Assessment of the
Impact of Rosemary Chitosan Microemulsion Effect on Escherichia coli and
Listeria monocytogenes Dipping in Chicken Meat Stored at 4˚C
Dalia Y. Youssef1†,
Nahed S.A. El-Shayeb2† and Dalia M.A. El-Masry3*†
1Department Food Hygiene, Animal Health
Research Institute (AHRI), Agricultural Research Center (ARC), Egypt
2Medicinal and Aromatic Plants Department,
Horticulture Research Institute, ARC, Dokki, Giza, Egypt
3Nanomaterials Research and Synthesis Unit,
Animal Health Research Institute, ARC, Dokki, Giza, Egypt
*For correspondence: dr_daliaelmasry@yahoo.com
†Contributed
equally to this work and are co-first authors
Received 02 September 2021;
Accepted 12 December 2021; Published 30 January 2022
Abstract
This article studies the potential use of the Rosemarry
(REO) microemulsion with/without chitosan to assess the growth inhibition of
Gram-positive and Gram-negative pathogenic bacteria represented by Listeria
monocytogenes and Escherichia coli. The chicken samples were divided
into four groups after inoculation of E. coli and L. monocytogenes
separately: control group (without treatment), 1% chitosan nanoparticle
treatment group, 0.5% REO microemulsion treatment group, and 0.5% REO + 1%
chitosan microemulsion treatment group. Chicken samples were dipped into the
treatments for 15 days of refrigerated storage at 4°C. All groups were kept
refrigerated, and bacterial counts were taken on days 0, 1, 3, 6, 9, 12 and 15.
Chitosan nanoparticle and REO microemulsion with/without chitosan were
spherical shape and showed a narrow size distribution of
23.98 ± 0.83, 34.24 ± 2.2 and
28.01 ± 1.36 nm with a polydispersity index (PDI) of 0.86,
0.33 and 0.54, respectively indicating that greater homogeneity was achieved.
REO chitosan microemulsion has 12 components detected by GC-Mass as follows:
pinene (22.21%), borneol (21.32%), 1-(4-methoxyphenyl) ethanoneoxime (3.48%),
oxocamphor (0.49%), camphor (2.99%), limonene (0.29%), cis-linalool oxide (0.87),
2-(5-chloro-methoxyphenyl) pyrrole (2.19%), homofarnesol (0.27%), levoverbenone
(0.45%), peruviol (0.73%) and campesterol (1.22%). The results showed a
substantial reduction in L. monocytogenes and E. coli count in
all treatment groups when compared to the control group, with the greatest
inhibitory efficacy in the 0.5% REO chitosan microemulsion group. A favorable
effect of chitosan treatment on chicken acceptability during refrigerated
storage was reported, with an improvement in the sensory qualities of the products.
Thus, REO chitosan microemulsion is advised to be used in chicken to enhance
resistance to harmful microorganisms.
© 2022 Friends Science Publishers
Keywords: Fillet; L. monocytogenes;
E. coli; Chitosan; Rosemary; Microemulsion; GC-Mass
Introduction
The chicken business is growing worldwide as chicken
remains one of the least expensive protein sources and white meat is considered healthier than red meat (Souza et al. 2018).
Poultry and poultry products
are among the foodstuffs that must be produced and stored safely and
sanitarily. Because of poor sanitary processing and storage procedures,
microbial contamination occurs, leading to safety and spoiling issues. Spoiled
chicken meat poses an economic hardship for farmers and necessitates new
techniques to increase shelf life and overall meat safety/quality, which is the
primary issue facing the poultry processing business (Petrou et al.
2012).
Food safety is one of the most
critical concerns in the food business. Therefore, new procedures and technologies
are being developed in the food industry to improve the quality and safety of
poultry meat. In the past few years, consumer demand for nutritious food devoid
of chemical preservatives has grown considerably (Petrou et al. 2012).
Accordingly, there is a growing trend toward using natural ingredients derived from plants and
animals, including antimicrobials, oxidants, coloring materials, and sweeteners (Mehdizadeh et al. 2020).
Listeria monocytogenes is a Gram-positive foodborne bacterium that results in serious food
safety problems, particularly in meat and poultry products (Malhotra et al.
2015), because of its capacity to survive and thrive at refrigeration
temperatures. Food surface treatments are crucial for food safety and quality
(FAO 2013). However, as people become more aware of the possible dangers of
synthetic preservatives, using a combination of natural antimicrobials and
antioxidants has received great attention. Escherichia coli have been
identified as a foodborne pathogen since 1982, which has greatly influenced the
food sector (McClure et al.
2000).
Rosemary plant and REO are
widely used as they are safe with no caveats. It is used as a flavoring agent
and prevents microbial growth and rancidity development in meat through the main
active compounds, such as rosmarinic acid and carnosic acid (Jongberg et al.
2013; JSMO 2016).
Rosemary oleoresin, extract,
and essential oils have been identified as possible antioxidants, which are
frequently utilized in the food sector (Hussain et al. 2010). The rosemary extract effectively delayed
lipid oxidation in meat. Furthermore, REO has potent antibacterial action
against Gram-negative and Gram-positive bacteria such as Staphylococcus
aureus, Bacillus cereus, Escherichia coli and Listeria monocytogenes (Keokamnerd
et al. 2008; Kahraman et al. 2015).
