International Journal of Biological Macromolecules 74 (2015) 289–296

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Development and characterization of biodegradable chitosan films

containing two essential oils
Zhu Shen, Donatien Pascal Kamdem ∗
School of Packaging, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI 48824, USA

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

Article history: Active biodegradable films from chitosan containing 10% to 30% w/w of citronella essential oil (CEO)
Received 8 September 2014 and cedarwood oil (CWO) were developed by casting and solvent-evaporation method, and their phys-
Received in revised form 4 November 2014 ical, mechanical and thermal properties were investigated. Possible interactions between the chitosan
Accepted 21 November 2014
chains and the essential oils were confirmed using Fourier-transform infrared spectroscopy (FTIR). Vari-
Available online 24 December 2014
ous amounts of CEO or CWO had significant effects on the films’ mechanical properties, with the exception
of 10% of CEO, which did not significantly affect the tensile strength of the films. The incorporation of
Keywords:
the two tested oils provoked a remarkable reduction in the water-vapor permeability properties, with a
Essential oils
Biodegradable film
decrease of about 63% when 30% CEO was added in chitosan films. Thermogravimetric analysis showed
Chitosan that degradation temperatures of the films containing CEO and CWO improved only slightly in compari-
SEM son to control films without essential oils. FTIR spectra analysis provided some insights on the possible
Tensile properties. interactions between chitosan and the two essential oils used. This study suggests that active films can
be developed by including CEO and CWO in a chitosan matrix. Such films can provide new formula-
tion options for packaging industries in developing active packaging with potential food-technology
applications.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction polymers combined with natural antimicrobial and/or antioxidant

Petroleum-based packaging is extensively used in various fields
due to its low density and high mechanical and barrier proper-
ties [1]. Although many types can be recovered and recycled, in the
United States, approximately 26 million metric tons of them end up
in landfills or the environment annually [2]. Given the increasing
environmental concerns created by excessive plastic accumulation,
as well as consumer demand for healthy, nutritive food products
and extended shelf life, the food and packaging industries have paid
increasing attention to biopolymer and edible films over the last
two decades [3]. Edible and biodegradable natural-polymer films
have been considered attractive alternatives to plastic packaging
due to their excellent biodegradability, biocompatibility and edibil-
ity, and the range of their potential applications [4]. Furthermore,
they may operate as carriers for incorporating a wide variety of
additives, such as antioxidants and antimicrobials agents. Since
food quality and safety are major concerns in the food industry,
development of bioactive edible coatings based on biodegradable
∗ Corresponding author. Tel.: +1 517 355 7735; fax: +1 517 353 8999.
E-mail addresses: shenzhu@msu.edu (Z. Shen), kamdem@msu.edu,
kamdem@anr.msu.edu (D.P. Kamdem).
compounds appears as one of the most interesting strategies in
the near future. Therefore, many researchers are seeking ways
to maintain food quality, minimize the growth of undesirable
microorganisms on the surface of food and provide extra protec-
tion against oxidative agents have studied the concept of “active
packaging” [5–7]. One of the current methods to design active food
packaging is to include active compounds, such as antioxidants
and antimicrobial agents, within the packaging material. However,
concerns about the potential health risks associated with the con-
sumption of synthetic additives that may be released into food
products have led investigators to develop active food packaging
based on biodegradable materials and natural substances.
Chitosan is a cationic natural polymer derived by the deacetyla-
tion of chitin, the major component of the shells of crustaceans. It
is a linear binary heteropolysaccharide composed of ␤-1,4-linked
glucosamine and N-acetylglucosamine, and has been proven to be
a non-toxic, biodegradable and biocompatible polymer [8]. It has
also been shown to have great potential as antimicrobial packaging
material owing to its antimicrobial activity [9,10]. The literature
has shown that chitosan can produce films with acceptable per-
meability to gases (CO2 and O2 ) and good mechanical properties
[11]. However, their uses are currently limited owing to their
hydrophilic character and weak water resistance. Many strategies

