Bio Diversiity and Cultivation Parameters, Mycochemical Analysis of Wild Mushrooms Collected from Western Ghats of Tamilnadu, India

Journal of Advances in Microbiology 2(2): 1-21, 2017; Article no.JAMB.30917

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M. Subbulakshmi1*, M. Kannan2 and S. Venketesan3

1Department of Microbiology, Bharathiyar University, Coimbatore, Tamilnadu, India. 2Department of Zoology, V.H.N.S.N. College, Virudhunagar, India. 3Department of Environmental Science, Periyar University, Salem, India.

Authors’ contributions

This work was carried out in collaboration between all authors. Author MS is the corresponding author, designed the study, performed the statistical analysis, wrote the protocol, managed literature search and wrote the manuscript. Author MK managed the analyses of the study. Authors MS, MK and SV read and approved the final manuscript.

Article Information

DOI: 10.9734/JAMB/2017/30917 Editor(s): (1) Pongsak Rattanachaikunsopon, Department of Biological Science, Faculty of Science, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand. Reviewers: (1) Djelloul Radia, University Chadli Bendjedid -El Tarf, Algeria. (2) Hesham Ali Elenshasy, Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia, Malaysia. (3) Eustaquio Souza Dias, University of Lavras, Brazil. (4) N. K. Kortei, University of Health and Allied Sciences, Ghana. (5) G. Krishnam, Kakatiya University, Warangal, India. (6) Blagoy Angelov Uzunov, Sofia University “St. Kliment Ohridski”, Bulgaria. (7) Shaiful Azuar B. Mohamad, Malaysian Nuclear Agency, Malaysia. Complete Peer review History: http://www.sciencedomain.org/review-history/18298

Received 7th December 2016 nd Original Research Article Accepted 2 March 2017 Published 22nd March 2017

ABSTRACT

Western ghats of Tamilnadu contains varieties of wild mushrooms. Macroscopic fungi were collected from Western Ghats of Tirunelveli District, especially Kalakad Mundanthurai Tiger Reserve & Range in Tamilnadu to document their distribution and diversity. Gilled mushroom are short lived, abundant soon after the monsoon where as the Polyporus species could be collected for a prolonged periods. The present survey includes 45 species from different habitats. Maximum number of species were belong to the family Polyporaceae followed by Ganodermataceae. The most common genera are Polyporus. This is the preliminary survey of collection and identification of macroscopic fungi as this has not been done in this part of Western ghats. This study also envisages further study on the predominant three mushrooms Calocybe indica, Laetiporous ______

*Corresponding author: E-mail: [email protected] *Co-author: E-mail: [email protected];

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sulphureus, Gleophyllum sepiarium were collected. Then Cultivation parameters, ptimization, separation, characterization and estimation of mycochemicals by TLC and GCMS studies were analysed for selected mushrooms.

Keywords: Calocybe indica; Laetiporous sulphureus; Gleophyllum sepiarium; TLC; GC-MS; antioxidant analysis.

1. INTRODUCTION the country. The habit of eating mushrooms differs from region to region and among the Fungi are present almost everywhere, in a different ethnic groups of the same region. spectacular array of shapes, sizes and colours, However, no clinical studies have been and performing a wide variety of different conducted to validate the medicinal claims activities. In 1991 David Hawksworth, a ascribed to this mushroom and no chemical mycologist estimated the world’s fungal diversity compound (biomarker) unique to the mushroom at 1.5 million species (equal to the estimated has been identified as a possible agent number of all known other living organisms). responsible for the mushroom's metabolic This was thought at the time to be a radical over effects. The field of metabolomics, aiming at estimate, but now other researchers have global analysis of numerous targeted or non- proposed figures in excess of 13 million. Fungi targeted low molecular compounds (metabolites) perform essential roles in every terrestrial, and in a biological sample, has recently found its many aquatic ecosystems, eg. decomposing application in diverse research areas, biomarker dead organic matter to release nutrients, discovery, functional genomics. supporting plant life on poor soils by improving the absorption of nutrients when they form 1.3 Gloeophyllum sepiarium mycorrhizal associations with roots, living inside plants as endophytes. Gloeophyllum sepiarium (Rusty gilled polypore) is a wood decay fungus that causes a brown 1.1 Calocybe indica rot. Gloeophyllum sepiarium grow in thin, dark

Calocybe indica is a tropical domesticated edible brown/green brackets on coniferous trees. mushroom; native to India. It is also known as Fruiting bodies can be seen throughout the dudh chatta / milky mushroom because of its year, but it sporulates in late summer to autumn. attractive milky whitish appearance with Gloeophyllum sepiarium is infrequent and excellent shelf life and large sized basidiocarp inedible. Decomposing the deadwood of with fleshy stipe and broadly adnate to conifers across North America, Gloeophyllum decurrently gills. The dried sporophores of this sepiarium is fairly easily recognized. When mushroom contain 17.69% protein, 4.1% fat, fresh and very young its cap is more or less 3.4% crude fiber and 64.26% carbohydrate. orange, but as it matures brown colors replace Mature sporocarp contains 4% soluble sugar, the orange from the center outwards. The 2.95% starch and 7.43% ash. In addition to this, underside of this polypore features gills, which it has most of the mineral salts such as is a bit odd, since polypores have pores rather potassium, sodium, phosphorus, iron, calcium than gills! (For help sorting through this mess, and amino acid namely alanine, aspartic acid, see the essay on the page for Lenzites betulina, glutamine, glutamic acid, glycine, another "gilled polypore.") Other distinguishing hydroxyproline, histidine, lysine, threonine, features include the brown flesh and the tyrosine, valine, arginine and proline [1,2]. Due black reaction to KOH, as well as microscopic to its alkaline ash and high fiber content it is features. highly suitable for the people with hyper acidity and constipation. It can be easily grown at a 2. MATERIALS AND METHODS temperature ranged between 25-35°C and its cultivation can be best fitted in early cropping 2.1 Study Area when no other mushroom can grown except Volvariella spp. at such a higher temperature. Tamilnadu encompasses diverse hills and plains in Western Ghats. These areas are 1.2 Laetiporous sulpureus rather treasure of diverse flora and fauna. Wild mushrooms were collected from different areas In Ethiopia, wild mushrooms are used as both of Western Ghats areas like Mundanthurai and food and medicine by various ethnic groups in Karayar regions in Tirunelveli district. Latitude

