Electrospinning Nanobres Of Pullulan Extracted From Phylloplane Fungus, Aureobasidium Pullulans

Aureobasidium pullulans isolated from the phylloplane of Peltophorum tree, produced pullulan, an extracellular polysaccharide. It was grown on three different carbon sources, sucrose, wheat bran and cotton stalk dust, for maximizing the pullulan yield. A. pullulans (67.4 gL -1 ) had the highest yield followed by A. pullulans MTCC 1991 (63.68 gL -1 ). Pullulan was characterized by X-ray diffractometer (XRD), Brunauer-Emmett-Teller (BET) surface area analyzer, DSC and NMR. Electrospinning of pullulan blended with poly (vinyl alcohol) (PVA) produced bead-less nanobres. The optimized parameters for electrospinning were 25 kV applied voltage, 0.5 mL/h ow rate, 18% polymer concentration (pullulan + PVA) and 150 mm tip-to-collector distance. The pullulan nanobre was characterized by SEM, AFM, BET, contact angle measurement, DSC and CIE color space analyzer. A maximum surface area of 183.4 m 2 /g while the minimum nanobre diameter (79 ± 19 nm by SEM) was obtained for the electrospun mat of commercial pullulan + 40% PVA. This work signies the importance of pullulan extracted from an isolate of Peltopohorum tree for conversion to high surface area nanobres by electrospinning process. used each of D-Xylose, D-Arabinose, D (+) Melibiose, α methyl-D-glucoside and D-Lactose whereas nitrogen source tested D-glucosamine Urease production Broth incubate for 48 h. Citrate utilization Growth tested for fungal the Illumination) color space coordinates determined. A color is dened by its RGB values which give the amount of red, green and blue in a particular color. In CIE L*a*b* color space values, L* stands for lightness, a* and b* for the green–red and blue–yellow color components and ΔL represents brightness difference between samples. The magnitude of color difference was quantied as ΔE. Structural characterization of pullulan was carried out by 1 H-NMR and 13 C-NMR spectroscopy and compared with that of commercial pullulan. Four chemical signals were displayed in the anomeric region between 4.4 to 5.3 ppm due to the four sugar repeating unit present in the pullulan polysaccharide. Signals were observed at 1.2 and 1.1 ppm was due to 6-deoxy-d-altrose present in the polysaccharide This conrms the presence = 0, and the brightest white at L* = 100. The L* (Lightness) values obtained for electrospun commercial pullulan was 91 whereas L* values for electrospun pullulan extracted from, A. pullulans MTCC 1991 and A. pullulans were 91 and 90 respectively When compared to standard white paper on which the electrospinning was carried out, ΔL* values for electrospun, commercial pullulan, pullulan extracted from A. pullulans MTCC 1991 and A. pullulans were 2.408, 1.394 and 1.367, respectively. The redness/greenness color component, a*, negative values indicate greenness while positive values indicate redness. The, a*, values obtained for electrospun commercial pullulan was 2.126 whereas for electrospun pullulan extracted from, A. pullulans MTCC 1991 and A. pullulans were -0.006 and 1.090 respectively. The blueness/yellowness color component, b*, negative values indicate blueness while positive values indicate yellowness. The, b*, values obtained for electrospun commercial pullulan was -3.051 whereas for electrospun pullulan extracted from, A. pullulans MTCC 1991 and A. pullulans were 4.556 and 1.078 respectively. When the magnitude of the color difference, ΔE*, is higher than 1, it indicates a visually detectable color difference and its value increases further for greater color changes. When compared to standard white paper on which the electrospinning was carried out, ΔE* values for electrospun, commercial pullulan, pullulan extracted from A. pullulans MTCC 1991 and A. pullulans were 6.995, 16.219 and 12.873 respectively. in turn could be helpful for application based on retaining ability of the nanobre mats at the nano level and thereby increasing the eciency of the product. The pullulan extracted from A. pullulans MTCC 1991 as well as isolate, A. pullulans could be electrospun into nanobres only when blended with PVA (40 -50%). The signicant change in melting temperature of the blended electrospun mats as observed in DSC thermograms could be attributed to the addition of PVA. This work provides newer isolates for production of pullulan using wheat bran as carbon source and also the process protocol for production of nanobre mat by electrospinning after blending with PVA.


