This research focused on optimizing cleaner and sanitizer concentration and contact time for several different chemicals and spoilage microorganisms relevant to the wine industry. Minimum inhibitory concentration (MIC) and minimum biocidal concentration (MBC) assays were performed, which expose the microbes to dilution series of antimicrobial agents to determine at which concentration different species are either inhibited (MIC) or inactivated (MBC) by exposure to the antimicrobial. As the MIC/MBC assay involves contact times for the microorganisms that are greater than would be reasonable for the wine industry (24 hrs), fluorescence spectroscopy was employed to provide complementary kinetic inactivation data. Peracetic acid was used at several different concentrations to determine the minimum contact time for inactivating S. cerevisiae cells in suspension. In a similar experimental design as the MIC/MBC assay, a minimum biofilm eradicating concentration (MBEC) assay was employed to assess whether sessile communities would require elevated concentrations in order to inactivate or remove the biofilm populations from the microtiter plates. While many of the chemicals did require higher concentrations to inactivate sessile communities, cleaners that contained surfactants and other detergents were effective at lower concentrations, possibly due to the fact that they physically removed the biofilm from the well plate regardless of whether the cells were inactivated. In combination with previous research efforts (Final Report 2017_2123) these results were used to develop an optimized cleaning and sanitation framework for assessment in the winery at the pilot scale (2000 L), which were assessed using ATP swabbing and traditional plate counts. Results from those trials indicate that cleaning and sanitizing contact times are less important beyond 5-minute exposure than proper attention to critical control points in the shadow of spray balls or mechanical agitation. Worker diligence in manually addressing and cleaning these sensitive areas may have a greater impact in cleaning and sanitizing success than increasing contact time several fold.
This project analyzed the ability of cleaners and sanitizers frequently used in the wine industry to inactivate microbial populations in solution (planktonic) and stationary (biofilm) physiologies. A screening of 20 different cleaner and sanitizer chemistries was conducted using 96-well plates and the crystal violet method. Next, biofilms were grown on 304 stainless steel coupons by incubating the coupons in an inoculated grape juice medium. These coupons were treated with chemicals at varying concentrations and contact times and then swabbed with ATP luminescence swabs and traditional plate count swabs to determine the microbial load and soil after treatment. Further trials were then conducted in the UC Davis teaching winery facility at the 200 L and 2000 L scale. For the 200 L fermentations, a custom device was constructed to prove 110 replicate soiled coupons that could be used for further treatment in the laboratory setting. These trials allowed for the development of an optimized protocol that could be tested against other similar treatments at the 2000 L-scale. For these larger scale trials, a five-step cleaning and sanitizing framework was employed, and again ATP and traditional plate count data were collected. The results of these experiments show that the vulnerable areas of tanks (gaskets and areas in the shadow of spray arms) have consistent microbial contamination, regardless of the cleaning protocol or contact time. These need to be areas of focus in any cleaning and sanitation protocol, and winemakers must be prudent to develop a system that exceeds the typical visual inspection protocol often employed in the winery environment.
This project analyzed the ability of cleaners and sanitizers frequently used in the wine industry to inactivate microbial populations in solution (planktonic) and stationary (biofilm) physiologies. Cleaning and sanitizing agents were used at manufacturer’s specified concentrations for planktonic cells grown in 96-well plates. The most effective treatments for the inactivation of planktonic cells were used in the biofilm trials. In these trials, biofilms were treated with the chemicals (cleansers and sanitizers) and subsequently stained to identify remaining biomass using crystal violet. Next, the same chemicals were tested for biofilms grown on stainless steel winery tank material. Stainless steel coupons were suspended vertically in pipet tip containers and incubated with various winery microbes in 50% water/50% grape juice medium for two weeks to develop biofilms. The coupons were cleaned with water, and then submerged in the cleaning or sanitizing agents for various time periods. The ability of the chemicals to eradicate the biofilms was measured using ATP swabs and cell culturing, the two techniques most frequently employed in wineries to examine microbial load in quality control applications. Remaining work involves scaling the stainless-steel trials to larger red wine fermentations, and the development of an optimized cleaning/sanitizing recommendation for the wine industry.