Confocal scanning laser microscopy (CLSM) (FV3000, Olympus, Japan) was employed on the microstructure of CaCO₃ S/O/W emulsions. Soybean oil (O phase) was pre-stained with Nile red (488 nm laser excitation source), NaCas and GEL were stained with Nile blue A (635 nm laser excitation source) before emulsification. The samples were magnified using a 10× eyepiece and a 60× objective lens (oil immersion) (23).

Cryo-scanning electron microscopy (Helios NanoLab G3 UC, FEI, USA) was employed on the cross-sectional and interfacial structure of CaCO₃ S/O/W emulsions. Samples were placed in liquid nitrogen and then transferred to a cryo-preparation chamber (PP3010T, Quorum Technologies, UK) under vacuum. After freeze-fracturing and high vacuum sublimation to sublimation at −95°C for 10 min to unbound water, and then the samples were sputter-coated with platinum and imaged using SEM. The observation was carried out at a distance between 3 and 5 mm with TLD detection at 2 kV (22).

The particle size distribution has a great influence on the stability of the emulsions system (26). The emulsion is a thermodynamically unstable system with two kinds of macroscopic instability, one is the migration of dispersed phase particles, and the other is the change of particle size of dispersed phase particles. Droplet size distribution is one of the most important characteristics of emulsions and is essential for system stability. In general, the emulsions are more stable with a narrower particle size distribution and smaller average particle size. The effects of different NaCas concentrations and S/O phase additions on the particle size of S/O/W emulsions were shown in Figure 1 and Table 1. The results show that when the S/O phase addition amount was constant, the NaCas-GEL-XG ternary composite stabilized S/O/W emulsion has a large distribution range and a relatively large average particle size with low NaCas concentration (2 wt%). In contrast, the S/O/W emulsions prepared by NaCas-GEL-XG as the W phase had a narrower particle size distribution range than prepared by NaCas, NaCas-GEL, and NaCas-XG, which was enhanced overall stability (22, 27). This was because the addition of GEL enhanced the emulsion stability performance of the system, and the addition of XG improved the viscosity and suspension stability of the system. With the NaCas concentration increased, the change of the particle size distribution was not obvious. When NaCas concentration was constant, the size distribution range became larger with the S/O phase addition increased, and the S/O/W emulsion system tended to be unstable.

The electrical properties of S/O/W emulsions are often characterized by Zeta-potential. The effects of different concentrations of NaCas and S/O phase addition amount on the Zeta-potential of S/O/W emulsions were shown in Figure 2 and Table 2. When the S/O phase added amount was constant, the absolute value of the Zeta-potential of microspheres was relatively small at low NaCas concentration, and slightly increases at high NaCas concentration. With the S/O phase addition increased, the absolute value of the microsphere potential decreased slightly and then increased. Compared with NaCas, NaCas-GEL, and NaCas-XG in phase W, the potential value of the microspheres was lower, which was due to electrostatic interaction in the mixed system. S/O/W emulsions (pH7.0) were negatively charged due to pH higher than the pI of NaCas, while GEL was positively charged, and electrostatic interaction between NaCas and GEL occurs, which was the result of interaction between GEL side groups (−NH³⁺) and negative ions dissociated from NaCas (28). The addition of XG increased the absolute value of Zeta-potential slightly due to the negative charge caused by the presence of anionic polysaccharide.

The stability analyzer can be used to directly measure the physical stability of the S/O/W emulsions. When the instability index is lower, the emulsions maintain good physical stability.

The instability index of S/O/W emulsions prepared with different NaCas concentrations and S/O phase addition amounts was shown in Figure 3. The results showed that when the NaCas concentration was low (2∼4 wt%), the transmittance of the microspheres increased as the S/O phase content increased, and the system became more and more unstable. The better stability of the microspheres with low S/O content is due to the special structure of the free phase combined with the droplet surface. On the other hand, it was also because of the strong steric effect between the droplets and the free molecules. When the S/O phase added amount increased, the free W phase in the system gradually decreased, so the structure became unstable. With the NaCas concentration increased (6∼10 wt%), the transmittance of microspheres during centrifugation decreased significantly with the increase of the S/O phase, because there were no free W phase molecules in the aqueous phase, which reduced the occurrence of lossy flocculation.

