3.4. X-ray analysis and rheology of the structured W/O
emulsions
To have additional evidence of the microstructure of the systems
developed, we obtained WAX
diffractograms for 1.5% and 3% CW oleogels and the corresponding 1.5%
and 3% CW W/O emulsions formulated with 40:60 and 60:40 water to
oleogel ratios. The corresponding diffractograms are shown in Figure 5.
As a reference to support the analysis of the diffractograms of the CW
oleogels and the W/O emulsions, Fig. 5 includes the WAX diffractograms
for deionized water (Figs. 5A and 5B) and CW (Fig. 5C). It is important
to note that, except for the CW and the water, the diffractograms of the
emulsions and the oleogels showed an amorphous signal with at peak at
scattering that peaks centered at d = 4.55 Å (≈19.5 2θ). This
amorphous signal was associated with the
liquid phase of triacylglycerols from the vegetable oil (Larsson, 1972).
On the other hand, the diffractogram for the CW diffraction peaks at 2θ
= 21.5° and 2θ = 23.8° corresponded to d values of 4.1 Å and 3.7
Å, respectively (Fig. 5C). These diffraction peaks are characteristic of
the orthorhombic perpendicular subcell packing of the n -alkanes
of the CW (Chopin-Doroteo et al., 2011; Dassanayake et al., 2009) and
were also present in the 1.5% and 3% CW oleogels (Fig. 5D) and in the
1.5% and 3% CW emulsions formulated with 40:60 and 60:40 water to
oleogel ratios (Fig. 5A and 5B). These results indicated that an oleogel
microstructure, developed mainly by the n -alkanes and long chain
esters of the CW, was present in the W/O emulsions. Additionally, the
characteristic amorphous broad signal of the water with a peak at 2θ ≈
29° corresponding to a d ≈ 3.15 Å (Maciel et al., 2016) observed
in the water diffractogram, was observed as a shoulder in the WAX
diffractograms for the W/O emulsions formulated with 1.5% and 3% CW
and water to oleogel ratios of 40:60 and 60:40 at 2θ ≈ 29° (d ≈
3.16; Figs. 4A and 4B). This shoulder was larger and, subsequently, more
evident in the emulsions formulated with the higher water proportion
(i.e., 60:40 water to oleogel ratio). We considered that these results
indicated the presence of a water phase confined throughout the
microstructure of the oleogel. Based on these results and the ones
obtained through PLM (Figs. 1 and 1SM) we consider that this water phase
was emulsified, tentatively by the triterpenic alcohols, esters of
triterpenic alcohols, aliphatic alcohols, and fatty acids. Therefore,
the system studied was a W/O emulsion structured (i.e., stabilized) by
an oleogel developed in the continuous oil phase by the n -alkanes
and long chain esters of the CW.
The f sweeps of the W/O
emulsions formulated with 1.5% and 3% CW concentrations at the
different water to oil ratios studied after 20 days of storage are shown
in Figure 3SM. Similar results were obtained with the W/O emulsions with
0.75% and 2.25% of CW (results not shown). All the emulsions studied
showed a f independent rheological behavior, i.e., a gel-like
rheological behavior. From the f sweeps of the emulsions, we
obtained the corresponding G’ value at an f of 1 Hz. From here we
evaluated the elasticity of the W/O emulsions at 0 and 20 days of
storage at 25°C as a function of the different water to oleogels ratios
and CW concentrations used (Fig. 6). The results showed that,
independent of the CW concentration, the G’ of the emulsions increased
as the water to oleogel ratio increased (P < 0.05), a behavior
directly associated with the increase in the volume fraction of the
emulsified water. Other studies also had shown that the increase in the
volume fraction of the dispersed phase resulted in an increase of the
emulsions’ elasticity (Farah et al., 2005; Pal, 2006; Poling-Skutvik et
al., 2020). Additionally, we observed that for the same water to oleogel
ratio, the G’ of the emulsions increased exponentially as a function of
the CW concentration in the emulsions (P < 0.05). The G’
increment was partly associated with a larger reduction in the water
droplet diameter achieved as the CW concentration increased, an effect
previously discussed regarding the WDD97.5% behavior as
a function of the CW concentration (Fig. 2). It is well-known that
emulsions with smaller droplet size have higher elasticity (i.e., higher
G’) than emulsions with larger droplet size (Pal, 2006, 1996). Another
factor associated with the G’ behavior observed in the emulsions
formulated at same water to oleogel ratio was that, as the CW
concentration increased the hardness of the oleogel phase ought to
increase. This behavior of the CW oleogels was previously reported by
our group (Toro-Vazquez et al., 2007). Because the systems developed
were W/O emulsions structured by the oleogel developed in the continuous
oil phase, as the CW concentration increased, we obtained emulsions with
a harder oleogel phase and, subsequently, emulsions of higher elasticity
(i.e., higher G’, Fig 6). It is important to note that at all CW
concentrations studied, after the 20 days of storage we observed a
decrease in the elasticity of all emulsions. Nevertheless, independent
of the CW concentration in the emulsion, the decrease in G’ was
significant just in the emulsions formulated with the 60:40 water to
oleogel ratio (P < 0.01; Fig. 6). In the 40:60 and the 50:50
emulsions the storage time effect on the emulsions’ G’ was not
significant at any of the %CW used (Fig. 6). These results corroborated
that at the highest proportion of water utilized (i.e., 60:40 water to
oleogel ratio), the amount of surface-active compounds present in the CW
was insufficient to achieve an efficient emulsification of the water
phase. Therefore, independent of the %CW used in the emulsions, we
obtained larger water droplet diameters in the 60:40 emulsions (Figs. 1,
Fig. 1SM, and Fig. 2) that resulted in emulsions with higher instability
when compared with the 40:60 and 50:50 emulsions (Fig. 4).
As indicated in the methodology section, the R10 s and
R300 s of the emulsions were determined from the
corresponding time-dependent recovery profiles of the emulsions. The
Fig. 4SM shows the time-dependent recovery master curves for the 1.5%
and 3% CW emulsions developed at the different water to oleogel ratios.
As a reference the Fig. 4SM-B indicates the points where
G’0 s, G’10 s, and the
G’300 s were determined to calculate, using the Eqs. 1
and 2, the corresponding R10 s and R300
s of the W/O emulsions stored 0 and 20 days at 25°C. The corresponding
R10 s and R300 s values were plotted as
a function of the %CW and the water to oleogel proportion in the
emulsions (Fig. 7). The results
showed that, independent of the storage time, the emulsions with the
highest R10 s and R300 s were those
formulated between 0.75% and 2.25% CW in the emulsions using water to
oleogel proportions of 40:60 and 50:50. In contrast, the emulsions
developed with CW concentrations between 0.75% and 3% at the 60:40
water to oleogel proportion always had the lowest R10 sand R300 s values. These results indicated that the
60:40 emulsions, the ones with the larger water droplet diameters (Figs.
1 and 2), showed lower recovery capacity after deformation than the
emulsions with a smaller water droplet diameter (i.e., the 40:60 and the
50:50 emulsions). From here and considering the previous results we
concluded that the W/O emulsions formulated with water to oleogel ratios
of 40:60 and 50:50 with CW concentrations between 1.5% and 3%,
provided the better rheological behavior and were the most stables, even
after two freeze-thaw cycles after storage for 20 days at 25°C.