Evaluation of a Novel Collagenous Matrix Membrane Cross-Linked

Subscriber access provided by Iowa State University | Library

Food and Beverage Chemistry/Biochemistry

Evaluation of a Novel Collagenous Matrix Membrane Cross-linked with Catechins Catalyzed by Laccase, a Sustainable Biomass Taotao Qiang, Liang Chen, Zhuan Yan, and Xinhua Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05810 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

Journal of Agricultural and Food Chemistry

236x134mm (150 x 150 DPI)

ACS Paragon Plus Environment


1

Evaluation of a Novel Collagenous Matrix Membrane

2

Cross-linked with Catechins Catalyzed by Laccase, a

3

Sustainable Biomass

4

Taotao Qiang a, b, *, Liang Chen a, b, Zhuan Yan a, b, Xinhua Liu a, b

5

a

6

Science &Technology, Xi’an 710021, China

7

b

8

Education ( Shaanxi University of Science & Technology ) , Xi’an 710021, China

9

Abstract

College of Bioresources Chemical and Materials Engineering, Shaanxi University of

National Demonstration Center for Experimental Light Chemistry Engineering

10

Collagen, a sustainable and biodegradable biomass material, has many

11

applications in different scope including applied in food packaging. However, owing

12

to its poor mechanical properties, this kind of application was limited. In this work,

13

the collagen was cross-linked with the catechin under the incubation of laccase to

14

improve the mechanical properties of collagen, and the cross-linked collagen

15

exhibited properties of excellent antioxidant capacity and lower swelling ratio.

16

Meanwhile, fourier transform infrared spectrometer (FTIR), X-ray diffraction (XRD),

17

and X-ray photoelectron spectroscopy (XPS) results provide evidence for the changes

18

in the structure of collagen after cross-linked with the catechin. From the aspects of

19

the thermal stability, tensile strength, elongation, antioxidant capacity, swelling,

20

solubility and morphological analysis, the cross-linked collagen has better physical

21

properties in comparison with natural collagen. This indicates that the physical


Page 2 of 37

Page 3 of 37


22

properties and antioxidant capacity of collagen after cross-linked with catechins were

23

improved significantly. Therefore, the cross-linked collagen can be used as green food

24

packaging materials.

25

Keywords: collagen; catechins; biocatalyst; laccase; food packaging



26

INTRODUCTION

27

Currently, renewable and biodegradable materials as well as natural additives are

28

considered as sustainable alternatives for food packaging applications1. Biomass

29

material is usually used as a potential replacement towards enhancing the natural

30

materials’ application efficiency and reducing the environmental negative issues

31

produced by the accumulation of short term plastic wastes. Collagen is one of the

32

most significant biomaterials with plenty of applications such as in food packaging,

33

prostheses, pharmaceutical, wound healing and cosmetics2-4. Thanks to the high

34

biodegradability, relatively good mechanical and self-aggregating properties of

35

collagen, it plays an important role in food packaging5, 6. However, collagen-based

36

materials have some defects such as inadequate mechanical strength, weak

37

thermostability, bad structural-stability and inoxidizability7-9, which limit their direct

38

application in food packaging. Hence, it is necessary for the collagen-based materials

39

to be modified to enhance their physicochemical performances and inoxidizability by

40

using some chemical or physical methods.

41

In previous research10-13, glutaraldehyde, 1-ethyl-3(-3 dimethyl aminopropyl)

42

carbodiimide hydrochloride (EDC), poly(γ-glutamic acid)-derivatives, formaldehyde,

43

acyl azide, carbodiimides and hexamethylene diisocyanate have been widely used as

44

modifying agent in collagen crosslinking. The introduction of these cross-linking

45

agents or additives could provide a potential and functional modification in

46

physicochemical performance of collagen materials. Generally, food packaging


Page 4 of 37

Page 5 of 37


47

materials were traditionally required to contain food products and protect them from

48

contamination, humidity and oxidations process14. These cross-linkers could afford

49

favourable physicochemical performances for collagen membrane. However, they

50

cannot provide good inoxidizability when collagen membrane is used as food

51

packaging. In addition to this, the safety issues and environmental concerns caused by

52

the usage of cross-linker should be also addressed. Catechins are well known as one

53

of the good plant antioxidants15 that are generally recognized as synonyms of

54

nutriceuticals16. Meanwhile, catechins are renewable and biodegradable and it present

55

extensively in vegetation or all winery residues from grapes17,

56

protecting point of view, catechin, a nonvolatile flavonoid, has gain superiority,

57

mainly because it allows reducing the original characters loss during storage process

58

such as reacting with nutrition compounds of foodstuffs (i.e.: essential oils)19. In this

59

sense, natural catechins are of special interest in the biomass food packaging not only

60

to protect food from oxidative deterioration processes, but also to afford favourable

61

physicochemical performances. Catechins as antioxidant was loaded into PU based on

62

PLLA-b-PCL-b-PLLA reported by Arrieta et al.20 In the same way, Arrieta et al.21

63

incorporated catechins with poly(ester-urethane) as food packaging that has shape

64

memory and antioxidant activity. These products all have good performances.

65

However, under the requirement of sustainable development and cleaner production,

66

collagen based material has better superiority that has a wide range of sources

67

including mammals’ skin and tendons and marine organism.


