Abstract
The object of the present study was to develop polycaprolactone (PCL)/casein (Cas) electrospun nanofibers incorporated with green tea essential oils (GTO) as an active food packaging to prevent spoilage and food loss. The obtained results show that increase in the weight concentration of Cas leads to increasing the viscosity and diameter of nanofibers up to the range of 237–298 nm, leading to enhance the mechanical strength owing to the creation of hydrogen bonds and increased surface wettability. However, increase of the PCL content led to the reduction of the fiber diameter (203 ± 108 nm), more porosity (around 80%), and high thermal stability. In addition, the nanofiber samples, with 5% (PC5), and 12% (PC6) concentrations of GTO exhibited a long-term sustained release with high antioxidant activity 39.85% and 58.56% for PC5 and PC6, respectively. These findings suggested the promising applications of the produced PCL/Cas/GTO electrospun fibers for active food packaging.
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Introduction
Recently, more research has focused on the development of suitable alternative materials for active food packaging to eliminate non-degradable oil-based packaging materials and increase the shelf life, quality, and safety of food products (Aghaei et al., 2018; Amjadi et al., 2020a; Amjadi et al., 2020b; Liu et al., 2021; Maroufi et al., 2022). The addition of antibacterial compounds into the polymer matrix of food packaging is a useful strategy for this end (Babitha et al., 2017; Duan et al., 2021; Liu et al., 2021; Pirsa et al., 2020; Riahi et al., 2021; Shao et al., 2019). Moreover, nanotechnology is one of the successful strategies to provide the packaging materials to encapsulate and sustaine release of functional substances which provided a new solution to improve their bioavailability (Akrami-Hasan-Kohal et al., 2020; Aman Mohammadi et al., 2021; Qin et al., 2019; Raeisi et al., 2019). Electrospinning, a single-stage encapsulation technology, has attracted more attention in the fabrication of polymeric nanofibers containing bioactive compounds due to their high porosity, high surface-to-volume ratio, flexibility, and high loading capacity (Akrami et al., 2020; Aman Mohammadi et al., 2019; Kumar et al., 2019). Moreover, electrospinning is an easy, highly versatile, and cost-effective technology that produces nanofibers using an external electric field, and the continuous drawing of a polymer solution from a syringe needle to the ground collector (Ahmadian et al., 2020; Sameen et al., 2022).
Polycaprolactone (PCL) is a type of aliphatic polyester and it has been extensively studied in the field of drug delivery, tissue engineering, and food packaging due to its good properties such as biocompatibility, biodegradability, and non-toxicity (Hajikhani et al., 2020; Li & Tan, 2014; Suwantong, 2016). It also has been extensively used in food packaging owing to its excellent mechanical characteristics (Cesur et al., 2018). Casein is a desirable hydrocolloid for using in food packaging system due to the great nutritional value, solubility in water, and emulsification capacity (Li et al., 2021). Also, owing to the distribution of polar amino acids along the chain of this protein, casein-containing systems display an excellent barrier to oxygen and other non-polar molecules, which makes them beneficial for the conservation of food products to oxidation (Aslaner et al., 2021; Picchio et al., 2018).
Essential oils (EOs) are a type of natural compound with high antimicrobial activity against a variety of foodborne bacteria (Bhavaniramya et al., 2019; Maroufi et al., 2022). However, their high volatility, hydrophobicity and special flavor limit their application (Wen et al., 2016a, b). Encapsulation of EOs into electrospinning nanofibers (NFs) can overcome these drawbacks (Bodbodak et al., 2021; Lin et al., 2017; Mohammadi et al., 2020; Nazari et al., 2019). Additionally, the electrospinning technique can generate NFs at ambient temperature which prevented from deterioration and volatilization of the EOs (Cerqueira et al., 2016; Dierings et al., 2021). Green tea oil (GTO), which is derived from the leaves of the green tea plant (Camellia sinensis), is used as a natural antioxidant and antimicrobial in a variety of food industry applications (Perumal et al., 2021). As reported in previous literature, GTO is mainly composed of phenolic compounds especially catechins (Yavari Maroufi et al., 2020). Recent studies have shown that green tea compounds play an important role in antioxidant and antimicrobial characteristics (Borzi et al., 2019; López de Dicastillo et al., 2013; Siripatrawan & Harte, 2010; Wrona et al., 2017).
