This is now a convenient point at which to discuss the use of sulfur dioxide and pure culture technique for wine making. As soon as the in tegrity of the grapes has been compromised by the crushing step, the sugars in the juice are liberated and made available for whatever microorganisms happen to be present. Ordinarily, the must is populated by epiphytic yeasts (that is, yeasts that reside on the surface of the grapes) and by yeasts that have "contaminated" the crushers, presses, and other wine-making equipment. Although the surface of a single grape may contain only about 102 to 104 yeast cells, after the grapes have been exposed to the contaminated equipment, the number of cells increases about 100-fold, to about 104 to 106 cells per ml. Whether this resident microflora actually commences a fermentation, however, depends on the intent of the wine maker.
Two options exist. First, a spontaneous or natural fermentation may be allowed to proceed ("natural" simply means that pure starter cultures are not added). In this case, except for temperature control, essentially no other restrictions are placed on the fermentation, and yeast (and bacterial) growth occurs with just a relatively short lag phase. The other option is to start the fermentation, under controlled circumstances, with a defined yeast starter culture selected by the wine maker. The latter option usually requires that the indigenous microflora be inactivated, so that it does not compete with and possibly interfere with the added culture. This is not, however, always the case, because it is still possible to add a starter culture even in the presence of the background flora. The use of wine starter cultures has now become commonplace, even among many traditional wine manufacturers. Moreover, recent research on yeast ecology suggests that there may be less diversity in wines made by spontaneous fermentations than previously thought and that these natural fermentations involve relatively few strains (Box 10-4).
When starter cultures are used, the naturally occurring or so-called wild yeasts are inactivated in one of two ways. First, the must can be
Box 10—4. Culture Wars and the Role of Diversity in Wine Fermentations
Like all of the fermented products described in this text,wines were made for thousands of years before scientists recognized that microorganisms were responsible for the fermentation. In contrast to dairy, meat, and other food fermentations, where first backslopping, and later pure culture techniques were applied, wine makers have long relied on the indigenous microflora to initiate and complete the wine fermentation. Even today, many of the wines produced by traditional manufacturers, especially those in Europe, are made by a "spontaneous" or natural fermentation carried out by the wild yeasts that are present on the grapes, in the must, or on equipment. Most large, modern wineries, however, have adopted yeast starter culture technology, and many have abandoned the traditional practice of allowing the wild yeasts to commence the fermentation.
Advocates of wine starter culture technology make several good arguments. First, they contend that using defined yeast strains results in wine having a "pure" or cleaner flavor. In addition, the availability of yeast with particular physiological characteristics such as osmotolerance or the ability to grow at low temperature allows the wine maker to select yeasts particularly suited to the grape characteristics one is using or the wine type one is making (Table 1).
Importantly, the wine maker can expect culture performance to be consistent in terms of fermentation times, flocculation properties, and flavor and end-product formation. In addition, culture yeasts can also be customized to provide traits and performance characteristics that suit the particular needs of the wine producer. Finally, there may be conditions that make pure cultures almost essential, such as when the grapes or musts contain very high sugar or acid concentrations.
Box 10—4. Culture Wars and the Role of Diversity in Wine Fermentations (Continued)
Table 1. Starter cultures versus natural fermentation for wine-making.
Starter cultures Natural
Cleaner flavor More complex flavor
Greater consistency Unique qualities
Low frequency of stuck fermentations Greater frequency of stuck fermentations
Can customize strains Cannot customize
Immune to killer yeasts May be sensitive to killer yeasts
Despite these advantages, it is undoubtedly true that naturally fermented wines have an excellent track record and many are highly regarded by wine experts. Even if one were to concede that the flavor, aroma, and other organoleptic properties are more variable in naturally fermented wines, is that such a bad thing? Certainly, one could argue that it is the variability itself that makes it possible to produce truly exceptional wines. Proponents of natural wine fermentations claim that such wines are more "complex" due to the more complicated metabolic processes that occur within a complex yeast flora. In other words, it is the diverse metabolic flora that leads to a more complex distribution of flavor components in the wine.
