Food Protection Trends - July/August 2022 - 285

TCS could only be displayed at ambient temperature for up to
4 h and would require a discard after 4 h (13). The FDA Food
Code has no references directly in it to evaluate the percentage
of ethanol as a food preservative or antimicrobial with the
exception of providing for the option of performing a product
assessment under the TCS definition in Chapter 1 (13).
Ethanol is the type of alcohol in beer, wine, and liquor and
has been used for many years as a disinfectant (3, 6). De Villiers
states that 15-17.5% ethanol based on free water (aw
) is an
effective preservative for oral pharmaceutical preparations
(4). Kalathenos and Russell, when reviewing ethanol as a
food preservative, stated that solutions containing <30% (vol/
vol) were rarely biocidal; however, bacterial growth could be
inhibited starting at 8-11% (vol/vol) (5). Waite and Daeschel
reported that log reductions of Staphylococcus aureus and
Escherichia coli O157:H7 occurred in 20 min in wine with
12.1-14.7% ethanol at pH 2.74-3.72 (14). Other studies have
established that ethanol at a 5% concentration produces a strong
bacteriostatic effect on the growth of pathogens like E. coli
O157:H7, Salmonella Typhimurium, and Listeria monocytogenes
(2, 8, 11).
This study was undertaken to evaluate the fate of E. coli
O157:H7, Salmonella, and L. monocytogenes inoculated into
alcoholic cocktail drinks prepared with ethanol concentrations
of 10, 20, 30, 40, and 50% in various fruit juices stored at
ambient temperature. The data can be used by regulators to
ascertain the relative risk of storing these beverages at ambient
temperature for more than 4 h.
MATERIALS AND METHODS
Cocktail preparation
Ethanol concentrations of 10, 20, 30, 40, and 50% (vol/
vol) were prepared using 190 proof ethyl alcohol and distilled
water. For each concentration, 1,000 ml of ethanol solution was
prepared. The 100-ml cocktail mixtures were prepared at each
concentration by mixing 50 ml of ethanol solution and 50 ml
of fruit juice or puree using banana puree, Asian pear puree,
orange juice (Pulp Free Original Orange Juice, Simply Orange
Juice Company, Apopka, FL), and apple juice (Fresh Pressed
Three Apple Blend 100% Juice, Tree Top Inc., Selah, WA). A
control for each concentration was also prepared by mixing 50
ml of ethanol and 50 ml of distilled water. The treatments for
cocktails for each ethanol concentration were control (ethanol
and distilled water), pear (ethanol and Asian pear puree), banana
(ethanol and banana puree), orange (ethanol and orange juice),
and apple (ethanol and apple juice).
Inoculum preparation
Five strains of E. coli O157 of vegetable origin or related to
vegetable outbreaks (H1730, EC4042, EC4045, EC4191, and
EC4206) were used; these were obtained from the culture
collection of Dr. Donald Schaffner at Rutgers University and
maintained at −80°C in Dr. Taylor Oberg's laboratory. Similarly,
five strains of Salmonella, Thompson FSIS 120 (chicken isolate),
Enteritidis H3502 (clinical isolate, phage type 4), Enteritidis
H3527 (clinical isolate, phage type 13a), Typhimurium
H3380 (clinical isolate, phage type DT104), and Heidelberg
F5038BG1 (ham isolate) were used; these were also obtained
from Schaffner's culture collection at Rutgers University and
maintained at −80°C in Oberg's laboratory. For L. monocytogenes,
five strains-J1-177 (serotype 1/2b, human isolate), C1-056
(serotype 1/2a, human isolate), N3-013 (serotype 4b, food
isolate), R2-499 (serotype 1/2a, sliced turkey isolate), and N1227
(serotype 4b, food isolate)-were obtained from Oberg's
culture collection at Utah State University. Cultures for each
pathogen strain were prepared from frozen stock maintained at
−80°C by transferring 0.1 ml of thawed frozen stock into 10 ml
of fresh tryptic soy broth (TSB; Neogen Corp., Lansing, MI)
and incubating at 37°C for 24 h. Individual strains were then
grown in TSB for 24 h at 37°C before inoculation. The fivestrain
mixture for each pathogen was prepared by combining
2-ml aliquots of each strain in a 15-ml conical centrifuge tube.
Cells were pelleted by centrifugation (1,509 × g for 15 min) and
resuspended in 10 ml of Butterfield phosphate buffer solution
(BPBS) 3 times.
Sample inoculation and incubation
The treatments prepared from the five varieties of cocktail
mixtures at 10, 20, 30, 40, and 50% ethanol concentrations
were transferred to sterile glass containers, loosely capped with
sterile caps, and stored at 25°C. The five-strain mixture of each
pathogen was then inoculated (10/100 ml) in the treatments
separately. All inoculated treatments were transferred to 25°C for
incubation for up to 48 h.
Microbial analysis, pH, and aw
measurement
Inoculated pathogens were first enumerated after approximately
30 min of inoculation into the treatments. Subsequent
enumerations were performed at 3, 6, 9, and 12 h, followed
by enumeration at 12-h intervals for up to 48 h. For enumeration,
1 ml of the sample was pipetted into 9 ml of BPBS, and
subsequent serial dilutions were performed. The samples were
then plated in duplicates. The treatments inoculated with E.
coli O157 were plated in Sorbitol-MacConkey agar (Neogen),
and colonies were enumerated after 24 h of incubation at 37°C.
For treatments inoculated with Salmonella, Salmonella Shigella
agar (Neogen) was used to enumerate colonies after 24 h of
incubation at 37°C. Similarly, for treatments inoculated with
L. monocytogenes, PALCAM agar base (Neogen) containing
PALCAM supplement (Neogen) was used to enumerate
colonies after 48 h of incubation at 37°C. For pH measurement,
approximately 10 ml of the sample was taken and the
pH was measured using a double-junction pH meter (pHTestr
30, Oakton Instruments, Vernon Hills, IL). Likewise, aw
was
measured by pipetting 3 ml of the sample into the sample cups
using a LabSwift portable water activity meter (Novasina AG,
Lachen, Switzerland).
July/August Food Protection Trends 285
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