Linear regression analysis were used to fit the particle size, potential value and instability index of S/O/W emulsions stabilized by different concentrations of NaCas and different S/O phase additions, and the results were shown in Figure 4.

Three models were constructed to analyze the relationship between instability index and particle size and Zeta-potential value (Model I), the relationship between instability index and particle size (Model II), and the relationship between instability index and Zeta-potential value (Model III). Model I was modeled using two variables: instability coefficient = −0.4612 × particle size−0.4591 × potential value. In terms of the absolute value of the coefficients of the regression equation, the coefficient of 0.4612 for particle size was greater, indicating a greater correlation between particle size and instability index, but the difference between the two was smaller. The linear regression model was established with a single feature in Models II and Model III, which further showed that grain size distribution was more correlated with stability, but the difference was small. The R² of model one (0.7262) was greater than the R² of Models II and III (0.6481, 0.6473), making it necessary to model using both variables, and considering both features simultaneously provides greater accuracy in determining the S/O/W emulsions instability coefficient.

In summary, although particle size has a greater degree of correlation with the instability coefficient, the difference in correlation degree between particle size and the instability coefficient and potential value and the instability coefficient was relatively small.

Confocal scanning laser microscopy can realize imaging from macro to micro and super-resolution microscopic observation. The effect of different NaCas concentrations on the microstructure of NaCas-GEL-XG ternary composite stabilized S/O/W emulsion was observed from a relatively large field of view by CLSM, and the results were shown in Figure 7A. With the NaCas concentration increased, S/O/W emulsions have a smaller particle size and a more uniform overall distribution, which was consistent with the results in Figure 1. At low NaCas concentrations (2∼4 wt%), the distribution of S/O/W emulsions was relatively uniform and the dispersion stability was good. With the NaCas concentration increased (6∼10 wt%), the viscosity of the system increased and even agglomeration occurred, but the stability was not good. Therefore, when the concentration of NaCas was too high and the ternary combination of GEL and XG, the stability of S/O/W emulsions became worse, which was consistent with the results in Figure 3.

Cryo-scanning electron microscopy showed the cross-sectional structure and attachment of S/O/W emulsions and observed the microstructure of S/O/W emulsions from a more microscopic perspective. The effects of different NaCas concentrations on the Cryo-SEM microstructure were shown in Figure 7B. When the NaCas concentration was low, S/O/W emulsions can form a 3D network structure. With the NaCas concentration increased, the microsphere honeycomb three-dimensional spatial network structure became more and more compact. With low NaCas concentration, the S/O/W emulsions could form a dense three-dimensional network space structure, indicating that the emulsion constructed with low NaCas concentration had good stability. In addition, low NaCas concentration could not form a three-dimensional network structure (22), while the binary compound of NaCas-XG formed a gelatinous network structure (28). The addition of GEL could effectively improve the gel-like properties of NaCas and XG, and promoted the formation of the honeycomb three-dimensional network structure of the S/O/W emulsion, and it is a benefit to improve the stability of the system.

X-ray diffraction analysis was used to characterize that the CaCO₃ was embedded in the S/O/W emulsions, and the results were shown in Figure 8A. XRD analysis showed that the CaCO₃ was calcite structure, and the diffraction peaks were 23, 29, 36, 39, 43, 47.5, and 48.5, respectively (31). The NaCas (W₁ phase) diffraction peaks were 10 and 20, respectively. The GEL (W₂ phase) crystal characteristic peaks were 7.2 and 20.2. The XG (W₃ phase) crystal characteristic peak was 20. The diffraction peaks of NaCas-GEL-XG (W phase) were 7.2 and 20.2, which was consistent with those of GEL. The XRD results of O/W emulsions were consistent with the W phase, indicating that a better oil-in-water structure can be formed without adding calcium carbonate. The diffraction peaks of S/O/W emulsion were 7.2, 20, 23, 29, 36, 39, 43, 47.5, and 48.5, respectively, indicating that the characteristic peak of CaCO₃ was partially covered and the intensity of the characteristic peak was weakened due to physical embedding. Compared with the S/O/W emulsions with stable W phases of NaCas, NaCas-GEL, and NaCas-XG, the S/O/W emulsions with a stable ternary mixture of NaCas-GEL-XG have enhanced the embedding effect of CaCO₃ (28, 32).

