Food Chemistry 299 (2019) 125109

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

The multi-layer film system improved the release and retention properties of T
cinnamon essential oil and its application as coating in inhibition to
penicillium expansion of apple fruit
Wanli Zhang, Chang Shu, Qiuyi Chen, Jiankang Cao, Weibo Jiang
⁎

College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, PR China

ARTICLE INFO ABSTRACT

Chemical compound studied in this article: The aim of this research is to develop, characterize and utilize a multi-layer antibacterial film using chitosan (CS)
Chitosan (PubChem CID: 129662530) and sodium alginate (SA) as biopolymers and cinnamon essential oil (CEO) as main antibacterial ingredients.
The dense cross-section of SA layer in the scanning electron microscopy (SEM) analysis verified that layer-by-
Keywords: layer method improved physical and mechanical properties of CS-CEO single layer film. The thermogravimetric
Multi-layer film (TGA) and fourier transform infrared (FT-IR) analysis indicated that the layer-by-layer method changed the
Layer-by-layer
intermolecular interaction and the thermal stability. Importantly, the multi-layer film exhibited more sustained
Chitosan
release and higher retention rate of CEO compared CS-CEO single layer film. The multi-layer coating showed a
Sodium alginate
Cinnamon essential oil more significant and lasting inhibition of penicillium expansion which further demonstrated that the layer-by-
layer method improved the release and retention of CEO in the multiphased system. To summarize, the mul-
tilayer film system is a promising controllable release system for loading essential oils.

1. Introduction
The loss of postharvest fruit during storage caused by fungal pa-
thogens has always been a problem to be solved. Due to the adverse
effects of synthetic fungicides on the environment and health, natural
antibacterial substances of plant origin are gaining increasing attention.
Plant-derived essential oils are a promising alternative to control the
fungal pathogens due to their natural antibacterial activity (Vilaplana,
Pérez-Revelo, & Valencia-Chamorro, 2018; Usall, Ippolito, Sisquella, &
Neri, 2016). The strong flavor and volatile nature of essential oils de-
termine that they are mostly incorporated into edible films and coatings
in practical applications (Hasheminya et al., 2019). As storage time
increases, the loss of essential oils in edible films and coatings directly
leads to a decrease in antimicrobial capacity (Xu et al., 2018). There-
fore, most recent research has been focused on how to increase the
retention rate of essential oils after their incorporation into polymer
matrices (Robledo et al., 2018; Luo et al., 2019).
Chitosan (CS), as a positively charged polysaccharide, has been
widely used in the preparation of edible coatings and films due to its
good biocompatibility, strong film forming ability and natural anti-
bacterial properties (Kadam, Shah, Palamthodi, & Lele, 2018; Zhang
et al., 2019). A plethora of research has reported that the incorporation
of essential oils can significantly improve the antibacterial ability and
affect the physical and chemical properties of chitosan films (Hafsa
et al., 2016; Hyeock, Yeon, & Jin, 2018; Peng & Li, 2014). In addition,
the application of chitosan coatings incorporated with essential oils can
maintain the quality of postharvest fruit and significantly enhance the
capacity of postharvest disease control to prolong the life during sto-
rage (Mohammadi, Hashemi, & Hosseini, 2015a; Mohammadi,
Hashemi, & Hosseini, 2015b).
Chitosan is mainly bonded by hydrogen bonding to terpenes in EOs
(essential oils) to retain the volatile antibacterial active substances
(Shojaee-Aliabadi et al., 2014). It has also been reported that the amino
group of chitosan can bind to the aldehyde group in EOs by Schiff-based
reactions (Chen et al., 2016). However, due to the large difference in
the polarity of CS and EOs, EOs and CS have low degree of crosslinking
between each other, it is difficult for the weak hydrogen bonds are
difficult to effectively retain the volatile antibacterial components in
EOs (Xu et al., 2018). The currently-used methods for increasing the
retention efficiency of EOs in CS include emulsification, nano-en-
capsulation, and the addition of biopolymers (Perdones, Chiralt, &
Vargas, 2016; Hasheminejad, Khodaiyan, & Safari, 2019).
In layer-by-layer electrostatic deposition method, polyelectrolyte
complexes are formed through electrostatic interaction based on op-
positely charged polymers, thereby improving the properties of edible
coatings and films (Ferreira et al., 2016; Yan et al., 2019). Recently, the

⁎
Corresponding author.
E-mail address: jwb@cau.edu.cn (W. Jiang).

