*3.1. Proof-of-Concept: Negative Template and Positive Replica*

This work intended to develop zein-based films incorporating licorice EO with potential applications in food packaging materials. Considering that these bio-based films are usually hydrophilic, and considering the "lotus effect", it was decided to try to mimic the hierarchical surface morphology of the lotus leaves in these films.

Firstly, the surface of the leaves was analyzed by SEM (Figure 1b), where it was possible to notice the common pattern of lotus leaves with its micro and nanostructures, measuring between 7 and 8 μm. Then, to prepare a negative template of the lotus leaves, a mixture of PDMS and its catalyzer was nano-casted on fresh lotus leaves. After solidification of PDMS, the negative template was lift off and visualized by SEM (Figure 1c). The PDMS-negative template presents the holes corresponding to the structures observed in the lotus leaves surface, which means that a complementary topographic surface structure of the original template (lotus leaves) was successfully obtained. Finally, the zein-based

films (with and without licorice EO) were casted on the PDMS-negative template to replicate the hierarchical surface morphology of the lotus leaves in the films. The films obtained were once again visualized by SEM (Figure 1d), ant it was possible to observe that the complex surface patterns of the lotus leaves were transferred to the surface of the zein-based films with high fidelity (positive replica), which validated the original concept behind this study. Moreover, the structures obtained in both types of zein-based films presented similar dimensions (≈7–8 μm), measured by SEM, indicating that the incorporation of the licorice EO in the films did not interfere with the nano-casting process. Other researchers had previously replicated the so-called lotus-leaf-like structures for biomedical and pharmaceutical applications [10,26–28].

**Figure 1.** Flowchart for negative template fabrication and films preparation (**a**). SEM images of lotus leaf surface (**b**), PDMS-negative template (**c**), and zein film-positive replica (**d**). (Magnification: 500×).
