Food Hydrocolloids 45 (2015) 150e157

Contents lists available at ScienceDirect

Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Characterization of physical, mechanical, and antibacterial properties

of agar-cellulose bionanocomposite films incorporated with savory
essential oil
Maryam Atef a, Masoud Rezaei a, *, Rabi Behrooz b
a
Department of Seafood Processing, Faculty of Marine Sciences, Tarbiat Modares University, P.O. Box 46414-356, Noor, Iran
b
Department of Wood and Paper Technology, Faculty of Natural Resource, Tarbiat Modares University, P.O. Box 46414-356, Noor, Iran

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

Article history: In this study, different concentrations of savory essential oil (0.5, 1, and 1.5%) were incorporated into
Received 25 February 2014 agar-based nanocomposite films as active packaging in order to evaluate their physical, mechanical, and
Accepted 25 September 2014 antimicrobial properties. X-ray diffractometry (XRD) and scanning electron microscopy (SEM) were
Available online 18 November 2014
performed to explain the structural properties and morphology of these films. Incorporation of savory
essential oil (SEO) into nanocomposite films decreased tensile strength and Young's modulus, but
Keywords:
increased the percent elongation at break. In addition, water solubility decreased when SEO was
Nanocomposite film
incorporated into the films, whereas it did not significantly affect water vapor permeability. The films
Agar
Savory essential oil
containing SEO were more effective against gram-positive bacteria (Listeria monocytogenes, Staphylo-
Antimicrobial activity coccus aureus and Bacillus cereus) than gram-negative bacteria (Escherichia coli). Results suggested that
Active packaging the agar-based nanocomposite films containing SEO can be used as active packaging for improving the
safety and shelf-life of foodstuff.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction
In recent years, consumers greatly demand for the higher
quality and longer shelf-life of foodstuff. One of the main ways for
protecting foods and extending their shelf-life during storage and
distribution is the usage of active packaging (Rhim, 2013). Various
natural components have been utilized for the preparation of edible
films; but, among these materials, polysaccharides, because of their
excellent mechanical and structural properties and poor barrier
capacity against moisture transfer, have been extensively used for
the development of biodegradable films (Phan, Debeaufort, Luu, &
Voilley, 2005).
Agar (AG) is an unbranched polysaccharide extracted from the
marine algae of class Rhodophyceae such as Gelidium sp. and Gra-
nez, Lo
cilaria sp. (Gime  pez de Lacey, Pe
rez-Santín, Lo
 pez-Caballero,
& Montero, 2013). The most important attribute of agar is its ability
to form hard gels at very low concentrations (0.04%); also, it has
been broadly utilized as a gelling agent in processed foods, phar-
maceutical products, cosmetics, biotechnology, and medicine (Li,
* Corresponding author. Tel.: þ98 122 6254986; fax: þ98 122 6253499.
E-mail address: rezai_ma@modares.ac.ir (M. Rezaei).
Yu, Jin, Zhang, & Liu, 2008; Wu, Geng, Chang, Yu, & Ma, 2009).
Due to its thermoplastic, biocompatible, biodegradable, and mod-
erate water resistance, agar has been tested as an alternative source
for petroleum plastic packaging materials (Rhim, 2011). However,
agar films have some poor physical, mechanical, and thermal
properties that are needed for food packaging applications (Rhim &
Ng, 2007). Nowadays, the application of nanotechnology to
biopolymer films may open new possibilities for improving not
only their properties but also the cost-price efficiency (Espitia et al.,
2012; Rhim & Ng, 2007). In this regard, different nanoparticles have
been used as the reinforcement for the preparation of nano-
composite films, among which, cellulose nanowhiskers or cellulose
nanocrystals, owing to their high tensile strength and modulus,
have attracted significant interest as potential nanoreinforcements
during the last decade (George & Siddaramaiah, 2012). Because of
the reinforcement provided by the nanoparticles dispersed in the
polymer matrix, nanocomposite-based films exhibit markedly
improved mechanical, physical, thermal, and optical properties
compared to pure biopolymer-based films (Rhim & Ng, 2007).
Another strategy for improving the functionality of biopolymer-
based films in packaging is their activation using various types of
additives such as antimicrobials in order to extend the shelf-life of
foods (Quilaqueo-Gutie rrez, Echeverría, Ihl, Bifani, & Mauri, 2012).