https://doi.org/10.1016/j.foodchem.2019.125109
Received 8 May 2019; Received in revised form 26 June 2019; Accepted 1 July 2019
Available online 02 July 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
W. Zhang, et al.
application of layer-by-layer coating based on carboxymethyl cellulose
and chitosan has notably improved quality of postharvest strawberry
during its storage time (Yan et al., 2019). In addition, the sodium al-
ginate-chitosan bilayer film assembled by the layer-by-layer electro-
static deposition method has also exhibited superior performance (Li,
Zhu, Guan, & Wu, 2019; Wang et al., 2019). However, up to now, rare
reports have investigated the effects of multi-layer film system on the
retention of essential oil.
In this research, chitosan (CS) and sodium alginate (SA) as biopo-
lymers and cinnamon essential oil (CEO) as main antibacterial in-
gredients were used to prepare multi-layer film and coating by layer-by-
layer electrostatic deposition technique. The aim of the research is to
prepare multi-layer antimicrobial edible film by electrostatic deposition
techniques, further exploring its potential in improving the retention of
cinnamon essential oil. The research also investigated the physical and
mechanical properties of multi-layer films. Moreover, the release
properties and retention rate of CEO in the composite film were com-
pared. Finally, the in vitro antifungal ability of multi-layer coating were
tested on apple fruits to verify the effect of multi-layer film system on
the retention characteristics of cinnamon essential oils.
2. Materials and methods
2.1. Material
2.1.1. Cinnamon essential oil and other reagents
Cinnamon essential oil (CEO) was purchased from Dingcheng Spices
Co., Ltd (Guangzhou, China). Chitosan, degree of deacetylation > 90%
and viscosity 100–200 cps (10 g L−1, 20 °C), was purchased from Sigma
Chemical Co (St. Louis, MO, USA). SA (AR, > 90%), viscosity 200–500
cps (1%, 20 °C), was procured from Tianjin Damao Chemical Reagent
Factory (Tianjin, China). All other chemical reagents used were of
analytical grade.
2.1.2. Fruit and fungus
Apple fruit of commercial maturity was purchased from local
market in Beijing, PR China. The fruit were transferred to our labora-
tory (Beijing, PR China) in 5 h via truck. The fruits with uniform size
and no physical damage were selected. The pathogen penicillium was
isolated from infected apple fruit and maintained on potato dextrose
agar (PDA). A spore suspension was obtained from penicillium cultured
for two weeks, and the concentration of the spore suspension was ad-
justed to1 × 105 spores per mL.
2.2. Preparation of film forming solutions and films
Preparation of films was adapted from previous reports with slight
modifications (Li et al., 2019). CS solution was prepared by adding 2%
(w/v) chitosan to 1% (v/v) acetic acid solution, 30% (w/w) glycerol
was added as a plasticizer. 2% (w/v) SA solution was prepared by
dissolving SA in distilled water and 30% (w/w) glycerol was added as a
plasticizer. In order to ensure the same amount of cinnamon essential
oil for each film, two concentrations of CS-CEO solution were prepared.
0.5% (v/v) and 1% (v/v) cinnamon essential oil were separately added
to the CS solution, and 20% (w/w) Tween 80 was added as an emul-
sifier, and stirring was continued for half an hour. All film forming
solutions were ultrasonicated (240 W, 40KHz) for 30 min for degassing
treatment. Finally, the CS-CEO single layer film forming solution con-
tained 30% (w/w) glycerol, 20% (w/w) Tween 80, 1% (v/v) acetic
acid, 2% (w/v) chitosan and 0.5% (v/v) CEO. The CS-CEO mixture
solution of SA/CS-CEO bilayer and SA/CS-CEO/SA trilayer film con-
tained 30% (w/w) glycerol, 20% (w/w) Tween 80, 1% (v/v) acetic
acid, 2% (w/v) chitosan and 1% (v/v) CEO, SA solution contained 2%
sodium alginate and 30% (w/w) glycerol.
The 20 mL CS, SA and 0.5% (v/v) CS-CEO film forming solutions
were separately cast onto polystyrene petri dishes (diameter of 9 cm)
2
Food Chemistry 299 (2019) 125109
and dried in a drying oven at 40 °C for 24 h to form pure CS, SA and CS-
CEO single layer film. 10 mL of SA solution was cast onto polystyrene
petri dishes (diameter of 9 cm) and dried in a drying oven at 40 °C until
a firm but adherent surface was obtained. Then 10 mL of 1% (v/v) CS-
CEO was cast onto top of the SA layer as the second layer, and the
bilayer was dried in a drying oven at 40 °C for 24 h to obtained SA/CS-
CEO bilayer film. The SA/CS-CEO/SA trilayer film was prepared in the
same manner as the two-layer film. When the surface of the second
layer 1% (v/v) CS-CEO was firm but adherent dried, 10 mL of the SA
solution was cast onto as a third layer, and the trilayer was dried in a
drying oven at 40 °C for 24 h to obtained SA/CS-CEO/SA trilayer film.
All films developed were peeled carefully and placed at 25 °C, 60%
relative humidity a desiccator containing saturated calcium nitrate for
48 h to balance moisture for further usage.
2.3. Physical and mechanical properties
2.3.1. Film thickness
The thickness of the films was measured with a digital micrometer
(Mitutoyo Absolute, Tester Sangyo Co. Ltd., Japan) with an accuracy of
0.001 mm. Each film was measured at six different positions. Ten re-
petitions of every kind of sample were conducted.
2.3.2. Transparency
The transparency of the films was calculated by the absorbance and
thickness of rectangular film at 600 nm by the following Eq. (1). Ten
repetitions of every kind of sample were conducted.
A
Opacity =
x (1)
where A was the value of absorbance at 600 nm and x was the thickness
of the film (mm).
2.3.3. Moisture content and water solubility
The moisture content was determined by drying the film sample at
105 °C to constant weight, and calculated according to the following
formula (2).
(Mi Md)
Moisture contents (% )= × 100
Mi (2)
where Mi (g) and Md (g) are the initial and after being dried of the film
sample. Three repetitions of every kind of sample were conducted.
The water solubility was determined according to Li et al. (2019).
Firstly, drying the film sample at 105 °C to constant weight (M0), then
the strips (1 cm × 1 cm) immersed in 100 mL of distilled water string at
50 rpm at 25 °C. After 12 h, the immersed pieces were extracted and
dried to constant weight (M1) at 105 °C. (3) was used to calculate the
water vapor permeability. Three repetitions of every kind of sample
were conducted.
M 0 M1
Water solubility (% )= × 100
M0 (3)
2.3.4. Water vapor permeability
The determination of the water vapor permeability of the film was
conducted according to the method of Kadam et al. (2018) with some
modifications. The film sample was sealed in a weighing cup mouth
(4 cm in diameter and 4 cm depth) containing 3 g of cacl2, and then the
cup was placed in a desiccator containing saturated potassium iodide
(20 °C, 70% relative humidity) and the weight of the cup was weighed
every 4 h for 2d. (4) was used to calculate the water vapor permeability.
