Fermentation

The Red Wine Diet

The Red Wine Diet

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The wine fermentation begins as soon as the grapes are crushed. However, when a starter culture is used and SO2 is added to control the indigenous organisms, limited ethanol fermentation will occur prior to addition of the culture.

In the case of white wine production, the culture is added to the must after pressing and clarification, whereas for red wine, culture addition is done prior to seed and skin removal.Thus, for red wines, fermentation occurs during maceration, just as it would for a natural fermentation. The amount, concentration, and form of the culture depends on the type of wine being produced, the composition of the grapes, and other considerations specific to the wine manufacturer. Of course, the culture's main responsibility is to produce ethanol from sugars (see below), but the criteria for culture selection actually includes many other properties (Table 10-3). For most wines, the culture inoculum, whether in a rehydrated dried or active liquid form, (Chapter 3), should provide about 106 cells per ml of must.

Traditionally, fermentations were performed in open barrels or vats with a capacity of 500 L or less. Such barrels still are used today; however, enclosed stainless steel tanks are now more common. The latter have several advantages. They are easy to clean and disinfect and often can be sterilized. Airborne microorganisms are less likely to contaminate the wine. Various control features, including temperature control and mixing and pumping activities (see below), are easily incorporated into the design, and are usually computerized. Finally, modern tanks can be quite large, with capacities of more than 250,000 L.

The temperature of incubation depends on the type of wine being produced. In general, white wines are fermented at lower temperatures than red wines. For example, many wineries control the temperature between 7°C and 20°C for white wine and between 20°C and 30°C for red wines. Some wineries prefer low incubation temperatures for all wines, because less ethanol is lost to evaporation and fewer of the volatile flavors are lost. In addition, low temperature incubations result in overall higher ethanol concentrations and less sugar remaining at the end of the fermentation (assuming time is not a factor). Although S. cerevisiae has a lower growth rate as the temperature decreases, the diversity of

Box 10—5. Molecular Archaeology and the Origin of Wine Yeast

How is it that the fermentation of beer, wine and bread all require yeasts with specific physiological and biochemical traits, yet all are made with Saccharomyces cerevisiae? Was there a progenitor S. cerevisiae strain that somehow evolved over thousands of years into specialized strains that were especially suited for these different fermented foods? If so, where did that progenitor strain arise and what were its properties? These are obviously questions that cannot be answered directly. However, it is possible, using molecular archaeology analyses, to make inferences regarding the evolution of these important fermentative yeasts (Cavalieri et. al., 2003).

As noted previously, wine making appears to have developed in the Caucasus and Zagreb Mountain areas of the Near East (the "Fertile Crescent") more than 7,000 years ago.Ancient clay jars (estimated date about 3150 B.C.E.) from these regions once contained wine, according to chemical and instrumental analyses. Furthermore, DNA residues were found when organic residues (presumably the lees, comprised of dead yeast cells) from these artifacts were analyzed. In fact, the amount of DNA present in the residue was high enough (it could even be seen in a gel) to eliminate the possibility that it had come from a stray microbial contaminant. Given the low water activity, low relative humidity (essentially zero), and overall ideal conditions for storing a biological material, it was also not surprising that the DNA was in such good shape.

The DNA was subsequently extracted and used as a template for PCR. Primers were based on S. cerevisiae rDNA sequences, such that the spacer regions between the 18S and 28S rRNA genes would be amplified.Three PCR products (540,580, and 840 base pairs) were initially sequenced. Based on a BLAST search, all of the sequences had homologies to existing GenBank se-quences.The sequence of the 580 bp PCR product was similar to various fungi, but remarkably, there was strong homology (nearly 90% identity) between the 540 bp sequence and a similarly derived sequence obtained from a fungal clone isolated from the clothing of "The Iceman Otzi." The latter dates back to 3300 B.C.E.The investigators suggest that both of the ancient fungi had been "buried" along with the wine.

Perhaps the most interesting finding from this analysis, however, concerned the 840 bp PCR product.This fragment, as well as smaller pieces obtained using internal PCR primers, had very high sequence similarity with a 748 bp region from chromosome 12 (part of the 5.8S rDNA) from contemporary strains of S. cerevisiae, Saccharomyces bayanus, and Saccharomyces para-doxus. Across this entire region there were only four nucleotide mismatches and no deletions or insertions between S. cerevisiae and the ancient wine sequence. This level of similarity should not be so surprising, perhaps, since the DNA regions represented by the PCR products are known to be quite stable.Thus, this stability could account, in part, for the lack of sequence alterations between the ancient and modern strains during the past 5,000 years of yeast evolution. Importantly, these findings suggest the yeast used to carry out the wine fermentation in ancient days was S. cerevisiae, just as it is today.

