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Immobilization of lysozyme on layer-by-layer self-assembled electrospun films: Characterization and antibacterial activity in milk
Journal Pre-proof Immobilization of lysozyme on layer-by-layer self-assembled electrospun films: characterization and antibacterial activity in milk Peng Wang, Cen Zhang, Yucheng Zou, Yang Li, Hui Zhang PII:
S0268-005X(20)32842-3
DOI:
https://doi.org/10.1016/j.foodhyd.2020.106468
Reference:
FOOHYD 106468
To appear in:
Food Hydrocolloids
Received Date: 22 September 2020 Revised Date:
3 November 2020
Accepted Date: 5 November 2020
Please cite this article as: Wang, P., Zhang, C., Zou, Y., Li, Y., Zhang, H., Immobilization of lysozyme on layer-by-layer self-assembled electrospun films: characterization and antibacterial activity in milk, Food Hydrocolloids, https://doi.org/10.1016/j.foodhyd.2020.106468. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Ltd. All rights reserved.
Author Statement Peng Wang: Conceptualization, Methodology, Investigation, Data curation, Writing-Original draft preparation. Cen Zhang: Formal analysis, Data curation, Software. Yucheng Zou: Formal analysis, Software. Yang Li: Methodology, Data curation, Software.
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Hui Zhang: Project administration, Supervision, Conceptualization, Review and
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Editing, Funding acquisition.
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Graphical Abstract
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Immobilization of lysozyme on layer-by-layer self-assembled
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electrospun films: characterization and antibacterial activity in milk
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Peng Wang a, Cen Zhang a, Yucheng Zou a, Yang Li a, Hui Zhang a,b *
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a
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Agro-Food Processing, Zhejiang University, Hangzhou 310058, China
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College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for
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Ningbo Research Institute, Zhejiang University, Ningbo 315100, China
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* Corresponding author. Tel.: +86-571-88982981; fax: +86-571-88982981. E–mail
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address: [email protected]
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Abstract In this study, the electrospun cellulose acetate (CA) films were alternately
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deposited by lysozyme and sodium alginate (SA) on the nanofiber surface by
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layer-by-layer self-assembly technique. With the increasing number of layers, the
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films presented an increased average fiber diameter from 364 to 611 nm. The
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enhanced thermal stability was observed due to the layer-by-layer electrostatic
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deposition, as well as the formation of hydrogen bonds in the case of the
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CA(Lys-SA)9 film. The immobilized lysozyme exhibited the improved pH and
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temperature resistance, excellent storage stability, and retained over 70% of its initial
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activity after reusing 4 cycles. Moreover, these self-assembled films resulted in a
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substantial decrease in Staphylococcus aureus colonies in ultra-high temperature milk
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at 4 °C and 25 °C, respectively. The results suggested that the immobilization of
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lysozyme on layer-by-layer self-assembled electrospun films with antibacterial
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activity showed promising applications in milk and dairy products.
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Keywords:
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lysozyme; antibacterial activity
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electrospinning;
layer-by-layer
self-assembly;
immobilization;
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1. Introduction Electrospinning is a facile fabrication technique that uses electrostatic force to
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produce polymer fibers with diameters ranging from nano- to micrometer. During
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electrospinning, a polymer solution is extruded by a syringe pump to form a droplet at
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the needle tip where an electric field is applied (Zhang, Li, Wang, & Zhang, 2020).
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When the electric field reaches a critical value, the electrically charged polymer jet is
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ejected from the droplet while the solvent evaporates rapidly, leading to the formation
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of randomly oriented polymer nanofibers deposited on the grounded collector in the
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form of a film (Wang, Li, Zhang, Que, et al., 2020). Due to the advantages of tailored
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morphology, high surface-to-volume ratio, porous and ultrafine structures, the
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electrospun films have been successfully applied in the area of biosensors, drug
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delivery, food packaging, and enzyme immobilization (Zhang, Feng, & Zhang, 2018).
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Recently, the layer-by-layer self-assembly method has been suggested powerful
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and effective to prepare multilayer ultra-thin nanofibrous films via electrospinning
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(Yoo, Kim, & Park, 2009). This method is based on the alternative deposition of
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polyanions and polycations on electrospun films through molecular interactions such
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as electrostatic interactions, hydrogen bonding, covalent bonding (Coustet, et al.,
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2014), resulting in the adjustable porosity, thickness and mechanical strength of the
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layer-by-layer self-assembled electrospun films (Park, Choi, & Hong, 2018). Tu, et al.
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(2019) prepared the electrospun silk fibroin films coated with amphoteric
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carboxymethyl chitosan by layer-by-layer self-assembly technique, and found that the
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fiber average diameter was increased with the increasing bilayers, leading to the
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improved thermal stability and mechanical properties of the multilayer films,
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compared to the silk fibroin film alone. Huang, et al. (2017) electrospun a cellulose
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acetate (CA) film as substrate, which was coated with the cationic naringinase and
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anionic sodium alginate (SA) via layer-by-layer self-assembly method, and found that
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the specific surface area and ratio of mesopores were lower than the electrospun CA
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film, and the activity of the immobilized naringinase increased after multilayer
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coating, leading to the debittering by removal of 22.72% naringin and 60.71%
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limonin from the grapefruit juice.
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Lysozyme is a natural enzyme which can damage bacterial cell walls by catalyzing
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hydrolysis of β(1-4) glycosidic bonds between N-acetylmuramic acid and
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N-acetyl-D-glucosamine in peptidoglycans (Cappannella, et al., 2016). A few
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techniques used for immobilization of lysozyme have been reported, including surface
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attachment, ionic binding, cross-linking, entrapment (Liu, Chen, & Shi, 2018). As
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well, it has been reported that electrospun nanofibrous films as a carrier for
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immobilization of lysozyme may effectively enhance the catalytic efficiency,
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reusability and stability of the enzyme (Siddiqui & Husain, 2019). Park, et al. (2013)
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immobilized lysozyme on the surface of the electrospun chitosan nanofibers which
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were then cross-linked with glutaraldehyde, and found that the immobilized lysozyme
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showed the improved pH and temperature resistance, and kept nearly 75.4% of its
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initial activity after 80 days of storage, compared to the free enzyme which lost all of
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its activity. Zhou, Li, Deng, Hu, and Li (2014) deposited lysozyme and gold
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nanoparticles (GNP) onto the CA film by layer-by-layer self-assembly technique, and
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found that the CA(lysozyme/GNP)5 film had a higher maximum degradation
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temperature than the CA film, and presented a higher inhibition rate against
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Staphylococcus aureus than Escherichia coli. In this work, it was hypothesized that the immobilization of lysozyme on
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layer-by-layer self-assembled electrospun films might affect the enzyme activity by
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modulation of the electrostatically deposited layers. Therefore, the anionic CA
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nanofibers were electrospun as substrate, and then the cationic lysozyme and the
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anionic SA were alternately deposited. The characterization of the films was
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conducted
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thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), Fourier
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transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS).
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The optimum pH and temperature, reusability, and storage stability of the
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immobilized lysozyme were investigated. In addition, the antibacterial activity of the
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films against S. aureus in milk were studied using colony counting method.
