International Journal of Food Microbiology 378 (2022) 109827

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Antimicrobial coating with organic acids and essential oil for the
enhancement of safety and shelf life of grape tomatoes
Tony Z. Jin *, Xuetong Fan, Sudarsan Mukhopadhyay
Food Safety and Intervention Technologies Research Unit, Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor,
PA 19038, United States

A R T I C L E I N F O A B S T R A C T

Keywords: This study investigated the antimicrobial efficacy of two coatings against populations of nalidixic acid-resistant
Coating Salmonella, Listeria monocytogenes and native microorganisms on whole grape tomatoes. Tomatoes were surface-
Safety coated in two chitosan-acid coating solutions. Solution 1 (Chitosan) consisted of 1 % chitosan and 2 % acetic,
Shelf life
lactic and levulinic acids. Solution 2 (Chitosan+AIT) consisted of Solution 1 plus 2 % allyl isothiocyanate (AIT).
Tomato
Chitosan
After the treatments, tomatoes were placed in PET containers and stored at 10 ◦ C for 21 days. Chitosan and
Acid Chitosan+AIT treatments reduced Salmonella populations from 3.65 to 1.28 and <0.70 log CFU/tomato on day 1,
Essential oil respectively. Both treatments reduced Salmonella populations to undetectable levels (<0.70 log CFU/tomato)
from Day 2 through Day 21. Similarly, Chitosan+AIT treatments caused a greater reduction in Listeria pop­
ulations than Chitosan treatment on day 1, but there were no significant differences between the two treatments
after day 2. Chitosan and Chitosan+AIT reduced native bacteria populations to an undetectable level after 2 days
and reduced the population of native yeasts & molds to an undetectable level after 1 day. The presence of mold
was only observed on control sample after 21 days. Quality analyses showed that samples which were subject to
coating treatment maintained their texture and color for 21 days at 10 ◦ C with less water loss compared to the
controls. This study suggests that chitosan-acid coating is applicable for extending the shelf-life and enhancing
the safety of grape tomatoes.

1. Introduction
Tomato is a versatile vegetable which possesses numerous beneficial
nutrients. However, multiple outbreaks of foodborne illness have been
attributed to the consumption of fresh tomatoes and recalls associated
with fresh tomatoes have been reported. Salmonella enterica was the
reported etiologic agent for a total of 56 multistate outbreaks associated
with fresh produce between 2010 and 2017. A total of 3778 illnesses
were linked to these outbreaks, which resulted in a 28.3 % known
hospitalization rate and 16 known deaths (Carstens et al., 2019). In
2019, there was an outbreak of Salmonella typhimurium associated with
the consumption of small tomatoes in Sweden (Colombe et al., 2019).
Leal-Cervantes et al. (2018) collected cherry tomato samples from four
supermarkets and four local markets in the central region of Mexico and
found that the incidence of Salmonella contamination was 14.1 % in
supermarkets and 7.8 % in local markets. The cost of foodborne illnesses
in the U.S. tops $15.6 billion was estimated by the USDA's Economic
Research Service (USDA-ERS, 2021); the cost of foodborne illnesses
associated with Salmonella are the most expensive and is estimated to be
$3.6 billion annually. Listeria monocytogenes is widely distributed in the
environment and is one of several foodborne pathogens responsible for
outbreaks of foodborne illness. In fact, outbreaks of foodborne disease in
hospitals in the Boston area which were associated with L. mono­
cytogenes were attributed to the consumption of raw tomatoes (Zhu
et al., 2017). Between 2010 and 2017, four multistate listeriosis out­
breaks associated with fresh produce occurred in the U.S. A total of 173
illnesses occurred due to these outbreaks, which led to a 99.4 % known
hospitalization rate and a 21.8 % known death rate (Carstens et al.,
2019). A nationwide outbreak of L. monocytogenes infection associated
with the consumption of packaged salad was also reported in the United
States in 2021 (FDA, 2022). Tomatoes could be contaminated with these
pathogens from contaminated irrigation water, contaminated wash
water, or via the food preparation environment and via animals.
Therefore, there are needs for intervention technologies and techniques
to inactivate the pathogen on tomatoes.
Various nonthermal techniques to eliminate or reduce populations of

