carboxymethyl chitosan biocomposite films incorporated with epigallocatechin gallate

Journal Pre-proof Preparation and characterization of multifunctional konjac glucomannan/ carboxymethyl chitosan biocomposite films incorporated with epigallocatechin gallate Jishuai Sun, Haixin Jiang, Mingwei Li, Yinzhu Lu, Yu Du, Cailing Tong, Jie Pang, Chunhua Wu PII:

S0268-005X(19)32545-7

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105756

Reference:

FOOHYD 105756

To appear in:

Food Hydrocolloids

Received Date: 29 October 2019 Revised Date:

11 January 2020

Accepted Date: 9 February 2020

Please cite this article as: Sun, J., Jiang, H., Li, M., Lu, Y., Du, Y., Tong, C., Pang, J., Wu, C., Preparation and characterization of multifunctional konjac glucomannan/carboxymethyl chitosan biocomposite films incorporated with epigallocatechin gallate, Food Hydrocolloids (2020), doi: https:// doi.org/10.1016/j.foodhyd.2020.105756. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author Statement Jishuai Sun: Conceptualization, Methodology, Investigation, Formal analysis, Software, Data curation, Writing-original draft. Haixin Jiang: Methodology, Investigation, Software, Writing-original draft. Mingwei Li: Investigation, Data curation, Software. Yinzhu Lu: Performed the experiments. Yu Du: Data curation. Cailing Tong: Formal analysis. Jie Pang: Validation, Funding acquisition. Chunhua Wu: Project administration, Supervision, Conceptualization, Writing-review & editing, Funding acquisition. All authors read and approved the final manuscript.

Graphical abstract

1

Preparation and characterization of multifunctional konjac

2

glucomannan/carboxymethyl chitosan biocomposite films incorporated

3

with epigallocatechin gallate

4

Jishuai Suna,c, Haixin Jianga,c, Mingwei Lic, Yinzhu Luc, Yu Dua,c, Cailing Tonga,c, Jie Panga,b,c,d,*, Chunhua

5

Wu a,b,c,d,*1 a

6

Engineering Research Centre of Fujian-Taiwan Special Marine Food Processing and Nutrition, Ministry

7

of Education, Fuzhou, Fujian, 350002, China

8

b

State Key Laboratory of Food Safety Technology for Meat Products, Xiamen, Fujian, 361100, China

9

c

College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China

10

d

Key Laboratory of Marine Biotechnology of Fujian Province, Institute of Oceanology, Fujian Agriculture

11

and Forestry University, Fuzhou, Fujian, 350002, China

12

Abstract

13

In this study, biocomposite films of konjac glucomannan (KGM)/carboxymethyl

14

chitosan (CMCS) with different epigallocatechin gallate (EGCG) concentrations (0%, 5%,

15

10%, 15%, and 20%, w/w, based on KGM dry weight) were prepared by the solution casting

16

method. To evaluate the effect of EGCG incorporation on the structural and physicochemical

17

properties of the KGM/CMCS matrix, the obtained composite film-forming solutions (FFS)

18

and films were systematically evaluated. The results from the Fourier transform infrared

19

spectroscopy (FT-IR) analysis showed that EGCG interacted with the KGM/CMCS matrix

20

through hydrogen bonds in the composite matrix, which corroborated with the rheological

21

results of FFS. The scanning electron microscopy (SEM) images revealed that the

22

incorporated EGCG (5–15%) was well-dispersed within the composite matrix, thereby

23

improving the final biocomposite films' physicochemical properties. The addition of EGCG

24

significantly enhanced the antioxidant and antibacterial activities of the films (P < 0.05),

25

while decreased the transmittance and elongation at break. In addition, appropriate content of

26

EGCG remarkably reduced water vapor permeability (WVP) and enhanced mechanical 1

*Corresponding author. E-mail addresses: [email protected] (J. Pang); [email protected] (C. H. Wu). 1

27

properties and thermal stability of the films. While the WVP of the films incorporated with 15%

28

EGCG reduced to 2.65 g·mm·m-2·day-1·KPa· -1, the tensile strength increased by 9.16 MPa.

29

Thus, the developed multifunctional KGM/CMCS biocomposite films when blended with

30

EGCG could have several potential applications as active packaging materials.

31

Key words: Konjac glucomannan; Carboxymethyl chitosan; Active films; Epigallocatechin

32

gallate; Bioactive activity

33

1. Introduction

34

In recent years, an increasing number of studies are focusing on fabricating active and

35

biodegradable food packaging films. Although synthetic petroleum-based packaging materials

36

are widely used in our daily life, the utilization of these packaging films has caused serious

37

ecological problems (S. S. K, M.P, & G.R, 2019; Tang, Zhang, Zhao, Guo, & Zhang, 2018).

38

Therefore, many researchers are committed to developing bioactive and bio-based packaging

39

materials to overcome these issues (Wang et al., 2019; Wu, Deng, Luo, & Deng, 2019).

40

Particularly, it has been shown that the use of natural biopolymers in active food packaging

41

can effectively extend shelf life, improve safety, and enhance the sensory properties of

42

packaged foods (Lei et al., 2019; Priyadarshi et al., 2018).

