Accepted Manuscript

Fabrication and characterization of multifunctional active food packaging from

chitosan-titanium dioxide nanocomposite as ethylene scavenging and antimicrobial
film

Ubonrat Siripatrawan, Patinya Kaewklin

PII: S0268-005X(17)32095-7
DOI: 10.1016/j.foodhyd.2018.04.049
Reference: FOOHYD 4416

To appear in: Food Hydrocolloids

Received Date: 21 December 2017

Revised Date: 30 April 2018
Accepted Date: 30 April 2018

Please cite this article as: Siripatrawan, U., Kaewklin, P., Fabrication and characterization of
multifunctional active food packaging from chitosan-titanium dioxide nanocomposite as ethylene
scavenging and antimicrobial film, Food Hydrocolloids (2018), doi: 10.1016/j.foodhyd.2018.04.049.

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 ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT

Synthesis and characterization of multifunctional active food packaging from chitosan-titanium dioxide

nanocomposite as ethylene scavenging and antimicrobial film

Ubonrat Siripatrawan and Patinya Kaewklin

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 ACCEPTED MANUSCRIPT
1 Fabrication and characterization of multifunctional active food packaging from chitosan-titanium

2 dioxide nanocomposite as ethylene scavenging and antimicrobial film

4 Ubonrat Siripatrawana, b, * and Patinya Kaewklin a

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5 Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

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6 The Novel Technology for Food Packaging & Control of Shelf Life Research Group, Faculty of Science,

Chulalongkorn University, Bangkok 10330, Thailand

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7

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9

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10 * Corresponding author: Email: ubonratana.s@chula.ac.th,
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11 Tel. +66 2 218 5536 Fax. 66 2 218 5516

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13 ACCEPTED MANUSCRIPT
Abstract

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15 Active packaging with multifunctional properties from nanocomposite of chitosan and nanosized

16 TiO2 to be used as ethylene scavenging and antimicrobial film was developed. Chitosan films containing

17 different TiO2 concentrations (0, 0.25, 0.5, 1 and 2 % w/w) were fabricated and characterized for structural,

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18 mechanical, optical and barrier properties. The scanning electron microscope images showed that TiO2

19 nanoparticles evenly distributed in the chitosan film matrix. However, the spontaneous agglomeration of the

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20 TiO2 was observed at high TiO2 concentration. The X–Ray diffraction patterns presented that crystallinity of

21 TiO2 in the nanocomposite films increased with TiO2 concentration. The Fourier transform infrared spectra

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22 indicated that there were hydrogen bonding and O-Ti-O bonding between TiO2 and chitosan, which affected

23 the tensile strength, elongation at break, and water vapor barrier properties of the films. The chitosan-TiO2

24
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nanocomposites exhibited ethylene photodegradation which increased with increasing TiO2 concentration.
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25 Based on tensile strength, water barrier and ethylene photocatalytic degradation properties, chitosan film

26 containing 1 % TiO2 (CT1) was found to be optimal and, hence, was selected for antimicrobial evaluation.
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27 The results suggested that the CT1 exhibited antimicrobial activity against Gram-positive (Staphylococus
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28 aureus) and Gram-negative (Escherichia coli, Salmonella Typhimurium, and Pseudomonas aeruginosa)

29 bacteria and fungi (Aspergillus and Penicillium).

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30
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31 Keywords: chitosan; titanium dioxide; nanocomposite; ethylene scavenging; antimicrobial

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33 1. Introduction ACCEPTED MANUSCRIPT

34 The major loss of fresh produce is generally due to the ripening process induced by ethylene and

35 contamination of spoilage and pathogenic microorganisms (de Chiara, Pal, Licciulli, Amodio, Colelli, 2015).

36 Active packaging which can suppress the negative effects of ethylene and inhibit microbial growth is

37 desirable to extend shelf life of fresh produce and might be good alternative to the use of chemical treatments

38 and disinfectants (Kaewklin, P., Siripatrawan, Suwanagul, & Lee, 2018; Maneerat et al., 2003).

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39 Titanium dioxide (TiO2) is an efficient photocatalyst and is useful as ethylene scavenger and

40 antimicrobial agent (Lin et al., 2015; Zhang et al., 2017). Titanium dioxide is considered as attractive

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41 photocatalyst because it is chemically stable, nontoxic, inexpensive and Generally Recognized as Safe

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42 (GRAS) (Lin et al., 2015). When exposed to ultraviolet (UV) light, TiO2 exhibits ethylene photodegradation

43 by generating strong reactive oxygen species (ROS) including hydroxyl radicals (OH•) and superoxide ions

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44 (O2−) on its surface which further react with ethylene and/or polyunsaturated phospholipid in the cell
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45 membrane of microorganisms (Lin et al., 2015; Hussain, Bensaid, Geobaldo, Saracco, & Russo, 2011; de

