3.3. Oleosome Structure
The organic moieties containing
O-H and bonds, such as phospholipid bilayer, glycosidic bond and
protein-phospholipid complexes, were analyzed by FTIR spectroscopy (Fig.
3). The stretching band of C=O at 1100 cm-1 and 1600
cm-1 was assigned to the polysaccharides and
phospholipid bilayers, respectively (Li et al., 2022). The H-OH
stretching around 2800-2900 cm-1 was assigned to
glycosidic bond between glycoprotein units, and the O-H stretching
between 3000-3500 cm-1 to the presence of
protein-phospholipid complexes (Qi et al., 2017).
The FTIR spectra was not affected
(P > 0.05 ) by
the solvent type but affected (P < 0.05 ) by the
extraction process. The intensity of the peak at around
1600 cm-1 was
lower with ball milling as compared to traditional extraction. This was
probably related to the amount of phospholipid compounds extracted and
potentially improve the stability of oil bodies. The intensity of the
peak around 2800-2900
cm-1 decreased with ball mill treatment. This can be
explained by protein-polysaccharide interaction. Ball mill treatment
increased the amount of released compounds into the solvent matrix.
Among these, soluble polysaccharides became more available for
intermolecular interactions with proteins. These interactions were seen
as reduced intensity of spectra around 2800-2900 cm-1representing glycosidic bond between amino group of protein and
carboxylic group of polysaccharides. This may prove the formation of
protein-polysaccharide complex (Guerrero et al., 2014).
Particle size and zeta potential are important factors that relate to
their dispersibility and stability. When oleoresins dispersed in an
aqueous environment without additional surfactants, they showed
remarkable homogeneity and stability. The particle size of oleoresins
ranged between 323-1760 nm (Figure 4A).
Both solvent type and ball mill
treatment significantly (P < 0.05 ) affected the
particle size. With traditional extraction, the particle size decreased
from a max of 1760 nm to 575 nm when dichloromethane and
IL2 used, respectively (Fig. 4). This showed that
charged groups in ionic liquids were anchored on the interface of oil
bodies that resulted in increased electrostatic repulsion and reduced
the average particle size (Liu et al., 2020). The ball mill process
reduced the average particle size of oleoresins. For instance, the
average particle size was reduced by nearly 50% when ball mill
treatment was applied using ethanol compared to conventional method. It
was observed that the friction and shear forces during ball milling
treatment reduced the particle size. This can be explained in
consideration of the FTIR analysis. For oleoresin dispersions of lower
particle size, the intensity of the FTIR band at 1100
cm-1, 1600 cm-1 and 2800-2900
cm-1 increased. In addition, the zeta potential was
measured as an indicator of the stability and extent of charged
polymers, mainly protein fraction of oleosome structure. The results
ranged from -8 mV to -35 mV (Fig 4B). In parallel to the particle size
data, small particles from ball mill extracts showed higher zeta
potential values, which demonstrate greater physical stability. The low
molecular weight oleosins as major protein fraction plays critical role
in the stability of the oleoresin dispersions. due to their hydrophobic
nature, oleosins can penetrate deep into the triacylglycerol core of
their emulsions with large detachment energies that reinforce their
integrity and stability (Wijesundera & Shen, 2014). Another protein
fraction, caleosins have shorter hydrophobic sequence and longer
hydrophilic unit as compared to oleosin with special N-terminal segments
bearing single Ca2+ ions responsible for the change in
the polarity (Hanano et al., 2023). The difference in the smaller
caleosin content of the oleoresins were probably responsible for the
formation of smaller particle size as expected with higher diffusion
rate of smaller polymers, and the higher zeta potential observed for
them.