Accepted Manuscript

Evaluations of physicochemical and biological properties of

pullulan-based films incorporated with cinnamon essential oil and
Tween 80

Yifu Chu, Tian Xu, ChengCheng Gao, Xiaoya Liu, Ni Zhang,

Xiao Feng, Xingxun Liu, Xinchun Shen, Xiaozhi Tang

PII: S0141-8130(18)33682-1
DOI: https://doi.org/10.1016/j.ijbiomac.2018.10.194
Reference: BIOMAC 10845
To appear in: International Journal of Biological Macromolecules
Received date: 18 July 2018
Revised date: 16 October 2018
Accepted date: 27 October 2018

Please cite this article as: Yifu Chu, Tian Xu, ChengCheng Gao, Xiaoya Liu, Ni Zhang,
Xiao Feng, Xingxun Liu, Xinchun Shen, Xiaozhi Tang , Evaluations of physicochemical
and biological properties of pullulan-based films incorporated with cinnamon essential oil
and Tween 80. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.10.194

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 ACCEPTED MANUSCRIPT

Evaluations of physicochemical and biological

properties of pullulan-based films incorporated with
cinnamon essential oil and Tween 80
Yifu Chu, Tian Xu, ChengCheng Gao, Xiaoya Liu, Ni Zhang, Xiao Feng,
Xingxun Liu, Xinchun Shen, Xiaozhi Tang*

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College of Food Science and Engineering/Collaborative Innovation Center for

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Modern Grain Circulation and Safety/Key Laboratory of Grains and Oils Quality

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Control and Processing, Nanjing University of Finance and Economics, Nanjing
210023, China
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*Corresponding Author: College of Food Science and Engineering, Nanjing

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University of Finance and Economics, Nanjing, 210023, China

Phone: 86-25-86718507; Fax: 86-25-86718507
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Email: warmtxz@nufe.edu.cn
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Abstract

The effects of cinnamon essential oil (CEO) and Tween 80 addition on the structural,

physical, antioxidant and antimicrobial properties of the pullulan-based edible films were

investigated in this study. The results demonstrated that the incorporation of CEO in

pullulan-based composite films decreased their tensile strength, transparency, water content

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and water vapor permeability, but considerably increased the antioxidant and antimicrobial

properties. The films with 12% CEO exhibited the strongest antioxidant and antimicrobial

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capacities. With regard to the influences of Tween 80 on the films, SEM micrographs

indicated that the submicroscopic micelle formed in the films by the addition of Tween 80.

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Tween 80 improved the stability of the composite films and reduced the loss of CEO, but

weakened the physical properties including transparency and water barrier properties.
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Keywords: pullulan; cinnamon essential oil; Tween 80; antioxidant properties; antimicrobial
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properties.
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1. Introduction

Edible films and coatings, referring to the materials used for coating various foods to

prolong their shelf life and to certain extent can be eaten together with the food, have

attracted more and more attention in recent years [1]. These materials are usually made up of

biopolymers such as polysaccharides, proteins and lipids. Among these, pullulan, a type of

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water-soluble microbial polysaccharide produced extracellularly by the fungus-like yeast

called Aureobasidium pullulans, has been widely studied in the food packaging field due to

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its excellent film-forming characteristics. Pullulan films exhibited several advantages over

other polysaccharides, including colorless, tasteless, odorless, transparent, heat-sealable and

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highly impermeable to both oil and oxygen [2-5].

Active packaging is an innovative packaging which performs several functions, such as

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generating/scavenging flavors, absorbing moisture and oxygen, inhibiting the growth of

microorganisms to preserve food or improve sensory properties of food. These functions are
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generally offered by the active substances incorporated inside or onto the surface of polymer

matrix. Current researches have focused on the incorporation of nature substances, such as
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essential oils, to enhance the antimicrobial and antioxidant abilities of the films [6, 7].
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Cinnamon essential oil (CEO) is a natural preservative containing several active components
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which have antimicrobial and antioxidant properties [8]. CEO has been used to improve the

antimicrobial and antioxidant properties of biopolymer-based films, such as chitosan [9, 10],
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starch [11], and protein-based films [10, 12]. However, the information on the effects of

cinnamon essential oil on the properties of pullulan-based films is deficiency.

