Cinnamon and clove essential oils to improve physical, thermal and antimicrobial properties of chitosan-gum arabic polyelectrolyte complexed films

Accepted Manuscript Title: Cinnamon and clove essential oils to improve physical, thermal and antimicrobial properties of chitosan-gum arabic polyelectrolyte complexed films Authors: Tian Xu, ChengCheng Gao, Xiao Feng, Meigui Huang, Yuling Yang, Xinchun Shen, Xiaozhi Tang PII: DOI: Reference:

S0144-8617(19)30356-X https://doi.org/10.1016/j.carbpol.2019.03.084 CARP 14753

To appear in: Received date: Revised date: Accepted date:

10 December 2018 20 February 2019 25 March 2019

Please cite this article as: Xu T, Gao C, Feng X, Huang M, Yang Y, Shen X, Tang X, Cinnamon and clove essential oils to improve physical, thermal and antimicrobial properties of chitosan-gum arabic polyelectrolyte complexed films, Carbohydrate Polymers (2019), https://doi.org/10.1016/j.carbpol.2019.03.084 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cinnamon and clove essential oils to improve physical, thermal and antimicrobial properties of chitosan-gum arabic polyelectrolyte complexed films Tian Xua, ChengCheng Gaoa, Xiao Fenga, Meigui Huangb, Yuling Yanga,

aCollege of Food Science

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Xinchun Shena, Xiaozhi Tanga*

and Engineering/Collaborative Innovation Center for Modern

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Grain Circulation and Safety/Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing University of Finance and Economics, Nanjing 210023, China

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of Food Science and Technology，College of Light Industry and Food

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bDepartment

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Engineering, Nanjing Forestry University, Nanjing, 210037, China

*Corresponding Author: College of Food Science and Engineering, Nanjing University

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of Finance and Economics, Nanjing, 210023, China Phone: 86-25-86718507

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Fax: 86-25-86718507

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Email: [email protected]

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Graphical abstract

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Highlights:    

CEO and CLO improved physical, thermal and antimicrobial properties of films. The addition of CEO showed better thermal stability and EO retention than CLO. Sustained release and enhanced antimicrobial activity were obtained as CEO% increased. Combined EOs decreased particle size and showed synergistic antimicrobial effects.

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EOs: essential oils, CEO: cinnamon essential oil, CLO: clove essential oil

Abstract

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Chitosan-gum arabic-based polyelectrolyte complexed films with cinnamon essential oil (CEO) and clove essential oil (CLO) were developed. The effect of EO concentrations, types

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and their combinations on the physical, thermal and antimicrobial properties of films were

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investigated. The results showed that the incorporation of EOs decreased the ζ-potential and

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viscosity, but increased the particle size of film-forming dispersions. Films incorporated with CEO and combined EOs exhibited better water barrier properties compared to those with CLO

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and single EO. Films containing CEO showed lower EO loss and higher thermal stability compared to those containing CLO, and the reason was attributed to the stronger interactions

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between chitosan, gum arabic and CEO. The combination of EOs resulted in higher retention and delayed release rate in food stimulant, resulting in stronger antimicrobial activities. The

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performance of films with the CEO and the combined EOs brought new formulation ideas in antimicrobial films.

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Keywords: chitosan; gum arabic; cinnamon essential oil; clove essential oil; polyelectrolyte complexed films; antimicrobial properties

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1. Introduction

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With growing concerns for food safety and environment, numerous efforts have aimed to

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develop active edible films instead of traditional fossil-based films to extend the shelf life of

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food. Edible films usually made of polysaccharides, proteins and some part of lipids (Chen &

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Liu, 2016; Li, Wu, et al., 2016). They have excellent biodegradability, biocompatibility and show great potential to carry active additives, such as antimicrobial agents and antioxidants

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(Dong, Xu, Ahmed, Li & Lin, 2018; Li, Mei, Xu, Lee, & Wu, 2016). Natural additives like essential oils (EOs), which are aromatic hydrophobic plant extracts,

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have exhibited well-documented antimicrobial activities (Ojagh, Rezaei, Razavi & Hosseini, 2010; Sánchez-González, Cháfer, Chiralt & González-Martínez, 2010). The structure,

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composition as well as the functional groups of EOs were the main factors influencing the antimicrobial effects (Ojagh, Rezaei, Razavi, & Hosseini, 2010). The antimicrobial effects also depend on the differences of microbial species. Studies reported that EOs exhibited more

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remarkable inhibition on Gram-positive bacteria than Gram-negative bacteria, which might be due to the characteristic differences of their outer membrane (Sun et al., 2014; Wang et al., 2011). Among all EOs, cinnamon EOs (CEO) and clove EOs (CLO), which were classified as Generally Recognized as Safe (GRAS), have gained great attention due to their better sensory performance and antimicrobial capacities (Peng & Li, 2014; Sun et al., 2014; Wang et al., 2011). The difference in antimicrobial spectrum of CEO and CLO were greatly attributed to their main 3

antimicrobial constituents: cinnamaldehyde and eugenol, respectively (Dong, Xu, Ahmed, Li, & Lin, 2018; Salgado, López-Caballero, Gómez-Guillén, Mauri, & Montero, 2013). Cinnamaldehyde and eugenol were regarded as two typical representatives of aldehydic and phnolice compounds for antimicrobial activity. Besides, the water solubility of eugenol was higher than that of cinnamaldehyde (Wang, et al., 2011), which may influence the efficiency of the application in films. Several studies have demonstrated their effective antimicrobial

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activities and application for the preservation of different types of food (Elbaroty, Elbaky, Farag, & Saleh, 2010; Ibrahium, Elghany, & Ammar, 2013).