Introduction
Pullulan (poly-α-1, 6-maltotriose) is a unique neutral water-soluble exopolysaccharide (EPS) produced in large quantities by microbial fermentation, mostly by Aureobasidium pullulans. Though sucrose and starch are traditional carbon sources, low-cost agricultural wastes like cassava starch residue, wheat bran and rice bran were also being evaluated for pullulan production to make it more economical as against petroleum-derived polymers (Ray et al. 2007). Pullulan is a promising biopolymer having a wide range of commercial and industrial applications in many elds like food science, health care, pharmacy and even in lithography (Singh et al. 2008). Other applications include bio-composite lms, oxygen-impermeable coatings, adhesives, plywood, medicine and as bre (Singh et al. 2008; Leathers et al. 2003; Knapp et al. 2010; Kong et al. 2014). For these special uses, polymer purity and molecular weight are important properties (Pollock et al. 1992; Punnapayak et al. 2003). So, research is also focused on developing an A. pullulans strain with enhanced pullulan productivity and raw material utilization ratio using a new method for genome shu ing of A. pullulans N3.387 (Kang et al. 2011).
A. pullulans is a yeast-like Ascomycete of Dothideales Order and Saccotheciaceae Family. They produce poly (β-L-malic acid), heavy oil liamocins, siderophore, and aubasidan-like β-glucan along with pullulan (Prasongsuk et al. 2018). A. pullulans var. pullulans, A. pullulans var. melanogenicum, and A. pullulans var. aubasidani are its three varieties based on the differences in its morphological and biochemical properties (Urzi et al. 1999; Yurlova et al. 1996; Zalar et al. 2008). The variety A. pullulans var. melanogenicum differs from the other varieties for its melanin production which occurs in the initial stages of growth and changes the colour of the colonies i.e. olive green to black color. The variety A. pullulans var. aubasidani shows negative response to the assimilation of methyl-α-D-glucoside and lactose and this feature is speci c to this variety differentiating it from the other varieties (Yurlova et al. 1996). This strain produces aubasidan-like Pullulan (glucans with α-1, 4-D-, ß-1, 6-D-and ß-1, 3-D-glycosidic bonds) (Yurlova et al. 1999).
Most of these strains were isolated from leaves of tropical plants. By in vitro digestibility study, it was reported that pullulan is less than 10% digestible and hence could act physiologically as dietary ber or residue (Kunkel et al. 1994).
Currently, the cost of pullulan is comparatively higher than the petroleum based polymers and hence, their use is limited. To enhance their use, apart from the attempts to reduce their production cost, newer methods of usage need to be explored. Electrospinning can be a good alternative method that produces continuous non-woven biopolymer nano bres with diameters from micron to nano scale range when an external electric eld is imposed on a spinneret containing the biopolymer solution (Liu et al. 2013). Thus produced nano bres nd potential applications in wound dressing, scaffold for tissue engineering, biosensors, drug delivery, gas barrier lms and nanocomposites. Since the beginning of the 21 st century, electrospun nano bres have been widely studied and a wide range of applications have been made, such as in energy storage, healthcare, environmental engineering, defense and security (Eslamian et al. 2019). Hence, this work explores the possibility of using cheaper carbon source for pullulan production and their electrospinning potential for production of nano bre mat and their characterization.
Sucrose was added to 1% (w/v). Agar was included at 15 gL -1 for solid plates.
A. pullulans MTCC 1991 was obtained from the culture collection of IMTECH Chandigarh, India. P2 medium with 5% sucrose was used to carry out submerged fermentation (SF). Wheat Bran (WB) and Cotton Stalk Dust (CSD) were obtained from the local market in Mumbai, India and used as solid substrate and sole carbon source for Solid State Fermentation (SSF). A basal production medium for SSF was prepared as follows: 3.5 g of sodium glutamate; 5 g of K 2 HPO 4 ; 2 g of KH 2 PO 4 , 2g of MgSO 4 ; 1g of NaCl and 0.5g of FeSO 4 .7H 2 O per liter and pH adjusted to 6.5 with 1N HCl. Solid substrate was prepared by taking 20 g of substrates in 250 mL Erlenmeyer asks and amended with 40 mL of basal medium (1:2 ratio, w/v) and thoroughly mixed. The prepared solid substrate was sterilized in an autoclave at 121 o C for 20 min and cooled to ambient temperature. All the chemicals used to make media were of analytical grade.
Isolation of pullulan producing fungi by selective enrichment Leaves (old and young) were removed from Peltophorum tree and soaked in sterile water for 3 days at 25 °C, and then 0.1 mL of soaked water was transferred to 10 mL of P2 minimal salts medium (pH 4) containing 1% (w/v) sucrose and 10 µg/mL of chloramphenicol. After 2 days of shaking of P2 medium broth at 25 °C, the turbid culture was allowed to stand undisturbed for 20 min to allow laments and aggregates to settle to the bottom. About 10 µL, from the upper partially clari ed phase that, was enriched for yeast-like cells were spread onto agar plates containing P2 medium (pH 5), 1% (w/v) sucrose and 10 µg/mL of chloramphenicol. After 4 days, independent colonies were puri ed by sub-culturing.

