Biosynthesis of silver nanoparticles using citrus sinensis peel extract and their application as antibacterial agent

Silver nanoparticles (Ag-NPs) have attracted huge importance due to their distinctive chemical, biological and physical properties. Silver nanoparticles are widely synthesized by the chemical method, which involves the use of toxic chemicals which affects its applications. The bio-reduction method, in comparison with chemical method is more economic and eco-friendly. In the present work, the bio-based production of Ag-NPs was done by using peel extract of orange (citrus sinensis), which played a role of reducing and stabilizing agent. The biosynthesis of silver nanoparticles was optimized by one factor at a time (OFAT) with respect to peel extract concentration, silver nitrate concentration and reaction temperature. The green synthesized silver nanoparticles were characterized by UV-visible spectroscopy, Fourier transforms infrared (FT-IR) spectroscopy, Scanning electron microscopy (SEM) and X-ray diffraction (XRD). Disk diffusion method was used for the study of antibacterial activity of the bio-synthesized silver nanoparticles against the bacteriaEscherichia coli and Staphylococcus aureus. The results showed that at a peel extract concentration of 6%, the temperature of 60C and silver nitrate concentration of 0.1M, the synthesis of Ag-NPs was effective. The orange peel synthesized Ag-NPs showed effective antibacterial activity against both bacteria. However better activity was observed against bacterium Staphylococcus aureus. The results con irmed the synthesis of Ag-NPs using peel extract of citrus sinensis and its role as antibacterial agent.


INTRODUCTION
Silver nanoparticles are helpful in today's situation due to its distinct properties such as optical, magnetic, electrical, shape and size (Skiba et al., 2019). These Ag-NPs has wide applications in the ield of electronic components, biosensor materials, complex ibers, aesthetic products and antimicrobial applications (Skiba et al., 2018). Ag-NPs are produced widely by Chemical reduction method under clement environment and large scale production. However, this technique makes use of harmful chemicals which have an unfavorable effect on the surroundings (Yang et al., 2019). This stops the usage of Ag-NPs in healthcare applications. Therefore green production of Ag-NPs has demonstrated to be more bene icial over other methods, as it is economical and environmentally friendly (Gudikandula and Maringanti, 2016). It does not require adverse conditions like usage of harmful chemi-cals, high pressure and temperature (Bhattarai et al., 2018;Manal et al., 2014). Hence, bio-based synthesis of Ag-NPs has been employed. In green synthesis, the Ag-NPs produced from microbial source are less stable than Ag-NPs produced from the plant source (Manal et al., 2014;Ocsoy et al., 2017). Therefore in the production of Ag-NPs, extract of fruit peel are being utilized. The peel extract plays a role of reducing and stabilizing agent (Dhand et al., 2016). Ag-NPs have shown suppressive effects against microbes (Karatoprak et al., 2017;Sierra et al., 2016). In this study, decayed orange peel extract was used to produce Ag-NPs and produced Ag-NPs were used for the study of antimicrobial activity. The effect of different parameters like concentration of silver nitrate, concentration of peel extract and temperature on the production of Ag-NPs was studied.

Materials
Silver Nitrate (AgNO 3 ) used in the synthesis was procured from SRL, India. Media components used were obtained from HiMedia. Cultures of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were obtained from the Department of Biotechnology, K.L.E. Technological University, Hubballi, India.

Preparation of peel extract
The orange peels were collected from Hubballi market and were washed with water. It was then air dried. 200 mL of distilled water was added to 100 g of dried orange peels. The mixture was boiled for 10 minutes at 100 o C. The iltered peel extract was stored for further use at 4 o C.

Green synthesis of silver nanoparticles
For the production of Ag-NPs, 0.1M and 0.01M stock solution of AgNO 3 and orange peel extract were taken. 20 mL of AgNO 3 solution and peel extract were mixed and allowed to react for 1h. The produced Ag-NPs were characterized by UV-Vis Spectrophotometer between the wavelength range 300-600 nm. The production of the Ag-NPs was studied by varying the factors like temperature (Room temperature, 40 o C and 60 o C), peel extract concentration (2%, 4%, and 6%) and silver nitrate concentration (0.1M and 0.01M). FT-IR, SEM and XRD techniques further characterized the biosynthesized Ag-NPs.

