Food Hydrocolloids 61 (2016) 662e671

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

Preparation, characterization, and properties of chitosan films with

cinnamaldehyde nanoemulsions
Huanle Chen a, 1, Xiaorong Hu e, 1, Enmin Chen a, Shan Wu a, David Julian McClements c, d,
Shilin Liu a, b, Bin Li a, b, Yan Li a, b, *
a
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
b
Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, China
c
Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA
d
Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
e
Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, China

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

Article history: Natural aldehydes, such as cinnamaldehyde, can be used to modify the properties of chitosan-based
Received 22 April 2016 materials by covalent cross-linking the chitosan molecules. Due to the non-polarity and hydrophobici-
Received in revised form ty of cinnamaldehyde, emulsification technique was used to prepare homogeneous cinnamaldehyde-
22 June 2016
chitosan films. Under optimized conditions, cinnamaldehyde nanoemulsions led to the successful
Accepted 24 June 2016
fabrication of uniform and smooth chitosan films. FTIR analysis of the films indicated the formation of
Available online 25 June 2016
chitosan-cinnamaldehyde Schiff base. The cross-linked films strongly absorbed UVevisible light and
inhibited the emission of fluorescent light, and may therefore provide good photo-protection. The
Keywords:
Chitosan
thickness (26e61 mm) and transparency (clear to turbid) of the chitosan films were modulated by varying
Cinnamaldehyde the aldehyde/amino group ratio. The mechanical properties and water vapor permeability of the films
Nanoemulsions also depended on the amount of cinnamaldehyde nanoemulsion utilized. Chitosan films showed good
Schiff base antimicrobial activity, but had better antifungal than antibacterial activity.
Films © 2016 Published by Elsevier Ltd.
Chemical compounds studied in this article:
Cinnamaldehyde (PubChem CID: 637511)
Tween 80 (PubChem CID: 6364656)

1. Introduction oxygen impermeability (Elsabee & Abdou, 2013). However, films

fabricated from pure chitosan tend to be rigid and brittle, and so
There is increasing interest in the development of sustainable plasticizers are needed to reduce the frictional forces between the
coating materials to protect the quality and safety of fruits, vege- polymer chains and thereby improve their mechanical properties
tables, fish, and meats (Kalia & Parshad, 2014; Pasha, Saeed, Sultan, (Aguirre, Borneo, & Leo
n, 2013; Thakhiew, Champahom,
Khan, & Rohi, 2014). Biopolymers, such as proteins and poly- Devahastin, & Soponronnarit, 2015). Alternatively, the functional
saccharides, are popular candidates for the fabrication of these groups on chitosan molecules (such as amino and hydroxyl groups)
coatings because they are non-toxic, biocompatible, biodegradable, can be chemically modified to enhance the mechanical attributes of
and renewable (Arora & Padua, 2010; Rhim & Ng, 2007; Vargas, chitosan films (Hu, Xin, Hu, Chan, & He, 2013; Yu, Song, Shi, Xu, &
Pastor, Chiralt, McClements, & Gonzalez-Martinez, 2008). Chito- Bin, 2011).
san is a cationic polysaccharide isolated from natural sources A common technique used to chemically modify chitosan films
(Croisier & Je
ro
^ me, 2013), which has been utilized in the food in- is based on the formation of a Schiff base between amino groups on
dustry for its film forming properties, antibacterial activity, and the chitosan chain and aldehyde groups on cross-linking agents
(Schiffman & Schauer, 2007). Schiff-base reactions have been
widely applied in the fields of biomedicine, chemistry, and food
science, due to mild reaction conditions and high reaction rates
* Corresponding author. No 1 Shizishan Road, Hongshan District, Wuhan 430070,
China.
(Xin & Yuan, 2012). Essential oils are aromatic oily liquids extracted
E-mail address: [email protected] (Y. Li). from certain fruits, vegetables and spices, which can be utilized as
1
These authors contributed equally to the work. natural antioxidant and antimicrobial substances. Previous

http://dx.doi.org/10.1016/j.foodhyd.2016.06.034
0268-005X/© 2016 Published by Elsevier Ltd.
 H. Chen et al. / Food Hydrocolloids 61 (2016) 662e671 663