To address this issue of metabolic diversity on a rational basis, several questions must first be raised. How many different strains are actually present during a natural wine fermentation? Are some strains more dominant than others? What are the phenotypic properties of these strains, relevant to wine flavor generation? Several recent studies have attempted to answer these questions. In the report by Cappello et al. (2004), between thirty and forty naturally-occurring yeasts were isolated from each of twelve different musts.Although the musts were made from different culti-vars (each obtained aseptically), the grapes were grown within the same vineyard.All of the yeast isolates were identified by classical methods and by genetic analyses as Saccharomyces cere-visiae, and, based on PCR amplification and mitochondrial restriction analyses, all of the strains within each of the twelve groups appeared to be genetically homogenous. Comparative analysis of representative strains from the twelve groups revealed that there were three genetically distinct groups. However,there were only minor differences in their physiological properties.
This study points out several important features. First, it suggests that the indigenous yeasts present in wine reflect the yeast population within the vineyard, rather than the grape. Second, it would appear that only a few strains, rather than a diverse collection of strains, dominate the wines produced within a vineyard. Finally, these dominant yeasts were well-adapted to the particular local environmental conditions and shared similar physiological properties relevant to wine making. However, because the yeasts were isolated at a single point in time (at the end of the fermentation), it is possible that other strains may have been present at the beginning or at some earlier time during the fermentation.
Another recent study addressed similar questions, but in these experiments the musts were analyzed at the beginning and end of the fermentation (Demuyter et al., 2004).Thus, strain diversity could be determined at two different stages. Over a three-year period, grapes from Alsace (used to produce Gewurztraminer wines) were obtained, crushed, and fermented under three different conditions. For each year, 100 yeast isolates obtained from the beginning and end of the three independent fermentations were identified and classified by polymerase chain reaction and pulsed field gel electrophoresis techniques. Grape and must handling conditions were slightly different for each of the three fermentations, such that the origins of the isolated yeasts could have been from the grapes, the press equipment, or the vat.
The results for the first-year grapes showed that nearly all of the grape-associated strains, both from the beginning and end, belonged to a single homogenous group of S. cerevisiae. In con-
Box 10—4. Culture Wars and the Role of Diversity in Wine Fermentations (Continued)
trast, the microflora from grapes exposed to crushing equipment or to the vat environment was more heterogenous and contained relatively few S. cerevisiae isolates. Instead,these musts contained mostly Saccharomyces bayanus subsp. uvraum. Results for the second-year grapes showed that S. bayanus subsp. uvraum was the dominant organism from all three environments at all sampling times.Third-year results for grapes or grapes exposed to crushing equipment indicated that S. cerevisiae was dominant at the beginning and end of the fermentation, but grapes exposed to the vat environment contained S. bayanus subsp. uvraum as the dominant organism (as was the case for the year one results).
Collectively, this study on Alsatian wine shows that a homogeneous group of S. cerevisiae strains appears to dominate the grape surface, yet other non-S. cerevisiae yeasts still show up in the wine.The latter are associated with both the crushing equipment or the vat environment. Moreover, under the conditions established in this study, those yeasts present in the vat environment may actually be the dominant yeasts in the wine fermentation, since they were present at the beginning and end of all three fermentations conducted over a three-year period.
Although results represent only two limited studies, they nonetheless suggest that the yeast population responsible for the wine fermentation had only modest diversity and that domination by a particular strain or strains may be common. Indeed, these findings are consistent with other ecological studies (Versavaud et al., 1995). Certainly, these reports also confirm previous reports that suggest wine crushing equipment and the wine environment serve as a major source of yeasts involved in the wine fermentation.Thus, it may well be that natural fermentations do not owe their special properties to a diverse population of wild yeasts that reside in the vineyard or on the surface of grapes, but rather to only a few yeasts that contaminate the wine-making equipment and that live in the confines of the winery environment.
Cappello, M.S., G. Bleve, F. Grieco, F. Dellaglio, and G. Zacheo. 2004. Characterization of Saccharomyces cerevisiae strains isolated from must of grape grown in experimental vineyard. J. Appl. Microbiol. 97:1274-1280.
