Formation and Stability of CoreâShell Nanofibers by Electrospinning

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Biofuels and Biobased Materials

Formation and stability of core-shell nanofibers by electrospinning of gel-like corn oil-in-water emulsions stabilized by gelatin Cen Zhang, and Hui Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04270 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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Journal of Agricultural and Food Chemistry

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Formation and stability of core-shell nanofibers by electrospinning of

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gel-like corn oil-in-water emulsions stabilized by gelatin

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Cen Zhang a, Hui Zhang a,*

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a

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National Engineering Laboratory of Intelligent Food Technology and Equipment,

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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

11 12 13 14 15 16 17 18 19 20 21

__________________________

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*

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Corresponding author. Tel.: +86-571-88982981; fax: +86-571-88982981. E–mail address: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT: The core-shell nanofibers were fabricated by electrospinning of

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gel-like corn oil emulsions stabilized by gelatin. The oil-in-water (O/W) emulsions

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satisfied the Herschel-Bulkley rheological model and showed shear-thinning and

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predominantly elastic gel behaviours. The increasing oil fractions (φ) ranging from 0

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to 0.6 remarkably increased the apparent viscosity, and then led to an increase in the

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average diameter and encapsulation efficiency of electrospun fibers. The core-shell

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structured fibers by emulsion electrospinning were observed in transmission electron

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microscopy (TEM) images. The encapsulated oil was found to randomly distribute as

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core, especially inside the beads. The binding of corn oil to gelatin was mainly driven

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by the non-covalent forces. These core-shell fibers at various φ values (φ = 0.2, 0.4,

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0.6 and 0.8) showed a high thermal decomposition stability upon heating to 250 °C,

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and the denaturation temperature were 85.32, 77.97, 82.99 and 87.25 °C, respectively.

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The corn oil encapsulated in emulsion-based fiber mats had good storage stability

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during 5 days. These results contributed to a good understanding on emulsion

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electrospinning of food materials for potential applications in bioactive encapsulation,

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enzyme immobilization and active food packaging.

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KEYWORDS: Gelatin; Corn oil; Gel-like emulsions; Emulsion electrospinning;

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Core-shell nanofibers

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44 2


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INTRODUCTION

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Electrospinning, as a simple and promising technique, has been used to fabricate

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micro- and nanofibers from a variety of polymers. During the electrospinning process,

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a polymer jet was charged and ejected in a high-voltage power system. The

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continuous fibers were obtained along with the evaporation of the solvent.1 Various

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structural advantages of the electrospun fibers (e.g., ultrafine fibrous structures, high

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surface area to volume ratio, high porosity, etc.) have good application potentials in

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food, cosmetics, pharmaceutical and biomedical industries.2 Currently, the

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electrospinning technique used to develop a controlled-release delivery are blending,

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coaxial or emulsion electrospinning, etc.,3,4,5 thus resulting in nanofibers with various

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shapes, including uniform, beaded and flat/ribbon fibers.6 Although the application of

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the blending electrospinning technique allowed the fabrication of polymer fibers

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loaded with bioactive compounds (e.g., vitamins, coumarins, and flavonoids), the

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bioactive compounds have to be dissolved or dispersed (if insoluble) in the solution

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prior to electrospinning. Then, the distribution of bioactive compounds inside the

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fibers is highly determined by the physicochemical properties of the solution and the

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interactions between the compounds and solution.7 Additionally, sensitive bioactive

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compounds (e.g., peptide, enzymes, and cytokines) may compromise their bioactivity

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due to the possible denaturation in the solvents. Coaxial and emulsion electrospinning

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are both available to fabricate core-shell structured fibers. Compared to coaxial

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electrospinning, emulsion electrospinning can be easily accomplished using only a

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single nozzle, and there is no need to precisely control the process variables (e.g., 3



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interfacial tension, feed rate, viscoelasticity of the two polymers). Therefore, emulsion

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electrospinning can provide a simpler approach to decrease the burst release of

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bioactive compounds inside the fibers.

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Core-shell nanofibers can be produced directly by electrospinning of either the

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oil-in-water (O/W) or water-in-oil (W/O) emulsions, which help to preserve the

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activity and bioavailability of encapsulated compounds. Cai et al. found that the

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electrospun Van/OA-MIONs-PLA nanofibers made from W/O Pickering emulsions

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possessed desirable mechanical and antibacterial properties.8 On the other hand, the

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nanofibers by electrospinning of O/W emulsions have been also developed as a

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delivery vehicle of hydrophobic compounds. Shin and Lee loaded phytoncide into an

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O/W emulsions stabilized by Tween 80, and then fabricated phytoncide/poly(vinyl

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alcohol) nanofibers by emulsion electrospinning. The obtained nanofibers had a

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core-shell structure and showed a sustained manner over 14 days as well as strong

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antimicrobial effects.9 To date, Tween 20 and Brij O10 have been small molecular

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surfactants commonly used in emulsion electrospinning.10,11 However, the toxicity

