Lauric Arginate

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Functional Structure/Activity Relationships

Electrospun chitosan/polyethylene oxide/lauric arginate nanofibrous film with enhanced antimicrobial activity Lingli Deng, Maierhaba Taxipalati, Aiping Zhang, Fei Que, Hewen Wei, Fengqin Feng, and Hui Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01493 • 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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Electrospun chitosan/polyethylene oxide/lauric

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arginate nanofibrous film with enhanced

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antimicrobial activity

4

Lingli Deng 1, Maierhaba Taxipalati 2, Aiping Zhang

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Fengqin Feng 1, Hui Zhang 1, *

1

,

Fei Que 3, Hewen Wei 4,

6 7

1

8

Key Laboratory for Agro-Products Postharvest Handling of Ministry of Agriculture,

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Key Laboratory for Agro-Products Nutritional Evaluation of Ministry of Agriculture,

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Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science,

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College of Biosystems Engineering and Food Science, Zhejiang University,

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Hangzhou 310058, China

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2

Turpan Vocational and Technical College, Turpan 838000, China

14

3

Department of Applied Engineering, Zhejiang Institute of Economics and Trade,

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Hangzhou 310018, China

16

4

17

* Corresponding author. E-mail: [email protected]. Phone: +86-571-88982981;

18

Fax: +86-571-88982981.

National Engineering Laboratory of Intelligent Food Technology and Equipment,

Jinhua Institute for Food and Drug Control, Jinhua 321000, China

19

1

ACS Paragon Plus Environment


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ABSTRACT

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In this study, the chitosan/polyethylene oxide (PEO)/lauric arginate (LAE) composite

22

nanofibrous films were fabricated via electrospinning. The addition of LAE did not

23

change the physical properties of chitosan/PEO in acetic aqueous solutions, but

24

increased the fluorescent intensity of chitosan by electrostatic interactions, resulting in

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uniform and beads-free nanofibers with an average diameter of 150 nm. The Fourier

26

transform infrared spectra and thermal analysis indicated that the LAE molecules

27

were homogenously dispersed within the chitosan/PEO nanofibers. The formation of

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electrostatic and hydrogen bonding interactions induced by the LAE addition changed

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the inter- and intramolecular interactions between PEO and chitosan, further affected

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the mobility of the polymer molecules, leading to the increased crystallinity and

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decreased melting point. The hydrophilicity of the nanofibrous films was significantly

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increased by the incorporation of LAE, as indicated by the decreasing water contact

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angle from 39o to 10o. Meanwhile, the chitosan/PEO/LAE nanofibrous films showed

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the LAE concentration dependent antimicrobial activity against Escherichia coli and

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Staphylococcus aureus, suggesting the enhanced antimicrobial activity. The

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fluorescent staining experiments demonstrated that the antimicrobial mechanism of

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the nanofibrous films was cell membrane damage.

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KEYWORDS: chitosan; lauric arginate; electrospinning; antimicrobial

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INTRODUCTION

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Antimicrobial food packaging has received more and more attentions since it

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may prolong the storage life of mildly preserved fresh food while maintaining the

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nutritional value and sensory characteristics. Currently the food-packaging industry

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depends mainly on plastics, including polyethylene and polypropylene materials,

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which are difficult to be degraded. However, the technology of food packaging is

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rapidly moving towards development of functionality and biodegradability

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Electrospinning is an electrostatic fiber fabrication technique to fabricate nanofibers

47

with diameters from 10 nm to 10 µm. When high voltage was applied to the

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electrospinning solution, the electrostatic force overcame the surface tension to stretch

49

the liquid droplet, resulting in the formation of the Taylor cone over a critical point.

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When the charged jet passed though the electrical field to collector, the jet solidified

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to ultrathin fiber as the rapidly evaporation of solvent 2.The fascinating features of the

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electrospun nanofibers such as high surface-to-volume ratio and porosity, ultrafine

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and oriented structures contribute to the enlarged contact area to the food surface 3. By

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electrospinning, antimicrobial agents could be directly incorporated in the nanofibers

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without further processing 4.

1

.