Chitosan is one of the natural
additives originating from animals with a wide range of applications in food
bio-preservation due to its biodegradability, biocompatibility, nontoxicity,
and antibacterial activity (No et al.
2007; Paparella et al. 2011; Grande-Tovar et al. 2018). Edible coatings and films have attracted further
attention because of their ability to carry food additives, including
antimicrobials, antioxidants, flavors, and colors and preserve the functionality
of such agents on the food surface (Ricci et
al. 2018).
Microemulsions are colloid
solutions and thermodynamically stable, single optically isotropic dispersions
composed of a water phase, oil phase, surfactant, and cosurfactant, with a
droplet size of 10–100 nm (Zhang et al. 2015). Microemulsions
can be specifically used in food products because of their unique features,
including ease of preparation, high-grade functions, and fine particle size;
these advantages facilitate the transfer of active compounds and enhance their
interactions with biomembranes (Moghimi et al. 2016). The work aimed to evaluate
the effect of REO and its microemulsion with/without chitosan on inhibiting the
growth of the pathogenic E. coli and L. monocytogenes inoculated
in chicken fillet stored for 15 days at 4˚C.
Materials and Methods
Preparation and extraction of REO
The fresh herb of Rosmarinus officinalis was
brought from the experimental farm at the Medicinal and Aromatic Plants
Research Department in Gezert El-Shaeer, El Qanatir El Khayriyah, Egypt. The
essential oil was extracted using the water-steam distillation method
(Cliventer system) on the whole fresh herb samples for 3 h in the Medicinal and
Aromatic Plants Lab, Dokki, Giza, according to Miller method (1963).
Preparation and Treatments of Chitosan Nanoparticles
The 1% chitosan nanoparticle was spontaneously
obtained upon adding 1 and 0.5% acetic acid chitosan acidic solutions,
respectively, to 0.7 mg/mL solutions of TPP aqueous basic solution. The ratio
of TTP to chitosan was 1:3 under magnetic stirring at room temperature for 1 h
according to Youssef and El-Masry (2018).
Preparation of Rosemary Microemulsion with or without
Chitosan
Rosemary oil (0.5%) was mixed with tween 80 (4.5%)
as an emulsifier and was stirred for 30 min. The 1% chitosan solution was added
to form and obtain a uniform, stable and clear emulsion, according to Rao and
McClements (2011). A 1% sodium chloride solution is used to
replace chitosan in the other treatment.
Characterization of Chitosan Nanoparticle and
Microemulsion
Zetasizer Malvern Instrument (Corp, Malvern, UK) was
used to measure surface charge (zeta potential), droplet size, size
distribution (polydispersity indexes) and electrical conductivity of the
microemulsion. High-resolution transmission electron microscopy (HRTEM)
observations were performed using a JEM 1400F HRTEM at beam energy of 300 keV.
The examined samples
determined using direct capillary column (30 m x 0.25 mm
x 0.25 µm film thickness) of Trace GC-TSQ mass spectrometer (Thermo
Scientific, Austin, TX, USA) for chemical composition. The components were
identified by comparing their mass spectra with those of WILEY 09 and NIST 14
mass spectral database according to El-Kareem et al. (2016).
Cell Culture
Green monkey cell line (Vero cell) purchasing from
Nawah Scientific, Inc. (Cairo, Egypt), was maintained in media supplemented
with 10% of fetal bovine heat-inactivated serum, streptomycin (100 mg/mL) and
penicillin (100 units/mL) at 37°C, 5% humidity and CO2 atmosphere.
Cytotoxicity and cell viability assays were performed using the SRB assay at
different solution concentrations (0.01, 0.1, 1, 10 and 100 µg/mL), according to Allam et al.
(2018).
Preparation of Microbial Inoculums
Strains from the Animal Health Research Institute of
E. coli (ATCC 8739) and L. monocytogens (ATCC 14028) were
acquired (Food Hygiene Department). Frozen crops were kept at −80°C and
activated in 9 mL of trypticase soy broth (TSB) during incubation at 35°C for
24 h with two successive passes. For individual strains, 1 mL of the inoculum
was introduced to 100 mL (TSB) of stock and the Oxoid Incubator Shaker was used
to obtain an estimated concentration of about 108 CFU/mL as
measured by a 0.5 McFarland standard using plating serial dilutions on ALOA and
TBX agar. Two serial dilutions of 1 mL of this inoculum have been added to a 9 mL
sterile saline to obtain the final concentration of around 106 CFU/g.
Dipping of chicken samples
Fresh chicken fillets (10 kg) from a market shop in
Cairo, Egypt, without skin were purchased and immediately brought to the
refrigerated lab. Then, they were divided into four groups of duplicates and
placed under running tap water for two minutes to remove foreign bodies or remains
or foreign bodies.
The chicken fillets were contaminated
with inoculated soaking solution for one minute and then with the L.
monocytogenes and E. coli strains for 20 min and were dried in
a laminar airflow (four groups for each strain) (Olaimat and Holley 2015).