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Chitosan (CS) is the deacetylated form of chitin, deriving from the crustaceans such as

crab and shrimp. It has been considered to be one of the most promising natural polysaccharides for elaborating edible films due to its biodegradation, antimicrobial activity and good film-

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forming ability (Noshirvani et al., 2017). Active additives, such as EOs, are usually

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incorporated into CS-based films to improve their physical, water barrier properties and

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antimicrobial activities. Ojagh et al (2010) have reported the influence of CEO on the antibacterial, physical and mechanical properties of CS films, indicating the decreased water

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vapor permeability and the cross-linking effect of cinnamaldehyde within the film network.

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Gum Arabic (GA), a negatively charged heteropolysaccharide derived from the branches or trunks of Acacia trees, have been regarded as the best gum in the use of oil-in-water emulsion systems due to its emulsification and encapsulation properties (Ali, Maqbool, Ramachandran,

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& Alderson, 2010). Previous studies have reported that the incorporation of GA exerted the emulsification effects on EOs in edible coatings, thus effectively controlled the postharvest

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anthracnose in papaya, banana (Mehdi Maqbool, et al., 2011) and avocado (Bill, Sivakumar, Korsten, & Thompson, 2014). Besides, GA is a polyelectrolyte due to the presence of uronic acid, and can be used as carriers in encapsulating active ingredients by simple or complex

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polyelectrolyte complexation (Fathi, Emam-Djomeh, & Sadeghi-Varkani, 2018). Polyanionic GA could form characteristic composites with polycationic polymers such as CS through electrostatic interactions, and these complexes exhibited different complexation behavior in different mass ratios (Malviya et al., 2009; Tsai et al., 2014). In our previous work, a polyelectrolyte complexed film based on CS, GA and CEO was developed to investigate the influences of CS/GA mass ratios on the retention and release 4

properties of antimicrobial films (Xu et al., 2018). However, the CEO concentration was fixed and their interactions with the polymer matrix were still unclear. Therefore, the present study describe the elaboration of CS-GA polyelectrolyte complexed films with different concentrations of CEO, CLO and their combinations. The objective is to study the influences of EO concentrations, types and their combinations on the physical, thermal and antimicrobial

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properties of polyelectrolyte complexed films.

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2. Materials and methods

2.1 Materials

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Chitosan (CS, 90.27% deacetylation degree, Mw=5.0104 g mol-1) was obtained from Jinan

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Haidebei Ocean Biological Engineering Co., Ltd (Shandong, China). Gum Arabic (Acacia

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senegal，GA, Mw=3.0105) was obtained from Shanghai Macklin Biotech Co., Ltd (Shanghai, China). Cinnamon essential oil (CEO, medical grade) and Clove essential oil (CLO, medical

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grade) was purchased from Dongshi Flavor Co., Ltd (Shanghai, China). Glycerol, hexane and

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acetic acid were all of analytical reagent grade.

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2.2 Preparation of the film-forming dispersions

CS (1.5% w/v) was dispersed in 1% v/v acetic acid solution at 40°C for 5 h. The impurities

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were removed by filtrating through a filter cloth of 200 mesh, and then 20% glycerol (g/g total solids) was added as a plasticizer. GA powder was dissolved in deionized water at room temperature until it was completely dissolved. The mass ratio of CS and GA was 1:1. After that,

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a certain amount of EOs (including CEO and CLO, g/g total solids) was added to the GA solutions, with vigorous stirring for 30 min. The film-forming dispersions (FFDs) were prepared by slowly adding GA-EO solutions containing various EO concentrations to CS solution. At the end, the FFDs were homogenized at 12000 rpm for 4 min with a rotor/stator homogenizer (IKA T18-Digital Ultra-Turrax, Staufen, Germany). The resulted CS-GA FFDs contained 0% CEO (pure CS-GA solutions), 5%CEO, 10%CEO, 15%CEO, 10%CLO, and 5

5%CEO+5%CLO.

2.3 Characterization of film-forming dispersions

2.3.1 pH values A PHS-3C pH meter (Jingke Equipment, Shanghai, China) was used to measure the pH

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value of FFDs in triplicate.

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2.3.2 ζ-potential and particle size distribution

ζ-potential and particle size distribution were analyzed with a ζ-potential and particle size analyzer (model Nano-ZS90, Malvern, UK). FFDs were first diluted 1:100 using ultra-pure

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water and agitated well. The Mie theory was performed for size distribution to analyze the

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optical properties of EOs, a refractive index of 1.456 and 0.001 absorption. Each FFD was

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2.3.4 Rheological behavior

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measured in triplicate.