investigations have demonstrated their antimicrobial properties solutions. All the other chemicals were of analytical grade. Escher-
and applications in the preservation of fruits, vegetables and meats ichia coli (E. coli) and Staphylococcus aureus (S. aureus) were ob-
(Ferna
ndez-Pan, Carrio
n-Granda, & Mate
, 2014; Perdones, tained from the culture collection in Department of Food Science
Sa
nchez-Gonza
lez, Chiralt, & Vargas, 2012; Severino et al., 2015). and Technology, Huazhong Agricultural University (Wuhan, China).
Cinnamon oil, which is an essential oil extracted from cinnamon Candida Albicans HG01 (C. albicans) was provided by the College of
bark, has been shown to be particularly effective (Burt, 2004). The Bioengineering in Hubei University of Technology (Wuhan, China).
main active constituent in cinnamon oil is cinnamaldehyde, which
is a hydrophobic aromatic aldehyde that has been approved by the 2.2. Formation of chitosan films
FAO/WHO Expert Committee on Food Additives (JECFA) for use as a
food-flavoring agent (Cocchiara, Letizia, Lalko, Lapczynski, & Api, 2.2.1. Cinnamaldehyde-loaded nanoemulsion preparation
2005). Cinnamaldehyde is a well-known natural antimicrobial The formation of cinnamaldehyde nanoemulsions was carried
compound that has been shown to be effective against a broad out using the spontaneous emulsification method described pre-
spectrum of food-borne pathogens (Burt, 2004; Holley & Patel, viously (Tian, Lei, Zhang, & Li, 2015). Briefly, an organic phase
2005). The aldehyde group in cinnamaldehyde can react with the (10 wt%) was added dropwise into an aqueous phase (90 wt%) at
amino group in chitosan through the Schiff base reaction. Studies ambient temperature while continuously stirring at 600 rpm using
have shown that cinnamaldehyde cross-linked chitosan nano- a magnetic stirrer for 15 min. The organic phase consisted of a
particles can be prepared using water-in-oil emulsions as templates mixture of cinnamaldehyde, MCT, and Tween 80, while the aqueous
(Nipun Babu & Kannan, 2012). Imino-chitosan films have also been phase consisted of distilled water. The mass ratio of MCT and cin-
fabricated using the Schiff-base reaction from cinnamaldehyde, namaldehyde was 1:1 and the concentration of Tween 80 was 7.5%.
vanillin, piperonal and other natural aldehydes (Marin, Simionescu,
& Barboiu, 2012; Marin et al., 2013; Marin et al., 2015). Typically, 2.2.2. Chitosan solution preparation
non-polar aldehydes need to be dissolved within water-miscible Powdered chitosan (2 wt%) was dispersed in 1 wt% acetic acid
organic solvents (such as methanol, ethanol or acetone) before solution at room temperature, and then agitated overnight. Finally,
they can be mixed with aqueous chitosan solutions. Then, high the solution was centrifuged to remove any air bubbles or other
reaction efficiency between aldehydes and chitosan can be greatly impurities.
achieved by the slow water removal. However, the efficiency of
chitosan cross-linking would be relatively low without the addition 2.2.3. Film preparation and cross-linking
of solvents, due to the poor water-solubility of the non-polar al- Cinnamaldehyde cross-linked chitosan films were prepared by
dehydes used. Our previous study showed that mixing cinna- mixing cinnamaldehyde nanoemulsions and chitosan solutions.
maldehyde oil with an aqueous chitosan solution did not promote The mixture was continuously stirred for 30 min to make sure the
gel formation, but mixing emulsified cinnamaldehyde led to gel thorough blending. Then, the air bubbles in the mixture were
