*3.2. FTIR and DSC Analyses of The Films*

The spectra of zein-based films with or without licorice EO, and both casted on simple polystyrene Petri dishes and on lotus negative template were recorded. It was observed that all the films without incorporation of licorice EO presented analogous FTIR spectra. Similar results were found for the films with licorice EO, indicating that the support material where the films were casted (polystyrene or PDMS) did not influence the chemical interactions between the components of the films. In addition, both sides of each film presented similar FTIR spectra, which means that the topographic surface structure did not affect the chemical composition of the films. Figure 2a shows the FTIR spectrum of the zein film without licorice EO, while Figure 2b presents the spectrum of the film incorporating the EO. The licorice EO is mainly constituted (71.8%) by isopropyl palmitate [13], also shown in Figure 2b. The large absorption at 2960 cm−<sup>1</sup> in the zein film with licorice EO was attributed to the stretching vibration of the aliphatic compounds, and the peak at 1740 cm−<sup>1</sup> was ascribed to the stretching vibration of the ester compounds. Isopropyl palmitate possesses an aliphatic chain with several methyl groups (CH), while being also an ester presenting a carbonyl group (C = O) [13]. These molecular features of the main compound of the EO are responsible for the peaks visible in the FTIR spectrum of the zein-based film incorporating licorice EO, when compared to the spectrum of the control film, indicating that the EO was successfully incorporated.

**Figure 2.** FTIR spectra of the films: control film (without EO) (**a**), film with EO (**b**), and films after 10 days of soil burial (**c**).

The thermal profiles of zein films with or without the licorice EO incorporated were evaluated by DSC. Similar to what was observed in FTIR analysis, the support material where the films were casted did not influence the DSC curves. Figure 3a shows the DSC curve of a zein film without EO and Figure 3b shows the DSC curve of a zein-based film incorporating licorice EO. Both thermograms show endothermic peaks in the range of temperature of 50 ◦C to 150 ◦C. The presence of such peaks is attributed to the loss of volatile components, like water, or the possibility of chain relaxation. Additionally, in this range of temperature, the breakdown of hydrogen bonds that are present in the zein structure and other molecular associations also happens [20]. Proteins have some features associated to their different tridimensional structure, such as the denaturation process. The DSC curve of zein-based film without EO (Figure 3a) exhibited endothermic peaks at 311 ◦C and 318 ◦C, which can be interpreted as the protein unfolding, similar to what was previously observed [20]. Moreover, through the DSC curve of the control film, at the 174 ◦C mark, a slight modification on the linear profile of the zein curve appears, which is linked to the zein glass transition temperature (Tg), since above this temperature the protein chains of zein enter in a flexible stage [20]. When incorporating licorice EO in the zein films, the DSC curve (Figure 3b) changed, having observed a lower value of Tg (165 ◦C), which is probably related to the loss of some flexibility in the films with the EO [20]. This fact can be explained by the interactions among zein and the compounds of licorice EO after the preparation of the films, as it was also observed by the FTIR analysis. Similar results were previously obtained by other authors dealing with zein-based films [29].

**Figure 3.** DSC curves of the films: control film (without EO) (**a**), and film with EO (**b**).