http://dx.doi.org/10.1016/j.ijbiomac.2014.11.046
0141-8130/© 2014 Elsevier B.V. All rights reserved.
290 Z. Shen, D.P. Kamdem / International Journal of Biological Macromolecules 74 (2015) 289–296
have been proposed to improve the barrier and mechanical proper-
ties of chitosan-based films. To improve water-barrier properties,
hydrophobic compounds, such as lipids, are usually included in the
polymer matrix. Over recent years different works have studied
the use of fatty acids as lipid components to be incorporated in
chitosan films to modulate the water vapor barrier [12–14]. The
incorporation of plant essential oils into these films represents an
interesting alternative to lipids. Essential oils extracted from plants
or spices are rich sources of phenolic compounds, and have been
demonstrated to have a wide spectrum of antimicrobial activity
[15]. Despite the essential oils’ potential for food preservation, their
usage is often restricted because of their intense aroma, toxicity
problems and possible changes in the organoleptic properties of
the food [16]. To avoid this problem, an interesting approach to
inhibit their diffusion into food products is to incorporate them
into the chemical structure of the film [17]. Furthermore, compared
with direct application, use of edible coatings to carry essential oils
could reduce the required doses of essential oils by the encapsula-
tion effect in the polymer matrix, thus reducing the oils’ negative
impact.
Citronella essential oil (CEO), from Cympobogon spp (Poaceae),
is popular in mosquito-repellent formulations [18]. Candles and
incense containing CEO have been reported to be sold as insect
repellents in several countries [19]. Pires et al. [20] demonstrated
that the main chemical components of CEO can be categorized as
38.3% sesquiterpenes hydrocarbons (␤-elemene, ␦-cadinene and
eremophilene), 27.6% monoterpene hydrocarbons (3 -carene and
␥-terpinene) and 22.5% oxygenated monoterpenes (␤-citronellal
and ␤-citronellol). Huang et al. [21] reported that citronellal is
the main component of CEO. Cedarwood oil (CWO) is an essen-
tial oil generally obtained from Juniperus species. The main
chemical components of CWO have been found to be cedrol
(13–15%), widdrol (9–12%), 8,14-cedranoxide (7–8%), 14-hydroxy-
(E)-caryophyllene (5–9%), cis-thujopsene (10–11%) and ␣-cedrene
(6–8%) [22]. CWO is reported to be produced in China (Cupres-
sus funebris) [23]. Studies have indicated inhibitory effects of
CEO on ectoparasites and pathogenic microorganisms, includ-
ing bacteria and fungi [24–26]. CWO has also been reported to
have antifungal, antitermite and anti-inflammatory effects [27,28].
These two essential oils have received approval from the FDA
for use as food additives, and could be considered as potential
alternatives to synthetic additives in antimicrobial food pack-
aging. However, there is as yet little information about their
possible application as components of edible films. Considering
that CEO and CWO are gaining importance for their consid-
erable antimicrobial activities, this study focuses on two main
specific objectives: (1) to develop an antimicrobial–antioxidant
film based on chitosan and two essential oils – CEO and
CWO; and (2) to evaluate some characteristics of these films,
such as their physical, mechanical, thermal and structural prop-
erties, to examine their potential applications as packaging
material.
2. Materials and methods
2.1. Materials
Chitosan (CH) used in this study was from shrimp shell with a
deacetylation degree of 75%. Tween 80 and 98% glacial acetic acid
were all purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Citronella essential oil (denoted as CEO) was supplied from Spec-
trum Chemical Corp. (Gardena, CA, USA) and cedarwood essential
oil (denoted as CWO) was purchased from Atomergic Chemicals
Corp. (Farmingdale, NY, USA) and stored in a closed glass container
at 23 ◦ C until further use.
2.2. Chitosan characterizations
The number average molecular weight (Mn) and weight average
molecular weight (Mw) were calculated by using data from the gel
permeation chromatography (GPC) analysis. The GPC system was
carried on an Agilent 1100 series HPLC equipped with two Waters
Styagel HR 4 Column Waters Hydrogel 250 and Waters Hydrogel
500, Milford, MA, USA. About 1% v/v acetic acid was used as eluent
at a flow rate of 0.65 mL min-1 at room temperature close to 30 ◦ C.
The sample concentration was 1% (w/v). Six standard references
with molecular weights ranging from 6000 to 2,000,000 gram per
mole (g/mole) were used for calibration following a method pre-
viously described by Stoklosa et al. [29]. Mn and Mw of chitosan
were calculated by using the elution time multiply by the flow rate
of the eluent obtained from GPC following the procedure reported
by Wu [30]. The Mn and Mw are expressed in g/mol.
2.3. Preparation of the film-forming dispersions
A chitosan solution with concentration of 1% (w/v) was prepared
by dissolving the chitosan powder in a 1% (w/v) aqueous acetic
acid while stirring at 250 rpm on a magnetic stirrer set at 45 ◦ C,
following the method of Ojagh et al. [31]. The chitosan solution
was stirred overnight at room temperature to achieve complete
dispersion. The emulsions were obtained by adding either CEO or
CWO to the chitosan solution to reach final concentrations of 10%,
20% and 30% w/w. Tween 80 was added as an emulsifier in quanti-
ties proportional to the essential oils (0.1%, 0.2% and 0.3% w/w) to
help distribute and completely incorporate the oil. Finally, emul-
sions were homogenized for 4 min at 2500 rpm with a Dual-Range
Mixer (IKA, Canada) and then degassed under vacuum to remove
dissolved air bubbles. The quantities of Tween 80, CEO and CWO
were defined according to our preliminary tests and based on liter-
ature data, taking into account the maximum levels of two essential
oils which could be incorporated into the matrix without oil phase
separation during film drying. Composite film without essential oil
and emulsifier was also produced and considered as control. The
suspension were then poured in polystyrene petri dishes measur-
ing 65 mm diameter, which was placed on a leveled surface and
allowed to dry for approximately 48 h at 30% RH and 22 ◦ C. Uni-
form film thicknesses were achieved by casting the same amount
of film-forming solution onto each plate. After drying, the films
were peeled off the casting surface and maintained at 22 ◦ C and 53%
relative humidity (produced with saturated Ca(NO3 )2 solution) in a
conditioning desiccator until further evaluation. For each test, three
different samples were prepared by taking three portions from each
film at different positions (two at the edges and one at the center)
with the exception of the water vapor permeability analysis, where
the whole sample was used, and replicates of each type of film were
evaluated.
2.4. Film characterization
2.4.1. Film thickness
Film thickness was determined using a hand-held digital
micrometer (Mitutoyo No. 293-766, Tokyo, Japan) with a precision