formation at pH > 4.6 (Lei et al., 2015). This study also showed that eliminated by the vacuum treatment. After this process, the pH of
gels formed using emulsified cinnamaldehyde and chitosan had the mixed solution was around 4.3. Aliquots (3 mL) of film-forming
higher antibacterial activities than solutions formed from cinna- solution were then cast on Petri dishes (polytetrafluoroethylene,
maldehyde oil and chitosan. 5.5 cm  5.5 cm) and dried for 10 h at 60
C in a vacuum oven. The
In the current study, we investigated the possibility of Schiff films were then peeled from the plates and placed in desiccators
base cross-linking of chitosan films at low pH by first converting containing silica gel before characterization. The preparation of a
cinnamaldehyde into a water-dispersible oil-in-water nano- pure chitosan film (control) was carried out using the same pro-
emulsion. The film was formed by the vacuum drying method to cedure but without including the nanoemulsion. The ratio and
remove water. Then, physicochemical properties and antimicrobial concentration of cinnamaldehyde nanoemulsion and chitosan so-
activity of the films were investigated. The results showed that the lutions were set based on the ratio of -CHO/-NH2 molar ratios. Here,
cross-linked chitosan films obtained in our study may be appro- -CHO and -NH2 refer to the carbonyl group on the cinnamaldehyde
priate for utilization as antimicrobial coatings for direct application molecules and the amino groups on the chitosan molecules,
to a variety of foodstuffs. respectively. For the sake of convenience, the films are referred to
by the abbreviation “CnN” according to their -CHO/-NH2 molar
2. Materials & methods ratios (n). For example, a film that has a molar ratio of 1.5 cinna-
maldehyde carbonyl groups to chitosan amino groups is referred to
2.1. Materials as “C1.5N”, while a pure chitosan film is referred to as “C0N”.
Finally, cinnamaldehyde-chitosan films were stored in an amber
Chitosan (degree of deacetylation of 85%; viscosity of 1% chito- glass desiccator with P2O5 at ambient temperature (around 23
C)
san solution at 20
C at 1250 mPa s; Mw: 2.78  105 Da by dynamic prior to use.
light scattering) was provided by Zhejiang Golden-Shell Pharma-
ceutical Co. (Zhejiang, China). Cinnamaldehyde (95%) and potas- 2.3. Characterization of chitosan films
sium bromide (KBr) were purchased from Aladdin Reagent Co.
(Shanghai, China). Medium chain triglycerides (MCT,  95%) were 2.3.1. Film thickness
obtained from Wuhan Boxing Chemical Co. (Wuhan, China). Agar Film thickness (T) was measured to the nearest 0.001 mm with a
was supplied from Shanghai Titan Scientific Co., Ltd (Shanghai, hand-held digital micrometer (QuantuMike Mitutoyo, Japan). The
China). Mueller Hinton broth (MHB) and Yeast Extract Peptone values obtained for each sample at three different locations were
Dextrose agar (YPD) were purchased from Qingdao Hope Biol- averaged.
Technology Co., Ltd (Qingdao, China). Tween 80, hydrochloric acid
(HCl), sodium hydroxide (NaOH), disodium hydrogen phosphate 2.3.2. Optical properties
(Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), sodium Transparency: The optical transmittance (Tm) of the films was
chloride (NaCl) and ethanol were purchased from Sinopharm measured using a UVevis spectroscopy from 200 to 800 nm using
Chemical Reagent Co. (Shanghai, China). Distilled water (electrical air as a reference material (F300s Ultraviolet lamp, Shimadu,
resistance z 18.2 MU$cm) was used to prepare all aqueous Tokyo). The transmittance was calculated by measuring the
664 H. Chen et al. / Food Hydrocolloids 61 (2016) 662e671