18

. From a food


68

Collagen has a large number of active groups, such as abundant amino and

69

carboxyl which can react with various types of cross-linkers. At the same time,

70

catechins have four phenolic hydroxyl groups. Previous study has confirmed that the

71

hydroxyl groups of catechins could mainly occur cross-link with amino and carboxyl

72

groups of collagen by hydrogen bonding22. Hence the physicochemical performances

73

of collagen film cross-linked with catechins still present poor durability during

74

washing and acid/alkali processes23. Laccase (EC1.10.3.2) is a promising biocatalyst

75

with many possible applications including cross-linking assistant in the process of

76

collagen cross-linking modifying. It is capable of oxidizing a variety of substrates

77

including polyphenols, polyamines and aromatic amines24, 25, further decarboxylate

78

them and attack their methoxy groups (demethylation)26. Laccase oxidize their

79

substrate via one electron hydrogen abstraction generating radicals3. Whilst these

80

radicals are prone to further react, leading to self-polymerization. Thus, relevant

81

oligomers or polymers are covalently coupled by C-C, C-O or C-N bonds27. Under the

82

presence of air, the o-hydroxylation of catechins will be catalyzed to o-diphenols and

83

the oxidation of o-diphenols will further be catalyzed to o-quinones28-30. The

84

schematic diagram of laccase catalyzed catechins is shown in Figure 1. Furthermore，

85

the o-quinones can undergo a complex set of non-enzymatic reactions with

86

nucleophilic amine groups of collagen. It has been shown that o-quinones are

87

covalently conjugated to free amine and mercapto groups through the Schiff’s base

88

and/or Michael-type reactions mechanisms in previous researches31-34. Based the


Page 6 of 37

Page 7 of 37


89

reaction, protein-flavonoid conjugates were used as modifier to improve flax fibre

90

surface properties in the research of Kim35. For food packaging area, the improvement

91

of mechanical properties and antioxidant capacity at the same time using biomaterial

92

is always a challenge. To realize this dream, we use the collagen extracted from

93

bovine tendon as the basic material for maintaining high mechanical performances

94

and further cross-linked with catechins under the assisting of laccase to improve the

95

mechanical properties and antioxidant capacity.

96

In this work, catechins were used as biocrosslinking agent and antioxidant at the

97

same time. Whilst, one useful method assisting catechins to form oligomers

98

(dehydrodycatechin) or o-quinones catechins by using laccase. Meanwhile, the

99

o-quinones groups on the catechins or dehydrodycatechin can further crosslink with

100

collagen though covalent bond. Moreover, the catechins, which were not oxidized by

101

laccase, also can cross-link with collagen by hydrogen bonding. Due to the covalent

102

cross-linking among catechins and collagen, the mechanical strength, thermostability,

103

structural-stability of collagen film is apparently improved. In addition, the

104

mechanical and chemical stability of the catechins cross-linked collagen film was

105

investigated as well. Therefore, this study provided an attractive route to produce

106

renewable and biodegradable collagen-based food packaging film exhibiting excellent

107

physicochemical performances and inoxidizability. In this way, the material provided

108

in this study can be considered as a potential alternative for addressing excessive

109

amounts of waste from synthetic packaging.



110 Figure 1. The schematic diagram of laccase catalyze catechins

111 112

MATERIALS AND METHODS

113

Materials. Collagen extracted from bovine tendon was self-prepared according to

114

reported work36. Catechins were supplied by Henan Hua Shang Food Additives Co.

115

Ltd., China. Laccase from White rot fungus was purchased from Suzhou Fu Lu Bio.

116

Technol. Co. Ltd., China. Unless noted otherwise, all of other reagents were analytical

117

grade.

118

Laccase Assay. Laccase activity was evaluated with the oxidation of 2,2’-Azobis

119

(3-ethylbenzothiazole-6-sulfonaic acid) (ABTS) to its cation radical (ABTS•+). The

120

laccase activity was measured by monitoring the oxidation of ABTS at 420 nm (ε420

121

=36000 M−1 cm−1) in 0.1 M citrate buffer at pH≈4.5 at 30 ± 1 ℃ using a UV BlueStar

122

A spectrophotometer.

123

Catalyzing of Catechins. 1 g of catechins and 160 U g-1 of laccase (on dry catechins

124

basis) were dissolved in 50 mL of citrate buffer at pH 5.5 in a 250 mL reaction vessel,

125

and the obtained solution were then agitated at 480 rpm at 40 ℃, with an incubation


Page 8 of 37

Page 9 of 37


126

time of 2 h. Meanwhile, air was supplied as O2 source and inlet slowly and

127

continuously through the air pump. After this, the reaction product were collected and

128

centrifuged for 3 min at 6000 T min-1, and the obtained supernatant liquid was

129

laccase-catalyzed catechin (LC).

130

UV-visible Spectroscopy (UV-vis) analysis of LC. The catechins catalyzed by

131

different dosage laccase (0, 20, 40, 60, 100, 160, 190 U g-1) was measured by

132

Keysight Cary-5000 UV−vis spectrophotometer (Agilent Technol. Inc., U.S.A). The

133

wavenumber range was carried out within 300 to 500 nm.

134

Collagen Film Cross-linked With Catalyzed Catechins. Firstly, the extracted soluble

135

collagen of 5 mg mL-1was diluted from 25 mL of initial concentration (20 mg mL-1)

136

by 75 mL of water. Subsequently, the diluted collagen was casted on circular teflon

137

plates (Yang Zhong Fuda Insulated Electrical Co. Ltd., China) (15×15 cm), and then it

138

was dried at ambient temperature. After this, collagen film was obtained, namely

139

Col-F. Sencondly, the Col-F was homogenized at different dosage (calculated from

140

LC mother solution) of LC (0 %, 5 %, 10 %, 15 %, 20 %, 25 %, w/w, based on Col-F)

141

that was diluted in 40 mL 0.1 M of citrate buffer with pH 5.5 at 30 ± 1 ℃, and the

142

incubation time is 5 h. Thirdly, the cross-linked Col-F were immersed in deionized

143

water for 30 min to reduce the content of redundant LC and then washed with PBS for

144

several times. Finally, the Col-F cross-linked by LC (LC-Col-F) after natural drying

145

was conditioned for 24 h at 25 ± 0.5 ℃ and 50 ± 2 % RH for further analysis.

