Green Synthesis and Characterization of Pullulan Mediated Silver Nanoparticles through Ultraviolet Irradiation

Nanoparticles (NPs) are, frequently, being utilized in multi-dimensional enterprises. Silver nanoparticles (AgNPs) have attracted researchers in the last decade due to their exceptional efficacy at very low volume and stability at higher temperatures. Due to certain limitations of the chemical method of synthesis, AgNPs can be obtained by physical methods including sun rays, microwaves and ultraviolet (UV) radiation. In the current study, the synthesis of pullulan mediated silver nanoparticles (P-AgNPs) was achieved through ultraviolet (UV) irradiation, with a wavelength of 365 nm, for 96 h. P-AgNPs were formed after 24 h of UV-irradiation time and expressed spectra maxima as 415 nm, after 96 h, in UV-vis spectroscopy. The crystallographic structure was “face centered cubic (fcc)” as confirmed by powder X-ray diffraction (PXRD). Furthermore, high resolution transmission electron microscopy (HRTEM) proved that P-AgNPs were covered with a thin layer of pullulan, with a mean crystalline size of 6.02 ± 2.37. The average lattice fringe spacing of nanoparticles was confirmed as 0.235 nm with quasi-spherical characteristics, by selected area electron diffraction (SAED) analysis. These green synthesized P-AgNPs can be utilized efficiently, as an active food and meat preservative, when incorporated into the edible films.


Introduction
Metal nanoparticles (NPs) have been frequently investigated by the researchers for their multi-dimensional applications after their synthesis in 1959 by Richard Feynman, with a size ranging from 1 nm to 100 nm, [1][2][3]. NPs exhibited a new behavior of efficiency at even very low concentration [4]. The recent trend of nanotechnology research has been diverted to antimicrobial food packaging, delivery of nano-medicines/drugs, gene delivery vectors, nano-imaging and Biosensors for cancer diagnosis, and polymeric nano-composite wound dressing [3,4].
Among metal NPs, the utilization of silver nanoparticles (AgNPs) in the biomedical sector, animal and human nutrition, electronic industry and food preservation is remarkable [5]. AgNPs, as compared to other metal NPs, exhibit very low volatility, with tolerance against high temperatures as compared to other nanoparticles [6]. Moreover, AgNPs can restrain the growth of the microorganisms (including Gram positive and Gram negative bacterial strains; fungi) from the initial contact and further penetration into the cellular membrane of the microbes [7,8].

Green Synthesis of Pullulan Mediated Silver Nanoparticles
Three concentrations of pullulan were prepared as per the procedure reported by Shameli et al. [13] with modifications. The 1.0, 2.0 and 4.0 g of pullulan powder, equal to 5, 10 and 20% (w/v),were stirred with 200 mL of deionized water for 1.0 h at 500 rpm at 24-25 • C.
A clear solution was obtained after 60 min of stirring. Three concentrations of AgNO 3 aqueous solution (0.02, 0.50, and 1.0 M) were produced by dissolving the calculated masses of AgNO 3 in deionized water. To synthesize P-AgNPs, 250 mL of each pullulan solution (5, 10, and 20 wt.%) was mixed with an equal volume of the aqueous solutions of AgNO 3 . The assorted solutions were stirred thoroughly for 1.0 h at 65 • C for 500 rpm to complete the chemical reaction.
After stirring, the obtained suspensions were clear but viscous. These suspensions were put under ultraviolet radiation with a wavelength of 365 nm and the magnetic stirring speed of 195 rpm. UV irradiation was done by an ultraviolet reactor (ChECA, UTM, Kuala Lumpure, Malaysia) with a power of 450 W. The feature of the UV-reactor bulb was UV-C Radiation (200 to 280 nm) (Philips, TUV 8W, G8 T5, Kuala Lumpure, Malaysia). The irradiation process was done for 1, 3,6,8,12,24,48,64, and 96 h (A1-A9). After UV irradiation, the suspensions were centrifuged 25 • C for 30 min to obtain the pellets of P-AgNPs.

