Adjustments Blending and Clarification

After the fermentation is complete, the wine will contain little or no sugar and about 12% to 14% ethanol. Still, because of differences in

Box 10-6. Killer Yeasts

Wine spoilage is often mediated by growth of wild yeasts that contaminate wine while it is aging in wooden barrels. In particular, species of the Brettanomyces and Dekkera groups are frequently involved in wine spoilage and are difficult to control.This is because these yeasts are naturally present not only on grapes and in musts, but also in the barrels in which the wine is aged (Comitini et al., 2004).

Growth of Dekkera and Brettanomyces may result in formation of ethylphenolic compounds that have unpleasant, "barny" or "wet-dog" off-odors. They are also a possible cause of the "mousy" off-odor defect (the smell of which, one can imagine). Control measures ordinarily include attention to cleaning and sanitation at the harvest and crushing steps and adequate sulfit-ing of the grapes.It is more difficult, however, to control Brettanomyces and Dekkera once the wine reaches the wooden barrels, which cannot be effectively sterilized.

Many yeast strains can produce and secrete exotoxins that can kill other yeast cells.There are numerous differences, depending on the producer, between the many so-called killer toxins that have been characterized (Magliani et al., 1997).They vary with respect to their genetic basis, how they are synthesized and exported, their size and structure, and their mode of action. Genes encoding for killer toxins, for example, may be located on linear plasmids, nuclear gene regions, or double-stranded, virus-like RNAs. In addition, the killer activity in sensitive cells may be due to pore formation and ion leakage, the arrest of G1 or G2 phases of cell growth, and the inhibition of cell wall synthesis (Magliani et al., 1997).

Among the yeasts that are known to produce killer toxins are several genera associated with the wine fermentation, including Saccharomyces, Kloeckera, Pichia, and Kluyveromyces.Al-though killer yeast strains are immune to the cognate toxin they produce, they may be sensitive to toxins produced by other strains. If wild killer yeast strains are present during the wine fermentation, and the wine starter yeast (whether part of a pure starter culture or are naturally-occurring) are susceptible to that toxin, a failed or stuck fermentation may result.Thus, it is essential that the killer sensitivity phenotype for wine cultures be known, since, in most natural wine fermentations, killer yeasts can be found (Vagnoli et al., 1993).

Box 10-6. Killer Yeasts (Continued)

In contrast to the detrimental effects killer yeasts may have during wine fermentations, they can also be exploited in wine fermentations. In fact, killer yeasts are now routinely used in the wine industry, just as they have been used in other industrial processes to control wild yeasts. Killer toxins have also been shown to be effective in clinical applications to treat fungal and bacterial infections (Buzzini et al., 2004; Conti et al., 2002; Palpacelli et al., 1991;Walker et al., 1995).Yeast cultures with a killer phenotype have the potential to dominate a fermentation. Provided that the killer strains have other desirable wine-making traits, the use of such cultures would be expected to produce a consistent quality wine, due to the inhibition of wild yeast competitors. Killer toxin genes can be introduced into non-killer wine-making strains by mating, protoplast fusion, transformation, or micro-injection techniques.

A wine preservation strategy based on the application of killer toxins also was recently reported (Comitini et al., 2004b).As noted above, spoilage of wine by Brettanomyces and Dekkera during barrel aging is a serious and difficult-to-control problem. In the study by Comitini et al. (2004b), however, it was shown that these yeasts can be controlled by specific killer toxins.Two toxins in particular, Pikt and Kwkt (produced by Pichia anomala and Kluyveromyces wicker-hamii, respectively), inhibited species of Brettanomyces and Dekkera. Their activities were stable within a typical wine pH range of 3.5 to 4.5.When the producer strains were grown in co-culture with Dekkera bruxellensis (at a 1:10 ratio), growth of the latter was delayed; however, when inoculated at higher rate (1:1), complete inhibition occurred. Finally, these researchers showed that direct addition of the killer toxins to wine effectively inhibited growth of D. bruxellensis for at least ten days.

