Journal of Food Engineering 111 (2012) 343–350

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Journal of Food Engineering

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A novel active bionanocomposite film incorporating rosemary essential oil

and nanoclay into chitosan
Mehdi Abdollahi a, Masoud Rezaei a,⇑, Gholamali Farzi b
a
Department of Fisheries, Faculty of Marine Sciences, Tarbiat Modares University, P.O. Box 46414-356, Noor, Iran
b
Department of Material and Polymer Engineering, Faculty of Engineering, Sabzevar Tarbiat Moallem University, Sabzevar, Iran

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

Article history: Montmorillonite (MMT) nanoclay and rosemary essential oil (REO) were incorporated into chitosan film
Received 9 August 2011 to improve its physical and mechanical properties as well as antimicrobial and antioxidant behavior. The
Received in revised form 4 February 2012 MMT weight percent relative to chitosan was varied from 1 to 5 and was activated by three REO levels
Accepted 7 February 2012
(0.5%, 1%, and 1.5% v/v), and their impact on physical, mechanical, and barrier properties of the chitosan
Available online 16 February 2012
films was investigated. Total phenolic and antimicrobial activity were also evaluated. Microstructure of
chitosan/MMT–REO nanocomposites was characterized through X-ray diffraction (XRD) and Fourier
Keywords:
transform infrared spectroscopy (FTIR). The results showed that incorporating MMT and REO into
Bionanocomposite
Chitosan
chitosan improves water gain, water vapor permeability, and solubility of the chitosan film by more than
Montmorillonite 50%. It was also shown that the combined effect of clay and REO improves significantly the tensile
Rosemary essential oil strength and elongation of chitosan (p < 0.05). The XRD and FTIR results confirmed that the improve-
Food packaging ments are related to the MMT exfoliation and good interaction between chitosan and MMT in the
presence of REO. Antimicrobial properties of the films also improved by REO incorporation in 1.5% v/v.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction based biodegradable films. These include the addition of plasticiz-

Biopolymers have been widely investigated over the last two
decades because they can be a viable solution to the waste disposal
of foods’ plastic packaging materials. Furthermore, biopolymer films
are excellent vehicles for incorporating a wide variety of additives,
such as antioxidants, antifungal agents, antimicrobials, colours,
and other nutrients (Rhim and Ng, 2007; Wong et al., 1994), and
so these biodegradable materials can also improve food quality
and extend shelf life by minimizing microbial growth in the product.
Chitosan, a deacetylated derivative of chitin, is the second most
abundant polysaccharide found in the nature after cellulose. It has
been found to be non-toxic, biodegradable, biofunctional, and bio-
compatible (Rinaudo, 2006; Shahidi et al., 1999; Srinivasa and
Tharanathan, 2007). Chitosan brings some advantages over other
biomolecule-based active polymers used as packaging materials be-
cause of its antibacterial behavior and bivalent minerals chelating
ability (Aider, 2010). However, poor mechanical and gas barrier
properties, and weak water resistance limit its application particu-
larly in the presence of water and humidity (Wang et al., 2005; Xu
et al., 2006).
As for other biopolymers, many strategies have been explored
to improve the barrier and mechanical properties of chitosan-
⇑ Corresponding author. Tel.: +98 122 6254986; fax: +98 122 6253499.
E-mail address: rezai_ma@modares.ac.ir (M. Rezaei).
ers and salt, chemical modification of hydroxyl groups, cross-link-
ing of polysaccharides, the use of suitable solvent, the change of
pH, the addition of different polysaccharides, and blending with
other polymers (Bourtoom and Chinnan, 2008; Ghosh et al.,
2010; Hoagland and Parris, 1996; Park and Zhao, 2004; Srinivasa
et al., 2007; Li et al., 2010; Gómez-Estaca et al., 2011).
Recently, polymer–clay nanocomposites have received signifi-
cant attention as an alternative to conventional filled polymers, be-
cause of their ability for nano-scale dispersion, which brings
significant improvement in mechanical and physical properties
compared to micro-scale polymer composites. Several studies have
reported amelioration of mechanical properties (Lavorgna et al.,
2010; Xu et al., 2006), thermal stability (Darder et al., 2003; Wang
et al., 2005), functional properties (Rhim et al., 2006), barrier prop-
erties (Casariego et al., 2009; Rhim et al., 2006), and water solubil-
ity (Casariego et al., 2009) of chitosan films via incorporation of
nanoclay into chitosan in the range of 1–5 wt.%. Montmorillonite
is the most widely studied type of clay which is a hydrated alu-
mina-silicate layered clay consisting of an edge-shared octahedral
sheet of aluminum hydroxide between two silica tetrahedral lay-
ers. The imbalance of the surface negative charges is compensated
by exchangeable cations (typically Na+ and Ca2+) (Azeredo, 2009).
On the other hand, antibacterial and antioxidant activities of
essential oils (EOs) have long been acknowledged, but the
food industry has recently been paying more attention to their

