Food Packaging and Shelf Life 19 (2019) 147–154

Contents lists available at ScienceDirect

Food Packaging and Shelf Life

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

Antioxidant and antimicrobial properties of carbohydrate-based films T

enriched with cinnamon essential oil by Pickering emulsion method
⁎
Hadi Fasihia,b, Nooshin Noshirvanic, , Mahdi Hashemid, Mohammad Fazilatie, Hossein Salavatie,
Véronique Comaf
a
Department of Medicinal and Aromatic Plants, University of Applied Science & Technology of Jihad-E Agriculture, Hamedan, Iran
b
Hamedan Agricultural and Natural Resources Research and Education Center, Hamedan, Iran
c
Department of Food Science and Technology, Tuyserkan Faculty of Engineering & Natural Resources, Bu-Ali Sina University, Hamedan, Iran
d
Faculty of Chemistry, Bu-Ali Sina University, Hamedan, Iran
e
Department of Biology, Payame Noor University, Tehran, Iran
f
University of Bordeaux, UMR 5629 CNRS, LCPO, 16 Avenue Pey Berland, F-33607 Pessac, France

A R T I C LE I N FO A B S T R A C T

Keywords: The overall aim of this research is to introduce a novel approach for maintaining a greater amount of cinnamon
Carboxymethyl cellulose essential oil (CEO) in the material. We used the Pickering stabilization method, in order to increase the anti-
Cinnamon essential oil oxidant and antifungal properties of carboxymethyl cellulose (CMC)-polyvinyl alcohol (PVA) based films. The
Pickering emulsion light transmission, antioxidant and antifungal properties of the films were studied. In addition, the effect of the
Bread packaging
packaging was studied on the shelf life of bread. The results showed great improvement of the antifungal and
antioxidant properties of the prepared films. The films containing 1.5 and 3% CEO were highly effective against
P. digitatum and showed complete inhibition in in vitro and in vivo tests. Moreover, the films exhibited a good UV
inhibitory effect. Consequently, the CEO Pickering emulsion was an ideal alternative for incorporation with
CMC-PVA based films for increasing the shelf life of bread.

1. Introduction
About one-third of the food produced in the world for human con-
sumption, equivalent to 1.3 billion tons is wasted annually (FAO,
2011). In this regard, microbiological food spoilage is a serious problem
which restricts the shelf life of foods and causes a considerable loss of
food products. Moreover, these contaminations have adverse effects on
human health due to the production of toxins and causing of various
diseases. Nan1 is the most eaten food in Iran, whereas its consumption is
approximately 2 times of the average world consumption. Poor quality
and low price of bread lead to a great loss of bread and bakery products
of about 30% in Iran with 300 million dollars a year (Ghanbari &
Shahedi, 2008). The shelf life of bread is generally restricted by two
factors of staling and fungal growth. According to the above-mentioned
items, finding a solution to these problems seems to be necessary for the
maintenance of limited food resources in the world.
A common method for preservation of bread and bakery products is
direct addition of weak organic acids such as propionic, benzoic, and
sorbic acid (Muhianldin, Hassan, & Saari, 2013). However, due to the
side effects of chemical preservatives to the human health, consumers
are demanding for preservative-free foods. Moreover, additional
amounts of the chemical additives have adverse effects on the sensorial
properties of food (Van Long, Joly, & Dantigny, 2016).
Alternatively, production of an active packaging system by addition
of antimicrobial agents into the packaging material and controlled re-
lease of the volatile compounds into the headspace of packaging during
their shelf life inhibits the fungal development in bread and bakery
products (Noshirvani, Ghanbarzadeh, Mokarram et al., 2017). Active
packaging involves interactions between food and packaging to in-
crease the shelf life of food; either by migration into the headspace or
direct contact with food (Manso, Cacho-Nerin, Becerril, & Nerín, 2013).
Plants containing beneficial phytochemicals, have antioxidant and
antimicrobial properties and therefore are suitable additives for bio-
based active packaging. A number of spices, extracts, and essential oils
(EOs) have been tested as antimicrobial compounds in recent years
(Burt, 2004; Noshirvani & Fasihi, 2018). Among different EOs,

⁎
Corresponding author.
E-mail address: n.noshirvani@basu.ac.ir (N. Noshirvani).
1
Common Iranian bread.