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and biodegradation of the surfactants can not be neglected, and the side bulging and

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rough morphology of nanofibers may be caused by the migration of some of the

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surfactants from the oil-water interface to the nanofiber surface during the

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electrospinning process.12

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Although many studies have focused on the fabrication of nanofibers by

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electrospinning of O/W emulsions, no report is available on gelatin used as

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emulsifying agents in emulsion electrospinning, in which gelatin could enhance the 4


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viscosity and stabilize O/W emulsions, independently of the surfactants used. As a

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safe and economic material, gelatin has been widely used to enhance the stability,

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elasticity and consistency of food products.13 Gelatin is also employed as emulsifiers

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in the stabilization of O/W emulsions because it has amphiphilic characteristics due to

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its amino acid composition. Additionally, gelatin can form time-dependent gel-like

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stabilizing layers at the surface of emulsion droplets, which may modify rheology

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properties to slow down the creaming process.14 The unique chemo-physical

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properties of gelatin such as emulsifying property, surface tension as well as its

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viscosity and conductivity contributed to the good spinnability.15 Recently, the

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gelatin-based electrospun fibers have drawn much attention in the field of controlled

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release of bioactive compounds due to its non-toxicity, biocompatibility and

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biodegradability. Li et al. incorporated vitamin A and E into the nanofibers by

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electrospinning of gelatin, with the aim of achieving their sustained release.16 Laha et

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al. fabricated the crosslinked gelatin nanofibers as a carrier for hydrophobic drug

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piperine,

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controlled-release properties at varying pH conditions.17

and

these

electrospun

fibers

showed

good

compatibility

and

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The aim of the current study was to fabricate core-shell structured fibers by

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electrospinning of corn oil-in-water emulsions stabilized by gelatin, which were

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expected to act as a carrier for hydrophobic compounds. The morphology of the

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core-shell nanofibers was analyzed by scanning electron microscopy (SEM),

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transmission electron microscope (TEM) and confocal laser scanning microscopy

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(CLSM). The thermal behavior and conformational changes of the nanofiber mats 5



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were evaluated using thermogravimetric analysis (TGA), differential scanning

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calorimetry (DSC) and Fourier transform infrared (FTIR). In addition, the rheology

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and microstructure of the emulsions were characterized by rheological measurements

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and optical microscopy.

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MATERIALS AND METHODS

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Materials. Gelatin (Type B, ~250 g Bloom, MW~100 kDa) from porcine skin and

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Nile Red were purchased from Aladdin, Inc. (Shanghai, China). Corn oil was obtained

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from a local supermarket. The other reagents obtained from Sinopharm Chemical

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Reagent Co. (Shanghai, China) were of analytical reagent grade. All the reagents were

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used without further processing, and the ultrapure water was used throughout the

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experiments.

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Preparation of the Oil-in-water (O/W) Emulsions. Initially, aqueous phases were

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prepared by dissolving gelatin (25%, w/v) in 40% (v/v) acetic aqueous solution. Corn

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oil was then added dropwise at the concentrations of 0.2, 0.4, 0.6, and 0.8 (v/v) with

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respect to the gelatin solution during stirring. The mixed solutions were homogenized

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using a high-speed homogenizer (IKA T18 digital ULTRA-TURRAX®, IKA GmbH,

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Germany) at 6,000 rpm for 2 min, and subsequently the emulsions at various oil

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fractions (φ = 0.2, 0.4, 0.6 and 0.8) were obtained by ultrasonic treatment (Model

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KQ-250DE, Ultrasonic Corporation, Kunshan, China) at 250 W for 3 min at room

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temperature (25 ± 5 °C).

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Optical microscopy. Optical microscopy images of the emulsions were recorded 6


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using a Leica ICC50 W optical microscope (Carl Zeiss, German) equipped with a

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built-in camera. An aliquot of sample was deposited on a microscope slide, and then

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photographed using a 40× objective lens and 10× eyepiece.

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Steady Shear Viscosities. Steady shear viscosities of the emulsions at different φ

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values were measured using a rotational rheometer (MCR 302, Anton Paar, Graz,

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Austria) equipped with a circulating water bath at 25 °C. A plate/plate measuring setup

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(PP50) was chosen, and the gap between two plates was set to 1.0 mm. the shear rate

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was continuously ramped from 1 to 100 s−1.

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The flow properties of the emulsions were evaluated using Herschel-Bulkley model: σ = σ0 + K·γn

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where σ and σ0 denote the shear stress and the apparent yield stress, respectively. K is

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the consistency index, which means apparent viscosity of the examined emulsions. γ

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is the shear rate, and n represents the flow behavior index.

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Viscoelastic Properties. Oscillatory frequency sweep experiments were performed

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at 1% strain within the identified linear viscoelastic region to investigate the

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viscoelastic properties of the gel-like emulsions. The frequency was oscillated from 1

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to 100 rad s−1. The elastic modulus (G′) and loss modulus (G″) were recorded using

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RheoCompass software.

