ostreatus was cultivated in substrates with seed cake added to different proportions of eucalypt sawdust, corncob, eucalypt bark and coffee husk. The purpose of adding these agroindustrial residues was to balance the carbon
and nitrogen ratio, which may stimulate the mycelial growth ( Nunes et al., 2012). The substrate compositions that were selected for this study were based on the results of these previous experiments were jatropha seed cake (Sc), Sc + 10 (g/100 g) of eucalypt sawdust (ScEs), Roxadustat molecular weight Sc + 10 (g/100 g) of eucalypt bark (ScEb) and Sc + 30 (g/100 g) of coffee husk + 30 (g/100 g) of rice bran (ScCh). In these substrates, the isolate Plo 6 had better biomass production and greater degradation rate of lignocellulosic compounds when compared to other tested substrates ( Da Luz, 2009). The substrates were humidified with water at 75% of the retention capacity and 1.5 kg of each substrate was placed in polypropylene bags. Next, the bags containing the substrates were autoclaved at 121 °C for 2 h. After sterilization, the substrates were inoculated with 70 g of spawn and incubated at 25 °C for 60 d. Samples for analyses
were taken at intervals of 15 d. Phytase activity (myo-inositol hexakisphosphate phosphohydrolase, EC 3.1.3.8) was determined using Taussky–Schoor PLX-4720 concentration reagent according to Harland and Harland (1980). To extract the enzyme, 3 g of the substrate was transferred to Erlenmeyer flasks (125 mL) containing 10 mL of sodium chloride (1 g/100 mL). The flasks were kept in a shaker for 1 h at 100 rpm, and the extracts were filtered through Millipore membranes (Whatman 1). The filtrate was centrifuged for 5 min at 2000 × g. The reaction to determine phytase activity contained 100 μL of the filtrate and 1 mL of sodium phytate solution (0.5 g/100 g, Sigma). This reaction was incubated in a water bath at 60 °C for 10 min, and then 1 mL of trichloroacetic acid (10 g/100 mL) and 5 mL of Taussky–Schoor
reagent were added. The phosphorus content was determined with a spectrophotometer (Thermo, Evolution 60) at 500 nm. The standard curve for phosphorus quantification was made using dibasic potassium phosphate (Sigma) with concentrations ADP ribosylation factor ranging from 0.004 to 0.02 g/100 mL. One unit of phytase was defined as the amount of enzyme required to release 1 μmol of inorganic phosphate per min from sodium phytate at 37 °C. To determine phytic acid content, 3 g of each substrate was transferred to Erlenmeyer flasks (125 mL) containing 25 mL hydrochloric acid (4 g/100 mL, Vetec). These flasks were kept in a shaker for 16 h at 220 rpm. The supernatants were transferred to centrifuge tubes (50 mL) containing 1 g of sodium chloride (Vetec), centrifuged at 1000 × g for 20 min and frozen at −20 °C for 30 min. After thawing, the supernatants were centrifuged under the same conditions and filtered through Millipore membranes (Whatman GF/D, 4.7 cm). The filtrate (1 mL) was diluted in 24 mL of deionized water.