Food Safety Magazine - April/May 2018

vive within barrels, even under “starvation” conditions. In fact, D./B. bruxellensis can survive within the porous wood structures of barrels not filled with wine for weeks or months. Brettanomyces has been isolated from used barrels in the Finger Lakes wine region of New York. 6 Since wood is porous and a source of wood sugars, barrels are difficult to clean and sanitize, and are vectors for Brettanomyces contamination. 7, 8

While the sanitation methods used in wineries are effective on surfaces such as stainless steel, plastic, and glass, sterilization of wood surfaces has proven more difficult. Steam sanitation of wood is a standard method used for wine cooperage. However, the temperatures required to inactivate spoilage microorganisms, and the times required to reach these temperatures within the wood, are poorly understood. Heat and mass transfer in capillary porous materials such as wood has been discussed previously, 9 particularly in regard to the thermal conductivity of wooden materials and the development of thermal inactivation regimes.

Wood is a natural polymer of complex chemical composition and microstructure. 10 Its hygroscopic and porous medium results in heat transfer by conduction, convection, and radiation. 11 Various methods of heat sterilization of wood are currently under investigation as means of killing exotic insects or pathogens within imported goods. One factor being studied is the amount of time (D) required to heat wood of various cross-sectional sizes and configurations to a temperature that will kill 90 percent of the insects or pathogens. 12, 13 The value of D is then used as a predictor of responses beyond the data to estimate the time required for disinfection ( 10-3 CFU/mL) or sterilization ( 10-6 CFU/mL). 14 The underlying assumption when utilizing this measure is that the relationship between the log10 number of survivors and time is linear. 14 If the logarithms of the D-values obtained at various temperatures are plotted against temperature, and the best straight line is drawn through the points, the reciprocal of the slope of this line is the value of z: the number of degrees by which the temperature has to be raised or lowered to bring about 90 percent reduction or tenfold increase in D. 13 In our research, steam treatment of barrels was used as a validation method after having obtained D- and z-values for different species of spoilage yeasts. The validation method was achieved in naturally contaminated barrels where we measured the temperatures reached at different times and depths of the staves. The understanding of temperature changes and times needed to achieve the sanitation as depth is increased, together with D- and z-values, will be useful to understand what parameters should be used when steam is the preferred sanitation method for wine cooperage.

Materials and Methods

Microorganisms
D./B. bruxellensis isolates (CE261, CE149), S. cerevisiae isolates (CE81, CE9, and
CE78) and Z. bailii (4A1) were obtained from the Department of Food Science col-
lection at Cornell University.

In vitro experiments

The yeasts were stored at –80 °C in 15% (w/v) glycerol, revitalized, and maintained on YPD agar (Difco™, Sparks, MD). All strains were grown until stationary phase (growth under agitation at 200 rpm, 30 °C) in YPD broth. Once the cultures reached stationary phase, the target inocula were verified via a viable count and injected in sterile glass capillary tubes; groups of five tubes were sealed with a direct flame and put into tubes with water already tempered in a water bath at the temperatures used for this study. The capillary tubes were removed at different times,

put into tubes with ethanol (70%) to decontaminate the exterior surface, and then left in ice until diluted to assess the residual microbial activity. The dilutions were plated on YPD agar and incubated at 30 °C for 48 to 72 hours for Z. bailii and S. cerevisiae, and up to 3 to 4 weeks for D./B. bruxellensis.

Microbiological enumeration for thermal inactivation

Plates were enumerated for total microbial counts. The counts were averaged and expressed in logs. The log reduction was then calculated for each strain and expressed in logs. Each experiment was performed until the best linear correlation coefficients were obtained (r2 = 0.9).

Microbiological enumeration from barrels

The samples were analyzed to determine the initial and final Dekkera/ Brettanomyces populations and general yeast populations, either by filtration (EZ-Fit™ manifold; EMD Millipore, Billerica, MA) using 0.22-µm disks and/ or pertinent dilutions of the samples, since the microbial loads differed for each barrel. If samples required dilution, 0.1% (w/v) buffered peptone water (Hardy Diagnostics, Santa Maria, CA) was used.

For the filtration method, 0.22-µm nitrocellulose membranes (GE, Pittsburgh, PA) were used, the samples were filtered twice, and the results were averaged. The maximum volume filtered was 100 mL, and the results were calculated as CFU/100 mL and transformed into log10 values. The membranes were placed onto WLD and YPD agar using sterile forceps. WLD agar (Oxoid Ltd., Basingstoke, Hampshire, England) was used to detect D./B. bruxellensis after incubation at 30 °C for 3–4 weeks. WLD agar containing 10 mg/L cycloheximide (Sigma-Aldrich, St. Louis, MO) was used for the selection of D./B. bruxellensis (dissolved in 50% ethanol and filter-sterilized); 150 mg/L biphenyl (Acros