Preparation, Characterization, and Antibacterial Effects of Chitosan

Subscriber access provided by Kaohsiung Medical University

Bioactive Constituents, Metabolites, and Functions

Preparation, Characterization and Antibacterial Effects of Chitosan Nanoparticles Embedding Essential Oil Synthesized in Ionic Liquid Containing System Jianan Wu, Qin Shu, Yongwu Niu, Yingchun Jiao, and Qihe Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01428 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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 40

Journal of Agricultural and Food Chemistry

1

Preparation, Characterization and Antibacterial Effects of Chitosan

2

Nanoparticles Embedding Essential Oil Synthesized in Ionic Liquid Containing

3

System

4

Jianan Wua, Qin Shua, Yongwu Niua, Yingchun Jiaob, Qihe Chen*a

5

a

Department of Food Science and Nutrition,Zhejiang University, Hangzhou 310058, China

6

b

College of Agriculture and Animal Husbandry, Qinghai University, Xining 810016, China

7 8

Running title:

9

Corresponding author:

10

Qihe Chen

11

Department of Food Science and Nutrition

12

Zhejiang University

13

Yuhangtang Rd.866

14

Hangzhou 310058

15

P. R. China

16

Email: [email protected]

17 18 19 20 21

22

ACS Paragon Plus Environment


23

Abstract

24

Several chitosan TPP nanoparticles embedded with Torreya grandis aril essential oils

25

(TEOs) were synthesized using emulsion-ionic gelation technique. Mannosylerythritol

26

lipid A (MEL-A), a type of biosurfactants, was selected as the emulsifier. Especially, to

27

replace the acetic acid, ionic liquid (IL) was employed for chitosan dissolving. The

28

physical properties, diameters, morphology, embedding rate and antibacterial effects of

29

those essential oils loaded chitosan nanoparticles were characterized. The results

30

demonstrated that chitosan nanoparticles can be successfully prepared in ionic liquid

31

containing system and the diameters for nanoparticles in acetic acid and ionic liquid

32

solutions are 144.1 ± 1.457 and 219.0 ± 4.045 nm. After loaded with essential oils, the

33

size increased to 349.6 ± 10.55 and 542.9 ± 16.74 nm, respectively. Antibacterial

34

properties were investigated by the observation of the inhibition zone against S. aureus.

35

The results revealed that TEOs loaded nanoparticles synthesized in acid and IL aqueous

36

systems have stronger antibacterial activities than CS nanoparticles.

37

Keywords: chitosan; essential oils; nanoparticles; ionic liquid; antimicrobial

38 39 40 41 42 43 44 45 46


Page 2 of 40

Page 3 of 40


47

1. Introduction

48

Torreya grandis is a species of evergreen conifer tree native in China (1). Its seeds

49

are edible and famous for their special flavor and anti-parasite activities according to

50

traditional Chinese medicine (2). The arils of Torreya grandis seeds, accounting for

51

50-60% weight of the fresh fruit, were always discarded. And as the arils have a strong

52

special smell, the disposal of them becomes an annoying issue. Recently, the extractive

53

from Torreya grandis arils was reported to consist of several bioactive compounds,

54

such as diterpenoids, essential oils (EOs), flavonoids and phenols (3). The essential oils

55

from Torreya grandis arils can be extracted by steam distillation extraction, vacuum

56

fractional distillation, solvent extraction and supercritical extraction (4). Like other

57

essential oils, TEOs are volatile and complex compounds with strong odor (5). A great

58

number of essential oils have been studied and characterized for their anti-bacterial,

59

antioxidant, antiparasitic, and insecticidal activities (5-7). TEOs were also been

60

reported to have antioxidant (1) and mosquito repellent activities (2). However, EOs

61

have several advantages including instability, intense odor and poor solubility. This

62

problems limited the applications of EOs in food and cosmetic industries. Thus, it is

63

vital to design and synthesize delivery systems for EOs.

64

Chitosan (CS), a cationic polysaccharide, is the second most abundant biopolymer

65

in the natural world after cellulose (8). It can be produced by deacetylation of chitin

66

which is ubiquitous in crustacea, fungi and other organisms. In addition, CS has

67

excellent properties such as biocompatible, biodegradable and mucoadhesive.

