Food Chemistry 396 (2022) 133650

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Investigation of physicochemical properties, antimicrobial and antioxidant

activity of edible films based on chitosan/casein containing Origanum
vulgare L. essential oil and its effect on quality maintenance of
cherry tomato
Narjes Roshandel-hesari , Majid Mokaber-Esfahani *, Akram Taleghani , Reza Akbari
Department of Chemistry, Faculty of Science, Gonbad Kavous University, P.O. Box 163, Gonbad Kavous, Iran

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

Keywords: Edible coatings prevent physicochemical and biological food deterioration. Using bioactive compounds like
Edible film essential oils can enrich films. In this study, edible films from chitosan (C), casein (Z) and oregano (OEO) were
Origanum vulgare L developed, and their physicochemical, barrier, antimicrobial, antioxidant, and structural properties (FTIR, SEM)
Antifungal activity
were investigated. The C1Z3 ratio had good mechanical and inhibitory properties, and OEO improves flexibility,
Chitosan
Casein
barrier, hydrophobic, antimicrobial, and antioxidant properties. The physicochemical and microbiological
Cherry tomato properties of cherry tomatoes were affected by C1Z3 and C1Z3O1.5 coatings. Coated fruits were stored at 4 ◦ C for
32 days. The best results for weight loss, shrinkage, and titratable acidity were found to be 17.88%, 31.12%, and
0.15% in C1Z3O1.5 coated cherry tomatoes, respectively. The TMAB of C1Z3O1.5 coated fruits was less than
detectable and the fungal growth was inhibited for 28 days. Accordingly, by adding OEO to chitosan/casein
coatings, the spoilage process of cherry tomatoes was delayed for long-term storage.

1. Introduction chitosan, pectin, starch, alginate, pullulan, carrageenan, and cellulose

In recent decades, there have been growing worries regarding the use
of synthetic polymers, commonly known as plastics, for the packaging of
food products. Since the majority of synthetic polymers are resistant to
biological degradation, they end up in landfills or the ocean. These two
behaviors are detrimental to the environment and pose a threat to
human health. Therefore, substantial studies have been conducted on
the manufacture of edible and biodegradable food packaging films and
coatings in order to reduce total solid waste (Pellá et al., 2020). Edible
packaging is a thin layer of edible material that is either formed directly
on the surface of the food (edible coating) or formed separately as a thin
film and then wrapped on the surface of the food (edible film).
Fruits and vegetables with edible film/coatings have a longer shelf
life because they reduce oxidative stress, limit oxygen penetration or
moisture loss (Aguilar-Sánchez et al., 2019), prevent color change,
minimize mechanical and microbiological damage, reduce shrinkage
and weight loss, and restrict the rate of respiration (Kong et al., 2019). It
is possible to make edible films or coatings using any of the various types
of commonly-used biopolymers. These include polysaccharides (like
derivatives), proteins (like casein, zein, whey, wheat gluten, gelatin,
soybean protein, and keratin), and lipids (like waxes and fatty acid es­
ters) (Jiang et al., 2021).
In recent studies, the development of edible and biodegradable films
and coatings by mixing polysaccharides, proteins, and lipids has been
investigated. The purpose of these investigations is to make use of the
characteristics of each component as well as the synergy that exists
between them. Therefore, it is essential to explore combinations of
biopolymers that provide improved functionality as a natural and edible
packaging material (Ponce, Roura, & Moreira, 2016). It has become
increasingly common in the food, pharmaceutical, and biotech in­
dustries to use polysaccharide-protein mixed systems. They could
demonstrate more desirable functional characteristics than proteins and
polysaccharides on their own (Pogaku, Seng, Boonbeng, & Kallu, 2007).
In this context, chitosan, a natural polycationic polysaccharide
consisting of (1,4)-linked 2-amino-deoxy-β-D-glucan obtained by the
deacetylation of chitin (Tokatlı & Demirdöven, 2020), has attracted
widespread interest in the field of biodegradable edible coating/film
packaging due to its biocompatibility, non-toxicity, biodegradability,

* Corresponding author.
E-mail address: m_mokaber@gonbad.ac.ir (M. Mokaber-Esfahani).

