International Journal of Biological Macromolecules 153 (2020) 846–854

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Application an edible active coating based on chitosan- Ferulago angulata

essential oil nanoemulsion to shelf life extension of Rainbow trout fillets
stored at 4 °C
Sajad Shokri a,c, Karim Parastouei a, Maryam Taghdir a, Sepideh Abbaszadeh a,b,⁎
a
Health Research Centre, Life Style Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
b
Department of Nutrition and Food Hygiene, Faculty of Health, Baqiyatallah University of Medical Sciences, Tehran, Iran
c
Department of Food Hygiene and Control, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

a r t i c l e i n f o a b s t r a c t

Article history: The present study was undertaken to evaluate the feasibility of nanoemulsification for improving the efficiency of
Received 25 January 2020 chitosan- Ferulago angulata essential oil coating (CH + EO) on extending the shelf life of Rainbow trout fillets dur-
Received in revised form 21 February 2020 ing 16 days storage at 4 °C. The average droplet size of coarse emulsions was dramatically reduced after
Accepted 10 March 2020
nanoemulsification which was accompanied by a significantly more positive ζ-potential and lower polydispersity
Available online 11 March 2020
index. In vitro antibacterial potential of CH + EO against two fish specific spoilage organisms, Shewanella
Keywords:
putrefaciens and Pseudomonas fluorescens, and its antioxidant activity improved significantly (p b 0.05) when
Chitosan coating nanoemulsions were fabricated. The CH + EO nanoemulsion (3% EO) treatment exhibited a significantly better
Ferulago angulata essential oil inhibitory effect on bacterial growth of refrigeration stored Rainbow trout fillets. Moreover, nanoemulsification
Nanoemulsion potentiated the efficacy of CH + EO in retarding the increase of TVB-N and lipid peroxidation in fish fillets. Tex-
Shelf life ture, colour, and overall acceptability of CH + EO nanoemulsion treated samples were significantly (p b 0.05) bet-
Rainbow trout ter than those of other samples. Results suggest nanoemulsification as a potential approach to enhance the
efficacy of the active coating.
© 2020 Published by Elsevier B.V.

1. Introduction
The genus Ferulago, a world wide spread plant, consists of 35–46
species, seven of which have been found in Iran, especially in the
areas above the heights 2800–3200 m from the sea level in Zagros
mountains in western provinces including Kermanshah, Ilam, Lorestan
and Kurdestan [1]. The leaves and stem of Ferulago angulata plant (is
famed as Chavir in Persian and Chenour in Kurdish languages) has
been used as a flavouring and aromatic agent by natives for a long
time in ghee for its preservation ability besides adding a pleasant taste
to it. To date, to the best of our knowledge, very little information can
be drawn from the literature on potentially using its essential oil in
food products, however, indicating its anti-oxidation, antibacterial
[2,3], and anti-diabetes properties [1]. Essential oils (EOs) are a group
of secondary metabolites naturally synthesized in different parts of
plants and characterized as oily aromatic liquid with strong odour.
Their flavouring, antioxidant and antimicrobial properties, and as well
as their therapeutic characteristics have led to employing them in
⁎ Corresponding author at: Health Research Centre, Life Style Institute, Baqiyatallah
University of Medical Sciences, Tehran, Iran.
E-mail address: abbaszade@ut.ac.ir (S. Abbaszadeh).
various fields; mainly in cosmetic, agriculture, health and food indus-
tries. Although using EOs in food is increasing due to consumer demand
for natural food additives, their application is limited because of the cost
and other disadvantages such as their intense aroma and in some cases
potential toxicity [4]. An approach to overcome these drawbacks could
be to incorporate EOs into coating formulation that lead to reduce
their doses with the same preservation effects [5].
Chitosan (polyβ-(1,4)-N-acetyl-D-glucosamine) has been well docu-
mented for its widespread applications in many areas including cos-
metics, carrier of drugs, waste water treatments as chelating agent,
wound healing, dietary supplement, food packaging, and many others,
too numerous to mention here [6]. These broad applications of chitosan
are due to its particular properties of biodegradability, biofunctionality,
biocompatibility, antimicrobial activities, film forming property, and
nontoxicity. Chitosan forms edible films and coatings which have been
successfully demonstrated to protect highly perishable foods against
contamination and microbial spoilage, thus maintaining their quality
and extending their shelf life. In addition, this natural biopolymer as a
matrix for carrying many active ingredients including antimicrobials,
antioxidants, and nutraceuticals such as EOs, have attracted consider-
able interest due to their potential biological activity [7]. However,
due to EOs poor water solubility, incorporating them into coating

