Novel chitosan films with laponite immobilized Ag nanoparticles for active food packaging

Accepted Manuscript Title: Novel chitosan films with laponite immobilized Ag nanoparticles for active food packaging Authors: Zhengguo Wu, Xiujie Huang, Yi-Chen Li, Hanzhen Xiao, Xiaoying Wang PII: DOI: Reference:

S0144-8617(18)30821-X https://doi.org/10.1016/j.carbpol.2018.07.030 CARP 13830

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

9-3-2018 29-6-2018 9-7-2018

Please cite this article as: Wu Z, Huang X, Li Y-Chen, Xiao H, Wang X, Novel chitosan films with laponite immobilized Ag nanoparticles for active food packaging, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.07.030 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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Zhengguo Wu, Xiujie Huang, Yi-Chen Li, Hanzhen Xiao, Xiaoying Wang*

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Novel chitosan films with laponite immobilized Ag nanoparticles for active food packaging

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State Key Laboratory of Pulp and Paper Engineering, South China University of Technology,

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Graphical abstract

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*E-mail address: [email protected]

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381 Wushan Road, Tianhe District, Guangzhou, 510640, China

Highlights 1

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Laponite immobilized AgNPs (LAP@AgNPs) were synthesized and added in chitosan films Only 5.6% of AgNPs were released from films, which hardly showed toxicity to cell

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The films with LAP@AgNPs exhibited good antimicrobial activity

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2% of laponite could induce good physicochemical properties of chitosan films

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The films with LAP@AgNPs could keep litchis fresher than commercial cling wrap

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Abstract

Silver nanoparticles (AgNPs) are a kind of excellent antimicrobial agent, but the application is limited in food field due to easy leakage. In this work, for the first time, laponite

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immobilized silver nanoparticles (LAP@AgNPs) were synthesized with quaternized chitosan

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as green reductant, in which AgNPs were embedded in the interlayer of laponite due to

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confinement effect. Subsequently, chitosan-based films with LAP@AgNPs were prepared for keeping litchis fresh. The results show that only about 5.6% of AgNPs were released from the

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films with laponite, which were much lower than those films without laponite (about 29.1%),

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and physicochemical properties of the films were improved due to the suitable addition of laponite. Furthermore, although the films showed very low toxicity to cells, they exhibited

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good antimicrobial activity and effectively extended the storage life of litchi as a packaging. Hence, the research provides the potential application for silver nanoparticles in food field.

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Keywords: chitosan, laponite, silver nanoparticle, immobilization, food packaging

1. Introduction Nowadays, people are looking for a food packaging with environment-friendly, good antimicrobial and barrier property to reduce the environment problems, extend the shelf-life 2

and improve the food storage environment (Cazón, Velazquez, Ramírez, & Vázquez, 2017). Films derived from biopolymer have been widely used in food packaging due to their edible, renewable, and biodegradable characteristics (Wang et al., 2015; Youssef & El-Sayed, 2018). Chitin, One of the most abundant natural polymers, can be obtained from exoskeletons

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of crustaceans and the cell wall of fugi (Tanodekaew et al., 2004; Zhang, Gao, Wang, Chen, & Ouyang, 2016). Its derivate chitosan (CS) offers intrinsic antimicrobial property, non-

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toxic, biodegradable and outstanding designability properties to be a suitable candidate for

the active packaging films (Siripatrawan & Vitchayakitti, 2016). However, chitosan films as

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food packaging face the demerits of low mechanical performance and not satisfactory

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antimicrobial capability (Nouri, Yaraki, Ghorbanpour, Agarwal, & Gupta, 2018; Zhang et al.,

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2017). The low mechanical property of composite films can be reinforced by cross-linking

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with nano-clay (Tang & Alavi, 2012), graphene (Ahmed, Mulla, Arfat, & Thai T, 2017; Fan

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et al., 2010), calcium silicate (El-Nahrawy, Ali, Abou Hammad, & Youssef, 2016), metal hydroxide (Wang & Zhang, 2014), cellulose (Coelho et al., 2017; Rubentheren, Ward, Chee,

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& Nair, 2015; Youssef, El-Sayed, El-Sayed, Salama, & Dufresne, 2016) and polyethylene

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glycol fumarate (Doulabi, Mirzadeh, Imani, & Samadi, 2013), and the incorporation of antimicrobial inorganic nanoparticles in chitosan films can enhance their antimicrobial features. Among the antimicrobial nanoparticles, silver nanoparticle (AgNP) has gained great