http://dx.doi.org/10.1016/j.foodhyd.2014.09.037
0268-005X/© 2014 Elsevier Ltd. All rights reserved.
 M. Atef et al. / Food Hydrocolloids 45 (2015) 150e157
Nowadays, there is a preference for the use of natural active anti-
microbials, instead of synthetics such as plant extracts, for the
purpose of food preservation.
Antibacterial activity of extracts and essential oils of herbs and
species can be attributed to their high content of phenolic com-
pounds (Alizadeh et al., 2010; Mihajilov-Krstev et al., 2009). Sat-
ureja hortensis (summer savory) belonging to the Lamiaceae family
is an annual and aromatic herb that is widely distributed in many
parts of the world. Summer savory is most often used as a culinary
herb; but, it also has marked medicinal benefits. The whole herb,
especially the flowering shoots, is antiseptic, aromatic, carminative,
digestive, expectorant, and stomachic (Hadian, Ebrahimi, & Salehi,
2010). Composition of oils from S. hortensis has been also investi-
gated. Major components of savory are carvacrol (>55%), p-cymene
(1.95% and 12.30%), and g-terpinene (0.71% and 20.94%). In addition
to these compounds, smaller amounts of a-terpinene and terpinene
have been determined (Tozlu et al., 2011).
So far, a vast number of studies have been published about the
preparation of nanocomposite films; but, only a few studies
(Abdollahi, Rezaei, & Farzi, 2012; Alboofetileh, Rezaei, Hosseini, &
Abdollahi, 2014; Tunc & Duman, 2011) have been performed on
the characteristic of functional bionanocomposite films. Further-
more, to the best knowledge of the present authors, no compre-
hensive research has studied the effect of savory essential oil (SEO)
concentrations on the properties of agar nanocomposites in the
film form for food packaging.
Thus, the focus of the present study was to prepare functional
nanocomposite films from agar reinforced with cellulose nano-
particles and comprehensively evaluate the effect of SEO on the
properties of agar-based nanocomposite films such as physical,
mechanical, microstructure, and optical attributes as well as anti-
microbial activity against Listeria monocytogenes, Staphylococcus
aureus, Bacillus cereus, and Escherichia coli bacteria.
2. Materials and methods
2.1. Materials
Food grade agar, Glycerol, and Tween 80 were obtained from
Merck Co. Germany. Savory essential oil (S. hortensis) supplied from
Barij Co. (Kashan, Iran) and stored in dark container at 4  C until
used. For antimicrobial assays, Brain Heart Infusion Broth (BHI) and
Tryptic Soy Agar (TSA) were purchased from Quelab, Canada.
2.2. Bacterial strains and maintenance
All the stock cultures such as L. monocytogenes (PTCC 1298), E.
coli (PTCC 1330), B. cereus (PTCC 1154), and S. aureus (PTCC 25923)
were obtained from Persian Type Culture Collection (Tehran, Iran).
All the strains were reserved in Brain Heart Infusion Broth (BHI)
with the supplement of 30% glycerol and stored at 20  C until use.
Subculturing was performed on a monthly basis to preserve bac-
terial viability.
2.3. Preparation of antimicrobial films
Agar film solution was prepared by dissolving 1.5 g of AG in
100 mL of distilled water under vigorous mixing at 95  C for 30 min.
Glycerol was used as a plasticizer at 33% content based on dry agar
film (Tian, Xu, Yang, & Guo, 2011). In order to reduce aggregation
and good dispersion of nanoparticles, the suspension of nano-
crystalline cellulose (NCC) was sonicated for 5 min, then dispersed
in the agar solution and stirred by a magnetic stirrer for 2 h. Af-
terwards, Tween 80, as an emulsifier, was added to the film solution
at the level of 0.2% of essential oil. After stirring at 50  C for 30 min,
151
SEO was added to the mixture at 0.5, 1, and 1.5% concentrations and
homogenized by Ultra-Turrax (Wiggen Hauser, D-500, Germany) at
7000 rpm for 2 min (Ojagh, Rezaei, Razavi, & Hosseini, 2010). In
order to remove the dissolved air, the solution was degassed by
slow stirring. Then, it was cast into Petri dishes and dried in an oven
(40  C) for 24 h. After the removal of excess water, the samples were
peeled off from the Petri dishes and stored in a closed reservoir
prior to characterization. All the films were preconditioned in the
desiccators containing saturated magnesium nitrate solution at
25  C and 52.89% relative humidity prior to testing.
2.4. Characterization
2.4.1. X-ray diffractometry (XRD)
XRD measurements were performed using Philips X'Pert MPD
diffractometer (Eindhoven, Netherlands) by Co Ka radiation and X-
ray wavelength of 1.54 nm at 40 kV and 30 mA. Nanocomposite
films were analyzed between 2q ¼ 5 and 40 with the angle size of
2q ¼ 0.02 at speed of 1 /min at room temperature.
2.4.2. Scanning electron microscopy (SEM) analysis
Scanning electron microscopy (Philips XL 30) was used to
examine the surface of agar/cellulose nanocomposite films with
and without SEO. The pieces were cut from the films, held by an
aluminum tape, and sputtered by gold in a BAL-TEC SCD 005 sputter
coater (BAL-TEC AG, Balzers, Liechtenstein). All the specimens were
examined by a scanning electron microscope (Philips, Eindhoven,
Netherlands) under high vacuum condition at the accelerating
voltage of 20.0 kV.
2.4.3. Film thickness
Film thickness was measured by a manual digital micrometer
(Mitutoyo, Mizonokuchi, Japan) with sensitivity of 0.001 mm, at 10
random positions for each film. The mean value was used in tensile
strength and water vapor permeability calculations.
2.4.4. Film solubility in water
To determine the percentage solubility of the films in water, the
procedure proposed by Tunc et al. (2007) was used. At first, the
specimens (4 cm  4 cm) were dried at 105  C for 24 h to measure
initial dry matter (Wi). Then, the dried nanocomposite films were
immersed in 50 mL of distilled water for 24 h at room temperature.
At the end of 24 h, the unsolubilized films were filtered using a
Whatman No. 1 filter paper. The remaining pieces of the samples
after immersion were dried at 105  C for 24 h to the constant final
dry weight (Wf). Values of water solubility (%) were determined in
triplicate for each treatment and calculated using the following
equation:

   .
Solubility in water % ¼ Wi  Wf  100 Wi (1)
2.4.5. Water vapor permeability (WVP)
WVP values of the nanocomposite films were determined using
a modified ASTM method E96-92, as described by Rhim (2011). The
samples were cut into circles and sealed on top of the permeation
cells containing 10 cc of distilled water (100% RH; 2.337  103 Pa
vapor pressure at 20  C). Then, the cells were stored in a desiccator
containing silica gel to provide constant RH at 20  C. Afterward, the
amount of water vapor transferred through the films was measured
using weight loss of the cell for the period of 8 h. WVP (1010 g/
ms Pa) of the films was measured by the following equation:
WVP ¼ W$X=A$t$DP (2)
152
Where W is weight gain of the cell (g), X is average film thickness
(m), and A, t, and DP are area of exposed film (m2), time (s) and
differential of partial water vapor pressure (Pa), respectively.
Average value of three replicates for each sample was also obtained.
2.4.6. Water contact angle (CA) measurement
Contact angle measurements of water on the surface of nano-
composite films were carried out using a PG-X goniometer (Model
PG-X, Switzerland). A water drop (5 mL) was placed on the film
surface with the dimensions of 2 cm  5 cm using a microsyringe at
room temperature. Reported values were the averages of five rep-
licates observed on different spots of the samples.
2.4.7. Swelling index
Kinetic test of swelling was evaluated by periodically measuring
weight increase of the sample films according to the method by
Lavorgna, Piscitelli, Mangiacapra, and Buonocore (2010). Film
specimens with the dimension of 2  2 cm were put into a desic-
cator (containing silica gel) for one week and weighed. Then, the
samples were immersed in the closed beakers containing 50 mL of
distilled water and stored at room temperature. Before each time of
weighing, the surface of all the film samples was dried by a filter
paper in order to remove the surface adsorbed water and weighing
was continued until reaching the equilibrium state at the selected
times. Swelling of the films was calculated using the following
equation:
Swelling ð%Þ ¼ ðWeight of wet film  Weight of dry filmÞ
 100=Weight of dry film
2.4.8. Visco analyzer
A Brookfield digital viscometer (Model LVDV-II þ P, USA) with
SC4-31 spindle was used to determine the rheological characteristic
of agar-based nanocomposite suspension containing various con-
centrations of savory essential oil at the speeds of 6, 10, 12, 20, 30,
50, 60, and 100 rpm before casting. Measurement was carried out in
about 15 mL of the film solution at 50  C.
2.4.9. Mechanical properties of the films
Tensile strength (TS), Young's modulus (YM), and elongation at
break (E%) of the nanocomposite films were evaluated by a Uni-
versal Testing Machine (Model TVT-300Xp, Perten, Sweden).
Measurements were performed according to ASTM standard
method D 882-02 (ASTM, 2002). The film samples were prepared in
rectangular pieces (2.54 cm  10 cm) and their thickness was
measured at 10 points. The films were then stretched with 50 mm
of initial grip separation and 5 mm min1 of cross-head speed.
Mechanical properties of the samples were also measured for five
times and average of the results was calculated.
2.4.10. Color, light transmission, and opacity measurements
Lightness (L), redness (a), and yellowness (b) color system was
used to evaluate the color of films by a colorimeter (BYK Gardner,
USA). Measurements were performed on white standard back-
grounds (L* ¼ 94.41, a* ¼ 0.78, b* ¼ 0.51) and color difference (DE),
whiteness index (WI), chroma (Cab * ), and hue (h* ) were calculated
ab
using the following equations:
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2 2
DE ¼ ðL*  LÞ þ ða*  aÞ þ ðb*  bÞ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
WI ¼ 100  ð100  L* Þ þ a*2 þ b*2
M. Atef et al. / Food Hydrocolloids 45 (2015) 150e157
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
*
2 2
Cab ¼ a* þ b* (6)
 