To the best of our knowledge, no published study was reported to fabricate NFs based on PCL and Cas containing GTO and its application as an active food packaging. Therefore, the present research fabricated the electrospun PCL/Cas nanofibers containing GTO, which is applicable for active food packaging. We assessed the properties and structure of the produced NFs through Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), mechanical properties, and water vapor transmission rate (WVTR) analyses. The optimum NFs were selected based on the SEM analysis. Then, the GTO was added as a powerful antioxidant and antibacterial to the optimized NFs in two different percentages. The DPPH free radical scavenging, and antibacterial activity were finally tested in the optimized NFs.
Material and Methods
Material
Polycaprolactone (PCL, Mn = 80 kDa), technical grade casein from bovine milk, DPPH (1,1-diphenyl-2-picrylhydrazyl), thiazolyl blue tetrazolium bromide (MTT), fetal bovine serum (FBS), Phosphate buffered saline (PBS), trypsin–EDTA, RPMI 1640 medium, and Muller Hinton Agar were supplied from Sigma-Aldrich (St. Louis, USA). Stock cultures, Escherichia coli (E. coli) (PTCC-1270), Staphylococcus aureus (S. aureus) (PTCC-1112) were obtained from Institut Pasture (Iran). The mouse embryonic fibroblast cell line (NIH-3 T3) was obtained from the Pasteur Institute of Iran. GTO was obtained from Barij Essence Company (Mashhad, Iran) in air-impermeable glass.
Preparation of the Electrospinning Solutions
Polymer solutions (total polymer weight 15% w/v) were prepared with dissolving the polymers at the four-weight ratio of PCL/Cas in acetic acid 90% (v/v) with stirring overnight at 25 °C. Moreover, GTO at concentrations of 5 and 12 (versus total polymer weight) was added to the selected optimal polymer electrospinning solutions.
Electrospinning
Polymer solutions at the four-weight ratio of PCL/Cas (95:5, 90:10, 85:15, and 80:20) were taken into a 10 mL syringe fitted with an 18-gauge steel needle. Next, these solutions were pumped at a flow rate (1 mL/h) via syringe pump. Electrospinning was conducted by applying the voltage of 16 kV, a receiving distance of 15 cm, a constant rate of 1 mL/min, at the room temperature, and the humidity of 50 ± 5%. Fibers were accumulated on an aluminum foil which is effective to take out NFs. Table 1 represents different electrospun NFs that were produced in this study.
Characterizations and Measurements
Scanning Electron Microscopy (SEM)
To determine the diameter, and morphology of the produced NFs, the scanning electron microscope (SEM) was utilized (Philips XL 30 FEG, Amsterdam, Holland) at the voltage of 26 kV. Before SEM, all produced NFs was coated by a thin layer of gold with a sputter coater. All images derived from SEM technique were investigated with ImageJ software to obtain average diameter of NFs.
Fourier Transform Infrared Spectra (FTIR)
FTIR measurements were carried out with a spectrometer (Tensor27, Bruker Co., Ettlingen, Germany) to determine the functional groups present in the PCL, Cas, and prepared PC nanofibers without, and with 5%, and 12% of GTO. The spectra were documented in the range of 500 to 4000 cm−1 at a resolution of 4 cm−1. The pellets of samples were prepared with KBr powder, so all samples were measured using a KBr-pellet method.
Thermal Properties
The thermogravimetric analysis (TGA) of formulated NFs was performed using thermogravimetric Analyzer (LINSEIS SPA PT 1600 device, Germany). This measurement was accomplished under a nitrogen atmosphere, and the NFs were heated up to 600 °C at a constant heating rate of 10 °C/min.
Porosity Assessment
The porosity of the produced NFs was evaluated with the liquid displacement method and calculated as follow (Salehi et al., 2020):
where, V1 and V2 are the initial volume of ethanol (96%) and the volume after immersing the NF samples, respectively. In addition, V3 is the volume of the ethanol after the NFs removed (after 5 min).