Pullulan production
The fungal inoculums of A. pullulans MTCC 1991 and the isolates were prepared as follows. A loop full of each culture was inoculated in basal medium and incubated at 30 °C for 48 h. About 0.5 mL culture was added to 40 mL of P2 (5% sucrose) liquid medium and the cultures were shaken at 200 rpm at 25 °C for 66 h for SF. While for SSF, 2 mL of 48 h old cultures grown on basal medium was inoculated in 20 g of solid substrates with 40 mL basal medium and incubated at 30 °C for 7 days. All the samples were analyzed in triplicates and the yields were expressed as average ± standard deviation (S.D.).

Biochemical Analysis of the substrates used for SSF
To determine the biochemical composition of WB and CSD, moisture content was determined by drying in hot air oven at 105 °C for 5 h, protein estimation (total nitrogen x 5.7 for WB and 6.25 for CSD) was determined by Kjeldahl method using Kelplus ® Instrument (Pelican Equipments, Chennai, India) and ash content was determined by heating in a mu e furnace at 550±25 °C for 3 h. Crude bre was determined by Weende's method of acid and alkali digestion using Fibraplus ® Instrument (Pelican Equipments, Chennai, India). All the parameters were analyzed in triplicates and were expressed as average ± (S.D.).

Downstream processing for extraction of EPS
The samples of SF, where 5% sucrose as carbon source was diluted with 1 volume of deionized water and centrifuged, and the EPS were recovered from the clari ed broth by precipitation with 1 volume of Isopropyl alcohol (IPA). The precipitate was removed and dried to constant weight in an oven at 80 °C. In case of SSF, the fermented mass was washed thoroughly with 5 volumes of deionized water by shaking at 250 rpm for 2 h. Cheese cloth was used for coarse ltration and centrifugation at 5488 x g for 45 min at 4 °C for ner particles separation. Pullulan was precipitated using 2 volumes of IPA after addition of 2% KCl in the aqueous extracts. The precipitated pullulan was collected on pre-weighed weighing bottle and dried at 80 °C till constant weight. The yield of pullulan was expressed as gL -1 of production medium in SF and as gL -1 of basal medium in SSF.

Screening of isolates for pullulan production
The extracted EPS from the isolates were tested for the presence of pullulan on the basis of the set of identi cation tests like solubility in water, pH of 10% solution in the range of 5-7, white precipitate formation with Polyethylene glycol 600 (PEG 600) and functional group analysis by Fourier transform infrared (FT-IR) (IR Prestige 21 ® model) by KBr pellet method. For PEG 600 test, 2 mL of PEG 600 was added to 10 mL of 2% aqueous EPS solution, development of white precipitate indicated a positive test for pullulan. FT-IR spectra were recorded with the following parameters: 64 scans; resolution, 4 cm -1 over the KBr pellet. Pullulan sample (1 mg) was manually well blended with 100 mg of KBr powder. These mixtures were then desiccated overnight at 50°C under reduced pressure prior to FTIR measurement. The pullulan extracted from A. pullulans MTCC 1991 and commercial pullulan were used for comparison. The cultures, that gave positive results for the above mentioned tests, were processed further for morphological and biochemical analysis for identi cation.