0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2012.02.012
344 M. Abdollahi et al. / Journal of Food Engineering 111 (2012) 343–350
application as natural antimicrobials and antioxidants (Du Plooy
et al., 2009; Sánchez-González et al., 2010a). The main advantage
of EOs’ application is their greater activity as compared with the ef-
fects of the individual active compounds, probably due to the syn-
ergistic effects (Bakkali et al., 2008; Sánchez-González et al.,
2010b; Burt, 2004). Among essential oils the largest number of re-
ports on the effective antioxidant properties pertains to extracts
from plants belonging to family Labiatae, particularly rosemary
(Rosmarinus officinalis L.) (Waszkowiak, 2008). Phenolic com-
pounds such as carnosol, carnosoic acid, rosmanol, rosmadial, epi-
rosmanol, rosmadiphenol, rosmarinic acid, etc. are considered to
have antioxidant ability (Bozin et al., 2007). Major components of
REO which have showed antimicrobial properties are a-pinene
(2–25% of composition), bornyl acetate (0–17%), camphor (2–
14%), and 1,8-cineole (3–89%) (Burt, 2004).
Although several reports on chitosan/clay nanocomposites have
appeared in the literature, to the best of our knowledge there are
no reported data on the combined effect of nanoclay and antioxi-
dant/antimicrobial compounds like EOs. It seems that the combi-
nation of nanoclay and an antimicrobial/antioxidant compound
in chitosan films can be suitable due to its acceptable structural
integrity and barrier properties imparted by the nanocomposite
matrix, and the antimicrobial/antioxidant properties contributed
by the natural antimicrobial/antioxidant agents impregnated with-
in. In addition, such nanocomposite films prepared based upon
natural biopolymers are environmentally friendly with all the ben-
efits one may expect from biopolymer and nanocomposite packag-
ing materials (Rhim and Ng, 2007). In the present study, the
combined effects of montmorillonite nanoclay and rosemary
essential oil on the physico-chemical and antimicrobial properties
of chitosan composite films have been investigated.
2. Materials and methods
2.1. Materials
Crab shell chitosan (medium molecular weight, 190–310 kDa,
75–85% deacetylated, Sigma–Aldrich Chemical Co., USA) and Na+-
montmorillonite (MMT) from Southern Clay Products, USA were
used as received. Glacial acetic acid and Tween 80 were purchased
from Merck, Germany. Rosemary essential oil was obtained from
Barij Essence Pharmaceutical Co., Kashan, Iran, and stored in dark
container at 4 °C until used.
2.2. Sample preparation
2.2.1. Preparation of chitosan/clay-rosemary solutions
Aqueous solution of chitosan was prepared by dissolving 20 g of
chitosan powder in 1000 mL of aqueous acetic acid solution (1%, v/
v) to obtain a 2% w/v chitosan solution, using a magnetic stirring
plate at 90 °C and 1250 rpm for 20 min and then cooled to room
temperature. Nanocomposite samples were prepared according
to methods reported by Xu et al. (2006). Selected amounts of clay
(1, 3, and 5 w% on solid chitosan) were dispersed in 100 mL of 1%
(v/v) aqueous acetic acid solution and vigorously stirred for 24 h.
Afterward 150 mL of chitosan solution was added to the clay solu-
tions and the mixture was stirred for 4 h. Then Tween 80 at a level
of 0.2% w/v of essential oil, as an emulsifier, was added to the mix-
ture and stirred at 40 °C for 30 min. Finally appropriate amounts of
rosemary essential oil (REO) were added to the solution, to reach a
final concentration of 0.5%, 1%, 1.5% v/v, on the basis of neat chito-
san solution, and homogenized with Ultra-Turrax (IKA T25-Digital
Ultra-Turrax, Staufen, Germany) at 7000 rpm for 2 min. After cool-
ing the resulting mixture at room temperature, it was degassed un-
der vacuum for 5 min in order to remove all bubbles.
2.2.2. Preparation of nanocomposite films
The chitosan/clay-rosemary solutions (160 mL) were cast in
simple cubic molds made from Teflon-coated steel with dimension
of 25.5  28.5 cm, then dried for 72 h at ambient conditions
(T = 25 °C and RH = 55% ± 2%) to prepare nanocomposite films.
Dried films were then peeled and stored in a desiccator containing
saturated magnesium nitrate solution at 25 °C and 52.89% relative
humidity until evaluation.
2.3. Sample characterization
2.3.1. X-ray diffraction (XRD) characterization
The structure of nanoparticle and its nanocomposites was evalu-
ated by XRD measurement. XRD patterns were taken with a Philips
X’Pert MPD Diffractometer (Eindhoven, Netherlands), with Co Ka
radiation at a wavelength of 1.544 nm, at 40 kV and 30 mA. Chito-
san/clay-rosemary films were scanned over the range of diffraction
angle 2h = 1–12°, with a scan speed of 1°/min at room temperature.
2.3.2. Fourier transform infrared (FTIR) spectra
FTIR spectra were collected in transmission mode by using a
Bruker Equinox 55 FTIR spectrometer (Ettlingen, Germany) in the
range of 400–4000 cm1 at a resolution of 4 cm1.
2.3.3. Measurement of film thickness
The thickness of the samples was determined with a manual
digital micrometer (0.001 mm, Mitutoyo, Mizonokuchi, Japan).
Measurements were repeated in ten different regions of each sam-
ple. Average values were calculated and used in water vapor per-
meability and tensile properties calculations.
2.3.4. Water resistance
The solubility of the film was determined as the percentage of
soluble matter to initial dry matter of film sample (Tunc et al.,
2007). The initial dry matter of samples (4  4 cm) was determined
by drying films at 105 °C for 24 h. The films were then immersed in
50 mL distilled water and then placed in a shaker incubator at
25 °C and stirred for 24 h at 250 rpm. Then, the samples were fil-
tered through Whatman No. 1 filter paper. The papers containing
any insolubilized film were dried at 105 °C for 24 h. The film solu-
bility (%) was calculated using the following equation:
Solubility in waterð%Þ ¼ ðInitial dry weight  Final dry weightÞ
 100=Initial dry weight ð1Þ
2.3.5. Calculation of water vapor permeability
The water vapor permeability (WVP) of the films was measured
gravimetrically according to the ASTM E96–92 method as described
