Effects of anthocyanin-rich purple and black eggplant extracts on the physical, antioxidant and pH-sensitive properties of chitosan film

Accepted Manuscript Effects of anthocyanin-rich purple and black eggplant extracts on the physical, antioxidant and pH-sensitive properties of chitosan film

Huimin Yong, Xingchi Wang, Xin Zhang, Yunpeng Liu, Yan Qin, Jun Liu PII:

S0268-005X(19)30263-2

DOI:

10.1016/j.foodhyd.2019.03.012

Reference:

FOOHYD 4991

To appear in:

Food Hydrocolloids

Received Date:

31 January 2019

Accepted Date:

06 March 2019

Please cite this article as: Huimin Yong, Xingchi Wang, Xin Zhang, Yunpeng Liu, Yan Qin, Jun Liu, Effects of anthocyanin-rich purple and black eggplant extracts on the physical, antioxidant and pHsensitive properties of chitosan film, Food Hydrocolloids (2019), doi: 10.1016/j.foodhyd. 2019.03.012

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pH 2

pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

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CS-PEE I film CS-PEE II film

PEE

CS-PEE III film

Purple eggplants

Antioxidant and pH-sensitive food packaging films

BEE

CS-BEE I film

CS film CS-BEE II film CS-BEE III film

Black eggplants CS: chitosan; PEE: purple eggplant extract; BEE: black eggplant extract

Graphical abstract

pH 12

pH 13

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Effects of anthocyanin-rich purple and black eggplant extracts on the physical,

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antioxidant and pH-sensitive properties of chitosan film

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Huimin Yong, Xingchi Wang, Xin Zhang, Yunpeng Liu, Yan Qin, Jun Liu*

4

College of Food Science and Engineering, Yangzhou University, Yangzhou 225127,

5

PR China

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* Corresponding author. E-mail: [email protected]

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Abstract

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Active and intelligent food packaging films were developed by mixing chitosan

9

(CS) with anthocyanin-rich purple eggplant extract (PEE) or black eggplant extract

10

(BEE). Results showed the anthocyanin contents in PEE and BEE were 93.10 and

11

173.17 mg/g, respectively. Besides, the anthocyanin compositions of PEE and BEE

12

were totally different. PEE and BEE increased the blueness, thickness, UV-vis light

13

barrier and mechanical properties of CS film. Nevertheless, PEE did not change the

14

moisture content of CS film and BEE did not change the water vapor permeability of

15

CS film. Microstructure observation showed low contents (1 and 2 wt%) of PEE and

16

BEE were well distributed in CS matrix. Fourier transform infrared spectroscopy

17

revealed the existence of intermolecular interactions between CS and extracts. X-ray

18

diffraction indicated PEE and BEE somewhat increased the crystallinity of CS film.

19

The antioxidant ability of CS film was remarkably enhanced by PEE and BEE.

20

Moreover, CS-PEE and CS-BEE films were pH-sensitive and showed remarkable

21

color changes in different buffer solutions, which could be used to monitor milk

22

spoilage. Our results suggested CS-PEE and CS-BEE films could be applied as active

23

and intelligent food packaging materials.

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Keywords: Anthocyanins; Antioxidant; Chitosan; Eggplant; Intelligent; Packaging

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film

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1. Introduction

27

Nowadays, active and intelligent packaging films have been widely applied to

28

extend food shelf-life and monitor food quality (Biji, Ravishankar, Mohan, & Gopal,

29

2015; Fang, Zhao, Warner, & Johnson, 2017; Janjarasskul & Suppakul, 2018). The

30

conventionally used plastic-based food packaging films are non-degradable and can

31

cause serious environmental pollution. Thus, biodegradable food packaging films

32

manufactured from natural resources, such as chitosan (CS) (Ashrafi, Jokar, & Nafchi,

33

2018), starch (Liu et al., 2017), agar (Choi, Lee, Lacroix, & Han, 2017) and gelatin

34

(Rasid, Nazmi, Isa, & Sarbon, 2018) have received increasing attention. CS, the

35

deacetylated product of chitin, is biodegradable and has excellent film forming ability.

36

CS is considered as a suitable material to develop biodegradable food packaging films

37

(Ashrafi et al., 2018; Genskowsky et al., 2015). Due to low antioxidant activity, plain

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CS film cannot meet the standards of active packaging. Therefore, many attempts

39

have been made to enhance the functionality of CS film by incorporating different

40

antioxidant agents (Yong et al., 2019; Zhang et al., 2019).

41

Polyphenols are abundant in nature, and contain several classes (e.g. phenolic

42

acids, flavonoids, stilbenes and lignans) (El Gharras, 2009; Ferrazzano et al., 2011).

43

Anthocyanins, one class of phenolic compounds, are the colorants of flowers and

44

friuts (Ongkowijoyo, Luna-Vital, & de Mejia, 2018). Anthocyanins are suitable

45

addictives to develop active and intelligent packaging films. On one hand,

46

anthocyanins possess potent antioxidant potential and can retard food oxidation

47

process (Kim, Baek, & Song, 2018). On the other hand, anthocyanins can change their

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chemical structures and colors in different pH values, which are suitable to be used as

49

pH indicators to monitor food spoilage (Uranga, Etxabide, Guerrero, & de la Caba,

50

2018). Till now, anthocyanins isolated from different plants, such as purple sweet

51

potato (Choi et al., 2017; Yong et al., 2019), red cabbage (Liang, Sun, Cao, Li, &

52

Wang, 2019; Musso, Salgado, & Mauri, 2019; Uranga et al., 2018), black soybean

53

seed coat (Wang et al., 2019), mulberry (Ma, Liang, Cao, & Wang, 2018) and

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blueberry (Nogueira, Soares, Cavasini, Fakhouri, & de Oliveira, 2019) have been used

55

to develop active and intelligent food packaging films. Existing studies have

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demonstrated the physical and functional properties of films are affected by the

57

content of anthocyanins. However, the effect of anthocyanin composition on the

58

physical and functional properties of films was seldom reported.

59

Eggplant (Solanum melongena L.) is an important and widespread food crop,

60

bearing different colors (Koley et al., 2018). Anthocyanins are the main phenolic

61

compounds in eggplant peels, which possess potent antioxidant activity (Jung, Bae, Jo,

62

Jo, & Lee, 2011). In this study, we aimed at developing antioxidant and intelligent

63

packaging films based on CS and anthocyanin-rich eggplant peel extract. First,

64

anthocyanins were isolated from the peels of different colored (purple and black)

65

eggplants. The obtained purple and black eggplant extracts (namely PEE and BEE)

66

were individually incorporated into CS film. Effects of PEE and BEE on the physical

67

and functional properties of CS film were compared for the first time.

68

2. Materials and methods

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2.1. Materials and chemical reagents

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Purple eggplants (cultivar of Zheqie 1) and black eggplants (cultivar of Bulita)

71

were purchased from local Auchan Supermarket (Yangzhou, China). CS (deacetylated

72

degree: 90%; average molecular weight: 1.5 × 105 Da) was purchased from Sangon

73

Biotechnology Co., Ltd. (Shanghai, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH)

74

was purchased from Sigma Chemical Co. (MO, USA). All other reagents were of

75

analytical grade.

76

2.2. Extraction and characterization of anthocyanins from purple and black eggplant

77

peels

78

Anthocyanins were extracted from purple and black eggplant peels according to

79

the method of Wang et al. (2019). First, peels were individually isolated from fresh

80

purple and black eggplants. Then, 100 g of peels were extracted in 500 mL of 80%

81

ethanol solution with 1% of HCl at 4 ºC for 1 day. The extract solution was

82

centrifuged (8000 × g) at 4 ºC for 15 min, condensed at 35 °C and vacuum dried to

83

afford PEE and BEE. The anthocyanin content in the extract was measured by

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pH-differential assay (Ge, Chi, Liang, & Gao, 2018).

85

Anthocyanins in PEE and BEE were characterized by Agilent 1200 HPLC

86

system equipped with Agilent 6460 triple quadrupole mass spectrometer (Agilent

87

Technologies, USA) and Agilent Zorbax C18 column (4.6 × 150 mm, 5 μm). The

88

HPLC testing conditions were based on the method of Wang et al. (2019). The eluent

89

A (0.1% (v/v) formic acid aqueous solution) and eluent B (acetonitrile) were used as

90

the mobile phases. The following elution conditions were used: 10−20% B (0−10

91

min); 20−25% B (10−20 min); 25−55% B (20−30 min); 55−80% B (30−45 min); and

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80−100% B (45−60 min). Mass spectrometry analysis was performed in positive

93

ionization mode with m/z in the range of 50−2000.