68

Specially, CS exhibited a wide antibacterial activities against both gram positive and

69

gram negative bacteria (9). Owning to its exceptional versatile, CS is used in solutions,

70

hydrogels and nano/microparticles in various of fields including food, cosmetic,

71

biomedical, agricultural and chemical industries (10). CS nano/microparticulate



72

systems, in particular, are employed to deliver genes, drugs, proteins, vaccines and

73

enzymes (11, 12). Using CS-based nano/microparticles to embed EOs was also

74

reported by several studies (13, 14). The EOs loaded CS nano/microparticles were

75

mainly used in food preservation including pork and fish preservation.

76

There are several ways to synthesize CS-based nanocapsules including in-situ

77

polymerization, interfacial polymerization emulsion-ionic gelation and multiple

78

emulsion-solvent evaporation techniques (13). For emulsion-ionic gelation, when the

79

core material is oil phase such as essential oils, adding emulsifiers with the core

80

material into the cliff material and then adding the coagulant, will improve the size

81

distribution and the encapsulation efficiency of the nanoparticles-forming solution.

82

Biosurfactants are extracellular amphiphilic compounds mainly produced by a

83

variety of microbes and have exhibited advantages over their chemical synthetic

84

counterparts, including biodegradability, excellent surface-activity, low toxicity and

85

stability at extreme temperature, acidity and salt concentration (15). Due to numerous

86

advantages, their applications not only in food, cosmetic and pharmaceutical industries

87

but in energy saving and environmental protection technology have been promoted

88

(16). A type of non-ionic glycolipid biosurfactants, Mannosylerythritol lipids (MELs),

89

was used in the present study. The amphiphilic properties of the MELs come from the

90

hydrophilic moiety 4-O-β-D-mannopyranosyl-meso-erythritol and hydrophobic

91

moiety fatty acids and acetyl groups. MELs were divided into four types according to

92

the differences in acetyl groups and fatty acids (Fig. 1). As emulsifiers, MEL-A and B

93

were reported to have remarkably low critical aggregate concentrations (CAC) at 4.0 ×

94

10-6 and 6 × 10-6 M, respectively. They also exhibited excellent surface and interfacial

95

tension-lowering activities (17). In addition to splendid emulsion properties, MELs

96

also exhibited versatile bioactivities. MELs were found to have apoptosis and cell


Page 4 of 40

Page 5 of 40


97

differentiation inducing capabilities against human promyelocytic leukemia cell HL-60

98

, rat pheochromocytoma and melanoma cell B16 (18). Furthermore, MEL-A and

99

MEL-B exhibited relatively low minimum inhibitory concentration against Gram

100

positive bacteria such as Bacillus subtilis, Micrococcus luteus, Mycobacterium

101

rhodochrous and Staphylococcus aureus, and gram-negatives like Pseudomonas

102

rivoflavina (16). Also, MEL-A used as wall material in liposomes significantly

103

improved membrane fusion and gene transfection efficiency (19). In the present study,

104

we use MEL-A as the emulsifier in CS-based nanoscale systems.

105

Ionic liquids, a kind of salt-like material that forms stable liquids below 100 oC

106

(20), have been reported to dissolve biopolymers and were regarded as green solvents

107

to replace organic compounds (21). After Swatloski et al. (22) first found that cellulose

108

can be dissolved in ionic liquid (IL) up to 25 wt% with heating in 2002, a series of

109

following studies were conducted on dissolving new biopolymers or synthesizing new

110

ILs. Up to now, several biological macromolecules including chitin (21), CS (23), wool

111

keratin (24), silk fibroin (25) and amylose (26). Zhang et al. (27) studied the hydrolysis

112

of CS in ILs and dissolved 0.255 mg CS in 4 g ILs. Also, a mixture of [DMIM]Cl and

113

[HMIM]Cl (9:1) was found to have considerable solubility for CS (23). Using IL,

114

instead of acetic acid, to dissolve CS can avoid strong acerbic odor and air pollution.

115

However, there are few reports on using IL containing system to prepare CS-based

116

nanoparticles.