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Electrospinning Process. Electrospinning of the emulsions was performed in

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horizontal alignment at an applied voltage of 15 kV and a tip-to-collector distance of

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100 mm. The feed rate of syringe pump (LSP02-1B, Baoding Longer Precision Pump 7



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Co., Ltd., China) loaded with the spinning solution was set at 0.5 mL h−1. The

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electrospun nanofibers were collected on a grounded cylindrical aluminium foil. The

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temperature and humidity throughout the electrospinning were kept at 25 °C and

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around 50%, respectively.

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Scanning Electron Microscopy (SEM). SEM (SU8010, Hitachi, Japan) was used

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to observe the morphology of the electrospun fibers at an acceleration voltage of 3 kV.

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The fiber diameters in the SEM images were measured using Nano Measure software

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by randomly selecting 200 data points for each image.

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Transmission Electron Microscope (TEM). TEM (JEM-1200EX, Jeol Ltd, Tokyo,

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Japan) was applied to confirm the core-shell structure of nanofibers, which were

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spread onto copper grid and observed at an accelerating voltage of 120 kV.

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Confocal Laser Scanning Microscopy (CLSM). To confirm the distribution of

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corn oil within the nanofibers, the electrospun fibers stained with Nile Red (0.01%

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w/w solution) were directly collected on microscope glass slides. The analysis was

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operated on CLSM (LSM780, Carl Zeiss Microscopy, Jena, Germany) using an EC

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Plan-Neofluar 40×/1.30 oil immersion objective at an excitation wavelength of 488

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nm.

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FTIR Spectroscopy. A FTIR spectrometer (Nicolet 170-SX, Thermo Nicolet Ltd.,

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USA) was used to record the infrared spectra of the nanofiber mats over the

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wavenumber range of 400 - 4000 cm−1 with a resolution of 4 cm−1 and 32 scans.

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Thermogravimetric Analysis. A TA-Q500 thermal gravimetric analyzer (TGA)

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was applied to investigate the thermal degradation behavior of the nanofiber mats. 8


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Samples (ca. 8 mg) were heated from 50 to 600 °C at a heating rate of 10 °C min−1

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under nitrogen atmosphere.

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DSC Measurements. The thermal properties of the electrospun fibers were

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evaluated on DSC (DSC-1, Mettler-Toledo corp., Switzerland) under a nitrogen

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atmosphere with a flow rate of 50 mL min−1. Samples were accurately weighed into

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40 mL aluminum pans. Thermal scans were conducted in a temperature range from 25

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to 250 °C with a scan rate of 10 °C min−1.

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Measurements of Encapsulation Efficiency and Storage Stability. The

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encapsulation efficiency of the electrospun mats were determined according to the

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method reported previously with modifications.18 Corn oil was dissolved in

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dichloromethane to prepare a series of standard solutions at different known

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concentrations, and were scanned in the wavelength range of 200 - 600 nm

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spectrophotometrically (SP-752 UV-visible spectrophotometer, Shanghai Spectrum

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Instruments Co. Ltd., China). The maximum absorbance was determined at 290 nm,

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and a standard curve of corn oil was obtained using a linear regression model. Then,

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10 mg of each fiber sample was added to 10 mL dichloromethane under stirring (100

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rpm) for 15 min. Subsequently, the suspensions were filtered through nylon syringe

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filter with a 0.45 μm pore size. The absorbance of resulting solutions at 290 nm was

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recorded. The blanks were the electrospun gelatin fibers without corn oil. The

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concentration of corn oil on the fiber surface was calculated using the standard curve

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(R2 = 0.9992). To investigate the storage stability of the encapsulated corn oil, the

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electrospun mats were kept in open culture dishes at relative humidity ranging from 9



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30 to 40%, and the change in the absorbance at 290 nm was recorded at 2 days

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interval. The encapsulation efficiency was determined by the following equation:

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Encapsulation efficiency (%) = (Wi − Ws)/ Wi × 100

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where Wi and Ws denote the initial weight of corn oil added for encapsulation and the

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weight of corn oil on fiber surface calculated according to the standard curve,

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respectively.

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Statistical Analysis. All the measurements were carried out in triplicate. Data were

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analyzed using Origin 8.0 and expressed as mean ± standard deviation (n = 3).

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One-way ANOVA was performed to determine the statistically significance at p
0.99) (Table 1). According to the

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parameters obtained by Herschel-Bulkley model, the non-linear relationship between

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shear stress and shear rate of the gelatin-stabilized O/W emulsions at various oil

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fractions demonstrated the shear-thinning behavior of the emulsions as a

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non-Newtonian fluid, which may be caused by breaking the network of entangled

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polymers during shearing. Then, the speed of disrupting intermolecular entanglement

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was faster than that of reformation, leading to lower apparent viscosity and less

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intermolecular resistance to flow.23 The σ0 was an indicator of the resistance of the

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emulsion droplets and network to gravitational separation. As the oil fractions were

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increased, the σ0 values of the emulsions were increased, and the low σ0 value of the

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emulsions with low oil fractions may be inclined to separate as described by Torres et