94

To determine the pH-sensitivity of extracts, PEE and BEE (2 mg) were dissolved

95

in 20 mL of different buffer solutions (pH 2−13). The UV–vis spectra of extract

96

solutions were recorded on Lambda 35 UV-vis spectrophotometer (PerkinElmer Ltd.,

97

USA).

98

2.3. Development of CS-PEE and CS-BEE films

99

CS-PEE and CS-BEE films were developed according to Yong et al. (2019).

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Firstly, 2 wt% of CS solution was obtained by dissolving CS in 1% of acetic acid

101

solution. Then, CS solution was mixed with different amounts (1, 2 and 3 wt%) of

102

PEE or BEE and 30 wt% of glycerol on CS basis. The film-forming solutions were

103

degassed and poured into Plexiglas plates (24 cm × 24 cm). Films were obtained by

104

drying the plates in a ventilated climatic chamber at 30 ºC and 50% relative humidity

105

for 2 days. Films containing 1, 2 and 3 wt% of PEE were termed as CS-PEE I,

106

CS-PEE II, CS-PEE III films, respectively. Likewise, films containing 1, 2 and 3 wt%

107

of BEE were named as CS-BEE I, CS-BEE II and CS-BEE III films, respectively. All

108

films were stored at 20 ºC in a desiccator with 50% relative humidity.

109

2.4. Characterization of CS-PEE and CS-BEE films

110

2.4.1. Physical appearance and color

111

The digital image of each film was taken by covering film sample on the half of

112

a printed white paper. Color parameters of film including L, a and b were obtained by

113

using SC-80C colorimeter (Kangguang Instrument Co., China), and the total color

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difference (ΔE) of film was calculated as follows (Wang et al., 2019):

E  ( L *  L) 2  (a * a) 2  (b * b) 2

(1)

116

where L* (91.25), a* (−1.11) and b* (2.70) were color parameters of white plate used

117

for calibration.

118

2.4.2. Thickness

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Film thickness was measured by Mitutoyo digital micrometer (Tester Sangyo

120

Co., Ltd., Japan) with the precision of 0.001 mm.

121

2.4.3. Moisture content

122 123

124

Moisture content was determined by drying film sample at 110 °C to a constant weight (Zhai et al., 2017). Moisture content (%) 

(M i  M t )  100 Mi

125

where Mi and Mt were the initial and final masses of film sample, respectively.

126

2.4.4. Water vapor barrier property

127

(2)

Water vapor barrier property of film was measured according to the method of

128

Wang et al. (2019). Film piece was sealed on test cup containing anhydrous silica gels.

129

Then, the cup was kept at 20 °C in a desiccator containing distilled water (100%

130

relative humidity). The cup was weighed every 24 h for 6 days. Water vapor

131

permeability (WVP) was calculated as follows:

132

WVP 

W x t  A  ΔP

(3)

133

where W was the weight gain of cup (g), x was film thickness (m), t was time (s), A

134

was the permeation area of film (m2), and ΔP was partial vapor pressure at 20 °C.

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2.4.5. UV-vis light barrier property 7

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UV-vis light barrier property of film was measured on Lambda 35 UV-Vis

137

spectrophotometer (PerkinElmer Ltd., USA) by scanning film sample from 200 to 800

138

nm.

139

2.4.6. Mechanical property

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Film piece (1 cm × 6 cm) was tested by TMS-Pro texture analyzer (Food

141

Technology Corp., USA) at crosshead speed of 6 cm/min (Wang et al., 2019). Tensile

142

strength and elongation at break (EAB) of film were calculated as follows: F x W

143

Tensile strength (MPa) 

144

Elongation at break (%) 

(4)

ΔL  100 L0

(5)

145

where F was the stress for film fracture (N), x was film thickness (mm), W was film

146

width (mm), ΔL and L0 was the elongated and initial lengths (mm) of film,

147

respectively.

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2.4.7. Scanning electron microscope (SEM)

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The surface and cross-section of film was observed by S-4800 scanning electron

150

microscope (Hitachi Ltd., Japan) at voltage of 5 kV.

151

2.4.8. Fourier transform infrared (FT-IR)

152

Attenuated total reflectance FT-IR spectrum of film was characterized by Varian

153

670 spectrometer (Varian Inc., USA).

154

2.4.9. X-ray diffraction (XRD)

155

XRD pattern of film was measured by D8 Advance X-ray diffractometer (Bruker

156

AXS GmbH, Germany) according to the method of Yong et al. (2019).

157

2.5. Determination of antioxidant ability of CS-PEE and CS-BEE films 8

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The antioxidant ability of film was determined by the method of Wang et al.,

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2019. Film sample was reacted with 4 mL of 100 μM DPPH methanol solution at 20

160

°C for 1 h. The absorbance of reaction solution was measured at 517 nm. The

161

antioxidant ability of PEE and BEE was also determined in the same way.

162

DPPH radical scavenging activity (%) 

163

where A0 and A1 were the absorbance of blank and reaction solution, respectively.

164

2.6. Determination of pH-sensitivity of CS-PEE and CS-BEE films

A0  A1 100 A0

(6)

165

Film sample was soaked in different buffer solutions (pH 2−13) for 15 min. The

166

color parameters of film sample were measured by SC-80C colorimeter (Kangguang

167

Instrument Co., China) (Wang et al., 2019).

168

2.7. Application of CS-PEE and CS-BEE films for monitoring milk spoilage

169

The application of CS-PEE and CS-BEE films for monitoring milk spoilage was

170

performed according to the method of Liang and Wang (2018) with some

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modifications. Briefly, fresh pasteurized milk was stored at 40 °C in a constant

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temperature incubator. The acidity and pH value of milk were measured every 2 h by

173

acid-base titration and FE20K digital pH meter (Mettler-Toledo Co., Greifensee,

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Switzerland), respectively. Meanwhile, film sample was immersed in the milk for 5

175

min and the color change of film sample was recorded.

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2.8. Statistical analysis

177

By applying SPSS 13.0 software, Duncan test and one-way analysis of variance

178

were carried out. Results were considered statistically different if p < 0.05.

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3. Results and discussion 9

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3.1. Characterization of anthocyanins in PEE and BEE

181

Anthocyanins were individually extracted from the peels of purple and black

182

eggplants. The extraction yields of PEE and BEE were 209 mg/100 g and 387 mg/100

183

g fresh eggplant peels, respectively. The pH-differential assay showed the

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anthocyanin content in BEE (173.17 mg/g extract) was 1.86 times as that in PEE

185

(93.10 mg/g extract). The anthocyanin compositions of PEE and BEE were further

186

analyzed by HPLC-MS. As illustrated in Table 1, PEE and BEE showed different

187

anthocyanin

188

cyanidin-3-O-glucosyl-rutinoside

189

(6.99%),

190

malvidin-3-O-glucoside (0.72%) were identified in the PEE based on their mass

191

spectra as well as the literatures (Paucar-Menacho, Martinez-Villaluenga, Dueñas,

192

Frias, & Peñas, 2017; Sánchez-Ilárduya et al., 2014; Zhao et al., 2017; Zhao, Wu, Yu,

193

& Chen, 2017). In contrast, delphinidin-3-O-rutinoside (97.32%) was the predominant

194

anthocyanin in BEE. Notably, the anthocyanin contents and compositions of PEE and

195

BEE in this study were different from those of previous reports (Azuma et al., 2008;

196

Sadilova, Stintzing, & Carle, 2006; Wu & Prior, 2005), which could be related to the

197

differences in the variety, cultivation region and growth condition of eggplants.

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3.2. Color variation of PEE and BEE in different buffer solutions

compositions.

Four (91.22%),

anthocyanins

including

delphinidin-3-O-glucoside-catechin

pelargonidin-3-O-(3'',6''-dimalonyl-hexoside)

(1.07

%)

and

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The color variation of PEE and BEE in different buffer solutions was measured

200

and shown in Fig. 1. Remarkable color change was observed in PEE and BEE

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solutions with different pH values. The color of PEE in pH ranges of 2–6, 7–10 and

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11–13 was pink, blue and yellow, respectively. In contrast, the color of BEE in the

203

same pH ranges was red/pink, purple and yellow, respectively. Corresponding with

204

the color change of PEE and BEE solutions, the maximum absorption peak of PEE

205

and BEE solutions shifted from 520 nm (pH 2) to 580 nm (pH 13) as presented in Fig.