b*
h*ab ¼ 180 þ arctg When a < 0 (7)
a*
Light barrier properties of the film samples were measured by
scanning the samples at wavelengths between 200 and 800 nm
using a UVevisible spectrophotometer. Also, the opacity of the
agar-based nanocomposite films (with the dimension of 1  4 cm)
was evaluated by measuring absorbance at 600 nm using a UV/VIS
spectrophotometer (Model UNIC 4802 UV/VIS Double Beam, China)
according to the method by Alboofetileh, Rezaei, Hosseini, and
Abdollahi (2013). Opacity of the samples was calculated using the
following equation:
Opacity ¼ Abs600 =X (8)
Where Abs600 is a value of absorbance at 600 nm and X is the film
thickness (mm).
2.5. Microbiological analysis
2.5.1. Evaluation of antimicrobial activity of essential oil,
nanocomposite films, and film solutions
For this purpose, 10 ml of 106 (CFU)/ml of each bacterial strain (L.
monocytogenes, S. aureus, B. cereus, and E. coli) was spread on tryptic
soy agar (TSA) by steel swab, which was cultured for 24 h in brain
heart infusion broth (Quelab, Canada) at 37  C (108 CFU/ml). 40 ml of
(3) SEO for well test (6 mm diameter) and sterile filter paper discs
(6 mm in diameter, Whatman no.1) impregnated with 10 ml of
essential oil were used for disc diffusion. For disc diffusion and
vapor-phase diffusion methods, circular pieces of different films
(1.5 cm in diameter) were placed on the surface of TSA and on the
lids of TSA plates, respectively. Also, for determining the antimi-
crobial activity of the film solutions, 40 ml of different film-forming
solutions was added to TSA wells (6 mm in diameter). All the plates
were incubated at 37  C for 24 h. After incubation, the microbial
growth and inhibition degree of essential oil were illustrated as
follows: þþþ: totally inhibited; þþ: partially inhibited; þ: slightly
inhibited; -: no inhibition (Go mez-Estaca, Lo pez de Lacey, Lo pez-
Caballero, Go mez-Guille n, & Montero, 2010). Also, the inhibition
zones surrounding the film areas were reported for disc diffusion
(Ojagh et al., 2010); but, for vapor-phase diffusion, the whole zone
area was measured as “zone of inhibition” according to Lo'pez-
Caballero, Go'mez-Guille n, Pe'rez-Mateos, and Montero (2005).
2.6. Statistical analysis
Analysis of variance (ANOVA) followed by Duncan's multiple
range test was used to determine any significant differences among
the treatments at 95% confidence level (p  0.05) and all the data
were reported in the form of mean ± SD.
3. Results and discussion
3.1. X-ray diffractometry (XRD)
XRD spectra of the agar/cellulose nanocomposite films with and
without essential oil are presented in Fig. 1. As can be seen from
XRD graph, pure agar film showed two characteristic crystallinity
2
(4) peaks at 2q ¼ 11.27 and 23.01 which was also observed in the
nanocomposites. When NCC incorporated in the agar matrix, a new
angle of diffraction peak was generated at 2q ¼ 26.27. This dif-
(5) fractograms suggested that NCC reinforced agar films as well as the
 M. Atef et al. / Food Hydrocolloids 45 (2015) 150e157 153