The position and intensity of absorption peak in the infrared spectrum reflect the characteristics of molecular structure and can be used to identify the structural composition of substances or determine their chemical groups. Figure 8B showed that the C-O asymmetric stretching and symmetric vibration peak were at 1796.99 and 1456.26 cm^–1, the out-of-plane and in-plane bending vibration peak were at 872.54 and 712.67 cm^–1.

The O-H and C-H bond stretching vibration of NaCas (W₁ phase) were at 3275.78 and 2930.05 cm^–1, the amide I band and amide II stretching vibration were at 1600–1700 and 1400–1550 cm^–1 (27). GEL (W₂ phase) has three distinctive absorption peaks of the amide band, which were the stretching vibration of −C = O at 1629.01 cm^–1 (amide I band), the -N-H bending vibration at 1521.34 cm^–1 (amide II bands), and the stretching vibration of C-H at 1235.44 cm^–1 (amide III bands) (33). The –COO– asymmetric and symmetric stretching vibrations of XG (W₃ phase) were at 1599.68 and 1405.46 cm^–1, and the C-O stretching vibration was at 1019.76 cm^–1. The -OH stretching vibration absorption peak shifted from 3275.78 to 3278.73 cm^–1, which was indicated that there was hydrogen bond interaction between NaCas, GEL, and XG molecules (34). The N-H bending vibration of NaCas disappears at 1513.56 cm^–1. The absorption peak of -N-H bending vibration at 1521.34 cm^–1 disappears, and the absorption peak at 1531.54 cm^–1 appears, which indicates that the –NH³⁺ vibration peak in the original absorption peak disappears, and the wave number is blue-shifted. This indicates the disappearance of the –NH³⁺ vibrational peak in the original absorption peak and the blue shift of the wave number; the absorption peaks of the –COO– stretching vibration of XG at 1599.68 and 1405.46 cm^–1 also disappeared; these results confirmed the electrostatic interaction between GEL and –NH³⁺, XG, and –COO–.

Molecular docking technology can effectively predict the binding mode between proteins and ligands, which can be compared with infrared spectroscopy and Raman spectroscopy analysis for verification (35). Computer simulation technology was first applied in the direction of rapid discovery of chemical entities with potential interaction between drugs and target proteins, and has been applied in the field of food in recent years (35, 36). Through the principles of energy matching and spatial matching, molecular docking technology screens the conformation with the lowest energy and the most reasonable spatial matching among all docking conformations according to the inherent structural information of proteins and small molecules (37). The affinity information and docking site information of the interaction between any two compounds can be obtained by molecular docking analysis. The interaction of NaCas, GEL, and XG was visualized by molecular docking technology, and the results were shown in Figure 9.

Table 3 showed the summary of molecular interactions among NaCas, GEL and XG. When NaCas was the receptor and XG + GEL was the ligand, the binding energy was −0.2 kJ/mol, and there was an electrostatic interaction. When GEL + XG was the receptor and NaCas was the ligand, the binding energy was −2 kJ/mol, and the hydrogen bond length of O5 on GEL + XG and O3 on NaCas was 2.93 Å, indicating hydrogen bond interaction between them. When XG + GEL was the receptor and NaCas was the ligand, the binding energy was −1.4 kJ/mol, and there was a Hydrophobic interaction. The molecular docking prediction model was consistent with the results of infrared spectroscopy analysis. Hydrogen bond interaction, electrostatic interaction and hydrophobic interaction exist among NaCas, GEL and XG ternary in phase W, which may help to further improve the stability of S/O/W emulsion system.