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attention due to strong antimicrobial activity against wide range of microorganism (Hartemann et al., 2015). But the researchers found that AgNPs had some toxicity for human’s body (Kaiser et al., 2017; Schluesener & Schluesener, 2013; Wang, Xia, & Liu, 2015). When AgNPs are directly used in food packaging as antimicrobial agents, the leakage 3

of AgNPs may cause potential damage to the people (Mackevica, Olsson, & Hansen, 2016). Therefore, it is necessary to find a material to immobilize silver nanoparticles, which can ensure low possibility of leakage. Recently, nanoclay has been used to fabricate functional nanocomposites because of its

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surface properties, such as binding metals via the surface charge effects (Ahmed, 2003). Laponite (LAP), a kind of typical synthetic clay with nano-size layered structure, has been

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applied to the various functional materials due to its unique physicochemical property (Chen et al., 2015; Yoo et al., 2014). For example, LAP is used to improve the tensile strength of

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cellulose composite films (Yuan et al., 2014). Moreover, laponite has special cationic

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exchange capacity due to the surfaces with a large amount of negative charges (per 100

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grams of nanocrystalline with 50-55 millimoles negative charges) (Li, Liu, Ye, Wang, &

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Wang, 2015). Hence, various positive-charged molecules and ions, such as doxorubicin (Li et

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al., 2014), chitosan (Shan, Han, Xue, & Cosnier, 2007) and metal ions (Tzitzios et al., 2010), could be fixed to the surface by electrostatic absorption or encapsulated within laponite via

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inserting in the layer to form a sandwich structure. Consequently, it indicates that laponite

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can be used as a template in the synthesis process of nanosilver which makes silver nanoparticles immobile on the surfaces of laponite. Furthermore, physical properties of composite films will be improved due to the interaction between laponite and

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biomacromolecule (Ghadiri et al., 2013; Li et al., 2015). According to our previous research (Chen, Shen, Luo, Wang, & Sun, 2015), quaternized chitosan (QCS) can be used as a green reducing agent and stabilizer for silver nanoparticles. So in the present work, firstly, LAP@AgNPs were synthesized with quaternized chitosan as 4

reducing and stabilizing agent, and then LAP@AgNPs were used as antimicrobial agent to prepare CS-based films for food packaging, as presented in Fig. 1. The releases of AgNPs from the films were performed under physiological conditions. Mechanical properties, water solubility, swelling behaviors and barrier capability of CS-based films were investigated.

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What’s more, the cytotoxicity of the nanocomposite films was evaluated. The antimicrobial activity of CS-based films against gram-negative Escherichia coli, gram-positive

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Staphylococcus aureus, fungi Aspergillus niger and Penicillium citrinum was also assessed.

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At last, CS-based composite films were applied to keep litchis’ freshness.

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2. Materials and methods

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2.1. Materials

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Chitosan was purchased from Jinan Haidebei marine bioengineering Co., Ltd. (Jinan, China). Its degree of deacetylation was 85%, and its weight average molecular weight (Mw)

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was 2.0×105 g/mol. Laponite (XLG, Na+0.7[(Si8Mg5.5Li0.3)O20(OH)4]-0.7) was obtained from

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Guangzhou ShengXin Chemical Technology Co., Ltd (Guangzhou, China). Litchis were picked from orchard (Guangzhou, China). The commercial polyethylene (PE) cling wrap was

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from Wal-Mart (Guangzhou, China). Escherichia coli, Staphylococcus aureus Aspergillus niger and Penicillium citrinum were supplied by Guangdong Institute of Microbiology

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(Guangzhou, China). All other chemicals were of analytical grade. 2.2. Synthesis of laponite immobilized silver nanoparticles LAP@AgNPs composite materials were synthesized by reactive template grain growth method on the laponite nanodisk. The typical synthetic procedure was described below. Laponite nanodisk (0.1 g) were dispersed in 20 mL distilled water and vigorously stirred 5

overnight to obtain a homogenous suspension, then added into a 250 mL flask in microwave reactor (800 W). Silver-ammonia solution (2 mL, 1 mol/L) was blended with laponite solution for 10 min at 70 oC. It may involve electrostatic interactions between Ag+ and the negative charge laponite nanodisks. Quaternized chitosan solutions (20 mL, 1%) were added

