Accepted Manuscript

Preparation and characterization of pullulan-chitosan and pullulan – carboxymethyl

chitosan blended films

Jia Wu, Fang Zhong, Yue Li, C.F. Shoemaker, Wenshui Xia

PII: S0268-005X(12)00077-X
DOI: 10.1016/j.foodhyd.2012.04.002
Reference: FOOHYD 1979

To appear in: Food Hydrocolloids

Received Date: 22 December 2011

Revised Date: 1 April 2012
Accepted Date: 11 April 2012

Please cite this article as: Wu, J., Zhong, F., Li, Y., Shoemaker, C.F., Xia, W., Preparation and
characterization of pullulan-chitosan and pullulan – carboxymethyl chitosan blended films, Food
Hydrocolloids (2012), doi: 10.1016/j.foodhyd.2012.04.002

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 ACCEPTED MANUSCRIPT

GRAPHICAL ABSTRACT

Preparation and characterization of pullulan-chitosan and pullulan

–carboxymethyl chitosan blended films

Jia Wu a, Fang Zhong *a, Yue Li a, C. F. Shoemaker b, Wenshui Xia a

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a
State Key Laboratory of Food Science and Technology, School of Food Science and

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Technology, Jiangnan University, Wu Xi 214122, PR China

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b
Department of Food Science and Technology, University of California, Davis, CA

95616, USA

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1 Preparation and characterization of pullulan-chitosan and pullulan

2 –carboxymethyl chitosan blended films

3 Jia Wu a, Fang Zhong *a, Yue Li a, C. F. Shoemaker b, Wenshui Xia a

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a
4 State Key Laboratory of Food Science and Technology, School of Food Science and

5 Technology, Jiangnan University, Wu Xi 214122, PR China

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b
6 Department of Food Science and Technology, University of California, Davis, CA

7 95616, USA

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∗
8 Corresponding author: Tel.:+8613812536912. Fax: +8651085329060.

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9 E-mail address: fzhong@jiangnan.edu.cn (F.Zhong).

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25 ABSTRACT

26 The characteristics of pullulan-chitosan and pullulan-carboxymethyl chitosan

27 (CMCH) blended films were investigated. The viscosity of the film-forming solutions,

28 mechanical properties, barrier properties (water and oxygen), water solubility, and

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29 color are reported. Fourier transform infrared spectroscopy (FTIR) and X-ray

30 diffraction (XRD) measurements were used to study the interactions and compatibility

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31 between the polysaccharides. The addition of the chitosan or CMCH to pullulan

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32 effectively modified the mechanical and oxygen barrier property of the film. The

33 improvements in the film properties have been related to strong interactions between

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34 chitosan/CMCH and pullulan polymers, which were observed with FTIR spectroscopy.

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36
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The ratio of pullulan and chitosan to achieve films with optimum mechanical

properties, oxygen and water barrier properties was identified to be 1:1. Increased
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37 concentrations of chitosan lead to the formation of inter-molecular hydrogen bonds

38 rather than the intra-molecular hydrogen bonds which were suggested from XRD
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39 measurements.
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41 Keywords: edible film, chitosan, pullulan, carboxymethyl chitosan

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42 1.Introduction

43 Edible films or coatings, refers to any type of material used for enrobing (i.e.,

44 coating or wrapping) various foods to extend their shelf life and may be eaten together

45 with the food, with or without further removal(Milda & Kerry, 2009). This has been

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46 an active research area during the past few decades. It has involved the use of several

47 types of materials, especially polysaccharide-based edible films due to their excellent

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48 biodegradability and unique gas barrier properties.

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49 Pullulan is a water-soluble microbial polysaccharide produced extracellularly by

50 the fungus-like yeast called Aureobasidium pululans. It has excellent film-forming

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51 properties. It produces films that are colorless, tasteless, odorless, transparent,

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53
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heat-sealable and highly impermeable to both oil and oxygen (Yuen, 1974). However,

its application in a pure form for films has been limited by the film’s poor mechanical
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54 properties (brittle, easy to break) and its high cost (Shibata, Nozawa, Teramoto, &

55 Yosomiya, 2002; Tong, Xiao, & Lim, 2008). One effective method to reduce the cost
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56 as well as to improve the film properties of pullulan-based films is to blend it with

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57 relatively cheaper compatible polymers with high-quality mechanical characteristics.

58 Blending is a common and potentially versatile way to develop new films with
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59 improved properties. Some reports have been focused on pullulan blend with proteins,

60 such as whey protein(Mahamadou, Shi, & Zhang, 2007), caseinate (Kristo, Biliaderis,
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61 & Zampraka, 2007), rice protein concentrate(F. F. Shih, 1996). The blending of
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62 pullulan with other polysaccharide-based edible films has been reported, including

63 starch (Biliaderis, Lazaridou, & Arvanitoyannis, 1999), alginate and

64 carboxymethylcellulose (Tong, et al., 2008). Lazaridou et al had studied the

65 thermophysical properties of chitosan-pullulan films near their glass transition

66 (Lazaridou & Biliaderis, 2002).

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67 Chitosan is a cationic polysaccharide biopolymer mainly composed of β

68 (1–4)-2-amino-2-deoxy d-glucopyranose repeating units. It is a deacetylated product

69 of chitin and the degree of acetylation varied in different chitosan samples, which is

70 the second most abundant naturally occurring biopolymer in nature. Chitosan has

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71 been shown to possess high-quality film-forming capabilities coupled with other

72 beneficial properties such as biodegradability, biocompatibility, low oxygen

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73 permeability coefficients (Butler, Vergano, Testin, Bunn, & Wiles, 1996; Suyatma,

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74 Copinet, Tighzert, & Coma, 2004) and desirable mechanical properties.

