Graphene fortified polyvinyl alcohol based nanofibre membranes for preserving perishable food

In recent years, graphene as a biomaterial has received considerable attention due to its outstanding physio-mechanical properties. In the present work, we found superior tensile strength, hydrophobic and antibacterial activities in graphene incorporated polyvinyl alcohol (PVA) based nanofibers, prepared by electrospinning. A series of ultrafine graphene-based ink (G-Ink) incorporated PVA nanofiber (GPN) with different concentrations of graphene (0, 0.008, 0.02, 0.04, 0.06, and 0.08% w/v) was fabricated. To overcome difficulty in direct dispersing graphene into the PVA solution, we have prepared graphene-based ink for dispersing into polymer solution. The morphology, composition, mechanical behaviour, and surface wettability of these membranes was investigated. The Fourier transform infrared spectra and the Raman spectra confirmed the successful incorporation of graphene into the GPN. Graphene when used as a nanofiller in polymers, provides excellent mechanical properties (814%), hydrophobicity (40%), and antibacterial properties. In the current study we tested GPN membranes for preserving two fast rotting foods like coriander leaves and tomato. We found that GPN membrane can be used safely for food packaging applications to increase the shelf life of perishing food items, such as up to 3 days for coriander leaves and 8 days for tomatoes.


Introduction
Electrospinning is the most efficient method for the fabrication of polymer nanofibers having diameters from nanometre to several micrometres [1,2]. Nanofibers (NF) are used for a wide range of applications such as insulation, filtration, drug delivery, wound healing, scaffolding, tissue regeneration, electronic devices, energy storage etc [3][4][5][6][7][8]. Nanofibers have a large surface area to volume ratio and have many fascinating properties, such as a high surface reactivity, high surface energy, and electrical conductivity either with conductive polymer or conductive filler [9,10]. Polyvinyl alcohol (PVA) is the Food & Drug Administration (FDA) approved biodegradable synthetic polymer that received much attention in recent years. PVA nanofibers have been widely used as a potential biomaterial attributed to its extraordinary properties such as hydrophilicity, inexpensive, biocompatible and water permeable with a superior electro-spinnability. It is also known for high thermal and mechanical properties [11][12][13][14]. These outstanding properties of PVA nanofibers are used in different areas such as a biosensor, antimicrobial fibers, composite films nanoporous films, and filtration membranes [15][16][17][18][19][20][21][22][23]. Researchers reported improved filtration efficiency by using electrospun nanofibers of mainly polyethylene oxide (PEO) and PVA nanofibers [24][25][26]. Vaisniene et al, reported electrospun PVA nanofibers to filter smoke from cigarette combustion [23]. Li and co-workers have shown the use of electrospun PVA nanofiber for filtration nano-sized sodium chloride (NaCl) aerosols [24]. PVA is known as a hydrophilic polymer with high biocompatibility, excellent chemical, and thermal stability [27].
Graphene is an allotrope of carbon, consisting of sp2 hybridized carbon atoms placed in a two-dimensional honeycomb lattice. Due to its excellent properties such as high mechanical properties, antibacterial activity, Any further distribution of this work must maintain attribution to the author-(s) and the title of the work, journal citation and DOI. hydrophobicity and electrical conductivity, graphene shows improvement in the overall performance of electrospun nanofibers. These results further enhance its application in graphene-based nanofibers membrane. Graphene-based nanofibers have achieved interest in biomedical applications such as drug delivery, cancer therapy, biological sensing and imaging, gene delivery, antimicrobial materials, and tissue engineering [28][29][30]. Wang and co-workers reported that some nanofillers including single-walled and multi-walled carbon nanotubes, montmorillonite, and graphene oxide, have been added to a polymer to prepare electrospun nanofibers which help in improving mechanical strength, electrical conductivity, and thermal stability [31]. Qiang's groups reported that a lower concentration of graphene oxide (GO) modified electrospun PVA NF scaffolds showed good biological activity such as haemolysis, wound dressing, however higher load increased the hydrophobicity that in turn increased the expression levels of the apoptosis, limiting cell proliferation and inhibiting cell viability [32][33][34]. Mukherjee and co-workers reported graphene added to PVA-Hydroxyapatite to produce electrospun nanofiber for air filter which help in increase mechanical properties, antibacterial activity, and particulate matter (pm) capturing efficiency [35]. In this work, we introduced graphene-based ink (G-Ink) as a modifier to improve the bioactivity of graphene-based ink (G-Ink) incorporated PVA nanofiber (GPN). It is expected that mechanical, electrical, antibacterial properties of PVA could be enhanced because of the addition of graphene or graphene derivatives.
In order investigate the effects of the G-Ink on the biological properties of electrospun nanofibers, we fabricated and intensively characterized a series of GPN membrane with different concentration of G-Ink. Many polymers such as silk fibroin, chitosan (CS) was used for food packaging application, but GPN is being studied for the first time for food packaging application. Introduction of properties like antibacterial and hydrophobicity which was absent in the control sample, gives a new horizon to this study.
According to the literature antimicrobial packaging is effective in inhibiting growth of microbes on food products and has the potential to extend the shelf-life of corresponding products [36][37][38]. As per previous studies, PVA electrospun nanofibers in combination with other polymers and some active substances with their antibacterial properties are shown to be promising for food packaging applications (cf, table 1). However, to the best of our knowledge there is no literature on graphene incorporated PVA nanofibers for preserving perishable food. To investigate the possibility of graphene in food packaging application, graphene-based ink was exploited as a functionalization agent for enhancing the properties of PVA membrane. A significant enhancement in hydrophobicity, tensile strength, and antibacterial activity as a function of dopant concentration was observed in GPN membranes. Also, we reported that with incorporation of graphene-based ink there was a significant (∼2-3 fold) increment in the tensile strength in comparison to previous studies. Furthermore, larger surface to volume ratio, higher strength and porosity of the electrospun nanofibers added additional advantages. Applications of these fibers as a temperature and humidity controller, oxygen scavenger, gas storage are some of the other areas which can be explored in the near future.