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by drop and the prepared mixtures were kept under 70 oC and stirred for 30 min. The color of

of silver nanoparticles. 2.3. Preparation of chitosan-based films with LAP@AgNPs

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the mixture was evolving from colorless to faintly yellow and sepia, indicating the formation

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Chitosan/Laponite/AgNPs nano-films were prepared by a casting-solvent evaporation

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technique. Laponite was dispersed in deionized water by using magnetic stirring. Chitosan

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was added to 40 mL of DI water containing 1% (v/v) acetic acid to obtain clear solution

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(2.5% w/v). Then the solution was mixed with the above prepared laponite solution (1%, 2%,

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5%, 10% (w/wcs)) and 25% (w/w) glycerol at 800 rpm for 1h. Finally, 5% (w/wcs) of LAP@AgNPs solution was added to the above mixing solution for 2 h with constant stirring.

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Then the mixture was casted into petri dishes (11 cm × 11 cm) and dried at 40 oC for 24 h.

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The dry film was treated by sodium hydroxide solution (5% w/v), then washed with DI water and dried at 40 oC. The chitosan with laponite and LAP@AgNPs film was named after CLn/LAP@AgNPs, and stored in a desiccator until used. The pure chitosan (CS) film and

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CS/AgNPs film (CA) were produced by the same method. 2.4. Characterization of LAP@AgNPs nanocomposite Transmission electron microscope (TEM) micrographs and energy-dispersive X-ray spectrometer (EDS) were obtained using a JEM 2100 microscope operated at 200 kV with a 6

point-to-point of 2.3 Å. Before measurements, the samples were dispersed in deionized water and suspension was treated in ultrasound for 30 minutes. A drop of very dilute suspension was placed on an ultrathin carbon-coated grid and dry at 40 oC. The X-ray diffraction (XRD) experiments were performed by a D8 Advance X-ray diffractometer (Bruker, Germany) with

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Cu Kα radiation (λ=0.154 nm) at 40 KV and 40 mA to step scan the diffraction angles (2θ) between 5° and 90°.

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2.5. Characterization of chitosan-based films

CS-based composite films were examined using an LEO1530VP (Zeiss, Germany)

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scanning electron microscope (SEM) with an Inca400 (Oxford, England) energy-dispersive

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X-ray spectrometer (EDS). All films were coated with a thin layer of Au. The surface

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morphology and cross section of CS-based composite films were observed by SEM at 5 KV.

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The structural interactions of chitosan and nanomaterials were detected by an attenuated total

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reflection-Fourier Transform Infrared spectrometer (ATR-FTIR, VERTEX 70, Bruker, Germany) and X-ray diffraction spectrometer (LEO1530VP, Zeiss, Germany). The ATR-

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FTIR spectra were recorded from 3700 to 560 cm-1, and the XRD patterns of films were

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obtained in the scan range of 2θ from 5o to 90o. 2.6. Mechanical properties Mechanical properties in terms of tensile strength and elongation at break of the

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chitosan-based films were detected by Universal Testing Machine (INSTRON 5565) at 25 oC. The films were cut into rectangle (1 cm × 5 cm) and strain rate of 10mm min-1 was applied. Three parallel samples were tested for each nanocomposite film and took the average. 2.7. Water solubility and swelling behaviors 7

The films were cut into 1 cm × 2 cm test sections, then dried in 40 oC for 12 h and weighted (M0). Afterward, the samples were soaked in 20 mL distilled water for 24 h. At fixed time, the films were weighted (M1) after removing the water from the surface with filter paper and dried in 40 oC for 12 h, then weighted again (M2).

Wsb =

M1 −M0 M0

× 100%

(1)

WWS =

M0 −M2 M0

× 100%

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Water solubility (Wws %) was calculated by using the equation:

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Swelling behaviors (Wsb %) was calculated from the following equation :

(2)

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2.8. Measurements of barrier properties

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Water vapor permeability (WVP) of chitosan-based films was determined according to

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ASTM E96 (ASTEM, 2010) procedure with minor modifications. A glass bottle containing

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anhydrous calcium chloride desiccant was covered by the nanocomposite films (26mm

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diameter in exchange area) under test and sealed using paraffin wax. The assembly was weighed 6 times at 1d intervals and kept in a humidity chamber maintaining 75±5% of

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relative humidity (T=25±1 oC) using standard solution of sodium chloride. The experiments

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were done thrice for each sample, and average was taken. WVP was obtained to the equation as below: WVP =

KL

S∆P

(3)

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where K is the slope of weight variations versus time linear function (g/ 24h). L is the thickness of the film (m), S is the vapor exchange area under test (m2), ∆𝑃 is the vapor pressure difference on both sides (Pa).

