Fabrication and characterization of antimicrobial hybrid electrospun polyvinylpyrrolidone/kafirin nanofibers activated by zataria multiflora essential oil

Nanofiber films were prepared using a polymer blend of kafirin and polyvinylpyrrolidone (PVP) by electrospinning. The zataria multiflora boiss. essential oil (ZEO) was encapsulated in the electrospun nanofibers, and the morphology, structural characteristics, thermal, antibacterial and release properties were investigated. The concentrations of ZEO selected for addition to the polymer solution were 7, 10 and 15% (v/v). It was found that the produced electrospun nanofibers, regardless of ZEO concentration, possessed a homogeneous morphology without beads and there was a positive correlation between ZEO addition and nanofiber diameter. Moreover, the electrospun nanofibers were found to be effective carriers of ZEO and were able to control the release of compounds. The nanofibers exhibited antibacterial activity against common foodborne bacteria (Listeria monocytogenes, Staphylococcus aureus, Escherichia coli 0157:H7, Salmonella enterica, and Pseudomonas aeruginosa). The fiber prepared with 15% (v/v) ZEO was the most effective against all microorganisms tested. The Fick model, was used to describe the release profiles of ZEO at different temperatures (4, 25 and 37 °C) and with different food simulants (50% and 98% ethanol). The findings revealed that diffusion phenomenon plays an increasingly important role in the delivery of ZEO. It is believed that the developed nanofibers have potential applications in active food packaging to reduce microbial contamination.


Introduction
Parallel with the public awareness and concern regarding the health and ecological risks associated with synthetic antimicrobial agents, the demand for other substitutes of safe and effective, natural, antimicrobials and preservatives in the food industry, is growing [1,2]. In this regard, the role of essential oils (EOs) as natural food preservatives cannot be ignored as they exert a broad range of antimicrobial activity Mohsen Esmaiili m.esmaiili@urmia.ac.ir 1 negatively affect their antimicrobial and sensory properties. To overcome these drawbacks, nanoencapsulation of EOs can be a practical and efficient approach to protect them against deterioration by direct exposure to adverse environmental conditions. The encapsulation improves controlled release of bioactive compounds, and even enhances their efficacy and bioavailability [10].
Electrospinning is a simple, easy to use, versatile, economic and novel nanoencapsulation method that allows the incorporation of different types of materials into a wide variety of polymers in the form of continuous nanofibers. Desirable elongation, large specific surface, porous structure and light weight are some advantages of electrospun nanofibers [11,12]. Some other advantages of electrospun nanofibers for encapsulation of bioactive compounds include firstly, the absence of heat, which is favorable to avoid thermal degradation of polymer and bioactive components and secondly, their high surface-to-volume ratio that enhance the ability of nanofibers to respond to changes in temperature and relative humidity of the surrounding environment. The release of bioactive compounds can be triggered by behaviors of these materials [13][14][15][16][17][18]. There are some reports about the incorporating natural compounds into electrospun nanofibers like encapsulation of ginger EO [19] and gallic acid [20] into protein electrospun nanofibers, jujube extract in PVA electrospun nanofibrous film [21] and Zenian EO in blend electrospun zein/ PLA/hydroxypropylmethylcellulose nanofibers [22]. According to all these reports, the EO/ electrospun nanofibers could prolong the shelf-life of food, suggesting a potential application in food active packaging.
With growing environmental awareness, environmentally friendly polymers and biopolymers are becoming more popular as a matrix for the preparation of electrospun antimicrobial films [23,24].
Sorghum grain has a large amount of annual production in the United States, but it is mainly used as animal feed due to its poor digestibility and low nutritional value. In the case of this highly underutilized bio-resource, creating value-added products has economic advantages. Kafirin is a natural prolamine protein derived from sorghum grain. Due to its similarity to zein in molecular weight ,amino acid composition and structure of polypeptides, being more hydrophobic than zein, as well as, its biodegradability, biocompatibility and solubility in common non-toxic polar solvents, kafirin was expected to show numerous application prospects in food and pharmaceutical industries [25,26]. However, in contrast to zein, kafirin is composed of thick rod-like assemblies, that act as globular protein bodies, when it is dissolved at high concentrations, and this is the major shortcoming of this protein for application in electrospinning [27]. This is why, research efforts into electrospinnability of kafirin are still lacking. In order to improve the properties of an individual polymer and finally fabricate a desirable scaffolding for particular application, the blending of polymers undoubtedly has a great importance [28,29]. For this reason, polyvinylpyrrolidone (PVP), a synthetic polymer widely used in medical, health-related domains, cosmetic, drug delivery and release systems, tissue engineering, wound dressing and bioactive packaging applications, was introduced to enhance the solvent stability and spinnability. PVP is non-toxic, biodegradable, biocompatible polymer with good conductivity, excellent film-forming properties, low surface tension, excellent spinnability and easy processability properties [30][31][32][33][34] but complete and immediate release of drugs from PVP fibers has limited its application in food packaging [35]. We expected that the addition of kafirin as a hydrophobic polymer to PVP may result a good mixture with overcoming the limitations of both of them which would be mutually beneficial for electrospinning purposes.
As far as we know, as of yet, there have been no reports in the literature regarding the development of a PVP/kafirin hybrid electrospun nanofibrous film loaded with ZEO as a potential active food packaging material to control foodborne bacterial pathogens. The purpose of this research therefore was to fabricate a novel hybrid nanofiber from the mixture of PVP, kafirin and ZEO via electrospinning method and evaluate the morphology, chemical interactions, thermal properties, antibacterial properties and in vitro ZEO release analysis of the fabricated nanofiber under different temperatures and food simulants to apply these nanofibers for food packaging and drug delivery applications.