https://doi.org/10.1016/j.ijbiomac.2020.03.080
0141-8130/© 2020 Published by Elsevier B.V.
 S. Shokri et al. / International Journal of Biological Macromolecules 153 (2020) 846–854
solutions requires a proper emulsifier to fully blend into an emulsion in
order to prevent their aggregation. The large particulate structures
within the emulsion causes visible light to scatter, resulting in increased
opacity, hence, affecting the appearance of coated food [8,9]. A number
of investigations have focused on improving the properties of these ac-
tive coatings.
Recently it has been shown that nanoemulsions with small droplet
size, typically from 10 to 100 nm, present several advantages over con-
ventional emulsions, due to their unique physicochemical and func-
tional characteristics [4]. It has been proved that nanoemulsions may
accelerate the active ingredients transportation through biological
membranes, hence amplifying their biological activities [4] i.e. enhanc-
ing the bactericidal activity of EOs. The enhanced biological activities
of nanoemulsions over coarse emulsions would allow reducing the con-
centration of EOs to be incorporated in foods (coating solutions). It is
hypothesised that the chitosan- Ferulago angulata essential oil
nanoemulsion would be more effective than a coarse emulsion of this
coating formulation as a potential approach for extending the shelf life
of foods. Therefore, the purpose of current study was to evaluate the ef-
ficiency of an edible active coating based on chitosan- Ferulago angulata
essential oil nanoemulsion to shelf life extension of Rainbow trout fillets
stored at 4 °C.
2. Materials and methods
2.1. Plant materials and essential oil extraction
The Ferulago angulata plants, were collected during their flowering
stage in June 2018 from Dalahoo Mountains, Kermanshah province,
Iran. After authentication at the Faculty of Agriculture, Razi University,
Kermanshah, Iran, the plants were dried for one week at ambient tem-
perature (about 25 °C) in a dark place. The dried parts were powdered
and 100 g of powdered materials poured into a flask containing
1200 mL distilled water and subjected to hydrodistillation for 4 h
using a Clevenger-type apparatus. The obtained oils were dried over an-
hydrous sodium sulphate and then filtered through 0.22 mm filter and
stored in sealed vials at 4 °C for the next steps.
2.2. Essential oil analysis
Chemical composition of the extracted essential oil (EO) was
analysed by gas chromatography–mass spectrometry (GC–MS; 6890N,
Agilent Technologies, Palo Alto, California, USA) equipped with a capil-
lary column (thickness: 0.25 μm; internal diameter: 0.25 mm; length:
30 m; HP-5MS Agilent Technologies, Palo Alto, California, USA) based
on the method used by Sadeghi, Mahtabani, Etminan and Karami [2]
with modification. The chromatographic conditions were as follows:
initial column temperature was set at 50 °C for 6 min, then gradually in-
creased to 240 °C in a range of 3.5 °C/min and held at this temperature
for 6 min followed by a final increase in temperature of 15 °C/min up to
300 °C and held at this temperature for 3 min, injection volume of 1 μL in
mode of split less, injector temperature of 290 °C, ionization voltage of
70 eV, and detector temperature of 250 °C. Helium was used as a carrier
gas with a constant velocity of 1 mL/min. The n- pentane was used for
diluting the samples. The individual compounds were identified by
comparing their retention indices with those of normal alkanes under
the same condition and their mass spectra with internal reference avail-
able from the library (Wiley 2001 data software).
2.3. Preparation of chitosan-EO coating solutions
Medium molecular weight chitosan (~450 kD) provided from
Sigma-Aldrich (St. Louis, MO, USA) with deacetylation degree of
75–85%. Chitosan solution (CH) was prepared by dissolving 1 g of chito-
san powder in 100 mL of 1% v/v acetic acid aqueous solution and was
stirred at room temperature for 3 h, followed by filtration through a
847
Whatman no. 3 filter paper. Coarse emulsions were prepared by mixing
the resulting chitosan solution with Ferulago angulata essential oil at 1,
2, and 3% which were previously mixed well with Tween 80 (v/v, with
respect to the volume of EO; HLB:15; Sigma, Aldrich, Germany) as sur-
factant and stirred using an T25 digital Ultra-Turrax mixer (IKA, Staufen,
Germany) at 13000 rpm for 5 min [10,11]. Nanoemulsion coating solu-
tions were prepared by subjecting coarse emulsions to ultrasonic emul-
sification using a 20 kHz SONOPULS ultrasonic homogenizer (HD3200,
150 W, Bandelin electronic GmbH & Co., Berlin, Germany) for 5 min.
This condition was chosen based on primary tests (data are not pub-
lished). To maintain the temperature of the coating solutions during
sonication, the flasks containing them were placed in a receptacle con-
taining ice.
2.4. Emulsions and nanoemulsions characterization
2.4.1. Particle size, ζ-potential, and polydispersity index
The average particle size (z-average values) and polydispersity
index of coating solutions were measured by dynamic light scattering
(DLS) using a Nanopartica Series Instruments (SZ-100, Horiba, Japan)
operating with a fixed scattering angle of 173° at 25 °C. The surface elec-
trical charge of droplets was determined by DLS (SZ-100 Nanopartica
Series Instruments, Horiba, Japan), and the electrophoretic mobility of
droplets was converted to ζ-potential (mV) using the Smoluchowski
equation. In order to prevent the multiple-scattering effects, samples
were diluted 10-times with double distilled water prior to analysis.
2.4.2. In vitro antimicrobial activity
Antimicrobial activity of chitosan emulsion and nanoemulsion coat-
ings containing different concentrations of Ferulago angulata EO was
assessed by determining the minimum inhibitory concentration (MIC)
and the minimum bactericidal concentration (MBC) of coating solutions
over two fish specific spoilage organisms (SSO), Shewanella putrefaciens
and Pseudomonas fluorescens [12]. These bacteria were purchased from
Scientific and Industrial Research Organization of Iran as freeze-dried
culture and revitalised according to supplier instructions. Different ra-
tios of coating solutions: BHI broth (1:19, 2:18, 3:17… 1:1 coating solu-
tion: BHI) equal to 5%, 10%, 15%… 50% v/v of coating solutions in BHI
broth were prepared in a 96 flat bottom microplates with a final concen-
tration of 106 of each bacterium. Contents of each well were thoroughly
mixed and the microplates were incubated for 48 h at 30 °C under shak-
ing. The MIC was then considered as the last well with no visible signs of
growth or turbidity. The MBC was determined by culturing wells with-
out any turbidity in BHI agar at 30 °C for 48 h that had killed 99.9% of
bacterium [13].
2.4.3. In vitro antioxidant activity
The ability of coating solutions to scavenge of 2,2-diphenyl-1-
picrylhydrazyl (DPPH) free radicals was used to determine their antiox-
idant activity [9]. Briefly, 500 μL of different coating formulations were
prepared in 1 mL of methanol and then added to 2 mL of methanolic so-
lution of DPPH (100 mmol/L−1). The resultant mixtures then were
shaken and kept at ambient temperature in the dark for 30 min, then
the absorbance was measured at 517 nm against a blank (pure metha-
nol). Antioxidant activity was calculated as follows [14]:
Antioxidant activity ð%Þ ¼ ½1−ðAðsampleÞ−AðDPPHÞ  100
where A(sample) is the absorbance of the mixture of coating solution
and DPPH solution, and A(DPPH) is the absorbance of DPPH solution
without coating.
2.5. Preparation and coating of Rainbow trouts fillets
Fresh Rainbow trouts varying from 225 g to 275 g were purchased
alive from a local market at Kermanshah (Kermanshah, Iran). The