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Chitosan, as biopolymer derived from partial deacetylation of chitin, is non-toxic,

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biodegradable, biocompatible and have broad-spectrum antibacterial activity for food

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packaging applications to prolong shelf life

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been shown to extend the shelf life of postharvest fresh-cut honeydew melon and

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prevent the deterioration of golden pomfret fillet 7, 8. Electrospinning of pure chitosan

5, 6

. For example, chitosan coating has

3



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was only possible by using toxic or highly concentrated acidic solvents due to its

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polycationic nature and chain stiffness 9. However, hybrid electrospinning of chitosan

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and other polymers in mild solvents, such as silk fibroin, polyethylene oxide (PEO)

64

and poly (vinyl alcohol) (PVA), has been well documented

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nanofibrous films have been also reported to exhibit a higher or broader antimicrobial

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activity by incorporation of silver nanoparticles 12, enzymes 13, 14, etc.

10, 11

. Moreover, these

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Lauric arginate (LAE) is a GRAS (generally recognized as safe) antimicrobial

68

agent, which has been reported to show a broad-spectrum antimicrobial efficiency

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against bacteria, fungi, and yeasts 15, 16. The application of LAE in food products as an

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antimicrobial agent has been reported 16-18. The efficient antimicrobial activity of LAE

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has been ascribed to the membrane disruption effect on microorganisms, where it

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altered the metabolic process without causing cellular lysis

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Visessanguan, Kruenate, Kingcha and Keeratipibul

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casted polylactic acid films as a food-contact antimicrobial packaging material, and

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found that the films prolonged the shelf life of cooked cured ham by decreasing the

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Listeria monocytogenes and Salmonella typhimurium from 6 logs to 2 logs after 7

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days storage. Higueras, et al.

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casting, and demonstrated that the LAE incorporated chitosan films showed more

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evidenced antimicrobial activity against various types of microorganisms, compared

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to the pure chitosan film.

19

17

15

. Theinsathid,

encapsulated LAE into the

incorporated LAE into the chitosan films by solvent

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In this work, the composite chitosan/PEO/LAE nanofibrous films were

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fabricated by electrospinning. Multiply characterizations were conducted including 4


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scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR),

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differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), X-Ray

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diffraction (XRD), and water contact angle. The antimicrobial activities were studied

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by disc diffusion method against Escherichia coli and Staphylococcus aureus and

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fluorescent staining observations were conducted to explore the antimicrobial

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mechanism.

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MATERIALS AND METHODS

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Chemicals

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Chitosan (Mw ~ 150 - 300 kDa, deacetylation degree ≥ 95%) and polyethylene

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oxide (PEO) (Mw ~ 900 kDa) were purchased from Aladdin, Inc. (Shanghai, China).

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Ethyl-Nα-lauroyl-L-arginate (LAE) was provided by Beijing HWRK Chemical Co.,

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LTD. Propidium iodide (PI) and 4’, 6-diamidino 2-phenylindole (DAPI) were

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purchased from Dojindo (Osaka, Japan). All the reagents were analytical grade and

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used as received.

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Solution preparation

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5% (w/v) Chitosan solutions without or with 0.1%, 0.25%, 0.5%, 0.75%, and 1%

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(w/v) PEO were prepared in 70% (v/v) acetic aqueous solution. Solutions with

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various LAE concentrations were prepared by dissolving 5% (w/v) chitosan, 0.5%

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(w/v) PEO without or with 0.1%, 0.25%, and 0.5% (w/v) LAE in 70% (v/v) acetic

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aqueous solution. Each solution was stirred over 12 h to ensure fully hydration of the 5



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polymers.

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Before electrospinning, the shear viscosity at 100 s-1, solution conductivity,

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surface tension, and fluorescent intensity of the chitosan/PEO/LAE solutions were

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measured according to our previous methods

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conducted using an Anton Parr MCR 302 rheometer with a cone-plate geometry

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(angle 1°, diameter 50 mm). The conductivity was measured by a DDS-307

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conductivity meter (Shanghai Precision & Scientific Instrument Co., Ltd, China). The

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surface tension was measured by the pendant drop method using a tensiometer

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(OCA20, Dataphysics Instruments, Germany). The fluorescence intensity at λ ex =

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313 nm was recorded by using Shimadzu RF-5301PC spectrofluorophotometer

113

(Shimadzu, Japan).

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Electrospinning

20

. The viscosity measurements were

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The electrospinning equipment consisted of a syringe pump (LSP02-1B, Baoding

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Longer Precision Pump Co., Ltd., China), a high voltage supplier (Gamma High

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Voltage, USA), and a grounded drum collector. Each solution was loaded in a 5 mL

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syringe and injected to the grounded collector through a 20 gauge nozzle. A positive

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voltage of 15 kV was set. The spinning solution was pumped at a flow rate of 1.0

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mL/h and the tip-collector distance was kept at 10 cm. During electrospinning, the

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temperature and relative humidity were kept at 25 °C and 50% RH, respectively.