Three repetitions of every kind of sample were conducted.
M×x
Water vapor permeability =
t×A× p (4)
where M is the weight difference (g), x is the film thickness (m), A is the
W. Zhang, et al.
film permeation area (m2), Δp is the difference of permeation area
vapor pressure (2339 Pa at 20 °C) and t is the permeation time (s).
2.3.5. Mechanical properties
The determination process of mechanical properties of film was
slightly modified according to the previous method (Shah, Vishwasrao,
Singhal, & Ananthanarayan, 2016). Tensile strength and elongation at
break were determined using a texture analyzer (CT3, Brookfield Ltd.,
USA). The film sample was cut into a rectangular shape
(60 mm × 8 mm), and then the ends were clamped with tensile grips.
The final effective length of the stretching was 30 mm, and the
stretching rate was 1.0 mm/s. The tensile strength and elongation at
break were calculated by the following Eqs. (5) and (6). Each film was
measured at least five times. Three repetitions of every kind of sample
were conducted.
F
Tensile strength (MPa) =
x×w (5)
x 2.6. Layer-by-layer coatings for the control of penicillium expansion of
Elongation at break (% )= × 100
x0 (6)
where F is the maximum force at rupture of the film (N), x is the film
thickness (mm), and W is the width of film piece (mm); Δx is the in-
crease in length at the breaking point (mm), and x0 is the initial length
of film piece (mm).
2.4. Characterization of films
2.4.1. Morphology by scanning electron microscopy (SEM)
The film sample (0.5 × 0.5 cm2) was fixed on a copper column with
double-sided tape, and then sprayed with gold to observe. For the cross-
section analysis, the film was frozen with liquid nitrogen and breaking
it with tweezers. The microscopic morphology of the surface and cross
section of the film was observed with a scanning electron microscope
(JSM-6700F, Jeol Ltd., Japan) at 3500× and 500× magnifications
with the accelerating voltage of 20 kV.
2.4.2. Fourier transform infrared (FT-IR) analysis
FT-IR spectrum of every kind of films was recorded on a Perkin
Eimer spectrum 1000 (Varian Inc., USA) using KBr dispersive method.
The film samples were ground to a powder after frozen in liquid ni-
trogen, and lyophilized under vacuum. FT-IR analysis was conducted in
the wavenumber ranging from 4000 to 500 cm−1 with a resolution of
4 cm−1.
2.4.3. Thermogravimetric analysis (TGA)
The thermal stability of film was conducted by thermogravimetric
analyzer (DTG-60 Shim ADZU, Japan). Approximately 4 mg of the
lyophilized film sample was placed in a ceramic crucible and heated
from 40 °C to 400 °C at a constant rate of 10 °C per minute under a
flowing nitrogen atmosphere.
2.4.4. X-ray diffraction analysis
Wide-angle X-ray diffraction measurements were performed by a
XRD diffractometer (D8-Advance, Bruker, USA) at a voltage of 40 kV
and 30 mA. The patterns with Cu Kα radiation were recorded in the
region of 2θ from 10 to 80° with a step speed of 2° min−1.
2.5. Release and retention properties of CEO in film
The release and retention properties of CEO in film during ambient
storage were determined with some modification from a previous
method (Xu et al., 2018). Specifically, the film was punched into reg-
ular small discs (6 mm) by punchers. Then each of the disc film had the
same area and was stored at room temperature. 10 pieces of disc film
were placed in 2 mL of distilled water, followed by the addition of 5 mL
3
Food Chemistry 299 (2019) 125109
of n-hexane and the shaking (120 rpm) at 25 °C for 24 h. After the
mixture was centrifuged, the absorbance value of the supernatant was
measured at 318 nm, and the CEO concentration of the supernatant was
quantified using a standard curve corrected from the known con-
centration of CEO n-hexane solution. 10 pieces of the disc of each film
were randomly selected every day for measurement until the seventh
day, as a release property study of the CEO in the film. In order to better
dissolve the film to determine the CEO content, 1 mL of acetic acid was
added to the solution containing 10 pieces of disc film, 2 mL of distilled
water and 5 mL of n-hexane. The retention of CEO was calculated using
the following Eq. (7). Three repetitions of every kind of sample were
conducted.
M10d
Retention of CEO (% )=
M1d (7)
Where M10d is the CEO content of tenth day, M1d is the CEO content of
first day.
wounded apples
The preparation of the 1% (w/v) CS and the 1% (w/v) SA coating
solution was the same as that of the film forming solution. According to
the experimental results of inhibition to penicillium in vitro (data not
shown), 4% (v/v) of the CEO was selected to be incorporated into the
CS solution to prepare a CS-CEO coating solution. Two varieties of
apples “Guoguang” and “Huangyuanshuai” were all divided into five
groups, i.e. CK, CS, CS-CEO, SA/CS-CEO and SA/CS-CEO/SA, with each
group containing 30 fruits. CS and CS-CEO fruits were immersed for
1 min into CS and CS-CEO solutions, respectively, and the CK fruit was
immersed in distilled water containing 0.1% (w/v) tween 80 for 1 min.
In the procedure of SA/CS-CEO bilayer coating, the fruit was immersed
in the SA solution first for 1 min, dried for 30 min, and then coated with
CS-CEO solution for 1 min. The procedure of SA/CS-CEO/SA trilayer
coating was performed with coating with SA solution for another 1 min
after the SA and CS-CEO coatings were dried for 30 min. All coatings of
the fruit were dried at room temperature for 30 min and then in-
oculated with penicillium expansion. The fruits were wounded at the
equator with a sterile needle (3 mm diameter × 3 mm deep), two
wounds per fruit, and 30 μL of spore suspension was injected into each
wound. All fruits were loaded into a uniform basket
(60 cm × 20 cm × 20 cm), covered with a PVB bag (0.03 mm), and
stored at a temperature of 20 ± 2 °C and relative humidity (RH) of
80–90%. After 15 days of incubation, data concerning lesion diameter
(LD) were measured. Three replicates were conducted for all assays.
2.7. Statistical analysis
The analyses of data performed by one-way analysis of variance
(ANONA) from SPSS 22.0 window. Significant differences were calcu-
lated by least significant difference (Duncan) test and defined as
P < 0.05.
3. Results and discussion
3.1. Thickness and transparency
According to our results, the thickness of the CS film significantly
increased with the incorporation of CEO (Table 1). This phenomenon
was in agreement with that reported for chitosan films incorporated
basil or thyme essential oil (Perdones, Chiralt, & Vargas, 2016). The
thickness of a control film made from 20 mL SA was significantly lower
than CS film made from 20 mL CS. Compared to the CS-CEO film, the
thickness of the SA/CS-CEO was significantly reduced, which was
mainly due to the less thickness of SA layer. The strong light barrier
ability of the packaging film is considered to be a superior property
W. Zhang, et al. Food Chemistry 299 (2019) 125109

