Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 35

Due to many changes in yeast classification and nomenclature since the beginning of taxonomic studies, wine‐related yeast names and their positions in the classification have often changed. This has inevitably resulted in some confusion for winemakers. Even the most recent enological textbooks (Fleet, 1993; Delfini, 1995; Boulton et al., 1995) use a number of species names (cerevisiae, bayanus, uvarum, etc.) attached to the genus name Saccharomyces to designate yeasts responsible for alcoholic fermentation. Although still in use, this enological terminology is no longer accurate to designate the species currently delimited by taxonomists.

The evolution of species classification for the genus Saccharomyces since the early 1950s (Table 1.3) has created this difference between the naming of wine yeasts and current taxonomy. By taking a closer look at this evolution, the origin of the differences may be understood.

In Lodder and Kregger‐Van Rij (1952), the names cerevisiae, oviformis, bayanus, uvarum, etc., referred to a number of the 30 species of the genus Saccharomyces. Ribéreau‐Gayon and Peynaud (1960) considered that two principal fermentation species were found in wine: S. cerevisiae (formerly called ellipsoideus) and Saccharomyces oviformis, which was encountered especially toward the end of fermentation and was considered more ethanol resistant. The difference in their ability to ferment galactose distinguished the two species: S. cerevisiae (Gal⁺) fermented galactose, whereas S. oviformis (Gal⁻) did not. According to the same authors, the species Saccharomyces bayanus was rarely found in wines. Although it possessed the same physiological fermentation and sugar assimilation characters as S. oviformis, its cells were more elongated, its fermentation was slower, and it displayed specific behavior toward growth factors. As for the species Saccharomyces uvarum, identified in wine by many authors, it differed from S. cerevisiae, S. oviformis, and S. bayanus because it could ferment melibiose.

In Lodder's following edition (1970), the number of species of the genus Saccharomyces increased from 30 to 41. Some species formerly grouped with other genera were integrated into the genus Saccharomyces. Moreover, several species names were considered to be synonyms and disappeared altogether. Such was the case of S. oviformis, which was moved to the species S. bayanus. Ribéreau‐Gayon et al. (1975) considered, however, that the distinction between S. oviformis and S. bayanus was of enological interest because of the different technological characteristics of these two yeasts. Nevertheless, by the early 1980s, most enology texts had abandoned the name S. oviformis and replaced it with S. bayanus.

TABLE 1.3 Evolution of the Nomenclature for the Saccharomyces Genus, 1952–2011 (Libkind et al. 2011)

Source : From Barnett et al. ( 2000), Lodder and Kregger‐Van Rij ( 1952), Lodder ( 1970), Kregger‐Van Rij ( 1984), and Kurtzman et al. ( 2011).

1952	1970	1984	2011
Saccharomyces cerevisiae ⇒ Saccharomyces pastorianus Saccharomyces bayanus ⇒ Saccharomyces oviformis Saccharomyces logos Saccharomyces chevalieri ⇒ Saccharomyces fructuum Saccharomyces lactis Saccharomyces elegans Saccharomyces heterogenicus Saccharomyces fermentati Saccharomyces mellis Saccharomyces italicus ⇒ Saccharomyces steineri Saccharomyces pastori Saccharomyces carlsbergensis Saccharomyces uvarum ⇒	Saccharomyces cerevisiae Saccharomyces aceti Saccharomyces bayanus Saccharomyces capensis Saccharomyces prostoserdovii Saccharomyces chevalieri Saccharomyces coreanus Saccharomyces diastaticus Saccharomyces globosus Saccharomyces heterogenicus Saccharomyces hienipiensis Saccharomyces inusitatus Saccharomyces italicus Saccharomyces norbensis Saccharomyces oleaceus Saccharomyces oleaginosuss Saccharomyces uvarum	Saccharomyces cerevisiae	Saccharomyces arboricolus Saccharomyces bayanus a Saccharomyces cariocanus Saccharomyces cerevisiae Saccharomyces eubayanus Saccharomyces kudriavzevii Saccharomyces mikatae Saccharomyces paradoxus Saccharomyces pastorianus a Saccharomyces uvarum
Saccharomyces acidifaciens Saccharomyces bailii ⇒ Saccharomyces fragilis Saccharomyces delbrueckii ⇒ Saccharomyces marxianus Saccharomyces exiguus ⇒ Saccharomyces veronae Saccharomyces florentinus ⇒ Saccharomyces bisporus ⇒ Saccharomyces willianus Saccharomyces microellipsodes ⇒	Saccharomyces incompspicuus Saccharomyces amurcae Saccharomyces bailii Saccharomyces cidri Saccharomyces dairensis⇒ Saccharomyces delbrueckii Saccharomyces eupagycus Saccharomyces exiguus ⇒ Saccharomyces fermentati Saccharomyces florentinus Saccharomyces bisporus Saccharomyces kloeckerianus Saccharomyces kluyveri ⇒ Saccharomyces microellipsodes Saccharomyces montanus Saccharomyces mrakii Saccharomyces pretoriensis	Saccharomyces dairensis Saccharomyces exiguus Saccharomyces kluyveri
Saccharomyces rosei ⇒ Saccharomyces rouxii ⇒	Saccharomyces rosei Saccharomyces rouxii Saccharomyces saitoanus Saccharomyces telluris ⇒ Saccharomyces transvaalensis Saccharomyces unisporus ⇒ Saccharomyces vafer	Saccharomyces telluris Saccharomyces unisporus Saccharomyces servazzii