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8° 25’ and 8° 53' N and longitude 77° 10' and collection place as well as in the laboratory 77° 35' E, about 45 km west of Tirunelveli. [3,4]. Colour status, overall shape, pileus shape Equator with an annual rainfall of 3,097.8 mm, from side, size, total height, head height, annual relative humidity of 78% and annual substrate attachment, habitat. mean temperature of 30.6 degree C). The preserved specimen and photographs of the Each specimen was carefully labelled before mushrooms were taken for identification the transporting to the laboratory. The specimens mushrooms were identified morphologically were air-dried and stored in transparent boxes mushrooms have been collected from a few that were loosely kept to allow for proper locations from various places of Western Ghats aeration of the specimens [5]. Those specimen (KMTR) of Tirunelveli District. were stored in the lab for further study. Identification of the specimens was carried out 2.2 Survey, Collection and Identification by standard microscopic methods and also considering various morphological and Regular survey and collection of macrofungi anatomical features into account. were carried out in Western Ghats of KMTR especially Karaiyar, Kannikatti, Kattalaimalai, 2.4 Sample Collection Pandiyankottai, Karuthalipudavu, Palodai, Poongulam, Seyaru, Ullaru, Thulukamattai, The selected two different species L. sulphureus Tunnel forest in the year of 2015. While and Gloeophyllum sepiarum were collected from collecting information with regard to the host, Western Ghats of TamilNadu. Pure cultures their habitat, colour of caps, associated features were isolated from internal tissues into potato were also noted down carefully. The specimens dextrose agar (PDA) and incubated at 30°C for were collected from different location from tree 14 days. After incubation, the agar surface was branches, moisture soil, and dead trees of fully covered with a white mycelium and the forest. According to the differences we learn the plate choosed for the spawn preparation after the mushrooms morphological characters from plate was stored at 4°C. selected area.

2.3 Macroscopic Examination 2.5 Spawn Preparation

Macroscopic characters of fresh specimens Sorghum is found to be the best substrates for were noted after the collection. Photographs of spawn production [6,7]. Spawn was prepared the fresh specimens were taken both in the according to Pandey et al. (2000).

Fig. 1. Western Ghats (KMTR)

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2.6 Substrate Optimization colonization, which was recorded daily. When the spawn run was complete and pinheads Most popular species under cultivation are started appearing, the bags were mouth opened naturally wood inhabiting fungi. So, growing on to facilitate the development of fruiting bodies. sawdust is a logical choice. Sawdust is mixed As soon as the fruiting bodies developed and with wheat bran (or another nitrogen source) at attained their full size, they were cut just above 5% and calcium carbonate (CaCO3) (a buffering surface of the substrate with sterile sharp knife agent) at 2-3%, and filled into autoclavable or blade. The harvesting was done in 4 flushes nd bags. The bags are sterilized for a minimum of of 1 week intervals. After the 2 flush, the 1 hour at 121°C (15 psi when at sea level). substrate was turned upside down and regularly rd th Note sterilization exposure times vary watered to harvest the 3 and 4 flushes. The depending on pressure and elevation. Finally, yields of mushroom were recorded. the substrate is cooled to at least 25°C then, inoculated with grain or liquid spawn and 2.9 Mycochemical Analysis incubated at appropriate temp for the species until colonization is complete. The selected mushroom extracts were subjected to qualitative chemical investigation to 2.7 Supplemented Substrate Used for test for the presence of various phytochemicals Cultivation in extracts.

Four different types of substrates and 2.10 Preparation of the Mushroom agricultural wastes, namely sorghum, corn cobs, Extract wheat grain and sawdust substrate, were used to determine which substrate is the best for Freshly-harvested whole mushrooms were cultivation of Calocybe indica, Laetiporous shade dried and finely powdered. Twenty five sulphureus and Gloeophyllum sepiarum. The grams of the powder were extracted with 250 ml spawn media were prepared as described by of 95% solvents like methanol, ethanol, ethyl Nwanze et al. (2005). The ingredients included acetate, hexane and aqueous using Soxhlet 1 kg of cereal grain/sawdust, 12 g of apparatus. The residue was filtered and CaSO4·2H2O, 3 g of CaCO3. The supplemented concentrated to a dry mass by vacuum cereal grain/sawdust was divided into two types distillation; the filtrate thus obtained was used as (Type 1: all ingredients were mixed with mushroom extract. gypsum. Each cereal grain/sawdust type was washed and soaked overnight, water was 2.10.1 Test for carbohydrates drained off, and boiled for15 minutes, and left to cool down for 20 minutes. Fifty grams of each 2.10.1.1 Molisch’s test cereal grain/sawdust were filled into 4-ounce media bottles, autoclaved at 121°C for 15 To 2 ml of extract 2-3 drops of alpha minutes and left to cool. Before mycelia of naphthalene solution in alcohol was added, Calocybe indica, L. sulphureus and shaken for 2 min and 1 ml of concentrated Gloeophyllum sepiarum were inoculated, the sulphuric acid was added slowly from the sides cereal grain/sawdust bottles were shaken to of the test tube. A deep violet colour at the prevent clump formation and to mix the mycelial junction of two layers indicates the presence of discs with the substrate. Spawn bottles were carbohydrates. incubated in dark condition at 30°C for 10 days. The linear mycelium length was measured 2.10.2 Test for reducing sugars every 2 days for 10 days to calculate the growth rate. 2.10.2.1 Fehling’s test