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In this study, the structural, physical, antimicrobial and antioxidant properties of

pullulan-based edible films incorporated with different concentrations of CEO were

investigated. To improve the essential oil solubility in film-forming solutions, a surfactant,

Tween 80 were also applied. Therefore, the combined effects of CEO and Tween 80 on the

properties of pullulan-based films were examined.

2. Materials and methods

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2.1 Materials

Pullulan with average molecular weight of 1×105 D was supplied by Shunfenghaideer

Biotechnology Co. Ltd (Zhejiang, China). Cinnamon essential oil was obtained from

Dongshi Flavor Co. Ltd (Shanghai, China). Tween 80 was purchased from Kermel Co. Ltd

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(Tianjin, China). All the other reagents were of chemical reagent grade.

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2.2 Methods

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2.2.1 Preparation of composite films

All films were prepared by solution casting method. Pullulan solution (2% w/v) was
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prepared by dissolving pullulan powder in deionized water at 45°C and stirring with a
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magnetic stirrer for 2 h at 800 rpm. After that, 15% glycerol (g/g total solids) was added as a

plasticizer to the pullulan solution and stirred for another half an hour. Different amount of

CEO was added to the solutions to reach concentrations of 0%, 4%, 8% and 12% (g/g total
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solids) and stirred for 10 min. To promote essential oil dissolution in the film-forming
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solutions, CEO was first dissolved in 2% or 4% (g/g total solids) Tween 80 solutions,
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respectively, at 8% CEO group. Finally, all solutions were homogenized for 4 minutes at
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12000 g with a rotor/stator homogenizer (IKA T18-Digital Ultra-Turrax, Staufen, Germany),

and then deaerated to obtain the film-forming solutions (FFDs). To obtain active films, 65

mL FFDs was casted over a polyethylene mold (12.5×12.5cm) and dried at 45oC for
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approximately 12 h in an oven. The dried films were peeled off and stored in a humidity

chamber at 25oC and 55% relative humidity for further test.

2.2.2 Fourier transform infrared Spectroscopy (FT-IR)

FT-IR tests were performed using FT-IR spectrometer (BRUKER Tensor 27, Germany)

in the wavelength ranging from 4000 to 600 cm-1 with a resolution of 4 cm-1. The films were

dried and then milled into powder using liquid nitrogen before measurement. Such film
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preparation might result in free CEO releasing while have no significant effect on the CEO

bounding with film matrix, thus would not influence FTIR data. The test specimens were

prepared by pelletizing with KBr.

2.2.3 Scanning electron microscope (SEM)

The micro-structure of the cross sections of the films was observed by a scanning

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electron microscopy (TM3000, Hitachi, Japan). For the assay, the samples were broken in

liquid nitrogen and then coated with gold and observed at different magnification.

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2.2.4 Film thickness

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Film thickness was determined using a thickness tester (CHY-C2A, Labthink, China),

which was taken at 10 random points on each film. Mean values were recorded.
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2.2.5 Film opacity
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The film opacity was determined by measuring the absorbance at 600 nm with an

ultraviolet spectrophotometer (Hitachi U-3900, Japan). The films were cut into strips (4.5×1
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cm), and then placed in a test cell. The opacity of the films was calculated by the following
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equation. Each sample was tested for three replicates.

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O=Abs600/L (1)
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Where O was referred to the opacity. Abs600 was the value of the absorbance at 600 nm and
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L was the film thickness (mm).

