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winemaking run further tests to determine) which microbes are likely to spoil that particular wine and which microbes are not. The presence of wine-tolerant microbes may result in holding back a bottling run, product recall, or even rebottling, but a well-trained wine microbiologist can advise a winemaker so wines are not held back unnecessarily. Depending on quantity and type, the presence of any microbes can indicate poor sanitation of the bottling line. However, the batch of wine usually can still be released if the microbes are intolerant to wine. While the ideal is the absence of any microbes at all in any bottles, the reality is that a culture of a newly bottled wine will often grow one or more colonies of incidental microbes intolerant to wine. Each winery must develop a standard to determine how many colonies per bottle of wine-intolerant microbes, or levels measured by an ATP tester on the bottling line, will trigger a review of bottling line sanitation procedures. In a wine bottled without sterile filtration (many reds and some whites), it is very important for the lab to distinguish microbes that can affect that wine negatively from those that cannot. For example, Candida cantarelli, a yeast very common in cellars and sometimes found in wine samples (though unable to grow in wine), closely resembles Brettanomyces in culture; both produce smooth, white, rounded colonies, but their cell morphology (Figure 2) and growth patterns are completely different. In another example, some vineyard sprays for powdery mildew contain Bacillus subtilis; the spores can persist in cellared wine for many months. It is not a threat to wine, but it grows alarminglooking, vertical, wrinkled colonies in culture (Figure 1). Winery microbiologists need to recognize these colonies so the wine is not unneccesarily reprocessed. How much microbiology should winery workers know? Whatever protocol is chosen, all wineries should follow a written HACCP plan tailored to their own situation. Suggestions by Dr. Bruce Zoecklein for a winery HACCP plan can be found at apps.fst.vt.edu/extension/enology/EN/115.html#2. Protocol A—All wine-production people are aware of which microbes grow in wine, which do not and what danger signals to look for. This protocol allows winery workers to focus attention on microbes and situations that could harm the wine at various stages, while also maintaining appropri- ate sanitation overall. Even staff who do not work in the lab know the danger signals in the cellar and during bottling. Protocol B—Only supervisors and/or microbiology staff (or commercial labs that are used) are aware of which microbes grow in wine and which do not. This protocol permits lab and supervisory personnel to evaluate winery procedures based on the microbes present, while not holding back wines containing microbes that are not a threat to the wine. More than occasional microbes in bottled wines, however, indicate that an overhaul of bottling line sanitation is needed. Cellar and bottling workers perform sanitation procedures intended to eliminate all microbes at bottling, and throughout production to reduce potential populations. Protocol C—No one in the winery is aware of which microbes grow in each type of wine and which do not (or if they are aware, that is not taken into account in designing lab or cellar procedures or releasing wine). With this protocol, there are distinctly different outcomes depending on what is produced. In food-processing plants, any "bug" in most products is a bad "bug," especially because there are so many foodborne illnesses. Food producers must eliminate human pathogens and try to eliminate microbes except those required for that product (such as Lactobacillus in yogurt or molds in certain cheeses), or those that cannot be eliminated because of the nature of the product (fresh meat, fruit and vegetables, for example). While food recalls show that lapses do occur, well-run plants keep pathogens out of the food, and the food does not spoil within its expected shelf life. In winemaking, however, this protocol can lead to difficulties, from overload of the lab's capacity to rebottling wines that do not contain wine-tolerant microbes. Pathogens (as far as we know now) are unable to survive, so public health is not an issue. Case histories for Protocol A A1—Selective vigilance A small winery whose staff each did several jobs, including sharing lab, cellar and other duties, requested seminars on wine microbes, where and how they grow, which protocols are needed to limit growth and what danger signals to look out for. It was very helpful for them to learn to watch closely for microbes that could cause wine problems and not to focus on microbes (such as on workers' muddy boots when it rained, or when someone sneezed) that would not infect the wine itself. A2—Brettanomyces management A medium-size winery had a serious problem with Brettanomyces, including one in-bottle spoilage resulting in the recall of a wine. The winemaker, an enology graduate, explained to all wineproduction personnel what the problem was, how it grew and how it was transferred from wine to wine. The incidence of infection dropped considerably, although it was years before they could manage Brettanomyces successfully. Case histories for Protocol B B1—Dirty bottling line but all microbes intolerant to wine A custom-bottling company routinely sent post-bottling samples to a commercial lab. The wines always grew a large number of colonies of many species (Figure 3). The lab determined that none could grow in the bottled wine, so the wines could be released. A consultant from the commercial lab was called in to investigate all parts of the bottling line extensively, making many suggestions about sanitation. The bottles now have only an occasional wine-intolerant microbe, and no wine microbes (yet). Figure 3—Petri dishes of "non-wine" microbes in bottled wine that triggered a bottling line sanitation audit. B2—Too many cultures A very large wine-bottling plant did around 250 cultures per day. Whenever a microbe grew in a culture, they held the wine back while they attempted to identify the microbe. Because there were so many colonies to examine, one colony of Zygosaccharomyces was missed, which caused a problem. The backlog of reserved wines was unmanageable. The wines were low-pH, no MLF, with sorbate added, and no fermentation or barreling occurred in the plant; the enemy was Zygosaccharomyces (insensitive to sorbate pr actica l win ery & vin eya rd JU NE 20 13 59