Table 1
Thicknesses, moisture contents, water solubility, opacity, water vapor permeability and mechanical properties of CS, CS-CEO, SA/CS-CEO and SA/CS-CEO/SA films.
Films Thickness (μm) Moisture contents Water solubility Water vapor permeability Opacity (A mm−1) Tensile strength Elongation at break
(%) (%) (10−5 g m−1 min−1 Pa−1) (MPa) (%)

CS 58.79 ± 8.70c 42.35 ± 2.61a 25.06 ± 1.36c 11.44 ± 0.51c 0.99 ± 0.24c 56.36 ± 7.85b 45.05 ± 5.92a
CS-CEO 67.70 ± 7.08b 30.24 ± 1.85b 17.28 ± 0.97d 10.21 ± 0.22c 2.59 ± 0.51ab 43.11 ± 6.39c 28.05 ± 2.91b
SA/CS-CEO 56.91 ± 7.49c 27.75 ± 0.42bc 33.71 ± 1.14b 14.22 ± 0.18b 2.90 ± 0.37a 78.58 ± 6.92a 5.28 ± 1.26c
SA/CS-CEO/ 86.83 ± 7.78a 25.73 ± 0.93c 41.81 ± 1.25a 22.31 ± 1.02a 2.78 ± 0.31a 73.43 ± 9.26a 4.2 ± 1.25c
SA

