Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 44

A large amount of research was focused on the description and ecology of wine yeasts. It concerned the distribution and succession of species found on the grape and then in wine during fermentation and conservation (Ribéreau‐Gayon et al., 1975; Lafon‐Lafourcade, 1983).

The ecological study of grape and wine yeast species represents a considerable amount of research. De Rossi began his research in the 1930s (De Rossi, 1935). Castelli (1955, 1967) pursued the study of yeast ecology in Italian vineyards. Peynaud and Domercq (1953) and Domercq (1956) published the first results on the ecology of wine yeasts in France. They described not only the species found on the grape and during alcoholic fermentation but also contaminating and spoilage yeasts. Among the many publications on this topic since the 1960s in viticultural regions around the world, the following works are worth noting: Brechot et al. (1962), Sapis‐Domercq (1970), Barnett et al. (1972), Minarik (1971), Cuinier and Guerineau (1976), Park (1975), Soufleros (1978), Belin (1979, 1981), Poulard et al. (1980), Poulard and Lecocq (1981), Bureau et al. (1982), Rossini et al. (1982), Fleet et al. (1984), Mills et al. (2002), Baleiras Couto et al. (2005), Hierro et al. (2006), and Nisiotou and Nychas (2007).

Yeasts are widespread in nature and are found in soils, on the surface of plants, and in the digestive tract of animals. Wind, insects, and birds disseminate them. Yeasts are distributed irregularly on the surface of the grapevine; found in small quantities on leaves, the stems, and unripe grapes, they colonize the grape skin during maturation. During the winter season, the main natural reservoir of yeasts is the trunk, while during the vegetative growth phase of the vine, it is found in the ground and in berries (Cordero‐Bueso et al., 2011). Observations under a scanning electron microscope have identified the location of yeasts on the grape. They are rarely found on the bloom, but multiply preferentially on exudates released from microlesions in zones situated around the stomatal apparatus. Botrytis cinerea spores and lactic acid and acetic acid bacteria also develop in the proximity of these peristomatic fractures (Figure 1.34).

The number of yeasts on the grape berry, just before harvest, is between 10³ and 10⁵, depending on the geographical location of the vineyard, weather conditions during maturation, the healthiness of the harvested grapes, and pesticide treatments applied to the vine. The most abundant yeast populations are obtained under warm weather conditions (lower latitudes and higher temperatures). Insecticide treatments and certain fungicidal treatments can make the native grape microflora less populous. Quantitative results available on this subject, however, are few. After the harvest, transport, and crushing of the crop, the number of cells capable of forming colonies on an agar medium generally reaches 10⁶ cells/ml of must.

The number of yeast species significantly present on the grape is limited. From fruit set to maturity, Aureobasidium pullulans, Cryotococcus, and Rhodotorula are the main genera and species encountered (Prakitchaiwattana et al., 2004; Martins et al., 2014). Their proportion then decreases in favor of Ascomycetes. Among the latter, the apiculate species (K. apiculata and its sporogenous form H. uvarum) are the most common. They comprise up to 99% of the yeasts isolated in certain grape samples. The following are generally found but in lesser proportions: Metschnikowia pulcherrima, C. famata, C. stellata, P. membranifaciens, Pichia fermentans, and Hansenula anomala. The increase of grape exudate sugar content may partly explain these changes in yeast populations (Martins, 2012).

FIGURE 1.34 Grape surface under scanning electron microscope, with detail of yeast peristomatic zones.

(Source: Photographs from B. Pucheu‐Plante and M. Mercier, Department of Electron Microscopy, Université de Bordeaux 1.)