117

In this work, IL 1-methylimidazolium-3-acetate chlorine ([AcMIM]Cl) aqueous

118

system was employed in dissolving CS and synthesizing CS-based nanoparticles

119

embedded with TEOs by emulsion-ionic gelation technique. The aim of this study is to

120

explore a green and environment-friendly way to synthesize CS-based nanoparticles in

121

IL containing system instead of acetic acid solution. MEL-A was added as the



122

emulsifier in the designed system. The precipitant salt concentration and its adding rate

123

was also investigated. To evaluated the quality of these CS-based nanoparticles, the

124

size distribution, zeta potential and the encapsulation efficiency were characterized.

125

The physical properties of nanoparticles were also investigated by X-ray diffraction

126

(XRD) and Fourier transform infrared spectrophotometry (FTIR). In addition, the

127

antibacterial effects against gram positive bacteria Staphylococcus aureus (S. aureus)

128

was determined by inhibition zone method. We hope that this study will be useful in

129

exploiting Torreya grandis sources and the reutilization of the Torreya grandis

130

industry waste. We also provide insights in synthesizing CS-based nanoparticles in IL

131

containing system.

132

2. Materials and Methods

133

2.1. Materials

134

Chitosan (Molecular weight: 8.0 × 104 Da, deacetylation rate: ≥ 95%) was

135

purchased from Cool Chemical Science and Technology (Beijing) Co., Ltd. China. The

136

Torreya grandis essential oil was provided by Shanghai Muzhaoxiangtang Co., Ltd.

137

China. Ionic liquid 1-methylimidazolium-3-acetate chlorine ([AcMIM]Cl) was

138

purchased from Shanghai Aichun Biological Technology Co., Ltd. China. Sodium

139

tripolyphosphate (TPP) was bought from Shanghai Macklin biochemical Co., Ltd.

140

China. Glacial acetic acid (analytical grade) was purchased from Sinopharm Chemical

141

Reagent Co., Ltd. China.

142

2.2. Chemical composition of Torreya grandis essential oils

143

The chemical composition of TEOs was analyzed by GC-MS (7890B/7000C,

144

Agilent Technologies, Palo Alto, USA). The column used was a silica capillary column

145

HP-5MS (30 m × 0.25 mm, 0.25 µm). The chromatographic conditions were referred

146

the method of Li et al. (13) with some modifications. In brief, the injection temperature


Page 6 of 40

Page 7 of 40


147

was fixed at 250 oC, 1 µL of sample solution was injected under helium carrier gas with

148

a flow rate of 1 mL/min (split ratio 15:1). The column was first hold at 50 oC for 2 min,

149

then gradually increased to 150 oC at a rate of 5 oC/min and hold for 1min, and finally

150

programmed to 280 oC at a rate of 8 oC and hold for 2min. For Mass Spectrometry, the

151

EI energy was set as 70 eV, ion source temperature was fixed at 280 oC and the mass

152

scan range was 30-550 amu. The results were analyzed in Masshunter software

153

(Agilent Technologies, Palo Alto, USA).

154

2.3. Production and purification of MELs

155

The MELs were produced and purified by the method of Fan et al. (18).

156

Pseudozyma aphidis was employed to manufacturing MELs. After 36 h of activation in

157

a medium containing 3% yeast exact, 3% malt extract, 10% glucose and 5% peptone

158

from soybean under 28 oC and 180 rpm, 1 mL of P. aphidis culture was inoculated into

159

seed medium consist of glucose 40 g/L, NaNO3 3 g/L, yeast extract 1 g/L,

160

MgSO4⋅7H2O 0.3 g/L, KH2PO4 0.3 g/L and distilled water. The seed culture was

161

incubated under same condition for 2 days and then centrifuged and washed by

162

physiological saline. The resting cells were inoculated into fermentation medium which

163

use 80 mL/L soybean oil to replace the glucose in seed medium. MELs were extracted

164

by vigorous mixing same volume of ethyl acetate with the culture suspension after 7

165

days of fermentation. The organic layer was separated by centrifuge and the ethyl

166

acetate was removed under reduced pressure. The sticky MELs were then washed with

167

methanol : cyclohexane (1:1, v/v) twice to remove the remaining oil and fatty acid.

168

Crude MELs were refined using silica gel column to obtain MEL-A were and the

169

refined production was confirmed by TLC (Silica gel GF254, chloroform : methanol :

170

water = 65:15:2, v/v).