https://doi.org/10.1016/j.foodchem.2022.133650
Received 1 January 2022; Received in revised form 13 June 2022; Accepted 5 July 2022
Available online 8 July 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
N. Roshandel-hesari et al.
film-forming ability, and antimicrobial and antifungal activities (Li &
Zhuang, 2020). The antimicrobial properties of chitosan occur through
damage to the structure of cell membranes and walls of microorganisms,
which causes leakage of components within the cells of microorganisms.
This means that metabolic activity is disrupted and leads to bacterial
death (Li & Zhuang, 2020). A semi-permeable film formed by chitosan
can alter the internal environment, reduce transpiration loss, delay fruit
and vegetable ripening (Tokatlı & Demirdöven, 2020), enhance the
appearance of fruit and vegetables by preventing the growth of micro­
organisms, reduce changes in firmness, decrease weight loss, and extend
the shelf life of food (Shao, Tu, Tu, & Tu, 2012). Numerous studies have
investigated the application of chitosan as an edible film coating, with
promising results for a variety of fruits and vegetables, including sweet
cherries, strawberries, mangos, pomegranates, pear, fresh cut broccoli,
and rose apple, which have all been effectively coated with chitosan as
an edible film coating (Tokatlı & Demirdöven, 2020).
Moreover, edible films based on casein as a protein are appealing for
use in the food industry due to their high nutritional value, water sol­
ubility, and emulsification capacity (Picchio et al., 2018). Casein films
provide an effective barrier to oxygen and other nonpolar molecules due
to the even distribution of polar amino acids throughout the protein
chain. This property makes it an excellent choice for the protection of
products that are susceptible to oxidation (Chevalier, Assezat, Pro­
chazka, & Oulahal, 2018). With the addition of glycerol, casein-based
films have good tensile strength and a moderate degree of elasticity
under normal conditions (Kurek, Galus, & Debeaufort, 2014).
Since casein peptides have a negative surface charge, they can
interact with chitosan molecules with the opposite charges and create a
three-dimensional network. Previous studies have also revealed
enhanced mechanical characteristics (tensile and impact strengths) and
decreased equilibrium moisture content in chitosan/casein composite
films compared to either chitosan or casein alone. This is because of the
strong electrostatic forces that are formed when the two macromole­
cules form a polyelectrolytic complex (Moreira, Pereda, Marcovich, &
Roura, 2011).
However, due to the fact that chitosan and casein are both water-
soluble biopolymers, their high hydrophilicity and high water vapor
permeability (WVP) significantly restrict the application of chitosan/
casein films in wet environments (Xie et al., 2020. This issue can be
remedied by incorporating hydrophobic components such as lipids into
chitosan/casein composite film formulations. As a lipid substance,
essential oil (EO) is an excellent candidate for improving the function­
ality of chitosan/casein-based films (Wu et al., 2014). Also, essential oils
have been found to be effective natural antimicrobial agents against a
wide variety of microorganisms, and their eco-friendliness and biode­
gradability have contributed to their acceptance by the public (Burt,
2004). The addition of this active ingredient to edible films help in the
inhibition of bacterial and fungal growth (Aguilar-Sánchez et al., 2019).
Origanum vulgare L. is a herbaceous plant in the Lamiaceae family.
Origanum vulgare L. essential oil (OEO) is one of the most common
essential oils for various therapeutic applications due to its antimicro­
bial and antioxidant activities. Some of the most bioactive compounds
found in OEO are carvacrol, thymol, γ-terpinene, and p-cymene. The
antioxidant and antibacterial capabilities of phenolic compounds in
OEO, such as carvacrol and thymol, can effectively enhance the shelf life
of food, making it an option to enrich food packaging (Yoncheva et al.,
2021).
The cherry tomato is one of the most popular vegetables in the So­
lanum genus. Despite being low in calories, it is rich in vitamin C,
lycopene, and beta-carotene, and its chemical composition also contains
vitamin E, carotenoids, chlorophyll, organic acids, flavonoids, and
phenols (Asensio, Sanvicente, Mallor, & Menal-puey, 2019). However,
cherry tomatoes, like other seasonal fruits, ripen quickly due to tran­
spiration and respiration, so they are rapidly decomposed by microor­
ganisms (Wu, Lu, & Wang, 2016). Therefore, it is necessary to provide
effective and practical solutions to extend its shelf life.
2
Food Chemistry 396 (2022) 133650
Based on the aforementioned issue, the ultimate objective of this
study was to evaluate the potential of a novel chitosan/casein composite
film to preserve cherry tomatoes. To the best of our knowledge, the
synthesis, characterization, and application of chitosan/casein blend
films with OEO for food preservation have not been studied or published
in the literature. The three main aspects of this study were OEO-enriched
chitosan/casein composite films characterization, antimicrobial activ­
ity, and fruit preservation, which were all carried out separately. The
optimal chitosan/casein ratio was found by analyzing the mechanical
and physicochemical properties of films. OEO has been added to the
films in order to improve their mechanical, physicochemical, and anti­
microbial activity. The mechanical, physicochemical, structural prop­
erties, antioxidant, antibacterial, and antifungal activities of the
composite films were evaluated. Finally, the ability of OEO-enriched
chitosan/casein edible coatings to maintain the quality of cherry to­
matoes was investigated in a 32-day storage study at 4 ◦ C.
2. Materials and methods
2.1. Materials and reagents
Cherry tomatoes harvested from greenhouses in the vicinity of
Gonbad Kavous were purchased at the city’s central market. The leaves
of three-year-old Origanum vulgare L. plant were collected in Amanabad
village, which is located in the center of Iran’s Markazi province (DMS:
34◦ 01′ 00′′ N, 49◦ 54′ 51′′ E; height; 1710 m). Medium molecular
weight chitosan (Sigma-Aldrich, 75%–85% deacetylation), casein
(Quelab), glycerol (Merck), distilled water, acetic acid (99–100%
glacial), magnesium nitrate (Merck), DPPH (2,2-diphenyl-1-picrylhy­
drazyl, Merck), sodium hydroxide (Merck), and for antibacterial activity
tests: Mueller Hinton Agar culture medium (MHA), The bacterial cul­
tures were grown on Mueller Hinton agar (Wasegh Sample Company,
Gorgan. Iran), Escherichia coli (1399 ATCC 25922), Staphylococcus aureus
(1113 ATCC 9144) were prepared by the Iran Scientific and Industrial
Research Center. The Botrytis cinerea strain (IBRC-M No. 30162)
deposited in the Iranian Biological Resource Center (IBRC) was cultured
on potato dextrose agar (PDA) and incubated at 25 ◦ C for 7 days.
2.2. Preparation of edible films
The general method solution casting is used to prepare films. Casein
protein solution with a concentration of 2.5% (w/v) was prepared by
dissolving casein powder (2.5 g) in 100 mL of distilled water after
continuous stirring for 3 h at room temperature. Glycerol was added as a
plasticizer with a 0.28 wt ratio of casein and the mixture was stirred
again for 15 min. Chitosan (2 g) was mixed in an aqueous solution of
acetic acid (%1 v/v, 100 mL) and stirred vigorously (1000 rpm) for 24 h
at room temperature until the completion of the solvation. Glycerol was
added with a weight ratio of 0.28 polysaccharides and stirred at 30 ◦ C
for 15 min. The chitosan/casein (CZ) film solution was prepared by
mixing two solutions in different ratios (v/v) (Scheme 1) (Pereda, Ara­
nguren, & Marcovich, 2008).
To determine the best ratio of chitosan/casein (CZ) in films, 9 solu­
tions of 1 mL:6mL (C1Z6), 1 mL:4mL (C1Z4), 1 mL:3mL (C1Z3), 1 mL:2mL
(C1Z2), 1 mL:1mL (C1Z1), 6 mL:1mL (C6Z1), 4 mL:1mL (C4Z1), 3 mL:1mL
(C3Z1), and 2 mL:1mL (C2Z1) were prepared. The final solutions were
heated and stirred at 30 ◦ C for 60 min to obtain a uniform blend. The
solution was degassed under a vacuum for 10 min to remove air bubbles
(Pereda et al., 2008). After performing physicochemical tests, the C1Z3
film was determined as the best edible film.
Thereupon, Tween 80 was added at 0.2% v/v OEO as an emulsifier to
help the dispersion of the essential oil and stirred for 30 min. The OEO
was added to the C1Z3 edible film solution at three different concen­
trations: 0.5% v/v (C1Z3O0.5), 1% v/v (C1Z3O1), and 1.5% v/v
(C1Z3O1.5). The mixture was stirred at room temperature for 30 min,
then 10 min under vacuum degasser. 20 mL of edible film solution was
N. Roshandel-hesari et al. Food Chemistry 396 (2022) 133650

