Fabrication of bio-nanocomposite films based on fish gelatin reinforced with chitosan nanoparticles

Accepted Manuscript Fabrication of bio-nanocomposite films based on fish gelatin reinforced with chitosan nanoparticles Seyed Fakhreddin Hosseini, Masoud Rezaei, Mojgan Zandi, Farhid Farahmandghavi PII:

S0268-005X(14)00302-6

DOI:

10.1016/j.foodhyd.2014.09.004

Reference:

FOOHYD 2710

To appear in:

Food Hydrocolloids

Received Date: 25 December 2013 Revised Date:

1 September 2014

Accepted Date: 1 September 2014

Please cite this article as: Hosseini, S.F., Rezaei, M., Zandi, M., Farahmandghavi, F., Fabrication of bionanocomposite films based on fish gelatin reinforced with chitosan nanoparticles, Food Hydrocolloids (2014), doi: 10.1016/j.foodhyd.2014.09.004. 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.

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FG-2%CSNPs

FG-6%CSNPs

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Fish gelatin film (FG)

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Graphical abstract

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Fabrication of bio-nanocomposite films based on fish gelatin reinforced with chitosan

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nanoparticles

3 Seyed Fakhreddin Hosseinia,*, Masoud Rezaeia, Mojgan Zandib, Farhid Farahmandghavic

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a

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P.O. Box 46414-356, Noor, Iran

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b

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Tehran, Iran

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Department of Seafood Processing, Faculty of Marine Sciences, Tarbiat Modares University,

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Box 14965/115, Tehran, Iran

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Department of Biomaterials, Iran Polymer and Petrochemical Institute, P.O. Box 14965/115,

Department of Novel Drug Delivery Systems, Iran Polymer and Petrochemical Institute, P.O.

12 Abstract

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The paper focuses on the synthesis of chitosan nanoparticles (CSNPs) by ionic gelation between

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chitosan (CS) and sodium tripolyphosphate (TPP) and subsequently its use as filler in a fish

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gelatin (FG) matrix to produce bio-nanocomposite films. The obtained particles exhibited a

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spherical shape with size range of 40-80 nm, and a positively charged surface with a zeta

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potential value of +10 mV. XRD results confirmed the cross-linking reaction between CS and

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TPP. SEM images showed that CSNPs could be well dispersed in FG polymer matrix at low

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content, while higher CSNPs loadings (8%, w/w) resulted in the aggregation of particles in the

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composites. FTIR spectroscopy results confirmed the interaction between CSNPs and FG

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through hydrogen bonding. The nucleating effect of the CSNPs was confirmed by DSC analysis.

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* Corresponding author. Tel.: +98 1144553101-3 ; fax: +98 1144553499 . E-mail addresses: [email protected], [email protected] (S. F. Hosseini)

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Results indicated that the addition of CSNPs caused remarkable increase in the tensile strength

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(TS) and elastic modulus (EM), which leading to stronger films as compared with individual FG

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films, but decreased the elongation at break (EAB). Furthermore, addition of CSNPs contributed

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to the significant decrease (p < 0.05) of water vapor permeability (WVP), leading to a 50%

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decline at 6% (w/w) filler. The light barrier measurements presented low values of transparency

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at 600 nm of the FG-based nanocomposite films, indicating that these films are very transparent

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(lower in transparency value) while they have excellent barrier properties against UV light. The

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results presented in this study show the feasibility of using bio-nanocomposite technology to

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improve the properties of biopolymer films based on FG.

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Keywords: Bio-nanocomposite, Edible films, Fish gelatin, Chitosan nanoparticles,

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Characterization

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35 1. Introduction

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Increasing and widespread environmental awareness, as well as efforts to reduce the volume

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flow of wastes and increase the use of renewable raw materials have placed emphasis on the

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disposal properties of different materials (Endres & Siebert-Raths, 2012). The non-degradable

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and non-renewable nature of plastic packaging has led to a renewed interest in packaging

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materials based on biopolymers derived from renewable sources. The use of biopolymer-based

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packaging materials can solve the waste disposal problem to a certain extent (Kumar, Sandeep,

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Alavi, Truong, & Gorga, 2010). The increscent interest in biopolymer based packaging has

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resulted in the development of protein-based films from soy protein, whey protein, casein,

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collagen, corn zein, gelatin, and wheat gluten (Cuq, Gontard, & Guilbert, 1998). Among all the

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protein sources, gelatin has also been extensively studied for its film forming capacity and

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applicability as an outer covering to protect food against drying, light and oxygen (Gómez-

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Guillén et al., 2009).

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Fish gelatin (FG) has gained great interest in recent years as the demand for non-bovine and non-

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porcine gelatin has increased, due to religious and social reasons, and also the bovine spongiform

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encephalopathy (BSE) crisis (Bae et al., 2009). Furthermore, fish skin, which is a major

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byproduct of the fish-processing industry, causing waste and environmental pollution, could

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provide a valuable source of gelatin (Badii & Howell, 2006). The elaboration of edible films

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from fish gelatin has been recently studied (Gómez-Estaca, Gómez-Guillén, Fernández-Martín,

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& Montero, 2011; Hosseini, Rezaei, Zandi, & Farahmandghavi, 2013; Núñez-Flores et al., 2012;

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Nur Hanani, Roos, & Kerry, 2012).

