portion of the bung), and then 70% ethanol was sprayed around the bunghole.
The samples were placed at 4 °C until
analysis. The samples were analyzed
to determine the initial and final
yeast populations, either by filtration
using discs or pertinent dilutions of
the samples, since the microbial loads
varied for each barrel. If samples needed
to be diluted, 0.1% (w/v) buffered peptone water was used.
The samples were divided into two
groups and filtered through cellulose.
The results of the two filters were then
averaged. The maximum volume filtered
was 100 mL, and the results were calculated as CFU/100 mL and then transformed into log10. The cellulose filters
were aseptically placed onto WL and
YPD agars. WL agar was used to detect
D./B. bruxellensis and incubated at 30 °C
for up to 3–4 weeks; colonies that grew
before 3 days were discarded. Incubation time was also used to demonstrate
growth, as nothing that grows before 3
days in WL with cycloheximide is D./B.
bruxellensis. The WL agar contained 10
mg/L cycloheximide to allow for selection of D./B. bruxellensis (dissolved in
50% ethanol and filter-sterilized), 150
mg/L biphenyl to prevent the growth
of mold, 30 mg/L chloramphenicol to
prevent the growth of lactic acid bacteria and 25 mg/L kanamycin sulfate to
inhibit the growth of acetic acid bacteria. YPD agar was used to detect general
yeast populations and was incubated at
30 °C for 48–72 hours. The YPD agar
contained all of the above selective
agents except cycloheximide.
The reductions in yeast were calculated from the initial concentration of
yeast cells (target inoculum) at time zero
minus the last concentration of yeast at
time 90 minutes for the in vitro experiments. All experiments, including the
controls, were performed in triplicate.
were obtained from the Department of Food Science collection at Cornell
Preparation of starter culture and inoculation
The yeasts, stored at -80 °C in 15% glycerol (w/v), were revitalized and maintained on YPD agar. All strains were grown until stationary phase (108 CFU/mL)
or at least 106 CFU/mL (growth under agitation at 200 rpm, 30 °C). The growth
time varied according to the strain. Each strain was grown in YPD broth adjusted
to different pH levels ( 3.0, 3. 2 and 3. 4) with 1 M HCl and/or 1 M NaOH. Once
the cultures reached 108 or 106 CFU/mL, the target inoculum was verified via a viability assessment. A 1-mL volume of culture was placed in sterile Eppendorf tubes
and centrifuged ( 4,500 rpm, room temperature); the supernatant was discarded and
resuspended in 1 mL McIlvaine buffer11 at the different pH levels stated above. A
1-mL culture was inoculated into 99 mL McIlvaine buffer at the different pH levels
mentioned above, reaching a concentration of 104–106 CFU/mL. Analyses were performed in triplicate.
Preparation of KMB solution
The solutions were prepared before each experiment by dissolving the required
amount of KMB in McIlvaine buffer at the different pH values. The final concentrations of KMB were 0 and 300 mg/L. Each KMB solution was spiked with 1 mL cells
to achieve a final volume of 100 mL and plugged with rubber stoppers.
Sampling and microbiological isolation
Each flask was aseptically sampled at different times. The flasks were incubated in
a water bath at 30 °C with no agitation and sampled at 0, 15, 30, 60 and 90 minutes.
At each sampling time, 1 mL was taken and properly diluted in 0.1% (w/v) buffered
peptone water, immediately plated in duplicate onto YPD agar and incubated at
30 °C for 48–72 hours for Z. bailii and S. cerevisiae, and up to 3–4 weeks for D./B.
bruxellensis. When necessary, direct plating from the flask was also performed to increase the level-of-detection threshold. The volume plated was 100 µL.
Plates were enumerated for total microbial count. The counts were averaged and
expressed as log values. The log reduction was then calculated for each strain and
expressed as log values. Each inactivation experiment and controls were performed
in triplicate for each strain.
Sulfur disc treatment of barrels
The 20 donated barrels used for this study were identified with
Dekkera/Brettanomyces by the wineries that donated them. These naturally contaminated barrels were
split in two groups of 10 barrels each to identify both Brettanomyces and general yeast
populations and were then treated with sulfur discs. Each barrel had an identification number. The treatments were conducted for 3 and 6 weeks. Briefly, 7 L distilled
water were added to each barrel, before and after burning the sulfur discs inside the
barrels. The barrels were rolled several times to enhance the contact of water with
the inner surface of the barrel, stored bung side up for 24 hours and then sampled
before and after burning the sulfur discs. Water samples were collected in sterile
bottles. The first portion of the water was discarded to “rinse” the bunghole (outer
“The purpose of the…study was to test SO2 at three pH levels to assess
its efficacy against wine-spoilage yeasts…”