Chemical composition
Analysis of the composition of the ZEO was conducted using a GC/MS Model Quadrupole, (Aglient, USA). The temperature of the injector was 150°C in the splitless mode (1:30). Helium gas with flow rate of 1 mL/min was selected as the carrier gas. Separations were performed using a HP-5MS capillary column with dimensions of 0.25 mm × 30 m × 0.25 µm (Agilent, USA). The oven temperature was reached from 70 to 240°C at a rate of 4 ℃/min and eventually held at the final temperature for 3 min. The 70 eV mass spectrometer was utilized in electronic ionization mode. Identification of the compounds were made by 20-i mass spectral library from NIST, 2011.

Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The microdilution broth assay in 96-well microtitre plates (eight rows marked from A-H and 12 columns marked from 1-12), was carried out to determine the minimum inhibitory concentration (MIC) of ZEO. Each well of the microplates was filled with a total volume of 100 µL containing MHB. According to clinical and laboratory standards institute (CLSI) recommendations (Performance Standards for Antimicrobial Susceptibility Testing), Sterile and saline suspensions at 0.5 McFarland standards (equivalent to 1.5 × 10 8 cfu/mL) were prepared from overnight cultures (37°C-24 h) of the S. aureus, E. coli, S. enterica, L. monocytogenes and P. aeruginosa bacteria. The prepared bacterial suspensions (1.5 × 10 8 cfu/mL) were diluted to 1.5 × 10 6 cfu/mL and then 100 µL of inoculums containing 1.5 × 10 6 cfu/ mL of test bacteria were added to each well. Various concentrations of ZEO were prepared in DMSO by serial dilution and 100 µL of each dilution was added to micro-wells. The final concentrations of ZEOs were 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%, 0.39%, 0.20% and 0.10% v/v in final volume. In the last rows of each microplate, negative and positive control wells were prepared, (MHB and ZEO for negative control in well H11 and bacterial inoculum and MHB, without ZEO for positive control in well H12). Afterwards, the microplates were incubated for 24 h at 37°C. The concentration of wells that represent no obvious turbidity was regarded as MIC. In order to determine the MBC, 10 µL of each MIC wells and two of the more MIC values were spot-inoculated on BHI agar plates. Following that, the plates were incubated for 24 h at 37°C. The concentration at which no growth was revealed was considered as MBC [36].

