3.2. Confocal Scanning Laser Microscopy (CSLM)
The bigels’ microstructure for 60:40 and 70:30 OG:HG ratios is shown in Fig. 2. The lipid and aqueous phases are depicted in red and green, respectively. The images shown were carefully chosen as representatives of what was observed across all the images taken for each sample. The 60:40(0) sample shows large oleogel regions dispersed in a hydrogel matrix, indicating this sample is an oleogel-in-hydrogel type bigel. In the literature, an oleogel-in-hydrogel type of bigel describes oleogel droplets dispersed in a hydrogel matrix, but in this formulation, the term “droplets” is not an accurate descriptor for the oleogel phase because the oleogel portion is quite large and irregularly shaped. When 0.5% (w/w) MDG is added to the 60:40 OG:HG formulation, there is a clear reduction in the size of the oleogel regions. This reduction in oleogel region size is evidence that MDG is working as an emulsifier at the lipid-aqueous interface. At 1% and 2% (w/w) MDG, the bigel transitions from an oleogel-in-hydrogel type (1% (w/w) MDG) to a bi-continuous bigel type (2% (w/w) MDG) in which neither phase forms droplets, but rather both phases create a bi-continuous matrix. The 60:40(3) bigel formulation shows a phase inversion where the hydrogel is now dispersed in an oleogel matrix. On the other hand, all 70:30 OG:HG formulations, regardless of the amount of MDG added, appear to be bi-continuous bigels where there is no clear distinction as to which phase is the continuous or dispersed phase (Lupi et al., 2016; Shakeel et al., 2018). A bi-continuous gel can also be referred to as a matrix-in-matrix type of bigel. As mentioned previously, the 70:30(3) formulation could not be stabilized and showed clear phase separation.
In addition to the microstructural changes, MDG also had an unexpected effect most evident in formulations with high MDG concentrations. Fig. 2 shows the formation of spherical structures that appeared in the lipid phase with sizes ranging from 10-μm to 40-μm. These spherical structures are evident in small amounts in 70:30(0), 70:30(0.5) and 70:30(1) bigels; nevertheless, they become very noticeable in 60:40(3) and 70:30(2) bigels. Observation under polarized light microscopy (PLM; not shown) confirmed that these spherical structures were not spherulites resulting from the crystallization of MDG. Therefore, it can be hypothesized that they are lipid droplets that were not incorporated into an oleogel region or matrix. Whether or not these droplets contain rice bran wax is still unclear, but PLM did not show rice bran wax crystals within these droplets. Additional studies are necessary to confirm the contents of these lipid droplets and why they form in greater numbers when MDG is present at high concentrations.