Gelatin Nanofibers Containing Zinc Oxide

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Food and Beverage Chemistry/Biochemistry

Hydrophobic Ethylcellulose/Gelatin Nanofibers Containing Zinc Oxide Nanoparticles for Antimicrobial Packaging Yuyu Liu, Yang Li, LingLi Deng, Lin Zou, Fengqin Feng, and Hui Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03267 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 25, 2018

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Journal of Agricultural and Food Chemistry

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Hydrophobic Ethylcellulose/Gelatin Nanofibers Containing Zinc Oxide

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Nanoparticles for Antimicrobial Packaging

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Yuyu Liu, Yang Li, Lingli Deng, Lin Zou, Fengqin Feng, Hui Zhang *

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College of Biosystems Engineering and Food Science, Fuli Institute of Food Science, Zhejiang Key

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Laboratory for Agro-Food Processing, Zhejiang R&D Center for Food Technology and Equipment,

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Zhejiang University, Hangzhou 310058, China

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* E-mail: [email protected]. Phone: +86-571-88982981. Fax: +86-571-88982981.

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ACS Paragon Plus Environment


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Abstract

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The ethylcellulose/gelatin solutions containing various concentrations of zinc oxide (ZnO)

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nanoparticles were electrospun, and the resultant nanofibers were characterized by scanning

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electron microscopy, energy dispersive X-ray, X-ray photoelectron spectrometer, X-ray diffraction,

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Fourier transform infrared spectroscopy, mechanical testing, water contact angle, and water stability.

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Results indicated that ZnO nanoparticles acting as fillers interacted with polymers, resulting in the

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enhanced surface hydrophobicity and water stability of nanofibers. The antibacterial assay showed a

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concentration-dependent effect of ZnO on the viabilities of Escherichia coli and Staphylococcus

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aureus. Notably, the antimicrobial efficiency of the 1.5 wt% ZnO-containing fibers against S.

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aureus was 43.7%, but increased to 62.5% after UV irradiation at 364 nm, possibly due to the

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significantly increased amounts of intracellular reactive oxygen species. These results suggested

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that the ZnO-containing nanofibers with excellent surface hydrophobicity, water stability and

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antimicrobial activity exhibited potential uses in food packaging.

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TOC Graphic

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Keywords

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electrospinning, ethylcellulose, gelatin, ZnO, antibacterial activity, UV irradiation

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Introduction

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Packaging has been widely used in food and beverages to maintain their quality and safety, and

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to reduce food waste.1 Except for the basic role in acting as inert barrier to external conditions, the

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ability to protect products from microbial contamination is preferred in food packaging, since

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microbial spoilage is responsible for shortened shelf life and degraded food quality.2-4 Antimicrobial

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packaging is reported as one of the most effective approaches to restrain multiplication of

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microorganisms by incorporating antimicrobial substances in the packaging systems.3 Due to the

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extensive antimicrobial activity, high specific surface area, and ability to improve mechanical

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properties of matrix, metal oxide nanoparticles are promising antibacterial agents in food

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packaging.5, 6 However, the potential toxicity of nanoparticles is an important obstacle in food

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applications.7 Zinic oxide (ZnO) nanoparticles as GRAS approved food additive have attracted an

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increasing interest in food packaging not only due to the excellent antimicrobial ability but also for

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the desired mechanical performance and the minimum impact on human cells.5, 8 Notably, as a

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photocatalytic material, UV illumination can induce the generation of reactive oxygen species (ROS)

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on the surface of ZnO nanoparticles, leading to enhanced bioactivity due to the fact that the ROS

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may penetrate into the cells and cause damage to bacteria.9 Li et al. detected the generation of ROS

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on surface of ZnO nanoparticles under UV (365 nm) irradiation, and indicated that the higher the

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total ROS concentration, the more significant the antibacterial activity.10

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Encapsulation of metal oxide nanoparticles into polymers is a promising approach to enhance

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the antimicrobial activity of nanoparticles due to the improved stability and sustained release.11

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Electrospinning has been thought to be of great suitability in encapsulating active compounds in

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food polymers and biopolymers, since it is a versatile and simple technique to fabricate functional 4


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fibrous membranes with high porosity and surface-to-volume ratio, and improved stability and

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mechanical strength.2 The enhanced dispersion of metal oxide nanoparticles was reported by

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Mayorga

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(3-hydroxybutyrate-co-3-hydroxyvalerate), and the resultant electrospun fiber mats showed the

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enhanced antimicrobial activity against Salmonella enterica and Listeria monocytogenes.11

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Augustine et al. observed that the addition of 5 wt% ZnO nanoparticles in the electrospun

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polycaprolactone nanofibers enhanced the mechanical stability as well as the antibacterial

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properties against Escherichia coli and Staphylococcus aureus.12

et

al.

through

incorporating

CuO

nanoparticles

into

poly

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Additionally, the encapsulation matrices for food packaging should be regarded as safe. The

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active loading based on gelatin nanofibers has been widely fabricated through electrospinning

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owing to its desirable biodegradability and biocompatibility.13 Ultrafine gelatin/nisin fiber mats with

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antimicrobial activity against S. aureus and L. monocytogenes were reported by Dheraprasart et al.13

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The gelatin-based electrospun fibrous membranes displaying remarkable antibacterial properties