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al..24 Additionally, the behavior of high viscosity was strongly influenced by the high

238

stabilizer concentration or dispersed phase volume in the examined systems. The

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increase in oil fractions of O/W emulsions led to the increased value of K and then the

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changes in the viscosity of continuous phase. The n value reflected the degree of 11



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non-Newtonian behavior. All the n values were less than 1 (Table 1), indicating the

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pseudoplastic fluid characteristics of the O/W emulsions (Figure 1B).24 Moreover, the

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increase in φ values of the O/W emulsions resulted in the decreased n value,

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indicating that the deviation from Newtonian behavior was increased.25

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Viscoelastic Properties of O/W Emulsions. The viscoelastic property of the corn

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oil emulsions stabilized by gelatin was determined by dynamic oscillation

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measurements. The emulsions at various φ values showed that G′ modulus was

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predominantly higher than G″ modulus at a given frequency over the range of 1 - 100

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rad s−1, indicating the presence of a gel-like structure in the O/W emulsion system.26

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The significant increase was observed regarding G′ modulus in emulsions with φ

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values increasing from 0.2 to 0.6. This may be attributed to the hydrocolloid-induced

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formation of an entanglement network between adsorbed and non-adsorbed proteins,

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and the oil droplets stabilized by proteins served as ‘active fillers’ in the gel network

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of the emulsions.27 On the other hand, both the tested modulus of these emulsions

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slightly but progressively increased with the increasing frequency (Figure 1C). A

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similar trend regarding the rise of G′ and G″ modulus with the increasing frequency

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has been also observed for the gel-like emulsions stabilized by texturised whey

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protein concentrate, which was ascribed to a predominantly solid behavior of the

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gel-like emulsions with long term stability.20

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Droplet Size and Microstructure of the Emulsions. The extensive dilution and

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ultrasonic treatment were necessary for the determination of average droplet size in

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the concentrated emulsions using a light-scattering instrument, which may not ensure 12


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that a sample examined actually properly represented the gel-like emulsions. Then a

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light microscope was available to assess the presence and aggregation extent of oil

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droplets in the emulsions as a straightforward and reliable way.

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As the φ value was increased from 0.2 to 0.6, the diameter of the oil droplets

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progressively increased. The average diameter of the oil droplets at φ = 0.2 was 5.82

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μm, and the emulsions consisted of aggregated oil droplets (Figure 2A). The

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inter-droplet attractive interactions may contribute to the formation of the gel-like

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network. Additionally, at the low φ value, the unabsorbed proteins would tend to form

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the aggregates driven by hydrophobic interactions after the emulsification, which may

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be helpful to the formation of the network.28 When the φ value was increased to 0.4,

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the increasing average diameter of the oil droplets might result in a closer packing and

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then a more compact gel network (Figure 2B). Chivero, Gohtani, Yoshii, and

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Nakamura also reported that an improved emulsion stability at higher oil fractions

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may be associated with a strong network formed among the close droplets. The oil

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droplet size became larger and came up to 14.67 μm at φ = 0.6 (Figure 2C), indicating

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the stronger interactions between protein-stabilized oil droplets and much less

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unabsorbed proteins present in the system.21,29 However, when the φ value rose up to

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0.8, some larger oil droplets were observed in the emulsions (Figure 2D). Since the φ

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value was increased, the volume of aqueous phase was relatively decreased. Then, the

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amount of gelatin emulsifier might be not sufficient to cover the O/W interface,

283

resulting in the formation of larger droplets.22 The changes of the oil droplets with the

284

increasing φ values were well in accordance with the observed flow behaviors under 13



285

precisely controlled conditions. Liu and Tang also found that the droplet size of

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Pickering emulsions basically increased along with the increasing oil volume fractions,

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even coarser emulsions were formed at higher oil fractions.30

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Morphology and Microstructure of Electrospun Nanofibers. The uniform and

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bead-free nanofibers could be obtained by electrospinning of the pure gelatin with an

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average diameter of 148.9 nm (Figure 3A). For the gelatin-stabilized emulsions, the

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increasing φ values from 0.2 to 0.8 appreciably affected the formation of the

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electrospun fibers. As shown in Figure 3B, the O/W emulsions yielded fibers with the

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average diameter of 198.4 nm at φ = 0.2, and a great number of beads were

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interspersed along the strings. It could be thus inferred that the oil droplets tended to

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accumulate in the center of the liquid along the direction of fluid due to the elongation

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effect in the expanding and bending process of fluid jets during electrospinning,

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which might contribute to the encapsulation of beads into fibers rather than on

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surfaces.31 When the φ value was increased to 0.4, the electrospun nanofibers had

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larger diameters, indicating that more corn oil was encapsulated in the fibers (Figure

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3C). Similarly, Gordon et al. reported that the increasing isohexadecane (oil phase)

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from 5.8 to 15% (w/w) resulted in the increasing average diameter of PVA fibers from

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246 to 268 nm.32 Although it was expected that the fibers produced from emulsions

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with higher φ values (e.g., at φ = 0.6) had a further increase in diameters (Figure 3D),

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the fibers tended to be merged together and lose the fiber morphology to some extent.