75 Carboxymethyl chitosan (CMCH) is a water-soluble chitosan derivative which has

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76 been used extensively in a wide range of biomedical applications due to its unique

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chemical, physical, and biological properties and especially its excellent

biocompatibility (Fan, et al., 2006). Davies et al (Davis, Elson, & Hayes,

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79 1988)reported the formation of strong films of CMCH. N, O-carboxymethyl chitosan

80 has been used as the main component of “Nutri-Save” (trademark) for the preserving
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81 of fruits in the United States and Canada.

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82 Other reports have shown that blending chitosan or CMCH with proteins or other

83 polysaccharides could effectively improve the properties of the resultant films. María
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84 et al (Garcia, Pinotti, & Zaritzky, 2006) reported that a chitosan and starch film had

85 improved water barrier properties. Thummanoon et al (Thummanoon, Soottawat, &

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86 Anuchit 2001) also reported that incorporating 10-40% chitosan into protein-based
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87 films increased the tensile strength of the blended film. Although numerous reports

88 are available regarding the properties of the pullulan and chitosan films as well as

89 their blends with starches and gums or proteins, that of pullulan-chitosan and

90 pullulan-CMCH blended films have been limited reports. Many potential applications

91 of pullulan have been hampered by its high cost. Blending chitosan or CMCH with

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92 pullulan has the advantage of not only reducing the cost, but could also enhance the

93 mechanical properties of the films. Both chitosan and CMCH possess antimicrobial

94 activity (Jeon, Kamil, & Shahidi, 2002), thus chitosan and CMCH may introduce

95 antimicrobial activity into pullulan-chitosan or pullulan-CMCH films.

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96 The present study was initiated to compare the mechanical and barrier properties

97 of the chitosan film, CMCH film and pullulan film. Furthermore the properties of the

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98 pullulan-chitosan and pullulan-CMCH blended films were investigated. FTIR (Fourier

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99 transform infrared spectroscopy) and XRD (X-ray diffractgrams) also were used to

100 study the interaction and compatibility between the polysaccharides.

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101 2. Materials and method

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103
2.1. Film preparation.
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Chitosan (85% deacetylated, 300,000 molecular weight) was obtained from
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104 Zhejiang Aoxing biotechnology Co.Ltd, China. N, O-Carboxymethyl chitosan (degree

105 of substitution is 96.5%) was bought from Shandong Haili biotechnology Co. Ltd,
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106 China. Pullulan (600,000 molecular weight) was purchased from the TCI (Japan).
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107 Films in this study were prepared by casting/solvent evaporation method. Film

108 solutions (9 different kinds) were prepared in different ratios (1:3; 2:2; 3:1, Table 1)
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109 by using chitosan, pullulan, and carboxymethyl chitosan. While film-forming

110 solutions which contain chitosan were dispersed in 100 mL of 2% acetic acid solution
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111 for chitosan only dissolve in diluted acid solution, the others were dispersed in
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112 distilled water. Glycerol was added at 15% (w/w with respect to the amount of

113 polysaccharide) in solution, followed by the continual mixing of the mixture for 10

114 min. The resulting dispersion was filtered to remove undispersed particles. The

115 dispersions were allowed to stay overnight to eliminate bubbles.

116 A dispersion (25 mL) was cast into flat and level bottom acrylic plates with the

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117 rim(11cm×11cm×1cm). After drying the films at 45°C, they were peeled from the

118 plates and conditioned in desiccator cabinet at 53% RH (Mg (NO3)2·5H2O) saturated

119 solution) at 25 °C for 72 h.

120 2.2. Rheology test of the film-forming solutions.

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121 The viscosity of the film-forming solutions was measured using a dynamic shear

122 rheometer (Rheometer AR-G2, TA Instruments, USA). The diameter of plate sensor

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123 geometry was 60 mm. All measurements were conducted at 25 °C. The steady shear

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124 measurements (flow curves) were performed in a shear rate range of 1–100 1/s. All

125 the tests were carried out in duplicate.

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126 2.3. Mechanical properties.

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128
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Film thickness measurements which were taken at 10 different points on each

specimen with a micrometer (Guilin, China) and mean values were computed.
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129 Tensile strength and percentage elongation of films were measured, after their

130 equilibration at 53% RH according to ASTM 828-88 (ASTM, 1989) with some
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131 modifications by using a texture analyzer (Stable Micro Systems, Surrey, UK). The
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132 films were initially cut into strips (2 cm × 8 cm) before testing. The initial grip

133 separation and crosshead speed used were set at 50 mm and 0.5 mm/s respectively.
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134 The results were the average of eight samples. Percentage elongation (E %) and

135 tensile strength of the film was calculated with equations (1-2) :
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l − l1
136 E % = 100% × (1)
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l1

maximum force( N )
137 Tensile strength = (2)
thickness( mm) × width( mm)

138 l1 is the initial length of the film, l is the length of the film when it break.

139 2.4. Water vapor permeability determination.

140 The water vapor permeability (WVP) and water vapor transmission rate (WVTR)
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141 were determined according to ASTM E96-00 with some modifications. After

142 equilibration at 53% RH for 72 h, films were sealed in cups containing silica gel (the

143 distance between the silica gel and the film was 9 mm). Test cups were placed in a

144 cabinet with a controlled relative humidity of 90% (BaCl2 saturated solution) at 25 °C.