https://doi.org/10.1016/j.fpsl.2018.12.007
Received 8 August 2018; Received in revised form 14 November 2018; Accepted 20 December 2018
Available online 02 January 2019
2214-2894/ © 2018 Elsevier Ltd. All rights reserved.
H. Fasihi et al.
cinnamon essential oil (CEO) is not harmful when consumed, as it is a
natural preservative and flavoring substance which considered as
GRAS2 by FDA3 (Kaskatepe et al., 2016). Cinnamon (Cinnamomum
zeylanicum) belongs to Lauraceae family and usually grows in South and
South-East Asia. The strong antimicrobial and antioxidant properties of
CEO have been proved by previous studies (Chao, Gary-Young, &
Oberg, 2000; Lopez, Sanchez, Batlle, & Nerin, 2005).
EOs are highly sensitive to light, oxidation, and thermal decom-
position (Cossu, Wang, Chaudhari, & Nitin, 2015). Moreover, due to
probable interactions between the compounds of EOs and the food in-
gredients, the antioxidant and antimicrobial effects of EOs in food
systems are lower than that of in vitro. All of the above-mentioned items
have limited the application of EOs as antioxidant and antimicrobial
agents in food preservation. As a solution, increasing the stability of
EOs in the films can help to protect the EOs and may lead to increase
their biofunctional effects during the shelf life of food.
Pickering emulsion is a novel way to this end. Pickering emulsion
involves the stabilization of O/W or W/O emulsions by solid particles
instead of surfactants. The stabilizing mechanism of Pickering emulsion
refers to the formation of a solid layer around the EOs and leading to
the prevention of the contact between oil and water phases, which, in
turn, causes higher emulsion stability regarding aggregation in com-
parison to those of conventional emulsions. Besides stabilizing effect of
the Pickering emulsions, they act as carriers of antimicrobial com-
pounds and protect these compounds from the effects of the outside
environment, especially oxidation (Dickinson, 2017).
Recently, we have shown that the Pickering emulsion can be used in
active packaging (Fasihi, Fazilati, Hashemi, & Noshirvani, 2017). Re-
garding the importance of bread as a vital food resource in Iran and
other Third World countries, the main idea of this study was to develop
a system to increase the shelf life of bread and therefore decrease a lot
of economic costs. In addition, we tried to introduce a chemical method
for increasing the effectiveness of the system by applying Pickering
emulsion for incorporation of CEO into the matrix of films. It is inter-
esting to note that the choice of cinnamon, in addition to its significant
antimicrobial properties, is related to the organoleptic compatibility
with bread's flavor, since it is usually used as a flavoring agent for
bakery products.
2. Material and methods
2.1. Materials
Cinnamon bark was purchased from the local market of Hamedan.
Sodium carboxymethyl cellulose (high viscosity, 1500–3000 cP), poly-
vinyl alcohol (degree of hydrolysis: 87–89%), tween 80, oleic acid,
glycerol, and sodium carbonate were provided from Sigma Aldrich
(USA). Potato dextrose agar (PDA) was obtained from Merck Millipore
(Darmstadt, Germany). Folin–Ciocalteu and 2, 2-diphenyl-1-picrylhy-
drazyl (DPPH) reagents were purchased from Sigma Aldrich (USA). All
other consumed materials were of analytical grade.
2.2. Preparation of Pickering emulsion and active films
Pickering emulsions were prepared by in situ hydrophobization
method by using oleic acid (OL) as a hydrophobic compound according
to previous reports (Binks, Muijwijk, Koman, & Poortinga, 2017; Ikem,
Menner, & Bismarck, 2009; Sadeghpour, Pirolt, & Glatter, 2013;
Santini, Guzman, Ferrari, & Liggieri, 2014). To this end, CMC (1% w/v)
and PVA (5% w/v) solutions were dissolved under magnetic stirring at
50 °C until complete dissolving in two separate flasks. Afterward, both
solutions were mixed together and stirred again for 15 min. Then, OL
2
Generally Recognized as Safe.
3
Food and Drug Administration.
148
Food Packaging and Shelf Life 19 (2019) 147–154
(0.3% v/v) was added to produce O/W emulsion after addition of tween
80 (10% wt. OL) for emulsification of OL into the film forming solutions
(FFS). Then, the solution was stirred for 15 min, and homogenized by
using a high-speed homogenizer (IKA-Ultraturrax T25, USA) for 15 min.
Afterward, CEO at different concentrations (0.5, 1.5 and 3%) was added
to the mixture and stirred for 15 min using a magnetic stirrer at
500 rpm, then sonicated using an ultrasound probe (Bandelin Sonopuls,
Germany) for 15 min. Glycerol (30 wt.% dry matter) was added to the
FFS and after string for 25 min, all FFSs were degassed under vacuum
pump until all air removal from the solutions. The control sample was
prepared as explained above without any addition of EOs and without
ultrasonic treatment. Finally, each FFS (50.0 mL for each film) was
poured into the Petri dishes and dried at 25 °C for 72 h. The films were
then gently peeled off from the Petri dishes and kept until tests. In
addition, for the microbial test (micro-atmosphere method) some Petri
dishes were prepared with pouring the FFS on the lids of the Petri
dishes and were not peeled off from the plates.
2.3. Characterization of suspension
The mean droplet diameter of the suspensions was measured by
dynamic light scattering (DLS) (Nano series, Malvern Instruments Ltd,
UK) at 25 ⁰C. The stability of emulsions over 8 weeks at 25 ⁰C was
measured by evaluating the changes that occurred at the height of the
emulsion phase compared to the height of the total test system.
2.4. Characterization of films
2.4.1. Light microscopy
A Leica DME (Leica Microsystems, United States) light microscope
was used with 10 X magnifications.
2.4.2. Scanning electron microscopy (SEM)
A SEM (TOPCON SM-200 SEM) microscopy was used to characterize
the surface microstructure of the films (under low vacuum at a voltage
of 20 kV). The films were prepared by depositing the samples on the
copper holder then coating by gold under vacuum.
2.4.3. UV–vis spectroscopy
The UV–vis spectra of the films were recorded by using a spectro-
photometer (UV-1800, Shimadzu, Kyoto, Japan) at two wavelengths of
280 and 600 nm. The air was used as a reference.
2.4.4. Antioxidant activity
2.4.4.1. DPPH radical scavenging assay. The antioxidant activity of the
films was evaluated by DPPH method modified according to the study
of Woranuch, Yoksan, and Akashi, (2015). Briefly, each film was cut
into thin strips (2 × 1.5 cm) and dissolved in 5 mL methanol, then
stirred (500 rpm) for 5 days. Afterward, 2 mL of the methanol solution
and 2 mL of the DPPH solution were mixed together and stirred for
2 min in a dark place. Then, the solution was kept at room temperature
in darkness for 1 h. The absorbance was read against pure methanol
(control) at 517 nm and the percentage of DPPH radical scavenging
activity was calculated using (Eq. (1)):
Ablank − Asample
DPPH scavenging activity (%) = × 100
Ablank (1)
Where, Ablank is the absorbance of the control, and Asample is the
absorbance of the sample.
2.4.4.2. Total phenolic content (TPC). The TPC of the films was
determined by using the Folin–Ciocalteu colorimetric method
according to the Slinkard and Singleton (1977). Briefly, 0.05 mL of
the obtained methanol solution (explained at 2.4.4.1 section) was
mixed with 0.5 mL of Folin–Ciocalteu reagent for 8 min in dark place.
Then, 1 mL of sodium carbonate (20%, w/v) solution and 8.45 mL of
H. Fasihi et al. Food Packaging and Shelf Life 19 (2019) 147–154