146

Fourier Transform Infrared Spectrometer (FTIR) Measurements. The structure



147

analysis of LC-Col-F was performed with a FTIR spectrophotometer (Vertex70,

148

Bruker Co., Germany) in the wavelength range of 4000~400 cm−1, and with 32 times

149

scanning and transmission mode at an interval of 4 cm−1. The samples were triturated

150

with KBr in the ratio of 1:100 (w/w) and then compressed into flake for FRIR

151

measurement.

152

X-ray Diffraction (XRD) Measurements. The samples were cut into small squares of

153

2×2 cm , and then measured by XRD instrument (D&Advance, Bruker Co., Germany)

154

in the 2θ range of 5~40°with Cu Kα1 radiation (λ = 0.1541 nm) at 40 kV and 40 mA.

155

The Cross-linking Degree of LC-Col-F. The content of amino slightly changes in the

156

Col-F can be directly reflected in the case of covalent cross-linking of collagen and

157

catechins. We determined the content of amino groups with ninhydrin assay37, 38. The

158

standard leucine of 5 μg mL-1 was used as the standard solution, and the standard

159

curve was derived from the absorbance at 570 nm in function of different amounts of

160

leucine. Before the measurement, these LC-Col-F samples were washed several times

161

with 0.5 % sodium bicarbonate, and catechins that bonded with collagen amino in the

162

form of hydrogen bonds were removed. The amino content of LC-Col-F (D1) and

163

Col-F (D0) were calculated according to the standard curve and the cross-linking

164

degree (Cd) was further obtained. The formula is as follows:

165

Cd =

166

X-ray Photoelectron Spectroscopy (XPS) Measurements. XPS (Kratos Analytical

167

Ltd designs, U.K.) was used to analyze the elemental composition of the sample.

D0 -D1 D0

×100% (1)


Page 10 of 37

Page 11 of 37


168

High-resolution spectra were obtained under ultra-high vacuum condition using

169

monochromatic Al Kα X-ray radiation at 12 kV and 25 mA and with an analyzer pass

170

energy of 10 eV. All the high-resolution spectra were corrected by a linear background

171

before fitting.

172

Thermostability Evaluation of LC-Col-F. Synchronous TG thermal analyzer

173

(STA409PC, NETZSCH-Gerätebau GmbH, Germany) was used for analyzing the

174

thermal stability of these LC-Col-F samples under the temperature ranging from 20 to

175

600 ℃ at a constant heating rate of 10 ℃ min-1 under nitrogen flow. Differential

176

scanning calorimetry (DSC) (DSC-Q2000, TA Instruments, USA) was used for

177

measuring the denaturation temperature (Td) of these samples, and scanned over a

178

temperature range from 30 to 140 ℃ at a heating rate of 10 ℃ min-1 under a nitrogen

179

flow of 40 mL min-1.

180

Mechanical Properties Evaluation of LC-Col-F. The thickness of LC-Col-F was

181

determined by ISO39 method using film thickness gauge (CHY-C2A, Labthink

182

Instruments Co., Ltd., China). Tensile strength and elongation at break were

183

determined by the standard ASTM D882 method40 using electronic universal testing

184

machine (Yangzhou Xintianhui Electronic Technol. Co. Ltd., China). The swelling of

185

Col-F and LC-Col-F were determined using the procedure of Figueiro41. Water

186

solubility measurements were carried out according to the method reported by Saiut42.

187

All of these measurements were performed three times, and the corresponding

188

averages have been illustrated along with the standard deviation.



189

Evaluation of Antioxidant Capacity. The LC-Col-F antioxidant potential was

190

evaluated by the method33, with which the scavenging activity of ABTS•+ free radicals

191

reflecting their antioxidant capacity was assayed. Here, the preformed ABTS•+ free

192

radicals were generated by the oxidation of 1.0 mM ABTS with 0.35 mM potassium

193

persulfate for 12 h in the dark at room temperature. Afterwards, the initial ABTS•+

194

solution was diluted with 0.2 M of PBS at pH≈7.4 to reach an appropriate

195

concentration. The absorbance of 4 mg samples mixed with 4 mL of working solution

196

was measured at 734 nm after standing at room temperature for 30 min in the dark.

197

The scavenging effect was calculated by equation (2). The absorbance of ABTS•+

198

solution was marked A0, the absorbance of samples was marked A1. All the results are

199

the averages of triplicate measurements.

200

scavenging effect=

201

Scanning Electron Microscopic (SEM) Analysis. A scanning electron microscopy

202

(MIRA II-XMH, TESCAN, Ltd, Czechia) was applied to observe the LC-Col-F

203

membranes’ morphology and the specimens were sputter coated with aurum and

204

carried out at accelerating voltage of 10 kV.

205

RESULTS AND DISCUSSION

206

UV-vis analysis of LC. UV-Vis spectroscopy analysis is an available method to

207

monitor the changes in the phenols structure such as oxidation and formation of

208

quinones43. The UV–vis spectra of catechins catalyzed by different dosage laccase are

209

shown in Figure 2, from which, significant changes were observed after the reactions

A0 -A1 A0

×100% (2)


Page 12 of 37

Page 13 of 37


210

processed at pH 5.5. The conspicuous broad-band between 320 and 410 nm is

211

detected in the spectra of catechins incubating with laccase belongs to the absorption

212

range of quinones 44. In addition, the absorbance from 320 to 410 nm also enhances

213

with the increase of the laccase dosage. However, a slight spectrum variation of

214

catechins was observed during the same time, which was attributable to the

215

auto-oxidation at a citrate buffer with pH of 5.5.

