Physical and Antioxidant Properties of Edible Chitosan Ascorbate Films

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Physical and Antioxidant Properties of Edible Chitosan Ascorbate Films Wenqiang Tan, Fang Dong, Jingjing Zhang, Xiang Zhao, Qing Li, and Zhanyong Guo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04567 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019

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Journal of Agricultural and Food Chemistry

Chitosan ascorbate films with many interesting physicochemical properties and excellent antioxidant activity can be applied as novel green food packaging material. 187x166mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Physical and Antioxidant Properties of Edible Chitosan Ascorbate

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Films

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Wenqiang Tan †, Fang Dong †, Jingjing Zhang †, ‡, Xiang Zhao §, Qing Li †, Zhanyong

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Guo †, ‡, *

5

†

6

Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, People’s

7

Republic of China

8

‡

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China

Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of

University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of

10

§

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People’s Republic of China

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Correspondence to: Prof. Zhanyong Guo, E-mail: [email protected]

College of Chemistry and Chemical Engineering, Yantai University, Yantai 264005,

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ABSTRACT

15

Chitosan ascorbates with different substitution degrees were synthesized based on

16

salification of chitosan and ascorbic acid at various molar ratios in water and were

17

successfully used to prepare antioxidative films by casting for the first time. FTIR and

18

1

19

meanwhile physicochemical property and antioxidant activity of the produced chitosan

20

ascorbate films were characterized and chitosan acetate film served as control were also

21

measured these properties for comparison. The results revealed that salification of

22

chitosan with ascorbic acid not only improved total color difference, chroma, opacity,

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the capacity for blocking UV-vis light, and water solubility of chitosan based films, but

24

also decreased water content, swelling degree, and water vapor permeability compared

25

with chitosan acetate film. Also, as was expected, the antioxidant activity assays

26

showed that incorporation of ascorbate into chitosan matrix enhanced effectively

27

scavenging activity against DPPH-radical and reducing power. Especially, Cs2Vc8 and

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Cs2Vc6 exhibited the strongest scavenging capacities against DPPH radical (EC50 0)

h* = tan-1(b/a) + 180

(2)

(if a < 0)

(3)

△E = ((L* - L)2 + (a* - a)2 + (b* - b)2)1/2

(4)

WI = 100 - ((100 - L)2 + a2 + b2)1/2

(5)

166

where L, a, and b are the color parameters of chitosan films, and L*, a*, and b* are the

167

color parameters of the standard white plate (L* = 93.49, a* = -0.25, and b* = -0.09).

168

2.5.4 Light Transmittance and Opacity

169

Optical transmittance of the produced films was measured using a TU-1810 UV –

170

vis spectrometer (General Instrument Co., Ltd. Beijing, People’s Republic of China) in

171

the scanning wavelength range of 200 - 800 nm. An empty test cuvette was used as the

172

blank. The opacity of the chitosan films was determined at 600 nm and calculated by

173

the following equation: 4 8



Opacity = A600 ⁄ d

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(6)

174

where A600 is the absorbance at 600 nm and d (mm) is the average thickness of film.

175

2.5.5 X-Ray Diffraction (XRD)

176

XRD patterns were obtained on Bruker D8 Advance X-ray diffractometer (Bruker

177

AXS GmbH, Germany) with Cu kα radiation (λ=1.541874 Å) at a scanning rate of

178

6°/min in the diffraction angle (2θ) range of 5 - 50°.

179

2.5.6 Thermal Analysis

180

Thermogravimetric analysis (TGA) and derivative thermogravimetric (DTG) were

181

measured by means of a STA 449F3 (ETZSCH-Gerätebau GmbH, Germany) system at

182

a heating rate of 10ºC/min from 25ºC to 600ºC under nitrogen atmosphere.

183

2.5.7 Water Content, Swelling Degree, and Water Solubility

184

Each film specimen was uniformly trimmed to a rectangle (2 cm × 2 cm) and

185

weighted (W0). Afterward, the square film was dried in a hot oven at 75ºC for 24 h to

186

obtain the initial dry weight (W1). Next, the dried sample was spread on a Petri dish

187

with 30 mL of deionized water covered with cling film, followed by storage at room

188

environment for 24 h. Filter paper was then used to remove carefully the surface water

189

and sample was weighed (W2). The residual film was desiccated at 75ºC for 24 h to

190

record the final dry mass (W3). Data were collected with three replications to calculate

191

water content, swelling degree, and water solubility according to Eqs (7) – (9): 15

192

Water content (%) = (W0 - W1) ⁄ W0 × 100

(7)

Swelling degree (%) = (W2 - W1) ⁄ W1 × 100

(8)

Water solubility (%) = (W1 – W3) ⁄ W1 × 100

(9)

2.5.8 Water Vapor Permeability (WVP)

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Water vapor transmission rate (WVTR) and WVP of chitosan based films were

194

gravimetrically determined by a modified method of ASTM E96-95. 32 The circular test

195

cups containing 18 mL of deionized water were sealed by test films, which were cut

196

into pieces (4 cm diameter). The cups were then placed in an airtight desiccator

197

containing dry silica gel and maintained at 25ºC. The changes in total weight of the test

198

cups were measured every 2 h in triplicate. The slopes of the linear portion in the curves

199

of weight loss as a function of time were used to calculate the WVTR (g/m2·s).

200

Equation (10) was used to calculate the WVP in g/m·Pa·s: 33 WVP = (WVTR × L) ⁄ △P

(10)

201

where L (m) is the average thickness of film and ΔP (Pa) is the difference in partial

202

water vapor pressure across the film.

203

2.6 Antioxidant Assay

204

The antioxidant activities of chitosan based films were measured by scavenging

205

assay against DPPH radical and reducing power assay. Firstly, the film sample (1% w/v)

206

was cut into smallest possible pieces and then immersed into deionized water. After

207

placed at the room environment for 24 h, insoluble residual film was removed from

208

each mixture by the centrifugation at 3000 rpm for 20 min. The supernatant were used

209

as stock solutions to evaluate the antioxidant activity and their concentrations were

210

regarded as 10 mg/mL.