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Nanofiber characterization

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Morphology

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A field emission scanning electron microscopy (SU8010, Hitachi, Japan) was

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used to observe the nanofiber morphology. The diameter distribution of fibers was

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analyzed by measuring 40 fibers in each SEM image using Nano Measure Software.

127

The autofluorescence of the nanofibrous films was detected with a Zeiss LSM 21

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700 confocal microscope (Carl Zeiss, Germany) as reported previously

. A 63x oil

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immersion objective was chosen, and images were analyzed with ZEN 2012 software.

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Thermal analysis

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The thermal properties of the nanofibrous films were analyzed using DSC (TA

132

Instruments Q200, U.S.A.) under nitrogen atmosphere (50 mL/min). The nanofibrous

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films were weighed into 40 µL aluminum pans. The heating were performed from -40

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to 200 °C with a heating rate of 10 °C/min.

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The thermal gravimetric analysis was carried out in a TGA Q500 instrument (TA

136

Instruments, Newcastle, USA) under nitrogen at a flow rate of 50 mL/min with a

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heating rate of 10 °C/min from 50 to 600 °C. The derivative thermograms were

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calculated using TA universal analysis software.

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FTIR analysis

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The Fourier transform infrared (FTIR) absorption spectra were recorded in the

7



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wavenumber range of 400 - 4000 cm-1 on a Nicolet 170-SX instrument (Thermo

142

Nicolet Ltd., USA) with the Attenuated Total Reflection (ATR) mode.

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X-Ray Diffraction

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The X-ray diffraction (XRD) analysis was conducted using the X’Pert Pro

145

diffractometer (PA Nalytical B.V., The Netherlands). A diffraction range of 2θ from 10°

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- 60° was selected.

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Water contact angle

148

The water contact angle measurements were conducted on a tensiometer

149

(OCA20, Dataphysics Instruments, Germany). A volume of 3 µL distilled water was

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deposited on the nanofibrous film, and then video recorded. The contact angles were

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obtained from the images at the time of 0 s and 3 s, respectively. The measurements

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were conducted five times for each sample at different locations on the surface.

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Antimicrobial activity

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The gram-negative Escherichia coli (ATCC 25922) bacteria and the

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gram-positive Staphylococcus aureus (CMCC 26003) were used to study the

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antimicrobial efficacy of the chitosan/PEO/LAE nanofibrous films using the disc

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diffusion method

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spread on the solidified nutrient agar plates. The films were punched into 5 mm

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diameter disks with thickness of 0.05 mm, and placed on the inoculated agar. The

20

. Briefly, the bacterial suspension (100 µL of 106 CFU/mL) was

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diameters of the inhibition zones were recorded in millimeters after 24 h incubation at

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37 °C.

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The inhibition mechanism of the chitosan/PEO/LAE nanofibrous films were

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studied by exposing the 5 mm diameter disks with thickness of 0.05 mm into the

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bacterial suspensions (1 mL of 1010 CFU/mL) for 1 h. The membrane

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permeabilization of S. aureus and E. coli after exposure to the chitosan/PEO/LAE

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nanofibrous films was determined using steady-state fluorescence. The bacterial cells

167

were stained with 4’, 6-diamidino 2-phenylindole (DAPI) (10 µg/mL) and propidium

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iodide (10 µg/mL) for 10 min in the dark. The images of the stained bacterial were

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obtained by a Zeiss LSM 700 confocal microscope (under a 63 x oil lens).

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RESULTS AND DISCUSSION

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Solution properties

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The solution properties including conductivity, surface tension, and viscosity of

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the chitosan/PEO/LAE solutions were summarized in Table 1. It was obvious that the

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viscosity of the chitosan/PEO in acetic aqueous solutions did not change after the

175

addition of LAE. Due to the protonation of the amine groups on chitosan, the

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viscosity of the chitosan in acetic acid solution was affected by the repulsive forces

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between the protonated -NH3+ groups. It was reported that once the acetic acid

178

concentration was higher than 50 wt%, the viscosity of chitosan solution remained

179

constant due to the fully protonation of the amine groups

180

conductivity with the addition of LAE was probably due to the ionic dissociation of 9


22

. The slight increase in


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the LAE molecules. Moreover, the surface tension of the chitosan/PEO solution was

182

not affected by the LAE addition.

