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
“Several microbial contaminants appear to survive on walls and other
interior surfaces of wineries…”