of 0.0001 mm. Measurements were carried out on at least five ran-
dom locations, and the mean thickness value was used to calculate
the permeability and mechanical properties of the films.
2.4.2. Film density
To determine film density, samples of 1 cm × 1 cm were main-
tained in a desiccator with calcium sulphate desiccant (0% RH) for
 Z. Shen, D.P. Kamdem / International Journal of Biological Macromolecules 74 (2015) 289–296
20 days and weighed. Dry-matter densities were calculated using
Eq. (1):
m calculated. Four replicates were used for the tensile testing.
s =
A×ı
where A is the film area (1 cm2 ), ı is the film thickness (cm),
m is the film dry mass (g) and s is the dry-matter density of the
film (g/cm3 ). The film density was expressed as the average of three
determinations.
2.4.3. Moisture uptake
Moisture uptake was measured as described in our previous
study [32]. Prior to testing all specimens were cut to a square piece
of 2.0 cm × 2.0 cm and conditioned using anhydrous calcium sul-
phate at 0% RH for 48 h. The films were accurately weighed to
give the dried film (Wi ). After weighing, they were conditioned at
23±1 ◦ C in a desiccator containing potassium sulphate saturated
solution to ensure a relative humidity of 97%. The samples were
removed from the desiccator after 48 h and weighed (W1 ). The
moisture uptake values of the samples were calculated using the
following equation (2):
(W1 − Wi )
% moisture uptake = × 100
Wi
where W1 is the weight of the film sample after exposure to 97%
RH for 48 h, and Wi is the initial weight of the sample. Each test con-
sisted of triplicate measurements and expressed as the mean value.
2.4.4. WVP (Water vapor permeability)
The WVP of films was determined gravimetrically in accordance
with ASTM E96/E96 M [33] with some modifications. Films with-
out pinholes or defects were cut into discs with a diameter slightly
larger than the diameter of special glass cups with a circular open-
ing of 0.000324 m2 . Each cup was completely filled with calcium
sulphate desiccant (0% RH) and covered with a disc, leaving an
air gap of 1 cm between the film underside and the desiccant. The
whole system was then placed in a desiccator containing a satu-
rated sodium chloride solution (75% RH). The RH inside the cell was
always lower than outside, and water-vapor transport was deter-
mined from the weight gain of the permeation cell at a steady state
of transfer. The cups were weighed to the nearest 0.0001 g. Changes
in the weight of the cup were recorded six times at 1 h interval
before a steady state was reached. The slope of each line was calcu-
lated by linear regression using Microsoft® Office Excel 2010 (the
lines’ regression coefficients were >0.998). The water vapor trans-
mission rate (WVTR) was obtained by dividing the slope (g/h) by
the effective film area (m2 ). This was multiplied by the thickness of
the film and divided by the pressure difference between the inner
and outer surfaces to obtain the WVP value, which was expressed
as [g m−1 s−1 Pa−1 ] and calculated according to Eq. (3):
m X
WVP =
At p
where m is the weight of moisture gain per unit of time (g/s),
t
X is the average film thickness (m), A is the area of the exposed
film surface (m2 ) and p is the water vapor pressure difference
between the two sides of the film (Pa). WVP was measured for three
replicated samples for each type of film.
2.4.5. Mechanical properties
The mechanical properties of the composite films were deter-
mined at 22 ◦ C and 30% RH with an Instron 5565 Universal Testing
machine (Instron, Canton, MA, USA) according to ASTM standard
method D882 [34]. Films were cut in rectangular strips of 50 mm
long and 6.35 mm wide. The films were fixed with an initial
grip separation of 25 mm and stretched at an extension speed of
291
0.8 mm/min. A microcomputer was used to record the stress-strain
curves. Tensile strength (TS and elongation at break (EAB) were
(1)
2.4.6. Thermogravimetric (TGA) analysis
Thermogravimetric (TGA) analysis was performed to evaluate
the thermal stabilities of composite films using a TGA 2950 running
with the Universal Analysis Software package V.3.9a (TA Instru-
ments, New Castle, DE, USA). Samples of approximately 5 mg were
heated from 50 to 500 ◦ C at 10 ◦ C/min heating rate under a nitrogen
flow of 70 mL/min. Weight losses of samples were measured as a
function of temperature. TGA was run in duplicate.
2.4.7. Differential scanning calorimetry (DSC)
A differential scanning calorimetry was performed using a DSC
Q100 (TA Instrument, New Castle, DE, USA) in accordance to ASTM
D3418 [35] with some modifications as described below. An empty
pan was used as an inert reference and the calibration was per-
formed using the indium standard. Samples were heated up at a
heating rate of 10 ◦ C/min between temperature ranges of 0 and
350 ◦ C under a nitrogen flow of 70 mL/min. The data obtained
(2) was then analyzed by using the Thermal Analysis Universal 2000
version 4.5A software (TA Instrument, New Castle, DE, USA). DSC
analysis was conducted in duplicate.
2.4.8. Fourier-transform-infrared spectroscopy (FT-IR)
FTIR spectra were obtained using ATR accessory. The used of ATR
to collect IR spectra on solids and liquids has been widely reported
by Subirade et al. [36]. Other advantages of ATR are the reduced
time and efforts for sample preparation.
The Fourier transformed infrared (FT-IR) spectra in the wave-
length range of 500–4000 cm−1 . were recorded with a Shimadzu
IR-Prestige 21 (Columbia, MD., USA) equipped with Miracle ATR
accessory from Pike Madison, WI., USA. ATR accessory was
equipped with a ZnSe crystal and a clamp to optimize the contact
between the sample and the crystal to control the spectrum qual-
ity. FT-IR was performed on films measuring about 25 ␮m thick by
5 mm width and 5 mm long with a resolution of 8 cm−1 on 64 scans
for each spectrum on reflectance mode. Prior to data analysis, all
the FTIR spectra were normalized by ratioing the intensity of peaks
to the intensity of the highest peak in the region between 2000 and
500 cm−1 .
2.4.9. Scanning electron microscopy (SEM)
A focused ion beam scanning electron microscope cross beam
(FIB-SEM) from Carl Zeiss Auriga (Tescan Mira 3 XMU, Cranberry
Township, USA) was used to scan the cross section of the film to
examine the microstructure and the morphology. Film was placed
in liquid nitrogen and hand fractured using a pair of plies to gen-
erate cross section. Cross sections were tungsten coated at 3 nm
(3)
thickness under vacuum level of 10−5 Torr. Imaging was collected
using a dual column focused ion beam with Gallium as ion source.
The ion and electron columns were set at a fixed angle of 36◦ . Film
samples were tilted to 54◦ so that the surface is perpendicular to
the ion beam. A 5 kV beam energy was used.
2.4.10. Statistical analysis
The results were presented as the mean ± standard deviation of
each treatment. The experiments were factorial with a completely
randomized design using analysis of variance (ANOVA), analyzed