Preparation of treatments
The previously dipped samples were treated using
three different immersing solutions: (1) 1% chitosan nanoparticles; (2) 0.5%
REO microemulsion; (3) 0.5% REO + 1% chitosan microemulsion for 1 min. Then,
the samples for drained for 15 min and stored at 4°C for 16 days. Finally, the
analysis was performed on days 0, 1, 3, 6, 9,12 and 15 of refrigerated storage (Sharifi et
al. 2017). Two groups were
assigned as positive controls for L. monocytogenes and E. coli (106
CFU/mL).
Enumeration of E. coli
With 0.1% sterile peptone water, chicken fillet
samples (10 g) were brought to a final volume of 90 mL. A stomacher was
used to homogenize the materials for 2 min. (Seward Medical, London). Following
the production of decimal dilutions, 1 mL of successive homogenate dilutions was cultivated onto TBX agar and incubated at 35°C for 24 h.
Enumeration of L. monocytogenes
25 grams of immunized fillets was stomached in 225 mL
of Listeria broth and serially diluted using maximal recovery to be counted on
selective medium (ALOA agar). Moreover, 1 mL of serial dilutions of homogenates
was cultivated onto duplicate plates and incubated at 35°C for 24 h.
All fillets were sealed in
plastic bags and stored on a refrigerator shelf for future examination. Each
inoculation group was regularly inspected for the inoculated strain count as
detect in the primary count at days 0, 1, 3, 6, 9, 12 and 15 of refrigerated
storage to assess the influence of the treatments on the viability of the
injected bacterial strains.
Sensory evaluation
For studying the effect of rosemary with/without chitosan
microemulsion on the sensory attributes of chicken fillets, four groups of
chicken fillet were prepared by dipping in solutions as follows: one group of
chicken fillet with 1% chitosan nanoparticles, 0.5% REO + 1%
chitosan microemulsion and 0.5% REO microemulsion, the last group without
treatment.
After 15 min of group treated
and then it was permitted to dry for another 15 min in the laminar flow before
being kept in the refrigerator at 4°C.
All samples of cooked chicken
fillets were organoleptically evaluated by seven panelists from the staff
members, according to Petrou et al. (2012). Only edible chicken
fillets from the control and treatment groups were cooked for 5 min in a
microwave oven set to high power (700 W). Using a nine-point
hedonic scale, seven panelists were asked to rate the
acceptability (total sensory assessment score) in terms of odor,
taste, and sight: 9, excellent; 8, very good; 7, good; 6, bad (initial
off-odor, off-taste development). A score of 6 was chosen as the bottom limit
of acceptability. After the emergence of the first off-odor or undesirable
color, the sample was deemed unsuitable.
Statistical analysis
Each test was done thrice, and standard deviation
mean value (SD) was given for each occurrence. All data were analyzed using
ANOVA on a single-way basis and mean separation was performed using Tukey’s
multiscope test (SPSS 19.0). Differences at P 0.05 level were considered
significant.
Results
Characterization of chitosan nanoparticle and REO microemulsion
(with or without coating chitosan)
Particle Size, Morphology, and Size Distribution. TEM was used to determine the size and morphology of the nanoparticles.
Three nanomaterials were spherical and showed no aggregation and narrow size
distribution of 23.98 ± 0.83, 34.24 ± 2.2 and
28.01 ± 1.36 nm (Fig. 1a–c) with a polydispersity index (PDI) of
0.86, 0.33 and 0.54, respectively, indicating that greater homogeneity can be
realized.
The zeta potential, which
indicates unstable and stable suspensions, is often measured via dynamic light scattering (DLS). The
zeta potential results for chitosan nanoparticle, rosemary chitosan
microemulsion, and rosemary microemulsion were
53.5 ± 5.14 mV, 9.69 ± 3.67 mV,
43.3 ± 6.23 mV, respectively, measured at pH 5.
The analysis of the rosemary
oil using GC-Mass showed the presence of terpineol (6.29%), camphor (37.82%),
isoborneol (25.96%), levoverbenone (17.92%), citronellol (0.90%), isopulegol
(1.53%), bornyl acetate (2.68%), sobrerol 8-acetate (1.09%), and caryophyllene oxide
(2.46%). On the other hand, rosemary chitosan microemulsion had 12 componentsL
1-(4-methoxyphenyl) ethanoneoxime (3.48%), oxocamphor (0.49%), α-pinene
(22.21%), camphor (2.99%), limonene (0.29%), borneol (21.32%), cis-linalool
oxide (0.87), 2-(5-chloro-methoxyphenyl) pyrrole (2.19%), homofarnesol (0.27%),
levoverbenone (0.45%), peruviol (0.73%) and campesterol (1.22%).
Fig. 1: TEM of (A) chitosan
nanoparticle and (B): rosemary
chitosan microemulsion and (C)
rosemary microemulsion
Fig. 2: Cell viability % of (A)
chitosan nanoparticle and (B):
rosemary chitosan microemulsion and (C)
rosemary microemulsion
On the confluent surface of
Vero cells, chitosan nanoparticles and rosemary with or without chitosan
microemulsion had different concentrations (0.01, 0.1, 1, 10, and 100 ug/mL)
after 3 days of inoculation. The cell viability% assessed by SRB assay was
87.43%, 80.69%, and 79.66%, respectively, in 100 µg/mL and IC50 > 100 µg/mL (Fig. 2a–c).
