# Hake Fish Preservation Using Plant-Based Impregnated Polylactic Acid Food Films as Active Packaging
## 3 Results and Discussion
### Characterization of Impregnated PLA Based-Film  
The impregnation conditions were selected according to a previous report where the highest antioxidant activity of the olive leaf extract impregnated PET/PP films was achieved at 400 bar and 35 \({}^{\circ}\)C [31]. In the present work, the impregnated PLA film presented an oxidation inhibition of 28.13 \(\pm\) 0.49%, indicating a concentration of 706 \(\mu\)g extract mg\({}^{-1}\) PLA. These results were higher than those found for the same extract using PLA filaments at 400 bar and 55 \({}^{\circ}\)C with an antioxidant activity of \(\sim\)15% and a loading of 0.5 mg g\({}^{-1}\) polymer [43]. These differences can be attributed to several factors: the film or filament form of the PLA, which affected the performance of supercritical impregnation, the higher CO\({}_{2}\) density employed in this work, which enhanced the solvent power (972.2 kg m\({}^{-3}\) at 400 bar and 35 \({}^{\circ}\)C compared to 906.85 kg m\({}^{-3}\) at 400 bar and 55 \({}^{\circ}\)C) as well as the slower depressurization rate used in this study (0.66 vs. 2 bar s\({}^{-1}\)) that decreased the dragging of the extract along with CO\({}_{2}\)[24; 46]. The morphology (form) of the polymer can affect the diffusion kinetics of bioactive compounds within the polymeric matrix. Specifically, in a film, a thinner structure allows for faster impregnation, while in a deeper matrix, diffusion occurs at a slower rate. Furthermore, the larger surface area exposed in a film compared to a filament provides more area for impregnation, resulting in potentially higher impregnation yields in films than in filaments. Moreover, changes in CO\({}_{2}\) density significantly impact the efficiency of impregnation. As observed in a previous study, the impregnation of bioactive compounds in olive leaf extract increases with CO\({}_{2}\) density [16]. Thus, a higher density results in a greater concentration gradient between the fluid phase and the polymer, which enhances the impregnation process. The depressurization rate also affects the impregnation efficiency as a faster pressure drop rate leads to the simultaneous release of soluble compounds along with CO\({}_{2}\) or their desorption from the polymer matrix, which can reduce the impregnation loading [46]. When the PET/PP films were impregnated with olive leaf extract, lower loadings were found at the same impregnation conditions (\(<\)100 ug extract mg\({}^{-1}\) PET/PP) [16; 47]. The differences in the PLA and PET/PP film loading could have resulted from the chemical interactions between the bioactive components that were soluble in the supercritical fluid phase and the polymers. The presence of hydroxyl moieties in the polyphenols can form strong H-bonds with carbonyl and ether groups present in the PLA and PET polyesters. In addition, weaker interactions, such as \(\pi\)-stacking and other Van der Waals forces, could occur between the aromatic rings of the main olive extract compounds and the terephthalate unit of PET. However, the presence of hydrophobic methyl groups in the PP layer of PET/PP films can reduce the affinity of polyphenols for this matrix, leading to considerably lower loadings [44]. Migration testing is used to evaluate the quality and safety aspects of packaging materials in food contact applications. This analysis helps to understand the interactions between ad ditives, packaging materials, and food products. Furthermore, migration is desirable when the incorporated additives are intended to prolong the product's shelf life. Since food products are complex, food simulants are used to mimic food systems. Ethanol 10% \(v/v\) (food simulant A) was designed to represent hydrophilic foods, and vegetable oil (food simulant D2) to simulate lipophilic foods. If the testing conditions are not technically feasible with food simulant D2, migration tests should be conducted using ethanol 95% \(v/v\) and isooctane [48]. In this study, the maximum amount of extract released from the PLA films into food simulant A was 57.305 \(\pm\) 2.3 mg dm\({}^{-2}\).