Mechanical Properties
Tensile strength, elongation at break, and Young’s modulus of produced NFs were determined at 25 °C with a Tensile Analyzer (Instron 5566, USA), according to ASTM D-882 (Zhang et al., 2021). The specimens were cut into pieces with dimensions of 10 mm × 30 mm, and the tests were carried out at room temperature and a moving crosshead speed of 50 mm/min.
Measurements of Surface Wettability
The surface wettability of produced NFs was measured with water contact angle (WCA) method (Data physics, OCA ES 149 r, Filderstadt, Germany). A drop of deionized water (2 µL) was dropped on the surface of each produced NFs (20 mm × 60 mm), and photographed instantly within 5 s.
Water Vapor Transmission Rate (WVTR)
The WVTR of the produced nanofibers was measured by the following method (Dadras Chomachayi et al., 2018; Fahimirad et al., 2021). The WVTR was measured with fixing the piece of NF samples on the round opening of a vial. The vial was filled with 2 mg of calcium chloride anhydride (RH = 0%). The vials were located in a desiccator comprising oversaturated sodium chloride solution at 25 °C for 24 h, and the WVTR was determined as follows:
In the above formula, ΔW is the weight changes of NF samples (g), A is the exposure area (m2) of NF samples, and t is the exposure time (24 h).
GTO Release Behaviors
For measurement of the in vitro release of GTO from produced NFs, each sample (20 mg) was immersed in 5 mL of PBS (pH = 7.4, at 37 °C) and shaken at 150 × g for 36 h. After the specified times, 1 mL of the solution was removed and analyzed with UV–vis spectroscopy at the wavelength of 275 nm. After that, the 1 mL was returned to the vial until the next time point. Then, the amount of GTO encapsulated in the electrospun NFs was detected (Karami et al., 2021).
Antioxidant Activity
The antioxidant activity of GTO-loaded PC fibers was calculated with 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. For this purpose, 2 mL of the NF samples extract was mixed with 3 mL of ethanolic DPPH solution (0.1 mM) and permitted to be kept in the dark condition for 30 min at 25 °C. At the end of the 30 min, the absorbance of the solution was evaluated at 517 nm by a UV–vis spectrophotometer (Spectrum SP-UV500DB) (Aytac et al., 2017; Borzi et al., 2019). The antioxidant activity (AA) of samples was calculated as follow:
Antimicrobial Activity
The antimicrobial properties of the produced PC/GTO nanofibers were assessed against two bacterial species (S. aureus and E. coli) through the agar diffusion assay. Initially, the suspensions of selected bacteria were prepared (1.5 × 106 CFU/mL). Then, the 100 µL of prepared bacteria suspension was spread onto solid media plates containing MHA for each selected bacterium. Next, the produced NFs were cut in circular shape, sterilized, and placed on inoculated petri dishes. These petri dishes were incubated for 24 h at 37 °C. The diameters of inhibition areas of the NF samples were calculated in millimeters (Ghorbani et al., 2020; Ngamakeue & Chitprasert, 2016; Zhang et al., 2020a, b).
Cytotoxicity Assessment
To investigate the cytotoxicity of the formulated NFs on NIH3T3 fibroblast cells, MTT test was performed (Ghorbani et al., 2016a, b; Golizadeh et al., 2019; Maroufi & Ghorbani, 2021). In short, after 1, 3, and 5 days, the NF samples with the attached NIH3T3 fibroblast cells were incubated in MTT solution (5 mg/mL) in RPMI-1640 at 37 °C under a 5% CO2 atmosphere (Sabzichi et al., 2017). Next, the specified amount of DMSO solution was added into each sample to dissolve the formed formazan crystals. Thereafter, the optical density (OD) of the supernatant solution was measured using a spectrophotometer at a wavelength of 570 nm (Shimadzu, Japan, UV-2550).
Statistical Analysis
All experiments were investigated in three replications, and were documented as mean ± standard deviation (SD). Statistical comparisons were carried out with ANOVA (Tukey’s test), and significance was defined at P < 0.05.