Inhibitory effect of different treatments on L.
monocytogenes and E. coli
Table 1 and Fig. 3 show the effectiveness of
different treatments on the behavior of L. monocytogenes during
refrigerated storage of chicken fillets samples. By comparing the treated
samples with the samples inoculated with the strains in the absence of
treatment (positive control) at zero days, the initial count of L
monocytogenes was 5.65 ± 0.55 log CFU/g. At 1 day after treatment,
the counts of the pathogens in treated samples with 0.5% REO microemulsion, 1%
chitosan nanoparticle, and 0.5% REO + 1% chitosan microemulsion
were reduced compared to control, whereas, in the control sample, the count
remained 5.77 ± 0.16 log CFU/g.
The count of treated chicken fillet
was reduced to 5.62 ± 0.34, 4.71 ± 0.27 and
4.18 ± 0.74 log CFU/g, respectively. Significant differences (P < 0.05)
were observed between means having different letters in the same row between
the three groups. Dipping chicken fillets into 0.5% REO microemulsion, 1%
chitosan nanoparticle, and 0.5% REO + 1% chitosan microemulsion
reduced the L. monocytogenes count to about 1.7, 2 and 3 log CFU/g,
respectively, during refrigerated storage up to 15 days. Chitosan coatings are
commonly mixed with essential oils and created in the form of microemulsions
during refrigerated storage at 4°C to intensify the impact of chitosan against
foodborne bacteria.
Table 1: Listeria monocytogenes count (Log CFU/g) of inoculated chicken
fillet stored at 4°C (Mean ± SD)
Storage days |
Control |
0.5% REO micro emulsion |
1% Chitosan nanoparticle |
0.5% REO + 1% Chitosan micro
emulsion |
Zero day |
6.17 a ± 0.72 |
5.58 a ± 0.25 |
5.51 a ± 0.44 |
5.83 a ± 0.29 |
1st day |
5.77 a ± 0.16 |
5.62 a ± 0.34 |
4.71 b ± 0.27 |
4.18 b ± 0.74 |
3rd day |
5.68 a ± 0.11 |
4.52 b ± 0.12 |
4.20 bc
± 0.69 |
3.84 c ± 0.11 |
6th day |
6.08 a ± 0.81 |
4.48 b ± 0.52 |
3.46 c ± 0.33 |
3.55 bc
± 0.42 |
9th day |
6.76 a ± 0.21 |
3.84 b ± 0.09 |
3.65 b ± 0.60 |
3.30 b ± 0.61 |
12th day |
6.81 a ± 0.06 |
3.54 b ± 0.21 |
3.04 b ± 0.92 |
3.27 b ± 0.63 |
15th day |
7.48 a ± 0.52 |
3.89 b ± 0.10 |
3.62 b ± 0.58 |
2.89 c ± 0.08 |
There are significance differences (P < 0.05) between means having different
letters in the same raw
Table 2: E. coli count (Log CFU/g) of inoculated
chicken inoculated chicken fillet stored at 4°C (Mean ± SD)
Storage days |
Control |
0.5% REO micro emulsion |
1% Chitosan nanoparticle |
0.5% REO + 1% Chitosan micro
emulsion |
Zero day |
6.19 a ± 0.55 |
6.02 a ± 0.75 |
5.70 a ± 0.02 |
4.43 b ± 0.70 |
1st
day |
6.26 a ± 0.64 |
5.64 ab ± 0.42 |
4.90 bc
± 0.14 |
4.54 c ± 0.53 |
3rd day |
6.46 a ± 0.45 |
4.16 b ± 0.54 |
4.06 b ± 0.37 |
3.69 b ± 0.36 |
6th day |
5.49 a ± 0.18 |
4.25 b ± 0.71 |
4.12 b ± 0.77 |
3.53 b ± 0.24 |
9th day |
5.85 a ± 0.17 |
3.72 b ± 0.24 |
3.67 b ± 0.27 |
2.88 c ± 0.36 |
12th day |
6.64 a ± 0.33 |
3.85 b ± 0.26 |
3.00 c ± 0.16 |
- |
15th day |
7.30 a ± 0.60 |
3.66 b ± 0.35 |
- |
- |
There are significance differences (P < 0.05) between means having
different letters in the same raw
Table 3: Overall Sensory Scores (Mean ± SD) of chicken fillet stored at 4°C
Storage period |
Control |
0.5% REO microemulsion |
1% chitosan nanoparticle |
0.5% REO+ 1% chitosan
microemulsion |
Zero day |
9.0 ± 0.0 |
9.00 ± 0.0 |
9.00 ± 0.0 |
9.00 ± 0.0 |
1st day |
8.8 a± 0.4 5 |
8.8 a± 0.4 5 |
8.8 a ± 0.4 5 |
8.8 a ± 0.4 5 |
3rd day |
8.50a ± 0.45 |
8.50a ± 0.45 |
8.50a ± 0.45 |
8.50a ± 0.45 |
6th day |
6.20a ± 0.45 |
8.20b ± 0.45 |
8.20b ± 0.45 |
8.20b ± 0.45 |
9th day |
4.45a ± 0.45 |
8.20b ± 0.45 |
8.20b ± 0.45 |
8.20b ± 0.45 |
12th day |
2.45a ± 0.45 |
6.67 a ± 0.45 |
6.20b ± 0.45 |
6.20b ± 0.45 |
There are significance differences (P < 0.05) between means having
different letters in the same raw
Fig. 3: Mean count of L. monocytogenes in different treatments during
storage
Fig. 4: Mean count of E. coli in different treatments during storage
Table 2 and Fig. 4 show the
effect of different treatments on the growth of E. coli during 15 days
of refrigerated storage. The initial count of E. coli was 6.00 ± 0.05
Log CFU/g in control samples and other treatments. The growth of E. coli
decreased at 4°C in all treatments. The maximum bacterial count was observed in
control samples on the 15th day of storage (7.45 ± 0.13
Log CFU/g), whereas the minimum count was observed in 0.5% REO + 1%
chitosan microemulsion samples (on 8th day of storage: 3.15 ± 0.21
log CFU/g; 12th and 15th the counts were less than 3 log
CFU/g). When compared to the control
group, the results demonstrated a significant drop in E. coli count in
all treatments with REO chitosan microemulsion having the strongest inhibitory
efficacy.