a
Values followed by the same letter in the column on the same day were not significantly different according to Duncan’s Multiple Range Test (P < 0.05). Data are
accompanied by standard errors of the means (n ≥ 3).

decreasing trend when chitosan films where incorporated with CEO

(Table 1). The lower moisture content and water solubility of CS-CEO
film were attributed to the hydrophobic nature of CEO (Peng & Li,
2014). However, the water solubility of the SA/CS-CEO and SA/CS-
CEO/SA films were significantly higher than that of CS and CS-CEO,
which is mainly caused by strong hydrophilicity of SA (Wang et al.,
2019). In addition, the water vapor permeability of the SA/CS-CEO and
SA/CS-CEO/SA films were significantly lower than that of CS-CEO and
the SA control film (data not shown), which may be attributed to the
compact network structure at the boundary of multi-layer films, and the
tortuous paths caused by different layer, resulting in more difficulty for
the water molecules to pass through (Li et al., 2019; Wang et al., 2019).

3.3. Mechanical properties

The mechanical properties of the film are primarily characterized by

Fig. 1. SEM micrographs of the surface (on the left) and cross-section (on the
right) of CS single layer film (A and E), CS-CEO single layer film (B and F), SA/
CS-CEO bilayer film (C and G), and SA/CS-CEO/SA trilayer film (D and H).
Magnification was × 3500 for A, B, C and D and was × 500 for E, F, G and H.
tensile strength (TS) and elongation at break, depending on the type of
polymer matrix, the type of additives and the interaction between them
(Ahmad, Dar, Abas, & Ahmad, 2018). The typical stress vs. deformation
curves exhibited mechanical properties of the film more clearly (Fig.
S1). Compared with the CS film, the incorporation of CEO significantly
reduced TS and elongation at break (Table 1). Likewise, the CS film
incorporated with zataria multiflora boiss essential oil had also been
observed to have lower TS and elongation at break, which resulted from
a heterogeneous film structure featuring discontinuities induced by
lipid incorporation into the chitosan molecules (Moradi et al., 2012).
However, the contrary result was reported by Ojagh, Rezaei, Razavi,
Mohamad, and Hosseini (2010), who found that the addition of EO to
CS films significantly increased the TS value, which may be attributed
to the composition difference of CEO and CS. Compared with the CS
and CS-CEO layer film, although the layer-by-layer structure sig-
nificantly reduced elongation at break, it had a higher level of TS
(Table 1). This was mainly because the layer-by-layer technology pro-
duced a strong interaction and friction at the boundary between CS-
CEO and SA layer, thereby making the film more rigid (Li et al., 2019).
In addition, the water of most hydrocolloid-based films is the most
ubiquitous plasticiser due to it could modify the structure of natural
polymers, therefor, the increase in TS and the decrease in elongation at
break may also be related to the reduced moisture content of mulity-
layer films (Ojagh et al., 2010).

3.4. Morphology by scanning electron microscopy (SEM)

because visible light can cause oxidation of food (Yong et al., 2019). As
shown in Table 1, in comparison with CS, the incorporation of CEO
remarkably increased the opacity of film, which potentially provide
better preservation ability for foods that are sensitive to white light
(Yang et al., 2015). However, for most edible films applied on the food
products, the decrease of shining and the transparency might reduced
acceptability of the customer for films.
3.2. Moisture content, water solubility and water vapor permeability
The trend of the moisture content and water solubility witnessed a
The microstructure of the film is determined by the arrangement of
the matrix and is closely related to its functional properties (Ahmad
et al., 2018). The surfaces and cross-section morphologies of each kind
of films are exhibited in Fig. 1. As shown in the figure, the CS and SA/
CS-CEO/SA films all had smooth surfaces, indicating that the SA as the
outer layer of the multilayer film also had excellent uniformity. It was
worth noting that small white spots appeared on the surface of the CS-
CEO and SA/CS-CEO films. Similar phenomena were observed on the
surface of the chitosan film incorporated with thyme EOs, which was
considered to be the result of EO droplet coalescence during the drying