Kafirin extraction
In this study, kafirin protein was extracted using Taylor's method [37] optimized by Pienaar [38] with a few modifications. In brief, the white whole grain sorghum samples were grinded into fine flour using a laboratory hammer mill equipped with a 0.5 mm mesh sieve. Glacial acetic acid (8.75 g) and sodium metabisulphite (12.5 g) were dissolved in water (728.75 g). This mixture was mixed with ethanol 96% (1750 g) and was then added to the sorghum flour (500 g). The extraction was conducted in a water bath for one hour at 70°C with vigorous stirring. The extract was separated through centrifugation at 3000 rpm (1000 g) at ambient temperature for 10 min. The supernatant was combined and the solvent was vaporized from the supernatant using vacuum evaporation. The protein was then washed in a minimum quantity of cold (< 10°C) distilled water. The pH was adjusted to about 5 with 1 M hydrochloric acid. The precipitated protein was recovered by filtration and freeze dried for 48 h and then stored at − 20°C for subsequent experiments.

Preparation of electrospinning solution
In this paper, glacial acetic acid was selected as a compatible solvent for kafirin and PVP. Firstly, 0.5 g PVP and kafirin were separately dissolved in 6 mL glacial acetic acid. The kafirin solution was dissolved at 70°C with magnetic stirring until having a homogeneous solution. The two solutions were then blended and examined in many blends of various ratios of PVP/kafirin to optimize the electrospinning process. The selected ratios of kafirin to PVP were 10.0%, 15.0%, and 25.0% w/w of kafirin. Before electrospinning, all polymer blends were stirred continuously overnight at room temperature to acquire a homogeneous and clear solution.
Once the concentration of PVP-kafirin required to obtain continuous, regular, bead-less and fine electrospun nanofibers was determined through scanning electron microscopy (SEM) analysis, different amounts of ZEO (7, 10, 15%v/v), were added to the polymeric solution and mixed for a further 24 h. Afterwards, all prepared solutions were subjected to electrospinning apparatus to produce nanofibers. The codes of the fabricated nanofibers are presented in Table 1.

Electrospinning process
Electrospinning process was conducted using a unit made by Fanavaran Nano-Meghyas (ES 1000, Tehran, Iran) utilizing the optimized processing parameters as follows: ambient conditions, plastic syringe 5 mL, stainless steel needle nitrogen at a flow rate of 20 mL/min. The TGA data analysis were done through OriginPro (Version 2018) software [39].

Antibacterial activity of nanofibers
Based on previous studies [41], disc diffusion method was used to evaluate the antibacterial activity of ZEO-containing nanofibers against S. aureus, E. coli, S. enterica, L. monocytogenes and P. aeruginosa one day after production of fibers. Suspensions containing (1.5 × 10 8 cfu/mL) colonies of the mentioned bacteria were prepared and spread onto the prepared MHA plate. The PVP/kafirin nanofiber (as control sample) and the PVP/kafirin nanofibers containing 7, 10 and 15% ZEO were cut into circular discs of 12 mm in diameter and were placed on the surface of MHA plate, and subsequently incubated at 37°C for 24 h. The inhibition zone diameters (mm) around the discs were estimated by Digimizer software. Experiments were done in triplicate.

Thickness measurement
To determine the thickness of the samples, a manual digital caliper Model Guanglu (China) with a precision of 0.01 mm was used. Measurements were taken at five randomly points. The average value was used as nanofiber thickness.

Release tests
Evaluation of ZEO release from nanofibers The in vitro release behavior of ZEO from kafirin/PVP nanofibers was assessed following the procedure of Tavassoli-Kafrani [42] with some modifications. First, free ZEO was scanned by UV-Vis spectroscopy (Hach-Dr5000 model, American company) at wavelengths from 190 to 800 nm and the maximum absorbance was observed at 288 nm. The calibration curve of ZEO was drawn as the plot of absorbance at 288 nm versus concentration (Abs = 0.506 × ppm ZEO -0.177 (R 2 = 0.992)). To determine the release of ZEO from electrospun nanofibers, squares of 2 × 2 cm 2 of the electrospun nanofibers loaded with 15% ZEO, were immersed in 25 mL of two different food simulants: ethanol (50% v/v) as a polar food simulant and ethanol (98% v/v) as a non-polar food simulant. Sample tubes were kept under 4, 25 and 37°C and stirred (20 rpm). At regular time intervals, 2 mL aliquot solution were withdrawn and analyzed spectrophotometrically at the wavelength of 288 nm. The removed solution (2 mL) was then replaced with fresh ethanol at each interval. of 18 G (gauge), voltage range from 24 to 28 kV, needle tip to collector distance of 15 cm, flow rate of 0.6 mL/h and the drum speed of 360 rpm. All nanofibers collected on aluminum foil and stored in sealed plastic bags at room temperature for further characterization tests [39].