by Casariego et al. (2009). The tested film was sealed on the top of a
glass permeation cell (internal diameter = 3 cm). The cell contained
distilled water (100% RH; 2.337  103 Pa vapor pressure at 20 °C)
placed in a desiccator. It was maintained at 20 °C and 1.5% RH
(28.044 Pa water vapor pressure) with silica gel, and air was stirred
in the desiccators. Weight loss of the permeation cell was deter-
mined at intervals of 2 h for 10 h. The weight loss of the cell was con-
sidered equal with the transferred water through the film and
adsorbed by the desiccant. The slope of weight loss vs. time was ob-
tained by linear regression. The WVP was then calculated as follows:
WVP ¼ ðWVTR  LÞ=DP ð2Þ
Where WVTR (water vapor transmission rate, kg/m2 s1) is the
measured slope, L is the mean film thickness (m), and DP is the
partial water vapor pressure difference (Pa) between the two sides
of the film. This test was repeated three times for each specimen to
confirm its repeatability.
 M. Abdollahi et al. / Journal of Food Engineering 111 (2012) 343–350
2.3.6. Mechanical properties
Tensile strength (TS) and elongation at break (E%) of the film
samples were determined according to ASTM standard method D
882–02 with an Instron Universal Testing Machine (Model 200,
Hiwa Engineering Co., Iran). The film samples were cut in rectangu-
lar specimens (2.54  10 cm). Initial grip separation was set at
50 mm, and cross-head speed was set at 50 mm/min. This test
was repeated five times for each specimen to confirm its
repeatability.
2.3.7. Surface colour measurement
Colour properties of films were measured using a colour meter
from BYK Gardner, USA. Measurements are expressed as L⁄ (light-
ness), a⁄ (red/green), and b⁄ (yellow/blue). The parameters were
determined by placing film samples on a standard plate
(L⁄ = 94.63, a⁄ = 0.88, and b⁄ = 0.65). Colour difference (DE) and
whiteness index (WI) were calculated with respect to standard
plate parameters by using following Eqs. (3) and (4), respectively.
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 using gallic acid in specific concentrations and the total phenolic
DE ðDa Þ2 þ ðDb Þ2 þ ðDL Þ2
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
WI ¼ 100  ð100  L Þ2 þ a2 þ b
2
2.3.8. Swelling
Swelling of the clay and REO containing chitosan films was eval-
uated by determining their water sorption according to the meth-
od explained by Lavorgna et al. (2010). The sample was cut into
small pieces (2  2 cm), desiccated overnight (containing silica
gel), and weighed to determine their dry mass. The weighed sam-
ples were placed in closed beakers containing 30 mL of water
(pH = 7) and stored at 25 °C. The kinetic of swelling was evaluated
by periodically measuring the weight increment of the samples.
The films’ wet surface was gently blotted with a tissue before
weighing with a balance accurate to 0.0001 g. The weighing was
continued until equilibrium state. The procedure was repeated
three times for each sample to confirm its repeatability. The water
gain of each sample was calculated as follows:
Water Gainð%Þ ¼ ðWeight of wet film  Weight of dry filmÞ
 100=Weight of dry film
2.3.9. Antibacterial activity of the essential oil, MMT, film solutions,
and film disks
Antibacterial properties of REO, film-forming solution and disks
were studied using the agar diffusion method (Gómez-Estaca et al.,
2010). Five different pathogenic and spoilage bacteria including
Listeria monocytogenes (Persian Type Culture Collection (PTCC)
1163), Pseudomonas putida (PTTC 1694), Streptococcus agalactiae
(PTCC 1768), Escherichia coli (PTCC 1533), and Lactococcus lactis
(PTCC 1336) were used for testing. Bacterial strains were cultured
overnight in Brain Heart Infusion Broth (Scharlua, Spain) at 37 °C.
30 lL of REO, MMT solution, and different film-forming solutions
were poured into Mueller Hinton (Scharlua, Spain) agar wells
(5 mm diameter), after their plates had been seeded with 0.1 ml
of inoculums by swab containing approximately 106–107 CFU/ml
of the indicated bacteria. In the same way, films were punched into
discs of 6 mm diameter, and then placed on the plates. Next, the
plates were incubated in chamber at 37 °C for 24 h. After incuba-
tion the microbial growth was observed and the degree of inhibi-
tion was expressed as follows; +++: totally inhibited; ++:
partially inhibited; +: slightly inhibited; : no inhibition as ex-
plained by Gómez-Estaca et al. (2010).
345
2.3.10. Total phenolic assay
Total phenolic content of the films was studied using the Folin–
Ciocalteu method as described by Siripatrawan and Harte (2010)
with some modification. Folin–Ciocalteu phenol reagent was used
to obtain a crude estimate of the amount of phenolic groups pres-
ent in the films. Phenolic compounds undergo a complex redox
reaction with phosphotungstic and phosphomolybdic acids pres-
ent in the Folin–Ciocalteu reaction. The color development is due
to the transfer of electrons at basic pH to reduce the phosphomo-
lybdic/phosphotungstic acid complexes (Curcio et al., 2009). 50 mg
of each film sample were dissolved in 3 ml of methanol, and 0.1 ml
of film extract solution was mixed with 7 ml distilled water and
0.5 ml of Folin–Ciocalteu reagent (Merck Company, Darmstadt,
Germany). After preserving the mixture for 8 min at room temper-
ature, 1.5 ml of saturated sodium carbonate solution and 0.9 ml of
distilled water were added to it. The mixture was stored in dark-
ness and at room temperature for 2 h. The absorbance values were
then measured at 765 nm using a spectrophotometer (Lambda 25,
Perkin–Elmer, Fremont, CA, USA). A calibration curve was drawn
ð3Þ
content of the films was expressed as Gallic acid equivalents. This
test was repeated five times for each specimen and the Gallic acid
ð4Þ equivalent value was reported as mean ± SD of triplicate.
2.4. Statistical Analysis
The difference between factors and levels was evaluated by the
analysis of variance (ANOVA). Duncan’s multiple range tests were
used to compare the means to identify which groups were signifi-
cantly different from other groups (p < 0.05). All data are presented
as mean ± SD.
3. Results and discussion