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However, these biodegradable fish gelatin films do have some limitations in their use, such as

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low tensile strength (TS) and high water solubility (Gómez-Estaca et al., 2011). In order to

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improve the mechanical property as well as barrier characteristic of gelatin films, recently, a new

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class of materials namely bio-nanocomposites (biopolymer matrix reinforced with nanoparticles)

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has introduced as a promising option (Bae et al., 2009). Fillers with at least one nano-sized

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dimension (nanofillers or nanoreinforcements) have better interfacial adhesion with the polymer

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matrices, when compared to the respective micro/macroscopic reinforcements. A uniform

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dispersion of nanofillers leads to a very large matrix/filler interfacial area, changing the

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molecular mobility, improve the relaxation behavior, and the consequent thermal and mechanical

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properties of the resulting nanocomposite (Ludueña, Alvarez, & Vasquez, 2007). Nanocomposite

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technology using nanofillers such as carbon nanotubes (CNTs) (Ma, Yu, & Wang, 2008),

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nanoclay (Bae et al., 2009; Casariego et al., 2009), and nanosilica (Ahmed, Varshney, & Auras,

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2010) has already proved to be an effective way to improve the mechanical, physical, and

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thermal properties of polymers. Newly, considering of the applications for edible films and/or

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food packing, much attention has been focused on polysaccharide nanofillers. Chitosan (CS) is a

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naturally occurring nontoxic, biocompatible, biodegradable, and cationic polysaccharide

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(Shahidi, Arachchi, & Jeon, 1999).

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Chitosan nanoparticles (CSNPs) which are composed of a natural material with excellent

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physicochemical properties, is environmentally friendly, and bioactive (Yang, Wang, Huang, &

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Hon, 2010). CSNPs can be prepared by the electrostatic interaction and resultant ionotropic

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gelation between CS polycation and sodium tripolyphosphate (TPP) polyanion (Calvo,

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Remũnán-López, & Vila-Jato Alonso, 1997; Yang et al., 2010). Using of these nanoparticles in

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edible films would be very promising, due to the food-grade properties of both components. De

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Moura et al. (2009) found that CS-TPP nanoparticles increases thermal and mechanical

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properties and decrease water vapor permeability of the hydroxypropyl methylcellulose (HPMC)

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films. In another study, these polysaccharide nanoparticles have been used as the reinforcing

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medium in glycerol plasticised-starch (GPS) matrices (Chang, Jian, Yu, & Ma, 2010). They have

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obtained an improvement of thermal stability, mechanical and barrier properties of GPS

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composites. More recently, Martelli, Barros, De Moura, Mattoso, and Assis (2013) was also

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reported that the incorporation of chitosan nanoparticles promoted noticeable improvement of

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the mechanical properties and acted in reducing the water vapor permeation rate in banana puree

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films.

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Hence, bio-nanocomposite films based on FG and CSNPs could be a good candidate for food

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packaging applications to extent the shelf life of foods and products. This research focused on

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fabrication and characterization of CSNPs as well as evaluation of the effects of incorporation of

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obtained nanoparticles on morphology, mechanical properties, water vapor permeability, light

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barrier properties, and thermal behavior of FG films.

94 2. Materials and methods

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2.1. Materials

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Gelatin from cold water fish skin, chitosan (CS) (medium molecular weight, 75-85%

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deacetylated) and sodium tripolyphosphate (TPP) were purchased from Sigma-Aldrich (St.

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Louis, MO, USA). Glycerol (analytical grade) and acetic acid were purchased from Merck

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Chemicals Co. (Darmstadt, Germany).

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2.2. Preparation of chitosan nanoparticles (CSNPs)

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CSNPs were prepared based on the ionotropic gelation between CS and TPP with some

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modifications (Calvo et al., 1997). CS solution (1% (w/v)) was prepared by agitating chitosan in

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an aqueous acetic acid solution (1% (v/v)) at ambient temperature (23-25 °C) overnight. The

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mixture was then centrifuged at 9000 rpm for 30 min; the supernatant was separated and filtered

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through 1 µm pore size filters. TPP solution (0.4% (w/v)) was added (weight ratios of CS: TPP

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was 2.5:1) drop wise to CS solution under vigorous stirring for 40 min. The formed particles

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were collected by centrifugation at 9000 rpm for 30 min at 4 °C, and subsequently washed

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several times with deionized water. Finally, ultrasonication (50 w) was applied by a sonicatior

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(Bandelin sonopuls HD3200, KE 76 probe, Germany) in an ice bath for 4 min with a sequence of

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0.7 s of sonication and 0.3 s of rest, resulting in a homogeneous suspension. The suspensions

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were immediately lyophilized at -35 °C for 72 h using freeze dryer (GAMMA 1-16 LSC, UK).

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2.3. Bio-nanocomposite film preparation

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The gelatin films were prepared according to the method described by Gómez-Estaca et al.

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(2011) with slight modifications. The FG solution was prepared by dissolving 4 g gelatin in 100

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ml distilled water for 30 min and then heated at 45 °C for 30 min under continuous stirring.

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Glycerol (0.3 g/g gelatin) was added as a plasticizer and solutions were again warmed and stirred

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at 45 °C for 15 min. For the preparation of bio-nanocomposite film forming dispersions,

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different levels of CSNPs (0, 2, 4, 6 and 8 %, w/w), based on dry FG) was first dispersed into

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distilled water (50 ml) and then sonicated for 10 min. Then, the CSNPs suspension were added to

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FG solution (50 ml) drop wise and gently stirred for 60 min. The film-forming dispersions were

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degassed under vacuum for 15 min to remove air bubbles. Finally, aliquots of 100 g of film-

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forming dispersions were poured in rectangular plastic dishes (24 × 12 cm) and dried at ambient

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temperature (23-25 °C) for 3 days. Dried films were peeled from the plate and conditioned at 25

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°C and a relative humidity (RH) of 50 ± 4% RH for 48 h for further analysis.