211

2.6.1 DPPH Radical Scavenging Activity Assay

212

The DPPH radical scavenging activities of chitosan based films were evaluated

213

using earlier reported method with some modifications. 34 Briefly, the extract solutions

214

of film samples were diluted with deionized water at 0.075, 0.15, 0.30, 0.60, 1.20, 2.40,

215

and 4.80 mg/mL. Afterwards, 1.0 mL of diluted solution was thoroughly mixed with 2.0

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216

mL of DPPH solution (180 μM, dissolved in absolute ethanol) followed by shaking for

217

10 s. The reaction mixture was then incubated in dark condition for 20 min before

218

triple-measuring the absorbance of each solution using UV–vis spectrophotometer at

219

517 nm. And deionized water displaced sample was used as blank. The scavenging

220

effect against DPPH radical was assessed using equation (11): Scavenging effect (%) = [1-(A1-A2)⁄A0] × 100

(11)

221

where A0, A1, and A2 are the absorbances of the blank group, the sample group, and

222

sample’s background (absolute ethanol instead of DPPH), respectively.

223

2.6.2 Reducing Power Assay

224

The reducing power of chitosan based films was estimated using a modified

225

version of a previously reported method. 35 The extract stock solutions were diluted with

226

deionized water at 0.60, 1.20, 2.40, 4.80, and 9.60 mg/mL. 1.0 mL of diluted solution

227

with various concentrations was added into 1.0 mL of potassium ferricyanide solution

228

(1%, w/v) and the mixture was reacted at 50°C for 20 min followed by the addition of

229

1.0 mL of trichloroacetic acid solution (10%, w/v). After centrifugation, a 1.5 mL

230

aliquot of the supernatant was mixed with 1.5 mL of ferric chloride solution (0.2‰, w/v)

231

and then incubated away from light for 10 min. The absorbance was recorded at 700 nm

232

and the reducing power of chitosan based films was calculated using the following

233

formula: Reducing power = A1 - A0

(12)

234

where A0 and A1 are the absorbances of the blank group and the sample group,

235

respectively.

236

2.7 Statistical Analysis

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237

Data were expressed as mean ± the standard deviation (SD, n ≥ 3). The significant

238

differences between sample means were determined using one-way analysis of variance,

239

where the level of significance was set at p < 0.05. Meanwhile multiple comparisons of

240

means were carried out by Duncan's test.

241

3. RESULTS AND DISCUSSION

242

3.1 Synthesis of Chitosan Ascorbates

243

As shown in Fig. 1, chitosan ascorbates were prepared in water based on the

244

electrostatic interaction between the reactive amino group in chitosan backbone and the

245

acidic hydroxyl group at C-3 (pKa 4.17) of ascorbic acid at different reaction molar

246

ratios (Cs2Vc1, Cs2Vc2, Cs2Vc3, Cs2Vc4, Cs2Vc6, and Cs2Vc8). Besides, chitosan acetate

247

(Cs2Ac4) was also obtained as control group in physical chemistry experiments and

248

antioxidant tests.

249 250

Fig. 1. Synthetic route for chitosan ascorbates.

251 252

3.1.1 FTIR Analysis

253

As shown in Fig. 2, the pristine chitosan (Cs) displays primary spectral peaks at

254

3428, 2877, 1646, 1600, 1380, and 1083 cm-1, which are assigned to N-H and O-H

255

stretching vibrations, C-H stretching vibration, C=O stretching vibration of residual

256

amide bond, N-H bending vibration, C-H bending vibration, and C-O stretching

257

vibration, respectively. 35 After salt-forming reaction, the major changes in the FTIR

258

spectra between chitosan and chitosan acetate show that the characteristic band at 1600

259

cm-1 disappears but new peaks at 1558 cm-1 and 1407 cm-1 are detected, which can be

260

attributed to –NH3+ bending vibration and –COO- symmetrical stretching vibration

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261

resulted from the electrostatic interaction. 8 In the spectra of chitosan ascorbates, the

262

difference in reaction molar ratios of chitosan to ascorbic acid shows less of an effect on

263

their FTIR spectra but the new characteristic peak is observed at 1716 cm-1, assigned to

264

the stretching vibration of carbonyl group of lactone in ascorbate molecule.

265

Additionally, the overlapping peak of C=C stretching vibration of ascorbate with –NH3+

266

bending vibration of chitosan is detected at about 1589 cm-1 and the weak peaks

267

appeared at 755 cm-1 and 713 cm-1 are assigned to C=C bending vibration of ascorbate.

30

268 269

Fig. 2. FTIR spectra of chitosan, chitosan acetate (Cs2Ac4), and chitosan ascorbates (CsxVcy).

270 271

3.1.2 1H NMR Analysis

272

1

H NMR spectra were further verified the successful preparation of chitosan

273

acetate and chitosan ascorbates. As shown in Fig. 3, the chemical shifts at δ = 3.10 ppm

274

and 3.50-4.00 ppm are assigned to H2 and H3-H6 of glucosamine monomer in chitosan.

275

36, 37

276

observed at δ = 1.78 ppm, which is attributed to methyl protons of acetate moiety.

277

Meanwhile, 1H NMR spectra of chitosan ascorbates apparently display new signals at δ

278

= 4.51 (a in the spectrum), 3.99 (b in the spectrum), and 3.71 ppm (c in the spectrum)

279

that can be assigned to the lactone CH proton next to glycol, the CH and CH2 protons of

280

glycol groups, respectively. 38 One notable thing is that all the chemical shifts of protons

281

in ascorbic acid are shifted towards high field after the salt-forming reaction with

282

chitosan in water. Additionally, the DS values of chitosan acetate and chitosan

283

ascorbates were evaluated based on the integral areas in the 1H NMR spectral analysis

284

and were calculated using the following formulas:

Compared with chitosan, a sharp peak (a in the spectrum) for chitosan acetate is

DS1 = I CH3 ⁄ (3 × I H2)

(13) 13


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DS2 = I Ha ⁄ I H2

(14)

285

where DS1 is the DS of chitosan acetate, DS2 is the DS of chitosan ascorbates, I CH3 is

286

the integral of the peak of methyl protons of acetate moiety, I H2 is the integral of the

287

peak of H2 proton in glucosamine rings, I Ha is the integral of the peak of lactone CH

288

proton next to glycol of ascorbate moiety.

