FŒB: Faculté d' Œnologie de l'Université de Bordeaux II, Talence, France. UTM: Université de Tras os Montes, Portugal. ŒC: Institut Œnologique de Champagne, France. CIVC: Comité Interprofessionnel des Vins de Champagne (Interprofessional Champagne Committee), Epernay, France. UB: Université de Bourgogne, Dijon, France. NA: not available.
TABLE 1.7 Characterization by PCR‐RFLP of the MET2 Gene of Various Species of Native Saccharomyces Isolated on the Grape and in Wine (Masneuf, 1996)
| Number of differentstrains analyzed | Origin | Collection | Enological designation | Species |
|---|---|---|---|---|
| 8 | Sauternes wines | FŒB | Saccharomyces cerevisiae | Saccharomyces cerevisiae |
| 2 | Dry white Bordeaux wines | FŒB | Saccharomyces bayanus | Saccharomyces cerevisiae |
| 9 | Sauternes wines | FŒB | Saccharomyces bayanus | Saccharomyces cerevisiae |
| 2 | Dry white Bordeaux wines | FŒB | Saccharomyces chevalieri | Saccharomyces cerevisiae |
| 1 | Sauternes wines | FŒB | Saccharomyces capensis | Saccharomyces cerevisiae |
| 36 | Unknown | Lallemand | Saccharomyces bayanus | Saccharomyces cerevisiae |
| 11 | Sauternes wines | FŒB | Saccharomyces uvarum | Saccharomyces uvarum |
| 1 | Sauternes wines | FŒB | Hanseniaspora uvarum | Saccharomyces cerevisiae |
| 10 | Sancerre and Pouilly Fumé (Loire) wines | FŒB | Hanseniaspora uvarum | Saccharomyces uvarum |
| 1 | Sancerre grapes | FŒB | Hanseniaspora uvarum | Hanseniaspora uvarum |
| 2 | Unknown | Lallemand | Hanseniaspora uvarum | Saccharomyces cerevisiae |
FŒB: Faculté dOEnologie de l'Université de Bordeaux II, Talence, France. Lallemand: Lallemand Inc., Montreal, Quebec, Canada.
Moreover, we have determined the species of 82 strains of native Saccharomyces isolated from fermenting wine or from grapes (Table 1.7). For the eight Gal+ Mel− strains analyzed, as for the 47 Gal− Mel−, respectively, called S. cerevisiae and S. bayanus by enologists, restriction profiles of the amplified fragment of the MET2 gene are characteristic of S. cerevisiae. The same goes for the two S. chevalieri strains, which ferment galactose but not maltose (Ma−), as well as for the S. capensis strain (Gal− Ma−). As for Mel+ strains, called S. uvarum until now, most of them (11 out of 12 for Sauternes isolates and 11 out of 11 for Sancerre isolates) belong to the S. uvarum species. However, some Mel+ strains are S. cerevisiae (one strain from Sauternes and two strains from the Lallemand collection). In short, regarding the classification of the main winemaking yeasts (Section 1.8.3), we can distinguish between three stages. First, we considered them to be several species: S. cerevisiae, S. bayanus, and/or S. oviformis, S. uvarum. Then, we decided they were different races of the S. cerevisiae species. The current taxonomy, based on molecular biology results, has made substantial changes. It has defined three species: S. cerevisiae, S. uvarum, and S. paradoxus. The involvement of S. paradoxus in the fermentation microflora of grapes remains to be confirmed.
All of the results of molecular taxonomy presented above show that the former phenotypic classifications, based on physiological identification criteria, are not suitable even for delimiting the small number of fermentative species of the Saccharomyces genus found in winemaking. Moreover, specialists have long known about the instability of the physiological properties of Saccharomyces strains. Rossini et al. (1982) reclassified a thousand strains from the yeast collection of the Microbiology Institute of Agriculture at the University of Perugia. They observed that 23 out of 591 S. cerevisiae strains conserved on malt agar had lost the ability to ferment galactose. The use of genetic methods is thus essential to identify winemaking yeasts.
1.8.4 Interspecific Hybrids
PCR‐RFLP associated with the MET2 gene can be used to demonstrate the existence of hybrids between the species S. cerevisiae and S. uvarum. This method has been used to prove the existence (Masneuf et al., 1998) of one such natural hybrid (strain S6U) among commercial dry yeasts sold by Lallemand Inc. (Montreal, Canada). Ciolfi (1992, 1994) isolated this yeast in an Italian winery, and it was selected for certain enological properties, in particular its aptitude to ferment at low temperatures, its low production of acetic acid, and its ability to preserve must acidity. The MET2 gene restriction profiles of this strain by EcoR1 and Pst1, composed of three bands, are identical (Figure 1.25). In addition to the amplified fragment, two bands characteristic of S. cerevisiae with EcoR1 and two bands characteristic of the species S. bayanus with Pst1 are obtained. The bands are not artifacts due to an impurity in the strain, because the amplification of the MET2 gene carried out on subclones (obtained from the multiplication of unique cells isolated by a micromanipulator) produces identical results. Hansen from the Carlsberg laboratory (Denmark) sequenced two of the MET2 gene alleles from this strain. The sequence of one of