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2.4. Characterization of CSNPs and bio-nanocomposite films

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2.4.1. Atomic force microscopy (AFM)

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Atomic force microscope (DualScopeTM DS95-50, DME, Denmark) was used for

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morphological characterization and nanoparticles size determination. A drop of diluted

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nanoparticle suspension (0.05 mg/ml) was spread on the clean glass surface, and dried at room

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temperature. The image measurement was performed in tapping mode using silicon probe

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cantilever of 230 micron length, resonance frequency of 150-190 kHz, spring constant of 20-60

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N/m and nominal, 5-10 nm tip radius of curvature. The scan rate was used as 1 Hz.

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2.4.2. Particle size and zeta potential

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The mean particle size, particle size distribution and zeta potential of the nanoparticle suspension

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were obtained using a Zetasizer Nano ZS 3300 (Malvern Instruments Ltd., United Kingdom) on

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the basis of dynamic light scattering (DLS) technique. Diluted samples were placed in glass

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cuvette with square aperture and the scatter intensity was measured at 25 °C.

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143 2.4.3. X-ray diffraction

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XRD patterns of CS powder and CSNPs were recorded over a 2θ range of 5-50° using an X-ray

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diffractometer (Siemens, model D5000) with a step angle of 0.04°/min.

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2.4.4. Fourier transform infrared (FTIR) spectroscopy

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FTIR spectra of CS powder, CSNPs and bio-nanocomposite films were recorded from wave

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number 400-4000 cm-1 by a Bruker Equinox 55 spectrometer (Bruker Banner Lane, Coventry,

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Germany). For CS powder and CSNPs, samples were prepared using KBr to form pellets. For

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each spectrum, 16 scans at a resolution of 4 cm−1 were obtained.

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2.4.5. Scanning electron microscopy (SEM)

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The morphology of the film samples was investigated by scanning electron microscopy (SEM)

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(VEGA II, TESCAN, Czech Republic) at an accelerating voltage from10 to 20 kV. Strips of dry

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films (obtained by maintaining in a desiccator with silica gel for 2 week) were immersed in

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liquid nitrogen and cryo-fractured manually. The specimens were stuck onto a cylindrical

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aluminum stub by a double-sided tape. Afterwards the film samples were sputtered with a thin

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layer of gold in an ion sputter coater (K-450X, EMITECH, England) and placed into SEM

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chamber to observe the surface and the cross-section of the films.

162 2.4.6. Differential scanning calorimetry (DSC) analysis

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Thermal properties of films were determined using a differential scanning calorimeter (DSC,

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Model 200-F3 Maia, Netzsch, Germany). Before analysing, films were conditioned in a

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desiccators containing dried silica gel for 2 weeks at room temperature (23-25 °C) to obtain

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dehydrated films. Aliquots of approximately 10 mg pre-dried samples were placed into

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aluminium pans, sealed and scanned over the range of 25 to 350°C with a heating rate of 10

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°C/min. The empty aluminum pan was used as a reference.

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170 2.4.7. Film thickness

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Thickness of the films was determined using a micrometer (Mitutoyo Manufacturing Co. Ltd.,

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Tokyo, Japan) to the nearest 0.001 mm at 9 random positions around the film, and average

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values were used in calculations.

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2.4.8. Mechanical properties

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A universal testing machine (SMT-20, Santam, Tehran, Iran) equipped with a 60 N load-cell was

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used to measure tensile strength (TS), elongation at break (EAB) and elastic modulus (EM)

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according to the ASTM standard method D 882-09 (2009). Films strips of 110 × 20 mm were

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conditioned at 23 ± 2 ºC and 53 ± 2% relative humidity for 48 h in an environmental chamber

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before testing. Initial grip separation and mechanical crosshead speed were set at 50 mm and 5

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mm/min, respectively. Each type of film was tested by at least five replicates.

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2.4.9. Water vapor permeability (WVP)

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The water vapor permeability of the films was measured gravimetrically according to the ASTM

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E96-05 method (2005) and adapted to hydrophilic edible films by McHugh, Avena-Bustillos and

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Krochta (1993). Circular test cups were made of glass with 49 mm internal diameter and 1.1 cm

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height. Films without pinholes or any defects were sealed to the cup mouth containing 6 ml

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distilled water (100% RH; 2.337 × 103 Pa vapor pressure at 20 °C), placed in a desiccators at 20

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°C and 0% RH (0 Pa water vapor pressure) containing silica gel. The water transferred through

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the film and adsorbed by the desiccant was determined from the weight loss of the permeation

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cell. The films were allowed to equilibrate for 2 h before the cells were initially weighed. The

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cells were weighted at intervals of 2 h during 10 h with an analytical balance (±0.0001g). The

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slope of weight loss versus time was obtained by a linear regression (r2 ≥ 0.99). The measured

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WVP of the films was determined using Eq. (1):

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WVP =

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Where WVTR is the water vapor transmission rate (g mm/kPa h m2) through a film, calculated

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from the slope of the straight line divided by the exposed film area (m2), L is the mean film

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thickness (mm), and ∆P is the partial water vapor pressure difference (kPa) across the two sides

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of the film. Three replicates of each film were tested.

