Accepted Manuscript

Title: Development of wheat gluten/nanocellulose/titanium

dioxide nanocomposites for active food packaging

Author: Nahla A. El-Wakil Enas A. Hassan Ragab E.

Abou-Zeid Alain Dufresne

PII: S0144-8617(15)00112-5
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2015.01.076
Reference: CARP 9669

To appear in:

Received date: 29-10-2014

Revised date: 22-1-2015
Accepted date: 30-1-2015

Please cite this article as: El-Wakil, N. A., Hassan, E. A., Abou-Zeid, R.
E., and Dufresne, A.,Development of wheat gluten/nanocellulose/titanium dioxide
nanocomposites for active food packaging, Carbohydrate Polymers (2015),
http://dx.doi.org/10.1016/j.carbpol.2015.01.076

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1 Development of wheat gluten/nanocellulose/titanium dioxide nanocomposites for
2 active food packaging

3 Nahla A. El-Wakila, Enas A. Hassana*, Ragab E. Abou-Zeida, Alain Dufresneb

4
a
5 Cellulose and Paper Department, National Research Centre, 33 Bohouth st. Dokki Giza.
6 Egypt.
b
7 Grenoble Institute of Technology (Grenoble INP) -The International School of Paper,

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8 Print Media and Biomaterials (Pagora), CS10065, 38402 Saint Martin d’Hères Cedex,

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9 France

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10 ABSTRACT

11 Bionanocomposites were developed by casting /evaporation of wheat gluten (WG),

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12 cellulose nanocrystals (CNC), and TiO2 nanoparticles. The effect of addition of different

13 percentages of CNC, and TiO2 on tensile strength (TS), Young’s modulus and water

14
an
sensitivity was studied. A significant improvement in the studied properties is observed

15 when 7.5% CNC and 0.6% TiO2 is added to WG. WG/CNC 7.5% /0.6% TiO2 blend
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16 suspension was chosen to coat commercial packaging unbleached kraft paper sheets via
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17 1, 2 and 3 coating layers. A significant enhancement of 56% and 53% in Breaking Length

18 and Burst Index, respectively, was achieved for 3 layers coated paper. The antimicrobial
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19 activity of the coated papers, against Saccharomyces cervisiae, Gram- negative bacteria

20 Escherichia coli and Gram- positive bacteria Staphylococcus aureus, was investigated
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21 and expressed in terms of reduction % of surviving number (CFU) of the tested

22 organisms. More than 98.5% reduction in CFU was observed against the organisms
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23 compared to TiO2 - free coated paper.

24 Keywords:

25 Bionanocomposites, cellulose nanocrystals, wheat gluten, TiO2 nanoparticle, active food

26 packaging
*
27 Corresponding author. E-mail address: [email protected]
1

Page 1 of 39
28 1. Introduction

29 Active and intelligent food packaging has become an integral part of food

30 production and distribution in most of the world countries. Packaging food keeps

31 freshness, avoids external contamination and can serve for marketing and as a source of

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32 information to consumers.

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33 Paper, due to its biodegradability and safety towards food, is widely used in food

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34 packaging applications. The porosity and hydrophilicity of paper can easily cause

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35 adsorption of water from the environment or from the food, so paper may lose its

36 physical and mechanical strength and become a suitable media for microorganisms

37
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growth. Coating of paper with other materials such as plastic materials and aluminum, to

38 overcome porosity and hygroscopicity, would result in losing its biodegradability and
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39 recyclability. To overcome this problem, biopolymers produced from natural resources

40 are regarded as a promising alternative since they are abundant, renewable, inexpensive,
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41 environmentally friendly and biodegradable (Bastioli, 2005; Belgacem & Gandini, 2008;

42 Yu, 2009). Polysaccharides are considered from the widely used biopolymers for paper
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43 coating and include starch (Matsui, Larotonda, & Paes, 2004), alginates (Rhim, Lee &
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44 Hong, 2006) and chitosan (Bordenave et al., 2007; Ho et al., 2003; Kjellgren et al., 2006;

45 Krasavtsev et al., 2002; Vartiainen et al., 2004). Lipid compounds, such as long-chain
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46 fatty acids and waxes, as well as long-chain alkanes can be used as coating matrix for

47 Paper and paperboard because of their hydrorepellency (Back, 1995; Despond et al.,

48 2005; Khwaldia et al., 2005; Khwaldia, 2010; Parris et al., 1998; Rodriguez, Batlle &

49 Nerin, 2007).

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50 Also, among biopolymers, plant proteins are the most attractive category. Proteins

51 are heteropolymers constituted of different amino acids which offer a large spectrum of

52 chemical functionalities and thus, various polymer network structures and sources,

53 display good film forming properties and can be used for coating applications. In addition

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54 to their large availability, full biodegradability and non-ecotoxicity, protein- based

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55 materials have interesting barrier and moderate cost for food packaging applications.

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56 Gällstedt, Brottman, & Hedenqvist (2005), Han and Krochta (1999), Han and Krochta

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57 (2001), studied the properties of whey protein coated paper. Soy protein isolate coated

58 paper was found to impart gas and oil barrier as well as adequate mechanical properties

59
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for extending the shelf life of food products (Park et al., 2000; Rhim, Lee & Hong, 2006).