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Oxygen transmission rates (OTRs) of the chitosan-based composite films were measured by Oxygen Permeation Analyzer (VAC-V1, China) under 50±1% RH at 25±1 oC in an area of 38.46 cm3 (70 mm diameter). 2.9. Cumulative release of AgNPs

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The release of AgNPs from chitosan-based films was carried out in 10 mL of TRIS-HCl buffer at pH 7.4 in an orbital shaker (25 oC). At fixed time, 500 μL of the media were

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withdrawn and fresh media was added to maintain initial conditions. The samples were diluted in HNO3 0.1M, then silver’s content was determined by atomic absorption

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spectrometer (Z-2000 HITACHI).

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2.10. Cytotoxicity evaluation

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The cytotoxic activity of CL/LAP@AgNP films was tested in comparison with CA films

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and the pure CS films. Firstly, the films were sterilized under the UV for 30 min, and then

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directly mixed with L02 cells (human normal hepatocyte line). Various quantities of the films (1, 5, 10 mg) were placed in the center of wells and exposed around the L02 cells in 24-well

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plates, then incubated at 37 oC in 5% CO2 atmosphere for 24 h. Subsequently, cell viability

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was estimated by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide) assay (Mynhardt et al., 2018; Tung, Ching, Ng, Tew, & Khung, 2017). 2.11. Antimicrobial test

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The prepared pure CS, CA and CL/LAP@AgNPs films were evaluated for their

antimicrobial activity, which is tested against Escherichia coli, Staphylococcus aureus Aspergillus niger and Penicillium citrinum by determining the inhibition zone diameters after exposure to the samples. Bacterial suspensions with a density about 105-106 CFU/mL were 9

prepared by using sterilized physiological saline. Subsequently, the nanocomposite films were cut into pieces with 10 mm in diameter and UV sterilized for 30 min each faces. 10 ml beef extract peptone agar medium or potato-dextrose agar (PDA) was added into the sterile Petri-dish, which was inoculated with 50 μL bacterial suspension, and then the films were

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stuck on the culture medium. The plates were incubated at 37 oC for 24 h and observed colonies growth. The fungi were incubated at 28 oC for 48 h.

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2.12. Storage study

Fresh litchi fruits were selected and washed with deionized water, then dried at room

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temperature. Subsequently, lychees were wrapped by CS composite films, and put into

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constant temperature humidity chamber (LHS-50CL, HengYi, Shanghai China) at 25±1 oC in

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75%±0.5 RH. We observed the change of litchis, and collected corresponding data at 1, 3, 5

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and 7 d. The commercial PE films and pure CS films were also investigated in these

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conditions.

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3. Results and discussion

3.1. TEM and XRD analysis of LAP@AgNPs

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In the TEM analysis, it is obvious that AgNPs (black solid) stably grew in the laponite

flake layers (gray) (Fig. 2a). Compared with the AgNPs without laponite (Fig. 2b), the size of

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LAP@AgNPs nano-structure was greater than only AgNPs, suggesting that the LAP@AgNPs nano-platelets spontaneously assembled to the sandwich structure because of the internal electrostatic interaction between laponite and QCS. To further ascertain the component of prepared nanomaterial, the element contents of LAP@AgNPs were analyzed by EDS (Fig. 2c). The LAP@AgNPs composites had high content of Ag and the ratio of Mg, Si and Na 10

basically met proportion of laponite chemical formula, which demonstrates that AgNPs have been grown well in the laponite. The crystalline structure of pure laponite, QCS and LAP@AgNPs were evaluated by XRD (Fig. 2d). The XRD pattern of LAP@AgNPs showed characteristic diffraction peaks of

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AgNPs at 2θ of 38.2°, 44.4°, 64.6°, 77.4° and 81.6°, which are in accordance with standard powder diffraction peak data of AgNPs (JCPDS no.89-3722). Furthermore, compared with

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original laponite, the diffraction peaks of laponite in the LAP@AgNPs disappeared because

AgNPs broke the surface lattice of laponite. It was also observed that the diffraction peaks of

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QCS in the LAP@AgNPs could not be found because Tollens reaction completely destroyed

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the ordered crystal structure of quaternized chitosan (Liu, Li, Zheng, Wang, & Sun, 2013).