intensity of transmitted light in the presence (I) and absence (I0) of The volume ratio of ethanol-to-water was set at 1:9 and 5:5 to
the film: Tm ¼ I/I0. simulate aqueous food and lipophilic (alcoholic >20%) food sys-
tems, respectively. Around 10 mg films were added to glass bottles
2.3.3. Scanning electronic microscopy (SEM) containing 20 mL of these simulated food solutions. The bottles
The morphology of the surface and the cross-section of the films were sealed and stored in a 37
C incubator with a shaking speed of
were examined using scanning electron microscopy (SEM, Hitachi 200 r/min. 100 mL of simulated solutions were collected at different
S-4500, Japan) at an accelerated electron energy of 30 kev. time intervals and the cinnamaldehyde concentration was deter-
mined by UVevisible spectroscopy (F300s Ultraviolet lamp). A
2.3.4. Atomic Force Microscopy (AFM) standard curve for cinnamaldehyde was prepared prior to each
The surface stiffness of chitosan-based films was investigated by experiment. The release of cinnamaldehyde from the cross-linked
Atomic Force Microscopy. AFM investigation was conducted on a chitosan films was calculated.
flat silicon substrate using a Multimode 8 AFM (Bruker, USA) in a
tapping mode. ~20 mL of filming solution was added on the silicon 2.5. Antibacterial assay
substrate and then the film was formed by a spin coater (Aurel
Technologies, USA) using a spin speed of 3000 r/min. The Oxford cup method was performed to evaluate the anti-
microbial activities of chitosan and chitosan-cinnamaldehyde films.
2.3.5. Fourier-transform infrared spectroscopy (FTIR) Gram-negative bacteria Escherichia coli (E. coli), gram-positive
Fourier transform infrared spectra of dried chitosan-based films bacteria Staphylococcus aureus (S. aureus) and fungus Candida
were recorded using a FTIR spectroscopy instrument (FT-IR 615, albicans (C. albicans) were selected as representative microorgan-
Japan). The dried samples were ground into powders, mixed with isms. An Oxford cup (a stainless cylinder, outer diameter
KBr, and then pressed to form a disk for the tests. 8.0 ± 0.1 mm, inner diameter 6.0 ± 0.1 mm and height
10.0 ± 0.1 mm) is used to limit the swelling of the membrane in the
2.3.6. X-ray diffraction (XRD) cup. 0.20 g film-forming solutions were added to an Oxford cup on
Wide-angle X-ray diffraction measurements were carried out Petri dishes (polytetrafluoroethylene, 5.5 cm  5.5 cm) and then
using a XRD diffractometer (D8-Advance, Bruker, USA). The pat- dried according to the methods described in section 2.2. Oxford
terns with Cu Ka radiation (the weighted average l ¼ 0.15406 nm) cups with films were then put on the surface of the nutrient agar or
at 40 kV and 40 mA were recorded in the region of 2q from 8 to 70
yeast extract peptone dextrose agar in Petri dishes. 100 mL micro-
with a step speed of 1
min1. organism suspensions taken from a diluted nutrient broth medium
were previously inoculated. The concentrations of E. coli and S.
2.3.7. Water vapor permeability (WVP) aureus suspension were approximately 108 CFU/mL and that of C.
The water vapor transmission rate (WVTR) of the films were albicans suspension was approximately 107 CFU/mL. The bacteria
measured according to a method described previously (Li, Wu, and fungus were incubated for 36 h at 37
C and 30
C, respectively.
Liang, Li, & Liu, 2014) with slight modification. Environments Then the inhibition zones were measured with a tolerance of 1 mm.
with different relative humidity (RH) were established inside the The experiment was repeated in triplicate for each sample. Saline
test chamber using distilled water (100% of RH at 15
C) and was evaluated as controls.
saturated aqueous solutions of NaCl (76% of RH at 15
C) and MgCl2
(33% of RH at 15
C). WVTR and water vapor permeability (WVP) 2.6. Statistical analysis
were calculated according to the following formulas:
All experiments were performed in triplicate on freshly pre-
WVTR ¼ Dw=Dt$A pared samples. The results were then reported as averages and
standard deviations of these measurements.
WVP ¼ WVTR$L=Dp
3. Results and discussions
where WVTR was in g/h$m2, Dw/Dt was rate of water gain in g/h, A
was the exposed area of the film in m2, WVP was in g/Pa$h$m, L was 3.1. Optical and morphological properties
the mean thickness of samples in m, and Dp was the difference in
partial water vapor pressure between the two sides of film samples The appearance of chitosan films is shown in Fig. 1a. Pure chi-
in Pa. The water vapor pressure on the high-stream side of the film tosan films were difficult to peel from the Petri dishes. All films
was 1.71, 1.29 and 0.57 kPa for distilled water, saturated NaCl so- were transparent and their surfaces appeared smoother as the
lution and saturated MgCl2 solution at 15
C, respectively, while the concentration of cinnamaldehyde nanoemulsion increased. The
low-stream side was assumed to be zero. incorporation of cinnamaldehyde nanoemulsions caused an in-
crease in the yellowish color of the chitosan films (Fig. 1a), which is
2.3.8. Mechanical properties indicative of the occurrence of the Schiff-base reaction according to
The mechanical properties of the films were characterized using previous studies (Marin et al., 2013). The results of the visual ob-
a universal tensile tester (CMT 6503, Shenzhen SANS Test machine servations of the chitosan films were supported by measurements
Co. Ltd, China) according to a standard method (ASTM/D638-91) at of their UVevis transmittance profile in the wavelength range from
a speed of 2 mm$min1 at room temperature. The tensile strength 200 to 800 nm (Fig. 1b). The pure chitosan films only gave limited
(TS), elongation at break (EAB) and Young’s modulus were deter- protection against UV light from 200 to 400 nm, which is consistent
mined. At least five replicates were tested for each sample. with previous reports (Leceta, Guerrero, & de la Caba, 2013). The
incorporation of cinnamaldehyde nanoemulsions into the films
2.4. Evaluation of cinnamaldehyde release in food simulants decreased the amount of UV light that could penetrate through
them regardless of the amount of nanoemulsion added. Luo et al.
Due to their different hydrophilic/hydrophobic properties, reported that the anti-ultraviolet performance of chitosan film was
ethanol-water solvents were utilized to simulate different food improved with the incorporation of xylan, due to the denser
environments according to a previous study (Balaguer et al., 2013). network structure and larger refractive index caused by the
 H. Chen et al. / Food Hydrocolloids 61 (2016) 662e671 665