^a Interspecific hybrids.

The new classification by Kregger‐Van Rij (1984), based on Yarrow's work on percentages of guanine and cytosine bases in yeast DNA, brought forth another important change in the designation of Saccharomyces species. Only seven species continued to exist, while 17 names became synonyms of S. cerevisiae. As with the preceding yeast classification, these yeast were differentiated by their sugar utilization profile (Table 1.4), and some authors considered them to be races or physiological varieties of S. cerevisiae. However, this was nothing more than an artificial taxonomy without biological significance. Enologists took the habit of adding the varietal name to S. cerevisiae to designate wine yeasts: S. cerevisiae var. cerevisiae, var. bayanus, var. uvarum, var. chevalieri, etc. In addition, two species, bailii and rosei, were removed from the genus Saccharomyces and integrated into two other genera to become Zygosaccharomyces bailii and T. delbrueckii, respectively.

Based on recent advances in genetics and molecular taxonomy, the latest yeast classification (Kurtzman et al., 2011) and recent studies (Libkind et al., 2011) have again thrown the species delimitation of the genus Saccharomyces into confusion. There are now 10 species (Table 1.3). The species Saccharomyces paradoxus, Saccharomyces arboricolus, Saccharomyces mikatae, and Saccharomyces eubayanus include strains initially isolated from tree exudates, insects, and soil (Naumov et al., 1998, 2000a; Wang and Bai, 2008; Libkind et al., 2011). Using genome analysis, Redzepovic has nevertheless identified a high percentage of S. paradoxus in Croatian grape microflora in Redzepovic et al. (2002).

TABLE 1.4 Physiological Races of S. cerevisiae Grouped Under a Single SpeciesS. cerevisiae by Yarrow and Nakase (1975)

	Fermentation
Ga	Su	Ma	Ra	Me	St
Saccharomyces
aceti	−	−	−	−	−	−
bayanus	−	+	+	+	−	−
capensis	−	+	−	+	−	−
cerevisiae	+	+	+	+	−	−
chevalieri	+	+	−	+	−	−
coreanus	+	+	−	+	+	−
diastaticus	+	+	+	+	−	+
globosus	+	−	−	−	−	−
heterogenicus	−	+	+	−	−	−
hienipiensis	−	−	+	−	+	−
inusitatus	−	+	+	+	+	−
norbensis	−	−	−	−	+	−
oleaceus	+	−	−	+	+	−
oleaginosus	+	−	+	+	+	−
prostoserdovii	−	−	+	−	−	−
steineri	+	+	+	−	−	−
uvarum	+	+	+	+	+	−

Ga, D‐galactose; Su, sucrose; Ma, maltose; Ra, raffinose; Me, melibiose; St, soluble starch.

