Antioxidant Activity Determination via Two-Step Extraction

For the analysis of the antioxidant activity, samples were prepared with a two-step extraction. The extraction procedure was developed based on a method described by Li et al. [72 (link)] and was used with the following modifications: approximately 500 mg freeze-dried powder was weighed into a tube containing 10 mL enzyme solution (0.4 mg lysozyme· mL⁻¹, Sørensen buffer; pH 7.4) and mixed for 45 min at 37 °C. The tube was centrifuged at 3226× g for 15 min at 15 °C. The residue was re-suspended with 80% ethanol (v/v), stirred for 1 min, and centrifuged under the same conditions as previously mentioned. Both supernatants were analyzed for total phenolic content (TPC), a well-known indicator of antioxidant activity, and Trolox equivalent antioxidant capacity (TEAC). The total amount was calculated as the sum of the two extracts.