2.3. Experimental conditions
Becker flasks (100mL) with 12 g of mediumwere used to obtain the enzymatic extract in the solid-state process. The flasks were inoculated with A. niger spores, in order to have an initial concen-tration of 1 107 spores/L, and incubated at 30 C in a humidity saturated environment, for 72 h. The extraction of the obtained enzymes by solid-state process was performed through suspension of humidified solid material (2.7 g) in distilled water (15 mL; pH 4), using 125 mL flasks under reciprocal agitation of 200 rpm, at 30 C. The obtained solutions were then centrifuged for 10 min at 5000 g.
The supernatant was kept at 4 C for using in posterior tests. Erlenmeyer flasks (500 mL) with 100 mL of substrate were used to obtain the enzymatic extracts by submerse process. The flasks were inoculated with a suspension of A. oryzae spores, aiming for an initial concentration of 1 106 spores/L, and being subsequently incubated at 28 C and 200 rpm, in a reciprocal shaker. After 96 h, the media was centrifuged for 10min, at 5000 g, the supernatant being kept at 4 C
for further tests (Malvessi & Silveira, 2004).