Page 305 - Demo

SECTION II. APPLIED BIOTECHNOLOGY IN THE PHARMACEUTICAL INDUSTRY... 3092.2. Solid-state fermentationUsing 250 ml Erlenmeyer flasks, seven fungal species were grown on pomegranate peel substrate in order to assess the flavonoids and antioxidant activity of solid-state fermentation (SSF). After autoclaving the flasks for 20 minutes at 121%u00b0C, water is added to the substrate to a moisture content of 60%u201370% (w/w). One disks with a diameter of 2 cm was used to inoculate the substrates using fungal colonies on PDA media. For 10 days, ascomycete and basidiomycete fungi were cultured at 25%u00b0C.2.3. Extracting enzymes For enzyme extraction, the culture was mixed with 50 ml of phosphate buffer (0.1 M, pH 6.5) and incubated in a rotary shaker at 200 rpm and 35%u00b0C for 1 hour. The mixture was then filtered through wet muslin cloth and centrifuged at 5,000 rpm for 15 minutes at 4%u00b0C, and the clear supernatant was collected for enzyme assays. For antioxidant estimation, 50 ml of 90% ethanol was added to the culture and incubated for 2 hours at room temperature with shaking at 250 rpm. The mixture was filtered, centrifuged at 5,000 rpm for 20 minutes, and the clear supernatant was used to measure flavonoids, and DPPH scavenging activity of the solid-state fermentation system (SSFS).2.4. Laccase activity assayThe spectrophotometric determination of laccase activity was carried out using ABTS as the substrate, following the method published by Slomczynski et al. (2021). The laccase reaction mixture, which contained 2.3 ml of enzyme in a 3 ml total volume, was diluted with 0.7 ml of ABTS substrate (0.02 M), 0.025 M succinic acid (pH 4.5) in the buffer [12]. One %u00b5mol of ABTS oxidised per minute is equivalent to one enzyme activity unit (U).