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w i inneeGM A K IN GG w R O WIN Photos: Left + middle, Lisa Van de water; right, nicola wiltoN microbes monitoring during fermentation BY Lisa Van de Water inemaking involves encouraging cooperation with microbes we want, and discouraging participation from microbes we do not want at all, or do not want at the time (for example, Saccharomyces in a bottled sweet wine). Levels of concern vary from wine to wine and from winery to winery, but there are some guidelines to start from. Monitoring microbes effectively and efficiently during the winemaking process requires coordinating several different techniques. Examination with a phase-contrast microscope, culturing on various media, genetic testing using polymerase chain reaction (PCR), usually quantitative PCR-based tests including Scorpions® and others, chemical tests, sensory cues, and others all have a place in today's wine microbe detection protocol. Especially during the harvest season, access to a good phase-contrast microscope is an extremely valuable tool, because samples can be examined directly without staining. Many problems can be detected in a few minutes, so winemakers can respond immediately. A brightfield microscope can be used to examine yeasts after staining, but not to detect wine bacteria reliably. Despite the W Please see "Spoilage microbe populations fluctuations during winemaking—causes, effects, solutions" by Robert Tracy, May/June 2009 PWV Journal. This text edited from first publication: Jan./Feb. 2010. cost (several thousand dollars), access to a high-level phase-contrast microscope is recommended for all wineries (groups of wineries could purchase one together). Even with such a microscope, the lower limit of detection by direct microscopic exam is around 2,000 cells/ml. Fortunately for the wine microbiologist, however, microbes involved in spoiling fermentations are present at higher levels — often much higher. before fermentation Microbes to watch out for: NonSaccharomyces yeast species, lactic acid bacteria, acetic acid bacteria Danger signals: Mold and/or rot, signs of spontaneous fermentation, acetic acid (vinegar) or ethyl acetate odor Monitoring methods: Sensory cues, microscopic exam, PCR-based genetic testing Mold/Rot If grapes arrive with mold and/or rot, take photos and save an uncrushed sample to freeze immediately as a record of the grapes' condition. Test laccase (responsible for the browning of white wines and discoloration of red wines) and volatile acidity (VA), and consider rejecting fruit if VA is more than 0.1 g/L in juice. Note that crushing of grapes may be considered legal acceptance of the grapes "as is." After grapes that were not completely healthy are processed, take a sample from the tank and examine it under the phasecontrast microscope for bacteria and yeast. Fortunately, molds are very intolerant to alcohol, so they die as soon as fer- Left: Non-Saccharomyces yeast in stuck wine; Center: Kloeckera/Hanseniaspora with two Saccharomyces cells; Right: Non-Saccharomyces (small) and Saccharomyces in stuck Chardonnay. mentation begins. It is normal to see a few yeasts, but no more than about 5 per 40x field. There should be no bacteria, or at most one rod-shaped bacterium in several 40x fields. Even one Acetobacter/ Gluconobacter cell seen under the microscope is too many! If any Lactobacilli are seen, add lysozyme to the must immediately, or continue to monitor the batch microscopically once per day, to see if the population increases. Also monitor growth of nonSaccharomyces yeasts, as discussed below. Non-Saccharomyces yeasts from grapes and in wineries There are many yeasts on grapes, though very few are Saccharomyces. This surprises most winemakers who assume that Saccharomyces is the dominant genus in vineyards, despite extensive scientific studies to the contrary. Kloeckera apiculata (teleomorph Hanseniaspora uvarum) is a major species, and there are about 100 other species in vineyards and wineries. Some species are very sensitive to alcohol, or are so aerobic (requiring air) that they cannot grow during fermentation, but they may grow before or during the start of fermentation. Other species may survive until middle to late in the fermentation process. Many yeast species — often originally from grapes — take up residence in winery cellars, and begin to colonize new grapes as soon as they arrive at the winery, even in the crusher/destemmer. When juice/must is not inoculated with cultured yeast, or if the cultured yeast has been mishandled, non-Saccharomyces yeasts may grow before or at the start of fermentation, sometimes at the expense of a healthy Saccharomyces population that can ferment to dryness. If odors of ethyl acetate (airplane glue/ nail polish remover), amyl acetate (banana skin), or other off-odors appear in juice or must, examine immediately under a phase-contrast microscope for yeasts that are not the round/ovoid shape of Saccharomyces. Non-Saccharomyces yeasts are sometimes round, but more often have other appearances: apiculate (pointy), elongated, or bizarrely shaped, and they may be unusually small or large. Kloeckera/Hanseniaspora is especially problematic for several reasons. Their SO2 sensitivity is comparable to that of Saccharomyces, and they are naturally pr actica l win ery & vin eya rd JANUARY 20 14 61