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w i inneeGM A K IN GG w R O WIN cold-tolerant so they grow readily in cool must (10 –15º C/50 –60º F). They can survive much farther into the fermentation than most other non-Saccharomyces species, occasionally all the way to the end. Sometimes a thick, slimy scum forms, or stringy clumps that fall out later as fluffy lees. Kloeckera can make prodigious amounts of ethyl acetate and amyl acetate, and is quite efficient in quickly depleting nutrients, particularly thiamine and other vitamins. Microscopically, it often resembles bowling pins or thin lemons with a knob on each end. Kloeckera is easy to culture because it is cycloheximide-resistant and grows in one to two days, unlike Brettanomyces (three to seven days), which Kloeckera resembles. There are also PCR primers for Kloeckera, and for Pichia membranifaciens, which could be used at this stage, although there are no primers for most other non-Saccharomyces vineyard species, so those will not be detected by PCR-based methods. However, the question is not whether there are Kloeckera and other vineyard yeasts in the must (there are), but how to monitor their population, and how to handle the must so that they do not grow too extensively. For this purpose, following the must microscopically, estimating percentages of non-Saccharomyces species, is more useful than any other test methods. Before fermentation begins, if more than 5 non-Saccharomyces yeasts are seen in a 40x field, a problem is likely to be developing. Examine some musts microscopically every day, especially any must over 10º C/50º F that are soaking on skins or warming up from soaking/settling, or musts that start fermenting spontane- Photo by patricia roca ously. Also examine microscopically if acetate or amyl acetate odor is noted. YEAST FERMENTATION Microbes to watch out for: NonSaccharomyces yeasts, lactic acid bacteria Danger signals: Spontaneous fermentation, ethyl acetate or other off-odor, sluggish fermentation, spontaneous malolactic fermentation (MLF), low viability of yeast inoculum, VA increase Monitoring methods: Microscopic exam, sensory cues, chemical tests, PCR-based genetic testing Saccharomyces When preparing dry yeast, check viability with methylene blue stain to make sure that enough survived transport, storage, a nd rehydrat ion i n t he w i ner y. Approximately half of the rehydrated yeasts should be viable, as evidenced by lack of blue staining. Counting yeasts with a hemocytometer also helps to monitor the population to be sure that it reaches 10 8 cells/ml, or a stuck ferment is likely. Non-Saccharomyces Photo by L ISA van de water Brettanomyces in stuck wine 62 p r acti c al w i ne ry & v i ne yard JANUARY 20 14 Some winemakers encourage the growth of some non-Saccharomyces vineyard species to contribute to aromatics, complexity and mouthfeel before Saccharomyces, which must become dominant for the fermentation to go to completion, takes over. Potential problems with this approach include depletion of vitamins and nutrie nt s (n at u ra l a nd adde d) t h at Saccharomyces will need, production of off-characters such as H2S, ethyl acetate and acetic acid, and possibly inhibition of Saccharomyces. It is difficult to over-emphasize the potential for problems that can result from too much growth by non-Saccharomyces yeasts. It is very important for winemakers encouraging participation by vineyard yeast species to guard vigilantly against their extensive growth. Many stuck fermentations have their origins early on, as growth of competing species did not allow Saccharomyces to achieve a large population of healthy yeasts. The winemaker may look for a cause late in fermentation, but what happens at the beginning can set the process in motion. Spontaneous fermentations, whether intentional or not, should be checked microscopically until Saccharomyces becomes the dominant yeast. If Saccharomyces does not take over within the first day of fermentation, a large inoculum of a vigorous active-dry yeast may be required (even if no yeast addition was originally planned). If non-Saccharomyces Yeast and Lactobacilli in stuck wine yeasts reach 10 6/ml or more, the population may need to be reduced by centrifuging or filtering, before inoculating with properly rehydrated dry yeast. Nutrient additions to uninoculated fermentations should be delayed until Saccharomyces is recognized under the microscope, or until a clean-smelling fermentation is under way. Fermentations with more than around 10% non-Saccharomyces yeasts should be considered at high risk and carefully watched for signs of sluggishness. Lactic acid bacteria Spontaneous growth of lactic acid bacteria during fermentation can sometimes be a positive occurrence, if the bacterium is Oenococcus oeni, MLF is desired, and no trouble in finishing the yeast fermentation is expected. For many years, beginning in the 1970s, red musts were often inoculated with Oenococcus once yeast fermentation was fully active, with very good success in completing both fermentations together (correctly called "co-fermentation" not "co-inoculation," because the yeast and bacteria are not added at the same time). But if certain hetero-fermentative Lactobacillus species (called "ferocious" by the late Dr. Ralph Kunkee) grow when there is fermentable sugar, the result can be a disaster, traditionally called "piqure lactique." This is more common in musts with higher pH (above 3.6). Besides converting malic to lactic acid, they metabolize glucose and fructose to acetic acid, raising VA to 1 g/L or higher. New research from Dr. Linda Bisson's lab at U.C. Davis has shown that some common wine spoilage bacteria and possibly other stresses can lead to a stressinduced "prion state." Saccharomyces harboring this prion have a reduced fermentative capacity and are less dominant