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At φ = 0.8, the nanofibers adhered to one another, and the fibrous structure could be

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hardly observed in SEM images (Figure 3E). These morphological changes may be 14


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attributed to the balance of the oil volume fraction and the viscosity of emulsions as a

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result of the amount of corn oil entrapped and the aggregation extent of oil droplets in

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the emulsions.33

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As shown in Figure 4A, TEM observations showed that the nanofibers by

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electrospinning of the gel-like emulsion at φ = 0.2 possessed an obvious core-shell

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structure. The encapsulated oil was not just located inside the beads as core, but a

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portion of corn oil was randomly distributed in the gelatin matrix. By taking a closer

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look at the core-shell structure of one single fiber (Figure 4B), the distinct boundaries

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between the inner dark and outer light region was found in TEM images, indicating

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the different transmissibility of electron beam through the center and side regions. It

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was evidence that corn oil was wrapped into the center of the bead of the shell

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material.34 Clearly, the outer diameter was about 400 nm while the inner diameter was

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around 290 nm. The W/O emulsion electrospinning was reported by Xu et al.,35 who

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suggested that the viscosity difference between the PEG-PLA/chloroform matrix and

321

the PEO-FITC/water droplets led to the inward movement of the emulsion droplets

322

and their mergence to some extent. In this study, the rapid evaporation of the acetic

323

aqueous solution led to the increasing viscosity of the outer layer (water phase), more

324

rapidly than that of the inner layer. Thus, the inward movement of emulsion droplets

325

from the surface to the center was caused by the viscosity gradient from the outer

326

layer to the inner layer, and the droplets were simultaneously condensed and stretched

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into elliptical shapes in the direction of the fibrous trajectory under the force of high

328

voltage electric field. Finally, the core-shell nanofibers were obtained on the grounded 15



329

collector.

330

To identify whether the corn oil was successfully encapsulated in the nanofibers,

331

the distribution of corn oil within the nanofibers was observed by CLSM. The

332

fluorescence emitted only from the fibers was clearly observed in CLSM images,

333

while no fluorescent intensity was detected in the space between the ﬁbers (Figure

334

4C). The corn oil stained with Nile Red (yellow core) were coated by gelatin (outer

335

dark layer), indicating the successful fabrication of core-shell fibers with corn oil in

336

the core. Dai, Niu, Liu, Yin, and Xu successfully encapsulated laccase into

337

PDLLA/F108 nanofibers by electrospinning of W/O emulsions, and identified that

338

laccase was located in the core of the core-shell fibers using CLSM.36 Yang, Li, Qi,

339

Zhou, and Weng showed the presence of the labeled lysozyme inside the composite

340

fibers, and confirmed that the emulsion-based electrospun fibers had a core-shell

341

structure under CLSM.34

342

Thermal Analysis. The thermal stability changes of the electrospun nanofibers

343

were monitored by TGA analysis. There were two stages involved in the

344

decomposition of the gelatin nanofibers on the TGA curves (Figure 5A). The initial

345

stage of weight loss began at 50 °C and ended up with around 100 °C, which could be

346

described as moisture vaporization of gelatin samples due to its hygroscopic property.

347

The second stage in the range of 250 - 600 °C was the major zone of weight loss,

348

corresponding to the main thermal degradation including protein chain breakage and

349

peptide bond rupture.37 Unlike the gelatin fibers, nanofibers fabricated by

350

electrospinning of the gelatin-stabilized emulsions showed a single stage of thermal 16


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degradation ranging from 250 to 450 °C. Therefore, the fibers at various oil fractions

352

(φ = 0 - 0.8) had a high thermal decomposition stability upon heating to 250 °C.

353

Below the onset decomposition temperature (250 °C), DSC measurements were

354

conducted to investigate the thermal properties of these fibers. The denaturation

355

temperature (TD) and the corresponding denaturation enthalpy (ΔHD) were obtained

356

from the characteristic endothermic peaks (Figure 5B), which were associated

357

with the conformational helix–coil transition of gelatin.38 The TD of the nanofibers by

358

gelatin electrospinning was about 89.67 °C, while the TD of the electrospun fibers

359

made from the gelatin-stabilized O/W emulsions (φ = 0.2, 0.4, 0.6 and 0.8) were 85.32,

360

77.97, 82.99 and 87.25 °C, respectively (Table 2). This moderate decrease in TD might

361

be due to a rearrangement of the triple helix into a random configuration.39

362

FTIR Analysis. The possible interactions between gelatin and corn oil in the

363

electrospun nanofibers were examined by FTIR spectra (Figure 6). Generally, the

364

characteristic bands of proteins at 3291 - 3298 cm−1 belonged to the stretching of N-H

365

and hydrogen bonding for the amide A. The amide I band (1636 - 1643 cm−1) was

366

assigned to the stretching of C=O. The amide II band at about 1541 cm−1 was ascribed

