Food Protection Trends - July/August 2021 - 382

mL of TSB with nalidixic acid and incubated at 37°C for 24
to 48 h. Then, 100 µL of enriched broth was spread onto TSA
plates with nalidixic acid and incubated at 37°C for 24 to 48
h. The plates were then checked for bacterial colonies (19).
The antibacterial effect of PAA at 3.13, 6.25, 12.5, 25, 50,
100, and 200 μg/mL was also determined using the method
described above.
To evaluate the antimicrobial interaction and any
synergism between NEW and PAA, 3.13, 6.25, 12.5, 25
and 50 μg/mL concentrations of each disinfectant were
prepared, and the same concentrations were mixed in equal
proportions and tested according to the previous method.
Sample preparation
Whole fresh chicken breasts with skin were obtained from
a local poultry processing plant. The breasts were transported
in ice, stored at 4°C, and used for treatments in < 2 h. The
average weight of each chicken breast sample was 400 ± 30 g.
Inoculation of pathogens onto chicken meat samples
Chicken breasts were randomly divided into three
batches, each containing 54 samples. Samples in each batch
were dipped for 10 min into separate bacterial suspensions
of L. monocytogenes, nalidixic acid-resistant Salmonella
Typhimurium, and nalidixic acid-resistant E. coli at 107
CFU/
mL in sterile normal saline. The ratio of chicken meat sample
to bacterial suspension volume was 1:1 (w/v). For draining
and bacterial attachment, the inoculated meat samples were
placed on a grid plate inside a laminar flow hood at room
temperature for 30 min (2). Inoculated untreated meat samples
were tested to determine the levels of the pathogenic bacteria.
Decontamination treatment
Inoculated chicken meat samples in each batch were
randomly divided into six groups, each containing nine
samples. Five treatments of NEW at 100 and 200 μg/mL,
PAA at 200 and 400 μg/mL, and NEW 100 μg/mL + PAA
200 μg/mL were prepared in a sterile chilled water container
(4°C). Sterilized chilled water was used to dip the control
samples. Inoculated chicken meat samples with pathogens
were dipped in treatment solutions for 10 min (7, 16, 42). The
ratio of chicken meat to treatment solution was 1:1 (w/v).
Following treatments, samples were drained for 10 min at
room temperature, packed individually into sterile reclosable
zippered polyethylene bags, and stored at 4 ± 0.5°C.
Noninoculated untreated chicken meat samples were
tested for background contamination with L. monocytogenes,
nalidixic acid-resistant Salmonella Typhimurium, and
nalidixic acid-resistant E. coli.
Microbiological analysis
Treated inoculated chicken meat samples were tested
immediately after treatment and on days 2 and 4 of storage.
Samples were transferred to sterile stomacher bags and
382 Food Protection Trends July/August
weighed, and sterilized 0.1% (w/v) buffered peptone water
(Merck) was added in at nine times the weight of each sample.
To ensure that the samples were washed well, bags were hand
massaged for 1 min. The rinsate was then collected, ten-fold
serial dilutions were made in sterile 0.1% buffered peptone
water. PALCAM agar, xylose lysine deoxycholate (XLD)
agar, and MacConkey agar, each supplemented with nalidixic
acid, were used to count healthy cells of L. monocytogenes,
Salmonella Typhimurium, and E. coli, respectively. A 100-μL
aliquot of each sample or appropriate diluent was surface
plated onto the appropriate medium and incubated at 37°C for
24 to 48 h.
After disinfectant treatment, some microorganisms may
be only injured rather than killed (48). For enumeration of
injured bacterial cells, the overlay method was used (27). For
each sample or appropriate diluent, 100 μL was surface plated
onto TSA and incubated at 37°C for 2 to 3 h to allow damaged
bacterial cells to resuscitate, then agar plates overlaid with 7 to
12 mL of the appropriate selective medium. After the overlay
solidified, the plates were incubated at 37°C for 24 to 48 h.
The difference in counts between selective (PALCAM agar,
XLD agar, and MacConkey agar) and nonselective (overlay
method) media was considered the count of injured cells. The
percentage of injured bacterial cells was calculated by using the
formula (20)
% injured cells =
CFUnonselective - CFUselective
CFUnonselective
× 100
Determination of residual chlorine
For the residue experiment, chicken breast meat samples
were dipped in 100 μg/mL NEW, 200 μg/mL NEW, the
combination treatment of 100 μg/mL NEW plus 200 μg/
mL PAA, and sterile water (control) and then placed in a grid
plate inside a laminar flow hood at room temperature for 4 h.
After drying, the meat samples were rinsed in distilled water
for 10 min at a 1:2 (w/v) ratio of samples:distilled water. The
rinse water was used for determination of residual chlorine
with a chlorine test kit (N,N-diethyl-p-phenylenediamine
method; Karizab Co., Tehran, Iran).
Statistical analysis
The experiment was replicated three times. The data were
analyzed with an analysis of variance in SPSS ver. 21 (SPSS
Inc., IBM, Armonk, NY). Differences between treatments
were assessed with Duncan's test and were considered
significant at P < 0.05.
RESULTS AND DISCUSSION
MIC determination for NEW and PAA
The bactericidal activity of NEW against pure cultures of
E. coli, Salmonella Typhimurium, and L. monocytogenes
during the 2 min of contact time is shown in Figure 1. At time
0, the initial populations of E. coli, Salmonella Typhimurium,
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