Distilled Acetic acid Distilled water Acetic acid

water solution solution

Casein Chitosan Casein Chitosan

Glycerol Glycerol, Tween-80 OEO

Continuous Continuous
stirring stirring

Casting Casting

Drying, Detaching Drying, Detaching

Edible film Edible film

A B
Scheme 1. Schematic of edible films preparations, A: C1Z3, B: C1Z3O.

cast in each 10 cm petri dish and dried at room temperature for 72 h
(Scheme 1). Peeled off dry edible films and stored in a desiccator con­
taining saturated magnesium nitrate Mg(NO3)2 solution at 50 ± 2%
relative humidity for 24 h prior to analysis. (Hosseini, Rezaei, Zandi, &
Faramandghavi, 2016).
2.3. Isolation of the essential oil
To prepare the essential oil of Origanum vulgare L, the aerial parts of
the crop were collected in June 2020 from Amanabad village in the
central part of the Markazi province of Iran. The plant was identified by
herbarium number GKU/NO.803895 and was kept in the herbarium of
Gonbad Kavous University, Iran. The plant was washed with water and
dried in the shadows for 7 days. 100 g of the chopped plant in water
(1200 mL) was subjected to water distillation for 4 h using a clevenger
device. Yellow oils with a yield of 2% were stored under nitrogen at­
mosphere at a cold temperature (4 ◦ C) (Shokri, Parastouei, Taghdir, &
Abbaszadeh, 2020).
2.4. Film thickness
(addition of weight as a function of time) were calculated and reported
by linear regression. The water vapor permeability (g mm/m2d kPa) was
calculated by Eq. (1).
WVP = WVTR/Δp (1)
Where WVTR is the transfer rate of water vapor (g/Cm2d), defined as the
slope (g/d) divided by the transfer area (Cm2). X (mm) is the thickness of
the film. Δp (kPa) is the difference in partial water–vapor pressure
across the film, calculated as Δp = p(RH2 − RH1) = 2.37 kPa, where p is
the saturation vapor pressure of water at 25 ◦ C. RH2 and RH1 the relative
humidity in the saturated desiccator and dry desiccator were 75% and
0%, respectively. Analyses were conducted in triplicate.
2.6. Moisture content (MC)
Square samples (2 cm × 2 cm) from each film were cut and weighed.
They were then placed in a drying oven (OMH 180, Heraterm, Germany)
at 105 ◦ C for 24 h. Their dried weights were obtained. The moisture
content (MC) of the film samples was calculated according to Eq. (2).
Analyses were conducted in triplicate.

The thickness of the films was measured by the electronic digital
caliper (Scala, 111-B, Mexico City, Mexico) with a sensitivity of 0.01
mm. Measurements were performed at five different locations in the
sample, and then the average value for each film was calculated. Ana­
lyses were conducted in triplicate.
2.5. Water vapor permeability (WVP)
The WVP was measured according to the ASTM E96/E96M standard
method with some modifications according to the Gheribi method
(2018). Edible films were placed in glass vials (with an average diameter
of 2.5 cm and a height of 7.5 cm) containing about 15 g of silica gel (to
adjust for approximately 10 mm distance between the sample and the
silica gel). The glass vials, well-sealed, were kept at an adjusted tem­
perature and humidity at 25 ◦ C and 75%RH. The transmitted water­
–vapor was defined by the weight addition of the cell, and slopes
%MC = 100 × (Wi − Wd )/Wi (2)
Where Wi (g) is the initial weight, and Wd (g) is the dry weight of the
samples.
2.7. Water solubility (WS)
The prepared films were dried in the oven at 105 ◦ C for 24 h to obtain
their initial dry weight. The samples were then soaked in water for 24 h
at room temperature. The film samples were dried again under the same
conditions to obtain their final dry weight. The water solubility of the
film samples was calculated according to Eq. (3). Analyses were con­
ducted in triplicate.
Water solubility = 100 × (W1 − W2 )/W1 (3)
Where W1 is the initial dry weight (g), and W2 is the final dry weight (g).

