Handbook of Enology: Volume 1. Pascal Ribéreau-Gayon

FIGURE 1.22 Mode of action of DNA polymerase.

FIGURE 1.23 Recognition site and cutting mode of an EcoR1 restriction endonuclease.

FIGURE 1.24 Identification principles for the S. cerevisiae and S. uvarum species by the MET2 gene PCR‐RFLP technique, after cutting the amplified fragment with EcoR1 and Pst1 restriction enzymes.

This PCR‐RFLP technique associated with the MET2 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: EcoR1 and Pst1 (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 MET2 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 MET2 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)

Strains	Commercial brand	Origin	Enological designation	Species
VL1	Zymaflore VL1	FŒB	Saccharomyces cerevisiae	Saccharomyces cerevisiae
VL3c	Zymaflore VL3	FŒB	Saccharomyces cerevisiae	Saccharomyces cerevisiae
WET 136	Siha levactif 3	Dormstadt	Saccharomyces cerevisiae	Saccharomyces cerevisiae
71B	Actiflore primeur	INRA Narbonne	Saccharomyces cerevisiae	Saccharomyces cerevisiae
F10	Zymaflore F10	FŒB	Saccharomyces bayanus	Saccharomyces cerevisiae
R2	Vitlevure KD	NA	Saccharomyces bayanus	Saccharomyces cerevisiae
BO213	Actiflore bayanus	Institut Pasteur	Saccharomyces bayanus	Saccharomyces cerevisiae
CH158	Siha levactif 4	NA	Saccharomyces bayanus	Saccharomyces cerevisiae
QA23	Lalvin QA23	UTM	Saccharomyces bayanus	Saccharomyces cerevisiae
IOC182007	IOC 182007	IŒC	Saccharomyces bayanus	Saccharomyces cerevisiae
DV10	Vitlevure DV10	CIVC	Saccharomyces bayanus	Saccharomyces cerevisiae
O16	Lalvin O16	UB	Saccharomyces bayanus	Saccharomyces cerevisiae
Epernay2	Uvaferm CEG	NA	Saccharomyces bayanus	Saccharomyces cerevisiae

      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)

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Number of differentstrains analyzed	Origin	Collection	Enological designation	Species
8	Sauternes wines	FŒB	Saccharomyces cerevisiae