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The rheological behavior of FFDs was carried out with a rheometer (MCR 302, Anton Paar, Graz, Austria) and the measuring geometry was set as 50 mm plate-plate. The shear stress (σ) was obtained as the function of shear rate (γ) ranging from 1-300 s-1 at 25°C. Rheological

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data were fitted to the power law model (equation (1)) to obtain the flow behavior index (n) and the consistency (k) of FFDs. (1)

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σ=k×γn

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2.4 Preparation of the films

Casting method was applied to obtain composite films. FFDs of 60 g were casted on

standardized polyethylene plates (12.5 cm×12.5 cm) and allowed to dry at 45°C for 12 h. Dried films were peeled from the plates and preconditioned at 25°C and 50% relative humidity in a desiccator before further measurements. 6

2.5 Physical and mechanical properties of the films

2.5.1 Visual appearances and the opacity Visual appearances of FFDs and films were obtained with a digital camera. The opacity of films was determined according to Shojaee-Aliabadi et al. (2014) by measuring the absorbance at 600 nm with an ultraviolet spectrophotometer (Hitachi U-3900, Japan). Films (4.5 cm×1 cm)

in triplicate. The film opacity was calculated by the following equation:

(2)

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Op= Abs 600/d

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were placed in the test cell and an empty cell was used as the reference. All tests were conducted

where Abs 600 is the value of absorbance at 600 nm, and d represents the average thickness

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of films (mm). According to this equation, high values of Op mean the lower transparency.

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2.5.2 The thickness and moisture content

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Film thickness was measured with a thickness tester (CHY-C2A, Labthink, China), and 10 random locations in each film were recorded for the average thickness. The moisture content

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(MC) of films was determined by drying the films (2 cm×2 cm squares, w1) at 105°C until the

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constant weight (w2) was reached. Triplicate tests were taken. Then, the following equation was applied to calculate MC:

(3)

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MC (%)=100%×(w1-w2)/w1

2.5.3 Water barrier property of the films

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Water vapor permeability (WVP) of films was measured according to the standard method

of ASTM E96-95 (ASTM, 2004). Film discs (ø=9 cm) were fixed onto permeability cups (LLY11, Yuanmore, China) containing silica gels. These cups were initially weighed and then placed

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in a humidity chamber at 25°C and 75% relative humidity. The weight of test cups was measured at 2 h intervals for at least 12 h. Water vapor transmission rate (WVTR) and water vapor permeability (WVP) were calculated by equations (4) and (5): WVTR=Δm/(A*t)

(4)

WVP=WVTR*d/ΔP

(5) 7

where, Δm is the weight change before and after the test (g); t is the test time (h), and A is the test area (m2); d is the thickness of films (mm), and ΔP is the partial pressure difference across the films (kPa). Each test was replicated three times.

2.5.4 Mechanical properties Prior to the test, films were equilibrated at 50% relative humidity and 25°C for at least 3

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days, then mechanical properties were determined using an auto tensile tester (XLW, Labthink,

China) based on the standard method ASTM D882-02 (ASTM, 2001). Samples were cut into

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strips (2.5 cm×9 cm), the initial gaps separation was 40 mm, and the test speed was 25 mm/min. At least 8 repetitions were performed for each sample.

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2.6 Fourier transform infrared Spectroscopy (FT-IR)

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FT-IR analysis was performed to observe the structural interaction in multiphased system, with a spectrometer (BRUKER Tensor 27, Germany) scanning in the range from 4000 to 600

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cm-1 and a resolution of 4 cm-1. Before the FT-IR measurement, films were milled into powders,

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mixing with KBr.

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2.7 Loss of EOs during storage time

The loss of EOs in films during storage was determined according to the method

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previously described (Chen & Liu, 2016). Each film (1.5 g) was first placed in centrifuge tubes with 5 mL deionized water for hydration swelling, then 25 mL hexane was added and continually stirred overnight at room temperature. The mixture containing CEO or CLO was

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centrifuged at 11,400 rpm for 10 min, and finally the supernatant was measured with the absorbance respectively at 318 nm and 265 nm by ultraviolet spectrophotometer (Hitachi U3900, Japan). The EO concentration was determined by a standard curve for CEO and CLO solutions in hexane. Loss of EOs was expressed as wt% with respect to the original amount of EOs in FFDs. All tests were carried out for a total of two weeks and triplicate measurements were taken. 8

2.8 Fluorescence microscopy

The fluorescence microscopy was employed for visually determining the distribution and retention of EOs in films, following the procedure previously described (Xu et al., 2018). Slices of dry film (1 cm1 cm) were dyed with the appropriate amount of lipophilic fluorescent dye

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(Nile red, in 0.1 mg/mL ethanol). The dyed film strips were put on microscope slides and

covered with coverslips. A fluorescence microscope (Axio Vert.A1, Carl Zeiss Microscopy

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GmbH, Jena, Germany) was used to capture the film images, with appropriate fluorescence intensity and focal length.

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2.9 Thermogravimetric/ Derivative thermogravimetric analysis (TGA/ DTG)

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Appropriate amount of film samples (3-5 mg) were tested under nitrogen atmosphere, with a heating rate of 10°C/ min from 30 to 600°C. TGA/ DTG tests were carried out with a

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thermogravimetric analyzer (Perkin Elmer Pyris 1, United States).

2.10 EO release in food simulant

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To determine the release kinetics of CEO and CLO, 60% glycerol solution was used as aqueous food simulation with a water activity around 0.6-0.7. Film samples (0.5 g) were added

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to the solution (30 mL) and constantly stirred at 30 rpm for 24 h. Finally, the solution aliquots was collected at specific time intervals and then dispersed in hexane. The concentration of CEO and CLO was calibrated by the absorbance respectively at 318 nm and 265 nm with the

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ultraviolet spectrophotometer (Hitachi U-3900, Japan). All tests were carried out in triplicate.