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against E. coli and S. aureus were produced by Padrao et al. with fish gelatin as structural matrix

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and bovine lactoferrin as the active antimicrobial agent.14 On the other hand, elements such as

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excellent hydrophobicity to block the entry of water and moisture are necessary for packaging.7 Our

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recent work suggested that due to the advantageous properties of safety, electrospinability and

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hydrophobicity, blending ethylcellulose with gelatin is an ideal approach to overcome the natural

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hydrophilicity of gelatin based fibers, rendering the improved water stability.15

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In this study, before electrospinning the ethylcellulose/gelatin solutions containing various

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levels of ZnO nanoparticles were evaluated by viscosity and zeta potential measurements. The

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resultant fibers were characterized by scanning electron microscopy (SEM), energy dispersive 5



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X-ray (EDX), X-ray photoelectron spectrometer (XPS), X-ray diffraction (XRD), Fourier transform

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infrared spectroscopy (FTIR), mechanical testing, water contact angle (WCA), and water stability.

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The antibacterial activities of the nanofibers were tested against E. coli and S. aureus based on disc

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diffusion technique. Subsequently, the fiber mat containing 1.5 wt% ZnO nanoparticles was selected

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for analyzing the impact of UV irradiation on the antimicrobial viability using culture turbidity

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method.

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MATERIALS AND METHODS

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Materials. Ethylcellulose (3-7 cP, MW, 20-30 kDa), gelatin of type B (Bloom 250, MW, 100 kDa)

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and zinc oxide (ZnO) nanoparticles with particle size of 30 nm were supplied by Aladdin, Inc.

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(Shanghai, China). Absolute ethanol (Sinopharm), acetic acid (99.8%, Sinopharm), and deionized

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water were used for solution preparations. All reagents were utilized as received.

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Solution preparation. Solutions were prepared by dissolving 15 wt% (w/v) ethylcellulose and 15

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wt% (w/v) gelatin into water/ethanol/acetic acid at a volume ratio of 2/2/6. Subsequently, ZnO

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nanoparticles were added at concentrations of 0 wt%, 1 wt%, 1.5 wt%, and 2 wt% (w/v), which

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were denoted as Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG, respectively. Thus, the theoretical mass

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fraction of ZnO regarded to polymers was 0%, 3.2%, 4.8% and 6.3%, respectively. The solutions

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were gently stirred for 1 h at 60 °C and then vigorously stirred for 2 h at 25 °C prior to

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electrospinning.

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Rheology and zeta potential. Rheology was characterized using an Anton Paar MCR302

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rheometer (Anton Paar, Austria, QC) at 25 °C with a scanning frequency from 1 to 100 rad/s using 1%

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strain. The complex viscosity at a shear rate of 1 rad/s was taken as the average solution viscosity.

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Zeta potential was measured using a dynamic light scattering instrument (Malvern, Zetasizer Nano 6


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ZS90, UK) at 25 °C. Triplicate measurements were performed.

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Electrospinning. The fabrication of nanofibers by electrospinning was according to our previous

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studies.15 4 mL solution was pumped into a 5 mL syringe with a 20-gauge needle. A high-voltage

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power supply (Gamma High Voltage) was employed to generate a voltage up to 15 kV. A pump

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(LSP02-1B, Baoding Longer Precision Pump Co., Ltd., China) provided a constant flow rate of 1.0

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mL/h. The resultant fibers were collected on a grounded cylindrical aluminum foil at a distance of

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100 mm away from the needle at 25 °C with a humidity of 50%. The thickness of the fibers was in

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the range of 200-300 µm measured by a thickness meter (C640, Labthink, China).

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Morphology and element mapping. The morphology observation was carried out using a field

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emission scanning electron microscope (SEM, SU8010, Hitachi, Japan). Nano Measure software

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was applied to measure the fiber diameter of 120 fibers at random from SEM images. To observe

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the distribution of Zn element, the fibers were analyzed by energy dispersive X-ray (EDX, SU8010,

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Hitachi, Japan) at a working depth of 5 µm.

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X-ray photoelectron spectrometer. The surface atomic composition of fibers was examined by

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X-ray photoelectron spectrometer (XPS, AXIS SUPRA, Kratos) using Al Kα X-ray source (1486.6

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eV) with a standard working depth of 10 nm. Wide scan spectra were recorded from 1000 to 0 eV.

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The scan steps of wide scan spectra and high-resolution spectra were 1.0 eV and 0.1 eV,

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respectively. The atom percentage was calculated using Speclab software, and the curve fitting of

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high-resolution C 1s spectra was analyzed by Origin pro9 software. The spectra calibration was

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based on setting the C-C/C-H contribution in the C 1s emission at 284.8 eV.16

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X-ray diffraction. The X-ray diffraction (XRD, Bruker D8 ADVACNCE, Germany) was

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performed using Cu Kα (1.5406 Å) radiation at a scanning rate of 1 °/min ranging from 4° to 80°. 7



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The average spacing of the peak was calculated as follow:17

d =

0.9λ (1) β cos

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where λ, β, and θ represent X-ray wavelength (1.541), full width of half maximum (FWHM) and

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the Bragg diffraction angle, respectively.

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Fourier transform infrared spectrometer. The Fourier transform infrared spectrometer (FTIR,

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AVA TAR 370) spectra were obtained by mixing nanofiber mats with KBr. The resolution of

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spectra was 4 cm−1 with 16 scans, and the measurement range was from 4000 to 400 cm−1.