60 Wheat gluten was used as a binder and its solutions showed good film-forming
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61 properties; the resulting coating having a strong adhesion to various substrates as

62 recorded by Derksen, Cuperus & Kolster (1995) and Gastaldi et al., (2007). Corn zein
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63 protein coatings are used as oxygen, moisture, and grease barriers for nuts, candies, and

64 other foods and no interference with paper recycling can be suggested (Trezza, Wiles &
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65 Vergano, 1998). When comparing to whey protein and corn zein, soy protein and wheat
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66 gluten have the advantage of the low cost in order to develop affordable packaging

67 materials (korchta & De Mulder-Johnston, 1997). In spite of this benefit, different

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68 approaches have been explored to overcome the other shortages of wheat gluten and to

69 enhance the mechanical and barrier properties, e.g. casting with montmorillonite (Tunc et

70 al., 2007), modification with hydrophobic materials (Gennadios et al., 1993; Gontard et

71 al., 1994), modification by irradiation (Lee, Lee & Song, 2005), incorporation of

72 carboxylated cellulose nanocrystals and hydroxyethyl cellulose (Rafieian et al., 2014;

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73 Song & Zheng, 2008). Crosslinking with formaldehyde, gluteraldehyde and glyoxal

74 showed significant increase in tensile strength of protein films. Nevertheless, due to the

75 toxicity of the chemicals used to cross-link proteins, the application of chemical

76 modification must raise the issue of the aldehyde residues remaining in the film and their

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77 migration in the event of these materials being used in direct contact with foods

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78 (Hernandez-Mun, Villalobos & Chiralt, 2004).

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79 In spite of the significant enhancement of the mechanical and barrier properties of

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80 wheat gluten – based biocomposites, they can still be easily attacked by microorganisms.

81 TiO2 photocatalyst forms have been developed for antimicrobial purposes since they are

82
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nontoxic and exhibit a notable disinfecting activity against a broad spectrum of

83 microorganisms, due to the high redox potential of the surface species formed by photo
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84 excitation (under UV illumination) affording non-selective oxidative attack of

85 microorganisms (Gumy et al., 2006). The FDA approved TiO2 as a "food contact
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86 substance" as well, meaning that it is safe to incorporate it into food package

87 (Martirosyan & Schneider, 2014). To the best of our knowledge, no report on the
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88 photocatalytic disinfection properties of kraft paper coated with wheat gluten, cellulose
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89 nanocrystals and TiO2 nanoparticles has been conducted.

90 The objective of the present study is to be a step towards using active biobased
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91 packaging in food applications instead of petrochemical based plastics through improving

92 hydrophobicity, mechanical and antimicrobial activity of commercial packaging kraft

93 paper. Nanocomposite films consisting of WG, glycerol, CNC and TiO2 nanoparticles

94 were processed by casting /evaporation. Morphology, tensile strength, water vapor

95 uptake, water vapor permeability and contact angle measurements of the nanocomposite

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 96 are discussed in relation to their ingredient concentration. Antimicrobial activity and

97 mechanical properties of coated unbleached kraft paper sheets with WG/CNC /TiO2,

98 chosen on the basis of tensile strength, was investigated as well.

99 2. Experimental

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100

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2.1 Materials

101 TiO2 718467 nanopowder (particle size ~ 20 nm) and gluten from wheat (~80%

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102 protein) were purchased from Sigma Chemical Co. All other reagents were of analytical

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103 grade. Bleached kraft bagasse pulp was kindly supplied by Qena Company for Pulp and

104 Paper, Qena, Egypt. The chemical composition of bagasse pulp was: α-cellulose 70.6%,

105
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pentosans 26.8%, ash 0.82% and degree of polymerization (number of glucose units in

106 cellulose chain) 1174.

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107 2.2. Preparation of cellulose nanocrystals (CNC) from bagasse pulp

108 CNC were prepared by acid hydrolysis using sulfuric acid (65 wt%) at 45 ◦C for
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109 45 min. The hydrolyzed pulp was centrifuged to remove the excess acid and dialyzed

110 against water for several days with changing water until the pH was similar to that of
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111 distilled water. The obtained CNC were sonicated for two min. under cooling using
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112 750W Sonix ultrasonic processor (USA). Some drops of chloroform were added to the

113 CNC suspension which was stored at 4◦C (Bras et al., 2010). The prepared nanocrystals
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114 were characterized by Atomic force microscopy (AFM) using an AFM Multimode (DI,

115 Veeco, Instrumentation Group) in tapping mode with Multi 130 tips and SEM FEG with

116 a Zeiss ultra 55 column Gemini.

117 2. 3. Preparation of WG and WG/CNC nanocomposites

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118 Wheat gluten films were prepared from a solution of gluten (9.0 g), absolute

119 ethanol (32.5 ml), glycerol (1.8 g) in 100 ml distilled water and ammonium hydroxide to
◦
120 pH 10. All components were mixed under magnetic stirring at 70 C and the solution was

121 centrifuged at 5856g for 6 min. at room temperature (Gontard, Guilbert & Cuq, 1993). To

t
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122 prepare WG/CNC nanocomposites, the aqueous dispersion of CNC was added to the WG

123 solution in different ratios ranging from 2.5 to 12.5% with 2.5 interval and 20% glycerol.

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124 CNC and glycerol percentages are based on wheat gluten content, and the mixture was

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125 stirred overnight under magnetic stirring. The suspension was poured and spread evenly

126 in a Teflon dish of diameter 10 cm and dried at 35 °C for 48 h.