46 Chiara et al., 2015).

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47 The nanosized TiO2 particles, due to their high surface area to volume ratio, have higher

48 photocatalytic activity than those in bulk form. The performance of TiO2 nanoparticles is enhanced because
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49 the nanosized TiO2 owes greater band gap energy when exposed to UV light and generates electron–hole
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50 pairs on its surface (Lian, Zhang, & Zhao, 2016; Maneerat et al., 2003; Onda et al., 2005). However, the

51 efficiency of the nanosized TiO2 is reduced by its tendency to agglomerate (Qian, Su, & Tan, 2011; Lin et
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52 al., 2015). The spontaneous agglomeration of TiO2 nanoparticles results in a decrease in specific surface

53 area, and consequently lessening the photocatalytic activity (Li et al., 2010; Lin et al., 2015). However, Al-
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54 Sagheer and Merchant (2011) and Qian et al. (2011) have reported that interactions between nanosized
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55 TiO2 and organic polymers can help to reduce the spontaneous agglomeration of the inorganic TiO2 in the

56 polymer matrix which can also have positive effect on the properties of the composite films.

57 A number of biopolymeric materials have recently been employed to incorporate with nanosized

58 TiO2 to enhance the functional properties of biopolymers and to exhibit the particular photocatalytic activity

59 of TiO2 (Kavitha et al., 2013; Lian et al., 2016; Qian et al., 2011). Among them, chitosan, a linear

60 polysaccharide of randomly distributed β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine, has

61 gained most interest for active food packaging applications due to its good film-forming ability and intrinsic

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antimicrobial and antioxidant properties MANUSCRIPT
(Cazón, Velazquez, Ramírez, & Vázquez, 2017; Crizel, Rios,

63 Alves, Bandarra, Moldão-Martins, & Flôres, 2018; Shankar, Wang & Rhim, 2017; Yuceer & Caner, 2014).

64 Nanocomposites prepared from TiO2 and chitosan or other biopolymers have been mostly studied for

65 their antimicrobial applications (Bodaghi et al., 2012; Lin et al., 2015; Zhang et al., 2017), while none has

66 been reported for the use of chitosan and TiO2 nanocomposite as ethylene scavenger together with

67 antimicrobial. Therefore, this study was aimed to fabricate and characterize multifunctional active food

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68 packaging from chitosan-TiO2 nanocomposite to be used as an ethylene scavenging and antimicrobial film

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69 aimed for postharvest applications of fresh produce.

70 2. Materials and Method

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71 2.1 Fabrication of nanocomposite film

72 Chitosan with 85% degree of deacetylation was obtained from Seafresh Industry Public Co., Ltd.,

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Thailand. Chitosan film forming solution (2 % w/v) was prepared using the procedure modified from the
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74 method of Siripatrawan and Vichayakitti (2016) by dissolving chitosan powder into 1% acetic acid solution,

75 and glycerol was used as plasticizer. The TiO2 nanopowders of 21 nm particle size (> 99.5%, Sigma-Aldrich
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76 Co., St. Louis, MO, USA) were added to the chitosan solution to obtain the final concentrations of 0, 0.25,
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77 0.5, 1 and 2 % w/w (designated as CS, CT0.25, CT0.5, CT1 and CT2, respectively). The resulting solution

78 was shaken at 90 rpm in a controlled-temperature water bath shaker (1083 GFL, Burgwedel, Germany) at 90
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79 C for 6 h. The solution was homogenized using a homogenizer (D-79282, Ystral GmbH, Ballrechten-

Dottingen, Germany) and subsequently degassed using a sonicator (Ultrasonic Processor, Cole-Parmer,
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81 Vernon Hills, Illinois, USA). The film-forming solution (190 mL) was casted on a 30 x 12 cm2 ceramic plate
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82 and dried. The obtained films were conditioned in an environmental chamber at 25 °C and 50 % relative
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83 humidity (RH) for 48 h before further analysis.

84 2.2 Scanning electron microscope

85 The TiO2 and cross sectional and surface morphologies of chitosan film, and chitosan-TiO2 nanocomposites

86 containing 0.25, 0.5, 1 and 2 % TiO2 were analyzed using scanning electron microscopy (JSM-5800 LV,

87 JEOL, Tokyo, Japan). The film samples were fractured in liquid Nitrogen and were sputtered with gold for

88 conductivity by a sputter coater before SEM analysis.

89

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91 Crystal structures of TiO2, chitosan, and chitosan-TiO2 nanocomposite films were analyzed using

92 X–Ray diffractometer (D8-Discover, Bruker AXS Inc., Madison, WI, USA) in the range of 2θ = 10°-80°

93 with a Cu Kα radiation source at 0.15 nm. The working parameters were voltage of 40 kV, current of 40 mA,

94 and scanning rate of 2 min-1.

