Practical Winery
65 Mitchell Blvd, San Rafael, CA 94903
phone: 415-453-9700 ext 102
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January/February 2010
Monitoring microbes during cellaring/bottling
Pediococcus and one Lactobacillus chain
Brettanomyces can use residual hexose sugars but do not require them, and can grow in wines with 0.2 g/L fermentable sugar (enzymatic glucose/ fructose test), which is considered well below dryness. They use a variety of carbohydrates, depending on strain, and some use alcohol as a sole carbon source.
All members of the widely varied group of Brettanomyces strains that have been studied can use arginine or proline (the two most plentiful amino acids in grapes), as a sole nitrogen source. Some strains are sensitive to higher levels of alcohol, but others have been found in wines over 15% alcohol. They do require the vitamins biotin and thiamine, but it is best to assume that they are able to grow in any infected wine.
“Brett” yeasts make significant amounts of acetic acid in culture, and can produce some in wine if enough oxygen is present, but a gradual rise in volatile acidity (VA) in a cellared wine could be attributable to Acetobacter, not necessarily to Brettanomyces.
At least some Brettanomyces strains are capable of forming the N-heterocycle compounds responsible for "mousy taint." However, lactic acid bacteria also produce these compounds and are more recently implicated as the culprits in many, if not most, instances. (See Lactic acid bacteria.)
There are seldom enough Brettanomyces cells in a wine to be seen directly with the phase-contrast microscope (approximately 2000 cells/ml), though it does occur. The population can range from less than 1/ml to more than 10 million/ml.
The traditional method to detect Brettanomyces is to culture the wine on media containing 50 ppmof the antibiotic cycloheximide to inhibit growth of most other yeasts, by interfering with protein synthesis. Bacteria are not affected, so sometimes bactericides such as chloramphenicol, penicillin, and streptomycin are added. Brettanomyces prefer media with 1.5% agar rather than 2%, and temperatures of 28° to 31°C. For some reason, they may grow poorly or not at all on WL Nutrient Medium produced in England or Europe.
Culturing detects the cells are alive and active. In optimal conditions at 30°C on WL Nutrient Medium, Brettanomyces forms white, hemispherical colonies in three to seven days. Recommendations for Brett management may be different if vigorous colonies are visible in three to four days than when small colonies struggle to grow after six or seven days.
Kloeckera apiculata (teleomorph Hanseniaspora uvarum), the major yeast on grapes, may grow in the must and cause fermentation problems, but it dies during or soon after fermentation. It is resistant to cycloheximide, and somewhat resembles Brettanomyces under the microscope, so it is sometimes mistaken for Brett. Some species of Candida and other genera, found on grapes and in cellars, are also resistant to cycloheximide.
Non-Brettanomyces yeasts resistant to cycloheximide tend to grow more quickly than Brettanomyces, with colonies visible after one to three days. In culture, Brettanomyces produces a strong acetic acid smell, while other cycloheximide-resistant yeasts do not. Recently, our laboratory group has also observed a commonly found cycloheximide-resistant cellar yeast, Candida cantarelli, and a few other yeasts in cellars and on grapes, to cross-react with some PCR primers previously thought to be specific for Brettanomyces. More cross-reaction studies would be valuable.
It is very important to keep track of SO2 additions when culturing for Brettanomyces, because they can quickly enter a “viable but not culturable” (VBNC) state for around one to three weeks after an SO2 addition, when they will not grow on a petri dish. These “VBNC” cells can be detected by epi-fluorescence or by PCR-based techniques.
Whether some PCR methods also detect dead cells in wine is controversial, as is the assumption that as soon as a cell dies in wine, its genetic material breaks down right away into undetectable pieces. Collaborative studies comparing methods are needed.
In summary, culturing, genetic techniques, and testing 4-EP/4-EG all provide different, and useful, information.
PCR-based techniques are much faster than culturing and detect VBNC cells which would otherwise go unnoticed. Testing 4-EP/4-EG, while lagging behind cell growth and continuing to rise after cells have declined, confirms whether or not Brettanomyces growth has occurred and provides a quantifiable measure of sensory effects, but does not assess viability of cells.
Film-forming yeast and bacteria
The wine surface in tanks should be checked visually at least once each week. If a film is present, sample the film by lowering a sample container on a string to the surface. A thin, slippery film is produced by Acetobacter, while a thick, chunky film is made by surface film yeasts such as Candida and Pichia. Acetobacter produces ethyl acetate (nail polish/airplane glue smell) and acetic acid from alcohol; surface film yeasts produce acetaldehyde (fino sherry smell), also from alcohol.
In barrels, check for a film at topping; resanitize the topping device before moving to the next barrel if a film is present.
With a phase-contrast microscope it is quite easy to distinguish film yeasts from Acetobacter bacteria. The yeasts tend to form clumps and chains. Acetobacter grows in rafts of small
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