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2. Materials and methods
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2.1. Materials
field-emission
scanning
electronic
microscopy
(FE-SEM),
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Lysozyme (20000 U/mg), CA (39.8% acetyl groups, MW ~ 30000 Da) and SA (MW
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~ 398.31 Da) were obtained from Aladdin, Inc. (Shanghai, China). Micrococcus
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lysodeikticus cells were purchased from Yuanye Biological Technology Co., Ltd.
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(Shanghai, China). Coomassie brilliant blue G-250 was obtained from Solarbio
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Science & Technology Co., Ltd. (Beijing, China). Other reagents were purchased
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from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the reagents were
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used without further purification, and deionized water was used throughout the
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experiments.
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2.2. Fabrication of layer-by-layer self-assembled films CA at a concentration of 18% (w/v) was dissolved in an acetone/dimethylacetamide
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(2/1, w/w) mixture solvent under a mild stirring for 3 h. Then the solution was loaded
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into a 5 mL syringe driven by a pump (LSP02-1B, Baoding Longer Precision Pump
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Co., Ltd., China) at a flow rate of 0.5 mL/h. A voltage of 16 kV was then applied at
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the needle tip of the syringe, and a tip-to-collector distance was kept at 10 cm. The
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electrospun nanofibers were collected on a cylindrical rotating drum (200 rpm). The
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electrospinning process was performed at 25 °C with a humidity of approximately 40
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- 50%. The CA films were dried at 60 °C for 24 h under vacuum to remove the trace
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solvent and then hydrolyzed in a 0.05 mol/L NaOH aqueous solution at ambient
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temperature for 7 days (Zhang, et al., 2015).
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The fabrication of layer-by-layer self-assembled films were based on the method
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described by Wu, et al. (2017) with modifications. Initially, the CA films (ζ-potential
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= - 25.2 mV) were immersed in the lysozyme solution (5 mg/mL, + 25.7 mV) for 20
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min, and then washed three times with NaCl solution (0.1 mol/mL). The washed films
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were then moved into the SA solution (1 mg/mL, - 55.9 mV) for 20 min, followed by
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washing three times with NaCl solution. The desired number of layers was obtained
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by repeating the immersing and washing steps. Here, (Lys/SA)n was used for labeling
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the layer-by-layer assembled films, where n was the number of the lysozyme/SA
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bilayers. When the outermost layer was lysozyme, n was 0.5, 4.5 and 8.5,
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respectively.
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2.3. Characterization of nanofibrous films
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2.3.1. Field-emission scanning electronic microscopy (FE-SEM) FE-SEM (SU8010, Hitachi, Japan) was used to observe the fiber morphology of the
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films. Samples were sputtered with a gold-palladium mixture prior to imaging. The
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average fiber diameter was analyzed by measuring 100 fibers in each image using
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Nano Measure software.
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2.3.2. X-ray photoelectron spectroscopy (XPS)
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The surface chemical composition of the films was analyzed on an X-ray
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photoelectron spectroscopy (Escalab 250Xi, Thermo scientific, UK) using an Al Kα
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X-ray source as the excitation source and C 1s as the energy reference.
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2.3.3. Thermal analysis
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Thermogravimetric analysis (TGA) was carried out in a TGA2 instrument
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(Mettler-Toledo, Switzerland) under a nitrogen gas flow (50 mL/min). The films
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(about 7 mg) were heated from 50 to 600 °C at a heating rate of 10 °C/min.
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The thermal properties were analyzed using differential scanning calorimetry (DSC,
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Mettler-Toledo, Switzerland) at a heating rate of 10 °C/min from 25 to 350 °C under
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dynamic nitrogen atmosphere (50 mL/min).
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2.3.4. Fourier transform infrared (FTIR) spectroscopy
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The films (about 3 mg) were mixed with KBr powders and pressed into a pellet for
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the FTIR analysis. The absorption spectra were recorded on a Nicolet iS50
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spectrometer (Thermo Scientific, USA) in the wavenumber range of 400 - 4000 cm-1
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with an average of 32 scans at 2 cm−1 resolution.
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2.4. Lysozyme content measurement The immobilized lysozyme on the films was quantified using a reported method
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(Muriel-Galet, Talbert, Hernandez-Munoz, Gavara, & Goddard, 2013). The films with
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a diameter of 12 mm were submerged in 5 mL of Coomassie brilliant blue G-250
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working reagent and shaken for 30 min. The absorbance was measured at 595 nm
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using a UV spectrophotometer (SP-752, Spectrum Shanghai, China), and a standard
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curve of bovine serum albumin (y = 0.9155x + 0.0137, R2 = 0.998) was used to
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calculate the protein mass per film area (μg of lysozyme per cm2).
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2.5. Lysozyme activity assay
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The lysozyme activity was measured according to the method described by Li, et al.
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(2014) with modifications. The free lysozyme (200 μL) was added into a 2.5 mL M.
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lysodeikticus cell suspension (0.8 mg/mL) in PBS solution (0.066 mol/L, pH 6.24).
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For the immobilized lysozyme, the film disks with a diameter of 6 mm were added
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into the cell suspension. The activities of the free and immobilized lysozyme were
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calculated using the following equations, respectively:
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(
)=
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(
)=
∆
∆
× 1000 (1) × 1000 (2)
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where ΔOD450 is the reduction of absorbance at 450 nm of the cell suspension per
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minute, m refers to the mass (mg) of free lysozyme in 200 μL solution, S is the area
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(cm2) of the film. Then the effects of pH and temperature were evaluated by
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determining the lysozyme activity after 1 h incubation at different pH conditions (3.0
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- 10.0) at room temperature and in the temperature range of 25 - 55 °C at pH 6.24,
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respectively.
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2.6. Storage stability of immobilized lysozyme The storage stability of the immobilized lysozyme was investigated by determining
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the residual activity. The free and immobilized enzymes were prepared in PBS
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solution (0.01 mol/L, pH 6.24) at 4 °C and 25 °C for 40 days, respectively. At
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intervals of five days, their activities with special substrate (M. lysodeikticus) were
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calculated on the same method as described above.
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2.7. Reusability of immobilized lysozyme
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The reusability of the immobilized lysozyme was measured according to the
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method reported by Park, et al. (2013) with modifications. As described above, the
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activity of the immobilized lysozyme was measured with the hydrolysis of M.
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lysodeikticus cells. After each cycle, the film was separated from the suspension and
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washed three times with PBS (0.1 mol/L, pH 6.24), and then placed into a fresh
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reaction solution for the next cycle.
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2.8. Antibacterial activity of lysozyme in milk
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The film cut into round disk with a diameter of 12 mm was sterilized by ultraviolet
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radiation for 15 min, before immersed in 5 mL ultra-high temperature (UHT) milk
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incubated with 1.0 × 106 CFU/mL S. aureus (CMCC 26003) at 4 °C for 72 h or 37 °C
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for 24 h. Then 1 mL of the bacteria suspension was pipetted onto a Petrifilm staph
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express count plate (3M Company, St Paul, Minnesota, USA) and incubated at 37 °C
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for 24 h. After incubation, the total number of colonies was recorded and reported as
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log CFU/mL.
