Changes in Particle Size and Zeta-Potential during In Vitro Digestion
Changes in particle size and zeta-potential throughout the various stages of the gastrointestinal (GIT) model are illustrated in Figure 4. Initially, the MF-prepared emulsion exhibited a significantly smaller mean particle size, primarily attributable to the different homogenization method employed. However, as the emulsions progressed through the oral phase, the particle size of the HPH-prepared emulsion experienced a notable increase, whereas the MF emulsion exhibited minimal changes. Subsequent to the gastric phase, the MF emulsion demonstrated a substantial increase in mean particle diameter, suggesting a propensity for significant droplet aggregation. This observation aligns with prior studies (Li et al., 2020), which have reported that protein-stabilized emulsions tend to aggregate under gastric conditions. This aggregation can be attributed to factors such as low pH, hydrolysis of adsorbed proteins, weakening of electrostatic repulsion, and the occurrence of depletion or bridging flocculation induced by mucin. The results obtained from confocal microscopy supported and reinforced these findings. Zeta-potential, a critical indicator of colloidal suspension stability, was continuously monitored as the emulsions progressed through the various stages of the GIT model to assess alterations in interfacial properties. Initially, the MF-prepared emulsion exhibited a higher absolute value of zeta-potential, signifying better stability, which aligns with the previously obtained results. However, following the oral phase, there was a noticeable decrease in the magnitude of the negative charge on the MF emulsion. This reduction could potentially be attributed to electrostatic screening caused by the presence of mineral ions in simulated saliva or interactions between mucin molecules and the surfaces of oil droplets.
Further reductions in the absolute value of zeta-potential were observed as the emulsions encountered simulated stomach conditions. The low pH and high ionic strength of simulated gastric fluids could have led to alterations in the electrical properties of the droplets. Subsequently, after incubation in the small intestine phase, all samples displayed negative charges. This phenomenon may be attributed to the presence of anionic species from various types of particles, including undigested lipids, undigested proteins, micelles, vesicles, and calcium salts. Notably, the negative charges of the MF-prepared emulsion remained relatively higher throughout the gastrointestinal model, indicating its superior stability in the small intestine phase. Therefore, the emulsion produced by MF demonstrated enhanced stability across the different stages of the GIT model, highlighting its potential as a robust delivery system throughout the gastrointestinal tract.