43

Konjac glucomannan (KGM), a natural polysaccharide and water-soluble dietary fiber, is

44

derived from the tubers of the Amorphophallus konjac plant (Wu et al., 2019b; Zhong et al.,

45

2018). KGM, which is non-toxic and possesses good film-forming ability and

46

biodegradability has been widely used in food, pharmaceutical, and cosmetic industries

47

(Zhong et al., 2018). Moreover, KGM has a relatively high viscosity in aqueous solutions, and

48

therefore the obtained active films could control the release of active compounds (Ni et al.,

49

2019). However, the innate shortcomings of single biopolymer-based films, such as low

50

mechanical strength and poor barrier properties are major restrictions to their industrial

51

applications (Wu et al., 2019b). Consequently, these limitations were overcome by chemically

52

modifying the biopolymer and/or by preparing biopolymer-based composite films (Kumar,

53

Kumar, & Pandey, 2018; Sun et al., 2019; Wu et al., 2012). In a recent study by Kumar,

54

Kumar, & Pandey (2018), the efficacy of a novel, antimicrobial binary grafted chitosan film 2

55

was tested and its potential use as a food packaging material was validated. Another study by

56

Sun et al., (2019) has demonstrated that the incorporation of TEMPO-oxidized chitin

57

nanocrystals in the konjac glucomannan (KGM)/chitosan (CS) matrix produced flexible and

58

transparent bionanocomposite films with enhanced mechanical and barrier properties. Further,

59

Wu et al., (2012) have shown that the presence of curdlan enhanced the moisture barrier

60

properties of the KGM/curdlan films, and that these blend films could have potential

61

applications as edible food films and coatings.

62

Carboxymethyl chitosan (CMCS), an amphoteric derivative of chitosan (CS), is prepared

63

by substituting carboxymethyl groups (COOH) into the CS amino and hydroxyl sites (Zimet

64

et al., 2019). CMCS has better water solubility compared with chitosan as it possesses

65

abundant carboxymethyl groups; it is a non-toxic, biodegradable, and a biocompatible

66

chitosan derivative (Bai et al., 2019). Further, Suriyatem, Auras, and Rachtanapun (2018)

67

have also shown that CMCS has antibacterial activity. However, CMCS films have poor

68

antioxidant activity and mechanical strength, deeming it necessary to improve the physical

69

and functional properties of CMCS films in order to broaden their scope of application in

70

food packaging.

71

In contrast to traditional packaging films, multifunctional biocomposite packaging films

72

can effectively maintain food safety and quality (Wang et al., 2018). Polyphenols, such as

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grape seed extract, gallic acid, tea polyphenols, and catechins are frequently used active

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compounds (Lei et al., 2019; Sogut & Seydim, 2019; Z. Wu et al., 2019d). Among various

75

polyphenols, epigallocatechin-gallate (EGCG) is one of the major phenolic compounds

76

extracted from tea leaves (Siripatrawan & Noipha, 2012). In recent years, many studies have

77

reported that EGCG can be used as an antioxidant and/or an antibacterial agent in food

78

packaging materials (Ni et al., 2019; Nilsuwan, Benjakul, Prodpran, & de la Caba, 2019).

79

EGCG possesses a large number of hydroxyl groups in its molecular structure. KGM and

80

CMCS also host abundant hydroxyl and/or carboxylate groups, respectively, in their

81

molecular chains. Therefore, it is evident that the hydrogen bonding interactions between

82

KGM, CMCS, and EGCG could lead to the formation of a dense and a strong biocomposite 3

83

matrix, thus improving the physical properties of the film. Moreover, EGCG acts as an

84

antioxidant and an antibacterial agent, thus enhancing the functional performance of the

85

biocomposite films (Lei et al., 2019; Ni et al., 2019). To the best of our knowledge,

86

multifunctional KGM/CMCS films containing EGCG have never been reported till date.

87

Therefore, the aim of this study was to fabricate and characterize the multifunctional

88

KGM/CMCS biocomposite films incorporated with EGCG. The effects of EGCG on the

89

rheological properties of the film-forming solutions (FFS), and physical and chemical

90

properties of film samples were evaluated. In addition, the functional properties of film

91

samples were also investigated.

92

2. Materials and methods

93

2.1. Materials

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Konjac glucomannan (KGM, MW: 1000 kDa) with 90% purity was purchased from San

95

Ai Konjac Food Co. Ltd. (Sichuan, China). Carboxymethyl chitosan (CMCS, MW: 260 kDa)

96

with 80% degree of substitution was purchased from Macklin Biotechnology Co. Ltd.

97

(Shanghai,

98

2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma Chemical Reagent Co.,

99

Ltd. (USA). Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were provided

100

by food microbiology laboratory in College of Food Science, Fujian Agriculture and Forestry

101

University (Fuzhou, Fujian, China). All the other chemical reagents were of analytical grade.

102

2.2. Preparation of film-forming solutions (FFS)

China).

Epigallocatechin-gallate

(EGCG,

98%

purity)

and

103

The films were prepared by casting method according to our previous study (Sun et al.,

104

2019). The KGM powder was dissolved in hot distilled water (60 °C) under magnetic stirring

105

for 2 h to obtain 1% (w/v) KGM solution. Subsequently, the CMCS powder was dissolved in

106

distilled water under magnetic stirring for 1 h to obtain 3% (w/v) CMCS solution. The

107

resulting CMCS solution was then added dropwise to the KGM solution at a KGM/CMCS

108

mass rate of 70/30 (w/w) to obtain the KGM/CMCS solution. Subsequently, EGCG was

109

dispersed into distilled water and sonicated for 5 min. For EGCG-incorporated films, EGCG

110

solution was added at the concentrations of 5%, 10%, 15%, and 20% (w/w, based on the 4

111

weight of the KGM) to the KGM/CMCS solutions, respectively. Subsequently, glycerol (0.1

112

g/g KGM/CMCS) was added to these solutions as a plasticizer. Finally, the mixtures were

113

stirred at 25 °C for 30 min and sonicated for 15 min to obtain the KGM/CMCS/EGCG

114

biocomposite FFS.