3
N. Roshandel-hesari et al.
2.8. Opacity
The opacity of the sample was determined by a UV–Vis spectro­
photometer (libera s 22, Shimadzu, Kyoto, Japan) with a wavelength of
600 nm. The opacity of the films was obtained by Eq. (4) (Marvdashti,
Koocheki, & Yavarmanesh, 2017). Analyses were conducted in
triplicate.
Opacity = Abs600 /L
Where Abs600 was the value of absorbance at 600 nm and L was the film
thickness (mm).
2.9. Mechanical properties
According to Wu (2014), Tensile strength (TS) and elongation at
break (EAB) were evaluated by using a texture analyzer (Zwick 25 kN,
Germany) according to D882-97 (ASTM, 1999) with some
modifications.
Three rectangular strips measuring 20 mm × 60 mm were cut from
each film to determine their mechanical properties. The average thick­
ness of each film strip was used to estimate the cross-sectional area of the
sample. The initial distance of the grips and head speed were adjusted to
30 mm and 1 mm/s, respectively. TS (MPa) was calculated by Eq. (5).
TS(MPa) = Fmax /A
Where Fmax is the maximum load (N) that is required to pull the sample
apart, A = cross-section area (square meters) of the sample. EAB was
calculated by the following equation.
EAB(%) = M/L0 × 100
Where M = edible film elongation (mm), L0 = primary grip length (mm)
of edible films.
2.10. Structural properties
2.10.1. Attenuated total reflectance-Fourier transforms infrared (ATR-
FTIR) spectroscopy
The Fourier transform infrared (FT-IR) spectra of the films were
investigated with a Bruker FT-IR spectrophotometer (Nicolet iS 10 Mid,
United States) equipped with a vertical ATR trough plate crystal cell
(ZnSe). All of the FT-IR spectra were taken with a resolution of 4 cm− 1 in
the range from 4000 to 400 cm− 1. They were then compared to a
background spectrum taken from a clean empty cell at room
temperature.
2.10.2. Field emission scanning electron microscopy (FE-SEM)
The cross-sectional and surface morphologies of the films were
observed with a scanning electron microscope (F16502, Phenom,
Eindhoven, Netherlands). The cross-sections of the samples were
exposed by fracturing the films in liquid nitrogen and sprayed with a
thin layer of gold and observed at 10 kV.
2.10.3. Gas chromatography–mass spectroscopy (GC–MS)
According to Ph. Eur. 9th edition, the analysis was carried out.
KONIK HRGC 5000C, Barcelona, spain, used an MS detector system
(running at 70 eV, 200 ◦ C for the ion source and 250 ◦ C for the interface
temperature) to perform the GC–MS analysis of diluted (1:10) OEO at an
injector temperature of 240 ◦ C. HP-5MS 5% phenylmethylsiloxane
capillary column (30 m × 0.25 mm, film thickness 0.25 µm) was used as
a capillary column. Helium was employed as a carrier gas, flowing at a
rate of 1.0 mL/min. KonikKrom Plus was used to collect the data. Data
were imported in the NIST Mass Spectral Search Program (Version 2.0 g)
and were compared to those in the NIST/EPA/NIH Mass Spectral
Database library (NIST 11). At long last, the Genesis algorithm, which is
Food Chemistry 396 (2022) 133650
part of the NIST Mass Spectral Search Program, was used to identify and
quantify the main peaks of this GC–MS spectrum.
2.11. Antioxidant activity
The antioxidant activity of edible films enriched with OEO was
determined using the DPPH free radical scavenging method. The end of
the reaction is marked by the purple discoloration of the DPPH free
(4)
radicals to the yellow color of hydrazine. 200 μL of each edible film
solution was added to 1 mL of methanol, which was then added to 2 mL
of a DPPH (0.1 mM) methanolic solution. Each solution was kept in the
dark for a half-hour, and the absorbance was recorded at 517 nm. DPPH
radical cation scavenging was expressed according to Eq. (7) (Shokri
et al., 2020). Analyses were conducted in triplicate.
A blank-A sample
DPPH Scavenging (%) = × 100 (7)
A blank
Where Ablank is the absorbance of blank, Asample is the absorbance of
edible film.
2.12. In-vitro antibacterial activity of the films
The antibacterial activity of the films was determined by the disk
diffusion method on Escherichia coli and Staphylococcus aureus bacteria.
(5) In brief, 100 μL of each bacterial suspension (106 CFU/mL) were spread
on MHA (Müller-Hinton Agar) plates. 10 mm diameter discs were cut
from each film, placed on the agar plates, and incubated for 24 h (37 ±
1 ◦ C). Finally, the diameters of the zone of inhibition (ZOI) were
measured by a digital caliper. Analyses were conducted in triplicate
(Khedri et al., 2021).
(6)
2.13. Coating of the cherry tomatoes
Cherry tomatoes were washed with distilled water for 5 min and left
to dry. Three groups of fruits were placed in plastic boxes (13 cm × 11
cm × 3 cm). Two cherry tomato groups were coated separately with
C1Z3 and C1Z3O1.5 film solutions for 3 min. In this study, uncoated
cherry tomatoes were considered the control group. All treated and
control cherry tomatoes were air-dried at room temperature for 30 min
before being stored at 4 ± 1 ◦ C and 45% RH for 32 days. The images of
the coated and control cherry tomatoes were taken from 0 to 32 days.
Each treatment consisted of three replicates of nine fruits each.
2.13.1. Shrinkage
The dimensions of cherry tomatoes were measured in two directions
of small and large diameters by an electronic digital caliper (Scala, 111-
B, Mexico City, Mexico) at different time intervals, and the shrinkage
percentage was obtained (Shakouri, Ziaolhagh, Sharifi-rad, Heydari-
majd, Tajali, Nezarat, & Da Silva, 2015). The percentage of fruit
shrinkage was expressed by Eq. (8).
Shrinkage (%) = 100 × (M0 − M1 )/M0 (8)
Where M0 is the geometric mean of the fruit dimensions at the initial
test, and M1 is the geometric mean of the fruit dimensions at each time.
2.13.2. Weight loss
The weight loss (WL) of cherry tomatoes was calculated by Eq. (9).
Weight loss = 100 × (M0 − M)/M0 (9)
M0 was the primary mass of cherry tomatoes at day 0 and M was the
mass of cherry tomatoes after storage time.
2.13.3. Titratable acidity (TA)
The cherry tomatoes were peeled off and minced to obtain a uniform
mixture. 5 g of crushed fruits were mixed with 50 mL of distilled water
4
N. Roshandel-hesari et al.
on a magnetic stirrer at 700 rpm for 30 min. 20 mL of supernatant was
titrated with NaOH (0.01 N) until pH 8.1. Titratable acidity was reported
as the percentage of citric acid.
2.14. Statistical analysis
Data was analyzed using SPSS 23 for Windows (Chicago, Illinois,
USA). In this study, all detection data was carried out in replicates (≥3)
and is reported as mean ± standard deviation. Statistical evaluations
were performed by one-way analysis of variance (ANOVA) and followed
by Duncan’s multiple tests. The overall significant differences were
considered p-value < 0.05.
Methods of analysis of in-vitro antifungal effects of the films,
microbiological analysis, and antifungal effect of coatings on
B. cinerea on cherry tomatoes are available in the Supplementary
Material file.
3. Results and discussion
3.1. Ratio selection of chitosan/casein/OEO
In this study, various ratios of CZ solutions (C1Z6, C1Z4, C1Z3, C1Z2,
C1Z1, C6Z1, C4Z1, C3Z1, and C2Z1) were prepared to make edible films
and tested to determine the best edible film. Based on the physico­