Sensory evaluation
The sensory analysis results are reported in Table
3. The overall acceptability in terms of appearance, color, odor and texture of
all samples started at a score of 9. Within 2 days of storage, no significant
changes in the samples were found (P 0.05). Three days later,
significant changes were observed, as with the scores of the control samples
were considerably recorded lower than those of any other treated samples (P 0.05). Based on these sensory scores,
especially overall acceptability, the 0.5% REO + 1% chitosan
microemulsion mixture yielded the highest acceptability scores between 6 and 9 days of storage.
Discussion
Previous studies have reported that the mean
rosemary nanoemulsion particle size ranges from 164 ± 9 to
676 ± 26 nm and the mean PDI was 0.230 ± 0.009 (Restrepo et al. 2018). REO is used to make a variety of products.
Ultrasound was used for 6 min to create a nanoemulsion with a droplet size of
139.9 nm. The lethal concentration (LC50) of spray application of
normal emulsion (EO) was 1,578.50 and 1,829.94 g/mL
for juvenile and adult female spider mites, respectively (Mossa et
al. 2019).
Nanocapsules containing
rosemary oil were of average size (145 ± 15 nm) with PDI
below 0.3 and negative zeta potential (−11, 0 ± 0.5 mV);
they were spherical nanocapsules with regular and homogeneous surfaces. The
following key components have been identified in REO using GC–MS: α-pinene
(16.07%), 1.8-cineol (13.99%), camphor (10.85%) and cis-verbenone (10.16%) (Khoobdel et
al. 2017).
α-Pinene is a major
constituent in the composition oil, which has antibacterial activity against
Gram-negative and Gram-positive bacteria. In the case of REO, major and minor
active components, such as borneol, 1,8 cineole, D-limonene, α-pinene,
L-linalool, γ-terpinene, D-camphor, p-cymene, α-terpineol, sabinene,
α-myrcene, a-thujenol, isocineole, α-phellandrene, α-terpinene,
myrtenol, α-terpinolene, 4-terpineol, terpinene-1-ol, γ-terpineol,
isopulegol acetate and geraniol have acaricidal activity against several
phytophagous mites (Mossa et al. 2019). GC/MS analysis of REO showed that γ-terpinene (3.92%), borneol
(11.07%), 1,8 cineole (31.45%), D-limonene (9.19%), α-pinene (10.91%),
L-linalool (8.86%), D-camphor (7.32%), α-terpineol (3.32%), linalyl
acetate (3.37%), and p-cymene (1.82%) were the major components (Gachkar
et al. 2007; Ebadollahi et al. 2014).
Therefore, the powerful synergistic antimicrobial activity of the rosemary
chitosan microemulsion against Gram-positive bacteria L. monocytogenes
resulted in a lower microbial count and inhibition of the microbiological
growth of L. monocytogenes by 3 log CFU/g. These results agreed with
those by Ismail et al. (2015), who reported
that the number of bacteria decreased in samples wrapped in bionano composites
(1.2–2.6 log CFU/g). Moreover, Souza et al. (2019) have stated similar results in fresh chicken
breast fillets coated with sodium caseinate incorporated with a nanoemulsion of
ginger essential oil. Moreover, Noori
et al. (2018) has shown the reduction of the population of
the inoculated meatballs covered with chitosan. The counts of L.
monocytogenes were around 2 log CFU/g lower, showing the inhibitory effect
of L. monocytogenes on chitosan growth. On the other hand, Antoniadou
et al. (2019) have studied edible chitosan film and claimed
that the growth of L. monocytogenes could not be avoided in RTE beef
dissolved at 0.5% (w/v), 0.5% (w/v), or 1% (w/v) and stored at 4°C. However, on
day 14, the number of L. monocytogenes for all chitosan
encapsulated samples was significantly distinct from that of control by 2–3 log
CFU/g; On day 15, it was significantly different. This could be attributed to
chitosan films having less antibacterial activity as an amino group is less
available on chitosan (Coma et al. 2002; Cagri et
al. 2004; Beverlya et al. 2008).