2.8 Cultivation Conditions and Fehling’s A and Fehling’s B solutions, each 1 ml Harvesting were mixed and boiled, for 1 ml and 2 ml of extracts were added heated in boiling water The inoculated bags were incubated in a bath for 10 min, appearance of yellow and then cultivation room and maintained at 25-30°C with brick red precipitate indicates the presence of relative humidity of 85 ± 5%, for substrate reducing sugars.

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2.10.3 Test for monosaccharide magnesium were added. Appearance of pinkcolour indicates the presence of flavonoids. 2.10.3.1 Barfoed’s test 2.10.10 Test for triterpenoids Equal volume (2 ml each) of Barfoed’s reagent and extract were mixed in a test tube heated in 2.10.10.1 Salkowaski test boiling water bath for 2 min and cooled. The appearance of red precipitate shows the To 2 ml of extract 5 drops of concentrated presence of monosaccharides. sulphuric acid was added, shaken and allowed to stand. Appearance of greenish blue colour 2.10.4 Test for pentose sugar indicates the presence of triterpenoids.

2.10.4.1 Bial’s test 2.10.10.2 Libermann burchard test

To 1 ml of boiling bial’s reagent, 1 ml of solution To 2 ml of the test solution, 10 drops of acetic extracts were added. The appearance of green anhydride was added and mixed well. To this 5 or purple colour shows the presence of pentose ml of concentrated sulphuric acid was added sugar. from the sides of the test tube, appearance of greenish blue colour indicates the presence of 2.10.5 Test for hexose sugars triterpenoids.

2.10.5.1 Seliwanoff’s test 2.10.11 Test for alkaloids

To 3 ml of seliwanoff reagent 1 ml of fungal To the 10 g of dry extracts, 20 ml of dilute extract was added and heated on water bath for hydrochloric acid was added, shaken well and 2 min. The change in colour to red indicates the filtered. The following tests were performed using presence of hexose sugar. the filtrate.

2.10.6 Test for proteins 2.10.11.1 Mayer’s test

2.10.6.1 Biuret’s test To 3 ml of the filtrates, 1 ml of Mayer’s reagent (potassium mercuric iodide) was To 3 ml of extract 1 ml of 4% w/v sodium added. The appearance of white precipitate hydroxide and 1 ml of 1% w/v copper sulphate indicates the presence of alkaloids. were added. The change in colour of the solution to violet or pink indicates the presence of 2.10.11.2 Wagner’s test proteins. To 3 ml of filtrate, 1 ml of Wagner’s reagent 2.10.7 Test for tannins and phenols (iodine in potassium iodide) was added. The appearance of reddish brown precipitate 2.10.7.1 Ferric chloride test indicates the presence of alkaloids.

To 3 ml of extract, 3 ml of 5% w/v ferric 2.10.11.3 Hager’s test chloride solution was added. The blue – black colour indicates the presence of tannins and To 3 ml of filtrate, 1 ml of Hager’s reagent phenols. (saturated picric acid solution) was added.

2.10.8 Test for saponins The appearance of yellow precipitate indicates the presence of alkaloids. 2.10.8.1 Foam test 2.10.11.4 Dragendroff’s test The extract (2 g) was shaken vigorously with 20 ml of water and observed for persistent foam, To 3 ml of the filtrate, 1 ml of Dragendroff’s which indicates the presence of saponins. reagent (potassium bismuth iodide) was added. The appearance of brick red precipitate indicates 2.10.9 Test for flavonoids the presence of alkaloids.

2.10.9.1 Shinoda test 2.10.12 Test for lipids

To the dry extract (2 g), 5 ml of ethanol (95% To 10 ml of extract 0.5N alcoholic potassium v/v), 5 drops of hydrochloric acid and 0.5 g of hydroxide was added along with a drop of

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phenolphthalein. This mixture was heated on The value for each compound as evident from water bath for 1 h and observed for the formation the blue and green florescent spots under UV of soap or partial neutralisation of alkali. was Calculated as the Rf value (retention factor) for that compound: 2.11 Identification of Phenol by Using TLC Rf value = Distance traveled by the compound / Distance traveled by the solvent 2.11.1 Thin layer chromatography (TLC) front The TLC Analysis result of three mushroom extract had the phenol and 2.11.1.1 Preparation of TLC plates flavonoid.

The glass plate, which measured 20 × 20 cm, 2.14 Moisture Analysis was prepared for TLC by coating it to a thickness of 0.25 mm with silica gel HF254 20 g of fresh mushroom was weighed into a (Article no. 7739, Merk) using a TLC spreader. weighed moisture box (A&D company ltd. N 92; P1011656; Japan) and dried in an oven at 2.11.1.2 Spotting the plates 100~105°C and cooled in a dessicator. The process of heating and cooling was repeated till The samples were spotted on the TLC plates a constant weight was achieved. with microcapillary tubes and the prepared (spotted) TLC plates were eluted using a solvent The moisture content was calculated as following equation: system comprising Chloroform: Methanol (27: 0.3). Moisture (%) = (initial weight − final weight) × 100/weight of sample (Raghuramulu et al. 2.11.1.3 Visualization of the TLC plates 2003 [8]).