chemical properties, the C1Z3 ratio had the best result. The results are
present in Tables S1 and S2. The use of plasticizers such as glycerol leads
to an increase in the elongation at break and flexibility of the edible
films. Plasticizers increase polymer chain mobility by decreasing inter­
molecular forces, so it improves film flexibility. They also cause an in­
crease in elongation at break and reduces the tensile strength of the films
(Kurek et al., 2014).
The tensile strength of the casein-rich edible films was lower (from
13.426 MPa to 1.832 MPa) than that of the chitosan-rich ones (18.227
MPa to 25.320 MPa), but their elongation at break was higher (from
14.24 to 21.86 %) than that of the chitosan-rich ones (from 12.66 to 9.5
%). The moisture content of the casein-rich films was lower than others.
The moisture content of edible films produced in this study showed that
it ranged from 9.56 to 29.45%. The more hydrophilic nature and func­
tional groups of chitosan would increase the moisture content and water
solubility of the films. Furthermore, the moisture content results may be
related to the less dense lattice as indicated by the low tensile strength of
the films. Casein-rich films had more flexible chains and a less dense
lattice than chitosan-rich films. The tensile strength of the casein-rich
film was lower than chitosan-rich content film. Thus, they had low
moisture content. The loose structure of the films with higher casein
volume ratios has lower water retention capacity due to their low tensile
strength (Cao, Yang, & Song, 2018). Because of this, water molecules
were quickly and easily removed from these films during the preparation
of the edible film and evaporated.
In this work, the C1Z3 and C1Z2 have good water vapor transfer rates
(WVTR), water vapor permeability (WVP), and the best physical prop­
erties (tensile strength). WVTR, and WVP were significantly different in
these two samples (p < 0.05). The C1Z3 had the lowest WVP, WVTR,
water solubility, and acceptable tensile strength, by 0.242 × 10− 4 (g.
mm/Cm2.h.KPa), 0.85 × 10− 3 (g/Cm2.h), 41.33% and 13.247(MPa),
respectively. The C1Z6 and C1Z4 had the lowest WVP, and WVTR, but
their mechanical strength (TS) was lower than the C1Z3 and C1Z2. As
shown in Table S2, a uniform and significant change in WVP is observed.
The permeability was increased in the treatments C1Z6 to C6Z1 by 0.165
to 0.572 (g.mm/Cm2.h.kPa × 10− 4) respectively. Chitosan poly­
saccharide is a hydrophilic polymer with low water resistance. There­
fore, reducing the WVTR and preventing WVP can be attributed to a
decrease in chitosan content or an increase in casein content (Cao et al.,
2018). Likewise, films with higher casein ratios showed lower water
solubility and MC than chitosan-rich films. A 39.17% to a 56.67% water
5
Food Chemistry 396 (2022) 133650
solubility range was found for films ranging in composition from C1Z6 to
C6Z1. The solubility of edible films with the various ratios of chitosan
and casein showed a significant difference (p < 0.05). Low water solu­
bility indicates that the film does not degrade quickly and could be used
as a suitable packaging, so the coated food is not easily damaged
(Marvdashti et al., 2017). High solubility makes the edible film easily
dissolve in water.
Increasing the thickness of the edible films can be due to the
increased amount of dry matter in the films. The thickness of the films
increased with increasing amount of casein or chitosan in the film
structure. The thickness of the casein-rich films from C1Z2 to C1Z6 was
0.056, 0.0633, 0.0667, 0.0753, and 0.0787 mm, respectively. The C2Z1,
C3Z1, C4Z1, and C6Z1 samples with more chitosan content were 0.0793,
0.0967, 0.103, and 0.113 mm thick, respectively. Chitosan’s hydrophilic
nature allows more water molecules to be incorporated into the film
matrix. This causes the film’s moisture content, solubility, and even
thickness to increase at higher ratios of chitosan. As a result, samples
with higher chitosan ratios have higher thickness and moisture content.
The opacity of the samples increased significantly with increasing the
amount of chitosan and casein. C6Z1, C4Z1, and C1Z6 had higher opacity
than others (p < 0.05) (Table S1).
Considering the parameters of WVP, WVTR, EAB, TS and opacity the
C1Z3 was selected as the best edible film. After determining the C1Z3 as
the best ratio, four different edible films with varying values of OEO
were made. C1Z3O0.5, C1Z3O1, and C1Z3O1.5 contained 0.5%, 1.0%, and
1.5% essential oil, respectively. In the following, the physicochemical
properties, structural properties, antioxidant, antibacterial, and anti­
fungal activity of the OEO-enriched edible films were investigated.
3.2. Physical properties
3.2.1. Film thickness
The thickness of the edible films containing various concentrations of
OEO is shown in Table S3. It is well known that the thickness of the films
is related to the total dry mass in the film-forming solution. Increasing
the concentration of OEO as an active ingredient in edible films caused a
significant increase in thickness. By increasing the OEO concentration
from 0 to 1.5%, the film thickness varied from 0.065 to 0.095 mm. The
sample thickness clearly increased with a significant difference (p <
0.05). This result is similar to chitosan edible films containing 0 to 5%
OEO that increased from 49.7 to 173.2 µm and also gelatin-chitosan
films incorporated with 0 to 4% OEO that raised from 27.04 to 29.63
µm (Wu et al., 2014).
3.2.2. Opacity
C1Z3 edible film with different concentration levels of OEO was more
opaque than edible film without OEO (Table S1). The opacity of the
films containing 0.5 to 1.5% of OEO was 0.917 to 1.31 nm/mm. The
opacity of the samples increased significantly after adding essential oil
(p < 0.05) (Supplementary Material Fig. S1). The increase in opacity
with the addition of EO may be due to EO globules in the film matrix
(Hosseini, Rezaei, Zandi, & Farahmandghavi, 2015). Increased opacity
in EO-containing films prevents light from passing through the film and
protects the food inside. Similar to our work, the opacity of edible films
increased from 1.6 to 2.14 (nm/mm) for gelatin–chitosan films by
addition of OEO (0%, 0.4%, 0.8%, 1.2% w/v) (Hosseini et al., 2015) and
also an increase opacity observed for Burdock Root Inulin/Chitosan
blend edible films containing 1%, 1.5% and 2% oregano and thyme
essential Oils (3.28–4.7(nm/mm)) (Cao et al., 2018).
3.2.3. Mechanical properties of the films
The effect of OEO concentration on mechanical properties of C1Z3
films containing OEO such as percentage of elongation at break (EAB)
and tensile strength (TS) is reported in Table S3. By increasing the OEO
concentration from 0 to 1.5%, the TS and EAB of biodegradable films
changed from 13.237 to 10.213 MPa and from 14.55% to 24.46%,
N. Roshandel-hesari et al.
respectively. As tensile strength is required to be greater than 3.5 MPa,
the mechanical strength of the films was acceptable. Significant differ­
ences were observed in TS and EAB values (p < 0.05). The inclusion of
essential oils such as OEO in polysaccharides or protein-based edible
films could increase the flexibility of the edible film by interfering with
polymer chain interactions and reducing polymer interactions. (Wu