water were added, then stirred and kept for 2 h at ambient temperature. serum (NaCl 0.9% w/v), then mixed under aseptic conditions for 5 min
The absorbance was read at 760 nm using a UV–vis spectrophotometer at 260 rpm. After that, serial 10-fold dilutions (10−1-10-5) were pre-
model SPECORD 210 (Analytik Jena, Germany). The results were pared and inoculated into the Petri dishes which containing about
expressed as mg gallic acid equivalent (GAE) per gram film (mg 15 mL of solidified PDA media. The Petri dishes were incubated at 25 °C
GAE/g film). for 5 days, then the number of yeasts and molds were counted and
expressed in a logarithm scale (log CFU/g).
2.4.5. In vitro antifungal activity of the films
The antifungal properties of the films were tested against the growth 2.4.8. Statistical analyses
of P. digitatum by both disc diffusion and micro-atmosphere methods. P. The statistical analysis of the data was performed through ANOVA
digitatum (PTCC 5251) was purchased from the Iranian type culture using SPSS software
collection (Tehran, Iran). (version16, SPSS Inc., Chicago, IL0 software). The differences
among mean values were detected by Duncan’s multiple range test
2.4.5.1. Disc diffusion method. Briefly, 100 μL of the fungal suspension (P < 0.05). All data were expressed as means ± standard deviation.
(adjusted to 106 spores/mL using physiological water and Neubauer Different letters indicated significant differences (P < 0.05) among
hemocytometer (Simax Kavalier)) was spread on the solidified PDA data.
culture media. Then, the films (diameter 4 mm) were deposited on the
surface of the PDA medium. Afterward, the films were sealed by
3. Results and discussion
Parafilm®, and incubated at 25 °C for 5 days. A digital ruler was used
to measure the diameter of the clear zone around the films. The
3.1. Characterization of suspensions
antifungal index (%) was calculated according to the Eq. (2)
(Balaguer, Lopez-Carballo, Catala, Gavara, & Hernandez-Munoz, 2013):
To evaluate the physical stability of the prepared emulsions, the
Antifungal index (%) = [(1 – Da)/Db] × 100 (2) mean droplet diameter, and emulsion stability of the CMC-PVA sus-
pension alone and incorporated with 0.5, 1.5 and 3% CEO over 8 weeks
Where, Da is the diameter of the growth zone of the film sample with EO
of storage were measured. The results are illustrated in Fig. 1 (A–B).
and Db is the diameter of the growth zone of the control sample.
The mean diameter of the particles increased during the storage time.
However, the control showed higher increase in the particle size com-
2.4.5.2. Micro-atmosphere method. The micro-atmosphere method was pared to the Pickering emulsions. Increasing the size of droplets in
used to investigate the antifungal efficacy of the films in no direct control may be due to the aggregation of dispersed phase over the time
contact between the film and fungal suspension according to the
Balaguer et al. (2013). The surface of PDA culture medium was
inoculated with 100 μL of the fungal suspension (106 CFU/mL). The
Petri dish lids containing the dried films (explained at Section 2.2) were
then assembled to the inoculated Petri dishes, and were carefully sealed
by Parafilm® to prevent any leakage of the volatile compounds. The
Petri dishes were then incubated at 25 °C, and the colony diameters
were measured after 5 days of incubation using a digital ruler. The Petri
dish without any film on its lid was served as a control. Three
replications were performed for each sample and the antifungal index
was calculated according to the Eq. (2).