115

2.3. Preparation of biocomposite films

116

The biocomposite films were prepared by casting method according to our previous

117

study (Wu et al. (2019a) with slight modifications. In brief, FFS (25 mL each) were poured

118

into plastic petri dishes (ΦA=90 mm) and subsequently, the dishes were dried at 45 °C for 24

119

h. After drying, the films were peeled from the surface of the petri dishes and conditioned at

120

50±1% relative humidity and 25±1 °C for at least 2 days in a controlled environment chamber

121

prior to characterization.

122

2.4. Characterization

123

2.4.1. Rheological measurements of FFS

124

The rheological behavior of FFS was measured by using an Anton paar MCR 301

125

rheometer (Anton Paar Instruments Inc., Austria) equipped with a parallel-plate geometry

126

having a diameter of 50 mm (PP50) at 25 °C and at a shear rate of 0.1–100 s-1. For dynamic

127

frequency sweep measurements, dynamic frequency sweeps were carried out by employing a

128

1% strain amplitude within the linear viscoelasticity range at a frequency of 0.01–100 rad·s-1

129

at 25 °C.

130

2.4.2. Fourier transform infrared spectroscopy (FT-IR)

131

The molecular structure of EGCG and KGM/CMCS/EGCG biocomposite films were

132

characterized by using a FT-IR spectrometer (Thermo Fisher Scientific Co., Ltd., MA, USA)

133

through KBr module. The FT-IR spectrometer worked at a resolution of 4 cm-1 and 32 scans

134

were performed in the range of 400–4000 cm-1.

135

2.4.3. Scanning electron microscopy (SEM)

136

The film samples were frozen in liquid nitrogen and snapped immediately to obtain the

137

cross-section. Finally, samples were mounted on a bronze stub and sputtered with a thin layer

138

of gold. The cross-section morphology of the film samples was observed by using a scanning 5

139

electron microscope (SEM, Japan Electron Optics Laboratory Co., Ltd, Tokyo, Japan) with an

140

acceleration voltage of 20 kV.

141

2.4.4. Thermal stability

142

The thermal stability of the KGM/CMCS/EGCG biocomposite films was determined

143

using a thermo-gravimetric analyzer (TGA, STA409-PC, Netzsch, Germany). The weight of

144

the film samples was about 5.0 mg. The scan was determined at a heating rate of 10 °C·min-1

145

from 25–600 °C under a nitrogen atmosphere at a scan rate of 30 mL·min-1.

146

2.4.5. Optical properties

147

The color of the KGM/CMCS/EGCG biocomposite films was measured by using a

148

colorimeter (CS-200, CHNSpec Technology Co., Ltd, Hangzhou, China). The parameters, L*

149

(lightness), a* (red/green), and b* (yellow/blue) were used to evaluate the color of the film

150

samples. The KGM/CMCS films were used as control films. The total color difference (△E)

151

was calculated as follows: ∆ = ∆ ∗  + ∆∗  + ∆ ∗ 

152

The light transmittance spectrum of the KGM/CMCS/EGCG films was measured by

153

using a UV-2600 spectrophotometer (Shimadzu Scientific Instruments, Inc., Kyoto, Japan). In

154

brief, these film samples were cut into 8
40 mm rectangular strips and placed into a

155

colorimetric apparatus using air as the reference. The spectra of these film samples were

156

obtained within the range of 200–800 nm at room temperature.

157

2.4.6. Film thickness

158

The thickness of the KGM/CMCS/EGCG biocomposite films was determined by using a

159

hand-held micrometer (Model MDC-25, Mitutoyo. Tokyo, Japan). The film samples were cut

160

into square strips (4 cm
4 cm) and were then measured at 5 different random locations. The

161

measurements were repeated for at least 5 times for each sample simultaneously.

162

2.4.7. Moisture content

163

The moisture content (MC) in the film samples was evaluated by employing a previously

164

method by R. K, G, Banat, Show, & Cocoletzi (2019) with some modifications. The film

165

samples (20
20 mm) were weighted as the initial weight (W1). Subsequently, they were 6

166

placed in a drying oven at 105 °C for 24 h and weighed again (W0). The MC in the film

167

samples was calculated as follows: MC % =

 −  × 100 

168

where,  and  are the initial and the final dry weights of each film sample.

169

2.4.8. Water vapor permeability

170

The water vapor permeability (WVP) of the KGM/CMCS/EGCG biocomposite films

171

was determined gravimetrically at 25 °C according to our previous method (Sun et al., 2019)

172

with slight modifications. Briefly, 3 g of anhydrous calcium chloride was dried for 24 h at

173

105 °C, placed into a weighing bottle, and then sealed with the obtained film samples. All the

174

film samples were balanced at a relative humidity of 75% at room temperature. All

175

measurements were tested at least 5 times simultaneously. The WVP was calculated as

176

follows: WVP =

× ×

177

where, WVP is the water vapor permeability (g·mm·m-2 day kPa), w is the mass change of the

178

weighing bottle (g) after 24 h, d is thickness of the film sample (mm), s is the effective film

179

area (m2), p is the partial pressure of water vapor over the film sample (kPa).