95 2.4 Fourier Transform Infrared spectroscopy

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96 Fourier Transform Infrared (FTIR) spectroscopy was carried out to observe the interactions between

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97 chitosan and TiO2. The FTIR spectra of chitosan-TiO2 nanocomposite films were recorded in the

98 wavenumber range of 4000-400 cm-1 with 4.0 cm-1 resolution of 64 scans using a FTIR spectrometer

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99 (PerkinElmer 1760X, PerkinElmer Life And Analytical Sciences, Inc., Waltham, Massachusetts, USA)

100 equipped with the Universal Attenuated Total Reflectance (ATR).

101 2.5 Mechanical properties

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102 Mechanical properties including tensile strength (TS) and elongation at break (EAB) were measured

103 with an Instron® universal materials testing machine (Instron Corporation, Canton, MA, USA) following the
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104 ASTM Standard Test Method D 882 (ASTM, 2010). The film samples, conditioned in an environmental

105 chamber at 25 °C and 50 % relative humidity (RH) for 48 h, were cut into strips with a test dimension of 15
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106 × 2.5 cm2. The film was mounted between grip heads with initial grip separation of 5 cm. The test was
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107 performed at a crosshead speed of 20 cm/min. TS (Eq.1) was calculated by dividing the maximum load
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108 (Fmax) by the initial cross-sectional area (φ) of the film sample and expressed as MPa. EAB (Eq.2) was

109 calculated as the ratio of the film extension (∆l) at the point of sample rupture to the initial length (l0) of a
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110 sample and expressed as a percentage. The measurements represent an average of at least nine samples.
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111 TS = (1)
Φ

∆l
112 % EAB = 100 (2)
l0

113 2.6 Optical transmittance

114 Optical transmittance of the chitosan-TiO2 nanocomposite film was measured at 800 nm using a UV-

115 Vis spectrophotometer (Thermo Scientific GENESYS 20, Thermo Fisher Scientific, Inc., Rochester, NY,

116 USA) following the method of Wang, Du, Luo, Lin, & Kennedy (2007). The films were cut into a rectangle

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piece and directly placed in a spectrophotometer testMANUSCRIPT
cell. Six replications were conducted for each sample

118 treatment.

119 2.7 Water vapor permeability coefficient

120 Water vapor transmission rate (WVTR) of the films per unit area was determined following the

121 ASTM Standard Test Method (ASTM, 2010). Film samples, previously equilibrated at 25 °C and 50% RH

122 for 48 h, were sealed to glass cups having 5 cm diameter containing silica gel. The film-covered cups were

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123 placed in an environmental chamber set at 25 °C and 75% RH using saturated solution of NaCl. The cups

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124 were weighed periodically until steady state was reached (±0.0001 g). The water vapor permeability (WVP)

125 coefficient of the films was calculated using Eq (3). At least six replications of each film treatment were

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126 tested for the WVP.

wx
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127 (3)
At∆p
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128 where WVP is the water vapor permeability coefficient (g mm m-2 d-1kPa-1), w is the moisture weight gain

129 transferred through a film area (A) during a definite time (t), x is the film thickness, ∆p is the partial water
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130 vapor pressure gradient between the inner (pin) and outer (pout) surface of the film in the chamber.
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131 Film thickness was measured using a digital micrometer (Mitutoyo Absolute, Tester Sangyo Co.,
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132 Ltd., Tokyo, Japan). Five measurements were taken at random positions around the film sample and the

133 mean values were calculated. Six replications were conducted for each sample treatment. The thickness of
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134 the films was 83 ± 4 µm in all treatments.

135 2.8 Ethylene photodegradation

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136 The ethylene photodegradation of the chitosan-TiO2 nanocomposite films was determined. Ethylene

137 concentration of 5 ppm was used in this study, comparable to ethylene concentration produced by various

138 fresh fruits (Barry et al., 2005; Burg & Burg, 1965). Five ppm of ethylene was separately prepared in a 1000

139 mL air-tight glass container previously inserted with CS, CT0.25, CT0.5, CT1 or CT2 films and stored at 25
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140 C and 85 % RH with exposure to ultraviolet light illuminated by two 15 W black light lamps (FL15 BLB,

141 Toshiba, Japan). The films were placed approximately 15 cm far from the lamps. The light intensity was

142 measured at 15 cm far from the lamps using a radiometer (Toshiba, Tokyo). The black light lamps

143 efficiently emit UV light at the wavelength range of 280-430 nm and ultraviolet radiation intensity of

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approximately 42 mw cm-2. The ability of the films toMANUSCRIPT
photodegrade ethylene gas was investigated by

145 measuring ethylene concentration remained in the containers after 15, 30, 45, 60, 90, 120, 150, and 180 min.