848 S. Shokri et al. / International Journal of Biological Macromolecules 153 (2020) 846–854
Rainbow trouts were killed by slurry ice and kept in an insulated foam
polystyrene box with ice and transferred to the laboratory. Then fish
were de-headed, eviscerated, and washed with tap water. Two fillets
were obtained from each fish. The fillet samples were randomly divided
into four groups consisting of uncoated samples as control (C), chitosan
(CH), chitosan + 3% Ferulago angulata EO (CH + 3%EO), and chitosan +
3% Ferulago angulata EO nanoemulsion (CH + 3%EO-NE) coated sam-
ples. Coating treatments were performed by immersing different groups
in corresponding coating solution twice each 60s with an interval of
2 min. Coated samples were then drained for 5 h at 10 °C and were
packed in polyethylene bags and tied off. The treated samples then
stored at 4 °C for 16 days and quality analyses were performed each
4 days as follows.
2.6. Microbiological analysis
Twenty five grams of samples were taken aseptically and homoge-
nized with a stomacher (STOMACHER®, UK) along with 225 mL of
0.1% peptone water for 1 min. From this dilution, serial dilution (1:10)
were prepared in 0.1% peptone water solution and then 0.1 mL of
each of them was spread on appropriate media. Plate count agar
(Merck, Darmstadt, Germany) was used for determining total viable
counts (48 h incubation at 37 °C). The psychrotrophic counts were car-
ried out using King Agar (Merck, Darmstadt, Germany) by incubating
cultured plates at 21 °C for 48 h. Results were expressed as log CFU/g.
2.7. Chemical analysis
2.7.1. Determination of total volatile basic nitrogen (TVB-N)
TVB-N was determined using direct distillation of the fillet homoge-
nates [15]. Ten grams samples was blended with 50 mL of distilled
water and transferred into a flask. 200 mL distilled water, 2 g MgO
and one drop silicon as antifoam were then added into the each flask.
The obtained mixtures were distilled into a flask containing 20 mL of a
3%aqueous solution of boric acid and a mixed indicator consisted of
0.1 g of methyl red and 0.1 g of methylene blue in 100 mL of ethanol. Af-
terward, distillates were titrated with 0.05 M sulphuric acid. Consumed
sulphuric acid was used for TVB-N calculation and results were
expressed as mg N/100 g flesh.
2.7.2. Determination of thiobarbituric acid reactive substances (TBARS)
The TBARS values were determined using a spectrophotometric
method as described in details by Cheng, Sun, Pu, Wang and Chen
[16]. Briefly, 5 g fish sample was minced and then mixed with 25 mL
of 20% trichloroacetic acid (w/v) following by 30 s homogenization in
a blender. 20 mL water was added to resultant mixture and mixed
well. This solution then was centrifuged for 10 min at 8000 rev min−1,
and 2 mL of supernatant was added to 2 mL of thiobarbituric acid re-
agent (0.02 M 2-TBA in 90% acetic acid) in test tubes. The test tubes
were then heated in boiling water bath for 15 min. Tubes were cooled
under piped water for 5 min and their absorbances were measured at
532 nm against a blank (5 mL of distilled water with 5 mL of TBA re-
agent). TBARS values were calculated from a standard curve of pure
malondialdehyde (Sigma, Aldrich, Germany) and results expressed as
mg of MDA eq/kg flesh.
2.8. Sensory analysis
Sensory attributes were evaluated by a panel of 6 semi-trained
members (2 females and 4 males) consisted of food science students
(22–35 years old). Five point scale scoring table was used for sensory
evaluation [10]. According to this scoring table, colour (5, no discolor-
ation; 1, extreme discoloration), odour (5, extremely desirable; 1, ex-
tremely unacceptable/off-odours), texture (5, firm; 1, very soft), and
overall acceptability (5, extremely desirable; 1, extremely unaccept-
able) were evaluated by panellists. Overall acceptability scores were
obtained as an average of colour, odour, and texture scores with similar
weight in calculation.
2.9. Statistical analysis
All experiments were replicated three times and data was analysed
using SPSS software (version 18.0 for Windows, SPSS, Inc., Chicago, IL,
USA). Analysis of variance (ANOVA) followed by Duncan's post hoc
test for parametric data and Kruskal Wallis test for sensory data, was
carried out to detect differences among mean values. The data was pre-
sented as mean ± standard deviation. Differences were considered sig-
nificant if p b 0.05.
3. Results and discussion
3.1. Chemical composition of Ferulago angulata essential oil
The essential oil from Ferulago angulata (EO) obtained in yield of
0.67% (w/w) as a light yellow oily liquid. GC–MS compositional analysis
of the obtained EO showed 57 different compounds, representing
94.72% of total the essential oil. As is presented in Table 1, the predom-
inant compounds were Cis-β-Ocimene (22.76%) and α-Pinene (22.59%)
belong to the group of monoterpene compounds [17]. Other major com-
pounds were trans-β-Ocimene (4.8%), γ-Terpinene (5.73%),
Germacrene-D (5.33%), Limonene (3.14%), Bornyl Acetate (4.89%),
Myrcene (4.49%), Camphene (2.18%), NOE-Allo-Ocimene (1.99%). Simi-
lar compounds have been reported by others [1,3,17], but with a differ-
ent concentrations. These differences could be due to difference in:
weather conditions, soil composition, season, geography, and geology,
in which the plants have grown, age and stage of maturity and as well
as processing of plant materials before EO extraction and method used
for extraction [18].
3.2. Coating solutions characterization
3.2.1. Particle size, ζ-potential, and polydispersity index
The average emulsion droplet size (z-average), ζ-potential, and
polydispersity index (PDI) of the different coating solutions are pre-
sented in Table 2. The z-average increased notably as the EO concentra-
tion increased; the highest size (462 ± 105 nm) was recorded for 3% EO
in combination with chitosan coating (CH + 3%EO). This could be due to
the promotion in droplet flocculation rate with increase in dispersed
phase concentration and decrease in the ratio between surfactant and
the dispersed phase [8,19]. Similar results are reported by Salvia-
Trujillo, Rojas-Graü, Soliva-Fortuny and Martín-Belloso [20]. Ultrasonic
emulsification of coarse emulsions exhibited a dramatic reduction of
their z-average to the nano-scale ranges (b100 nm), regardless of EO
concentrations. This is probably related to the disruption of non-
covalent interactions due to sonochemical and sonophysical effects as
a consequence of acoustic cavitation upon sonication, thereby, form
smaller particles from large aggregates. However, the increase in EO
concentration increased z-average significantly (p b 0.05) (in
nanoemulsions); the lowest droplet size was observed in ultrasound
emulsificated chitosan solution containing 1% EO (68 ± 14 nm). Similar
behaviour has been observed by Bonilla, Atarés, Vargas and Chiralt [21]
in chitosan-based film-forming dispersions incorporated with basil and
thyme essential oils. This would be related to the increased surface area
with decrease in droplet size; more surface area needs more surfactant
to cover the complete increased interfacial area of the smaller droplets
[19], to prevent bridging flocculation. Another suggested hypothesis
for this observation is, with incorporation surfactant (Tween 80) in
coating solution prior to sonication, it adsorbed in the oil surface,
thereby stabilizing the dispersed oil droplets, thus it is more difficult
to reduce the droplet size [20].
The ζ-potential of chitosan coating solution was +56.2 ± 3 eV,
which can be explained by the positive charge the amino groups of
 S. Shokri et al. / International Journal of Biological Macromolecules 153 (2020) 846–854 849