using SAS software (version 9.3; Statistical Analysis System Insti-
tute Inc., Cary, NC, USA). Duncan’s multiple range tests were used
to compare the differences among the mean values for the films’
properties at the level of 0.05.
292 Z. Shen, D.P. Kamdem / International Journal of Biological Macromolecules 74 (2015) 289–296
3. Results and discussion
3.1. Molecular weight of chitosan
The molecular weight of chitosan was determined by using
gel permeation chromatography (GPC) to be 199,000 g/mol with
a number-average (Mn) of 138,000 g/mol. Islam et al. [37] reported
chitosan molecular weight of 165,000 g/mol using viscosity and the
Mark–Houwink equation. The difference in the molecular weight
may be due to chitosan origin and method of preparation. Tsai et al.
[38] reported a wide variation of the molecular weight of chitosan
from 108,000 to 491,000 g/mol functions of the method of prepara-
tion. Kasaai et al. [39] also noted the variation of chitosan molecular
weight due to solvent used in the preparation and the pH. The
molecular obtained in our work is within the variation described
in the literature [39].
3.2. Physical properties of the films
The composite films incorporating CEO and CWO were visu-
ally homogeneous with no brittle areas or bubbles, and could be
easily peeled from the casting plates. To identify the highest con-
centration of either CEO or CWO that could be incorporated into
the CH matrix, increasing amounts (up to 50% w/w) were tested in
the film-forming solution. As we expected, a stronger aroma was
found when the concentration of essential oil increased. No lipid
separation occurred in composite films even when the essential-
oil content was high. Generally, the effects of additives on the
properties of the films depended on the type of compounds and
concentrations and their inherent hyrophilicity and hydrophobic-
ity indices. The composite CH film thickness varied between the
range of 22 and 27 ␮m, and the incorporation of two essential oils
(CEO and CWO) did not significantly (P > 0.05) affect the result-
ing film thickness (data not shown). Control CH films generally
were very hydrophilic, and swelled when they absorbed water
quickly. As shown in Table 1, CH films without any essential oil
had significantly higher (P < 0.05) moisture uptake than did CH
films incorporating either CEO or CWO. The moisture uptake of
both CEO and CWO incorporated films decreased as the concentra-
tion of essential oil increased from 10% to 20% w/w, although this
reduction was not found to be significant (P > 0.05) in films incorpo-
rating either CEO or CWO. Moisture uptake decreased significantly
(P < 0.05) to 41.1% and 38.8% for the films containing 30% w/w CEO
and CWO, respectively. This was in accordance with our expec-
tation that increasing the concentration of essential oils would
decrease the films’ hydrophilicity. The lower moisture content of
CWO-containing films compared to those containing CEO may be
attributed to more hydrogen and covalent interactions between
CH chains and CWO components, which reduced the availability of
hydrogen groups to form hydrophilic bonds with water molecules.
This would result in a decrease in the tendency of CH film to absorb
water [40]. These findings were similar to those of other authors
when they incorporated a hydrophobic agent such as ␣-tocopherol
or cinnamon essential oil into CH films [41,42].
3.3. Water vapor barrier properties
The WVP of CH films containing CEO or CWO is shown in Fig. 1.
In the present study, the WVP of the CH composite films was
significantly (P < 0.05) affected by the incorporation of CEO or
CWO compared to control films. The addition of either CEO or
CWO significantly (P < 0.05) reduced the WVP of the films, hav-
ing attained a maximum permeability reduction of 63% in the
film prepared with 30% w/w CWO. Films containing CWO had
lower WVP than those with an equal concentration of CEO. In
the present study, CH films containing CEO showed no significant
4.5
CWO
4
CEO
WVP (g. s-1m-1Pa)× 1010
3.5
3
2.5
2
1.5
0 10 20 30
Essenal Oil Con. (%)
Fig. 1. Comparison of the water vapor permeability (WVP) of the chitosan com-
posite films including different concentration of citronella essential oil (CEO) and
cedarwood oil (CWO).
differences (P > 0.05) at concentrations of 10% and 20% (w/w),
although the data showed a general downward trend. The lower
concentration of essential oil might not have been high enough
to induce structural changes in the CH structure. However, a sig-
nificant decrease (P < 0.05) was observed when 20% CWO was
added (2.7 ± 0.2 g s−1 m−1 Pa−1 × 10−10 ), much lower than that for
films emulsified with CEO (3.2 ± 0.2 g s−1 m−1 Pa−1 × 10−10 ). As
explained previously, the lower WVP of the CH films with either
CEO or CWO may be due to the hydrogen and covalent interac-
tions between the CH network and these polyphenolic compounds.
These interactions may reduce the availability of the hydrophilic
groups to form hydrophilic bonds and diminish their interactions
with water, leading to a more water-resistant film. Moreover, the
presence of a hydrophobic disperse phase, even at small ratios,
introduces discontinuities in the hydrophilic phase that increase
the tortuosity factor for mass transfer, thus decreasing the WVP.
The effect of CEO or CWO addition observed in the present study
was in agreement with Shojaee-Aliabadi et al. [43], who found
that adding 3% (w/w) Satureja hortensis essential oil into kappa-
carrageenan-based films resulted in a considerable reduction in
the films’ WVP. Similarly, Salarbashi et al. [40] observed a signifi-
cant decrease in the WVP of soluble soybean polysaccharide films
incorporated with Zataria multi-flora Boiss or Mentha pulegium. The
present study also confirmed the outcome of earlier research by Ali-
heidari et al. [44], who had concluded that incorporating Matricaria
recutita essential oil in casein-based films decreased its WVP.
3.4. Mechanical properties
Table 1 shows the influence of essential oil incorporation on the
mechanical properties of CH-based films. The addition of CEO and
CWO in the considered concentration range significantly affected
(P < 0.05) the mechanical properties of the CH-based films. The TS of
CH films slightly decreased (P < 0.05) when CEO content increased
from 10% (33.0 MPa) to 20% (29.4 MPa); this behavior was more evi-
dent at the highest concentration (30%). Our findings suggest that
an increase in the concentration of the CEO significantly increased
the EAB. Nonetheless, the EAB showed an opposite trend in CH
films containing 30% CEO, exhibiting a reduction of 36% compared
to the control films. On the other hand, in the case of CWO, TS
did not significantly change when the essential oil concentration
was increased from 0% to 10%, but significantly changed when con-
centration was increased to 30%. The EAB of CH film containing
10% CWO increased. However, a significant difference (P < 0.05)
 Z. Shen, D.P. Kamdem / International Journal of Biological Macromolecules 74 (2015) 289–296 293

