ORAC Assay of Fish Protein Hydrolysates

The ORAC assay, based on the scavenging of peroxyl radicals generated by AAPH, was performed following the Santos-Sánchez et al. method [27 (link)]. Briefly, 25 µL of FPH (at the final concentrations of 0.1, 0.5 and 1.0 mg/mL) was added to 50 µL sodium fluorescein (2.934 mg/L) and incubated for 15 min at 37 °C. Therefore, 25 µL of AAPH (60.84 mM) was added, and the fluorescence read at 485 nm ex. and 528 nm em. every 5 min for 120 min using a Synergy H1 microplate reader. The area under the curve (AUC) was calculated for each sample subtracting the AUC of the blank. The results were calculated using a Trolox calibration curve (2–50 µM).