Materials
Polyvinyl alcohol (Mwt = 1,15000 KD) was procured from Loba Chemie Pvt. Ltd, Carboxy Methyl Cellulose (CMC) was purchased from Loba chemie pvt. Ltd, Byk 190 was purchased from BYK-Chemie GmbH. Graphene flakes was used as produced in Tata Steel Limited and the same reported elsewhere [42]. The graphene received was used for G-Ink preparation. Before making the ink, cytotoxicity of the graphene was also evaluated by extraction method on L-929 cell according to ISO 10993-5.

Materials and methods
2.2.1. Preparation of PVA solution 10 g of PVA was added into 100 ml of DI water. The mixture was then heated at 80°C with a constant stirring at 300 rpm to achieve a clear dispersion.

Preparation of G-Ink
For preparation of G-Ink, 1.6 g of CMC was dissolved in 100 g of DI water to make a clear solution. The CMC solution was then first poured in a 700-watt kitchen blending pot and 4 g of graphene was added to it. The resulting mixture was blended for 15 times at 18000 rpm followed by sonicated using a 40 kHz bath sonicator for two hours during which 0.5 g of Byk 190 was gradually added. The mixture was finally sonicated for another eight hours.

Preparation of G-Ink incorporated PVA solution
A series of G-Ink doped PVA solutions were prepared by adding different %w/v of G-Ink in PVA solution. The amounts of G-Ink were taken as 0 g, 0.2 g, 0.5 g, 1 g, 1.5 g and 2 g per 100 ml of PVA solution in each beaker (cf, figure 1). This resulted in the final PVA electro-spinnable solution with graphene concentration as 0, 0.008 g, 0.02 g, 0.04 g, 0.06 g, and 0.08 g per 100 ml. These mixtures were stirred further for 30 min for further dispersion. The above mixtures were then sonicated for 15 min to ensure complete mixing of the two solutions and thereby uniform dispersion of graphene in the final mixures.