Characterization of pullulan
Extracted pullulan was subjected to X-ray diffractometer (XRD, X'pert Pro, PANalytical ® ) to study the crystallinity. Wide angle X-ray diffraction patterns were obtained with nickel ltered Cu Kα (λ = 1.54 Å) radiation and analyzed using automatic powder diffraction (APD) software. The diffracted intensities were recorded from 5 to 80 2θ angles. The crystallite height (I 002 ), measured at the peak around 20 2θ angle, and amorphous height (I am ), measured at the valley around 11 2θ angle, were used to calculate the apparent crystallinity index (apparent Cr.I.) using the empirical method (Choudhury et al. 2013) as per the equation (1).
The average pore size, total pore volume and speci c surface area were determined by Brunauer-Emmett-Teller (BET) method. Samples were degassed at 160 °C for 3.5 h under vacuum before analysis. BET analyses were performed using Quantachrome Nova model and Novawin software and using the equation (2).
Where, P = partial vapor pressure of adsorbate gas in equilibrium with the surface at 77.4 K (boiling point of liquid nitrogen), in Pascals; Po = saturated pressure of adsorbate gas, in Pascals; Va = volume of gas adsorbed at standard temperature and pressure (STP) [273.15 K and atmospheric pressure (1.013 × 10 5 Pa)], in mL; Vm = volume of gas adsorbed at STP to produce an apparent monolayer on the sample surface, in mL; C = dimensionless constant that is related to the enthalpy of adsorption of the adsorbate gas on the powder sample. A value of Va is measured at each of not less than 3 values of P/Po. Then the BET value calculated as given in equation (2) is plotted against P/Po according to the equation (3). This plot should yield a straight line usually in the approximate relative pressure range 0.05 to 0.3. All the parameters were analyzed in triplicates and were expressed as average ± standard deviation (S.D.).
The thermal properties of the extracted pullulan were evaluated by a differential scanning calorimeter (Mettler Toledo TC-15 TA).
Samples were placed onto aluminum crucibles and then the runs were carried out from room temperature to 450 °C at a heating rate of 10 °C min -1 under nitrogen atmosphere.
For 1 H-NMR spectroscopy, the solutions were prepared by dissolving 5 to 9 mg of commercial as well as lyophilized pullulan samples per mL of D 2 O (Sigma Aldrich, USA) and analyzed using Agilent 600 MHz AR Spectrophotometer, USA. The raw data was processed using the Vnmrj ® software.

Electrospinning of pullulan
The extracted pullulan from A. pullulans MTCC 1991 and A. pullulans were dissolved in distilled water with the help of heating and magnetic stirring. Final concentration was adjusted to 18% for extracted pullulan and 12% for commercial pullulan for electrospinning.
For the electrospinning solution, pH was measured by pH meter (EI Model -1012E), particle size and zeta potential by using Zeta Sizer Nicomp TM 380 ZLS (Santa Barbara, California, USA), conductivity by using Conductivity meter (Eutech Instruments, Con 2700) and viscosity by Brooke eld DV III Ultra Programmable Rheometer. All the parameters were analyzed in triplicates and were expressed as average ± S.D. The electrospinning was carried out with varying proportions PVA and pullulan for bead-less nano bre formation. The inhouse assembled electrospinning system consisted of a fume hood housing the equipment, syringe pump (New Era Pump Systems, Inc.) with a syringe having a metallic needle, a controllable high-voltage source (0 to 50 kV) connected to the needle, and a grounded copper foil wrapped collector plate for collection of the brous mats. Optimization experiments were carried out by varying the following process conditions: 15-20 cm tip to collector distance (TCD); 0.2 to 1.0 mL/h ow rate; and 15 to 25 kV voltages to obtain the bead-less nano bres.

Characterization of pullulan nano bre mats
The morphology of electrospun nano bres was studied using a scanning electron microscope (SEM), Philips XL-30, with an accelerating voltage of 12 kV. Each sample was coated with a thin layer of conducting material (gold/palladium) using a sputter coater before SEM analysis. The SEM images were analyzed in Image J ® software to determine the average diameter of nano bres. The morphology of nano bres was observed and bre diameter, roughness and surface area were measured using Atomic Force Microscope (AFM), (diInnova® SPM Veeco, Santa Barbara, CA, US), equipped with a 90 μm scanner by tapping mode in ambient condition. The silicon nitride cantilever with a spring constant of 40 Nm −1 was used for scanning. The scan rate of 1.0 Hz and 512 lines per 10 μm were used to optimum contrast. No ltering was done during scanning. The average pore size, total pore volume and speci c surface area were determined by BET analyzer. Samples were degassed at 160 °C for 3.5 h under vacuum before analysis. All the parameters were analyzed in triplicates and were expressed as average ± standard S.D.
Water contact angle was measured using GBX ILMS Version 3.6 instrument to study the hydrophilic nature of the electrospun nano bre mats. Using a microsyringe, 5 μL deionized water was dropped perpendicularly to each surface of the mat placed on a horizontal glass sheet. Then, the images of water drops on the surface of the mat were recorded and analyzed. The thermal properties of the nano bre mats were evaluated by a differential scanning calorimeter (Mettler Toledo TC-15 TA ® ). Samples were cut into small pieces and placed onto aluminum crucibles and then the runs were carried out from room temperature to 450 °C at a heating rate of 10 °C min -1 under nitrogen atmosphere. For determining the grey values of developed electrospun nano bre mats, the CIE (International Commission of Illumination) color space coordinates were determined. A color is de ned by its RGB values which give the amount of red, green and blue in a particular color. In CIE L*a*b* color space values, L* stands for lightness, a* and b* for the green-red and blue-yellow color components and ΔL represents brightness difference between samples. The magnitude of color difference was quanti ed as ΔE.