* Corresponding author.
E-mail address: tony.jin@usda.gov (T.Z. Jin).

https://doi.org/10.1016/j.ijfoodmicro.2022.109827
Received 16 May 2022; Received in revised form 28 June 2022; Accepted 1 July 2022
Available online 6 July 2022
0168-1605/© 2022 Published by Elsevier B.V.
T.Z. Jin et al.
pathogenic and spoilage microorganisms on fresh produce have been
considered and investigated. Among them, food surface treatments and
coatings with organic acids have been widely studied. Fan et al. (2018)
and Gurtler et al. (2012) used acid washes to reduce populations of
Salmonella enterica on the stem scar area of grape tomatoes. Jin et al.
(2017) developed an edible chitosan-acid coating solution and they
found that the combined treatment of edible coating with sanitizer
washing could extend the shelf life of fresh ginseng roots to 6 months.
The chitosan- acid coating solution reduced Salmonella populations
artificially inoculated on tomato stem scars by 6.0 log CFU and
completely inactivated mold and yeast populations on day 1 with no
growth reoccurrence (Jin and Gurtler, 2012; Mukhopadhyay et al.,
2018).
Recently, essential oils used as antimicrobials have attracted a great
deal of attention from the food industry. Innovations in food packaging
have also been focused on the incorporation of these active additives in
polymer matrices with the purpose of extending the shelf-life of foods
and enhancing food safety. Allyl isothiocyanate (AIT) is a popular
essential oil used for foods and is a main component of mustard oil. It is
permitted for use as a food preservative in Japan and is regarded by the
FDA as a GRAS flavoring agent in the U.S. (Chen et al., 2012). AIT has
been shown to have strong antimicrobial activity in liquid media as well
as in its vapor form. Several studies have shown that AIT can be incor­
porated in antimicrobial packaging to improve the microbiological
safety of fresh produce and extend their shelf life. Chen et al. (2012) and
Mukhopadhyay et al. (2018) investigated the antimicrobial effect of a
chitosan coating+AIT against Salmonella populations artificially inocu­
lated on the surface of whole fresh cantaloupes and tomatoes. Guo et al.
(2018) developed a chitosan-bio-fiber gum-AIT coating solution for
fresh strawberries. All those studies reported that the coatings with AIT
were very effective for the simultaneous reduction of pathogenic and
spoilage microorganisms on produce. However, the unpleasure odor of
AIT from coated food samples was readily noticeable immediately after
coating, although the odor mostly disappeared during the first day
drying after coating treatments (Yun et al., 2015). It is worthy to
compare the antimicrobial effectiveness of these coatings with and
without addition of AIT.
Many previous studies investigated the antimicrobial efficacy of
surface treatments against non-acid-resistant Salmonella strains and non-
pathogenic Listeria innocua. Gorman and Adley (2003) reported that 3.9
% of human Salmonella strains and 2.2 % of animal Salmonella strains
were nalidixic acid resistant. During the last two decades, increased
resistance to nalidixic acid has become a cause of global concern, as
nalidixic acid-resistant strains of Salmonella showing decreased suscep­
tibility to Fluoroquinolones and ciprofloxacin that are used to treat
salmonellosis (Bhagra et al., 2017). In addition, although strains of
L. innocua have been used as surrogates, pathogenic Listeria mono­
cytogenes may have different resistance to acid-coating treatments as
non-pathogenic L. innocua does. However, there were limited literatures
on the use of coating treatments against nalidixic acid-resistant Salmo­
nella strains and pathogenic Listeria strains (L. monocytogenes) on whole
tomatoes. Therefore, the objective of the present study was to investi­
gate the efficacy of acid-chitosan coatings with or without AIT on the
inactivation of a nalidixic acid-resistant Salmonella enterica cocktail