Silver nanoparticles antibacterial activity
Antibacterial activity of Ag-NPs was checked by Agar disk diffusion method. A suspension of the microorganism (Escherichia coli and Staphylococcus aureus) was spread by a glass spreader on nutrient agar. 20µl of an aqueous solution of biosynthesized silver nanoparticles and 20µl positive and negative control (tetracycline and de-ionized water respectively) were added to 5 mm diameter ilter paper disks and were located on the inoculated plates. The plates were incubated at 37 • C for 24h. The zone of inhibition was measured in millimeters.

UV-vis analysis
The orange peel extract was used to react with 0.1M and 0.01M of AgNO 3 at different orange peel concentration and temperature. Figure 1 showed a color change from yellow color to dark brown for mixtures containing silver nitrate and orange peel extract showing the production of Ag-NPs. It may be due to Vitamin C content of citrus fruit peel. It acts as reducing agent which reduces Ag+ of AgNO 3 to Ag o (Durán et al., 2016). The color intensity increased with the incubation period indicating the formation of more number of nanoparticles (Ouay and Stellacci, 2015). The results of UV-vis spectroscopy con irmed the production of Ag-NPs using orange peel extract. It also showed the surface Plasmon band of Ag-NPs between 400-480nm which showed the development of Ag-NPs. The wavelength did not alter much with the change in parameter. But variation in absorption was observed. The increase in absorption indicated the production of numerous nanoparticles (Li et al., 2010). The Plasmon resonance peak varied to longer wavelengths and broadened, as the diameter of the particle enlarged. From Figures 2 and 3, it was observed that the orange peel extract concentration of 6% and temperature of 60 o C had maximum effect, whereas 2% of peels extract concentration and room temperature had minimum impact on the biosynthesis of silver nanoparticles.

FT-TR and SEM analysis
To know the role of functional groups in the orange peel in the formation of Ag-NPs, FT-IR analysis was done. The FT-IR results are shown in Figures 4 and 5. The shift in the following peaks at 3374, 1625, 1410, 1058, 872 in orange peel extract indicated its participation in the process of nanoparticles synthesis. The composition of orange peels consisted of pectin, carbohydrates, hemicelluloses and cellulose (Shet et al., 2015b). The reduction of particle to nanoscale is due to the interaction of biological compounds with metal salts through functional groups (Shet et al., 2015b).
SEM was done to analyze the shape and structure of  Deionized water 0 0 Tetracycline 10.1 9.6 Ag-NP sample synthesized using orange peel and 0.01M AgNO 3 8.0 7.6 Ag-NP sample synthesized using orange peel and 0.1M AgNO 3 8.2 7.9 the produced Ag-NPs. The results of SEM indicated the presence of Ag-NPs, as shown in Figure 6. It was observed that the Ag-NPs are relatively spherical.

XRD analysis
The produced Ag-NPs indicated face-centered cubic crystal structure which was con irmed by XRD pattern as shown in Figure 7. The results of XRD ensured that the peaks at 38.04 • , 44.08 • , 64.36 • and 77.22 • attributed to 111, 200, 220, and 311 crystalline structures of the face-centered cubic synthesized Ag-NPs. In addition, the pattern indicated that Ag-NPs were mainly present in the nanocomposites with no contamination peaks.

Antibacterial activity
Zone of inhibition was used to study the role antibacterial agent of produced Ag-NPs. The inhibition zone of synthesized Ag-NPs is summarized in Table 1. From Figure 8, it was observed that the negative control (Deionized water) had no inhibition zone and positive control (tetracycline) had clear zone inhibition. There was a clear zone inhibition of silver nanoparticles against Gram-negative (E. coli) and Gram-Positive (S. aureus) bacteria. But more activity was observed against S. aureus. This may be due to the in iltration of Ag-NPs into the cells, causing intracellular loss leading to cell death. E. coli and S. aureus are highly sensitive to silver nanoparticles due to the high lipopolysaccharide and thick peptidoglycan layer of the microorganisms (Shet et al., 2015a).

CONCLUSION
The reported work exhibited an easy, environmental friendly and economical method for Ag-NPs synthesis. Orange peel extract played a role of reducing agent and stabilizing agent. Results showed that the reaction time, concentration of peel extract, concentration of AgNO 3 and temperature affected the yield and particle size of the produced silver nanoparticles. The production of Ag-NPs was veri ied by the analysis of UV-vis spectroscopy, FT-IR, SEM and XRD results. The antibacterial activity of Ag-NPs was determined by disk diffusion method, which showed activity against the bacterial strains E. coli and S. aureus. However better activity was shown against bacterium S. aureus.

Future Scope
The anticancer and antioxidant activity of the synthesized silver nanoparticles can be determined.