Table 1 chitosan polymer (pKa NH3 + /NH2 ≈ 6.5) [8] at coating solution pH
Chemical composition of Ferulago angulata essential oil. (3.98). Incorporation EO in chitosan coating solution significantly
No. Compound RI Percentage (p b 0.05) decreased ζ-potential. These decreases were in accordance
1 Cis-β-Ocimene 1033 22.76
with increase in EO concentration (Table 2). Given that the negative
2 α-Pinene 945 22.59 charge nature of EOs have reported in several previous studies [9,22]
3 Trans-β-Ocimene 1038 4.8 due to their hydroxyl and carboxyl groups from chemical compositions
4 γ-Terpinene 1060 5.73 of EO, the reduced ζ-potential in this study can be explained by electro-
5 Germacrene-D 1484 5.33
static interaction between chitosan and Ferulago angulata essential oil.
6 Limonene 1024 3.14
7 Bornyl Acetate 1277 4.89 Similarly, a reduction in the chitosan nanoparticle surface charge by
8 Myrcene 987 4.49 adding essential oils, such as thyme, basil, and, carvacrol was also de-
9 Camphene 952 2.18 scribed by others [8,21]. Coating solutions were emulsified by Tween
10 NOE-Allo-Ocimene 1117 1.99 80, a non-ionic emulsifier, which would expect a natural electrical
11 β-Phellandrene 1021 0.98
12 α-Terpinolene 1072 0.18
charge (close to zero) in the obtained emulsions, but a positive ζ-
13 Bicyclogermacrene 1517 0.92 potential was observed for all the coating solutions. This can be ex-
14 δ-Cadinene 1524 0.86 plained by the adsorption of chitosan molecules dispersed in the contin-
15 δ-3-Carene 1010 1.12 uous phase, due to their cationic nature. When coating solutions
16 Germacrene-B 1118 0.72
containing different EO concentrations were treated by ultrasound,
17 p-Cymene 1023 1.63
18 Spathulenol 1577 0.32 their ζ-potential increased significantly (p b 0.05), regardless of EO con-
19 α-Terpinene 1012 0.66 centration. However, the highest ζ-potential was recorded for ultra-
20 α-Phellandrene 1002 1.04 sound treated chitosan solution containing 1% EO, represented net
21 α-Copaene 1365 0.28 charge of 72.5 ± 2.03 eV. An explanation for these results could be a
22 Geranyl Isovalerate 1641 0.25
break in the chitosan chains exposed to sever mechanical stress [23],
23 Methyl Eugenol 1364 0.44
24 Trans-Caryophyllene 1422 0.19 namely sonication in this study, thus more low molecular chitosan
25 β-Bourbonene 1390 0.23 chains can be available for adsorption on the surface of EO droplets,
26 Linalool 1080 0.48 resulting in increased surface charge [8]. It has been suggested that a
27 β-Cubenone 1379 0.3
ζ-potential of ±30 mV could provide enough electrostatic repulsion
28 α-Amorphene 1281 0.26
29 Terpinene-4-ol 1160 0.1
among droplets, to maintain the stability of nanoemulsion system
30 β-Elemene 1355 0.18 [24]. In light of this, it can be concluded that the chitosan coating solu-
31 α-Cadinol 1558 0.13 tions in this study were stable. Polydispersity index (PDI) shows hetero-
32 β-Bisabolene 1029 0.21 geneity in the droplet size distribution; ranging from 0 to 1: a higher PDI
33 Borneol 1144 0.18
indicate more heterogenous size distributions [22]. The PDI for chitosan
34 Epi-α-Cadinol 1258 0.17
35 Cis-Jasmone 1098 0.12 coating solution was 0.1 ± 0.02 and increased up to 0.69 ± 0.1 when
36 γ-Elemene 962 0.1 EOs were added (Table 2). Ultrasound emulsification decreased PDI of
37 Cuparene 1020 0.12 coating solutions notably (p b 0.05) to 0.19 ± 0.02, 0.28 ± 0.03, and
38 β-Pinene 978 1.09
0.26 ± 0.04 in chitosan coating solutions containing 1, 2, and 3% EO, re-
39 α-Terpineol 1188 0.11
40 Tricyclene 925 0.08
spectively, indicating the performance of ultrasound emulsification to
41 Verbenene 975 0.08 form a uniformly size distributed emulsion.
42 Trans-Sabinene hydrate 982 0.09
43 α-Muurolene 1483 0.06 3.2.2. In vitro antimicrobial activity
44 Pinocarvey Acetate 1680 0.06
The efficiency of coating solutions on inhibiting two fish specific
45 Iso-Spathulenol 1302 0.08
46 α-Humulene 1466 0.08 spoilage organisms (SSO), Shewanella putrefaciens and Pseudomonas
47 Butanoic Acid, 3-methyl 1450 0.08 fluorescens [12], is shown in Table 2. Chitosan coating solution showed
48 γ-Curcumene 1477 0.07 similar MIC and MBC values for both SSO; 20% and 40% of coating solu-
49 Trans-β-Farnesene 1119 0.08
tion in BHI broth, respectively. Combination EO at 1% with CH had no
50 α-Cedrene 1418 0.05
51 Elemol 1532 0.07
additional effect on MIC and MBC values for both SSO; while at higher
52 α-Campholeneal 1440 0.04 concentrations (2% and 3%) MIC and MBC values notably were de-
53 p-Cymene-8-ol 992 0.04 creased. These effects of EO were similar on MIC and MBC for both
54 trans-Verbenol 1142 2.36 SSO, except MBC for Shewanella putrefaciens at 3% EO concentration;
55 Carvacrol 1080 0.05
25% of coating solution in BHI broth for Shewanella putrefaciens vs 30%
56 α-Cubebene 995 0.04
57 Myrtenal 1280 0.03 of coating solution in BHI broth for Pseudomonas fluorescens. Leakage
Total 94.72 of cell membrane due to interactions between the positively charged
chitosan and negatively charged bacterial cell surface, chelating of