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2. Generally, anthocyanins presented different structures at different pH values:

207

flavylium cation (red, at strong acidic conditions), carbinol pseudobase (colorless, at

208

weak acidic conditions), quinoidal base (blue, at weak alkaline conditions) and

209

chalcone (yellow, at strong alkaline conditions) (Zepon et al., 2019; Zhai et al., 2017).

210

Therefore, the color change and corresponding bathochromic shift in maximum

211

absorption peak were mainly caused by structure transformation of anthocyanins at

212

different pH values (Ma et al., 2018). Similar results were reported in studies for other

213

anthocyanin-rich plant extracts (Wei, Cheng, Ho, Tsai & Mi, 2017; Zhai et al., 2017).

214

3.3. Physical appearances and colors of CS-PEE and CS-BEE films

215

Color is an important property reflecting the appearance of films. As shown in

216

Fig. 3A, the colors of CS-PEE and CS-BEE films were significantly different from

217

that of CS film. CS film was pale yellow and transparent, whereas CS-PEE and

218

CS-BEE films were blue. Moreover, the colors of CS-PEE and CS-BEE films

219

deepened when PEE and BEE contents increased from 1 to 3 wt%. Notably, CS-BEE

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films were darker than CS-PEE films at the same extract incorporation levels.

221

Color parameters of CS, CS-PEE and CS-BEE films were summarized in Table

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2. CS-PEE and CS-BEE films both showed lower L, a and b values, however, higher

223

ΔE values as compared with CS film (p < 0.05), which indicated CS-PEE and

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CS-BEE films turned to darkness, greenness and blueness. The blue color of CS-PEE

225

and CS-BEE films could be attributed to anthocyanins in the extracts. CS-PEE films

226

showed a significant decrease in L, a and b values when PEE content increased from 1

227

to 3 wt% (p < 0.05), indicating CS-PEE films became darker, greener and bluer.

228

Similar decrease in L and b values was observed in CS-BEE films when BEE content

229

increased from 1 to 3 wt% (p < 0.05). At the same extract incorporation levels,

230

CS-PEE films showed higher L and b values, however, lower ΔE values than CS-BEE

231

films (p < 0.05). This confirmed CS-BEE films were darker and bluer than CS-PEE

232

films when the extract content was the same, which could be due to the differences in

233

the composition and content of anthocyanins in PEE and BEE (Kurek et al., 2018).

234

Similar color changes were observed when CS film was incorporated with

235

anthocyanin-rich bluberry extract (Kurek et al., 2018).

236

3.4. Thicknesses of CS-PEE and CS-BEE films

237

Film thickness is an important parameter that directly affects mechanical strength,

238

water vapor permeability, light transmittance and opacity of film (Toro-Márquez,

239

Merino, & Gutiérrez, 2018; Wang et al., 2019). As presented in Table 3, the

240

thicknesses of CS-PEE film and CS-BEE films were in ranges of 0.062−0.068 mm

241

and 0.065−0.074 mm, respectively. Compared with CS film, CS-PEE II and CS-PEE

242

III films were significantly thicker (p < 0.05). However, CS-PEE I and CS film

243

presented no significant difference in film thickness (p > 0.05). This suggested that

244

the thickness of CS-PEE films was affected by PEE content. A low content of PEE (1

245

wt%) could be well distributed in CS matrix. By contrast, higher contents of PEE (2

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and 3 wt%) could create more complex matrices, resulting in the increase of film

247

thickness (Yong et al., 2019). For CS-BEE films, similar increase in film thickness

248

was observed when BEE content increased from 1 to 3 wt% (p < 0.05). In addition,

249

CS-PEE I and CS-BEE I film showed no remarkable difference in film thickness (p >

250

0.05). However, CS-BEE films were significantly thicker than CS-PEE films at the

251

same extract incorporation levels of 2 and 3 wt% (p < 0.05). The differences in film

252

thickness between CS-PEE and CS-BEE films could be related to different

253

anthocyanin compositions and contents in the extracts (Lozano-Navarro et al., 2017).

254

Similar results were found when CS film was incorporated with purple sweet potato,

255

blueberry and cranberry extracts (Lozano-Navarro et al., 2017; Yong et al., 2019).

256

3.5. Moisture contents of CS-PEE and CS-BEE films

257

As presented in Table 3, there was no significant difference in moisture content

258

between CS and CS-PEE films (p > 0.05). Moreover, the moisture content of CS-PEE

259

II film was not significantly different from that of CS-PEE I film (p > 0.05). However,

260

significant increase in moisture content was observed when PEE content increased to

261

3 wt% (p < 0.05). The relatively lower moisture contents in CS-PEE I and CS-PEE II

262

films could be attributed to the interactions between amino and hydroxyl groups of CS

263

and anthocyanins in PEE, which could limit the CS-water interactions (Wang et al.,

264

2019; Yong et al., 2019). Different from CS-PEE films, three CS-BEE films showed

265

significantly higher moisture contents as compared with CS film (p < 0.05). The

266

increase in the moisture contents of CS-BEE films could be related with the

267

hydrophilic nature of anthocyanins in BEE (Rubilar et al., 2013). With the addition of

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BEE, more polar sites were available to absorb moisture from surroundings (Gutiérrez,

269

Toro-Márquez, Merino, & Mendieta, 2019).). An increase in the moisture content was

270

also observed in CS film incorporated with anthocyanin-rich grapefruit seed extract

271

(Rubilar et al., 2013). Among three CS-BEE films, CS-BEE II film exhibited the

272

highest moisture content (p < 0.05). Notably, CS-PEE films showed lower moisture

273

contents than CS-BEE films at the same extract incorporation levels (p < 0.05).

274

Above results implied the moisture contents in CS-PEE and CS-BEE films were

275

greatly influenced by the composition and content of anthocyanins in the extracts.

276

3.6. WVP of CS-PEE and CS-BEE films

277

WVP is a vital barrier parameter reflecting the ability of film against water vapor

278

(Huang et al., 2019). As presented in Table 3, the WVP of CS-PEE II and CS-PEE III

279

films was not significantly different from that of CS film (p > 0.05). Nevertheless,

280

CS-PEE I film showed the lowest WVP among three CS-PEE films (p < 0.05). Above

281

results indicated a small amount of PEE (1 wt%) could decrease the WVP of CS film,

282

which could be due to intermolecular interactions established between PEE and CS

283

had decreased the affinity of CS film towards water molecules (Kurek et al., 2018).

284

By contrast, higher amounts of PEE (2 and 3 wt%) could cause less dense and

285

compact structure in CS film (Liu et al., 2018; Yong et al., 2019). Notably, there was

286

no obvious difference in WVP between CS film and CS-BEE films (p > 0.05).

287

However, CS-BEE I and CS-BEE II films showed relatively lower WVP than

288

CS-BEE III film (p < 0.05). Above results indicated the WVP of CS film was not

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significantly affected by the incorporation of BEE. Similar phenomena were reported

290

in CS-blueberry extract and CS-blackberry extract films (Kurek et al., 2018).

291

3.7. Light transmittance of CS-PEE and CS-BEE films

292

Light transmittance is a vital parameter reflecting the barrier ability of film

293

against light, since UV-vis light is harmful for food storage (Han, Yu & Wang, 2018).

294

UV-vis light transmittance of different films was shown in Fig. 3B. In comparison

295

with CS film, CS-PEE and CS-BEE films all showed remarkably decreased light

296

transmittance, which was due to anthocyanins in the films could absorb UV-vis

297

radiation (Peralta, Bitencourt-Cervi, Maciel, Yoshida & Carvalho, 2019). Moreover,

298

the light transmittance of CS-PEE and CS-BEE films gradually decreased with the

299

increase of extract contents. At the same extract incorporation levels, CS-BEE films

300

showed significantly lower light transmittance than CS-PEE films. This was because

301

BEE contained a higher polypheol content than PEE, and BEE could absorb more

302

UV-vis radiation. When CS was incorporated with other anthocyanin-rich extracts

303

(e.g. purple sweet potato, black soybean seed coat and mulberry extracts), the

304

enhanced UV-vis light barrier property of films was also observed (Ma et al., 2018;

305

Wang et al., 2019; Yong et al., 2019). Our results indicated that CS-BEE films

306

possessed better UV-vis light barrier properties than CS-PEE films.