127 2.4. Preparation of WG/CNC/TiO2 nanocomposites

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128 TiO2 nanoparticles (0.2, 0.4, 0.6, 0.8 and 1% based on WG) were added to the
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129 WG/CNC 7.5% suspensions very slowly with intense stirring which therefore may

130 prevent those nanoparticles to form colloid composite with WG molecules in the film
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131 solution. The WG/CNC 7.5% suspension with dispersed TiO2 nanoparticles was poured

132 and spread evenly in a Teflon dish of diameter 10 cm and dried at 35°C for 48 h. All
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133 films used for experiments were equilibrated at 50% RH at 25 °C for 48 h before being
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134 tested.

135 2.5. Scanning electron microscopy (SEM)

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136 Scanning electron microscopy (surfaces and cross sections) was performed for

137 WG, WG/CNC 7.5%, WG/CNC 12.5% and WG/CNC 7.5% / 0.6% TiO2 films using a

138 Jeol JXA 840A system running at 5- 10 keV. Before scanning, samples were coated with

139 gold using a sputter coater system (Edwards Sputter Coater, UK).

140 2.6. Differential Scanning Calorimetry (DSC)

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141 Thermal characterization of the WG, WG/CNC and WG/CNC/TiO2

142 nanocomposites was carried out via DSC using DSC Q100 TA Instruments, USA.

143 Around 10 mg of sample was placed in a DSC cell in glove box. Each sample was heated

144 from -08 to 100 °C at a heating rate of 10 °C min-1. Experiments were carried out under

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145 nitrogen cooling system. Sealed aluminum pans were used to prevent mass loss during

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146 the experiment.

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147 2.7. Water Sensitivity

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148 2.7.1. Contact angle

149 The dynamic contact angle of distilled water drop on the WG, WG/CNC and

150
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WG/CNC/TiO2 nanocomposites was measured with an OCA20 automated and software-

151 controlled Video-Based Contact Angle Meter (Data Physics Instruments GmbH, Filder-
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152 stadt, Germany). Changes in the drop shape over time were recorded and averaged for

153 each sample. All measurements were conducted at 25 °C and 58% RH. At least three
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154 measurements per film were performed.

155 2.7.2. Water vapor uptake test (WVU)

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156 Water vapor uptake of WG, WG/CNC and WG/CNC/TiO2 nanocomposites was
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157 carried out by keeping the films in a closed container having relative humidity of about

158 98% at 25 ◦C. Squared samples (15mm×15mm×0.2 mm) were first dried at 50 ˚C for 24h.
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159 After being weighed, dry samples were stored over K2SO4 saturated salt (98% RH) and

160 weighed after 2, 4, 6, 24 and 48h. WVU was expressed as:

161

Page 7 of 39
162 Where m0 is the weight of the dry sample and mt is the weight of the samples after time.

163 2.7.3. Water vapor permeability (WVP)

164 WVP of WG, WG/CNC and WG/CNC/TiO2 nanocomposites were determined

165 according to Tunc et al., (2007). WVP was gravimetrically determined at 20 ◦C using a

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166 modified (ASTM Standards, 1995) procedure. Samples (discs 40 mm in diameter) were

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167 hermetically sealed (with Teflon seals and silicone grease) in a glass permeation cell

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168 containing distilled water. The permeation cell was placed at 20 ◦C in desiccators

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169 containing silica gel, thus obtaining a RH gradient equal to 100%. Cells were weighed

170 every 2h over a 48h period. At least three samples of each type of film were tested and

171 WVP was calculated from the following equation: an

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172
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173 Where w is the slope of the weight loss versus time (g.s−1), x is the film thickness (mm),

174 Δt is the time of weight loss (s), A is the area of exposed film (mm2) and Δp is the water
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175 vapor pressure differential across the film (at 20 ◦C, Δp = 2.33×103 Pa, assuming that the

176 RH on the silica gel is negligible).

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177 2.8. Coating of WG/CNC/TiO2 on paper

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178 Coating of commercial unbleached paper sheets with WG/ CNC 7.5% and

179 WG/ CNC 7.5%/ TiO2 0.6% suspensions was carried out using a thin film applicator

180 (film applicator M360, ERICHSEN). The coating was performed via 1, 2 and 3 layers.

181 Coating of paper was performed firstly with the first layer; after drying the second one is

182 applied and finally the third layer.

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183 2.9. Mechanical testing

184 Tensile tests for the nanocomposites were carried out using a RSA3 (TA

185 Instruments, USA) equipment working in tensile mode. The measurements were

186 performed at room temperature (~25°C), with a distance between jaws of 10 mm, cross

t
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187 head speed of 0.6 mm.min-1 for the first 250 s, then 1.5 mm.min-1 up to 2000 s, and

188 finally 3 mm.min-1 up to the break; five replicates were tested for each film. The sample

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189 dimensions were 20 mm long and 5 mm wide. Film thickness was measured to the

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190 nearest 0.001 mm with a hand-held digimatic micrometer (QuantuMike Mitutoyo). Four

191 thickness measurements at different positions were taken on each specimen, being in the

192 range of 0.15 to 0.2 mm.