289 290

Fig. 3. 1H NMR spectra of chitosan, chitosan acetate (Cs2Ac4), and chitosan ascorbates (CsxVcy).

291 292

The DS values of chitosan acetate and chitosan ascorbates are shown in Table 1. It

293

is very clear that with the increase of the reaction molar ratios of ascorbic acid to

294

chitosan, the DS values of chitosan ascorbates increase and can be reached a maximum

295

of about 0.95 at a molar ratio of 4:1. In consideration of 95% of the deacetylation degree

296

of pristine chitosan, it is reasonable to deduce that all of amino groups of pristine

297

chitosan have been involved in the salt-forming reaction with ascorbic acid.

298 299

Table 1. Reaction molar ratio and degree of substitution of chitosan acetate and chitosan ascorbates.*

300 301

3.2 Characterization of Chitosan Ascorbate Films

302

3.2.1 Thickness, Density, and Mechanical Properties

303

The chitosan films were formed with the solution/solvent casting method. The

304

thickness of the chitosan films ranges between 66 ± 6 μm and 71 ± 6 μm (Table 2) and

305

this difference is not statistically significant between each film. This result indicated

306

that the content of ascorbate had no significant influence (p > 0.05) on the average

307

thickness of chitosan based films. However, film densities decrease along with the

14



308

incorporation of ascorbate compared with chitosan acetate film but no significant

309

differences (p > 0.05) are found in densities of six chitosan ascorbate films.

310 311

Table 2. Thickness, density, and mechanical properties of chitosan acetate film and chitosan

312

ascorbate films.*

313 314

Table 2 shows the effect of the category and content of organic acid salt on tensile

315

strength and elongation at break of chitosan based films. Firstly, for chitosan acetate

316

film, the tensile strength and elongation at break values are found to be 43 ± 1% and 31

317

± 6%, respectively. It can be seen that the salt-forming reaction of chitosan with

318

ascorbic acid produced significant effect in their mechanical properties. A significant

319

drop (p < 0.05) in tensile strength and elongation at break values of chitosan ascorbate

320

films resulting in tougher films was observed compared with chitosan acetate film. The

321

mechanical property of the film could be greatly influenced by the systematic

322

composition and intermolecular forces.

323

elongation at break could be ascribed to the stronger destructive capacity of ascorbic

324

acid to crystalline structure as well as intramolecular and intermolecular hydrogen

325

bonding of chitosan matrix than that of acetic acid. 16 Moreover, the introduction of

326

ascorbate with an increasing substitution degree from 0.50 to 0.95 decreases the tensile

327

strength values from 27 ± 4 MPa to 21 ± 4 MPa and elongation at break values from 16

328

± 2% to 10.0 ± 0.4%. Similar findings have been reported previously by Sun et al.. 12

329

Above result showed that an increasing content of ascorbate in chitosan matrix resulted

330

in an accelerating trend towards the decrease in crystalline structure and intermolecular

331

forces between chitosan chains. 40

332

3.2.2 Surface Color, Opacity, and Light Transmittance

10, 39

The decrease in tensile strength and

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333

Significant differences between the chitosan based films in the color parameters,

334

including L, a, b, C*, h*, △E, WI, and opacity, are observed in Table 3. As the control

335

group, chitosan acetate film was colorless and transparent. However, chitosan ascorbate

336

films had a fawn-colored appearance compared to chitosan acetate film and changes

337

occurred in all the color parameters with the incorporation of ascorbate into chitosan

338

films. Moreover, as the substitution degrees of ascorbate increase from 0.50 to 0.95, a

339

decrease in L values from 59.03 ± 0.05 to 51.53 ± 0.01, and increases in b values (14.48

340

± 0.02 to 25.57 ± 0.01) and C* values (14.8 ± 0.8 to 26 ± 3) indicate that the films lose

341

clarity and become darker, yellower, but increased color saturation. Chitosan ascorbate

342

films show higher redness as growing a values are noted when the substitution degrees

343

of ascorbate are 0.50-0.80, while the decrease in redness is also observed from a values,

344

which decline from 2.50 ± 0.14 to 1.99 ± 0.01 when the substitution degrees of

345

ascorbate reach 0.80-0.95. The increasing △E values (37.52 ± 0.04 to 49.23 ± 0.01), the

346

decreasing WI values (56.44 ± 0.05 to 45.17 ± 0.01), and the decreasing h* values (102

347

± 2 to 84.1 ± 0.4) indicate that the more colored films are obtained. The transparency is

348

an important property for the film and is generally expressed as opacity. Higher value of

349

opacity indicated the lower transparency and vice versa. The increasing opacities of

350

chitosan ascorbate films from 0.78 ± 0.07 to 1.31 ± 0.01 A600/mm are observed in Table

351

3 with the growing substitution degrees of ascorbate.

352 353

Table 3. Optical properties of chitosan acetate film and chitosan ascorbate films.*

354

Fig. 4. UV – vis spectra of chitosan acetate (Cs2Ac4) film and chitosan ascorbate (CsxVcy) films.

355 356

Ultraviolet and visible light can catalyze the oxidation process of packaged food

357

and too much exposure to UV – vis light can accelerate the food deterioration and 16



358

nutrient loss. 13, 15 Thus, the capacity for blocking UV – vis light to extend shelf life of

359

foodstuffs is an important characteristic for food packaging materials. Fig. 4 shows the

360

light transmittance of chitosan based films in the wavelength range of 200-800 nm. The

361

transmittance values of chitosan acetate film in the wavelength range of 200-400 nm are

362

4.29-78.02%. The introduction of ascorbate into chitosan matrix significantly exhibits

363

transmittance values of below 35% compared with chitosan acetate film, this higher

364

blocking capacity might be due to the absorption capacity of ascorbate towards UV light.