Fig. 1. Influence of the addition of cinnamaldehyde nanoemulsions on the optical properties of chitosan films: (a) visual photos of chitosan films; (b) UVeVisible absorption from
200 to 800 nm.

formation of xylan ball nodules (Luo, Pan, Ling, Wang, & Sun, 2014). (Acevedo-Fani, Salvia-Trujillo, Rojas-Graü, & Martín-Belloso, 2015).
On the other hand, there was a minimum in the transparency at Electron microscopy indicated that at the microscopic level the
600 nm for the C0.4N and C0.6N systems, indicating that they were chitosan films were compact and had relatively smooth surfaces
the most effective at protecting against visible light. The physico- without pores or cracks (data not shown). Further insights into the
chemical origin of these optical effects can be attributed to two surface morphology of the films were obtained using AFM topo-
phenomena: (i) light scattering by the small lipid droplets resulting graphic images (Fig. 3). The pure chitosan film (control) had a
from the nanoemulsions; (ii) light absorption by the Schiff base relatively low level of surface roughness, whereas the chitosan
products. films containing the cinnamaldehyde nanoemulsions had rougher
Electron microscopy images of the cross section of the films surfaces and contained some holes (dark regions). These holes were
revealed some cracks at low cinnamaldehyde levels (CS and C0.1N), probably the result of defects formed in the films caused by cross-
bubble-like structures at intermediate levels (C0.2N to C1.5N), and linking of the chitosan molecules in the presence of cinnamalde-
smooth structures at high levels (C2.0N) (Fig. 2). These bubble-like hyde. Previous studies have reported that the incorporation of
structures may have been due to the presence of nanoemulsion vanillin into chitosan films also leads to an increase in their surface
droplets within the films. At the high concentration of cinna- roughness and to hole formation (Marin et al., 2013). In our study,
maldehyde, the interaction between chitosan and cinnamaldehyde at low cinnamaldehyde contents, the chitosan films remained
became stronger. When the ratio of aldehyde group to amino group relatively smooth and thin, but contained numerous holes. At
was higher than 2.0, some of nanodroplets leached out from the higher cinnamaldehyde contents, the films became rougher and
films after the vacuum drying process used to prepare the samples. thicker, and there were more numerous and larger holes present.
During the preparation of films, it was found that the evaporation of This latter effect may have occurred because of aggregation of the
water was slower at higher nanoemulsion contents, due to the oil droplets at the surfaces of the films. An increase in cinna-
rapid formation of a cross-linked network between the cinna- maldehyde content would also lead to faster cross-linking of the
maldehyde and chitosan, which could hinder the movement of the chitosan, which may have altered the nature of the biopolymer
water molecules. Previous studies have also reported that the network formed. The maximum particle size changed from around
structure of pure chitosan films is smoother and more homogenous 8 nme300 nm, indicating that the chitosan films were structured at
than that of films formed by mixing the polymer with pure the nanometer range (Marin et al., 2013). The AFM observations
essential oils (Perdones et al., 2012) or with nanoemulsions were consistent with the appearance and transmittance of the films

Fig. 2. Cross-section morphology of vacuum-dried chitosan films by SEM observation.

666 H. Chen et al. / Food Hydrocolloids 61 (2016) 662e671

Fig. 3. Three-dimensional phase images by AFM of chitosan films and cross-linked chitosan films.