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145 Periodical weightings monitored the weight changes for every 2 h over 24 h. The

146 gradient was calculated from the slope of a linear regression of the weight increase vs.

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147 time. WVP values were calculated according to the correction method suggested by

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148 Gennadios et al (Gennadios, Weller, & Gooding, 1994) and McHugh et al (McHugh,

149 Avena-Bustillos, & Krochta, 1993):

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150 WVTR = (3)
Film area

151 WVTR =
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P × D × Ln ( P − PA3 ) / ( P − PA2 ) 
(4)
R ×T × Z
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WVTR × thickness (mm)
152 WVP = (5)
PA1 − PA2
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153 Where P = Total pressure; D = Diffusivity of water through air at 25°C; R = Gas
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154 law constant; T = Absolute temperature (298 °K); Z = Mean stagnant air gap height

155 (9 mm). PA1 = Water vapor partial pressure at film outer surface in the cabinet; PA2 =
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156 Corrected water vapor partial pressure at film inner surface in cup; PA3 = Water

157 vapor partial pressure at silica gel surface.

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158 2.5. Water solubility.

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159 Films were preserved in a desiccator (0% RH) for 10 days before the water

160 solubility test. The weight of the 20 mm × 20mm dry sheet film (W0) was measured

161 directly, and then the film was soaked in 5 ml deionized water in 25 ± 1 °C oscillating

162 water bath for 5 min at 50 rpm, followed by a careful separation of the insoluble film.

163 The separated films were first dried in a vacuum drying oven (100 °C) until constant

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164 weights (W1) were obtained. The water solubility (WS) was calculated with equation

165 (6). All the tests were carried out in triplicate.

166 WS = (W 0 − W 1) / W 0 × 100% (6)

167 2.6. Oxygen barrier properties.

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168 A 60 mL jar was filled with 30.0 ml of fresh soybean oil, covered with different

169 films, sealed using bungee, and stored at a controlled temperature (60 °C) for 10 days.

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170 The peroxide value of the soybean oil samples was determined by sodium thiosulfate

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171 titration (Ou, Wang, Tang, Huang, & Jackson, 2005). All the tests were carried out in

172 triplicate.

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173 2.7. Color and film transparency.

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Color of the film was determined by using UltraScan Pro1166 (Hunterlab, USA)

175 and expressed as L*, a* and b*. Film transparency was determined according to the
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176 method of Ubonrat Siripatrawanand by using the UV spectrophotometer (UV-2802H,

177 Unico, Shanghai) and calculated according to equation(7) (Siripatrawan & Harte,
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178 2010):
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log T 600
179 T =− (7)
thickness
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180 Where T600 is the fractional transmittance at 600 nm and thickness is the film

181 thickness (mm). According to this equation, the high values of T indicate lower
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182 transparency and higher degree of opacity. All the tests were carried out in triplicate.
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183 2.8. Scanning electron microscopy (SEM)

184 Scanning electron microscopy pictures of the film samples was recorded by a

185 scanning electron microscope (Quanta-200, FEI, Holland). Films were conditioned in

186 a desiccator at 25±1 before measurement. Samples were attached to double-sided

187 adhesive tape and then mounted on the specimen holder. The samples were sputter

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188 coated with 10 µm thickness of gold under vacuum. The sputtered coated film

189 samples were scanned with an accelerating beam voltage of 10 kV.

190 2.9. FTIR analysis.

191 FTIR spectra of the films were recorded in attenuated total reflection (ATR)

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192 mode using a FTIR spectrometer (Nicolet Nexus 470, Thermo Electron, USA). The

193 films were scanned from 700 to 4000 cm-1 at a 4 cm-1 resolution. Each spectrum

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194 represented an average of 64 consecutive scans.

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195 2.10. XRD analysis.

196 X-ray patterns of films were analyzed using an X-ray diffractometer (D8

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197 Advance, Bruker AXS Germany) with Cu Kα (λ=0.154 06 nm) radiation at a voltage

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199
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of 40 kV and 40 mA. The samples were scanned between 2θ = 2–40° with a scanning

speed of 4° min−1. Prior to testing, the samples were stored in a desiccator (0 % RH).
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200 2.10. Statistics.

201 Mean values and standard deviations were obtained using the GLM procedure in
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202 SAS Software (Release 8.0, SAS Institute Inc.). Significantly (P<0.05) different
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203 means were separated using Duncan’s multiple range tests.

204
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205 3. Results and discussion

206 3.1. Rheological properties of the film-forming solutions.

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207 The presence or absence of defects in thin liquid films after coating depends
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208 partly on the rheological properties of film-forming solutions (Peressini, Bravin,

209 Lapasin, Rizzotti, & Sensidoni, 2003). A positive character of the film forming

210 solutions is the presence of a moderate viscosity for high or low viscosity will lead to

211 nonuniform film.

212 The viscosities of the 9 film-forming dispersions of pullulan, chitosan, CMCH

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213 and their blends were measured, Figure 1. The pullulan solution had a significantly

214 (p≤0.05) lower viscosity (0.0064 Pa.s) compared with that of chitosan (0.37 Pa.s) and

215 CMCH, which has been ascribed to flexible linear structure of the pullulan polymer.

216 CMCH dispersion showed shear-thinning behavior under steady state shear flow, its

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217 viscosity decreased from 0.54 to 0.44 Pa.s when shear rate increased from 1 to 100 1/s.

