## 3 Results and Discussion
### Bioactivity Characterisation
#### Identification of Phenolic Compounds  
A detailed analysis of free and bound phenolic compounds is required to elucidate which compounds are responsible for the antioxidant activity of the edible fractions (liquid and pulp fractions). Table 2 shows the free phenolic and bound phenolic profile of the crude olive pomace, liquid and pulp fractions, identified by LC-ESI-UHR-QqTOF-MS. The distribution (in percentage) of the phenolic compounds from C-OP, L-OP and P-OP are given in the Supplementary Material (Figure S1). The HPLC quantification of phenolic compounds for which a standard is available is presented in Table 3. In agreement with TPC results, the amounts of phenolic compounds identified in the free phenolic extract were higher than in the bound phenolic extract for all samples. The main phenolic classes identified in free phenolic extracts were (1) hydroxytyrosol/tyrosol and its derivatives, followed by (2) flavonoids class (largely luteolin), (3) comselogosid and derivatives class (essentially comselogoside, verbascoside and caffeoyl-6-secologanoside) and (4) phenolic acids (mainly caffeic acid and \(p\)-coumaric acid). The liquid fraction exhibits a rich composition in hydroxytyrosol and its derivatives (\(625.76\pm 51.33\) and \(513.61\pm 27.85\) mg/100 g DW in OM 1 and OM2, respectively) owing to the hydrophilic nature of this phenolic class [56]. Previous works also supported the efficiency of centrifugation to recover these compounds [18]. Hydroxytyrosol and its derivatives are one of the principal phenolic compounds present in olive fruits and by-products. They have been characterised to have potent biological activities as an antioxidant, anti-inflammatory and antimicrobial, with promising applications in foods, but also in cosmetics and medicine [5; 8; 57]. Moreover, the European Food Safety Authority (EFSA) has issued a favourable scientific opinion regarding health claims for the dietary consumption of hydroxytyrosol and its derivatives (protection of blood lipids from oxidative damage) [58]. As a result, in the last years, hydroxytyrosol application and recovery studies have increased exponentially. Comselogoside, verbascoside, caffeoyl-6-secologanoside and caffeic acid, which are also associated with the prevention of cardiovascular diseases, were also identified in a slightly higher amount in liquid than in pulp fraction. Luteolin was the predominant phenolic compound of the pulp fraction. The reduced water solubility of luteolin could explain the retention of this compound in the pulp fraction [59]. Studies reported that luteolin has antioxidant, anti-microbial, anti-inflammatory and anti-diabetic activity [60]. The pulp fraction was also characterised by a significant amount of bound phenolic compounds, namely vanillin, hydroxytyrosol, protocatechuic acid and caffeic acid. These bound phenolic compounds represented a significant amount of the total phenolic compounds of the pulp fraction (18-27%). The highest amount of bound phenolics in the pulp fraction could be linked to its higher fibre content and with the possible linkage of vanillin, caffeic and protocatechuic acids to structural components of the cell wall (cellulose, lignin and protein) [61]. Caffeic acid was detected in a much higher amount in bound form, and protocatechuic acid and vanillin were only noticed in bound form. Caffeic acid has been related to the reduction or complete inhibition of \(\alpha\)-glucosidase activity (potential anti-diabetic activity) [62]. Vanillin and protocatechuic acid have been described to have potent antioxidant activity and antimicrobial activity [63; 64]. Other compounds like hydroxybenzoic and ferulic acid were only identified in bound phenolics extracts. Overall, the liquid fraction allowed the recovery of a considerable amount of hydroxytyrosol and its derivatives related to the prevention of cardiovascular diseases, and the pulp fraction exhibited a significant quantity of free (mainly luteolin) and bound phenolics.