Result and Discussion
Fiber Morphology
The SEM results and the mean diameters of the electrospun NFs with different weight ratios of PCL/Cas, and PC containing different concentrations of GTO (5 and 12%) are shown in Fig. 1, and Fig. 2, respectively. Since the processing parameters and the environmental conditions were constant during electrospinning process, so the only parameter which was affected on the morphology and diameter distribution of the synthesized NFs was the different weight ratios of the PCL, Cas, and the GTO. First, the different weight ratios of PCL and Cas were investigated on the morphology of produced NFs. Four different weight ratios of PCL/Cas (95:5, 90:10, 85:15 and 80:20) were electrospun. All samples displayed a porous, free-beads, and uniformity structure. However, with increasing the weight ratio of Cas, the average fiber diameter increased (Fig. 1). So, the diameter of PCL/Cas fibers increased from 203 ± 108 nm with ratio of 95:5 to 298 ± 49 nm with ratio of 80:20. As known, the viscosity of the electrospinning solution is one of the most important factors that effects on the morphology of NFs (Panzavolta et al., 2011). Therefore, increasing the viscosity of polymer solution by enhancing the content of protein (Cas) leads to the formation of electrospun NFs with a high average diameter. As reported in previous studies, in the electrospinning process, increasing the viscosity of the solution owing to the creation of stronger resistance of the viscoelastic force to the electric field force leads to the production of thicker nanofibers with a high average diameter (Aydogdu et al., 2018; Colín-Orozco et al., 2015; Drosou et al., 2018). Besides, the encapsulation of GTO into the selected sample (PC1 95:5), significantly increased the average diameter of the fabricated NFs (Fig. 2). This result may be due to the effect of GTO in reducing the electrical conductivity of the electrospinning PCL/Cas solution, because EOs have low ionic conductivity (Wen et al., 2016a, b). Therefore, the lower electrical conductivity may result in lower elongation of a jet by the electrostatic force to form fibers of thick diameter (Bhagwan et al., 2019). A similar result based on the decrease in the electrical conductivity of electrospinning solutions by addition of EOs has been reported in other studies (Ardekani et al., 2019; Tavassoli-kafrani et al., 2018). These researchers reported that the encapsulation of orange essential oil, and zataria multiflora essential oil into the gelatin (Tavassoli-kafrani et al., 2018), and poly (vinyl alcohol) (Ardekani et al., 2019) nanofiber resulted in the formation of thicker nanofibers due to reducing the electrical conductivity of electrospinning solutions. Moreover, with increasing GTO concentration, the diameter of the fiber also increases. In addition to the reseason mentioned above, as reported by Hosseini et al. (2021), increase in the nanofiber diameter can be due to the greater resistance of the high concentration of GTO to be stretched toward the collector (Heydari-Majd et al., 2019; Miri et al., 2021).
Morphology, and the average fiber diameter of Polycaprolactone/Casein (PCL/Cas) nanofibers with different weight ratio of PCL/Cas (95:5 (PC1), 90:10 (PC2), 85:15 (PC3), and PC4 (80:20))
Morphology, and the average fiber diameter of selected of Polycaprolactone/Casein (PCL/Cas) nanofibers with weight ratio of 95:5 containing 5, and 12% concentration of green tea oils that coded PC5, and PC6, respectively
FTIR Spectroscopy
To study the molecular interactions between the compound and polymers, FTIR analysis was performed. The FTIR spectra of all samples are revealed in Fig. 3. The FTIR of PCL demonstrated the specific peaks at 2949 cm−1, 2865 cm−1, 1727 cm−1, 1293 cm−1, 1240 cm−1, 1190 cm−1, 1170 cm−1, and 1157 cm−1, which could be attributed to the asymmetric CH2 stretching, symmetric CH2 stretching, carbonyl stretching, C–O and C–C stretching in crystalline phase, asymmetric COC stretching, CC–O stretching, symmetric COC stretching, and C–O, C–C stretching in amorphous phase, respectively (Salehi et al., 2020). The main peaks at 1510–1580 cm−1, 1661 cm−1, 3100 cm−1, and 3455 cm−1 were seen in Cas spectrum, which are consistent with that previously reported by Singh et al., (2015a, b) that may be related to the amide II (N–H bending vibration), amide I (C = O stretching vibration), amide B, and amide A, respectively. The shifts of peak positions in the FTIR spectrum of produced PCL/Cas fibers indicate a successful interaction between the PCL, and Cas (hydrogen bonding). In the FTIR spectrum of produced PCL/Cas fibers containing 5 and 12% concentration of GTO, no new peaks were observed in these samples with and without GTO due to the similar chemical composition between PCL (C6H10O2)n and GTO catechins C15H4O6. However, a minor change in peak intensity was observed due to the formation of a new hydrogen bond between the compounds. In the previous study reported by Mohd Rashid et al. (2021), it was confirmed that the encapsulation of GTO in the polymer structure of PCL does not generate a novel peak in the IR spectrum of the final sample due to the similar structure.