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Mechanical testing. The tensile properties were measured at 25 °C using an electron testing

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machine (Zwick GmbH & Co.KG, China). The fiber mats were cut into 5 mm × 30 mm stripes,

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with thickness of 200 ± 50 µm measured by a thickness meter (C640, Labthink, China). The

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stress-stain curves of the mats were recorded to determine the elastic modulus, tensile strength, and

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elongation at break. Five tests were performed for each sample.

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Water contact angle. The water contact angle (WCA) was measured at 25 °C using a surface

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tension meter (DCAT-21, Delta Phase, Germany). Milli-Q water was dropped on the fiber surface,

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and the images were captured by a camera system. Three random spots were measured for each

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sample.

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Water stability. The water stability measurements of the fibers were conducted by following the

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procedure of our previous method.15 The disc-shaped fiber mats (D0=11 mm) were first determined

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(W0) before immersed in water at 25 °C with a shaking speed at 150 rpm for 24 h. After immersion,

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the fiber diameter (DT) was measured, followed by weighing the mats before (Ww) and after (WD)

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drying at 60 °C for 1 h, respectively. The surface swelling (SW), water-uptake capacity (WT), and

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weight loss (WL) were calculated by the equations: 8


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D −D (2) D W − W W = (3) W W − W W = × 100% (4) W S =

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Inhibitory zone evaluation. The antibacterial activities of the fibers with different ZnO levels

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against Escherichia coli (ATCC 25922) and Staphylococcus aureus (CMCC 26003) were measured

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using a disc diffusion method.18 The cells (~105 CFU/mL) of each pathogen were prepared by the

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serial dilution method. 0.1 mL diluent was spread on agar media using a triangular metal rod. The

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disc-shaped fibers with a diameter of 11 mm (2-3 mg) were sterilized by UV for 1 h before being

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adhered to the medium. After incubation for 24 h at 37 °C, the inhibitory zone was measured to

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estimate the sensitivity of bacteria to fibers. Three replications were performed for each sample.

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Bactericidal kinetics after UV irradiation. The Z1.5-EG fibrous mat was selected to analyze the

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antibacterial activity after UV irradiation (364 nm, 250 W), using a culture turbidity method.10, 18 11

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mg of the sterilized mat was added into tubes containing 20 mL culture medium. One set of tubes

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was exposed to UV for 1 h, and the other set was protected from light for 1 h. After treatment, 0.2

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mL of each bacterial suspension (106 CFU/mL) was added, and then incubated in the shaker

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platform (150 rpm, 37 °C). The growth of bacteria was monitored at an interval of 2 h for 24 h by a

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microplate reader (MDS ANALYTICAL, America). Medium with only inoculum or the fibrous mat

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without ZnO (Z0-EG) was used as negative control or positive control, respectively. All

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experiments were performed in triplicate. The inhibition efficiency (IE) was calculated as follows:

IE =

OD#" − OD$" OD#"

× 100% (5)

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where OD#" and OD$" were the OD600 values of culture medium without and with the fiber mat

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at time t, respectively. 9



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In order to estimate the generation of intracellular reactive oxygen species (ROS) caused by

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ZnO nanoparticles under UV irradiation, the above E. coli suspensions after 24 h of exposure to

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fibers were used to quantify the ROS level within the cells by the method provided by Najim et al.19

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After incubation, the cells were collected by centrifugation and suspended with PBS. The resultant

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solutions were mixed with 2,7-dichlorofluorescein diacetate (DCFH-DA, sigma, USA) at a volume

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ratio of 2000:1 for 45 min at 37 °C under dark conditions. The fluorescence intensity of DCFH was

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measured by fluorescence spectrophotometer (Molecular Devices, China) at an excitation and

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emission wavelength of 488 nm and 535 nm, respectively.

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Statistical analysis. The experimental results were presented as mean plus or minus standard

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deviation. The statistical evaluation was carried out using Origin pro9 software via one-way

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ANOVA with analysis of variance at the 0.05 significance level.

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Results and Discussion

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Solution rheology and zeta potential. As listed in Table 1, the apparent viscosities of the

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ethylcellulose/gelatin solutions without (Z0-EG) and with 1 wt% (Z1-EG), 1.5 wt% (Z1.5-EG), and

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2 wt% (Z2-EG) ZnO nanoparticles were 2.05, 1.77, 1.30 and 1.78 Pa s, respectively. Obviously, the

173

solutions became less viscous after the addition of ZnO nanoparticles, while the viscosity of the

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Z2-EG solution gave a significant (p < 0.05) rise compared with that of the Z1.5-EG solution. A

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viscosity decrease after adding AgNO3 into poly (vinyl pyrrolidone)/cellulose nanocrystals systems

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was observed by Huang et al., who owed the changes to the competition between polymers and Ag+,

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leading to destruction of some intramolecular or intermolecular hydrogen bonds in polymers.20 An

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interpenetrating network among ethylcellulose and gelatin through interactions such as hydrogen

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bonds has been proposed previously.15 Therefore, it could be assumed that the ZnO nanoparticles 10


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probably acting as fillers were dispersed in the network by blocking interactions among polymers,

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resulting in the decreased viscosity. However, the increased viscosity of the Z2-EG solution could

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be an indicator of aggregation of ZnO nanoparticles at a higher concentration. Augustine et al.