Fabrication of GPN membrane using electrospinning technique
All the GPN membranes were electrospun using electrospinning technique. The electrospinning apparatus (ESPIN-NANOTECH, Kanpur India) consisted of high voltage supply, a syringe infusion pump and a ground electrode. The polymeric solutions were loaded into a plastic syringe (5 ml), and the applied voltage was 20 KV. Flow rate of 1.2 ml h −1 was used and the spinning distance between the needle and the collector was maintained at 12 cm. Collector was covered with A4 size plain white paper to collect the nanofibers membrane. All the parameters were kept constant for all other sets of the experiments. For the GPN-0 membrane, 20 ml of 10% PVA solution is taken in a syringe for fabrication, for the GPN-0.2 membrane, 20 ml of 0.2 wt% G-Ink doped PVA solution is taken for fabrication. For GPN-0.5 membrane, 20 ml of 0.5 wt% G-Ink doped PVA solution was taken for fabrication. Same process was repeated for the fabrication of GPN-1, GPN-1.5 and GPN-2. Here G-Ink added in PVA solution act as a functionalization agent and is expected to enhance variations in properties of the fibre. The fibre deposited on the collector (on A4 size paper) is carefully peeled out and used for further characterizations.

Characterizations
The size and morphology of the electrospun fibers were investigated by the field scanning electron microscopy (SEM, Thermo Fisher, Netherlands) with an Electron Dispersive x-ray unit (EDX). To study the surface morphology of GPN, the membranes were cut into 1 cm 2 size and fixed on the mould using carbon tape. Gold coating was done for 100 s. After coating the samples were used for SEM and EDAX analysis. Porosity was calculated using Image J software. Fourier Transmission-Infrared Spectrophotometer (FTIR-Thermo Fisher Scientific, USA) with a resolution of 2 cm −1 was used to explore the change in the functional groups after functionalization of the nanofibers with G-Ink. The spectrum was recorded in the wave number range of 4000-500 cm −1 . To investigate the chemical composition as well as graphene signature, GPNs were characterized by a fully automated laser Raman spectroscope (Witec alpha 300 ACCRSS, Germany) with a 633 nm excitation wavelength and 50X magnification at room temperature. To estimate the surface wettability of the prepared membrane, the water contact angle of GPNs were measured by contact angle system (Kyowa Interface Science Co. Ltd, Japan). The droplet size was set at 3 μl, and three readings were recorded for each sample. The mechanical properties of prepared samples were measured using the testing machine Instron Elecroplus E1000K6301, UK. Equipped with a dynamic capacity of 1000 N at a tensile rate of 0.1 mm min −1 . Each sample with dimensions of 10 mm * 25 mm length * 0.02 mm thickness were analyzed. The tensile strength and elongation at the break of the samples were recorded. Antibacterial Test was done using a qualitative method AATCC: 147-2016 using two tests organisms' Gram-positive bacteria Staphylococcus aureus ATCC 6538 and Gram-negative bacteria Klebsiella pneumoniae ATCC 4352. This method is also known as a parallel streak method and it is designed to qualitatively evaluate the antimicrobial activity of the diffusible agents on a treated surface. This method allows to evaluate the residual efficacy of the electrospun fibres. Since PVA is water soluble polymer and it is unstable in water, the above method was chosen for the experiment.

Results and discussion
Cytotoxicity of graphene was done on L-929 cell line. A stock of 200 mg ml −1 of test item has been prepared in Dulbecco's Modified Eagle Medium (DMEM) with 5% Fetal Bovine Serum (FBS) for 100% concentration. Based on observation at 24 h, 100% and 50% concentration of extracts of the graphene were found to be causing moderate reactivity (Grade 3: greater than 50% to less than 70% of the cell layers contain rounded cells or are lysed) and none (Grade 0: discreate intracytoplasmic granules, no cell lysis). Hence it is concluded that DMEM extracts of test item graphene at 100% and 50% concentrations were found to be cytotoxic and non-cytotoxic to L-929 cells, by both qualitative and quantitative analysis as per ISO 10993. Maximum amount of graphene used for these studies was 8 mg ml −1 . Therefore, it may be concluded that GPN membrane were safe for food packaging applications.