365

In particular, the UV light transmittances of all the chitosan ascorbate films attain

366

almost zero in the range of 230-295 nm. Additionally, the light transmittances of

367

chitosan ascorbate films are further reduced as the increasing content of ascorbate in the

368

range of 380-600 nm. All the data indicated that chitosan ascorbate films had great

369

capacity to block UV-vis light.

370

3.2.3 X-Ray Diffraction

371 372

Fig. 5. XRD patterns of chitosan acetate (Cs2Ac4) film and chitosan ascorbate (CsxVcy) films.

373 374

The crystal structures of various samples including pristine chitosan powder,

375

chitosan acetate film, and chitosan ascorbate films were characterized by XRD. As

376

shown in Fig. 5, pristine chitosan shows semi-crystalline morphology with two peaks, a

377

flat one at 11.82° signified the hydrous polymorphs characteristics of chitosan lattice

378

bound with water and a broad one at 19.99° related to anhydrous crystal feature. 1, 41 The

379

peak intensity of chitosan acetate film at around 20° is declined compared to chitosan

380

powder in the XRD patterns. After ascorbate is formed in the chitosan matrix,

381

significant changes are observed from the XRD profiles, in which the peak at 11.82°

382

disappears and the diffraction peaks at about 20° become more flattened and less 17


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383

discernible. These data indicated that salt-forming reaction with ascorbic acid led to an

384

amorphous structure of chitosan films. The breaking of intramolecular and

385

intermolecular hydrogen bonding of chitosan backbones could lead to this lower

386

crystallinity, which was also in agreement with the result of mechanical property of

387

chitosan based films. However, no significant difference in crystal structures of chitosan

388

ascorbate films could be identified from XRD patterns with an increment of ascorbate

389

contents.

390

3.2.4 Thermal Analysis

391 392

Fig. 6. TGA (a) and DTG (b) curves of chitosan acetate (Cs2Ac4) film and chitosan ascorbate

393

(CsxVcy) films.

394 395

TGA and DTG variation trends of pristine chitosan powder, chitosan acetate film,

396

and chitosan ascorbate films are presented in Fig. 6. Chitosan powder displays two

397

thermal decomposition stages, the first one ( ≤ 100°C) with about 6 wt% weight loss

398

mainly goes through the loss of residual water, and the second one (200 - 400°C)

399

remained 36 wt% solid residue corresponds to the decomposition of chitosan backbone.

400

39, 42

401

first mass loss at 25 - 100°C with about 5 wt% degradation could be also ascribed to the

402

evaporation of bound or free moisture. The second step occurs at about 100 - 225°C

403

with about 25 wt% degradation and it can be assigned to the decomposition of glycerol.

404

16

405

accompanied with a massive weight loss (40 wt%). The thermal degradation of chitosan

406

backbones mainly happens in this step and the temperatures at maximum decomposition

407

rate of chitosan films are observed at 271-288°C, which are below that of pristine

However, for all the chitosan films, degradations are recorded in three steps. The

The onset temperature of the main mass loss is also observed at nearly 225°C

18



408

chitosan at 296°C. Given the above, the much lower degradation temperature and bigger

409

weight loss in the decomposition process of chitosan backbone displayed poor thermal

410

stability of chitosan based films compared with pristine chitosan. This reduced thermal

411

stability might be related to the lower crystallinity of chitosan films. But it was found

412

that the increment of ascorbate contents did not exert substantially influence on the

413

thermal stability of chitosan ascorbate films.

414

3.2.5 Water Content, Water Solubility, Swelling Degree, and Water Vapor

415

Permeability

416 417

Table 4. Water content, water solubility, swelling degree, water vapor permeability, and EC50 value

418

against DPPH radical of chitosan acetate film and chitosan ascorbate films.*

419 420

Chitosan acetate film as the control group shows the highest moisture content and

421

swelling degree but the lowest solubility in water. Trends toward the decreases on

422

moisture content and swelling degree and an increase on the water solubility are

423

detected in Table 4 for chitosan ascorbate films. This result might be ascribed to the

424

salt-forming reaction and hydrogen-binding interaction between chitosan and ascorbic

425

acid molecules, which could cause the lack of free hydrophilic groups such as amino

426

and hydroxyl groups in chitosan backbones to bind with water by forming hydrogen

427

bonds due to the competitive binding effect. 15, 40 Meanwhile, the hydrophilic property

428

of ascorbate moiety in chitosan backbones and the disruption of the intra- or inter-

429

molecular hydrogen bond networks might take responsibility for the higher solubility in

430

water of chitosan ascorbate films. Similar result was found for chitosan-PA

431

(protocatechuic acid) composite films.

13

Besides, these obvious variation tendencies

19


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432

can be further strengthened with an increment on the substitution degrees of ascorbate

433

groups.

434

For edible films, the blocking of water-exchanging between packaged foodstuffs

435

and external environment can efficiently extend their shelf life. 17, 43 Table 4 shows the

436

influence of the salt-forming reaction on the WVP values of chitosan based films. The

437

WVP values of chitosan ascorbate films are significantly lower (p < 0.05) than that of

438

chitosan acetate film. But, no significant differences (p > 0.05) in WVP values are

439

detected with the increasing on ascorbate content. Past research has demonstrated that

440

the permeation of water vapor for chitosan-based films involved two processes,

441

including adsorption and diffusion steps. 4, 44 Moisture could be easily adsorbed into the

442

polymeric matrix by free hydrophilic groups of chitosan backbones followed by the

443

improved diffusion step.

444

ascorbate molecule greatly occupied a large amount of hydrophilic groups of chitosan,

445

which ultimately led to the decreasing on water affinity of chitosan ascorbate films.

446

3.3 Antioxidant Activity

45

The electrovalent bond and hydrogen bond in chitosan

447 448

Fig. 7. DPPH radical scavenging effect (a) and reducing power (b) of chitosan acetate (Cs2Ac4) film

449

and chitosan ascorbate (CsxVcy) films.

