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.