146 The ethylene concentration was analyzed using gas chromatography (GC) (Shimadzu GC-9A, Japan) with

147 porapack Q column (Mesh 60/80) and a flame ionization detector (FID). Six replications were determined

148 for this study. Ethylene degradation (%) was calculated using Eq.(4) following the method of Hussain et al.

149 (2011).

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150 C 2 H 4 deg(%) = 100 (4)
C t =0

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151 where Ct=0 and Ct=t are the concentration of ethylene at an initial time and at time t, respectively.

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152 2.9 Antimicrobial activity

153 The optimal CT nanocomposite film selected from the previous sections was used in this study. The

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154 antimicrobial activity of the CT film in comparison to CS film and control (cell suspension without CT or
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155 CS films) with and without UV illumination was test against Gram-negative and Gram-positive bacteria and

156 fungi following the method of Shankar et al. (2017) with slight modification. Gram-positive bacteria
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157 (Staphylococus aureus TISTR 517), Gram-negative bacteria including Escherichia coli (TISTR 780),

158 Samonella Typhimurium (TISTR 292), and Pseudomonas aeruginosa (TISTR 1467), and fungi including
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159 Aspergillus oryzae (TISTR 3018) and Penicillium roqueforti (TISTR 3511) were obtained from Thailand
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160 Institute of Scientific and Technological Research, Thailand. Non-pathogenic fungi strains were used in this

161 study for safety considerations for the laboratory environment in order to avoid the exposure of the
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162 researchers to hazardous mycotoxigenic fungi.

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163 Before use, the test bacteria were grown in nutrient broth (HiMedia Laboratories Pvt. Ltd., Bombay,

India) and then aseptically transferred to 100 mL of 0.85% sodium salt solution to give a final density of
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165 107 CFU/mL in a flask containing 10×10 cm2 of CT or CS film and incubated at 37 °C with shaking at 170

166 rpm in a water bath shaker (1083 GFL, Germany). Inoculum suspension of fungi strains (107 spores/mL)

167 was prepared from 5-day-old cultures grown on potato dextrose agar at 25°C following the method of

168 Siripatrawan and Makino (2015). All tested samples were separated into half. The first half was not

169 exposure to UV light. The second half was exposure to UV light illuminated by two 15 W black light lamps

170 (FL15 BLB, Toshiba, Japan). The samples were placed approximately 15 cm far from the lamps. The

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inhibitory effect was estimated periodically every 2 hMANUSCRIPT
for 6 h by measuring the viable cell count by plating

172 the samples on agar plates and incubated at 37°C for 24 h for bacteria, and at 30 °C for 48 h for fungi. The

173 antimicrobial activity was performed in triplicate and expressed as growth inhibition (%) using Eq. (5).

174 ℎ ℎ % = (5)

175 where Nt=0 and Nt=t are the number of microbial cells at an initial and at time t, respectively.

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176 2.10 Statistical analysis

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177 A completely randomized design (CRD) was used as an experimental design for mechanical, optical,

178 barrier and ethylene phtodegradation properties, where different TiO2 concentrations in the nanocomposite

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179 films were applied as treatments. For antimicrobial activity evaluation, the optimal nanocomposite film and

180 chitosan film with and without UV illumination and control with UV exposure were used as treatments. The

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181 experimental data were subjected to one-way analysis of variance (ANOVA). The statistical significance of
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182 differences between mean values was established at P ≤ 0.05 and the Duncan’s Multiple Range Test was

183 applied for all statistical analyses.

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184 3. Results and Discussion

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185 3.1 Structural properties of nanocomposite films

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186 The SEM images of cross-sectional and surface of chitosan and chitosan-TiO2 nanocomposite films

187 as well as TiO2 are depicted in Fig. 1. TiO2 nanoparticles distributed uniformly in the film matrix as
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188 evidenced in the cross-sectional images of the films. However, the agglomerates of the TiO2 nanoparticles in

189 the film matrix were observed at high TiO2 concentration. As shown in Fig. 1, the nanocomposite film
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190 containing 2% TiO2 exhibit a larger cluster size of agglomerated TiO2 nanoparticles than the lower TiO2
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191 concentrations. The TiO2 agglomeration is expected to affect the mechanical properties of the composite

192 materials (He et al., 2016; Li et al., 2011).