Several previous studies (Raeisi et
al. 2012; Shahbazi et al. 2015; Ehsani et al. 2016) have confirmed the above finding.
Nevertheless, chitosan films may have antagonistic, synergistic, or additive
effects based on the type of antimicrobial agent and microorganism. The present
study confirmed that the application of coating treatments could eliminate the
bacterial count to an undetectable (103 CFU/g) level. As mentioned,
this could be due to the use of coating solutions containing REO as
microemulsions.
The effects of 1% chitosan are
similar to those found in previous research (Youssef and El-Masry
2018), which indicated the significant
antimicrobial activity of 0.5, 1 and 2% chitosan nanoparticles in eradicating
foodborne pathogens and maintaining an acceptable sensory quality of chicken
meat.
The primary components found
in prepared REO microemulsions, such as α-pinene, 1,8-cineol, camphor,
myrcene, camphene, borneol and verbenone, significantly contributed to the high
antioxidant and antibacterial activities of REO against L. monocytogenes,
E. coli, Salmonella indiana, and Listeria innocua
(Abdullah et al. 2015).
Previous research
(Hassanzadazar et al. 2019) had found that in comparison to
Gram-negative bacteria, REO and REO nanoemulsion have more important
antibacterial properties and effects on Shewanella spp., L.
monocytogenes x Staphylococcus aureus, S. enteritidis, E. coli,
and P. aeruginosa. The REO nanoemulsion mechanism of action in
Gram-positive bacteria is explained by the fact that (Aminzare et al.
2017) the cell membrane was improved by the ion's permeability due to direct
interaction between the phospholipid layer of the cell membrane and the
lipophilic components of EO and the lack of an external phospholipid
membrane–aided intraexcretion.
Our findings did not agree with
those by Ntzimani et al. (2010), who found that applying REO (0.2%) to
cooked chicken produced an acceptable odor and taste. The obtained results
revealed higher sensorial scores in 0.5% REO + 1% chitosan
microemulsion samples, which indicate the effects of chitosan coating on
preserving sensory characteristics of chicken meat. The results were in line
with those of Hassanzadeh et al. (2017). Color influences the customer’s preferences and choice of food. Food
color is determined by the chemical, biochemical, physical, and microbial
changes occurring during storage. Accordingly, chitosan's antioxidant qualities
and its capacity to function as a metal ion transition chelator that catalyzes
myoglobin oxidation can cause redness in muscle food (Yen et al.
2008).
Conclusion
This study has demonstrated the counts of E. coli
and L. monocytogenes, which were considerably lowered after treatment
for cold chicken fillets with the REO chitosan microemulsion. Compared to the
control samples, the sensory characteristics were improved and the storage
quality of the chicken breast muscle was retained in the cooling phase.
Naturally retaining the storage quality of the chicken breast muscle in
conjunction with the microemulsion of chitosan could be a viable technique in
the food industry.
Acknowledgements
The authors are grateful to Dr. Kalid Tolba and Dr. Khaled El
Khawas, AHRI's members for their scientific review and recommendations.
Author Contributions
All authors designed, coordinated, and conducted the
experiment, analyzed the data, and wrote the manuscript. All authors read and
approved the final manuscript.
Conflicts
of Interest
All
authors declare no conflicts of interest.
Data
Availability
Data
presented in this study will be available on a fair request to the
corresponding author.
Ethics
Approval
Not
applicable in this paper.
References
Abdullah BH, SF Hatem, WA Jumaa (2015). Comparative study of the
antibacterial activity of clove and rosemary essential oils on multidrug
resistant bacteria. Pharm Biosci J 3:18–22
Allam RM, AM Al-Abd, A Khedr, OA Sharaf (2018). Fingolimod interrupts the
cross talk between estrogen metabolism and sphingolipid metabolism within
prostate cancer cells. Toxicol Lett 291:77‒85
Aminzare M, M Hashemi, H Hassanzadazar, E Amiri, Z Abbasi (2017).
Antibacterial activity of corn starch films persicum essential Oils. Annu
Res Rev Biol 19:1‒9
Antoniadou D, A Govaris, I Ambrosiadis, D Sergelidis (2019). Effect of
chitosan coating on the shelf life of ready-to-eat bovine meatballs and the
control of Listeria monocytogenes growth on their surface during refrigeration
storage. J Hell Vet Med Soc 70:1495‒1502
Beverlya RL, ME Janes, W Prinyawiwatkula, HK No (2008). Edible chitosan
films on ready-to-eat roast beef for the control of listeria monocytogenes. Food
Microbiol 25:534‒537
Cagri A, Z Ustunol, ET Ryser (2004). Antimicrobial edible films and
coatings. J Food Prot 67:833‒848
Coma V, A Martial-Gros, S Garreau, A Copinet, F
Salin, A Deschamps (2002). Edible antimicrobial films based on chitosan matrix.