2.2.6 Water content

To determine the water content of films, films were cut into 2×2 cm squares and

weighed (w1). The water content of the films was measured by drying the film samples at

105oC until the constant weight (w2) was reached. Three film replicates prepared from each

formulation were tested.

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Water content=100%×(w1-w2)/w1 (2)

2.2.7 Water vapor permeability

Water vapor permeability (WVP) was determined according to ASTM E96-00

(ASTM2000). The films, which had been equilibrated at 55% relative humidity (RH) for 72

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h, were fixed on the top of equilibrated cups containing desiccants (40 g silica gel, dried in

105oC for 2 h before being used). Test cups were placed in a cabinet with a controlled 75%

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RH at 25oC. The weight changes were monitored for every 2 h over 12 h. The gradient was

calculated from the slope of a linear regression of the weight increase vs. time. Water vapor

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transmission rate (WVTR) and WVP were calculated according to the following equations:
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WVTR=Δm/(A×t） g/(h•m2) (3)
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Where Δm was weight changes of test cups (g), A was the test area (m2) and t was test time

(h).
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WVP=WVTR×d/ΔP g•mm/(kPa•h• m2) (4)

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Where d was the thickness of films (mm) and ΔP was the partial pressure difference across
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the films (KPa).

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2.2.8 Mechanical properties

To determine the tensile strength (TS) and elongation at break (E) of films, samples

were cut into strips (25×80 mm) and then mounted into the auto tensile tester (XLW,

Labthink, China). Grip separation and cross-head speed were set at 40 mm and 25 mm/min,

respectively. At least eight measurements were performed for each sample.

2.2.9 Thermogravimetric analysis (TGA)

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Thermogravimetric analysis was performed to evaluate the degradation characteristics

and thermal stability of films with a thermogravimetric analyzer (PerkinElmer Pyris 1,

United States). The films were dried and milled into powder with liquid nitrogen, then the

samples of approximately 3-5 mg were measured at a heating rate of 10oC /min from 30oC to

600oC under nitrogen. Grinding the films into powder might lead to partial release of free CEO

during sample preparation, but it brought little impact on the CEO bounding with film matrix and

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would not effect the degradation temperature of the films.

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2.2.10 Loss of CEO during film formation

The loss of CEO during the film formation was quantified following a procedure

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described previously [13]. The fresh film (1.5 g) was placed in a 50 mL centrifuge tube with

5 mL ultrapure water. Twenty five mL hexane was added and vigorously stirred overnight at
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25oC. The solution was centrifuged at 11,400 g for 10 min and the upper solution was

analyzed by measuring the absorbance at 318 nm with an ultraviolet spectrophotometer

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(Hitachi U-3900, Japan). The CEO concentration was then calculated by a standard curve

corrected from different CEO concentrations in hexane. The loss of CEO was estimated
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based on the difference of the CEO concentration in the fresh film and the desired
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concentration in the FFDs. All measurements were run in triplicate.

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2.2.11 Antioxidant properties

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The antioxidant properties of films were evaluated using 2, 2-diphenyl-1-picrylhydrazyl

(DPPH) free radical scavenging method described previously [14] with a slight modification.
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Fifty mg of each film was dissolved in 5 mL of acetic ether and then centrifuged at 2300 rpm

for 10 min. One mL of film extract solution was mixed with 2 mL of 40 mg/L methyl

alcohol solutions of DPPH (Fluka Chemical Company, St. Louis, MO, USA). The

absorbance was measured at 517 nm after the mixture had been incubated in the dark at

ambient temperature for 30 min. The assay was performed in triplicate. The percentage of

DPPH radical-scavenging activity was calculated using following equation:

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RSA (%) = (1-AS/AC) ×100% (5)

Where RSA (%) was the percentage of DPPH radical-scavenging activity, AC and AS were

the absorbance of control and samples, respectively.