2.4.6. Antimicrobial effect of active films on bread slices

The antifungal effectiveness of all films on the shelf life of wheat
bread was tested according to Noshirvani, Ghanbarzadeh, Mokarram
et al. (2017). The wheat bread slices without any chemical pre-
servatives were purchased from a local supermarket (Hamedan, Iran).
Briefly, the bread slices were sandwiched between two pieces of films
and were packaged in polyethylene bags (30 × 17 cm2). All the pack-
aged slices of bread were kept to the incubator (25 °C) until visual
observation of fungal growth. For each sample, three replications were
prepared. The control sample was a packaged bread slice without any
film.

2.4.7. Determination of antifungal properties of active coatings in sliced

wheat bread
In order to have the maximum contact between the active com-
pounds and bread slices, active coatings were prepared as a model
system to better estimate of the microbial growth in bread slices.
Briefly, 50.0 mL of each FFS was coated on each bread pieces by a
sterilized paint brush and the coats were then dried under ventilation in
aseptic conditions for 1 h. The prepared coated bread slices were
packed under plastic bags (30 × 17 cm2) and kept at 25 °C for 15 days.
The numbers of yeasts and molds in bread samples were counted ac-
cording to Otoni, Pontes, Medeiros, and Soares, (2014) with some
modifications. Briefly, 10.0 g of each sliced bread was aseptically Fig. 1. Mean droplet diameter (A) and emulsion stability (B) of the CMC-PVA
weighted and poured into a flask containing 90.0 mL physiological and those suspensions incorporated with 0.5, 1.5 and 3% CEO.

149
H. Fasihi et al. Food Packaging and Shelf Life 19 (2019) 147–154

Fig. 2. Optical images (10X) of the CMC-PVA (A) and, CMC-PVA-CEO 0.5% (B) films; and surface SEM images of the CMC-PVA (C) and CMC-PVA-CEO 0.5% (D–E)
films.

by means of aggregation or coalescence of droplets. Comparison of
Pickering suspensions indicated a slight increase in the mean droplet
size with increasing the concentration of CEO. The results of emulsion
stability (Fig. 1-B) suggesting higher stability for all Pickering emulsion
suspensions in comparison to the common emulsion suspensions since
they exhibit relatively constant mean droplet of particles over the sto-
rage time. The higher stability of Pickering emulsion compared to that
of the control can attribute to the formation of a rigid layer around the
EOs which prevents the contact between oil and water phases. This can
lead to more stability of emulsion against aggregation in compared to
that of conventional emulsions. According to the Hashemi, Ziaee,
Eskandari, and Hosseini, (2017), the particle size of the emulsified EOs
has a significant effect on their functional properties. Thus, the higher
emulsion stability and the smaller droplet size over the time in the
Pickering emulsion solutions probably have a significant effect on the
antioxidant and antimicrobial activities of the prepared films.

150
H. Fasihi et al. Food Packaging and Shelf Life 19 (2019) 147–154

Table 1
Light transmittance of the CMC-PVA film and those incorporated with 0.5, 1.5
and 3% CEO.
Film type T 280 nm (%) T 600nm (%)

CMC-PVA 43.32 ± 1.08a 73.28 ± 0.46a

CMC-PVA-CEO 0.5% 14.12 ± 1.03b 59.15 ± 1.25b
CMC-PVA- CEO 1.5% 10.36 ± 2.21c 45.35 ± 1.28c
CMC-PVA-CEO 3% 8.05 ± 1.32d 34.12 ± 2.98d

Averages in each column with different letters indicate a significant difference

(P < 0.05).

3.2. Characterization of films

3.2.1. Films microstructure

Fig. 2 illustrates the optical (Fig. 2: A–B) and SEM (Fig. 2: C–E)
images of the pure CMC-PVA film and those incorporated with 0.5%
CEO. The control film (CMC-PVA) showed a very smooth surface (Fig. 2
A and C) without pores or cracks, endorsing excellent compatibility
between CMC and PVA (Fig. 2 A and C). After the incorporation of CEO
to the film matrix, a very regular structure with a good distribution of
droplets was observed. The droplets exhibited almost the same size
which corresponded to be CEO (Fig. 2 B and D). Also, the formation of
Pickering emulsion and the presence of a layer around the CEO globules
can be clearly seen (Fig. 2-E). These observations are fully correlated
with the obtained results from the emulsions properties discussed be-
fore.