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2.9. Statistical analysis All the measurements were performed at least in triplicate (n = 3). Data were
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expressed as mean ± standard deviation (SD), and analyzed by one-way ANOVA
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followed by Tukey's post-hoc test using DPS software version 7.05. A value of p <
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0.05 was considered statistically significant.
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3. Results and discussion
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3.1. Morphology
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The fiber morphology of the films is shown in Fig. 1. The CA film had smooth and
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bead-free fibers with an average diameter of 364 nm, which increased to 611 nm
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when the number of layers was 9. It could be seen that the self-assembled films
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exhibited a rough fiber surface with irregular protuberances and some cracks,
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indicating the inhomogeneous deposition of lysozyme and SA on the CA fibers (Jiang,
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et al., 2015). These results are in agreement with the findings of Pan et al (2015), who
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reported
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N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) and soy
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protein isolate (SPI) showed an increase in the average fiber diameter from 555 to 808
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nm in the case of the CA(HTCC-SPI)10.5 film.
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3.2. XPS
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that
the
electrospun
CA
film
with
alternate
assembly
of
The XPS spectra of the films are shown in Fig. 2. For the CA film, the binding
energies at 539.58 eV and 292.08 eV represented oxygen (O) and carbon (C),
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respectively. For the self-assembled films, the presence of nitrogen (N, 403.58 eV)
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and sulfur (S, 69.58 eV) were attributed to the positively charged nitrogen atoms and
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four disulfide bonds in lysozyme, suggesting the deposition of the enzyme on the
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films (Li & Peng, 2015). The N ratio of the CA(Lys-SA)8.5 film with lysozyme as the
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outermost layer (13.02%) was higher than that of the CA(Lys-SA)9 film with the SA
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outmost layer (9.50%), indicating the successfully coating of SA on the lysozyme
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layer (Zhou, Hu, Li, & Li, 2014).
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3.3. Thermal analysis
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The TGA curves of the films are shown in Fig. 3A, and the thermal parameters are
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listed in Table 1. Generally, the films exhibited three thermal degradation stages: the
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first stage (0 - 100 °C) of weight loss was related to the water loss, the second stage
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(240 - 400 °C) was associated with the decomposition of the polymeric chains, and
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the third stage (400 - 600 °C) was attributed to the carbonization of the degraded
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products to ash (Zhou, Hu, et al., 2014). The maximum degradation temperature (Tdm)
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of the self-assembled films was lower than that of the CA film, which was mostly due
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to the earlier degradation of lysozyme and SA (Carneiro-da-Cunha, et al., 2010).
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However, the char residue of the CA film at 600 °C was 18.14%, which increased to
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36.28% when the layer number was increased to 9 (Table 1), indicating that the
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enhanced thermal stability of the films by coating with lysozyme and SA (Pan, et al.,
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2015).
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The DSC curves of the films are shown in Fig. 3B, and the corresponding
denaturation temperature (TD) and denaturation enthalpy (ΔHD) are presented in Table
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1. The degradation temperature of the CA film was decreased upon coating, but then
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increased greatly to 306.33 °C in the case of the CA(Lys-SA)9 film, indicating the
250
enhanced thermal stability after the layer-by-layer deposition. Wu, et al. (2008)
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reported that the melting temperature of the [poly(acrylic acid)/graphite oxide]13 film
252
(136.1°C) was higher than that of the poly(acrylic acid)/graphite oxide]3 film
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(133.9 °C), indicating that the interaction between the carbon layer and the
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poly(acrylic acid) layer was enhanced.
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3.4. FTIR analysis
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The FTIR spectra of the films are shown in Fig. 4. The characteristic bands of the
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CA film at 3416, 1383, 1263, and 1066 cm-1 were due to the stretching of −OH, the
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stretching of −C−CH3, the asymmetric stretching of C−O−C bond, and the stretching
259
of C–O, respectively (Babar, et al., 2018; Wang, Li, Zhang, Feng, & Zhang, 2020).
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For the self-assembled films, the absorption bands at 1647 - 1657 cm−1 (amide I) and
261
at 1536 cm-1 (amide II) belonged to the stretching vibration of C=O and the stretching
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of C−N and bending of N−H of lysozyme, respectively. In the case of the
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CA(Lys-SA)9 film, the absorption band at 3416 cm−1 shifted to 3424 cm−1, and a
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broadening band with an enhanced intensity in the range of 3340 - 3600 cm−1 (the
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−OH and −NH stretching vibrations) was observed, indicating the formation of
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hydrogen bonds (Aceituno-Medina, Mendoza, Lagaron, & López-Rubio, 2013). Luo,
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et al. (2012) fabricated the multilayer chitosan (CS)/lignosulfonate (LGS) film by
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layer-by-layer self-assembly method, and found that the peak at 3410 cm-1 of the
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(CS/LGS)22 film was broader than that of CS and LGS, indicating the formation of
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hydrogen bonds between CS and LGS.
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3.5. Lysozyme content and activity The lysozyme content and activity of the films are shown in Table 2. With the
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increasing bilayer number, the lysozyme content was increased from 97.10 to 822.55
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μg/cm2, but the enzymatic activity of the films with SA as the outermost layer was
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lower than that of the films with the lysozyme outermost layer, which could be related
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to the reduced molecular flexibility of lysozyme as well as the hindrance and
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destruction of some active sites during the self-assembly process (Bayazidi, Almasi,
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& Asl, 2018). Huang, et al. (2012) reported that lysozyme-CS-organic rectorite
279
(OREC) and SA were deposited on the CA films by layer-by-layer self-assembly
280
technique, and found that the CA(lysozyme-CS-OREC/SA)10.5 film had a higher
281
lysozyme activity than the CA(lysozyme-CS-OREC/SA)10 film, indicating that
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lysozyme was easier to leach from the films when it was on the outermost layer.
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3.6. Effect of pH and temperature
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The residual activities of the free and immobilized lysozyme at varying pH
285
conditions are shown in Fig. 5. The optimum pH value of free lysozyme was 6, while
286
the immobilized lysozyme on the films was 7. However, the residual activities of the
287
lysozyme immobilized on the CA(Lys-SA)8.5 (94.81%) and CA(Lys-SA)9 films
288
(92.76%) at pH 8 were significantly higher than that of free lysozyme (81.44%),
289
indicating that the immobilization improved the enzyme stability in alkaline
290
environments (Lian, Ma, Wei, & Liu, 2012).
The effect of temperature on the lysozyme activity is shown in Fig. 5. The optimum
292
temperatures for the free and immobilized lysozymes were all 40 °C, but the residual
293
activities of the immobilized lysozyme were higher than that of free lysozyme at the
294
temperature ranging from 40 to 55 °C. This might be ascribed to the decreased
295
conformational flexibility of lysozyme by immobilization on the film, leading to the
296
reduced ability of thermal denaturation (Xue, Chen, Li, Liu, & Li, 2019).