180

2.4.9. Mechanical properties

181

The mechanical properties of the KGM/CMCS/EGCG biocomposite films were tested by

182

using an AG-IC50kN Texture Analyzer (Shimadzu, Tokyo, Japan) according to a previously

183

described method by Sun et al. (2019) with slight modifications. Briefly, film samples were

184

cut into 10ⅹ50 mm2 rectangular strips and conditioned at approximately 50% relative

185

humidity for 24 h. The strips were clamped between two tensile grips of the machine and

186

were pulled using a crosshead speed of 10 mm·min-1. All measurements were tested at least 6

187

times simultaneously. The mechanical properties of film samples namely, the tensile strength

188

(TS, MPa) and elongation at break (Eb, %) were calculated as follows: Tensile strength TS, MPa =

7

. ×/

189 Elongation at breakEb, % =

−  × 100% 

190

where, F is the maximum force used for the strips (N), W is the width (mm) of the film

191

sample, T is the thickness (mm) of the film sample, L is the final length (mm) of the film

192

sample, and L0 is the initial length (mm) of the film sample.

193

2.4.10 Antioxidant test

194

The antioxidant properties of the KGM/CMCS/EGCG films were evaluated by

195

employing the DPPH free radical scavenging assay, according to a previously described

196

method by Chunhua Wu et al. (2020) with slight modifications. In brief, the film samples (20

197

mg) were individually immersed in 4 mL of 100 µM DPPH ethanol solution. The solutions

198

were then placed for 12 h in darkness at room temperature. Then, the absorbance of the

199

solutions was measured at 517 nm. The DPPH scavenging activity was calculated as follows: DPPH scavenging ability % =

9:;;< − 9= × 100% 9:;;<

200

where, ADPPH is the absorbance of 95% ethanol with DPPH solution, AS is the absorbance of

201

the film sample solution with DPPH solution.

202

2.4.11 Antibacterial test

203

The antibacterial properties of the film samples were evaluated according to a previously

204

described method by Wu et al. (2020) with slight modifications. The KGM/CMCS/EGCG

205

biocomposite films were evaluated for antibacterial properties against the gram-positive S.

206

aureus and the gram-negative E. coli bacteria via the agar disk diffusion method by assessing

207

the inhibition zones (mm). One hundred microliters of bacterial suspension from the

208

inoculums (~105 CFU·mL-1) was seeded onto LB agar. Then, the film samples were cut into

209

10 mm discs and placed on the inoculated LB agar. Thereafter, the petri dishes were incubated

210

at 37 °C for 24 h and the inhibition zones were measured with a caliper.

211

2.5. Statistical analysis

212

Statistical data were analyzed using Origin 9.0 software and SPSS software statistical

213

analysis system (SPSS 25.0 for windows, SPSS Inc., Chicago, IL). Least significant

214

differences (LSD) multiple comparison tests were used to determine significance of the 8

215

obtained data (P < 0.05). All obtained data were presented as mean ± standard deviation.

216

3. Results and discussion

217

3.1. Characterization of the FFS

218

3.1.1. Rheological properties of the FFS

219

It is necessary to evaluate the rheological properties of the FFS, as they are directly

220

related to the structure, the spreadability, and mechanical properties of biopolymer solutions

221

(Ma, Du, Yang, & Wang, 2017; Wu et al., 2019b). The apparent viscosities of all sample

222

solutions at different shear rates are shown in Fig. 1a. The shear-thickening behavior observed

223

at low shear rates may be due to a newly formed entanglement structure that developed

224

through interactions between CMCS, KGM, and/or EGCG. At high shear rates, the apparent

225

viscosities of all the sample solutions decreased as the shear rates increased, which indicated

226

the presence of pseudoplastic properties or existence of a shear thinning region in these FFS

227

(Liang, Sun, Cao, Li, & Wang, 2018; Sun et al., 2019). Similar results have been observed in

228

other biopolymer-based FFS (Silva-Weiss, Bifani, Ihl, Sobral, & Gómez-Guillén, 2014; Wu et

229

al., 2019b). As the EGCG concentration increased from 5% to 15%, the overall apparent

230

viscosities of the KGM/CMCS/EGCG FFS increased gradually when compared with that of

231

the pure KGM/CMCS FFS. Therefore, in accordance to a previous study, our results suggest

232

the formation of new hydrogen bonds among KGM, CMCS, and EGCG (Sun et al., 2019).

233

More specifically, the hydrogen bonds could have formed between the hydroxyl and/or

234

carboxylate groups of the KGM, CMCS, and EGCG, thus resulting in the formation of a

235

dense and compact matrix with improved properties (Lei et al., 2019). However, the

236

incorporation of 20% (w/w) EGCG decreased the apparent viscosity of the FFS. Collectively,

237

these findings indicate that EGCG promotes the biopolymer reassembly by crosslinking the

238

biopolymer molecules on one hand, and subsequently retards it by competitively binding to

239

the –OH, –NH2, and –COOH bonds of the biopolymers, on the other. Therefore, these

240

findings indicate that 15% (w/w) EGCG is the optimal concentration, and that it is more

241

suitable for the casting process at room temperature.