3.2. Determination of nanocomposite microstructure using SEM

Fig. 2 shows SEM micrographs of the surface nanocomposite

films with different SEO contents in their formulation. The control
film had a homogeneous, smooth, and continuous microstructure
without irregularities, pores, or cracks. This indicated cellulose
nanoparticles were uniformly dispersed in the agar matrix (Fig. 2a).
Presence of SEO in the nanocomposite films affected the structure
of the samples and caused discontinuities in the matrix polymer
(Fig. 2bec) so that, at high concentration of the SEO (1.5%), cracks
and accumulation of oil particles to the coarser shape were
observed at the surface of films (Fig. 2d). Also, the images showed
that the number of oil droplets increased at the highest SEO content
that was related to the flocculation of oil particles on the surface of
films. In addition, when oil concentration increased in the formu-
lation, the polymer chains might be weakened because of greater
molecular contact between the film solution and SEO, making the
matrix more open, and consequently creating cracks in the sam-
ples. This could increase of water vapor permeability and decrease
the tensile strength of the films (Bonilla, Vargas, Atare s, & Chiralt,
Fig. 1. XRD patterns of agar-based nanocomposite films with different concentration of
2012). This observation was in agreement with the previous
SEO.
study (Ahmad, Benjakul, Prodpran, & Agustini, 2012), which has
incorporated the bergamot and lemongrass essential oils into
gelatin films.
crystallinity of the agar films was increased by incorporating of
NCC. Previous studies have reported the same behavior for
3.3. Water solubility (WS)
alginate-NCC (Abdollahi, Alboofetileh, Rezaei, & Behrooz, 2013) and
chitosan-NCC nanocomposite films (Khan et al., 2012). However,
WS is considered an indicator for the resistance of the film
addition of savory essential oil at different concentrations did not
sample to water. Generally, higher water solubility indicates lower
effect on the structure of nanocomposite films (Fig. 1a, b, c). Similar
water resistance. For food packaging applications where water ac-
result was reported by Abdollahi et al. (2012) who indicated that
tivity is high, or when the film must be in contact with water and
the structure of chitosan/clay nanocomposite films is not affected
act as food protective, low solubility of films is one of the most
by the presence of the rosemary essential oil.
important requirements. As well WS is also related to the

Fig. 2. Scanning electron microscopic images of surface of agar/cellulose nanocomposite films: (a) Control, (b) agar/cellulose nanocomposite with 0.5% (v/v) SEO, (c) agar/cellulose
nanocomposite with 1% (v/v) SEO and (d) agar/cellulose nanocomposite with 1.5% (v/v) SEO.
154 M. Atef et al. / Food Hydrocolloids 45 (2015) 150e157

Table 1
Physical and mechanical properties of agar-based nanocomposite films with SEO.

SEO content Thickness Solubility WVP Young Tensile Elongation Contact

(%) (mm) in water (%) (1010 g/ms Pa) Modulus (MPa) strength (MPa) at break (%) angle (q) ( )

Control 0.073 ± 0.00 29.68 ± 1.30b 1.60 ± 0.01c 55.76 ± 4.20b 31.21 ± 0.80a 51.73 ± 2.27a 62.00 ± 2.79c
SEO 0.5% 0.088 ± 0.01 29.67 ± 1.97b 1.53 ± 0.13c 62.99 ± 3.72a 28.26 ± 0.82b 46.17 ± 2.83b 66.13 ± 0.65b
SEO 1% 0.089 ± 0.01 24.80 ± 2.84ab 1.82 ± 0.12b 57.02 ± 2.45b 28.13 ± 1.64b 49.38 ± 3.08ab 72.87 ± 2.61a
SEO 1.5% 0.107 ± 0.00 23.54 ± 3.96a 2.34 ± 0.13a 46.50 ± 4.24c 20.38 ± 1.67c 51.67 ± 4.87a 69.35 ± 1.45ab

Values within each column with different letters are significantly different (p < 0.05).