297
3.7. Storage stability of immobilized lysozyme
of
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The storage stability of the free and immobilized lysozyme is shown in Fig. 6. The
299
activity loss of the immobilized lysozyme was less than 66% and 46% after storage
300
for 40 days at 4 °C and 25 °C, respectively, compared with free enzyme which lost
301
almost all of its activity. This suggested that the storage stability of lysozyme was
302
enhanced by the immobilization process, and the immobilized lysozyme retained a
303
high residual activity possibly due to the minimal distortion effect on its active site
304
(Park, et al., 2013). Guedidi, et al. (2010) immobilized trypsin on the
305
polyacrylonitrile-based films by layer-by-layer self-assembly technique, and found
306
that the free enzyme kept nearly 30% of its initial activity after 100 days while the
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self-assembled film had 60% of the initial activity, indicating the increased stability
308
by immobilizing the enzyme.
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3.8. Reusability of immobilized lysozyme
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The reusability of the immobilized lysozyme is shown in Fig. 7. After 4 cycles, the
311
residual lysozyme activities of the CA(Lys-SA)8.5 and CA(Lys-SA)9 films were 73.90%
312
and 72.53%, which were decreased to 21.78% and 19.80% after 10 cycles,
313
respectively.
314
Sarma, Islam, Miller, and Bhattacharyya (2017) immobilized laccase on the
315
polyacrylic acid/functionalized polyvinylidene fluoride films by layer-by-layer
316
self-assembly technique, and found that the enzyme lost 14% of its initial activity
317
after 4 cycles, indicating that the oxidation products produced by the degradation of
318
the substrate led to the decrease in the lysozyme activity after repetitive operation. Liu,
319
Wang,
320
catalase/poly(diallyldimethylammonium
321
layer-by-layer electrostatic self-assembly deposition, and found that the residual
322
activity of the immobilized catalase was nearly 30% after 6 cycles, indicating good
323
reusability of the immobilized enzyme.
324
3.9. Antibacterial activity in milk
Huang
(2013)
fabricated
of
and
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chloride)-assembled
wool
fabrics
the via
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Fan,
The antibacterial activities of the films against S. aureus in UHT milk are shown in
326
Fig. 8. Obviously, the CA film showed no inhibition activity, but the self-assembled
327
films exhibited substantial antibacterial activities against S. aureus due to the
328
contact-killing capability of lysozyme. As the layer number was increased to 9, the
329
bacteria colonies in UHT milk at 4 °C and 25 °C were decreased from 7.06 and 7.67
330
to 2.17 and 2.31 log CFU/mL, respectively. Zhang, et al. (2015) fabricated the
331
electrospun CA films coated with lysozyme/pectin bilayers by layer-by-layer
332
self-assembly technique, and found that the growth inhibition of the films against S.
333
aureus was increased with the increasing number of bilayers, in proportional to the
334
quantity of the immobilized lysozyme. Huang, et al. (2012) fabricated the CA films
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335
deposited with lysozyme-CS-OREC and SA by layer-by-layer self-assembly
336
technique, and found that the multilayer films exhibited excellent antibacterial
337
activities against E. coli and S. aureus, and could prolong the shelf life of fresh pork
338
up to 12 days.
339
4. Conclusions In this work, the layer-by-layer self-assembled CA(Lys-SA)n films were prepared
341
based on the electrospun CA nanofibers as substrate. Generally, the increasing number
342
of layers resulted in the increased average fiber diameter with enhanced thermal
343
stability. In the case of the CA(Lys-SA)9 film, the average fiber diameter was 611 nm,
344
and a higher degradation temperature was obtained due to the formation of hydrogen
345
bonds. It was observed that the enzymatic activity of the films with the SA outermost
346
layer was lower than that of the films with lysozyme as the outermost layer. However,
347
the immobilized lysozyme exhibited better pH and temperature resistance, excellent
348
storage stability, acceptable reusability, and antibacterial activity against S. aureus in
349
UHT milk. These results suggested the potentials of immobilized lysozyme on
350
layer-by-layer self-assembled electrospun films to extend the shelf-life of milk and
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dairy products, but more work is needed to carefully assess the practical applications
352
as part of food antimicrobial packaging in future studies.
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Acknowledgement
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The research was supported by the National Natural Science Foundation of China
(Grant No. 31772013).
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Figure captions
479
Fig. 1. FE-SEM images of the (A) CA, (B) CA(Lys-SA)0.5, (C) CA(Lys-SA)1, (D)
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CA(Lys-SA)4.5, (E) CA(Lys-SA)5, (F) CA(Lys-SA)8.5, and (G) CA(Lys-SA)9 films.
481
Fig. 2. XPS spectra of the films.
482
Fig. 3. (A) TGA and (B) DSC curves of the films.
483
Fig. 4. FTIR spectra of the films.
484
Fig. 5. Effect of pH (A) and temperature (B) on the activities of the free and
485
immobilized lysozyme. Values with different lowercase letters represent significant
486
differences (p < 0.05).
487
Fig. 6. Storage stability of the free and immobilized lysozyme at 25 °C (A) and 4 °C
488
(B).
489
Fig. 7. Reusability of the immobilized lysozyme on the CA(Lys-SA)8.5 and
490
CA(Lys-SA)9 films.
491
Fig. 8. Bacterial count (log CFU/mL) of S. aureus in UHT milk after storage at 25 °C
492
and 4 °C.
493
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Figures
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Fig. 1.
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Fig. 2.
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Fig. 3.
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(A)
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Fig. 4.
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Fig. 5.
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Fig. 6.
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Fig. 7.
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Tables
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Table 1. TGA and DSC data of the films.
TGA
534
TD (°C)
ΔHD (J/g)
CA
349.50
18.14
321.67
- 55.72
CA(Lys-SA)0.5
327.67
27.77
271.67
- 25.81
CA(Lys-SA)1
329.38
32.56
CA(Lys-SA)4.5
329.73
CA(Lys-SA)5
329.50
CA(Lys-SA)8.5
329.33
CA(Lys-SA)9
329.67
274.98
- 83.25
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Residue at 600 °C (%)
275.01
- 75.59
35.24
275.15
- 28.81
35.27
280.70
- 37.06
36.28
306.33
- 5.68
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533
Tdm (°C)
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DSC
Table 2. Lysozyme content and activity of the films.
Lysozyme activity (U/cm2)
CA(Lys-SA)0.5
104.53 ± 20.40c
10.04 ± 0.41c
CA(Lys-SA)1
97.10 ± 28.53c
6.46 ± 0.30d
CA(Lys-SA)4.5
254.87 ± 99.23b
11.5 ± 0.62b
CA(Lys-SA)5
264.90 ± 35.53b
9.83 ± 0.15c
CA(Lys-SA)8.5
786.97 ± 20.69a
CA(Lys-SA)9
822.55 ± 70.43a
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Different letters in each column indicate significant differences (p < 0.05).
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Lysozyme content (μg/cm2)
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535
15.40 ± 0.79a
12.42 ± 0.28b
Highlights
Layer-by-layer self-assembled films were prepared based on electrospun nanofibers. The deposition induced the increased fiber diameter with enhanced thermal stability.
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The immobilized lysozyme showed good storage stability and acceptable
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reusability.
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The self-assembled films exhibited strong antibacterial activity in UHT milk.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0268-005X(20)32842-3
DOI:
https://doi.org/10.1016/j.foodhyd.2020.106468
Reference:
FOOHYD 106468
To appear in:
Food Hydrocolloids
Received Date: 22 September 2020 Revised Date:
3 November 2020
Accepted Date: 5 November 2020
Please cite this article as: Wang, P., Zhang, C., Zou, Y., Li, Y., Zhang, H., Immobilization of lysozyme on layer-by-layer self-assembled electrospun films: characterization and antibacterial activity in milk, Food Hydrocolloids, https://doi.org/10.1016/j.foodhyd.2020.106468. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Ltd. All rights reserved.
