3.2. Stabilizing mechanism of the W/O emulsions developed by the CW
The PLM photographs of the W/O emulsions included in Figs. 1 and 1SM showed that the water droplets were surrounded by a birefringent material. This birefringent material, indicated in the Fig.1 with black arrows, was uniformly distributed around the surface of all the water droplets. It is important to point out that none of the PLM photographs of the W/O emulsions showed the presence of wax crystals on the water-oil interface, indicating that the water droplets were not stabilized through the Pickering effect. These results contrast with the microstructure of the W/O emulsions formulated with 5% carnauba wax (40% water) or with 5% beeswax (20% water) developed through a pilot scale two-step process, consisting of a pre-emulsification step (90°C) followed by dynamic crystallization step (5°C) (Penagos et al., 2023). These authors showed through PLM and confocal laser scanning microscopy, that the water droplets of the carnauba wax and beeswax W/O emulsions were stabilized by wax crystals surrounding the droplets (i.e., Pickering effect) and by a crystal network developed in the oil phase by the corresponding wax (Penagos et al., 2023). On the other hand, as previously indicated pentacyclic triterpenes like the ursolic acid, can develop W/O emulsions tentatively stabilized also through the Pickering effect (Liu et al., 2022). Within this context it is important to note that the different stabilizing mechanisms observed in the emulsions developed in the present study with that reported by Penagos et al. (2023), might be associated with the different conditions used to develop the W/O emulsions and with the differences in wax composition. Carnauba wax consists mostly of long chain (C26 to C30) aliphatic esters (≈40%) and diesters of 4-hydroxycinnamic acid (≈21.0%), and a significant amount of long chain ω-hydroxycarboxylic acids (≈ 13.0%) and fatty alcohols (≈12%) (Wolfmeier et al., 2016). Beeswax consists of ≈71% esters (mainly including ≈35% monoester, ≈14%diesters, ≈3% triesters, and ≈12% of hydroxymono- and hydroxypoly-esters), ≈14% n -alkanes, and ≈13% free fatty acids and alcohols (Tulloch, 1980). In the study of Penagos et al. (2023) the emulsification step was done at temperature conditions where the vegetable wax components were soluble in the oil phase. Under these conditions the surface-active molecules (i.e., fatty acids and fatty alcohols) of carnauba wax (≈ 25%) and beeswax (≈ 13%) would be adsorbed at the oil-water interface through their polar groups with their aliphatic chains pointing toward the oil. We consider that because the molecular compatibility between the aliphatic chains of the adsorbed surface-active molecules and the long chain esters of the wax still in the oil solution, their nucleation and further crystallization on the oil-water droplet surface could occur during the cooling stage, followed by the additional crystallization in the continuous oil phase of the remaining long chain esters. The overall results would be that under the emulsifying and crystallization conditions used by Penagos et al. (2023) the carnauba wax and the beeswax developed O/W emulsions stabilized by Pickering and by a network of long chain ester crystals distributed through the continuous oil phase. In contrast, in the present study the emulsification was done at 25°C using mixtures of water and CW oleogels (i.e., 40:60, 50:50 and 60:40). The cooling thermograms included in Fig. 3 showed that, although most of the components of the CW were already crystallized at 25°C (temperature indicated with a dotted line in the thermograms of Fig. 3), still some CW components remained in the oil solution (i.e., required lower temperatures to crystallize in the oil phase). Within this context, the thermograms included in Fig.3 indicate the % of solid content achieved at 25°C (%SFC25°C, determined by NMR) in the corresponding CW oil solution. With the values of %SFC25°C we calculated the percentage of the CW that crystallized at 25°C in the oleogels (%CW25°C). The corresponding statistical analysis showed that the %CW25°C was statistically the same in all the CW oleogels, i.e., the %CW25°C was the same independent of the CW concentration in the oleogel. The corresponding mean value of the %CW25°C was 73.6% (± 5.0%). The %CW25°C value mainly included the crystallization of the n -alkanes and long chain esters, components mainly involved in the development of the tridimensional crystal network of CW oleogels (Chopin-Doroteo et al., 2011; Morales-Rueda et al., 2009; Romero Regalado, 2013; Toro-Vazquez et al., 2007). However, the %CW25°C value indicated that ≈26% of the CW components remained in the oil phase at 25°C. We considered that these CW components included the surface-active compounds involved in developing the W/O emulsion and, subsequently, forming the birefringent material present around the surface of all the water droplets (Fig. 1). Given the composition of the CW above reported (see section of “Materials”), these compounds tentatively involved the triterpenic alcohols, esters of triterpenic alcohols, aliphatic alcohols, and fatty acids. The PLM photographs of the CW emulsion also showed the presence of highly birefringent crystals in the oil phase (shown in Figs. 1 and 1SM with dashed arrows), particularly evident in the W/O emulsions with a final CW concentration of 3%. These birefringent microstructures, also present in CW oleogels, are characteristics of the crystals developed mainly by the co-crystallization of n -alkanes with long-chain esters. As a reference the Fig. 2SM includes PLM photographs of 1.5% and 3% CW oleogels developed following the same time-temperature and shearing conditions used in the development of the oleogels used for the development of the emulsions. The PLM photographs show the characteristics crystals found in CW oleogels (Fig. 2SM). From here we concluded that under the conditions used the systems developed by the CW were structured W/O emulsions where, tentatively, the triterpenic alcohols, esters of triterpenic alcohols, aliphatic alcohols, and fatty acids acted as surface-active agents at the oil-water interface, while the n -alkanes and long chain esters gelled the continuous oil phase.