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2.4.10. Water solubility

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Water solubility of the films was measured according to the method of Gontard, Guilbert and

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Cuq (1992). Three pieces (1 × 4 cm2) of the films were weighed (±0.0001 g) and subsequently

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dried in an air-circulating oven at 105 ºC for 24 h. After this time, films were recovered and re-

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weighed (±0.0001 g) to determine their initial dry weight (Wi). Afterwards, the samples were 9

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immersed in 30 ml of distilled water and the system gently shaken (100 rpm) for 24 h at room

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temperature (22-25 ºC). The samples were then passed through a weighed filter paper (Whatman

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1). The filter paper together with unsolubilized fraction was dried in a forced-air oven (105 ºC,

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24 h) and weighed (Wf). The film solubility (FS %) was calculated using Eq. (2):

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FS% =

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Wi= initial dry film weight (g)

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Wf= final dry film weight (g)

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213 2.4.11. Light transmission and transparency

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The barrier properties of the films against ultraviolet (UV) and visible light were measured at

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selected wavelength between 200 and 800 nm, using a UV-1650 spectrophotometer (Model PC,

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Shimadzu, Kyoto, Japan). Film strips of 10 × 40 mm were placed in a spectrophotometer test

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cell. An empty test cell was used as the reference. The measurement was done in triplicate and

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the average of three spectra was calculated. Transparency of the films was calculated as in the

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following equation (Han & Floros, 1997):

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Transparency = A600/x

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where A600 is the absorbance at 600 nm, and x is the film thickness (mm). The greater

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transparency value represents the lower transparency.

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2.4.12. Statistical analysis

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The statistical analysis of the data were performed using analysis of variance (ANOVA) and the

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differences between means were evaluated by least significant difference multiple range test at p

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≤ 0.05. SPSS statistic program (SPSS 17.0 for window, SPSS Inc., Chicago, IL, USA) was used

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for data analysis.

230 3. Results and discussion

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3.1. Characterization of CSNPs

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AFM imaging is an effective method to provide the surface morphology and accurate size and

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size distribution of the particles. AFM images confirmed the spherical shape and nanosize

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structure of CSNPs (Fig. 1a). The size distribution obtained by AFM indicated that most of

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CSNPs were distributed in the range of 40-80 nm (Fig. 1b). However, the size of the CSNPs is

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smaller than those obtained by De Moura et al. (2009), using the same process but different CS

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and TPP concentration, describing particles with size range of 85-221 nm. These authors

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reported that the nanoparticles with lowest particle size (85 nm) were obtained when the lowest

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CS and TPP concentration was used. Chang et al. (2010) observed when commercial CS was

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used to prepare CSNPs at a concentration of 1% (w/v) of CS and TPP, the particle size

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distribution was in the range of 50-100 nm. The effect of CS/TPP ratio on particle size was

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previously studied. Calvo et al. (1997) reported that the particle size is dependent on both CS and

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TPP concentrations.

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Dynamic light scattering (DLS) technique was also applied to investigate the mean particle size

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and particle size distribution of the nanoparticles. Fig. 2a shows a typical size distribution profile

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of the nanoparticles with a mean diameter of 259 ± 17.9 nm in a narrow size distribution

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(polydispersity index <1). The size measured in hydrated state is reported to be slightly higher

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than the size measured in dried state (Yoksan et al., 2010). It should be pointed out that the

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particle size determined by DLS technique is a hydrodynamic diameter. The larger diameter thus

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might be a result of the swelling of the chitosan layer surrounding the individual particles, and/or

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the aggregation of single particles while dispersed in water (Yoksan et al., 2010).

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Particle aggregation can be prevented by electrostatic repulsions according to DLVO theory

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(Derjaguin and Landau, 1993; Verwey and Overbeek, 1948). Electrostatic repulsion between

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particles depends on the value of zeta potential. The higher the zeta potential, the stronger the

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repulsion, the more stable the system becomes. CSNPs showed zeta potential values about +10

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mV (Fig. 2b). The positive zeta potential found on the surface of the particles is due to the

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cationic characteristics of the chitosan chains (Lorevice, De Moura, Aouada, & Mattoso, 2012).

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Dudhani and Kosaraju (2010), and Liu et al. (2011), also reported the positively charged surface

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of chitosan-TPP particles. Lorevice et al. (2012) showed that the zeta potential of chitosan

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nanoparticles can vary between 14.50 to 37.58 mV, depending on chitosan concentrations used.

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The XRD patterns of CS powder and CSNPs are presented in Fig. 3. CS exhibits one

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characteristic peak at 2θ of 25° (Fig. 3a), indicating the high degree of crystallinity (Ali,

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Rajendran, & Joshi, 2011; Jingou et al., 2011). After ionic cross-linking with TPP, no peak is

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found in the diffractogram of chitosan nanoparticles, reflecting the destruction of the native

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chitosan packing structure (Yoksan et al., 2010) (Fig. 3b). It is well-known that the width of X-

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ray diffraction peak is related to the size of crystallite, the broadened peak usually results from

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imperfect crystal (Jingou et al., 2011). So the broad peak of CSNPs may be caused by the cross-

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linking reaction between CS and TPP, which may destroy the crystalline structure of CS

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(Rokhade et al., 2006).

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The FTIR experiments of CS and CSNPs were performed to evaluate the chemical structure of

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nanoparticles. Fig. 4 shows FTIR spectra of CS powder and CSNPs. In general, CS powder

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shows characteristic peaks at 3433 (-OH and -NH2 stretching), 2920 (-CH stretching), 1647

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(amide I), 1088 (C-O-C asymmetric stretching) and 591 cm-1 (pyranoside ring stretching

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vibration) (Fig. 4a). The sharp peaks at 1380 cm−1 and 1419 cm−1 can be assigned to CH3

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symmetrical stretching band (Dudhani & Kosaraju, 2010). After formation of CSNPs (Fig. 4b),

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the peak of amide I (-NH2 bending) was shifted from 1647 to 1651cm-1, and a new peak

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appeared at 1555cm-1 (amide II), implying the complex formation via electrostatic interaction

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between NH3+ groups of CS and polyphosphoric groups of TPP to enhance both the

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intermolecular and intramolecular interaction in CSNPs (Jingou et al., 2011; Qi, Xu, Jiang, Hu,

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& Zou, 2004; Yoksan et al., 2010 Dudhani & Kosaraju, 2010; Jingou et al., 2011). Moreover, the

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shoulder at 1258 cm−1 reduced dramatically or disappeared and a new peak was formed at 1238

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cm−1 (C-O-C stretch).