Table 1
Physical and mechanical properties of chitosan films containing CEO and CWO essential oils.

Film type Essential oil conc. (% w/w) Moisture uptake (%) Density (g/cm3 ) Tensile strength (MPa) Elongation at break (%)

Control 0 55.71 ± 9.94 a

1.04 ± 0.04a 39.68 ± 1.85a 13.00 ± 2.74a
CWO 10 50.04 ± 7.15ab 1.10 ± 0.02ab 36.54 ± 3.78ab 25.80 ± 1.53b
20 45.93 ± 2.43ab 1.13 ± 0.01b 28.47 ± 1.42c 18.33 ± 2.17c
30 38.78 ± 3.05b 1.21 ± 0.05c 22.29 ± 0.83d 5.07 ± 1.01d

CEO 10 51.93 ± 5.78ab 1.12 ± 0.03b 33.00 ± 1.94b 14.50 ± 0.50a

20 45.61 ± 5.87b 1.14 ± 0.02b 29.42 ± 0.47c 24.50 ± 0.50b
30 41.11 ± 5.75b 1.17 ± 0.03bc 17.12 ± 2.25e 8.25 ± 1.92e

Values represent the mean ± standard deviation of four replicates for tensile strength and three replicates for other test
Values in a column having different superscript letters are significantly different (P < 0.05).

in EAB was shown after adding 30% CWO; the greatest reduc- 100
tion of EAB was when 30% of CWO was incorporated. This effect
could primarily be explained by the partial replacement of stronger (a)
polymer–polymer interactions by weaker polymer–oil interactions
80
in the film network when essential oil components are present,
which may reduce the cohesion of the polymer network forces, thus

Weight (%)
decreasing the TS. Values of mechanical properties obtained for CH
films were similar to those found by some investigators [41,45], 60
but differed from those obtained in other works [46,47]. Due to the
differences in reactivity in binding or interacting with the polymer
network, different essential oils affected the mechanical properties Chitosan
40
of the films differently. Moreover, the source of chitosan, the acid CWO 10 %
medium used to disperse the polymer and the experimental condi- CWO 20 %
tions (pH, concentration, RH used during equilibration of the film, CWO 30 %
presence of emulsifiers) can explain the observed differences [47]. 20
50 150 250 350 450 550
3.5. Thermal stability Temperature (oC)
100
TGA thermograms showing thermal degradation behavior of CH
films with or without CEO and CWO are shown in Fig. 2. (b)
The films’ corresponding degradation temperatures (Td), weight
80
loss (w) and residue are presented in Table 2. The control CH
film generally exhibited three main stages of weight loss. Previ-
Weight (%)

ous studies have also shown that the thermal degradation of CH

films follows a three-step weight loss [48]. However, five main 60
stages of weight loss were observed in films incorporated with
both CEO and CWO. The first weight loss (w1 = 5.90–9.18%),
observed at an approximate temperature (Td1 ) of 100–122 ◦ C, is Chitosan
40
generally due to the evaporation of residual water and the acetic CEO 10 %
acid in the film. It was found that CH films incorporated with CEO 20 %
CEO had lower weight loss than those containing CWO. This is CEO 30 %
mainly attributed to the lower amount of water in films incorpo- 20
rated with CEO, which could be partially explained by the more 50 150 250 350 450 550
hydrophobic nature of the main components of this essential oil. Temperature (oC)
The weight loss (w2 = 15.00–20.30%) in the second stage, which
Fig. 2. DSC curves of chitosan composite films made of chitosan and various con-
corresponds to the degradation of lower molecular weight frac-
centration of (a) cedarwood oil (CWO) and (b) citronella essential oil (CEO). Please
tions or structurally bound water in the film network, occurred at see online edition for color images.
258–286 ◦ C. For the third stage of weight loss, w3 of 21.76–33.22%
and Td3 of 334–342.48 ◦ C were observed for all film samples, pos-
sibly associated with a complex process including the dehydration for control CH films, the fourth stage of weight loss (w4 ) disap-
of the saccharide rings, depolymerisation and decomposition of the peared. This fourth stage in films containing either CEO or CWO
acetylated and deacetylated units of the polymers. A higher ther- was possibly associated with the loss of thermally stable compo-
mal degradation temperature of the second and third stages was nents in the essential oils incorporated into the film matrix [47].
observed in films incorporated with both CEO and CWO compared The fifth stage of weight loss (w5 = 7.73–12.49%) appeared at Td5
with the control film. Our results indicated that both essential oils of 465–480 ◦ C, and was most likely due to the presence of highly
at a level of 20% did not improve the matrix’s thermal degradation. stable substances in the film containing essential oils or Tween 80
However, it was found that when the essential oil content reached that might possess higher heat resistance than CEO or CWO. Overall,
30%, CH films began thermal degradation at lower temperatures. films with CWO had lower residual mass than other films, consis-
The higher thermal stability of the films with essential oils could tent with their weaker film network, as indicated by the lowered
be associated with the interaction between the CH and oil, which TS. Amongst all films, those containing 30% CEO had the highest
might delay the thermal degradation of the composite films. The residual. TGA study clearly showed that CEO and CWO at various
fourth stage of weight loss (w4 of 6.38–11.83%) was obtained for concentrations led to different thermal stability of the resulting CH
films incorporated with each of the two essential oils. Nevertheless, films.
294 Z. Shen, D.P. Kamdem / International Journal of Biological Macromolecules 74 (2015) 289–296

Table 2
Thermal degradation temperature (Td, ◦ C) and weight loss (w, %) of chitosan films incorporated different concentrations of citronella essential oil (CEO) and cedarwood oil
(CWO).