Fourier transform infrared (FTIR) spectra of Polycaprolactone (PCL), Casein (Cas), produced PCL/Cas fibers, and PCL/Cas nanofibers containing 5, and 12% concentration of green tea essential oils (GTO)
Thermal Stability
The thermal stability of produced NFs with different weight ratio of PCL/Cas (95:5 (PC1), 90:10 (PC2), 85:15 (PC3), and PC4 (80:20)) was investigated with TGA analysis, and the detailed thermal data are summarized in Table 2. As can be seen, the first stage of weight loss occurred at around 25–150 °C, which was related to the volatilization of residual water. The second stage occurred at around 250–400 °C, which could be associated to the heating degradation. The last one happened at 400–600 °C that can be due to the progress in heating degradation of polymeric chains (Hadad & Goli, 2018). According to these results, the weight loss of the produced NFs increased slightly with increasing Cas content. In fact, the highest residual weight (6.54%) is related to PC1 sample with the high PCL (95%) content, presenting PCL has an effective role in increasing thermal stability. A similar result was observed in previous studies in which electrospun nanofibers with the highest content of PCL showed the lowest weight loss and the highest thermal stability (Aynali et al., 2021; Hivechi et al., 2019; Zou et al., 2020).
Porosity
The porosity of the formulated NFs was revealed in Fig. 4. The porosity value of the produced fibers was found as follow (Fig. 4): 82%, 74%, 70%, and 63% for PCL/Cas with weight ratios of 95:5, 90:10, 85:15, and 80:20, respectively. According to these findings, by increasing the content of Cas, the viscosity and the diameter of produced NFs increased, resulting in the reduction of the porosity of formulated NFs reduced. The highest porosity (82%) was related to PCL/Cas fibers with a weight ratio of 95:5 that could be associated to the geometry of their fibrous structure, and formed hydrogen bond between polymer chains. Previous studies have shown that thinner electrospun fibers formed as a result of low viscosity lead to higher porosity (Ahmadian et al., 2020; Ghorbani et al., 2020).
The porosity of the Polycaprolactone/Casein (PCL/Cas) nanofibers with different weight ratios of PCL/Cas (95:5 (PC1), 90:10 (PC2), 85:15 (PC3), and PC4 (80:20)). The labels of **p, and ***p, show significant difference (P < 0.05) between data of PC1, PC2, PC3, and PC4
Mechanical Properties
Mechanical characteristics of the electrospun fibers are considered as a major factor in food packaging applications. The mechanical parameters (Tensile stress, Young’s modulus, and Elongation at break) are summarized in Table 3. As a general trend, increasing the weight ratio of Cas led to the increasing of tensile stress (TS) and Young’s modulus (YM), as well as reducing the value of elongation at break (EAB). Mechanical properties of the electrospun fibers can be affected by a series of parameters such as composition of fibers, average diameter of fibers, and morphology of fibers (Fadaie et al., 2018). Bölgen et al. (2005) demonstrated that thinner PCL fibers have lower TS, and YM than thicker fibers. Therefore, by increasing the weight ratio of Cas, the average diameter of NFs increased which caused the two parameters of YM and TS to improve. It can be related to the increase in intermolecular interactions of Cas and PCL molecular chains (Fadaie et al., 2018). For instance, the TS of the PC1 (95:5) sample was 46.31 ± 0.34 MPa, while the TS of the PC2 (90:10), PC3 (85:15), and PC4 (80:20) samples with increasing weight ratio of Cas, significantly enhanced to 53.4 ± 0.42, 56.11 ± 0.17, and 58.2 ± 0.11 MPa, respectively. In the case of the EAB parameter, as a general trend, this parameter showed a decreasing trend with increasing the weight ratio of Cas. Thus, the PC1 sample elongated around 136.14 ± 2.83% of its initial length while the elongation at break was reduced to 98.14 ± 11.21% upon increasing Cas weight ratio up to 20%. The decrease in EAB with increasing Cas content can be related to the interactions between PCL and Cas, which contribute to create a stiffer but brittle network within the electrospun nanofibers. As reported in previous literature, the intermolecular hydrogen bonds formed between polymer chains (PCL and Cas) may restrict the movement of the polymer chains and promoted