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193 The mechanical properties of coated kraft paper were determined by measuring
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194 the basis weight, thickness, tensile strength and burst according to standards ISO 536,

195 ISO 534, ISO 1924-3 and ISO 1974. The measurements were the average of 5 readings.
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196 2.10. Antimicrobial activity of coated paper

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197 To assess the antimicrobial activity of WG/CNC (control) and

198 WG/CNC/TiO2 coated paper, Gram-positive bacteria (Staphylococcus aureus) and Gram
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199 negative bacteria (Escherichia coli), as well as yeast (Saccharomyces cervisiae), were

200 used as test organisms. A preculture of bacteria and yeast were grown in Tryptic Soy
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201 Broth medium with 0.6% yeast extract overnight at 37 and 30◦C for bacteria and yeast,

202 respectively. The inoculums were prepared by diluting an overnight culture grown in

203 Tryptic Soy Broth medium to 103 times. The method was performed as reported by

204 Chawengkijwanich & Hayata, (2008). Test pieces (a square of 5 cm) were placed in

205 sterilized Petri dishes under aseptic conditions. One ml of each inoculum was distributed
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Page 9 of 39
206 all over the surface of each test piece in its Petri dish and the Petri dish was covered with

207 a lid. Petri dishes containing the test piece inoculated with organisms were placed under

208 ultraviolet lamp (VILBER LOURMAT of wavelength 315–400 nm). Samples were taken

209 in three replicates for 30, 60 and 120 min. After the sample test piece was removed from

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210 the light, it was immediately washed, placed in a sterile cup with 9 ml of sterile distilled

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211 water, and shaken for 10 min. One milliliter of solution was withdrawn at each sampling

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212 and diluted to 1/10,000 with sterile distilled water. Finally, 0.1 ml of the 1/10,000 diluted

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213 solution was plated onto Tryptic Soy agar medium. The plates were incubated for 24 h at

214 37 and 30°C, for the bacteria and yeast, respectively. The colony-forming units (CFU)

215
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were then counted. The percentage reduction was calculated using the following

216 equation:
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217
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218 Where A is the number of surviving cells (CFU- colony forming units) for the flasks

219 containing the treated substrate and B the number of surviving cells from the control.
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220 3. Results and discussion

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221 3.1. Atomic Force Microscopy

222 Bagasse cellulose nanocrystals isolated from bleached kraft bagasse pulp had
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223 length ranging from 143.43 to 202.5 nm and width of 7.5 to 16.56 nm as shown in the

224 AFM image (Fig. 1). The average length and width were obtained using imageJ program.

225 The aspect ratio of the obtained bagasse CNC is therefore around 15.

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226 Figure 1

227

228 3.2. Mechanical properties of WG, WG/CNC and WG/CNC/TiO2 films

229 CNC were blended with wheat gluten and the mixture was cast / evaporated to

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230 prepare bionanocomposites. Scanning electron microscope images (surfaces and cross

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231 sections) of neat WG, WG/ CNC 7.5%, WG/ CNC 12.5% and WG/CNC 7.5%/ 0.6%

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232 TiO2 nanoparticles are shown in Fig. 2. Heterogeneous surface is clear for WG/ CNC

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233 12.5% (images e, f) due to agglomeration of the high concentration of the CNC during

234 the processing. Whereas the images of the other films show a homogeneous surface with

235
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absence of visible CNC and TiO2 nanoparticles aggregates.

236 Figure 2
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237

238 Typical stress–strain curves for WG and WG -based composites loaded with
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239 different CNC percentages are presented in Fig. 3(a) and the corresponding modulus in

240 Fig. 3(c). A gradual increase in the modulus was observed when WG is loaded with
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241 different percentages of CNC. On the other hand, the increase in the CNC content from
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242 0% to 12.5% led to a significant gradual decrease in the elongation at break (EB) from

243 644 % to 76 %, respectively. It is known that the addition of rigid fillers to the soft
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244 matrix causes generally higher stiffness but simultaneously has an embrittling effect on

245 the composite in comparison with the neat polymer matrix [(Majer, Hutaˇr, & Nahlik,

246 2013).

247 Figure 3

248

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249 Filling of WG with 2.5% CNC resulted in an increase in TS from 1.14 × 106 to

250 1.51× 106 Pa respectively. Further filling of CNC content (10 wt %) increased TS to

251 2.16× 106 Pa. The increase in TS is ascribed to the high reinforcing effect which is

252 assigned, as already reported, to a mechanical percolation phenomenon of cellulose

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253 nanocrystals which form a stiff continuous network of cellulosic nanoparticles linked

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254 through hydrogen bonding (Azizi Samir et al., 2004; Dufresne, 2008a,b). The formation

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255 of this continuous network is supposed to be formed above the percolation threshold of

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256 the rod-like nanoparticles. It strongly depends on the aspect ratio of the rod-like

257 reinforcing particles and therefore on the origin of cellulose. For rod-like nanorods with

258
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an aspect ratio of 15, the percolation threshold is 4.7 vol %, i.e.8.9 wt%. However, the

259 composite loaded with more than 10 wt. % CNC (12.5%) exhibited a slight decrease in
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260 the TS (1.94 × 106 Pa). This decrease presumably results from slight agglomeration and

261 non-uniform dispersion of the filler at high filler concentrations as shown in Fig. 2 (e, f).
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262 The same trend has been reported with the addition of nanocellulose (more than 5 %) to

263 alginate (Abdollahi et al., 2013).