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suggested that with the concentration of ZnO nanoparticles up to 2 wt% in polycaprolactone

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solution, particle agglomeration encountered, leading to high solution viscosity and apparently

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increased fiber diameter.12

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The solutions of 0%, 1%, 1.5%, and 2% ZnO nanoparticles without polymers displayed zeta

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potential values of 3.89, 6.98, 5.73, and 3.94 mV, respectively. The lower zeta potential value of 2%

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ZnO solution might be due to the formation of ZnO aggregates. Meanwhile, the zeta potential

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values of the Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG solutions were 1.47, 1.08, 0.87, and 1.00 mV,

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respectively. Matusiak et al. reported that in the case of polymer and oxide systems, adsorption of

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the polymer on oxide surface could change the zeta potential of the bulk.21 Yu et al. ascribed the

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zeta potential changes of ZnO-containing matrix to the interaction effect between nanoparticles and

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polymers.22 Thus, the observed decrease in zeta potential values of ZnO nanoparticles after

194

introduced into the ethylcellulose/gelatin solutions could be a result of interactions between ZnO

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and the absorbed polymers. Additionally, the zeta potential of Z2-EG solution was slightly higher

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than that of the Z1.5-EG solution, probably resulting from the reduced interactions caused by

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aggregates of ZnO nanoparticles at a higher concentration.

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Nanofiber morphology and element mapping. SEM images in Figure 1 (A-D) showed that the

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fibers without ZnO nanoparticles (Z0-EG) were smooth with an average diameter of 500 nm. The

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diameters of fibers with ZnO nanoparticles (Z1-EG, Z1.5-EG and Z2-EG) were 588, 640 and 804

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nm, respectively, without any visually observed morphology changes, indicating the homogenous 11



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distribution of ZnO nanoparticles within the fibers.23 It is universally accepted that the high zeta

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potential value is responsible for strong electrostatic force.20 During electrospinning, the

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electrostatic force acting as a strong drag force fights on the jet, leading to strong elongation of jet

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and generation of uniform and thin fibers.15 Thus, the reduced zeta potential values of solutions

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after the ZnO introduction played a major role in the formation of thicker fibers. Figure 1 (E-J)

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presented the EDX analysis and Zn element mapping of the Z0-EG, Z1-EG and Z2-EG fibers,

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respectively. Obviously, all the fibers showed the presence of C, N, O, while the signals of Zn

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element were detected in the ZnO-containing fibers. Figure 1 (H) exhibited a highly Zn dispersion

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in the Z1-EG fibers, indicating good system compatibility.24 On the other hand, the increased Zn

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distribution density and slight aggregates were observed for the Z2-EG fibers in Figure 1 (J),

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validating the interpretation of viscosity changes proposed in solution rheology characterization.

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X-ray photoelectron spectrometer. To evaluate the chemical composition of the fiber surface, the

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XPS analysis was conducted and the results were displayed in Figure 2 and Table 2. The wide scan

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spectra showed the presence of C, N and O atoms in each fiber mat, and the typical peak of Zn was

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observed except the Z0-EG fibers. The high resolution scan of Zn element exhibited two peaks at

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1020 and 1043 eV assigned to Zn (2p3/2) and Zn (2p3/2),25 confirming the existence of Zn element in

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the Z1-EG, Z1.5-EG and Z2-EG fibers, respectively. The high resolution XPS spectra for C 1s

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revealed that two peaks at 284.8 eV and 286.4 eV corresponding to C-C and C-O existed in all the

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fiber samples, but another small peak at 288.1 eV related to C=O was only observed for the Z2-EG

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fibers.26

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As summarized in Table 2, the N atom percentage values for the Z0-EG, Z1-EG, Z1.5-EG and

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Z2-EG fibers were 7.3%, 6.9%, 5.8% and 5.2%, respectively, indicating the decreased N content on 12


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the fiber surface with the increasing ZnO concentration. The calculated ratio of surface Zn regarded

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to theoretical content was 47.6% for the Z1-EG fibers, 46.9% for the Z1.5-EG fibers but 71.4% for

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the Z2-EG fibers, implying a higher ZnO content on the Z2-EG fiber surface. The decreased N but

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increased Zn content on the fiber surface suggested the replacement of hydrophilic groups by

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hydrophobic ZnO nanoparticles, contributing to the improved surface hydrophobicity.27

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X-ray diffraction. The XRD spectra of the fabricated nanofibers as well as the ZnO nanoparticles

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were shown in Figure 3 and Table 3. It was evident that there was an extra peak at 31.82° in the

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case of the fibers containing 2 wt% ZnO nanoparticles (Z2-EG), consistent with the typical

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crystalline peak of ZnO nanoparticles assigned to the (100) plane.28 This implied the presence of

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ZnO aggregates in the Z2-EG fibers, which was in agreement with our EDX analysis. Generally,

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ethylcellulose possesses two crystalline reflections at 2θ = 7.8° and 2θ = 20.3°, and gelatin has a

235

broad diffraction peak in the 2θ range around 20°.29, 30 Figure 3 indicated that the second reflection

236

at around 20° became less prominent after the addition of ZnO nanoparticles, and the calculated d

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spacing value of the fibers without ZnO nanoparticles (Z0-EG) in Table 3 was increased from 0.222

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to 0.289, 0.292 and 0.336 after the addition of 1 wt% (Z1-EG), 1.5 wt% (Z1.5-EG), and 2 wt%

239

(Z2-EG) ZnO nanoparticles, respectively. Li et al. demonstrated that the decreased peak intensity

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and increased d value implied a less compact structure of the composite system.31 Thus, these

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results suggested that the higher amounts of ZnO nanoparticles, the less compact structure of the

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polymer network, which might be due to some destroyed intramolecular or intermolecular

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interactions of polymers caused by the ZnO addition.