J Food Sci 67:1162‒1169
Ebadollahi A, JJ Sendi, A Aliakbar,
J Razmjou (2014). Chemical composition and acaricidal effects of essential oils
of Foeniculum vulgare Mill. (Apiales:
Apiaceae) and Lavandula angustifolia
Miller (Lamiales: Lamiaceae) against Tetranychus
urticae Koch (Acari: Tetranychidae). Psyche 2014:1–6
Ehsani A, M Hashemi, SS Naghibi, S Mohammadi, SK
Sadaghiani (2016). Properties of Bunium persicum essential oil and its application in Iranian white
cheese against Listeria monocytogenes and Escherichia coli O157:
H7. J Food Saf 36:563‒570
El-Kareem MMA, MAEF Rabbih, ETM Selim, EAEM Elsherbiny, AY El-Khateeb
(2016). Application of GC/EIMS in combination with semi-empirical calculations
for identification and investigation of some volatile components in basil
essential oil. Intl J Anal Mass Spectr Chromatogr 4:14‒25
FAO (2013). Poultry Development Review; FAO: Rome, Italy, ISBN
978-92-5-108067-2
Gachkar L, D Yadegari, MB Rezaei, M Taghizadeh, SA Astaneh, I Rasooli
(2007). Chemical and biological characteristics of Cuminum cyminum and Rosmarinus
officinalis essential oils. Food Chem 102:898‒904
Grande-Tovar C, C Chaves-López, A Serio, C Rossi, A Paparella (2018).
Chitosan coatings enriched with essential oils: Effects on fungi involve in
fruit decay and mechanism of action. Trends Food Sci Technol 78:61‒71
Hassanzadazar H, SYA Ghafari, M Fathollahi, M Aminzare
(2019). Antimicrobial effects of the nanoemulsion of rosemary essential oil
against important foodborne pathogens. J Hum Environ Health Prom
5:79‒85
Hassanzadeh P, H Tajik, SMR Rohani, M Moradi, M Hashemi, J Aliakbarlu
(2017). Effect of functional chitosan coating and gamma irradiation on the
shelf-life of chicken meat during refrigerated storage. Radiat Phys Chem 141:103‒109
Hussain AI, F Anwar, SAS Chatha, A Jabbar, S Mahboob, PS Nigam (2010). Rosmarinus officinalis essential oil: Antiproliferative,
antioxidant and antibacterial activities. Braz J Microbiol 41:1070‒1078
Ismail MS, MF Soliman, AH Abo-Ghalia, MM Ghallab (2015). The acaricidal
activity of some essential and fixed oils against the two-spotted spider mite
in relation to different temperatures. Intl J Pest Manage 61:121‒125
JSMO - Jordan Standards and Metrology Organization (2016). List and
publications: List of allowed additives in food products. Codex Standard nº 192
and European Directives. JSMO, Jordan
Jongberg S, MA Torngren, A Gunvig, LH Skibsted, MN Lund (2013). Effect of
green tea or rosemary extract on protein oxidation in Bologna type sausages
prepared from oxidatively stressed pork. Meat Sci 93:538‒546
Kahraman T, G Issa, EB Bingol, BB Kahraman, E Dumen (2015). Effect of rosemary
essential oil and modified-atmosphere packaging (MAP) on meat quality and
survival of pathogens in poultry fillets. Braz J Microbiol 46:591‒599
Keokamnerd T, JC Acton, IY Han, PL Dawson (2008). Effect of commercial
rosemary oleoresin preparations on ground chicken thigh meat quality packaged
in a high-oxygen atmosphere. Poult Sci
87:170‒179
Khoobdel M, SM Ahsaei, M Farzaneh (2017). Insecticidal
activity of polycaprolactone nanocapsules loaded with Rosmarinus officinalis essential oil in Tribolium castaneum (Herbst). Entomol Res 47:175‒184
Malhotra P, L Vig, G
Shroff, P Agarwal (2015). Long short-term memory networks for anomaly detection
in time series. In: Proceedings European Symposium on Artificial Neural
Networks, Computational
Intelligence and Machine Learning. 22-24 April, 2015. Bruges
Belgium
McClure P (2000). The impact of E.
coli O157:H7 on the food industry. World J Microbiol Biotechnol
16:749‒755
Mehdizadeh T, H Tajik, AM Langroodi, R Molaei, A Mahmoudian (2020).
Chitosan starch film containing pomegranate peel extract and Thymus kotschyanus essential oil can
prolong the shelf life of beef. Meat Sci 163:108073
Miller LC (1963). The British pharmacopoeia 1963. J Pharm Pharmacol
15:766‒768
Moghimi R, L Ghaderi, H Rafati, A Aliahmadi, DJ McClements (2016).
