Food Protection Trends - July/August 2021 - 381

ganisms (4, 11). The disruption of physiological processes,
cellular membranes, or other cellular constituents are the
main antimicrobial activities of chemical compounds (34).
However, most of these compounds have limitations, which
include chemical residues, discoloration of meat, negative
health effects on food handlers, corrosion of machinery, environmental
pollution, and high costs (40, 42). Red meat and
poultry decontamination technologies have been applied for
years in the United States (6, 24). Electrolyzed water, a safe
sanitizer, has been increasingly used in the food industry to
reduce and control of microorganism on foods and food contact
surfaces (23). Near-neutral electrolyzed water (NEW),
with a pH of 6.0 to 6.8, with an oxidation-reduction potential
(ORP) of 800 to 900 mV, and containing 95% hypochlorous
acid, 5% hypochlorite ion, and trace amounts of Cl2
, is
generated by passing an aqueous salt solution (ca. 1% NaCl)
through a nonmembrane electrochemical cell (21, 33). Hypochlorous
acid is the most active of the chlorine compounds
and has high bactericidal activity (36). In several studies,
the efficacy of NEW has been evaluated for reduction of
bacterial population in fresh meat (12, 35, 42, 43), eggshells
(44), seafoods (28), and vegetables (1). If NEW were to be
combined with other chemical disinfectants such as organic
acids, its antimicrobial efficacy might increase. Peroxyacetic
acid (PAA) is produced from the reaction of acetic acid with
hydrogen peroxide (31). Its disinfection efficiency is due to
the strong oxidizing potential, which can result in disruption
of the cell membrane and blockage of enzymatic and transport
systems in microorganisms (32, 46). Application of PAA
for meat decontamination has been studied (3, 30). The aim
of the present study was to evaluate the efficacy of individual
treatments with NEW and PAA and their combined effect on
reduction of the foodborne pathogens Listeria monocytogenes,
Salmonella Typhimurium, and Escherichia coli in vitro and in
fresh chicken meat.
MATERIALS AND METHODS
Bacterial cultures
The bacterial strains used in this study were L. monocytogenes
(PTCC 1297), Salmonella enterica subsp. enterica serovar
Typhimurium (ATCC 14028), and E. coli (ATCC 35218)
(School of Veterinary Medicine, Shiraz University, Shiraz,
Iran). To confirm L. monocytogenes, cultures were streaked on
PALCAM (polymyxin acriflavine lithium chloride ceftazidime
aesculin mannitol) agar (Merck, Darmstadt, Germany)
supplemented with nalidixic acid (50 μg/mL), amphotericin
B (10 μg/mL), and acriflavin (30 μg/mL). For easy
differentiation of E. coli and Salmonella Typhimurium from
background bacteria of chicken breast meat, these pathogens
were cultured to be resistant to nalidixic acid. Nonresistant
cultures were inoculated into 100 mL of trypticase soy
broth (TSB; Merck) and incubated at 37°C for 24 h, 100
mL of TSB containing 200 µg/mL nalidixic acid (1451000,
Sigma-Aldrich, St. Louis, MO) was added, and this culture
Preparation of disinfectant solutions
NEW with a pH of 6.8, an ORP of 830 ± 5 mV, and
available free chlorine (AFC) concentration of 800 μg/mL
was obtained from Khosro Medisa Teb Co. (Envirolyte,
Tehran, Iran). NEW was produced by electrolysis of a salt
solution (ca. 1% NaCl in tap water) with an electrolysis
device. Solutions with various AFC concentrations were
prepared with sterile distilled water. PAA was obtained from
Behban Shimi Co. (Percidine 15%, Golestan, Iran), and
various concentrations were prepared with sterile distilled
water (v/v).
MIC determination for NEW and PAA
The AFC concentration of NEW was measured with a
chlorine test kit (Karizab Co., Tehran, Iran) immediately
before use. The original NEW was then diluted with sterile
distilled water to obtain AFC concentrations of 3.13, 6.25,
12.5, 25, 50, 100, and 200 μg/mL. A 0.1-mL aliquot of
each bacterial suspension was combined with 9.9 mL of
each concentration of NEW and vortexed for 5 s and then
incubated at room temperature. The final level of each
test pathogen per treatment was approximately 106
CFU/
mL. Sterile distilled water was used as a control for each
experiment. To evaluate the effect of treatment time on the
reduction of bacterial cells, treatment tubes were sampled at
0, 1, 2, 3, and 4 min after inoculation. The results indicated
that no additional reduction was observed after 2 min;
therefore, 2 min of contact time at room temperature was
chosen for the experiment. Following 2-min treatments,
0.1 mL of each sample was transferred to 9.9 mL of sterile
neutralizing buffer (0.5% sodium thiosulphate + 0.03 M
phosphate buffer solution), and the tubes were shaken. After
neutralization, 0.1 mL of each treatment was plated on TSA
with nalidixic acid and incubated at 37°C for 24 to 48 h. The
colonies were enumerated with the plate count method.
When necessary, 10-fold serial dilutions were made from
samples before plating. Using this method, the presence
of low numbers of surviving cells may not be detected. In
parallel, a 100-µL aliquot of each treatment was added to 10
July/August Food Protection Trends 381
was subsequently incubated at 37°C for a further 24 h. A
0.1-mL aliquot of this culture was spread on the surface of a
trypticase soy agar (TSA; Merck) plate containing 100 µg/
mL nalidixic acid and incubated at 37°C for 24 h. A single
colony was selected and restreaked onto another TSA plate
to confirm resistance to nalidixic acid (45). Each strain of
pathogens was separately transferred into TSB with nalidixic
acid (50 μg/mL) and incubated at 37°C for 24 h, and 10 mL
of each culture was then centrifuged at 4,000 × g for 10 min.
The supernatant was discarded, the cell pellet was washed
twice with normal saline solution, and the final cell pellet
was resuspended with the same solution. The density of the
suspensions was compared to the 0.5 McFarland turbidity
standard and adjusted to 1.5 × 108
CFU/mL.
Food Protection Trends - July/August 2021

Table of Contents for the Digital Edition of Food Protection Trends - July/August 2021