3.1. XRD analysis of chitosan nanocomposites
Wide-angle X-ray diffraction is a classical method for determin-
ing the gallery height (d-spacing distance) in clay particles. During
intercalation, the insertion of polymer into the organoclay galleries
forces the platelets apart and increases the d-spacing, resulting in a
shift of the diffraction peak to lower angles (Xu et al., 2006). Re-
ð5Þ sults of XRD characterization of chitosan and chitosan/MMT nano-
composite are shown in Fig. 1. in the text. The crystalline structure
of chitosan is strongly dependent on its processing treatment, as
well as its origin and molecular constitution, such as its degree
of deacetylation and molecular weight (Rhim et al., 2006). How-
ever, the data reported in Fig. 1 show lower intensity for the char-
acteristic crystallinity peak of chitosan for composite films at
2h = 10.18°, which means the crystallinity of chitosan is slightly re-
duced by the addition of the MMT. This agrees with previously re-
ported results of Lavorgna et al. (2010). MMT had a characteristic
peak at 2h = 8.56° (d0 0 1 = 11.93 Å). With the addition of 1 and
3 wt.% MMT to chitosan, the reflection peak disappeared, indicat-
ing formation of an exfoliated structure, which was disordered
and not detectable by XRD. Further increasing the amount of
MMT to 5 wt.% gave a broad peak at 2h = 5.82° (d0 0 1 = 17.59 Å).
This was much lower than that of pristine MMT, suggesting that
intercalation occurs together with some exfoliation (Xu et al.,
2006). Fig. 1 shows that the structure of nanocomposites is not af-
fected by the presence of the REO. These results are in good agree-
ment with those obtained by other investigators (Lavorgna et al.,
2010; Rhim et al., 2006; Xu et al., 2006). It was shown that
inorganic and hydrophilic MMT in low concentration (1–5%) could
easily be dispersed in the chitosan solution to form a favorable
interaction with hydrophilic chitosan. Strong polar interactions,
346 M. Abdollahi et al. / Journal of Food Engineering 111 (2012) 343–350
Fig. 1. XRD patterns of MMT powder and chitosan-based nanocomposite films.
especially hydrogen bonding, critically affect the formation of
intercalated and exfoliated hybrids (Vaia and Giannelis, 1997).
3.2. FTIR analysis of chitosan/clay nanocomposites
Fig. 2 shows the FTIR spectra of MMT, chitosan, and chitosan/
MMT nanocomposite films. The peak of Al–Al–OH stretching vibra-
tion at 3626 cm1 found in the spectrum of MMT is typical for
smectites with high amount of Al in the octahedral layer. The peaks
at 3426 and 1639 cm1 were the H–O–H stretching and bending
vibrations of the adsorbed water, respectively. The sharp peak at
1034 cm1 was attributed to Si–O stretching frequency (Tunç and
Duman, 2008) which could act as hydrogen-bonding sites for func-
tional groups of chitosan. These characteristic peaks have shifted to
lower frequencies in nanocomposites which recommend some
interaction between functional groups of chitosan and MMT (Katti
et al., 2008). It is also important to note that the peaks between
3500 and 3000 cm1 correspond to the stretching vibration of free
hydroxyl and to asymmetric and symmetric stretching of the N–H
bonds in the amino group (Siripatrawan and Harte, 2010), respec-
tively. These are stronger in neat chitosan film (Fig. 2b) compared
to those incorporated with clay (Fig. 2c) and REO (Fig. 2d). The
bands appearing between 2750 and 3000 in the spectrum of chito-
san film are due to stretching vibrations of C–H bond in –CH2
(m = 2930 cm1) and –CH3 (m = 2870 cm1) groups (Paluszkiewicz
et al., 2010) respectively. In addition, two strong bands at 1541
Fig. 2. FTIR spectra of MMT (a), chitosan (b), chitosan-MMT3% (c), and chitosan-
MMT3%–REO1% (d) films.
and 1403 cm1, associated with OH in-plane bending, are less dis-
cernible in the films incorporated with clay and REO. These peaks
flattened more with incorporating clay and REO. This may be due
to hydrogen bonding between the –OH group in MMT and func-
tional groups in REO ingredients and the –NH and –OH groups in
chitosan (Wang et al., 2008).
In the spectra of the nanocomposites, the N–H bonded to the O–
H vibration band at 3209 cm1 in chitosan, and shifted towards
lower frequency (m = 3202 cm1) in nanocomposite with 3%
MMT. This fact indicates that NH2 and OH groups of chitosan
formed hydrogen bonds with the OH group of MMT (Wang
et al., 2006). It is further shifted to lower frequency
(m = 3130 cm1) for chitosan/clay 1% nanocomposite containing
1% REO, which shows that hydrogen-bonding interaction between
chitosan and clay is enhanced in the presence of REO in accordance
with other results (Tang et al., 2009).
A shift towards lower frequency in the absorption bands at
3209 in the FTIR spectra was coincidental with the decrease in
peaks at 3500–3000, 1541, and 1403 cm1. This observation pre-
sents an assumption that there could be a particular arrangement
in the films due to the interactions of MMT and REO ingredients’
functional groups with hydroxyl and amino groups in chitosan ma-
trix (Siripatrawan and Harte, 2010).
3.3. Film appearance characterization
The colour of the packaging is an important factor in terms of
general appearance and consumer acceptance (Bourtoom and
Chinnan, 2008). The result of colour measurements for chitosan
films are shown in Table 1. All of the films were transparent, with
a slight yellowish tint according to the amounts observed for b⁄ of
the films. The results were in good agreement with the results of
Rhim et al. (2006). Chitosan film lightness increased significantly
by incorporating MMT but REO did not affect the nanocomposites’
lightness. Casariego et al. (2009) reported a similar result about
incorporating MMT in chitosan film. Red/green (a⁄), yellow/blue
(b⁄) coordinates, and total colour differences (DE) significantly in-
creased by incorporating REO and increased as REO increased.
Whiteness (WI) increased in the presence of MMT and decreased
when REO was incorporated. The results are in agreement with
the results of Ojagh et al. (2010) and Siripatrawan and Harte
(2010).
 M. Abdollahi et al. / Journal of Food Engineering 111 (2012) 343–350 347