2.11 Antimicrobial activity of the films

Antimicrobial efficiency of the films against Gram-positive bacteria (Staphylococcus aureus ATCC 25923, S.aureus) and Gram-negative bacteria (Eschericha coli ATCC 25922, 9

E.coli) was assessed by the optical density (OD) measurement (Salleh et al., 2010). Each film strip (1 g) firstly swelled in nutrient broth. The media were carefully inoculated with 60 μL bacterial target suspension, in which the microbial concentration was 108 CFU/mL. The same volume of broth was used as a reference. The mixtures were then transferred to a temperaturecontrolled incubator and shaken at 150 rpm, and all films were ensured to be immersed completely while shaking. During the rotation, the mixture aliquots were taken out from the

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readings with a microplate reader (Versamax–M2e, Molecular devices, China).

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tube periodically. The microbial growth profiles (S.aureus and E.coli) were obtained by OD600

2.12 Statistical analysis

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Experimental analysis of variance (ANOVA) was performed by means of SPSS 17.0

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(SPSS Inc. 2008). Differences in properties of films were determined with Duncan’s multiple

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range at 0.05.

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3. Results and discussion

3.1 Properties of film forming dispersions (FFDs)

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The pH, ζ-potential, particle size and rheological data of FFDs with different contents of CEO and CLO were shown in Table 1. The pH is an important parameter impacting the

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conformation of components in film formulation and the stability of emulsions (Campelo et al., 2017). The pH values of FFDs ranged from 4.29 to 4.32 and was nearly not affected (p>0.05) by the incorporation of EOs.

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Table 1

The pH, ζ-potential, particle size and rheological parameters of film-forming dispersions. FFDs

pH

ζ-potential (mV)

Particle size (nm)

n

k (Pa•s)n

R2

0% CEO

4.32±0.01a

50.20±2.10a

214.4

0.90698

0.08788

0.992

5% CEO

4.30±0.01a

47.94±1.02b

228.8

0.91698

0.0792

0.997

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10% CEO

4.29±0.00a

44.57±1.60c

269.4

0.93050

0.07291

0.996

15% CEO

4.29±0.01a

41.23±0.44d

341.6

0.93441

0.0692

0.996

10% CLO

4.31±0.00a

45.28±0.56c

278.3

0.91023

0.0802

0.997

5% CEO+5% CLO

4.31±0.01a

45.09±0.87c

260.6

0.91431

0.0799

0.993

Data are mean values ± standard deviation. Different letters (a-d) in each column indicate significant

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differences among formulations (p<0.05)

The increase in CEO content (0%-15%) significantly (p<0.05) decreased the ζ-potential and increased the particle size of FFDs, implying bigger droplets with reduced surface charge

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appeared. These changes are in agreement with those reported by other authors for chitosanbergamot essential oil FFDs (Sánchez-González, Cháfer, Chiralt, & González-Martínez, 2010; Sanchez-Gonzalez, Chiralt, Gonzalez-Martinez, & Chafer, 2011). The decreased ζ-potential

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(lower surface charge of droplets) could be explained that the increased amount of CEO led to

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their stronger electrostatic interactions with the amino groups of CS at the pH around 4.30, thus

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reducing the electrical net charge (Sanchez-Gonzalez, Chiralt, Gonzalez-Martinez, & Chafer,

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2011). The increased particle size was related to the decreased weight ratio of GA to EOs, inducing EOs droplets easy to aggregate. It was worth noting that no significant difference was

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observed between ζ-potential of FFDs contained CEO and CLO, but the latter had a larger particle size, which was due to the differences in affinity grades, oil polarity and chemical

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composition of EOs (Acevedo-Fani, Salvia-Trujillo, & Martín-Belloso, 2015). The combination of two types of EOs led to the decreased particle size, which might be related to

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the change of aggregation state of single EOs due to the combination (Peng & Li, 2014). Furthermore, the typical size distributions of FFDs were measured and plotted in Fig.1A. The distributions were multimodal except for the formulation containing 0% CEO and 10% CLO FFDs. In this multiphased system, CEO brought by GA with its emulsifying effects was

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embedded in the core of an entangled structure, which was formed by the entanglement of CS and GA through electrostatic interactions (Xu et al., 2018). The occurrence of small peak around 450 nm indicated the presence of bigger droplets, which was likely related to the presence of bigger EO droplets, EO droplets flocculation and/or aggregation.

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Fig.1. The particle size distribution (A) and rheogram (B) of film-forming dispersions analyzed at 25°C.