Characterization of Pullulan Mediated Silver Nanoparticles
Pullulan mediated silver nanoparticles (P-AgNPs) were characterized [9,12,13] through ultraviolet-visible spectroscopy (UV-vis) by a U-5100, UV-vis spectrophotometer (Hitachi, High Technologies Co., Tokyo, Japan) with a wavelength range of 220 nm to 800 nm. The analysis of the structure was conducted through powder X-ray diffraction (PXRD) (PANanalytical EMPYREAN, Eindhoven, The Netherlands), Xpert High score Plus (V.14.0; PANanalytical EMPYREAN) at a lower angle of 30 • -80 • and scan with a speed of 2 • /min. The topographic analysis was performed by field emission scanning electron microscopy (FESEM) using a JSM 7600 F FESEM (JEOL Ltd., Tokyo, Japan). High-resolution transmission electron microscopy (HRTEM) was conducted through a JEM 2100F filed emission electron microscope (JEOL Ltd.) with a high resolution of 200 kV using a LaB 6 electron gun. Zeta potential of P-AgNPs was confirmed by a Litesizer 500, particle analyzer (Anton paar USA Inc., Ashland, VA, USA) while Fourier transform infrared spectroscopy (FT-IR) was conducted, with the spectral range of 500 cm −1 to 4000 cm −1 , by using an IRTracer 100 Fourier transform infrared spectrophotometer (SHIMADZU Corp., Kyoto, Japan). The colloidal solution was centrifuged through a refrigerated micro-centrifuge 5810 R (Sigma-Aldrich) to obtained P-AgNPs pellets.
Most of the characterization was performed with the colloidal solution dissolved in distilled water, while P-AgNPs powder was used for PXRD and FESEM analyses. Statistical analysis of the data was done by using SPSS software to measure the mean crystalline size (V.20.0, IBM Corp., Armonk, NY, USA).

Results and Discussion
In the present study, AgNPs were synthesized by UV irradiation successfully and pullulan was used as "stabilizer". Further, the impact of irradiation time on the formation of NPs along with the size of NPs was also considered. Pullulan 10% (w/v) solution along with 0.50 M AgNO 3 aqueous solution showed the formation of P-AgNPs. The formation of P-AgNPs was confirmed by the dark color of the colloidal suspension, after 96 h of UV irradiation [13] (Figure 1). Technologies Co., Tokyo, Japan) with a wavelength range of 220 nm to 800 nm. The analysis of the structure was conducted through powder X-ray diffraction (PXRD) (PANanalytical EMPYREAN, Eindhoven, The Netherlands), Xpert High score Plus (V.14.0; PANanalytical EMPYREAN) at a lower angle of 30°-80° and scan with a speed of 2°/min. The topographic analysis was performed by field emission scanning electron microscopy (FESEM) using a JSM 7600 F FESEM (JEOL Ltd., Tokyo, Japan). High-resolution transmission electron microscopy (HRTEM) was conducted through a JEM 2100F filed emission electron microscope (JEOL Ltd.) with a high resolution of 200 kV using a LaB6 electron gun. Zeta potential of P-AgNPs was confirmed by a Litesizer 500, particle analyzer (Anton paar USA Inc., Ashland, VA, USA) while Fourier transform infrared spectroscopy (FT-IR) was conducted, with the spectral range of 500 cm −1 to 4000 cm −1 , by using an IRTracer 100 Fourier transform infrared spectrophotometer (SHIMADZU Corp., Kyoto, Japan). The colloidal solution was centrifuged through a refrigerated micro-centrifuge 5810 R (Sigma-Aldrich) to obtained P-AgNPs pellets.
Most of the characterization was performed with the colloidal solution dissolved in distilled water, while P-AgNPs powder was used for PXRD and FESEM analyses. Statistical analysis of the data was done by using SPSS software to measure the mean crystalline size (V.20.0, IBM Corp., Armonk, NY, USA).