193 The XRD pattern of TiO2 represented the distinct crystalline peaks at 2θ = 25.29°, 27.42°, 36.08°

194 and 48.04°, which correspond to the characteristic peaks of anatase (101), rutile (110), anatase (101), and

195 anatase (200) crystal phase, respectively. The characteristic peak of CS appears at 20.40°. These results

196 coincide with those of Li, Zhang, Yang, Qui, and Sun, (2016) and Lin et al. (2015) who reported the highest

197 peaks of crystallinity of TiO2 and pure chitosan could be observed at 25.3° and 19.7°, respectively. The

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diffraction peaks of TiO2 at 2θ = 25.50°, MANUSCRIPT
25.29°, 25.39°, and 25.33° attributed to the characteristic peak of

199 anatase (101) crystal phase also appeared in the XRD patterns of CT0.25, CT0.5, CT1, and CT2,

200 respectively, but with lower TiO2 intensities and minor shifts. An increase in the diffraction peak intensity of

201 the TiO2 was observed with increasing TiO2 concentration in the nanocomposite films. The crystalline peak

202 of chitosan at 2θ = 20.40o in the chitosan-TiO2 nanocomposite films was broader but with lower intensities

203 than that in the pattern of chitosan film probably because an addition of the nanosized TiO2 affected

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204 crystallinity of the chitosan and may result in an amorphous structure of the polymer (Ahn et al., 2001; Lin et

205 al., 2015). These results are consistent with those of Wang et al. (2007) and Lin et al. (2015) who reported

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206 that hydrogen bonds between TiO2 and functional groups (–OH and –NH groups) of chitosan interrupt the

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207 chitosan network in the film matrix and the incorporation of TiO2 in the chitosan matrix takes place mainly

208 in the amorphous region of chitosan.

209

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The FTIR transmittance spectra of TiO2, chitosan film and chitosan-TiO2 nanocomposite films
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210 containing 0.25, 0.5, 1, and 2% TiO2 (w/w) in the wavenumber range of 400 – 4,000 cm-1 are shown in Fig.

211 3. Chitosan spectrum exhibits two major peaks at 1518 cm-1 (N-H bending) and 1645 cm-1 (C-O stretching),
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212 respectively, consistent with existing literature (Lin et al., 2015; Liu et al., 2017; Siripatrawan &

213 Vitchayakitti, 2016). These two peaks shifted respectively to 1516 cm-1 and 1692 cm-1, probably due to the
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214 attachment of titanium to the amide functional groups of chitosan (Lin et al., 2015). The band at 1650 cm-1
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215 region is associated with C=O stretching vibration of the acetamido groups of chitosan and the bands in the

216 600-750 cm-1 region indicate the absorption of O-Ti-O for the chitosan-TiO2 interactions (Kavitha et al.,
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217 2013; Wiącek, Gozdecka, & Jurak, 2018). As the concentration of TiO2 in the chitosan film increased, so as

the peaks around 530-720 cm-1 which indicate bending vibrations of Ti-O-Ti. The bands at 1029 cm-1 and at
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219 1152 cm-1 indicate bending vibrations of Ti-O-C and stretching vibrations of C-O-C, respectively, suggesting
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220 that there is hydrogen bonding between -OH groups of chitosan and Ti ( Al-Sagheer & Merchant, 2011;

221 Kavitha et al., 2013). The red shift of the bands in the 3351-3292 cm-1 region might be caused by the

222 interactions between Ti and -OH and -NH functional groups of chitosan through hydrogen bonding (Lin et

223 al., 2015; Qian et al., 2011). Similarly, peaks of C-H at 1406 cm-1 and C-N at 1576 cm-1 of chitosan

224 decreased when TiO2 was added probably due to hydrogen bonding between –NH groups of chitosan and Ti

225 (Al-Sagheer & Merchant, 2011).

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3.2 Mechanical, optical and water vapor MANUSCRIPT
barrier properties

227 The variations of the tensile strength and elongation versus TiO2 concentration in the nanocomposite

228 films are shown in Fig. 4a and 4b, respectively. TS of the films significantly increased (P < 0.05) with

229 increasing TiO2 concentration and reached a maximum value of 16.43 MPa at 1 % TiO2, and then decreased

230 at 2% TiO2. The TiO2 concentration plays an important role in the particle agglomeration which affects the

231 TS of the nanocomposite films. At 0.25-1% TiO2 concentration, the TiO2 nanoparticles could uniformly

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232 disperse in the chitosan matrix, and may perform as a reinforcing filler by strengthening the film network

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233 (Lian et al., 2016). At high TiO2 concentration (>1% TiO2), the TiO2 nanoparticles tended to agglomerate

234 probably due to the decreasing distance between the suspended particles (during film-forming preparation),

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235 which results in the increased probability of collision between the TiO2 nanoparticles (Sillanpää, Paunu, &

236 Sainio, 2011). A decrease in TS of the CT2 was probably because the agglomerates of the excessive TiO2, as

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237 evidenced on the cross-sectional SEM image, may disrupt the chitosan network (He et al., 2016). These
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238 results are consistent with those of Li et al. (2011) who found that tensile strength of whey protein isolate

239 and TiO2 nanocomposite films increased when low TiO2 concentration was added but decreased at high TiO2
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240 concentration due to an interference of agglomerated TiO2 in the whey protein network. Similarly, Zhou,