242

The dynamic rheological properties of FFS were also determined by an oscillatory test to

243

enhance our understanding of the delicate network structure of the polymer system. The

244

storage modulus (G´, Pa) and the loss modulus (G´´, Pa) of FFS are shown in Fig. 1b. The

245

value of G′ and G″ of FFS increased as the frequency increased. It was observed that at low

9

246

frequencies, the value of G´´ was higher than that of G´, which indicated that FFS was in the

247

liquid form and no gelation was observed. In contrast, at higher frequencies, the value of G´´

248

was lower than that of G´ implying the formation of a close non-covalent entangled network

249

between KGM, CMCS, and EGCG (Zhang, Liu, Han, Sun, & Wang, 2019b). Thus, G″ value

250

and G´ value of FFS increased upon the addition of 5% (w/w) EGCG, indicating that EGCG

251

blended well in the KGM/CMCS matrix. Further, we found that G″ and G´ values of FFS

252

significantly increased with an increase in EGCG concentration. Meanwhile, a solid-like flow

253

behavior of FFS was also observed. Nevertheless, it was found that the G´ value of FFS

254

incorporated with 20% EGCG decreased at low frequency. However, the addition of 15%

255

EGCG showed the highest G′ value at the lowest frequency, suggesting the existence of a

256

strong entanglement and hydrogen bonding between KGM, CMCS, and EGCG that in turn

257

enhanced the mechanical strength of the resulting films (Sun et al., 2019; Wu et al., 2019b).

258

This finding was further confirmed in the next structural characterization.

259

3.2. Characterization of the KGM/CMCS/EGCG biocomposite films

260

3.2.1. FT-IR spectra

261

Fig. 2 shows the FT-IR spectra of EGCG, KGM/CMCS biocomposite films, and

262

KGM/CMCS biocomposite films incorporated with different concentrations of EGCG. As

263

shown in Fig. 2a, the major bands of EGCG were observed at 3358 cm-1, 1697 cm-1, 1621

264

cm-1, and 1461 cm-1, which corresponded to O-H stretching, C-H stretching, C=O stretching,

265

and C=C stretching, respectively (Nilsuwan, Benjakul, & Prodpran, 2018; Ruan et al., 2019).

266

In Fig. 2b, the FT-IR spectra of the KGM/CMCS biocomposite films showed a relatively

267

broad peak at around 3433 cm-1, which was associated with O-H stretching. The peak around

268

2926 cm-1 was attributed to the C-H vibrations. The peak visible at 1636 cm-1 was assigned to

269

intermolecular hydrogen bonds. These results were similar to our previous reports (Wu et al.,

270

2012; Wu et al., 2019b). Notably, there were no additional peaks after incorporating EGCG

271

into the KGM/CMCS matrix, suggesting that no covalent bonds were formed between KGM,

272

CMCS, and EGCG. However, a few peak intensities were found to rise upon the

273

incorporation of EGCG. For instance, the peak intensity increased to 1636 cm-1 following the

274

addition of EGCG into the KGM/CMCS matrix. These changes indicated that EGCG was

275

successfully dispersed into the KGM/CMCS film matrix owing to the formation of 10

276

intermolecular hydrogen bonds (Gomes Neto et al., 2019; Lei et al., 2019). Collectively, these

277

findings indicate that incorporation of EGCG into the KGM/CMCS films is useful for

278

enhancing the mechanical and barrier properties of the biocomposite films.

279

3.2.2. Film micromorphology analysis

280

Cross-sectional micromorphology images of the films are shown in Fig. 3. There were

281

noticeable differences in the cross-section micromorphology between the KGM/CMCS and

282

the KGM/CMCS/EGCG biocomposite films. The cross-section of KGM/CMCS biocomposite

283

films showed smooth and homogeneous surfaces, which indicated that both KGM and CMCS

284

had good compatibility and film-forming ability (Lei et al., 2019). The cross-section

285

microstructure of the films became rougher with the increasing EGCG content, possibly due

286

to the formation of new hydrogen bonding interactions and destruction of original hydrogen

287

bonding interactions between the KGM, CMCS, and EGCG (Ruan et al., 2019; Zhang et al.,

288

2019a). After 15% EGCG was incorporated into the FFS matrix, the cross-section

289

microstructure of the films had relatively a neat microstructure, without any noticeable pores

290

or cracks, demonstrating that EGCG was well distributed in the film-forming matrix.

291

However, the incorporation of 20% EGCG exhibited an undesirable microstructure, which

292

could be attributed to the excessive amounts of EGCG that in turn resulted in agglomerate

293

formation in the film-forming matrix. These findings were found to be in agreement with the

294

observed rheological properties of the FFS. Similar changes to the surface of the films have

295

also been reported in previous studies (Lei et al., 2019; Ruan et al., 2019). As shown in Fig. 3,

296

the cross-section morphology of the KGM/CMCS/EGCG 15% film was much smoother than

297

that of the other films. The relatively tighter inner structure of the KGM/CMCS/EGCG 15%

298

film was beneficial to the improvement of the mechanical and barrier properties of the

299

biocomposite films.

300

3.2.3. Thermogravimetric analysis

301

To understand the intermolecular structural interaction between KGM, CMCS, and

302

EGCG, it is necessary to evaluate the thermal stability of the biocomposite films. The TGA

303

curves of EGCG and film samples are shown in Fig. 4. Notably, EGCG was more thermally

11

304

stable than KGM/CMCS/EGCG films. It was found that KGM/CMCS films incorporated with

305

EGCG enhanced the thermal stability. The TGA curves of the film samples were divided into

306

three main stages between 25 °C and 600 °C. The first stage between 25 °C and 230 °C

307

mainly corresponded to the evaporation of physically weak and chemically strong bound

308

water (Shankar, Reddy, Rhim, & Kim, 2015). In the second stage between 230 °C and 350 °C,

309

a large loss of weight occurred (~ 50%), which may be related to the polysaccharide pyrolytic

310

decomposition of the polysaccharide (Lei et al., 2019). In the final stage between 350 °C and

311

600 °C, the reduced weight loss could be ascribed to the thermal decomposition of char (Lei

312

et al., 2019). It was found that the mass loss of the KGM/CMCS/EGCG biocomposite films

313

was lesser than that of the KGM/CMCS films at the tested temperature, indicating that the

314

incorporation of EGCG enhanced the thermal stability of the biocomposite films. Similar

315

results have been observed in the pectin-konjac glucomannan composite edible films

316

incorporated with tea polyphenol (Lei et al., 2019).