(Fig. 1a and b), since the intensity of light scattering is known to attributed to the consumption of amino groups on the chitosan
depend on the dimensions of any heterogeneities within a material. chain (Chen, Shi, Neoh, & Kang, 2009). With increasing cinna-
maldehyde content, the absorption peak at 1567 cm1 became
3.2. FTIR and XRD analyses weaker and weaker. The cross-linked films had some distinctive
peaks at 688, 748, 1458 cm1, which corresponded to the phenyl
There were appreciable changes in the FTIR spectra of the chi- group of cinnamaldehyde. It has been reported that the signal at
tosan films as the amount of cinnamaldehyde nanoemulsion added 688 cm1 is for CH¼CH bending out-of-plane in alkenes, 748 cm1
to the system increased (Fig. 4), which suggests that imine bond for -CH bending out-of-plane in aromatic ring and 1458 cm1 for
formation and other structural modifications occurred. For the pure -CH stretching in aromatic ring (combined with -CH bending of
chitosan film: the peaks in the range 3600e3200 cm1 were aliphatic compounds). This result strongly suggests the formation
assigned to different eOH stretching vibrations corresponding to of imino bonds after the addition of cinnamaldehyde nano-
intra- and inter-molecular H-bonds; the peak at 3430 cm1 was emulsions (Chen & Chen, 2009; Marin et al., 2015). The new peak
assigned to -NH and -OH stretching vibrations; and the peaks observed at 1744 cm1 can be assigned as a carboxylate group or
observed at 2925 and 2856 cm1 were assigned to symmetric and carbonyl group (Shi et al., 2014), which became sharper with higher
antisymmetric -CH2 stretching vibrations. The peaks observed in nanoemulsion content. From 2600 to 3800 cm1, no new peak was
the spectra of the films at wavenumbers of 1638, 1567 and observed, but the intensities of the peaks at 2921 and 2856 cm1
1409 cm1 were assigned to -C¼O (amide I), NH (amide II), and HN- increased with higher cinnamaldehyde content.
CO (amide III) stretching vibrations, respectively (Brugnerotto et al., Information about the effect of cinnamaldehyde on chitosan was
2001; Lagaron, Fernandez-Saiz, & Ocio, 2007; Ziani, Oses, Coma, & obtained by using X-ray diffraction. XRD profiles of the chitosan
Mate
, 2008), while the broad absorption peak at 1073 cm1 was films exhibited a broad-band between 7 and 35
(Fig. 5), with a
assigned to C-O stretching. maximum around 23
and a shoulder around 12
, which is indic-
In comparison to the spectra of the pure chitosan films, the new ative of two overlapping reflections. This type of XRD pattern is
peak at 1637 cm 1 corresponded to the C¼N vibrations charac- characteristic of the presence of hydrated H-bonded chitosan
teristic of imines, indicating the occurrence of Schiff-base reaction ~ as, & de
crystallized phase form II (Leceta, Guerrero, Ibarburu, Duen
(Wang, Lian, Wang, Jin, & Liu, 2012). The imine formation could be la Caba, 2013). The XRD patterns of the cross-linked chitosan films
further confirmed by the decreasing intensity ratio of amide II indicated that there were some changes in the degree of crystal-
stretching/amide I stretching (1567 cm1/1637 cm1), which can be linity. When the ratio of aldehyde to amino group was less than 1,
 H. Chen et al. / Food Hydrocolloids 61 (2016) 662e671 667

Fig. 4. FTIR spectra of blank chitosan film and cross-linked chitosan films in the regions (A) 3800e2600 cm1 and (B) 1800e600 cm1.

preponderant intermolecular distances among the newly formed

imine units. When the ratio of aldehyde to amino groups was
greater than 1, there was a shoulder peak with an intensity
maximum around 11.3
, indicating the separation of chitosan
chains by the introduction of cinnamaldehyde. The absence of a
peak around 5
indicated that there was no self-organized cinna-
maldehyde layer (Marin et al., 2014; Marin et al., 2015), because
cinnamaldehyde was encapsulated in the core of the nanoemulsion
droplets. FTIR also showed that hydrogen bonding among chitosan
molecular chains decreased with increasing cinnamaldehyde
content.

3.3. Mechanical and barrier properties

The mechanical properties of films determine their integrity and

performance during utilization, storage and handling. Young’s
modulus (E), tensile strength (TS) and elongation at break (EAB)
were measured and are shown in Table 1. The tensile strength and
Young’s modulus of the chitosan films decreased with increasing
cinnamaldehyde nanoemulsion concentration, whereas the elon-
gation at break had a maximum value at intermediate concentra-
tions (around C0.2N). The tensile strength of the pure chitosan film
Fig. 5. XRD patterns of blank chitosan film and cross-linked chitosan films. was 98 MPa, which is much higher than that the reported previ-
ously (Ojagh, Rezaei, Razavi, & Hosseini, 2010), which may be due
the reflection intensity in the low-angle XRD patterns of the cross- to differences in chitosan type, film formation method, and
linked films (C0.2N, C0.4N, C0.6N) disappeared, indicating non- analytical methods used. The XRD results (Fig. 5) indicated that the
periodic incorporation of cinnamaldehyde onto the chitosan back- inter-chain distances of the chitosan increased with increasing
bones (Marin et al., 2015). Compared to the pure chitosan films cinnamaldehyde nanoemulsion concentration. This change would
(23
), the d-spacing values shifted into higher values (20
) and the be expected to decrease the entanglement of the chitosan chains
reflection peaks became sharper with increasing cinnamaldehyde and increase their molecular mobility, which may have caused the
amount. These results suggest that the inter-chain distances observed decrease in tensile strength and Young’s modulus. When
increased and the H-bonding network of crystalline chitosan was the content of cinnamaldehyde nanoemulsion was relatively low
disturbed in an irregular manner (Marin et al., 2015), which is in (C0.1N and C0.2N), the increment of elongation at break may have
agreement with the results of FTIR. The sharper reflection peak been caused by the change in mobility of the chitosan molecules.
indicated a higher density of imine linkages which lead to the However, at higher cinnamaldehyde contents, the cross-linking
668 H. Chen et al. / Food Hydrocolloids 61 (2016) 662e671

Table 1
Thickness, Tensile strength (TS), elongation at break (EAB) and Young’s modulus of
chitosan films with increasing content of cinnamaldehyde nanoemulsion.