241 Wang, and Gunasekaran (2009) suggested that the possible reason for the decrease in TS might be
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242 inhomogeneous distribution of TiO2 and aggregation of TiO2 in the film matrix when certain concentration is
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243 reached. The results of tensile strength obtained in our study may be summarized by the fact that by adding

244 sufficient amount of TiO2 nanoparticles, the matrix of the nanocomposite could be reinforced through
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245 electrostatic interactions, hydrogen bonding, or O–Ti–O bonding. However, this equilibrated nanocomposite

246 system may be disturbed by an inhomogeneous distribution of the agglomerated TiO2 which may lead to a
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247 matrix disruption and a decrease in film tensile strength (He et al., 2016; Zhou et al., 2009). Shidfar,
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248 Tavangarian, Nemati, and Fahami (2017) also suggested that the higher TiO2 concentration can be

249 considered as molecular spacer which prevents the homogeneous dispersion in polymer matrix and thus

250 leads to the lower tensile strength.

251 The elongation at break of the nanocomposite (Fig. 4b) showed somewhat different characteristics.

252 The EAB significantly decreased (P ≤ 0.05) when TiO2 was incorporated into film, but unchanged (P > 0.05)

253 with different TiO2 concentrations. A decrease in EAB is probably because the hydrogen bonds between –

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Ti lowered the stretchability of the resulting film (Al-Sagheer &

255 Merchant, 2011).

256 The optical transmittance of chitosan and chitosan-TiO2 nanocomposite films is shown in Fig. 4c.

257 Chitosan film appeared transparent as its transmittance was about 90%. The transparency of the

258 nanocomposite films of low TiO2 concentration was well-retained because of the uniform dispersion of the

259 nanosized TiO2 in the chitosan matrix. The optical transmittance of the nanocomposite films significantly

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260 decreased (P<0.05) with an increase in TiO2 concentration. The optical transmittance of the films

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261 corresponds to the crytallinity of the film. As shown on the XRD patterns, films with higher TiO2

262 concentration show higher TiO2 crystallinity so as the opacity of the nanocomposite films. A very low

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263 optical clarity of the film with 2% TiO2 may be attributed to the agglomerates of TiO2 as evidenced on the

264 SEM image. Similar effect of the TiO2 agglomeration on the optical transmittance of the films was also

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265 observed in chitosan/organic rectorite nanocomposite films (Wang et al., 2007).
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266 The water vapor permeability at 25 oC of the nanocomposite films versus the concentration of TiO2

267 is shown in Fig 4d. A significant decrease in WVP (P ≤ 0.05) was observed when TiO2 was added but
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268 increasing the TiO2 concentration from 0.25 to 2 % showed no significant effect (P > 0.05%) on the WVP.
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269 These results suggested that the water-insoluble TiO2 may block the micro-paths of the water vapor in the

270 film network microstructure (He et al., 2016; Lian et al., 2016). It is also possible that TiO2 may interact
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271 with hydrophilic -OH and -NH groups of chitosan and thus lowering the available hydrophilic groups for

sorption of water vapor on the film surface (Al-Sagheer & Merchant, 2011). These results are in line with
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273 those of Li et al. (2011) who found that WVP of TiO2 and whey protein isolate nanocomposite decreased
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274 with an addition of TiO2 due to the ability of TiO2 to hinder water vapor to diffuse through film. Similarly,
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275 Qian et al. (2011) found that an addition of TiO2 lowered the WVP of chitosan-TiO2 nanocomposites due to

276 the modification of the film backbone structure with the substituent of TiO2.

277 3.3 Ethylene photodegradation ability

278 Fig.5. depicts the percentage of ethylene photocatalytic degradation of the chitosan-TiO2

279 nanocomposite films at 25 oC versus UV light illumination time. The ethylene photocatalytic degradation of

280 the chitosan-TiO2 nanocomposite films significantly increased (P ≤ 0.05) with increasing TiO2 concentration.

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281 However, no significant increase (P ACCEPTED MANUSCRIPT
> 0.05) in ethylene photocatalytic degradation of the nanocomposite

282 films was observed when the TiO2 concentration increased from 1% to 2 %.

283 The mechanism of titanium dioxide nanoparticles in ethylene photodegradation has been described

284 by Hussain et al. (2011) and Song et al. (2017). As illustrated in Fig. 6, when activated by UV light, the

285 electrons on the TiO2 surface are promoted and transferred from the valence band to the conductance band.

286 This produces electron-hole pairs in the valence band. The positive hole (h+) reacts with water or hydroxide

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287 ions (OH−) adsorbed on the TiO2 surface to produce the highly reactive hydroxyl radicals (OH•). The

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288 electron (e-) reduces oxygen to produce superoxide ions (O2−). These O2− and OH• are strong reactive

289 oxygen species (ROS) which further oxidize ethylene to carbon dioxide and water as end products (Song et

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290 al., 2017).

