# Increasing the Bioactive Compound Content of Olive Oil by Acidification of Olive Paste
## 4 Discussion
### Phenolic Compounds and Antioxidant Capacity  
Table 3 demonstrates that acid treatments generally elevated hydroxytyrosol levels. Specifically, increasing concentrations of acetic and citric acids resulted in progressively higher hydroxytyrosol content, with the 'High' concentration treatments approaching a doubling of the control value (2.20 \(\pm\) 0.21 mg/kg). Initially, it might be hypothesized that a reduction in paste pH promotes hydroxytyrosol formation, thereby enhancing its partitioning into the oil phase via equilibrium. However, correlation analysis between hydroxytyrosol content and pH revealed no significant trend, indicating that pH is not the primary determinant of hydroxytyrosol enrichment in the oils. Notably, ascorbic acid at the 'Low' concentration yielded the highest hydroxytyrosol level among all treatments (3.03 \(\pm\) 0.35 mg/kg), suggesting a distinct mechanism of action. Regarding tyrosol, acetic and citric acids exhibited a dose-dependent increase in oil concentration, whereas ascorbic acid resulted in a reduction, independent of the applied dose. An alternative method for obtaining oils rich in specific bioactive compounds, employing supercritical CO\({}_{2}\) extraction, yielded hydroxytrosol and tyrosol concentrations of 13 and 29 mg/kg, respectively [43]--markedly higher than our findings. In a study comparing pharmacologically relevant phenolic compounds in various Omani olive oil cultivars, the Picual variety exhibited mean values (mg/kg) of approximately 4.2 for hydroxytrosol and 1.6 for tyrosol [44]. Notably, the hydroxytrosol content in our oils produced with the highest citric and acetic acid doses exceeded this value, and several of our acidified conditions also demonstrated higher tyrosol concentrations. In the flavonoid and lignan columns of Table 3, luteolin is shown to be the predominant flavonoid in all oils extracted from acidified pastes. However, its concentration only increased compared to control oils when ascorbic acid was applied. On the contrary, the apigenin content increased with all acids, with the most significant increase being observed at low ascorbic acid concentrations, tripling the content in the oils with respect to the control. For lignan pinoresinol, all concentrations of citric acid led to an increase compared to the control oils. In contrast, only low concentrations of the other two acids resulted in higher pinoresinol levels than the control oils. In none of the cases did the pH variation due to the different doses of acids seem to have a significant influence on the content of these compounds in the oils. Table S2 shows the phenolic acids and their derivatives quantified in the oils. Regarding phenolic acids, vanillic acid was identified as the most abundant, followed by \(p\)-coumaric acid. Vanillic acid concentrations were elevated across all acid treatments compared to the control. However, the dose-response varied among acids. Acetic acid exhibited no discernible dose-dependent effect, citric acid showed an inverse relationship between dose and vanillic acid concentration, and ascorbic acid demonstrated a positive correlation. Notably, despite this positive correlation, ascorbic acid resulted in the smallest overall increase in vanillic acid levels. \(p\)-Coumaric acid concentrations were elevated in oils treated with all acids, with no significant variation observed across acid types or dosages. Table S2 indicates that acetic and citric acids did not significantly alter \(t\)-ferulic acid concentrations in the oils. In contrast, ascorbic acid elicited an increase, exhibiting an inverse dose-response relationship, with the highest \(t\)-ferulic acid concentration, approximately threefold greater than the control, observed at the lowest dose. Considering the pH of the vegetation waters, these acids are expected to be predominantly in their molecular form, thus exhibiting increased solubility in the oils. However, this expected trend is not consistently observed in the results.