A pre-typified wild L. edodes was obtained from Mushroom Research Centre, Himachal Pradesh, India. This was reproduced by tissue culturing and maintained by sub-culturing monthly and stored at 4°C on Potato Dextrose Agar (PDA) slants to maintain viability.

The mutagenesis process was carried out by irradiating 14-day actively growing culture of the wild fungus on PDA plate (90 mm) with ultraviolet irradiation (254 nm, 15 cm) between the ranges of 5 to 90 mins. Mutants were obtained at 5, 10, 15, 20, 25, 30, 45, 60, 75 and 90 mins and were instantly sub-cultured and incubated in the dark at 26 ± 2°C for 2 weeks. Based on improved (EPS) productivity (assessed using the method of Dubois et al. [32Dubois, M.; Gilles, K.; Hamilton, J.; Rebers, P.; Smith, F. Colorimetric method for determination of sugar and related substances. Anal. Chem., 1956, 28, 350-356.
[http://dx.doi.org/10.1021/ac60111a017] ]), a resultant viable mutant obtained at 10 mins of UV irradiation (UV10) was carefully chosen. This culture was incubated at 25 ± 2°C for 70 hours and subsequently re-introduced onto the newly prepared PDA slant, incubated for 2 weeks to obtain fully ramified UV10 strain. The wild and UV10 strains were re-evaluated for quality enhancement and used for the production of EPS using combined methods of Adebayo et al. [33Adebayo, A.E.; Oloke, J.K.; Achana, Y.; Bora, T. Improvement of Laccase production in Pleurotus pulmonarius LAU 09 by mutation. J. Microbiol., 2012, 2, 11-17.
[http://dx.doi.org/10.5923/j.microbiology.20120201.03] ] and Majolagbe et al. [34Majolagbe, ON; Oloke, JK; Adebayo, EA; Adewoyin, AG; Ayandele, A; Bamigboye, C Study on the antimicrobial activity of exopolysaccharides of Lentinus subnudus using swiss albino rats as animal model. Am Eurasian J of Sci Res, 2013, 8, 47-52.].

Exactly 14 day old UV10 strain was used to inoculate 50 ml of production media in 250 ml conical flask consisting in g/L of glucose (10.0), KH₂PO₄ (0.5), MgSO₄ (0.5), peptone (2.0) and yeast extract (1.0). Other fermentation conditions include agitation (100 rpm), temperature (27 ± 2^oC), and incubation time of 7 days. Crude EPS was obtained as cell free extract after filtration and centrifugation. Extraction of EPS was carried out by mixing cell free supernatant from the respective production medium with 2 volumes of cold absolute ethanol (v/v) and held at 4^oC overnight for EPS precipitation. EPS precipitate was collected as crude fraction after centrifugation at 4000 rpm for 15 min (AG 5702, Eppendof, Germany) using the protocol of Majolagbe et al. [34Majolagbe, ON; Oloke, JK; Adebayo, EA; Adewoyin, AG; Ayandele, A; Bamigboye, C Study on the antimicrobial activity of exopolysaccharides of Lentinus subnudus using swiss albino rats as animal model. Am Eurasian J of Sci Res, 2013, 8, 47-52.] and re-suspended in 20 ml of sterilised distilled water. The quantity of EPS produced per millilitre of distilled water was evaluated by UV spectrophotometry. Absorbance at 490 nm of digested EPS was obtained per extract using phenol-sulphuric acid method of Dubois et al. [32Dubois, M.; Gilles, K.; Hamilton, J.; Rebers, P.; Smith, F. Colorimetric method for determination of sugar and related substances. Anal. Chem., 1956, 28, 350-356.
[http://dx.doi.org/10.1021/ac60111a017] ] and calibrating to total sugar contents using glucose as standard. EPS sugar monomer compositions of the obtained polysaccharides were subsequently assessed using GC-MS (GC2010, Shimadzu, Japan) [35Varinder, K; Bera, MB; Panesar, PS; Chopra, HK Production and characterization of exopolysaccharide produced by Alcaligenes faecalis B14 from indigenous soil. Int. J. Biotechnol Bioengineering Res, 2013, 4, 365-374.].

