2.6. Fat and fatty acid
Fat content was determined by Soxhlet extraction procedure (AOAC, 1984). 1 g of dried mycelia was homogenized first with 10 ml methanol and followed by 20 ml chloroform in presence of butylated hydroxytoluene (BHT) as antioxidant. After filtration, the solid residue was re-suspended in 30 ml chloroform: methanol (2:1) and homogenized. The procedure was repeated thrice. To the total filtrate 30 ml water with 0.88% KI was added. The extracted lipid was washed thrice with 1:1 methanol and water and nitrogen gas was passed over till the solvent evaporated out. Anhydrous Na2SO4 was added to remove water. Samples were kept sealed in a tube in nitrogen atmosphere until further study. Methyl esters of lipid extract were prepared by using acid based transesterification (Wollenweber, Seydel, Lindner, Lüderitz, & Rietschel, 1984). Solvents used contained BHT as an antioxidant and C17:0 fatty acid as internal standard. Methyl esters were analyzed on a Hewlett-Packard Model 6890 Gas Chromatograph using a DB 23 column. Chromatograms were analyzed using HP Chemstation software.