Demuyter, C., M. Lollier,J.-L. Legras, and C. Le Juence. 2004. Predominance of Saccharomyces uvarum during spontaneous alcoholic fermentation, for three consecutive years, in an Alsatian winery. J. Appl. Microbiol. 97:1140-1148.
Versavaud,A., I? Courcoux, C. Roulland, L. Dulau, and J.-N. Hallet. 1995. Genetic diversity and geographical distribution of wild Saccharomyces cerevisiae strains from the wine-producing areas of Charentes, France.Appl. Environ. Microbiol. 61:3521-3529.
heat treated, usually via a high-temperature, short-time (or flash) pasteurization process. Although very effective against most organisms found in musts, even moderate heating is often detrimental to the juice and to the wine.Thus, this process is rarely used. The preferred method is to chemically pasteurize the must by adding sulfites. It should be noted that even naturally fermented wines, especially white wines (see below), are often sulfite-treated to control undesirable organisms.The most common sulfit-ing agents are SO2 gas and potassium bisulfite salts. Sulfites are cheap, effective, and multi-func tional. In addition to their effectiveness against wild yeast, these agents also inhibit growth of acetic acid-producing microorganisms, malolac-tic bacteria, and various fungi. Sulfites can thus be considered as serving a preservative function in wine. Importantly, they also control several deleterious chemical reactions, particularly oxidation and browning reactions.
The amount of sulfite added and when it is added varies depending on the condition of the grapes, the microbial load, must pH and acidity, and the type of wine (red or white). Musts from mature grapes that often contain high levels of wild yeast require more SO2, but in general, about 80 mg/L is sufficient.Also, the lower the pH, the less sulfite is necessary for antimicrobial activity. Due to human health concerns, however, there are also regulations that dictate how much sulfite can be present in wine. In France, most red and white wines must contain less than 160 mg/L or 210 mg/L, respectively. In the United States, the limits are 350 mg/L for both red and white wines. Usually, SO2 or sulfite salts are added to the must just after crushing.
When pure yeast cultures are used, they are usually obtained from commercial sources. So-called house strains, although common for beer, are infrequently used for wine making, due primarily to the expertise and equipment required for strain maintenance and propagation. In contrast, commercial cultures are easy to use and require only modest technical ex-pertise.The main advantage of commercial cultures, however, is that they provide the wine manufacturer with many culture options.This is because a variety of strains is available, and the choice of strain can be selected based on the needs of the customer (Table 10-3). For example, grapes with consistently high sugar concentrations may require an osmotolerant yeast culture. Yeast used to perform the secondary fermentation that occurs in Champagne manufacture must sediment well to facilitate its removal during the disgorgement step (described later in this chapter).
Table 10.3. Desirable properties of wine cultures.
Able to produce high levels of ethanol Does not produce off-flavors Capable of producing unique flavors Ethanol tolerant Osmotolerant
Ferment sugars to completeness Sediments or flocculates well (especially for
Champagne yeasts) SO2 and sulfite resistant Cold resistant Rapid fermentation rates Able to grow at temperatures below 10°C Predictable and consistent and genetically stable
Yeasts are usually supplied in the form of active dry yeast, not unlike that used by the bread industry (Chapter 8). Culture suppliers ordinarily recommend inoculum levels sufficient to provide a starting cell density of about 106 cells per ml of must. An increase of only about two log-cycles occurs during the fermentation. The manner in which the yeast cultures are produced and preserved is important, however, because how the cells are handled during the culture production steps may influence how they grow later in the juice.
When culture companies produce yeast cultures, cells are grown under highly aerobic conditions to achieve the maximum amount of biomass. These high biomass fermentations also elicit a general stress response that enhances survival and viability during the subsequent drying steps. It appears that resistance to drying is promoted by the synthesis of the disaccharide trehalose by wine yeast (similar to the cryoresis-tance mechanism by bakers' yeast, described in Chapter 8). Thus, following culture production, yeast cells are induced for aerobic metabolism and anabolic activities, whereas, when these cells are inoculated into the must, fermentative metabolism, with a minimum of biomass, is desired. Therefore, rapid adaptation to the new wine environment is also important.
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