216 217

Figure 2. UV–vis spectra of the laccase catalyzed catechins

218

Characterization of LC-Col-F. The FTIR spectra of Col-F cross-linked with different

219

content of LC are displayed in Figure 3. As previously reported45,

220

collagen possesses a special triple helix conformation, which can be directly

221

correlated with its characteristic amide bands. The collagen III band absorption peak


46

, the intact


222

appeared at 1246 cm-1, which was caused by the C-N stretching vibration and the N-H

223

bending vibration of the amide bond in the collagen molecular chains. Furthermore,

224

the amide I, II and III bands are closely indicative of the degree of molecular order of

225

collagen47. The characteristic absorption peaks of the amide bands with LC-Col-F

226

were all presented, but the peaks intensity slightly decrease compared with that of

227

Col-F. This indicates that the quite stable Schiff’s base C=N cross-linking bond

228

formed between LC and collagen fiber could destroy the hydrogen bonds between the

229

collagen molecular chains. It is noted that after LC cross-linked with collagen, the

230

peaks attributed to amide A and B band were broadened in varying extent, and a slight

231

blue shift appeared, which shows that the molecular order of collagen increases after

232

the introduction of LC, and the collagen molecules might occur self-aggregation to a

233

certain extent7, 48.


Page 14 of 37

Page 15 of 37


234 235

Figure 3. FTIR spectra of the different dosage LC cross-linked Col-F (A: 0%, B: 5%, C: 10%, D:

236

15%, E: 20%, F: 25%)

237

X-ray diffraction can be used to directly analyze the crystal structure of collagen.

238

As shown in Figure 4 for X-ray diffraction patterns of Col-F and LC-Col-F, there is a

239

relatively sharp diffraction peak in the range of 5° to 10°, which represents stacking

240

distance between each other or axial spiral according to the non-covalent interaction

241

between three α-chains of collagen or its aggregates through the electrostatic and/or

242

hydrogen bonds between the main chain or side chain units. According to the Bragg

243

equation (2dsinθ=λ), the stacking distances between each other or axial spiral in the

244

dosage of 0 %~25 % cross-linked Col-F are 1.189 nm, 1.170 nm, 1.163 nm, 1.160 nm,

245

1.117 nm and 1.109 nm, which indicated that the variation was slightly decreased.



246

This suggests that the crystalline phase of CL-Col-F presents stable with the LC

247

cross-linked with collagen molecules in axial helices. A diffuse scintillation peak

248

appearing at 15° to 20° is associated with “core-shell” structure of collagen and

249

belongs to the diffuse reflection of collagen amorphous domains. The absorption peak

250

around 30° implies that the helical pitch of the adjacent amino acids in the α-helical

251

chain of the collagen molecule in the helix axis can reflect the integrity of the

252

collagen helix structure to some extent. Whilst the axial projection pitch between

253

adjacent amino acids in the dosage of 0 %~25 % cross-linked Col-F molecule is 0.299

254

nm, 0.298nm, 0.294 nm, 0.295 nm, 0.293 nm and 0.293 nm, respectively, the changes

255

was not large. It also indicates that the collagen helix chain of Col-F is more stretched,

256

and it may be related to its multiple forces between lysine and collagen molecules

257

during fibril formation.


Page 16 of 37

Page 17 of 37


258 259

Figure 4. XRD spectra of different dosage LC cross-linked Col-F (A: 0%, B: 5%, C: 10%, D: 15%,

260

E: 20%, F: 25%)

261

As mentioned before, LC can be covalently cross-linked with the free amino

262

group in the side chain of Col molecule through Schiff’s base reaction. Therefore, the

263

changes of free amino group content of Col-F before and after cross-linking with LC

264

can be determined by ninhydrin method to reflect the different doses of LC

265

cross-linking with Col-F situation. Figure 5 shows the free amino content of LC-Col-F.

266

Apparently, it is seen that with the increasing of LC content, the amount of free amino

267

in the LC-Col-F decreases correspondingly, which indicates that the cross-linking rate

268

of Col-F cross-linked with LC increases due to the formation of covalent cross-linking

269

shield the free amino group of Col-F. The cross-linking degree of LC-Col-F was



Page 18 of 37

270

calculated by using mentioned formula. As shown and seen in Table 1, with the

271

increasing content of LC, the cross-link density between collagen and LC is much

272

improved. In particular, when the content of LC achieved 15 %, with the dosage of

273

LC continued to increase, the degree of crosslinking increases slightly, which was

274

caused by the both combining sites were close to saturation.

275 276

Figure 5. The curve of free amino in Col-F and Col-F cross-linked with different LC contents.

277

Table 1. Cross-linking Degree of LC-Col-F LC/%

5

10

15

20

25

Cross-linking degree/%

40.20

50.20

68.48

75.75

80.39

278

Figure 6a shows that the survey XPS spectrum of Col-F contains signals for the

279

elements of C, N and O, and O/C ratio was 0.49. These elements together with


Page 19 of 37


280

hydrogen chemically constitute the amino acid residues of collagen49. Similarly, in the

281

survey spectra of LC-Col-F surface, the signals for the elements of C, N and O were

282

also detected. Nevertheless, the ratio of elements has changed markedly seen from

283

Table 2. After introducing catechins, the overall carbon content on the surface

284

increases sharply while the O/C ratio was 0.37 and decreases slightly. This indicates

285

that the reaction of collagen with catechins under laccase incubation conditions leads

286

to the decline of O/C ratio, which confirms that the catechins reacts with collagen. Table 2. Surface elemental composition from wide-scan

287

Atomic (ratio) Sample

O/C ratio O 1s

N 1s

C 1s

Col-F

40.69

13.35

45.96

0.89

catechin

45.43

n/Aa

54.57

0.83

LC-Col-Fb

30.01

9.72

60.28

0.50

288

an/A

means “not available”.

289

bThe

dosage of LC is 15 %.