Results And Discussion
Isolation of pullulan producing fungi by selective enrichment Selective enrichment of the extract from Peltophorum leaves resulted in ve isolates namely OL1, OL2, OL3 obtained from old leaf and YL1 and YL2 obtained from young leaf respectively.

Discussion:
All isolates were maintained on P2 as well as PDA media. P2 medium was used as it contained minimal salts medium plus sucrose and low pH, which acted as basis for selective enrichment. Chloramphenicol was added to prevent bacterial growth during enrichment and isolation. After incubation for 2 days at 30 °C, the color of the colony ranged from off-white or light beige to pale pink to green and black appeared.

Production of pullulan and screening of isolates for pullulan production
The ve isolates were subjected to screening for pullulan production by SF and SSF. Basal medium consisting of trace elements, mineral nutrients and buffering agents was used to supplement the solid substrate for pullulan production. Pullulan yield was higher for WB by SSF as compared to 5% Sucrose in SF. CSD could not be utilized to produce pullulan, as the precipitates extracted from the cultures post fermentation were insoluble in water which is a preliminary con rmatory qualitative test for identi cation of pullulan (Table 1).  As per the results in Table 1, the yield obtained for A. pullulans MTCC 1991 using WB in SSF was more than the reported commercial production rate ( OL3 gave more yield than A. pullulans MTCC 1991 using WB in SSF. Thus, WB could be a promising economic substrate for production of pullulan in SSF. Thus the above results support the nding that WB has more than 50% nutritional value ( Table 2) as compared to CSD, making it a good solid substrate for pullulan production by SSF. Pullulan content and color depends on the strain of microorganisms used and the chemical composition of the substrate. Earlier work reported the preference of glucose over sucrose for production of pullulans by A. pullulans (Punnapayak et al. 2003).
As seen in Fig. 1, the FTIR spectrum of the commercial grade pullulan matches well with the EPS produced by A. pullulans MTCC 1991 and the isolate, thereby con rming the production of pullulan.

Morphological and biochemical analysis for identi cation
The selected fungal isolate for pullulan production, OL3 was subjected to identi cation based on morphological and nutritional physiological characteristics. Fungal growth in standard conditions (PDA at 30 °C) occurred within 48 h that were not pigmented or light pink at the beginning, later became olive green-black. As per Fig. 2, A. pullulans MTCC 1991 and OL3 had septate hyphae of diameter more than 2 µm. The conidiophore bearing conidia were observed for A. pullulans MTCC 1991 and prototunicate asci and blastic conidiogenesis of OL3 were also observed. Growth on different cultural media (PDA, MEA and YMA) affected both the morphology and pigment production. Variation in colony color was also detected when the fungi were cultivated on MEA for seven days.
Following the identi cation keys provided earlier (de Hoog et al. 1994) and morphological appearance, the isolate namely OL3 was identi ed as Aureobasidium pullulans (de Bary & Lowenthal) G. Arnaud. This has also been con rmed by the morphological characterization carried out by National Fungal Culture Collection of India (NFCCI)-A National Facility, Agharkar Research Institute, Pune, India. As per the sequencing results done by Internal transcribed spacer (ITS) method, it was found that the strain showed 99% similarity with Aureobasidium melanogenum CBS 105.22, carried out at NCMR-NCCS, Pune, India. The same strain has been deposited at NCCS, Pune, with the accession number MCC 1868.