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Fourier transform infrared spectrometer. It can be seen from Figure 4 that some characteristic

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absorption peaks of the fibers without ZnO nanoparticles (Z0-EG) were observed at 3385 cm−1, 13



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1651 cm−1, 1538 cm−1, and 1446 cm−1, related to O-H bending, C=O stretching (amide I), N-H/C-N

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stretching (amide II), respectively.29 In contrast, the ZnO-containing fibers (Z1-EG, Z1.5-EG and

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Z2-EG) showed a new peak at 667 cm−1 assigned to the metal-oxygen (M-O) stretching mode,28

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indicating the existence of ZnO nanoparticles. Additionally, the fibers containing ZnO nanoparticles

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exhibited the decreased intensities of the amide II bands, especially an almost nonexistent peak at

251

1446 cm−1. Generally, the higher the intensity of amide band, the more number of the triple helix

252

structures gelatin chains have.32 Therefore, the observed changes indicated the loss of triple helix

253

state linked by hydrogen bonds, which evidenced the destruction of gelatin molecular interactions

254

after introduction of ZnO nanoparticles.

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Mechanical testing. The tensile properties of the fiber mats including average elastic modulus (E),

256

tensile strength (σy) and elongation at break (εb) were listed in Table 4. The fiber mats without ZnO

257

(Z0-EG) possessed E, σy and εb of 148.10 MPa, 1.70 MPa and 1.30%, respectively. After the ZnO

258

addition ranging from 1 - 1.5 wt%, these values increased with the highest E (203.24 MPa), σy (2.42

259

MPa) and εb (1.57%) observed for the Z1.5-EG fibers. It was reported that dispersion of fillers and

260

filler-matrix interactions were the dominant factors in determining mechanical performance of

261

polymer composites. The enhancement in tensile properties was indicators of good nanoparticle

262

dispersion and strong interfacial interactions.4 However, the fibers containing 2 wt% ZnO presented

263

a slight decrease in E (134.28 MPa), σy (1.65 MPa) and εb (0.98%) compared with the Z0-EG fibers.

264

In the case of ZnO loaded cellulose films reported by Zhao et al., the 7.4 wt% ZnO addition led to

265

decreased εb values compared with pure cellulose films, which was attributed to the poor dispersion

266

of ZnO in cellulose matrix caused by formation of agglomerates at higher concentrations.8 Thus,

267

our results indicated that the addition of ZnO at a concentration below 1.5 wt% might improve the 14


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mechanical properties of fibers, while a tendency of formation of agglomerates at a higher

269

concentration (2 wt%) could lead to a poor mechanical performance.

270

Water contact angle. The results of water contact angle (WCA) recorded at 1 s, 5 s and 15 s were

271

displayed in Figure 5. The WCA of the fibers without ZnO nanoparticles (Z0-EG) was 119.3° at 1 s,

272

lower than the values of the Z1-EG (129.7°), Z1.5-EG (134.0°) and Z2-EG (128.5°) fibers. Anitha

273

et al. observed that the wetting property of the cellulose acetate fibers changed from hydrophilic

274

(47°) to hydrophobic (124°) after impregnating ZnO.33 Hydrophobicity is largely dependent on the

275

surface morphology and chemical structure.34 Considering the reduced N atom percentage on

276

nanofiber surface by introduction of ZnO nanoparticles from the XPS analysis, the improved

277

surface hydrophobicity could be a result of the decreased number of gelatin chains distributed on

278

surface, since N element was the characteristic element of gelatin in the fiber matrix. Additionally,

279

the presence of the hydrophilic group (C=O) of the Z2-EG fibers observed in the XPS spectrum for

280

C 1s might give an explanation to the decreased WCA value, compared to the other ZnO-containing

281

nanofibers. The Z0-EG, Z1-EG, Z1.5-EG and Z2-EG fibers gave the WCA values of 109.5°, 117.5°,

282

130.0° and 122.1° at 15 s showing a 9.8°, 12.2°, 4.0° and 5.4° reduction compared to the initial

283

values, suggesting the higher water stability at higher ZnO levels. Thus, the introduction of ZnO

284

nanoparticles into fibers effectively strengthened the surface hydrophobic property, which was

285

suitable for packaging materials to inhibit water penetration.7

286

Water stability. As presented in Table 5, the fibers without ZnO nanoparticles (Z0-EG) gave the

287

highest water-uptake capacity (WT) value of 536.41%, compared with the Z1-EG (297.9%),

288

Z1.5-EG (209.31%) and Z2-EG (203.61%) fibers, respectively. The lower gelatin content on the

289

fiber surface supported the remarkably decreased WT values of the ZnO-containing fibers due to the 15



290

fact that the lower the gelatin content, the less the hydrophilic groups to absorb water molecules.35 It