Table 1
Surface colour parameters of chitosan-based nanocomposite films.

Concentration L⁄ a⁄ b⁄ DE WI
MMT (%w/w chitosan) REO (%v/v)
Control 82.51b ±0.28 0.17d ± 0.04 7.54f ± 0.90 13.96abc ± 0.66 80.94abcd ± 0.59
1 0 82.63b ± 1.95 0.67c ± 0.11 6.44f ± 0.54 10.38d ± 1.31 84.62a ± 1.52
1 0.5 81.95b ± 1.64 1.09b ± 0.21 9.42de ± 1.70 16.26a ± 2.23 80.21bcd ± 1.96
1 1 82.12b ± 1.74 1.22b ± 0.15 9.17de ± 1.37 14.60abc ± 2.20 80.33bcd ± 2.16
1 1.5 82.70b ± 0.96 1.35b ± 0.24 11.22c ± 1.43 16.15a ± 1.65 79.16cd ± 1.57
3 0 85.21b ± 1.02 0.70c ± 0. 07 7.86def ± 0.77 12.01bcd ± 0.77 82.81abc ± 0.57
3 0.5 85.42a ± 1.61 1.11b ± 0.15 10.71ce ± 2.60 15.43a ± 1.33 79.56 cd ± 1.43
3 1 87.50a ± 1.14 2.10a ± 0.49 11.22bce ± 0.20 15.56a ± 0.68 81.90abcd ± 1.25
3 1.5 85.67a ± 1.68 1.93ab ± 0.36 12.12abce ± 1.18 16.24a ± 0.99 82.61abcd ± 0.99
5 0 85.94a ± 1.04 0.87bc ± 0.20 8.54abc ± 0.30 11.76cd ± 0.75 83.51ab ± 0.85
5 0.5 84.28a ± 1.02 1.96ab ± 0.36 13.37a ± 0.90 16.50a ± 1.45 80.01cd ± 1.15
5 1 86.09a ± 2.24 1.57ab ± 0.14 11.17bce ± 1.01 13.60abc ± 2.19 82.10abcd ± 2.4
5 1.5 86.19a ± 2.04 2.51a ± 0.51 13.00a ± 1.03 15.20ab ± 1.65 80.77bc ± 2.10
a-e
Different superscripts within the same column indicate significant differences among formulations (p < 0.05).Montmorillonte (MMT), Rosemary Essential oil (REO), Colour
difference (DE) and Whiteness index (WI).