Essential Oil Concentration 1 2 3 4 5 Residues (%)

oil type (% w/w)
Td1,onset w1 Td2,onset w2 Td3,onset w3 Td4,onset w4 Td5,onset w5

Control – 122.79 7.84 258.86 17.26 338.56 33.22 – – – – 41.68

CEO 10 100.11 7.42 277.62 16.35 336.56 22.71 424.31 11.83 465.23 7.97 33.72
20 105.36 6.75 284.94 15.89 337.6 26.11 438.38 8.56 472.82 7.73 34.96
30 112.67 5.90 286.39 15.00 342.21 25.39 442.33 8.90 475.36 8.30 36.51

CWO 10 117.67 8.94 278.67 18.99 334.43 21.76 423.74 9.75 472.95 9.36 31.19
20 103.04 9.18 274.33 17.22 338.43 24.71 443.16 6.38 480.04 12.46 30.05
30 119.87 6.40 260.51 20.30 342.31 22.73 440.96 9.84 477.86 12.49 28.24

1, 2, 3, 4 and 5 indicate the first, second, third, fourth and fifth stage weight loss, respectively, of film during heating scan

3.6. Thermal properties exothermic peak at the higher temperature was associated with
polymer decomposition. As shown in Fig. 3, the exothermic tem-
Typical DSC thermograms (Fig. 3) show that all CH film (with peratures of those films containing 10% w/w CEO and CWO were
or without essential oils) produces one broad endothermic peak higher than the control films. This clearly showed that both CEO
at approximately 140 ◦ C and one exothermic peak at 290 ◦ C. As and CWO contributed to an improvement in the thermal stability
we commented in the TGA results, the endothermal effect can be of the CH films. However, adding more of either CEO or CWO did
related to the evaporation of residual solvent used in the prepa- not further improve the films’ thermal stability.
ration of the films. With the 10% w/w increase of either CEO or
CWO, no difference was found in this endothermic peak. However, 3.7. Structural properties
the peak tended to shift to lower temperatures with the increase
of oil concentration to 30% w/w. This change could be explained To identify possible molecular interactions between CH func-
by the crystallinity of film samples. The results suggested that tional groups and the two essential oils studied in this work, FTIR
adding high concentrations of CEO or CWO into CH film changes the spectroscopy analysis was performed. The FT-IR spectra of control
film’s intermolecular interactions, leading to the observed changes
in the degree of crystallinity. Similar behavior was observed 1022
Chitosan
when ␣-tocopheryl acetate adding into chitosan film [49]. The (a) 1060
CWO 10 % 894
CWO 20 % 1149

3352 CWO 30 % 1562

(a) 2870
1257
Absorbance

1647
Endo

Chitosan
CWO 10 %
CWO 20 %
CWO 30 % 3600 3100 2600 2100 1600 1100 600
Wave number (cm-1)
0 50 100 150 200 250 300 350
Temperature (oC)
1149
Chitosan 894
(b) CEO 10%
(b) CEO 20% 1562
3352
2870 CEO 30%
Endo

1647
Absorbance

Chitosan
CEO 10 % 1022
CEO 20 % 1257
CEO 30 %

0 50 100 150 200 250 300 350 3600 3100 2600 2100 1600 1100 600
Temperature (oC) Wave number (cm-1)