Results and Discussion
In the present study, AgNPs were synthesized by UV irradiation successfully and pullulan was used as "stabilizer". Further, the impact of irradiation time on the formation of NPs along with the size of NPs was also considered. Pullulan 10% (w/v) solution along with 0.50 M AgNO3 aqueous solution showed the formation of P-AgNPs. The formation of P-AgNPs was confirmed by the dark color of the colloidal suspension, after 96 h of UV irradiation [13] (Figure 1). The formation of NPs was due to the reduction of AgNO3, changing the color of clear colloidal solution into light gray, then gray, and finally darker gray [13,32]. Our results are in agreement with the results as reported by Kanmani and Lim [32], Shameli et al. [13], and Saravanan et al. [24] for the formation of AgNPs by the reduction of AgNO3. The chemical structure of pullulan consists of repeating units of malto-triose connected with ά-(1-6) linkages, providing solubility and flexibility to pullulan [33]. During the reduction process, the produced AgNPs were attracted by the amino The formation of NPs was due to the reduction of AgNO 3 , changing the color of clear colloidal solution into light gray, then gray, and finally darker gray [13,32]. Our results are in agreement with the results as reported by Kanmani and Lim [32], Shameli et al. [13], and Saravanan et al. [24] for the formation of AgNPs by the reduction of AgNO 3 . The chemical structure of pullulan consists of repeating units of malto-triose connected with

18
-(1-6) linkages, providing solubility and flexibility to pullulan [33]. During the reduction process, the produced AgNPs were attracted by the amino radicals (NH 2 ) and hydroxyl (OH) ends of malto-triose units of pullulan [13]. As a result, pullulan acts as a strong protective layer and a highly functional coating for AgNPs, with better stability in the solution over the time [30].
The impact of pullulan, as reducing and capping agent, on the formation of P-AgNPs was proven by ultraviolet visible (UV-vis) spectroscopy after various irradiation times ( Figure 2). The UV-vis spectroscopic peaks were recorded from wavelengths 300-800 nm, where NPs formed a broad surface plasma resonance ultraviolet absorption band in the wavelength range of 380-480 nm [24]. It was possible to control the synthesis, size, and the shape of AgNPs surrounded by pullulan, by irradiation time as expressed by UV-vis spectra [12]. UV irradiation induced the reduction of the AgNPs precursor in the presence of pullulan solution [12,13]. Photo-chemical induced reduction of AgNO3 into AgNPs, in the presence of a polysaccharide capping agent, can not only control the sizes of NPs, but also their shapes [35].The prolonged exposure of AgNO3 colloidal solution containing silver cation ions causes the heating of AgNPs, resulting in their breakdown into smaller sized NPs [35]. As the irradiation time increased, the size of the synthesized AgNPs was reduced. This can be considered as the irradiation time dependent factor to control the synthesis, size, and shape of nanoparticles [36,37].
Our results are in agreement with Pandey et al. [34], Esumi et al. [35], Kuthirummal et al. [36], and Huang et al. [37] with respect to irradiation time controlling the size and shape of NPs. The crystalline nature along with the topography of P-AgNPs, synthesized after 96 h of irradiation, was further confirmed by powder X-ray diffraction (PXRD). It can be observed that the crystalline nature of P-AgNPs was confirmed as crystalline silver (Figure 3). The nanoparticles exhibited four silver peaks in PXRD analysis at 2 theta/degree, i.e., 111 (38.17°), 200 (44.33°), 220 (64.49°), and 311 (77.44°). These peaks are consistent with Ag° Peak Ref. No. 00-004-0783 (99.9% pure silver; fcc) confirming their topographic nature as "face centered cubic (fcc)" [30]. From PXRD results, it is possible to estimate the average crystalline size of P-AgNPs by using the Debye-Scherrer's equation which is given as shown below; where n is the mean crystalline size, K is Sherrer's constant (shape factor) with value 0.94 for spherical crystallites with cubical symmetry, λ is X-ray wavelength (1.54 Å; for powder samples only), θ is the Bragg angle and β is the line broadening at half of the XRD peak [38]. The confirmation of actual crystalline size was conducted by high resolution transmission electron microscopy It was also observed that P-AgNPs, formed in the colloidal solution after 96 h of irradiation, expressed a wavelength peak at 415 nm. It was due to the fact that the AgNPs absorb UV radiation in the visible range of electromagnetic spectrum of 380 nm to 450 nm, because of surface plasmon resonance (SPR) transition of AgNPs [17,34].