rigidity (Zhang et al., 2020a, b). By adding the GTO to the selected sample (PC1 95:5), TS values were decreased from 46.31 ± 0.34 to 45.3 ± 0.14 MPa, and 40.7 ± 0.22 MPa, which were, respectively, related to fibers containing 5 and 12% GTO. On the other hand, EAB values were increased from 136.14 ± 2.83 to 140.45 ± 6.45%, and 145.14 ± 8.5%, which were respectively related to fibers containing 5 and 12% GTO. Previous studies have confirmed that mechanical properties (TS, and YM) of composite nanofibers/films were decreased by incorporating hydrophobic compounds such as EOs (Ardekani et al., 2019; Ge et al., 2019; Karami et al., 2021). On the other hand, EAB of composite nanofibers/films were increased by incorporating EOs due to their plasticizing effect, so the interaction between polymer chains and EOs caused the mobility of polymer chains to increase and improve the flexibility (Imran et al., 2012).
Surface Wettability
PCL has been identified as a biocompatible synthetic polymer for a number of food industry applications (Joseph et al., 2011; Lukic et al., 2020). However, its hydrophobic nature is a limited parameter for a design of active food packaging, because hydrophilicity is a required parameter to achieve the effective release of active compounds encapsulated in the polymeric matrix of active packaging (Choi et al., 2006). Accordingly, the synthesis of composite electrospun NFs consisting of PCL and Cas can be a reliable strategy to overcome this limitation. For this purpose, water contact angle (WCA) measurement was performed to determine the surface wettability of produced PCL/Cas electrospun NFs. The images of the WCA evaluation are shown in Fig. 5. The hydrophobic nature of PCL is related to the presence of CH2 groups along the main polymer chain (Qian et al., 2014). As shown in this figure, the average WCA values for PC1 (95:5), PC2 (90:10), PC3 (85:15), and PC4 (80:20) fiber samples were 95 ± 2.1°, 87 ± 1.3°, 80 ± 2°, and 76 ± 1.2°, respectively, that were in correlation with the weight ratio of PCL, and its hydrophobic property. In fact, increasing the weight ratio of Cas as the hydrophilic segment in the PCL/Cas electrospun NFs (due to the presence of carboxyl and amine groups along the main polymer chain), increased the surface wettability.
The water contact angle (WCA) of Polycaprolactone/Casein (PCL/Cas) nanofibers with different weight ratios of PCL/Cas (95:5 (PC1), 90:10 (PC2), 85:15 (PC3), and 80:20 (PC4)). The labels of NS (No Significance), *p, and **p, show significant difference (P < 0.05) between data of PC1, PC2, PC3, and PC4
Water Vapor Transmission Rate (WVTR)
The WVTR of the produced PCL/Cas fibers with different weight ratios of PCL and Cas is exposed in Fig. 6. It is known that WVTR is the one of main parameter for design of food packaging materials that is attributed to the moisture transfer between the environment and food product (Zou et al., 2020). From WVTR examinations, the average WVTR values for PC1 (95:5), PC2 (90:10), PC3 (85:15), and PC4 (80:20) fiber samples were about 4100 ± 10.12, 5980 ± 45.61, 6000 ± 46.21, and 6237 ± 85.46 g/m2.24 h, respectively. Regarding the results, the WVTR of produced fibers was significantly increased with increment of Cas weight ratio. The most important factors affecting WVTR are porosity and hydrophilicity of fibers (Martins et al., 2018). As explained in previous studies, the WVTR is inversely related to the porosity of fibers caused by fibers average diameter (Ahmadian et al., 2020; Lu et al., 2017; Rashidi et al., 2021). The incorporation of GTO at concentration of 5 and 12%, resulted in a decrease of the WVTR with respect to the fiber samples with high content of Cas (PC2, PC3, and PC4), but they did not show a significant difference with PC1. In consistence with the results of the present study, Nazari et al. (2019) (Nazari et al., 2019) found that the WVTR of poly(vinyl alcohol) nanofibers loaded with cinnamon EO decreased significantly. Reduced WVTR values of resulting nanofibers loaded with GTO can be a consequence of the reduction in the hydrophilic nature of the nanofibers and crosslinking interactions between hydrophobic constituents and biopolymers, which may lead to a scarcity of hydroxyl groups for interaction with water molecules (Ardekani et al., 2019; Nazari et al., 2019; Pourhojat et al., 2017).
