Electrospinning water permeability, low toxicity and susceptibility

Electrospinning is a novel cost effective technique for manufacturing
nanofibers from a broad range of materials likely to be used as a coating film. In this project, pectin and
chitosan solutions containing polyvinyl alcohol (PVA) were prepared and
simultaneously electrospun with separate syringes for the first time. The antimicrobial and physical
properties of the novel chitosan/PVA-pectin/PVA nanofibrous film were evaluated
using different analysis techniques such as disc diffusion assay, scanning
electron microscopy (SEM) and transmission electron microscopy (TEM), viscosity and
conductivity tests, and fourier-transform infrared spectroscopy (FTIR). The
images of SEM and TEM could prove the formation of the films in the scale of
nano. The results showed that simultaneously electrospun dispersion of
chitosan/PVA (50:50) with pectin/PVA (50:50) leading to the formation of thin
nanofibers with the least beads. The results of FTIR proposed the
dispersion of chitosan and PVA in nanofiber mats and the interaction of
chitosan with pectin and PVA with pectin. Disc diffusion assay showed that nanofilm
could possess a significant antibacterial activity against S. aureus at
37?C but showed no effects against E. coli. Based on the
results of this study, the novel chitosan/PVA-pectin/PVA nanofibrous film can be
considered as a novel coating film for promising application in future food


Keywords: Electrospinning; Pectin; Chitosan; PVA; Nanofiber.

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1.     Introduction

In recent years, studies and developments in the area of food
packaging have focused on bio-based functional packaging materials, sometimes
incorporating natural active compounds and ingredients. Bio-based packaging
materials consisting antioxidants and antimicrobials have become popular since
oxidation, deterioration and microbial contamination are the major phenomena
threaten food quality and safety.

Chitosan (1–4 linked 2-amino-deoxy-?-D-glucan) is a
functional biopolymer possesses significant antimicrobial and antioxidant properties
with high potential to develop biodegradable active packages. This
biopolymer is a linear cationic polysaccharide prepared from chitin, found in
shells of marines such as shrimp, lobster and crab. It
has widespread application in biomedical, chemical and food industries due to
its antimicrobial activity, biocompatibility, biodegradability, high water
permeability, low toxicity and susceptibility to chemical modifications. Chitosan
could prevent the growth of wide varieties of fungi, pathogenic bacteria and
spoilage microorganisms. The molecular weight (MW) and the degree of deacetylation
determine the antimicrobial activity of chitosan. Chitosan may dissolve in
various organic acids, then undergo drying process, being prepared to form
flexible, clear and tough films with compatible oxygen barrier properties. In
2001, chitosan has been classified as a safe component by FDA, thus being
extensively used in food industry. For example, chitosan has been used for
direct surface coating of meat and fruit products to reduce food deterioration
and water loss, in addition to postpone the ripening of fruits.

Pectin, on the other hand, is an anionic natural
polysaccharide that is found in ripened fruits such as apples and plums. It has
been used as a cross-linking agent for cationic polymers, e.g. chitosan,
to form a polyelectrolyte complex. Chitosan cross-linked with pectin has been
shown to possess improved hydrophilicity, biocompatibility, and mechanical
strength, thus very efficient for drug delivery and tissue engineering
purposes. Incorporation of chitosan with pectin has resulted in a
polyelectrolyte complex (PEC) at pH values in the range of 3 to 6. Pectin
and chitosan may also interact via hydrogen bonding at low pH