Physical characterization of pullulan
The XRD patterns of pullulan extracted from A. pullulans MTCC 1991 and A. pullulans (Fig. 3) had a very distinct broad valley and peak, indicating the amorphous and crystalline nature of the material. The deepest point of the valley was considered for calculation purpose of amorphous region. The apparent crystallinity indices of commercial pullulan, A. pullulans MTCC 1991 and A. pullulans were calculated as 29.0%, 45.8% and 18.3%, respectively.
As seen in Table 4, the average pore radius of commercial pullulan and that of extracted pullulan from A. pullulans MTCC 1991 as well as isolates was the same around 24-30 Å. Similarly the total pore volume was found to be in the range 0.02 to 0.075 cc g -1 . 13.66 ± 2.5 As seen in the thermograms (Fig. 4), T g values were 41, 43 and 43 for commercial pullulan, pullulans from A. pullulans MTCC 1991 pullulan and A. pullulans respectively. The range of melting temperature was found to be from 107 to 152 °C.
Structural characterization of pullulan was carried out by 1 H-NMR and 13 C-NMR spectroscopy and compared with that of commercial pullulan. Four chemical signals were displayed in the anomeric region between 4.4 to 5.3 ppm due to the four sugar repeating unit present in the pullulan polysaccharide. Signals were observed at 1.2 and 1.1 ppm was due to 6-deoxy-d-altrose present in the polysaccharide as shown in Fig. 5a. This con rms the presence of the major components of the pullulan structure in the extracted polymer as compared with the commercial in 1 H-NMR spectra. For all the extracted pullulans (Fig. 5b), it was found that there was splitting of C-6 (peaks between 60 to 62 ppm) and C-4 (peaks between 71 to 79 ppm) were due to C-1 of (1 → 4) linked glucose unit. C-6 signals arrived at around 60 ppm are due to two kinds of 1, 4 linked α-D glucose, whereas the signal at around 69 ppm corresponds to C-6 of the 1, 6 linked α-D glucose. There was a single peak between 82 and 88 ppm at around 84ppm which explained that very few of the sugar residues were available in furanose form and most of them were in pyranose form in biopolymer. Anomeric α (1 → 6) appeared by peak at around 99 ppm. Signals were detected in the 57.7-64.7 regions indicating that some C-6 positions were non-glycosylated.
Anomeric α (1 → 6) was indicated by peak resonance at 99.73 ppm whereas anomeric α (1 → 4) was indicated by peak resonance at 100-101 ppm. None of the peaks were obtained between 93 and 97 ppm indicating the absence of oligomers. The technique is named after its inventors; BET is the most frequent method for determination of speci c surface area of porous materials. The measurement is based on physical adsorption of gas on the surface of the sample. As seen in Table 4, the average pore radii and total pore volume of the commercial pullulan and extracted samples were similar. But, the surface area for extracted pullulan was more than twice the surface area of commercial pullulan. It is also evident that particle size, charge, viscosity and conductivity, of the lyophilized pullulans from A. pullulans MTCC 1991 and A. pullulans, were signi cantly larger than commercial pullulan. The increased amount of charge as seen from the values of zeta potential corresponds to the increased conductivity of the extracted pullulans. This increased conductivity in turn would help in the process of electrospinning thereby giving larger surface area, pore size and pore volume.

Discussion
The glass transition temperature (T g ) was taken as the mid-point of the change of slope in the DSC curves. As per the literature depending on different sources of pullulan, the range of T g of pullulan was from 38 to 59 °C (Xiao et al. 2015). As seen in the thermograms (Fig. 4)

Electrospinning of pullulan
The pH of the solution was found to be on the acidic side for commercial pullulan but neutral or slightly alkaline for the extracted pullulan. The zeta potential and conductivity of extracted pullulans were higher than that of commercial pullulan. The viscosity of extracted pullulans was higher than that of commercial pullulan. All the values are given in Table 4. Taking into account, pH, zeta potential, conductivity and particle size of the extracted pullulans, there was a need for a carrier polymer to aid the process of electrospinning to produce nano bers for which PVA was chosen.
Throughout the experiment the applied voltage, ow rate, polymer concentration and TCD were xed at 20kV, 0.5 mL h -1 , 18% pullulan and 15 cm, respectively. The proportion of PVA required to produce nano bres was found to be 50% for pullulan extracted from A. pullulans where as it was 40% for pullulan extracted from A. pullulans MTCC 1991. But, commercial pullulan gave beaded nano bres with PVA (Fig. 6).