290 291 292

Figure 6. XPS analysis showing survey spectra for Col-F (a), catechin (b) and LC-Col-F (c)

Furthermore, the high-resolution XPS spectra was permitted to direct quantify



293

the chemical species, which could investigate the state or binding configuration in the

294

catechins by inducing is present on the surface. High resolution spectra of the C 1s

295

and N 1s peak were acquired as shown in Figure 7. We observed three peaks (287.90,

296

286.06 and 284.21 eV) in Figure 7a corresponding to the different chemical bonds

297

(C=O, C-O&C-N, C-H&C-NH2) of carbon in Col-F. The C 1s spectrum of LC-COL-F

298

in Figure 7a' could also accommodate these components, and one corresponding to

299

the catechinic carbon at the lower binding energy at 283.12 eV to carbon (C=C&C-C)

300

was observed. The Figure 7b depicted three components that is the binding energy

301

(BE) component at 398.43 eV corresponds to nitrogen atoms (=N-R), at 399.80 eV

302

and 401.52 eV assigned to the amide (R-NH-R', N-C). Analogously, as shown in

303

Figure 7b', the nitrogen binding energy of different chemical bonds (=N-R, R-NH-R',

304

N-C) at 398.79 eV, 399.44 eV, and 401.25 eV, respectively. Meanwhile, Table 3

305

presents the binding energy ratio of Col-F and LC-Col-F from high resolution scan of

306

C 1s and N 1s peak. For instance, the relative area of C-O&C-N increases from 15.75

307

to 17.72 for LC-Col-F, and the ratio of C-H&C-NH2 declines from 78.11 to 60.98. It

308

is also observed that the ratio of R-NH-R' and N-C has a slight drop with the

309

introduction of LC. On the contrary, the ratio of =N-R has an obvious increase from

310

2.54 to 7.76. The relative ratio shift of the C-O&C-N, C-H&C-NH2 and =N-R area is

311

indicative of the LC had cross-linked with collagen according to Schiff’s base.


Page 20 of 37

Page 21 of 37


312 313

Figure 7. High resolution C 1s XPS spectra of Col-F (a) and LC-Col-F (a') and N 1s XPS spectra

314

of Col-F (b) and LC-Col-F (b')

315

Table 3. Binding energy ratio of Col-F and LC-Col-F from high resolution XPS scan of C 1s peak

316

and N 1s peak binding energy ratioa (%) sample C=O

C-O&C-N

C-H&C-NH2

C=C&C-C

=N-R

R-NH-R'

N-C

Col-F

6.14

15.75

78.11

n/A

2.54

90.78

6.68

LC-Col-Fb

7.47

17.72

60.98

13.83

7.76

86.01

6.32

317

aThe

values are ratios of peak ares calculated from given XPS spectrum.

318

bThe

dosage of LC is 15 %.

319

Thermostability Evaluation of LC-Col-F. The thermo-gravimetric (TG) curves of the

320

Col-F and Col-F cross-linked with different contents of LC are presented in Figure 8.

321

Both of Col-F and LC cross-linked Col-F had three major stages of weight loss. At



322

temperatures ranging from 30 to 110 ℃, these samples were weightless gradually, but

323

the weightlessness was not obvious. The reason for the weight loss in this temperature

324

domain is that the physical adsorption of free water and chemical binding moisture in

325

Col-F and LC-Col-F destroys the combined hydrogen bonds between these

326

membranes due to the increase in temperature, resulting in a large amount of steaming

327

out and thus causing these samples lose weight. Additionally, the weight loss of 15 %

328

LC cross-linked Col-F is lower than that of 20 % and 25 % LC cross-linked Col-F,

329

which was due to the introduction of excess LC exerting a slight destructive effect on

330

the natural helical structure of collagen. Afterwards, with the temperature increasing

331

to 250 ℃, the weight loss of LC-Col-F tended to be unchanged, while the weight loss

332

for Col-F is going on with increasing the temperature, and the weightless rate of

333

Col-F was significantly higher than that of LC-Col-F. This is directly related to the

334

cross-linking of Col aggregates and LC cross-linked with the collagen. Under the

335

action of heating, it destroys the interactions between collagen molecules in collagen

336

aggregates and further destroys the triple helix structure of collagen molecules. The

337

introduction of LC allows an effective covalent cross-linking between collagen,

338

therefore, the weightless rate is relatively small. In the temperature range of

339

250~450 ℃, the weight loss of Col and LC-Col is quite obvious. This stage is usually

340

considered to be a thorough thermal decomposition of the natural structure of collagen,

341

and the helix structure is completely disintegrated into the random coil structure

342

polypeptide with minimal molecular weight. When the temperature was more than


Page 22 of 37

Page 23 of 37


343

450 ℃, Col and LC-Col were both carbonized, and there is a process to eliminate

344

carbon deposition. The total weight loss at this stage tends to be stable. From the

345

above observation, the response to heat and the ability to resist thermal decomposition

346

of 15 % LC cross-linked Col-F are obviously higher than that of other content LC

347

cross-linked Col-F and Col, hence it has the highest thermal stability, while the

348

thermal weight loss process of Col and LC-Col-F is almost the same.

349 350

Figure 8. TG curves of Col-F and LC-Col-F

351

Generally, when collagen was heated, the triple-helical structure within collagen

352

was separate from one another and its α-chains tend to be looser and more relaxed.

353

And with the temperature arriving to the denaturation temperature (Td), collagen

354

would unwind its triple helix and completely dissociate into the three randomly coiled



355

peptide α-chains. Figure 9 shows the diverse endothermic peaks of Col-F in the

356

presence of different LC contents: 0 %, 5 %, 10 %, 15 %, 20 %, 25 %, and the Td of

357

the composite sponges are 57.03 ℃, 62.15 ℃, 66.99 ℃, 74.71 ℃, 79.14 ℃ and

358

81.22 ℃, respectively. As expected, the Td of Col-F increases to different degrees

359

with increasing contents of LC, manifesting the enhanced thermostability of Col-F

360

and the existence of interactions between Col-F and LC. It is noteworthy that a

361

conspicuous jump in Td occurs when the content of LC increases to 15 %, which

362

confirms that Col-F may have a higher crosslinking degree with LC under this content.