All research confirms the extreme rarity of S. cerevisiae on grapes (Mortimer and Polsinelli, 1999). However, these yeasts are not totally absent. Their existence cannot be proven by streaking diluted must on a solid medium prepared under aseptic conditions, but their presence on grapes can be proven by analyzing the spontaneous fermentative microflora of grape samples placed in sterile bags, then aseptically crushed and vinified in the laboratory in the absence of all contamination. Red and white grapes from the Bordeaux region were treated in this manner. At mid‐fermentation in the majority of cases, S. cerevisiae represented almost all of the yeasts isolated. In some rare cases, no yeast of this species developed and non‐Saccharomyces yeasts began the fermentation. Nevertheless, growth of S. cerevisiae under these conditions from healthy grapes is not guaranteed. Thus, based on 134 samples of grapes collected in the Bordeaux region, 31% of samples were positive for S. cerevisiae at the two‐thirds mark of alcoholic fermentation (Börlin, 2015). Under the same operating conditions, 28–38% of positive fermentation was obtained for grape samples from the Douro region in Portugal (Schuller et al., 2012). The frequency of isolation of S. cerevisiae may approach 100% on damaged grapes, which are a very favorable environment for the development of fermentation yeasts (Mortimer and Polsinelli, 1999).

Ecological surveys carried out at the Bordeaux Faculty of Enology from 1992 to 1999 (Naumov et al., 2000a) demonstrated the presence of S. uvarum yeasts on grapes and in spontaneously fermenting white musts from the Loire Valley, Jurançon, and Sauternes. The frequency of the presence of this species alongside S. cerevisiae varies from 4 to 100%. On one estate in Alsace, strains of S. uvarum were identified on grapes, in the press, and in tanks, where they represented up to 90% of the yeasts involved throughout fermentation in three consecutive years (Demuyter et al., 2004). More recently, other authors (Naumov et al., 2002; Zhang et al., 2015) have shown that S. uvarum, identified on grapes and in fermenting must, is involved in making Tokaji and New Zealand wines.

The adaptation of S. uvarum to relatively low temperatures (6–10°C) certainly explains its presence in certain ecological niches: northerly vineyards, late harvests, and spontaneous “cold” fermentation of white wines. In contrast, this strain is sensitive to high temperatures and has not been found in spontaneous fermentations of red Bordeaux wines.

Between two harvests, winery walls, floors, equipment, and sometimes even the winery building itself are colonized mostly by the various non‐fermentation species previously cited. Winemakers believe, however, that spontaneous fermentations are more difficult to initiate in new tanks than in tanks that have already been used. This empirical observation leads to the supposition that S. cerevisiae can also survive in the winery between two harvests. Moreover, this species was found in non‐negligible proportions in the wooden fermentors of some of the best vineyards in Bordeaux during the harvest, just before they were filled.

Recent studies relying on global approaches to sequencing of DNA extracted from a biological sample demonstrate the presence of the main yeast species in fermenting must (H. uvarum, S. bacillaris, and S. cerevisiae) in the various zones of winemaking cellars (pressing, fermentation, and storage) before, during, and after the harvest period (Bokulich et al., 2013).

In the first hours of spontaneous fermentations, the first tanks filled have a very similar microflora to that of the grapes, with a large proportion of H. uvarum, S. bacillaris (formerly known as Candida zemplinina), and M. pulcherrima species. After about 20 hours, S. cerevisiae develops and coexists with the grape yeasts (Zott et al., 2008). Non‐Saccharomyces yeasts quickly disappear at the start of spontaneous fermentation (Figure 1.35). The microflora of tanks inoculated with yeast is very similar to that of tanks undergoing spontaneous fermentation (Figure 1.36). The major difference resides in the nature of S. cerevisiae strains that conduct fermentation: there is a dominant clone in the case of adding yeast. Meanwhile, in the case of spontaneous fermentation, several clones coexist (Section 1.10.2). Thus, the practice of adding yeast does not eliminate populations of non‐Saccharomyces yeasts at the start of alcoholic fermentation. In red winemaking in the Bordeaux region, as soon as must specific gravity drops below 1.070–1.060, the colony samples obtained by plating diluted must on a solid medium generally isolate S. cerevisiae (10⁷–10⁸ cells/ml) exclusively. This species plays an essential role in the alcoholic fermentation process. Environmental conditions influence its selection. This selection pressure is exhibited by four main parameters: anaerobic conditions, must or grape sulfiting, sugar concentration, and the increasing presence of ethanol. The increase in temperature, especially in the case of red winemaking, also favors the development of S. cerevisiae to the detriment of non‐Saccharomyces yeasts (Goddard, 2008). In winemaking, where no sulfur dioxide is used, such as white wines for the production of spirits, the dominant grape microflora can still be found. It is largely present at the beginning of alcoholic fermentation (Figure 1.35). Even in this type of winemaking, the presence of apiculate yeasts is limited at the midpoint of alcoholic fermentation.