Fiber morphology by SEM test
The morphological properties and diameter of the resulting fibers were investigated using a scanning electron microscope (SEM, ChamScan MV2300). Samples were cut into 1 cm × 1 cm squares for sputter coating with a thin layer of gold (5 min) (DST1, nanostructured coating, Tehran, Iran) and were taken at 15 kV acceleration voltage. The average fiber diameter was calculated using the Digimizer software version 5.4.5 (Medcalc Software Ltd., Belgium), by measuring 50 random readings from different nanofibers from each micrograph [40].

Fourier transforms infrared (FTIR) spectroscopy
FTIR spectroscopy (Tensor 27, Bruker, Germany) was used to determine the various chemical groups and the structural interactions of nanofiber mats. FTIR spectra were obtained in a wavenumber range of 400 to 4000 cm − 1 with a resolution of 4 cm − 1 using KBr pellets under controlled ambient conditions [36].

Thermogravimetric analysis (TGA)
The thermal stability of the electrospun samples was studied by means of a thermogravimetric analyser (TGA) (Linseis-L81A1750, Germany). The samples (2-5 mg) were prepared and placed in aluminum cups, then scanned from 25 to 500°C at 10°C/min under an inert atmosphere purged with where V s and V n symbolize the molar volume of the simulant and nanofiber, respectively, and K n,s stands for the partition coefficient of ZEO between stimulant and nanofiber and is calculated as follows: where C n,∞ and C s,∞ represent the concentrations of ZEO in PVP/kafirin nanofibers and the stimulant at equilibrium, respectively. If the amount of solvent can be regarded as infinite (i.e. α >> 1 since V s > > V n and/or K n,s < < 1) Eq. (1) can be simplified as: Due to the long release time, the diffusion coefficient is calculated from the first term of Eq. (4), i.e .m = 0 Equation (5) was applied to fit the M t /M ∞ and t data. For each sample, D was computed at a variety of experimental temperatures. By minimizing the sum of the squares errors (SSE) of the measured and estimated values, the D was determined. In order to determine the fit of the experimental data to Eq. (5), the nonlinear regression function (nlinfit) in MATLAB® R2013a (MathWorks, Natick, MA, USA) was applied to the data [43].

Statistical analysis
Statistical analyses were conducted using SPSS software (version 19, SPSS Inc., Chicago, IL, USA). Significance of differences between samples was determined by one-way ANOVA and Tukey's multiple range test (P < 0.05) was used to detect differences among mean values. Microsoft OriginPro 2018 was used to generate TGA and FTIR plots, and MATLAB R2013a was used to fit the release data.

Chemical composition of ZEO
GC-MS analysis results of ZEO are presented in Table 2.

Calculation of the diffusion coefficient (D)
Diffusion coefficients (D) of ZEO were derived from release data over time. Some assumptions were taken into account: (1) the resistance of ZEO mass transfer from the nanofiber surface to the simulated food media was negligible; (2) ZEO concentration in the simulated food media was zero at the beginning; (3) there were no concentration gradients of ZEO in the simulated food media; (4) at a given temperature, both partition coefficients and diffusion coefficients were constant; and (5) interactions between food simulant and nanofibers were not taken into consideration. Having regard to these assumptions, D was calculated using Fick's second law equation for diffusion in one dimension and limited volumes of packaging and food: where M t represents the amount of ZEO diffused at time t, M ∞ illustrates the amount of ZEO diffused at equilibrium; l indicates the thickness of the nanofiber (0.06 ± 0.01 mm); D stands for the diffusion coefficient (cm 2 .s − 1 ); and t is an abbreviation for time. q n q n represents non-zero positive roots of tan q n = − αq n ; and α is stated as follows: compound that shows great antibacterial activity against a variety of bacteria [9]. Therefore, it is thought that the antibacterial activity of nanofibers containing ZEO is derived from thymol.