The water vapor transmission rate (WVTR) of Polycaprolactone/Casein (PCL/Cas) nanofibers with different weight ratios of PCL/Cas (95:5 (PC1), 90:10 (PC2), 85:15 (PC3), PC4 (80:20)), and selected optimal (PC1) nanofibers containing 5 and 12% concentrations of green tea essential oils named PC5, and PC6, respectively. The label of **p shows significant difference (P < 0.05)
Release Studies
The in vitro release study of GTO from the optimal PCL/Cas fibers was performed for 36 h in PBS (pH = 7.4, at 37 °C). Experimental curves are demonstrated in Fig. 7. The degradation of the polymer matrix in an aqueous medium may occur for longer periods of exposure. During the 36-h analysis period, it can be confirmed that there is no degradation in the polymer matrix, and the diffusion process is solely responsible for the oil diffusion (da Costa et al., 2020). In the present study, all samples revealed a gradual release of the GTO. As shown in Fig. 7, the maximum release rate of GTO (around 68.21%) corresponds to a PC6 sample containing 12% GTO at 37 °C and an incubation time of 36 h. Thus, the release rate of GTO enhanced with the increment of GTO concentration up 12% in the samples, and over time. Moreover, these findings illustrated that the formulated PCL/Cas fibers with GTO had the long-term release behavior during 36 h for PC5 and PC6. Because during 36 h, the perfect release of GTO was not observed for both samples. These results agreed with the report of Shetta et al. (2019).
The release rate of GTO from optimal Polycaprolactone/Casein (PC) nanofibers containing 5 (PC5) and 12% (PC6) concentrations of green tea essential oils for 36 h. The labels of NS, **p, and ***p show significant difference (P < 0.05) between data of PC5, and PC6
Antioxidant Activity
Green tea polyphenols such as catechins, tannins, and gallic acid, which are present in GTO, propose high activity in inhibiting DPPH free radicals owing to the presence of phenolic hydroxyl groups (Siripatrawan & Harte, 2010). Therefore, the encapsulation of GTO into polymeric matrix of food packaging could considerably improve the antioxidant activity of packaging to protect food product from an oxidative spoilage. In this regard, the antioxidant activity of the formulated NFs with different concentrations of GTO (5 and 12%) was investigated with free radical (DPPH) scavenging technique, and the findings are exposed in Fig. 8. As expected, the pure NFs (PC1) illustrated negligible activity against DPPH free radicals. On the other hand, the antioxidant properties of fibers containing GTO enhanced significantly (P < 0.05) due to the capability of phenolic groups of green tea to provide hydrogen to stabilize free radicals. Green tea is one of the most effect antioxidant compounds that has a high capability to inhibit free radicals and has been proven in many studies to have strong antioxidant function (Dou et al., 2018; Wen et al., 2020; Yavari Maroufi et al., 2020). The antioxidant activity for formulated PCL/Cas fibers without and with 5%, 12% concentration of GTO was estimated to be around 10.21%, 39.85%, and 58.46%, respectively.
