2.8. Antioxidant Tests

The DPPH method previously reported by [31,32] was used to evaluate the antioxidant activity of haloarchaea strains. Briefly, 150 μL of a prepared solution of 400 μM DPPH solution was mixed with 50 μL of standards or extracts, (curve from 10–250 μg/mL). The mixture was homogenized using a vortex, allowed to react in the dark at room temperature for 20 min, after which time absorbance was measured at 517 nm. Astaxanthin was used as a reference standard (curve from 10 to 250 μg/mL). The results were expressed in Trolox micromoles per gram of extract (μmol Trolox/g extract) and as IC₅₀ in μg/mL, (concentration of infusion or standard in μg/mL required to inhibit 50% of DPPH radical present in solution, as reported in [32,33]). A Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments Inc., Winoosky, VT, USA) was used.

The capturing capacity of the ABTS^•+ radical was evaluated by the decolorization method developed by Re et al. (1999) and modified by Kuskoski et al. (2004) [34]. A solution of 7 mM ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) in deionized water and a solution of 2.45 mM potassium persulfate in water was prepared. Both solutions were mixed in a 1:1 ratio (v/v) and incubated for 16 h in the dark for the formation of the radical ABTS^•+ by the oxidation of ABTS with potassium persulfate at room temperature. Then, to 20 μL of the extract or standard to be measured (curve from 10–250 μg/mL), 200 μL of the previously ABTS^•+ solution (adjusted with 80% methanol to an absorbance of 0.70 ± 0.02) was added, mixed using a vortex mixer and subsequently allowed to react in darkness at room temperature [35]. The absorbance was then measured at 765 nm after 6 min of reaction, and astaxanthin was used as a reference standard (curve from 10–250 μg/mL). The results were expressed as TEAC, in Trolox micromoles per gram of extract (μmol Trolox/g extract) and also as IC₅₀ in μg·mL⁻¹, (concentration of infusion or standard in μg·mL⁻¹ required to inhibit 50% of DPPH radical present in solution) [32,33]. A Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments Inc., Winoosky, VT, USA) was used.

For the FRAP test, the methodology described by Benzie and Strain [32,36] was performed with some modifications. Briefly, a buffer solution made of CH₃Nax₃H₂O 3.1%/CH₃COOH (glacial) 16% dissolved in water plus 20 mM FeCl₃ in aqueous solution HCl 0.02 M and 10 mM TPTZ dissolved in absolute ethanol was prepared. The working solution corresponded to a mixture of one volume of buffer with one volume of FeCl₃ and 11 volumes of ethanol. The working solution (290 μL) was mixed with 10 μL of the standard Trolox (from 10 to 250 μg/mL) in a well of the microplate in quintuplicate, and a curve was prepared measuring the absorbance of the colored Fe-TPTZ complex at 593 nm after 5 min. Then, absorbance values (in quintuplicate) of strain extracts (10 μL, at a concentration: 2 μg/mL) were replaced in the Trolox standard curve equation [32,35]. The results were expressed as Trolox equivalents (TE) in Trolox micromoles per gram of extract (μmol Trolox/g extract). A Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments Inc., Winoosky, VT, USA) was used.