218 When pullulan was blended with CMCH, all the blended dispersions showed

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219 shear-thinning behaviors at varying degree. It was shown that with the increase of the

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220 chitosan or CMCH amount in blended solutions, the viscosity of the blend increased

221 sharply (pul: chi=3:1, 0.042 Pa.s; pul: chi=2:2, 0.096 Pa.s; pul: chi=1:3, 0.21 Pa.s).

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222 The low-viscosity pullulan dispersion could be easily effected by the environment

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224
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during the drying process and eventually resulting nonuniform film. With the

increasing addition of the chitosan or the CMCH, the viscosity of the blend solution
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225 both increased. In the end, by changing the proportion of the chitosan or CMCH, the

226 suitable processing viscosity of blend solutions could be reached.

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227 3.2. Mechanical properties.

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228 Film thickness, tensile strength and elongation at the break of the pullulan,

229 chitosan, CMCH and blended films were measured, Table 2. The tensile strength
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230 measured film strength, and the elongation at the break was an indicator of the film

231 flexibility. It was found that chitosan film had higher film thickness (69.8 µm) and E%
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232 (19.2%) than the pullulan (47.3 µm, 1.27%) and CMCH film (48.1 µm, 5.64%) while
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233 the CMCH film (65.2 Mpa) had the highest tensile strength, which suggested that the

234 chitosan film was much more flexible than the others and the CMCH film was the

235 toughest one (p ≤ 0.05).

236 The results showed for the blends, the E% increased with the increased of the

237 chitosan/CMCH to pullulan ratio, suggesting that the addition of the chitosan or the

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238 CMCH modified the flexibility of the pullulan film. In the case of the

239 pullulan-chitosan blended film, film thickness increased with the increase of the

240 chitosan content, while there was no significant difference between the

241 pullulan-CMCH blended films (p≤0.05).

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242 For the pullulan-CMCH blended films, when the CMCH to pullulan ratio

243 reached 3:1 the blended film showed higher tensile strength than that of CMCH and

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244 pullulan films. The observed increase in tensile strength following the increase of

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245 CMCH content could have been caused by the formation of inter-molecular hydrogen

246 bonds between the COO- group of the CMCH backbone and hydroxyl group of the

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247 pullulan. The observed differences in tensile strength as a function of the chitosan to

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249
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pullulan ratio were not simple. The tensile strength of the pullulan-chitosan film

initially increased upon increasing the content of chitosan. This may have been related
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250 to a raising level of the chitosan in the film would increase the number of amino

251 groups, resulting into the strengthening of the inter-molecular hydrogen bonds and
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252 consequently, led to an improved tensile strength. The maximum tensile strength
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253 occurred at the pullulan to chitosan ratio of 2:2 suggesting that equal amount of the

254 both materials (pullulan and chitosan) could have provided the favorable formation of
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255 inter-molecular hydrogen bonds between amino groups of the chitosan backbone and

256 hydroxyl group of the pullulan. However, further increase of the ratio above 2:2 led to
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257 a reduction in tensile strength of film, possibly because the higher ratio favored the
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258 chitosan intra-molecular hydrogen bond formation at the expense of the

259 inter-molecular hydrogen bonds resulting in poor tensile strength of the film. Xu (Xu,

260 Kim, Hanna, & Nag, 2005) reported that at the higher starch to chitosan ratios, a phase

261 separation between the two main components has been observed which lead to the

262 decrease of the tensile strength. Shih Chao Ming (C.-M. Shih, Shieh, & Twu, 2009)

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263 also proved that for the cellulose/chitosan blended film, when the chitosan content

264 reach 5%, phase separation has been observed.

265 In other words, the pullulan-CMCH blended film showed higher tensile strength

266 with lower flexibility than that of pullulan-chitosan ones, which could be ascribe to

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267 the properties of the chitosan (highest elongation at break among the three

268 films,19.2%, Table 2) and CMCH film (toughest film among the three films, 65.2

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269 Mpa, Table 2). The addition of the chitosan/CMCH into the pullulan film could

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270 effectively modify the strength and the flexibility of the pullulan film. Experimental

271 results showed that when chitosan and pullulan were mixed in the same proportion,

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272 the blended film exhibited the best mechanical performance.

273

274
3.3. Color and film transparency.
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Film color is an important index in terms of general appearance and consumer
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275 acceptance. L*, a* and b* values of chitosan, pullulan, CMCH, pullulan-chitosan, and

276 pullulan-CMCH blended films were shown in Table 2. There were no significant
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277 differences (p≤ 0.05) between all these films in L* value. In general, both the chitosan
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278 and CMCH film had a slight yellow appearance, and darken as thickness increased.

279 Chitosan films were transparent with slight distortions caused by shrinkage as
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280 reported by Butler et al (Butler, et al., 1996). The blended films had increases in a*

281 and b* values with increasing chitosan or CMCH levels indicating that the film
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282 became more yellowish following the incorporation of chitosan or CMCH. This
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283 observation was in agreement with the findings of Thummanoon et al (Thummanoon,

284 Soottawat, & Anuchit, 2007). The differences observed in the films’ color could be

285 ascribed to non-enzymatic browning reactions that may have occurred during the film

286 drying process and storage. It has been previously reported that glycerol added in the

287 chitosan film may be oxidized causing the products probably to react with chitosan

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288 through the Maillard reaction (Zhong, Song, & Li, 2011). The increase in chitosan to

289 pullulan ratio would result in the increase in the numbers of the free amino group.