Table 2
Physicochemical, antimicrobial, and antioxidant characterization of chitosan- Ferulago angulata essential oil coating emulsions and nanoemulsions.

z-Average (nm) ζ-Potential (eV) PDI Pseudomonas fluorescens Shewanella putrefaciens DPPH (%)

MIC (%)⁎ MBC (%) MIC (%) MBC (%)

CH 38 ± 7a +56.2 ± 3a 0.1 ± 0.02a 20 40 20 40 6.15 ± 0.12a

CH + 1%EO 232 ± 42b +51.01 ± 2.3ab 0.69 ± 0.1b 20 40 20 35 7.2 ± 0.28b
CH + 2%EO 295 ± 64b +50.8 ± 1.89b 0.38 ± 0.08c 15 35 15 35 14 ± 0.83c
CH + 3%EO 462 ± 105c +44.14 ± 1.6c 0.53 ± 0.04d 10 30 10 25 22 ± 0.19d
CH + 1%EO-NE 68 ± 14d +72.5 ± 2.03d 0.19 ± 0.02e 15 30 15 25 12 ± 0.92e
CH + 2%EO-NE 96.7 ± 21de +67.1 ± 0.9e 0.28 ± 0.03f 10 10 5 10 29.14 ± 0.16f
CH + 3%EO-NE 99.5 ± 17e +61.72 ± 1.94f 0.26 ± 0.04f 5 10 5 5 30.17 ± 0.44g