The plates were examined under UV at 254 and 2.15 Determination of Total Protein 365 nm. The spots were marked with a pencil. The value for each compound as evident from Five gram of grinded mushroom was taken with the blue and green fluorescent spots under UV 50 ml of 0.1 N NaOH and boiled for 30 min. The was calculated as the Rf value (retention factor) solution was cooled in room temperature and for that compound: centrifuged at 1000 × g by a DSC-200T tabletop centrifuge (Digisystem Laboratory Instruments, Rf value = Distance traveled by the Taipei, Taiwan). The supernatant was collected compound / Distance traveled by the solvent and total protein content was measured front according to the method of Lowry et al. [9]. For the determination of protein content from fresh 2.12 Identification of Flavonoid by Using mushroom, 5 g was taken with 50 ml phosphate TLC buffer and homogenized with a tissue homogenizer (Polytron, Lucerne, Switzerland). 2.12.1 Preparation of TLC plates Five milliliter of homogenized was taken with 50

The glass plate, which measured 20 × 20 cm, ml of 0.1 N NaOH and protein content was was prepared for TLC by coating it to a determined as mentioned above. thickness of 0.25 mm with silica gel HF254 2.16 Determination of Crude Fiber (Article no. 7739, Merk) using a TLC spreader. Ten grams of moisture and fat-free sample was 2.12.2 Spotting the plates taken in a beaker and 200 ml of boiling 0.255 The samples were spotted on the TLC plates N H2SO4 was added. The mixture was boiled with microcapillary tubes and the prepared for 30 minutes keeping the volume constant by (spotted) TLC plates were eluted using a solvent the addition of water at frequent intervals. The system comprising chloroform and methanol mixture was then filtered through a muslin cloth (19:1). and the residue washed with hot water till free from acid. The material was then transferred to 2.13 Visualization of the TLC Plates the same beaker, and 200 ml of boiling 0.313 N NaOH added. After boiling for 30 minutes The plates were examined under UV at 254 and (keeping the volume constant as before) the 365 nm. The spots were marked with a pencil. mixture was filtered through a muslin cloth and

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the residue washed with hot water till free from measured spectrophotometricallly at 670 nm. alkali, followed by washing with some alcohol Gallic acid was used as the standard in order to and ether. It was then transferred to a crucible, create a calibration curve by plotting absorbance dried overnight at 80~100°C and weighed versus concentration. TP content was (We). The crucible was heated in a muffle standardized against gallic acid and the data was furnace (Nebertherm: Mod-L9/ 11/c6; Germany) expressed as Gallic Acid Equivalent (GAE) per at 600°C for 5~6 hours, cooled and weighed dry weight of the mushroom. again (Wa). The difference in the weights (We- Wa) represents the weight of crude fiber. Crude 2.20 Estimation of Flavonoid fiber (g/100 g sample) = (100 − (moisture + fat) × (We-Wa)/Wt of sample Raghuramulu et al. [8]. The concentrations of total flavonoid in the extracts were determined as described 2.17 Determination of Total Ash previously [11].

One gram of the sample was weighed accurately Mushroom extract (0.2 ml) was added to 0.15 ml into a crucible. The crucible was placed on a of NaNO and the mixture was incubated at clay pipe triangle and heated first over a low 2 room temperature for 6 min. Next, 0.15 ml of flame till all the material was completely AlCl .6H O (10% w/v) was added to the mixture, charred, followed by heating in a muffle 3 2 which was then left at room temperature for 6 furnace for about 5~6 hours at 600°C. It was min. Next, 0.8 ml of NaOH (10% w/v) was added then cooled in a dessicator and weighed. To and the absorbance of the mixture was read at ensure completion of ashing, the crucible was 510 nm after standing at room temperature for then heated in the muffle furnace for 1 h, cooled 15 min. For the blank, the extracts were and weighed. This was repeated till two replaced with water. To correct for background consecutive weights were the same and the ash absorbance, a blank was prepared for each was almost white or grayish white in color. Then sample in which the AlCl .6H O was replaced total ash was calculated as: 3 2 with water. A standard curve was prepared from Ash content (g/100 g sample) = weight of 0 – 500 mg/ml quercetin dissolved in 80% ash × 100/weight of sample taken [8]. ethanol. Total flavonoid content was expressed in mg quercetin equivalents/g dry matter. 2.18 Mineral Analysis 2.21 Estimation of Protein Total ash was taken for the analysis of mineral contents. Two ml of conc. HNO3 was added to  In a series of clean test tube pipetted out the ash and heated for 2 minutes. One drop of 0.2, 0.4, 0.6, 0.8 and 1.0 ml of BSA stock hydrogen peroxide was added into the solution. standard solution. Unknown solution (1 The solution was then transferred into a ml) was taken in another test tube. volumetric flask and total volume was made  All the test tubes were made upto 1 ml 50 ml by adding deionized distilled water. This with distilled water. To all the test tubes was then used to analyze the contents of are added with 5 ml of Lowry’s reagent calcium (Ca), iron (Fe), manganese (Mn), followed by addition of 0.5 ml of Folin’s magnesium (mg), zinc (Zn), Selenium (Se) and reagent and vortex the tubes immediately arsenic (As) by flame and graphite method with and let it for 20 minutes at room atomic absorption spectrophotometer (Perkin temperature. Elmer: AS 80).  The blue colour developed was read at 2.19 Estimation of Phenol 650 nm against the blank.  The standard graph was drawn by taking The amount of total phenolics (TP) in the concentration of BSA on x-axis and their extracts was determined by the modified Folin- corresponding optical density on y-axis Ciocalteau method [10]. Mushroom extract of from the graph the amount of protein was 100 μl was added to 4 ml of 20% sodium calculated and measured the optical carbonate mixed thoroughly and allowed to density. stand for 2 minutes; to this mixture 100 μl of Folin-Ciocalteau and 500 μl of distilled water 2.22 Estimation of Carbohydrate were added. The mixture was vortexed for 15 sec and allowed to stand for 30 min at 40°C The series of standard solution of 0.2, 0.4, 0.6, for color development. Absorbance was then 0.8 and 1.0 ml were taken in a series test