Therefore, based on the achieved results, pullulan with 10% concentration showed the formation of P-AgNPs, confirmed by the UV-vis spectra, indicating the impacts of pullulan concentrations, as well as UV irradiation time, on AgNPs formation (Figure 2). Pullulan mediated silver nanoparticles (P-AgNPs) formed after 24 h from UV-irradiation time, and the peak intensities were observed after 48, 64 and 96 h (Figure 2). This long-term study was related to evaluate the effect of radiation on the size and distribution of AgNPs. During that period we witnessed two phenomena at different times: contained aggregation at the beginning, and disaggregation at the end of radiation time. It was confirmed that the size of NPs was also reduced as the irradiation time increased from 0 h to 96 h. The power consumption (power × time), during irradiation, was recorded as 10.80 (at 24 h) to 43.20 (at 96 h) kWh/500 mL of colloidal solution for P-AgNPs synthesis (Table 1, Figure 2C). The UV irradiation induced reduction process has been provided below: n Ag + + Ag + 2 → Ag + n (4) Ag + is the nano-cluster of the silver obtained from silver precursor (AgNO 3 ) and e − (eq) are the electrons in the aqueous solution. After UV irradiation of pullulan/AgNO 3 colloidal solution, Ag + was, first, reduced to (Ag) • , then Ag + n , leading towards the formation of (Ag) n by the large number of aqueous electrons produced during the process [13]. Ultraviolet light covers a wave length spectrum from 100 to 380 nm and is sub-divided into three regions by wavelength including UV-A (320-400 nm), UV-B (280-320 nm), and UV-C (200-280 nm). UV-C is considered as the most energetic ultraviolet radiation [11]. Therefore, in this study, UV-C was used for the reduction of Ag + to (Ag) n .
It was possible to control the synthesis, size, and the shape of AgNPs surrounded by pullulan, by irradiation time as expressed by UV-vis spectra [12]. UV irradiation induced the reduction of the AgNPs precursor in the presence of pullulan solution [12,13]. Photo-chemical induced reduction of AgNO 3 into AgNPs, in the presence of a polysaccharide capping agent, can not only control the sizes of NPs, but also their shapes [35].The prolonged exposure of AgNO 3 colloidal solution containing silver cation ions causes the heating of AgNPs, resulting in their breakdown into smaller sized NPs [35]. As the irradiation time increased, the size of the synthesized AgNPs was reduced. This can be considered as the irradiation time dependent factor to control the synthesis, size, and shape of nanoparticles [36,37].
Our results are in agreement with Pandey et al. [34], Esumi et al. [35], Kuthirummal et al. [36], and Huang et al. [37] with respect to irradiation time controlling the size and shape of NPs.
The crystalline nature along with the topography of P-AgNPs, synthesized after 96 h of irradiation, was further confirmed by powder X-ray diffraction (PXRD). It can be observed that the crystalline nature of P-AgNPs was confirmed as crystalline silver (Figure 3). The nanoparticles exhibited four silver peaks in PXRD analysis at 2 theta/degree, i.e., 111 (38. No. 00-004-0783 (99.9% pure silver; fcc) confirming their topographic nature as "face centered cubic (fcc)" [30]. From PXRD results, it is possible to estimate the average crystalline size of P-AgNPs by using the Debye-Scherrer's equation which is given as shown below; n = Kλ/βcos θ where n is the mean crystalline size, K is Sherrer's constant (shape factor) with value 0.94 for spherical crystallites with cubical symmetry, λ is X-ray wavelength (1.54 Å; for powder samples only), θ is the Bragg angle and β is the line broadening at half of the XRD peak [38]. The confirmation of actual crystalline size was conducted by high resolution transmission electron microscopy (HRTEM). Nevertheless, PXRD patterns are the main characteristic of P-AgNPs, also in concordance with the findings of Coseri et al. [30]; Bankura et al. [17]; Kanmani and Lim, [32] and Roopan [20].
