Electrospun gelatin nanofibers encapsulated with peppermint and

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Electrospun gelatin nanofibers encapsulated with peppermint and chamomile essential oils as potential edible packaging Yadong Tang, Ying Zhou, Xingzi Lan, Dongchao Huang, Tingting Luo, Junjie Ji, Zihui Mafang, Xiaomin Miao, Han Wang, and Wenlong Wang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 4, 2019

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Journal of Agricultural and Food Chemistry

1

Electrospun gelatin nanofibers encapsulated with peppermint and chamomile

2

essential oils as potential edible packaging

3 4

Yadong Tang a,b*, Ying Zhou a, Xingzi Lan a, Dongchao Huang a, Tingting Luo a,

5

Junjie Ji a, Zihui Mafang a, Xiaomin Miao a, Han Wang c, and Wenlong Wang d*

6 7

a

Department of Pharmaceutical Engineering, Guangdong University of Technology, Guangzhou, 510006, China

8 b

9

School of Biotechnology and Health Sciences, Wuyi University, Jiangmen, 529020, China

10 c

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Guangdong Provincial Key Laboratory of Micro-nano Manufacturing Technology

12

and Equipment, School of Electromechanical Engineering, Guangdong University of

13

Technology, Guangzhou, 510006, China

14 15

d

School of Mechanical and Electric Engineering, Guangzhou University, Guangzhou, 510006, China

16 17

E-mail: [email protected]; [email protected]

18 19 20

ACS Paragon Plus Environment


21

Abstract

22

Natural and edible materials have attracted increasing attentions in food

23

packaging, which could overcome the serious environmental issues caused by

24

conventional non-biodegradable synthetic packaging. In this work, gelatin nanofibers

25

incorporated with two kinds of essential oil (EO), peppermint essential oil (PO) and

26

chamomile essential oil (CO), were fabricated by electrospinning for potential edible

27

packaging application. Electron microscopy showed that smooth and uniform

28

morphology of the gelatin/EOs was obtained, and the diameter of nanofibers was

29

mostly enlarged with the increase of EO content. 1HMR spectrum confirmed the

30

existence of PO and CO in nanofibers after electrospinning. The addition of EOs led

31

to an enhancement of the water contact angle of nanofibers. The antioxidant activity

32

was significantly improved for the nanofibers loaded with CO, while the anti-bacteria

33

activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was

34

better for the fibers with PO addition. The combination of half PO and half CO in

35

nanofibers compensated their respective limitations and exhibited optimum

36

bioactivities. Finally, the MTT assay with NIH-3T3 fibroblasts demonstrated the

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absence of cytotoxicity of the gelatin/EOs nanofibers. Thus, our studies suggest that

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the developed gelatin/PO/CO nanofiber could be a promising candidate for edible

39

packaging.

40 41

Keywords:

Electrospinning, Gelatin, Essential oils, Edible packaging

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1. Introduction

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Food packaging is essential for protecting food from surrounding environment

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and extending the shelf-life of food products 1. However, materials currently used for

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food packaging are mostly non-biodegradable petrochemical-based plastics, which is

47

one of the main causes of environmental issues

48

materials are drawing more and more attentions in food packaging field due to their

49

environment-friendly and biodegradable characteristics, as well as the effective

50

controlling of the surface microbial by applying directly on the surface of food 6.

2-5.

Nowadays, natural and edible

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The materials used for edible must be chosen carefully. Gelatin, one of the

52

versatile biomaterials, has been widely used in edible packaging, wound dressing and

53

tissue engineering, and it can be formed by hydrolysing collagen, the most abundant

54

biopolymer in animals

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bioactivities, such as the protective effects against microbial and oxidative damage,

56

which are the main factors of food spoilage 7-9. Especially for many refrigerated food

57

products, such as fresh meat, in addition to isolating them from the outside bacteria,

58

controlling the surface microbial growth on food is also crucial since it is the main

59

source of contamination 9, 10. Besides, the high hydrophilic nature is another weakness

60

of gelatin as food packaging, due to its sensitivity to moisture

61

have been studying blending gelatin with other materials possessing bioactivities or

62

hydrophobicity to alleviate these drawbacks in food packaging applications 12, 13.

2, 7.

However, sole gelatin packaging does not have enough

11.