Fig. 3. Results of thermal stability of chitosan composite films containing various Fig. 4. FTIR spectra of chitosan composite films including various concentrations
concentrations of (a) cedarwood oil (CWO) and (b) citronella essential oil (CEO) of (a) cedarwood oil (CWO) and (b) citronella essential oil (CEO). Please see online
analyzed through TGA. Please see online edition for color images. edition for color images.
 Z. Shen, D.P. Kamdem / International Journal of Biological Macromolecules 74 (2015) 289–296
CH films and those incorporated with CEO and CWO at different
concentrations are shown in Fig. 4.
All FTIR spectra showed quite similar general features, with
most of the peaks characteristic of CH films as reported by Silva-
Weiss et al. [50]. The peaks at 894, 1022, 1060 (C O stretching)
and 1149 cm−1 (asymmetric stretching of C O C bridge, glycosidic
linkage) were associated with the structure of pure CH, and were
common to all films. The band located at 1378 cm−1 is assigned to
the acetamide groups, indicating that CH is not completely deacety-
lated [51]. The absorption peak at 1647 cm−1 was attributed to
C O stretching (amide I), and the one at 1554 cm−1 corresponded
to N H bending (amide II) [52]. At the same time, these bands
showed stronger peak intensity in those films containing oils com-
pared with those of pure CH. The bands appearing at 2930 and
2870 cm−1 in the spectrum of CH film occur because of stretching
vibrations of the C H bond in CH2 and CH3 groups, respec-
tively [53]. These peaks became more flattened when 30% essential
oil was included. This may be due to hydrogen bonding between
the OH group in functional groups in oil ingredients and the
NH and OH groups in CH. Moreover, the CH film exhibited a
strong absorption band between 3190 and 3352 cm−1 that cor-
responded mainly with the stretching vibration of free hydroxyl
and to the asymmetric and symmetric stretching of the N H bonds
[46]. When the band intensities of the spectra of 10% and 20% w/w
Fig. 5. Scanning electron microscopy micrographs of the cross section of films (a) chitosan film, (b) film containing 10% essential oil, (c) film containing 20% essential oil, and
(d) film containing 30% essential oil viewed at a magnification of 6500×.
295
of CEO and CWO-containing films were analysed and compared,
it was hard to find any difference; this that little chemical inter-
action had taken place during the film preparation. However, the
addition of more CEO (30% w/w) resulted in a marked increase in
the intensity of the CH stretching peak at around 2870 cm−1 , sug-
gesting an increase in the content of ester groups, which might
come from the CEO molecule. A new peak at 1257 cm−1 was
observed at higher concentrations of CEO, which can be related
to the bending vibration of the OH groups from the phenol group
present in the essential oil structure [40]. Besides, the new peak
appeared at 1562 cm−1 which might be due to the interaction of the
amine group with a functional group of the essential oil. Another
important change was found with a new peak at 1647 cm−1 , indi-
cating an ester linkage, which could be attributed to the more
intensive interaction between CH and essential oil. The absorp-
tion intensity increased with increasing oil concentration. A shift
of the peak at 1026 cm−1 to 1022 cm−1 was noted for the 10%
w/w CEO-containing film; this could be ascribed to C O stretching
of hydroxyl and ether bonds. Such a shift toward lower wave-
number values indicates weaker interactions with these essential
oils. All these changes led to an assumption that intermolecular
interaction and molecular compatibility between the essential-oil
ingredients’ functional groups and hydroxyl and amino groups in
the CH matrix could exist. This information may explain the weaker
296 Z. Shen, D.P. Kamdem / International Journal of Biological Macromolecules 74 (2015) 289–296
mechanical properties of these films reported in the previous sec-
tion.
3.8. Microstructure
Fig. 5 shows SEM micrographs of the cross-sections of the films
containing various essential oil concentrations. Films containing
CWO and CEO show a similar microstructure. While a continuous
structure was observed for the chitosan film (Fig. 5a), the pres-
ence of essential oil (Fig. 5b–d) caused a heterogeneous structure in
which oil droplets were entrapped in the continuous carbohydrate
network. The number and size of the lipid droplets were increased
with the essential oil concentration. Little creaming was observed
in the emulsified films at 10% and 20% w/w essential oil concentra-
tion, probably due to the reduction of oil droplet mobility induced
by the highly viscous effect of chitosan, which, increased when dry-
ing progressed [52]. Nevertheless, the great oil phase concentration
(30% w/w) contributes to visible creaming in the micrograph; it was
possibly due to the high concentration of essential oil facilitate the
aggregation of lipid, which led to flocculation of essential oil in the
polymer.
4. Conclusion
This study was the first to explore the physical, thermal and
structural properties of CH-based films containing CEO or CWO.
The addition of either of two essential oils at the lowest concen-
tration did not significantly affect the film properties. However,
an improvement was observed for the highest concentration of
either essential oil particularly CEO, resulting in a CH film with bet-
ter barrier properties than the control film. TGA analysis clearly
showed that CEO and CWO at various concentrations led to differ-
ent thermal stabilities for the resulting CH films. The FTIR spectra
analysis indicated that a strong interaction occurred between CH
and each essential oil. This may answer the question of how to pro-
duce extremely flexible CH films when essential oil is included in
the matrix. Overall, this study suggests that it is possible to formu-
late CH films having CEO or CWO. Nevertheless, in vivo studies are
