2.3.3. Antioxidant Activity (ABTS+ Method)

The ABTS assay was performed following the method described by Contreras et al. 2011 [21]. Briefly, 100 μL of sample (diluted appropriately with water) were mixed with 1 mL of ABTS+ solution. The degraded color was read after 30 min at 730 nm using a spectrophotometer (UV-1700, Shimadzu®, Kyoto, Japan). A Trolox calibration curve was conducted for quantification purposes and the results are expressed as Trolox equivalents (TE) or μmol TE/L.

#### 2.3.4. Ferric Reducing Antioxidant Power (FRAP)

The FRAP was measured as previously described by Benzie and Strain (1996) with modifications [22]. Briefly, 90 μL of deionized water were mixed with 30 μL of sample (diluted appropriately with water) and added to 900 μL of the FRAP reagen<sup>t</sup> (pre-warmed at 37 ◦C). The sample was incubated for 30 min at 37 ◦C. Subsequently, the sample absorbance at 593 nm was measured using a spectrophotometer (UV-1700, Shimadzu®, Kyoto, Japan). A Trolox® calibration curve was employed for quantification purposes and the results are expressed as μmol TE/L.

#### 2.3.5. Antioxidant Activity (DPPH Method)

The DPPH assay was performed as previously described [23]. Briefly, 2 mL of 0.5 mM DPPH reagen<sup>t</sup> was mixed with 2 mL of methanol and 0.2 mL of sample (diluted appropriately with water). The absorbance peak at 517 nm was recorded after 30 min of incubation under darkness (UV-1700, Shimadzu®, Kyoto, Japan). A Trolox® calibration curve was done for quantification purposes and the results are expressed as μmol TE/L.

#### 2.3.6. Antimicrobial Activity of Annatto Seed Extracts

The antibacterial activity was determined employing the colorimetric microdilution method with broth incubated with *Bacillus cereus* (ATCC 11778) and *Staphylococcus aureus* (ATCC 6538). Bacterial strains were incubated for 24 h at 37 ◦C in a Mueller–Hinton broth and adjusted to a final density of 10<sup>6</sup> CFU/mL before inoculation. Samples were dissolved in dimethylsulfoxide (DMSO) to reach a final concentration of 4.096 mg/L. Double serial dilutions were made at a concentration ranging from 4 to 4096 mg/L. Then, 96-well microplates were prepared by adding 20 μL of sample on the respective dilution medium followed by addition of 220 μL of Mueller-Hinton broth. The last row containing only 220 μL of Mueller–Hinton broth and 10 μL of inoculum was used as a negative control. The final volume in each well was 250 μL. Once the samples were homogenized, they were incubated for 5 h at 37 ◦C. After incubation, 25 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (Alfa Aesar, Germany), dissolved in DMSO (800 mg/L) were added to each well and incubated for 1 h to allow viable microorganisms to metabolize the yellow MTT dye in formazan. The minimum inhibitory concentration (MIC) was considered as the concentration of the first well that did not show any color change (from yellow to purple). The procedure was repeated three times for each microorganism and pH (4, 7, and 11) [1].