(Salmonella Panama 19,454, Salmonella Poona 953, Salmonella Stanley
H0558) and a L. monocytogenes cocktail (H7762 serotype 4b, H7764
serotype 4b, F4249 serotype 1/2a, and F4561 serotype 1/2a) on the
surface of grape tomato. The reduction of spoilage microorganisms and
impact on food quality after these coating treatments were also evalu­
ated throughout storage for 21-days at 10 ◦ C.
2. Materials and methods
2.1. Materials
Nalidixic acid, levulinic acid, sodium pyruvate, chitosan (low
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International Journal of Food Microbiology 378 (2022) 109827
molecular weight, 75–85 % deacetylation) and allyl isothiocyanate (95
% purity) were purchased from Sigma-Aldrich Chemical Co. (St. Louis,
MO, USA). Palcam agar, plate count agar (PCA), Tryptic soy broth (TSB),
tryptic soy agar (TSA), and peptone water (PW) were purchased from
Difco (Becton Dickinson, Sparks, MD, USA). Dichloran rose bengal
chloramphenicol agar (DRBC) was purchased from Remel Inc. (Lenexa,
KS). Palcam selective supplement was purchased from Oxoid Co.
(Basingstoke, UK). Lactic acid was purchased from Spectrum Chemical
Mfg. Co. (New Brunswick, NJ, USA), and acetic acid was purchased from
Avantor Performance Materials Inc. (Phillipsburg, NJ, USA). Grape to­
matoes were purchased from local supermarkets.
2.2. Preparation of tomato samples
Grape tomatoes were purchased at local grocery stores and stored at
4 ◦ C until used. Tomatoes were divided into two groups. For the
reduction of pathogens, tomatoes were washed in tap water to remove
any debris present and disinfected with 70 % ethanol to remove any
background bacteria present on the surface. For the reduction of natural
microflora and quality evaluation, tomatoes were neither washed nor
disinfected with 70 % ethanol.
2.3. Preparation of inoculum
Preparation of Salmonella inoculum was done according to the
method of Jin and Gurtler (2012). A three-strain cocktail of Salmonella
was used in this study. The three serovars of Salmonella enterica in this
cocktail were Salmonella Panama 19,454, Salmonella Poona 953, and
Salmonella Stanley H0558. These three strains were obtained from the
ERRC culture collection. Selection of these strains was based on their
association with fresh produce related outbreaks and their resistance to
100 ppm of nalidixic acid. These serovars were incubated for 24 h at
37 ◦ C in Tryptic Soy Broth +100 ppm nalidixic acid (TSBN), centrifuged
for 10 min at 1800 ×g, concentrated 10-fold by re-suspending in 10 % of
the original volume with sterile 0.1 % peptone water, and combined in a
single test tube. The inoculum was maintained at 22 to 24 ◦ C and applied
to tomatoes within 1 h of preparation. Four L. monocytogenes strains
(H7762 serotype 4b, H7764 serotype 4b, F4249 serotype 1/2a, and
F4561 serotype 1/2a) were also obtained from the ERRC culture
collection. The strains were propagated on tryptic soy agar at 37 ◦ C and
maintained at 4 ◦ C until use.
2.4. Procedure for inoculation
Disinfected tomatoes were held at 21 ◦ C for 2 h immediately pre­
ceding inoculation with Salmonella and L. monocytogenes. 100 μl of the
inoculum prepared above was transferred to a jar containing 50 ml of
0.1 % sterile Peptone Water. The diluted inoculum was placed on a
stirrer for one minute to allow for the inoculum to mix throughout. The
jar was removed from the stirrer after one minute and each grape tomato
was dip-inoculated in the diluted inoculum with gentle shaking of the jar
for one minute. Each inoculated grape tomato was dried under the hood
for two hours to allow for the cells to adhere to the surface before the
coating treatments.
2.5. Preparation of coating solutions, coating treatments and sample
storage
The two coating solutions used in this experiment were prepared as
previously described by Jin and Gurtler (2012) with some modifications.
Coating solution 1 was prepared by adding 9 g of chitosan powder to