291

was obvious that the surface swelling (SW) and weight loss (WL) displayed a similar trend with the

292

WT value, the higher the ZnO concentration the lower the values. Notably, the SW and WL values of

293

the Z0-EG and Z1-EG fibers were much higher than those of the Z1.5-EG and Z2-EG fibers. By

294

comparing the surface dimensional stability of pure chitosan or gelatin fibers with its binary

295

scaffolds containing hydrophobic polycaprolactone, Gomes et al. reported much lower SW values

296

for binary scaffolds due to the increased surface hydrophobicity caused by polycaprolactone.36 Our

297

results indicated that the enhanced hydrophobicity of the ZnO-containing fibers provided by WCA

298

analysis contributed to the improved water stability. These parameters were related to dislocations

299

or blistering and sensitivity to water, which was necessary for using as food package and coating

300

films.35, 37

301

Inhibitory zone evaluation. Table 6 summarized the inhibitory diameters of the nanofibers against

302

E. coli and S. aureus. The fibers without ZnO (Z0-EG) as control showed absence of antimicrobial

303

activity, while the fibers containing 1 wt%, 1.5 wt% and 2 wt% ZnO nanoparticles showed the

304

inhibitory diameters of 0.69, 1.30, 1.61 mm/mg against E. coli and 0.75, 1.17, 1.33 mm/mg against

305

S. aureus, respectively. These results indicated that the bacteria inhibition displayed a dose-effect

306

manner, and E. coli was observed to be more susceptible than S. aureus. Xu et al. revealed that the

307

addition of 2 wt% AgNO3 endowed the gelatin fibers with an inhibition zone of 1.6 mm against S.

308

aureus and 2.5 mm against Pseudomonas aeruginosa, and attributed the antimicrobial activity

309

difference to the thinner cell wall of P. aeruginosa.18 The similar results were also reported by

310

Kanmani et al., who suggested that the thicker and complex peptidoglycan structure of Gram

311

positive bacterial obstructed the nanoparticles penetration, while a negatively charged and thinner 16


Page 16 of 37

Page 17 of 37


312

peptidoglycan layer facilitated the nanoparticles transport.38

313

Bactericidal kinetics after UV irradiation. The growth curves of E. coli and S. aureus in Figure 6

314

(A) consisted of three phases: lag phase, exponential phase, and stabilization phase. Under exposure

315

to the different fibers, no reduction in cell viability was observed between the positive (Z0-EG

316

fibers) and negative control, while substantial decrease at exponential phase was obvious for the

317

fibers containing 1.5 wt% ZnO nanoparticles (Z1.5-EG). Notably, the Z1.5-EG fibers after UV

318

irradiation for 1 h (denoted as UV Z1.5-EG) was found to exhibit a higher antimicrobial activity

319

than the light protection for 1 h (denoted as Dark Z1.5-EG), as indicated by the lower OD600 value

320

at each time interval. From Figure 6 (B), the inhibition efficiency of the Z1.5-EG fibers against

321

either E. coli or S. aureus firstly increased, and then decreased significantly as the incubation time

322

prolonged. Finally, no significant difference of the inhibition efficiency between the Z1.5-EG fibers

323

and negative control was observed at stationary phase. The highest inhibition efficiency values of

324

the UV Z1.5-EG and Dark Z1.5-EG were 98.4% and 98.0% against E. coli, and 62.5% and 43.7%

325

against S. aureus, respectively, suggesting a more efficient antibacterial activity after UV irradiation.

326

This suggested that UV irradiation was an effective way to enhance the bacteriostatic performance

327

of the prepared ZnO-containing nanofibers.

328

It was proposed that the increased reactive oxygen species (ROS) production from ZnO under

329

UV irradiation resulted in the enhanced antibacterial activity due to the high reactivity and

330

oxidizing property of ROS.9 Thus, the E. coli suspensions after exposure to fibers for 24 h were

331

selected to detect the ROS concentration, and the results expressed as percentages of the value

332

against negative control were presented in Figure 6 (C). A significant difference of fluorescence

333

intensity was observed after exposure with the highest value (166%) for the UV Z1.5-EG fibers 17



334

while the lowest value (130%) for the Z0-EG fibers, indicating the higher intracellular ROS levels

335

of the ZnO-containing fibers after exposure to UV light. The similar results were also reported by Li

336

et al., who demonstrated that under UV irradiation (365 nm), ZnO nanoparticles generated three

337

types of ROS, and the more the total ROS concentration, the higher the antibacterial activity against

338

E. coli.10

339

In summary, the present work demonstrated that ZnO nanoparticles were successfully

340

encapsulated in the ethylcellulose/gelatin nanofibers through electrospinning. Generally, ZnO

341

nanoparticles were homogeneously dispersed and did not change the fiber morphology, due to the

342

fact that ZnO nanoparticles acting as fillers destroyed intramolecular or intermolecular interactions

343

among polymers and instead formed new interactions with polymers. It was confirmed that the

344

ZnO-containing fibers exhibited the enhanced surface hydrophobicity and water stability owing to

345

the decreased gelatin distribution on fiber surface caused by the ZnO addition. The introduction of

346

ZnO nanoparticles endowed the ethylcellulose/gelatin nanofibers with the improved mechanical

347

properties and concentration-dependent antibacterial properties against E. coli and S. aureus.