183

From Figure 1, the fluorescent intensity of the chitosan/PEO solution increased

184

with the increasing addition of LAE. It is known that the autofluorescence of

185

polymers is attributed to the π-π∗ or n−π∗ transitions of the unsaturated bonds. The

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C=O bond from acetyl groups can exhibit the n−π∗ transition, which is blue shifted by

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the formation of hydrogen bonds 23. Therefore, the OH and NH2 auxochrome groups

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from LAE could favor the autofluorescence of chitosan by hydrogen bonding

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Chitosan in acetic acid carries strongly positive charge, and is expected to interact

190

with ionic surfactants electrostatically. The LAE micelles with positive charge could

191

repel the chitosan molecules, leading to the increased mobility of the chitosan

192

molecules and the increased vibration of the double bonds, as reflected by the

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increased fluorescent intensity of the solution. Dibbern-Brunelli, et al. 26 reported that

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the fluorescent intensity of fluorescein dye could be altered by hydrogen bonds

195

formed between the fluorescein and poly (vinyl alcohol), resulting in mobility change

196

of the polymer molecules.

197

Nanofiber morphologies

24, 25

.

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Figure 2a showed the morphology of the chitosan/PEO nanofibers at various

199

weight ratios. Clearly, thin nanofibers with beads were fabricated from the pure

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chitosan in 70% acetic aqueous solution. With the addition of 0.25 wt% PEO, smooth

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nanofibers were observed. The PEO molecules may produce “links” between chitosan 10


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molecules through the formation of hydrogen bonds to increase the chain

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entanglements in solution, leading to higher electrospinnability

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of PEO from 0.25 to 1.0 wt%, the average diameter of nanofibers increased from 114

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nm to 170 nm. The similar trend was observed by Pakravan, Heuzey and Ajji 22, who

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found that the decrease in the chitosan/PEO weight ratio from 90/10 to 50/50 caused a

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significant fiber diameter increase from 63 to 123 nm. As shown in Figure 2b, the

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incorporation of LAE did not affect the morphology of the nanofibers with an average

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diameter of approximately 150 nm (Table 1) from the solution composed of 5%

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chitosan and 0.5% PEO. The CLSM images confirmed that the autofluorescence of

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the nanofibers was attributed to the chitosan molecules. It was reported that solid

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chitosan showed weak fluorescence, which has been applied to monitor lipid

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oxidation in food sample 27, 28.

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FTIR analysis

22

. With the addition

215

Figure 3a showed the FTIR spectrum of the chitosan/PEO/LAE nanofibrous

216

films in comparison with chitosan, PEO, and LAE powders. In the FTIR spectrum of

217

chitosan, a peak observed at 1649 cm-1 was related to the vibration of C=O, while the

218

peak at 1597 cm-1 was attributed to the bending of N-H. The band at 1380 cm-1 was

219

ascribed to CH2 deformation, and the absorption at 1077 cm-1 referred to the

220

stretching of C-O-C bonds 9. The absorption band of PEO at 2885 cm-1 was related to

221

the CH2 stretching vibration, which overlapped with chitosan at this region. Since

222

PEO with a relatively high molecular weight (900 kDa) was used, the OH absorption 11



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bands resulted from PEO could be negligible in the FTIR spectra of the nanofibrous

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film 29.

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The FTIR spectra chitosan/PEO/LAE nanofibrous films showed six distinct

226

peaks in Figure 3b. The broad band between 3500 - 3100 cm-1 resulted from the O-H

227

and N-H stretching, which could be affected by the hydrogen bonding interactions

228

among chitosan, PEO, and LAE 30. The weak bands at 2877 cm-1 and 2922 cm-1 were

229

related to the C-H stretching. The peak centered at 1634 cm-1 was attributed to the

230

C=O bond, and the peak at 1557 cm-1 was associated with the bending of N-H.

231

However, the peak around 1597 cm-1 of the NH2 group from chitosan was not shown,

232

which might be due to the NH3+ formation, as suggested by Nista, et al.