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The measurement of rheological behavior was generally considered as one of the most

significant parameters to characterize the system stability and the internal structure of polymerbased suspensions (Acevedo-Fani, Salvia-Trujillo, & Martín-Belloso, 2015). From Table1, all

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rheological data of FFDs were fitted to the Ostwald de Waele model (R2>0.990), showing less

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marked shear-thinning behavior (n<1) after the addition of EOs, which was consistent with the

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decreased viscosity in Fig.1B. Besides, the consistency index (k) also decreased when CEO content increased from 0% to 15%. These results could be explained that with the increasing

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content of CEO, the viscous contribution, or the thickening effects of biopolymers reduced due

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to the relatively decreased effective concentration at continuous phase (Peng & Li, 2014; Sánchez-González, Cháfer, Chiralt, & González-Martínez, 2010). The reduction of electroviscous effects due to the decreased ζ-potential should also be considered (Perdones,

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Chiralt, & Vargas, 2016; Sanchez-Gonzalez, Chiralt, Gonzalez-Martinez, & Chafer, 2011). Compared to the FFDs containing CEO, the FFDs incorporated with CLO showed higher

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viscosity at 10% level. The viscosity of blended oil solutions displayed just between those of the FFDs containing two types of EOs, achieving balance of a single essential oils. EOs are complex mixtures with different components, so different interaction strength between oil

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droplets and biopolymers might occur, resulting in the differences of viscosity (Acevedo-Fani, Salvia-Trujillo, & Martín-Belloso, 2015).

3.2 Physical and mechanical properties of films

All films showed smooth and homogeneous surface and the aroma was found stronger 12

with increasing CEO contents (Fig.2a1-d1). With the increasing CEO, the color of FFDs obviously changed from yellowish (Fig.2a) to milky white (Fig.2d). The visual observations were supported by the opacity results revealed in Table 2. The opacity was increased from 0.71 to 2.53 as the CEO increased from 0% to 15%, and this could be attributed to the increased intensity of light-scattering induced by the increasing size of oil droplets within film matrix (Acevedo-Fani, Salvia-Trujillo, & Martín-Belloso, 2015). This results were in agreement with

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those films incorporated with different amount of EOs (Noshirvani et al., 2017; Perdones,

Chiralt, & Vargas, 2016; Sanchez-Gonzalez, Chiralt, Gonzalez-Martinez, & Chafer, 2011). As

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to oil type, the films containing CLO were the most transparent (lower opacity), which could be related to the nature, state and internal amount of EOs during drying. Meanwhile, the

incorporation of CEO increased the yellowish color of control films (Fig.2a1), which inferred

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the occurrence of Schiff-base reaction between CS and CEO (Chen et al., 2016; Mariana Pereda,

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Amica, & Marcovich, 2012). It was reported that the Schiff-base reaction between CS and CEO

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would help the compatibility of the CS-CEO film (Chen et al., 2016; Wang et al., 2011).

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Fig.2. Visual appearances of FFDs (A)and films (B) with different amount of CEOs at 25°C: a,a1-0% CEO; b,b1-5% CEO; c,c1-10% CEO; d,d1-15% CEO.

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The influences of EOs on the physical, water barrier and mechanical properties of different

films were shown in Table 2. Results showed that the incorporation of EOs significantly (p<0.05) increased the thickness of films (varied from 60.01 to 69.52 mm), indicating the looser microstructure formed due to the addition of EOs (Wang et al., 2011). Two types of EOs (CLO and CEO) presented similar results, as well as their combination. Moisture content (MC) indicates the void volume occupied by water molecules in film network (Xu, Kim, Hanna, & 13

Nag, 2005). Water vapor permeability (WVP) represents the ability of moisture in films exchanging with the surrounding environment. Lower WVP means a better water vapor barrier performance of films (Atef, Rezaei, & Behrooz, 2015; Peng & Li, 2014). As expected, the addition of oil phase led to the decreased MC and WVP of films. It has been reported that the high content of hydrophobic EOs could increase the discontinuities of film network and the tortuosity factor for water transport, thus enhancing the water barrier properties (Peng & Li,

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2014; Perdones, Chiralt, & Vargas, 2016; Shojaee-Aliabadi et al., 2014). In addition, films

containing two types of EOs showed similar value of WVP, but the mixture of EOs decreased

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the WVP of films. The reason was likely related to the decreased oil droplet size (Table 1), which increased the interface area between EOs and matrix and then hindering the formation of the hydrogen bonding between polymer and water (Peng & Li, 2014).

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TS represents the maximum tensile stress that the film can endure, and E represents the

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maximum variation of a sample before fracture (Zhang et al., 2018). Regardless of the type of

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EOs, the incorporation of EOs significantly (p<0.05) reduced the TS, while increased the E of films. These changes were more dramatic as higher concentrations of EOs were added. When

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the CEO content reached 15%, the TS reduced by 45% and the E increased by 41% compared

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to those of the control film. The decreased TS could primarily be attributed to the partial replacement of the stronger intermolecular polymer interactions and polymer-polymer interactions (complex interactions between CS and GA) by the weaker polymer-oil interactions

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in film matrix (Shen & Kamdem, 2015; Shojaee-Aliabadi et al., 2014). The increased E could be attributed to the strong plasticizing effect of EOs, which improved the polymer chain

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mobility in film stretching. Trends of mechanical properties were in agreement with the studies of Ma et al. (2016) and Hosseini, Razavi, & Mousavi (2009) on chitosan-based films incorporated with CEOs.

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Table 2

Film thickness, moisture content, WVP, opacity and mechanical properties of films. Data are mean values ± standard deviation. Different letters (a-e) in each column indicate significant differences among formulations (p<0.05).