et al., 2014).
The TS and EAB outcomes are comparable to gelatin–chitosan
biodegradable biocomposite films containing varying quantities of OEO,
whose values changed from 44.35 to 18.49 MPa and from 33.97 to 41.25
%, respectively (Hosseini et al., 2015). Burdock root inulin/chitosan
blend edible films containing 1, 1.5, and 2 % OT essential oils changed
from 26.85 to 17.08 MPa and from 19.98 to 25.42 % in TS and EAB,
respectively (Cao et al., 2018) These results confirm that the application
of essential oils such as OEO can increase the flexibility of the edible
film.
3.2.4. Water vapor permeability (WVP)
One of the most important functions of edible films is water vapor
permeability. The film should reduce the water exchange between the
edible product and the environment. Food quality degrades and spoils
faster due to the transfer of mass and moisture between the food and the
environment (Gheribi et al., 2018). Utilization of a film with hydro­
phobic components such as oils, fats, waxes, emulsifiers, and essential
oils could be applied to increase the separation of food moisture from the
environment (Hosseini et al., 2015). The WVP and WVTR of the chito­
san/casein edible films containing various concentrations of OEO are
shown in Table 1.
The WVP and WVTR of edible films decreased from 0.241 × 10− 4 to
0.187 × 10− 4 (g.mm/Cm2.KPa.h) and 0.884 × 10− 3 to 0.469 × 10− 3 (g/
Cm2.h) with the increase in concentration of the EO from 0 to 1.5%
respectively (p < 0.05). Due to the hydrophobic compounds of OEO,
such as monoterpenes, the hydrophobicity of OEO-enriched edible films
is increased. Increasing the hydrophobicity of the edible film reduces
water absorption (Wu et al., 2014).
Furthermore, the transfer of water vapor occurs through the hydro­
philic part of the edible film network and depends on the hydrophobic/
hydrophilic ratio of the film components. Similar behavior were also
observed for WVP of gelatin-chitosan films incorporated with 0 to 4% of
OEO (11.66 × 10− 11 to 9.58 × 10− 11 g/m.Pa.s) and in burdock root
inulin/chitosan blend films containing OT essential oils decreased from
3.74 × 10− 9 to 3.15 × 10− 9 g/m.Pa.s (Wu et al., 2014, Cao et al., 2018).
Accordingly, a hydrophobic phase in a small proportion, limits water
vapor permeability and water vapor transmission rate in edible films
(Tongnuanchan, Benjakul, & Prodpran, 2012).
3.2.5. Water solubility and moisture content
The effects of OEO concentration on moisture content and water
solubility of films are reported in Table 1. The results showed significant
Table 1
Physical properties of the C1Z3O edible films.
C1Z3O Edible MC (%) WS (%) WVP × 10− 4 (g. WVTR × 10− 3 A Clevenger apparatus was used to distill OEO with a yield of 2%.
films mm/Cm2.h.KPa) (g/Cm2.h)
C1Z3 14.18 ± 41.36 ± 0.241 ± 0.008c 0.884 ± 0.012d
0.098a 0.28d
C1Z3O0.5 13.81 ± 37.80 ± 0.222 ± 0.007c 0.667 ± 0.059c
0.16b 0.61c
C1Z3O1 12.58 ± 34.14 ± 0.203 ± 0.016b 0.565 ± 0.042b
0.13c 0.023b
C1Z3O1.5 10.95 ± 31.79 ± 0.187 ± 0.009a 0.469 ± 0.035a
0.12d 0.080a
MC: Moisture Content; WS: Water Solubility; WVP: Water Vapor permeability;
and WVTR stands for Water Vapor Transmission Rate.
Means in the same column with different letters are significantly different (p <
0.05). Data (n = 3) reported as mean values ± SD.
6
Food Chemistry 396 (2022) 133650
differences in the values of water solubility and moisture content (p <
0.05). Low solubility indicates that edible film does not degrade quickly
and can be used as a suitable packaging so the coated food is not easily
damaged. High solubility makes the edible film easily dissolve in water
and reduces its ability to hold water. The water solubility and moisture
content of edible films containing 0 to 1.5% of OEO were from 41.36 to
31.79% and 14.18 to 10.95%, respectively.
In this test, the C1Z3O1.5 had the lowest water solubility and moisture
content. The incorporation of OEO in the chitosan/casein film decreased
the water solubility and moisture content of the films. The increased
hydrophobicity of edible films was attributed to terpenoid and phenolic
compounds of the essential oil. Burdock root inulin/chitosan blend
edible films have similar water solubility and moisture content results.
The water solubility and moisture content of edible films containing 1,
1.5, and 2% OT essential oils ranged from 35.15 to 32.41 % and 17.38 to
12.23 %, respectively (Cao et al., 2018).
3.3. Structural properties
3.3.1. FT-IR analysis
The FT-IR spectra of the film samples were merged in Fig. 1. Chito­
san/casein edible films showed designated peaks at 3288 cm− 1 related
to the OH stretching vibration of the hydroxyl group in the carbohy­
drate. This peak is also demonstrated in the edible films containing OEO.
The absorption peak at 2924 cm− 1 corresponds to the asymmetric
stretching vibration of aliphatic CH from casein and OEO. The absorp­
tion peak of about 1650 cm− 1 corresponded to the stretching vibration
of amide carbonyl in the casein structure. The peak of about 1410 cm− 1
corresponds to the OH bending vibration of the hydroxyl group in the
chitosan and OEO structures. The absorption peak of about 1030 cm− 1 is
related to the stretching vibration of the C–O–C bond in chitosan. The
stretching vibration of the NH and NH2 bonds of casein protein in the D
was also observed at 3400 and 3550 cm− 1.
3.3.2. FE-SEM analyses
Surface and cross-section SEM images of C1Z3 edible films and C1Z3
edible films incorporated with OEO were illustrated in Fig. 2. The C1Z3
edible film without OEO had a homogeneous surface and showed a
smooth and compact cross-section. OEO microdroplets increase with
increasing of concentration in the film and its effect could be seen on the
FE-SEM surface and cross-section images.
The presence of microdroplets in the FE-SEM images is due to the
difference in the fat-like structure of OEO compared to chitosan and
casein molecules. This difference in the properties of the film compo­
nents causes a discontinuous system. Changes in the hydrophobic
properties of OEO constituents relative to chitosan and casein cause
changes in the microstructure of the edible film, as well as a reduction in
WVP and TS of the films containing OEO compared to the C1Z3. The EO
microdroplets can reduce water transfer through the edible film matrix
(Wu et al., 2014).
3.3.3. Volatile compounds in oregano essential oil
Gas chromatography–mass spectrometry (GC–MS) was used to identify
the essential oil’s chemical constituents. The relative concentrations of
the main compounds in the oil were determined after calculating the
area under the curve using the Genesis algorithm in the NIST program
(Table S4). The major components of OEO was carvacrol, γ-terpinene
and o-cymene by 57.8, 13.5 and 5.8% respectively. Yoncheva et al.
(2021) found that o-cymene/m-cymene, carvacrol, terpinolene, and
γ-terpinene accounted for 39.44, 29.80, 20.82, and 4.05% of their OEO,
which is in line with our findings. Variation in EOs compounds is
influenced by a number of variables, including plant species, harvesting
seasons, and geographical origins. These results are in accordance to
those previously published (Radünz et al., 2021).
N. Roshandel-hesari et al. Food Chemistry 396 (2022) 133650