Author Statement Peng Wang: Conceptualization, Methodology, Investigation, Data curation, Writing-Original draft preparation. Cen Zhang: Formal analysis, Data curation, Software. Yucheng Zou: Formal analysis, Software. Yang Li: Methodology, Data curation, Software.
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Hui Zhang: Project administration, Supervision, Conceptualization, Review and
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Editing, Funding acquisition.
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Graphical Abstract
1
Immobilization of lysozyme on layer-by-layer self-assembled
2
electrospun films: characterization and antibacterial activity in milk
3 4
Peng Wang a, Cen Zhang a, Yucheng Zou a, Yang Li a, Hui Zhang a,b *
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a
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Agro-Food Processing, Zhejiang University, Hangzhou 310058, China
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b
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College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for
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Ningbo Research Institute, Zhejiang University, Ningbo 315100, China
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* Corresponding author. Tel.: +86-571-88982981; fax: +86-571-88982981. E–mail
11
address: [email protected]
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Abstract In this study, the electrospun cellulose acetate (CA) films were alternately
15
deposited by lysozyme and sodium alginate (SA) on the nanofiber surface by
16
layer-by-layer self-assembly technique. With the increasing number of layers, the
17
films presented an increased average fiber diameter from 364 to 611 nm. The
18
enhanced thermal stability was observed due to the layer-by-layer electrostatic
19
deposition, as well as the formation of hydrogen bonds in the case of the
20
CA(Lys-SA)9 film. The immobilized lysozyme exhibited the improved pH and
21
temperature resistance, excellent storage stability, and retained over 70% of its initial
22
activity after reusing 4 cycles. Moreover, these self-assembled films resulted in a
23
substantial decrease in Staphylococcus aureus colonies in ultra-high temperature milk
24
at 4 °C and 25 °C, respectively. The results suggested that the immobilization of
25
lysozyme on layer-by-layer self-assembled electrospun films with antibacterial
26
activity showed promising applications in milk and dairy products.
27
Keywords:
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lysozyme; antibacterial activity
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electrospinning;
layer-by-layer
self-assembly;
immobilization;
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1. Introduction Electrospinning is a facile fabrication technique that uses electrostatic force to
35
produce polymer fibers with diameters ranging from nano- to micrometer. During
36
electrospinning, a polymer solution is extruded by a syringe pump to form a droplet at
37
the needle tip where an electric field is applied (Zhang, Li, Wang, & Zhang, 2020).
38
When the electric field reaches a critical value, the electrically charged polymer jet is
39
ejected from the droplet while the solvent evaporates rapidly, leading to the formation
40
of randomly oriented polymer nanofibers deposited on the grounded collector in the
41
form of a film (Wang, Li, Zhang, Que, et al., 2020). Due to the advantages of tailored
42
morphology, high surface-to-volume ratio, porous and ultrafine structures, the
43
electrospun films have been successfully applied in the area of biosensors, drug
44
delivery, food packaging, and enzyme immobilization (Zhang, Feng, & Zhang, 2018).
45
Recently, the layer-by-layer self-assembly method has been suggested powerful
46
and effective to prepare multilayer ultra-thin nanofibrous films via electrospinning
47
(Yoo, Kim, & Park, 2009). This method is based on the alternative deposition of
48
polyanions and polycations on electrospun films through molecular interactions such
49
as electrostatic interactions, hydrogen bonding, covalent bonding (Coustet, et al.,
50
2014), resulting in the adjustable porosity, thickness and mechanical strength of the
51
layer-by-layer self-assembled electrospun films (Park, Choi, & Hong, 2018). Tu, et al.
52
(2019) prepared the electrospun silk fibroin films coated with amphoteric
53
carboxymethyl chitosan by layer-by-layer self-assembly technique, and found that the
54
fiber average diameter was increased with the increasing bilayers, leading to the
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improved thermal stability and mechanical properties of the multilayer films,
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compared to the silk fibroin film alone. Huang, et al. (2017) electrospun a cellulose
57
acetate (CA) film as substrate, which was coated with the cationic naringinase and
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anionic sodium alginate (SA) via layer-by-layer self-assembly method, and found that
59
the specific surface area and ratio of mesopores were lower than the electrospun CA
60
film, and the activity of the immobilized naringinase increased after multilayer
61
coating, leading to the debittering by removal of 22.72% naringin and 60.71%
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limonin from the grapefruit juice.
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Lysozyme is a natural enzyme which can damage bacterial cell walls by catalyzing
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hydrolysis of β(1-4) glycosidic bonds between N-acetylmuramic acid and
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N-acetyl-D-glucosamine in peptidoglycans (Cappannella, et al., 2016). A few
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techniques used for immobilization of lysozyme have been reported, including surface
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attachment, ionic binding, cross-linking, entrapment (Liu, Chen, & Shi, 2018). As
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well, it has been reported that electrospun nanofibrous films as a carrier for
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immobilization of lysozyme may effectively enhance the catalytic efficiency,
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reusability and stability of the enzyme (Siddiqui & Husain, 2019). Park, et al. (2013)
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immobilized lysozyme on the surface of the electrospun chitosan nanofibers which
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were then cross-linked with glutaraldehyde, and found that the immobilized lysozyme
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showed the improved pH and temperature resistance, and kept nearly 75.4% of its
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initial activity after 80 days of storage, compared to the free enzyme which lost all of
75
its activity. Zhou, Li, Deng, Hu, and Li (2014) deposited lysozyme and gold
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nanoparticles (GNP) onto the CA film by layer-by-layer self-assembly technique, and
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found that the CA(lysozyme/GNP)5 film had a higher maximum degradation
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temperature than the CA film, and presented a higher inhibition rate against
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Staphylococcus aureus than Escherichia coli. In this work, it was hypothesized that the immobilization of lysozyme on
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layer-by-layer self-assembled electrospun films might affect the enzyme activity by
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modulation of the electrostatically deposited layers. Therefore, the anionic CA
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nanofibers were electrospun as substrate, and then the cationic lysozyme and the
84
anionic SA were alternately deposited. The characterization of the films was
85
conducted
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thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), Fourier
87
transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS).
88
The optimum pH and temperature, reusability, and storage stability of the
89
immobilized lysozyme were investigated. In addition, the antibacterial activity of the
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films against S. aureus in milk were studied using colony counting method.
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2. Materials and methods
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2.1. Materials
field-emission
scanning
electronic
microscopy
(FE-SEM),
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Lysozyme (20000 U/mg), CA (39.8% acetyl groups, MW ~ 30000 Da) and SA (MW
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~ 398.31 Da) were obtained from Aladdin, Inc. (Shanghai, China). Micrococcus
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lysodeikticus cells were purchased from Yuanye Biological Technology Co., Ltd.
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(Shanghai, China). Coomassie brilliant blue G-250 was obtained from Solarbio
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Science & Technology Co., Ltd. (Beijing, China). Other reagents were purchased
98
from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the reagents were
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used without further purification, and deionized water was used throughout the
100
experiments.