Thus, researchers

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Essential oils (EOs) are extracted from plants and exhibit natural bioactivities,

64

which have been categorized as GRAS (Generally Recognized as Safe) by the Food



14-17.

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and Drug Administration (FDA)

Thus, EOs are eligible to be added in edible

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food packaging and have been proved to improve the bioactivities as well as the

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hydrophilicity of gelatin-based packaging

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essential oil （ PO ） possesses outstanding antimicrobial activity, which has been

69

well-studied and applied in food preservation, pharmaceutical and wound dressing

70

18-20.

71

medicinal tea, cosmetics perfume and food industry due to its calming and

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anti-bacteria properties, and in particular, the excellent antioxidant activity, which has

73

been widely studied 21-24.

1.

Among many others, peppermint

On the other hand, chamomile essential oil (CO), has been largely used in

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In this study, PO and CO were chosen as bioactive agents to be added in gelatin

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edible packaging to improve the antibacterial, antioxidant and hydrophobic properties.

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Particularly, different from most previous reports 1, 2, 25, electrospinning technique was

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used in this work, but not the commonly used casting method for film formation of

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gelatin, to avoid the heating process resulted rapid evaporation loss of volatile EOs.

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Electrospinning is a simple and versatile technique used to form homogeneous and

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porous nanofibers

81

of electrospun nanofibers, have been proved to be beneficial for the sustained release

82

of bioactive agents from nanofibers to food surface, and amplify the bioactive effects

83

29, 30.

84

well as their controllable tensile modulus and strength by morphology and diameter of

85

nanofibers, make them promising food packaging materials

86

electrospinning of EOs has been reported mostly for wound dressing 32, 33, Han et al 30

26-28.

The high surface area to volume ratio and the nano-structure

Besides, the excellent mechanically flexibility of electrospun nanofiber mats, as


31.

Although the

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34

87

and Lin et al

have also reported the electrospun nanofibers of essential oils and

88

synthetic polymers for active food packaging. However, electrospun nanofibers with

89

EOs and natural polymers for edible packaging and the joint effect of different EOs in

90

nanofibers were rarely studied.

91

In this work, gelatin nanofibers incorporated with PO and CO for potential edible

92

packaging were fabricated via electrospinning, and characterized by scanning electron

93

microscopy (SEM), 1NMR and water contact angle (WCA) measurement. The

94

antibacterial property and the barrier function of nanofibers against Escherichia coli

95

(E. coli) and Staphylococcus aureus (S. aureus) were investigated by dynamic contact

96

method and microbial penetration test, respectively. DPPH radical scavenging assay

97

and MTT assay were performed to evaluate the antioxidant activities and cytotoxicity

98

of the gelatin/EO nanofibers, respectively.

99 100

2. Materials and methods

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2.1 Materials

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EOs of peppermint and chamomile were purchased from Jiangxi Cedar Natural

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Medicinal Oil Co., Ltd. Gelatin (G2625), 2,2-diphenyl-1-picrylhydrazyl hydrate

104

(DPPH), 3-[4,5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) and

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dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. High glucose

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Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), phosphate

107

buffer solution (PBS), Trypsin-ethylenediaminetetraacetic acid (EDTA) solution and

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Penicillin-Streptomycin were purchased from Gibco. Nutrient agar and mueller hinton



109

broth (MH) were obtained from Guangdong Huankai Microbiology Technology Co.

110

Ltd.

111 112

2.2 Preparation of electrospinning solutions

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The process of electrospinning solution preparation was illustrated in Fig. 1(a).

114

Gelatin solution (12% w/v) was prepared by dissolving gelatin powder in a solvent

115

mixture of acetic acid and distilled water with a volume ratio of 22:3. Then a certain

116

percentage (0, 3%, 6%, 9% v/v) of essential oils (peppermint oil (PO) or chamomile

117

(CO) or 50% v/v PO and 50% v/v CO (PO/CO)) was added into the mixture

118

mentioned above and stirred at room temperature for 3 h using a magnetic stirrer

119

(C-MAG HS7 digital) to get homogeneous solutions used for electrospinning.

120 121

2.3 Fabrication of nanofibers

122

Gelatin/EOs nanofibers were fabricated via electrospinning, as shown in Fig.

