*2.1. Chemicals*

Tara gum, pectin esterified from citrus fruits with a degree of methoxylation of 55–70%, gallic acid, (+)-hydrated catechin, L-ascorbic acid, hydrogen peroxide (H2O2, 30% *v*/*v*), Folin–Ciocalteu reagent, carbonate sodium (Na2CO3) radical 2,2-diphenyl -1- picrylyhydrazyl (DPPH), radical 2,2-azino-bis (3-ethylbenzothiazolin-6-sulfonic) (ABTS), potassium persulfate (K2S2O8), ammonium molybdate tetrahydrate ((NH2)2MoO4), sodium molybdate (Na2MoO4), sodium nitrite (NaNO2), sodium phosphate (Na3PO4), aluminum chloride (AlCl3), hydrochloric acid (HCl), sodium hydroxide (NaOH), absolute ethanol, methanol, Whatman No. 3 filter paper, dialysis membrane (MWCO: 12,000–14,000 Da), were purchased from Sigma Aldrich (Sigma Chemical Co., St. Louis, MO, USA). HPLC grade water, formic acid and acetonitrile were purchased from VWR (Chromasolv, VWR International Srl, Milano, Italy).

### *2.2. Sample Collection and Preparation*

Green olives of Roggianella cultivar were harvested (October 2019) in the Northern of Calabria and processed on-site the next day (Oil mill Vinciprova srl in San Vincenzo la Costa (Cosenza, Italy)) using a semi-continuous Enorossi 150 traditional olive oil pressing system (Enoagricola Rossi, Calzolaro di Umbertide, Perugia, Italy) standardized to press a maximum of 150 kg of olives at a time. Olive mill wastewater (OMW) sample was collected and stored in 1.0 L low density polypropylene airtight containers at −18 ◦C until use. OMW were filtered and, subsequently, the filtrate was centrifugated (10 min at 10,000 rpm). Finally, the solution was frozen and dried by freeze-drying providing a dark colored vaporous solid (LOMW).

### *2.3. Chemical Characterization of Lyophilized Olive Mill Wastewaters* 2.3.1. HPLC-MS Analysis of Lyophilized Olive Mill Wastewaters

HPLC 1100 system composed of a degasser, quaternary pump solvent delivery, thermostatic column compartment, auto-sampler, single wavelength UV-Vis detector and MSD triple quadrupole QQQ 6430 in a series configuration (Agilent Technologies, Palo Alto, CA, USA) was employed for the polyphenols analysis. LOMW was resuspended in 2 mL ethanol/water (1:1, *v*/*v*) to a final concentration of ~1.2 mg mL−<sup>1</sup> and filtered through 0.2 μm pore size regenerated cellulose filters (VWR International Srl, Milano, Italy) and injected onto a reversed stationary phase column, Luna C18 (150 × 2 mm i.d., particle size 3 μm, Phenomenex, Torrance, CA, USA) protected by a C18 Guard Cartridge (4.0 × 2.0 mm i.d., Phenomenex). HPLC separation was carried out through a binary gradient consisting in (solvent A) H2O/formic acid 0.1% (*v*/*v*) and (solvent B) acetonitrile: 0 min, 10% B; 1 min, 10% B; 15 min, 30% B; 22 min, 50% B; 28 min, 100% B; 34 min, 100% B; 36 min, 10% B, followed by washing and re-equilibrating the column (with ~20 column volume). The column temperature was controlled at 20 ◦C and the flow was maintained at 0.4 mL min−1. UV-Vis detection wavelength was set at 280 nm.

Because polyphenols contain one or more hydroxyl and/or carboxylic acid groups, MS data were acquired in negative ionization mode with capillary voltage at 4000 V, using nitrogen as drying (T = 350 ◦C; flow rate = 9.0 L min−1) and nebulizing gas (40 psi). MS and MS/MS spectra were acquired in the range between *m*/*z* 50 and 1200. All data were processed using Mass Hunter Workstation software (version B.01.04; Agilent Technologies). UV absorption, retention times (RT), elution order and mass spectra (MS and MS/MS) were compared with those from pure standards (3-hydroxytyrosol, caffeic acid and *p*-coumaric acid) and/or matched with those already reported in the literature [15–17]. Then, the main revealed compounds were quantified by multiple reaction monitoring (MRM) as 3-hydroxytyrosol (R<sup>2</sup> = 0.99923). Mass Hunter Optimizer software (version B.03.01; Agilent Technologies) (Table S1).

### 2.3.2. H-NMR Analysis of Lyophilized Olive Mill Wastewaters

The sample LOMW (16.7 mg) was dissolved in 0.6 μL of D2O (99.9% D). 1H-NMR spectrum was performed at 25 ◦C using a Brucker Advance 200 spectrometer of 300 MHz equipped with 13C/1H dual probe. The NMR experiments were recorded with a spectral width of 6983.240 Hz, an acquisition time of 10.20 s, a number of 64 scans, a relaxation time of 2 s and a pulse width of 7 s. The spectra were processed by XWIN-NMR.

### 2.3.3. Polyphenols Total Content

Folin–Cicocalteu method was employed to evaluate available phenolic groups (APG) [18]. Different concentrations of an aqueous solutions of LOMW (6.0 mL) were added to 1.0 mL of Folin–Ciocalteu reagen<sup>t</sup> and after 3 min, 3.0 mL of Na2CO3 (2.0 % *w*/*v*) was also added keeping the solution under stirring in the dark (time = 2 h) and spectrophotometrically measuring at 760 nm. APG was expressed as milligrams of catechin (CT) per gram of LOMW (mg CT/g LOMW), by using an equation obtained from a calibration curve of CT at different concentrations (8.0, 16.0, 24.0, 32.0 and 40.0 μM). The method of least square

was used to calculate a calibration curve. Each measure was performed in triplicate and data expressed as means (±SD). UV-Vis absorption spectra were recorded with a Jasco V-530 UV/Vis spectrometer (Jasco, Tokyo, Japan).

### 2.3.4. Phenolic Acid Content

Arnov test, with some modifications, was used to evaluate the phenolic acids content (PAC), expressed in milligrams of CT per gram of LOMW (mg CT/g LOMW) [19]. In a volumetric flask (10.0 mL) 1.0 mL of an aqueous solution of LOMW, 1.0 mL of HCl (0.5 mol <sup>L</sup>−1), 1.0 mL of Arnov's reagen<sup>t</sup> (sodium molybdate and sodium nitrite 0.1 mg mL−1), 1.0 mL of NaOH (1.0 mol <sup>L</sup>−1) and purified water were mixed. The absorbance of the solutions was measured by a spectrophotometer at 490 nm. Each measurement was performed in triplicate and data expressed as means (±SD).