Materials 2019, 12, x FOR PEER REVIEW 6 of 12 (HRTEM). Nevertheless, PXRD patterns are the main characteristic of P-AgNPs, also in concordance with the findings of Coseri et al. [30]; Bankura et al. [17]; Kanmani and Lim, [32] and Roopan [20]. Similar findings have been reported for the typical PXRD spectra of AgNPs with four diffraction peaks (111, 200, 220, 311) by Coseri et al. [30]. A four diffraction peak pattern of AgNPs confirms the "fcc" topographic nature of NPs [17,30,32]. Bankura et al. [17] and Roopan [20] had previously reported that the four peak pattern at 2-theta/degree could be due to the crystallization of reducing and capping agent used for the synthesis of Ag NPs.
The topographic structure and shape of P-AgNPs was confirmed as "spherical" by field emission scanning electron microscopy (FESEM) ( Figure 4A,B). The FESEM micrographs of P-AgNPs revealed the presence of spherical shaped P-AgNPs at 10,000×. Our study was in agreement with the findings of Saravanan et al. [24], for the distribution of spherical shaped "bacterial exo-polysaccharide AgNPs. The mean crystalline size of NPs was observed as 42.27 ± 12.2 nm after 48 h of UV irradiation ( Figure 4C) and 6.02 ± 2.37 nm after 96 h of UV irradiation ( Figure  4D), as confirmed by high resolution transmission electron microscopy (HRTEM). The decrease in crystalline size of P-AgNPs from 48 h to 96 h of UV irradiation, was proven by HRTEM analysis, along with "quasi-spherical" characteristics, as per reports by Quester et al. [39]. The typical "quasi-spherical" nature is one of the prominent shape characteristics of AgNPs for their application in various fields [40]. By HRTEM observation at 5 nm, it was found that P-AgNPs were delimited by a thin layer of an organic material ( Figure 4E) which is supposed to be the reducing and the capping agent pullulan [40]. The top to top distance, between the surface lattice of AgNPs, was 0.235 nm ( Figure 4E) in selected area electron diffraction (SAED) analysis ( Figure 4F) [41].
The possible involvement of the functional groups of pullulan for the synthesis of P-AgNPs was studied by FT-IR spectra. Pullulan aqueous solution (10% w/v, without UV irradiation) was compared with pullulan + AgNO3 colloidal solution (with 96 h of UV irradiation, A9). It can be observed that P-AgNPs expressed their characteristic peaks from wave numbers 3925 cm −1 to 1325 cm −1 (Figure 5a). The first strong and broader peak was at 3359 cm −1 (Figure 5a) indicating the stretching and involvement of hydroxyl (O-H) group of pullulan [31]. Similar findings have been reported for the typical PXRD spectra of AgNPs with four diffraction peaks (111, 200, 220, 311) by Coseri et al. [30]. A four diffraction peak pattern of AgNPs confirms the "fcc" topographic nature of NPs [17,30,32]. Bankura et al. [17] and Roopan [20] had previously reported that the four peak pattern at 2-theta/degree could be due to the crystallization of reducing and capping agent used for the synthesis of Ag NPs.