290 Thus, the browning via Maillard reaction could be enhanced as chitosan levels

291 increased, causing an increase in yellowness (b* value). Moreover, the presence of

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292 free amino groups in the structure may not be entirely underrated regarding their

293 contribution to the yellow color intensity of CMCH film.

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294 The transparency of chitosan, pullulan, CMCH, pullulan-chitosan, and

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295 pullulan-CMCH films were summarized in Table 2. The transparency of the pullulan

296 film was low indicated by the high value of T, which could be linked to the drying

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297 process where the low viscosity of pullulan film forming solution was easily

298

299
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influenced by the wind in the oven indicated by the visibility of ripples on the surface

of the pullulan film which undoubtedly, finally increased the T value. There were no
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300 significant differences between the pullulan-chitosan/CMCH films in T values (p≤

301 0.05) which indicated the amount of chitosan or CMCH did not affect the
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302 transparency of the films.

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303 3.4. Water vapor permeability.

304 Water vapor permeability is an important criterion for many practical

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305 applications of edible films. Low WVP or WVTR values widen the application of a

306 composite packaging film, especially in a highly humid environment. The WVP and
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307 WVTR values of chitosan, pullulan, CMCH, pullulan-chitosan, and pullulan-CMCH

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308 blended films were measured and shown in Figure 2. Pullulan films had a lower WVP

309 value (0.38 g.mm/m2.h.kpa) compared to those of both chitosan (0.65 g.mm/m2.h.kpa)

310 and CMCH (0.85g.mm/m2.h.kpa) films. Addition of chitosan or CMCH to pullulan

311 films resulted in increased WVP values of the films, probably due to the increased

312 free-volume of the composite matrix caused by the bulkier side groups of chitosan and

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313 CMCH.

314 The variation in the WVTR values of the pullulan-CMCH films showed similar

315 trends with their WVP values. However, the WVTR values of the pullulan-chitosan

316 films as a function of chitosan to pullulan ratio did not

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317 show a similar trend as the WVP values. The WVTR values of the films first

318 decreased with the increase of chitosan content until 2:2, reaching 18.52 g/m2.h, due

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319 to interactions between chitosan and pullulan molecules which might have prevented

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320 water molecules from diffusing through the films. Increasing the chitosan to pullulan

321 ratio above 2:2, saw an increasing WVTR value owing to the increase of the chitosan

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322 intra-molecular hydrogen bonds formation instead of the inter-molecular hydrogen

323

324 the film thickness.

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bonds. The different trend of the WVP and the WVTR values was mainly attributed to
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325 The influence of thickness on the pullulan and chitosan film was also critically

326 examined, Figure 3. The thickness of both pullulan and chitosan films exhibited
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327 similar WVP values from 20 µm thickness to 50 µm thickness. Chitosan film had
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328 better WVP value compared with pullulan film with the same film thickness when the

329 thickness above 50µm.

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330 WVP and WVTR values of films are commonly measured using the ASTM E96

331 standard method (cup method). However, a shortcoming of this method is that a
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332 stagnant air layer exists between the underside of the film mounted on the cup and the
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333 surface of the desiccant, saturated salt solution or distilled water contained in the cup,

334 thereby increasing risk of introducing errors in the measurement of the WVP of the

335 hydrophilic films (Gennadios, et al., 1994). Mchugh et al (McHugh, et al., 1993) has

336 proposed a correction method for the WVP calculation and this was adopted to correct

337 the errors of WVP measurements accordingly.

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338 Using the proposed correction method of Mchugh to treat the data from this

339 study did not yield the desired results since the WVP and film thickness showed a

340 slope relationship (chitosan film: R2=0.9937, Y=0.0049X+0.1481; pullulan film

341 R2=0.9893, Y=0.0087X+0.0041). Ghorpade et al (Ghorpade, Li, Gennadios, & Hanna,

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342 1995) studied soy protein films which showed the similar relationship (R2=0.99).

343 Mchugh and Bertuzzi (Bertuzzi, Castro Vidaurre, Armada, & Gottifredi, 2007) also

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344 got a similar result with starch based edible films. As film thickness increased, the

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345 film provided an increased resistance to mass transfer across it, and the equilibrium

346 water vapor partial pressure at the inner film surface changed. So different film

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347 thickness will result in different test RH condition, under different RH conditions,

348

349
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attractive forces between films and water became different, causing film swelling

owing to the varying film structures, as well as the thickness effect (McHugh, et al.,
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350 1993).

351 3.5. Water solubility.

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352 The water solubility of edible films was determined in order to establish film
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353 performance and, therefore, possible applications in the frame of food applications.

354 The water solubility of the pullulan, chitosan, CMCH, pullulan-chitosan and
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355 pullulan-CMCH blended films were measured, Figure 4. The solubility data showed

356 that pullulan and CMCH films dissolved more easily in water than the chitosan film.
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357 The observed differences in solubility may be related to the structures of the materials
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358 where the chitosan film has a crystalline structure, different from that of the

359 amorphous structure of pullulan and CMCH films. When the chitosan film or

360 pullulan-chitosan blended films were mixed with water, both films exhibited a slow

361 dissolution since they have to go through the steps including water absorption,

362 swelling and softening before the dissolution process. The CMCH film appeared to

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363 possess better water solubility, this might as a result of the loss of crystallization in the

364 chitosan film caused by the modification of the chitosan to CMCH making it easier

365 for water to permeate into the CMCH film. In the case of the pullulan film, a direct

366 dissolution occurred in water. Film swelling has been reported to be associated with

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367 water diffusion, ionization of amino or carboxyl groups, dissociation of hydrogen and

368 ionic bonds, and polymer relaxation(Mathew, Brahmakumar, & Abraham, 2006b).

