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.