biodegradability of films when used as packaging materials. 3.6. Swelling

Therefore, reducing the water solubility of film sample is aimed at
studies. Effects of incorporating SEO on the water solubility of agar-
based nanocomposite films are presented in Table 1. In the present
study, agar-NCC film showed a low solubility value (29.68 ± 1.30)
after 24 h. When SEO was added to the agar-cellulose nano-
composite matrix, the solubility of the film significantly decreased
(p  0.05). By increasing SEO concentration from 0.5 to 1.5% of film
solutions, the WS further decreased and the lowest water solubility
was observed for nanocomposite films containing 1.5% SEO
(23.54 ± 3.96). Such increase in water resistance of agar-NCC films
probably occurred due to i) the hydrophobic property of SEO and ii)
formation the interaction between savory essential oils compo-
nents and hydroxyl groups of polymer matrix. This behavior has
also been reported in other biopolymer-based films such soybean,
carrageenan, corn starch, gelatin and chitosan by other authors
(Ahmad et al., 2012; Ghasemloua et al., 2013; Ojagh et al., 2010;
Salarbashi et al., 2013; Shojaee-Aliabadi et al., 2013).
3.4. Water vapor permeability (WVP)
Barrier properties of a polymer are main parameters for esti-
mating and predicting the product package shelf-life and WVP is
one of the most important features in food packaging applications.
Because water vapor passes from the polymer package wall,
consequently, it can be effective in the product quality (Kanatt, Rao,
Chawla, & Sharma, 2012).
WVP value of the film samples with different SEO concentra-
tions is presented in Table 1. The WVP of the agar-NCC film was
1.60 ± 0.01  1010 g/msPa, which decreased about 46% for the
nanocomposites containing 1.5% SEO. As previously observed in the
SEM images, the use of high concentrations of essential oil creates
several cracks or fractures in the nanocomposite films. This
enhanced moisture passing through the films and thereby in-
creases the WVP values of the prepared films. Similar results were
reported by Jouki, Tabatabaei Yazdi, Mortazavi, and Koocheki
(2014) who found that increase in oregano essential oil concen-
tration resulted in increase WVP of quince seed mucilage films.
Swelling ratio of edible films is an important parameter for
determining the resistance of material packaging to water, espe-
cially at moist environments. Water sorption measurements of the
agar/cellulose nanocomposite films with different SEO contents at
ambient temperature are shown in Fig. 3. According to the obtained
results, the lowest rate of water uptake was related to the nano-
composite films containing 0.5% SEO concentration, whereas the
highest values of swelling was observed for the nanocomposite
with 1.5% SEO content. This finding might be due to the intermo-
lecular interaction of the nanocomposite solution and higher mo-
lecular weight of phenolic compounds of SEO, which can cause
fractures in the film samples (Moradi et al., 2012), as was supported
by SEM images. Moradi et al. (2012) also showed that the incor-
poration of Zataria multiflora and grape seed extract into chitosan
films increased the swelling index of films.
3.7. Rheological behavior of the nanocomposite film solutions
Viscosity curve of the film solutions for different concentrations
of SEO at 50  C is shown in Fig. 4. Viscosity values increased with
the addition of SEO in the film solution and this behavior was
similar for all the concentrations at 50  C. These results can be
attributed to the aggregation of oil droplets and also the interaction
between polymer and solvent by hydrogen bonding. Atare s, Bonilla,
and Chiralt (2010) observed the similar effect of essential oil
addition in the film solution on the viscosity value.
Also, with increasing shear rate, viscosity values decreased,
because by the increase of shear rate, stiff polymer molecules were
quickly aligned in the direction of flow and consequently the
physical interaction between nearby polymer chains was reduced
(Hosseini-Parvar, Matia-Merino, Goh, Razavi & Mortazavi et al.,
2010).

3.5. Surface hydrophobicity of nanocomposite films

Contact angle procedure is a simple way for demonstrating the

superficial hydrophilicity of films. In addition, it is one of the basic
wetting attributes of packaging materials, which is an indicator of
the hydrophilic or hydrophobic properties of materials (Han &
Krochta, 1999). Contact angle (CA) of different nanocomposite
films is shown in Table 1. Results exhibited that the contact angle
values significantly increased by adding SEO concentrations from
62 for control film to 72.87 for 1% SEO (p < 0.05). This improve-
ment was due to the hydrophobic nature of the essential oil that
prevented the entering of water into the structure of nano-
composite films. These observations were similar to the findings by
other researchers such as the incorporation of cinnamon essential
oil into chitosan films (Ojagh et al., 2010) and olive oil into chitosan Fig. 3. Water gain of the agar/cellulose nanocomposite films formulated with different
films (Pereda, Amica, & Marcovich, 2012). concentration of SEO.
 M. Atef et al. / Food Hydrocolloids 45 (2015) 150e157 155

(2010), Benavides, Villalobos-Carvajal, and Reyes (2012), Shojaee-

Aliabadi et al. (2013), Ghasemloua et al. (2013), Salarbashi et al.
(2013), and Jouki et al. (2014).