2.2.12 Antibacterial activities

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The antibacterial properties of films were evaluated using liquid culture test [15]. One g

of the pullulan films was immersed in 20 mL nutrient broth for 1 h up to swell. The medium

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was inoculated with 60 μL of Eschericha coli (E. coli) and Staphylococcus aureus (S. aureus)

(108 CFU/mL), respectively and then the mixture was transferred to an incubator rotating

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with 150 rpm at 37oC. During the incubation, 150 μL of the above mixture was pipetted

every 1 h for the measurement. The same volume of bacterial suspension was used as a
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blank control, while the broth was used as a reference. Optical density was measured using a

microplate reader (SpectraMax–M2e, Molecular devices, China) at a wavelength of 650 nm

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to obtain microbial growth profiles. All samples were tested in triplicate.

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2.3 Statistic analysis

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Statistical analysis of the data for each test was performed using SPSS 17.0 (SPSS Inc.,

2008). Statistical significance of differences in means was calculated using ANOVA and Du
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ncan’s multiple range tests at p < 0.05.

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3 Results and discussions

3.1 Fourier transform infrared spectroscopy analysis

The FT-IR spectra of the composite films with different content of CEO and Tween 80

were presented in Fig.1. Remarkable differences of spectra profiles were detected between

pure pullulan films and composite films whereas a slight difference among the different

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concentrations of CEO was observed. With the incorporation of CEO, absorption band at

3426 cm-1, which was assigned to the characteristic absorption band of stretching vibration

of –OH and the association of hydrogen bonds, was enhanced indicating the interactions

between pullulan and CEO through hydrogen bonds. The absorption bands observed in the

spectra of the composite films at 2920 cm-1, 1639 cm-1 and 853 cm-1 were assigned to -CH2,

-C=O (amide Ι) and C-O stretching vibrations respectively [16], which also showed

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significant changes in the peak area and intensity in comparison with pure pullulan films. In

addition, an increase in the absorption bands at 2323 cm-1-2375 cm-1 was observed, which

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could be ascribed to the characteristic peaks of CEO. For films incorporated with Tween 80,

absorption band at 1732 cm-1 was assigned to the stretching vibration of carbonyl group in

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Tween 80 [17]. NU
3.2 Scanning electron microscopy
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The microstructure of the composite films was affected by the structural arrangement of

the different components (Pullulan, CEO and Tween80). SEM micrographs revealed the
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microstructure of cross-sections of the films straightforwardly. As shown in Fig.2, when

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adding lower amount of (4% and 8%) of CEO, the composite films showed a continuous and
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homogeneous structure, as a result of a low level of oil droplet flocculation and coalescence,

and the high compatibility among these formulations [18]. It had been reported that there
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existed interactions between CEO and biopolymers by hydrogen bonding [19, 20], which

was also confirmed by our FTIR results. However, the further addition of CEO (12%)
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increased the roughness of the cross-section slightly, indicating that the compatibility of the

films had been weakened, due to the root polarity differences of the hydrophilic pullulan and

hydrophobic CEO.

The film microstructure was significantly affected by incorporating surfactant (Tween

80). Submicron CEO droplets were encapsulated in small surfactant micelles, which were

embedded in the pullulan network after adding Tween 80. The encapsulation of CEO in

nonionic surfactant micelles might improve the solubility and stability of CEO components

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in the aqueous phase, thus increased their effectiveness in antioxidant and antimicrobial

properties of the films [21]. However, their size were increased with the increase of Tween

80 concentration (Fig.2e and f)，which was probably due to excess Tween 80 on the interface

or Tween 80 aggregation in the aqueous phase.