Sample Thickness (mm) TS (MPa) EAB (%) Young’s modulus

(GPa)

CS 0.026 ± 0.0086 98.26 ± 5.69 4.16 ± 0.47 5.41 ± 0.85

C0.1N 0.027 ± 0.0020 62.29 ± 3.47 16.12 ± 2.63 3.74± 0.85
C0.2N 0.025 ± 0.0011 51.78 ± 4.70 24.50 ± 0.58 2.67± 0.21
C0.4N 0.030 ± 0.0059 44.90 ± 4.11 12.17 ± 3.45 2.07± 0.34
C0.6N 0.042 ± 0.0088 37.42 ± 4.02 14.53 ± 2.86 1.42 ± 0.04
C0.8N 0.053 ± 0.0068 38.84 ± 4.74 14.57 ± 2.54 1.31 ± 0.11
C1.0N 0.050 ± 0.0143 29.57 ± 4.21 11.41 ± 2.59 1.05 ± 0.06
C1.5N 0.056 ± 0.0043 17.44 ± 3.48 14.91 ± 3.69 0.55 ± 0.05
C2.0N 0.061 ± 0.0038 7.57 ± 1.34 12.61 ± 2.21 0.26 ± 0.06

interaction between the cinnamaldehyde and chitosan molecules

would get stronger, thereby limiting the motion of the chitosan
chains. Some studies have reported the reduction of all tensile
parameters (strength, modulus, and elongation at break) after the
incorporation of liquid oils into chitosan films (Sa
nchez-Gonza
lez, Fig. 6. Release rate profile of cinnamaldehyde after equilibrium under 10 v/v% and
Chiralt, Gonza
lez-Martínez, & Cha
fer, 2011; Sa
nchez-Gonza
lez, 50 v/v% alcohol.