450 451

Antioxidant activities of chitosan films were measured using the scavenging

452

activity assay on DPPH radical and reducing power assay. Meanwhile, the EC50 value in

453

mg/mL (namely the mass concentration of antioxidants produced 50% scavenging effect

454

against active free radicals) against DPPH radical (shown in Table 4) is also calculated

455

as an indicator of the antioxidant capacity of chitosan based films. Naturally, the lower

20



Page 22 of 43

456

the EC50 values, the stronger the scavenging abilities against free radicals of the

457

compounds.

458

As illustrated in Fig. 7, chitosan acetate film shows poor capacity for scavenging

459

DPPH radical with an EC50 value far exceed 1.60 mg/mL and negligible reducing power

460

within the tested dosage. However, chitosan ascorbate films exhibit powerful DPPH

461

radical scavenging capacity and reducing power with a dose-dependent manner in the

462

range of selected concentration. Continuous enhancements in the scavenging activities

463

against DPPH radical are detected for Cs2Vc1, Cs2Vc2, Cs2Vc3, Cs2Vc4, Cs2Vc6, and

464

Cs2Vc8 with scavenging values of 63.73 ± 0.82%, 68.75 ± 2.61%, 74.89 ± 1.58%, 83.56

465

± 2.17%, 87.43 ± 2.61%, and 88.67 ± 2.56% respectively at the testing concentration of

466

0.10 mg/mL compared with chitosan acetate film with 1.22 ± 0.16% scavenging rate.

467

But at 0.40 mg/mL or higher concentrations, their scavenging values against DPPH

468

radical remain above 96% and in fact there are no meaningful differences between their

469

scavenging activities. However, the obtained values for EC50 reflect that the increase in

470

ascorbate content enhanced the scavenging efficiency of chitosan ascorbate films

471

against DPPH radical. Meanwhile, the significant difference in reducing power at the

472

concentration of 0.40 mg/mL for chitosan ascorbate films is observed and the reducing

473

power of Cs2Ac4, Cs2Vc1, Cs2Vc2, Cs2Vc3, Cs2Vc4, Cs2Vc6, and Cs2Vc8 are measured as

474

0.01 ± 0.01, 2.87 ± 0.16, 3.12 ± 0.12, 3.45 ± 0.18, 3.74 ± 0.17, 3.85 ± 0.18, and 3.86 ±

475

0.14, respectively. This result demonstrated that with the increment of ascorbate content,

476

the antioxidant activities of chitosan ascorbate films were further enhanced. This

477

wonderful effect might mainly get the benefit of the function of ascorbate, which can be

478

available to act as a one-electron donor to potentially damage active oxidizing radicals

479

by a reversible and thermodynamically favorable oxidation.

21


46

Meanwhile, ascorbyl

Page 23 of 43


480

radical can readily generate in this one-electron oxidation of ascorbate (Fig. 8). Two

481

molecules of the ascorbyl radical can also undergo a pH-dependent disproportionation

482

reaction, resulting in one molecule of ascorbate and the two-electron oxidized form,

483

dehydroascorbate. 47

484 485

Fig. 8. Redox forms and the antioxidative mechanism of ascorbate.

486 487

In conclusion, for the first time, chitosan ascorbates were synthesized in water

488

without any organic reagent based on salt-forming reaction of chitosan and ascorbic

489

acid at different reaction molar ratios to prepare chitosan-based antioxidative films for

490

food preservation. Besides, chitosan acetate film was also obtained used as control

491

group in physicochemical experiments and antioxidant tests. As a result, chitosan

492

ascorbate films showed improved the surface color, reduced light transmittances, lower

493

water content and swelling degree, decreased water vapor permeability, and higher

494

water solubility, whereas reduced mechanical properties, lower crystallinity and thermal

495

stability than chitosan acetate film. Those changes in physical property were due to the

496

stronger destructive capacity of ascorbic acid to crystalline structure of chitosan matrix

497

as well as intramolecular and intermolecular hydrogen bonding between chitosan chains.

498

In vitro antioxidant activity studies demonstrated that the incorporation of ascorbate

499

could greatly enhance scavenging capacity against DPPH radical and reducing power of

500

chitosan matrix. Besides, the antioxidant activities of chitosan ascorbate films were

501

further enhanced with the increase of ascorbate content. Current results converge to

502

promise the potential of chitosan ascorbate films as a suitable candidate in the field of

503

food packaging.

504

AUTHOR INFORMATION 22



Page 24 of 43

505

Corresponding Authors

506

*E-mail: [email protected] (Z.G.)

507

ORCID

508

Wenqiang Tan: 0000-0001-6075-2456

509

Zhanyong Guo: 0000-0002-2143-6933

510

Notes

511

The authors declare no competing financial interest.

512

Funding

513

The authors thank the financial support from the National Natural Science Foundation

514

of China (41576156), the seed project of Yantai Institute of Coastal Zone Research,

515

Chinese Academy of Sciences (Grant No. YIC Y755031011), Natural Science

516

Foundation of Shandong Province of China (ZR2017BD015), and Science and

517

Technology Service Network Initiative of Chinese academy of sciences (KFJ-STS-

518

ZDTP-023).

519

References

520

(1) Galvis-Sánchez, A. C.; Sousa, A. M. M.; Hilliou, L.; Gonçalves, M. P.; Souza, H.

521

K. S. Thermo-compression molding of chitosan with a deep eutectic mixture for

522

biofilms development. Green Chem. 2016, 18, 1571-1580.

523 524 525

(2) Wang, H.; Qian, J.; Ding, F. Emerging chitosan-based films for food packaging applications. J. Agric. Food Chem. 2018, 66, 395-413. (3) Liu, K.; Lin, X.; Chen, L.; Huang, L.; Cao, S.; Wang, H. Preparation of

526

microfibrillated

527

enhancing antibacterium and strength of sodium alginate films. J. Agric. Food

528

Chem. 2013, 61, 6562-6567.

cellulose/chitosan-benzalkonium

23


chloride

biocomposite

for

Page 25 of 43


529

(4) Liu, J.; Meng, C.-g.; Liu, S.; Kan, J.; Jin, C.-h. Preparation and characterization of

530

protocatechuic acid grafted chitosan films with antioxidant activity. Food

531

Hydrocolloid. 2017, 63, 457-466.

