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.