Food Protection Trends - September/October 2021 - 466

18 ± 1°C; and condensing, −85 ± 1°C. GS and PP were then
reduced to nano size according to Khataee et al. (35). Briefly,
GS and PP were fine ground (model MC300, Moulinex
France) to micro size (110 to 150 μm) and then crushed by
a high-energy planetary ball-mill (model PM 2400, Iran) to
obtain nano size. The transfer process was performed using
a ball mass-to-powder mass ratio of 10:1 and rotation rate of
320 rpm for 2 h under atmospheric conditions. GS-NPs and
PP-NPs were measured by a Zetasizer Nano ZS instrument
(NanoSight NS300, Malvern Panalytical, Malvern, UK),
exhibiting with an average size of 65 ± 2 and 80 ± 2 nm,
respectively. Both NPs were packed in dark glass bottles and
stored until the experiments.
Antimicrobial activity of antimicrobial strategies (in vitro)
Antimicrobial activity of GS-NPs and PP-NPs was estimated
using a plate overlay assay (55). In brief, plates were overlaid
with 10 mL of semisoft TSA (0.5% [w/v] agar) seeded
with 100 μL of an overnight broth culture of E. coli O157:H7
and Salmonella Typhimurium (~5 log CFU/mL). An aliquot
of 20 μL of each GS-NPs or PP-NPs at different concentrations
(0.1, 0.2, 0.3, 0.5, 1, and 1.5% [w/v]) was spotted onto
seeded lawns and dried in the laminar air hood for 5 min.
Pu coatings incorporated with GS-NPs or PP-NPs were
cut into pieces (1 by 1 cm). The antimicrobial activity was
evaluated using plate overlay assays (44). Plates were scored
for inhibition zones after 48 h of incubation at 37°C.
The antimicrobial activity of the EO vapor phase was
determined using the inverted petri dish method (66). TSA
plates were inoculated with 100 μL of E. coli O157:H7 and
Salmonella Typhimurium suspension (~5 log CFU/mL) and
allowed to dry 5 min. Sterile filter paper disks (Whatman
No. 1, Whatman, Maidstone, UK) were positioned on the
center of the lid of the petri dish, and 0, 10, 50, 100, 250, and
500 μL of thyme or oregano oil was poured. The plates were
tightly sealed with Parafilm to avoid vapor fugues and were
incubated at 37°C for 48 h. Blanks (controls) were performed
by adding 10 μL of ethanol to the filter paper disks, which
was shown to have no effect on the viability of the E. coli
O157:H7 and Salmonella Typhimurium. Inhibition zones
were measured with a Vernier caliper, and the concentration
of EOs was expressed as microliters per liter of air (μL/Lair
).
Strawberries challenge study (in vivo)
Before each experiment, strawberries were surface treated
with UV under a laminar flow hood for 15 min to reduce
background microflora before artificial contamination (52).
Subsequently, the strawberries were spot inoculated with
overnight and diluted cultures of E. coli O157:H7 and Salmonella
Typhimurium at ~5 log CFU/cm2
and dried for 20 min
to allow for cell attachment. Strawberries not inoculated were
used as controls.
Washing solutions
Inoculated strawberries were washed with 0.5% CA,
0.5% LA, 0.5% AA, 100 ppm C, 0.1% SC, and 0.1% ASC by
immersion and spray for 2 min (30). The spray method was
performed using a multifunction manual pressure sprayer 2L
(Ningbo Synkemi Co., Ltd., Zhejiang, China) with a flow rate
of 6.5 mL/s. The nozzle was placed vertical at 20 cm over the
fruits twice each side for 2 min (30).
EO vapor phases
Strawberries were placed in 500-mL hermetic seal transparent
glass jars (Middel East of Glass Co., Cairo, Egypt).
Sterile filter paper disks (Whatman No. 1) were taped on the
container, and thyme or oregano oil at 500 μL/Lair
(EO volumes
were adjusted to the total volume of the container) was
poured on disks. The strawberries were placed on the containers'
lids, and after pouring the EOs, the containers were closed
(upside down) and sealed with Parafilm to avoid vapor fugues.
A blank sample (control) was prepared by adding 10 μL of
ethanol to the filter paper discs, which was revealed to have no
impact on the viability of the E. coli O157:H7 and Salmonella
Typhimurium. The jars were kept at 4 ± 1°C for 18 days (67).
The concentration of thyme or oregano EO was expressed as
microliters per liter of air. The experiment was performed in
triplicates. The jars were kept at 4 ± 1°C for 18 days (66).
Pu coatings
Strawberries were coated with pullulan solution (5%
[w/v]) one by one and then allowed to dry aseptically in
a biological safety cabinet at ambient temperature. Several
treatments were performed: uncoated (control), water wax
with thiabendazole as positive control (67), pullulan (Pu),
Pu with GS-NPs (Pu-GS-NPs), Pu with thyme oil vapor and
GS-NPs (Pu-TOV-GS-NPs), Pu with PP-NPs (Pu-PP-NPs),
and Pu with TOV and PP-NPs (Pu-TOV-PP-NPs) (58). The
coated strawberries were placed in a container and kept at 4 ±
1°C for 18 days.
Microbiological assay
On days 0, 3, 6, 9, 12, 15, and 18, 10 ± 1 g of strawberries
was aseptically transferred into 90 mL of 0.1% buffered
peptone water (Himedia, Rabat, Morocco) and stomached
for 2 min (model G560E mixer, Scientific Industries, Inc.,
Bohemia, NY). Next, serial dilutions (10-fold) were made.
An aliquot of 100 µL of each dilution was spread plated in duplicates
onto sorbitol MacConkey agar (Himedia) for E. coli
O157:H7 and xylose lysine desoxycholate agar (Himedia) for
Salmonella Typhimurium. All plates were incubated at 37°C
for 24 to 48 h, and the remaining colonies were counted and
expressed as log CFU per gram (44).
Statistical analysis
Data from the challenge study were statistically analyzed
for remaining bacteria by using one-way analysis of variance
466 Food Protection Trends September/October
Food Protection Trends - September/October 2021

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