363

With the dimension of catechinics formed oligomer or polymer to some degree, the

364

adjacent collagen molecules bind firmly in a block owing to the crosslinking reaction

365

by LC, which can fruitfully enhance the thermal stability of Col-F. Furthermore, the

366

enhancement in Td is favorable for holding the triple helix structure of collagen which

367

plays a significant role in the physicochemical properties of collagen based

368

biomaterials.


Page 24 of 37

Page 25 of 37


369 370

Figure 9. DSC evaluation of different LC content cross-linked Col-F.

371

Mechanical Properties Evaluation of LC-Col-F. Table 2 summarizes the results of

372

mechanics properties of LC-Col-F. It is concluded from this table that the thickness of

373

Col-F and LC-Col-F is concentrated around 0.015 mm indicating that the introduction

374

of catechins has no obvious effect on the thickness of Col-F. Overall, the tensile

375

strength (TS) of LC cross-linked Col-F increases from 6.21 to 10.13 MPa with

376

increasing different dosages of LC which is because the catechin molecules form an

377

effective covalent cross-linking is formed between the collagen aggregate molecules.

378

However, the LC-Col-F showed a slight decrease in TS when LC was used at 25 %,

379

due to the slight damage to the collagen's natural structure caused by the introduction

380

of excess LC. However, the elongation of LC-Col-F has different trend, with content



Page 26 of 37

381

of LC increased the elongation of LC-Col-F decreased gradually. It can be seen that

382

with the degree of covalent cross-linking increasing between catechins and collagen

383

molecules, the gaps between the collagen fibers become smaller, at the same time, the

384

fibers weaving become denser. The macroscopic performance showed that the

385

elongation becomes weaker as the LC increases. Additionally, we observed the

386

swelling of LC-Col-F had a conspicuous downward trend with the introduction of

387

catechins. The decreased swelling of LC-Col-F might be due to the high degree of

388

chemical covalent cross-linking of the o-quinone of LC with the amine of Col-F that

389

increases the hydrophobicity of LC-Col-F. As expected, the solubility of both Col-F

390

and LC-Col-F is quite low showing their insolubility seen from Table 2. The

391

insolubility of Col-F was mainly due to the structural organization within the fibril,

392

where the axial and lateral organization and topology of the collagen molecules

393

ensured strong intermolecular interactions and crosslinkage37. Furthermore, the

394

introduction of catechins which are slightly soluble in water makes this interactions

395

and crosslinkage between collagen fibers more stable. As a result, there is not much

396

effect on the solubility of Col-F. To conclude, considering these excellent mechanical

397

properties, the LC-Col-F might apply in foodstuffs storage progress. Table 4. Results of mechanics properties of LC-Col-F

398 samples

thickness/mm

tensile strength/MPa

elongation/%

swelling/%

solubility/%

0%

0.015±0.0005

4.01±0.19

41.54±2.67

273.82±8.26

2.14±0.62

5%

0.014±0.0006

6.21±0.42

74.52±2.83

212.53±4.12

2.32±0.12


Page 27 of 37


10%

0.014±0.0007

7.37±0.72

64.63±3.51

180.48±12.90

2.63±0.86

15%

0.015±0.0008

9.33±0.72

57.87±2.32

169.91±4.46

2.87±0.36

20%

0.014±0.0007

10.13±0.37

42.96±1.95

151.73±10.35

1.96±0.55

25%

0.015±0.0006

8.13±0.32

37.65±2.49

148.40±9.81

2.65±0.83

399

Evaluation of Antioxidant Capacity. The effect of LC dosage on the antioxidant

400

activity of Col-F was evaluated by ABTS assay. As shown in Figure 10, all the

401

LC-Col-F present relatively favorable scavenging activity towards the ABTS cation

402

radicals. The ABTS•+ scavenging capacities of LC-Col-F prepared by using 0 %, 5 %,

403

10 %, 15 %, 20 % and 25 % LC are 38.06 %, 53.34 %, 76.46 %, 97.86 %, 99.19 %

404

and 99.20 %, respectively. This result suggests that the ABTS•+ scavenging capacity

405

of LC-Col-F is appreciably improved via introducing a large amount of phenolic

406

hydroxyl groups when conjugated with LC. Combining this result and the excellent

407

mechanical properties exhibited above, it is concluded that LC-Col-F has better

408

beneficial for the applications in food packaging, especially during high lipid-based

409

foods storage process.



410 411

Figure 10. The scavenging effect of different content LC cross-linked Col-F

412

SEM Analysis. Figure 11 provides the scanning electron microscope (SEM) images of

413

and cross-section of Col-F incorporated with different content of LC. As it shown,

414

homogeneous macrostructures with a relatively rough surface are presented,

415

especially with high dosage, whereas no apparent phase separation was detected. Seen

416

from the cross-section micrographs, more compactness in the cross-linked collagen

417

membrane structure is exhibited comparison with that of untreated one. Specifically,

418

the micro-roughness of LC-Col-F at 15 % of the LC was observed to be promoted to

419

some extent, which is associated with introduction of the large content of LC and the

420

drastic interaction between Col-F and LC governs the aggregation in the membrane

421

matrix via Schiff’s base or/and Michael addition reactions. Similarly, Hoque et al.50


Page 28 of 37

Page 29 of 37


422

has also reported such rough surface in the case of gelatin membrane incorporated

423

with star anise extracts.

424 425

Figure 11. SEM surface images of Col-F (a), 5 % LC-Col-F (b), 15 % LC-Col-F (c) and 25 %

426

LC-col-F (d) and cross-section images of Col-F (a'), 5 % LC-Col-F (b'), 15 % LC-Col-F (c') and

427

25 % LC-col-F (d')

428

AUTHOR INFORMATION

429

Corresponding Author

430

*E-mail: [email protected]

431

Funding

432

The authors acknowledge the financial support from National Science and

433

Technology Major Project (2017YFB0308500).