Exactly 0.1 g of anhydrous glucose, as obtained from Sigma Aldrich (South Africa), was used for preparation of working standard solution and dispensed into Erlenmeyer flask, liquefied in 19 ml of distilled water, and then made up to 100 ml, making dilution factor of 10^-2 (1 mg/ml). Then, 2, 3, and 5 ml of the stock solution was diluted to 100 ml to give working standards of 0.02, 0.03, and 0.05 mg per ml respectively. Subsequently, 1 ml was taken from the working solutions into separate 50 ml tube and 1 ml of 5% phenol was added, followed by the addition of 5 ml of concentrated H₂SO₄ added rapidly and then allowed to cool. The absorbances of admixtures were then read at 490 nm on a UV-visible spectrophotometer (Genesys 10UV scanner, Thermo electron corporation, UK), and used to plot a calibration curve for the determination of sugar content.

Specifically, about 1 ml of the re-dissolved EPS in distilled water (stock) was measured in a test tube and 1 ml of 5% phenol solution was thereafter added, followed by the rapid addition of 5 ml of concentrated sulphuric acid and then allowed to cool. The absorbance for each EPS product of all the UV mutants were therefore obtained at 490 nm and quantity determined using the calibration curve obtained.

Silver NPs were synthesised by reacting crude EPS of UV10 with 1 milli molar solution of silver nitrate. Exactly 1 ml of EPS solution of the Wild and UV10 was dispensed into 5 ml 1mM AgNO₃ in a 15 ml capacity test tube. A control was set up in a third test tube which contained 6 ml of 1 mM AgNO₃. The 3 tubes were strongly shaken for about 10 minutes and examined for colour development at room temperature (28 ± 2°C) during intermittent shaking. The formation of colour as a result of AgNPs development was monitored visually and the absorbance spectrum of the reaction solution was measured and recorded on a UV-visible spectrophotometer (Genesys 10 UV, Thermoelectron Corporation, UK). Morphology of the biosynthesised silver nanoparticles was elucidated by Scanning Electron Microscopy (SEM). SEM images were collected using an ASPEX 3020 at an accelerating voltage of 15 kV in bright field mode.

The exopolysaccharide productivity study was according to the method of Dubois et al. [32Dubois, M.; Gilles, K.; Hamilton, J.; Rebers, P.; Smith, F. Colorimetric method for determination of sugar and related substances. Anal. Chem., 1956, 28, 350-356.
[http://dx.doi.org/10.1021/ac60111a017] ] and Majolagbe et al. [34Majolagbe, ON; Oloke, JK; Adebayo, EA; Adewoyin, AG; Ayandele, A; Bamigboye, C Study on the antimicrobial activity of exopolysaccharides of Lentinus subnudus using swiss albino rats as animal model. Am Eurasian J of Sci Res, 2013, 8, 47-52.]. UV Visible and colour development pattern studies for AgNPs production were carried out to observe the surface plasmon resonance and typical silver nanoparticle colouration respectively. The results obtained were assessed for comparative efficiencies of the resultants EPSs and nanoparticle produced.

UV10 culture resulting from 10 minutes of exposure to ultraviolet irradiation gave EPS productivity of 2.783 mg/ml at 7 days of fermentation using the sugar calibration curve in Fig. (1). This was done by inserting the absorbance reading obtained from UV-vis spectroscopy of UV10 into the regression equation. The monosaccharide sugar composition of the produced EPS was identified as shown in Table 3. The results indicated the presence of monomer sugars that were characteristic of exopolysaccharides including arabinose (50.65%), mannose (19.20%), mannitol (15.58%), fructose (7.96%), trehalose (6.49%), and glucoronic acid, xylose, galactose and glucose with low percentages of ≤ 0.11. The results therefore indicate that the EPS produced is mainly made of arabinose, mannose and mannitol. It confirms the potentials of L. edodes for applications in production of polysaccharides with industrial usefulness. According to Sutherland [36Sutherland, I.W. Structure-function relationships in microbial exopolysaccharides. Biotechnol. Adv., 1994, 12(2), 393-448.
[http://dx.doi.org/10.1016/0734-9750(94)90018-3] [PMID: 14545899] ], monosaccharide components most classically found in EPS are sugars such as amino sugars (D-Galactosamine and D-Glucosamine), hexoses (D-Mannose, D-Glucose, D-Galactose, D-Allose, L-Rhamnose, L-Fucose), pentoses (as D-arabinose, D-Ribose, and D-Xylose), or uronic acids (D-Galacturonic acids and D-Glucuronic acids). Thus, these results are mostly in accordance with those previously reported [36Sutherland, I.W. Structure-function relationships in microbial exopolysaccharides. Biotechnol. Adv., 1994, 12(2), 393-448.
[http://dx.doi.org/10.1016/0734-9750(94)90018-3] [PMID: 14545899] -38Carbonero, E.; Gracher, A.; Komura, D.; Marcon, R.; Freitas, C.; Baggio, C.; Santos, A.; Torri, G.; Gorin, P.; Locomini, M. Lentinus edodes heterogalactan: Antinociceptive and anti-inflammatory effects. Food Chem., 2008, 111, 531-537.
[http://dx.doi.org/10.1016/j.foodchem.2008.04.015] ].

