Multiskan uv vis spectrometer

Total polyphenolic content was determined by spectrophotometry according to Nunes et al., 2018 [37 (link)], with minor modifications. Briefly, 30 μL of the sample was mixed with 150 μL of Folin-Ciocalteu reagent 0.2 N (1:10) and 120 μL of Na2CO3 (7.5%). The mixture was incubated at 45 °C for 15 min, protected from light, and for 30 min more at room temperature. Finally, absorbance was measured at 765 nm in a UV–Vis Multiskan spectrometer (Thermo Scientific). The content of total polyphenols was determined by means of a standard curve of gallic acid 0–110 μM/mL. All measurements were carried out five times.

Total carotenoid content was determined by spectrophotometry according to Biswas et al., 2011 [38 (link)] and Sánchez-Camargo et al., 2019 [39 (link)] with minor modifications. 100 mg of the sample were mixed with 5 mL of acetone at 4 °C, vortexed for 1 min and allowed to set for 30 min at 4 °C, vortexed again for 5 min, and centrifuged at 1370 g for 10 min. This procedure was repeated 3 times, the supernatants were mixed, and absorbance was measured at 450 nm in a UV–Vis Multiskan spectrometer (Thermo Scientific). The total carotenoid content was determined by means of a standard curve β-carotene (32 μg β-carotene/mL −0.031 μg β-carotene/mL). All measurements were carried out three times.

In 96-well microplates, 35 μL of sample and 265 μL of FRAP reagent (buffer 0.3 M: TPTZ 10 mM: Ferric chloride 20 mM (10:1:1)) were mixed, incubated in the dark at 37 °C for 30 min. Absorbance was measured in a Multiskan UV–Vis spectrometer (Thermo Scientific) at 595 nm. The antioxidant activity was determined by means of a standard Trolox curve 0–500 μM (r² = 1) [37 (link)]. All measurements were carried out five times.