**3. Results and Discussion**

#### *3.1. Effect of Polysaccharides on Ternary Nanoparticles Fabrication*

The effects of varying concentrations and types of polysaccharide on particle size of S/Z were illustrated in Figure 1. S/Z was approximate 82.81 nm particle size. The particle size of the ternary nanoparticles was differentially altered from that of S/Z after introduction with CMC and ALG into the binary system. After supplementation with CMC, the particle size of CMC/S/Z stepwise grew as the concentration of CMC increased (Figure 1A). Moreover, the incorporation of ALG resulted in a significant increase in particle size (*p* < 0.05). And in comparison to CMC/S/Z, ALG/S/Z showed a greater particle size at the identical polysaccharide level (Figure 1B). The above phenomenon might be attributed to the distinctive molecular structure of two studied polysaccharides and interaction force among molecules. The changes in particle size of zein/sophorolipid/polysaccharide ternary nanoparticles are possibly a result of sufficient anionic CMC and ALG being absorbed on the surface of S/Z through the electrostatic attractions. The changes in PDI value of CMC/S/Z and ALG/S/Z was analogous to that of particle size. As the concentration of CMC and ALG increased, the PDI value of CMC/S/Z and ALG/S/Z gradually increased as well. However, ALG/S/Z showed a higher PDI value at the same polysaccharide level.

**Figure 1.** Particle size, PDI, zeta potential and turbidity of CMC/S/Z and ALG/S/Z with different zein to polysaccharide mass ratios. Particle size and PDI (**A**,**B**). Zeta-potential and turbidity (**C**,**D**). The photograph of each group appearance (**E**,**F**).

The zeta-potential of S/Z was −40.63 mV. When CMC and S/Z were combined, their charge values became more negative as the fraction of present CMC increased (Figure 1C). On the other hand, the negative charges of ALG/S/Z initially declined and grew whereafter when the level of ALG was increased (Figure 1D). When the mass ratio of polysaccharide to zein was above 1:10, the negative charges of ALG/S/Z were higher than that of CMC/S/Z. This difference is potentially caused by the different molecular structures of the two polysaccharides. The zeta-potential of zein/sophorolipid/polysaccharide ternary nanoparticles had been dominated by negatively charged CMC and ALG. It is reported that electrostatic attraction played a critical role in the formation of protein-surfactantpolysaccharide complexes, for instance, zein-rhamnolipid-propylene glycol alginate [29], and zein-lecithin-propylene glycol alginate [30]. Meanwhile, the uniform trend of turbidity in ternary nanoparticles was consistent with that of PDI value and particle size, as the increasing trend of turbidity was primarily caused by micro-aggregation of complexes [31].

#### *3.2. CMC and ALG Prevents S/Z Ternary Nanoparticles Precipitation at pH = 4*

Our previous work confirms that S/Z exhibits weak stability in low pH conditions. To overcome this deficiency, we explore the influence of polysaccharide type (CMC and ALG) and concentration on S/Z stability at pH = 4. As shown in (Figure 2A), S/Z ternary nanoparticles were extremely unstable and aggregated together as expected. This phenomenon could be elucidated by the lack of appropriate electrostatic repulsion, which is required to conquer attractive interactions (e.g., van der Waals) among nanoparticles. Furthermore, at low pH values, the pKa of hydrophilic sugar residues present on sophorolipid molecules were reported to promote this instability [32]. After CMC was added, the PDI value, particle size, and turbidity of CMC/S/Z initially declined and subsequently jumped until a mass of zein to CMC was 5:1, the CMC/S/Z were possessed the lowest values for particle size, PDF, and turbidity (particle size ≈ 341.50 nm, PDI ≈ 0.335 and turbidity ≈ 2.257) (Figure 2A,C). Moreover, the incorporation of ALG similarly decreased and then increased PDI value, particle size and turbidity of ALG/S/Z. The particle size for ALG/S/Z was smallest at the 10:1 (353.62 nm), while the PDI value (0.335) and turbidity (2.509) were lowest at 5:1 mass ratio of zein to ALG (Figure 2B,D). As seen from photograph (Figure 2E,F), no floccules were observed at the bottom of the container at varying zein to polysaccharide mass ratios (10:1, 5:1, 3:1, and 2:1). A reason for lacking floccules at these mass ratios might be a result of appropriate CMC and ALG participation, which potentially increased the steric or electrostatic repulsions for S/Z against aggregation. Nevertheless, at a zein to polysaccharide mass of 1:1, there were sediments in both CMC/S/Z and ALG/S/Z. The underlying mechanism for these observations is bridging flocculation, i.e., the ability of a single CMC or ALG molecule to adsorb to the surfaces of two or more S/Z. Therefore, CMC or ALG adsorption results in the formation of clusters [33]. Furthermore, with the CMC and ALG concentrations raised, the charge values of CMC/S/Z and ALG/S/Z became increasingly negative. The intensification of these charges revalidates the idea that CMC/S/Z and ALG/S/Z experience sufficient electrostatic repulsion to avoid precipitation at varying mass ratios of 10:1~2:1. Based on these results, we subsequently selected CMC/S/Z and ALG/S/Z at a mass ratio of zein to polysaccharide at 5:1 as the optimum ratio to study in the following research.

**Figure 2.** *Cont.*

**Figure 2.** Particle size, PDI, zeta potential and turbidity of CMC/S/Z and ALG/S/Z with different zein to polysaccharide mass ratios at pH = 4. Particle size and PDI (**A**,**B**). Zeta-potential and turbidity (**C**,**D**). The photograph of each group appearance (**E**,**F**).

#### *3.3. Physical-Chemical Stability Study on Ternary Nanoparticles*

The optimum delivery system for food nutraceuticals must accommodate flexible pH and ionic environments during beverages processing, storage, and gu<sup>t</sup> passage. Therefore, pH and salt stability testing of delivery systems is necessary to evaluate colloidal particles' functionality.