Table 3
EPS sugar monomer compositions.

Fig. (1) Calibration curve for polysaccharide content determination.

Aqueous silver nitrate ions were reduced during exposure to the EPS extract of UV10. The colour of the reaction mixture changed from colourless to purple with observed colour pattern getting stable after 24 hours as shown in Fig. (2) (Wild in-set). Purple and yellowish-brown colouration have been reported in relation with nanoparticle synthesis and due to excitation of surface plasmon vibration in metal nanoparticle [39Vanmathi, K.; Sivakumar, T. Isolation and characterization of silver nanoparticles from Fusarium oxysporum. Int. J. Curr. Microbiol. Appl. Sci., 2012, 56-62.]. Characteristic AgNPs colouration earlier reported include yellowish brown [40Paulraj, K.; Seung, T. Synthesis and structural characterization of silver nanoparticles using bacterial exopolysaccharide and its antimicrobial activity against food and multi-drug resistant pathogens. Process Biochem., 2013, 48, 1099-1106.
[http://dx.doi.org/10.1016/j.procbio.2013.05.011] ], yellow and reddish brown [41Mirunalini, S.; Arulmozhi, V.; Deepalakshmi, K.; Krishnaveni, M. Intracellular biosynthesis and antibacterial activity of silver nanoparticles using edible mushrooms. Not. Sci. Biol., 2012, 4, 55-61.
[http://dx.doi.org/10.15835/nsb448051] ], light-gray [42Chan, Y.; Mat-Don, M. Instantaneous biosynthesis of silver nanoparticles by selected macrofungi. Aust. J. Basic Appl. Sci., 2012, 6, 222-226.], and dark brown [43Karthika, R; Sevarkodiyone, SP Synthesis and characterization of silver nanoparticles using aqueous extract of goat faecal pellets. Int J Current Sci Res, 2015, 1, 2454-5422.]. Observed variation in colour pattern has been attributed to the differences in composition of biomolecules used in nanoparticle synthesis and the excitation of surface Plasmon vibrations in metal nanoparticles [44Mulvaney, P. Surface plasmon spectroscopy of nanosized metal particles. Langmuir, 1996, 12, 788-800.
[http://dx.doi.org/10.1021/la9502711] ]. Time for development of AgNPs reported by some authors include 10 hrs [40Paulraj, K.; Seung, T. Synthesis and structural characterization of silver nanoparticles using bacterial exopolysaccharide and its antimicrobial activity against food and multi-drug resistant pathogens. Process Biochem., 2013, 48, 1099-1106.
[http://dx.doi.org/10.1016/j.procbio.2013.05.011] ], 24 hrs [43Karthika, R; Sevarkodiyone, SP Synthesis and characterization of silver nanoparticles using aqueous extract of goat faecal pellets. Int J Current Sci Res, 2015, 1, 2454-5422.], and 48 hrs [41Mirunalini, S.; Arulmozhi, V.; Deepalakshmi, K.; Krishnaveni, M. Intracellular biosynthesis and antibacterial activity of silver nanoparticles using edible mushrooms. Not. Sci. Biol., 2012, 4, 55-61.
[http://dx.doi.org/10.15835/nsb448051] ]. In this study EPS of L. edodes rapidly reacted with AgNO₃ solution to form AgNPs in 24 hrs.

Fig. (2) AgNPs Colour patterns of Control (A) and UV10 mutant (B) after 24 hours. Inset is the colour pattern of AgNPs of Wild (C).