233

absorption peak observed at 1409 cm-1 indicated the stretching of C-N, and the peak

234

at 1084 cm-1 was related to the vibration stretching of the ether (C-O-C) group. It was

235

obvious that the intensity of the peak centered at 1634 cm-1 increased with the

236

increasing concentration of LAE, which might be ascribed to the hydrogen bonds

237

formed between LAE and chitosan. The increased intensity of 1634 cm-1 could

238

confirm that the autofluorescence of the chitosan/PEO/LAE nanofibrous films was

239

derived from the n−π∗ transitions of the C=O bonds 28.

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XRD analysis

31

. The

241

The nanofibrous films showed a narrow peak 16.9° (2θ) and a broad peak at

242

22.7°(2θ) (Figure 4), corresponding to the inter-planar spacing of 5.23 Å and 3.96 Å,

243

respectively. It could be seen that the diffraction intensity of the main crystalline plane 12


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of the chitosan/PEO nanofibrous films after the introduction of LAE was gradually

245

increased, indicating the increased crystallinity. It was reported that pure PEO has

246

very strong reflections at 19° and 23°

247

assigned to the crystal structures in PEO and chitosan, respectively. However, the

248

addition of LAE affected the crystallization of chitosan and PEO, as the amino groups

249

from LAE molecules could form hydrogen bonds with PEO, while the carboxyl

250

groups from LAE might form hydrogen bonds with the amino groups of chitosan as

251

well. This interference of LAE changed the interactions between amino groups from

252

chitosan and ether groups from PEO, leading to a higher mobility of polymers during

253

electrospinning, which produced nanofibers with higher crystallinity.

254

Thermal analysis

32

. The peaks of 22.7° and 16.9° could be

255

Figure 5 showed the DSC and TGA curves of the chitosan/PEO/LAE

256

nanofibrous films, and the thermal data of DSC and TGA were shown in Table 2. The

257

DSC curves of the films exhibited three endothermic peaks. The first peak

258

corresponding to the melting of PEO 31, increased from 43.48 °C to 49.20 °C with the

259

increasing concentration of LAE. It has been reported that the increase in crystallinity

260

will induced a higher value of Tm

261

molecules such as inter-molecular hydrogen bonding may retard the mobility of the

262

PEO molecules, and thus result in the increase in the Tm1 value. The second peak

263

around 100 °C is normally due to the evaporation of water. Due to the hydrogen

264

bonding interaction of polysaccharides, the majority of them including chitosan don’t

33

. In addition, the strong interactions among

13



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have a specific melting process but a degradation process during heating 34. The third

266

endothermic peaks around 150 °C were associated to the degradation of chitosan.

267

With the increasing concentration of LAE, the melting point of Tm2 decreased, and the

268

entropy of the peak decreased accordingly, indicating less hydrogen bonds formed

269

after the introduction of LAE in the nanofibers. It should be also mentioned that the

270

melting of LAE was not observed, suggesting that LAE has been homogeneous

271

incorporated within the nanofibers. The TGA thermograms of the nanofibrous films showed three regions of weight

272

35

273

loss (Figure 5b). The first step is due to loss of adsorbed water on the film

. The

274

second region is related to the degradation temperature of chitosan at around 300 °C

275

36

276

Nista, Bettini and Mei

277

curve corresponding to the maximum weight loss rate is 288.88 °C and 284.2 °C for

278

the chitosan/PEO and chitosan/PEO with 5% LAE nanofibrous films, respectively.

279

The decreased thermal stability of chitosan might be attributed to the lower chain

280

entanglement of chitosan molecules due to the interactions between chitosan and LAE.

281

In contrast, the temperature of peak 3 (Table 2) was increased by the LAE addition.

282

The improved thermal stability of PEO might be ascribed to the high chain

283

compactness of PEO due to the interactions among the polymer molecules and LAE

284

37

. The third region around 380 °C is related to the degradation of PEO, as reported by 31

. The peak 2 temperatures (Table 2) of the TGA derivative

.

14


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Surface wetting

286

The water contact angles of the chitosan/PEO/LAE nanofibrous films at 0 s and

287

3 s were shown in Figure 6, respectively. The water drop could not maintain a certain

288

shape until it was fully absorbed by the nanofibrous films. The chitosan/PEO film

289

gave water contact angles of 52.6 o at 0 s and 39.1o at 3 s, respectively. In contrast, the

290

water drop deformed very quickly on the LAE incorporated films, which had smaller

291

water contact angles. Interestingly, the rate by which the water drop was absorbed into

292

the nanofibrous films was significantly increased by the addition of LAE. Stephansen,

293

et al.