307

3.8. Mechanical property of CS-PEE and CS-BEE films

308

As shown in Table 4, the tensile strength of three CS-PEE films was higher than

309

that of CS film (p < 0.05). In addition, CS-PEE films exhibited gradually increased

310

tensile strength with the increase of PEE content (p < 0.05). The tensile strength of

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CS-BEE I and CS-BEE II films was significantly higher than CS and CS-BEE III

312

films (p < 0.05). However, there was no significant difference in tensile strength

313

between CS-BEE III and CS films (p > 0.05). The increase in tensile strength of films

314

containing PEE and BEE could be due to hydrogen bonds formed between

315

hydroxyl/amino groups of CS and polypheols in the extracts (Koosha & Hamedi,

316

2019; Mushtaq, Gani, Gani, Punoo & Masoodi, 2018). As for CS-BEE III film, the

317

reduction in tensile strength was probably because a high amount of BEE could

318

interrupt the interactions between BEE and CS matrix (Shankar & Rhim, 2016).

319

Table 4 also indicated that PEE and BEE could significantly increase the

320

flexibility of CS film. The EAB of CS-PEE films gradually increased with the

321

increase of PEE content (p < 0.05). In addition, the EAB of CS-PEE and CS-BEE

322

films showed a similar trend with their tensile strength. However, when BEE content

323

reached 3 wt%, phonelic compounds in BEE could exert anti-plasticizing effect and

324

limit the motion of polymer chains, leading to the decrease in flexibility of CS-BEE

325

III film (Mushtaq et al., 2018). Notably, the flexibility of CS-BEE films was

326

significantly higher than CS-PEE films at the same extract incorporation levels of 1

327

and 2 wt% (p < 0.05). Similar changes of mechanical property were observed in

328

agar-nanocellulose and zein-pomegranate peel extract films (Mushtaq et al., 2018;

329

Shankar & Rhim, 2016).

330

3.9. Microstructure of CS-PEE and CS-BEE films

331

Microstructure including surface and cross-section of different films was

332

observed by SEM. As shown in Fig. 4, all films showed smooth surfaces, indicating

16

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333

the compatibility of all the film components (e.g. CS, PEE/BEE and glycerol) (Wang

334

et al., 2019). By contrast, the cross-sections of CS-PEE and CS-BEE films were

335

rougher than CS film. Notably, the cross-section of CS-PEE III film was significantly

336

rougher than those of CS-PEE I and CS-PEE II films. This indicated that low contents

337

of PEE (1 and 2 wt%) could be well distributed in CS matrix, which was beneficial to

338

enhance the mechanical property and water vapor barrier ability of films (Liu et al.,

339

2018). Similar changes were observed in the cross-sections of CS-BEE films. Other

340

researchers also reported similar results when CS film was incorporated with hibiscus

341

and black soybean seed coat extracts (Peralta et al., 2019; Wang et al., 2019).

342

3.10. FT-IR spectra of CS-PEE and CS-BEE films

343

FT-IR spectra of different films were investigated to confirm intermolecular

344

interactions between CS and extracts. As shown in Fig. 5, the FT-IR spectrum of PEE

345

was similar to that of BEE. PEE and BEE exhibited a wide band from 3700 to 3000

346

cm−1 (O–H stretching), several bands at about 1634, 1600 and 1514 cm−1 (C=C

347

stretching of aromatic ring), and a strong band at about 1028 cm−1 (C–H deformation

348

of aromatic ring) (Choi et al., 2017; Ma & Wang, 2016). For CS film, its FT-IR

349

spectrum presented a broad band around 3327 cm−1, assigned to O−H and N–H

350

stretching (Halász & Csóka, 2018). Two bands at 2924 and 1406 cm−1 were attributed

351

to C–H stretching and C–H bending, respectively (Priyadarshi, Kumar, Deeba,

352

Kulshreshtha, & Negi, 2018). The bands at 1633 and 1551 cm−1 were characteristic

353

C=O stretching and N−H bending, respectively (Yong et al., 2019). Other bands at

354

1151 cm−1 (asymmetric stretching of C–O–C) and 1026 cm−1 (skeletal stretching of

17

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355

C–O) corresponded to saccharide structure (Liu, Liu et al., 2017). However, the FT-IR

356

spectrum of CS film was not significantly changed after PEE and BEE were added,

357

which was because of low contents of extracts in the films (Yong et al., 2019). For

358

CS-PEE and CS-BEE films, their bands around 3260 cm−1 were broader than CS film

359

due to abundant hydroxyl groups from anthocyanins in the extracts (Yong et al., 2019).

360

Besides, the band shifts around 1551 and 1335 cm−1 in CS-PEE and CS-BEE films

361

could be caused by intermolecular interactions (e.g. hydrogen bonds) between CS and

362

extracts (Choi et al., 2017). Many researchers also suggested the band shifts in

363

anthocyanin-rich films were related to interactions between film components (Choi et

364

al., 2017; Halász & Csóka, 2018; Liang & Wang, 2018; Liu et al., 2018; Ma & Wang,

365

2016).

366

3.11. XRD patterns of CS-PEE and CS-BEE films

367

As shown in Fig. 6, XRD patterns of PEE and BEE both presented a broad peak

368

around 20.0°, suggesting PEE and BEE were amorphous. XRD pattern of CS film

369

exhibited five diffraction peaks at 8.3°, 11.3°, 16.2°, 18.0° and 23.0°, corresponding

370

to semi-crystalline nature of CS film (Liu, Meng, Liu, Kan, & Jin,

371

CS-PEE and CS-BEE films showed the same diffraction peaks as CS film, which

372

indicated these two extracts were well dispersed in CS matrix (Huang et al., 2019).

373

Besides, the diffraction peak intensities of CS-PEE and CS-BEE films were

374

significantly higher than those of CS film. This indicated the incorporation of PEE

375

and BEE somewhat increased the crystallinity of films. However, many other

376

researchers found the diffraction peak intensities of films were decreased after the

18

2017). Notably,

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377

addition of anthocyanin-rich extracts (Pourjavaher, Almasi, Meshkini, Pirsa, &

378

Parandi, 2017; Wang et al., 2019; Zhai et al., 2017), which was due to the

379

incorporation of extracts disrupted the ordered structures of polymeric matrix. Our

380

results suggested the crystallinity of anthocyanin-rich films could be related to the

381

content and composition of anthocyanins added.

382

3.12. Antioxidant ability of PEE, BEE, CS-PEE and CS-BEE films

383

Free radicals can lead to food spoilage and nutritional loss. Thus, antioxidant

384

ability is important for active food packaging (Priyadarshi et al., 2018). As presented

385

in Fig. 7, the DPPH radical scavenging ability of CS film was remarkably enhanced

386

by PEE and BEE (p < 0.05). Similar enhancement in antioxidant activity was

387

observed when anthocyanin-rich roselle and black soybean seed coat extracts were

388

added in CS film (Wang et al., 2019; Zhang et al., 2019). Notably, the DPPH radical

389

scavenging ability of CS-PEE and CS-BEE films significantly increased with the

390

increase of extract content (p < 0.05). Moreover, CS-BEE films showed higher

391

antioxidant ability than CS-PEE films (p < 0.05) at the same extract incorporation

392

levels, which was because BEE had higher anthocyanin content than PEE. Our results

393

indicated the antioxidant ability of films was affected by the composition and content

394

of anthocyanins in the extracts.

395

3.13. pH-sensitivity of CS-PEE and CS-BEE films

396

The pH-sensitivity of CS, CS-PEE and CS-BEE films were presented in Fig. 8.

397

CS film was not pH-sensitive due to the lack of anthocyanins. By contrast,

398

anthocyanin-rich CS-PEE and CS-BEE films were all pH-sensitive and showed

19

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399

remarkable color changes in different buffer solutions. The color of CS-PEE and

400

CS-BEE films changed from purple to green/blue with the increase of pH value. At

401

the same extract incorporation levels, CS-BEE films were darker than CS-PEE film.