3.3 The thickness and opacity of the composite films

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The physical properties, including thickness, opacity, water content, WVP, and

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mechanical properties of the composite films, were shown in Table 1. The thickness of the

films significantly increased from 46.42 to 53.77 μm when the concentration of CEO was

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increased from 0% to 8%. However, the thickness was decreased remarkably when CEO

was increased to 12%. For composite films containing lower concentration of CEO, the
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intermolecular reactions among pullulan molecules were attenuated due to the good

compatibility between pullulan and CEO through hydrogen bonding, leading to loosed film
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structure and increased film thickness. Nevertheless, the compatibility between pullulan and

CEO was impaired, as shown in SEM images (Fig.1d), with high concentration of CEO
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(12%) being added, resulting in the decrease of film thickness [22]. Interestingly, it can be
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seen that the presence of Tween 80 remarkably increased the thickness of pullulan films. The
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possible reasons could be that large size of Tween 80 micelles with encapsulation of CEO

disrupted the continuous phase of pullulan matrix as shown in SEM images (Fig.1e and f),
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leading to the less dense film structure.

Opacity of the films has a direct impact on the appearance of the coated product.
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Compared with control group (0% CEO), the opacity of the films was significantly increased

with the addition of different concentration of CEO (Table 1). This probably was caused by

the natural color of CEO and the formation of CEO droplets throughout the films. The

addition of surfactants led to further increase in the opacity properties (e.g., from 0.90±0.34

at 8% CEO without Tween 80 to 1.69±0.75 at 8% CEO with 8% Tween 80, p<0.05), which

may be attributed to the increase in oil droplet size formed in the films, as shown in SEM

images (Fig.2). In general, the intensity of light scattering depends on the concentration and

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size of the dispersed phase. The higher the droplet concentration, the greater the intensity of

light scattering. However, the effect of the size of dispersed phase on optical properties was

difficult to predict because of the complexity of the interaction between the size of dispersed

phase and light scattering [23].

3.4 Water content and water vapor permeability

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The water content of pullulan-based composite films was decreased significantly from

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25.21% to 9.88% when 12% of CEO was incorporated. Previous studies have shown that the

interactions between the functional groups of CEO and pullulan impaired the pullulan–water

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interactions, leading to a decrease of water content [10, 24]. It may be also due to the

hydrophobic nature of CEO [10]. However, the incorporation of Tween 80 did not change
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the water content significantly (Table 1).

WVP plays a critical role in evaluating practical applications of edible films. Our study
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showed that the WVP of the films was decreased with the increase of CEO concentration.

The hydrophobic nature of CEO was known to contribute to the decreased WVP [10]. The
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oil phase increased the distance travelled by water molecules diffusing through the film,
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which could be further improved by the increase of oil concentration and the decrease of oil
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particle size [25]. However, the WVP of the films increased dramatically after combining

with Tween 80 (Table 1), suggesting that surfactant weakened the water barrier properties of
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the films. Our SEM data showed that Tween 80 increased the oil droplets size (Fig.2e and f),

which may destroy the continuous polymeric structure. In addition, Tween 80 has been
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shown to improve the water content due to its hydrophilic nature, and thus increases WVP of

the films [26].

3.5 Mechanical properties

The mechanical properties of films should be highlighted because of their significance

in evaluating food packaging. Elongation at break (E) and tensile strength (TS) of all films

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were examined in this study. With the increase of CEO concentration, the TS of the film was

significantly decreased while the E of the film was remarkably increased (Table 1). However,

no significant difference was detected in these two parameters when Tween 80 was added

into the film. The incorporation of CEO has been shown not only to attenuate the

inter-molecular hydrogen bonding between pullulan-pullulan molecules but also to loose the

film structure by allowing the oil droplet occupancy, leading to the decrease of TS in the

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composite films [27, 28]. Previous study has shown that CEO functioned as a plasticizer to

enhance the polymer chain movements through hydrogen bonding [18], which may

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contribute to the increase of E in CEO loaded films.