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264 Based on this test, WG film loaded with 7.5% CNC was chosen as a control for
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265 the forthcoming sample series (addition of TiO2 nanoparticles) where the stress for this

266 nanocomposite is 1.89× 106 Pa while that for nanocomposite loaded with 10% was 2.02×
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267 106 Pa. This slight enhancement is not imperceptible when 33% CNC is saved. It should

268 be noted that it is not necessary to fill the host matrix with high quantities of filler to

269 achieve high mechanical properties; in other words the properties of the nanocomposites

270 must be compromised for the development of high performance multifunctional

271 composites.

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Page 12 of 39
272 Different percentages (0.2, 0.4, 0.6, 0.8 and 1%) of TiO2 nanoparticles were

273 added to WG/CNC 7.5% blend suspension and the corresponding stress- strain curves

274 and the modulus are shown in Fig. 3(b and d) respectively. The results indicate that the

275 addition of 0.2 and 0.4% TiO2 nanoparticles showed no significant improvement in TS

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276 and decreased the elongation at break when compared to WG/CNC 7.5% nanocomposite.

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277 TS values were increased to 2.67× 106 and 2.50× 106 Pa when 0.6 and 0.8% TiO2

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278 nanoparticles were added to WG/CNC 7.5% respectively; this increase in TS is

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279 accompanied by a decrease in elongation at break (192 % and 172% ). Maximum

280 modulus (3.47 × 107 Pa) was obtained for WG/ CNC 7.5% / 0.6 % TiO2. The

281
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enhancement in mechanical properties of nanocomposites can be attributed to the high

282 compatibility between WG/CNC matrix and dispersed TiO2 nanoparticles through
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283 interfacial interaction as it is clear from SEM images. Further increase of TiO2

284 nanoparticles addition (1%) resulted in a slight decrease in TS to 2.26× 106 Pa which is
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285 still higher than that of WG/CNC 7.5% film (1.89× 106 Pa). This result is in agreement

286 with Zolfi, et al., (2014) who found that addition of 1, 3 and 5% TiO2 nanoparticles to
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287 kefiran-whey protein isolate films significantly decreased the tensile strength and
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288 Young’s modulus, while it increased the elongation at break. Therefore, it can be

289 concluded that the addition of 1% TiO2 nanoparticles and more could decrease the
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290 resistance of the polymer network against fracture. So, we can report that ~ 1% TiO2

291 nanoparticles could be the maximum content of loading protein matrix to avoid

292 agglomeration of the nanoparticles and consequently decrease TS.

293 3.3. Water Sensitivity:

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Page 13 of 39
294 Water sensitivity of protein-based materials is an important character, for the

295 substitution of traditional plastics, especially for packaging applications. The poor

296 resistance of WG films to water is due to the hydrophilic nature of the protein (presence

297 of polar peptides) and to the substantial amount of hydrophilic plasticizer (glycerol)

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298 added to impart adequate film flexibility (Angellier-Coussy et al., 2013). Water

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299 sensitivity was evaluated in this study in terms of final contact angle, water vapor uptake

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300 and water vapor permeability.

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301 3.3.1. Contact angle

302 Water contact angle for neat WG film and WG films loaded with different CNC

303
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contents are tabulated in table 1. As indicated in the table, the contact angle of neat WG

304 is 45.10⁰ which revealed the poor resistance of WG films to water due to the previously
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305 mentioned reasons. Contact angle values increased from 58.88⁰ to 78.48⁰ as CNC content

306 increased from 2.5 to 12.5 % respectively.

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307 Table 1
308
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309 The same trend was obtained when WG/CNC 7.5% blend suspension was loaded
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310 with 0.2 to1% TiO2 with interval 0.2% and the results are also collected in Table 1. A

311 considerable increase of the contact angle is observed when increasing the TiO2 ratio.
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312 40.07% is the increase in contact angle due to addition of 1% TiO2 compared to

313 WG/CNC 7.5% film. The results indicate that increasing the amount of CNC and TiO2

314 tended to prevent the spreading of the water drop over the film surface resulting in an

315 increasing of the surface hydrophobicity.

316 3.3.2. Water vapor uptake (WVU)

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Page 14 of 39
317 In order to secure food safety it is advised to test the packaging materials under worst

318 case conditions (98% RH for WVU and 100% RH for WVP) for the full- anticipated

319 shelf- life (Rhim, and Wang, 2013 & Rhim, 2011).

320 WVU of WG loaded with different amounts of CNC, was studied and compared with

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321 neat WG; the results are presented in Fig.4 (a). The results demonstrated that the presence

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322 of CNC in wheat gluten matrix modifies the WVU property, i.e. the values for all

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323 nanocomposites decreased with increasing CNC content. Gradual decrease of all filled

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324 samples, which reached 50% for WG/CNC 12.5% after 48h of exposure to water vapor,

325 compared to neat WG is obtained. When TiO2 nanoparticles loaded into WG/CNC 7.5%

326
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from 0.2 to 1%, WVU values of the films were also decreased [Fig. 4(b)]. WVU of WG/

327 CNC 7.5 % with 0.2, 0.6 and 1% TiO2 nanoparticles was 10.5, 19.7 and 40.8%,
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328 respectively compared to WG/CNC 7.5% after 48h of exposure to water vapor.

329
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330 Figure 4
331
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332 3.3.3 Water vapor permeability (WVP)

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333 As one of the most important functions of food packaging is often to avoid or at

334 least to decrease the moisture between the food and the surrounding atmosphere; the
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335 WVP of the packaging material should be as low as possible (Han, & Krochta, 1999).