Compared to CEO, the plasticizing effect of CLO in reducing TS were pronounced. In reality, different types of EOs influenced the mechanical properties differently due to their 14

capability to interact with polymer matrix (Shen & Kamdem, 2015). It was reported that there existed Schiff-base reaction between CS and cinnamaldehyde, which helped the compatibility of the CS-CEO film (Chen et al., 2016; Wang et al., 2011) and finally manifested as the higher TS. The particle size of FFDs should also be taken into account. Smaller particle size of oil droplets (Table 1) in films could facilitate the formation of continuous network of whole matrix and increase the intermolecular polymer interactions and polymer-polymer interactions, thus

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leading to higher TS.

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3.3 FT-IR analysis of films

FTIR spectroscopy was studied to investigate the interactions between CS, GA, and EOs

Moisture content

WVP

(μm)

(%)

0% CEO

60.01±2.95c

14.99±0.01a

5% CEO

63.63±3.47b

13.50±0.01b

b

b

TS

E

(10 g•m •KPa •h )

(%)

(MPa)

(%)

0.36±0.01a

0.71±0.11d

30.03±4.35 a

32.03±1.97d

0.31±0.00b

0.96±0.32bc

26.66±3.62b

38.92±2.17c

c

b

d

42.56±2.30b

-1

-1

0.27±0.00

-1

10% CEO

64.67±3.02

15% CEO

69.52±5.28a

12.09±0.02c

0.26±0.00cd

2.53±0.17a

16.53±1.65e

45.04±3.87a

10% CLO

66.07±4.16b

13.36±0.01b

0.28±0.01c

0.78±0.45cd

20.31±2.31c

37.43±2.11c

5% CEO+5% CLO

65.79±2.88b

13.42±0.02b

0.23±0.01d

0.89±0.42bc

24.06±2.58c

41.03±4.86b

1.02±0.35

23.27±3.95

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M

13.51±0.01

Opacity

-3

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Thickness

A

Films

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in the multiphased system, as presented in Fig.3. In pure CS film, the broad band at 3445 cm-1

was the -OH stretching and the peak at about 1657 cm-1 corresponded to the stretching vibration

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of C=O in amide I. Similar spectrum for CS film has been reported by Li, Deng, Deng, Liu, & Xin (2010). In the case of GA, the characteristic peaks at 1610 cm-1 and 1413 cm-1 corresponded

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to the symmetrical and asymmetric stretching vibration of -COO-, respectively. For CS-GAbased films, peaks at 1657 cm-1 and 1598 cm-1 of chitosan gradually merged with the peak at 1610 cm-1 (-COO-) of GA into one intensive peak at 1642 cm-1 (Fig.3a), indicating the

A

occurrence of electrostatic interactions between the amino groups of the chitosan and the carboxyl groups of GA (Sakloetsakun et al., 2016).

15

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Fig.3. FT-IR of CS-GA based films containing 0% CEO (pure CS-GA film, a), 5%CEO (b), 10%CEO

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(c), 15%CEO (d), 10%CLO (e), and 5%CEO+5%CLO (f).

Great changes had taken place when EOs were added to the CS-GA system. In comparison

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to the spectra of CS-GA films (Fig.3a), the peak at 1626 cm-1 was observed from the spectra of

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the CS-GA-CEO films (Fig.3b), which might be corresponded to the C=N vibrations

A

characteristic of imines, indicating the occurrence of Schiff-base reaction between CS and CEO

M

(Chen et al., 2016; Wang, Ziru Lian, Hedong Wang, Jin, & Liu, 2011). In addition, films had the new peaks at 1260 cm-1 and 1560 cm-1, which respectively represented the symmetric

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expansion of C-O-C from acid ester in phenolic components and the C=C vibration from aromatic rings in aromatic components (Jeyaratnama et al., 2016). The new peak at 1430 cm-1,

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which might present the -CH stretching in aromatic ring (Chen et al., 2016), became stronger with increasing CEO content (Fig.3a-d). Some new characteristic bands appeared at 2370-2320

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cm-1, which could be associated with the characteristic absorption peaks of EOs (Zhang et al., 2018). These results suggested the development of imine bonds, accompanying other structural modifications. As to the CS-GA-CLO films (Fig.3e), the peak at 1560 cm-1, which represented

A

the C=C vibration from aromatic rings in aromatic components, became more flattened. Moreover, the film with combined use of CLO and CEO showed the balanced spectra of those containing a single essential oil.

3.4 The loss of EOs during film storage

16

Many authors have reported the great loss of volatile antimicrobial substances during film formation and subsequent storage (Kurek, Descours, Galic, Voilley, & Debeaufort, 2012; Ma et al., 2016; Sánchez-González, Cháfer, González-Martínez, Chiralt, & Desobry, 2011). The loss of EOs involved two mass transport phenomena: the diffusion from the film interior to surface and from the surface to atmosphere (Gahruie, Ziaee, Eskandari, & Hosseini, 2017). Fig.4 showed the loss of EOs from CS-GA based films in two weeks. Results indicated that all film

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formulation showed the gradually increased loss trend during ambient storage. The films

incorporated CEO showed lower loss compared to the films incorporated with CLO but the

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films with combined EOs presented the increased loss. In addition, the loss of EOs greatly increased with increasing EO contents. The loss in fresh films (day 0) was 41.25% for films containing 5% CEO and increased significantly to 50.56% for films containing 15% CEO,

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A

N

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which increased 1.23 times.

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Fig.4. The loss of EOs in films during storage time at 25°C.