317

3.2.4. Optical properties

318

The optical properties of food packaging films directly influenced food acceptability and

319

preservation (R. K et al., 2019; Lei et al., 2019). The transparency of the film samples is

320

shown in Fig. 5. The incorporation of EGCG into the KGM/CMCS matrix noticeably reduced

321

the light transmittance of the films, providing good UV-Vis light barrier properties.

322

Particularly, the UV-Vis light barrier properties of the films may be beneficial to the

323

preservation of fat-rich food. As shown in Fig. 5, the KGM/CMCS/EGCG biocomposite films

324

exhibited lower UV-Vis light transmittance compared with that of the KGM/CMCS film. The

325

UV-Vis light barrier properties of the KGM/CMCS films incorporated with EGCG gradually

326

increased as the EGCG content increased. Moreover, the UV-Vis light transmittance of the

327

KGM/CMCS film incorporated with EGCG was nearly 0% between 200 nm and 400 nm,

328

which indicated that the KGM/CMCS/EGCG biocomposite films could effectively protect

329

food from UV-Vis light. The color parameters of each film sample are listed in Table 2. The

330

value of L* (P < 0.05) decreased, indicating that the incorporation of EGCG into the

331

KGM/CMCS matrix reduced the brightness of the films. On the other hand, the value of ∆E

332

increased significantly (P < 0.05), which suggested that the KGM/CMCS/EGCG films had a

333

bright color. Overall, the KGM/CMCS films incorporated with EGCG had better light barrier

334

properties, indicating that they could be used to prevent food spoilage when employed as food 12

335

packaging materials in the future.

336

3.2.5. Film thickness and moisture content

337

The thickness and MC of the biocomposite films with various concentrations of EGCG

338

are shown in Table 2. Incorporation of EGCG significantly increased (P < 0.05) the thickness

339

of the KGM/CMCS/EGCG biocomposite films, which may be due to the increase in the

340

amount of solid content in FFS with an increase in the EGCG content (Lei et al., 2019; Wu et

341

al., 2019b). As shown in Table 2, the KGM/CMCS biocomposite film presented the highest

342

MC value (P < 0.05), which may be attributed to the hydrophilic groups in KGM and CMCS

343

that formed intermolecular interactions with moisture (Liu et al., 2019). The MC value of the

344

KGM/CMCS film was found to be 16.52%. Notably, the MC values of the KGM/CMCS

345

biocomposite films incorporated with 5%, 10%, 15%, and 20% (w/w) EGCG decreased to

346

13.40%, 13.21%, 13.59%, and 12.36%, respectively. Therefore, the significantly decrease in

347

the MC values of the KGM/CMCS/EGCG biocomposite films could be possibly due to the

348

hydrogen bonding interactions between the KGM, CMCS, and EGCG in the matrix of the

349

biocomposite films (P < 0.05). Similarly, Lei et al. (2019) also demonstrated a significant

350

decrease in the moisture content of the films when tea polyphenol was incorporated into the

351

pectin-KGM biocomposite films. In addition, we noticed that the thickness of the film

352

samples was also affected by the moisture content inside the films.

353

3.2.6. Water vapor permeability

354

The effect of EGCG on the WVP of the film samples is presented in Table 2. As it can be

355

seen that with an increase in the EGCG content, the values of WVP decreased from 5.70

356

g·mm·m-2 ·day-1·kPa-1 (for the KGM/CMCS biocomposite film) to 2.65 g·mm·m-2·day-1·kPa-1

357

(for the KGM/CMCS/EGCG film with 15% EGCG (w/w)), and 3.91 g·mm·m-2·day-1·kPa-1

358

(for the KGM/CMCS/EGCG film with 20% EGCG (w/w)). The KGM/CMCS/EGCG films

359

exhibited lower WVP values than the KGM/CMCS film (P < 0.05). The decrease in WVP

360

could be due to the existence of strong hydrogen bonding between KGM, CMCS, and EGCG

361

in the matrix, which was consistent with the FT-IR and the SEM studies. These findings were

362

similar to those of the previous studies where a significant decrease was observed in the WVP

363

values of gelatin films incorporated with green tea extract (Lei et al., 2019; Wu et al., 2013).

364

Nevertheless, excess amounts of EGCG decreased the WVP, which further indicated that

365

EGCG could promote the biopolymer reassembly by crosslinking the biopolymers molecules,

366

and subsequently could also retard the biopolymer reassembly by competitively binding to the

367

–OH, –NH2, and –COOH bonds of the biopolymers. Notably, the WVP of the film samples 13

368

may also be affected by the moisture content inside film. Therefore, the EGCG content

369

directly affects the WVP of the film samples.

370

3.2.7. Mechanical properties

371

Mechanical properties are useful parameters for evaluating the attributes of the food

372

packaging materials (Priyadarshi et al., 2018). Particularly, tensile strength (TS) and

373

elongation at break (Eb) are two important elements for evaluating the mechanical properties

374

of these films. The effect of different concentrations of EGCG on the mechanical properties of

375

the KGM/CMCS films are shown in Fig. 6. The presence of EGCG in the KGM/CMCS films

376

showed significant differences in the TS and Eb values (P < 0.05). The TS value of the

377

KGM/CMCS film was found to be 12.56 MPa. In contrast, the TS values of the incorporated

378

EGCG films with 5%, 10%, 15% and 20% (w/w) EGCG increased to 16.10, 16.98, 21.72, and

379

17.00 MPa, respectively. On the other hand, the Eb value of the KGM/CMCS film was found

380

to be 131.06%, whereas the Eb values of the incorporated EGCG films with 5%, 10%, 15%

381

and 20% (w/w) EGCG decreased to 75.04%, 70.66%, 56.77% and 57.46 %, respectively.