values (pH < 2). At this condition, pectin is unionized and the intensity of electrostatic interactions is weakened. It has been reported that pure pectin/chitosan film have high permeability and swelling ratio. Its permeability has been found to be lowered by addition of nanoclay, thus can be used for food packaging and prevents absorption or desorption of moisture content. Using nanomaterials to create packaging with different methods has been demonstrated to possess some functions including improved barrier properties, and extension the shelf life of food products. In recent years, the development of electrospinning technique in nanoscience has been increasingly explored. Electrospinning is a simple, inexpensive technique with the capability to control the morphology of ultrafine fibers. The films produced by this method possess exceptional characteristics, such as very large surface-to-volume ratio and high porosity with small pore size. To enhance the electrospinnability of the polymers and to preserve individual fiber morphology on the collector, polyvinyl alcohol (PVA) has often been added to the chitosan solution. In this project, pectin and chitosan solutions containing PVA were prepared and simultaneously electrospun with separate syringes for the first time. The physical and antimicrobial properties of the novel chitosan/PVA-pectin/PVA nanofibrous film were analyzed.   2. Materials and methods 2.1. Materials PVA (MW = 94–120 kDa) and acetic acid (100 %) were purchased from Merck, Germany. Chitosan (MW = 1600 kDa, 86.2% deacetylation) and pectin (MW = 30–100 kDa, DM 30%) were purchased from Sigma, UK. Ethanol (96%) was obtained from Zakaria Co., Iran. Gram-positive bacteria S. aureus and gram-negative bacteria E. coli were purchased from the Persian Type Culture Collection (Tehran, Iran). Plate Count Agar (PCA) media was purchased from Merck, Germany. 2.2. Sample preparation Pectin (2 g) was mixed firstly with ethanol (30 mL) and then with water (70 mL), after that stirred for 24 h to achieve complete dissolution. Chitosan sample (1.5% w/v) was dissolved in acetic acid solution (1%) and stirred for 24 h to achieve complete dissolution. PVA solution (5%) was prepared in distilled water and stirred for 24 h at 60°C to achieve complete dissolution, then added into pectin and chitosan solutions with two ratios (50:50, 25:75). All the samples then stirred for 3 h. 2.3. Viscosity and electrical conductivity of fiber-forming dispersions The viscosity of the dispersions was measured using a rotational viscometer (Brookfield, DV-II + Pro, USA). The measurement was carried out in continuous ramp mode at 10°C using the cone and plate geometry. The correlations between the viscosity and the shear stress with shear rate were measured. The electric conductivity of the polymer dispersions was determined using a benchtop conductivity meter (Mettler-Toledo Co., Ltd., USA). 2.4. Electrospinning setup and procedure The electrospinning system (Asian Nanostructure Technology Company, Iran) was adjusted on a constant voltage at 18.0 kV applied to the positive electrode using direct current supply. The spinneret was connected to the positive electrode, while the stainless steel collector plate was grounded. The distance between the spinneret and the collector was adjusted to 10 cm. Electrospinning was conducted under ambient conditions (22°C; 15% RH). The final solutions were simultaneously electrospun with two separate syringes. The flow rate was adjusted on 0.4 mL/h. Table 1 shows the experimental conditions for preparing different fibers with combination of pectin/PVA + chitosan/PVA solutions. The schematic of the setup is shown in Figure 1. 2.5. Fiber analysis with scanning electron microscopy (SEM) A Philips XL 30 scanning electron microscope (Vega3, TESCAN, Brno, Czech) was used for fiber size estimation and analysis the morphology. The sample was mounted on the specimen holder with aluminum tape and then coated with gold in BAL-TEC SCD 005 putter coater. Morphologic pattern of the surface of the fibers was observed under high vacuum condition with an accelerating voltage of 20.0 kV. 2.6. Fiber analysis with Transmission electron microscopy (TEM) The morphology of the fibers was checked out by transmission electron microscope (Philips CM-10, Eindhoven, Netherlands). Fibers were mounted on metal stubs by double-faced tape and the surface was coated by carbon. Diluted nanofiber suspension (5 µL) was deposited on the carbon-coated grids and allowed to dry at room temperature. Morphological observation of the fiber was performed at an accelerating voltage of 80.0 kV. 2.7. Fourier transform infrared (FTIR) spectroscopy FTIR analysis (Nicolet Magna-IR 560) was performed to study the possible interaction among PVA, pectin and chitosan. FTIR spectra were recorded at wave numbers ranging from 4000 to 400 cm-1 by FTIR system (Perklin Elmer, spectrum RXI, USA).  