The topographic structure and shape of P-AgNPs was confirmed as "spherical" by field emission scanning electron microscopy (FESEM) ( Figure 4A,B). The FESEM micrographs of P-AgNPs revealed the presence of spherical shaped P-AgNPs at 10,000×. Our study was in agreement with the findings of Saravanan et al. [24], for the distribution of spherical shaped "bacterial exo-polysaccharide AgNPs. The mean crystalline size of NPs was observed as 42.27 ± 12.2 nm after 48 h of UV irradiation ( Figure 4C) and 6.02 ± 2.37 nm after 96 h of UV irradiation ( Figure 4D), as confirmed by high resolution transmission electron microscopy (HRTEM). The decrease in crystalline size of P-AgNPs from 48 h to 96 h of UV irradiation, was proven by HRTEM analysis, along with "quasi-spherical" characteristics, as per reports by Quester et al. [39]. The typical "quasi-spherical" nature is one of the prominent shape characteristics of AgNPs for their application in various fields [40]. By HRTEM observation at 5 nm, it was found that P-AgNPs were delimited by a thin layer of an organic material ( Figure 4E) which is supposed to be the reducing and the capping agent pullulan [40]. The top to top distance, between the surface lattice of AgNPs, was 0.235 nm ( Figure 4E) in selected area electron diffraction (SAED) analysis ( Figure 4F) [41].  The possible involvement of the functional groups of pullulan for the synthesis of P-AgNPs was studied by FT-IR spectra. Pullulan aqueous solution (10% w/v, without UV irradiation) was compared with pullulan + AgNO 3 colloidal solution (with 96 h of UV irradiation, A9). It can be observed that P-AgNPs expressed their characteristic peaks from wave numbers 3925 cm −1 to 1325 cm −1 (Figure 5a). The first strong and broader peak was at 3359 cm −1 (Figure 5a) indicating the stretching and involvement of hydroxyl (O-H) group of pullulan [31].
The O-H group of pullulan is considered as an efficient coordinator with silver ions of P-AgNPs during the reduction process [34]. The prominent peaks were at wave numbers 2418, 2167, 1950, 1637, and 1535 cm −1 (Figure 5a), proving the stretching frequencies of carbonyl (C=O) and C=C groups of pullulan [31,32,42]. For the involvement and the stretching frequencies of C=O and C=C functional groups, the standard wave numbers range from 1000 cm −1 to 1900 cm −1 [31]. The FT-IR spectral results were in agreement with the reports of Ganduri et al. [31], Kanmani and Lim [32] and Kumar et al. [41] for the involvement of O-H, C=O and C=C groups of pullulan during the synthesis of P-AgNPs.
The zeta potential of P-AgNPs was confirmed as 3.27 + (Figure 5b), proving "stability" of the NPs in the colloidal solution at 25 °C and pH 6.34 [34]. Pullulan has an "uncharged nature" in the colloidal solution when used as the reducing and capping agent [30]. Due to the positive charge at the core, P-AgNPs exhibited positive zeta potential in the solution, with better stability [30,34]. Pullulan concentration, in the formation of P-AgNPs, can also govern zeta potential (ZP) with positive or negative charges at room temperature [30].

Conclusion
An environment friendly, economical, safer and green physical technique has been introduced for the synthesis of P-AgNPs. Pullulan was used as a "green" capping, stabilizing and reducing agent without any addition of toxic or harmful chemicals. The effect of UV irradiation time was also recorded as the main factor for controlling the synthesis, size, shape and distribution of the nanoparticles. Green synthesized P-AgNPs contained "quasi-spherical characteristics" showing 415 nm as spectra maxima with mean crystalline size of 6.02 ± 2.37 nm after 96 h of UV irradiation. The crystallographic nature of P-AgNPs was recorded as "face centered cubic" surrounded by a thin layer of pullulan. The involvement of O-H, C=O, and C=C functional groups of pullulan, during the The O-H group of pullulan is considered as an efficient coordinator with silver ions of P-AgNPs during the reduction process [34]. The prominent peaks were at wave numbers 2418, 2167, 1950, 1637, and 1535 cm −1 (Figure 5a), proving the stretching frequencies of carbonyl (C=O) and C=C groups of