532

(5) Nunes, C.; Maricato, É.; Cunha, Â.; Rocha, M. A. M.; Santos, S.; Ferreira, P.;

533

Silva, M. A.; Rodrigues, A.; Amado, O.; Coimbra, J.; Silva, D.; Moreira, A.;

534

Mendo, S.; Silva, J. A. L.; Pereira, E.; Rocha, S. M.; Coimbra, M. A. Chitosan-

535

genipin film, a sustainable methodology for wine preservation. Green Chem. 2016,

536

18, 5331-5341.

537

(6) Pérez-Córdoba, L. J.; Norton, I. T.; Batchelor, H. K.; Gkatzionis, K.; Spyropoulos,

538

F.; Sobral, P. J. A. Physico-chemical, antimicrobial and antioxidant properties of

539

gelatin-chitosan based films loaded with nanoemulsions encapsulating active

540

compounds. Food Hydrocolloid. 2018, 79, 544-559.

541

(7) Marin, L.; Stoica, I.; Mares, M.; Dinu, V.; Simionescu, B. C.; Barboiu, M.

542

Antifungal vanillin–imino-chitosan biodynameric films. J. Mater. Chem. B 2013, 1,

543

3353-3358.

544

(8) Mauricio-Sánchez, R. A.; Salazar, R.; Luna-Bárcenas, J. G.; Mendoza-Galván, A.

545

FTIR spectroscopy studies on the spontaneous neutralization of chitosan acetate

546

films by moisture conditioning. Vib. Spectrosc. 2018, 94, 1-6.

547

(9) Rui, L.; Xie, M.; Hu, B.; Zhou, L.; Yin, D.; Zeng, X. A comparative study on

548

chitosan/gelatin composite films with conjugated or incorporated gallic acid.

549

Carbohydr. Polym. 2017, 173, 473-481.

550

(10) Liang, J.; Yan, H.; Zhang, J.; Dai, W.; Gao, X.; Zhou, Y.; Wan, X.; Puligundla, P.

551

Preparation and characterization of antioxidant edible chitosan films incorporated

552

with epigallocatechin gallate nanocapsules. Carbohydr. Polym. 2017, 171, 300-306.

24



553

(11) Aguirre-Loredo, R. Y.; Rodriguez-Hernandez, A. I.; Morales-Sanchez, E.; Gomez-

554

Aldapa, C. A.; Velazquez, G. Effect of equilibrium moisture content on barrier,

555

mechanical and thermal properties of chitosan films. Food Chem. 2016, 196, 560-

556

566.

557

(12) Sun, L.; Sun, J.; Chen, L.; Niu, P.; Yang, X.; Guo, Y. Preparation and

558

characterization of chitosan film incorporated with thinned young apple

559

polyphenols as an active packaging material. Carbohydr. Polym. 2017, 163, 81-91.

560

(13) Liu, J.; Liu, S.; Wu, Q.; Gu, Y.; Kan, J.; Jin, C. Effect of protocatechuic acid

561

incorporation on the physical, mechanical, structural and antioxidant properties of

562

chitosan film. Food Hydrocolloid. 2017, 73, 90-100.

563

(14) Sun, X.; Sui, S.; Ference, C.; Zhang, Y.; Sun, S.; Zhou, N.; Zhu, W.; Zhou, K.

564

Antimicrobial and mechanical properties of beta-cyclodextrin inclusion with

565

essential oils in chitosan films. J. Agric. Food Chem. 2014, 62, 8914-8918.

566

(15) Souza, V. G. L.; Fernando, A. L.; Pires, J. R. A.; Rodrigues, P. F.; Lopes, A. A. S.;

567

Fernandes, F. M. B. Physical properties of chitosan films incorporated with natural

568

antioxidants. Ind. Crop. Prod. 2017, 107, 565-572.

569

(16) Kaya, M.; Khadem, S.; Cakmak, Y. S.; Mujtaba, M.; Ilk, S.; Akyuz, L.; Salaberria,

570

A. M.; Labidi, J.; Abdulqadir, A. H.; Deligöz, E. Antioxidative and antimicrobial

571

edible chitosan films blended with stem, leaf and seed extracts of Pistacia

572

terebinthus for active food packaging. RSC Adv. 2018, 8, 3941-3950.

573

(17) Kalaycioglu, Z.; Torlak, E.; Akin-Evingur, G.; Ozen, I.; Erim, F. B. Antimicrobial

574

and physical properties of chitosan films incorporated with turmeric extract. Int. J.

575

Biol. Macromol. 2017, 101, 882-888.

25


Page 26 of 43

Page 27 of 43


576

(18) Pastor, C.; Sánchez-González, L.; Chiralt, A.; Cháfer, M.; González-Martínez, C.

577

Physical and antioxidant properties of chitosan and methylcellulose based films

578

containing resveratrol. Food Hydrocolloid. 2013, 30, 272-280.

579

(19) Talón, E.; Trifkovic, K. T.; Nedovic, V. A.; Bugarski, B. M.; Vargas, M.; Chiralt,

580

A.; González-Martínez, C. Antioxidant edible films based on chitosan and starch

581

containing polyphenols from thyme extracts. Carbohydr. Polym. 2017, 157, 1153-

582

1161.

583

(20) Ge, L.; Zhu, M.; Li, X.; Xu, Y.; Ma, X.; Shi, R.; Li, D.; Mu, C. Development of

584

active rosmarinic acid-gelatin biodegradable films with antioxidant and long-term

585

antibacterial activities. Food Hydrocolloid. 2018, 83, 308-316.

586 587

(21) Hu, Q.; Luo, Y. Polyphenol-chitosan conjugates: Synthesis, characterization, and applications. Carbohydr. Polym. 2016, 151, 624-639.