4
W. Zhang, et al.
Fig. 2. FT-IR spectra (A), X-ray diffraction pattern (B), thermogravimetric curves (C) and first derivative curves (D) of CS single layer film (a), CS-CEO single layer
film (b), SA/CS-CEO bilayer film (c) and SA/CS-CEO/SA trilayer film (d).
period (Peng & Li, 2014). Although the CS-CEO was the outer layer of
the SA/CS-CEO film, the SA/CS-CEO film had less white spots on its
surface than the CS-CEO film, which indicated that the layer-by-layer
technology reduced the coalescence of the CEO in the chitosan film. At
small magnifications (500×), the cross-section of the CS film showed a
clear wrinkle. However, due to the CEO droplet coalescence, the cross-
section of CS-CEO film was rough and showed the cavitation phenom-
enon (Fig. 1). It was evident in the SA/CS-CEO and SA/CS-CEO/SA
films that SA had a dense and continuous cross-section and was tightly
bonded to CS-CEO in a layered structure (Fig. 1). This phenomenon
could also be reflected in the mechanical properties that compared with
the CS-CEO film, the SA/CS-CEO and SA/CS-CEO/SA films had im-
proved TS (Table 1).
3.5. Fourier transform infrared (FT-IR) analysis
FT-IR spectroscopy is used to characterize the functional groups and
interactions in experimental samples. When two components are mixed,
the chemical interaction between them can be effectively reflected in a
specific change in the spectral band (Matta, Tavera-Quiroz & Bertola,
2019). As shown in Fig. 2A, according to previous work (Yong et al.,
2019), the characteristic peaks of CS films appeared at 1708 cm−1, and
1564 cm−1 due to the C]O stretching of amide group (amide I) and
NeH bending (amide II) respectively. The broad peak of the CS film
appearing at 3396 cm−1 corresponded to the overlapping stretching
vibration of OeH and NeH, and peaks at 1156 cm−1 and 1040 cm−1
were related to the characteristic saccharide structure (Liu et al., 2017).
With the incorporation of the CEO, the peak around 2876 cm−1 was
bathochromic shifting to 2856 cm−1 and became stronger, which might
be related to the CeH stretching (eCH2) of the CEO. In addition, the
new absorption peak appeared at 1740 cm−1, indicating the formation
of ester bond between CS and CEO molecules (Li et al., 2019). Com-
pared with the CTS-CEO film, two changes were observed in the
5
Food Chemistry 299 (2019) 125109
infrared spectra of SA/CS-CEO and SA/CS-CEO/SA films (Fig. 2A).
First, it was detected that the peak at 1564 cm−1 disappeared, which
might be due to the binding between the SA and the CS molecules at the
boundary of multi-layer films, resulting in a decrease in free amino
groups in the CS. In addition, the new absorption peak appeared at
1632 cm−1, indicating the formation of amide bond between the SA
and CEO molecules or between the SA and CS molecules (Liu et al.,
2017).
3.6. X-ray diffraction analysis (XRD)
The crystal structures of the films are revealed by XRD. According to
Fig. 2B, the CS film showed characteristic diffraction peaks at around
14° that reflected the amorphous structure of chitosan (Liu et al., 2017).
As the incorporation CEO, the new sharp and narrow crystalline peaks
at 32.5, 45.5° in the CS-CEO film were detected. This might be due to
the formation of new crystals by the CEO and CS molecules. The films
prepared by the layer-by-layer method exhibited a weaker crystalline
peaks at 32.5, 45.5°, which indicated an enhancement in hydrogen
bonding (Li et al., 2019). The hydrogen bonding and electrostatic in-
teraction formed by CS and SA at the boundary of multi-layer films,
resulting in more molecular states of the amorphous (Liu, Cai, Jiang,
Wu, & Le, 2015).
3.7. Thermogravimetric analysis (TGA)
The thermal stability of activity packaging films is frequently ana-
lyzed by TGA. The weight loss of 0–150 °C is mainly due to the loss of
bound water inside the film (Chu et al., 2019). As shown in Fig. 2C, The
decrease of weight loss at this stage was presented in TGA curve of the
CS film incorporated of CEO, which was consistent with the lower
moisture content of CS-CEO film (Table 1). The weight loss of the
second stage occurred at 150–400 °C, mainly including the
W. Zhang, et al.
volatilization of glycerol, degradation of CS and SA molecules (Yong
et al., 2019). Similar to the weight loss trend of the first stage, com-
pared with the CS film, as the incorporation of CEO, the weight loss of
CS-CEO film was reduced at the second stage (Fig. 2C). This may be
because the formation of ester bond between CS and CEO molecules,
which can be reflected in the infrared spectra of CS-CEO film (Fig. 2A).
The stages of weight loss could be observed better in the DTG curves
(Fig. 2D). The rapidest weight loss rate of CS and CS-CEO films ap-
peared at 172.8 °C and 186.9 °C respectively, while the SA/CS-CEO and
SA/CS-CEO/SA films 243.2 °C and 236.7 °C. This is not only related to
the moisture content of different films, but also indicates that the layer-
by-layer method increased the intermolecular interaction of films (Li