Fig. 1. FT-IR spectroscopy of C1Z3 (A), C1Z3O0.5 (B), C1Z3O1 (C), and C1Z3O1.5 (D) edible films.

Fig. 2. FE-SEM images of surface and cross-section of C1Z3 (A), C1Z3O0.5 (B), C1Z3O1 (C), and C1Z3O1.5 (D) edible films.

3.4. Antioxidant activity
The antioxidant activity of the edible films was evaluated based on
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and is
shown in Table S5. The range of DPPH radical scavenging ability was
17.32% to 70.40%. DPPH radical scavenging of the chitosan/casein
edible film was only 17.32%, and for the C1Z3O0.5, C1Z3O1, and C1Z3O1.5
films, it clearly increased by 38.03, 50.98, and 70.40%, respectively (p
< 0.05). OEO-treated quince seed mucilage films showed comparable
antioxidant activity, as demonstrated by Jouki, Yazdi, Mortazavi, &

7
N. Roshandel-hesari et al. Food Chemistry 396 (2022) 133650

Koocheki, 2014. The DPPH radical scavenging activity of quince seed

Samples Control C1Z3 C1Z3O1.5
mucilage films was 18.39 %. Higher activity was observed in films
containing 0, 1, 1.5, and 2% OEO by 45%, 56%, and 61%, respectively.
The antioxidant activity of the films showed a growing trend by
increasing the OEO content loaded in the chitosan/casein films. The Day 0
higher antioxidant activity of essential oil-enriched film is due to the
phenolic compounds and terpenoids such as carvacrol and thymol in the
OEO (Yoncheva et al. (2021). The results showed that the edible films
containing OEO acted as stronger donors of hydrogen atoms, electrons, Day 4
and reduced the purple DPPH• radical to yellow DPPH-H. The antioxi­
dant activity of the films was illustrated in (Fig. S2).

Day 8
3.5. In-vitro antibacterial activity of the films

The antibacterial activity of chitosan/casein edible films containing

various concentrations of OEO was tested against Staphylococcus aureus
Day 12
and Escherichia coli as a gram-negative and gram-positive bacteria.
(Table 2 and Fig. S3). Chitosan/casein film did not show antibacterial
activity; however, all chitosan/casein edible films containing OEO
showed suitable antibacterial activities against both the E. coli and
Day 16
Staphylococcus aureus bacteria. Inhibition zones with diameters of 14.7,
22.0, and 22.5 mm were observed on E. coli for edible films with 0.5%,
1%, and 1.5% OEO, respectively. Furthermore, inhibition zones with
diameters of 13.9, 17.1, and 19.3 mm were observed on Staphylococcus
Day 20
aureus for edible films with 0.5%, 1%, and 1.5% OEO, respectively (p <
0.05). However, the positive control tetracycline antibiotics show inhi­
bition zones of 18.5 and 14.7 mm on E. coli and Staphylococcus aureus
bacteria, respectively. The results show that the edible films with 1%
Day 24
and 1.5% OEO had more potent antibacterial activity than tetracycline.
Antibacterial activity against E. coli and Staphylococcus aureus bacteria is
mainly ascribed to thymol and carvacrol, which could disturb the bac­
terial cell membrane (Langeveld, Veldhuizen, & Burt, 2014). Cell walls
Day 28
of Gram-negative bacteria are surrounded by an exterior lipopolysac­
charide membrane. Due to the hydrophobic qualities of essential oils,
which allow them to permeate the lipid membrane of bacterial cells,
OEO-containing films displayed increased antibacterial effectiveness in
preventing the growth of gram-negative bacteria. This feature disrupts Day 32
the structure of the bacterial membrane and eventually leads to leakage
of the contents out of the bacterial cell. It has been reported that the
number of phenolic components in an essential oil increases its anti­ Fig. 3. The visual appearance of edible coated (C1Z3, C1Z3O1.5) and uncoated
bacterial activity (Burt, 2004). cherry tomatoes (control group) during storage at 4 ◦ C (0–32 days).