Discussion:
Acidic solution tend to give better electrospinning due to presence of H + ion concentration aiding in attraction of polymer solution droplet from the tip of the collector towards the negative electrode when electric eld is applied. So, the pullulan with neutral to alkaline pH was not getting electrospun without any carrier polymer. Higher zeta potential and conductivity let to increasing the charge carrying ability of the polymer jet which subjected to higher tension under the electric eld, resulted in poor bre formation. The viscosity range of different polymer solutions at which electrospinning is done is a major variable. Higher viscosity of extracted pullulans aided them to give continuous smooth bres when electrospun whereas commercial pullulan having lower viscosity would give beaded bres. PVA was used as a carrier polymer as it is relatively inexpensive, chemically and thermally stable, and not degradable under most physiological conditions. An earlier work reported the production of gelatin/polyurethane blended nano ber by electrospinning having the potential application for use as a wound dressing, wherein the polyurethane was used to reduce the hydrophilicity of gelatin (Kim et al. 2009). Yet another work reported the production of a composite electrospun nano bre using pullulan, PVA and montmorillonite clay having enhanced thermal stability and mechanical property (Islam et al. 2012).
The morphology of electrospun nano bres can be affected by electrospinning instrument parameters including applied voltage, tip to collector distance (TCD), ow rate and solution parameters such as polymer concentration. Earlier work reported the electrospinning of chitosan from its solutions in 2% aqueous acetic acid by adding PVA as a "guest" polymer (Zhang et al. 2007).

Characterization of nano bre mats
The SEM images showed smooth bead free nano bres for electrospun pullulan extracted from A. pullulans and A. pullulans MTCC 1991 but commercial pullulan gave beaded nano bres (Fig. 6). As analyzed in Image J ® software, the average diameter range of nano bres was found to be 130 to 179 nm for commercial pullulan blended with PVA whereas the diameter range was 180 to 220 nm for pullulan extracted from A. pullulans and A. pullulans MTCC 1991 blended with PVA (see Table 5). The AFM images (Fig. 7) as analyzed in the SPM lab software showed that the bres obtained on electrospinning of commercial pullulan and PVA were larger in height and diameter as compared to their blended bres (Table 6). Also, the extracted pullulans blended with PVA not only gave thinner bres than commercial polymer but also gave bres in the nano-size range. The BET analysis data measuring the average pore radius, total pore volume are given in the table 7. The average pore radius (8.664 Å) was smallest in case of the electrospun nano bre mat produced from commercial pullulan + 40 % PVA. The total pore volume (0.08785 cc/g) was lowest in case of the electrospun nano bre mat produced from A. pullulans pullulan + 50% PVA. The surface area (1302 m 2 /g) was the highest in case of the electrospun nano bre mat produced from PVA alone. The surface area for commercial pullulan alone was found to be 183.4 m 2 /g. But the surface areas of their blended solutions were more than 183.4 m 2 /g, this is attributed to the high surface area of PVA nano bres. In the DSC analysis, the electrospun pullulan mat without PVA did not show any endothermic peak around 220 °C, which is attributed to melting temperature of PVA (Fig. 8). This peak was prominent in all the electrospun blended mats of pullulan and PVA at around 220 °C.
As per the CIE colour space analysis, the lightness value, L*, represents the darkest black at L* = 0, and the brightest white at L* = 100. The L* (Lightness) values obtained for electrospun commercial pullulan was 91 whereas L* values for electrospun pullulan extracted from, A. pullulans MTCC 1991 and A. pullulans were 91 and 90 respectively When compared to standard white paper on which the electrospinning was carried out, ΔL* values for electrospun, commercial pullulan, pullulan extracted from A. pullulans MTCC 1991 and A. pullulans were 2.408, 1. obtained for electrospun commercial pullulan was -3.051 whereas for electrospun pullulan extracted from, A. pullulans MTCC 1991 and A. pullulans were 4.556 and 1.078 respectively. When the magnitude of the color difference, ΔE*, is higher than 1, it indicates a visually detectable color difference and its value increases further for greater color changes. When compared to standard white paper on which the electrospinning was carried out, ΔE* values for electrospun, commercial pullulan, pullulan extracted from A. pullulans MTCC 1991 and A. pullulans were 6.995, 16.219 and 12.873 respectively.