367

to the stretching and bending of C-N. The spectra showed a peak at around 1456 cm−1,

368

which was assigned to N-H bending and C-N stretching combination band as well as

369

NH3+ symmetric deformation. The amide III band between 1241 and 1243 cm−1 was

370

associated with the stretching of C-N and bending of N-H.40 For the corn oil, the peak

371

at around 3008 cm−1 represented C-H stretching vibration of the cis-double bond

372

(=CH). The 2925 and 2854 cm−1 bands were assigned to symmetric and asymmetric 17



373

stretching vibration of the aliphatic CH2 group. The peak at 1745 cm−1 could be

374

explained by the presence of ester carbonyl groups of the triglycerides. The bands at

375

1464 and 1377 cm−1 belonged to bending vibrations of the CH2 and CH3 aliphatic

376

groups as well as CH2 groups. In the fingerprint region, the band at around 1162 cm−1

377

denoted the stretching vibration of the C-O ester groups.41 The characteristic bands of

378

both the gelatin fibers and corn oil were found in all the spectra of the nanofiber mats

379

by emulsion electrospinning (φ = 0.2 - 0.8), and no obvious change in the intensity

380

and position of these peaks was observed, indicating that the binding of corn oil to

381

gelatin may be mainly driven by the non-covalent interaction forces.

382

The amide I band may provide the information of the secondary structures of

383

proteins. The curve-fitting analysis of the amide I region (1700 - 1600 cm−1) was used

384

to quantitatively determine the secondary structural changes of gelatin.42 The spectral

385

bands ranging from 1610 to 1640 cm−1, 1640 to 1650 cm−1, 1650 to 1660 cm−1, 1660

386

to 1680 cm−1 and 1680 to 1692 cm−1 were assigned to β-sheet, random coil, α-helix,

387

β-turn and β-antiparallel, respectively.43 Compared to the nanofibers by gelatin

388

electrospinning, a trend in the decreasing α-helix content and the increasing random

389

coil occurred in the fibers electrospun by the gelatin-stabilized emulsions at various φ

390

values (Table 3), indicating the conformational helix–coil transition of gelatin, in

391

good agreement with our DSC results. The β-sheet structure was predominant at a

392

high percentage in the electrospun fibers, which seemed to be inconsistent with the

393

previous work that the α-helix structure dominated the secondary structure of

394

gelatin.44 The difference may be caused by the acidic solvents used herein to dissolve 18


Page 18 of 39

Page 19 of 39


395

gelatin, which had some adverse effect on the structural properties of protein.45 In

396

addition, the electrospinning process may also have a great impact on the protein

397

conformation. In general, the protein/peptide aggregation mainly involved β-sheet due

398

to a weaker dipole moment of β-sheet than that of α-helix.34 When the φ value was

399

increased to 0.4 or 0.6, the increase in the oil volume fraction led to a significant

400

increase in the β-sheet content, indicating the increased aggregation of gelatin

401

molecules in the electrospun fibers with higher oil fractions. This aggregation at the

402

interface was helpful to the formation of continuous fibers coating corn oil.46

403

However, a lower aggregation of gelatin may occur at φ = 0.8, which may be

404

associated with the insufficient coverage of gelatin molecules on the interface, and

405

then result in the loss of fiber morphology.

406

Encapsulation Efficiency and Storage Stability. The encapsulation efficiency of

407

the emulsion-based electropun fibers was significantly increased with the increasing φ

408

value (Table 4). As the φ value increased to 0.6, the encapsulation efficiency reached

409

up to 86.86%, indicating that the emulsion-based electropun fibers have a great

410

potential to be used as a delivery vehicle. Similar results were reported by

411

Tavassoli-Kafrani, Goli, and Fathi,18 who fabricated the electrospun gelatin and

412

gelatin-crosslinked nanofibers loaded with orange essential oil, respectively. They

413

found an increase in the encapsulation efficiency of both gelatin and

414

gelatin-crosslinked nanofibers with the increasing amount of essential oil. However,

415

the highest encapsulation efficiency of the gelatin and gelatin-crosslinked nanofibers

416

was observed at the ratios of 1:1 and 1:0.74, respectively, probably due to the 19



417

insufficient coverage of gelatin crosslinked by tannic over the surface of oil droplets

418

at a 1:1 ratio during the emulsion formation.

419

As expected, there was no significant change in the encapsulation efficiency at

420

various φ values during 5 days. The encapsulation efficiency of fresh nanofibers (φ =

421

0.2, 0.4, 0.6, and 0.8) were 80.41, 85.85, 86.86, and 87.16%, while it became 79.89,

422

85.28, 87.16, and 87.75% after 5 days, respectively. Especially, the encapsulation

423

efficiency at φ = 0.6 showed no significant difference during 5 days. The results

424

showed the excellent storage stability of the encapsulated corn oil and the good

425

barrier effect of emulsion-based electropun fibers as wall materials. Tavassoli-Kafrani,

426

Goli, and Fathi also reported that the storage stability of orange essential oil

427

encapsulated in the electrospun gelatin fibers was more than 120 h.18

428

In this work, core-shell nanofibers were fabricated by electrospinning of corn

429

oil-in-water emulsions stabilized by gelatin. For the gelatin-stabilized O/W emulsions,

430

gelatin could rapidly diffuse to the newly formed water-oil interface and form the

431

steric protein-based barrier. The higher oil fraction contributed to the higher apparent

432

viscosity and modulus of elasticity, resulting in the gel-like appearance of emulsions.