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3.2. Structure characterization of chitosan-based films

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The micromorphology of CS-based films (Fig. 3) were examined by AFM (Fig. 3a-c)

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and SEM (Fig. 3d-i), and surface compositions of nano-films were confirmed by EDS analysis (Fig. 3j-l). The surface structure of CL/LAP@AgNPs films (Fig. 3c, f) appeared

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denser and more uniform than pure CS films (Fig. 3a, d) and CA films (Fig. 3b, e). In

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addition, the cross section of CL/LAP@AgNPs films (Fig. 3i) was smoother and more homogeneous. This was because that the interaction between negative-charged LAP@AgNPs nanoplatelets and positive-charged chitosan enabled the chitosan molecules to be a stable

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ordered structure. The other possible reason is that the LAP@AgNPs nanoplatelets with a nano-size scale clay can fill interspace of films (Yoo et al., 2014). To further analyze intermolecular interaction of chitosan composite films, FT-IR and XRD have been employed. The FT-IR and ATR-FT-IR spectra of CS powder, LAP, CS 11

films, CA and CL/LAP@AgNPs films are shown in Fig. 4a and b, respectively. The characteristic peaks for CS were presented at 3440 cm-1 (O-H and N-H stretching vibration), 1658 cm-1 (amideⅠ, C=O stretching vibration) and 1598 cm-1 (amideⅡ, -NH2 bending vibration) (Theapsak, Watthanaphanit, & Rujiravanit, 2012; Wang et al., 2015). The main

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characteristic peaks of LAP consisted of 1001 and 661 cm-1 (Si-O stretching vibration and bending vibration) (Tan et al., 2015). Compared with CS films, the peaks of CA

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nanocomposite films had hardly changed, but the peak at 3500-3100 cm-1 became wide and weak, suggesting that AgNPs interacted with chitosan. However, in the spectrum of

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CL/LAP@AgNPs nanocomposite films, changes occurred at 1555 and 992 cm-1, the

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absorption peaks of amides and Si-O have shifted to a lower wavenumbers, and the peak

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intensity of 3500-3100 cm-1 became wide and weak. These indicate that electrostatic and

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hydrogen bond interactions between laponite and chitosan may have happen (Ordikhani,

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Dehghani, & Simchi, 2015; Roozbahani, Kharaziha, & Emadi, 2017). The findings can further be supported by XRD analysis (Fig. 4c and d). The main diffraction peaks of chitosan

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were observed at 10.53o and 19.9o. After fabrication of CL/LAP@AgNPs, the crystallization

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peak of AgNPs and laponite can be observed in the spectrogram, and the half-peak width and its intensity in the XRD of chitosan diminished. The results show that the interaction between nanoparticles and chitosan have happened (Chen, Xu, Li, Wang, & Zhang, 2011; Liu, Pang,

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Xu, & Zhang, 2016).

3.3. Properties of chitosan-based films Mechanical properties of CS-based food packaging films are very important factors to affect their applications. In order to evaluate the reinforcing effect of laponite in CS films, 12

mechanical properties of the composite films (Fig. 5a) were characterized and compared with the neat CS film. From Fig. 5a, with the increase of laponite concentration, tensile strain increased gradually and then decreased, as well as the tensile strength. It can be explained that negative-charged laponite contributed an electrostatic interaction to positive-charged

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chitosan, making the structure of CS-based films tighter, more compact and layered to obviate the brittleness (as confirmed in Fig. 3), and then improved the mechanical strength of

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CL/LAP@AgNPs films. However, over-loading with laponite caused excessive accumulation of negative charge, which disrupted the structure of nano-films and form discontinuous

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internal structures, weakening the mechanical strength of the composite films.

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The swelling and solubility are the indicators of water resistance of composite materials

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(Pena, Caba, Eceiza, Ruseckaite, & Mondragon, 2010; Roger, Talbot, & Bee, 2006). It is

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worthy to note that controlled solubility and swelling of CS-based composite films can

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extend the application in food packaging field. The results are depicted in Fig. 5b. Compared to neat CS films, the solubility and swelling of the composite films reduced rapidly in the

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presence of laponite and AgNPs, this was because there was interaction between laponite and

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chitosan, and the filling of laponite and AgNPs made films denser and more sealing. The results show the content of laponite had a significant effect on water solubility but not on swelling. CS nanocomposite films with proper addition (e.g. 2%) of laponite had relatively