Discussion:
Results of SEM and measurement of average diameter range of nano bers indicated that the addition of PVA helps to reduce the diameter of nano bres out of pullulan. Earlier work reported the diameter of composite pullulan-whey protein nano bers made by electrospinning to be 231 nm (Drosou et al. 2018).
As per the results of AFM, the height and diameter of nano bres increased by 40% and 70% for pullulan extracted from A. pullulans whereas it decreased by 40% and 35% for pullulan extracted from A. pullulans MTCC 1991, respectively, when compared to the nano bres of commercial pullulan / PVA blend. The roughness of pullulan nano bres increased by 45% and 10% for pullulans extracted from A. pullulans and lamentous yeast, respectively whereas it decreased by 35% for pullulan extracted from A. pullulans MTCC 1991 as compared to the commercial pullulan / PVA blend. In case of measurement of surface area, the difference in the extracted pullulan and commercial were less than 5%. Overall surface area calculated for all the samples were not signi cantly different.
As per the BET results in Table 7, the average pore radius electrospun nano bres from pullulan of A. pullulans MTCC 1991 + 40% PVA was 80% more than its comparable commercial pullulan with 40% PVA. But, the average pore radius electrospun nano bres from pullulan of A. pullulans + 50% PVA was 35% lesser than its comparable commercial pullulan with 50% PVA. The total pore volume of electrospun nano bres from pullulan of A. pullulans + 50% PVA is 56% lesser than its comparable commercial pullulan with 50% PVA. On the other hand, the total pore volume of electrospun nano bres from pullulan of A. pullulans MTCC 1991 + 40% PVA was 14.5% more than its comparable commercial pullulan with 40% PVA. Thus, extracted pullulan gave better average pore radius and total pore volume as compared to commercial when same amount of PVA carrier polymer was added. The surface area of electrospun nano bres from pullulan of A. pullulans MTCC 1991 + 40% PVA was 36.4% less than its comparable commercial pullulan with 40% PVA. The surface area of electrospun nano bres from pullulan of A. pullulans + 50% PVA was 32% less than its comparable commercial pullulan with 50% PVA. This proves that nest nano bres were produced from extracted pullulan from A. pullulans + 50% PVA. The surface area obtained in this work is very much higher than the reported value of 17.6 m 2 /g in case of electrospun gelatin nano bres (Jakub et al. 2012) and 19.49 m 2 /g in case of β-Cyclodextrin based electrospun nano bers (Zhao et al. 2015). Similar result was reported earlier wherein a facile route for fabrication of novel microporous material based on chitosan and PVA nano bres resulted in high speci c surface area (1680 m 2 /g), considerably small, pore volume (0.061 cc/g), and small pore radius (0.08 Å), as proved by BET analysis (Sargazi et al. 2018).
The wettability of a material surface is an important property that is described as the contact angle between the liquid and the material surface. It clearly showed that as the presence of PVA imparted hydrophobic effect to the electrospun pullulan mat. In all the cases, the contact angle of the blended polymer electrospun mat was in between that of pure pullulan and pure PVA. In an earlier report, the uorinated silane functionalized superhydrophobic pullulan/poly(vinyl alcohol) blend membrane with water contact angle larger than 150° was prepared by the electrospinning method and characterized (Karim et al. 2011). Another study was reported earlier wherein the hydrophobicity was introduced in the biodegradable lms using PVA (Karim et  As per the CIE colour space analysis, no signi cant difference in whiteness (L*) was observed among various pullulan samples. The electrospun mat of extracted pullulan was darker than that of commercial pullulan as per the values of ΔL*. The commercial pullulan and one extracted from A. pullulans were found be having redness color component whereas pullulan extracted from A. pullulans MTCC 1991 was found to be having greenness color component as per the values of a*. The commercial pullulan was found to be having blueness color component whereas pullulan extracted from A. pullulans MTCC 1991 and A. pullulans MTCC 1991 were found to be having yellowness color component as per the values of b*. According to the values of ΔE*, mat from electrospun extracted pullulan is having signi cant color difference than that of commercial pullulan.

Conclusion
The isolate, A. pullulans yielded more pullulan (67.4 gL -1 ) as compared to A. pullulans MTCC 1991 (63.68 gL -1 ) in solid state fermentation. Wheat Bran, a low-cost and easily available waste material from wheat our production, could provide an economic advantage as a solid substrate as well as sole carbon source for production of the pullulan by A. pullulans isolated from the Peltophorum species. Even though the speci c surface area of the commercial pullulan was higher than the extracted pullulan nano bres, the average pore size and total pore volume for extracted pullulan nano bres were lesser than the commercial as calculated by BET analysis, indicating ner nano bre formation. This in turn could be helpful for application based on retaining ability of the nano bre mats at the nano level and thereby increasing the e ciency of the product. The pullulan extracted from A. pullulans MTCC 1991 as well as isolate, A. pullulans could be electrospun into nano bres only when blended with PVA (40 -50%). The signi cant change in melting temperature of the blended electrospun mats as observed in DSC thermograms could be attributed to the addition of PVA. This work provides newer isolates for production of pullulan using wheat bran as carbon source and also the process protocol for production of nano bre mat by electrospinning after blending with PVA. Availability of data and materials All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

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