Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 34

Yeast taxonomy (from the Greek taxis: putting in order), and the taxonomy of other microorganisms for that matter, includes classification and identification. Classification groups organisms into taxa according to their similarities and/or their ties to a common ancestor. The basic taxon is species. A species can be defined as a collection of strains having a certain number of morphological, physiological, and genetic characteristics in common. This group of characteristics constitutes the standard description of the species. Identification compares an unknown organism to individuals already classified and named that have similar characteristics.

TABLE 1.1 Classification of Grape and Wine Yeast Genera (Kurtzman et al., 1994)

Phylum	Subphylum	Order	Families	Genera
Ascomycota	Saccharomycotina	Saccharomycetaceae	Saccharomycetales	Saccharomyces Kluyveromyces Torulaspora Zygosaccharomyces Kazachstania Lachancea
Pichiaceae	Pichia Brettanomyces Dekkera
Metschnikowiaceae	Metschnikowia
Debaryomycetaceae	Debaryomyces Lodderomyces
Saccharomycodaceae	Hanseniaspora Kloeckera Saccharomycodes
Saccharomycetales Incertae sedis	Wickerhamo‐mycetaceae	Candida

FIGURE 1.19 Observation of two winemaking yeast species having an apiculate form

(Source: Photographs from M. Mercier, Department of Electron Microscopy, Université de Bordeaux 1.)

(a) H. uvarum. (b) S. ludwigii.

Taxonomists first delimited yeast species using morphological and physiological criteria. The first classifications were based on the phenotypic differences between yeasts: cell shape and size, spore formation, cultural characteristics, fermentation and assimilation of various sugars, assimilation of nitrates, growth‐factor needs, and resistance to cycloheximide. Ribéreau‐Gayon et al. (1975) described the application of these methods to wine yeasts in detail. Since then, many rapid, ready‐to‐use diagnostic kits have been proposed to determine yeast response to different physiological tests. Lafon‐Lafourcade and Joyeux (1979) and Cuinier and Leveau (1979) designed the API 20 C system for the identification of winemaking yeasts. It contains eight fermentation tests, 10 assimilation tests, and a cycloheximide resistance test. For a more complete identification, the API 50 CH system contains 50 substrates for fermentation (under paraffin) and assimilation tests. Heard and Fleet (1990) developed a system that uses the various tests listed in the work of Barnett et al. (1990).

FIGURE 1.20 Binary fission of S. pombe.

(Source: Photographs from M. Mercier, Department of Electron Microscopy, Université de Bordeaux 1.)

Due to the relatively limited number of yeast species significantly present on grapes or in wine, these phenotypic tests identify winemaking yeast species in certain genera without difficulty. Certain species can be identified by observing growing cells under the microscope alone. Small apiculate cells, with small lemon‐like shapes, are indicative of the species H. uvarum and its imperfect form Kloeckera apiculata (Figure 1.19). Saccharomycodes ludwigii is characterized by much larger (10–20 μm) apiculate cells. Since most yeast multiply by budding, the genus Schizosaccharomyces can be recognized because of its vegetative reproduction by binary fission (Figure 1.20). In modern taxonomy, this genus only contains the species Schizosaccharomyces pombe. Finally, the budding of Candida stellata (formerly known as Torulopsis stellata) occurs in the shape of a characteristic star.

According to Barnett et al. (1990), the physiological characteristics listed in Table 1.2 can be used to distinguish between the principal grape and wine yeasts. Yet some of these characteristics (for example, fermentation profiles of sugars) vary within the species and are even unstable for a given strain during vegetative reproduction. Taxonomists have realized that they cannot differentiate species based solely on phenotypic discontinuity criteria. They have progressively established a delimitation founded on the biological and genetic definition of a species.

In theory, a species can be defined as a collection of interfertile strains whose hybrids are themselves fertile—capable of producing viable spores. This biological definition runs into several problems when applied to yeasts. First of all, many Ascomycetes yeasts are homothallic; hybridization tests are thus especially tedious and difficult for routine identification. Finally, certain wine yeast strains have little or no sporulation aptitude, which makes the use of strain infertility criteria even more difficult.

TABLE 1.2 Physiological Characteristics of the Principal Grape and Wine Yeasts (Barnett et al., 1990)

	Fl D‐Glucose fermentation	F2 D‐Galactose fermentation	F3 Maltose fermentation	F4 α‐D‐glucoside fermentation	F5 Sucrose fermentation	F6 α,α‐Trehalose fermentation	F7 Melibiose fermentation
Candida stellata	+	–	–	–	+	–	–
Candida vini	v	–	–	–	–	–	–
Candida famata	v	v	v	–	v	v	v
Dekkera anomala	+	v	v	v	+	v	–
Dekkera bruxellensis	v	v	v	+	+	+	–
Hanseniaspora uvarum	+	–	–	–	–	–	–
Melschnikowia pulcherrima	+	v	–	–	–	–	–
Pichia anomala	+	v	v	v	+	v	–
Pichia fermentans	+	–	–	–	–	–	–
Pichia membranifaciens	v	–	–	–	–	–	–
Saccharomyces cerevisiae	+	v	v	v	v	v	v
Saccharomycodes ludwigii	+	–	–	–	+	–	–
Kluyveromyces thermolerens	+	v	v	v	+	v	–
Schizosaccharomyces pombe	+	v	v	v	+	–	v
Zygosaccharomyces bailii	+	–	–	–	v	v	–
	C30 Galactitol growth	C31 myo‐Inositol growth	C32 D‐Glucono‐1,5‐lactone growth	C33 2‐Keto‐D‐gluconate growth	C34 5‐Keto‐D‐gluconate growth	C35 D‐gluconate growth	C38 DL‐Lactate growth
Candida stellata	–	–	–	–	–	–	–
Candida vini	–	–	–	v		–	v
Candida famata	v	–	v	+		v	v
Dekkera anomala	–	–	v	–	–	v	v
Dekkera bruxellensis	–	–	v	v	–	–	v
Hanseniaspora uvarum	–	–	+	+	–	v	–
Melschnikowia pulcherrima	–	–	+	+		+	v
Pichia anomala	–	–	+	–	–	v	+
Pichia fermentans	–	–	v	–	–	–	+
Pichia membranifaciens	–	–	v	–	–	–	v
Saccharomyces cerevisiae	–	–	v	–	–	–	v
Saccharomycodes ludwigii	–	–	v	–	–	–	v
Kluyveromyces thermolerens	–	–	v	v	–	v	–
Schizosaccharomyces pombe	–	–	v	v	–	v	–
Zygosaccharomyces bailii	–	–	v	v	–	–	–

To overcome these difficulties, researchers have developed a molecular taxonomy based on the following tests: DNA–DNA reassociation, the similarity of DNA base composition, the similarity of enzymes, ultrastructure characteristics, and cell wall composition. The development of DNA sequence analysis has profoundly changed the way in which yeasts are identified. More specifically, the comparison of gene sequences coding for ribosome RNA now resolves the taxonomic position of closely related species, as well as the most distantly related taxa. The sequence analysis of variable domains 1 and 2 (D1/D2) has been successfully applied to the identification of ascomycetous yeasts (Kurtzman and Robnett, 1998). Then, Fell et al. (2000) developed the method by bringing in an additional database to identify basidiomycetous yeasts. Moreover, analysis of internal transcribed spacer (ITS) sequences is often used to identify species (Granchi et al., 1999). Currently, the sequencing of all genomes is used to refine the taxonomy of yeasts in phylogenomic studies.