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low solubility, because laponite at low concentration helped nanocomposite films to form more ordered structure to prevent dissolution. However, the increase of laponite content led to the weight loss of CS nanocomposite films. This could be attributed to the solubility of laponite clay. The swelling of the films usually represented by water absorption, and the pure 13

CS films had high water absorption due to the formed hydrogen bonding between hydrophilic groups and water (Nayak, Jyothi, Balakrishna, Padaki, & Ismail, 2015). When laponite, AgNPs and LAP@AgNPs were added to the chitosan matrix, the swelling capability of CS nanocomposite films greatly decreased. This may be due to the interactions between

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nanomaterials and chitosan which interrupted the intermolecular motion, disentangled the chitosan chain and enclosed the hydroxyl groups. Furthermore, with the increase of laponite

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content, the swelling of nanocomposite films increased slightly then decreased on account of the swelling property of laponite clay.

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To maintain the freshness and extend the food preservation, it is important to prevent

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dehydration of the food, meanwhile, hinder water and oxygen infiltration from the outside,

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and then suppress internal damage and improve the internal storage environment of food

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(Atarés & Chiralt, 2016; Duncan, 2011; Kurek, Ščetar, Voilley, Galić, & Debeaufort, 2012).

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Therefore, water vapor permeability (WVP) and oxygen transmission rates (OTR) of CSbased composite films were investigated, as shown in Fig. 5c and d, respectively. As shown

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in Fig. 5c, the certain addition of nanomaterials changed the WVP of composite films, less

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moisture were absorbed from outside into internal environment through the films, confirming that introducing AgNPs and laponite into the CS matrix was effective way to decrease the WVP of pure CS film. This indicates that compared to pure chitosan films, the

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nanocomposite films structures were more compact and tight. The reason can be the following: on one hand, there were the electrostatic interactions between laponite and CS; on the other hand, nanoparticles can fill the nanoscale defects and cracks of the composite films. These interactions inside the films may enclose inner hydrogen groups to limit them to form 14

hydrophilic bonding with water and then decrease the diffusion of water vapor through the films (Siripatrawan & Vitchayakitti, 2016). However, the WVP of nanocomposite films with the high concentrations of laponite were raised, even more than the pure CS films. It may be ascribed to excessive amount of laponite which induced the disorder and loose of

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microstructures in CL/LAP@AgNPs films so that more water vapor can go through the films. Moreover, high levels of laponite increased the films solubility and prompted it to absorb

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more water vapor from outside environment. The films with high WVP cannot keep food fresh and may induce some damage in the long storage due to the loss of moisture on the

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surface of the fruit and vegetables or the invasion of moisture and microorganisms. Hence,

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from the perspective of ensuring the freshness and extending the storage of food, the films

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with low WVP should be chosen as food packaging films.

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Generally, food packaging materials require low oxygen permeability to maintain

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freshness and qualities of food, the breathability of the films depended on film microstructure such as void volume and porous structure (Siripatrawan & Vitchayakitti, 2016). As shown in

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Fig. 5d, the OTR of CS-based films, CS, CA and CL/LAP@AgNPs were examined. The

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OTR values of CA and CL/LAP@AgNPs films were 22.154 and 3.951 cm3/m2·d·atm, respectively, which were extremely low by comparing with pure CS films (959.092 cm3/m2·d·atm), indicating that the nanocomposite films had a good oxygen barrier property.

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Inorganic nanoparticles were an impermeable oxygen barrier substance (Bikiaris & Triantafyllidis, 2013; Yoo et al., 2014) which can improve the OTR of nanocomposite films. In our case, AgNPs were effective in oxygen barrier to fill micro-pore and reduce void volume. As shown in analysis of micromorphology structure and WVP, the suitable addition 15

of laponite changed the nano-sized channels of films by tightening the CS chain-to-chain junction for denser and more compact films, so it can also make the gas hardly pass through the CS nanocomposite films. Based on the above analysis, CS-based films with 2% laponite has the optimal

films were chosen for the next study.