Values are means ± standard deviations. Means with different lowercase letters within the same column are significantly different (p b 0.05).
⁎ % of coating solution in BHI broth.
850 S. Shokri et al. / International Journal of Biological Macromolecules 153 (2020) 846–854
nutrients and essential metals, and penetration of chitosan into the bac-
terial cells thereby preventing DNA transcription [25] are suggested as
possible mechanisms for antimicrobial activity of chitosan. In compari-
son with other essential oils such as cinnamon [10], lemongrass [26],
and Apricot (Prunus armeniaca) kernel [14], the synergic effect of
Ferulago angulata EO on antimicrobial activities of chitosan was lower.
Similar moderate antimicrobial activity of Ferulago angulata EO against
different microorganisms has been reported by others [3,17]. This may
probably be related to the lower concentrations of phenolic compounds
such as carvacrol and thymol, as the major antimicrobial agents in EOs
[6], in Ferulago angulata EO and a higher concentration of monoterpene
compounds such as α-Pinene and Cis-β-Ocimene with a weak antimi-
crobial activity [17]. However, nanoemulsification increased the anti-
bacterial activity of coating solutions (Table 2). This increase was
depending on coating formulation; the higher concentrations of EO
more influenced by nanoemulsification. Higher water dispersibility ac-
companied with a smaller droplets size after ultrasound emulsification
provide more easier and faster penetration of antimicrobial compounds
through the membrane of the bacterial cells, thus increased their effi-
ciency [9,27]. Additionally, severe mechanical stress [23] break chitosan
molecular chains into lower chains, thereby they are able to pass easier
the bacterial membrane, resulting in increased antibacterial activity.
3.2.3. In vitro antioxidant activity
The DPPH method was used to evaluate the antioxidant activity of
the coating solutions, and the results were expressed as a percentage
of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging capac-
ity (Table 2). Chitosan solution with no EO showed a 6.15 ± 0.12% scav-
enging activity on DPPH. The antioxidant activity of chitosan has been
previously shown by others [10,28], mainly due to the reaction of resid-
ual free amino (NH2) groups of chitosan with free radicals to form sta-
ble macromolecular radicals and ammonium groups [29]. This is based
on absorbing a hydrogen ion from the solution [29]. The antioxidant ac-
tivity of chitosan increased to 7.2 ± 0.28, 14 ± 0.83, and 22 ± 0.19% by
adding 1, 2, and 3% EO, showing the scavenging activities of chitosan so-
lution dependent on EO concentration. Similar to antibacterial activity;
the antioxidant activity of Ferulago angulata EO was also low compared
to other incorporated EOs in chitosan; this may be due to lower concen-
trations of phenolic compounds and a high amounts of monoterpene
hydrocarbons such as α-Pinene and Limonene with low antioxidant ac-
tivities in Ferulago angulata essential oil [3,17].
However, higher antioxidant activities were observed after ultra-
sound nanoemulsification, representing 66.66, 108.14, and 37.13% in-
crease for nanoemulsified chitosan solution incorporated with 1, 2,
and 3% EO, respectively. A possible explanation for these results could
be a more uniform distribution of Ferulago angulata EO, resulting in a
greater stability of chitosan-EO thereby more and better scavenging ac-
tivity of coating solution [26]. In addition, droplet size reduction of
chitosan-EO emulsion after nanoemulsification by ultrasound leads to
increased specific surface of chitosan-EO, hence an easier and more
free radical absorption would be achieved [9]. In line with these results,
Noori et al. [9] reported a significant increase in antioxidant efficiency of
sodium caseinate coating containing ginger essential oil by ultrasonic
nanoemulsification.
3.3. Microbiological, chemical, and sensory analysis of the coated Rainbow
trout fillets
According to the results of antimicrobial and antioxidant activities of
different coating solutions (see above), incorporation of 3% of EO into
chitosan was selected as the best coating formulation for Rainbow
trout fillets treatment.
3.3.1. Microbiological analysis
Changes in total viable counts (TVC) and total psychrotrophic counts
(TPC) of the Rainbow trout fillets are presented in Fig. 1a–b. The initial
values of TVC and TPC (in control) were 3.95 ± 0.18 log CFU/g and
3.21 ± 0.23 log CFU/g, respectively. These values decreased significantly
(p b 0.05) in CH + 3%EO and CH + 3%EO-NE immediately after treat-
ment. Afterward, both TVC and TPC increased gradually within each
group throughout the storage for 16 days, but with a lower increasing
rate in treatments. The TVC in control exceeded the maximal recom-
mended limit of 7 log CFU/g in raw fish [30] by day 8, whereas it was
lower than this acceptable limit in CH and CH + 3%EO until the 12th
day of storage. Nanoemulsion coated fillets, CH + 3%EO-NE lot, showed
the lowest TVC during the storage; 4.65 log CFU/g lower than control
and 2.57 log CFU/g lower than CH + 3%EO at the end of study.
Psychrotrophic bacteria are the major group of microorganisms respon-
sible for spoilage of fresh fish stored at low temperatures (4 °C) [30],
therefore, enumeration of these bacteria could be a good reflection of
the quality of cold stored fish flesh. The TPC changes showed a similar
behaviour as that of TVC, so that the lowest and the greatest counts
were observed in CH + 3% EO-NE and control, respectively, at the end
of storage period. The increase in TPC of fillets treated by chitosan- 3%
EO nanoemulsion was notably less than that treated by conventional
chitosan- 3% EO emulsion or EO-free chitosan coating at the 8th, 12th,
and 16th day of storage. As a consequence of these results, it can be pos-
tulated that the chitosan coating retards the microbial growth in cold
stored Rainbow trout fillets. This inhibitory effect of chitosan was pro-
moted when Ferulago angulata EO added, and was more effective after
nanoemulsification. Similar synergistic antibacterial effects with the in-
corporation of EOs with chitosan coating solution have been previously
reported. Ojagh, Rezaei, Razavi and Hosseini [10] reported an extended
shelf life of fresh Rainbow trout during the refrigerated storage by ap-
plying chitosan coatings enriched with cinnamon oil, as a consequence
of inhibiting the microbial growth. Wang et al. [11] also showed that
carvacrol-chitosan coating significantly inhibited the growth of total vi-
able count of Pacific white shrimp by about 3 log CFU/g lower than un-
coated samples during iced storage. One other important result
concluded from microbial assessment is that the inhibitory effect of
EO-chitosan coating nanoemulsion was more than its coarse emulsion.
As PDI results showed, a higher diffusion rate of antibacterial in EO-
chitosan nanoemulsion can increase and enhance the antibacterial ef-
fectiveness of EO-chitosan coating. In addition, the reduced particle
size in the nanoemulsion resulting in an increased surface-to-volume
ratio of EO droplet, which would allow antibacterial compounds to pen-
etrate easier and at a faster rate in the bacterial cell membrane, thereby
improved the EO's antibacterial effectiveness [27].
3.3.2. Chemical analysis
3.3.2.1. TVB-N. Analysis of volatile nitrogenous compounds such as
trimethylamine, dimethylamine, and ammonia, known as TVB-N
index, has been considered as an indicator of spoilage in meat and
meat products. These compounds come from the degradation of nitrog-
enous compounds due to microbial and enzymatic activities in flesh. Ac-
cordingly, as it was expected, the TVB-N results showed a good
correlation with microbial changes in samples, r = 0.98 (this value is
the correlation between average amounts of TVB-N and TVC in control
as an example to showing how TVB-N changes are related to microbial
changes). The initial amount of TVB-N in control was 9.15 ± 0.25 mg N/
100 g, with no significant difference (p N 0.05) among the different
batches at the beginning of storage (Fig. 2a). Parallel to increases in bac-
terial counts in all groups, the TVB-N was gradually increased during the
storage time. However, the TVB-N in treated samples was significantly
lower than control, in all sampling times. Regarding the amount of
25 mg N/100 g, as the maximal acceptable level in fish flesh [31], the
TVB-N limit was reached by the control by the eighth day, while this
limit in chitosan coated and chitosan-3% EO coated samples were
reached by the 12th and 16th days, whereas in chitosan- 3% EO
nanoemulsion remained lower than this limit throughout the entire
storage time. The TVB-N for chitosan- 3% EO nanoemulsion was 27.5,
 S. Shokri et al. / International Journal of Biological Macromolecules 153 (2020) 846–854 851

a
a
Control CH CH+3%EO CH+3%EO-NE
9 a
Maximum acceptable limit

Total viable counts (log CFU/g )

8 b
a c
7 b
c
6 b
a c
5 a d
a b d
4 ab d
bc c c
3

2
0 4 8 12 16
Storage time (days)

b
9 Control CH CH+3%EO CH+3%EO-NE
a a
Maximum acceptable limit
Psychrotrophic counts (log CFU/g)

8
b
a
7
b
6
a b c

5 b
c
d
4 c
d
a a c
ab d
3 c
b

2
0 4 8 12 16
Storage time (days)

Fig. 1. Effects of chitosan- Ferulago angulata essential oil coating emulsion and nanoemulsion on total viable counts (a) and psychrotrophic counts (b) of Rainbow trout fillets during
16 days storage at 4 °C. Letters above each bar indicate the results of Duncan's test (p b 0.05); values with same letters in the same group are not significantly different.