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3.6 Thermal stability NU
TGA curves of pullulan-based composite films were presented in the Fig.3, which

showed the similar curves with three stages of weight loss. The first stage of weight loss was
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shown at approximate 50-120oC, which could be attributed to the loss of adsorbed and

bound water and small part of volatile components in CEO. A similar degradation
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temperature of CEO (e.g. 63-119oC) was reported by a prior study [29]. The weight loss in the
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second stage occurred at 120-256oC for all samples, which was probably associated with the
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numerous decomposition of lower molecular weight fractions and glycerol. For the third

stage, these pullulan-based films were suffered a main weight loss at 256-357oC, which was
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caused by de-crosslinking of polymer networks and decomposition of pullulan. TGA

analysis showed that the addition of CEO did not change the thermal stability of the films.
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However, the addition of Tween 80 in the CEO contained films had lower weight loss and

higher amount of residues in the samples exposed to extra high temperature (300-600oC) due

to formation of surfactant micelles. The encapsulated oil droplets exhibited higher thermal

stability and Tween 80 might be complex with pullulan due to its hydrophilic nature. To be

noted, the composite film with 4% Tween 80 had higher weight loss and lower amount of

residues after thermal decomposition than that of 2% Tween 80. This probably was caused

by excess Tween 80 on the interface or Tween 80 aggregation in the aqueous phase.

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3.7 Loss of CEO

In general, essential oil loss of the films was mainly due to the volatilization of a large

amount of CEO during the film drying process [30]. Our results showed that CEO loss

increased gradually from 43.78% to 52.12% with the increase concentration of CEO (Fig. 4),

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which was consistent to a previous study [31]. That study concluded that the lower quantity

of polymer indicated a reduction in its encapsulating capacity to the oil, which caused the oil

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to volatilize more easily. In addition, according to their study, the encapsulating ability of

pullulan through hydrogen bonding and endowed viscosity were affected when its

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concentration reached a critical viscosity at a determined level of water evaporation in the

film and, at this moment, a large part of the free oil has been volatilized when there was a
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lower pullulan/CEO ratio in the film forming solution. Nevertheless, after combining Tween

80 into the films, the loss of essential oil reduced remarkably (Fig. 4), indicating that the
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Tween 80 increased the stability of oil in the films, probably due to CEO droplets were

encapsulated in small surfactant micelles and embedded in the pullulan network.

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3.8 Antioxidant activity

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The antioxidant activity of the films was evaluated by DPPH free radical-scavenging
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capacity. Fig.5 clearly showed that incorporation of CEO in the pullulan-based films

significantly increased DPPH free radical-scavenging capacity, and films with 12% CEO
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exhibited the highest radical-scavenging activity (52%). Similar results were observed by

Arancibia et al. [32] and Salmieri and Lacroix [33], who incorporated CEO into

alginate-based films. Arancibia et al. [32] domenstrated that eugenol was the main phenolic

component relating to the radical-scavenging capacity of CEO. With continuous addition of

CEO, the content of phenolic compounds increased, thus enhancing the antioxidant activity

of the composite films. Moreover, by combining Tween 80 into the blended films, the

antioxidant capacity of the composite films got slightly improved. This could be due to less

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CEO loss when Tween 80 was incorporated.

3.9 Antibacterial activity

The antimicrobial activity of the pullulan-based composite films was investigated with

E. coli and S. aureus as test microorganisms. Fig.6(a) showed the liquid culture results of

composite films inhibiting the growth of E. coli at 37oC. At the stationary growth phase,

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OD650nm value of the control (1.4013) was about 10 times more than that of films containing

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12% CEO. Meanwhile, Fig.6(b) represented a stronger inhibition of S. aureus by the 12%

CEO films (OD650nm at 0.1545) compared with the control group (OD650nm at 0.9768).