291 The enhancement of the ethylene photodegradation of the nanocomposite films with an increase of

292
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TiO2 concentration from 0.25 to 1% probably because the higher concentration of TiO2 particles yield more
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293 adsorption sites and generate more ROS, which play an important role in the photocatalytic degradation of

294 ethylene. These results are in accordance with those of Maneerat et al. (2003) who found hydroxyl radicals
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295 increased as the TiO2 concentration increased. CT2 showed the highest tend of ethylene degradation but not
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296 significantly different (P > 0.05) from CT1, probably because at 2% TiO2concentration the TiO2
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297 nanoparticles may spontaneously agglomerate as evidenced on the SEM image. The agglomeration of the

298 TiO2 nanoparticles causes a decrease in effective surface area of the photocatalyst which leads to a lower
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299 photocatalytic activity (Maneerat et al., 2003). These results are in line with those of Shih and Lin (2012)

300 who found the azo dye photocatalytic degradation of TiO2 decreased with larger secondary particle sizes
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301 because of the higher intercrystalline diffusional resistance across the agglomerated TiO2 particles. Li et
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302 al. (2010) also found the similar effect of the agglomeration of TiO2 nanoparticles on their photocatalytic

303 performance.

304 The results suggested that CT1 nanocomposite film exhibited optimal mechanical, barrier and

305 ethylene photocatalytic degradation characteristics. The CT1 film was, hence, selected for further

306 antimicrobial evaluation.

307

308

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310 The CT1 and CS films were evaluated with and without UV irradiation for antimicrobial activity

311 against Gram-positive (S. aureus) and Gram-negative (E. coli, P. aeruginosa and S. Typhimurium) bacteria

312 and fungi (Aspergillus and Pennicillium). The microorganisms under UV illumination were also

313 investigated. The effect of each treatment (UV, CS, CS+UV, CT1, and CT1+UV) on the percentage growth

314 inhibition of all tested microorganisms are displayed in Fig. 7.

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315 The results suggested that CT1+UV had significantly higher (P ≤ 0.05) microbial growth inhibition

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316 than CS+UV, CT1, CS, and UV, respectively. The CS film, due to the intrinsic antimicrobial properties of

317 chitosan (Cazón et al., 2017; Crizel et al., 2018; Vilela et al., 2017), showed growth inhibition against all

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318 tested bacteria and fungi. When exposure to UV light, CT1 and CS films showed higher antimicrobial

319 activity against all tested bacteria and fungi because UV light not only activates the photocatalysis of TiO2

320
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but is known to possess antimicrobial effects (Akgün & Ünlütürk, 2017).
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321 CT1+UV exhibited the most effective antimicrobial activity against all tested microorganisms
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322 probably attributed to the combined effects of the TiO2, chitosan and UV light. The TiO2 in the CT film

323 exhibited antimicrobial activity when exposure to UV light (Foster, Ditta, & Varghese, 2011) and chitosan
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324 has been reported to have intrinsic antimicrobial activity against both Gram-positive and negative bacteria
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325 (Cazón et al., 2017; Crizel et al., 2018; Vilela et al., 2017). When exposure to UV light as illustrated in

326 Fig.6, TiO2 in the nanocomposite undergoes the photocatalytic reaction and generates the ROS including O2−
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327 and OH• which play a key role in the inhibition of bacteria and fungi by oxidizing polyunsaturated

328 phospholipids of microbial cell membranes causing changes in the cell membrane and cytoplasmic leakage
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329 (Foster et al., 2011). Moreover, it has been reported that TiO2 nanoparticles by producing the ROS increase
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330 the permeability of microbial cell membrane and induce destructive effects inside the microbial cells,

331 oxidation of intra cellular Coenzyme A, and peroxidation of lipids which decrease respiratory activity and

332 subsequently cause cell death (Wang, Hu, & Shao, 2017; Zhu, Cai,& Sun, 2018).

333 All treatments were more effective against Gram-positive bacteria than the Gram-negative bacteria

334 and fungi. The different susceptibilities of Gram-negative and Gram-positive bacteria could be related to the

335 differences in cell wall structure, cell physiology, metabolism, or surface charge of bacterial cell (Nikaido,

336 2009). Gram-negative bacteria, apart from the cell membrane, possess an additional outer layer membrane,

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337 consisting of phospholipids, proteinsACCEPTED MANUSCRIPT
and lipopolysaccharides, which is impermeable to most molecules

338 (Nikaido, 2009; Siripatrawan & Vichayakitti, 2016). Whereas, Gram-positive bacteria have no outer

339 membrane although they have a thicker cell wall (Foster et al., 2011; Kiwi & Nadtochenko, 2005). Fungi

340 are more resistant than bacteria because they possess thick cell wall consisting of glucan and chitin (Inoue,

341 1986; Wang et al., 2017).