434

Notes

435 436 437

The authors declare no competing financial interest.

ACKNOWLEDGMENTS And the authors also thank the Institute of Biomass and Functional Materials



438

(http://www.leather420.com) for help during the experiment.

439

REFERENCES

440

(1) Etxabide A, Uranga J, Guerrero P, et al. Development of active gelatin films by

441

means of valorisation of food processing waste: a review. Food Hydrocolloids.

442

2017, 68, 192-198.

443

(2) Kaewprachu P, Osako K, Rungraeng N, et al. Characterization of fish myofibrillar

444

protein film incorporated with catechin-Kradon extract. Int. J. Biol. Macromol.

445

2017, 13, 56-65.

446 447

(3) Jus S, Stachel I, Schloegl W, et al. Cross-linking of collagen with laccases and tyrosinases. Mater. Sci. Eng., C. 2011, 31(5):1068-1077.

448

(4) Elango J, Bu Y, Bao B, et al. Effect of chemical and biological cross-linkers on

449

mechanical and functional properties of shark catfish skin collagen films. Food

450

Biosci. 2017, 17, 42-51.

451

(5) Jeevithan E, Zhang J, Wang N, et al. Physico-chemical, antioxidant and intestinal

452

absorption properties of whale shark type-II collagen based on its solubility with

453

acid and pepsin. Process Biochem. 2015, 50(3):463-472.

454

(6) Jeevithan E, Wu W, Wang N, et al. Isolation, purification and characterization of

455

pepsin soluble collagen isolated from silvertip shark (Carcharhinus albimarginatus)

456

skeletal and head bone. Process Biochem. 2014, 49(10):1767-1777.

457

(7) Liu X H. Feasibility Study of the Natural Derived Chitosan Dialdehyde for

458

Chemical Modification of Collagen. Int. J. Biol. Macromol. 2016, 82(4):63-64.


Page 30 of 37

Page 31 of 37

459 460


(8) Tian Z, Liu W, Li G. The microstructure and stability of collagen hydrogel cross-linked by glutaraldehyde. Polym. Degrad. Stab. 2016, 130, 264-270.

461

(9) Utrera M, Estévez M. Impact of trolox, quercetin, genistein and gallic acid on the

462

oxidative damage to myofibrillar proteins: the carbonylation pathway. Food Chem.

463

2013, 141(4):4000-4009.

464

(10) Barbani N, Giusti P, Lazzeri L, et al. Bioartificial materials based on collagen: 1.

465

Collagen cross-linking with gaseous glutaraldehyde. J. Biomater. Sci., Polym. Ed.

466

1995, 7(6):461-469.

467 468

(11) Powell H M, Drexler J W. Dehydrothermal crosslinking of electrospun collagen. Tissue Eng., Part C. 2011, 17(1):9.

469

(12) Zhang M, Yang J, Ding C, et al. A novel strategy to fabricate water-soluble

470

collagen using poly(γ-glutamic acid)-derivatives as dual-functional modifier.

471

React. Funct. Polym. 2018, 122:131-139.

472

(13) Liu T, Shi L, Gu Z, et al. A novel combined polyphenol-aldehyde crosslinking of

473

collagen film-Applications in biomedical materials. Int. J. Biol. Macromol. 2017,

474

101:889-895.

475

(14) Arrieta M P, Fortunati E, Dominici F, et al. Bionanocomposite films based on

476

plasticized PLA-PHB/cellulose nanocrystal blends. Carbohydr. Polym. 2015,

477

121:265-275.

478

(15) Selvi I M K, Nagarajan S. Separation of catechins from green tea (Camellia

479

sinensis, L.) by microwave assisted acetylation, evaluation of antioxidant potential



480

of individual components and spectroscopic analysis. LWT. 2018, 91, 391-397.

481

(16) Grzesik M, NaparåO K, Bartosz G, et al. Antioxidant properties of catechins:

482 483 484

Comparison with other antioxidants. Food Chem. 2018, 241, 480-492. (17) Gadkari P V, Balaraman M. Catechins: Sources, extraction and encapsulation: A review. Food Bioprod. Process. 2015, 93:122-138.

485

(18) Teixeira A, Baenas N, Dominguezperles R, et al. Natural bioactive compounds

486

from winery by-products as health promoters: a review. Int. J. Mol. Sci. 2014,

487

15(9):15638-15678.

488

(19) Lopez de D C, Castro-Lopez M M, Lasagabaster A, et al. Interaction and release

489

of catechin from anhydride maleic-grafted polypropylene films. ACS Appl. Mater.

490

Interf. 2013, 5(8):3281-3289.

491

(20) Arrieta M P, Peponi L. Polyurethane based on PLA and PCL incorporated with

492

catechin: Structural, thermal and mechanical characterization. Eur. Polym. J. 2017,

493

89, 174-184.

494

(21) Arrieta M P, Sessini V, Peponi L. Biodegradable poly(ester-urethane)

495

incorporated with catechin with shape memory and antioxidant activity for food

496

packaging. Eur. Polym. J. 2017, 94, 111-124.

497 498

(22) Shi B. Gelatin-polyphenol interaction. J. Am. Leather Chem. Assoc. 1994, 89, 96-102.

499

(23) Kim H W, Song D H, Choi Y S, et al. Effects of Soaking pH and Extracting

500

Temperature on the Physicochemical Properties of Chicken Skin Gelatin. Korean


Page 32 of 37

Page 33 of 37

501 502 503 504 505 506


J. Food Sci. An. 2012, 32(3):57-65. (24) Galli C, Madzak C, Vadalà R, et al. Concerted electron/proton transfermechanism in the oxidation of phenols by laccase. Chembiochem. 2013, 14, 2500. (25) Mate D M, Alcalde M. Laccase engineering: from rational design to directed evolution. Biotechnol. Adv. 2015, 33, 25-40. (26) Wesenberg D, Kyriakides I, Agathos S N. White-rot fungi and their enzymes for

507

the treatment of industrial dye effluents. Biotechnol. Adv. 2003, 22(1-2):161.