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2.2. Fabrication of layer-by-layer self-assembled films CA at a concentration of 18% (w/v) was dissolved in an acetone/dimethylacetamide
103
(2/1, w/w) mixture solvent under a mild stirring for 3 h. Then the solution was loaded
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into a 5 mL syringe driven by a pump (LSP02-1B, Baoding Longer Precision Pump
105
Co., Ltd., China) at a flow rate of 0.5 mL/h. A voltage of 16 kV was then applied at
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the needle tip of the syringe, and a tip-to-collector distance was kept at 10 cm. The
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electrospun nanofibers were collected on a cylindrical rotating drum (200 rpm). The
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electrospinning process was performed at 25 °C with a humidity of approximately 40
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- 50%. The CA films were dried at 60 °C for 24 h under vacuum to remove the trace
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solvent and then hydrolyzed in a 0.05 mol/L NaOH aqueous solution at ambient
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temperature for 7 days (Zhang, et al., 2015).
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The fabrication of layer-by-layer self-assembled films were based on the method
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described by Wu, et al. (2017) with modifications. Initially, the CA films (ζ-potential
114
= - 25.2 mV) were immersed in the lysozyme solution (5 mg/mL, + 25.7 mV) for 20
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min, and then washed three times with NaCl solution (0.1 mol/mL). The washed films
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were then moved into the SA solution (1 mg/mL, - 55.9 mV) for 20 min, followed by
117
washing three times with NaCl solution. The desired number of layers was obtained
118
by repeating the immersing and washing steps. Here, (Lys/SA)n was used for labeling
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the layer-by-layer assembled films, where n was the number of the lysozyme/SA
120
bilayers. When the outermost layer was lysozyme, n was 0.5, 4.5 and 8.5,
121
respectively.
122
2.3. Characterization of nanofibrous films
123
2.3.1. Field-emission scanning electronic microscopy (FE-SEM) FE-SEM (SU8010, Hitachi, Japan) was used to observe the fiber morphology of the
125
films. Samples were sputtered with a gold-palladium mixture prior to imaging. The
126
average fiber diameter was analyzed by measuring 100 fibers in each image using
127
Nano Measure software.
128
2.3.2. X-ray photoelectron spectroscopy (XPS)
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The surface chemical composition of the films was analyzed on an X-ray
130
photoelectron spectroscopy (Escalab 250Xi, Thermo scientific, UK) using an Al Kα
131
X-ray source as the excitation source and C 1s as the energy reference.
132
2.3.3. Thermal analysis
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Thermogravimetric analysis (TGA) was carried out in a TGA2 instrument
134
(Mettler-Toledo, Switzerland) under a nitrogen gas flow (50 mL/min). The films
135
(about 7 mg) were heated from 50 to 600 °C at a heating rate of 10 °C/min.
Jo
133
136
The thermal properties were analyzed using differential scanning calorimetry (DSC,
137
Mettler-Toledo, Switzerland) at a heating rate of 10 °C/min from 25 to 350 °C under
138
dynamic nitrogen atmosphere (50 mL/min).
139
2.3.4. Fourier transform infrared (FTIR) spectroscopy
140
The films (about 3 mg) were mixed with KBr powders and pressed into a pellet for
141
the FTIR analysis. The absorption spectra were recorded on a Nicolet iS50
142
spectrometer (Thermo Scientific, USA) in the wavenumber range of 400 - 4000 cm-1
143
with an average of 32 scans at 2 cm−1 resolution.
144
2.4. Lysozyme content measurement The immobilized lysozyme on the films was quantified using a reported method
146
(Muriel-Galet, Talbert, Hernandez-Munoz, Gavara, & Goddard, 2013). The films with
147
a diameter of 12 mm were submerged in 5 mL of Coomassie brilliant blue G-250
148
working reagent and shaken for 30 min. The absorbance was measured at 595 nm
149
using a UV spectrophotometer (SP-752, Spectrum Shanghai, China), and a standard
150
curve of bovine serum albumin (y = 0.9155x + 0.0137, R2 = 0.998) was used to
151
calculate the protein mass per film area (μg of lysozyme per cm2).
152
2.5. Lysozyme activity assay
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The lysozyme activity was measured according to the method described by Li, et al.
154
(2014) with modifications. The free lysozyme (200 μL) was added into a 2.5 mL M.
155
lysodeikticus cell suspension (0.8 mg/mL) in PBS solution (0.066 mol/L, pH 6.24).
156
For the immobilized lysozyme, the film disks with a diameter of 6 mm were added
157
into the cell suspension. The activities of the free and immobilized lysozyme were
158
calculated using the following equations, respectively:
Jo
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153
159
(
)=
160
(
)=
∆
∆
× 1000 (1) × 1000 (2)
161
where ΔOD450 is the reduction of absorbance at 450 nm of the cell suspension per
162
minute, m refers to the mass (mg) of free lysozyme in 200 μL solution, S is the area
163
(cm2) of the film. Then the effects of pH and temperature were evaluated by
164
determining the lysozyme activity after 1 h incubation at different pH conditions (3.0
165
- 10.0) at room temperature and in the temperature range of 25 - 55 °C at pH 6.24,
166
respectively.
167
2.6. Storage stability of immobilized lysozyme The storage stability of the immobilized lysozyme was investigated by determining
169
the residual activity. The free and immobilized enzymes were prepared in PBS
170
solution (0.01 mol/L, pH 6.24) at 4 °C and 25 °C for 40 days, respectively. At
171
intervals of five days, their activities with special substrate (M. lysodeikticus) were
172
calculated on the same method as described above.
191
2.7. Reusability of immobilized lysozyme
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The reusability of the immobilized lysozyme was measured according to the
193
method reported by Park, et al. (2013) with modifications. As described above, the
194
activity of the immobilized lysozyme was measured with the hydrolysis of M.
195
lysodeikticus cells. After each cycle, the film was separated from the suspension and
196
washed three times with PBS (0.1 mol/L, pH 6.24), and then placed into a fresh
197
reaction solution for the next cycle.
198
2.8. Antibacterial activity of lysozyme in milk
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199
The film cut into round disk with a diameter of 12 mm was sterilized by ultraviolet
200
radiation for 15 min, before immersed in 5 mL ultra-high temperature (UHT) milk
201
incubated with 1.0 × 106 CFU/mL S. aureus (CMCC 26003) at 4 °C for 72 h or 37 °C
202
for 24 h. Then 1 mL of the bacteria suspension was pipetted onto a Petrifilm staph
203
express count plate (3M Company, St Paul, Minnesota, USA) and incubated at 37 °C
204
for 24 h. After incubation, the total number of colonies was recorded and reported as
205
log CFU/mL.
206
2.9. Statistical analysis All the measurements were performed at least in triplicate (n = 3). Data were
208
expressed as mean ± standard deviation (SD), and analyzed by one-way ANOVA
209
followed by Tukey's post-hoc test using DPS software version 7.05. A value of p <
210
0.05 was considered statistically significant.