Because not all killer toxin-producing yeasts grow well in wine (e.g.,P. anomala and K.wick-erhamii), and it is unlikely that killer toxin preparations could be added directly to wine, other means of applying killer toxin technology must be considered. As noted above, killer toxin genes could be genetically introduced into other wine-adapted yeast strains, but it would be necessary for these strains be present during the aging step. It is also conceivable that the toxins could be used to treat the interior surfaces of wine barrels and thereby inactivate the Bret-tanomyces and Dekkera that reside there.

References

Bizzini, P, L. Corazzi, B.Turchetti,M. Bratta, and A.Martini. 2004. Characterization of the in vitro antimycotic activity of a novel killer protein from Williopsis saturnus DBVPG 4561 against emerging pathogenic yeasts. FEMS Microbiol. Lett. 238:359-365. Comitini, F., N. Di Pietro, L. Zacchi, I. Mannazzu, and M. Ciani. 2004a. Kluyveromycesphaffii killer toxin active against wine spoilage yeasts: purification and characterization. Microbiol. 150:2535-2541. Comitini, F.,J.I. De, L. Pepe, I. Mannazzu, and M. Ciani. 2004b.Pichia anomala and Kluyveromyces wicker-hamii killer toxins as new tools against Dekkera/Brettanomyces spoilage yeasts. FEMS Microbiol. Lett. 238:235-240.

Conti, S.,W. Magliani, S.Arseni, R. Frazzi,A. Salati, L. Ravanetti, and L. Polonelli. 2002. Inhibition by yeast killer toxin-like antibodies of oral streptococci adhesion to tooth surfaces in an ex vivo model. Mol. Med. 8:313-317.

Magliani,W., S. Conti, M. Gerloni, D. Bertolotti, and L. Polonelli. 1997.Yeast killer systems. Clin. Microbiol. Rev. 10:369-400.

Palpacelli,V., M. Ciani, and G. Rosini. 1991.Activity of different killer' yeasts on strains of yeast species undesirable in the food industry. FEMS Microbiol. Lett. 84:75-78. Vagnoli, P., R.A. Musmanno, S. Cresti,T. di Maggio, and G. Coratza. 1993. Occurrence of killer yeasts in spontaneous wine fermentation from the Tuscany region of Italy.Appl. Environ. Microbiol. 59:4037-4043. Walker, G.M.,A.H. McLeod, and VJ. Hodgson. 1995. Interaction between killer yeasts and pathogenic fungi. FEMS Microbiol. Lett. 127:213-222.

grape composition, microflora, and wine manufacturing practices, variations in wine composition and sensory quality are to be expected. Therefore, adjusting the wine after fermentation (and sometimes before) is a normal step. The pH and acidity, in particular, can vary markedly, as can the color and flavor.Therefore, some wineries adjust the acidity of wine by acidification or deacidification steps (e.g., by adding acids or neutralizing agents).

When acidity of wine or must is due to malic acid, deacidification is managed via the malo-lactic fermentation, which is discussed in detail later in this chapter. Adjustment to wine color and flavor can also be done, if legally permitted, by filtration and enzyme treatments, respectively. Filtration techniques, for example, are most often used to "decolorize" wine by removing undesirable pigments.

Except for very small wineries, which may have only a few vats of wine, most modern wineries have many individual vats of wine. Each one is unique, in that a particular vat may contain wine made from grapes harvested at a time or place different from the grapes in a neighboring vat.Wines within a single winery may be made from different grape varieties. Therefore, another common procedure, especially for premium wines, is to blend different wines to optimize or enhance the organoleptic properties. Blending also produces wines with consistent flavor, aroma, and color from year to year. Perhaps more so than any other wine-making step, however, blending is a tricky business, and is a highly subjective process. Success relies on the imagination, creativity, and skill of the wine blending specialist.

At the end of the fermentation, the wine contains non-soluble proteins and proteintannin complexes, as well as living and dead microorganisms.These materials give the wine a cloudy, hazy, undesirable appearance. The clarification step removes these substances from the wine without removing desirable flavor and aroma components. It is particularly important that the cells are removed. If left in the wine, these cells can lyse, releasing enzymes that may catalyze formation of off-

flavors and odors (although an exception to this rule exists, as described below). Inducing precipitation of tartrate salts and tanninprotein complexes is also commonly done to facilitate their removal before they precipitate later during aging. It is important to recognize that, in some cases, cell lysis may be a good thing. Intracellular constituents released during cell lysis include amino acids and nu-cleotides, providing nutrients that are later used by bacteria in secondary fermentations or that contribute to the sensory properties.

