## 2 Results and Discussion
### Shelf-Life Study
#### 2.3.1 Evolution of Chemical Characteristics over Time  
**Legal quality parameters**: Figure 1A-D shows the evolution of key chemical quality parameters (i.e., free acidity, peroxide value, spectrophotometric indices) over time, while Table 6 summarizes the statistical significance of the effect of each factor and their interactions, as evaluated using 3-way ANOVA. Detailed data are also provided in Table S4 of the Supplementary Material. Free Aidity: Table 6 shows that free acidity varied significantly with oil type, storage time, and their interaction. No significant effects were observed in relation to bottle size or its interaction with other factors. Free acidity increased from an initial value of 0.22% to 0.31% in the 250 mL bottle and to 0.28% in the 500 mL bottle for EVOO 1, and from an initial value of 0.21% to 0.28% in the 250 mL bottle and to 0.29% in the 500 mL bottle for EVOO 2. These values are considered acceptable for high-quality oils after one year of storage. Across various combinations of oil type and bottle size, a general trend of increasing free acidity values was observed. This increase is due to the spontaneous, though slow, hydrolysis of triglycerides, resulting in the formation of free fatty acids and partial glycerides [38]. As expected, bottle size had no significant effect on this parameter, as hydrolysis is not influenced by oil mass or surface-to-volume ratio. 250 mL bottle and to 8.6 \(\mathrm{meq}_{02}/\mathrm{kg}_{\mathrm{oil}}\) in the 500 mL bottle for EVOO 2. These values are still acceptable for high-quality oils after one year of storage. The increase in peroxide value during oil storage is well-documented, often showing an initial rise, and a subsequent decline in sealed containers with limited oxygen availability [45; 46; 47]. For both EVOOs, Figure 1B shows a continuous and relatively steady increase in the 250 mL bottles, while a rise followed by a plateau is observed for the 500 mL bottles. This suggests that the greater oil mass in the larger bottles consumes oxygen more quickly, thereby slowing peroxide formation. These findings confirm the better performance of larger bottles in slowing oxidation processes. K\({}_{232}\): Table 6 shows that K\({}_{232}\) values were significantly influenced by storage time, EVOO sample, and their interaction, as well as the interaction between time and bottle size. The latter significance indicates that K\({}_{232}\) increased over time to varying extents depending on bottle size. K\({}_{232}\) values ranged from an initial value of 1.65 to 2.25 in the 250 mL bottle and to 2.19 in the 500 mL bottle for EVOO 1, and from 1.62 to 2.16 in the 250 mL bottle and to 1.98 in the 500 mL bottle for EVOO 2. These values are still within acceptable limits for high-quality oils. A consistent increase was observed under all conditions (Figure 1C), more pronounced in the 250 mL bottles. The rise in K\({}_{232}\) is primarily associated with the formation of conjugated dienes in the early stages of lipid oxidation. These results further confirm that larger bottles more effectively limit oxidation. In comparison with previous research that demonstrated improved protection in larger bottles under light exposure [48], these findings suggest that even in the dark, larger bottles better preserve EVOO's quality from oxidation. K\({}_{270}\): Table 6 indicates that the K\({}_{270}\) was significantly influenced by storage time, oil sample, and bottle size. Additionally, significant effects for the interaction between oil sample and storage time, as well as between storage time and bottle size, were observed. K\({}_{270}\) values ranged from an initial value of 0.13 to 0.17 in the 250 mL bottle and to 0.18 in the 500 mL bottle for EVOO 1, and from 0.13 to 0.15 in both bottle sizes for EVOO 2. These values are also considered acceptable for high-quality oils after one year of storage. A consistent increase in K\({}_{270}\) was observed for EVOO 1, while in EVOO 2, the increase occurred primarily in the final months of storage (Figure 1D).