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369 When the chitosan to pullulan ratio increased in the blended film, the number of the

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370 amino groups increased accordingly causing WS decreased. It is note worthy to

371 mention that all the pullulan-CMCH blended films rapidly and completely dissolved

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372 in water suggesting that such blended films could be potentially used as better edible

373

374 3.6. Oxygen barrier properties.

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films for enrobing products directly consumed by consumers.
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375 Edible films were used to cover fresh soybean oil-filled jars to test their oxygen

376 barrier properties in the prevention of the oxidation of soybean oil. The PV (peroxide
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377 value) of the oil covered by chitosan, pullulan, CMCH, pullulan-chitosan, and
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378 pullulan-CMCH films were measured, Figure 5. The PV of the oil without the

379 coverage of film was 156.92 ± 1.56 meq/kg. The results showed a distinct degree of
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380 variations in oxidation prevention capacity of the various films. The oxidation

381 prevention ability of the chitosan film was apparently greater than (91.82 ± 4.59
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382 meq/kg) those of both pullulan (114.69 ± 0.32 meq/kg) and CMCH film (102.33 ±
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383 1.67 meq/kg).

384 The PV decreased with the increase of the chitosan to pullulan ratio and CMCH

385 to pullulan ratio until a ratio of 2:2 was achieved, which fostered the inter-molecular

386 bonding necessary to strengthen the chitosan-pullulan and pullulan-CMCH hydrogen

387 bonds. However further increases of the chitosan to pullulan and CMCH to pullulan

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388 ratios, led to increase in the PV values indicating that chitosan and CMCH

389 intra-molecular hydrogen bonds became stronger at the expense of the inter-molecular

390 hydrogen bonds. Film thickness also affects the film oxygen barrier properties.

391 Pullulan and CMCH films which were much thinner than the chitosan film had worse

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392 performance than the chitosan film in this test.

393 On the whole, the addition of the chitosan or CMCH into the pullulan film

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394 strengthens the oxygen resistance ability of the film, especially when chitosan or

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395 CMCH to pullulan ratio reached equal levels. The blended films could therefore be

396 used to extend the shelf life of foods by decreasing the oxygen permeability.

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397 3.7. Scanning electron microscopy

398

399
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The morphology of polymer films at the surface can be investigated by SEM.

The scanning electron micrograph of the surfaces of the films and the blend films are
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400 shown in Fig.6. All the films were found to be smooth, homogenous except when the

401 blend ratio of the pullulan-chitosan film was 1:3. The smooth and compact structure
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402 of the blend suggested the high miscibility between the pullulan and chitosan/CMCH.
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403 However, for the pullulan-chitosan blend film high miscibility occurred when the

404 blend ratios were 3:1 and 2:2. And with increasing chitosan content, the morphology
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405 of the blend film surfaces changed from smooth to rough, which may be related to the

406 micro-phase separation of the blend film.

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407 3.8. FTIR analysis.

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408 FTIR spectroscopy was used to examine the interactions between pullulan and

409 chitosan or pullulan and CMCH. The infrared spectra of pullulan, chitosan, CMCH,

410 pullulan-chitosan, and pullulan-CMCH blended films were measured, Figure 7 and

411 figure 8.

412 For the pullulan film (Figure 7 and figure 8), a broad peak located around 3331

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413 cm-1 was assigned to O–H stretching which was affected by the intermolecular or

414 intramolecular hydrogen bonds. The peak around 2923 cm-1 corresponded to the C–H

415 stretching, while the peak between1200-1030 cm-1 was assigned to the C-O stretching

416 vibration. The peak located at 1150 cm-1 on other hand, appeared to be the

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417 polysaccharide (1→4) glycosidic bond stretching vibration. The chitosan spectrum

418 (Figure 7a) was similar to previous reports (Martínez-Camacho, et al., 2010; Wang,

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419 Dong, Du, & Kennedy, 2007). The broad band at 3249 cm−1 was the O–H stretching,

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420 which overlaps the N–H stretching in the same region and also affected by the

421 intermolecular/ intramolecular hydrogen bonds. The bands from 1025 to 1152 cm−1

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422 corresponded to the C–O bond stretching. The unique spectral feature of chitosan film

was observed around 1550 cm-1, which corresponded to the N-H bending (amide II).
423

424
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The carboxymethyl chitosan film (Figure 8) exhibited peaks around 1588 cm−1 and
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425 1407 cm−1, which have been assigned to antisymmetric and symmetric vibrations for

426 COO- group, respectively.

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427 When two or more substances are mixed, physical blends versus chemical
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428 interactions are reflected by changes in characteristic spectra peaks (Guan, Liu, Zhang,

429 & Yao, 1998). In the spectrum of pullulan-chitosan blended film (Figure 7), the amino
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430 peak of chitosan shifted from 1550 to around 1559 cm−1 when mixed with pullulan

431 (pul: chi=3:1, 1559.5 cm−1; pul: chi=2:2, 1558.7 cm−1; pul: chi=1:3, 1558 cm−1). This
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432 result indicated that strong interactions were present between the hydroxyl groups of
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433 pullulan and the amino groups of chitosan confirming our early results in this study

434 regarding the change of the properties of the blended film. Xu (Xu, et al., 2005)

435 reported that when the chitosan blend with the corn starch, the amino peak of chitosan

