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.