3.4. Water resistance
Water resistance is an important property of biodegradable or
edible films for applications in food protection where water activ-
ity is high, or when the film must be in contact with water perma-
nently as long as it acts as food protective film, e.g., to avoid
exudation of fresh or frozen products (Bourtoom and Chinnan,
2008). As the results presented in Table 2. indicates, chitosan film
showed a low solubility value of (17.97 ± 0.6) similar to that re-
ported by Ojagh et al. (2010). When MMT was added to the film,
a significant (p < 0.05) decrease in water solubility was observed
(Table 2). This can be justified by the fact that MMT nanoclay
brings significant barrier properties due to its disk-like morphol-
ogy and there are of course some specific interactions between
chitosan and MMT that stabilize the film structure. In the other
word, water could not break hydrogen bonds between MMT layers
and chitosan sufficiently which caused the lower solubility in the
nanocomposites (Tunç and Duman, 2010). Similar results were re-
ported by Casariego et al. (2009). When REO was added to the
chitosan clay nanocomposite matrix the solubility of the film fur-
ther decreased. The lowest water solubility was observed for nano-
composite films containing 3% clay and 1% REO (9.55 ± 0.78). This
fact is highly attributable to cross-linking effects of REO
components to esters and/or amide groups (Ojagh et al., 2010). It
was supported by FTIR spectra that showed the interaction of
chitosan-functional groups with MMT and further interactions in
the presence of REO. Higher cross-linking density in chitosan leads
to a matrix with lower water solubility results, which is beneficial
when product integrity and water resistance are intended (Hosse-
ini et al., 2009).
3.5. Swelling
Knowledge of the swelling is also important for predicting stabil-
ity and quality changes during packaging and storage of food product
(Srinivasa et al., 2007b). Swelling of chitosan and its nanocomposites
are displayed in Fig. 3. As can be seen in these figures, chitosan film
absorbed large amounts of water (about 1400%) in the first few
minutes, and swelled and crumpled before reaching equilibrium
(Fig. 3 A). But all nanocomposites reached equilibrium and the
amount of sorbed water was less than chitosan film. The lowest
amount of water sorption relates to chitosan nanocomposite contain-
ing 3% MMT. The result was in contrast with result of Lavorgna et al.
(2010) that showed chitosan/clay nanocomposite had more water
sorption compared to neat chitosan film. Fig. 3 B, C, and D show swell-
ing of REO-incorporated chitosan nanocomposites. As can be seen
from this figure, water sorption of REO containing nanocomposites is
85% less than films not containing REO. In nanocomposites containing
1 and 5% MMT, the lowest amount of water sorption was observed in
1.5% REO (Fig. 3B and D) but in films with 3% MMT it was detected in
1% REO (Fig. 3 B). As explained previously, the hydrophobic nature of
REO may increase film hydrophobicity. Moreover, this low water sorp-
tion is also most likely to be due both to the formation of the cross-link
network induced by the hydrogen bonds between the chitosan and
REO and good intercalation of chitosan molecules into the silicate
galleries (Lavorgna et al., 2010), as confirmed by FTIR analysis.