123

1(b). In brief, the above electrospinning solution was filled into a 1 ml syringe with an

124

18-G needle and mounted on a syringe pump with a preset flow rate of 0.3 ml/h. The

125

needle was connected to the anode of high potential power supply with a bias voltage

126

of 15 kV. The cathode was connected to a grounded roller covered with a piece of

127

aluminum foil for nanofiber collection. The distance between needle and aluminum

128

foil was 10 cm. After electrospinning, the collected fibers were placed in a vacuum

129

dryer overnight to completely remove residual solvent and set aside for experiment.

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2.4 Scanning electron microscopy (SEM) observation

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The morphology of nanofibers was characterized by SEM (TM3030, Hitachi,

133

Tokyo, Japan) at an accelerating voltage of 5 kV. Nanofiber’s diameter was calculated

134

directly from the SEM images using Image Pro Plus 6.0 soft imaging system, and

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each sample was measured by randomly selecting 150 fibers from the SEM image.

136

Then, the diameter of sample was presented by average diameter (AD) ± standard

137

deviation (SD).

138 139 140

2.5 Chemical characterization 1H

NMR (DPX-400, Bruker) spectra was recorded at 400 MHz by dissolving 20

141

mg nanofiber into 500 μL of DMSO-d6 to prove the existence of essential oils in

142

nanofibers 35. The chemical shifts were reported in parts per million (ppm) relative to

143

residual DMSO-d6 (δ=2.50, 1H).

144 145

2.6 Water contact angle measurement

146

The hydrophilicity of nanofibers was evaluated by water contact angle

147

measurement with an automatic contact angle system (OCA100, Dataphysics,

148

Germany) 2. 2 μL of distilled water was dropped on the surface of each nanofiber

149

sample and photographed immediately (t = 0). In this manner, each sample was

150

measured at least three times at different locations for average.

151 152

2.7 Antioxidant activity study



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The antioxidant activity of nanofibers was analyzed in terms of DPPH radical

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scavenging activity 36. In brief, nanofiber samples were placed in a six-well plate and

155

soaked with 3 ml DPPH solution (0.1 mmol/L) prepared by dissolving DPPH powder

156

in alcohol. After 30 min of incubation at room temperature in dark, the absorbance of

157

the solution was recorded at 517 nm by using an UV-Vis spectroscopy (Lambda 25,

158

PerkinElmer, America). Then, the efficiency of scavenging free radicals was

159

calculated by the following formula:

160

Radical scavenging activity (%) =

𝐶𝑜𝑛𝑡𝑟𝑜𝑙𝑂𝐷 ― 𝑆𝑎𝑚𝑝𝑙𝑒𝑂𝐷 𝐶𝑜𝑛𝑡𝑟𝑜𝑙𝑂𝐷

× 100

161

Where, ControlOD is the absorbance of the DPPH aqueous solution without fiber,

162

while, SampleOD is the absorbance of the DPPH aqueous solution with nanofiber

163

sample.

164

The ultraviolet (UV) light transmittance (%) of gelatin/EOs nanofibers was

165

determined by quantifying the transmittance of light at selected wavelengths between

166

200 nm and 400 nm and using an UV-Vis spectrophotometer (Lambda 25,

167

PerkinElmer, America) 1. Gelatin film prepared by casting was regarded as control.

168

Firstly, gelatin solution (12% (w/v)) was prepared by dissolving gelatin powder in

169

distilled water, then the mixture was heated at 70 ℃ for 30 min with continuous

170

stirring to obtain completely solubilize gelatin. Next, the gelatin solution was casted

171

on a plastic plate and placed in a laboratory fume hood at room temperature for

172

gelation 2. During experiment, the thickness of gelatin films was controlled to 0.2 mm

173

by controlling the casting volume of gelatin solution.

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2.8 Antibacterial assay

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The antimicrobial activity of nanofibers against E. coli and S. aureus was

177

evaluated by dynamic contact method 37.The frozen strains were removed from - 80℃

178

and incubated overnight at 37 ℃ on nutrient agar plate to obtain viable strains. Then,

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a certain amount of bacteria was extracted from the plate with an inoculating ring and

180

diluted to a density of 105 CFU/ml in physiological saline. Secondly, 100 μL of the

181

diluted bacterial solution was added into MH to obtain a bacteria solution of 104

182

CFU/ml. Subsequently, UV sterilized nanofibers were added into the solution and

183

incubated for 6 h at 37 ℃ in a shaker with an oscillation frequency of 200 r/min. For

184

comparison, the group without nanofiber was regarded as control. Then, 100 μL of the

185

bacterial solution after incubation was serially diluted 10-fold in saline, and 20 μl of

186

diluent was spread on nutrient agar plate. After overnight incubation at 37 ℃,

187

surviving colonies on each plate were counted. Three parallel experiments were

188

performed for each sample, and the average value of CFU was recorded.