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tubes. Then the test tubes were made upto 1 ml 2.24.2 Determination of DPPH radical with distilled water. 1 ml of distilled water on scavenging activity another test tube served as blank. 4 ml of anthrone reagent was added to all test tubes The 1, 1- diphenyl-2- picryl- hydrazyl (DPPH) and incubated for 5 to 10 minutes in a boiling assay was performed by using the method. water bath. The colour was burned from green Various concentrations of mushrooms extract to dark green. The intensity of colour formed (100-600 μg/ml) were added to 1 ml of the was read by using colorimeter at 625 nm. A 0.004% methanol solution of DPPH, and the standard graph was drawn by taking the mixture was vortexed vigorously. The tubes were concentration of glucose on x-axis and then incubated at room temperature for 30 absorbance on y-axis. A straight line was minutes in dark, and the absorbance was taken obtained from this standard graph. The amount at 517 nm. Lower absorbance of the reaction of glucose in the unknown solution was mixture indicates higher free radical scavenging calculated. activity. Catechin was taken as known free radical scavenger. Percentage inhibition activity 2.23 GC/MS Analysis was calculated by using the formula:

2.23.1 Gas chromatography/ mass spectro- metry analysis

Qualitative and quantitative GC/MS analyses Where A0 was the absorbance of the control and were carried out on a Hewlett-Packard 5890 A1 was absorbance in the presence of Series II Plus gas chromatograph interfaced to mushrooms extract/ known antioxidant. an HP 5989B mass spectrometer. Separation 2.25 Methodology for Antimicrobial was done on a 25 m x 0.25 mm HP5 –MS capillary column coated with 0.50 μm 5% Activity phenyl 95% methylpolysiloxane. Temperature 2.25.1 Microorganisms programming was set at 70-250°C, at a rate of 3°C/min. The carrier gas used was helium at a Escherichia coli MTCC 1687, Bacillus subtilis constant flow rate of 1.9 ml/min. Injector and MTCC 441, Salmonella MTCC 3858, Shigella interface temperature were adjusted to 250°C MTCC 11947, Pseudomonas aeruginosa MTCC and 280°C, respectively. EI mass spectra were 1688, Streptococcus pyogenes MTCC 2608, recorded at 70 eV ionization voltage (source MRSA MTCC 2940, Proteus mirabilis MTCC temperature 250°C). Compounds were identified 2813, Klebsiella MTCC 5328. by mass spectral comparison with a commercial database (Wiley8 and NISTO5 mass spectral The bacteria were subcultured in nutrient agar library) and the laboratory’s own database. medium and the culture of each bacterium was Spectral data were compared with linear preserved on the same medium at 4°C. The retention indices published in the literature. cultures were subcultured periodically on the same medium at 37°C ± 2°C. 2.24 Bioactivity of Selected Mushrooms 2.25.2 Antimicrobial screening 2.24.1 Antioxidant activity In vitro antibacterial activity of the different The Phenol and Flavonoid extracts were compounds was studied against seven bacterial prepared by the following methods. The 0.5 g of strains by the agar well diffusion methodd by sample was immersed in 50 ml of methanol. Perez et al. (1990) with certain modifications. Then the sample was filtered by using whatman Nutrient agar (HiMedia, India) was used as the filter paper. The filtered extract was used for bacteriological medium. The antibacterial activity antioxidant activity. The 0.1 g of sample was of different compounds was taken at different immersed in 10 ml of methanol. The methanol concentrations (500 and 250 μg/well). The nutrient agar was melted and cooled to 48-50°C extract for waterbath for 5 min. The water and 6 ethylacetate ratio (10:1) was added to the and a standardized inoculum of 1 × 10 CFU/ml sample. Then the sample was filtered by using was then added aseptically to the molten agar whatman filter paper. The filtered extract was and poured into sterile Petri dishes to give a used for antioxidant activity. Phenol extract s o lid plate. Wells were prepared in the seeded (1 ml) and 1 ml of flavonoid extract was mixed agar plates. The test compound was introduced and it was used for antioxidant activity. in the well (5 mm). The plates were incubated

