Pascal Ribéreau-Gayon - Handbook of Enology - Volume 1

FIGURE 1.22 Mode of action of DNA polymerase.

FIGURE 1.23 Recognition site and cutting mode of an Eco R1 restriction endonuclease.

FIGURE 1.24 Identification principles for the S. cerevisiae and S. uvarum species by the MET 2 gene PCR‐RFLP technique, after cutting the amplified fragment with Eco R1 and Pst 1 restriction enzymes.

This PCR‐RFLP technique associated with the MET 2 gene has been developed and adapted for rapid analysis. The whole cells are simply heated in water to 95°C for 10 minutes before amplification. Only two restriction enzymes are used: Eco R1 and Pst 1 ( Figure 1.24; Masneuf et al ., 1996a,b).

By applying this relatively simple and quick technique to different wine yeast strains studied by Naumov et al . (1993), we obtained perfect concordance between the MET 2 gene PCR‐RFLP and hybridization tests in order to delimit S. cerevisiae and S. uvarum species.

We extended this type of analysis by PCR‐RFLP of the MET 2 gene to different yeast strains selected from the market and often used in winemaking. Depending on their ability to ferment galactose or not, they are still sometimes called S. cerevisiae or S. bayanus by wine professionals around the world at the time this handbook is being written ( Table 1.6). For all of these strains, we have obtained the same characteristic restriction profiles of the species S. cerevisiae .

TABLE 1.6 Characterization by PCR‐RFLP of the MET2 Gene of Various Commercial Strains of the S. cerevisiae Species Used in Winemaking (Masneuf, 1996)

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)

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 MET 2 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.