Green Synthesis and Characterization of Silver NPs Using Oyster Mushroom Extract For Antibacterial Efficacy

Since historic time silver is well known for antimicrobial noble and coinage metal and is being deliberately used to store potable water of daily use to improve silver content. In recent decade silver NPs shown its significance of antimicrobial and antifungal nature compared to its bulk counterpart because of increased surface to volume ratio (Davies & Etris, 1997; Bhardwaj et al., 2019b). Silver NPs got popularity in the incorporation of a diverse range of commercial products, including plastics, soaps, pastes, food and textiles, increasing their market value due to broad spectrum bactericidal and fungicidal activity (Ahamed et al., 2010; Bhardwaj et al., 2019a). The rapidly emerging tendency of drug resistant and infectious microbes through fast evolution by mutation has created thrust need to develop advance formulation for the modification or inactivation of such microbes (Mandal et al., 2014). In recent few years several attempts of green synthesis of nanoparticles using the microorganisms such as bacteria, fungi and algae as well as various higher plants part have been made (Maurya et al., 2016; Sanjivkumar et al., 2019; Tomer et al., 2019). The biogenic extract have diversity of phytochemical compounds (alkaloids, amino acids flavonoids, inositol, phenolic compounds, resins, saponins, tannins, terpenes, and vitamins), particularly, secondary metabolites available in plant and animal tissues with unique potential of NPs synthesis (Abbasi et al., 2018). Such natural components have ability to act in reducing and stabilizing of metal ions into zerovalent ions. These green approaches are always nontoxic, biocompatible, simple process and environmentally benign. Mushrooms extract have various unique metabolites and enzymes able to reduce metal ions under extracellular or intracellular environment. Vigneshwaran et al. (2007) reported effectiveness of P. sajor-caju mushroom based Green Synthesis and Characterization of Silver NPs Using Oyster Mushroom Extract For Antibacterial Efficacy


Introduction
Since historic time silver is well known for antimicrobial noble and coinage metal and is being deliberately used to store potable water of daily use to improve silver content. In recent decade silver NPs shown its significance of antimicrobial and antifungal nature compared to its bulk counterpart because of increased surface to volume ratio (Davies & Etris, 1997;Bhardwaj et al., 2019b). Silver NPs got popularity in the incorporation of a diverse range of commercial products, including plastics, soaps, pastes, food and textiles, increasing their market value due to broad spectrum bactericidal and fungicidal activity (Ahamed et al., 2010;Bhardwaj et al., 2019a). The rapidly emerging tendency of drug resistant and infectious microbes through fast evolution by mutation has created thrust need to develop advance formulation for the modification or inactivation of such microbes (Mandal et al., 2014). In recent few years several attempts of green synthesis of nanoparticles using the microorganisms such as bacteria, fungi and algae as well as various higher plants part have been made (Maurya et al., 2016;Sanjivkumar et al., 2019;Tomer et al., 2019). The biogenic extract have diversity of phytochemical compounds (alkaloids, amino acids flavonoids, inositol, phenolic compounds, resins, saponins, tannins, terpenes, and vitamins), particularly, secondary metabolites available in plant and animal tissues with unique potential of NPs synthesis (Abbasi et al., 2018). Such natural components have ability to act in reducing and stabilizing of metal ions into zerovalent ions. These green approaches are always nontoxic, biocompatible, simple process and environmentally benign.
Mushrooms extract have various unique metabolites and enzymes able to reduce metal ions under extracellular or intracellular environment. Vigneshwaran et al. (2007) reported effectiveness of P. sajor-caju mushroom based silver NPs against pathogenic bacteria K. pneumonia and S. aureus. Aygun et al. (2020) utilized reishi mushroom for the preparation of silver NPs and proved antimicrobial activity against various gram-positive and gram-negative bacteria; S. aureus, E. hirae, B. cereus, E. coli, P. aeruginosa, L. pneumophila sub sp. Pneumophila, C. albicans. Moreover, Maurya et al. (2016) demonstrated synthesis of silver NPs using oyster mushroom extract with the successful employment in bacterial deactivation particularly against to S. aureus and E. coli.
In the present study, silver NPs were prepared using mushroom extract of oyster mushroom Pleurotus sajor-caju through a very fast, simple, economical and environmental friendly process. The aliquots of synthesized silver NPs were further used for the antibacterial activity against Escherichia coli.

Procurement of Chemicals and Culture Media
All chemicals used in the present work were of analytical grade. The silver nitrate (AgNO 3 99.98%), and Milli-Q grade water were purchased from Merck Pvt. Ltd. However, the nutrient agar culture media (NA) was procured from Himedia Pvt. Ltd.

Preparation of Mushroom Extract
The fresh and healthy fruiting bodis of oyster, Pleurotus sajorcaju ( Figure 1) were achieved from the mushroom cultivation facility of Mushroom Training Research Laboratory (MTRC), Veer Bahadur Singh Purvanchal University, Jaunpur, India. The fruiting bodies were washed properly using double distilled water and were oven dried at 40˚C for 2 hours and powdered for the exercise of NPs synthesis. For this 6g of powder was added into 50 mL of hot and boiled (100˚C) distilled water and kept for cooling at room temperature, which was then filtered using Whatman-4 filter paper, thus obtained filtrate was used as mushroom extract.