211
3. Results and discussion
212
3.1. Morphology
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The fiber morphology of the films is shown in Fig. 1. The CA film had smooth and
214
bead-free fibers with an average diameter of 364 nm, which increased to 611 nm
215
when the number of layers was 9. It could be seen that the self-assembled films
216
exhibited a rough fiber surface with irregular protuberances and some cracks,
217
indicating the inhomogeneous deposition of lysozyme and SA on the CA fibers (Jiang,
218
et al., 2015). These results are in agreement with the findings of Pan et al (2015), who
219
reported
220
N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) and soy
221
protein isolate (SPI) showed an increase in the average fiber diameter from 555 to 808
222
nm in the case of the CA(HTCC-SPI)10.5 film.
223
3.2. XPS
224
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213
that
the
electrospun
CA
film
with
alternate
assembly
of
The XPS spectra of the films are shown in Fig. 2. For the CA film, the binding
energies at 539.58 eV and 292.08 eV represented oxygen (O) and carbon (C),
226
respectively. For the self-assembled films, the presence of nitrogen (N, 403.58 eV)
227
and sulfur (S, 69.58 eV) were attributed to the positively charged nitrogen atoms and
228
four disulfide bonds in lysozyme, suggesting the deposition of the enzyme on the
229
films (Li & Peng, 2015). The N ratio of the CA(Lys-SA)8.5 film with lysozyme as the
230
outermost layer (13.02%) was higher than that of the CA(Lys-SA)9 film with the SA
231
outmost layer (9.50%), indicating the successfully coating of SA on the lysozyme
232
layer (Zhou, Hu, Li, & Li, 2014).
233
3.3. Thermal analysis
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The TGA curves of the films are shown in Fig. 3A, and the thermal parameters are
235
listed in Table 1. Generally, the films exhibited three thermal degradation stages: the
236
first stage (0 - 100 °C) of weight loss was related to the water loss, the second stage
237
(240 - 400 °C) was associated with the decomposition of the polymeric chains, and
238
the third stage (400 - 600 °C) was attributed to the carbonization of the degraded
239
products to ash (Zhou, Hu, et al., 2014). The maximum degradation temperature (Tdm)
240
of the self-assembled films was lower than that of the CA film, which was mostly due
241
to the earlier degradation of lysozyme and SA (Carneiro-da-Cunha, et al., 2010).
242
However, the char residue of the CA film at 600 °C was 18.14%, which increased to
243
36.28% when the layer number was increased to 9 (Table 1), indicating that the
244
enhanced thermal stability of the films by coating with lysozyme and SA (Pan, et al.,
245
2015).
246
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234
The DSC curves of the films are shown in Fig. 3B, and the corresponding
denaturation temperature (TD) and denaturation enthalpy (ΔHD) are presented in Table
248
1. The degradation temperature of the CA film was decreased upon coating, but then
249
increased greatly to 306.33 °C in the case of the CA(Lys-SA)9 film, indicating the
250
enhanced thermal stability after the layer-by-layer deposition. Wu, et al. (2008)
251
reported that the melting temperature of the [poly(acrylic acid)/graphite oxide]13 film
252
(136.1°C) was higher than that of the poly(acrylic acid)/graphite oxide]3 film
253
(133.9 °C), indicating that the interaction between the carbon layer and the
254
poly(acrylic acid) layer was enhanced.
255
3.4. FTIR analysis
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of
247
The FTIR spectra of the films are shown in Fig. 4. The characteristic bands of the
257
CA film at 3416, 1383, 1263, and 1066 cm-1 were due to the stretching of −OH, the
258
stretching of −C−CH3, the asymmetric stretching of C−O−C bond, and the stretching
259
of C–O, respectively (Babar, et al., 2018; Wang, Li, Zhang, Feng, & Zhang, 2020).
260
For the self-assembled films, the absorption bands at 1647 - 1657 cm−1 (amide I) and
261
at 1536 cm-1 (amide II) belonged to the stretching vibration of C=O and the stretching
262
of C−N and bending of N−H of lysozyme, respectively. In the case of the
263
CA(Lys-SA)9 film, the absorption band at 3416 cm−1 shifted to 3424 cm−1, and a
264
broadening band with an enhanced intensity in the range of 3340 - 3600 cm−1 (the
265
−OH and −NH stretching vibrations) was observed, indicating the formation of
266
hydrogen bonds (Aceituno-Medina, Mendoza, Lagaron, & López-Rubio, 2013). Luo,
267
et al. (2012) fabricated the multilayer chitosan (CS)/lignosulfonate (LGS) film by
268
layer-by-layer self-assembly method, and found that the peak at 3410 cm-1 of the
Jo
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256
269
(CS/LGS)22 film was broader than that of CS and LGS, indicating the formation of
270
hydrogen bonds between CS and LGS.
271
3.5. Lysozyme content and activity The lysozyme content and activity of the films are shown in Table 2. With the
273
increasing bilayer number, the lysozyme content was increased from 97.10 to 822.55
274
μg/cm2, but the enzymatic activity of the films with SA as the outermost layer was
275
lower than that of the films with the lysozyme outermost layer, which could be related
276
to the reduced molecular flexibility of lysozyme as well as the hindrance and
277
destruction of some active sites during the self-assembly process (Bayazidi, Almasi,
278
& Asl, 2018). Huang, et al. (2012) reported that lysozyme-CS-organic rectorite
279
(OREC) and SA were deposited on the CA films by layer-by-layer self-assembly
280
technique, and found that the CA(lysozyme-CS-OREC/SA)10.5 film had a higher
281
lysozyme activity than the CA(lysozyme-CS-OREC/SA)10 film, indicating that
282
lysozyme was easier to leach from the films when it was on the outermost layer.
283
3.6. Effect of pH and temperature
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284
The residual activities of the free and immobilized lysozyme at varying pH
285
conditions are shown in Fig. 5. The optimum pH value of free lysozyme was 6, while
286
the immobilized lysozyme on the films was 7. However, the residual activities of the
287
lysozyme immobilized on the CA(Lys-SA)8.5 (94.81%) and CA(Lys-SA)9 films
288
(92.76%) at pH 8 were significantly higher than that of free lysozyme (81.44%),
289
indicating that the immobilization improved the enzyme stability in alkaline
290
environments (Lian, Ma, Wei, & Liu, 2012).
The effect of temperature on the lysozyme activity is shown in Fig. 5. The optimum
292
temperatures for the free and immobilized lysozymes were all 40 °C, but the residual
293
activities of the immobilized lysozyme were higher than that of free lysozyme at the
294
temperature ranging from 40 to 55 °C. This might be ascribed to the decreased
295
conformational flexibility of lysozyme by immobilization on the film, leading to the
296
reduced ability of thermal denaturation (Xue, Chen, Li, Liu, & Li, 2019).
297
3.7. Storage stability of immobilized lysozyme
of
291
The storage stability of the free and immobilized lysozyme is shown in Fig. 6. The
299
activity loss of the immobilized lysozyme was less than 66% and 46% after storage
300
for 40 days at 4 °C and 25 °C, respectively, compared with free enzyme which lost
301
almost all of its activity. This suggested that the storage stability of lysozyme was
302
enhanced by the immobilization process, and the immobilized lysozyme retained a
303
high residual activity possibly due to the minimal distortion effect on its active site
304
(Park, et al., 2013). Guedidi, et al. (2010) immobilized trypsin on the
305
polyacrylonitrile-based films by layer-by-layer self-assembly technique, and found
306
that the free enzyme kept nearly 30% of its initial activity after 100 days while the
307
self-assembled film had 60% of the initial activity, indicating the increased stability
308
by immobilizing the enzyme.
