Preparation, Characterization and Antibacterial Effects of Chitosan

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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

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Journal of Agricultural and Food Chemistry

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Preparation, Characterization and Antibacterial Effects of Chitosan

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Nanoparticles Embedding Essential Oil Synthesized in Ionic Liquid Containing

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System

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

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a

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

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b

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

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Running title:

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Corresponding author:

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Qihe Chen

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Department of Food Science and Nutrition

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Zhejiang University

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Yuhangtang Rd.866

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Hangzhou 310058

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P. R. China

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Email: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Several chitosan TPP nanoparticles embedded with Torreya grandis aril essential oils

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

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

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

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

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those essential oils loaded chitosan nanoparticles were characterized. The results

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demonstrated that chitosan nanoparticles can be successfully prepared in ionic liquid

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

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

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

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

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

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systems have stronger antibacterial activities than CS nanoparticles.

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

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1. Introduction

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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vital to design and synthesize delivery systems for EOs.

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

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

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

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excellent properties such as biocompatible, biodegradable and mucoadhesive.

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

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

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

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



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

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

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

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mainly used in food preservation including pork and fish preservation.

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

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polymerization, interfacial polymerization emulsion-ionic gelation and multiple

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

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

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

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

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Biosurfactants are extracellular amphiphilic compounds mainly produced by a

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variety of microbes and have exhibited advantages over their chemical synthetic

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

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

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

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but in energy saving and environmental protection technology have been promoted

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

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

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

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

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

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

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

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

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


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differentiation inducing capabilities against human promyelocytic leukemia cell HL-60

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

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MEL-B exhibited relatively low minimum inhibitory concentration against Gram

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positive bacteria such as Bacillus subtilis, Micrococcus luteus, Mycobacterium

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rhodochrous and Staphylococcus aureus, and gram-negatives like Pseudomonas

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

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

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

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

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

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

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

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following studies were conducted on dissolving new biopolymers or synthesizing new

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

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

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

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

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

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

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nanoparticles.

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

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

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

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

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



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

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

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

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

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

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

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

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exploiting Torreya grandis sources and the reutilization of the Torreya grandis

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

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containing system.

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2. Materials and Methods

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2.1. Materials

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

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

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

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

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

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

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

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Reagent Co., Ltd. China.

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2.2. Chemical composition of Torreya grandis essential oils

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

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

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

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


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

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

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

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

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

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

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(Agilent Technologies, Palo Alto, USA).

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2.3. Production and purification of MELs

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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2.4. Preparation and characterization of nanoparticle-forming solutions



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

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

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

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

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

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

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

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

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

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

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

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

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

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[AcMIM]Cl aqueous solution.

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2.5. Characterization of the nanoparticles

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2.5.1.Size distribution and zeta potential

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

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

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

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

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

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o

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

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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


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

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EE was calculated by the following equation:

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

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

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

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

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

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

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

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2.5.4. Morphology observation

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

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

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

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KV.

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2.6. Antibacterial effects of TEOs loaded CS nanoparticles

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

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

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

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

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

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

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2.7. Statistical analysis



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

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

Preparation, Characterization and Antibacterial Effects of Chitosan

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