*3.5. Contact Angle and Water Solubility*

Understanding the wettability of the films is often carried out by measuring the contact angle formed between a liquid drop and the film [32]. Additionally, the water contact angle measurements provide information about the hydrophobicity/hydrophilicity of the surface of the films. A hydrophobic surface is a surface in which the water contact angle is higher than 90◦ [5]. Table 3 summarizes the water contact angle measured in both sides of the films (with and without the licorice EO), casted on polystyrene and on lotus negative template. The addition of licorice essential oil increases significantly

(*p*-values < 0.05) the contact angle of the films (Table 3). In films casted on simple polystyrene Petri dishes, the contact angle raises from 48.27◦ (without EO) to 71.80◦ (with EO). These results may be explained by the chemical composition of the licorice EO, mainly constituted by isopropyl palmitate, which presents an octanol/water partition coefficient (logP) of 8.16, an indicator of its strong hydrophobic character [13]. Licorice is usually taken as a hydrophobic component of edible films and coatings [33]. More interestingly was the effect observed in the films casted on lotus negative template. The upper side of the films (without the hierarchical surface morphology of the lotus leaves), presented contact angles of 65.15◦ (without EO) and 58.04◦ (with EO); contrariwise to what was verified in the lower side of the films (with the hierarchical surface morphology of the lotus leaves) that presented significantly higher (*p*-value < 0.05) contact angles than the ones obtained for the films casted on polystyrene (Table 3). The film without EO that presents the complex surface patterns of the lotus leaves (positive replica) had a contact angle of 81.95◦; significantly higher (*p*-value < 0.05) contact angle was obtained for the film with EO, which is also a lotus positive replica (112.50◦) (Table 3). Here, the strong hydrophobic effect of licorice/isopropyl palmitate was noticed, in addition to the "lotus effect", which confirms the initial idea behind this work. Combining the "lotus effect" with the incorporation of the licorice EO resulted in a near superhydrophobic surface (water contact angle superior than 150◦) [5]. To the best of our knowledge, it was the first time that zein-based films were obtained with such high water contact angle.

Water solubility is a measure of the resistance of the films to water [24]. The results obtained showed that all the films presented a water solubility of about 20% (Table 3), indicating the lower affinity to water of these films, which also corroborates the water contact angles obtained for the films.

#### *3.6. Antioxidant and Antibacterial Properties*

Figure 4 shows the antioxidant activity of the films measured by DPPH scavenging assay over time. All the films presented the ability to scavenge the free radicals in a time-dependent manner, as demonstrated by the linear regressions (*p*-values < 0.05). After 5 h of reaction, the films presented near 90% of inhibition of DPPH free radicals (Figure 4). Although the licorice EO is a potent antioxidant [13], the films without it also presented the capacity to scavenge free radicals, which is due to zein. The antioxidative nature of zein was previously reported, being frequently used in food packaging (edible films and coatings) without adding any other antioxidant during processing [34]. Some studies have reported the benefits of using zein films as packaging material for cooked turkey and fresh broccoli [34]. The capacity of the films to inhibit lipid peroxidation was also evaluated (Table 4). The results showed that the films with licorice EO have significantly higher (*p*-value < 0.05) percentages of inhibition than the control films. Both types films (polystyrene and lotus negative template) incorporating the EO presented similar capacity to inhibit the lipid peroxidation measured by the β-carotene bleaching test (20–30%), contrariwise to what was obtained with the control films (3–5%) (Table 4). This capacity can be attributed to isopropyl palmitate, the major compound of licorice EO, which is generally used in cosmetic and food industries due to its binder and fragrant properties, together with skin-conditioning and emollient activities. The Food and Drug Administration (FDA) considers that this compound is not ecotoxic and classifies it as not expected to be potentially toxic or harmful, presenting a low human health priority [35].

Also, licorice is generally recognized as safe (GRAS) by the FDA, indicating that there is no evidence, in the available information on licorice, that identifies a hazard to the public when it is used at levels that are now current and in the manner now practiced [36].



*Microorganisms* **2019**, *7*, 267

highlighted in bold.

**Figure 4.** Antioxidant activity of the films measured by DPPH scavenging assay.

**Table 4.** Antioxidant activity (β-carotene bleaching assay) and diameters of inhibition zones obtained with the films.


(Results expressed as mean ± SD) a, b, c and d correspond to each type of film; Significant *p*-values are highlighted in bold.

The antibacterial properties of the films were evaluated against two well-known foodborne pathogens (*E. faecalis* ATCC 29212 and *L. monocytogenes* LMG 16779). *E. faecalis* is a commensal of the human gastrointestinal tract that can persist in the external environment and is a leading cause of several infections. Given its diverse habitats, the organism has developed numerous strategies to survive a multitude of environmental conditions [37]. *L. monocytogenes* is a foodborne pathogen responsible for a disease called listeriosis, which is potentially lethal in immunocompromised people and can provoke septicemia, meningitis and fetal infection or abortion in infected pregnant women [38].

The results of the antibacterial activity studied by solid diffusion assay are presented in Table 4. Both types of zein films incorporating licorice EO presented significantly higher (*p*-value < 0.05) diameters of inhibition zones for the two bacterial species. Moreover, the anti-biofilm potential of zein films against the same foodborne pathogens was evaluated by SEM, forming the bacterial biofilms directly on the surface of the films (results not shown). It was possible to verify that in the films presenting the hierarchical surface morphology of the lotus leaves, the bacterial adhesion did not occur. More than inhibiting the bacterial growth, the films inhibited the adhesion. The anti-bio adhesion of surfaces with lotus-leaf-like rugosities is well described in the literature [39].

Since zein-based films incorporating licorice EO were able to scavenge free radicals to inhibit lipid peroxidation and the growth of foodborne pathogens, they can potentially be used as alternative food packaging systems, particularly in foods with high contents of lipids.