2.8. Antimicrobial properties of nanofibers For determination of antimicrobial activity of nanofibers, E. coli (as a representative G-negative bacterium) and S. aureus (as a representative G-positive bacterium) were used. The bacteria were cultivated in nutrient broth at 37?C for 24 h, then 0.1 mL of suspensions was spread plated on PCA for both E. coli and S. aureus. The film prepared on the aluminum foil was cut to make a certain size, and the antimicrobial activity was tested using disc diffusion assay (disc test). The plates were examined for possible clear zones after incubation at 37?C for 2 days. The presence of any clear zone was recorded as the indication of growth inhibition of microbial species.  2.9. Statistical analysis All experiments were performed at least in triplicates (n=3). Analysis of variance (ANOVA) was performed to determine significant differences between the means. Duncan multiple range test (P < 0.05) was used to compare the means. SAS program (ver. 9.1 SAS Inst. Inc., Cary, NC, USA) was used to perform the statistical analysis. 3. Results and discussion 3.1. Viscosity and conductivity Viscosity of fiber-forming dispersions is a key parameter affecting electrospinning process. The amount of beads in the final fiber is also affected by the viscosity of the dispersions. Table 2 shows the viscosity of the sample dispersions at ambient temperature. The high MW of chitosan and more importantly, water absorption of pectin and PVA active groups are the main reasons for increasing the viscosity. In this study, the viscosity was not influenced by addition of PVA to pectin and chitosan solutions. Normally, higher viscosity is preferred to create suitable electrospun fibers with low amounts of beads over the structure. Of course very high viscosity may also possess some issues, for example it may prevent withdrawing of liquids from the syringe by forming gel like materials, thus avoiding Taylor cone formation, which is necessary for electrospinning process. Conductivity is another important parameter which directly affects the entanglement properties and the thickness of electrospun fibers. The result of conductivity measurement is presented in Table 1. In the case of chitosan + PVA polymer, the conductivity value was not affected by changing the ratio, nevertheless, addition of PVA to pectin reduced the conductivity, thus expecting thicker fibers.     3.2. SEM imaging Figure 2 shows the SEM images of electrospun fibers, which states that simultaneously electrospun dispersion of chitosan/PVA (50:50) with pectin/PVA (50:50) resulted in thin fibers with less beads than that of chitosan/PVA (75:25) with pectin/PVA (75:25). The viscosity and the conductivity of the dispersions increased with increasing pectin content. The number of beads on the fibers were also increased. Expectedly, reduction of PVA content in dispersions has decayed the electrospinnability of the polymers (Li & Hsieh, 2006; Zhou et al., 2008). Given the results of morphological analysis, we concluded that the fibers generated by PVA/pectin + PVA/chitosan (50:50 + 50:50) was the best combination among tested in this study for fabrication of nanofiber using electrospinning. 3.3. TEM Figure 3 shows the TEM image of electrospun fibers from simultaneously electrospun dispersions of chitosan/PVA (50:50) with pectin/PVA (50:50). This image approved the production of the highly porous nanofibers with insignificant beads and a large specific surface area which can promote fibroblast adhesion, migration, and proliferation. To overcome this problem, it is suggested to add nanofillers as for instance nanoclay to the dispersion before performing the electrospinning. This may reduce the permeability of the produced nanofilm, providing a good structure to be used as a packaging film. 3.4. FTIR spectroscopy Different functional groups of polymers can be determined using FTIR spectra. Figure 4 shows the FTIR spectra of pure PVA, pectin, chitosan powders and nanofiber mats of pectin/PVA + chitosan/PVA (50:50 + 50:50). The PVA powder spectrum showed the dominant absorption peaks at 3436, 2934, 2400, 1251, 1094 and 836 cm?1, which were attributed to the O–H, CH2, CH2, C–H, C–O and C–O, respectively. These bands are the characteristic bands of PVA, reported previously in literature (Figure 4a). On the FTIR spectrum of pectin (Figure 4b), the bands related to C?O stretching of the carboxyl group could be observed at 1606 cm-1 (Doner, 1986). The absorption bands, which were in the wavelengths area 800-1200 cm-1, are the finger-print regions of the typical pectin polymers. The absorption band at 1606 cm-1 and the absence of any band at 1700-1800 cm-1 