Morphology of nanofibers
SEM image and the diameter distributions of the pure PVP and PVP/kafirin nanofiber mats with and without different amounts of ZEO (7, 10, 15%) are illustrated in Fig. 2.
The SEM images revealed that all mats were composed of bead-free, uniform and continuous fibers. Among all PVP/ kafirin nanofiber mats without ZEO (Fig. 2b, c and d), the nanofiber with 75% PVP and 25% kafirin (Fig. 2d) with the highest amount of kafirin, had the lowest mean fiber diameter (341.405 nm ± 135.23) and was selected as the optimal nanofiber for the addition of various amounts of ZEO. The uniform nanofibers of the PVP/kafirin/ZEO (7, 10, and 15%) (Fig. 2e, f and g), with mean fiber diameters (n = 50) in a range of 595.814 nm ± 169.54, 700.578 nm ± 219.06 and 846.470 nm ± 310.12 were attained, respectively. The results obtained from this research showed that the addition of ZEO increased the mean fiber diameter and the average diameter increased by increasing the amount of ZEO, which were in line with previous studies [6,49,50]. This could be related to the effect of ZEO and its concentration in increasing the viscosity and decreasing the conductivity of the polymer solution, leading to less stretching of the jet during electrospinning, and thus resulting in thicker nanofibers with larger mean fiber diameters [9,48,51,52]. Moreover, the addition of ZEO to the fibers and increasing chemical compositions but different component concentrations in their reports. Variations may occur depending on the geographical location, soil type, plant's age, harvesting time, drying methods and EO extraction method used in the studies [8].

Antibacterial assessment of ZEO and nanofibers
According to Table 3, ZEO demonstrated striking antimicrobial activity against gram-positive and gram-negative bacteria. In accordance with previous studies [6,36,46,47], ZEO inhibited bacterial growth at concentrations of 0.2-0.39%. It also exhibited MBCs against the tested bacteria at concentrations equivalent to the corresponding MICs. Inhibition zone diameters of the nanofibers against the five tested strains of foodborne pathogens (S. aureus, L. monocytogenes, E. coli, P. aeruginosa and S. enterica) are shown in Table 4 and Fig. 1. On the basis of Table 4, the ZEO free PVP-KAF1 nanofiber mat revealed no antibacterial activity. Incorporation of ZEO into PVP-KAF1 nanofibers showed antibacterial activity against all tested bacteria even at the lowest concentration of 7%. This means that the antibacterial activity was attributed to the existence of ZEO. As shown by the results of the antibacterial tests, the inhibition zones increased with increasing ZEO content in the electrospun nanofibers. These results are in accordance with previous research [3], which indicated that the antimicrobial activity of cinnamon essential oil (CEO) against S. aureus and E. coli increased as a result of the increase in CEO concentration in PVA/CEO/β-cyclodextrin electrospun mats. Moreover, the inhibition zone was greater for gram-positive bacteria (S. aureus and L. monocytogenes) than gramnegative (E. coli, P. aeruginosa and S. enterica) ones. The largest zone diameters were obtained with PVP-KAF1-Z3 that measured as 25.13 ± 1.17 and 21.55 ± 0.95 mm for L. monocytogenes and S. aureus, respectively. This might be due to differences in the composition of gram-negative and gram-positive bacteria's cell walls. Gram-negative bacteria have a thick membrane of lipopolysaccharide covering their cell walls, whereas gram-positive bacteria have only a peptidoglycan layer [48]. Therefore, gram-negative bacteria are more resistant to hydrophobic antibacterial agents such as ZEO. The main component of ZEO, thymol, is a phenolic  Table 4 Inhibition zone diameter of the nanofibers against five foodborne pathogens. Different letters in each column show significantly difference (p < 0.05).
For each fiber, three replications were applied. Data were exhibited as mean ± standard deviation group. Peaks at 2962 cm − 1 and 2872 cm − 1 are correlated with the asymmetric and symmetric methyl C-H stretching, respectively. Moreover, the C═C-C ring-related vibration peaks are seen near 1600 to 1400 cm − 1 . There is also an apparent peak at about 811 cm − 1 (C-H wagging vibrations), which is characteristic of thymol [6,55,56]. The FTIR spectra of PVP/kafirin nanofiber mat proved peaks at 3457, 2928, 1685, 1541 and 1281 cm − 1 indicating the presence of O-H and N-H, C-H, C═O (amide Ι), N-H and C-H (amide ΙΙ) and C-N stretch plus N-H (amide ΙΙΙ) bands, respectively. Therefore, the nanofiber mat is comprising all PVP and kafirin compounds. According to Fig. 4, the PVP/kafirin blend fiber exhibits a carbonyl stretch profile distinct (at 1685 cm − 1 ) from those of the two individual components (at 1653 cm − 1 for PVP and 1652 cm − 1 for kafirin), providing further evidence that kafirin and PVP form a compatible blend. Moreover, the FTIR spectra of the PVP/kafirin nanofiber containing 15% ZEO was investigated to determine whether ZEO is still present in the nanofibrous film structure after electrospinning. On the basis of the obtained results, it can be concluded that besides the peaks at 3297, its concentration, improved the spinnability of the fibers (Fig. 2). This result was in good agreement with the literature on nanofibers made from PVP and CEO [32].  [25][26][27]. The FTIR spectra of ZEO revealed a peak at 3418 cm − 1 corresponding to a phenolic process started, which could be duo to the moisture loss absorbed by the material and the evaporation of the residual solvent during electrospinning [57]. The main weight loss occurred between 220°C and 470°C, with degradation peaks at about 320°C and 422°C, probably due to the decomposition of the structural polymers.