UV-visible absorption spectroscopy has proved to be a versatile technique in the studies of AgNPs [45Sastry, M.; Mayya, K.S.; Patil, V.; Paranjape, D.V.; Hegde, S.G. Langmuir−Blodgett films of carboxylic acid derivatized silver colloidal particles: Role of subphase pH on degree of cluster incorporation. J. Phys. Chem. B, 1997, 101, 4954-4958.
[http://dx.doi.org/10.1021/jp964087f] ] and provides useful information about morphology, size and stabilisation of AgNPs [46Kanmani, P.; Lim, S.T. Synthesis and structural characterization of silver nanoparticles using bacterial exopolysaccharide and its antimicrobial activity against food and multidrug resistant pathogens. Process Biochem., 2013, 48, 1099-1106.
[http://dx.doi.org/10.1016/j.procbio.2013.05.011] ]. Fig. (3) illustrates the UV-visible spectra of biosynthesised AgNPs of UV10 strain after 24 hours (Wild inset). The result shows a broad Surface Plasmon Resonance (SPR) with peak attained at 421 nm. The UV spectrophotometry reported in this study falls within the range of a usual and well reported pattern of SPR peaks of silver nanoparticles production [47Kim, J.S.; Kuk, E.; Yu, K.N.; Kim, J.H.; Park, S.J.; Lee, H.J.; Kim, S.H.; Park, Y.K.; Park, Y.H.; Hwang, C.Y.; Kim, Y.K.; Lee, Y.S.; Jeong, D.H.; Cho, M.H. Antimicrobial effects of silver nanoparticles. Nanomedicine (Lond.), 2007, 3(1), 95-101.
[http://dx.doi.org/10.1016/j.nano.2006.12.001] [PMID: 17379174] -51Kannan, R.R.R.; Arumugam, R.; Ramya, D.; Manivannan, K.; Anantharaman, P. Green synthesis of silver nanoparticles using marine macroalga. Chaetomorphalinum Appl. Nanosci., 2013, 3, 229-233.
[http://dx.doi.org/10.1007/s13204-012-0125-5] ]. These characteristic AgNPs absorption peaks justified the formation of AgNPs. Several authors have also reported peaks on spectra of AgNPs in the range of 391-650 nm [50Thirumurugan, A.; Neethu, A.; Hema, P.; Prakash, P. Biological synthesis of silver nanoparticles by Lantana camara leaf extracts. Int. J. Nanomater. Bioresource, 2011, 1, 22-24.-52Brennan, M.; Armstrong, G; Kelly, J; Whelan, A. Patent publication: Sensors for detecting an analyte using silver nanoparticles. Patent Application Publication, 2006, 1, 1-39.]. According to some established protocol by Brennan et al. [52Brennan, M.; Armstrong, G; Kelly, J; Whelan, A. Patent publication: Sensors for detecting an analyte using silver nanoparticles. Patent Application Publication, 2006, 1, 1-39.], silver particles which displays single absorption band and occur between the range of 410 and 450 nm are spherical in nature. The silver particles produced by UV10 in the wavelength of 421 nm can therefore be presumed to be spherical in nature. Peaks in this range are further supported by other reports to be characteristics of spherical or somewhat spherical shaped nanoparticles [39Vanmathi, K.; Sivakumar, T. Isolation and characterization of silver nanoparticles from Fusarium oxysporum. Int. J. Curr. Microbiol. Appl. Sci., 2012, 56-62., 42Chan, Y.; Mat-Don, M. Instantaneous biosynthesis of silver nanoparticles by selected macrofungi. Aust. J. Basic Appl. Sci., 2012, 6, 222-226., 53Philip, D. Biosynthesis of Au, Ag and Au-Ag nanoparticles using edible mushroom extract. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2009, 73(2), 374-381.
[http://dx.doi.org/10.1016/j.saa.2009.02.037] [PMID: 19324587] ]. These further substantiates the thought that extracts of UV10 produced nanoparticles that are somewhat of spherical morphology under the conditions investigated. The outcome of nanoparticles produced in this work is similar to those previously reported by different authors [21Mittal, A.K.; Chisti, Y.; Banerjee, U.C. Synthesis of metallic nanoparticles using plant extracts. Biotechnol. Adv., 2013, 31(2), 346-356.
[http://dx.doi.org/10.1016/j.biotechadv.2013.01.003] [PMID: 23318667] , 42Chan, Y.; Mat-Don, M. Instantaneous biosynthesis of silver nanoparticles by selected macrofungi. Aust. J. Basic Appl. Sci., 2012, 6, 222-226., 53Philip, D. Biosynthesis of Au, Ag and Au-Ag nanoparticles using edible mushroom extract. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2009, 73(2), 374-381.
[http://dx.doi.org/10.1016/j.saa.2009.02.037] [PMID: 19324587] -55Selvi, K.; Sivakumar, T. Isolation and characterization of silver nanoparticles from Fusarium oxysporum. Int. J. Curr. Microbiol. Appl. Sci., 2012, 1, 56-62.] with respect to the production of silver nanoparticles using whole cells and extracts of mushrooms and other plants.