402

This could be due to the difference in the composition and content of anthocyanins in

403

the extracts (Kurek et al., 2018). Color parameters (ΔE) of different films at pH 2−13

404

were compared and summarized in Table 5. CS-PEE and CS-BEE films both showed

405

higher ΔE values as compared with CS film at same pH conditions (p < 0.05). Overall,

406

the ΔE values of CS-PEE and CS-BEE films first increased and then gradually

407

decreased with the increase of pH value. Meanwhile, the ΔE values of CS-BEE films

408

were higher than those of CS-PEE films at same pH conditions and the same extract

409

incorporation levels (p < 0.05). The pH-sensitivity has been also found in other

410

anthocyanin-rich films, such as agar/potato starch-purple sweet potato extract,

411

Artemisia sphaerocephala Krasch. gum-red cabbage extract, CS-blackberry pomace

412

extract films (Choi et al., 2017; Kurek et al., 2018; Liang et al., 2019). Our results

413

suggested CS-PEE and CS-BEE films could be further developed as pH indicators to

414

monitor food freshness.

415

3.14. Application of CS-PEE and CS-BEE films for monitoring milk spoilage

416

The pH-sensitive CS-PEE and CS-BEE films were further used to monitor milk

417

spoilage. As shown in Table 6, the acidity of pasteurized milk gradually increased

418

from 13.25 to 17.73 °T in the first 10 h of storage (p < 0.05). Based on the Chinese

419

Standard of GB 25190-2010, milk is drinkable when its acidity is lower than 18 °T.

420

However, the acidity of milk significantly increased from 23.38 to 69.00 °T when the

20

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421

storage time extended from 12 to 16 h. This indicated milk was seriously deteriorated

422

after storage at 40 °C for 10 h (Liang & Wang, 2018). Meanwhile, the pH value of

423

milk significantly decreased after storage at 40 °C for 8 h (p < 0.05). When used to

424

monitor milk spoilage, CS-PEE I, CS-PEE II and CS-BEE I films did not show

425

significant color change because these films had low anthocyanin contents. By

426

contrast, CS-PEE III, CS-BEE II and CS-BEE III films showed obvious color change

427

when immersed in the spoiled milk (storage at 40 °C for over 10 h). Notably, CS-BEE

428

films showed more significant color change than CS-PEE films at the same extract

429

incorporation levels. Above results suggested CS-PEE III, CS-BEE II and CS-BEE III

430

films could be used to monitor milk spoilage.

431

4. Conclusion

432

Active and intelligent packaging films were successfully developed by

433

incorporating CS with anthocyanin-rich PEE and BEE. PEE and BEE could increase

434

the blueness, thickness, and UV-vis light barrier, mechanical, antioxidant and

435

pH-sensitive properties of CS film. Notably, the UV-vis light barrier, antioxidant and

436

pH-sensitive properties of CS-PEE and CS-BEE films gradually increased with the

437

increase of extract content. At the same extract incorporation levels, CS-BEE films

438

showed bluer colors, and higher thicknesses, moisture contents, UV-vis light barrier,

439

antioxidant and pH-sensitive properties than CS-PEE films, which could be related to

440

different compositions and contents of anthocyanins in the extracts. Due to high

441

anthocyanin contents, CS-PEE III, CS-BEE II and CS-BEE III films could be used to

442

monitor milk spoilage. Our results suggested CS-PEE and CS-BEE films could be

21

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443

used as active and intelligent packaging films in future.

444

Acknowledgements

445

This work was financed by National Natural Science Foundation of China (Nos.

446

31571788 and 31101216), Natural Science Foundation of Jiangsu Province (No.

447

BK20151310), Qing Lan Project of Jiangsu Province and High Level Talent Support

448

Program of Yangzhou University.

449

Reference

450

Ashrafi, A., Jokar, M., & Nafchi, A. M. (2018). Preparation and characterization of

451

biocomposite film based on chitosan and kombucha tea as active food packaging.

452

International Journal of Biological Macromolecules, 108, 444−454.

453

Azuma, K., Ohyama, A., Ippoushi, K., Ichiyanagi, T., Takeuchi, A., Saito, T., &

454

Fukuoka, H. (2008). Structures and antioxidant activity of anthocyanins in many

455

accessions of eggplant and its related species. Journal of Agricultural and Food

456

Chemistry, 56, 10154−10159.

457

Biji, K. B., Ravishankar, C. N., Mohan, C. O., & Gopal, T. S. (2015). Smart

458

packaging systems for food applications: a review. Journal of Food Science and

459

Technology, 52, 6125−6135.

460

Choi, I., Lee, J. Y., Lacroix, M., & Han, J. (2017). Intelligent pH indicator film

461

composed of agar/potato starch and anthocyanin extracts from purple sweet

462

potato. Food Chemistry, 218, 122−128.

463 464

El Gharras, H. (2009). Polyphenols: food sources, properties and applications–a review. International Journal of Food Science & Technology, 44, 2512−2518.

22

ACCEPTED MANUSCRIPT

465 466

Fang, Z., Zhao, Y., Warner, R. D., & Johnson, S. K. (2017). Active and intelligent packaging in meat industry. Trends in Food Science & Technology, 61, 60−71.

467

Ferrazzano, G., Amato, I., Ingenito, A., Zarrelli, A., Pinto, G., & Pollio, A. (2011).

468

Plant polyphenols and their anti-cariogenic properties: a review. Molecules, 16,

469

1486−1507.

470

Ge, J., Yue, P., Chi, J., Liang, J., & Gao, X. (2018). Formation and stability of

471

anthocyanins-loaded nanocomplexes prepared with chitosan hydrochloride and

472

carboxymethyl chitosan. Food Hydrocolloids, 74, 23−31.

473

Genskowsky, E., Puente, L. A., Pérez-Álvarez, J. A., Fernandez-Lopez, J., Muñoz, L.

474

A., & Viuda-Martos, M. (2015). Assessment of antibacterial and antioxidant

475

properties of chitosan edible films incorporated with maqui berry (Aristotelia

476

chilensis). LWT-Food Science and Technology, 64, 1057−1062.

477

Gutiérrez, T. J., Toro-Márquez, L. A., Merino, D., & Mendieta, J. R. (2019).

478

Hydrogen-bonding interactions and compostability of bionanocomposite films

479

prepared from corn starch and nano-fillers with and without added Jamaica

480

flower extract. Food Hydrocolloids, 89, 283−293.

481

Halász, K., & Csóka, L. (2018). Black chokeberry (Aronia melanocarpa) pomace

482

extract

immobilized

in

chitosan

for

colorimetric

483

application. Food Packaging and Shelf Life, 16, 185−193.

pH

indicator

film

484

Han, Y., Yu, M., & Wang, L. (2018). Preparation and characterization of antioxidant

485

soy protein isolate films incorporating licorice residue extract. Food

486

Hydrocolloids, 75, 13−21.

23

ACCEPTED MANUSCRIPT

487

Huang, S., Xiong, Y., Zou, Y., Dong, Q., Ding, F., Liu, X., & Li, H. (2019). A novel

488

colorimetric indicator based on agar incorporated with Arnebia euchroma root

489

extracts for monitoring fish freshness. Food Hydrocolloids, 90, 198−205.

490

Janjarasskul, T., & Suppakul, P. (2018). Active and intelligent packaging: the

491

indication of quality and safety. Critical Reviews in Food Science and

492

Nutrition, 58, 808−831.

493

Jung, E. J., Bae, M. S., Jo, E. K., Jo, Y. H., & Lee, S. C. (2011). Antioxidant activity

494

of different parts of eggplant. Journal of Medicinal Plants Research, 5,

495

4610−4615.

496

Kim, S., Baek, S. K., & Song, K. B. (2018). Physical and antioxidant properties of

497

alginate films prepared from Sargassum fulvellum with black chokeberry extract.

498

Food Packaging and Shelf Life, 18, 157−163.

499

Koley, T. K., Tiwari, S. K., Sarkar, A., Nishad, J., Goswami, A., & Singh, B. (2018).

500

Antioxidant potential of Indian eggplant: comparison among white, purple and

501

green

502

10.1007/s40003-018-0347-1.

genotypes

using

chemometrics.

Agricultural

Research,

Doi:

503

Koosha, M., & Hamedi, S. (2019). Intelligent chitosan/PVA nanocomposite films

504

containing black carrot anthocyanin and bentonite nanoclays with improved

505

mechanical, thermal and antibacterial properties. Progress in Organic

506

Coatings, 127, 338−347.