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overnight at 37°C. The antimicrobial spectrum of 4.2 Effect of Media on Mycelial Growth the compounds was determined for the bacterial species in terms of zone sizes around each well. Our results are similar to those of Siwulski et al. [12]. C. indica grew well on PDA medium. 3. RESULTS 4.3 Effect of Temperature on Mycelial According to survey results fourty five different Growth varieties of wild mushrooms were found in the Western Ghats of KMTR. These belongs to 10 The Calocybe indica, L. sulphureus and and 40 generas, and 45 species (Fig. 2 (1- 45)). Gloeophyllum sepiarum strains were tested for After survey the most common 4 species was the suitable temperature for promoting mycelial collected, it was introduced in to cultivation growth on PDA medium. Temperatures of 20°C, parameters analysation .then the mycochemical 25°C, 30°C and 35°C were used and the test was taken. According to this test Results mycelium grew well between 25°C and 30°C, GC-MS test was taken. Antimicrobial tests were while the most unfavourable was 35°C. Even taken. though the mycelial growth of L. sulphureus occurred at 30°C [13], growth of Gloeophyllum 4. DISCUSSION sepiarum was observed at 25–30°C (Jayasinghe et al. 2008). In this survey results are fourty five different varieties of wild mushrooms are found in the 4.4 Effect of pH for Mycelial Growth Western Ghats of KMTR. These mushrooms belongs to 10 families like Polyporaceae (Large All pH from 5–8 were suitable for growth of aged trees), Gleophyllaceae (large trees), mycelium of C. indica. The optimal pH on Schizophyllaceae (decayed woods), Xylariaceae mycelia growth of L. sulphureus was in the (dead woods), Hygrophoropsidaceae (decayed range of pH 7, however. The optimal pH for trees), Hymenochaetaceae (large trees) Gloeoophyllum sepiarum was in the range of Tricholomataceae (treebarks), Umblicariaceae pH7 [13,14]. (tree barks), Fomitopsidaceae (tree barks), Schizoporaceae, Pluteaceae, Ganodermataceae 4.5 Supplemented Substrates Used for (moisture trees), Tremellaceae (decayed Cultivation woods), Amanitaceae (soil), Agaricaseae (soil), Marasmiaceae (soil), Auriculariaceae (moisture Our study examined the use of different cereal containing dead woods), Nidulariaceae, grain/sawdust media for promoting mycelia Bondarzewiaceae, (dead woods) growth and spawn production. After 10 days of Schizophyllacea (tree barks), and 40 generas incubation, the mycelium was able to colonize with 45 species (Fig. 2 (1- 45)). Karaiyar hills all the cereal grains and agricultural waste contains ever green forest. It includes Large, medium (sawdust). The data for mycelium and aged trees. Mostly Polyporaceae family growth on different cereal grain media/sawdust mushrooms like big conks, and honey conks are was investigated. Therefore, the results showed found in this trees. that the various cereal grain media can be used in order to promote mycelia growth of Calocybe 4.1 Mushroom Morphology and Isolation indica, L. sulphureus and Gloeophyllum sepiarum. Basidiocarps of Calocybe indica vary considerably in size (6–12 cm wide and 10–24 4.6 Nutritional Studies cm across). The distinctive characteristics of the fruiting bodies are the milky white colour, large The results of the studies carried out on the sized basidiocarps with fleshy stipe and broadly three species of Calocybe indica, Laetiporous adnate to decurrently gills. The pores of the sulpureus and Gloeopyllum sepiarum on dry basidiocarps are milky white in colour. The weight basis is depicted. In these mushrooms internal tissues of Calocybe indica fruiting Crude fibres were found to be maximum in C. bodies were cut and placed on PDA plates and indica (14.7) while lowest in L. sulpureus (13.4) incubated at 30°C. After 2 months, the mycelia and G. sepiarum (10.9) ash content in C. indica were fully covered the plates and produced (9.3), L. sulpureus (8.7) and G. sepiarum (6.9). young primordial fruiting bodies, observed after The average moisture content in dried samples incubating the fully mycelia covered PDA plates was highest in C. indica (88.6) and minimum in for 10 days. L. sulpureus (84.3) and G. sepiarum (76.1).

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1 2 3 4

5 6 8 7

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9 10 11 12

13 14 15

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20 21 22 23

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28 29 30 31

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32 33 34 35

36 37 38 39

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40 41 42 43

44 45

Fig. 2. Mushroom biodiversity in KMTR 1. Ganoderma crusae, 2. Gleophyllum sepiarium, 3. Cerena unicolor, 4. Tremetes gibbosa, 5. Schizophylum commune, 6. Daldinia concentrica, 7. Hygrophoropsis aurantiaca, 8. Phellinus torulosus, 9. Tricholoma magnivelare, 10. Umbilicaria esculenta, 11. Leotiphorous sulphurus, 12. Inonotus obliquous, 13. Xylariapolymorpha, 14. Fomes conatus, 15. Daldinia concentrica, 16. Volvariella pusilla, 17. Ganoderma applanatum, 18. Fomitopsis officinalis, 19. Tremella mesenterica, 20. Amanita virosa, 21.Ganoderma sp 22. Ganoderma lucidum, 23. Agaricus sp, 24. Coprinus comatus, 25. Bridgeporous nobilissimus, 26. Pleurotus ostreatus, 27. Coprinus atramentarius, 28. Lycoperdon perlatum, 29. Exidia recisa, 30. Chlorophylum rachodes, 31. Bovista plumbea, 32.Coprinus fimatarius, 33. Cyathus striatus, 34. Pleurotus eryngii, 35. Nolanea strictia, 36. Pholiota sp, 37. Coprinus microcarpus, 38. Auricularia aurantia, 39. Mycena sp, 40. Spongipellis pachydon, 41. Pheolus schweinitzii, 42. Tremetes gibbosa, 43. Ceratiomyxa fruticulosa, 44. Ganoderma carnosum, 45. Bonderzewia berkeley

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Table 1. Mushroom collection details