3.3. UV–vis spectroscopy

The light transmission at selected wavelengths (UV: 280 nm and
visible: 600 nm) are shown in Table 1. The percentage of transmittance
at 280 nm decreased from 43.32 to 14.12, 10.36 and 8.05% for the pure
CMC-PVA and those incorporated with 0.5, 1.5 and 3% CEO, respec-
tively. This seggested substantially UV absorbance effect of the films
added CEO. This could be due to the strong absorbance of cinna-
maldehyde as the main component of CEO at 287 nm, which resulted in
a notable UV absorbance properties (Wu et al., 2015). Similarly, the
percent of transmittance at 600 nm decreased with increasing the CEO
content. This could be attributed to the interruption of light passage by
the CEO droplets distributed in the polymer matrix. Generally, the high
UV absorbance is a good factor in food packaging in which restricts
lipid oxidation in food products. The results suggested that this
packaging material could be used for light-sensitive food products.
3.4. Total phenolic content (TPC) and antioxidant activity
The TPC and DPPH scavenging activity of the CMC-PVA film and
those incorporated with CEO are shown in Fig. 3 (A–B). As expected,
the CMC-PVA film exhibited no significant TPC and DPPH scavenging
activity. However, the addition of CEO to the FFS increased the TPC (to
6.99 mg GAE /g film) and DPPH scavenging effect (to 67%). Indeed, the
higher the CEO content in the dry film, the higher the DPPH scavenging
value and so, the greater the antioxidant power. CEO has been widely
known to possess good antioxidant activity (Singh, Maurya, & Catalan,
2007; Singh et al., 2007; Atarés, Bonilla, & Chiralt, 2010). The anti-
oxidant effect of CEO is mainly due to the cinnamaldehyde as its main
constituent (> 70%) according to our previous study (Noshirvani,
Ghanbarzadeh, Gardrat et al., 2017). Nonetheless, it is worth men-
tioning that EOs are a complex combination of different components,
and their biological activity correspond to a synergistic effect of dif-
ferent molecules (Singh et al., 2007).
Taking into account previous studies, EOs may undergo rapid de-
pletion within foods, due to physical diffusion within the food system or
degradation. Therefore, the antioxidant properties of the EOs is less
than that of pure state, when used inside the film. Accordingly,
Fig. 3. TPC (mg GAE /g film) and DPPH radical scavenging activity (%) of the
pure CMC-PVA film and those incorporated with 0.5, 1.5 and 3% CEO.
Perdones, Vargas, Atarés, and Chiralt, (2014) reported higher anti-
oxidant effect of pure CEO compared to the CEO added to the chitosan
films. They attributed this behavior to the loss of CEO added to the films
during film preparation, drying, storage, and oxidation. It seems that
the encapsulation of EOs within a solid coating by Pickering emulsion
preparation method has a protective effect against adverse external
factors. Moreover, according to the study of Jiménez, Domínguez,
Pascual-Pineda, Azuara, and Beristain, (2017), the particle size of the
emulsion droplets has a significant effect on the antioxidant activity of
cinnamon added films. The reduction of the droplets size leads to an
increase of the surface area to react with the oxygen present in the
system, and therefore the antioxidant activity increases. In fact, the
mechanisms of increasing the biological activity of the emulsions with
relatively smaller droplet size correspond to i) decreasing the ag-
gregation of droplets, and increasing the stability of the emulsion; and
ii) increasing the surface area for biological interactions.
3.5. Antifungal effects of active films (In vitro test)
3.5.1. Disc diffusion method
The results of the antifungal activity of films as a function of CEO
content by the disc diffusion method are shown in Fig. 4-A. As expected,
the control film (CMC-PVA) exhibited no antifungal activity against P.
digitatum. The results revealed that the addition of CEO to the film
matrix had a remarkable antifungal activity. 48% of fungal inhibition
was observed for the films containing 0.5% CEO. The higher the CEO
content, the greater the diameter of the inhibition zone. Complete
fungal inhibition of P. digitatum were obtained for the films with 1.5
and 3% CEO. The antimicrobial compounds of CEO mainly cinna-
maldehyde diffuse into the culture medium and result in a clear zone
surrounding the films. Efficient antifungal effect of cinnamon oil
against Aspergillus and Penicillium sp. were found by Hossain, Follett,
Vu, Harich, and Salmieri, (2016). Damage of the cell wall, plasma
membrane destruction, loss of cytoplasm and mitochondria, cell