507

Kurek, M., Garofulić, I. E., Bakić, M. T., Ščetar, M., Uzelac, V. D., & Galić, K.

508

(2018). Development and evaluation of a novel antioxidant and pH indicator film

24

ACCEPTED MANUSCRIPT

509

based on chitosan and food waste sources of antioxidants. Food Hydrocolloids,

510

84, 238−246.

511

Liang, T., Sun, G., Cao, L., Li, J., & Wang, L. (2019). A pH and NH3 sensing

512

intelligent film based on Artemisia sphaerocephala Krasch. gum and red cabbage

513

anthocyanins anchored by carboxymethyl cellulose sodium added as a host

514

complex. Food Hydrocolloids, 87, 858−868.

515 516

Liang, T., & Wang, L. (2018). A pH-sensing film from tamarind seed polysaccharide with litmus lichen extract as an indicator. Polymers, 10, 13.

517

Liang, Z., Wu, B., Fan, P., Yang, C., Duan, W., Zheng, X., Liu, C., & Li, S. (2008).

518

Anthocyanin composition and content in grape berry skin in Vitis germplasm.

519

Food Chemistry, 111, 837−844.

520

Liu, B., Xu, H., Zhao, H., Liu, W., Zhao, L., & Li, Y. (2017). Preparation and

521

characterization of intelligent starch/PVA films for simultaneous colorimetric

522

indication and antimicrobial activity for food packaging applications.

523

Carbohydrate Polymers, 157, 842−849.

524

Liu, J., Liu, S., Wu, Q., Gu, Y., Kan, J., & Jin, C. (2017). Effect of protocatechuic

525

acid incorporation on the physical, mechanical, structural and antioxidant

526

properties of chitosan film. Food Hydrocolloids, 73, 90−100.

527

Liu, J., Meng, C. G., Liu, S., Kan, J., & Jin, C. H. (2017). Preparation and

528

characterization of protocatechuic acid grafted chitosan films with antioxidant

529

activity. Food Hydrocolloids, 63, 457−466.

25

ACCEPTED MANUSCRIPT

530

Liu, J., Wang, H., Wang, P., Guo, M., Jiang, S., Li, X., et al. (2018). Films based on

531

κ-carrageenan incorporated with curcumin for freshness monitoring. Food

532

Hydrocolloids, 83, 134−142.

533

Liu, X., Mu, T., Sun, H., Zhang, M., & Chen, J. (2013). Optimisation of aqueous

534

two-phase extraction of anthocyanins from purple sweet potatoes by response

535

surface methodology. Food Chemistry, 141, 3034−3041.

536

Lozano-Navarro, J. I., Díaz-Zavala, N. P., Velasco-Santos, C., Martínez-Hernández,

537

A. L., Tijerina-Ramos, B. I., García-Hernández, M., Rivera-Armenta, J. L.,

538

Tijerina-Ramos, B. I., & Reyes-de la Torre, A. I. (2017). Antimicrobial, optical

539

and mechanical properties of chitosan-starch films with natural extracts.

540

International Journal of Molecular Sciences, 18, 997.

541

Ma, Q., Liang, T., Cao, L., & Wang, L. (2018). Intelligent poly (vinyl

542

alcohol)-chitosan nanoparticles-mulberry extracts films capable of monitoring

543

pH variations. International Journal of Biological Macromolecules, 108,

544

576−584.

545

Ma, Q., & Wang, L. (2016). Preparation of a visual pH-sensing film based on tara

546

gum incorporating cellulose and extracts from grape skins. Sensors and

547

Actuators B: Chemical, 235, 401−407

548

Mushtaq, M., Gani, A., Gani, A., Punoo, H. A., & Masoodi, F. A. (2018). Use of

549

pomegranate peel extract incorporated zein film with improved properties for

550

prolonged shelf life of fresh Himalayan cheese (Kalari/kradi). Innovative Food

551

Science & Emerging Technologies, 48, 25−32.

26

ACCEPTED MANUSCRIPT

552

Musso, Y. S., Salgado, P. R., & Mauri, A. N. (2019). Smart gelatin films prepared

553

using red cabbage (Brassica oleracea L.) extracts as solvent. Food

554

Hydrocolloids, 89, 674−681.

555

Nogueira, G. F., Soares, C. T., Cavasini, R., Fakhouri, F. M., & de Oliveira, R. A.

556

(2019). Bioactive films of arrowroot starch and blackberry pulp: Physical,

557

mechanical and barrier properties and stability to pH and sterilization. Food

558

Chemistry, 275, 417−425.

559

Ongkowijoyo, P., Luna-Vital, D. A., & de Mejia, E. G. (2018). Extraction techniques

560

and analysis of anthocyanins from food sources by mass spectrometry: An

561

update. Food Chemistry, 250, 113−126.

562

Paucar-Menacho, L. M., Martinez-Villaluenga, C., Dueñas, M., Frias, J., & Peñas, E.

563

(2017). Optimization of germination time and temperature to maximize the

564

content of bioactive compounds and the antioxidant activity of purple corn (Zea

565

mays L.) by response surface methodology. LWT-Food Science and

566

Technology, 76, 236−244.

567

Peralta, J., Bitencourt-Cervi, C. M., Maciel, V. B., Yoshida, C. M., & Carvalho, R. A.

568

(2019). Aqueous hibiscus extract as a potential natural pH indicator incorporated

569

in natural polymeric films. Food Packaging and Shelf Life, 19, 47−55.

570

Pourjavaher, S., Almasi, H., Meshkini, S., Pirsa, S., & Parandi, E. (2017).

571

Development of a colorimetric pH indicator based on bacterial cellulose

572

nanofibers and red cabbage (Brassica oleraceae) extract. Carbohydrate

573

Polymers, 156, 193−201.

27

ACCEPTED MANUSCRIPT

574

Priyadarshi, R., Kumar, B., Deeba, F., Kulshreshtha, A., & Negi, Y. S. (2018).

575

Chitosan films incorporated with Apricot (Prunus armeniaca) kernel essential oil

576

as active food packaging material. Food Hydrocolloids, 85, 158−166.

577

Rasid, N. A. M., Nazmi, N. N. M., Isa, M. I. N., & Sarbon, N. M. (2018). Rheological,

578

functional and antioxidant properties of films forming solution and active gelatin

579

films incorporated with Centella asiatica (L.) urban extract. Food Packaging and

580

Shelf Life, 18, 115−124.

581

Rubilar, J. F., Cruz, R. M., Silva, H. D., Vicente, A. A., Khmelinskii, I., & Vieira, M.

582

C. (2013). Physico-mechanical properties of chitosan films with carvacrol and

583

grape seed extract. Journal of Food Engineering, 115, 466−474.

584

Sadilova, E., Stintzing, F. C., & Carle, R. (2006). Anthocyanins, colour and

585

antioxidant properties of eggplant (Solanum melongena L.) and violet pepper

586

(Capsicum annuum L.) peel extracts. Zeitschrift für Naturforschung C, 61,

587

527−535.

588

Sánchez-Ilárduya, M. B., Sánchez-Fernández, C., Garmón-Lobato, S., Abad-García,

589

B., Berrueta, L. A., Gallo, B., & Vicente, F. (2014). Detection of non-coloured

590

anthocyanin–flavanol derivatives in Rioja aged red wines by liquid

591

chromatography-mass spectrometry. Talanta, 121, 81−88.

592

Shankar, S., & Rhim, J. W. (2016). Preparation of nanocellulose from

593

micro-crystalline cellulose: The effect on the performance and properties of

594

agar-based composite films. Carbohydrate Polymers, 135, 18−26.

28

ACCEPTED MANUSCRIPT

595

Toro-Márquez, L. A., Merino, D., & Gutiérrez, T. J. (2018). Bionanocomposite films

596

prepared from corn starch with and without nanopackaged Jamaica (Hibiscus

597

sabdariffa) flower extract. Food and Bioprocess Technology, 11, 1955−1973.

598

Uranga, J., Etxabide, A., Guerrero, P., & de la Caba, K. (2018). Development of

599

active fish gelatin films with anthocyanins by compression molding. Food

600

Hydrocolloids, 84, 313−320.