Fig. (3) UV-Visible spectra of AgNPs of UV10 (A) after 24 hours. Inset, the spectra of UV-Vis spectra of AgNPs of Wild (B).

The scanning electron microscopy implemented to confirm the morphometric physiognomies of shape and size of the bio-nanoparticles showed that nanoparticles produced contained aggregates of particles with spherical morphology (Fig. 4). The size ranges between 50-100 nm. Spherical shaped AgNPs of varying sizes have been earlier described [51Kannan, R.R.R.; Arumugam, R.; Ramya, D.; Manivannan, K.; Anantharaman, P. Green synthesis of silver nanoparticles using marine macroalga. Chaetomorphalinum Appl. Nanosci., 2013, 3, 229-233.
[http://dx.doi.org/10.1007/s13204-012-0125-5] , 56Lateef, A.; Azeez, M.A.; Asafa, T.B.; Yekeen, T.A.; Akinboro, A.; Oladipo, I.C.; Beukes, L.S. Cola nitida-Mediated Biogenic Synthesis of Silver Nanoparticles Using Seed and Seed Shell Extracts and Evaluation of Antibacterial Activities. Bionanoscience, 2015, 5, 196-205. a
[http://dx.doi.org/10.1007/s12668-015-0181-x] , 57Lateef, A.; Ojo, S.A.; Akinwale, A.S.; Azeez, L.; Gueguim-Kana, E.B.; Beukes, L.S. Biogenic synthesis of silver nanoparticles using cell-free extract of Bacillus safensis LAU 13: antimicrobial, free radical scavenging and larvicidal activities. Biologia, 2015, 70, 1295-1306. b
[http://dx.doi.org/10.1515/biolog-2015-0164] ]. Nanoparticles have been reported to possess high reduction potentials, good solution chemistry as well as extremely small particle size with large surface area [58Rivero-Huguet, M.; Marshall, W.D. Reduction of hexavalent chromium mediated by micron- and nano-scale zero-valent metallic particles. J. Environ. Monit., 2009, 11(5), 1072-1079.
[http://dx.doi.org/10.1039/b819279k] [PMID: 19436867] ]. The large surface area of nanoparticles as a result of nano-sizes provides a greater number of active sites for pollutants binding. In addition, the particles demonstrate unique characteristics like catalytic potential and high reactivity, which make them better adsorbing materials [10Ali, I. New generation adsorbents for water treatment. Chem. Rev., 2012, 112(10), 5073-5091.
[http://dx.doi.org/10.1021/cr300133d] [PMID: 22731247] ]. Moreover, high density of low coordinated atoms at the surface edges of nanomaterials further makes them very reactive [59Sánchez, A.; Recillas, S.; Font, X.; Casals, E.; González, E.; Puntes, V. Ecotoxicity of, and remediation with, engineered inorganic nanoparticles in the environment. TrAC. Trends Analyt. Chem., 2011, 30, 507-516.
[http://dx.doi.org/10.1016/j.trac.2010.11.011] ]. These distinctive properties can be employed in the adsorption and degradation of water and air pollutants. The successful synthesis of biologically mediated nanomaterial using EPS of L. edodes in this study may therefore herald a simple novel technique of biosynthesis and modulation of nanoparticles of varied sizes and properties. Nanoparticle of varied sizes such as synthensised can be applied in diverse industries, especially, in the field of hydrology as highlighted in this report.

Comparing the EPS productivity of the Wild and UV10 mutant strains of L. edodes, UV10 obtained at 10 minutes of exposure to Ultraviolet irradiation gave the highest exopolysaccharide productivity of 2.783 mg/ml while the Wild strain has a productivity of 1.044 mg/ml after 7 days of fermentation (Table 4). The 10 minutes UV improved strain has an enhanced capacity of 2.7 fold over the Wild strain, which confirms the effectiveness of UV irradiation for EPS productivity enhancement in the mushroom strain. The synthesised nanoparticle of UV10 has a purple colouration and detected around 421 nm while that synthesised by wild strain possesses dark brown colouration with absorbance of 394 nm which are significant nanoparticles formation characteristics [19Zielińsk, A.; Skwarek, E.; Zaleska, A.; Hupka, M.G. Preparation of silver nanoparticles with controlled particle size. Procedia Chem., 2009, 1, 1560-1566.
[http://dx.doi.org/10.1016/j.proche.2009.11.004] ].

Fig. (4) Silver Nanoparticle synthesised from UV10.

Table 4
Comparative activities of UV10 and Wild.