Table 2
Summary of water vapor permeability, and mechanical and physical properties for chitosan-based nanocomposite films.

Concentration Thickness (mm) Solubility (%) WVP (g/msPa) 1010 TS (MPa) E (%)
MMT (%w/w chitosan) REO (%v/v)
Control 0 0.049 ± 0.005 17.97a ± 0.60 0.71a ± 0.01 60.80d ± 8.60 3.79 cd ± 0.54
1 0 0.049 ± 0.004 11.70defg ± 1.66 0.43c ± 0.01 63.74cd ± 3.38 3.56cd ± 0.54
1 0.5 0.050 ± 0.006 12.37def ± 1.02 0.36f ± 0.01 72.02bc ± 6.98 4.28bc ± 0.53
1 1 0.051 ± 0.003 11.11efg ± 1.23 0.42c ± 0.00 74.66ab ± 2.90 3.91bc ± 0.74
1 1.5 0.051 ± 0.005 10.75efg ± 1.18 0.41cd ± 0.02 64.86bcd ± 1.47 5.47a ± 0.71
3 0 0.050 ± 0.004 15.03bc ± 0.96 0.39de ± 0.01 72.54bc ± 1.46 3.44bcd ± 0.22
3 0.5 0.050 ± 0.003 12.53de ± 1.38 0.47b ± 0.01 73.48abc ± 4.39 4.11bc ± 0.11
3 1 0.051 ± 0.004 9.55g ± 0.78 0.30g ± 0.01 71.45bcd ± 3.78 4.31bc ± 0.27
3 1.5 0.052 ± 0.005 12.31def ± 0.75 0.31g ± 0.02 71.33bcd ± 5.34 4.83ab ± 0.49
5 0 0.051 ± 0.004 15.50b ± 1.00 0.45b ± 0.00 69.33bcd ± 5.22 3.01d ± 0.52
5 0.5 0.051 ± 0.006 10.40fg ± 1.47 0.36ef ± 0.01 83.00a ± 3.47 4.10bc ± 0.61
5 1 0.052 ± 0.005 12.83cde ± 0.54 0.39d ± 0.02 65.71bcd ± 7.48 3.42cd ± 0.09
5 1.5 0.052 ± 0.005 13.90bcd ± 0.29 0.35f ± 0.01 74.71ab ± 4.84 4.31bc ± 0.47
a-g
Different superscripts within the same column indicate significant differences among formulations (p < 0.05).
Montmorillonte (MMT), Rosemary Essential oil (REO), Water Vapor Permeability (WVP),Tensile Strength (TS), Elongation €.
348 M. Abdollahi et al. / Journal of Food Engineering 111 (2012) 343–350

Fig. 3. Water sorption profiles of (A) chitosan film without REO: (j) CS, () CS/MMT 1%, (N) CS/MMT 3%, and (d) CS/MMT 5%, (B) film containing 1% MMT: (j) CS/MMT 1%,
() CS/MMT 1/REO 0.5%, (N) CS/MMT 1/REO 1%, and (d) CS/MMT 1/REO 1.5%, (C) film containing 3% MMT: (j) CS/MMT 3%, () CS/MMT 3/REO 0.5%, (N) CS/MMT3/ REO 1%,
and (d) CS/MMT3/REO 1.5%, (D) film containing 5% MMT: (j) CS/MMT 1%, () CS/MMT5/REO 0.5%, (N) CS/MMT 5/REO 1%, and (d) CS/MMT 5/REO 1.5%.

3.6. Water vapor permeability
WVP is one of the most important parameters for biodegrad-
able films. This parameter was studied to evaluate the combined
effect of clay and REO on the barrier properties of the chitosan
film. The WVP of chitosan-based films is shown in Table 2. As
can be seen from this table, when 1 or 3 w% MMT was incorpo-
rated into chitosan, WVP decreased about 40 or 45%, respectively.
The presence of REO resulted in further decreasing of WVP. It was
demonstrated that for chitosan nanocomposite containing 3 w% of
clay, and 1% or 1.5% REO, WVP decreases by 58%. As mentioned
previously, it is believed that clay improves the barrier properties
of polymers due to its disk-like morphology and its creation of a
tortuous path for the molecule diffusion (Rhim et al., 2006). This
is the case for decreasing the WVP of chitosan/clay nanocompos-
ites compared to pure chitosan. A significant decrease of WVP re-
lates to the presence of ordered dispersed nanoparticle layers
with relatively large aspect ratios in chitosan (Cussler
et al.,1988; Yano et al., 1997). This can be seen from the XRD pat-
tern of the chitosan/MMT.
Further decreasing WVP in the presence of REO relates to the
hydrophobic nature of REO, which could affect the hydrophilic/
hydrophobic property of the films. It may be that the hydrogen
and covalent interactions between the chitosan network and REO
ingredients limit the availability of hydrogen groups to form
hydrophilic bonding with water, subsequently leading to a de-
crease in the affinity of chitosan film to water and decreasing
WVP (Siripatrawan and Harte, 2010). This hypothesis was sup-
ported by FTIR spectra in which the interaction of chitosan func-
tional groups with MMT in the presence of REO is shown.
Despite the low percentages (1 and 3 w%) of MMT could de-
crease WVP of chitosan/MMT nanocomposite, when the amount
of MMT increased to 5 w%, WVP of chitosan/MMT, nanocomposite
did not improve. This can be attributed to the quality of clay dis-
persion in chitosan. It seems that orientation of nanoclay changes
once its concentration reaches a certain level and/or some aggrega-
tion of nanoclay probably occurs (Casariego et al., 2009). As the
XRD pattern showed under this condition, exfoliation of chitosan
MMT occurs together with some intercalation (Fig. 1).
3.7. Mechanical properties
Mechanical properties of chitosan-based films are presented in
Table 2. The result showed that the tensile strength (TS) of chitosan
film increased significantly by incorporating MMT into chitosan up
to 3 w% but adding more MMT could not improve it. As can be seen
from Table 2, TS increased about 20% and 15% when the concentra-
tion of MMT was 3 and 5 w%, respectively. However, for similar
samples Elongation (E%) decreased slightly. Xu et al. (2006) and
Lavorgna et al. (2010) also found that the TS of the chitosan film
containing various concentrations of MMT was significantly higher
than that of chitosan. This improvement in the TS of nanocompos-
ite in a low amount of MMT may be attributed to the uniform dis-
persion of MMT in chitosan matrix and a strong interaction
between chitosan and MMT. The decrease in TS at high concentra-
tion of MMT may be caused by some aggregation of MMT particles
with high surface energy (Xu et al., 2006).
The TS of nanocomposite improved significantly by incorporat-
ing REO, even at a low amount of REO. The highest TS was observed
for nanocomposite with 5% of MMT and 0.5% of REO. TS value for
 M. Abdollahi et al. / Journal of Food Engineering 111 (2012) 343–350 349