588

(22) Tan, W.; Li, Q.; Dong, F.; Wei, L.; Guo, Z. Synthesis, characterization, and

589

antifungal property of chitosan ammonium salts with halogens. Int. J. Biol.

590

Macromol. 2016, 92, 293-298.

591 592

(23) Ren, J.; Liu, J.; Li, R.; Dong, F.; Guo, Z. Antifungal properties of chitosan salts in laboratory media. J. Appl. Polym. Sci. 2012, 124, 2501-2507.

593

(24) Patel, P.; Parmar, K.; Patel, D.; Kumar, S.; Trivedi, M.; Das, M. Inhibition of

594

amyloid fibril formation of lysozyme by ascorbic acid and a probable mechanism

595

of action. Int. J. Biol. Macromol. 2018, 114, 666-678.

596

(25) Arumugam, N.; Kim, J. Quantum dots attached to graphene oxide for sensitive

597

detection of ascorbic acid in aqueous solutions. Mater. Sci. Eng. C 2018, 92, 720-

598

725.

26



599

(26) Sha, R.; Badhulika, S. Facile green synthesis of reduced graphene oxide/tin oxide

600

composite for highly selective and ultra-sensitive detection of ascorbic acid. J.

601

Electroanal. Chem. 2018, 816, 30-37.

602 603

(27) Meng, H.; Yang, D.; Tu, Y.; Yan, J. Turn-on fluorescence detection of ascorbic acid with gold nanolcusters. Talanta 2017, 165, 346-350.

604

(28) Hafsa, J.; Charfeddine, B.; Smach, M. A.; Limem, K.; Majdoub, H.; Sonia, R.

605

Synthesis, characterization, antioxidant and antibacterial proprieties of chitosan

606

ascorbate. Inter. J. Pharm. Chem. Biol. Sci. 2014, 4, 1072-1081.

607

(29) Rossi, S.; Marciello, M.; Sandri, G.; Bonferoni, M. C.; Ferrari, F.; Caramella, C.

608

Chitosan ascorbate: a chitosan salt with improved penetration enhancement

609

properties. Pharm. Dev. Technol. 2008, 13, 513-521.

610

(30) Elbarbary, A. M.; El-Sawy, N. M.; Hegazy, E. A. Antioxidative properties of

611

irradiated chitosan/vitamin C complex and their use as food additive for lipid

612

storage. J. Appl. Polym. Sci. 2015, 132, 42105-42112.

613

(31) Jouki, M.; Yazdi, F. T.; Mortazavi, S. A.; Koocheki, A. Physical, barrier and

614

antioxidant properties of a novel plasticized edible film from quince seed mucilage.

615

Int. J. Biol. Macromol. 2013, 62, 500-507.

616

(32) ASTM. Standard test methods for water vapor transmission of materials. Standard

617

Designations: E96-95 Annual book of American society for testing materials, West

618

Conshohocken, PA, USA. 1995.

619

(33) Germadios, A.; Weller, C. L.; Gooding, C. H. Measurement errors in water vapor

620

permeability of highly permeable, hydrophilic edible films. J. Food Eng. 1994, 21,

621

395-409.

27


Page 28 of 43

Page 29 of 43


622

(34) Tan, W.; Li, Q.; Li, W.; Dong, F.; Guo, Z. Synthesis and antioxidant property of

623

novel 1,2,3-triazole-linked starch derivatives via 'click chemistry'. Int. J. Biol.

624

Macromol. 2016, 82, 404-410.

625

(35) Tan, W.; Zhang, J.; Zhao, X.; Dong, F.; Li, Q.; Guo, Z. Synthesis and antioxidant

626

action of chitosan derivatives with amino-containing groups via azide-alkyne click

627

reaction and N-methylation. Carbohydr. Polym. 2018, 199, 583-592.

628

(36) Liu, X.; Xia, W.; Jiang, Q.; Yu, P.; Yue, L. Chitosan oligosaccharide-N-

629

chlorokojic acid mannich base polymer as a potential antibacterial material.

630

Carbohydr. Polym. 2018, 182, 225-234.

631

(37) Rui, L.; Xie, M.; Hu, B.; Zhou, L.; Saeeduddin, M.; Zeng, X. Enhanced solubility

632

and antioxidant activity of chlorogenic acid-chitosan conjugates due to the

633

conjugation of chitosan with chlorogenic acid. Carbohydr. Polym. 2017, 170, 206-

634

216.

635

(38) Zhang, J.; Tan, W.; Wang, G.; Yin, X.; Li, Q.; Dong, F.; Guo, Z. Synthesis,

636

characterization, and the antioxidant activity of N,N,N-trimethyl chitosan salts. Int.

637

J. Biol. Macromol. 2018, 118, 9-14.

638

(39) Bilbao-Sainz, C.; Chiou, B. S.; Williams, T.; Wood, D.; Du, W. X.; Sedej, I.; Ban,

639

Z.; Rodov, V.; Poverenov, E.; Vinokur, Y.; McHugh, T. Vitamin D-fortified

640

chitosan films from mushroom waste. Carbohydr. Polym. 2017, 167, 97-104.

641

(40) Riaz, A.; Lei, S.; Akhtar, H. M. S.; Wan, P.; Chen, D.; Jabbar, S.; Abid, M.;

642

Hashim, M. M.; Zeng, X. Preparation and characterization of chitosan-based

643

antimicrobial active food packaging film incorporated with apple peel polyphenols.

644

Int. J. Biol. Macromol. 2018, 114, 547-555.

28



645

(41) Moghadas, B.; Dashtimoghadam, E.; Mirzadeh, H.; Seidi, F.; Sadrabadi, M. M. H.

646

Novel chitosan-based nanobiohybrid membranes for wound dressing applications.

647

RSC Adv. 2016, 6, 7701-7711.