508

(27) Claus H. Laccases: structure, reactions, distribution. Micron. 2004, 35(1-2):93.

509

(28) Silva C, Matamá T, Kim S Y, et al. Antimicrobial and antioxidant linen via

510

laccase-assisted grafting. React. Funct. Polym. 2011, 71, 713-720.

511

(29) Nady N, Schroën K, Franssen M C R, et al. Mild and Highly Flexible

512

Enzyme-Catalyzed Modification of Poly (ethersulfone) Membranes. ACS Appl.

513

Mater. Interfaces. 2011, 3, 801.

514

(30) Bark K M, Yeom J E, Yang J I, et al. Spectroscopic Studies on the Oxidation of

515

Catechin in Aqueous Solution Spectroscopic Studies on the Oxidation of Catechin

516

in Aqueous Solution. Bull. Korean Chem. Soc. 2011, 32(9):462-4.

517

(31) Sampaio S, Taddei P, Monti P, et al. Enzymatic grafting of chitosan onto Bombyx

518

mori silk fibroin: kinetic and IR vibrational studies. J. Biotechnol. 2005, 116(1):

519

21-33.

520

(32) Muzzarelli R A A, Littarru G, Muzzarelli C, et al. Selective reactivity of

521

biochemically relevant quinones towards chitosans. Carbohydr. Polym. 2003,



522

Page 34 of 37

53(1): 109-115.

523

(33) Božič M, Gorgieva S, Kokol V. Laccase-mediated functionalization of chitosan

524

by caffeic and gallic acids for modulating antioxidant and antimicrobial properties.

525

Carbohydr. Polym. 2012, 87, 2388-2398.

526

(34) Qiang TT, Chen L, Zhang Q, et al. A Sustainable and Cleaner Speedy Tanning

527

System Based on Condensed Tannins Catalyzed by Laccase. J.

528

2018, 197, 1117-1123.

529 530

Cleaner

Prod.

(35) Kim S, Cavaco-Paulo A. Laccase-catalysed protein-flavonoid conjugates for flax fibre modification. Appl. Microbiol. Biotechnol. 2012, 93(2):585-600.

531

(36) Hu Y, Liu L, Dan W, et al. Evaluation of 1-ethyl-3-methylimidazolium acetate

532

based ionic liquid systems as a suitable solvent for collagen. J. Appl. Polym. Sci.

533

2013, 130(4):2245-2256.

534

(37) Ming-kung Yeh, Yan-ming Liang, Kuang-ming Cheng, et al. A novel cell support

535

membrane for skin tissue engineering: Gelatin film cross-linked with

536

2-chloro-1-methylpyridinium iodide. Polym. 2011, 52(4):996-1003.

537

(38) Hu Y, Liu L, Dan W, et al. Synergistic effect of carbodiimide and dehydrothermal

538

crosslinking on acellular dermal matrix. Int. J. Biol. Macromol. 2013,

539

55(2):221-230.

540 541 542

(39) ISO 4593-1993. Method 630A: Determination of thickness by mechanical scanning of flexible sheet. 1993. (40) ASTM D882. Standard Test Method for Tensile Properties of Thin Plastic


Page 35 of 37

543


Sheeting. 2001.

544

(41) Figueiro S D, Goes J C, Moreira R A, et al. On the physico-chemical and

545

dielectric properties of glutaraldehyde crosslinked galactomannan-collagen films.

546

Carbohydr. Polym. 2004, 56(3):313-320.

547

(42) Saiut S, Benjakul S, Rawdkuen S. Retardation of lipid oxidation using gelatin

548

film incorporated with longan seed extract compared with BHT. J. Food Sci.

549

Technol. 2015, 52(9):5842-5849.

550

(43) Torreggiani A, Jurasekova Z, Sanchez-Cortes S, et al. Spectroscopic and pulse

551

radiolysis studies of the antioxidant properties of (+) catechin: metal chelation and

552

oxidizing radical scavenging. J. Raman Spectrosc. 2008, 39(2):265-275.

553 554

(44) dSL Melo. Enzymatic polymerisation in situ of depolymerised mimosa tannin applied to stabilisation of collagen. The University of Northampton. 2018.

555

(45) Liu X, Dan W, Ju H, et al. Development and analysis of a novel collagen

556

composite encapsulating a natural pharmic amino acid, dencichine. Polym.

557

Compos. 2016, 37(7):399-426.

558 559 560

(46) Prystupa D A, Donald A M. Infrared study of gelatin conformations in the gel and sol states. Polym. Gels Networks. 1996, 4(2): 87-110. (47) Liu X, Dan W, Ju H, et al. Preparation and Evaluation of a Novel pADM-derived

561

Micro-and

562

5(64):52079-52087.

563 564

(48) Ding

C,

nano

Zhang

Electrospun

M,

Li

Collagen

G.

Membrane.

Preparation

and

RSC

Adv.

2015,

characterization

of

collagen/hydroxypropyl methylcellulose (HPMC) blend film. Carbohydr. Polym.



565

2015, 119:194-201.

566

(49) Sionkowska A, Wisniewski M, Kaczmarek H, et al. The influence of UV

567

irradiation on surface composition of collagen/PVP blended films. Appl. Surf. Sci.

568

2006, 253(4): 1970-1977.

569

(50) Hoque M S, Benjakul S, Prodpran T. Properties of film from cuttlefish (Sepia

570

pharaonis) skin gelatin incorporated with cinnamon, clove and star anise extracts.

571

Food Hydrocolloids. 2011, 25: 1085-1097.

572


Page 36 of 37

Page 37 of 37

573


Table of content

574


Evaluation of a Novel Collagenous Matrix Membrane Cross-Linked

Recommend Documents