348

Additionally, UV irradiation enabled the increased antibacterial efficiency of the fibers containing

349

1.5% ZnO nanoparticles from 43.7% to 62.5% against S. aureus. These results suggested that the

350

ZnO-containing nanofibers with excellent hydrophobicity, water stability and antibacterial

351

performance may have promising applications as food packaging materials.

352

Acknowledgement

353

This work was supported by the National Natural Science Foundation of China (Grant No.

354

31772013).

355

Conflict of interest 18


Page 18 of 37

Page 19 of 37


The authors declare no competing financial interest.

356 357

References

358

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Oxygen Species Induced by Zinc Oxide Nanoparticles. Aro 2015, 3, 30-34.

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Properties of Electrospun Poly (Vinyl Pyrrolidone)/Cellulose Nanocrystal/Silver Nanoparticle

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the Chitosan/Silica System. Colloid Surf. A-Physicochem. Eng. Asp. 2018, 554, 245-252.

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of Particle Size on Zno-Protein Corona. Nanomaterials 2017, 7, 14.

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M. R.; Ramakrishna, S., Protein Adsorption on Electrospun Zinc Doped Hydroxyapatite Containing

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Nylon 6 Membrane: Kinetics and Isotherm. J. Colloid Interface Sci. 2015, 443, 143-152.

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Cellulose Composite Nanofiber and Its Drug Release Properties. Mater. Sci. Eng. C. 2017, 74,

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Nanofibrous Zinc Oxide Film Surface by Electrospinning. Thin Solid Films 2008, 516, 2495-2501.

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Swine Gastrointestinal Tract Using Gelatin Microcapsules Coated with Aluminum Carboxymethyl

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Cellulose and Beeswax. J. Agric. Food Chem. 2014, 62, 12641-12648.

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Degradation of Biodegradable Blends of Polyethylene with Cellulose and Ethylcellulose.

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of Blend Membranes of Polyvinylpyridine with Ethylcellulose. Polymer. 2001, 42, 6859-6869.

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Bactericidal and Water Repellent Properties of Electrospun Nano-Composite Membranes of

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Cellulose Acetate and Zno. Carbohydr. Polym. 2012, 87, 1065-1072.

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Controlled Release in Bicomponent Electrospun Fibers. Int. J. Pharm. 2010, 397, 50-58.

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Enhancement of the Mechanical Properties and Film Coating Efficiency of Shellac by the

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Formation of Composite Films Based on Shellac and Gelatin. J. Food Eng. 2012, 108, 94-102.

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455

Scaffolds Obtained from Blends of Chitosan, Gelatin and Polycaprolactone for Skin Tissue

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Engineering. Int. J. Biol. Macromol. 2017, 102, 1174-1185.

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of Nanofibrous Cellulose Acetate/Gelatin Skin Substitutes for Variety Wound Treatment

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Applications. J. Biomater. Appl. 2014, 28, 909-921.

460

(38) Kanmani, P.; Rhim, J. W., Physicochemical Properties of Gelatin/Silver Nanoparticle

461

Antimicrobial Composite Films. Food Chem. 2014, 148, 162-169.

462 463

23



464

Figure captions

465

Figure 1 SEM images (A-D) of the ethylcellulose/gelatin fibers with ZnO levels of 0 wt%, 1 wt%,

466

1.5 wt%, and 2 wt% (denoted as Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG, respectively), and EDX

467

analysis (E, G, I) and Zn mapping (F, H, J) of the Z0-EG, Z1-EG and Z2-EG fibers.

468 469

Figure 2 XPS spectra of wide scan (A), high resolution of Zn scan (B) and C1s scan (C-F) of the

470

ethylcellulose/gelatin fibers with ZnO levels of 0 wt%, 1 wt%, 1.5 wt%, and 2 wt% (denoted as

471

Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG, respectively).

472 473

Figure 3 XRD patterns of ZnO nanoparticles and the ethylcellulose/gelatin fibers with ZnO levels

474

of 0 wt%, 1 wt%, 1.5 wt%, and 2 wt% (denoted as Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG,

475

respectively).

476 477

Figure 4 FTIR spectra of the ethylcellulose/gelatin fibers with ZnO levels of 0 wt%, 1 wt%, 1.5

478

wt%, and 2 wt% (denoted as Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG, respectively).

479 480

Figure 5 Water contact angles recorded at 1 s, 5 s and 15 s of the ethylcellulose/gelatin fibers with

481

ZnO levels of 0 wt%, 1 wt%, 1.5 wt%, and 2 wt% (denoted as Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG,

482

respectively).

483 484

Figure 6 Growth curves (A), inhibition efficiency (B) of E. coli and S. aureus, and DCFH

485

fluorescence intensities in E. coli (C) exposed to the ethylcellulose/gelatin fibers containing 1.5 wt% 24


Page 24 of 37

Page 25 of 37


486

ZnO with and without UV light (denoted as UV Z1.5-EG and dark Z1.5-EG, respectively).

487

Lowercases indicated statistical significance (p < 0.05).

