# Edible Films Based on Ovine Second Cheese Whey with Oregon Essential Oil
## 3 Results and Discussion
### Microstructure  
Scanning electron microscopy was used to analyze the film microstructure. Figure 5 shows the surface images of experimental films. The WPI film surface was smooth, uniform and dense, while SCW films displayed a rough surface with aggregates of different sizes, probably due to the presence of denatured proteins and lipids. The WS films showed pinholes and cavities probably caused by lipid droplets from the SCW powder used in film-forming solutions; these droplets increased their size, and their distribution was more irregular proportionally to the amount of SCW in the mixture. When EO was added, more parts of the images appeared in a darker color compared with other sites of the film images; similar observations have been reported in WPI films containing free thymme extract [46].  
Figure 6 presents the cross-sectional images of the films. The WPI films exhibited a rough and compact image due to the protein aggregates that formed the film matrix. The SCW films  
Figure 5: Surface image of the films described in the materials and methods section. Scale: 100 μm; magnification \(\times\)200.  
\begin{table}
\begin{tabular}{c c c c c} \hline
**Film** & **Stage I** & **Stage II** & **Stage IIIa** & **Stage IIIb** \\  & **32–120 °C** & **120–235 °C** & **235–370 °C** & **235–370 °C** \\ \hline WS 1:1O1 & 62.2 & 197.0 & 247.2 & 315.8 \\ WS 1:2O1 & 65.4 & 193.1 & 249.9 & 316.8 \\ WS 2:1O2 & 61.2 & 192.6 & 251.3 & 314.4 \\ WS 1:1O2 & 64.4 & 193.4 & 251.7 & 314.4 \\ WS 1:2O2 & 66.1 & 192.3 & 250.3 & 312.5 \\ \hline \end{tabular} nd, not detected.  
\end{table}
Table 10: _Cont._showed a coarser appearance with cavities, due to the presence of highly denatured proteins. The WS 2:1 samples had a compact structure with small, sinuous creases. The increase of SCW content made the cross-sectional morphology of films rougher with larger aggregates, particularly in the WS 1:2 films. This phenomenon may be associated with the diminution of tensile strength and elongation values in films with higher proportions of SCW. In the case of the WS 1:2 films, the large aggregates were likely formed by denatured proteins from SCW, thereby impairing the mechanical properties of these films.  
The addition of EO modified the microstructure of the films. Larger aggregates and an increase in void sizes were observed in the WS 2:1 O2 and in the WS 1:1 and WS 2:1 films at both EO concentrations. The increase in roughness in protein films added with essential oils has been attributed to the attraction among the matrix components and EO, which at high concentrations can lead to the formation of agglomerates [44]. However, the nature of these interactions is likely to vary depending on the structural characteristics of the proteins involved. In the WS 2:1 O2 films, the presence of trabeculae that intersect voids was observed. These structures are likely the result of the interaction between WPI proteins and EO molecules, potentially contributing to enhanced mechanical properties, such as tensile strength and elongation, in comparison with the WS 2:1 and WS 2:1 O1 films. As previously documented by other researchers, an enhancement of these properties was observed when  
Figure 6: Cross-sectional images of the films described in the materials and methods section. Scale: 20 μm; magnification \(\times\)700.  
increasing concentrations of essential oil were added [44]. The aforementioned effect was not observed in films with lower WPI content, likely due to the EO concentration exceeding the limit over which WPI proteins can interact with EO components. It is possible that denatured SCW proteins exhibited a diminished capacity for binding EO compounds due to changes in their structure.