189 190

2.9 Microbial penetration assay

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To evaluate the barrier function of nanofibers against microbial penetration, 5 ml

192

of sterile nutrient broth was added into each test tube, then the test tubes were capped

193

with nanofiber samples

194

regarded as control group. After 14 days, the growth of microbial in each test tube

195

was revealed by the absorbance value of the nutrient broth, measured with a

196

microplate reader (Multiskan FC, Thermo, USA) at 620 nm.

38.

During experiment, the test tubes without any cap was



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2.10 Cytotoxicity test The cytotoxicity of nanofibers was measured by MTT assay

35.

NIH-3T3 cells

200

were cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v)

201

penicillin/streptomycin at 37 ℃ and 5% CO2. 10 mg of each sample was sterilized for

202

2 h under UV, then soaked in 2 ml of DMEM and incubated for 24 h at 37 ℃. Then

203

the extract solutions of samples were filtered via sterile disposable filter (0.22 μm,

204

Merck, Darmstadt, Germany). NIH-3T3 cells were seeded in 96-well plates at a cell

205

density of 5×103 per well and incubated for 24 h, then the culture medium was

206

exchanged with 100 μL of the extract solution. After 48 h of culture, the extract

207

solution was removed and 100 μL of MTT solution was added. After 4 h of

208

incubation in dark, 100 μL of DMSO was added to change the MTT solution and

209

dissolve the dark blue formazan crystals. Finally, the absorbance of formazan solution

210

was measured by a microplate reader at a wavelength of 570 nm.

211 212

2.11 Statistical analysis

213

The statistical analysis of the data was carried out using one-way analysis of

214

variance (ANOVA) with GraphPad Prism (V.7). To evaluate the statistically

215

significance difference between groups, Tukey’s post-hoc test was applied, and p

348

0.05). The result demonstrates that the gelatin/EOs nanofibers are not cytotoxic which

349

is eligible for edible packaging application.

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In conclusion, gelatin nanofibers encapsulated with PO and CO were

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successfully fabricated via electrospinning with homogeneous and smooth

353

morphology. The incorporation of EOs was confirmed by 1H-NMR. The surface

354

hydrophobicity of nanofibers was enhanced with the addition of EOs. All the gelatin

355

nanofibers

356

concentration-dependent antibacterial property against E. coli and S. aureus, as well

357

as certain antioxidant property. Especially, the addition of PO resulted into better

358

antibacterial activity, while that of CO showed better antioxidant property. The joint

359

of PO and CO in gelatin nanofiber showed overall optimum bioactivities compared

360

with single PO or CO. Finally, the MTT assay indicated the non-cytotoxicity of

361

gelatin nanofibers incorporated with PO and CO, demonstrated the qualification of the

362

gelatin/EOs nanofibers as potential edible packaging. Furthermore, the addition of EO

363

has been reported to strengthen the films by inducing the rearrangement of protein

364

network, and some compounds in EO might also cross-link polymer chains to

365

enhance the tensile property of film 5. Therefore, the impact of EO on nanofiber’s

366

mechanical property in food packaging application would be of concern in our future

367

research.

containing

PO,

CO

or

PO/CO

exhibited

improved

EO

368 369

Acknowledgement

370

This work was financially supported by National Natural Science Foundation of

371

China (Grant No. 81801830), and Project of Innovative Research Teams of Jiangmen

372

(2017TD02).



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oil/beta-cyclodextrin inclusion complex for antibacterial packaging. Carbohydr. Polym. 2018, 179,

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360-369.

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35. Rieger, K. A.; Schiffman, J. D. Electrospinning an essential oil: cinnamaldehyde enhances the

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antimicrobial efficacy of chitosan/poly(ethylene oxide) nanofibers. Carbohydr. Polym. 2014, 113,

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561-8.