14.47, and 11 mg N/100 g lower than control, chitosan coated and 3.3.2.2. TBARS. The TBARS index is a widely used indicator to evaluate
chitosan-3% EO coated samples, respectively, at the end of 16 days stor- the level of lipid oxidation by measuring second stage auto-oxidation
age. As mentioned before, since TVB-N are produced mainly due to bac- products, particularly aldehydes such as malondialdehyde. The perceiv-
terial degradation of protein and non-protein nitrogenous compounds, able level of TBARS in food as objectionable odour has been recom-
lower amounts in coated samples could be attributed to microbial in- mended about 1–2 mg MDA eq/kg [32], however, Raeisi, Tajik,
hibitory effects of treatments, resulting in lower TVB-N formation. In Aliakbarlu, Mirhosseini and Hosseini [33] suggested 5 mg MDA eq/kg
line with these results Ojagh et al. [10] found that coating of Rainbow as the maximal acceptable level in Rainbow trout without having any
trout fillets with 2% chitosan solution enriched with 1.5% cinnamon oil adverse effect on quality and safety. As the results show (Fig. 2b), the
could retard the increasing rate in the TVB-N index. Ramezani et al. amounts of TBARS remained below the maximal acceptable level
[28] after comparison the effectiveness of chitosan and nanochitosan during16-day storage. Similar observations have been reported by
coatings on the quality of refrigerated Silver carp fillets, reported a others [10,32,33]. However, the initial amount of TBARS ranged from
stronger ability of nanochitosan to inhibit the TVB-N formation than 0.11 ± 0.015 to 0.13 ± 0.014 MDA eq/kg which is in agreement with
conventional chitosan coating. To the best of our knowledge, there are those reported by others for fresh Rainbow trout [10,32,33]. The
no reports on the effect of chitosan-essential oil nanoemulsion on the TBARS values increased in all samples up to day 4 and after that showed
formation of TVB-N in Rainbow trout fillets; however, the lower TVB- a constant or decreasing trend up to 8th day, depending on treatment
N in chitosan-EO nanoemulsion coated samples in current study could type. From the 8th day until the end of storage period TBARS again in-
be probably due to facilitated antibacterial efficiency of chitosan- 3% creased in all groups. The increase in TBARS indicates developing the ox-
EO coating after nanoemulsification as it has been discussed above. idation of unsaturated fatty acids in fish flesh. Decrease in TBARS
852 S. Shokri et al. / International Journal of Biological Macromolecules 153 (2020) 846–854

a a
45 Control CH CH+3%EO CH+3%EO-NE

40 Maximum acceptable limit

a

TVB-N (mg N/100 g)

35
b
30 b c
a
25 b c
20 c d
a d
15 a b
d
c
10 a a a a

5
0 4 8 12 16
Storage time (days)

0.3
b
Control CH CH+3%EO CH+3%EO-NE

a
a
0.25
TBARS (MDA eq/kg)

a a b

0.2
b c
a a c
a
a
0.15 ab a b
b b b
ab
b
0.1

0.05
0 4 8 12 16
Storage time (days)

Fig. 2. Changes in TVB-N (a) and TBARS(b) valuse of treated Rainbow trout fillets by chitosan- Ferulago angulata essential oil coating emulsion and nanoemulsion during 16 days storage at
4 °C. Letters above each bar indicate the results of Duncan's test (p b 0.05); values with same letters in the same group are not significantly different.

between days 4 and 8 could be due to the reaction between formed mal-
onaldehyde with proteins, amino acids, and glycogen in flesh, leading to
decrease its amount [34]. Changes in TBARS were group dependent, so
that a notable lower TBARS were observed in chitosan- 3% EO
nanoemulsion coating and chitosan- 3% EO coating, respectively, than
control and EO-free chitosan. The slightly lower oxidation rate in chito-
san coated samples could be due to both antioxidant activity (see the
DPPH results) and oxygen barrier properties of chitosan coating which
have been well documented previously [10,28]. Furthermore, chitosan
can acts as a chelator on transition metal ions in muscle foods, which
have a main role in the induction of lipid peroxidation [25]. As is men-
tioned above, a more antioxidant activity of CH + 3%EO than free EO-
chitosan could be due to presence of antioxidant compounds such as
γ-Terpinene and α-Terpinolene in Ferulago angulata EO [17]. However,
the interactions effects between minor and major compounds of essen-
tial oil should not be neglected. Another important result in this study
was the effectiveness of nanoemulsification on enhancing the antioxi-
dant activity role of EO-chitosan coating to prevent lipid oxidation in
coated samples. This may probably be related to the higher PDI for
EO-chitosan coating nanoemulsion, resulting in a higher distribution
rate of active compounds in the coating solution, thereby coating the
surface of samples properly and more uniform, hence, exhibition a bet-
ter efficiency in the control of lipid oxidation [26]. Furthermore, droplet
size reduction of EO-chitosan coating after nanoemulsification leads to
increased specific surface of EO-chitosan; hence a fast and efficient
free radical absorption would be achieved. These results were opposite
to what was reported by Noori et al. [9] who observed no significant dif-
ferent in TBARS index between emulsion and nanoemulsion coated
chicken breast fillets. This difference could be due to difference in coat-
ing formulations as well as the method used for nanoemulsion
preparation.
3.3.3. Sensory analysis
Changes in sensory quality of Rainbow trout fillets during 16 days
storage at 4 °C are given in Table 3. As can be seen, in general, the un-
coated samples received the lowest scores by panellists throughout
the entire storage time except for that at day 0, following by chitosan
coating, EO-chitosan coating, and EO-chitosan nanoemulsion coating
samples, respectively. Regarding to the acceptable sensory score for
consumption, 4 and above it [10], the control samples received scores
lower than this limit at the 8th day, while this time for chitosan and
EO-chitosan coated samples were the 12th and 16th days, and for EO-
 S. Shokri et al. / International Journal of Biological Macromolecules 153 (2020) 846–854 853