S. no Botanical name Family Vernacular name Collected place Habit 1 Ganoderma crusae Polyporaceae Reishi mushroom, Lingzhi mushroom, spirit All areas Trees plant 2 Gleophyllum sepiarium Gleophyllaceae Rusty gilled polypore Kattalai malai, karuthali pudavu Trees 3 Cerena unicolor Polyporaceae Mossy maze polypore Karaiyar Dead woods 4 Tremetes gibbosa Polyporaceae Lumpy bracket fungus Karaiyar, tunnel forest Trees 5 Schizophylum commune Schizophyllaceae common split gill Karaiyar Decayed trees 6 Daldinia concentrica Xylariaceae Earth balls, King Alfred's Cake, cramp balls, Upper part of the Puliodai Trees and coal fungus. 7 Hygrophoropsisaur antiaca Hygrophoropsidaceae False chanterelle Kannikatti Decayed trees 8 Phellinus torulosus Hymenochaetaceae Tufted bracket fungi Ullaru, Karuthalipudavu Trees 9 Tricholoma magnivelare Tricholomataceae White matsutake, gilledmusroom Karaiyar Trees 10 Umbilicaria esculenta Umblicariaceae Lichen Panatheertham falls areas Trees 11 Leatiporous sulphurus Fomitopsidaceae Chicken of the woods Seyaru, Kattalai malai Soil 12 Inonotus obliquous Hymenochaetaceae Cinder conk Kannikatti Long living trees 13 Xylaria polymorpha Xylariaceae Dead man’s fingers Karuthali pudavu Decayed trees 14 Fomes conatus Schizoporaceae Hedgehog mushrooms,beardedhed ge hog,or Karaiyar Trees bearded tooth 15 Daldinia Xylariaceae Tree mushrooms King Alfred's Cakes / Cramp Karaiyar Trees Balls,carbonballs 16 Volvariella pusilla Pluteaceae Small mushroom Mundanthurai Soil 17 Ganoderma applanatum Ganodermataceae Reishi mushroom Kannikatti Trees 18 Fomitopsis officinalis Fomitopsidaceae Honey conk Rain falling areas, Kannikatti,karaiyar Aged trees 19 Tremella mesenterica Tremellaceae Gellyfungus,transulentf ungus,yellowbrain,witc Ever green forest Woods hes’butter karaiyar 20 Amanita virosa Amanitaceae Destroying angel Kannikatti Soil 21 Ganoderma sp Ganodermataceae Lingzhi mushroom Deap forest areas Decayed woods 22 Ganoderma lucidum Ganodermataceae Tree mushrooms Lacquered Bracket Karaiyar Trees 23 Agaricus sp Agaricaseae Food mushroom Karaiyar Soil

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S. no Botanical name Family Vernacular name Collected place Habit 24 Coprinus comatus Agaricaceae Shaggy mane, Inkcap Lawyer's Wig Mushroom Karaiyar, Mundanthurai Soil 25 Bridgeporousnobili ssimus Polyporaceae Noble polypore or fuzzy sandozi Deap forests in Karaiyar. Decayed woods 26 Pleurotus ostreatus Pleurotaceae Oyster mushrooms Most part of Mundanthurai and karaiyar Trees and woods 27 Coprinopsis variegata Agaricaceae Powder mushroom Karaiyar hills and its surroundings Soil 28 Lycoperdon perlatum Lycoperdaceae Skull shaped puff ball, earth ball ,buff balls. Karaiyar hills Soil 29 Exidia recisa Auriculariaceae Willow brain, amber jelly roll Puliodai areas Decayed trees 30 Chlorophylum rachodes Agaricaceae Shaggy parasol Karaiyar Soil 31 Bovista plumbea Agaricaceae Small puffball Pandiyan kottai hills Soill 32 Coprinus fimatarius Psathyrellaceae Gray shag Karaiyar Soil 33 Cyathus striatus Nidulariaceae Splash cups Kattalaimalai Deacayed woods 34 Pleurotus eryngii Pleurotaceae King oyster mushroom Karaiyar Soil 35 Nolanea strictia entolomataceae Poisonous mushroom Karaiyar Soil 36 Pholiota sp strophariaceae Scally caps Karaiyar Soil 37 Coprinus microcarpus Agaricaceae Powdered mushroom Karaiyar Soil 38 Auricularria aurantia Auriculariaceae Orange peel fungus Tunnel forest Decayed trees 39 Marasimus sp Marasmiaceae Poisonous mushroom Karaiyar Soil 40 Spongipellis pachydon Polyporaceae Tree mushroom Thuluka mattai Dead trees 41 Pheolus schweinitzii Polyporaceae Veivet top fungus Kannikatti Large trees 42 Tremetes gibbosa Polyporaceae Sponge fungus Kattalai malai Decayed trees 43 Ceratiomyxa fruticulosa Ceratiomyxaceae Slime mold Puliodai Dead trees 44 Ganoderma carnosum Ganodermatac eae Tree mushrooms Karaiyar Trees 45 Bonderzewia bekeleyi Bondarzewiaceae Yellow brown shelf fungus Karaiyar dam surroundings Dead woods

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4.7 Evaluation for Mineral Elements results of mineral values of the three edible species of mushrooms clearly indicate the During the present investigation minerals potential for their use as sources of good elements evaluation was estimated on dry quality food. weight basis and results obtained are documented in respectively. Out of 3 wild 4.8 Evaluation for Nutraceutical samples of C. indica, L. sulpureus and C o m p onents G. sepiarum examined for mineral estimation, Fe content of mushrooms had 258 mg/100 g in T h e pr esent study indicates the presence of C. indica, 242 mg/100 g in L. sulpureus and p h e n olics, flavonoids, proteins and G. sepiarum. Cu content of mushrooms had 8 carbohydrates in the extracts of selected mg/100 g in C. indica, 6 mg/100 g in mu s h ro o m s . The selected mushroom extract of L. sulpureus and 3 mg/100 g in G. sepiarum. Calocybe indica has the presence of saponins, Mn content in C. indica, L. sulpureus and ta n n i ns , p henols, flavonoids, proteins, G. sepiarum were 3, 2 and 1 mg/ 100 g, carbohydrate, steroids and sterols. Laetiporous respectively. Mg content in C. indica, su lp u r e u s has the presence of phenols, L. sulpureus and G. sepiarum were 247, 204 proteins, flavonoids and carbohydrate. and 132 mg/ 100 g, respectively. Ca content in Gloeophyllum sepiarium has the presence C. indica, L. sulpureus and G. sepiarum were of phenols, proteins, carbohydrate and 13, 21 and 3 mg/ 100 g, respectively. The flavonoids.