Antioxidant activity of selected optimal Polycaprolactone/Casein (PC1) nanofibers containing 5 and 12% concentrations of green tea essential oils named PC5, and PC6, respectively. The label of **p shows significant difference (P < 0.05) between data of PC1, PC5, and PC6
Antimicrobial Activity
Antimicrobial activity of the optimal PCL/Cas fiber containing GTO with concentrations of 5 and 12 wt.% was examined against two pathogenic bacteria namely S. aureus (gram positive) and E. coli (gram negative). The obtained results are showing in Fig. 9 and Table 4. It is evident from the results that the formulated fiber without GTO did not prevent the growth of the mentioned bacteria. While, the fibers containing GTO exposed a notably (P < 0.05) inhibitory effect against both bacteria. Moreover, the formulated fibers with GTO illustrated the concentration-dependent antibacterial ability against bacteria. Many previous studies have confirmed that green tea catechins are responsible for the antimicrobial activity of green tea. These compounds attach to bacterial membranes and inhibit bacterial activity by affecting cellular processes such as apoptosis and mitochondrial function (Belščak-Cvitanović et al., 2015; Dou et al., 2018; Shetta et al., 2019; Singh et al., 2015a, b; Wang et al., 2020; Wrona et al., 2017; Yavari Maroufi et al., 2020). The findings of this study showed that although it has caused the inhibition of both types of bacteria, but its effect on gram-positive bacteria is more than gram-negative. It is noteworthy that the antimicrobial properties of pure GTO on gram-negative bacteria such as E. coli depend on the interaction of lipophilic oil and the phospholipid membrane which induce permeability of the GTO molecules (Singh et al., 2015a, b).
Antimicrobial activity of the optimal Polycaprolactone/Casein (PC1) nanofibers containing 5 and 12% concentrations of green tea essential oils named PC5, and PC6, respectively, against S. areus, and E. coli bacteria
Cell Viability
The effects of the formulated PCL/Cas fibers without and with different concentrations of GTO on the viability of NIH3T3 fibroblast cells are exposed in Fig. 10. According to the MTT results, no significant reduction in the number of viable cells was observed in the presence of fiber samples with and without GTO, after 1, 3, and 5 days. As revealed in this figure, all the values of cell viability were higher than 90%. Moreover, these results illustrate that fiber samples have great biocompatibility and ability to support cell proliferation. Additionally, the presence of GTO improved the fiber hydrophilicity due to the presence of green tea polyphenols. As reported in previous studies, increasing the hydrophilicity of scaffolds improves adhesion and cell proliferation (Dziadek et al., 2021; Raja et al., 2021; Zadeh et al., 2019).
Cell viability of the optimal Polycaprolactone/Casein (PC1) nanofibers containing 5 and 12% concentration of green tea essential oils named PC5, and PC6, respectively
Conclusion
In the present study, the PCL/Cas nanofibers containing GTO were developed by electrospinning technique as a suitable material for active food packaging. Physico-chemical and biological characterization of produced nanofibers were assessed. Morphological study exposed that the average fiber diameter was enhanced with increment of Cas weight content. In addition, surface wettability, WVTR, and mechanical properties gradually enhanced with the increment of Cas weight content, notably, for the PC4 sample. Moreover, the antioxidant and antimicrobial activities of the fibers containing GTO were investigated and the results exposed that the fiber with high concentration of GTO revealed the good performance. In addition, the cell cytotoxicity studies presented excellent cell viability and proliferation of NIH3T3 fibroblast cells in the presence of fiber samples with and without GTO. In conclusion, the fabricated novel nanofiber with significant biocompatibility and antioxidant/microbial activity possesses promising potential for active food packaging.
Data availability
All data generated or analyzed during this study are included in this published article.
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The authors gratefully acknowledge the Nutrition Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
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This research has received financial support from the Nutrition Research Center, Tabriz University of Medical Sciences, Tabriz, Iran (69797).
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Leila Yavari Maroufi: Investigation; Formal analysis; Methodology; Data curation; Writing—Original draft. Sepideh PourvatanDoust: Formal analysis; Methodology; Writing—Original draft. Fatemeh Naeijian: Formal analysis; Data curation. Marjan Ghorbani: Project administration; Conceptualization; Methodology; Investigation; Data curation; Supervision; Funding acquisition; Review & editing.
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Yavari Maroufi, L., PourvatanDoust, S., Naeijian, F. et al. Fabrication of Electrospun Polycaprolactone/Casein Nanofibers Containing Green Tea Essential Oils: Applicable for Active Food Packaging. Food Bioprocess Technol 15, 2601–2615 (2022). https://doi.org/10.1007/s11947-022-02905-1
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DOI: https://doi.org/10.1007/s11947-022-02905-1