488

25



489

Figure 1

26


Page 26 of 37

Page 27 of 37


Figure 2

490

27



Figure 3

491

28


Page 28 of 37

Page 29 of 37


Figure 4

492

29



Figure 5

493

30


Page 30 of 37

Page 31 of 37


Figure 6 (A)

(B)

494 495

(C)

496 497 498

31



Page 32 of 37

499

Tables

500

Table 1. The apparent viscosities and zeta potential values of the ethylcellulose/gelatin solutions

501

with ZnO levels of 0 wt%, 1 wt%, 1.5 wt%, and 2 wt% (denoted as Z0-EG, Z1-EG, Z1.5-EG, and

502

Z2-EG, respectively), and zeta potentials of the ZnO solutions at levels of 0 wt%, 1 wt%, 1.5 wt%,

503

and 2 wt% (denoted as Z0, Z1, Z1.5, and Z2, respectively).

504

Type

Apparent viscosity (Pa·s)

Zeta potential (mV)

Type

Zeta potential (mV)

Z0-EG

2.05 ± 0.06 a a

1.47 ± 0.21 a

Z0

3.89 ± 0.64 c

Z1-EG

1.77 ± 0.07 a

1.08 ± 0.16 ab

Z1

6.98 ± 0.37 a

Z1.5-EG

1.30 ± 0.12 b

0.87 ± 0.10 b

Z1.5

5.73 ± 0.26 b

Z2-EG

1.78 ± 0.03 a

1.00 ± 0.12 ab

Z2

3.94 ± 0.25 c

a


505

32


Page 33 of 37


506

Table 2. Atomic concentration percentages of C, O, N and ZnO examined by XPS of the

507

ethylcellulose/gelatin fibers with ZnO levels of 0 wt%, 1 wt%, 1.5 wt%, and 2 wt% (denoted as

508

Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG, respectively). Surface element composition Type

509 510

a

Zn (Theoretical) (%)

Zn ratio (%)a

C (%)

O (%)

N (%)

Zn (%)

Z0-EG

65.7

27.0

7.3

-

0

0

Z1-EG

66.9

25.2

6.9

1.0

2.1

47.6

Z1.5-EG

65.8

27.0

5.8

1.5

3.2

46.9

Z2-EG

66.5

25.2

5.2

3.0

4.2

71.4

Zn ratio = Zn (surface)/Zn (Theoretical)×100%.

33



Page 34 of 37

511

Table 3. The 2θ value, FWHM value and crystal area of the ethylcellulose/gelatin fibers with ZnO

512

levels of 0 wt%, 1 wt%, 1.5 wt%, and 2 wt% (denoted as Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG,

513

respectively). Type

2θ (°)

Area

FWHM (β) (°)a

d (Å)b

Z0-EG

19.08

1376.46

6.32

0.222

Z1-EG

19.94

411.73

4.87

0.289

Z1.5-EG

20.04

538.50

4.82

0.292

Z2-EG

20.09

251.00

4.19

0.336

514

a

FWHM is the angular peak width at half maximum.

515

b

d = 0.9λ/(β cos(θ)), where λ is X-ray wavelength (1.541).

516

34


Page 35 of 37


517

Table 4. The tensile parameters of the ethylcellulose/gelatin fibers with ZnO levels of 0 wt%, 1 wt%,

518

1.5 wt%, and 2 wt% (denoted as Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG, respectively).

519

Type

Elastic modulus (E, MPa)

Tensile strength (σy, MPa)

Elongation at break (εb, %)

Z0-EG

148.10 ± 14.33 a a

1.70 ± 0.16 b

1.30 ± 0.11 ab

Z1-EG

188.77 ± 32.67 a

2.39 ± 0.17 a

1.39 ± 0.22 ab

Z1.5-EG

203.24 ± 43.06 a

2.42 ± 0.24 a

1.57 ± 0.31 a

Z2-EG

134.28 ± 45.17 a

1.65 ± 0.18 b

0.98 ± 0.17 b

a


520 521

35



Page 36 of 37

522

Table 5. The surface swelling, water uptake and weight loss of the ethylcellulose/gelatin fibers with

523

ZnO levels of 0 wt%, 1 wt%, 1.5 wt%, and 2 wt% (denoted as Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG,

524

respectively) after immersed in water for 24 h.

525

a

Type

Surface swelling (SW, %)

Water uptake (WT, %)

Weight loss (WL, %)

Z0-EG

-13.98 ± 0.63 a a

536.41 ± 21.46 a

13.78 ± 0.67 a

Z1-EG

-13.22 ± 0.33 a

297.90 ± 23.19 b

12.44 ± 0.97 ab

Z1.5-EG

-12.55 ± 0.13 b

209.31 ± 14.38 c

9.64 ± 0.86 b

Z2-EG

-12.08 ± 0.43 b

203.61 ± 20.71 c

9.33 ± 0.97 b


526

36


Page 37 of 37


527

Table 6. The inhibition zone of the ethylcellulose/gelatin fibers with ZnO levels of 0 wt%, 1 wt%,

528

1.5 wt%, and 2 wt% (denoted as Z0-EG, Z1-EG, Z1.5-EG, and Z2-EG, respectively).

529

a

Type

E. coli (mm/mg)

S. aureus (mm/mg)

Z0-EG

0 ± 0 da

0±0d

Z1-EG

0.69 ± 0.03 c

0.75 ± 0.020 c

Z1.5-EG

1.30 ± 0.04 b

1.17 ± 0.04 b

Z2-EG

1.61 ± 0.05 a

1.33 ± 0.06 a


37


Gelatin Nanofibers Containing Zinc Oxide

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