336 The effect of CNC concentration (0, 2.5, 5, 7.5, 10, and 12.5%) based on WG on WVP as

337 a function of time is shown in Fig. 4(c). Loading of WG with 2.5% CNC decreased WVP

338 from 1.63 × 10−9 g.m−1 s−1 KPa−1 for the WG film to 1.28 × 10−9 g.m−1 s−1 KPa−1. Further

339 increase in CNC content (5, 7.5 and 10%) resulted in a slight increase in WVP but still

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Page 15 of 39
340 less than that of WG film. A considerable decrease in WVP was obtained upon filling

341 WG with 12.5% CNC (1.02 × 10−9 g.m−1 s−1 KPa−1).

342 Fig. 4(d) shows the dependence of WVP, upon addition of different percentages

343 TiO2 nanoparticles, on WG/ CNC 7.5%. The results indicated that an increase in TiO2

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344 nanoparticles content decreased the WVP of the film by 10% when filled with 1% TiO2

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345 nanoparticles. The results are in agreement with Zolfi et al., (2014) where kefiran-whey

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346 protein isolate filled with TiO2 nanoparticles reduced WVP, moisture content, moisture

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347 absorption and water solubility by increasing the TiO2 nanoparticles content.

348 From the previous results, water sensitivity of WG was greatly enhanced due to

349
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loading with CNC. This enhancement is clear from increasing contact angle, decreasing

350 WVU and WVP with increasing CNC content. Improvement of the hydrophobic
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351 character of WG, due to increased CNC content, is ascribed to the formation of a rigid

352 nanocrystal network, probably linked by hydrogen bond. Also, the good barrier properties
ed

353 of the prepared nanocomposites, even at concentrations less than the percolation

354 threshold, are ascribed to the high crystallinity of CNC. These results are in accordance
pt

355 with previous observations on adding carboxylated nanocellulose to alginate and chitosan
ce

356 biopolymer matrix. The enhancement in water resistivity of the nanocomposites is

357 ascribed to the crystallinity index of carboxylated nanocellulose and the formation of
Ac

358 hydrogen bond by a dipole–dipole force between the matrix and hydroxyl groups of

359 carboxylated nanocellulose and consequent improvement of the biopolymer matrix

360 cohesiveness (Abdollahi et al., 2013; Li, Zhou & Zhang, 2009).

361 Additional enhancement in water sensitivity as a result of TiO2 nanoparticles

362 addition can be explained due to the low hydrophilicity of TiO2 nanoparticles compared

16

Page 16 of 39
363 with WG and CNC, suggesting the network structure formed by combining TiO2

364 nanoparticles with WG/CNC composite. This combination could prevent the water

365 molecules from absorption and thus, improves the hydrophililic / hydrophobic nature of

366 the film. Also this improvement in water sensitivity could be attributed to enhancement

t
367 of the chains irregularity due to the presence of TiO2 nanoparticles and its low aspect

ip
368 ratio; these results were claimed by Zolfi et al., (2014).

cr
369 3.4 Thermal properties

us
370 The analysis of thermal properties of materials is important to determine their

371 processing temperature range and use. It is possible to determine the main thermal

372
an
characteristics of polymeric systems such as glass transition temperature (Tg) through

373 DSC experiments. For polymers similar to that under investigation DSC tool is the best
M
374 one to get and analyze this criterion. Fig. 5 (a) shows the DSC thermograms for WG and

375 WG filled with 5 wt % and 7.5 wt % CNC as representative examples and their Tg
ed

376 temperatures; a single Tg was observed for each sample. The results indicate that no

377 significant change in the Tg values was observed. Previous studies by Rafieian et al.,
pt

378 (2014) showed no significant influence of carboxylated CNC content on the Tg of WG.
ce

379 Also, several authors did not observe important changes in the Tg of nanocomposites

380 reinforced with cellulose nanofillers (Angles & Dufresne 2000; Azizi Samir et al., 2004;
Ac

381 Grunert & Winter, 2002; Lu, Wang & Drzal, 2008). It means that the molecular mobility

382 of amorphous WG chains is not impacted by the presence of the cellulosic nanofiller.

383 Figure 5

384

17

Page 17 of 39
385 The thermal properties of WG and WG/CNC 7.5 % nanocomposites with

386 different TiO2 nanoparticle contents are also illustrated in Fig. 5 (b). The Tg of the

387 nanocomposite was shifted to higher temperatures by increasing the TiO2 nanoparticle

388 content even for low TiO2. The Tg value for the WG/CNC 7.5 % film was 9.3◦C, whilst

t
389 that of WG/CNC 7.5% / 0.2 % TiO2 nanoparticles was 14.8◦C. By increasing the TiO2

ip
390 nanoparticle content, to 1% Tg increased 18.0˚C. The increment in Tg can be ascribed to

cr
391 the strong interfacial interaction between the TiO2 nanoparticles and the WG/CNC matrix

us
392 without disruption in the regularity of the WG chains. This interfacial interaction was

393 confirmed when Xian-liang et al., (2011) doped titanium dioxide nanoparticles into wheat

394
an
gluten and infrared spectrum analysis was conducted.