601

Wang, X., Yong, H., Gao, L., Li, L., Jin, M., & Liu, J. (2019). Preparation and

602

characterization of antioxidant and pH-sensitive films based on chitosan and

603

black soybean seed coat extract. Food Hydrocolloids, 89, 56−66.

604

Wei, Y. C., Cheng, C. H., Ho, Y. C., Tsai, M. L., & Mi, F. L. (2017). Active gellan

605

gum/purple sweet potato composite films capable of monitoring pH variations.

606

Food Hydrocolloids, 69, 491−502.

607

Wu, X., & Prior, R. Yong, H., Wang, X., Bai, R., Miao, Z., Zhang, X., & Liu, J.

608

(2019). Development of antioxidant and intelligent pH-sensing packaging films

609

by incorporating purple-fleshed sweet potato extract into chitosan matrix. Food

610

Hydrocolloids, 90, 216−224.

611

Wu, X., & Prior, R. L. (2005). Identification and characterization of anthocyanins by

612

high-performance liquid chromatography-electrospray ionization-tandem mass

613

spectrometry in common foods in the United States: Vegetables, nuts, and grains.

614

Journal of Agricultural and Food Chemistry, 53, 3101−3113.

615

Yong, H., Wang, X., Bai, R., Miao, Z., Zhang, X., & Liu, J. (2019). Development of

616

antioxidant and intelligent pH-sensing packaging films by incorporating

29

ACCEPTED MANUSCRIPT

617

purple-fleshed sweet potato extract into chitosan matrix. Food Hydrocolloids, 90,

618

216−224.

619

Zepon, K. M., Martins, M. M., Marques, M. S., Heckler, J. M., Morisso, F. D. P.,

620

Moreira, M. G., Ziulkoskic, A. L. & Kanis, L. A. (2019). Smart wound dressing

621

based on κ-carrageenan/locust bean gum/cranberry extract for monitoring

622

bacterial infections. Carbohydrate Polymers, 206, 362−370.

623

Zhang, J., Zou, X., Zhai, X., Huang, X., Jiang, C., & Holmes, M. (2019). Preparation

624

of an intelligent pH film based on biodegradable polymers and roselle

625

anthocyanins for monitoring pork freshness. Food chemistry, 272, 306−312.

626

Zhao, Y., Wu, X., Yu, L., & Chen, P. (2017). Retention of polyphenols in blueberries

627

(Vaccinium

corymbosum)

after

different

cooking

methods,

using

628

UHPLC–DAD–MS based metabolomics. Journal of Food Composition and

629

Analysis, 56, 55−66.

630

Zhao, Z., Wu, M., Zhan, Y., Zhan, K., Chang, X., Yang, H., & Li, Z. (2017).

631

Characterization and purification of anthocyanins from black peanut (Arachis

632

hypogaea L.) skin by combined column chromatography. Journal of

633

Chromatography A, 1519, 74−82.

634

Zhai, X., Shi, J., Zou, X., Wang, S., Jiang, C., Zhang, J., et al. (2017). Novel

635

colorimetric films based on starch/polyvinyl alcohol incorporated with roselle

636

anthocyanins for fish freshness monitoring. Food Hydrocolloids, 69, 308−317.

30

ACCEPTED MANUSCRIPT

637

Figure captions:

638

Fig. 1. Color variations of PEE (A) and BEE (B) in different buffer solutions (pH 2 to

639

13).

640

Fig. 2. UV-vis spectra of PEE (A) and BEE (B) in different buffer solutions (pH 2 to

641

13).

642

Fig. 3. Physical appearances (A) and UV-vis light transmittance (B) of CS, CS-PEE

643

and CS-BEE films.

644

Fig. 4. SEM micrographs of surfaces (on the left) and cross-sections (on the right) of

645

CS (A and H), CS-PEE I (B and I), CS-PEE II (C and J), CS-PEE III (D and K),

646

CS-BEE I (E and L), CS-BEE II (F and M), and CS-BEE III (G and N) films.

647

Fig. 5. FT-IR spectra of PEE (a), BEE (b), CS (c), CS-PEE I (d), CS-PEE II (e),

648

CS-PEE III (f), CS-BEE I (g), CS-BEE II (h) and CS-BEE III (i) films.

649

Fig. 6. XRD patterns of PEE (a), BEE (b), CS (c), CS-PEE I (d), CS-PEE II (e),

650

CS-PEE III (f), CS-BEE I (g), CS-BEE II (h) and CS-BEE III (i) films.

651

Fig. 7. DPPH radical scavenging activity of CS, CS-PEE I, CS-PEE II, CS-PEE III,

652

CS-BEE I, CS-BEE II and CS-BEE III films. Each value represents mean ± standard

653

deviation (SD) of triplicates.

654

Fig. 8. Appearances of CS, CS-PEE and CS-BEE films after being immersed in

655

different buffer solutions (pH 2 to 13) for 15 min.

31

(A) pH 2 pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

pH 9

pH 10 pH 11 pH 12 pH 13

(B) pH 2

pH 4

pH 5

pH 6

pH 7

pH 8

pH 9

pH 10 pH 11 pH 12 pH 13

pH 3

Fig. 1

32

ACCEPTED MANUSCRIPT (A) 2.5 pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 pH 11 pH 12 pH 13

Absorbance

2

1.5

1

0.5

0 450

500

550

600

650

700

Wavelength (nm)

(B) 2.5 pH2 pH3 pH4

2

pH5

Absorbance

pH6 pH7

1.5

pH8 pH9 pH10

1

pH11 pH12 pH13

0.5

0 450

500

550

600

Wavelength (nm)

Fig. 2

33

650

700

(A)

CS film

CS-PEE I film

CS-PEE II film

CS-PEE III film

CS-BEE I film

(B) 100

Transmission (%)

80

60

CS-BEE III film CS-PEE I film CS-PEE II film CS-PEE III film CS-BEE I film CS-BEE II film CS-BEE III film

40

20

0

-20 200

300

400

500

600

700

Wavelength (nm)

Fig. 3

34

800

CS-BEE II film

CS-BEE III film

ACCEPTED MANUSCRIPT (A)

(H)

(B)

(I)

(C)

(J)

(D)

(K)

(E)

(L)

(F)

(M)

(G)

(N)

Fig. 4

35

a

b 1632 1514 1601 1442

3257

c

808 1259 1194

1634 1514 1600 1444

3259

1028 804

1189 1026

d

1633

1335 1551 1406

2924

Relative intensity

3327

2878

1151

925

649

925

649

1026

e 3279

1634

1151

1634

1151

1336 1551 1406

2922 2878

1026

f 2926 3265

g

1547

2879

925

649

1023 1633

1336 1550 1406

2925 3277

1338 1405

2879

1151

925

649

1026

h

1634

1337 1549 1405

2927 3272

2879

649

1023

i

1634

2927 3266

3267

4000

925

1151

925

1151

649

1547 1405

2879

1024 1634

1338 1549 1406

2927 2879

3000

1338

925

1151

649

1024

2000

1000 −1

Wavenumber (cm )

Fig. 5

36

400

a b Relative intensity

c d e f g h i 0

10

20

30

40

50

60

70

2θ (º)

Fig. 6

37

80

ACCEPTED MANUSCRIPT 50 CS film CS-PEE I film CS-PEE II film CS-PEE III film CS-BEE I film CS-BEE II film CS-BEE III film

Scavenging activity (%)

40

30

20

10

0 0

1

2

3

Film equivalent (mg/mL)

Fig. 7

38

4

5

pH 2

pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

pH 9

CS film CS-PEE I film

CS-PEE II film

CS-PEE III film

CS-BEE I film CS-BEE II film CS-BEE III film

Fig. 8

39

pH 10

pH 11

pH 12

pH 13

ACCEPTED MANUSCRIPT Highlights Anthocyanin-rich purple and black eggplant extracts (PEE and BEE) were obtained. PEE and BEE increased physical, antioxidant and pH-sensitive properties of CS film. Film property was related to the composition and content of anthocyanins in extracts. CS-BEE films had better physical and functional properties than CS-PEE films. CS-PEE and CS-BEE films could be used as antioxidant and intelligent packaging films.