et al., 2019). The films prepared by the layer-by-layer method exhibited
a better thermal stability than the CS and CS-CEO films at 0–240 °C
(Fig. 2C). The SA/CS-CEO/SA film had a weight loss of approximately
3% until 240 °C and a CS of approximately 20%. However, the SA/CS-
CEO and SA/CS-CEO/SA films had higher weight loss after 240 °C,
which might be attributed to the more volatilization of glycerol in SA/
CS-CEO and SA/CS-CEO/SA films.
3.8. The release properties and retention rate of CEO in the multi-layer film
The release properties of the essential oil incorporated into the
composite film directly affect its antibacterial ability. In this situation,
the essential oil could be released at a stable rate for a certain period of
time, which is beneficial to the shelf life of the food in practical ap-
plication (Xu et al., 2018). Fig. 3A shows the effect of layer-by-layer
method on the release properties of CEO incorporated into the com-
posite film. On the first day, the CS-CEO film released a large amount of
CEO, which was significantly higher than SA/CS-CEO and SA/CS-CEO/
SA films did. However, on the second day, the release content of CEO in
the CS-CEO film drastically decreased, significantly lower than the SA/
CS-CEO and the SA/CS-CEO/SA films. The CEO in the CS-CEO film was
almost completely released in the previous two days, and in the next
five days, it had been a very little release content. Although the amount
of CEO release in the SA/CS-CEO and SA/CS-CEO/SA films on the first
day was lower than the CS-CEO film, it was significantly higher than CS
during the subsequent storage period. The amount of CEO release in the
SA/CS-CEO film also showed a sharp drop on the second day, but
showed a steady release rate of CEO over the next five days. The SA/CS-
CEO/SA film exhibited the most stable CEO release properties and all
showed a significantly higher CEO release content than CS-CEO and
SA/CS-CEO films during storage time other than the first day. There-
fore, our results indicated that the layer-by-layer method improved the
release properties of the CEO in the multi-layer film.
In addition to the loss caused by the evaporation of the CEO during
Fig. 3. The release properties (A) and retention rate (B) of CEO in composite films. The vertical bars indicate the standard deviations for each treatment (n ≥ 3). Bars
with different letters represent statistical differences (P < 0.05).
6
Food Chemistry 299 (2019) 125109
film drying, the CEO incorporated into the edible active film would
suffer a great loss during the room-temperature storage, further re-
sulting in a decrease in the antibacterial ability of the composite film
(Chu et al., 2019). Fig. 3B shows the retention rate of the CEO after ten
days of ambient storage in different films. According to our results, the
CS-CEO film exhibited the CEO loss of up to 70%. It has been reported
that CS and CEO molecules are mainly bonded through weak hydrogen
bonds, and due to the differences in molecular polarity, the CEO mo-
lecules in CS films are difficult to retain (Shojaee-Aliabadi et al., 2014;
Chen et al., 2016). It was worth noting that our results indicated that
the retention rate of the CEO in the SA/CS-CEO and SA/CS-CEO/SA
films was significantly higher than that of the CS-CEO film (Fig. 3B).
This was consistent with a previous research, which concluded that the
CS-CEO film incorporated into GA (gum arabic) formed a network-
spherical intertwined structure due to intermolecular interactions, al-
lowing the CEO to be embedded therein, thus increasing the retention
of the CEO in the composite film (Xu et al., 2018). In addition to the
network-intertwined structure formed by SA and CA molecules, the
improvement in retention of CEO in multi-layer films might also result
from that the CEO was covered by the SA film, so that it was converted
from the surface of the film to the deep position.
3.9. Layer-by-layer coatings for the control of penicillium expansion of
wounded apples
Chitosan coating incorporated essential oils is a promising method
for controlling the fungal decay in postharvest fruits. Previous research
has shown that CEO not only has a spectral inhibitory effect on bacteria,
but also has a significant inhibitory effect on fungi (Yuan, Chen, & Li,
2016). In this work, in vitro experiments have verified that the CEO has
significant inhibition of Penicillium expansion (data not shown). Fig. 4
shows the photographs of two varieties of apples (“Guoguang” and
“Huangyuanshuai”) coated by different film forming solutions after
inoculated with Penicillium expansion for 15 days. After 15 days of in-
oculation, the infection status of fruit coated by CS did not improve and
coated by CS-CEO had a slight improvement compared to the uncoated.
However, the fungal spore of the fruit coated by SA/CS-CEO/SA wound
was still immature white, which indicated that the layer-by-layer
coating significantly inhibited the infection and growth of Penicillium