1000 ml of an acid solution containing 2 % acetic, lactic and levulinic
acids. This solution was stirred with a magnetic stir bar on a stir plate
until the polymer was completely dissolved. Coating solution 2 consisted
of 9 g chitosan powder, 1000 ml of the abovementioned three-acid so­
lution, and 20 ml of AIT.
T.Z. Jin et al.
Tomato samples were dipped in the abovementioned coating solu­
tions for one minute. Coated tomatoes were allowed to dry under the
biosafety hood for 4 h before being placed in a PET clam shell container
(2.0 × 16.5 × 13.5 cm3) (Dart Container Corporation, Mason, MI, USA).
Each container contained 30 tomatoes and each treatment had three
containers. All containers were stored at 10 ± 2 ◦ C for 21 days. Non-
coated tomatoes served as the controls.
2.6. Microbiological analysis
On each sampling day, two tomatoes were randomly selected from
each box and were placed in an individual Whirl-Pak bag containing 10
ml D/E Neutralizing Broth. Tomatoes were hand-massaged for thirty
seconds to facilitate the release of remaining microbial cells from the
surface of each grape tomato. Macerated samples in filter bags were then
pummeled in a stomacher for 2 min. A 1 ml sample of each homogenate
was diluted up to five times in 9 ml of 0.1 % Peptone Water. To deter­
mine surviving Salmonella populations, 0.1 ml of each dilution was
spread-plated in duplicate onto Tryptic Soy Agar +100 ppm nalidixic
acid +0.1 % sodium pyruvate (TSAPN) to assist in the recovery of
injured cells. All plates were incubated at 37 ◦ C for 24 h before colonies
were counted. To determine surviving L. monocytogenes populations, the
samples were serially diluted in BPB (10-fold dilutions), and 1 ml of the
appropriate dilutions sample were spread-plated using Listeria-specific
Palcam Agar with Palcam selective supplement. Three 1-ml aliquots
were plated per dilution. The plates were then incubated for approxi­
mately 48 h at 37 ◦ C before enumeration. Experiments were repeated 3
times for each treatment.
To determine surviving populations of native microflora, total aer­
obes were enumerated by plating on plate count agar (PCA) with incu­
bation at 35 ◦ C for 24–48 h, and mold and yeast were enumerated on
dichloran rose bengal chloramphenicol agar (DRBC) at 25 ◦ C for 5 days.
Experiments were repeated 3 times for each treatment.
2.7. Quality analysis
Non-inoculated tomatoes were subject to coating treatment as
described previously and used for quality analyses. Each container
contained 30 tomatoes and each treatment had three containers. All
containers were stored at 10 ± 2 ◦ C for 21 days. Changes in weight loss,
firmness, and color of tomatoes after coating treatments and during
storage were determined.
2.7.1. Weight loss
Weight of tomato samples in an individual container (three con­
tainers per treatment) was recorded on Day 0 and after 21 days' storage.
Cumulative weight losses were expressed as percentage loss of original
weight. Data from three containers were averaged.
2.7.2. Firmness measurements
Firmness was evaluated using a TA-XT2i Texture Analyzer (Texture
Technologies Corp., Scarsdale, NY, USA). A 3-mm diameter probe was
used to penetrate the center of the surface of each grape tomato to a
depth of 10 mm at a speed of 10 mm/s. Three tomatoes from each of 3
replicated containers were randomly selected for firmness measure­
ments, and there was a total of 9 measurements per treatment.
Maximum force (Newton) was recorded using the Texture Expert soft­
ware, Version 1.22 (Texture Technologies Corp).
2.7.3. Color analysis
The color of grape tomato surfaces was measured with a Hunter
UltraScan® VIS colorimeter (Hunter Associates Lab, Reston, VA, USA)
using a 1.3 cm measuring aperture. D65/10◦ was used as the illuminant-
viewing geometry. The colorimeter was calibrated using the standard
black and white plates. Measurements were made at the middle point of