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284 3.2. Film microstructure

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Permeability of films can be affected by the structure, morphology, and homogeneity of the

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matrix (McHugh & Krochta, 1994). SEM micrographs of the surfaces of neat FG and prepared

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nanocomposite films are shown in Fig. 5. Pure FG film displays smooth and homogeneous

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surfaces, without pores and with excellent structural integrity (Fig. 5a). Different surface

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morphology was observed after CSNPs was added into FG films. At low CSNPs content (2%,

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w/w), most of CSNPs are dispersed uniformly in the FG matrix without obvious aggregation

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(Fig. 5b). As illustrated in Fig. 5c, when 6% (w/w) CSNPs was added to the FG matrix, slightly

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aggregation was observed, whereas at high filler content (i.e. 8%, w/w) agglomeration of CSNPs

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was obvious (Fig. 5d). Nevertheless, in comparison with neat fish gelatin film, relatively dense

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structure was observed after adding 2, 6 and 8% CSNPs. This explained by the improved

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mechanical and barrier properties of the nanocomposite films due to good bonding strength

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between nanoparticles and polymer. Similar observation was reported by Chang et al. (2010) that

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worked with starch-chitosan nanoparticles composites.

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Fig. 6 represents the cross-section morphology of FG and FG-based nanocomposite films. It is

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clear that addition of CSNPs caused changes in the film microstructure, since the non-reinforced

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films (Fig. 6a) exhibited a smooth and continuous surface, as expected for a homogeneous

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material. SEM micrographs obtained from the samples containing 2% (w/w) CSNPs showed a

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good distribution of particles in the FG matrix, without aggregates formation (Fig. 6b). However,

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addition of 6% (w/w) CSNPs led to a rough surface, with a small aggregates of particles in which

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is possible to see (Fig. 6c). In the case of FG films with 8% (w/w) CSNPs, obvious aggregation

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was observed in part of the film matrix (Fig. 6d). The similar microstructure has been reported in

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alginate film incorporated with nanocrystalline cellulose (Huq et al., 2012).

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308 3.3. FTIR analysis

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the structure of the FG matrix, and the results are shown in Fig. 7. The spectrum of FG film (Fig.

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7a) showed characteristic bands at approximately 3291 cm-1 (amide-A, N-H stretching), 1679

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cm-1 (amide-I, C=O stretching), 1540 cm-1 (amide-II, N-H bending) and 1242 cm-1(amide-III, C-

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N and N-H stretching) (Pereda, Ponce, Marcovich, Ruseckaite, & Martucci, 2011). The

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characteristic peak appeared at 2935 cm-1 corresponds to the C-H stretching vibration (Kanmani

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& Rhim, 2014). The band situated at the wavenumber of ~1046 cm-1 was found in all film

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samples, corresponding to the glycerol (-OH group) added as a plasticizer (Bergo & Sobral,

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2007). The FTIR peaks of FG-based nanocomposite films are relatively similar to those of FG

320

film as control. As it could be observed, some of the peaks are shifted to higher or lower wave

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numbers with increasing CSNPs loading. For example, the peaks of amide-A and amide-II

322

shifted to higher wavenumbers as shown in Fig.7 b-d, indicating a possible formation of

323

hydrogen bonding between nanoparticles and polymer matrix (Khan et al., 2012). The shift of

324

the amide-A band indicated that the hydrophilicity of gelatin was reduced, because the free -OH

325

group of gelatin was involved in hydrogen bonding association with CSNPs and less susceptible

326

to hydration (Le Tien et al., 2000). In gelatin -OH, the -COOH end groups and -NH2, and the

327

side groups are capable of forming hydrogen bonds with the -OH and the -NH2 groups of

328

chitosan (Sionkowska, Wisniewski, Skopinska, Kennedy, & Wess, 2004). Khan et al. (2012)

329

reported the fabrication of chitosan based nanocomposite films reinforced with varying

330

concentrations of nanocrystalline cellulose (NCC) (1-10%, w/w). These authors represented that

331

the shift of amide-A peak to higher wavenumber was associated with formation of hydrogen

332

bonding between chitosan and NCC. In another study, the shift of amide-II peak in gelatin/clay

333

composite films to higher wavenumber was also attributed to the strong interaction between

334

nanoclay and polymer matrix through the hydrogen bonding (Kanmani & Rhim, 2014).

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3.4. Thermal properties

337

DSC thermograms of the pure FG and fabricated nanocomposites, are shown in Fig. 8. Melting

338

points (Tm) of the samples were evaluated from the position of the maximum in the endothermic

339

peaks of DSC thermograms. As shown in Fig. 8a, FG exhibited two sharp endothermic peaks at

340

243.5 ºC and 264.9 ºC, implying Tm of the material possessing two main different crystal

341

structures. The crystal melted at lower temperature might be assigned to the devitrification of

342

blocks rich in α-amino acids; while the one melted at higher temperature might be attributed to

343

the devitrification of blocks rich in imino acids such as proline and hydroxyproline (Zheng, Li,

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Ma, & Yao, 2002). By addition of 2 and 4% (w/w) CSNPs, melting peaks was alleviated and

345

shifted to lower temperatures (Fig. 8b and c). The temperature position of the melting peaks

346

decreased from 239.7 to 237.5 ºC (first endothermic peak) and 258.8 to 255.8 ºC (second

347

endothermic peak) as CSNPs content increased from 2 to 4% (w/w), respectively. The lower Tm

348

observed in the heating run can be an indication of faster crystallization induced by CSNPs

349

which act as nucleating agents for FG. CSNPs allow heterogeneous nucleation mechanism which

350

induces a decrease of the free energy barrier and fastens the crystallization. This observation is in

351

agreement with the results of Frone, Berlioz, Chailan, and Panaitescu (2013) related to the effect

352

of nucleating agents of cellulose nanofibers on the thermal properties of polylactic acid (PLA)

353

composites. However, by increasing of CSNPs contents (i.e. 6 and 8%, w/w), only a sharp

354

endothermic peak was observed (Fig. 8d and e), reflecting that the obtained bio-nanocomposite

355

films possessed sole-structured crystals.

