Aims: The purpose of this study was to determine the origin of the yeasts involved in the spontaneous alcoholic fermentation of an Alsatian wine. Methods and Results: During three successive years, must was collected at different stages of the winemaking process and fermented in the laboratory or in the cellar. Saccharomyces yeasts were sampled at the beginning and at the end of the fermentations. Saccharomyces cerevisiae clones were genetically characterized by inter-d PCR. Non-S. cerevisiae clones were identified as Saccharomyces uvarum by PCR-RFLP on MET2 gene and characterized at the strain level by karyotyping. The composition of the Saccharomyces population in the vineyard, after crushing and in the vat was analyzed. This led to three main results. First, the vineyard Saccharomyces population was rather homogeneous. Second, new non-resident strains had appeared in the must during the winemaking process. Finally, the yeast population in the vat only consisted in S. uvarum strains. Conclusion: This 3-year study has enabled us to show the involvement of indigenous S. uvarum in the alcoholic fermentation. Significance and Impact of the Study: This study gives a first insight into the polymorphism of S. uvarum strains involved in a spontaneous alcoholic fermentation.

Medium-chain fatty acids (octanoic and decanoic acids) are well known as fermentation inhibitors. During must fermentation, the toxicity of these fatty acids is enhanced by ethanol and low pH, which favors their entrance in the cell, resulting in a decrease of internal pH. We present here the characterization of the mechanisms involved in the establishment of the resistance to these fatty acids. The analysis of the transcriptome response to the exposure to octanoic and decanoic acids revealed that two partially overlapping mechanisms are activated; both responses share many genes with an oxidative stress response, but some key genes were activated differentially. The transcriptome response to octanoic acid stress can be described mainly as a weak acid response, and it involves Pdr12p as the main transporter. The phenotypic analysis of knocked-out strains confirmed the role of the Pdr12p transporter under the control of WAR1 but also revealed the involvement of the Tpo1p major facilitator superfamily proteins (MFS) transporter in octanoic acid expulsion. In contrast, the resistance to decanoic acid is composite. It also involves the transporter Tpo1p and includes the activation of several genes of the beta-oxidation pathway and ethyl ester synthesis. Indeed, the induction of FAA1 and EEB1, coding for a long-chain fatty acyl coenzyme A synthetase and an alcohol acyltransferase, respectively, suggests a detoxification pathway through the production of decanoate ethyl ester. These results are confirmed by the sensitivity of strains bearing deletions for the transcription factors encoded by PDR1, STB5, OAF1, and PIP2 genes.

On the basis of the comparison of the nucleotide sequences of the histidine decarboxylase genes (hdcA) of Lactobacillus 30A and Clostridium perfringens and the amino acid sequences of these histidine decarboxylases and those of Lactobacillus buchneri and Micrococcus, oligonucleotides unique to the hdcA genes were synthesized and used in PCR. All histidine-decarboxylating lactic acid bacteria gave a signal with primer set JV16HC/JV17HC in PCR. In addition to this primer set, CL1/CL2 and CL1/JV17HC were also useful for the detection of histamine-forming Leuconostoc aenos strains in PCR. The 150 base pair amplification product of the decarboxylating Leuc. aenos strain generated with primer set CL1/CL2 was sequenced. Alignment studies showed a high degree of relatedness among the hdcA gene products of Gram-positive bacteria. The amplification products of the hdcA genes from Lac. buchneri and Leuct. aenos were used to serve as a DNA probe in hybridization studies. All histidine-decarboxylating lactic acid bacteria gave a hybridization signal with the DNA probes. In hybridization only one false-positive signal with a Lactobacillus lindneri strain was observed, which was anticipated to contain a truncated hdcA gene. In addition to these DNA probe tests, a simple and reliable activity test is presented, which can be used during starter selection to test strains for histidine decarboxylase activity.

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Nine yeast strains were isolated from spontaneous fermentations in the Alsace area of France, during the 1997, 1998 and 1999 grape harvests. Strains were characterized by pulsed-field gel electrophoresis, PCR-restriction fragment length polymorphism (RFLP) of the MET2 gene, delta-PCR, and microsatellite patterns. Karyotypes and MET2 fragments of the nine strains corresponded to mixed chromosomal bands and restriction patterns for both Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum. They also responded positively to amplification with microsatellite primers specific to both species and were demonstrated to be diploid. However, meiosis led to absolute nonviability of their spores on complete medium. All the results demonstrated that the nine yeast strains isolated were S. cerevisiaexS. bayanus var. uvarum diploid hybrids. Moreover, microsatellite DNA analysis identified strains isolated in the same cellar as potential parents belonging to S. bayanus var. uvarum and S. cerevisiae.