The mechanism of polysaccharides to retain the aroma compounds is mainly due to the

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molecular interactions between macromolecules and aroma compounds like entrapment and hydrogen bonds (Kurek, Descours, Galic, Voilley, & Debeaufort, 2012). In the current system, CS and GA were entangled through electrostatic interaction. EOs was caught by GA due to its

A

emulsifying property and then embedded in the core of the CS-GA intertwined structure, leading to the high stability and retention of EOs in the polymer matrix. The increasing contents of CEO led to the relatively lower weight ratio of intertwined macromolecules to CEO, which reduced the matrix encapsulation capacity to CEO and made CEO easier to volatilize. Perdones, Chiralt, & Vargas (2016) reported the EO loss showed good correlation coefficients with some parameters of FFDs, such as the EO loss increased when the droplet particle size rose but ζ17

potential decreased.

3.5 Fluorescence microscopy of films

From Fig 5, we can visually evaluate the relative amount and distribution of oil droplets in the film, as they greatly affected the properties of films. It was obvious that with the

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increasing ratios of CEO from 5% to 15% (Fig.5A-C), more and more oil droplets appeared in the picture. Meanwhile, the particle size became larger, which was consistent with the results

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of the particle size of oil droplets in FFDs (Table 1). In current system, the intertwined structure

by CS and GA and emulsification effects of GA on EOs played a major role in restraining oil droplets from aggregating. However, with oil concentration increased, higher probability of

U

contact between oil molecules increased, leading to more aggregation of oil droplets and larger

N

particle size during film formation and drying stage. As to the types of EOs, the fresh films

A

incorporated with CEO exhibited higher retention than that with CLO, which was also manifested by the lower loss in day 0 (Fig.4). On the other hand, the mixture of CEO and CLO

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caused synergistic effects, leading to the larger intermolecular force with CS and finally helped

A

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the distribution and retention of mixed EOs.

Fig.5. The fluorescent images of CS-GA based films containing 5%CEO (A), 10%CEO (B), 15%CEO (C), 10%CLO (D), and 5%CEO+5%CLO (E).

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3.6 Thermal properties of films

Both TG and DTG thermograms reveal the thermal degradation behavior of films, but the former illustrates the weight loss as the function of temperature and the latter is obtained by the first derivative of TG curves (Xu et al., 2018). Fig.6 displayed TG (A) and DTG (B) curves for all samples, showing similar thermal behaviors with slight differences. It was clear that all films

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incorporated with CEO and CLO showed the decreased weight loss compared to the control

film (Fig.6 A), indicating the enhanced thermal stability. This result was similar to the studies

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in gelatin films incorporated with bergamot oil (Ahmad, Benjakul, Prodpran, & Agustini, 2012)

and polyethylene films containing rosemary essential oil (Dong, Xu, Ahmed, Li & Lin, 2018). The increased thermal stability was probably attributed to a stronger film network favored by

U

the interaction between CS and EOs (Ahmad, Benjakul, Prodpran, & Agustini, 2012).

N

Furthermore, the lower amount of CEO (5% and 10%) led to an increased thermal stability, but

A

the continued incorporation (15%) immediately resulted in a decreased thermal stability. The explanation could be that the addition of higher concentrations of CEO increased the

M

discontinuity of film matrix and thus resulted in the decreased compatibility of multiphased

ED

system, which brought the films with lower heat resistance (Noshirvani et al., 2017). On the other hand, films incorporated with CEO showed lower weight loss compared to those containing CLO, which might be related to the stronger interactions between CS and

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cinnamaldehyde (main components in CEO). The films with mixed EOs showed the balanced thermal stability compared to those with single EO. All films showed higher amount of residual

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mass (31.50%-35.62%) at 600°C except the one incorporated with CLO, which displayed lower residue compared to control film. This phenomenon could be ascribed to the increased plasticizing effect of CLO as the temperature rose, which hindered the intermolecular polymer

A

interactions and polymer-polymer interactions in film network (Hosseini, Rezaei, Zandi, & Farahmandghavi, 2016). As seen from DTG curves, the control film (Fig.6 B, a) exhibited three weight loss sections. However, four main sections were observed in the films containing EOs (Fig.6 B, b-f). The first one around 30-115°C was mainly attributed to the loss of free water and some volatile components in EOs. The second one between 115-200°C corresponded to the decomposition 19

of stable volatile/ non-volatile components in EOs (Feng et al., 2017; Xu et al., 2018). The third ozne, appeared between 200-274°C, was related to the degradation of great mass of GA macromolecules. The last with the major weight loss of about 46.20-51.31%, took place between 274-600°C. In this section, weight loss was mostly associated to the degradation of polymers (CS and GA), and the loss of structurally bound water in film matrix (Shen & Kamdem, 2015). Three peaks at 75°C, 245°C and 294°C in Fig.6 B, a presented the degradation

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of free water, GA and CS, respectively. The addition of EOs resulted in the presence of new peak at 165°C (Fig.6 B, b), corresponding to the decomposition of stable components in EOs

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(Xu et al., 2018).

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A

N

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A

A

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B

Fig.6. TG (A) and DTG (B) curves of CS-GA based films containing 0% CEO (a), 5%CEO (b), 10%CEO (c), 15%CEO (d), 10%CLO (e), and 5%CEO+5%CLO (f).