Superior antibacterial activity of nanoemulsion of Thymus daenensis essential oil against E. coli. Food Chem 194:410‒415
Mossa AH, SL Afia, SM Mohafrash, BA Abou-Awad (2019). Rosemary essential
oil nanoemulsion, formulation, characterization and acaricidal activity against
the two-spotted spider mite Tetranychus
urticae Koch (Acari: Tetranychidae). J Plant Prot Res 59:102‒112
No HK, SP Meyers, W Prinyawtwatkul, Z Xu (2007). Applications of chitosan
for improvement of quality and shelf life of foods: A review. J Food Sci
72:87‒100
Noori S, F Zeynali, H Almasi (2018). Antimicrobial and antioxidant efficiency
of nanoemulsion-based edible coating containing ginger (Zingiber officinale) essential oil and its effect on safety and
quality attributes of chicken breast fillets. Food Contr 84:312‒320
Ntzimani AG, VI Giatrakou, IN Savvaidis (2010). Combined natural
antimicrobial treatments (EDTA, lysozyme, rosemary and oregano oil) on semi
cooked coated chicken meat stored in vacuum packages at 4°C: Microbiological
and sensory evaluation. Innov Food Sci Emer Technol
11:187‒196
Olaimat AN, RA Holley (2015). Control of Salmonella on fresh chicken breasts by
κ-carrageenan/chitosan-based coatings containing allyl isothiocyanate or
deodorized oriental mustard extract plus EDTA. Food Microbiol 1:83‒88
Paparella A, A Serio, FD Pasquale, LD Nicola, C Chaves-López (2011).
Antimicrobial activity of chitosan in pork loins. Ital J Food Sci 23:131‒134
Petrou S, M Tsiraki, V Giatrakou, I Savvaidis (2012).
Chitosan dipping or oregano oil treatments, singly or combined on modified
atmosphere packaged chicken breast meat. Intl J Food Microbiol 156:264‒271
Raeisi M, H Tajik, RS Razavi, M Maham, M Moradi, B Hajimohammadi (2012).
Essential oil of tarragon (Artemisia
dracunculus) antibacterial activity on Staphylococcus aureus and Escherichia coli in culture media and
Iranian white cheese. Iran J Microbiol 4:30‒34
Rao J, DJ McClements (2011). Formation of flavor oil microemulsions, nanoemulsions and emulsions: Influence of composition and preparation
method. J Agric Food Chem 59:5026‒5035
Restrepo AE, JD Rojas, OR Garcia, LT Sanchez, MI Pinzon, CC Villa (2018).
Mechanical, Barrier, and Color Properties of Banana Starch Edible Films
Incorporated with Nanoemulsions of Lemongrass (Cymbopogon citratus) and Rosemary (Rosmarinus officinalis) Essential Oils. Food Sci Technol 24:705‒712
Ricci A, A Allende, D Bolton, M Chemaly, R Davies, PSF Escámez,
R Girones, L Herman, K Koutsoumanis, B Nørrung, L Robertson, G Ru, M Sanaa, M Simmons,
P Skandamis, E Snary, N Speybroeck, BT Kuile, J Threlfall, H Wahlström, J Takkinen,
M Wagner, D Arcella, MTDS Felicio, M Georgiadis, W Messens, R Lindqvist (2018).
Listeria monocytogenes contamination of ready-to-eat foods and the risk for
human health in the EU. EFSA J
16:e05134 https://doi.org/10.2903/j.efsa.2018.5134
Shahbazi Y, N Shavisi, E Mohebi (2015). Effects of Ziziphora clinopodioides essential oil and nisin, both separately
and in combination, to extend shelf life and control Escherichia coli O
157: H 7 and S. taphylococcus aureus
in raw beef patty during refrigerated storage. J Food Saf 36:227‒236
Sharifi F, S Khanzadi, M Hashemi, M Azizzadeh (2017). Control of Listeria
monocytogenes and Escherichia coli O157: H7 inoculated on fish fillets
using alginate coating containing lactoperoxidase system and Zataria multiflora boiss essential oil. J
Aquat Food Prod Technol 26:1014‒1021
Souza VG, JR Pires, ÉT Vieira, IM Coelhoso, MP Duarte, AL Fernando
(2019). Activity of chitosan-montmorillonite bionanocomposites incorporated
with rosemary essential oil: From in
vitro assays to application in fresh poultry meat. Food Hydrocoll
1:241‒252
Souza VG, JR Pires, ÉT Vieira, IM Coelhoso, MP Duarte, AL Fernando
(2018). Shelf-life assessment of fresh poultry meat packaged in novel
bionanocomposite of chitosan/montmorillonite incorporated with ginger essential
oil. Coatings 8:177
Yen MT, JH Yang, JL Mau (2008). Antioxidant properties of chitosan from
crab shells. Carbohydr Polym 74:840‒844
Youssef
DY, DMA ELMasry (2018). Effect of chitosan-nanoparticles on the shelf life of
chilled chicken meat and decontamination of Staphylococcus
aureus and Salmonella typhimurium.
Anim Health Res J 6:9‒18
http://animalhealth.ahri.gov.eg/Home/Publication?id=1
Zhang J, Y Lv, B Wang, S Zhao, M Tan, G Lv
(2015). Influence of microemulsion-mucin interaction on the fate of
microemulsions diffusing through pig gastric mucin solutions. Mol Pharm
12:695‒705