395 3.6. Antimicrobial activity of coated paper

M
396 It is expected that paper will stay an important biobased packaging material due to

397 its excellent mechanical properties. However, their water permeability and being a good
ed

398 media for microbial growth are the main drawbacks for many food applications. In this

399 study kraft paper was coated with different layers of WG/ CNC 7.5% /0.6 % TiO2 blend
pt

400 suspension.
ce

401 Antimicrobial activity of WG/ CNC 7.5% as well as WG/ CNC 7.5% / 0.6 %

402 TiO2 coated paper sheets against S. cervisiae yeast, Gram- negative bacteria E. coli and
Ac

403 Gram- positive bacteria S. aureus were tested using the viable cell counting method after

404 UVA light illumination for ½ , 1 and 2h. Regarding the antimicrobial activity of S.

405 cervisiae, 1, 2 and 3 layers coated paper with WG/ CNC 7.5% / 0.6 % TiO2 showed

406 complete inactivation i.e. 100 % reduction after exposure to UVA light illumination for

18

Page 18 of 39
407 half an hour compared to paper coated with WG/ CNC 7.5%. These data are not shown in

408 Table 2. A similar result was explored by Bodaghi et al., (2013).

409 Table 2 shows the colony-forming units (CFU/ml) and the reduction % of

410 surviving number of the tested bacteria of the coated paper with and without TiO2

t
411 nanoparticles. The reduction % depends on the number of coated layers and the time of

ip
412 exposure to UVA light. Upon exposure to UVA light for ½ h the reduction % was 58.3,

cr
413 84.6 and 87.9 % for S. aureus and 21.9, 40.6 and 53.1% for E. coli for 1, 2 and 3 layers,

us
414 respectively.

415
Table 2
416
an
417 Increasing the exposure time to UVA light for 1 and 2h improve the reduction%
M
418 of surviving number of cells. Complete reduction of S. aureus was achieved for 1, 2 and

419 3 layers coated paper sheets after 2h of UVA light illumination. In the case of E. coli,
ed

420 98.5 reduction % was recorded for 3 layers coated paper sheets after 2h exposure to UVA

421 light. These results showed that 2h exposure to UVA light is sufficient for inactivation of
pt

422 S. aureus and E. coli due to its photoactivation effect. No inhibition was observed upon
ce

423 testing the antimicrobial activity of coated paper sheets with WG/ CNC 7.5% /0.6 % TiO2

424 against all the used microorganisms without UVA light illumination. This result is in
Ac

425 accordance with (Chawengkijwanich & Hayata, 2008; Kikuchi et al., 1997; Shieh Li et

426 al., 2006; Yu et al., 2011). The inactivation effect of TiO2 nanoparticles can be explained

427 on the basis of being in an oxygenated aqueous environment and exposed to UVA light

428 with wavelengths above 250 nm, reduction of O2 to form superoxide (O2•) takes place

429 whereas the electropositive holes oxidize water to form hydroxyl radicals (OH•) which
19

Page 19 of 39
430 are generated on the illuminated TiO2 film surface causing oxidation of the membrane

431 lipids and disruption of the outer and cytoplasmic membranes of the bacteria by lipid

432 peroxidation. This leads in turn to the death of the bacterial cells (Blake et al., 1999;

433 Maness et al., 1999; Saito, Iwase & Morioka, 1992).

t
434 After evaluating the antimicrobial activity of the coated paper, it was necessary to

ip
435 ensure that the other paper properties, specially the mechanical properties, were not

cr
436 affected. Coating of paper sheets (1, 2 and 3 layers) with WG/ CNC7.5% /0.6% TiO2

us
437 resulted in an increase 3, 7.4 and 10.7 µm, of the thickness and consequently an increase

438 3.4, 6.4 and 7.7 g/m2 in the basis weight respectively, compared to uncoated paper. A

439
an
strong correlation was observed between the number of coating layers and the mechanical

440 properties (Breaking Length and Burst Index) as observed in Table 3.

M
441 Table3

442
ed

443 A significant enhancement of 56 and 53% in Breaking Length and Burst Index,

444 respectively, was achieved for 3 layers coated paper. This enhancement, in addition to
pt

445 reasons mentioned in sections 3.2, is ascribed to filling the paper mat with the blend
ce

446 suspension and the possible hydrogen bonding interaction between cellulosic nanofibers

447 and the blend suspension ingredients.

Ac

448 4. Conclusion

449 Wheat gluten – based films can act as efficient matrix for incorporating various

450 additives including reinforcing agents and antimicrobial agents. Incorporation of CNC

451 with and without TiO2 nanoparticles, by casting and evaporation, into WG furnished

452 bionanocomposites that have the potential to be used in active food packaging
20

Page 20 of 39
453 applications. An optimal CNC content (7.5%) and TiO2 nanoparticles (0.6%) were

454 approved for improving the functional properties of WG based materials on the basis of

455 TS and water resistance of the bionanocomposites. Moreover, the coated kraft paper with

456 WG/ CNC 7.5%/ 0.6% TiO2 exhibited excellent antimicrobial activities i.e. 100, 100 and

t
457 98.5% against S. cervisiae, E. coli, and S. aureus respectively, for 3 layers coated paper

ip
458 after 2h of exposure to UVA light illumination.

cr
459 Further work is needed to evaluate O2 and CO2 barrier properties of the coated paper

us
460 and to demonstrate the real potential of the prepared paper for packaging applications. Its

461 validity should be proven with food systems under real storage and release conditions.