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3.4. Cumulative release of silver nanoparticles from chitosan-based films

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mechanical, water solubility, swelling, WVP and OTRs properies. Hence, CL2/LAP@AgNPs

As shown in Fig. 6a, the releases of AgNPs from CL2/LAP@AgNPs films and CA films

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were investigated. According to the Fig. 6a, there were two stages for the release of the

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nanocomposite films -- explosive and slow release stages. In the explosive stage (i.e. within

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12 hrs), the release of AgNPs from CA films (26.5%) is significantly faster than that from

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CL2/LAP@AgNPs films (4.4%). Interestingly, during the slow release stage, the release of

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AgNPs from CA and CL2/LAP@AgNPs films are towards a plateau phenomenon. The AgNPs release from CL2/LAP@AgNPs films (from 4.4% to 5.6%) is 3 times less than that

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from CA films from26.5% to 29.1%. It suggests that the reduction of the amount of AgNPs’

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leakage may be attributed to the immobilization of AgNPs on the laponite. In addition, the existence of laponite in the CS matrix can prompt the immobilization effect and reduce the leakage of AgNPs from films.

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3.5. Cytotoxicity analysis The cytotoxicity of chitosan composite films was evaluated by MTT assays and cell viability was used as a measuring parameter. It was observed that all of the films showed high cell viability when 1 mg of samples was added into the medium. However, with 16

increasing content of the films, the significant difference of the cell viability can be observed for various films from the Fig. 6b. Noteworthily, the cell viability of CL2/LAP@AgNPs films and pure CS films were 85.81% and 90.37%, respectively, whereas CA films’ was 41.51%. It can be seen that CL2/LAP@AgNPs films have lower toxicity for L02 cells in

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comparing with CA films. The results confirm that the cytotoxic effect was greatly reduced by the immobilization of AgNPs on laponite. Moreover, the chitosan might hinder the

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interaction between bare nanoparticles with cellular components (Regiel-Futyra et al., 2015) and reduce the cytotoxicity of the films including AgNPs comparing to other materials

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containing nanosilver (Sarhan, Azzazy, & El-Sherbiny, 2016).

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3.6. Antimicrobial activity of chitosan-based films

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To evaluate the potential antimicrobial activity of CS-based films as food packaging,

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four representative food pollution microorganisms (S. aureus, E. coli, A. niger and P.

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citrinum) were tested, because they usually invaded the surface or interior of vegetable and fruit and caused them putrefy and decompose. The results are expressed via size of inhibition

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zone and shown in Fig. 7.

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Fig. 7 represents the antimicrobial effects of the produced CL2/LAP@AgNPs films in comparison with the pure CS films and CA films. Obviously, the CS films exhibited a good antimicrobial activity against S. aureus, E. coli, A. niger and P. citrinum, whereas the pure

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CS films displayed scarcely any antimicrobial activity against S. aureus, E. coli, A. niger and P. citrinum. The antimicrobial activity of CL2/LAP@AgNPs films was slightly lower than CA films due to decreasing release of AgNPs by laponite immobilization. Nevertheless, the

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addition of LAP@AgNPs could enhance the antimicrobial activity of CS-based films (Fig. 7e). The antimicrobial effect of the films may be mainly attributed to the synergistic effect of CS, AgNPs and laponite. Some researchers (Regiel-Futyra et al., 2015; Sarhan et al., 2016)

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proved that polycationic CS had the antimicrobial activity. The amino groups of chitosan interacted with the negatively-charged bacterial cell wall, which could increase cell

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membrane penetrability and cause intracellular components to leak. On the other hand, AgNPs can interact with sulfur-containing proteins in the cell membrane to change its

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permeability and make cell leaking (Duran et al., 2016). Moreover, AgNPs can infiltrate into

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the interior of the cell and disrupt it normal metabolism (Duran et al., 2016). In addition,

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laponite as a two-dimensional layered material, like montmorillonite and rectorite, may have

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the ability of adsorption and fixing microorganism. Therefore, compared with the pure CS

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films, the antimicrobial activity of CA and CL2/LAP@AgNPs films has been enhanced. However, compared with CA films, the antimicrobial activity of CL2/LAP@AgNPs films

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reduced because the addition of laponite decreased AgNPs release. In addition, release

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experiments showed that the release of AgNPs from CL2/LAP@AgNPs films is much fewer than CA, but its antimicrobial activity was not much weaker than the CA, this is because laponite can adsorb and fix microorganism.

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In addition, it can be concluded that the antimicrobial activities of CS nanocomposite

films against S. aureus (gram-positive bacteria) were superior to E. coli (gram-negative bacteria). This may be attributed to the difference of their cell wall structures (Siripatrawan & Vitchayakitti, 2016). The cell walls of gram-negative bacteria are multilayer structure, which 18

contain thin peptidoglycan layer, lipoprotein layer, phospholipids or lipopolysaccharide layer. These complex layer structures weakened the interaction between chitosan/laponite and cells and also prevented AgNPs into the cell (Siripatrawan & Vitchayakitti, 2016). The cell walls of gram-positive bacteria are composed of a thick but single peptidoglycan layer, enabling the

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easier entry of AgNPs into the cell. Noteworthy, the CS-based films have good antifungal activity against A. niger and P.