the surface of tomato. Three tomatoes from each of 3 replicated
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International Journal of Food Microbiology 378 (2022) 109827
containers were randomly selected for color measurements, and there
was a total of 9 measurements per treatment. The color parameters L*,
a*, and b* were recorded.
2.8. Statistical analysis
All experiments were replicated two times using separate prepara­
tions of bacteria and batches of fresh produce conducted on different
days. Data were pooled and analyzed using Analysis of Variance with
SAS version 9.1 software (SAS Institute, Cary, NC). Duncan's multiple
range test was used to determine the significant differences of mean
values. Significance was defined at p < 0.05.
3. Results
3.1. Antimicrobial activity
Fig. 1 shows populations of Salmonella on the tomato surface
throughout storage at 10 ◦ C for 21 days. The initial Salmonella popula­
tion on the surface of all samples before treatments was 3.65 log CFU/
tomato. Salmonella populations in the control tomatoes and in tomatoes
subject to treatment with chitosan were reduced to 3.51 and 1.28 log
CFU/tomato, respectively, on day 1. Salmonella populations in tomatoes
subject to treatment with Chitosan + AIT were reduced to undetectable
levels (< 0.70 log CFU/tomato) on Day 1. Both coating treatments
reduced Salmonella populations to undetectable levels (< 0.70 log CFU/
tomato) from Day 2 through Day 21. Salmonella populations in the
Control tomatoes decreased to 2.15 log units after 21 days.
Changes in the population of L. monocytogenes on the surface of grape
tomato throughout storage are presented in Fig. 2. The initial L. mono­
cytogenes population on the tomato surface was around 3.0 log CFU/
tomato. The L. monocytogenes populations on the controls increased to
approximately 9 log after 1 days while the L. monocytogenes populations
were reduced to undetectable levels in samples which were subject to
coating treatment after 6 days. Hence, monitoring L. monocytogenes
populations was discontinued.
Changes in the population of native microflora on the surface of
tomato throughout storage are presented in Fig. 3. TPC population in
control samples increased slightly throughout storage, reaching a
maximum level of 4.91 log CFU/tomato after 21 days. Both the Chitosan
and Chitosan + AIT coating treatments reduced these populations from
3.01 to 1.22 and 1.12 log units, respectively, on Day 1 (Fig. 3A). Both
coating treatments reduced these populations to an undetectable level
after 2 days. Both coating treatments reduced the populations of mold
and yeast from 3.14 to an undetectable level after 1 day while the
populations on Control increased to 5.45 log units after 21 days
(Fig. 3B). The images shown in Fig. 5 clearly indicate that the tomatoes
in the control group were moldy after 21 days.
The abovementioned chitosan-3 acid coating solution was used to
treat tomatoes. Jin and Gurtler (2012) and Mukhopadhyay et al. (2018)
investigated the efficacy of these antimicrobial coatings for the inacti­
vation of Salmonella on the surface of tomato stem scars. The chitosan-3
acid coating solution was applied to the stem scar area of inoculated
tomatoes. They found that this coating solution reduced Salmonella
populations by 6.0 log CFU per stem scar. The treatment also completely
inactivated mold and yeast populations on day 1 with no growth
reoccurrence.
3.2. Quality evaluation
Table 1 presents the color change of tomatoes during storage at
10 ◦ C. The L* values indicate the brightness/darkness of the tomato
surface. The a* and b* values indicate the red/green bias and the yel­
low/blue deviation of the tomato surface, respectively. During storage,
control samples had significantly (p < 0.05) lower L* and b* values than
coated samples (Table 1). The a* values of the coated samples were
T.Z. Jin et al. International Journal of Food Microbiology 378 (2022) 109827