3.6. Coating of the cherry tomatoes mold-free and smooth. After 8 days of storage, shrinkage was observed
in control fruits. The C1Z3 and C1Z3O1.5 coated cherry tomatoes seemed
In this part of study, the use of C1Z3 and C1Z3O1.5 as edible coatings identical for the first 12 days.
to increase the shelf-life of cherry tomatoes was investigated. The results During the fruit storage period, uncovered cherry tomatoes showed
showed that C1Z3 and C1Z3O1.5 coatings were able to delay changes in the most shrinkage, weight loss, and mold growth. At the end of storage,
titratable acidity (TA), shrinkage percentage, and weight loss compared cherry tomatoes with the C1Z3O1.5 coating showed the least shrinkage
to control group (uncovered cherry tomatoes) during storage at 4 ◦ C (p and weight loss. Therefore, the C1Z3O1.5 layer maintained the quality
< 0.05). and firmness of the cherry tomatoes for 32 days. In contrast, the
In general, titratable acidity (TA), weight loss, shrinkage percentage, apparent quality of the fruits of the C1Z3 coating group was acceptable
and fruit appearance photographs were performed every 4 days in three for up to 20 days of refrigerated storage.
replications. Fig. 3 shows the appearance of cherry tomatoes stored at 4◦
C for 32 days. From the beginning, the skin of the cherry tomato was 3.6.1. Titratable acidity (TA)
Ascorbic acid and citric acid make up the majority of the cherry to­
Table 2 mato fruit’s titratable acidity Barreto et al., 2016(. The acidity of control
Antibacterial activity of C1Z3O edible films. group, cherry tomatoes covered with C1Z3, and C1Z3O1.5 decreased from
0.34%, 0.35% and 0.35% to 0.09%, 0.12% and 0.15%, respectively,
Edible film Escherichia coli (mm) Staphylococcus aureus (mm)
after 32 days of storage. Increased storage time considerably decreased
C1Z3 0 ± 0a 0 ± 0a the titratable acidity of uncovered cherry tomatoes. There was a sig­
C1Z3O0.5 14.7 ± 0.051b 13.9 ± 0.11b
C1Z3O1 22 ± 0.040c 17.1 ± 0.092c
nificant difference between group treatments in the tests on the same
C1Z3O1.5 22.5 ± 0.039d 19.3 ± 0.050d days (p < 0.05) (Table S6, Fig. 4). Metabolic biochemical activities such
Antibiotic (Tetracycline) 18.5 ± 0.070e 14.7 ± 0.049e as ATP synthesis can be carried out using organic acid breakdown.
Means in the same column with different letters are significantly different (p < Higher titratable acidity is consistent with a decrease in the metabolism
0.05). Data (n = 3) reported are mean values ± SD. of organic acids (Barreto et al., 2016). A similar result has been reported

8
N. Roshandel-hesari et al.
Fig. 4. Weight loss (A), Shrinkage (B), and titratable acidity (C) of edible coated (C1Z3, C1Z3O1.5) and uncoated (control) cherry tomato fruits in 32 days.
for other chitosan/essential oil edible films in cherry tomatoes and to­
matoes (Dovale-Rosabal, Casariego, Forbes-Hernandez, & Garcia,
2015).
3.6.2. Weight loss and shrinkage
Weight loss is an important parameter for the consumer and could be
directly related to reduced fruit quality and fungal infections (Pobiga,
Przybyl, Zubernik, & Gniewosz, 2020). Shrinkage is the deformation of
food volumes such as fruits or vegetables during storage. Evaporation
and transpiration significantly lower the water content of fruit. Due to
the transfer of moisture of natural pores and respiration, fruits and
vegetables can shrink and lose weight. Hydrophobic essential oils, on the
other hand, help improve the semi-permeable membrane coating’s
characteristics by preventing water from passing through, which re­
duces sample weight loss and shrinkage (Perdones, Sanchez-Gonzalez,
Chiralt, & Vargas, 2012). The weight loss of coated and uncoated
cherry tomato fruits at different times is shown in Table S6 and Fig. 4.
Weight loss during the storage time increased for all cherry tomato fruits
(p < 0.05). However, C1Z3 and C1Z3O1.5 coated fruits showed less weight
loss than the uncoated fruits. In this study, at the end of storage (32
days), the weight loss in control group was 42.78%, but the C1Z3 and
C1Z3O1.5 coatings were 29.18% and 17.88%, respectively. There was a
significant difference between the group treatments on the same day test
(p < 0.05). At the end of storage time, the uncoated cherry tomatoes
showed the highest shrinkage, 66.84%, while the C1Z3, C1Z3O1.5 coat­
ings exhibited lower shrinkage, 45.1% and 31.12%, respectively
(Table S6 and Fig. 4). Similar results for peach gum polysaccharides as
an edible coating to prevent cherry tomato weight loss have been
observed (Pobiga et al., 2020).
Result and discussion of in-vitro antifungal effects of the films,
microbiological analysis, and antifungal effect of coatings on
B. cinerea on cherry tomatoes are available in the Supplementary
Material file.
4. Conclusion
In this study, due to the high antibacterial, antioxidant, and anti­
fungal properties of OEO, it was used as a bioactive agent in combina­
tion with chitosan and casein to produce efficient, biodegradable edible
films to increase food shelf life. Due to mechanical and physical
9
Food Chemistry 396 (2022) 133650
properties, the best ratio of chitosan and casein (C1Z3) was determined
to prepare the base or the film. Then, based on physicochemical prop­
erties, antibacterial, antioxidant, and antifungal activity tests, it was
determined that 1.5% of essential oil had the greatest effect on the films.
OEO-enriched films showed strong inhibitory activity against Escher­
ichia coli and Staphylococcus aureus, as well as significantly increased
DPPH radical scavenging activity. Films containing essential oils and
somewhat non-essential oils also showed good antifungal activity
against the growth of B. cinerea. Along with antioxidant, antibacterial,
and antifungal activities, other properties of edible films were also
studied. These included mechanical properties (tensile strength (TS) and
elongation at break (EAB)), physicochemical properties (film thickness,
opacity, water solubility, moisture content, and water vapor perme­
ability), and structural properties (FTIR, SEM, GC–MS).
Study of coatings with and without essential oil on cherry tomatoes
and storage at 4 ◦ C confirmed the bioactive properties of OEO to
maintain fruit quality. The coatings were able to delay changes in
titratable acidity (TA), shrinkage percentage, and weight loss compared
to control fruits during storage. The shelf life of the cherry tomatoes at a
cold temperature was increased to 32 days. In the end, we can say that
chitosan-casein-OEO edible films can be used in the food industry as an
effective biodegradable packaging material for long-term fruit storage.
CRediT authorship contribution statement
Narjes Roshandel-hesari: Formal analysis, Investigation, Method­
ology, Software, Visualization. Majid Mokaber-Esfahani: Conceptual­
ization, Supervision, Writing – review & editing. Akram Taleghani:
Formal analysis, Software, Investigation. Reza Akbari: Validation.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
The authors do not have permission to share data.
N. Roshandel-hesari et al. Food Chemistry 396 (2022) 133650

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