433

Under high-voltage electric field, the O/W emulsions could be electrospun to form

434

core-shell nanofibers. The average diameter of electrospun fibers was increased from

435

148.9 to 311.4 nm by the increasing oil fraction (φ = 0.2 - 0.6), and the encapsulation

436

efficiency was also increased from 80.41 to 86.86%. Moreover, the nanofiber mats

437

had good storage stability and thermal decomposition stability, and thus were expect

438

to encapsulate thermosensitive or hydrophobic bioactive compounds as a 20


Page 20 of 39

Page 21 of 39


439

controlled-release delivery vehicle.

440

ACKNOWLEDGMENTS

441

We sincerely acknowledge the financial support by the National Natural Science

442

Foundation of China (Grant No. 31772013).

443

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444 445 446

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by oscillatory rheology. Colloid Surf. A-Physicochem. Eng. Asp. 2007, 302(1-3),

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2012, 29(1), 185-192.

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(26) Miao, M.; Huang, C.; Jia, X.; Cui, S. W.; Jiang, B.; Zhang, T.

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Physicochemical characteristics of a high molecular weight bioengineered α-D-glucan

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from Leuconostoc citreum SK24.002. Food Hydrocolloids 2015, 50, 37-43.

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Fabrication of gel-like emulsions and their potential as sustained-release delivery

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systems for β-carotene. Food Hydrocolloids 2016, 56, 434-444.

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emulsion electrospinning: Morphology characterization and preliminary release

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structural integrity of lysozyme encapsulated in core–sheath structured poly

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(DL-lactide) ultrafine fibers prepared by emulsion electrospinning. Eur. J. Pharm.

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Biopharm., 2008, 69(1), 106-116.

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(35) Xu, X.; Zhuang, X.; Chen, X.; Wang, X.; Yang, L.; Jing, X. Preparation of

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core-sheath composite nanofibers by emulsion electrospinning. Macromol. Rapid

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Commun. 2006, 27, 1637-1642.

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Enhancing water repellence and mechanical properties of gelatin films by tannin

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addition. Bioresour. Technol. 2010, 101, 6836-6842.

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(39) Bigi, A.; Cojazzi, G.; Panzavolta, S.; Rubini, K.; Roveri, N. Mechanical and

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thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking.

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Biomaterials 2001, 22, 763-768.

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(40) Muyonga, J. H.; Cole, C. G. B.; Duodu, K. G. Fourier transform infrared

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(FTIR) spectroscopic study of acid soluble collagen and gelatin from skins and bones

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of young and adult Nile perch (Lates niloticus). Food Chem. 2004, 86, 325-332.

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Psaroudaki,

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Chatzilazarou, A.; Tegou, E. Applications of fourier transform-infrared spectroscopy

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to edible oils. Anal. Chim. Acta, 2006, 573, 459-465.

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(42) Baltacıoğlu, H.; Bayındırlı, A.; Severcan, F. Secondary structure and

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conformational change of mushroom polyphenol oxidase during thermosonication

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treatment by using FTIR spectroscopy. Food Chem. 2017, 214, 507-514.

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(43) Wang, X.; Xie, X.; Ren, C.; Yang, Y.; Xu, X.; Chen, X. Application of

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molecular modelling and spectroscopic approaches for investigating binding of

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vanillin to human serum albumin. Food Chem. 2011, 127, 705-710.

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(44) Prystupa, D. A.; Donald, A. M. Infrared study of gelatin conformations in the 26


Page 27 of 39

569


gel and sol states. Polym. Gels Networks 1996, 4, 87-110.

570

(45) Song, J. H.; Kim, H. E.; Kim, H. W. Production of electrospun gelatin

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nanofiber by water-based co-solvent approach. J. Mater. Sci.: Mater. Med. 2008, 19,

572

95-102.

573

(46) Lefèvre, T; Subirade, M. Formation of intermolecular β-sheet structures: a

574

phenomenon relevant to protein film structure at oil–water interfaces of emulsions. J.

575

Colloid Interface Sci., 2003, 263(1), 59-67.

576

27



577

Figure captions

578

Figure 1. (A) Shear-rate dependence of the viscosity of the gelatin-stabilized O/W

579

emulsions at various oil fractions (φ = 0.2 - 0.8). (B) Plots of shear stress versus shear

580

rate for the emulsions. The solid lines are the lines of best fit to the Herschel-Bulkeley

581

model. (C) Dynamic frequency sweep of the emulsions at various oil fractions (φ =

582

0.2 - 0.8). G′, solid line; G″, dashed line.