indicated the free carboxyl groups with low degree of esterification. For pure chitosan powder (Figure 4c), the significant peaks were observed in the spectrum at wavelengths 3439, 2924, 1608 and 1090 cm-1. The absorption peak at 3439 cm-1 corresponds to _OH and _NH stretching vibrations. The absorption band at 2924 cm?1 is the characteristic absorbance peak of _CH. The _NH bending (amide II) band at 1608 cm-1 region was observed in the IR spectrum of chitosan. The absorption peaks at around 1090 cm?1 were attributed to asymmetric stretching vibration of the C–O–C skeleton (Yang et al., 2017; Nunthanid et al.,2001). Spectrum of nanofilm showed clear bands at 1637 and 1429 cm-1, which were absent on the PVA spectrum. The peak at 1637cm?1 can be attributed to the C?O band corresponded to a primary amide from chitosan. The absorption peaks at 1637 and 1428 cm?1 suggested the formation of hydrogen bond between the chitosan and PVA molecule. The results illustrate that chitosan and PVA were dispersed in nanofiber mats. Furthermore, the absorption at 1637 cm-1 is a clear evidence of the interaction of the amino and carboxyl groups of chitosan and pectin. The broadening of the peak at 3414 cm-1 in nanofilm spectrum compared to the peak at 3436 cm-1 related to PVA and the peak at 3434 cm-1 related to pectin, may be attributed to formation of hydrogen bonds over the fiber between hydroxyl groups of PVA and pectin. 3.5. Antimicrobial effect An example of antibacterial action of PVA/pectin + PVA/chitosan (50:50 + 50:50) nanofibers on E. coli and S. aureus in cultures is shown in Figure 4. Nanofibers could possess significant antibacterial activity against S. aureus at 37?C but had no effects against E.coli. As shown in Figure 4, when nanofilm was present in a culture of S. aureus, a zone of inhibition was observed indicating efficient bactericidal activity of nanofibers against the gram positive rather than the gram negative bacterium. Antibacterial efficacy of nanofibers corresponds to the existence of chitosan biopolymer in electrospun nanofibers. Chitosan possesses a broad spectrum antimicrobial activity to the Gram-negative bacteria, Gram-positive bacteria and fungi. Antimicrobial property of chitosan is dependent on its structural conditions such as MW, degree of deacetylation, derivative form, its concentration, and original source. The critical MW for antibacterial properties of chitosan is above 10 kDa, which is effective on Gram positive as well as Gram negative bacteria. With increasing the MW of chitosan, antibacterial activity is increased, especially against E. coli. The MW over 100 kDa have been shown to reduce the antibacterial activity of chitosan against E. coli, while this trend is not observed against Gram positive bacteria. Here, it can be proposed that the antibacterial activity of nano-chitosan created by electrospinning technique on Gram negative bacteria is suppressed. This on the other hand may be very promising in the case of protection of Gram negative bacteria. Given this, a layer of nanofilm created by chitosan may inhibit damaging the outer membrane of Gram negative bacteria, which in the case of beneficial bacteria as for instance Acetic acid bacteria provides a suitable protection. Nevertheless, it is known that most of the Gram negatives are the source of infections for humans. 4. Conclusion In this work, electrospun nanofibers were successfully produced from simultaneously electrospun dispersion of chitosan/PVA (50:50) with pectin/PVA (50:50).  The nanofibers containing chitosan/PVA (50:50) and pectin/PVA (50:50) were thinner and more homogeneous having less beads in the structure in comparison with the nanofibers containing chitosan/PVA (75:25) and pectin/PVA (75:25). The results of FTIR spectroscopy proposed the dispersion of chitosan and PVA in nanofiber mats and the interaction of chitosan with pectin and PVA with pectin. Antibacterial efficacy of nanofibers corresponds to the existence of chitosan biopolymer in electrospun nanofibers. The generated nanofibers had significant antibacterial activity against S. aureus but had no effects against E. coli proposing using this combination  for protection of beneficial Gram negative bacteria. Antibacterial activity of nanofibers against Gram positive bacteria can be connected to structural conditions of chitosan. In conclusion, the novel chitosan/PVA-pectin/PVA nanofibrous film is proposed as promising materials for applying to the active packaging technology.