Table 2. Effect of media on mycelial growth by using three selected mushrooms

Culture media Growth rate C. indica L. sulpureus G. sepiarum PDA 1.08 0.03 0.06 MEA 1.10 0.01 0.04

Table 3. Effect of temperature on mycelial growth by using three selected mushhrooms

Temperature Mycelial growth rate C. indica L. sulpureus G. sepiarum 20 0.98 0.12 0.18 25 1.65 0.46 0.36 30 0.84 0.32 0.28 35 0.62 0.21 0.24

Table 4. Qualitative mycochemical screening of selected three mushrooms

Test Calocybe indica Leotiphorous sulpheurus G. sepiarum Alkaloids - - - Glycosides - - - Saponins + - - Steroids and Sterols + - - Phenols + + + Tannins + - - Proteins and aminoacids + + + Fixed oils and fats - - - Carbohydrates + + + Gums and Mucilage - - - Flavonoids + + +

Table 5. Name of species and mineral elements (mg/100 gm) of dry samples

S. no Fe Mg Cu Mn Ca C. indica 258±0.76 247±1.93 8±0.28 3±0.26 13±0.53 L. sulpureus 242±1.59 204±0.39 6±0.64 2±0.48 21±0.33 G. sepiarum 225±1.41 132±0.87 3±0.71 1±0.16 3±0.72 (mean ± SD; n = 3)

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Table 6. TLC Analysis of phenol in three selected mushrooms

Samples Distance traveled by the Distance traveled by the Rf value compound (cm) solvent front (cm) Rf value C. indica 17.2 18 0.955 L. sulpheurus 14.8 18 0.822 G. sephiarium 15.6 18 0.866

4.9 GCMS Analysis purpose. So the sample of B was used with mixed extracts had the percentage of 82.31% Qualitative and quantitative GC/MS analyses was present. were done in Laetiporous sulpureus and Gloeophyllum sepiarum. 4.11 Antibacterial Activity

4.10 DPPH Radical Scavenging Activity In the present study five different extract of (Water, Hcl, Chloroform, Phenol and Flavonoid) During the present investigation antioxidant three mushroom extract showed activity against activity was estimated on dry weight basis and the organisms of E. coli, Salmonella typhi, results obtained are documented in respectively. Staphylococcus aureus, Bacillus sp, Out of 3 wild samples of C. indica, L. sulpureus Pseudomonas sp, Klebsiella sp, Salmonella, and G. sepiarum examined for antioxidant Proteus and Streptococcus were documented in activity of phenol extract had 71.42% in b e lo w . T h e s e results confirm that bioactive C. indica, 49.66% in L. sulpureus and 30.61% in component of mushroom may differ in their G. sepiarum. Flavonoid extract of mushrooms solubility depending on the extractive solvents. had 70.06% in C. indica, 63.26% in L. Antimicrobial activity in natural source extracts sulpureus and 36.05% in G. sepiarum. From depends not only on the presence of phenolic above activity the more activity was present in compounds but also on the presence of various sample B. The sample A has the reference secondary metabolites [15].

Contol plates Inhibitory activity

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Fig. 3. Antimicrobial activity of Calocybe indica

5. CONCLUSION Madasamy Retired Forestor, My Mother Mrs. M. Madathiammal and My life Partner Mr. N. The present investigation can conclude that the Thangaselvin for their sacrifice and constant extract of 3 edible mushrooms showed encouragement for my research. biopharmaceutical potentiality. However whether such extracts will act as effective COMPETING INTERESTS therapeutic agents remain to be investigated, the identification of the bioactive compounds Authors have declared that no competing and study of mechanisms of actions are interests exist. necessary prior to application. REFERENCES ACKNOWLEDGEMENT

My sincere thanks to Bharathiyar Univesity for to 1. Tripathi DP. Cultivation of specialty do this research. mushroom. In Mushroom Cultivation. Oxford & IBH Pub. CO. PVT. LTD. 2005; I thank Dr. Mr. Melkani IFS The Principal Chief 250-253. Conservator of Forests, Tamilnadu and Mr. 2. Bhatt P. Kushwaha KPS, Singh RP. Venkatesh IFS The Chief Conservator of Evaluation of different substrate and casing Forests and Field Director, Kalakad mixture for production of Calocybe indica. Mundanthurai Tiger Reserve, Tirunelveli. Indian Phytopath. 2007;60(1):128-130. 3. Atri NS, Kaur A, Kaur H. Wild mushrooms: My sincere thanks to My Guide and also my Collection and identification. Chambaghat, well wisher Dr. M. Kannan, Assistant Professor Solan. 2003;16. in Zoology for his constant encouragement. 4. Kaviyarasan V, Ravindran C, Senthilarasu Finally my heart felt thanks to my family G, Narayanan K, Kumerasan V, Kumar M. members, especially my father Mr. M. A field guide to South India Agaricales

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