151
H. Fasihi et al.
Fig. 4. Antifungal effects of the pure CMC-PVA film, and those incorporated
with CEO (0.5, 1.5 and 3%) against P. digitatum in disc diffusion (A) and micro-
atmosphere methods (B).
folding, and inhibitory effects on enzymatic systems are of the main
mechanisms for antifungal activity of cinnamon oil (Manso et al., 2014;
Xing et al., 2014).
Taking into account the study of Wen et al. (2016), entrapment of
CEO into cyclodextrin would improve the release of CEO into the agar
medium by affecting its solubility. Based on this, it seems that the
covering of CEO by a solid layer through the formation of a Pickering
emulsion, may protect the CEO from environmental effects and im-
proves its effectiveness as an antimicrobial and antioxidant agent. In
addition, showing low particle size of the CEO droplets in Pickering
emulsion is another critical factor which increases the contact surface
between CEO and fungi.
3.5.2. Micro-atmosphere method
The micro-atmosphere method was performed, in order to evaluate
the indirect effects of active films against P. digitatum (Fig. 4-B). As
already shown above by disc diffusion results, no fungal inhibition was
found for CMC-PVA film. Addition of 0.5% CEO, led to 12% inhibition
of fungal growth. Higher antifungal effects were obtained by adding
higher amounts of CEO. 28 and 50% fungal growth inhibition were
observed for the films incorporated with 1.5 and 3% CEO, respectively.
The films exhibited higher antifungal effects in disc diffusion test
compared to the micro-atmosphere assays. This could be attributed to
the fact that in disc diffusion test both direct contact and migration of
active compounds from the film to the outside caused antimicrobial
effects, while only the migration of the volatile compounds to the
152
Food Packaging and Shelf Life 19 (2019) 147–154
Table 2
The number of days after visual observation mold growth in bread slices at
25 °C.
Packaging type Growth delay (days)
Control (bread without film) 4 ± 0.46 d
CMC-PVA 6 ± 0.5 c
CMC-PVA-CEO 0.5% 30 ± 2.45 b
CMC-PVA-CEO 1.5% NG ± 0.0 a
CMC-PVA-CEO 3% NG ± 0.0 a
N.G: No mold growth detected.
Averages with different letters indicate a significant difference (P <
0.05).
headspace, may cause this effect in micro-atmosphere method.
3.6. Antifungal effects of active films on bread shelf life (In vivo test)
In order to evaluate the performance of active packaging on food
products, in vivo antifungal effects of the pure CMC-PVA film and those
containing CEO (0.5, 1.5 and 3%) were investigated on bread slices
(Table 2 and Fig. 5). The control showed the earliest mold growth after
4 days of storage at 25 °C. Longer shelf life was found for the bread kept
in active films. Fungal growth was observed after 6 days when the CMC-
PVA film was used and more for films containing CEO. The higher the
CEO content, the greater the shelf life of sliced bread. Thus, the films
enriched with 1.5 and 3% CEO showed no fungal growth over the 60
days of incubation. Exhibiting complete inhibition from mold growth
over a long storage time in bread slices may be attributed to the sta-
bilization of the CEO in the film matrix by the Pickering emulsion
method. This method may preserve the sufficient concentrations of
active compounds in the headspace of packaged bread by slowing down
the release of active compounds from the film. Generally, the release of
antimicrobial compounds from a film or coating has a critical effect on
their efficiency, i.e. the controlled release of active compounds required
to ensure the availability of a certain concentration of a compound on
the food surface. Moreover, application of Pickering emulsion may in-
crease the stability of CEO by reducing the destructive effects of the
environment on CEO, resulting in an increase of its efficiency. The re-
sults are in agreement with the in vitro tests results (disc diffusion and
micro-atmosphere). In addition, the antimicrobial results followed the
same tendency than antioxidant activity.
3.7. Antifungal effects of active coatings on bread shelf life
The number of yeasts and molds over 15 days of incubation at 25 °C
are shown in Table 3. An increase in the number of yeasts and molds
over the storage time were observed for the control and the bread
stored with CMC-PVA coating. However, no fungal growth was ob-
served for active coatings at studied concentrations (0.5, 1.5 and 3%),
suggesting a complete fungal growth inhibition on bread slices. The
results are fully consistent with the results of the in vitro tests. Ob-
servation of high antimicrobial activity in coatings could be attributed
to the high stability of the film forming emulsion through the Pickering
emulsion, which increases its performance by maintaining a greater
amount of CEO against environmental degradation effects. Moreover,
slowing down the release of CEO from the film to the headspace of
packaging may lead to maintain sufficient concentrations of active
compounds in the packaging over the storage time of bread samples.
The comparison of the films and coatings indicates the higher effec-
tiveness of coatings than the films in controlling the fungal develop-
ment in bread slices. It may probably attribute to the more contact
surface area between bread slices and active compounds in the coatings
in comparison to the films. In addition, the presence of lower air be-
tween the coatings and bread slices in coated slices of bread compared
to the films may lead to lower mold growth, as mold growth only occur
H. Fasihi et al. Food Packaging and Shelf Life 19 (2019) 147–154

Fig. 5. Slices of bread stored at different packaging (A): without film, (B): the CMC-PVA film, and the films incorporated with 0.5 (C), 1.5 (D) and 3% (E) CEO after 30
days storage at 25 °C.