171

2.4. Preparation and characterization of nanoparticle-forming solutions



172

The nanoparticle-forming solution (NFS) without TEOs was prepared by ionic

173

gelation technique. In brief, CS (0.1%,w/v) was dissolved in 1% (v/v) acetic acid

174

solution under magnetic stirring for 4 h. Then 10 mL TPP solution of different con

175

centration (0.025, 0.05, 0.075, 0.1 and 0.125%) was added drop wise into 20 mL of CS

176

solution by a peristaltic pump (HL-2B, Shanghai Qingpu-Huxi Instruments Factory,

177

China) under magnetic stirring at 1800 rpm. Different adding speed of TPP (2, 5, 7, 10,

178

13, 15 and 18 rpm) was also studied. TEOs loaded nanoparticle-forming solution was

179

prepared by emulsion-ionic gelation technique (28). Different amount (1, 5, 10, 15, 20,

180

25 and 30 mg) of MEL-A was added into 20 mL CS solution and stirred for 1 h to

181

obtain homogeneous solution. Subsequently, certain amount of TEOs was added and a

182

homogenizer (ULTRA-TURRAX T18, IKA, Staufen, Germany) was employed for 5

183

min for emulsification. Then TPP solution was slowly dropped into the above emulsion

184

with constant magnetic stirring. For IL group, the CS was dissolved in 2% (w/v)

185

[AcMIM]Cl aqueous solution.

186

2.5. Characterization of the nanoparticles

187

2.5.1.Size distribution and zeta potential

188

The mean particle size and the zeta potential of the CS-based nanoparticles were

189

determined by dynamic light scattering (DLS) via a Zetasizer Nano ZS90 (Malvern

190

Instruments, UK). The NFS was directly measured without any dilution. The

191

measurement angle and the temperature were set at 90o and 25 oC, respectively.

192

2.5.2. Encapsulation efficiency The nanoparticles were collected by centrifuging the NFS at 9643 g for 20 min at 4

193 194

o

195

supernatant was used for determination of encapsulation efficiency by GC-MS. The

196

amount of unencapsulated TEOs was determined by the comparison of 3-Carene

C and lyophilized (FreeZone 2.5, Labconco, Kansas city, MO, USA) for 2 days. The


Page 8 of 40

Page 9 of 40


197

concentration between sample of certain TEOs concentration and the supernatant. The

198

EE was calculated by the following equation:

199 200

EE (%) = (Initial TEOs conc.-unencapsulated TEOs conc.)/ Initial TEOs conc. 2.5.3. X-ray diffraction and Fourier transform infrared spectrometry

201

The physical properties of nanoparticles were analyzed by XRD and FTIR. The

202

X-ray diffraction data was collected by a diffractometer (D8 A25 Advanced, Bruker,

203

Karlsruhe, Germany). All samples were scanned from 5 to 90o of 2θ.

204

The Fourier transform infrared spectra of four samples were collected at a

205

wavelength range from 4000 to 400 cm-1 on a FTIR spectroscopy (Nicolet Avatar 370,

206

Madison, USA). Samples were prepared by KBr pellet method.

207

2.5.4. Morphology observation

208

The morphology of the nanoparticles were studied using a JEM-1230 transmission

209

electron microscope (JEOL, Akishima, japan). In brief, the sample was first absorbed

210

on a cooper grid and then stained with uranyl acetate. The acceleration voltage was 120

211

KV.

212

2.6. Antibacterial effects of TEOs loaded CS nanoparticles

213

The inhibition zone against Gram positive bacteria S. aureus was investigated.

214

The measurements were conducted by cylinder plate method. First, 100 µL of 107

215

CFU/mL bacteria suspended in Luria-Bertani Broth (LB) was spread on LB agar.

216

Sterilized oxford-cups were inserted into the inoculated plated. Then, each cup was

217

added with 150 µL of sample solution. The HAc solution was used as control. After

218

incubation under 37 oC for 24 h, the diameters of the inhibition zone were measured.

219

2.7. Statistical analysis



220

All experiments were carried out in triplicate. Differences between means were

221

processed by Ducan test using SPSS Statistics 20 and considered to be significant at P

Preparation, Characterization, and Antibacterial Effects of Chitosan

Recommend Documents