Cory Marx, Paul van dar Merwe, Lucy Joseph, and Anita Oberholster*
*University of California, Davis, Department of Viticulture and Enology, 595 Hilgard Ln, Davis, CA 95616 (aoberholster@ucdavis.edu)

Cleaning and sanitizing are essential aspects of wine production. Winery spoilage microbes can exist in planktonic physiologies or as biofilms, making the organisms variably resistant to antimicrobial agents. Commonly used winery cleaning and sanitizing chemistries were screened for effective inactivation of seven winery spoilage organisms (Acetobacter pasteurianus, Lactobacillus casei, Oenococcus oeni, Pediococcus parvulus, Brettanomyces bruxellensis, Saccharomyces cerevisiae, and Zygosaccharomyces bailii) in 96-well microtiter plates. Effective cleaners were determined via plating for planktonic treatments and with crystal violet staining for biofilm biomass remaining in the microtiter plates. The most effective treatments from these trials were used to analyze the inactivation and removal of biofilms grown on 2B mill stainless steel (SS) coupons. Biofilms remaining on the coupons were measured using ATP swabs and tip plating for enumeration of viable colonies. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined for treatments that were effective in the coupon trials. Live/dead fluorescence staining was used to analyze minimum effective contact times. These results were used in red wine fermentation trials to create an optimized SOP for SS cleaning. Caustic-based cleaning agents are not only most effective at removing biofilms and soil in well plates and SS, but also provide high levels of microbial inactivation and serve as dual cleaner/sanitizers. Biofilm communities were not effectively removed from SS by agents marketed as sanitizers, suggesting a cleaning step is necessary for surfaces, even in visually clean states. MBC and MIC values varied among organisms and are lower than manufacturer’s recommended levels for caustic treatments. Fluorescence staining was a useful tool to determine viability of microbe populations and for observing biofilms with confocal microscopy.

Funding Support: American Viticulture Foundation