Morphological study of GPN membrane
The morphological studies of the synthesized nanofibers were performed using Scanning electron microscope (SEM). Since the morphology of the GPN were determined by various parameters such as the applied voltage, distance between the needle tip to the collector and concentration of the solution prior to electrospinning, parameters were optimized to attain well-oriented bead-less fibres [30]. The fibres seemed to be uniform with clear strips with no obvious impurities. The average diameter of control (GPN-0) was found to be 157 ± 43 nm ( figure 2). Upon addition of G-Ink at different concentration i.e., 0.2%, 0.5%, 1%, 1.5%, 2% into 10 wt% of PVA solution, the diameters increased to i.e., 212 ± 17 nm, 213 ± 18 nm, 215 ± 20 nm, 215 ± 21 nm and 217 ± 17 nm, respectively. Average pore size of GPN-0 was 95 to 150 nm whereas GPN-1 was 400 to 800 nm. With the increase in the dopant concentration the average fiber diameter shows an increasing tendency with average diameter reaching maximum value when the concentration is 2%. This result also correlates well with the mechanical testing data as explained in the later section. As the percentage of G-Ink loading in PVA solution was increasing, viscosity of these solution was also increasing i.e., 400 cps, 420 cps, 420 cps, 450 cps, 500 cps and 520 cps. Most importantly, no beaded structures were observed, indicating that PVA solution containing G-Ink as nano filler was electro-spinnable.

FTIR spectra of GPN membrane
Fourier transform infrared (FTIR) spectroscopy was used to identify the functional groups that appears on the GPN membrane. The FTIR spectrum of GPN-0 shows absorption peaks near 3292 and 2915 cm −1 , which are related to stretching vibration of O-H and attributed to symmetric stretches of C-H groups (figure 3). The broad band at 3292 cm −1 also corresponds to stretching vibration of -OH in PVA polymer chain, broad band formation. The band at 1664 cm −1 is assigned to stretching vibration of free carbonyl group present in PVA. The peak observed at 1640 and 1412 cm −1 in GPN-0.2 to GPN-2 could be attributed to alkene (C=C), which is the backbone of the graphene structure. Peak observed at 1086 cm −1 is due to the symmetric stretching vibrations of C-O from the carboxyl group.

Raman spectra of GPN membrane
Raman spectra were used to confirm the incorporation of G-Ink as shown in figure 4. The Raman spectra of the GPNs showed the characteristic G band and 2D band of G-Ink at 1580 and 2700 cm −1 respectively, suggesting the presence of G-ink in GPN-0.2, GPN-0.5, GPN-1, GPN-1.5, GPN-2. I 2D /I G ratio of GPN 0.2, GPN 0.5, GPN 1, GPN 1.5, and GPN 2 were 0.63, 0.65, 0.69, 0.72 and 0.64. The sharp 2D peak and higher I 2D /I G ratio, might be because there is exfoliation of graphene during electrospinning due to G-Ink based formulation and thus showing spectral evidence of few layers of graphene.

Surface resistivity of GPN membrane
The mechanism of electrical conductivity of the nanofibers is based on two factors. One is the intrinsic electrical conductivity of nanofiller and another is the conductivity of the polymer (if the polymer is conductive). The surface resistivity study of the films was 10 12 ohms/sq in case of GPN-0, 10 11 ohms/sq in GPN-0.2 and GPN-0.5 and 10 10 ohms/sq in case of GPN-1, GPN-1.5 and GPN-2 as shown in table 2. This study revealed that control film (GPN-0) is insulating, and graphene incorporated films (GPN-0.2 to GPN-2) were antistatic and static dissipative which is also helpful for food and electronic packaging [43].   GPN-0, GPN-1, GPN-1.5 and GPN-2 were evaluated for antibacterial activity. These nanofibers were cut into sample size: 25 × 50 mm and placed over the agar plate then incubation at 37°C for 24 h. There were many bacterial colonies seen over the sample GPN-0 whereas GPN-1, GPN-1.5 and GPN-2 was colony free, but zone of inhibition was absent around all the samples. After removal of samples GPN-1, GPN-1.5 and GPN-2 there was no bacterial growth at lower side. Mechanisms behind the antibacterial activity of graphene towards specified bacteria involves oxidative stress, membrane stress, and electron transfer. Graphene can also physically damage the bacterial membranes by direct contact of its sharp edge [44]. Graphene can generate reactive oxygen species (ROS) which damages the proteins and lipids content of bacterial membrane and therefore microorganisms can no longer proliferate [45]. The degradation of cell membranes of E. coli caused by graphene was reported by various authors.