FIGURE 1.35 Comparison of yeast species present at the start of alcoholic fermentation (d = 1.06). A, in a tank of sulfited red grapes in Bordeaux (Frezier, 1992); B, in a tank of unsulfited white must, for the production of Cognac (Versavaud, 1994).

During dry white winemaking, the separation of the pomace after pressing, combined with clarification by racking, greatly reduces yeast populations, at least in the first few days of the harvest. The yeast population of a severely racked must rarely exceeds 10⁴–10⁵ cells/ml.

A few days into the harvest, the S. cerevisiae yeasts colonize the harvest equipment, grape transport machinery, and especially the grape receiving equipment, the crusher, stemmer, the wine press, and cellar atmosphere (Grangeteau et al., 2015). For this reason, S. cerevisiae is already widely present at the time of filling the tanks (around 50% of yeasts isolated during the first homogenization pump‐over of a red grape tank). Fermentations are initiated more rapidly as harvest goes on. In fact, the last tanks filled often complete their fermentations before the first ones. Similarly, static racking in dry white winemaking becomes more and more difficult to achieve, even at low temperatures, from the second week of the harvest onward, especially in hot years. The entire facility inoculates the must with a sizeable fermentation yeast population. General weekly disinfection of the pumps, piping, wine presses, settling tanks, etc., is therefore strongly recommended.

During the final part of alcoholic fermentation (the yeast decline phase), the population of S. cerevisiae progressively decreases while still remaining greater than 10⁶ cells/ml. Under favorable winemaking conditions, characterized by a rapid and complete exhaustion of sugars, no other yeast species significantly appears at the end of fermentation. Under poor conditions, spoilage yeasts can contaminate the wine. One of the most frequent and most dangerous contaminations is due to the development of B. bruxellensis, which is responsible for serious off‐odors (Volume 2, Section 8.4.5).

FIGURE 1.36 Dynamics of total yeasts and non‐Saccharomyces yeasts during red winemaking monitored by (a) culture/RFLP‐ITS‐PCR and (b) specific quantitative PCR applied on a DNA pellet extracted directly from fresh must. Hour 0/day 0, time of inoculation with commercial yeast; − hours/days, cold soaking; + hours/days, alcoholic fermentation (Zott et al., 2010).

In the weeks that follow the completion of alcoholic fermentation, the viable populations of S. cerevisiae drop rapidly, falling below a few hundred cells/ml. In many cases, other yeast species (spoilage yeasts) can develop in wines during bulk or bottle aging. Some yeasts have an oxidative metabolism of ethanol and form a veil on the surface of the wine, such as Pichia or Candida, or even certain strains of S. cerevisiae—sought after in the production of specialty wines. By topping up regularly, the development of these respiratory metabolism yeasts can be prevented. Some other yeasts, such as Brettanomyces or Dekkera, can develop under anaerobic conditions, consuming trace amounts of sugars that have been incompletely or not fermented by S. cerevisiae. Their population can attain 10⁴–10⁵ cells/ml in a contaminated red wine in which alcoholic fermentation is otherwise completed normally. These contaminations can also occur in the bottle. Lastly, refermentation yeasts can develop significantly in sweet or botrytized sweet wines during aging or bottle storage. The principal species found are S. ludwigii, Z. bailii, and also some strains of S. cerevisiae that are particularly resistant to ethanol and sulfur dioxide.