*2.3. Chemical Characterization of Extracts*

#### 2.3.1. Olive Wastewater (OWW) and Olive Pomace (OP)

The total phenol content (TPC) and antioxidant activity (ABTS, 2,2 -azino bis(3 ethylbenzothiazoline-6-sulfonic acid)) were determined spectrophotometrically following the method described by De Bruno [19] with some modifications. TPC, was quantified on the obtained extracts by FolinCiocalteau method, 0.1 mL of the phenolic extract (OWW and OP), were placed in a 25 mL volumetric flask and mixed with 20 mL of deionized water and 0.625 mL of the FolinCiocalteau reagent. After 3 min, 2.5 mL of a saturated solution of Na2CO3 (20%) were added. The content was mixed and diluted to volume with deionized water. Thereafter the mixture was incubated for 12 h at room temperature and in the dark. The absorbance of the samples was measured at 725 nm against a blank using a double-beam ultraviolet-visible spectrophotometer (Perkin-Elmer UV-Vis λ2, Waltham, MA, USA) and comparing with a gallic acid calibration curve (concentration between 1 and 10 mg·L<sup>−</sup>1). The results were expressed as mg of GAE mL−1. For total antioxidant activity determination, ABTS assay was used. The reaction mixture was prepared by mixing 2990 μL of ABTS and 10 μL of extracts (OWW and OP), and the absorbance was measured after 6 min at 734 nm. The quenching of initial absorbance was plotted against the Trolox concentration (from 1.5 to 24 mmol L−1) and the TEAC value was expressed as μmol TE mL−<sup>1</sup> of PE. Identification and determination of the main bioactive phenolic compounds were performed by UHPLC-DAD of the phenolic extract, following the method described

by Romeo et al. [18] with some modifications. The UHPLC system consisted of an UHPLC PLATINblue (Knauer, Berlin, Germany) equipped with a binary pump system using a Knauer blue orchid column C18 (1.8 μm, 100 × 2 mm) coupled with a PDA-1 (Photo Diode Array Detector) PLATINblue (Knauer, Berlin, Germany). The used software was Clarity 6.2 (Clarity-DataApex, Prague, The Czech Republic). The samples were filtered with a 0.22 μm nylon syringe filters (diameter 13 mm) and then injected in the system with a volume of 5 μL. The mobile phases were (A) water acidified with acetic acid (pH 3.10) and (B) acetonitrile; the gradient elution program consisted of 0–3 min, 95% A; 3–15 min, 95%–60% A; 15–15.5 min, 60%–0% A; finally, returning to the initial conditions was achieved during analysis keeping the column at 30 ◦C and the injection volume 5 μL. External standards (concentration between 1 and 100 mg·L<sup>−</sup>1) were used for the quantification and the results were expressed as mg·mL<sup>−</sup>1.