3.9. Optical properties of films

Color changes due to the addition of SEO to the films can be

Fig. 4. Viscous flow curves of agar-based nanocomposite films with different con-
 (b*), color difference (DE), and chroma (Cab) significantly increased
centrations of savory essential oil at 50 C.
3.8. Mechanical properties of the films
Influence of various SEO concentrations on the mechanical
properties (tensile strength, elongation at break, and young's
modulus) of the nanocomposite films is shown in Table 1. Incor-
poration of SEO into film solutions caused a significant reduction in
the TS and YM of the agar-based nanocomposites (p < 0.05). The TS
and YM of the control film was (31.21 ± 0.80) and (55.76 ± 4.20)
which decreased about 35% and 30% for the nanocomposites con-
taining 1.5% SEO, respectively. In contrast, E value of the nano-
composite films did not significantly increase by the incorporation
of SEO (p > 0.05), because the essential oil acted as a plasticizer and
increased the flexibility of the polymer chains. Presence of essential
oil in the films can lead to the formation of a weak network
structure. As a result, TS may reduce and E% may increase. Also, by
adding high contents of SEO to the solutions, a heterogeneous and
irregular structure was observed in the film matrix, which was
confirmed by SEM images. This result was in agreement with the
outcome of the works by Du et al. (2009), Lim, Jang, and Song
described by other color parameters such as total color difference
(DE), whiteness index (WI), chroma (Cab * ), and hue (h* ). Hunter Lab
ab
color values (L, a, b) and color measurements are presented in
Table 2. The obtained results indicated that the nanocomposite
films without SEO were more transparent and clearer (the highest L
value) than the samples with essential oil. Adding savory essential
oil affected the appearance, color, and transparency of films; so
that, by incorporating SEO, whiteness index (WI) and lightness (L*)
significantly decreased (p < 0.05); but, red/green (a*), yellow/blue
(p < 0.05). No significant difference (p > 0.05) in hab values was
detected among the nanocomposite films with different concen-
trations of essential oil.
Presence of essential oil in the nanocomposite films had a direct
influence on the appearance and light barrier properties of the film
samples. Opacity of all the nanocomposite films is reported in Fig. 5.
As shown in this figure, the opacity values of control films were
1.70 ± 0.26, whereas, by adding SEO concentration to the formu-
lation of films, their opacity significantly increased to 4.12 ± 0.52.
These observations showed that the films without SEO were more
transparent than the samples with SEO, because thickness of the
films affected their transparency. Shojaee-Aliabadi et al. (2013)
reported the opacity of carrageenan film which was significantly
increased by adding SEO.
Light transmission of UV and visible ranges at the selected
wavelengths of 200e800 nm of the nanocomposite films contain-
ing various concentrations of savory essential oil is shown in Fig. 5.
Transmission range of ultraviolet light was between 200 and
280 nm and the film samples containing savory essential oil
exhibited low transmission in UV light, especially at the wave-
lengths of about 280 nm. Decreased light transmission can be due

Table 2
Color parameters of nanocomposite films with SEO.

SEO content (%) L* a* b* DE WI Cab hab

Control 90.30 ± 0.21a 1.10 ± 0.04a 5.00 ± 0.16d 6.10 ± 0.26c 89.03 ± 0.25a 5.12 ± 0.15d 178.65 ± 0.01a
SEO 0.5% 88.60 ± 0.32ab 1.17 ± 0.12b 7.68 ± 0.38c 9.24 ± 0.40b 86.20 ± 0.37b 7.77 ± 0.38c 178.58 ± 0.01a
SEO 1% 88.85 ± 0.06a 1.33 ± 0.06a 8.54 ± 0.05b 9.78 ± 0.02b 85.89 ± 0.02b 8.65 ± 0.06b 178.58 ± 0.01a
SEO 1.5% 88.09 ± 0.50b 1.64 ± 0.03b 10.66 ± 0.78a 11.99 ± 0.92a 83.93 ± 0.88c 10.79 ± 0.77a 178.58 ± 0.01a

Means (n ¼ 3) with different letters within the same column indicate significant differences (p < 0.05).

Fig. 5. Effect of SEO on (a) opacity and (b) light transmission of agar/cellulose nanocomposite films.
156 M. Atef et al. / Food Hydrocolloids 45 (2015) 150e157