TABLE 1.5 DNA/DNA Reassociation Percentages Between the Four SpeciesBelonging to the Saccharomyces Genus in the Strict Sense (Vaughan Martini and Martini, 1987)

	Saccharomyces cerevisiae	Saccharomyces bayanus	Saccharomyces pastorianus	Saccharomyces paradoxus
Saccharomycescerevisiae	100
Saccharomycesbayanus	20	100
Saccharomycespastorianus	58	70	100
Saccharomycesparadoxus	53	24	24	100

Saccharomyces pastorianus replaces the former name Saccharomyces carlsbergensis, given to brewery yeast strains, used for low‐temperature fermentations (lager) and until then included in the cerevisiae species. The placement of S. bayanus, S. uvarum, and S. pastorianus within the Saccharomyces genus was the subject of controversies among yeast scientists for years. The latest works, based on DNA sequencing and the recent discovery of the S. eubayanus species, have definitively determined the taxonomic positions of S. bayanus and S. pastorianus (Tables 1.4 and 1.5; Nguyen and Gaillardin, 2005; Libkind et al., 2011; Nguyen and Boekhout, 2017). It has been clearly established that S. bayanus and S. pastorianus refer to hybrid individuals, composed of S. eubayanus, S. uvarum, and S. cerevisiae genomes. On the other hand, S. uvarum and S. eubayanus are considered as being of genetically pure lineage.

Thus, S. bayanus is now considered a distinct hybrid species of S. cerevisiae and S. uvarum according to taxonomists. Nevertheless, enologists and winemakers use the name of bayanus (ex oviformis) to designate a physiological race of S. cerevisiae that does not ferment galactose. It possesses a greater resistance to ethanol than S. cerevisiae. The implementation of molecular biology methods, based on DNA analysis, has helped establish the position of the winemaking yeasts formerly designated as var. bayanus and var. uvarum in agreement with the current taxonomy.

For some 30 years, fragment amplification of the genome by the polymerase chain reaction (PCR) has provided an excellent discrimination tool for winemaking yeast species.

Since its discovery by Saiki et al. (1985), PCR has often been used to identify different plant and bacteria species. This technique consists in enzymatically amplifying one or several gene fragments in vitro. The reaction is based on the hybridization of two oligonucleotides that frame a target region on a double strand of DNA or template. These oligonucleotides have different sequences and are complementary to the DNA sequences that frame the strand being amplified. Figure 1.21 illustrates the various stages of the amplification process. The DNA is first denatured at a high temperature (95°C). The reaction mixture is then cooled to a temperature between 37 and 55°C, enabling the hybridization of these oligonucleotides on the denatured strands. The strands serve as primers from which a DNA polymerase enables the step‐by‐step addition of deoxyribonucleotide units in the 5′–3′ direction. The DNA polymerase (Figure 1.22) requires four deoxyribonucleoside‐5Π‐triphosphates (dATP, dGTP, dTTP, and dCTP). A phosphodiester bond is formed between the 3Π–OH end of the primer and the innermost phosphorus of the activated deoxyribonucleoside. Pyrophosphate is thus released. The newly synthesized strand is elongated on the template. A heat‐resistant enzyme, TAQ DNA polymerase, comes from the heat‐resistant bacteria Thermus aquaticus. It is used to conduct a large number of amplification cycles (25–40) in vitro without having to add DNA polymerase after each denaturation. In this manner, the DNA fragment amplified during the first cycle serves as the template for the following cycles. In consequence, each successive cycle doubles the target DNA fragment, which is thus amplified by a factor of 10⁵ to 10⁶ during 25–30 amplification cycles.

FIGURE 1.21 Principle of the polymerization chain reaction (PCR).

Hansen and Kielland‐Brandt (1994) proposed MET2 gene PCR amplification to differentiate between S. cerevisiae and S. uvarum (formerly S. bayanus), when working on strain types of these two species. This gene, which codes for the synthesis of homoserine acetyltransferase, has different sequences in the two species. Part of the gene is initially amplified by using two complementary oligonucleotides of the sequences bordering the fragment to be amplified. The fragment obtained (about 600 bp) is the same size for typical strains of the S. cerevisiae and S. uvarum species. Different restriction endonucleases, which recognize certain specific DNA sequences, then digest the amplified fragment. Figure 1.23 gives an example of the mode of action of the EcoR1 restriction endonuclease. This enzyme recognizes the base sequence GAATTC and cuts at the location indicated by the arrows. Electrophoresis is used to separate the obtained fragments. As a result, restriction fragment length polymorphism (RFLP) can be assessed. The restriction profiles obtained differ between S. cerevisiae and S. uvarum.

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)

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.