20

3.7 Release to food simulant

The release properties of antimicrobial compounds was a crucial feature related to the antimicrobial capability of films. Release of EOs in CS-GA based films were shown in Fig.7. Glycerol solution with 60% concentration was chosen as food simulant, simulating food system with the water activity of 0.6-0.7. Overall, all films presented initially rapid release and

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subsequently slower release till a constant level. The release profile illustrated concentration dependent: at lower concentration of EOs (5%), about 58.90% CEO was released at 40 min and

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about 99.42% at 300 min. However, about 40.68% from the films containing 15% CEO was

released at 40 min and reached the highest value at 500 min. The slower release of CEO for the films containing higher levels of EOs in liquid food simulant seemingly contradicted with the

U

results in atmosphere (Fig.4), but in reality this could be explained by the difference between

N

gas-solid balance and liquid-solid balance. Release of EOs depended on the concentration

A

gradient of mass transfer. For the films stored at atmosphere, the constant high concentration gradient acted as great driving force, promoting the fast release of EOs in films. However, for

M

the films emerged in food simulant, the release of EOs rendered the increasing concentration in

ED

the simulant, and thus reduced the concentration gradient between two phases, which made the diffusion of EOs to the simulant difficult. High levels of EOs led to the higher amount of EOs accumulating in simulant, then reduced the concentration gradient between film matrix and

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simulant, and finally contributed to the lower release from film network.

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Fig.7. Release properties of CS-GA based films containing CEO and CLO.

Compared to the films with 10% CEO, the films incorporated with 10% CLO showed slightly increased release rate, but the films with the combined EOs displayed the decreased release, indicating the synergistic effect occurred. The larger intermolecular force between CS and combined EOs might be the reason. As is well known, the slow release of EOs resulted in

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its controlled concentration on the surface of food, which will facilitate the antimicrobial

activity of films and prolong the shelf life of food (Xu et al., 2018). Furthermore, the rapid

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release in a short time (600 min) were observed for all films. Antimicrobial release is relevant to the characteristics of food, thus the substance migration in different simulation systems will make great distinction. The employment of 60% glycerol solution as the simulation brought

U

higher polarity, helping the penetration of EOs from polar CS/GA network, and finally leading

N

to the higher release rate. The gradient effect caused by slow stirring during the operation should

M

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3.8 Antimicrobial activity

A

also be mentioned (Xu et al., 2018).

The antimicrobial properties of films incorporated with EOs have been studied against Gram-negative bacteria (E.coli) and Gram-positive bacteria (S.aureus) by liquid culture test

PT

(Fig. 8). The OD values, which illustrated the growth of bacteria, significantly decreased with the increasing CEOs, indicating stronger antimicrobial activity was obtained for films.

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Furthermore, the OD reduction for the films containing CEO was higher than that containing CLO. Compared to the control film (0% CEO), 10% CEO films could reduce the OD values regarding to E.coli and S.aureus to 0.21 and 0.13 at 15 h, respectively, while films containing

A

10% CLO reduced to 0.26 and 0.14, respectively. The observations were in favor with previous study, who reported that the minimum inhibitory concentrations (MICs) of CEO and CLO against E.coli was 18 and 27 mg/L, respectively (Goñi et al., 2010). The well-documented antimicrobial activities of CEO and CLO could be attributed to the cinnamaldehyde (main active constituents of CEO) and eugenol (main active constituents of CLO), separately. The hydrophobic nature of them aided their abilities of disrupting and 22

destabilizing of bacteria membrane, leading to the cytoplasmatic leakage, cell lysis and eventually its destruction (Peng & Li, 2014; Wang et al., 2011). Dong, Xu, Ahmed, Li & Lin (2018) reported the synergistic effect occurred in combined EOs, which could explain the

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greater antimicrobial effects of the films incorporated with 5% CEO and 5% CLO.

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Fig.8. Antimicrobial activities of CS-GA based films containing CEO and CLO. A represents E.coli and

A

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B represents S.aureus.

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4. Conclusion

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The incorporation of CEO and CLO showed great influences on the properties of the FFDs and the resulted polyelectrolyte complexed films based on CS and GA. The incorporation of

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EOs led to the significant decrease in ζ-potential and apparent viscosity, whereas increased the particle size of FFDs. FFDs with combined EOs showed smaller particle size but no significant

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differences in ζ-potential and apparent viscosity compared to those containing single EOs. With the addition of EOs, films especially the films incorporated with CEO, exhibited lower tensile strength but higher elongation, better water barrier properties and thermal stability, which could

A

be attributed to their stronger interactions with CS matrix. Higher EO loss during storage was found with the increasing amount of EOs for the films, but the initial higher concentration incorporated in FFD rendered the slower release properties of films. In addition, films containing the combined EOs showed lower release rate of EO in simulant, which benefited the antimicrobial capabilities of the composite films against S.aureus and E.coli. It could be very promising to develop excellent CS-GA-based antimicrobial films incorporated with the 23

combined EOs (CEO and CLO). Further study will be focused on the effects of these films on the biochemical and physiological responses of fresh produce.

Acknowledgements

We would like to thank National Natural Science Foundation of China for funding this

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project (Grant no. 31801493). The project was also funded by the Priority Academic Program

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Development of Jiangsu Higher Education Institutions (PAPD). References

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