Comparative Study of Five Pleurotus Species Cultivated in Warm Temperature on Non-sterilized Rice Straw

Das, et al.: Comparative study of five pleurotus Spp. 750 Emir. J. Food Agric ● Vol 27 ● Issue 10 ● 2015 MATERIALS AND METHODS Mushroom species Five Pleurotus spp. were used in the present study. Pleurotus flabellatus (MTCC 1799), Pleurotus ostreatus (MTCC 1802), Pleurotus pulmonarius (MTCC 1805) and Pleurotus floridanus (MTCC 6315) were obtained from Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India whereas Pleurotus florida (ITCC 3308)) was obtained from Society for Rural Industrialization, Ranchi, India. All the species were maintained in PDA medium (Das and Mukherjee, 2007). Spawn preparation About 1000 gm of wheat grains has been taken for spawn production. The grains were boiled for half an hour then washed in flowing water. Excess water present was drained off and the grains were spread on the surface of clean blotting paper and air dried. 10 gm of calcium sulphate and 5 gm of calcium carbonate were mixed with the grains. Then about 100 gm of grain was placed in conical flask (250 ml) and sterilized in autoclave at 121 ̊C for 15 min and inoculated with the respective mushroom strains and kept at 30°C for 15 days (Das et al., 2010). Substrate preparation Dried rice straw has been collected from a local farm at Barasat, West Bengal, India. The chopped rice straws (1 cm/5 cm) are weighed and soaked in water for overnight. Excess water present in the substrate was drained off and the substrate is air dried for 15 min. There is no heat treatment of the substrate. About 750 gm wet substrate was mixed with either 10% or 20% spawn (wet wt./wet wt.). These spawned substrates were then put into 30 cm x 42 cm polythene bags. The bags were closed tightly with pin holes on the surfaces and kept in 30±2 oC (Das et al., 2010). Biological efficiency (BE) BE was calculated according to Das et al. (2010). BE (%) = (fresh weight of mushroom ×100)/dry weight of substrate Microscopical studies Microscopical studies and measurement were done using Carl Zeiss Microscope, Germany and measurement were done using Axio Vs 401E V4.3.0.101 (2004) software. Preparation of fruiting body extract for determination of protein and carbohydrate Fresh fruiting bodies (30 g) were disrupted by being crushed with acid washed sea sand in mortar and pestle. Then tissues were extracted with 100 ml of 20 mM imidazole buffer containing 1 mM EDTA, 2 mM PMSF (pH 7.8). Unbroken cells and cell debris were removed after centrifugation at 10000 rpm for 30 min and the supernatant was used for determination of protein and carbohydrate (Das and Mukherjee, 2007). Protein determination Protein concentration was determined by the method of Lowry et al. (1951) with slight modification. At first Lowry A (2% sodium carbonate mixed with 0.1 N NaOH solution) and Lowry B (1.56% copper sulphate solution mixed with 2.37% sodium potassium tartarate) reagents were prepared. Lowry C reagent was made by mixing 2 ml of (Lowry B) with 100 ml of (Lowry A). 20 μl mushroom extract was mixed with 980 μl of distilled water and incubated for 15 min after addition of 2 ml Lowry C reagent. After incubation 1 ml 1N Folin Ciocalteau reagent solution (Diluted 2N commercial reagent with an equal volume of water on the day of use) was mixed and incubated for 30 min in dark condition. The OD values were taken at 660 nm and the protein values of mushroom samples were determined by comparing the standard curve of BSA. Carbohydrate determination Total carbohydrate was determined using anthrone reagent according to Pons et al. (1981) with some modifications. 100 μl of the sample was taken in a test tube and 5 ml of 2.5 N HCl was added with it. Hydrolyzed the mixture in a boiling water bath for three hours and neutralized it with solid sodium carbonate until the effervescence ceased. Made up the volume to 100 ml and centrifuged. Supernatant was collected. One ml of sample was mixed with 4 ml of anthrone reagent. The mixture was boiled for eight minutes in a boiling water bath. The OD values were taken at 630 nm after rapid cooling. The carbohydrate content of mushroom samples were determined by comparing the standard curve of glucose. Polyphenol determination Oven dried mushroom powder (500 mg) was mixed with 2 ml of 80% methanol. It was centrifuged at 10,000 rpm for 30 min. This process is repeated thrice by same way mixing with 80% methanol extract with pellet. Finally supernatant is collected and volume made up to 5 ml by adding 80% methanol. 2 ml supernatant was taken in a petriplate, evaporated to dryness then dissolved the residue in 2 ml distilled water. 1 ml sample has been taken and mixed with 2 ml Na2CO3 (2%), after 2 min incubation 1 ml Folin Ciocalteau reagent is added. After 30 min incubation at room temperature OD720 has been taken and compared with gallic acid standard. Das, et al.: Comparative study of five pleurotus Spp. Emir. J. Food Agric ● Vol 27 ● Issue 10 ● 2015 751 Determination of moisture content and dry matter of mushroom Fresh mushroom fruiting bodies were weighed and kept in oven at 60 ̊C for overnight until the constant weight was gained. The moisture content and dry matter of Pleurotus spp. were calculated according the following formulas given by Khan et al. (2013). Moisture content (%) = 100 X (W1W2)/W1 Dry matter (%) =100 X W2/W1 Where W1= weight of fresh sample W2= weight of dry sample Determination of ash content One gram of oven dried sample was taken in cleaned and previously weighed china crucible. After ignition on a flame, crucible was placed in a muffle furnace at (550±50) oC for three hours. After that crucible was cooled in desiccator and then weighed. Ash contents were calculated according to the formula of Khan et al. (2013). Ash (%) = 100 X (W2-W1)/weight of sample Where W1=weight of crucible W2= weight of crucible ± material Determination of crude fiber content The crude fibre content was determined according to Khan et al. (2013). Sample (10 g) was heated at simmering temperature (80 oC) with 200 ml H2SO4 and kept about half an hour. Through frequent addition of hot water the medium volume was kept constant. After addition of 500 ml cold water, the boiling was stopped. The content was filtered immediately under vacuumed condition. The residues were washed for several times with hot water and digested. The digested sample was then filtered until become neutral after addition of acetone. The residues were properly washed and transferred to crucible. The sample was then dried in constant weight. Crucible was placed in the muffle furnace at 65 oC for ignition. The ash and crude fibre was calculated according to the following formula Crude fibre(%)=a-(b/w)×100 Where a=Dry weight after digestion.