Table 3 Acknowledgments
Numbers of yeasts and molds (log CFU/g) during the storage time at 25 °C.
Days Control CMC-PVA CMC-PVA- CMC-PVA- CMC-PVA-
The authors are gratefully acknowledging the Bordeaux Imaging
CEO 0.5% CEO 1.5% CEO 3% Center, a service unit of the CNRS-INSERM and Bordeaux University,
and kind effort of Isabelle Svahn.
0 2.35 ± 0.02d 1.92 ± 0.02c NDa NDa NDa
3 5.0 ± 0.03c 4.33 ± 0.03b NDa NDa NDa
References
7 6.26 ± 0.03b 6.11 ± 0.03a NDa NDa NDa
15 7.46 ± 0.04a 6.71 ± 0.04a NDa NDa NDa
Atarés, L., Bonilla, J., & Chiralt, A. (2010). Characterization of sodium caseinate-based
N.D: No total counts detected. edible films incorporated with cinnamon or ginger essential oils. Journal of Food
Engineering, 100(4), 678–687.
Averages in each column with different letters indicate a significant difference
Balaguer, M. P., Lopez-Carballo, G., Catala, R., Gavara, R., & Hernandez-Munoz, P.
(P < 0.05). (2013). Antifungal properties of gliadin films incorporating cinnamaldehyde and
application in active food packaging of bread and cheese spread foodstuffs.
in the surface of bread due to the presence of enough air in the surface International Journal of Food Microbiology, 166, 369–377.
Binks, B., Muijwijk, K., Koman, H., & Poortinga, A. T. (2017). Food-grade Pickering
compared to the inner parts of bread. stabilization of foams by in situ hydrophobisation of calcium carbonate particles.
Food Hydrocolloids, 63, 585–592.
Chao, S. C., Gary-Young, D., & Oberg, C. J. (2000). Screening for inhibitory activity of
essential oils on selected bacteria, fungi and viruses. Journal of Essential Oil Research,
4. Conclusion
12, 639–649.
Cossu, A., Wang, M. S., Chaudhari, A., & Nitin, N. (2015). Antifungal activity against
The Pickering emulsion method was used to increase the anti- Candida albicans of starch Pickering emulsion with thymol or amphotericin Bin sus-
pension and calcium alginate films. International Journal of Pharmaceutics, 493,
oxidant and antimicrobial activities of active packaging enriched with
233–242.
CEO. The films showed superior anti-UV properties which is a good Dickinson, E. (2017). Biopolymer-based particles as stabilizing agents for emulsions and
factor for food packaging application. Moreover, the results of anti- foams. Food Hydrocollioids, 68, 219–231.
oxidant and antifungal properties of the prepared films demonstrated FAO (2011). Global food losses and food waste. Extent, causes and prevention. Rome1–29.
Fasihi, H., Fazilati, M., Hashemi, M., & Noshirvani, N. (2017). Novel carboxymethyl
promising findings. The active films stabilized by the Pickering emul- cellulose-polyvinyl alcohol blend films stabilized by Pickering emulsion incorpora-
sion method displayed complete antifungal activity in vitro test at 1.5 tion method. Carbohydrate Polymers, 167, 79–89.
and 3% of CEO. In addition, the application of these packagings for Ghanbari, M., & Shahedi, M. (2008). Effect of baking time and temperature on Taphtun
bread quality and staling. Science and Technology of Agriculture and Natural Resources
bread preservation showed 100% antifungal activity over the storage Journal, 43, 327–333.
time at 1.5 and 3% of CEO. Our results indicated efficient antimicrobial Hashemi, G. H., Ziaee, E., Eskandari, M. H., & Hosseini, S. M. (2017). Characterization of
and antioxidant properties of active films, possibly due to the Pickering basil seed gum-based edible films incorporated with Zataria multiflora essential oil
nanoemulsion. Carbohydrate Polymers, 166, 93.
stabilization effect on the CEO by several mechanisms: i) generation of Hossain, F., Follett, P., Vu, K. D., Harich, M., & Salmieri, S. (2016). Evidence for sy-
a uniform and regular structure of dispersed phase throughout the nergistic activity of plant-derived essential oils against fungal pathogens of food. Food
matrix of the films leading to an increase of the contact between CEO Microbiology, 53, 24–30.
Ikem, V. O., Menner, A., & Bismarck, A. (2009). High internal phase emulsions stabilized
and fungi; ii) controlled and regular release of CEO from the film to the
solely by functionalized silica particles. Angewandte Chemie International Edition,
outside, which could help to maintain sufficient antimicrobial and an- 48(4), 632.
tioxidant agents in the headspace; and iii) protection of CEO from Jiménez, M., Domínguez, J. A., Pascual-Pineda, L. A., Azuara, E., & Beristain, C. I. (2017).
Elaboration and characterization of O/W cinnamon (Cinnamomum zeylanicum) and
oxidation against external undesirable effects, which could increase its
black pepper (Piper nigrum) emulsions. Food Hydrocolloids, 77, 902–910.
efficiency as an active compound. The obtained results suggest that Kaskatepe, B., Kiymaci, M. E., Suzuk, S., Erdem, S. A., Cesur, S., & Yildiz, S. (2016).
active films containing CEO stabilized by Pickering emulsion method Antibacterial effects of cinnamon oil against carbapenem resistant nosocomial
have a good potential to preserve bread. Acinetobacter baumannii and Pseudomonas aeruginosa isolates. Industrial Crops and
Products, 81, 191–194.
Lopez, P., Sanchez, C., Batlle, R., & Nerin, C. (2005). Solid- and vapor phase antimicrobial