FTIR spectroscopy analysis
For the ZEO-loaded electrospun nanofibers, the main degradation temperature with the highest weight loss was at 432°C, 435°C, and 435°C for 5, 7, and 10% ZEO-containing nanofibers, respectively, indicating that increasing the ZEO content did not significantly affect the thermal degradation. Compared to PVP/kafirin nanofibers, ZEO slightly enhanced the thermal stability of the nanofibers from 71 to 83°C in the first weight loss step and from 422 to about 435°C in the main weight loss step. The addition of ZEO also decreased the percent of weight loss, indicating that the thermal stability of the nanofibers increased. Hydrogen bonds between the polymer chains and ZEO molecules could explain this increment [58,59]. Several studies have reported similar results, including Nazari et al [1], Fonseca et al [60] and Paulaa et al [61]. In general, the high heat 2955, 1666, 1540, 1428 and 1288 cm − 1 which are the manifestation of the presence of PVP and kafirin in the nanofiber, three new peaks at 2869, 944 and 809 cm − 1 were appeared which are due to symmetrical methyl groups [6], carboxylic acid groups and thymol [56], respectively, confirming the incorporation of ZEO in the nanofiber mat. Furthermore, spectral changes were found due to the addition of ZEO into the PVP/kafirin nanofiber. The peaks at 3457cm − 1 in PVP/ kafirin nanofiber were shifted to 3301 in the nanofiber containing ZEO. In this case, the hydrogen bonding between PVP/kafirine polymer and ZEO is probably responsible for this phenomenon [36].

Thermal analysis
Thermogravimetric analysis (TGA) and differential thermal gravimetric (DTG) curves of the PVP/kafirin electrospun nanofiber and the ZEO-loaded electrospun nanofibers are shown in Fig. 4. The initial weight loss (about 4.7%) of PVP/ kafirin nanofiber occurred between 35°C and 100°C, with an endothermic peak around 71°C before the degradation

Release studies
The release behavior was expressed by the amount of ZEO released from the nanofibers over time. The release profiles of ZEO from PVP/kafirin nanofibers containing 15% ZEO in 50% ethanol and 98% ethanol at three temperatures of 4, 25 and 37°C are depicted in Fig. 5. As can be seen, the tolerance of ZEO-loaded PVP/kafirin composite nanofibers (up to 200°C) makes them a good candidate for use in food packaging [39].