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citrinum, because AgNPs with unique nano-size and surface effect can easily infiltrate into the interior of mold spore or mycelium, then damage the cell wall and disrupt its normal

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metabolism (Balashanmugam, Balakumaran, Murugan, Dhanapal, & Kalaichelvan, 2016;

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Dananjaya et al., 2017; Jannoo, Teerapatsakul, Punyanut, & Pasanphan, 2015).

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3.7. Storage study

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Here, to show the food packaging application of the CL2/LAP@AgNPs films, we

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applied it as a food packaging for the storage of litchis. The results of litchis’ storage are shown in Fig. 8. After 5 days, the control sample overgrew with mycete, and some of litchis

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wrapped in the pure CS films were decay and grew with mycete, as well as litchis wrapped

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with commercial PE films. Noticebly, litchis wrapped by CL2/LAP@AgNPs films still maintained its freshness. Moreover, unrotten litchis were still observed in the CL2/LAP@AgNPs films after 7 days of storage. The results indicate that CL2/LAP@AgNPs

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films as a food packaging could prolong the storage time of food.

4. Conclusions In the study, the CS films with flexibility, special functional performance and low cytotoxicity were easily produced by the solution casting of CS incorporated with laponite 19

and LAP@AgNPs. The synergistic effects of laponite and AgNPs obviously improve the mechanical, water solubility and OTR properties of CS-based films. Importantly, laponite exhibited a significant ability to prevent the release of AgNPs from in CS-based films that could reduce the cytotoxic effects of AgNPs. In particular, CL/LAP@AgNPs films possess a

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high antimicrobial activity and thus could keep litchis fresher than PE films (commercial cling wrap). Therefore, this study on laponite as an AgNP immobilization agent may solve

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the bottleneck problem of nano-silver used in food preservation. Acknowledgements

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This work was financially supported by the National Natural Science Foundation of China

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(No. 31622044), Science and Technology Planning Project of Guangzhou City (No.

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201707010190), Youth science and technology innovation talent of Guangdong Special Fund

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SCUT (No. 2017ZX003).

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Plan (No. 2016TQ03Z904) and the Fundamental Research Funds for the Central Universities,

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Figure captions

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Fig. 1. Schematic illustration for the fabrication of CL/LAP@AgNPs films.

Fig. 2. TEM images of LAP@AgNPs and AgNPs (a, b), EDS of LAP@AgNPs (c) and XRD pattern of QCS, LAP and LAP@AgNPs (d).

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Fig. 3. AFM (a, b,c) and SEM micrographs of CS, CA and CL/LAP@AgNPs films surface

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(d, e, f) and cross section (g, h, i). Surface of each film’s EDS (j, k, l).

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Fig. 4. ATR-FTIR (a) and XRD (c) analysis of chitosan composite films, FTIR (b) and XRD

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(d) analysis of LAP and CS powder.

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Fig. 5. Mechanical properties (a), water solubility and swelling behavior (b), WVP (c) and oxygen transmission rates (OTR) (d) of CS-based films. CLn/LAP@AgNPs: L1 (1% laponite), L2 (2% laponite), L5 (5% laponite), L10 (10% laponite). Data show mean ±

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standard error (n=3).

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Fig. 6. Release of AgNPs from CA and CL/LAP@AgNPs films in Tris-HCl buffer solution (a). Data show mean ± standard error (n=3). L02 cell viabilities after incubating with

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different contents of chitosan-based films (b). Data are expressed as the mean ± standard

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error (n=5), * indicates significant difference (**P<0.01, *** P<0.001).

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Fig. 7. Antimicrobial activities of CS-based films against S. aureus (a), E. coli (b), A. niger (c) and P. citrinum (d) and inhibition zone diameter of the films (e). Data show mean ±

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standard error (n=3), ▼no significant difference (P﹥0.05).

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Fig. 8. The storage result of litchis for using CS-based films as the packaging, from left to

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right in turn for commercial PE cling wrap (1), CL2/LAP@AgNPs films (2), pure CS films

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(3) and control (4): test condition was in constant temperature humidity chamber at 25 oC

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(75% RH).

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