Table 1 The compositions of anthocyanins in PEE and BEE as analyzed by HPLC-MS. [M]+ (m/z) Relative content (%)

Extract Retention time (min) Anthocyanins PEE

BEE

8.68

Pelargonidin-3-O-(3'',6''-dimalonyl-hexoside) 605

1.07

9.34

Malvidin-3-O-glucoside

493

0.72

11.30

Cyanidin-3-O-glucosyl-rutinoside

757

91.22

12.50

Delphinidin-3-O-glucoside-catechin

755

6.99

4.27

Malvidin-3-O-glucoside-4-vinyl-catechin

805

1.36

5.15

Delphinidin-3-O-rutinoside

611

97.32

6.47

Delphinidin-3-O-rutinoside-5-O-glucoside

773

1.18

15.62

Pelargonidin-3-O-acetyl-glucoside

475

0.13

40

Table 2 Color parameters including L, a, b and ΔE of CS, CS-PEE and CS-BEE films. Films

L

CS film

b

ΔE

91.25 ± 0.14a –1.11 ± 0.07a

2.70 ± 0.65a

3.48 ± 0.61f

CS-PEE I film

74.84 ± 0.60b –5.93 ± 0.17b

–1.81 ± 0.13b

16.89 ± 0.61e

CS-PEE II film

64.88 ± 0.02c –10.52 ± 0.02c –9.45 ± 0.08c

29.31 ± 0.01d

CS-PEE III film

57.39 ± 3.08d –13.22 ± 0.28d –12.42 ± 0.69d 37.80 ± 3.03c

CS-BEE I film

56.07 ± 0.49d –8.23 ± 2.20c

–12.30 ± 0.47d 37.56 ± 0.13c

CS-BEE II film

40.86 ± 3.15e –8.85 ± 0.03c

–16.87 ± 0.47e 53.22 ± 2.80b

CS-BEE III film 32.45 ± 1.49f

a

–4.04 ± 1.23b

–15.54 ± 1.36e 60.92 ± 1.00a

Values are given as mean ± SD (n = 3). Different lower case letters in the same column indicate significantly different (p < 0.05).

41

Table 3 Thicknesses, moisture contents and water vapor permeability of CS, CS-PEE and CS-BEE films. Films

Film thickness (mm) Moisture content (%) Water vapor permeability (10−11 g m−1 s−1 Pa−1)

CS film

0.060 ± 0.002e

31.71 ± 0.08cd

1.14 ± 0.09ab

CS-PEE I film

0.062 ± 0.001de

31.00 ± 0.4d

0.96 ± 0.01c

CS-PEE II film

0.064 ± 0.001d

31.07 ± 0.17d

1.24 ± 0.01a

CS-PEE III film

0.068 ± 0.002bc

32.05 ± 0.01c

1.24 ± 0.01a

CS-BEE I film

0.065 ± 0.002cd

33.12 ± 0.14b

1.07 ± 0.01b

CS-BEE II film

0.070 ± 0.001b

34.53 ± 0.60a

1.10 ± 0.08b

CS-BEE III film 0.074 ± 0.001a

33.38 ± 0.62b

1.22 ± 0.17a

Values are given as mean ± SD (n = 10 for film thickness, and n = 3 for moisture content and water vapor permeability). Different lower case letters in the same column indicate significantly different (p < 0.05).

42

Table 4 Tensile strength and elongation at break of CS, CS-PEE and CS-BEE films. Films

Tensile strength (MPa) Elongation at break (%)

CS film

24.27 ± 3.02d

27.34 ± 5.54d

CS-PEE I film

29.42 ± 3.31c

30.97 ± 4.91d

CS-PEE II film

30.60 ± 3.80bc

38.23 ± 4.54c

CS-PEE III film

39.78 ± 4.42a

57.38 ± 5.30a

CS-BEE I film

33.91 ± 4.68b

60.26 ± 5.87a

CS-BEE II film

33.99 ± 4.17b

61.03 ± 7.21a

CS-BEE III film 24.74 ± 4.02d

48.96 ± 5.72b

Values are given as mean ± SD (n = 6). Different lower case letters in the same column indicate significantly different (p < 0.05).

43

Table 5 The ΔE values of CS, CS-PEE and CS-BEE films after being exposed to different buffer solutions for 15 min. pH values CS film

CS-PEE I film

CS-PEE II film

CS-PEE III film CS-BEE I film

CS-BEE II film CS-BEE III film

pH 2

3.85 ± 0.08ef F

9.44 ± 1.41ef E

16.60 ± 3.61f D

21.00 ± 0.24e C

22.29 ± 0.57f BC 25.20 ± 1.77h B

39.88 ± 0.05e A

pH 3

4.67 ± 0.01cd E 5.98 ± 0.07g E

15.66 ± 1.18f D

24.31 ± 0.27de C

23.01 ± 0.04ef C

29.57 ± 2.55g B

54.49 ± 0.15b A

pH 4

5.22 ± 0.12bc F

8.94 ± 1.11fg E

17.52 ± 0.48f D

29.01 ± 0.69c C

29.75 ± 4.60cd C 37.75 ± 0.06f B

56.49 ± 1.63a A

pH 5

5.12 ± 0.41bc F

18.08 ± 5.18ab E

31.93 ± 0.90a D

38.19 ± 0.66a CD 35.28 ± 0.62ab C 49.62 ± 2.31ab B 56.47 ± 1.16a

pH 6

5.63 ± 1.28b F

17.13 ± 0.01abc E 33.44 ± 0.86a D

41.13 ± 0.18a D

34.62 ± 1.84ab C 46.16 ± 0.99cd B 56.74 ± 0.08a A

pH 7

3.83 ± 0.19ef F

15.80 ± 1.19bc E

32.22 ± 2.24a D

34.34 ± 0.43b C

34.97 ± 1.47ab C 47.63 ± 0.84bc B 57.78 ± 0.72a A

pH 8

4.80 ± 0.21cd G 19.47 ± 0.34a F

33.22 ± 0.75a E

28.32 ± 3.73cd D

38.31 ± 0.26a C

44.91 ± 2.00d B

53.54 ± 0.58bc A

pH 9

6.76 ± 0.13a D

11.54 ± 0.53def C 25.95 ± 0.82c B

26.97 ± 2.92cd B

27.77 ± 0.38de B 50.25 ± 0.43a A

51.21 ± 1.37d A

pH 10

3.18 ± 0.20f F

14.08 ± 0.05cd E

23.05 ± 0.45de D 28.97 ± 1.52c C

28.81 ± 0.81cd C 41.24 ± 0.02e B

50.42 ± 1.46d A

pH 11

3.26 ± 0.03f F

12.38 ± 0.61ed E

29.00 ± 0.92b D

34.56 ± 4.18b CD 32.25 ± 4.94bc C 46.17 ± 0.08cd B 50.74 ± 0.31d A

pH 12

3.59 ± 0.36ef F

17.44 ± 0.18ab E

20.92 ± 0.36e D

28.52 ± 2.92c C

27.12 ± 0.43de C 35.47 ± 0.22f B

51.24 ± 1.21d A

pH 13

4.21 ± 0.24de G 19.42 ± 1.58a F

24.86 ± 1.01a E

28.43 ± 0.35c D

34.72 ± 0.35ab C 36.36 ± 0.17f B

51.90 ± 0.46cd A

A

Values are expressed as mean ± SD (n = 3). Different lower case letters in the same column indicate significantly different (p < 0.05). Different capital letters in the same row indicate significantly different (p < 0.05).

44

Table 6 The change of acidity and pH values in the milk as well as the color response of CS-PEE and CS-BEE films at different storage times. Time Acidity (°T)

pH value

(h)

Color response of CS-PEE and CS-BEE films CS-PEE I film

0

13.35 ± 0.05g

6.68 ± 0.02a

2

13.90 ± 0.20fg

6.54 ± 0.01b

4

14.53 ± 0.13f

6.54 ± 0.01b

6

15.71 ± 0.01e

6.53 ± 0.01b

8

16.05 ± 0.05e

6.52 ± 0.01b

10

17.73 ± 0.18d

6.46 ± 0.04c

12

23.38 ± 0.08c

6.18 ± 0.01d

14

31.40 ± 0.39b

6.00 ± 0.01e

16

69.00 ± 1.50a

4.47 ± 0.05f

CS-PEE II film

CS-PEE III film

CS-BEE I film

CS-BEE II film

Values are given as mean ± SD (n = 3). Different letters in the same column indicate significantly different (p < 0.05).

45

CS-BEE III film