2.4. Determination of the Antioxidant Activity

The best detoxifying fungi were selected according to residual phorbol ester or FG on SSF treatments. Fermentations were repeated with the selected macrofungi to obtain the antioxidant activity profil. The SSF flasks were sacrificed every three days for analysis.

Extraction of total phenolic compounds (TPC) was performed as reported by Asolini et al. [11], with modifications. Each dry sample was weighed (4 g) in Falcon tubes, added 35 mL of 80% (v/v) ethanol acidified with 0.5% (v/v) of HCl. Tubes were placed in boiling water for 30 min, the supernatant was separated, and the precipitate was used for another extraction. Second supernatants were removed and placed together with the supernatants from the first extraction. The extracts were centrifuged for 30 min at 6000 rpm and stored at 2 °C in the absence of light.

Total antioxidant activity (TAA) was quantified by the ABTS+ [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] method and DPPH (2,2-Difenil-1-picril-hidrazila) method. ABTS method was carried out according to Rufino et al. [12]. The ABTS+ radical, generated during the oxidation of ABTS with potassium persulfate, captures hydrogen (electron donor of the antioxidant substance) by the radical, promoting discoloration of the solution. Extract color does not interfere with analysis, and the reagent is soluble in both polar and nonpolar solvents [13].

DPPH method uses a purple and stable free radical, which after receiving the hydrogen atom, is reduced and acquires a yellow color, measured by spectrometry [14]. First, 150 µmol/L DPPH solution, 2000 µmol/L Trolox solution (a synthetic antioxidant analogue to vitamin E), extract dilutions at 1 mg/mL, 0.5 mg/mL and 0.1 mg/mL and a Trolox curve (40–360 µmol/L) were prepared. TAA analyses were performed in wells of a microplate, with 22 µL of each diluted extract or each point of the curve, together with 200 µL of the DPPH solution. Methanol was used as a blank, and absorbance (520 nm) was taken every 15 min, for 1 h, and then every 1 h for more 5 h. The ideal time of activity in the Trolox equivalent was determined. DPPH oxidation inhibition by the time was plotted on a graph to determine the ideal time of antioxidant activity.