Table 3
Effects of chitosan- Ferulago angulata essential oil coating emulsion and nanoemulsion treatments on sensory changes of Rainbow trout fillets during 16 days storage at 4 °C.

Sensory parameter Treatments Storage time (days)

0 4 8 12 16

Texture Control 5 ± 021a 4.5 ± 0.26a 3.76 ± 0.19a 1 ± 0.4a 1 ± 0.0a

CH 5 ± 0.14a 5 ± 0.30a 4.1 ± 0.12b 3.23 ± 0.52b 2.9 ± 0.24b
CH + 3%EO 5 ± 0.31a 5 ± 0.16a 4.6 ± 0.35c 4.2 ± 0.19c 4.1 ± 0.46c
CH + 3%EO-NE 5 ± 0.18a 4.95 ± 0.24a 4.96 ± 0.13c 4.9 ± 0.49d 4.7 ± 0.35c
Odour Control 4.98 ± 0.11a 4.6 ± 0.22a 2.9 ± 0.19a 1 ± 0.12a 1 ± 0.00a
CH 4.95 ± 0.14a 4.8 ± 0.16a 4.4 ± 0.24b 3.9 ± 0.22b 3.14 ± 0.12b
CH + 3%EO 4.02 ± 0.18b 4.01 ± 0.17b 3.8 ± 0.31c 3.6 ± 0.35b 3.2 ± 0.08b
CH + 3%EO-NE 4.15 ± 0.12b 4.21 ± 0.02c 4.32 ± 0.17b 4.2 ± 0.14c 4.01 ± 0.42c
Colour Control 5 ± 0.04a 4.65 ± 0.16a 4.12 ± 0.13a 2.84 ± 0.24a 1.24 ± 0.06a
CH 4.8 ± 0.1b 4.85 ± 0.12a 4.29 ± 0.14ab 3.89 ± 0.16b 2.99 ± 0.39b
CH + 3%EO 4.56 ± 0.12c 4.46 ± 0.23a 4.59 ± 0.19b 4.25 ± 0.12c 4.12 ± 0.22c
CH + 3%EO-NE 4.46 ± 0.23c 4.73 ± 0.25a 4.5 ± 0.21b 4.29 ± 0.18c 4.19 ± 0.15c
Overall acceptability⁎ Control 4.99 ± 0.02a 4.55 ± 0.15a 3.59 ± 0.19a 1.61 ± 0.18a 1.08 ± 0.02a
CH 4.9 ± 0.08a 4.88 ± 0.19a 4.28 ± 0.08b 3.67 ± 0.26b 3.09 ± 0.22b
CH + 3%EO 4.52 ± 0.14b 4.55 ± 0.32a 4.33 ± 0.16bc 4.01 ± 0.09c 3.8 ± 0.42c
CH + 3%EO-NE 4.59 ± 0.11b 4.63 ± 0.21a 4.6 ± 0.18c 4.43 ± 0.18d 4.3 ± 0.3d

Different lower case super index letters within a column are significantly different (p b 0.05). Scores are given as mean ± SD.
⁎ Overall acceptability scores were calculated as an average of colour, odour, and texture scores with same weight in calculation.

chitosan nanoemulsion coated samples were above than this acceptable
limit even after 16 days storage. These results indicate an extension of
the shelf life of Rainbow trout fillets up to at least 8 days using the EO-
chitosan nanoemulsion coating. Sensory scores are in agreement with
the results of microbial and chemical analysis; therefore, it can be con-
cluded that the treatments kept the Rainbow trout fillets sensory quality
due to their antimicrobial and antioxidant properties. It should be noted
that samples coated with chitosan coating containing EO received a
lower score for odour until the 4th day; however the scores for EO-
chitosan coating were significantly (p b 0.05) lower than EO-chitosan
nanoemulsion coating. These results indicate the possibility of using ac-
tive coating in form of nanoemulsion in order to extending the shelf life
of stored products and/or reducing the EO concentration without reduc-
ing their efficiency. Similar results have been reported by Noori et al. [9]
and Ramezani et al. [28].
4. Conclusion
The results of this study showed that nanoemulsification has poten-
tial to improve the functional properties of chitosan coating solution
containing Ferulago angulata essential oil including: a dramatically re-
duced of average droplet size of coating solution which was accompa-
nied by a significantly more positive ζ-potential and lower PDI and
improved its antioxidant activity and antibacterial potential against
two fish specific spoilage organisms. Application of EO-chitosan
nanoemulsion (3% EO) could successfully retard the bacterial growth
of refrigeration stored Rainbow trout fillets. Moreover, increase in
TVB-N and lipid peroxidation in Rainbow trout fillets were notably con-
trolled by this approach. Based on sensory results, the minimum shelf
life of 16 days could be suggested for coated samples with chitosan-3%
Ferulago angulata essential oil coating nanoemulsion.
Funding
This research did not receive any specific grant from funding agen-
cies in the public, commercial, or not-for-profit sectors.
Ethical statement
This article does not contain any studies with human participants
performed by any of the authors.
Declaration of competing interest
The authors declare that they have no conflict of interests.
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