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3.5. Film thickness

358

Film thickness is a crucial parameter in calculation of mechanical properties and water vapor

359

permeability values. Final thickness strongly depends on preparation method and drying

360

conditions. The film thickness increased from 50.5 ± 2.8 µm for the control FG film to 64.8 ± 3.3

361

µm for bio-nanocomposite film with 8% (w/w) CSNPs (Table 1). This behavior could be as a

362

result of the increase in dry matter content. Those values are lower than those presented in the

363

literature for nanocomposite films based on fish gelatin and montmorillonite (MMT) nanoclay

364

(Jeya Shakila, Jeevithan, Varatharajakumar, Jeyasekaran, & Sukumar, 2012). Moreover, the

365

values were lower than those reported by Pereda et al. (2011) for chitosan-gelatin composite

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366

films with thickness of 100 ± 6 µm. This was due to the differences in film forming solution

367

formulations and in the used film-making procedures.

368 3.6. Mechanical properties of films

370

It is highly desirable that an edible film maintains its integrity during processing, shipping, and

371

handling. Mechanical properties are important in edible films, because adequate mechanical

372

strength ensures the integrity of the film and its freedom from minor defects such as pinholes

373

(Murillo-Martínez, Pedroza-Islas, Lobato-Calleros, Martínez-Ferez, & Vernon-Carter, 2011).

374

Representative stress-strain curves of the films are shown in Fig. 9. These curves exhibited the

375

typical plastic deformation behavior; at low strains (< 10%) the stress increased rapidly with an

376

increase in the strain and the slopes were in the elastic region defining the elastic modulus. At

377

strains >10%, the stress decreased slowly until failure occurred.

378

Table 1 shows the mechanical properties of the films, in terms of tensile strength (TS),

379

elongation at break (EAB), and elastic modulus (EM). The TS of pure FG film was 7.44 ± 0.17

380

MPa, which increased to a maximum value of 11.28 ± 1.02 MPa when the CSNPs content was

381

8% (w/w) (Table 1). However, the increase was not significant (p >0.05) below 4% (w/w)

382

CSNPs content. Addition of CSNPs resulted in a stiffer material confirming the reinforcing

383

effect of the nanoparticle in the polymeric matrix, which is consistent with previously reported

384

results (Chang et al., 2010; De Moura et al., 2009). In these ways, Chang et al. (2010) reported

385

that the addition of CSNPs to glycerol plasticised-starch matrix results in improvement of TS in

386

the films. Compared with CSNPs, addition of chitosan into walleye pollock-skin gelatin was

387

found to have obvious effect of improving the TS property of resultant materials (Liu et al.,

388

2011). The TS of packaging film must be more than 3.5 MPa, according to conventional

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standards (Kim, Lee, & Park, 1995). Thus, the value of over 11 MPa for FG-CSNPs (8%, w/w)

390

film is a super value for its use as a disposable packaging film. For elongation at break (EAB), a

391

decreasing trend was found. The EAB value decreased from 102.04 ± 28.38% to 32.73 ± 7.38%

392

by increasing in the content of CSNPs. Decrease in EAB is an indication of an increase in the

393

brittleness of the gelatin films. A decrease in elongation of fish gelatin by incorporation of

394

chitosan was also observed by Gómez-Estaca et al. (2011). This has been reported to be also a

395

result of decreases in the free volume between polymer chains caused by an increase in

396

intermolecular attractive forces, making the polymer network high dense and thus less

397

permeable. Therefore, direct interaction and proximity between protein chains gets reduced,

398

which led to the decrease of EAB (Jeya Shakila et al., 2012). Elastic modulus (EM), a measure

399

of intrinsic film stiffness, is the slope of the linear range of the stress-strain plot (Mauer, Smith,

400

& Labuza, 2000). As can be seen from Table 1, the EM of the control FG film was 287.03 ±

401

14.25 MPa. With increasing content of CSNPs, the EM was found to be elevated from 287.03 ±

402

14.25 to 467.2 ± 49.63 MPa, which was an improvement by ~60%. Similarly, De Moura et al.

403

(2009) reported that the blending of CS-TPP nanoparticles with HPMC films exhibited increased

404

EM of the films. Moreover, EM values of the nanocomposite films in the present study were

405

higher than those for chitosan incorporating grouper bone gelatin films, which have been

406

reported 378 MPa (Jeya Shakila et al., 2012). Increased elastic modulus can significantly be

407

serviceable in the food packaging application.

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3.7. Water solubility

410

Solubility is an important property of edible films as they are used as protective layers on food.