Gonz
alez-Martínez, Chiralt, & Cha
fer, 2010). Other studies have
reported that incorporation of cinnamon oil enhanced the tensile
hydrophilic and hydrophobic food systems, respectively. Fig. 6
strength, but decreased the elongation at break of chitosan films
shows the release profile of cinnamaldehyde from films in the
(Ojagh et al., 2010). The microstructure and rheology of the films
two model media. In general, there was an initial rapid increase in
therefore appears to depend on the type and structure of cross-
the amount of cinnamaldehyde released, followed by a more
linking agent used.
gradual release until a constant level was reached. Overall, the
Water vapor permeability of the films was measured using pure
amount of cinnamaldehyde released was higher in the samples
water, NaCl and MgCl2 solutions (Table 2). In general, incorporation
with the higher alcohol content, which can be attributed to the
of increasing levels of the cinnamaldehyde nanoemulsions into the
relatively non-polar nature of the essential oil. The time required to
chitosan films resulted in a significant increase in their WVP at
reach a constant cinnamaldehyde level increased with increasing
different humidities studied. Lower humidity caused lower WVP
nanoemulsion level, which may be attributed to differences in the
values. For example, the WVP value of the C2.0N film was about
structure and properties of the chitosan films caused by the pres-
3.9  107 g$m1$h1$Pa1 at a humidity of 100% while that was
ence of the cross-linking agent. The release kinetics of essential oils
around 3.5  107 g$m1$h1$Pa1 at a humidity of 76%. At low
from polypropylene films has previously been reported to follow a
relative humidity, films had lower WVP values
Fickian model (Ramos, Beltra
n, Peltzer, Valente, & Garrigo
s, 2014).
(2.9  107 g$m1$h1$Pa1 at a humidity of 33% for C2.0N film).
This model was therefore used to calculate the release rate of the
The observed increase in WVP of the chitosan films at high cinna-
cinnamaldehyde from the films used in our study (Fig. 7). The
maldehyde levels might have been caused by an increase in the
release rate initially increased with nanoemulsion content (up to
number and size of the holes appearing in the films, as seen in the
C1N) and then decreased. These results suggest that high levels of
AFM images (Fig. 3).
cinnamaldehyde may have led to changes in the microstructure and
properties of the chitosan films that slowed down cinnamaldehyde
3.4. Cinnamaldehyde release from chitosan films diffusion.
The morphology of films (Fig. 8) after dipping in the 50% (v/v)
The release of cinnamaldehyde from the chitosan films was alcohol was significantly different from that of the original films
monitored using two model food systems: 10% and 50% (v/v) (Fig. 2). Compared to the original films, the bubble-like surface
alcohol solutions were used as release media, corresponding to structure disappeared and a more striated laminar structure was
formed. This effect can be attributed to the ability of the alcohol to
Table 2 dissolve the cinnamaldehyde nanoemulsion droplets. Fig. 9 shows
WVP values measured with a relative humidity (RH) gradient of 33%, 76% and the surface structure after dipping into alcohol solutions. In the
100% (external to internal) of chitosan and cross-linked chitosan films. Values absence of cinnamaldehyde, the smooth surface structure of the
within the same experimental group containing the same marked small letter
are not significantly different at the level of 5%.
chitosan films was greatly altered due to the dissolution of the oil
droplets by the alcohol solution. In the presence of cinnamalde-
Sample Distilled water NaCl (RH 76%) MgCl2 (RH 33%) hyde, the chitosan films maintained their compact and even surface
(RH 100%)
structure after coming into contact with the alcohol solutions,
WVP WVP WVP although a few granular structures were observed. These results
(g/Pa$h$m)  107 (g/Pa$h$m)  107 (g/Pa$h$m)  107
suggest that cross-linking the chitosan films with cinnamaldehyde
CS 1.42 ± 0.29efg 1.33 ± 0.37ef 1.26 ± 0.49dg increased the resistance of the chitosan films to disruption by
C0.1N 1.31 ± 0.36fg 1.17 ± 0.18ef 1.03 ± 0.12f
alcohol solutions.
C0.2N 1.15 ± 0.09g 0.93 ± 0.30f 0.83 ± 0.25f
C0.4N 1.69 ± 0.05def 1.29 ± 0.07ef 1.12 ± 0.08ef
C0.6N 1.74 ± 0.14de 1.44 ± 0.31de 1.24 ± 0.41de
C0.8N 2.01 ± 0.27cd 1.84 ± 0.40cd 1.55 ± 0.37d 3.5. Antibacterial assay
C1.0N 2.24 ± 0.17c 2.16 ± 0.43c 2.04 ± 0.34 c
C1.5N 3.33 ± 0.47b 2.92 ± 0.45b 2.28 ± 0.44 b The antibacterial/antifungal activity of the cross-linked chitosan
C2.0N 3.91 ± 0.59a 3.49 ± 0.61a 2.88 ± 0.50 a
film was evaluated against Gram-positive bacteria (S. aureus),
 H. Chen et al. / Food Hydrocolloids 61 (2016) 662e671 669

Fig. 7. Kinetically release amount of cinnamaldehyde from alcohol medium at the concentration of 10 v/v% and 50 v/v%.

Fig. 8. Cross-section morphology of air-dried chitosan films by SEM observation after dipping in the 50 v/v% alcohol.

Fig. 9. Surface morphology of air-dried chitosan films after dipping into 50 v/v% alcohol. A, B, C were chitosan films incorporated with blank MCT nanoemulsion, corresponding to
NM0.2, NM1.0 and NM2.0, respectively. A-1, B-1, C-1 were chitosan films incorporated with cinnamaldehyde-loaded nanoemulsion corresponding to C0.2N, C1.0N and C2.0N,
respectively.

gram-negative bacteria (E. coli), and fungus (C. albicans) (Fig. 10). concentrations (MICs) of cinnamaldehyde against E. coli and S.
Cinnamaldehyde has been reported to have broad-spectrum anti- aureus have been reported to be 98 and 195 mg/mL, respectively
microbial activity. For example, the minimum inhibitory (Tian et al., 2015), and 4.62 mg/mL for C. albicans (Lo

pez, S

anchez,
670 H. Chen et al. / Food Hydrocolloids 61 (2016) 662e671

Conflict of interest

The authors have no declared conflict of interest.

Acknowledgments

This work was financially supported by National Natural Science

Foundation of China (No. 31401528), supported by Fundamental
Research Funds for the Central Universities (No. 2662016PY066)
and National Science-technology Support Plan Projects (No.
2014BAK19B03). We appreciate Prof. Yumin Du from Wuhan Uni-
versity for providing chitosan powder. We also appreciate Prof.
Weiping Wang from college of Bioengineering in Hubei University
of Technology, for providing Candida Albicans HG01.

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