382

Therefore, it is evident that the incorporation of 15% EGCG (w/w) resulted in a significant

383

increase in TS (21.72 MPa) and a significant decrease in Eb (56.77%), which corresponded to

384

an increase of 72.93% and a decrease of −56.68% compared with those of the KGM/CMCS

385

films, respectively (P < 0.05). Collectively, these findings indicate that the mechanical

386

properties of the films were significantly enhanced, especially the Eb. Consistent with the

387

results of this study, a study by Nilsuwan et al. (2018) has reported that TS and Eb

388

significantly increase decrease, respectively, in fish gelatin-based films incorporated with

389

EGCG. Moreover, as seen in the SEM images (Fig. 3), the cross-section microstructures of

390

the KGM/CMCS/EGCG films with 15% EGCG (w/w) had relatively a neat microstructure,

391

without any noticeable pores or cracks, thus demonstrating an enhancement of structural

392

properties in the incorporated films. Notably, the internal moisture content of the film samples

393

was also found to have a certain effect on the mechanical properties of the film samples.

394

3.2.8. Antioxidant properties

395

Oxidative reactions have a negative impact on the appearance, quality, and nutritional

396

value of food at all stages of food processing and transportation (Liu et al., 2019). Therefore,

397

it is necessary to assess the antioxidant properties of active packaging films. The antioxidant

398

activity of the KGM/CMCS/EGCG biocomposite films was determined by the DPPH radical

399

scavenging assay. As shown in Table 3, the DPPH radical scavenging activity of the 14

400

biocomposite films significantly increased with increasing concentrations of EGCG (P <

401

0.05). The DPPH radical scavenging activity of the KGM/CMCS biocomposite film was

402

found to be 20.00%, which could be attributed to the antioxidant nature of the hydroxyl

403

groups present in KGM and CMCS (Wang, Chen, Zhong, & Xu, 2007; Zhang, Chen, & Yang,

404

2014). By contrast, the DPPH radical scavenging activity of the KGM/CMCS/EGCG

405

biocomposite films significantly increased (P < 0.05) with increasing concentrations of

406

EGCG, which could be possibly due to the strong antioxidant nature of EGCG (Nilsuwan et

407

al., 2018; Wang, et al., 2019a). Similar results related to the DPPH radical scavenging activity

408

of the biocomposite films have been observed when green tea extracts were incorporated into

409

other biopolymer FFS (Lei et al., 2019; Ruan et al., 2019; Wang et al., 2018). The above

410

results indicated that the KGM/CMCS/EGCG biocomposite films could be used in

411

antioxidant packaging.

412

3.2.9. Antibacterial properties

413

Agar diffusion assays were used to evaluate the antibacterial activity of the

414

KGM/CMCS/EGCG biocomposite films. The antibacterial activities of the KGM/CMCS

415

biocomposite films in the presence and absence of EGCG are shown in Table 2. It was found

416

that the KGM/CMCS films did not exert a significant antibacterial effect on the gram-positive

417

S. aureus and the gram-negative E. coli bacteria. However, the diameter of the inhibition zone

418

against the tested bacteria significantly increased (P < 0.05) as the concentration of EGCG

419

increased. Previous studies have demonstrated that EGCG could have a negative effect on the

420

growth rate of a wide variety of bacteria, and is especially more effective against the

421

gram-negative E. coli bacteria than the gram-positive S. aureus bacteria (Wang, et al., 2019a).

422

The antibacterial activity of the biocomposite films observed in this study suggested that the

423

films could be applied as antibacterial packaging materials in the food industry.

424

4. Conclusions

425

In this study, EGCG was used as an active agent that was incorporated into the

426

KGM/CMCS-based FFS in order to prepare EGCG-loaded KGM/CMCS biocomposite films.

427

The effect of EGCG concentration on different properties of the biocomposite films was

428

systematically evaluated. The FT-IR and rheological results indicated that EGCG interacted

429

with KGM and CMCS through intermolecular hydrogen bonding. The SEM images revealed

430

that EGCG blended well into the KGM and CMCS film matrix. The addition of EGCG 15

431

improved antioxidant and antibacterial properties of the KGM/CMCS films. Moreover, the

432

KGM/CMCS/EGCG biocomposite films served as good water vapor barriers possessing a

433

high thermal stability and good UV-light barrier properties, and demonstrated better

434

mechanical properties than those films lacking EGCG. Nevertheless, excessive amounts of

435

EGCG might have negative effects on the properties of these biocomposite films, thus

436

indicating that EGCG can promote the biopolymer reassembly by crosslinking the

437

biopolymers molecules, on one hand, and can also retard the biopolymer reassembly by

438

competitively binding with the –OH, –NH2, and –COOH bonds of the biopolymers, on the

439

other. In conclusion, the KGM/CMCS biocomposite films incorporated with EGCG could

440

have potential applications as active food packaging materials.

441

Declaration of interest

442

The authors declare that they have no conflict of interest in the publication of this

443

manuscript. This study is original research that has not been published previously, and not

444

under consideration for publication elsewhere.