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Consistently, the inhibition ability of the growth of both bacteria was highly correlated with

the concentration of CEO being incorporated. Previous studies have shown that CEO
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inhibited the growth of both gram-positive and gram-negative bacteria [12, 29]. In addition,

antibacterial ability of CEO has been attributed to its noticeable effects on the surface
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structure and functional properties of cell membrane and this ability was mainly determined

by the concentration of the essential oils and their release rate in the films [34, 8]. It was also
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worth noticing that the antimicrobial activity of the composite films containing 2% Tween
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80 got slightly improved compared to its counterparts (without Tween 80). This
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phenomenon may be related to the fact that the surfactant reduced CEO loss and

consequently improved the sustained release of antimicrobial agents. However, the film
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containing 4% Tween 80 did not improve antimicrobial activity. One possible reason was

excess Tween 80 on the interface or Tween 80 aggregates in the aqueous phase prevented the
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interactions of CEO with mycelia, losing the ability to enhance the bioactivity of CEO.

4. Conclusion

In this study, the addition of CEO and Tween 80 resulted in significant changes in the

physicochemical and biological properties of the pullulan-based films. By incorporating

CEO, the TS, transparency, water content and WVP of the films were significantly decreased,

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while the antioxidant and antimicrobial capacities of the films were dramatically enhanced.

The addition of Tween 80 in the presence of 8% CEO significantly increased WVP, stability

of the composite films and reduced the loss of CEO. Our study provided valuable

information on the interactions between pullulan, essential oils and surfactants. Further

study will be performed to obtain the optimal proportion of the mixtures and optimize the

maximal practicability for commercial application of the pullulan-based active films.

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Acknowledgements

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The study was funded by the Priority Academic Program Development of Jiangsu Higher

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Education Institutions (PAPD). We would like to thank Dr. Gang Xi for his kind help for the

preparation of the manuscript.

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Figure Captions
Fig.1 FTIR spectra of pullulan films with different concentrations of CEO and Tween80.

Fig.2 Scanning electron microscopy micrographs of the cross-sections of the pullulan-based

films: (a)0%CEO; (b)4%CEO; (c)8%CEO; (d)12%CEO; (e)8%CEO+2%Tween 80;

(f)8%CEO+4%Tween 80.

Fig.3. TGA curves of pullulan-based composite films.

Fig.4 CEO loss of pullulan-based composite films.

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Fig.5 DPPH scavenging activity of pullulan-based composite films.

Fig.6 Antimicrobial activity of pullulan-based composite films: (a) E.coli. (b) S.aureus.

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Table 1

Thickness, opacity, water content, water vapour permeability (WVP), tensile strength (TS), elongation at break (E) of the

films.

Formulation Thickness Opacity Water content WVP TS E

(μm) (%) -1 -1
(g·m ·KPa ·h ) -1
(MPa) (%)
e d a a a
2.28±1.53d

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0%CEO 46.42±2.45 0.52±0.23 25.21±0.09 0.082±0.00 55.1±2.2

4%CEO 50.25±2.80d 0.60±0.21d 20.95±0.09b 0.069±0.00c 51.0±2.6b 4.74±2.17c

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8%CEO 53.77±3.66c 0.90±0.34c 14.08±0.02c 0.060±0.00cd 46.4±2.6c 5.22±2.30bc

47.66±4.57e 1.25±0.54b 9.88±0.01d 0.057±0.01d 40.1±1.8d 7.89±3.13a

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12%CEO

8%CEO+ 56.86±3.02b 1.17±0.48b 14.67±0.02c 0.078±0.01b 45.6±2.2c 5.66±2.11b

2%Tween80
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8%CEO+ 59.99±4.78a 1.69±0.75a 14.12±0.03c 0.080±0.01ab 45.6±1.1c 5.35±4.86bc
4%Tween80
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Different superscripts (a-e) within a column indicate a significant difference by the Duncan test (p<0.05)
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Highlights:

a. Pullulan-based edible films incorporated with cinnamon essential oils

(CEO) were prepared.

b. CEO considerably enhanced antioxidant and antimicrobial properties

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of the composite films

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c. The submicroscopic micelle formed in the films by the addition of

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Tween 80.

d. Tween 80 improved the stability of the composite films and reduced

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the loss of CEO.
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6