Green Synthesis of Silver Nanoparticles (AgNPs)
The synthesis of silver NPs was done according to the standard process (Bhardwaj et al., 2018). The 25 mL aliquot of prepared extract was added into 25 mL (20 mM) silver nitrate solution and maintained final volume up to 50 mL. Thereafter, solution was kept on hot plate with rotating magnetic stirrer for 2 hrs at 50 o C. In consequence, colourless solution was turned into wine red colour that indicated initial confirmation of silver NPs synthesis.

Characterization of Silver NPs
Qualitative imaging of synthesized Ag NPs samples was done by standard photography which shows the change in colour colourless to wine red of Ag NPs. The concentration and surface Plasmon resonance (SPR) of silver NPs was measured through UV-Visible absorption double beam spectrophotometer (PerkinElmer Lambda 35). Furthermore, transmission electron microscopy (TEM) was used to determine size and shape of silver NPs.

Antibacterial Assay
The antibacterial susceptibility assay of silver NPs was carried out by bacterial broth test. In this assay, E. coli was selected as the model bacteria, which was cultured in nutrient broth for overnight at 37 º C. The initial cell concentration of bacterial inoculants was 10 7 cfu/mL. The silver NPs with 50 ppb concentration was used as bactericidal, where as the control set contained only silver nitrate and mushroom extract. Thereafter, NPs treated broth was poured on nutrient agar plates to check the viability of E. coli cells.

Results and Discussion
Mushroom assisted green synthesis is a one of the most promising method with rapid simple and environmentally benign scope (Maurya et al., 2016;Owaid et al., 2019). The green synthesis of silver NPs cannot be possible without the presence of functional materials in the mushroom extract and other biologicals. The presence of such functional group play a significant role in the reduction of silver ions (Ag + ) into zerovalent silver (Ag 0 ), after that the nucleation process starts and finally reaction completes within 2 hrs to finish the synthesis of nanomaterials. Pleurotus mushroom is recognized to serve an excellent amount of riboflavin (Imran et al., 2011) in their fruiting bodies. The riboflavin acts as a catalyst for various reduction-oxidation reactions and functions in the bound coenzyme forms; flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD) (Bhat et p.15 al., 2011;Khatun et al., 2015;Bhardwaj et al., 2018). According to several observations flavins are known for their strong light sensitivity and excellent oxidizing properties (Eichler et al., 2005). In accordance to these facts it appears that flavoproteins with appreciable redox potential, play a significant role in the production of NPs. Hence, flavins (flavoproteins) existing in the aliquots of mushroom extract accomplish reduction of Ag + ion into Ag 0 NPs once exposed to sunlight (Bhardwaj et al., 2018). In our study the colour of transparent solution was turned into wine red which was the indication of NPs synthesis and this whole reaction was completed within a period of two hours. Change in colour of mixture and the green synthesis of silver NPs represented in Figure 2. Similar observations were also made by other authors also (Owaid et al., 2019) Figure 2: Reduction of silver nitrate into silver NPs in the combination of mushroom extract.

UV-Visible Spectroscopy of the Synthesized Silver NPs
The wine red colour of synthesized silver NPs was the initial confirmation of the presence of silver particles ofnano-range in the prepared colloidal solution. The UV-VIS spectra of mushroom extract and colloidal solution of synthesized silver NPs has been illustrated in Figure 3. The highest peak of the absorbance spectra of the nanoparticle sample was obtained in between the range of 430-450 nm are due to the characteristic peaks of surface plasmon resonance phenomenon; the collective oscillation of the conducting electrons on the surface of the nanosized particle absorbs visible electromagnetic waves which confirms silver particles are nano-sized (Tan et al., 2013;Bhardwaj et al., 2017a;Bhardwaj et al., 2018).

Transmission Electron Microscopy (TEM) of NPs
The colloidal aqueous silver NPs synthesized using mushroom was characterized by transmission electron microscopy (TEM) for the shape and size measurements. According to the observations it was found that synthesized spherical, small, and mono-dispersed silver NPs particles are uniformly present (Figure 4). The average particle size of NPs was recorded as 18 nm with a standard deviation of 8.13 nm. The TEM image recorded found fully supported with the data obtained through UV-VIS spectrophotometer which have shown the broad peak due to range of particle distribution. Our results are also in accordance to the findings of other studies (Parmar et al., 2020;Oyanedel et al., 2008).

Antibacterial Efficacy of Silver NPs
It has been found that the prepared silver NPs demonstrated high bactericidal effect against E. coli. Control was tested using mixture of silver nitrate, bacteria, and mushroom extract but does not contained. The growth of tested bacteria was completely inhibited at the lesser concentration 50 ppb of NPs shown in Figure 5. Study clearly shows that the smaller concentration is much effective for complete inhibition of the growth of bacteria using mushroom assisted silver NPs. The significant inhibition observed might be due to the smaller size of Ag NPs and their surface volume ratio would be break integrity of cell wall (Srinivasan et al., 2013). Bhardwaj et al., (2018) reported that the mushroom assisted silver NPs synthesis and inhibitory nature against E. coli. Moreover, Nadafan et al., (2016) also reported effective antibacterial property of silver nanoparticles.

Conclusion
Based on the results of the present study AgNPs synthesized through green synthesis method exhibited strong bactericidal properties. The green method of synthesis found rapid, simple, environmental friendly and highly cost effective. The silver NPs observed by the TEM were well dispersed and with small size and round shape. Silver NPs employed against E. coli successfully deactivated the cells at the level of 100% using smaller concentration of 50 µg/L. The results of the present study suggests that the prepared NPs can be used in the field of water disinfection and safe disinfectants, safe medical equipment, wound healing, and also for the purpose of preservations, safe mask and the hand sanitization.