648

(42) Bao, Y.; Zhang, H.; Luan, Q.; Zheng, M.; Tang, H.; Huang, F. Fabrication of

649

cellulose nanowhiskers reinforced chitosan-xylan nanocomposite films with

650

antibacterial and antioxidant activities. Carbohydr. Polym. 2018, 184, 66-73.

651

(43) Ren, L.; Yan, X.; Zhou, J.; Tong, J.; Su, X. Influence of chitosan concentration on

652

mechanical and barrier properties of corn starch/chitosan films. Int. J. Biol.

653

Macromol. 2017, 105, 1636-1643.

654

(44) Liu, J.; Liu, S.; Chen, Y.; Zhang, L.; Kan, J.; Jin, C. Physical, mechanical and

655

antioxidant properties of chitosan films grafted with different hydroxybenzoic

656

acids. Food Hydrocolloid. 2017, 71, 176-186.

657

(45) Ma, Y.; Xin, L.; Tan, H.; Fan, M.; Li, J.; Jia, Y.; Ling, Z.; Chen, Y.; Hu, X.

658

Chitosan membrane dressings toughened by glycerol to load antibacterial drugs for

659

wound healing. Mater. Sci. Eng. C 2017, 81, 522-531.

660 661 662 663

(46) Du, J.; Cullen, J. J.; Buettner, G. R. Ascorbic acid: chemistry, biology and the treatment of cancer. BBA-Rev. Cancer 2012, 1826, 443-457. (47) Harrison, F. E.; May, J. M. Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2. Free Radical Bio. Med. 2009, 46, 719-730.

664

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Figure captions Fig. 1. Synthetic route for chitosan ascorbates. Fig. 2. FTIR spectra of chitosan, chitosan acetate (Cs2Ac4), and chitosan ascorbates (CsxVcy). Fig. 3. 1H NMR spectra of chitosan, chitosan acetate (Cs2Ac4), and chitosan ascorbates (CsxVcy). Fig. 4. UV – vis spectra of chitosan acetate (Cs2Ac4) film and chitosan ascorbate (CsxVcy) films. Fig. 5. XRD patterns of chitosan acetate (Cs2Ac4) film and chitosan ascorbate (CsxVcy) films. Fig. 6. TGA (a) and DTG (b) curves of chitosan acetate (Cs2Ac4) film and chitosan ascorbate (CsxVcy) films. Fig. 7. DPPH radical scavenging effect (a) and reducing power (b) of chitosan acetate (Cs2Ac4) film and chitosan ascorbate (CsxVcy) films. Fig. 8. Redox forms and the antioxidative mechanism of ascorbate.

30



Fig. 1.

31


Page 32 of 43

Page 33 of 43


Fig. 2.

32



Fig. 3.

33


Page 34 of 43

Page 35 of 43


Fig. 4.

34



Fig. 5.

35


Page 36 of 43

Page 37 of 43


Fig. 6.

36



Fig. 7.

37


Page 38 of 43

Page 39 of 43


Fig. 8.

38



Page 40 of 43

Table 1. Reaction molar ratio and degree of substitution of chitosan acetate and chitosan ascorbates.*

*

Chitosan derivative

Reaction molar ratio

Degree of substitution

Cs2Ac4

chitosan: 10 mM, glacial acetic acid: 20 mM

0.60

Cs2Vc1

chitosan: 10 mM, ascorbic acid: 5 mM

0.50

Cs2Vc2


0.65

Cs2Vc3


0.74

Cs2Vc4


0.80

Cs2Vc6


0.88

Cs2Vc8


0.95

CsxAcy: chitosan acetate, CsxVcy: chitosan ascorbate.

39


Page 41 of 43


Table 2. Thickness, density, and mechanical properties of chitosan acetate film and chitosan ascorbate films.* Tensile strength

Elongation at

(MPa)

break (%)

1.16 ± 0.02a

43 ± 1a

31 ± 6a

68 ± 7a

1.06 ± 0.04b

27 ± 4b

16 ± 2b

Cs2Vc2

71 ± 6a

1.03 ± 0.03b

26 ± 5bc

13 ± 2bc

Cs2Vc3

68 ± 5a

1.07 ± 0.03b

24 ± 2bc

12.4 ± 0.9bc

Cs2Vc4

66 ± 6a

1.08 ± 0.05b

23 ± 5bc

12.2 ± 1.5bc

Cs2Vc6

67 ± 6a

1.06 ± 0.01b

22 ± 5bc

12.0 ± 1.5bc

Cs2Vc8

68 ± 8a

1.04 ± 0.03b

21 ± 4c

10.0 ± 0.4c

Film

Thickness (μm)

Density (g/cm3)

Cs2Ac4

70 ± 4a

Cs2Vc1

*Values a, b, c

are given as mean ± standard deviation (SD).

Different superscripts within the same column indicate significant differences among films (p
1.60a

Cs2Vc1

11.6 ± 0.9b

36.3 ± 1.0c

18 ± 2b

6.6 ± 0.2bc

0.084 ± 0.004b

Cs2Vc2

11.4 ± 1.9b

41.5 ± 0.4b

16.8 ± 1.7b

7.0 ± 0.1b

0.071 ± 0.006c

Cs2Vc3

11.4 ± 0.7b

41.5 ± 0.4b

14.5 ± 1.8bc

6.7 ± 0.2bc

0.062 ± 0.001d

Cs2Vc4

10.9 ± 0.1bc

41.7 ± 1.6b

12 ± 5cd

6.3 ± 0.1c

0.046 ± 0.002e

Cs2Vc6

10.7 ± 0.4bc

43.0 ± 1.6ab

9 ± 2de

6.3 ± 0.1c

< 0.025f

Cs2Vc8

9.0 ± 0.2c

44.0 ± 0.5a

7.0 ± 0.3e

6.1 ± 0.2c

< 0.025f

*Values

are given as mean ± standard deviation (SD).

a, b, c, d, e, f

Different superscripts within the same column indicate significant differences among films

(p < 0.05). CsxAcy: chitosan acetate, CsxVcy: chitosan ascorbate.

42


Physical and Antioxidant Properties of Edible Chitosan Ascorbate Films

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