4.5
A A A
4.0 A
A A
3.5
A
3.0 A
A
Log CFU/tomato

2.5

2.0
B
1.5

1.0
C B B B B B B B B B B

0.5

0.0
0 1 2 6 9 14 21
Storage me (day)

Control Chitosan Chitosan+AIT

Fig. 1. Survival of Salmonella during storage at 10 ◦ C after two coating treatments. Bars with different letters on the same day are significantly different (p < 0.05).

11.0

10.0 A A A

9.0

8.0

7.0
Log CFU/tomato

6.0

5.0

4.0 A A A

3.0 B
C
B B
2.0

1.0 B B

0.0
0 1 2 6
Storage me (day)

Control Chitosan Chitosan+AIT

Fig. 2. Survival of L. monocytogenes during storage at 10 ◦ C after two coating treatments. Bars with different letters on the same day are significantly different (p
< 0.05).

4
T.Z. Jin et al. International Journal of Food Microbiology 378 (2022) 109827

6 A
A
5

4 A
A
A A
A A A A
Log CFU/tomato

2
B C

1 B B B B B B B B B B

0
0 1 2 6 9 14 21
Storage me (day)

Control Chitosan Chitosan+AIT

6 B A

A
4 A
A A A A A
A
Log CFU/tomato

1 B B B B B B B B B B B B

0
0 1 2 6 9 14 21
Storage me (day)
Control Chitosan Chitosan+AIT

Fig. 3. Survival of aerobic bacteria (A) and Mold & Yeast during storage at 10 ◦ C after two coating treatments. Bars with different letters on the same day are
significantly different (p < 0.05).

similar to that of the controls. The images shown in Fig. 5 indicate that
the treated tomatoes had a slightly yellow color. There were no signif­
icant differences in color between two coating treatments. After 21 days
of storage, the a*/b* values were higher in the control samples (1.29),
whereas the a*/b* values of treated samples were around 1.0. The re­
sults indicated the tomatoes which were subject to coating treatment
could maintain their color after 21-day storage. The USDA has recom­
mended that the color values at the range of red stage should be from
0.95 to 1.21 (Batu, 2004).
The results shown in Fig. 4 indicate that the firmness of tomato
samples with skin fluctuated around 6.35 N throughout storage, and
samples treated with chitosan+AIT had slightly lower firmness than the
other two samples after 21 days. The firmness of tomato flesh was
maintained at around 120 g throughout storage.
Water is the largest component of tomato fruit and maintains the cell
turgor of fruit during growth and postharvest storage. Our results (data
not presented) show that the control group presented the highest weight
loss (1.75 %), whereas samples coated with chitosan+AIT and with
chitosan had 1.62 % and 1.58 % weight loss, respectively. The chitosan
coating on the surface of the grape tomato likely provided a moisture
barrier that resulted in delayed migration of moisture from fruit to the
environment.
Similar to the observation reported by Yun et al. (2015), the odor of
AIT from coated food samples was noticeable immediately after coating,

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T.Z. Jin et al. International Journal of Food Microbiology 378 (2022) 109827

Table 1
Color changes of tomatoes during storage at 10 ◦ C.
Parameters Sample Storage time (day)

0 1 2 6 9 14 21

L* Control 34.09 ± 0.98a 34.09 ± 0.99b 34.50 ± 1.09b 33.77 ± 0.91b 33.27 ± 1.07b 33.13 ± 1.14b 33.00 ± 1.76b
Chitosan 34.09 ± 0.98a 36.44 ± 1.02a 36.32 ± 0.66a 35.94 ± 0.96a 35.85 ± 1.70a 36.28 ± 0.99a 35.89 ± 1.09a
Chitosan+AIT 34.09 ± 0.98a 36.38 ± 1.59a 36.12 ± 1.07a 35.56 ± 0.91a 35.11 ± 0.49a 35.93 ± 1.00a 35.57 ± 0.76a
a* Control 20.58 ± 2.60a 20.58 ± 2.61a 21.19 ± 2.02a 21.36 ± 1.12a 21.91 ± 2.15a 22.31 ± 1.99a 21.99 ± 2.14a
Chitosan 20.58 ± 2.60a 17.77 ± 2.91b 17.15 ± 1.66b 18.22 ± 1.67b 18.26 ± 2.47b 21.16 ± 1.78a 20.20 ± 2.31a
Chitosan+AIT 20.58 ± 2.60a 17.19 ± 2.89b 17.19 ± 1.52b 18.39 ± 2.14b 20.03 ± 1.73a 19.47 ± 1.94b 20.79 ± 1.06a
b* Control 18.39 ± 1.51a 18.39 ± 1.52b 19.00 ± 1.87a 18.01 ± 1.45b 18.22 ± 2.25a 18.07 ± 2.45b 16.96 ± 2.86b
Chitosan 18.39 ± 1.51a 20.42 ± 2.16a 19.10 ± 1.45a 21.12 ± 1.42a 20.93 ± 1.82a 22.93 ± 2.33a 19.61 ± 2.21a
Chitosan+AIT 18.39 ± 1.51a 20.79 ± 2.71a 20.20 ± 2.01a 20.33 ± 1.93a 19.01 ± 1.18a 22.09 ± 1.22a 20.79 ± 0.71a
a*/b* Control 1.11 ± 0.07a 1.11 ± 0.08a 1.11 ± 0.07a 1.18 ± 0.05a 1.20 ± 0.09a 1.23 ± 0.09a 1.29 ± 0.12a
Chitosan 1.11 ± 0.07a 0.87 ± 0.11b 0.90 ± 0.06b 0.86 ± 0.11b 0.87 ± 0.18b 0.92 ± 0.09b 1.03 ± 0.09b
Chitosan+AIT 1.11 ± 0.07a 0.82 ± 0.09b 0.86 ± 0.08b 0.90 ± 0.06b 1.05 ± 0.08b 0.88 ± 0.10b 1.00 ± 0.05b