Contact angle measurement of GPN membrane
Hydrophilicity/hydrophobicity assessment of membrane is a very important parameter in the field of different biological application. In general, hydrophilicity was favourable for cell adhesion, attachment and growth of cells need scaffolds with a relatively hydrophilic surface [46]. Whereas, hydrophobic nanofibers use for filtration application, oil filtration, fog water collection, mechanism reported by Knapczyk-Korczak et al that polyamide 11(PA11) allows a higher water collection rate to be obtained due to faster removal of water droplets [47]. Same  GPN-0 and GPN-0.2, GPN-0.5, GPN-1, GPN-1.5, GPN-2 with the concentration of G-Ink loading 0% 0.2%, 0.5%, 1%, 1.5% and 2%.   Control (coriander leaves) Dry but green in colour Dry and turned into brownish colour Leaves turned into completely black Coriander leaves wrapped in the tissue paper (control), perished. Coriander leaves inside GPN-0 Partially dry but green in colour Partially dry but green in colour Leaves started decaying GPN-0: weight reduced, and coriander leaves started degradation Coriander leaves inside GPN-1 Green in colour Some leave became dry, and rest remains green in colour Some leaves remain greenish, and some were started decaying GPN-1: weight was same, but the membrane become moist due to trapped moisture mechanism might be applicable in our case i.e., removal of moisture water droplet from GPN which were used in food packaging of vegetables. Therefore, to determine the hydrophilicity and hydrophobicity of the nanofibers, the water contact angle of GPNs were measured using a contact angle meter analysis system ( figure 6). GPN-0 exhibited the contact angle of 71.63 ± 1.60°, which indicates that these fibres have good hydrophilicity. Whereas the contact angle values of 75.25 ± 3.50°, 81.86 ± 6.74°, 92.57 ± 4.15°, 94.95±2.02°and 100.26 ± 7.59°were observed for GPN-0.2, GPN-0.5, GPN-1, GPN-1.5 and GPN-2 respectively. G-Ink reduces the hydrophilicity of the membranes, and it was observed that the higher the percentage of graphene content, the lower is the hydrophilicity. The loaded GPNs showed an increase in contact angle compared to control PVA nanofibers indicating that graphene can improve and regulate the moisture content of nanofibers. This might be due to the agglomeration of graphene particles on the surface of nanofiber. These graphene agglomeration on the surface of nanofibers might be the reason behind the hydrophobicity of these nanofibers. This property endowed by the graphene has been explored for food packaging application that can help regulate the moisture penetration keeping food/vegetables fresh for a longer duration.

Tensile strength measurement of GPN membrane
The addition of G-Ink into PVA polymer is expected to improve the mechanical properties because of numberless interfacial interactions. It is confirmed from the results, that addition of graphene does increase the tensile strength of the nanofibres. GPN-0 has a tensile strength of 1.3 MPa. There is a significant increase in tensile strength as the percentage of G-Ink dopant increases. Mukherjee et al also reported that after incorporation of graphene the tensile strength increased significantly. After incorporation of 1 wt% G-Ink to the PVA solution, the tensile strength was 9.8 MPa whereas with 2 wt% incorporation, the tensile strength increased up to 814% i.e., 10.6 MPa ( figure 7). This result implies that incorporation of 1 wt% G-Ink is almost stabilised, and the curve showed the saturation state and there was no huge increment with incorporation of 2 wt% G-Ink.