153
H. Fasihi et al.
activities of six essential oils: Susceptibility of selected foodborne bacterial and fungal
strains. Journal of Agricultural and Food Chemistry, 53, 6939–6946.
Manso, S., Cacho-Nerin, F., Becerril, R., & Nerín, C. (2013). Combined analytical and
microbiological tools to study the effect on Aspergillus flavus of cinnamon essential
oil contained in food packaging. Food Control, 30(2), 370–378.
Muhianldin, B. J., Hassan, Z., & Saari, N. (2013). Lactic acid bacteria - R & D for food,
health and livestock purposes. In M. Kongo (Ed.). Lactic acid bacteria in biopreservation
and the enhancement of the functional quality of bread (pp. 155–172). Rijeka, Croatia:
Intech.
Noshirvani, N., & Fasihi, H. (2018). Control of Aspergilus niger in vitro and in vivo by
three Iranian essential oils. International Food Reserach Journal, 25(4), 1745–1752.
Noshirvani, N., Ghanbarzadeh, B., Gardrat, C., Rezaei, M. R., Hashemi, M., Le Coz, C.,
et al. (2017). Cinnamon and ginger essential oils to improve antifungal, physical and
mechanical properties of chitosan-carboxymethyl cellulose films. Food Hydrocolloids,
70, 36–45.
Noshirvani, N., Ghanbarzadeh, B., Mokarram, R. R., & Hashemi, M. (2017). Novel active
packaging based on carboxymethyl cellulose-chitosan-ZnO NPs nanocomposite for
increasing the shelf life of bread. Food Packaging and Shelf Life, 11, 106–114.
Otoni, C. G., Pontes, S. F., Medeiros, E. A., & Soares, N. D. F. (2014). Edible films from
methylcellulose and nanoemulsions of clove bud (Syzygium aromaticum) and oregano
(Origanum vulgare) essential oils as shelf life extenders for sliced bread. Journal of
Agricultural and Food Chemistry, 62(22), 5214–5219.
Perdones, A., Vargas, M., Atarés, L., & Chiralt, A. (2014). Physical, antioxidant and an-
timicrobial properties of chitosan–cinnamon leaf oil films as affected by oleic acid.
Food Hydrocolloids, 36, 256–264.
154
Food Packaging and Shelf Life 19 (2019) 147–154
Sadeghpour, A., Pirolt, F., & Glatter, O. (2013). Submicrometer-sized Pickering emulsions
stabilized by silica nanoparticles with adsorbed oleic acid. Langmuir, 29, 6004–6012.
Santini, E., Guzman, E., Ferrari, M., & Liggieri, L. (2014). Emulsions stabilized by the
interaction of silica nanoparticles and palmitic acid at the water-hexane interface.
Colloids Surface A, 460, 333–341.
Singh, G., Maurya, S., & Catalan, C. A. (2007). A comparison of chemical, antioxidant and
antimicrobial studies of cinnamon leaf and bark volatile oils, oleoresins and their
constituents. Food and Chemical Toxicology, 45(9), 1650–1661.
Slinkard, K., & Singleton, V. L. (1977). Total phenol analyses: Automation and compar-
ison with manual methods. American Journal of Enology and Viticulture, 28, 49–55.
Van Long, N. N., Joly, C., & Dantigny, P. (2016). Active packaging with antifungal ac-
tivities. International Journal of Food Microbiology, 220, 73–90.
Wen, P., Zhu, D. H., Wu, H., Zong, M. H., Jing, Y. R., & Han, S. Y. (2016). Encapsulation of
cinnamon essential oil in electrospun nanofibrous film for active food packaging.
Food Control, 59, 366–376.
Woranuch, S., Yoksan, R., & Akashi, M. (2015). Ferulic acid-coupled chitosan: Thermal
stability and utilization as an+ antioxidant for biodegradable active packaging film.
Carbohydrate Polymers, 115, 744–751.
Wu, J., Liu, H., Ge, S., Wang, S., Qin, Z., Chen, L., et al. (2015). The preparation, char-
acterization, antimicrobial stability and in vitro release evaluation of fish gelatin
films incorporated with cinnamon essential oil nanoliposomes. Food Hydrocolloids,
43, 427–435.
Xing, F., Hua, H., Selvaraj, J. N., Zhao, Y., Zhou, Y., & Liu, X. (2014). Growth inhibition
and morphological alterations of fusarium verticillioides by cinnamon oil and cinna-
maldehyde. Food Control, 46, 343–350.