The different letter in the same column represents the significance difference (p < 0.05).

Skin and Fresh Firmness

800

700

600

500
Force (g)

400

300

200

100

0
0 1 2 6 9 14 21
Storage days

Skin Control Skin Chitosan Skin Chitosan+AIT

Flesh Control Flesh Chitosan Flesh Chitosan+AIT

Fig. 4. Changes in texture firmness of tomatoes during storage at 10 ◦ C after two coating treatments.

then disappeared during the first day after coating treatments.
4. Discussion
Chitosan is nontoxic, can be dissolved in weak acid solution, and has
excellent film-forming properties with some antimicrobial activity.
Organic acids in the coating solutions did not only help to dissolve the
chitosan powder but also enhanced its antimicrobial activities when
released from the coating. Organic acids are known to exhibit general
antimicrobial activities against a wide range of foodborne pathogens
and spoilage microorganisms and have a long history of being used as
food additives and preservatives in preventing food deterioration and
extending the shelf life of perishable foods. Organic acids can be added
directly or indirectly to the food products. Several studies have shown
that 1–2 % of three organic acids (acetic, lactic, and levulinic acids) can
be used to produce antimicrobial chitosan films and coatings which
could be potentially used as antimicrobial packaging (Guo et al., 2015;
Jin and Gurtler, 2012; Jin et al., 2020; Wang et al., 2015). Organic acids
inactivate microorganisms via several mechanisms, one of which in­
volves the diffusion of undissociated forms of acids into the cells via the
cytoplasmic membrane. The alkaline environment of the cytoplasm
causes the acids to become ionized, leading to the release of hydrogen
ions. The released hydrogen ions create an acidic pH environment
within the cell and cause structural deformation to proteins, DNA
structures, and the extracellular membrane. This results in significant
damage to the normal enzymatic activities of the cell (Mani-Lopez et al.,
2012).
For produce surface treatments, organic acids can be used in washing
(Fan et al., 2018; Jin and Gurtler, 2012) or incorporated in chitosan
coating (Chen et al., 2012). Both methods show antimicrobial efficacy
for tomatoes. However, compared to the acid wash method, chitosan-
acid coating provides additional benefits due to the formation of film
on the food surface. The films reduced the amount of water and acids
used to sanitize tomatoes, reduced water loss in tomatoes, and prevented