445

Acknowledgements

446

This work was supported by Fujian Province Natural Science Foundation (Grant No.

447

2019J01390), the National Natural Science Foundation of China (Grant No. 31801616 and

448

31772045) and 13th Five-year Plan on Fuzhou Marine Economic Innovation and

449

Development Demonstration City Project (Grant No. FZHJ17).

450

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19

Table caption Table 1 Color parameters of the biocomposite films. Table 2 Physical properties of the biocomposite films. Table 3 Antioxidant and antibacterial properties of the biocomposite films. Table 1. Color parameters of the biocomposite films. Sample

L*

a*

b*

∆E

KGM/CMCS

56.29 ± 0.18a

-4.57 ± 0.06d

0.82 ± 0.16e

------

KGM/CMCS/EGCG 5%

43.20 ± 0.15b

3.56 ± 0.03c

17.60 ± 0.05a

22.25 ± 0.08d

KGM/CMCS/EGCG 10%

34.86 ± 0.67c

5.89 ± 0.59a

13.02 ± 0.62b

26.81 ± 0.29c

KGM/CMCS/EGCG 15%

29.81 ± 0.19d

4.48 ± 0.34b

7.30 ± 0.49c

28.09 ± 0.30b

KGM/CMCS/EGCG 20%

26.92 ± 0.38e

3.36 ± 0.13c

6.66 ± 0.13d

30.28 ± 0.48a

Photographs

All data are shown as mean ± standard deviation (SD). The superscripts different letters in a column indicate significant differences (P < 0.05).

Table 2. Physical properties of the biocomposite films. Sample

Thickness (μm)

MC(%)

WVP (g mm/m2 day kPa)

KGM/CMCS

87.80 ± 1.57b

16.52 ± 0.26a

5.70 ± 0.56a

KGM/CMCS/EGCG 5%

92.40 ± 0.71a

13.40 ± 0.15b

3.56 ± 0.17b

KGM/CMCS/EGCG 10%

94.33 ± 1.23a

13.21 ± 0.24b

3.49 ± 0.30b

KGM/CMCS/EGCG 15%

94.37 ± 0.83a

13.59 ± 0.37b

2.65 ± 0.28c

KGM/CMCS/EGCG 20%

95.27 ± 1.07a

12.36 ± 0.10c

3.91 ± 0.22b

All data are shown as mean ± standard deviation (SD). The superscripts different letters in a column indicate significant differences (P < 0.05).

Table 3. Antioxidant and antibacterial properties of the biocomposite films. Diameter of inhibition zone (mm)

DPPH radical scavenging activity(%)

S. aureus (+)

E. coli (−)

KGM/CMCS

20.00 ± 0.32e

10.53 ± 0.21e

10.30± 0.16e

KGM/CMCS/EGCG 5%

53.82± 0.13d

13.03 ± 0.25d

14.17 ± 0.29d

KGM/CMCS/EGCG 10%

66.67 ± 0.80c

14.70 ± 0.33c

15.60 ± 0.51c

KGM/CMCS/EGCG 15%

72.76 ± 0.63b

16.17 ± 0.29b

17.07 ± 0.42b

KGM/CMCS/EGCG 20%

75.58 ± 0.67a

17.10 ± 0.16a

17.73 ± 0.33a

Sample

All data are shown as mean ± standard deviation (SD). The superscripts different letters in a column indicate significant differences (P < 0.05).

FIGURE CAPTIONS Fig. 1. Steady (a) and dynamic (b) rheological properties of biocomposite film-forming solutions. Fig. 2. FT-IR spectra of EGCG (a); KGM/CMCS films (b); KGM/CMCS/EGCG 5% films (c); KGM/CMCS/EGCG 10% films (d); KGM/CMCS/EGCG 15% films (e); and KGM/CMCS/EGCG 20% films (f). Fig. 3 SEM micrographs of films cross-sections of KGM/CMCS films (A, B); KGM/CMCS/EGCG 5% films (C, D); KGM/CMCS/EGCG 10% films (E, F); KGM/CMCS/EGCG 15% films (G, H); KGM/CMCS/EGCG 20% films (I, J). Fig. 4 TGA curves of the biocomposite films. Fig. 5 UV-vis light transmittance of the biocomposite films. Fig. 6 Effect of EGCG content in the tensile strength and elongation at break (%) of the biocomposite films.

Fig. 1. Steady (a) and dynamic (b) rheological properties of biocomposite film-forming solutions.

Fig. 2. FT-IR spectra of EGCG (a); KGM/CMCS films (b); KGM/CMCS/EGCG 5% films (c); KGM/CMCS/EGCG 10% films (d); KGM/CMCS/EGCG 15% films (e); and KGM/CMCS/EGCG 20% films (f).

Fig. 3. SEM micrographs of films cross-sections of KGM/CMCS films (A, B); KGM/CMCS/EGCG 5% films (C, D); KGM/CMCS/EGCG 10% films (E, F); KGM/CMCS/EGCG 15% films (G, H); KGM/CMCS/EGCG 20% films (I, J).

Fig. 4. TGA curves of the biocomposite films.

Fig. 5. UV-vis light transmittance of the biocomposite films. .

Fig. 6. Effect of EGCG content in the tensile strength and elongation at break (%) of the biocomposite films.

Highlights 1. Epigallocatechin gallate (EGCG) was well dispersed in the KGM/CMSC matrix. 2. EGCG was incorporated into KGM/CMCS films to develop active food packaging. 3. Mechanical and barrier properties of the films were enhanced by EGCG. 4. The films showed good antioxidant and antibacterial activity.