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36. Yuan, Y.; Zhang, J.; Fan, J.; Clark, J.; Shen, P.; Li, Y.; Zhang, C. Microwave assisted extraction

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of phenolic compounds from four economic brown macroalgae species and evaluation of their

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antioxidant activities and inhibitory effects on alpha-amylase, alpha-glucosidase, pancreatic lipase and

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tyrosinase. Food Res Int 2018, 113, 288-297.

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37. Park, J.-A.; Kim, S.-B. Preparation and characterization of antimicrobial electrospun poly(vinyl

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alcohol) nanofibers containing benzyl triethylammonium chloride. React. Funct. Polym. 2015, 93,

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420-432.

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39. Celebioglu, A.; Yildiz, Z. I.; Uyar, T. Thymol/cyclodextrin inclusion complex nanofibrous webs:

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2018, 106, 280-290.

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Tessaro, I. C. Effect of chitosan addition on the properties of films prepared with corn and cassava

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starches. Journal of food science and technology 2018, 55, 2963-2973.

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41. Khazaei, N.; Esmaiili, M.; Djomeh, Z. E.; Ghasemlou, M.; Jouki, M. Characterization of new

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biodegradable edible film made from basil seed (Ocimum basilicum L.) gum. Carbohydr. Polym. 2014,

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102, 199-206.

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42. Sánchez Aldana, D.; Andrade-Ochoa, S.; Aguilar, C. N.; Contreras-Esquivel, J. C.;

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Nevárez-Moorillón, G. V. Antibacterial activity of pectic-based edible films incorporated with

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Mexican lime essential oil. Food Control 2015, 50, 907-912.

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Immobilization of Cinnamaldehyde on Chitosan Films via Schiff Base Formation and Their

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Figure captions

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Figure 1. Schematic illustration for preparation of gelatin/EOs nanofibers. (a)

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Preparation of gelatin/EOs solutions for electrospinning; (b) Electrospinning process

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for gelatin/EOs nanofiber fabrication; (c) Photograph of gelatin/EOs nanofibrous mat;

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(d) SEM image of gelatin/EOs nanofibers.

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Figure 2. SEM images and fiber diameter distribution of gelation/EOs nanofibers: (a)

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gelatin nanofibers; (b-d) gelatin/PO nanofibers with PO ratio at 3%, 6%, 9% (v/v),

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respectively; (e-g) gelatin/CO nanofibers with CO ratio at 3%, 6%, 9% (v/v),

521

respectively; and (h- j) gelatin/PO/CO nanofiber with PO/CO ratio at 3%, 6%, 9%

522

(v/v), respectively. AD and SD refer to average diameter and standard deviation,

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respectively.

524 525

Figure 3. 1H NMR spectra of (a) peppermint essential oil; (b) chamomile essential oil;

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(c) gelatin nanofiber; and (d−f) gelatin/EOs nanofibers containing PO, CO and

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PO/CO respectively with EOs ratio at 9% (v/v) dissolved in DMSO-d6. Protons used

528

to prove the existence of gelatin, PO, and CO are shown by star sign; purple and blue,

529

PO; orange and black, CO; green and red, gelatin.

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Figure 4. (a-d) Images of water droplets in contact angle measurements on the surface

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of (a) gelatin nanofibers, (b) gelatin/PO nanofibers, (c) gelatin/CO nanofibers, and (d)

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gelatin/PO/CO nanofibers; (e) Water contact angles of nanofibers contained different



534

EOs (black, PO; blue, CO; red, PO/CO).

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Figure 5. Antimicrobial activity of gelatin/EOs nanofibers against E. coli (a) and S.

537

aureus (b). *p < 0.05 versus the control group. Results are mean ± SD (n=3).

538 539

Figure 6. DPPH radical scavenging activity of nanofiber incorporated with different

540

concentration (0%, 3%, 6%, 9%) of EOs (PO, CO, PO/CO) and the corresponding

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photographs of DPPH solution containing different nanofibers. *p < 0.05 versus the

542

control group. Results are mean ± SD (n=3).

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Figure 7. Cytotoxicity of gelatin/EOs nanofibers. NIH-3T3 cells were cultured in the

545

extract solutions of different nanofibers for 48 hours, and cell viability was quantified

546

by MTT assay. The content of EOs has little effect on the biocompatibility of

547

nanofibers (p > 0.05 between groups). Results are mean ± SD (n=4).

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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TOC graphic:

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Electrospun gelatin nanofibers encapsulated with peppermint and

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