6
T.Z. Jin et al.
Fig. 5. Appearance of grape tomatoes after 21-day storage at 10 ◦ C. A: Control,
B: Chitosan C: Chitosan+AIT.
recontamination of tomatoes during the transportation, storage, and
retail phases (Chen et al., 2012).
Salmonella can adapt to and survive acidification from organic acids.
This is due to the presence of lipopolysaccharides in the outer membrane
as well as other mechanisms (Ricke, 2003; Dittoe et al., 2019). Over 1
million cases of non-typhoidal S. enterica occur each year in the U.S., of
which an estimated 27.2 % result in hospitalizations (Hoffmann et al.,
2012). Ciprofloxacin is frequently used for the treatment of
7
International Journal of Food Microbiology 378 (2022) 109827
salmonellosis. There have been reports of treatments failure with cip­
rofloxacin, when Salmonella species showed nalidixic acid resistance
(Hwang et al., 2010). The significant reduction in population of the
nalidixic acid-resistant Salmonella cocktail on the surface of tomatoes
(Fig. 1) indicates that the coating treatments could reduce the chance of
outbreaks and also the risk of antibiotic-treatment failure. Furthermore,
the coating treatments effectively reduced the populations of L. mono­
cytogenes on the surface of tomatoes (Fig. 2), which enhanced the food
safety.
AIT is a broad-spectrum antimicrobial agent. In the present study,
chitosan-AIT reduced populations of pathogens and spoilage microor­
ganisms effectively. The addition of AIT to chitosan-acid coating was
able to achieve higher reductions in populations of Salmonella, Listeria
and TPC after 1 day storage. However, there were no differences be­
tween the two treatments in microbial reductions after 2 days (Figs. 1, 2,
and 3). The results from the present study suggest that it may not be
necessary to add AIT in chitosan-acid coating for tomatoes. In our pre­
vious study (Jin and Gurtler, 2012), acid-chitosan coating treatment
reduced the populations of nalidixic acid-resistant Salmonella from 7.7
log CFU to 2.2 log CFU per stem scar after 1 day and then reduced the
pathogen populations to undetectable levels at all storage times longer
than 1 day. Other study (Mukhopadhyay et al., 2018) also showed a
chitosan-acid coating treatment was able to reduce populations of non-
nalidixic acid-resistant Salmonella on tomato stem scars by up to 6 log
CFU/g. Future studies should investigate the synergistic effect of lower
concentrations of the three acids and AIT on higher population levels of
Salmonella artificially inoculated on the tomato surface.
The chitosan-acid coatings did not only effectively reduce pop­
ulations of the pathogens, but also maintained the quality of tomatoes
throughout storage at 10 ◦ C. Color, texture and water loss are important
indices of the quality of fresh produce, such as tomatoes. The USDA
recommends that a*/b* values for tomatoes should be in the range of
0.95–1.21 at the red stage (Batu, 2004). In the present study, there were
no significant (p > 0.05) differences in the a*/b* values among all the
treatments (Table 1) after 21 days of storage. The a*/b* values of all the
samples were between 0.95 and 1.05, except for the control after 21 days
(0.91). Our results are in agreement with those obtained by Fan et al.
(2018). The thick cuticle layer protects them from the effects of chem­
icals, especially acids. In addition, the firmness of grape tomatoes
treated with both coatings was maintained throughout the 21 day-
storage period and had lower rates of water loss.
In conclusion, two coating treatments effectively reduced the pop­
ulations of nalidixic acid-resistant Salmonella, L. monocytogenes, and
native microorganisms on the surface of whole grape tomatoes. The
coating treatments did not cause quality deterioration on tomatoes
during 21 days of storage at 10 ◦ C. Incorporation of AIT in chitosan-acid
coating may not be necessary for pathogenic contaminants present at
low levels on the whole surface of grape tomatoes. Therefore, the
chitosan-acids coating can be applied to enhance the microbial safety of
tomatoes while maintaining their quality.
Declaration of competing interest
We, the authors declares no conflict of interest for this research work.
Acknowledgement
The authors wish to thank Wenxuan Chen of Zhejiang Academy of
Agricultural Science, China and Anita Parameswaran for their excellent
technical assistance. This study was also funded by the USDA-ARS CRIS
project 8072-41420-026-00D through ARS National Program 108.
Mention of trade names or commercial products in this publication is
solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the U.S. Department of
Agriculture (USDA). The USDA is an equal opportunity provider and
employer.
T.Z. Jin et al. International Journal of Food Microbiology 378 (2022) 109827

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