According to traditional practices, wine is clarified by simply allowing the sediment, containing the yeasts and bacterial cells, as well as precipitated material, to settle naturally in barrels or vats.The wine could then be removed from the sediment (or "lees") by decantation. This process, called "racking", is usually done for the first time after three to six weeks following the end of the fermentation. Racking can be repeated several times over a period of weeks or months until the wine is nearly crystal clear. During the racking step, the wine is also aged (see below). Racking can now be done in enclosed tanks using automated transferring systems.

Filtration is another method used to clarify wine. This can be especially effective if fining agents, such as bentonite, albumin, or gelatin, are used as filtration aids. If micropore filtration membranes are used, it is even possible to sterilize wines. Clarification may occur after racking or after aging.

In contrast to removing the sediment shortly after the fermentation has ended, some wines are intentionally left in contact with the lees for an extended time before the first racking oc-curs.This traditional maturing practice, known as "sur lies" enhances the flavor, character, mouth feel, and complexity of the wine. It is more common for white wines than red. Aging

Aging actually begins just after fermentation. Thus, aging occurs when the wine is racked, as well as beyond. Aging conditions vary con siderably. Some wines are aged for several years, whereas others are "aged" for only a few weeks. Some wines are aged in expensive oak barrels, others in stainless steel, and yet others depend on bottle-aging, or a combination of all of the above.Whether a wine is aged for a long time in oak barrels or is quickly bottled and sent to market depends, in part, on marketing considerations, but also on the original composition of the grapes and how they are made into wine. Thus, long, careful aging should be reserved for only premium wines made from high quality grapes. By analogy, Cheddar cheese manufactured for the process cheese market cannot be expected to develop into a flavorful, two-year Cheddar, no matter how carefully it may have been aged.

Of course, some excellent quality wines, like excellent cheeses, are meant to be consumed in a "fresh" or un-aged state, so whether or not a wine is aged does not distinguish wine quality, per se. For example, Beaujolais nouveau, a popular wine from the Burgundy area in France, is meant to be drunk after only a few weeks after the grapes are harvested. These wines are fruity and "gulpable"; no amount of aging will lead to their improvement.

To say that the actual events that occur during aging are complicated would be quite the understatement. Hundreds of enzymatic, microbiological, and chemical reactions occur, and as many as 400 to 600 volatiles, including esters, aldehydes, higher alcohols, ketones, fatty acids, lactones, thiols, and other compounds are formed (Table 10-4).The wine interacts with the wood and wood constituents in the barrel, oxygen in the air, and even the cork. It is important to recognize that not all of these reactions are beneficial in terms of wine quality, and some wines may actually deteriorate during aging. In fact, long aging is not good for most wines.

Ordinarily, in large wineries, fermentations occur in large tanks (exceeding 250,000 liters), and then the wine is moved into wooden 200 liter barrels for aging. However wine can also be aged, at least initially, directly in tanks, and then later moved into oak barrels for final aging.

In many European and other traditional wineries, in contrast, the entire aging period is conducted in oak barrels. The oak barrel or "cooperage" is so important to wine quality that entire industries devoted to oak tree production and cooperage construction have de-veloped.This is because the oak barrels are not inert containers used simply to store wine, but rather they are a source of important flavor and aroma compounds. In fact, one of the major steps in barrel construction involves heating or "toasting" the barrels to promote pyro-lysis. This generates a number of flavor and aroma volatiles. In the presence of wine, these compounds, along with tannins, phenolics, lignins, and lactones, are extracted from the wood and solubilized in the wine. Some of these compounds impart unique flavor notes, including vanilla and coconut. Aging wine in oak cooperage is not, however, without a downside.Oak barrels are expensive, and, even if carefully maintained, do not last forever. Loss of wine volume (and hence profit) due to evaporation can also occur. Thus, alternative

Table 10.4. Effects of aging on wine.

Reaction or step

Effects

Tannin precipitation

Color darkens; astringency increases initially,

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