expansion after 15 days of inoculation. Fig. 5 shows the lesion diameter
of apples after 15 days of inoculation. The two varieties of apples
showed consistent results. There was no significant difference in the
lesion diameter between the fruit coated by CS and the uncoated, yet
the fruits coated by incorporating into the CEO were all significantly
reduced. Sánchez-González, Cháfer, Chiralt, and González-Martínez
(2010) also found that pure CS film has no inhibitory effect on
W. Zhang, et al.
Penicillium, which was attributed to Penicillium is not sensitive to the
antibacterial activity of chitosan. Although chitosan itself has anti-
fungal activity, it mainly inhibits spore germination in the early stage of
infection, and the coating method cannot directly act on pathogens,
therefor, the antifungal function of chitosan coating in postharvest fruit
is mainly achieved by inducing resistance (Shi, Wang, Lu, & Deng,
2018; Grande-Tovar, Chaves-Lopez, Serio, Rossi, & Paparella, 2018).
This may be the reasons that no significant difference in the lesion
diameter was observed at late stage of infection between uncoated fruit
with chitosan-coated fruit.
Importantly, the inhibition effects of both fruits coated by SA/CS-
CEO and SA/CS-CEO/SA on fungal infections were significantly higher
than coated by CS-CEO. Li, Kong, and Wu (2013) indicated that the
antibacterial ability of the CEO depended on the concentration of cin-
namaldehyde and eugenol that were easily lost. Therefore, the layer-by-
layer method improved CEO retention of coating and extended re-
sistance to Penicillium expansion, which was consistent with the results
of CEO release and retention in the multi-layer film, further demon-
strating that the layer-by-layer method was a viable CEO controlled
release system. In addition, the multi-layer coating has an increased
thickness, limit the across of oxygen, which may also hinder the growth
of Penicillium in apple.
Fig. 5. The lesion diameter of two varieties of apples “Guoguang” (A) and “Huangyuanshuai” (B) after inoculated with Penicillium expansion for 15 days with different
coatings. The vertical bars indicate the standard deviations for each treatment (n ≥ 3). Bars with different letters represent statistical differences (P < 0.05).
7
Food Chemistry 299 (2019) 125109
Fig. 4. Appearance of two varieties of apples
“Guoguang” (on the left) and “Huangyuanshuai” (on
the right) after inoculated with Penicillium expansion
for 15 days: (A1 and A2) uncoated apples, (B1 and
B2) apples coated with CS, (C1 and C2) apples
coated with CS-CEO, (D1 and D2) apples coated
with SA/CS-CEO and (E1 and E2) apples coated with
SA/CS-CEO/SA.
4. Conclusion
In this work, the multi-layer antibacterial films based on chitosan
(CS) containing cinnamon essential oil (CEO) and sodium alginate (SA)
were developed. The incorporation of CEO led to the changes of chit-
osan film in physical and mechanical properties. The moisture content,
water vapor permeability, tensile strength and elongation at break of
the CS-CEO composite film were significantly lower than those of the
CS film, indicating that the incorporation of CEO changed the original
bond between the CS-CS and CS-water. This was further supported by
the results of FT-IR analysis, TGA and X-ray diffraction analysis. The
SA/CS-CEO bilayer film and SA/CS-CEO/SA trilayer film prepared by
layer-by-layer solvent-casting method had higher tensile strength and
lower water vapor permeability compared to the CS-CEO single layer
film. The dense and continuous cross-section of the SA layer observed
by SEM analysis confirmed the improved mechanical properties and
water vapor permeability of the multi-layer film. The test of release
properties and retention rate of CEO in the different films showed that
the multi-layer film had higher release stability and retention of CEO
compared to the CS-CEO single layer film. Moreover, the multi-layer
coating based on the layer-by-layer method showed a more significant
and lasting inhibition of penicillium expansion than the CS-CEO single
layer coating on both varieties of apples. This has further demonstrated
that the layer-by-layer method improved the release and retention of
CEO in the multiphased system. In summary, the multi-layer film
W. Zhang, et al.
system based on CS and SA is a promising controlled release system for
loading essential oils, which could improve the efficiency of essential
oils in practical applications.
Acknowledgement
This research was supported by the National key R & D projects (No.
2018YFD0401302) of china.
Declaration of interest statement
The authors declared that they have no conflicts of interest to this
work. We declare that we do not have any commercial or associative
interest that represents a conflict of interest in connection with the
work submitted.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.125109.
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