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342 Many studies also reported that Gram-negative bacteria are more resistant to photocatalytic

343 inactivation because Gram-negative bacteria have a triplelayer cell wall structure which contains a

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344 cytoplasmic inner membrane (IM), a thin peptidoglycan (PG) middle layer and an outer membrane (OM) and

345 exhibited the ability to restrict many molecules passing through the cell membrane , while Gram-positive

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346 bacteria could be more susceptible to destruction by ROS due to the lacking of outer membrane (Foster et

347 al., 2011; Zhu, Cai, & Sun, 2018).

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348 The results suggested that the developed CT nanocomposite film not only exhibited ethylene

349 degradation property but also exhibited antimicrobial activity against bacteria and fungi. It can be concluded
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350 that the antimicrobial inactivation efficiency of the composite film depends on microorganism.

351 4. Conclusion
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352 An addition of TiO2 into chitosan films improved tensile strength and water vapor barrier properties,
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353 but lowered optical transmittance of the film. The nanocomposite films possessed ethylene

354 photodegradation activity. However, an agglomeration of TiO2 at 2% TiO2 affected mechanical and ethylene
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355 photodegradation of the nanocomposites. The CT film also exhibited antimicrobial activity against Gram-
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356 positive and Gram-negative bacteria and fungi. Based on mechanical, barrier, ethylene photodegradation,
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357 and antimicrobial properties, the CT nanocomposite film has potential to be used as active packaging for

358 prolonging storage life of fresh produce. The CT film is expected to be able to reduce microbial load or

359 inhibit their growth and may provide solutions to delay ripening and senescence of fresh produce. Moreover,

360 this bionanocomposite film is considered environmentally friendly and would not cause waste problems after

361 use.

362

363

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364 Acknowledgement ACCEPTED MANUSCRIPT

365 This research was partly supported by the 90th Anniversary of Chulalongkorn University Fund

366 (Ratchadaphiseksomphot Endowment Fund) under Grant No. GCUGR11255725088M. The authors would

367 like to thank Dr. Anawat Suwanagul, Director, Postharvest Technology Division, Thailand Institute of

368 Scientific and Technological Research, for providing facility on ethylene measurement.

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Caption of Figures

Fig. 1. SEM images of (a) TiO2 nanoparticles and cross sectional film and (b) surface of chitosan

(CS) and chitosan-TiO2 nanocomposite films containing 0.25, 0.5, 1, and 2% TiO2 (CT0.25, CT0.5,

CT1, CT2).

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Fig. 2. XRD patterns of TiO2, chitosan (CS) and chitosan-TiO2 nanocomposite films containing 0.25,

0.5, 1, and 2% TiO2 (CT0.25, CT0.5, CT1, CT2).

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Fig. 3. FTIR spectra of TiO2, chitosan (CS) and TiO2-chitosan nanocomposite films containing 0.25,

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0.5, 1, and 2% TiO2 (CT0.25, CT0.5, CT1, CT2).

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Fig. 4. Tensile strength (a), elongation at break (b), optical transmittance (c) and WVP (d) of chitosan
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(CS) and chitosan-TiO2 nanocomposite films containing 0.25, 0.5, 1, and 2% TiO2 (CT0.25, CT0.5,

CT1, CT2). Data are reported as mean ± standard deviation. Different lowercase letters indicate mean
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values with significant difference (P ≤ 0.05).

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Fig. 5. Ethylene photocatalytic degradation of chitosan (CS) and chitosan-TiO2 nanocomposite films
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containing 0.25, 0.5, 1, and 2% TiO2 (CT0.25, CT0.5, CT1, CT2). Data are reported as mean ±

standard deviation of six replicates.

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Fig. 6. A schematic diagram of the possible mechanism of photocatalytic degradation of ethylene and

antimicrobial activity of the chitosan-TiO2 nanocomposite film.

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Fig.7. Growth inhibition of UV light, CS, and CT1 without and with UV illumination (UV, CS,

CS+UV, CT1, and CT1+UV) against Gram-positive (S. aureus) and Gram-negative (E. coli, S.

Typhimurium, P. aeruginosa) bacteria, and fungi (Aspergillus and Penicillium). Data are reported as

mean ± standard deviation of six replicates. Different lowercase letters indicate significant differences

between treatments for each microorganism according to Duncan's multiple range test (P ≤ 0.05).
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Highlight:

• Chitosan- TiO2 nanocomposite film exhibited ethylene photocatalytic degradation

• Chitosan-TiO2 film exhibited antimicrobial activity against bacteria and fungi

• Functional properties of chitosan film were improved by incorporating with TiO2

• Chitosan-TiO2 film has potential to be used as active packaging for fresh produce

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