Handbook of Enology: Volume 1. Pascal Ribéreau-Gayon
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.
1.10.2 The Ecology of S. cerevisiae Strains
The ecological study of the clonal diversity of yeasts, and in particular of S. cerevisiae during winemaking, was inconceivable for a long time because of a lack of means to distinguish yeast strains from one another. Such research has become possible with the development of molecular yeast strain identification methods (Section 1.9). This section focuses on recent advances in this field.
The alcoholic fermentation of grape must or grapes is essentially carried out by a single yeast species, S. cerevisiae. Therefore, an understanding of the clonal diversity within this species is much more important for the winemaker than investigations on the partially or non‐fermentative grape microflora.
The analysis of S. cerevisiae strains under practical winemaking conditions in particular is intended to answer the following questions:
Is spontaneous fermentation carried out by a dominant strain, a small number or a very large number of strains?
Can the existence of a succession of strains during alcoholic fermentation be proven? If so, what is their origin: grapes, harvest equipment, or winery equipment?
During winemaking and from one year to another in the same winery or even the same vineyard, is spontaneous alcoholic fermentation carried out by the same strains?
Can the practice of inoculating with selected strains modify the wild microflora of a vineyard?
During recent research conducted in the Bordeaux region (Dubourdieu and Frezier, 1990; Frezier, 1992; Masneuf, 1996), many samples of yeast microflora were taken in the vineyard and the winery from batches of white and red wines spontaneously fermenting or inoculated with ADYs. Several conclusions can be drawn from this research, carried out on several thousand wild strains of S. cerevisiae.
In the majority of cases, a small number of major strains (one to three) representing up to 70–80% of the colonies isolated, carry out the spontaneous fermentations of red and dry white wines. These dominant strains are found in comparable proportions in all of the fermentation tanks from the same winery from the start to end of alcoholic fermentation. This phenomenon is illustrated by the example given in Figure 1.37, describing the native microflora of several tanks of red must from a Pessac‐Léognan vineyard in 1989. The strains of S. cerevisiae, possessing different karyotypes, are identified by an alphanumeric code comprising the initial of the vineyard, the tank number, the time of the sampling, the isolated colony number, and the year of the sample. Two strains, Fzlb1 (1989) and Fzlb2 (1989), are encountered in all of the tanks throughout the entire alcoholic fermentation process.
The spontaneous fermentation of dry white wines from the same vineyard is also carried out by the same dominant yeast strains in all of the barrels.