436 shifted from 1578 to 1584 cm−1. Mathew’s (Mathew, Brahmakumar, & Abraham,

437 2006a) research proved that the characteristic peak of starch and the amide peaks of

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438 chitosan shifted to a higher frequency, indicating the interaction between the NH3+ of

439 the chitosan and hydroxyl groups of starch. In the case of the pullulan-CMCH blended

440 film (Figure 8) , the antisymmetric and symmetric vibrations for COO- group located

441 at the 1588 cm−1 and 1407 cm−1 shifted to 1591 cm−1 and 1411 cm−1 (pul: CMCH=3:1,

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442 1591 cm−1 ,1411 cm−1; pul: CMCH=2:2, 1591 cm−1 ,1411 cm−1; pul: CMCH=1:3,

443 1590 cm−1 ,1411 cm−1), This result indicated that interactions were present between

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444 the hydroxyl groups of pullulan and the COO- groups of CMCH; however, the

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445 interaction between the pullulan and the CMCH was not as strong as the interaction

446 between the pullulan and the chitosan.

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447 This FITR result indicated that strong interactions were present between the

448

449
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hydroxyl groups of pullulan and the amino groups of chitosan which lead to the

change of the properties of the blended film.

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450 3.9. XRD analysis.

451 X-ray diffractgrams of chitosan, pullulan, CMCH, pullulan-chitosan, and

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452 pullulan-CMCH blended films were measured, Figure 9. Kozo ogawa et al (Ogawa,
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453 Yui, & Okuyama, 2004) while studying structures of several kinds of chitosan

454 solubilized in different acids found different conformations. These authors proposed
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455 three forms: noncrystalline, hydrated (“tendon”), and anhydrous (“annealed”)

456 crystalline. The hydrated crystalline structure reflects at 2θ = 10◦ (or peaks at around
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457 8◦ and 12◦), while the anhydrous structure at 2θ=15◦. In this study, chitosan film
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458 exhibited a similar behavior. Chitosan film (Figure 9a) exhibited three crystal peaks

459 that appeared at 2θ = 8.5◦, 11.5◦ and 18◦. According to Rivero (Rivero, García, &

460 Pinotti, 2010) the first two peaks corresponded to the hydrated crystalline structure,

461 while the broad peak around 2θ = 23◦ indicated the existence of an amorphous

462 structure.

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463 The pattern of pullulan, Figure 9, had a very weak broad peak around 2θ = 13.4◦,

464 indicating that pullulan was an amorphous material. If chitosan and pullulan have very

465 low compatibility, each polymer would have its own crystal region in the mixtures.

466 Accordingly, X-ray diffraction patterns would be expressed as simple mixed patterns

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467 of chitosan and pullulan with the same ratio as those for blending. When these two

468 film-forming components were mixed, the three peaks of chitosan film could not be

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469 observed. This could be explained by the strong interaction between chitosan and

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470 pullulan which might have caused the destruction of the close packing of the chitosan

471 molecules necessary for the formation of regular crystallites. However, the peak

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472 around 2θ = 23◦ still existed in the X-ray diffractgrams of the pullulan-chitosan

473

474
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blended film (pullulan: chitosan=1:3) suggesting that the action of stronger

inter-molecular hydrogen bonds of chitosan come into play to maintain the structure
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475 compared with that of the pullulan-chitosan blended film with other ratios. This was

476 consistent with the results of the WVTR and oxygen barrier property (PV value) as
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477 both increased noticeably as well as the decrease of the tensile strength when the
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478 pullulan to chitosan ratio reach 1:3 due to change of the interaction between the

479 chitosan and pullulan.

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480 Compared with the chitosan film, the CMCH film, Figure 9b, only exhibited two

481 weak board peaks located at 2θ = 13.8◦ and 2θ = 21.4◦, indicating when the chitosan
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482 was modified, the structure of the CMCH underwent destruction resulting into
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483 amorphous structure. When pullulan was mixed with CMCH, the resultant films all

484 had very weak broad peaks around 2θ = 13.6◦. The peak around 2θ = 21.4◦ apparently

485 disappeared, probably due to the hydrogen bond interactions between CMCH and

486 pullulan.

487 The results of X-ray diffraction reinforced the existence of good compatibility

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488 between the CMCH and pullulan due to the strong hydrogen bonds interactions, and

489 the pullulan and chitosan showed good compatibility with ratios of 3:1 and 2:2.

490

491 4. Conclusion

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492 In conclusion, all of these three kinds of polysaccharides showed good

493 film-forming properties, chitosan film was much more flexible than the others and the

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494 CMCH film was the toughest one while the pullulan film showed the best water

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495 barrier property and water solubility. The optimum ratio of blended pul-chitosan film

496 lead to strong, stable, and flexible film was 1:1 (tensile strength was increased by

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497 29.2% and the oxygen permeability rate has been decreased by 23% compared with

498

499
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pullulan film). Strong interactions between functional groups of chitosan/CMCH and

pullulan have been verified by FTIR and XRD analysis. Both of the pul-Chitosan and
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500 pul-CMCH blended film show potential to be used as edible film.

501
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502 Acknowledgment
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503 We gratefully acknowledge the financial support of the Nature Science

504 Foundation 30871744, 30901000. This work was also supported by the
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505 SKLF-MB-200805, 111 project-B07029 and 125 project 2011BAD23B02.

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506 References

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582 Yuen, S. (1974). Pullulan and its applications. Process Biochem, 9(9), 7-9.