583

Figure 2. Optical micrographs for the gelatin-stabilized O/W emulsions at (A) φ = 0.2,

584

(B) 0.4, (C) 0.6 and (D) 0.8.

585

Figure 3. SEM micrographs of nanofibers by electrospinning of (A) gelatin, and the

586

gelatin-stabilized O/W emulsions at (B) φ = 0.2, (C) 0.4, (D) 0.6 and (E) 0.8.

587

Figure 4. (A), (B) TEM and (C) CLSM micrographs of the core-shell nanofibers by

588

emulsion electrospinning at φ = 0.2.

589

Figure 5. (A) TGA thermogram and (B) DSC curves of nanofibers by electrospinning

590

of gelatin and the gelatin-stabilized O/W emulsions at various oil fractions (φ = 0.2 -

591

0.8).

592

Figure 6. ATR-FTIR spectra of the gelatin-based nanofibers by emulsion

593

electrospinning at various oil fractions (φ = 0 - 0.8).

594

28


Page 28 of 39

Page 29 of 39


Table 1. Rheological parameters of the gelatin-stabilized O/W emulsions by Herschel-Bulkley model.

Oil fractions

σ0 (Pa)

K (Pa Sn)

n

R2

φ = 0.2

0.231

0.968

0.998

0.999

φ = 0.4

1.707

2.787

0.885

0.999

φ = 0.6

5.972

9.901

0.754

0.999

φ = 0.8

5.643

8.207

0.702

0.999

29



Page 30 of 39

Table 2. The denaturation temperature (TD) and the corresponding denaturation enthalpy (ΔHD) of nanofibers by electrospinning of gelatin and the gelatin-stabilized O/W emulsions at various oil fractions. Gelatin

φ = 0.2

φ = 0.4

φ = 0.6

φ = 0.8

TD (°C)

89.67 ± 0.58a

85.32 ± 2.21b

77.97 ± 1.67c

82.99 ± 3.21c

87.55 ± 2.48a

ΔHD° (J g−1)

289.84 ± 31.62a

179.78 ± 15.72b

109.66 ± 10.54c

100.39 ± 22.16c

64.75 ± 6.89d

30


Page 31 of 39


Table 3. The contents of secondary structures of nanofibers by electrospinning of gelatin and the gelatin-stabilized O/W emulsions at various oil fractions. α-helix/%

β-sheet/%

β-turn/%

Random coil /%

β-antiparallel/%

Gelatin

22.62 ± 0.38a

27.22 ± 0.21b

22.40 ± 0.57b

13.57 ± 0.21b

14.19 ± 0.40a

φ = 0.2

17.65 ± 0.21b

28.51 ± 0.72b

23.09 ± 0.14ab

17.65 ± 0.46a

13.10 ± 0.98ab

φ = 0.4

14.15 ± 0.11c

45.02 ± 0.17a

18.63 ± 0.51c

18.20 ± 0.22a

4.0 ± 0.66c

φ = 0.6

14.42 ± 0.29c

44.69 ± 0.13a

19.63 ± 0.33c

17.21 ± 0.19a

4.05 ± 0.51c

φ = 0.8

17.30 ± 0.45b

28.13 ± 0.27b

24.94 ± 0.19a

17.47 ± 0.86a

12.16 ± 0.79b

Results are expressed as mean values ± standard deviation of 3 replicates. Different letters in each column indicate significant difference (p < 0.05).

31



Page 32 of 39

Table 4. The encapsulation efficiency and storage stability of the emulsion-based electrospun fiber mats at various oil fractions. Encapsulation efficiency (%) 0 day

1 day

3 day

5 day

φ = 0.2

80.41 ± 0.12Aa

79.92 ± 0.05Ba

79.89 ± 0.08Ba

79.89 ± 0.08Ba

φ = 0.4

85.85 ± 0.07Ab

86.10 ± 0.05Ab

85.27 ± 0.14Bb

85.28 ± 0.07Bb

φ = 0.6

86.86 ± 0.05Ac

87.13 ± 0.12Ac

86.93 ± 0.06Ac

87.16 ± 0.03Ac

φ = 0.8

87.16 ± 0.01Ac

87.12 ± 0.07Ac

86.86 ± 0.08Ac

87.75 ± 0.11Ac

The same superscript capital letters in each row represent no significant difference at p < 0.05. The same superscript small letters in each column represent no significant difference at p < 0.05.

32


Page 33 of 39


Figure 1. (A)

(B)

(C)

33



Figure 2. (A)

(B)

(C)

(D)

34


Page 34 of 39

Page 35 of 39


Figure 3. (A)

(B)

(C)

(D)

(E)

35



Figure 4. (A)

(B)

(C)

36


Page 36 of 39

Page 37 of 39


Figure 5. (A)

(B)

37



Figure 6.

38


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TOC Graphic

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Formation and Stability of CoreâShell Nanofibers by Electrospinning

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