294

increase the hydrophilicity of the fish sarcroplasmic protein electrospun fibers. The

295

dramatically decreased water contact angle of the PCL (polycaprolactone)

296

nanofibrous film by introduction of various surfactants was also reported by Hu, et al.

297

39

298

interactions between LAE and chitosan might facilitate the increased hydrophilicity of

299

the nanofibers, due to the presence of LAE on the fiber surface, contributing to the

300

surface wettability. Similar results have been observed by Muriel-Galet, et al. 40, who

301

fabricated an antimicrobial ethylene-vinyl alcohol copolymer/LAE casting film, and

302

found that the water contact angle was deceased by the addition of LAE. It has been

303

reported that hydrophilic surfaces tend to have lower tendency of fouling than

304

hydrophobic surfaces

305

decrease the bacteria attachment

306

nanofibrous films desirable to create an effective anti-infection coating for food

38

found that Triton X-100 and benzalkonium chloride could significantly

. The decreased water contact angle caused by the LAE addition indicated that the

41

. Some studies showed that the surface hydrophilicity could 42, 43

, which might make the chitosan/PEO/LAE

15



307

products.

308

Antimicrobial activity

309

The antimicrobial activities of the chitosan/PEO/LAE composite nanofibrous

310

films were studied against Gram-negative E. coli and Gram-positive S. aureus by disc

311

diffusion method. Figure 7a showed the good antimicrobial activity of the films

312

against E. coli, and the inhibition zone increased from 13.6 mm to 20.7 mm for

313

chitosan/PEO nanofibrous films without and with 0.5% LAE, respectively. As shown

314

in Figure 7b, the nanofibrous films showed larger inhibition zones against S. aureus

315

compared to those against E. coli. It has been reported that the cell wall

316

lipopolysaccharides in Gram-negative bacteria have barrier effect against the

317

antimicrobial compounds 44. The inhibition zone against S. aureus increased gradually

318

from 15.1 mm to 29.2 mm with the increasing concentration of LAE from 0 to 0.5%.

319

To further prove the antimicrobial mechanism of the chitosan/PEO/LAE

320

nanofibrous films, CLSM was used to observe the physiological status of bacteria.

321

The bacteria after 1 h exposure to the films were fluorescent stained and observed by

322

CLSM (Figure 8). The bacteria with disrupted bacterial membrane were stained by PI

323

with red fluorescence, while the live bacteria were stained by DAPI with blue

324

fluorescence

325

chitosan/PEO film exhibited few amounts of the PI labeled bacteria, indicating that a

326

large proportion of the bacteria were alive. However, a significantly higher proportion

327

of S. aureus or E. coli labeled with PI was observed after treatment by the films

45

. The CLSM images of the S. aureus or E. coli treated by the

16


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containing LAE. These results demonstrated that the antimicrobial mechanism of the

329

chitosan/PEO/LAE nanofibrous films was cell membrane damage. Moreover, the E.

330

coli bacterial aggregates were observed after treatment by the chitosan/PEO/LAE

331

nanofibrous films. It was reported that the possible antimicrobial mechanism of

332

chitosan against the gram-negative E. coli was that the electropositive chitosan could

333

adsorb the electronegative substances on the cell and then flocculate them 46.

334

In summary, we successfully fabricated the chitosan/PEO/LAE nanofibrous films

335

with ultrafine 3D porous structures. The formation of hydrogen bonds and

336

electrostatic interactions induced by the LAE addition changed the inter- and

337

intramolecular interactions between PEO and chitosan in nanofibers, leading to the

338

increased crystallinity and hydrophilicity of the nanofibrous films. As well, the

339

antimicrobial activities of the films against S. aureus and E. coli were significantly

340

enhanced after the incorporation of LAE. This work suggested that the

341

chitosan/PEO/LAE nanofibrous films have potential applications in the field of

342

antimicrobial food packaging.

343

ACKNOWLEDGEMENT

344

This work was supported by the National Natural Science Foundation of China

345

(Grant No. 31471622), the National Science & Technology Pillar Program of China

346

(Grant No. 2015BAD16B03), Jinhua Science and Technology Projects of Zhejiang

347

Province (Grant No. 2017-2-017), and Zhejiang Provincial Public Welfare

348

Technology Research Program of China (Grant No. 2017C32078).

349

CONFLICT OF INTEREST 17



The authors declare no competing financial interest.