required to evaluate the possible interactions between the film con-
stituents and the food matrix. Additional work is in progress in our
research group to study the antimicrobial and antifungal activity of
these films.
Acknowledgments
The authors gratefully acknowledge the financial assistance pro-
vided by the School of Packaging, Michigan State University. We are
also thankful to Muyang Li for GPC analysis.
References
[1] R. Tharanathan, Trends Food Sci. Technol. 14 (2003) 71–78.
[2] G. Robertson, in: E. Chiellini (Ed.), Environmentally Compatible Food Packaging,
Woodhead Publishing, Cambridge, UK, 2008, pp. 3–28.
[3] A. Lopez-Rubio, E. Almenar, P. Hernandez-Muñoz, J.M. Lagarón, R. Catalá, R.
Gavara, Food Rev. Int. 20 (2004) 357–387.
[4] S. Shojaee-Aliabadi, H. Hosseini, M.A. Mohammadifar, A. Mohammadi, M.
Ghasemlou, S.M. Hosseini, R. Khaksar, Carbohydr. Polym. 101 (2014) 582–591.
[5] D. Salarbashi, S. Tajik, S. Shojaee-Aliabadi, M. Ghasemlou, H. Moayyed, R. Khak-
sar, M.S. Noghabi, Food Chem. 146 (2014) 614–622.
[6] E. Almenar, R. Catala, P. Hernandez-Muñoz, R. Gavara, LWT-Food Sci. Technol.
42 (2009) 587–593.
[7] M. Ghasemlou, N. Aliheidari, R. Fahmi, S. Shojaee-Aliabadi, B. Keshavarz, M.J.
Cran, R. Khaksar, Carbohydr. Polym. 98 (2013) 1117–1126.
[8] M. Rinaudo, Prog. Polym. Sci. 31 (2006) 603–632.
[9] M.N. Ravi Kumar, React. Funct. Polym. 46 (2000) 1–27.
[10] P. Dutta, S. Tripathi, G. Mehrotra, J. Dutta, Food Chem. 114 (2009) 1173–1182.
[11] I. Leceta, P. Guerrero, K. de la Caba, Carbohydr. Polym. 93 (2013) 339–346.
[12] Y. Ruiz-Navajas, M. Viuda-Martos, E. Sendra, J. Perez-Alvarez, J. Fernández-
López, Food Control 30 (2013) 386–392.
[13] Á. Perdones, M. Vargas, L. Atarés, A. Chiralt, Food Hydrocoll. 36 (2014) 256–264.
[14] Y. Peng, L. Yin, Y. Li, Int. J. Food Sci. Technol. 48 (2013) 44–50.
[15] S. Burt, Int. J. Food Microbiol. 94 (2004) 223–253.
[16] M. Viuda-Martos, Y. Ruiz-Navajas, J. Fernández-López, J.A. Pérez-Álvarez, Int. J.
Food Sci. Technol. 43 (2008) 526–531.
[17] A.G. Ponce, S.I. Roura, C.E. del Valle, M.R. Moreira, Postharvest Biol. Technol. 49
(2008) 294–300.
[18] L.S. Nerio, J. Olivero-Verbel, E. Stashenko, Biores. Technol. 101 (2010) 372–378.
[19] M. Arancibia, A. Rabossi, P.A. Bochicchio, S. Moreno, M. López Caballero, G.
Guillén, P. Montero García, Food Control 44 (2014) 7–15.
[20] C. Pires, C. Ramos, B. Teixeira, I. Batista, M. Nunes, A. Marques, Food Hydrocoll.
30 (2013) 224–231.
[21] X.-w. Huang, Y.-c. Feng, Y. Huang, H.-l. Li, J. Essent. Oil Res. 25 (2013) 315–323.
[22] Z. Tunalier, N. Kirimer, K. Baser, J. Essent. Oil Res. 16 (2004) 233–235.
[23] R.P. Adams, S. Li, J. Essent. Oil Res. 20 (2008) 235–242.
[24] M.Y. Arancibia, M.E. López-Caballero, M.C. Gómez-Guillén, P. Montero, Food
Control 44 (2014) 7–15.
[25] Q. Chen, S. Xu, T. Wu, J. Guo, S. Sha, X. Zheng, T. Yu, J. Sci. Food Agric. 94 (2014)
2441–2447.
[26] P. Tongnuanchan, S. Benjakul, T. Prodpran, Int. J. Food Sci. Technol. 48 (2013)
2143–2149.
[27] F. Eller, C.A. Clausen, F. Green, S. Taylor, Ind. Crops Prod. 32 (2010) 481–485.
[28] I. Tumen, I. Süntar, F.J. Eller, H. Keleş, E.K. Akkol, J. Med. Food 16 (2013) 48–55.
[29] R.J. Stoklosa, J. Velez, S. Kelkar, C.M. Saffron, M.C. Thies, D.B. Hodge, Green Chem.
15 (2013) 2904–2912.
[30] C.-S. Wu, Handbook of Size Exclusion Chromatography and Related Techniques:
Revised and Expanded, CRC Press, Boca Raton Florida, 2003.
[31] S.M. Ojagh, M. Rezaei, S.H. Razavi, S.M.H. Hosseini, Food Chem. 122 (2010)
161–166.
[32] Z. Shen, M. Ghasemlou, D.P. Kamdem, J. Appl. Polym. Sci. (2014).
[33] ASTM Standard E96, Standard Test Methods for Water Vapor Transmis-
sion of Materials, ASTM International, West Conshohocken, PA, 2013,
http://dx.doi.org/10.1520/C0033-13, www.astm.org.
[34] ASTM Standard D882, Standard Test Method for Tensile Properties of
Thin Plastic Sheeting, ASTM International, West Conshohocken, PA, 2009,
http://dx.doi.org/10.1520/C0033-09, www.astm.org.
[35] ASTM Standard D3418, Melting Point by DSC Testing Services, ASTM Inter-
national, West Conshohocken, PA, 2003, http://dx.doi.org/10.1520/C0033-03,
www.astm.org.
[36] M. Subirade, I. Kelly, J. Guéguen, M. Pézolet, Int. J. Biol. Macromol. 23 (1998)
241–249.
[37] M. Islam, S.M. Masum, M.M. Rahman, M. Molla, A. Islam, A. Shaikh, S. Roy, Int.
J. Basic Appl. Sci. 11 (2011).
[38] G.J. Tsai, W.H. Su, H.C. Chen, C.L. Pan, Fish. Sci. 68 (2002) 170–177.
[39] J.Z. Knaul, M.R. Kasaai, V.T. Bui, K.A. Creber, Can. J. Chem. 76 (1998) 1699–1706.
[40] D. Salarbashi, S. Tajik, M. Ghasemlou, S. Shojaee-Aliabadi, M. Shahidi Noghabi,
R. Khaksar, Carbohydr. Polym. 98 (2013) 1127–1136.
[41] J.T. Martins, M.A. Cerqueira, A.A. Vicente, Food Hydrocoll. 27 (2012) 220–227.
[42] M. Hosseini, S. Razavi, M. Mousavi, J. Food Process. Preserv. 33 (2009)
727–743.
[43] S. Shojaee-Aliabadi, H. Hosseini, M.A. Mohammadifar, A. Mohammadi, M.
Ghasemlou, S.M. Ojagh, S.M. Hosseini, R. Khaksar, Int. J. Biol. Macromol. 52
(2013) 116–124.
[44] N. Aliheidari, M. Fazaeli, R. Ahmadi, M. Ghasemlou, Z. Emam-Djomeh, Int. J.
Biol. Macromol. 56 (2013) 69–75.
[45] W. Thakhiew, S. Devahastin, S. Soponronnarit, J. Food Eng. 119 (2013) 140–149.
[46] M. Vargas, A. Albors, A. Chiralt, C. González-Martínez, Food Hydrocoll. 23 (2009)
536–547.
[47] L. Sánchez-González, M. Cháfer, A. Chiralt, C. González-Martínez, Carbohydr.
Polym. 82 (2010) 277–283.
[48] D. Altiok, E. Altiok, F. Tihminlioglu, J. Mater. Sci.: Mater. Med. 21 (2010)
2227–2236.
[49] S.-i. Park, Y. Zhao, J. Agric. Food Chem. 52 (2004) 1933–1939.
[50] A. Silva-Weiss, V. Bifani, M. Ihl, P. Sobral, M. Gómez-Guillén, Food Hydrocoll.
31 (2013) 458–466.
[51] I. Leceta, P. Guerrero, I. Ibarburu, M. Dueñas, K. De la Caba, J. Food Eng. 116
(2013) 889–899.
[52] J. Brugnerotto, J. Lizardi, F. Goycoolea, W. Argüelles-Monal, J. Desbrieres, M.
Rinaudo, Polymer 42 (2001) 3569–3580.
[53] M. Abdollahi, M. Rezaei, G. Farzi, Int. J. Food Sci. Technol. 47 (2012) 847–853.