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June 2018 WINES&VINES 61 PRACTICAL WINERY & VINEYARD WINEMAKING for extended periods of time. This would be particularly true during malolactic fermenta- tion, when free sulfur dioxide levels are low or non-existent." Carter suggests that winemakers could use the information to make decisions on the basis of wine style. If Brett is an issue in a winery, winemakers could opt to have higher alcohol (15% or more) in their wines as a means to prevent Brett. Bottom line: Cold storage temperatures can help lower the risk of Brett. Wines with low alcohol have a higher risk of Brett than high-alcohol wines and should be kept in the coldest rooms of the cellar, when possible. Knowledge that high-alcohol wines have lower risk of Brett could potentially be part of a winemaker's decision-making process if Brett is a problem in the winery. Study methods The study used two strains of B. bruxellensis, F3 and I1a, in Merlot wines obtained from a commercial winery. Total sulfur dioxide was measured and removed. The wines of five al- cohol concentrations (12%, 13%, 14%, 15% and 16%) were held at four temperatures (54°, 59°, 64° and 70° F). Volumes of wine were fixed. Variable proportions of ethanol:water mixtures were added to yield wines with dif- ferent alcohol percentages. After supplement- ing the wines with glucose, fructose and a yeast extract, pH was adjusted to 3.75. The wines were sterile-filtered and incubated for 24 hours under the four temperature treatments before inoculation with the same amount of the two Brett strains (10 4 cfu/mL, the number of col- ony forming units per milliliter). The wines were monitored for 100 days by sampling twice per week for the first four weeks and then once per week thereafter. After 100 days, concentrations of volatile acidity (primar- ily acetic acid), 4-ethylphenol (4-EP) and 4-eth- ylguaiacol (4-EG) were quantified. 4-EP is the compound responsible for contributing earthy and rustic sensory characteristics, also de- scribed as animal, smoky or medicinal aromas. 4-EG is the compound associated with bacon, spice and clove aromas. Both compounds are associated with the earthy and barnyard odors resulting from Brett-tainted wines. Temperature and alcohol interaction A synergistic response from the interaction of storage temperature and alcohol concentration impacted the growth and production of acetic acid, 4-EP and 4-EG in both strains of Brett. The combination of low storage temperatures (54° F or less) and higher alcohol concentra- tions of 14% or more resulted in populations at levels below the detection limit of 30 cfu/ mL for up to 100 days. At the lowest tempera- ture of 54° F or less, neither strain of Brett grew well, regardless of the alcohol level. However, when temperatures were 64° F or higher, both strains grew well in the wines containing 12%, 13% and 14% alcohol and reached peak populations of 10 6 cfu/mL in those wines in 40 days or less. Growth of Brett was inhibited in the 15% and 16% alcohol wines, and the high alcohol wines also showed longer lag phases at the lower temperatures. Strain I1a reached a population of 10 6 cfu/ ml within 20 days of inoculation in the 12% v/v alcohol wine maintained at 64° F, but it required 40 to 70 days to reach the same popu- lation in the 13% and 14% alcohol wines maintained at the same temperature. In the low-alcohol wines of 12%, stored at the cold- est temperature of 54° F, both strains showed a decrease in culturability, and recovery was slowed or nonexistent. Additionally, growth of the two strains was retarded in wines above 14% alcohol. Both strains could not be recovered from the 16% alcohol wines regardless of temperature. Strain differences The two strains generally displayed similar growth patterns under the various temperature and alcohol treatments, except for the 15% alcohol wines, where strain differences were observed. Here, F3 appeared to be more inhib- ited and showed less growth and reduced concentrations of acetic acid, 4-EP and 4-EG at the higher temperatures than strain I1a. The F3 strain never reached populations above 10 4 cfu/mL in the experiment, even when wines were held at 70° F. The different responses of the two strains in the 15% alcohol wines reinforces the con- cept of genetic diversity within the Brettano- myces genus. "What that suggests is that you have to be very careful about what strain you have in your winery, because there are situa- tions where they behave differently," Edwards says. "The diversity of Brettanomyces bruxel- lensis, a single species, seems to be quite large. We do not know much about Brett strains from a genetic standpoint or how they behave in different matrices." What about cellar humidity? Humidity was not part of Edwards' study. Humidity of a cellar does affect the loss of water and alcohol through the wood. Humidity is often added to barrel rooms to control top- ping losses, and there is some cooling effect from humidity, but humidification is done in addition to using a refrigeration system for temperature control. From a temperature point of view, temperature affects humidity but not vice versa, Edwards explains. "Humidity is crucial to maintain during stor- age because you are trying to balance the ethanol and amount of water passing through the wood," he says. "But this study looked for whether or not there was a relationship that winemakers could use between the amount of ethanol present in the wine and storage temperature." Wines in the study were not stored in wood, and Edwards did not try to learn if exterior humidity has an effect on the growth of micro- organisms inside a wood barrel. If there is any effect from humidity, he believes it would be indirect. Defenses and barriers Defense technology is a risk-management concept used in food manufacturing. It is based on the theory that you should not rely on just one approach to limit the growth of spoilage microorganisms but should use a series of bar- riers or defenses that the organism must over- come to still remain active or in large enough concentrations to be an issue. If enough de- fenses are used, the organism is eliminated or eradicated. In the case of Brett, the goal is to control levels below sensory thresholds. Several methods to control B. bruxellensis previously evaluated by the Edwards labora- tory include the use of sulfur dioxide, dimethyl dicarbonate, chitosan and filtration. These are all helpful barriers that have been shown to be useful to manage Brett. With today's trend of higher alcohol wines ranging from 14% to 15%, winemakers may be able to take advantage of cellar temperature to head off potential issues with this particular yeast, Edwards notes. "Each winemaker has to consider all the parameters of a given wine that pertain to Brett, such as history, pH, sulfur dioxide and filtration," he adds. "In short, what is the risk to that particular lot? Then the winemaker has to find a balance that uses enough risk man- agement barriers without breaking the bank." Keep it cold For wineries that have multiple cellar rooms, keeping the lowest alcohol wine in the coldest room may be a viable option. For those with one cellar room, Edwards suggests wine be kept at the lowest temperature that is eco- nomically feasible. He recognizes that energy costs for refrigeration can be expensive in some wine regions, but where feasible, cold temperatures can be used as another barrier against Brett. Melissa Hansen, research program manager for the Washington State Wine Commission, works to make viticulture and enology research supported by the Washington wine industry more accessible to the state's growers and winemakers. Hansen was a journalist for nearly 20 years for Good Fruit Grower, a Washington- based magazine, and was involved with California's table grape and tree fruit industries for 15 years. To see acknowledgments and references for this article, go to winesandvines.com and search under Magazine › Features › June 2018.