411

Potential applications may require water insolubility to enhance product integrity and water

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resistance (De Moura, Lorevice, Mattoso, & Zucolotto, 2011). As can be seen in Table 1, the

413

solubility of pure FG films in distilled water was around 72%, that was in accordance with the

414

values reported by Jiang, Liu, Du, and Wang (2010) for films elaborated from catfish skin gelatin

415

and were considerably lower than those reported for cod skin gelatin films of around 20%

416

(Núñez-Flores et al., 2012). By addition of CSNPs to the film matrix, the observed decrease in

417

water solubility can be attributed to the formation of strong hydrogen bonds between gelatin

418

matrix and the nanoparticles, which confirmed by FTIR results. Water molecules are not able to

419

break this bond sufficiently hence the solubility of FG-CSNPs film with 6% (w/w) CSNPs was

420

the lowest (Table 1). Our results are comparable to a previous report by De Moura et al. (2011)

421

who have reported a reduction in the water solubility on addition of chitosan nanoparticles to the

422

methyl cellulose films. Voon, Bhat, Easa, Liong, and Karim (2012) also found that the addition

423

of the nanoclay in bovine gelatin film matrix decreased the water solubility, and they explained

424

that this could be due to the formation of strong hydrogen bonds between gelatin matrix and the

425

nanoparticles. In comparison with CSNPs effect on FG film in the present study, the water

426

solubility of tuna-skin gelatin film was significantly reduced by adding chitosan biopolymer

427

(Gómez-Estaca et al., 2011). This improvement was attributed to the formation of polyelectrolyte

428

complexes (PECs) between the ammonium groups of chitosan and the negatively charged side-

429

chain groups in the gelatin (i.e. carboxylate groups) through electrostatic interaction (Yin, Li,

430

Sun, & Yao, 2005).

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3.8. Water Vapor Permeability (WVP)

433

The problem in composite films used in food industry is the relatively high-water vapor

434

permeability of the edible films. Permeability in films is controlled by the diffusivity and

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solubility of water molecules within the film matrix (Gontard et al., 1992). Thus using

436

nanoscience, new forms of tightly linked three dimensional networks can be developed to

437

prevent migration of water in food products (De Moura et al., 2009). The effect of nanoparticle

438

contents on water vapor permeability (WVP) of FG films is given in Table 1. The WVP of the

439

control FG film with no particle inclusions was 1.421 ± 0.087 g mm/ kPa h m2, which is

440

comparable to those for films based on fish gelatin as reported in literature (Avena-Bustillos et

441

al., 2006; Ninan, Joseph, & Abubacker, 2010). The addition of CSNPs significantly decreased

442

the WVP of FG film (Table 1), as this decline for 6% (w/w) CSNPs has been of about 50% (p <

443

0.05). According to Chang et al. (2010), the addition of CSNPs produces a tortuous path that

444

hinders the passage of water molecules through the film matrix. Again, the decrease in WVP of

445

films could be attributed to the restricted mobility of protein molecules due to the incorporation

446

of nanoparticles and its interaction with gelatin (Vanin et al., 2014). On the other hand, De

447

Moura et al. (2009) reported that formation of hydrogen bonding between CS-TPP nanoparticles

448

and the HPMC film matrix decreased the WVP of films to a considerable extent. In another

449

study, the effectiveness of the reinforcing effect of chitosan nanoparticles in banana puree films

450

was described by Martelli et al. (2013). The results from this study demonstrated that the

451

incorporation of nanoparticles acted in reducing the water vapor permeation rate, which has been

452

attributed to the diffusion of chitosan nanoparticles during solvent evaporation and filling empty

453

spaces in the film matrix. When more than 6% (w/w) CSNPs was added to the FG matrix, WVP

454

values slightly increased. This trend can be explained to this way that at low levels, CSNPs could

455

disperse well in the FG matrix (as shown by SEM), thereby blocking the water vapor. However,

456

as illustrated by SEM micrographs, additional CSNPs (i.e. 8%, w/w) aggregated easily, which

457

actually decreased the effective content of CSNPs and facilitated water vapor permeation.

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458

Similar effects on the water vapor behavior of glycerol plasticised-starch/CSNPs composites

459

have been reported by Chang et al. (2010).

460 3.9. Light transmission and transparency

462

Transparency of films is greatly relevant to the films functionality due to their great impact on

463

the appearance of the products (Bilbao-Sainz, Bras, Williams, Sénechal, & Orts, 2011). Usually,

464

higher transparency is considered to be desirable for food packaging films and coatings, since

465

consumers prefer to see foods. Light transmission and transparency of control FG film and FG-

466

based nanocomposite films at selected wavelengths are shown in Table 2. The transmission of

467

UV light is found very low at 200-280 nm for all the films. This result is in agreement with

468

previous reports on fish gelatin based films (Jiang et al., 2010; Hosseini et al., 2013). According

469

to Aitken and Learmonth (2000), barrier properties of proteins against UV radiation are

470

associated with the presence of UV-absorbing chromophore, especially aromatic amino acids-

471

tyrosine and tryptophan and in a less extent, phenylalanine and disulfide bonds. Moreover, the

472

addition of CSNPs led to the decline of light transmission in the UV region; depend on the

473

amount of particles added as shown in Table 2. Since CSNPs are not dissolved in the film

474

solution, its addition to the film matrix causes a decrease in the transparency of the films. As a

475

consequence the opaque appearance of the FG-CSNPs composite films reflects the UV light,

476

thereby hinders light transmission through the films. Therefore, FG-CSNPs films have excellent

477

barrier properties against UV light, which induces lipid oxidation in the food system (Coupland

478

& McClements, 1996). When comparing bio-nanocomposite prepared films with composite

479

films based on fish gelatin and chitosan (Hosseini et al., 2013), the better light barrier properties

480

of FG films containing CSNPs than those containing chitosan have been found. As shown in

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Table 2, in visible range (350-800 nm), all bio-nanocomposite films showed the lower light

482

transmission than the control. As a result of the increased light blocking properties (as discussed

483

above), films with CSNPs also had lower transparency values than pure gelatin films (Table 2).