References

Cavalieri, D., P.E. McGovern, D.L. Hartl, R. Mortimer, and M. Polsinelli. 2003. Evidence for S. cerevisiae fermentation in ancient wine. J. Mol. Evol. 57:S226-S232.

metabolic end products may actually increase, enhancing flavor development. In addition, lower temperatures may favor growth of K. apiculata and other wild yeasts (at least when they are not inactivated by SO2) that produce various volatile compounds, making the wine aroma and flavor appear more complex.

It is critical to recognize that the wine fermentation is exothermic and a considerable amount of heat may be generated. Some of this heat is gradually lost or dissipated into the environment without ill effect. However, in large volume fermentations, in particular, much of this heat is retained, raising the temperature of the wine. For example, if the initial temperature starts at 20°C, the temperature can increase 10°C or more. If the temperature were to rise above 30°C, the yeast may become inhibited or stop growing altogether. The wine will contain less ethanol and more residual sugar.

Such fermentations are said to be "stuck." Although other factors may cause a wine fermentation to become stuck (see below), high temperature is the most common reason. It is, therefore, essential that the appropriate temperature is maintained. For fermentations conducted in modern, stainless steel, jacketed vats, coolant solutions can easily be circulated, externally.Alternatively, internal cooling coils can also achieve the same effect. The cooling requirement can also be met, especially for white wines, by simply locating fermentation barrels in cold rooms or cellars. However, the need for adequate cooling has led even some traditional wine manufacturers to abandon oak barrels and casks in favor of stainless steel vats.

The actual fermentation period is not long. After culture addition, the yeasts enter a short lag phase (from a few hours up to a day or two) that is then followed by a period of active growth (log phase) that lasts for three to five days. If the fermentation is conducted at lower temperatures (10°C to 15°C), the lag and log phases can be extended for several days. Conversely, if the yeast culture is highly active at the outset, by virtue of having been previously propagated under ideal growth conditions, the cells will almost immediately enter log phase.Al-though one might expect that a natural fermentation would take longer, in fact, growth of the indigenous yeast begins so soon after crushing, that the lag phase is barely noticeable.

During the log phase of growth, when an active fermentation is occurring, a layer of CO2 forms across the surface. In red wine production, some of the pomace will float to the top and be trapped within this CO2 layer, forming a dense blanket or cap. Since the pigments and tannins are present in this thick cap layer, a mixing step is required to return these substances back into the fermenting must.The temperature in the cap can also become elevated, supporting growth of undesirable thermophilic bacteria; thus, mixing serves to maintain a more uniform temperature. Various techniques exist for this mixing step (called pigeage). In the "pumping over" technique, a portion of the must is periodically removed from the vat and pumped onto the cap. Alternatively, the cap can be "punched down," either manually or via mechanical means. In modern wineries, automated pumping and punching systems are used to mix the pomace cap into the fermenting must.

The fermentation of white wine occurs after the must is pressed and clarified. After about the seventh or eighth day of fermentation, cell numbers may begin to decline, representing the end of the primary ethanolic fermentation. Some yeast strains will flocculate or clump together, causing them to settle to the bottom of the tank.This is a desirable property that enhances their removal later during the racking and clarification processes. The fermentation is considered complete when all or most of the sugars are depleted, as determined by a decrease in the Brix value. For red wines this may take as long as five to six weeks. If less than about 0.5% sugar is present, and there is no apparent perception of sweetness,the wine is considered to be dry. Of course, not all wines are intended to be dry. If some sugars are present in the wine after fermentation (or if sugars are added), a sweet wine results. Specialized techniques for the manufacture of sweet wines will be described later.

For red wines, much (if not all) of the fermentation occurs in the presence of the pomace. When the extraction of pigments, tannins, flavor compounds, and other materials is considered sufficient, or when the desired ethanol concentration is reached, the free run juice is separated from the pomace and moved into another tank. The fermentation is then completed (if not already). In the meantime, the pomace is pressed and is either fermented separately from the free run or is mixed with the free run for the final fermentation. Since the pressed wine is rich in pigments and tan nins, adding a portion back to the free run wine makes the final product richer in color and flavor.

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