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REFERENCES

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heat-management structures for smart food packaging. Food Hydrocoll. 2013, 30,

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182-191.

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24


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495

Figure captions

496

Figure 1 The fluorescent intensity of the 5% chitosan/0.5% PEO solutions without or

497

with 0.1%, 0.25%, and 0.5% LAE.

498 499

Figure 2 (a) SEM images of the nanofibrous films of 5% chitosan without or with

500

0.1%, 0.25%, 0.5%, 0.75%, and 1% PEO; (b) SEM and CLSM images of the 5%

501

chitosan/0.5% PEO nanofibrous films without or with 0.1%, 0.25%, and 0.5% LAE.

502 503

Figure 3 FTIR spectra of (a) the chitosan, PEO, and LAE powders and (b) the

504

chitosan/PEO nanofibrous films without or with addition of 0.1%, 0.25%, and 0.5%

505

LAE.

506 507

Figure 4 X-ray diffraction patterns of the chitosan/PEO nanofibrous films without or

508

with 0.1%, 0.25%, and 0.5% LAE.

509 510

Figure 5 (a) DSC curves and (b) TGA and TGA derivative curves of the

511

chitosan/PEO nanofibrous films without or with 0.1%, 0.25%, and 0.5% LAE.

512 513

Figure 6 Water contact angles of the chitosan/PEO nanofibrous films without or with

514

0.1%, 0.25%, and 0.5% LAE at 0 s and 3 s, respectively.

515 516

Figure 7 Inhibition zones of the chitosan/PEO nanofibrous films without or with 25



517

0.1%, 0.25%, and 0.5% LAE against (a) E. coli and (b) S. aureus.

518 519

Figure 8 CLSM images of the DAPI/PI stained E. coli and S. aureus after 1 h

520

exposure to the chitosan/PEO nanofibrous films without or with 0.1%, 0.25%, and 0.5%

521

LAE.

522

26


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Page 27 of 37


523

Figures

524

Figure 1

525

526 527

27



528

Figure 2

529 530

(a)

531 532 533

(b)

28


Page 28 of 37

Page 29 of 37


534

Figure 3

535 536

(a)

537 538 539

(b)

29



540

Figure 4

541 542

30


Page 30 of 37

Page 31 of 37


543

544 545

546 547 548

Figure 5

(a)

(b)

31



549

Figure 6

550 551

32


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Page 33 of 37


552

Figure 7

553 554

(a)

555 556

(b)

557

33



558

Figure 8

559 560

34


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Page 35 of 37


561

Tables

562

Table 1. Conductivity, viscosity, surface tension of 5% chitosan and 0.5% PEO in 70%

563

acetic acid added without or with 0%, 0.1%, 0.25%, 0.5% LAE, and the average fiber

564

diameter of the corresponding nanofibers. Sample

Conductivity

Viscosity

Surface tension

Average fiber

(ms/cm)

(mPa·s)

(mN/m)

diameter (nm)

2.15 ± 0.01

4.71 ± 0.21

33.32 ± 0.06

140.9 ± 24.8

0.1% LAE

2.25 ± 0.02

4.65 ± 0.21

33.46 ± 0.21

157.6 ± 36.4

0.25% LAE

2.28 ± 0.01

4.73 ± 0.23

32.84 ± 0.26

146.8 ± 26.8

0.5% LAE

2.26 ± 0.01

4.54 ± 0.18

32.82 ± 0.09

152.4 ± 41.6

Without LAE

565

35



566

Page 36 of 37

Table 2. DSC and TGA data of the chitosan/PEO nanofibrous films without or with 0.1%, 0.25%, and 0.5% LAE. DSC

TGA

Tm1 (°C)

Tm2 (°C)

∆Hm2 (J/g)

Tm3 (°C)

Without LAE

43.48

94.23

-217.39

156.86

0.1% LAE

46.47

85.95

-149.46

0.25% LAE

45.64

90.90

0.5% LAE

49.20

86.81

∆Hm3

(J/g)

Peak 1 (°C)

Peak 2 (°C)

Peak 3 (°C)

Residue at 600 °C (%)

-13.10

110.27

288.88

382.35

30.49

155.58

-18.21

117.75

287.16

385.47

33.06

-173.77

157.72

-18.61

104.84

287.35

382.23

32.90

-141.43

149.95

-8.35

107.70

284.20

391.04

33.36

567

36


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568


Graphic abstract

569

37


Lauric Arginate

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