462 Acknowledgments
an
463 The authors acknowledge the French Ministry of Foreign Affairs (project No. 28656PE)
M
464 and Academy of Scientific Research in Egypt (project No. EGY-FR 9/005) for the

465 financial support.

ed

466 References
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469 nanoparticles. International Journal of Biological Macromolecules, 54, 166–173.

470 Angles, M.N., & Dufresne, A. (2000). Plasticized starch/tunicin whiskers

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471 nanocomposites. 1. Structural analysis. Macromolecules, 33, 8344-8353.

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478 Belgacem, M.N., & Gandini, A., (2008). Materials from vegetable oils: major sources,

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619 casting. Journal of Membrane Science, 289, 159–168.

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621 Chitosan-coated paper: effects of nisin and different acids on the antimicrobial activity.

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623 Xian-liang, S., Sheng-ying, Z., Ming-jun, Z., & Jia-hua, H. W. (2011). Effect of Doping

624 Nano-Titania on Mechanical Properties of Wheat Gluten Films and the Characterization

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626 533-537.

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627 Yu, B., Leung, K. M., Guo, Q., Lau, W. M., & Yang, J. (2011). Synthesis of Ag-TiO2

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628 composite nano thin film for antimicrobial application. Nanotechnology, 22, 115603 (9

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629 pp).

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632
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634 International Journal of Biological Macromolecules, 65, 340–345.

635
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635 Fig.1: Atomic Force Microscopy of hydrolyzed bagasse pulp.

636

637 Fig. 2: SEM micrographs of surfaces and cross sections of neat WG (a, b), WG/CNC
638 7.5% (c, d), WG/CNC 12.5% (e, f) and WG/CNC 7.5% / 0.6%TiO2 (g, h).

639 Fig. 3: Typical stress-strain curves obtained from tensile tests for neat WG & WG /CNC

t
640 (a) and WG /CNC 7.5% /TiO2 (b), Young’s modulus for neat WG &WG / CNC (c) and

ip
641 WG /CNC 7.5% / TiO2 (d). Vertical bar indicates the standard error (SE).
642
Fig. 4: Dependence of WVU and WVP on CNC content (a & c) and on TiO2 content (b &

cr
643
644 d).
645

us
646 Fig. 5: DSC thermograms of WG, WG/CNC 5% & WG/CNC 7.5% (a) and WG/CNC 7.5
647 % / TiO2 (b).

648

649
an
650
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650 Table 1: Contact angle of WG, WG / CNC and WG/CNC 7.5% / TiO2
651

WG/CNC WG/CNC 7.5%/ TiO2

t
Sample Contact angle Sample Contact angle

ip
WG 45.10 ± 1.18 WG /CNC 7.5% 64.04 ± 2.84

cr
2.5 % CNC 58.88 ± 0.74 0.2 % TiO2 67.10 ± 0.11

us
5 % CNC 62.00 ± 0.38 0.4 % TiO2 68.86 ± 0.41

7.5 % CNC 64.04 ± 2.84 0.6 %TiO2 74.63 ± 0.66

10 % CNC 75.19 ± 0.25

an
0.8% TiO2 85.6 ± 2.31

12.5 % CNC 78.48 ± 1.18 1.0% TiO2 89.70 ± 0.14

M
652
ed

653

654
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 654 Table 2: Reduction % of coated paper against, S. aureus and E. coli.

S. aureus E. coli
Time of UVA light exposure (h)

1/2 1 2 1/2 1 2

t
ip
Sample CFU/ml R (%) CFU/ml R (%) CFU/ml R (%) CFU/ml R (%) CFU/ml R (%) CFU/ml R (%)

cr
Blank 2.4×105 - 3.7×105 - 3.1×105 - 3.2×105 - 6.0×105 - 6.6×105 -

us
4 5 5 5
I 1.0×105 58.3 8.0×10 78.4 0 100 2.5×10 21.9 2.9×10 51.7 1.0×10 84.9
layer

II
layers
3.7×104 84.6 1.0×104 97.3 0 100
an
1.9×105 40.6 1.0×105 83.8 3.0×104 95.5

2.9×104 3.0×103 1.5×105 4

1.0×104
M
III 87.9 98.2 0 100 53.1 6.0×10 90.0 98.5
layers

655
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656

657
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658
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658 Table 3: Mechanical properties of coated paper.

Coated Paper
Property
Blank 1 Layer 2 layers 3 layers

Thickness (µm) 112.6 115.6 120.0 123.3

t
ip
Basis Weight (g/m2) 60.0 63.4 66.4 67.7

Breaking Length (km) 6.18 7.18 8.25 9.65

cr
Burst Index (kPa.m2/g) 1.73 2.1 2.43 2.65
659

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660

661

662
an
663
M
664

665
ed

666

667
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668
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669
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669

670 Highlights

671 - New nanocomposite from gluten, cellulose nanocrystals, and titanium dioxide
672 nanoparticles was prepared.

t
673 - Cellulose nanocrystals and titanium dioxide nanoparticles improves mechanical are

ip
674 water barrier properties of gluten films.

675 - Paper sheets coated with the aforementioned nanocomposite.

cr
676 - Coated paper sheets showed good antimicrobial activity against G +ve and G -ve.
677 Bacteria and yeast.

us
678

679
an
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Figure(s)

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Figure(s)

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ip
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