Table 3
Antibacterial activity of REO, MMT, film-forming solutions (FFS), and film discs.

Clay con. (%) REO con. (%) L. lactis P. putida S. agalactiae E. coli L. monocytogenes
Pure REO + + ++ ++ ++
MMT 1 0     
MMT 3 0     
MMT 5 0     
FFS well Control     
3 0.5   +  
3 1  + + + +
3 1.5 + + ++ + ++
Film disk Control     
3 0.5     
3 1   +  +
3 1.5   + + +

: No inhibited; +: slightly inhibited; ++: partially inhibited.

this nanocomposite is about 36.5% higher than pure chitosan film.
The E% increased significantly by incorporating REO up to 44%, and
it seems that REO has shown a plasticizing ability. As described for
swelling, perhaps the presence of REO in low amounts changes the
hydrogen-bonding network within the material and allows better
interaction between nanofiller and matrix.
3.8. Antibacterial activity and total phenolic content
Antibacterial activity of REO, MMT, film forming solutions, and
film discs is shown in Table 3. Regarding their extra protective out-
er membrane, gram-negative bacteria are usually considerably
more resistant to antibacterial agents than their gram-positive
counterparts. In this regard, the essential oil showed its best
antibacterial activity in the disc diffusion test on gram-positive
bacteria (i.e., L. monocytogenes, S. agalactiae). The results are in
agreement with the results of other published data (Bozin et al.,
2007; Gómez-Estaca et al., 2010). However, its inhibitory activity
on gram-negative bacteria (especially on E. coli) was also notable
and interesting. Bozin et al. (2007) also reported good inhibitory
activity for REO against E. coli strain. As explained before, the high
antimicrobial properties of REO are mainly related to phenol diter-
penes, such as carnosic acid, carnosol, rosmanol, isorosmanol, and
rosmarinic acid (Bozin et al., 2007; Türe et al., 2008). MMT did not
show antibacterial activity in the well test but we could not find
reported results about this test for comparison.
Neat chitosan did not show antimicrobial properties in neither
solution form nor in film form. These results coincide with the re-
sults of Ojagh et al. (2010) and Zivanovic et al. (2005) about chito-
san film. This effect of chitosan may be related to the fact that
chitosan does not diffuse through the adjacent agar media in the
agar diffusion test method, so that only organisms in direct contact
with the active sites of chitosan are inhibited (Coma et al., 2002).
Chitosan/clay nanocomposite without REO did not show antibacte-
rial properties, which was in agreement with the results of Rhim
and Ng (2007). Films solution and discs containing 1% and 1.5%
REO could show antibacterial activity in comparison to neat
chitosan.
Total phenolic content of chitosan-based films was shown in
Fig. 4. As expected, total phenol content of chitosan film increased
significantly by incorporating REO and the highest value (2.23 mg
galic acid/g sample) was for the film formulated with clay 3% and
REO1.5% which was in agreement with other reported results
(Gómez-Estaca et al., 2009; Norajit et al., 2010; Siripatrawan and
Harte, 2010). In general, it has been showed in many recent studies
that the antioxidant activity of plants is caused mainly by phenolic
compounds. The effect of phenolic compounds on lipid molecules
may depend on structural factors, like the number of phenolic hy-
droxyl or methoxyl groups, flavone hydroxyl, keto groups, free car-
boxylic groups, and other structural features (Jayabalan et al.,
2008).
4. Conclusions
It was shown that incorporation of very low amounts of nano-
clay into chitosan significantly improves its physical and mechan-
ical properties. XRD pattern showed MMT exfoliation and FTIR
spectra demonstrated good interaction between chitosan and
MMT. The compatibility of REO with chitosan/MMT nanocompos-
ite was confirmed in order to produce an active bionanocomposite
for food packaging. Good interaction between chitosan and MMT
could improve WVP, water sensitiveness, and mechanical proper-
ties of the chitosan films. In addition, chitosan nanocomposites
containing REO exhibit antimicrobial properties and more phenol
content, which make them very interesting for food preservation.
In the near future, our group will report the results of the antimi-
crobial and antioxidant aspects of chitosan nanocomposites.

Acknowledgments

We are grateful to Dr. Badiei for preparing some experimental

facilities for this research. We also thank to Dr. Zarei for graciously
providing some raw materials.

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