Application of GPN membrane to save food from spoilage
Food packaging materials play a critical role in preserving the perishable food items. Without proper packaging, food may become compromised by physical, chemical or biological means. In view of this, fast-perishing food like coriander leaves and tomatoes were selected. An experimental study was carried out to investigate the efficiency potential of GPN-1 membrane -based food packaging by comparing with GPN-0 and with tissue paper. Fresh tomato and corianders leave were considered for the experiment and were packed in three different containers. One container for each type was packed by tissue paper, second one by GPN-0 membrane and other was packed by GPN-1. All samples were placed in same environmental condition. Tomatoes and coriander leaves were examined for 7 days and 3 days respectively. Table 3 shows details of the experiment on food packaging application of G-Ink membranes (GPN-1) to control spoilage of coriander leaves. And figure 8  The membrane started to shrink At one side it started decaying and there were 5 scars Tomatoes wrapped in the tissue paper (control), perished. Tomato inside GPN-0 As it is Became red but damage free There were 5 scars and one black spot GPN-0: weight reduced, and the tomatoes started degrading Tomato inside GPN-1 As it is Same as fresh one There were 5 yellow scars GPN-1: weight was same, but the membrane become moist due to trapped moisture showed the results obtained by the experiment. Table 4 demonstrates the details of experiment on food packaging application of G-Ink membranes (GPN-1) to control spoilage of tomatoes and figure 9 illustrates the results obtained by the experiment. It can be concluded that tomatoes and coriander leaves packed in GPN-1 -based packaging were having better freshness than the packed tissue paper followed by GPN-0. By virtue of submicron to nano-scale diameter fibers has large surface area, electrospun nanofibers may offer many additional advantages compared to film and sheet carriers, which might change due to the surrounding atmosphere such as relative humidity and temperature. Electrospun nanofibers have been exploited by many researchers to incorporate bioactive substances due to advantages of their large surface area, for example poly (lactic acid) (PLA) fibers were employed to carry silver nanoparticles for antibacterial properties [48]. There may be several reasons behind these results, primary reason could be, GPN-1 have excellent air permeability due to high pore size and secondly this membrane may possess excellent breathability due to the diameter of fibers in nano range, high surface area, antibacterial property, high tensile strength and lastly moisture management due to moderate contact angle as compared to tissue paper. Therefore, it can be concluded that GPN-1 with high tensile strength, antibacterial and hydrophobicity-based food packaging is superior to conventional food packaging application, and it significantly increases the shelf life of these vegetable and reduces the chance of spoilage.

Conclusions
GPN-0 to GPN-2 was successfully produced by electrospinning. The incorporation of G-Ink into the nanofiber was confirmed via FTIR and Raman spectroscopy. The comparison study of the fiber diameters of the different doping percentages of G-Ink on the PVA solution i.e., GPN-0.2, GPN-0.5, GPN-1, GPN-1.5, GPN-2 indicated that there was continuous increase in diameter up to 1% doping of G-Ink whereas fibers' diameters were not significantly increased when the doping percentage of G-Ink was increased to 1.5 and 2. The presence of G-Ink lead to a relative improvement in mechanical properties, including the enhanced tensile strength as the doping percentage of graphene was increased. GPN-0 membrane had not shown any antibacterial activity whereas GPN-1, GPN-1.5, GPN-2 showed antibacterial activity. Further, high surface area to volume ratio of anofibers, allowed humidity and temperature modulation. This membrane can be used for moisture absorption and oxygen scavenger. While exploring the application of theses nanofibers for food packaging, coriander leaves remained fresh for three days in GPN-1 membrane whereas, in GPN-0, wilting of leaves were observed in just 8 h. Similarly, tomatoes also retained their freshness for seven days inside the GPN-1 membrane unlike GPN-0 where some yellow patches were observed. Thus, it may be concluded that graphene plays a crucial in preserving the shelf life of perishable food like these. Additionally, the antimicrobial property of these nanofibers further preserves the food by preventing microbial infestation. GPN-1 membrane with enhanced properties might be useful in different application but here we have shown promise in food packaging application within the scope of this study. Detailed experiment of these membrane for food packaging application will be performed in near future.