484

The transparency of the FG-based films decreased significantly (p < 0.05) when the film was

485

blended with the CSNPs. This decrease was found to depend on the amount of CSNPs added as

486

shown in Table 2. The reason that the films lost transparency when adding the CSNPs at higher

487

contents (i.e. 8%, w/w) might be due to the aggregation of nanoparticles which, in turn, obstruct

488

the transmission of light. The effect of CSNPs on reducing the transparency of FG films is

489

similar to the effect of cellulose nanoparticles on transparency of HPMC films (Bilbao-Sainz et

490

al., 2011).

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491 4. Conclusions

493

In this research, bio-nanocomposite films based on FG and CSNPs have been successfully

494

developed. The particles were spherical in shape with size range 40-80 nm. The incorporation of

495

CSNPs to FG films improved their water vapor barrier, as well as TS and elastic modulus,

496

indicating that the nanoparticles improve the film applicability as edible packaging. SEM images

497

revealed that CSNPs was dispersed evenly in the FG matrix at lower loading levels. These

498

findings indicate that use of nanotechnology can improve functionality to edible films for food

499

applications. However, relatively high WVP and film solubility values of these films as

500

compared to those of LDPE and PVDC might limit the application of these bio-nanocomposite

501

films to packaging of high moisture foods. Hence, there is a need to further improve the

502

properties of these nanocomposite films for commercial application.

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Acknowledgement

505

Authors would like to thank the Iran National Science Foundation (INSF) (Project No.

506

90000234) and Iran Nanotechnology Initiative council (INIC) for financial support of this

507

research.

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& Gómez-Guillén, M. C. (2012). Role of lignosulphonate in properties of fish gelatin films.

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ACCEPTED MANUSCRIPT Figure captions Fig 1. (a) AFM images (3D) and (b) size distribution obtained from the height of CSNPs. Fig 2. (a) Size distribution and (b) zeta potential distribution of CSNPs determined by DLS technique.

Fig 4. FTIR spectra of (a) CS powder and (b) CSNPs.

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Fig 3. XRD patterns of (a) CS powder and (b) CSNPs.

Fig 5. SEM micrographs of the surface of (a) FG and FG-based nanocomposite films containing (b) 2%, (c) 6%, and (d) 8% (w/w) CSNPs content.

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Fig 6. SEM micrographs of the cross-section of (a) FG and FG-based nanocomposite films containing (b) 2%, (c) 6%, and (d) 8% (w/w) CSNPs content.

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Fig 9. Typical stress-strain curves of FG-based nanocomposite films with different CSNPs content.

ACCEPTED MANUSCRIPT Table 1 Tensile strength (TS), elongation at break (EAB), elastic modulus (EM), film solubility and water vapor permeability (WVP) of FG-based nanocomposite films with different CSNPs content.

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TS (MPa) EAB (%) EM (MPa) Film solubility (%) WVP (g mm/kPa h m2) a a a 7 .44 ± 0.17 102.04 ± 28.38 287.03 ± 14.25 71.80 ± 1.51a 1.421 ± 0.087a 7.99 ± 1.46a 70.09 ± 11.93ab 371.93 ± 4.61b 68.55 ± 2.67a 1.006 ± 0.170b ab b bc b 8.77 ± 1.11 64.72 ± 24.59 392.25 ± 6.43 63.79 ± 0.15 0.832 ± 0.038bc b b c b 10.57 ± 0.19 44.71 ± 11.80 453.46 ± 65.46 62.63 ± 1.14 0.717 ± 0.036c bc b c ab 11.28 ± 1.02 32.73 ± 7.38 467.2 ± 49.63 65.19 ± 2.32 0.884 ± 0.127bc standard deviation. Values means followed by the same letter are not significantly (p > 0.05) different

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CSNPs content (%) Thickness (µm) 0 50.58 ± 2.80a 2 54.52 ± 4.10a 4 61.16 ± 2.48b 6 62.14 ± 1.67b 8 64.87 ± 3.36b Reported values for each film are means ± according LSD test.

ACCEPTED MANUSCRIPT Table 2 Light transmission (%) and transparency of FG-based nanocomposite films with different CSNPs content. Transparency 800 83.24 ± 0.95a 0.97 ± 0.03a 82.53 ± 1.32a 0.99 ± 0.01a 81.43 ± 0.89a 1.29 ± 0.18a 76.94 ± 3.57b 1.61 ± 0.23b 57.24 ± 1.02c 3.65 ± 0.08c significantly (p > 0.05) different

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CSNPs content Light transmission at different wavelengths (%) 200 280 350 400 500 600 (%) 0 0 ± 0.00a 14.52 ± 0.36a 69.94 ± 1.37a 77.79 ± 1.62a 80.54 ± 1.06a 82.51 ± 1.02a a b a a a 2 0 ± 0.00 11.30 ± 0.57 68.34 ± 1.47 75.03 ± 2.40 80.07 ± 1.07 81.56 ± 1.37a 4 0 ± 0.00a 10.83 ± 0.44bc 66.67 ± 2.57a 73.7 ± 1.48a 78.88 ± 2.48a 80.26 ± 1.51a a cd b b b 6 0 ± 0.00 9.58 ± 1.12 54.05 ± 3.82 63.95 ± 4.38 72.48 ± 2.44 73.84 ± 4.15b a e c c c 8 0 ± 0.00 7.76 ± 0.91 43.01 ± 0.63 49.4 ± 1.00 53.44 ± 1.12 55.34 ± 1.12c Reported values for each film are means ± standard deviation. Values means followed by the same letter are not according LSD test.

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Highlights CSNPs had spherical shape and size range about 40-80 nm.

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FG-based nanocomposite films were prepared using solution casting method.

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Incorporation of CSNPs improved the mechanical properties of the FG film.

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6% (w/w) CSNPs loading decreased the WVP of the FG film by 50%.

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