309
3.8. Reusability of immobilized lysozyme
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310
The reusability of the immobilized lysozyme is shown in Fig. 7. After 4 cycles, the
311
residual lysozyme activities of the CA(Lys-SA)8.5 and CA(Lys-SA)9 films were 73.90%
312
and 72.53%, which were decreased to 21.78% and 19.80% after 10 cycles,
313
respectively.
314
Sarma, Islam, Miller, and Bhattacharyya (2017) immobilized laccase on the
315
polyacrylic acid/functionalized polyvinylidene fluoride films by layer-by-layer
316
self-assembly technique, and found that the enzyme lost 14% of its initial activity
317
after 4 cycles, indicating that the oxidation products produced by the degradation of
318
the substrate led to the decrease in the lysozyme activity after repetitive operation. Liu,
319
Wang,
320
catalase/poly(diallyldimethylammonium
321
layer-by-layer electrostatic self-assembly deposition, and found that the residual
322
activity of the immobilized catalase was nearly 30% after 6 cycles, indicating good
323
reusability of the immobilized enzyme.
324
3.9. Antibacterial activity in milk
Huang
(2013)
fabricated
of
and
ro
Sun,
chloride)-assembled
wool
fabrics
the via
na
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re
-p
Fan,
The antibacterial activities of the films against S. aureus in UHT milk are shown in
326
Fig. 8. Obviously, the CA film showed no inhibition activity, but the self-assembled
327
films exhibited substantial antibacterial activities against S. aureus due to the
328
contact-killing capability of lysozyme. As the layer number was increased to 9, the
329
bacteria colonies in UHT milk at 4 °C and 25 °C were decreased from 7.06 and 7.67
330
to 2.17 and 2.31 log CFU/mL, respectively. Zhang, et al. (2015) fabricated the
331
electrospun CA films coated with lysozyme/pectin bilayers by layer-by-layer
332
self-assembly technique, and found that the growth inhibition of the films against S.
333
aureus was increased with the increasing number of bilayers, in proportional to the
334
quantity of the immobilized lysozyme. Huang, et al. (2012) fabricated the CA films
Jo
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325
335
deposited with lysozyme-CS-OREC and SA by layer-by-layer self-assembly
336
technique, and found that the multilayer films exhibited excellent antibacterial
337
activities against E. coli and S. aureus, and could prolong the shelf life of fresh pork
338
up to 12 days.
339
4. Conclusions In this work, the layer-by-layer self-assembled CA(Lys-SA)n films were prepared
341
based on the electrospun CA nanofibers as substrate. Generally, the increasing number
342
of layers resulted in the increased average fiber diameter with enhanced thermal
343
stability. In the case of the CA(Lys-SA)9 film, the average fiber diameter was 611 nm,
344
and a higher degradation temperature was obtained due to the formation of hydrogen
345
bonds. It was observed that the enzymatic activity of the films with the SA outermost
346
layer was lower than that of the films with lysozyme as the outermost layer. However,
347
the immobilized lysozyme exhibited better pH and temperature resistance, excellent
348
storage stability, acceptable reusability, and antibacterial activity against S. aureus in
349
UHT milk. These results suggested the potentials of immobilized lysozyme on
350
layer-by-layer self-assembled electrospun films to extend the shelf-life of milk and
351
dairy products, but more work is needed to carefully assess the practical applications
352
as part of food antimicrobial packaging in future studies.
353
Acknowledgement
354
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of
340
The research was supported by the National Natural Science Foundation of China
(Grant No. 31772013).
356
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for antibacterial application. Carbohydrate Polymers, 117, 687-693. Zhou, B., Hu, Y., Li, J., & Li, B. (2014). Chitosan/phosvitin antibacterial films
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fabricated via layer-by-layer deposition. International Journal of Biological
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Macromolecules, 64, 402-408.
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Zhou, B., Li, Y., Deng, H., Hu, Y., & Li, B. (2014). Antibacterial multilayer films
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fabricated by layer-by-layer immobilizing lysozyme and gold nanoparticles on
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nanofibers. Colloids Surfaces B: Biointerfaces, 116, 432-438.
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Figure captions
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Fig. 1. FE-SEM images of the (A) CA, (B) CA(Lys-SA)0.5, (C) CA(Lys-SA)1, (D)
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CA(Lys-SA)4.5, (E) CA(Lys-SA)5, (F) CA(Lys-SA)8.5, and (G) CA(Lys-SA)9 films.
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Fig. 2. XPS spectra of the films.
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Fig. 3. (A) TGA and (B) DSC curves of the films.
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Fig. 4. FTIR spectra of the films.
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Fig. 5. Effect of pH (A) and temperature (B) on the activities of the free and
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immobilized lysozyme. Values with different lowercase letters represent significant
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differences (p < 0.05).
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Fig. 6. Storage stability of the free and immobilized lysozyme at 25 °C (A) and 4 °C
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(B).
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Fig. 7. Reusability of the immobilized lysozyme on the CA(Lys-SA)8.5 and
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CA(Lys-SA)9 films.
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Fig. 8. Bacterial count (log CFU/mL) of S. aureus in UHT milk after storage at 25 °C
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and 4 °C.
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Figures
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Fig. 1.
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Tables
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Table 1. TGA and DSC data of the films.
TGA
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TD (°C)
ΔHD (J/g)
CA
349.50
18.14
321.67
- 55.72
CA(Lys-SA)0.5
327.67
27.77
271.67
- 25.81
CA(Lys-SA)1
329.38
32.56
CA(Lys-SA)4.5
329.73
CA(Lys-SA)5
329.50
CA(Lys-SA)8.5
329.33
CA(Lys-SA)9
329.67
274.98
- 83.25
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Residue at 600 °C (%)
275.01
- 75.59
35.24
275.15
- 28.81
35.27
280.70
- 37.06
36.28
306.33
- 5.68
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Tdm (°C)
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DSC
Table 2. Lysozyme content and activity of the films.
Lysozyme activity (U/cm2)
CA(Lys-SA)0.5
104.53 ± 20.40c
10.04 ± 0.41c
CA(Lys-SA)1
97.10 ± 28.53c
6.46 ± 0.30d
CA(Lys-SA)4.5
254.87 ± 99.23b
11.5 ± 0.62b
CA(Lys-SA)5
264.90 ± 35.53b
9.83 ± 0.15c
CA(Lys-SA)8.5
786.97 ± 20.69a
CA(Lys-SA)9
822.55 ± 70.43a
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Different letters in each column indicate significant differences (p < 0.05).
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Lysozyme content (μg/cm2)
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15.40 ± 0.79a
12.42 ± 0.28b
Highlights
Layer-by-layer self-assembled films were prepared based on electrospun nanofibers. The deposition induced the increased fiber diameter with enhanced thermal stability.
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The immobilized lysozyme showed good storage stability and acceptable
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reusability.
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The self-assembled films exhibited strong antibacterial activity in UHT milk.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: