference was that several genes implicated in cytosolic fatty acid synthesis were downregulated, while those involved in mitochondrial short-chain fatty acid synthesis were upregulated in the fermentation process conducted at 13 degrees C with respect to that at 25 degrees C. These transcriptional changes were qualitatively correlated with improved resistance to ethanol and increased production of short-chain (C(4)-C(8)) fatty acids and their corresponding esters at 13 degrees C as compared to 25 degrees C. While this increase of ethyl esters may account in part for the improved sensory quality of wine fermented at 13 degrees C, it is still unclear how the esterification of the short-chain fatty acids takes place. On the basis of its strong upregulation at 13 degrees C, we propose a possible role of IAH1 encoding an esterase/ester synthase in this process.

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Interest in the use of non-Saccharomyces yeasts in winemaking has been increasing due to their positive contributions to wine quality. The non-Saccharomyces yeast Hanseniaspora vineae is an apiculate yeast that has been associated with the production of wine with good aromatic properties. However, little is known about the fermentation dynamics of H. vineae in natural must and its interaction with autochthonous yeasts. In the present study, we performed semi industrial fermentations of Macabeo and Merlot musts inoculated with either H. vineae or S. cerevisiae. The yeast population dynamics were monitored by plate culturing, PCR-DGGE and massive sequencing techniques. The results obtained with these techniques show that H. vineae was able dominate the autochthonous microbiota in Macabeo must but not in Merlot must, which exhibited a larger, more diverse yeast population. The presence of H. vineae throughout most of the Macabeo fermentation resulted in more fruity and flowery wine, as indicated by the chemical analysis of the final wines, which demonstrated a strong presence of phenyl ethyl acetate at concentrations higher than the threshold of perception and approximately 50 times more than that produced in wines fermented with S. cerevisiae. This compound is associated with fruity, floral and honey aromas.