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584 starch-chitosan composite films as a function of acid solvent types. Carbohydrate Polymers,

585 84(1), 335-342.

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586 Table 1. Composition of film-forming dispersions

Weight of dry matter (g) in dispersion (100 mL)

Chitosan Pullulan CMCH

Chitosan 4 0 0

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chi :pul=3:1 3 1 0
chi :pul=2:2 2 2 0
chi :pul=1:3 1 3 0
Pullulan 0 4 0

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CMCH :pul=3:1 0 1 3
CMCH :pul=2:2 0 2 2
CMCH :pul=1:3 0 3 1
CMCH 0 0 4

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587 Table 2. Mechanical properties, color and transparency of pullulan, chitosan, CMCH films and their blended film a

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Film thickness Tensile strength
E% L* a* b* T
(µm) (Mpa)

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A D A A C A
Chi 69.8± 4.8 48.8 ±3.0 19.2%± 1.8% 84.3± 0.12 -1.32± 0.12 3.96± 0.43 0.65± 0.02 D
B C C A C A
Chi: pul= 3:1 62.8±3.1 58.4± 3.1 3.61 %± 0.52 % 84.7±0.24 -1.53± 0.07 3.79± 0.29 0.66± 0.15 D
B BC CD A C B
Chi: pul =2:2 60.3± 5.5 61.5± 4.5 2.78 %± 0.28 % 85.0± 0.08 -1.49± 0.13 3.31± 0.47 0.82± 0.03 CD

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C D D A D C
Chi: pul =1:3 56.1± 2.3 47.9±2.4 2.30 %± 0.36 % 85.2± 0.06 -1.50± 0.09 2.83± 0.25 0.77± 0.10 CD
DE D E A A G
pul 47.3± 4.7 47.6±4.3 1.27 %± 0.18 % 85.3± 0.04 -0.83± 0.08 0.75± 0.06 1.78± 0.11 A
D D DE A B F
CMCH: pul =1:3 51.4± 2.3 45.9±1.5 1.85 %±0.27 % 84.8± 1.25 -1.02± 0.06 1.21± 0.08 0.91± 0.04 BC
DE B DE A B EF
0.81± 0.01 CD

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CMCH: pul =2:2 49.8± 4.3 65.0±5.8 2.03 %±0.44 % 85.3± 0.10 -1.09± 0.06 1.57± 0.18
E A D A B E
CMCH: pul =3:1 45.9±1.5 73.6±4.6 2.43 %±0.48 % 85.2±0.16 -1.13± 0.02 1.64± 0.17 1.08± 0.05 B

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DE B B A B D
CMCH 48.1± 2.5 65.2±4.2 5.64%±0.85% 85.0± 0.11 -1.12± 0.005 2.07± 0.20 0.87±0.08 CD
a
588 Values are given as mean ± standard deviation. Different letters in the same column indicate significantly different (p ≤ 0.05)

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589 Figure caption:

590 Fig.1. Viscosity of pullulan-chitosan blended dispersions (a), pullulan-CMCH blended

591 solutions (b).

592 Fig.2. WVP and WVTR values of pullulan-chitosan blended films (a), and

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593 pullulan-CMCH blended films (b).

594 Fig.3. The thickness effect of pullulan and chitosan film.

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595 Fig.4. Water solubility of the pullulan-chitosan and pullulan-CMCH blended films.

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596 Fig.5. PV of the oil samples protect by pullulan-chitosan and pullulan-CMCH blended

597 films.

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598 Fig.6. Scanning electron microscopy pictures of the film samples.

599

600 partial enlargement (b).

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Fig.7. FTIR spectroscopy of the pullulan-chitosan blended films (a), drawing of
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601 Fig.8. FTIR spectroscopy of the pullulan-CMCH blended films (a), drawing of partial

602 enlargement (b).

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603 Fig.9. X-ray diffraction patterns of pullulan–chitosan blended films (a) and
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604 pullulan-CMCH blended films (b).

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Figure 1

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Figure 2

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Chitosan pullulan CMCH

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Chitosan: pullulan=1:3 Chitosan: pullulan=2:2 Chitosan: pullulan =3:1
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CMCH: pullulan=1:3 CMCH: pullulan=2:2 CMCH: pullulan =3:1

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Figure 6
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 Absor bance

0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08

-0.04
-0.03
-0.02
-0.01

Pullulan
1800
Chitosan

Pul:chi=3:1
Pul:chi=2:2
Pul:chi=1:3
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C

1600
1559.48 1558.70 1557.91 1550.07
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1410.24 1409.86 1408.49 1407.59

1400
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1362.48
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(b)

Figure 7
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Wav enumbers (cm-1)

1200
1152.51
1150.84 1151.53 1151.99 1152.34
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1079.16 1078.68 1070.37
1079.05
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1020.91
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1000
1023.19
1023.93
930.59 929.37 928.11 1025.00 1025.50
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852.81

800
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1056.84
0.10

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1588.42

1040.13
0.09
1406.94

1023.90
0.08 AN
1322.66

1022.42
0.07
1589.94

1410.86

CMCH
0.06
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Absorbance

1020.91
0.05 Pul: CMCH=1:3
1410.98
1591.19

0.04

927.96
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Pul: CMCH=2:2
0.03
1410.82
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930.59 927.61
1590.88

0.02 Pul: CMCH=3:1

1362.48

0.01
Pullulan
0.00
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1700 1600 1500 1400 1300 1200 1100 1000 900

Wav enumbers (cm-1)
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