## Materials and Methods
### Bioactive Characterisation  
#### 2.6.1 Free and Bound Phenolic Compounds  
The extracts of free phenolic compounds were obtained according to the [PERSON] et al. [20] with some modifications. Samples (1.5 g) of crude olive pomace, liquid and pulp fraction samples were extracted on an orbital shaker (250 RPM) using methanol (25 mL) for 60 min (twice), followed by centrifugation (4000 RPM, 10 min) and supernatant recuperation. The extraction of bound phenolic compounds was achieved according to [PERSON] et al. [28] with some adaptations. The extraction residue was hydrolys for 4 h (20 mL of 4 M NaOH on an orbital shaker at 250 RPM) followed by acidification to pH 1.5-2.0 using 6 M hydrochloric acid (HCI). After 30 min of centrifugation (8000 RPM), the supernatant was extracted 5 times with ethyl acetate (30 mL). The ethyl acetate was dried using anhydrous Na\({}_{2}\)SO\({}_{4}\) and evaporated to dryness using a rotary vacuum evaporator at 30 \({}^{\circ}\)C. The resulting residue was subsequently dissolved in methanol to a final volume of 10 mL. The free and bound phenolic extracts obtained were stored at \(-\)20 \({}^{\circ}\)C until use.  
The total phenolic content (TPC) of free and bound phenolic extracts was determined according to the [PERSON]'s method [29]. Briefly, 50 \(\upmu\)L of the methanolic extract was mixed with 50 \(\upmu\)L [PERSON]'s reagent, 1 mL of Na\({}_{2}\)CO\({}_{3}\) (75 g/L) and 1.4 mL of ultra-pure water. The reaction tubes were incubated for 60 min at room temperature in the dark. The absorbance of the reaction mixtures was measured at 750 nm using gallic acid as a standard. All analyses were performed in triplicate. Results were expressed as mg gallic acid equivalents (GAE)/g DW.  
To identify the phenolic compounds, the extracts were analysed in an liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-UHR-QqTOF-MS) system (Bruker Daltonics, Billerica, MA, USA) following the methodology of [PERSON], [PERSON] [30], with some modifications in the gradient elution program (mobile phase A: 0.1% aqueous formic acid and mobile phase B: acetonitrile with 0.1% formic acid) to obtain a good separation of phenolic compounds: 0-5 min (5% B), 5-25 min (15% B), 25-35 min (30%), 35-40 min (95% B), 40-41 (5% B) and 41-42 min (0% B). Identification of the main phenolic compounds was based on standard solutions retention time, UV-Vis and mass spectra, when available. The other peaks were tentatively identified based on the literature, and their elemental composition was confirmed according to accurate mass (within 5 mDa of the assigned elemental composition) and isotope rate calculations designated mSigma (mSigma values < 20). The main phenolic compounds identified were quantified by HPLC (Waters e2695 separation module system interfaced with a Photodiode array UV/V is detector) using external calibration curves constructed based on their maximum UV signal (280, 320 and 360 nm) following [PERSON] et al.'s [31] procedure. The separation was performed in a reverse-phase column (COSMOSIL 5 C1 8-AR-II packed column--4.6 mm Inner Diameter. \(\times\) 250 mm; Dartford, UK).  
Sample and standard solutions were analysed using a solution of water/acetonitrile (95:5 \(\upmu\)V) with 0.2% trifluoroacetic acid (TFA) was used as mobile phase A, while mobile phase B was constituted of acetonitrile (100%) with gradient program at a flow rate of 1 mL/min: 0-2 min (100% A), 2-28 min (60% A) and 28-30 min (100% A). [31]. Identification of the main phenolic compounds (3-hydroxyprosol, protocatechuric acid, tyrosol, oleuropein, luteolin-7-O-glucose, caffe acid, rutin hydrate, p-coumaric acid, luteolin and quercetin) in methanol was performed by comparison of retention times, spectra and peak areas at maximum absorption wavelength. The results of the main phenolic compounds were expressed as mg/100 g DW.