Fig. 5
Experimental curves of ZEO release from PVP/kafirin electrospun films incorporating 15% ZEO into 50% Ethanol (-■-) and 98% Ethanol (-•-) at different temperatures this behavior. In that research, the diffusion of linalool and methylchavicol from low-density polyethylene increased with a rise in temperature from 4 to 25 °C.
The results indicate that temperature and food simulant type could definitively affect ZEO release from nanofibers. However, further investigation is necessary to determine the relationship between the amount of ZEO released and the extension of shelf life of particular foods.

Conclusion
This study was performed for the production and characterization of antibacterial, physiochemical and release of ZEO-loaded PVP/kafirin nanofibers. Loading of ZEO in a polymeric matrix of PVP-kafirin was successfully accomplished by electrospinning process under optimal conditions. As stated in the SEM results, generally uniform and bead-free nanofibers were produced, and increasing the ZEO concentration resulted in an increase in the nanofiber diameter. A GC-MS analysis revealed that thymol is the main component of ZEO, and could contribute to the antimicrobial activity of nanofiber films against gram-positive and gram-negative pathogens. Based on ATR-FTIR spectroscopy, encapsulation of ZEO into nanofibers caused some interactions between ZEO and polymer chains; also, ZEO enhanced the thermal stability of mats. During the release study, it was found that the amount of migrated ZEO was affected by its chemical affinity for the release medium and its diffusion increased with temperature increasing. The obtained results highlight that the encapsulation of EOs with nanofiber films may be a promising method for fabrication of food active packaging and deserves more attention from researchers. Future studies should be conducted to evaluate the antimicrobial effect of electrospun nanofibers loaded with EOs in food models such as meat, fish, chicken and other perishable foods.
release curves followed a two-stage pattern: at the beginning, the release of the compound in the electrospun mats was rapid, followed by a plateau or gradual release of the remaining compound. Apparently, this phenomenon is due to the release of surface oil. Clearly, some of the EO cannot be trapped by electrospinning and is present on the surface of the nanofibers. Therefore, it is possible to release the surface EO quickly by placing the nanofibers in the release medium. At all studied temperatures, increasing the ethanol content in the medium from 50-98% resulted in an increase in the amount of ZEO released. This could be due to the fact that the affinity of the EO for the medium increased. A Villasante et al. [62] and Lamarra et al. [63] reported similar results. They worked with films of poly(lactic) acid loaded with α-tocopherol as a hydrophobic compound and Cabreuva EO encapsulated in PVA/chitosan nanofibers, respectively. As the temperature increased, the release also increased. Basically, a relative higher temperature can increase the diffusion coefficient of EO molecules [64].
Based on Fick's second law (Eq. 5), the release kinetics of ZEO incorporated into electrospun PVP/kafirin mats, was described. The diffusion coefficient of the nanofibers was determined by fitting the experimental obtained data to Eq. 5 using Matlab software. The apparent diffusion coefficients in 50% ethanol and 98% ethanol at 4, 25 and 37°C, are presented in Table 5. Due to the higher R 2 (> 0.97) and lower sum of squared error estimate of errors (SSE) and root mean square error (RMSE) for all samples, the results of fitting with the Fick model are satisfactory. The PVP-kafirin-ZEO nanofiber showed the highest D value at 37°C in 98% ethanol. Across all temperatures, the D coefficient of the PVP-kafirin-ZEO electrospun film of 50% ethanol simulant was lower than that of the 98% ethanol simulant, which can be attributed to the different polarity values of the two media. This result agrees well with the results of Tampau et al. [65], who analyzed the release rate of carvacrol encapsulated in electrospun polycaprolactone mats deposited on multilayer starch films. Higher release rates of carvacrol were achieved in the less polar food simulants such as 10% ethanol and 3% acetic acid. In 50% ethanol and isooctane as polar simulants, carvacrol was released from the fibers in a controlled manner.
The diffusion coefficient for ZEO increased with increasing temperature. A report published in [66], also suggested