# Increasing the Bioactive Compound Content of Olive Oil by Acidification of Olive Paste
## 4 Discussion
### Phenolic Compounds and Antioxidant Capacity
The mechanism by which low-dose ascorbic acid enhances secoiridoid content in oils remains unclear, with a paucity of supporting literature. We hypothesize that, in addition to pH reduction and consequent facilitation of \(\beta\)-_glucosidase_ activity, ascorbic acid may modulate the antioxidant capacity, thereby attenuating phenol oxidase activity. This attenuation could mitigate the conversion of oleuropein to oxidized forms that are not the target compounds of this study. Due to the diversity of agronomic, meteorological, and technological factors that influence the composition of olive oil, no two oil samples are exactly alike [3; 46]. Therefore, it is not possible to claim that one olive oil inherently contains more or fewer phenolic compounds than another, as their composition is highly dependent on external factors. Rather, it is only feasible to determine whether a given factor influences a particular parameter or response when tested on a single, standardized olive sample. Consequently, this study exclusively manipulated the type and dosage of acid, utilizing a single olive sample across all trials, while maintaining all other process parameters constant to isolate the impact on the responses under investigation. [PERSON] et al. [7] analyzed 51 Italian and Spanish extra virgin olive oils (EVOOs), reporting total phenolic contents ranging from 29.5 to 316.3 mg/kg, depending on the cultivar, values lower than those found in this study. [PERSON] et al. [47], using the Picaul cultivar, investigated oils processed at temperatures below 27 \({}^{\circ}\)C with 30 min malaxation times, reporting a maximum phenolic content of 296 mg/kg at the optimal fruit ripening stage. Similarly, [PERSON] et al. [46] analyzed the phenolic profile of virgin olive oil (VOO) from 40 cultivars across three growing seasons, emphasizing the impact of fruit moisture on phenolic content. For the Picaul cultivar, average oleacein and oleocanthal contents were reported as 97.3 and 67.1 mg/kg, respectively. Some oils produced in this study through acidification exceeded these reported values by more than 100%. Oman-grown Picaul gives yield oils with average oleacein and oleocanthal concentrations of 43.5 and 58.4 mg/kg, respectively (total: 101.9 mg/kg) [44]. Our control oils demonstrated a combined average of 212.1 mg/kg for these two compounds, and the 1% ascorbic acid-treated oils reached an impressive 329.0 mg/kg. This represents a remarkable increase over the Omani Picaul variety (more than threefold) and approximately doubles the reported values for phenolic-rich cultivars like Coratina (181.2 mg/kg) and Kalamata (137.2 mg/kg). In contrast, a study examining the impact of adding fresh leaves from the Arbequina and Santulhana varieties during oil extraction reported a reduction in phenolic content, attributing this to the partial inhibition of \(\beta\)-_glucose_[48]. This finding contradicts another study, which, using Arbequina leaves, demonstrated the effectiveness of this method in enhancing phenolic content [49]. From the data in Table 4, the total secoiridoid content in the oils can be determined. Comparing this value with the total phenolic compound content reveals that secoiridoids account for more than 75% of the total phenols, with oils obtained using low doses of ascorbic acid exhibiting the highest secoiridoid percentage (79.4%). It was observed that the trend in antioxidant capacity paralleled the phenolic and secoiridoid content, with the highest values documented at the lowest ascorbic acid concentrations, specifically 1% (Figure S1, Supplementary Material). While minor compounds in olive oil, such as tocopherols, also contribute to its antioxidant capacity, their relatively low concentrations suggest that the variations observed in this study are primarily attributable to changes in the content of oleupreein-derived secoiridoids--3,4-DHPEA-EA and 3,4-DHPEA-EDA--and the final phenolic alcohol, 3,4-DHPEA, as proposed by [PERSON] et al. [3].