Table 3 3.11. Antimicrobial effectiveness of the nanocomposite films

Antimicrobial activity of the savory essential oil against gram-positive and gram-
negative bacterias.
Results of the experimental inhibition areas of the films against
Type of test L. monocytogenes S. aureus B. cereus E. coli gram-positive bacteria (L. monocytogenes, S. aureus, and B. cereus)
(Gram þ) (Gram þ) (Gram þ) (Gram ) and gram-negative bacteria (E. coli) are shown in Table 4. Agar/
Disc diffusion þþþ þþþ þþþ þþþ cellulose nanocomposite films without SEO were considered the
Well test þþ þþ þþ þ control samples that did not show any antibacterial properties
þþþ: totally inhibited; þþ: partially inhibited; þ: slightly inhibited; -: no inhibited. against the microorganisms.
Incorporation of SEO (0.5% concentration) into agar-based
nanocomposite films exhibited a weak inhibitory effect; but, in
to light scattering at the interface of essential oil droplets in the film the vapor-phase test, this process did not inhibit the growth of the
matrix (Tongnuanchan, Benjakul, & Prodpran, 2012). This phe- four pathogenic bacteria. As the content of savory essential oil
nomenon is able to retard lipid oxidation and prevent nutrient increased, the zone of inhibition also significantly increased in all
losses, discoloration, and off-flavors in food systems. These obser- three tests (p < 0.05) and the films containing 1.5% SEO demon-
vations were in agreement with the finding by Ahmad et al. (2012) strated the highest zone of inhibition (p < 0.05). This phenomenon
in terms of the improvement of light barrier properties after adding might be due to the relatively high concentration of some of the
essential oils to gelatin films. components such as carvacrol, g-terpinene, and p-cymene (Hadian
et al., 2010).
3.10. Antimicrobial assay of essential oil In two disc film methods (direct contact and vapor-phase),
L. monocytogenes and B. cereus were the most sensitive bacteria
Antimicrobial activity of savory essential oil against the four to the films containing savory essential oil, while E. coli was the
microbial species which was assessed by well test and disc diffusion most resistant one. Also, in the well test of film solutions, the
method is presented in Table 3. The data obtained from disc same results were observed with disk film methods and E. coli was
diffusion method indicated that gram-positive bacteria were more the more resistant bacteria to SEO. This phenomenon can be
sensitive microorganisms to the action of antibacterial agents than attributed to the fact that gram-negative ones are in general more
gram-negative ones, which can be due to the cell wall lipopoly- resistant owing to the external lipopolysaccharide wall sur-
saccharides of gram-negative organisms that will prevent the rounding the peptidoglycan cell wall (Burt, 2004). These obser-
permeating active component of essential oil from cytoplasmic vations were in agreement with the results by Shojaee-Aliabadi
membrane (Ouattara, Canh, Vachon, Mateescu, & Lacroix, 2002). It et al. (2013).
is worth mentioning that essential oils are a useful source of anti-
bacterial compounds and the antimicrobial nature of savory
4. Conclusions
essential oil (SEO) is related to its phenolic components such as
carvacrol, g-terpinene, and p-cymene (Burt, 2004). There is a syn-
This study showed that an active film from agar-based nano-
ergism activity between carvacrol and p-cymene. P-cymene is
composite film could be achieved by incorporation with SEO, as a
much weaker antibacterial compound than carvacrol that can cause
natural antimicrobial. This incorporation influenced microstruc-
the swelling of bacterial cell membrane. Consequently, carvacrol is
ture, physical, mechanical, color, and antimicrobial properties of
more easily transported to the cell and leads to cell death (Ultee,
the nanocomposite films. Experimental results indicated that
Bennink, & Moezelaar, 2002). These results were in agreement
with small amount of savory essential oil incorporated, SEO
 mez-Estaca et al., 2010;
with the previously published literature (Go
dispersed in the film matrix homogeneously. However, at high
Iturriaga, Olabarrieta, & Mart‫ﻱ‬nez de Mara;n, 2012).
concentrations (1.5%), cracks and accumulation of oil particles
occurred. Addition of SEO decreased tensile strength, young's
modulus, and water solubility, while increased the percent
Table 4 elongation, WVP and opacity of the nanocomposite films.
Antibacterial activity of agar/cellulose nanocomposite films incorporated with Furthermore, the films' light transmission at UV light, which can
different percentage of savory essential oil. be desirable for the food packaging industry, was decreased. The
Edible film Inhibition zone (mm) obtained results indicated that the antimicrobial properties of the
agar-based nanocomposite film could be enhanced by incorpo-
L. monocytogenes E. coli S. aureus B. cereus
(Gram þ) (Gram ) (Gram þ) (Gram þ)
ration with savory essential oil. Overall, this report demonstrates
that biodegradable films containing SEO present a good potential
Direct contact
for their utilization in food packaging. Nevertheless further
control N.D.c N.D.c N.D.b N.D.c
SEO 0.5% 38.48 ± 0.36b N.D.c N.D.b N.D.c studies are required to evaluate their performance in different
SEO 1% 103.87 ± 0.70ab 15.9 ± 0.41b 316.52 ± 0.02a 86.59 ± 0.68b foodstuffs. For this purpose in near future, our group will report
SEO 1.5% 288.53 ± 0.28a 113.10 ± 0.54a 233.71 ± 0.76a 306.35 ± 0.85a the results of the wrapping of present nanocomposite film on fish
Vapor-phase
fillet.
control N.D.c N.D.c N.D.c N.D.c
SEO 0.5% N.D.c N.D.c N.D.c N.D.c
SEO 1% 415.48 ± 0.32b 50.27 ± 0.93b 1075.21 ± 0.35a 338.16 ± 0.48b Acknowledgments
SEO 1.5% 816.86 ± 0.58a 481.11 ± 0.55a 649.18 ± 1.31b 907.92 ± 0.29a
Well test
control N.D.b N.D.c N.D.c N.D.b
We gratefully acknowledge Mehdi Alboofetileh for his valuable
SEO 0.5% N.D.b 3.98 ± 0.45b 7.07 ± 0.12bc N.D.b guidance and scientific editing.
SEO 1% 82.52 ± 0.42ab 30.68 ± 0.05ab 122.72 ± 0.25a 33.18 ± 0.06ab
SEO 1.5% 306.35 ± 0.04a 99.40 ± 0.1a 117.86 ± 0.33ab 70.88 ± 0.21a
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