*3.6. Micromorphology*

The microstructural features of loaded composite nanoparticles were analyzed via TEM. As observed in (Figure 5A), the diameter of DHF-S/Z was roughly 100 nm, this finding was in agreemen<sup>t</sup> with the results of dynamic light scattering. After adding CMC and ALG, DHF-CMC/S/Z and DHF-ALG/S/Z showed a similar spherical structure that was comparable to the DHF-S/Z. However, the diameter of DHF-CMC/S/Z and DHF-ALG/S/Z was larger than that of DHF-S/Z, exhibiting a particle size of 100~200 nm (Figure 5B,C). These findings demonstrate that CMC and ALG were absorbed on the surface of DHF-S/Z. Additionally, the diameter of DHF-CMC/S/Z was larger than that of DHF-ALG/S/Z, which was consistent with the results of DLS.

**Figure 5.** TEM images of DHF-S/Z ( **A**), DHF-CMC/S/Z (**B**) and DHF-ALG/S/Z ( **C**), 30,000× magnification times.

Furthermore, FE-SEM was applied to further observe the differences in surface microscopic morphology among individual composite nanoparticles (Figure 6). As seen from (Figure 6A), the surface morphology of DHF-S/Z expressed an irregular and rough shape consisting of many interlinked independent complexes. This morphological change was possibly attributed to sophorolipid adsorption on the surface of zein particles. The microphotograph of CMC and ALG depicted a shape similar to that of a silk ribbon (Figure 6D,E). When CMC and ALG were incorporated into DHF-S/Z, the micromorphology of DHF-CMC/S/Z and DHF-ALG/S/Z were spherical with uniform size and smooth surface (Figure 6B,C). The particle sizes of DHF-CMC/S/Z and DHF-ALG/S/Z were larger than that of DHF-S/Z. The polysaccharide coating on the outer layer of DHF-S/Z nanoparticles via electrostatic attraction possibly caused this size difference [49]. The above results were consistent with DLS measurements.

**Figure 6.** FE-SEM images of DHF-S/Z (**A**), DHF-CMC/S/Z (**B**), DHF-ALG/S/Z (**C**) (50,000× magnification times), individual CMC (**D**) and ALG (**E**) (2000× magnification times).

#### *3.7. A Graphic Illustration for the Formation and Stability Mechanism of Nanoparticles*

Diverse technologies and measurements including EE, PDI, particle size, zeta potential, turbidity, CD, TEM, FE-SEM, DSC, XRD, and FTIR were used to make clear the formation and stability mechanism of zein/sophorolipid/polysaccharide ternary delivery system (Figure 7). After zein and sophorolipid were rapidly added into polysaccharide (polysaccharide: zein mass ratio ≤ 1:2), sufficient CMC or ALG acted as a shielding effect (electrostatic repulsion) and steric hindrance stabilizer was coated onto the surface of S/Z particles to prevent their sedimentation at low pH 3~4 range condition, showing a homogeneous PDI, turbidity and size based on DLS and UV. Certain internal drives (electrostatic interaction, hydrogen bonding and hydrophobic effect) participated in the formation of DHF-CMC/S/Z and DHF-ALG/S/Z, and CMC and ALG had no significant impact on secondary structure of zein in S/Z according to CD, FTIR, and FE-SEM. Upon employing EE, XRD, DSC, TEM analysis, 7,8-DHF was shown to be successfully encapsulated in CMC/S/Z and ALG/S/Z with relatively uniform sphericity, displaying a good entrapment efficiency.

**Figure 7.** An illustration of the formation and stability mechanism of DHF-CMC/S/Z and DHF-ALG/S/Z.

#### *3.8. Storage Stability of 7,8-DHF*

During storage, preventing food neutraceuticals from heat or light exposure is challenging, but critically necessary for mitigating degradation. To meet this application end, short and long term storage were investigated under varying environments for 7,8-DHF, loaded binary and ternary nanoparticles. As shown in (Figure 8A), free 7,8-DHF was mostly degraded at 25 ◦C with light exposure for 15 days post-storage. Encapsulation of the 7,8-DHF in S/Z nanoparticles strengthened storage stability of 7,8-DHF (26.26 %). The addition of CMC and ALG further enhanced the stability of encapsulated 7,8-DHF, in particular DHF-CMC/S/Z (58.75%). At 50 ◦C under dark conditions (Figure 8B), a similar effect was observed. The active groups of 7,8-DHF were possibly protected within the hydrophobic lumen of DHF-S/Z, DHF-CMC/S/Z, and DHF-ALG/S/Z nanoparticles as a mechanism [50], besides, due to the different chemical structures of CMC and ALG, DHF-CMC/S/Z have higher EE, which caused more 7,8-DHF to be protected in the hydrophobic part. These results are in agreemen<sup>t</sup> with previous studies that introduced curcumin being embedded in zein and quaternized chitosan complexes [51], along with work showing that quercetagetin was loaded using the zein-hyaluronic acid binary complexes [52].

**Figure 8.** Storage stability of free 7,8-DHF, DHF-S/Z, DHF-CMC/S/Z and DHF-ALG/S/Z at 25 ◦C under light (**A**), at 50 ◦C under dark (**B**).

#### *3.9. In Vitro Simulated Gastrointestinal Digestion*

A gastrointestinal tract (GIT) model was applied to study the digestive fate and bioaccessibility of 7,8-DHF in different formulations. Particle size changes were monitored at a specific digestion time (30, 60, 120, and 180 min), and the results were presented in (Figure 9A). The mean particle size of DHF-S/Z was significantly increased after 60 min SGF digestion (*p* < 0.05). This finding was possibly due to the fact that the S/Z were exposed to ionic strength along with low pH and partially digestion via pepsase. The low pH and ionic strength exposure likely weakened electrostatic repulsion forces among the nanoparticles [52]. Particle size reduction in DHF-S/Z post-SIF-exposure was attributed to the fact that SIF contains bile salt with strong emulsifying ability. Bile salt can bind many biopolymer molecules and induce bridging flocculation [21]. The particle size of DHF-CMC/S/Z was increased after exposure to the stomach phase but remained relatively constant during incubation in SIF. This behavior suggested that the existence of CMC strengthened intestinal stability of DHF-CMC/S/Z. However, the particle size of DHF-ALG/S/Z remained fairly steady throughout simulated GIT, only showing a large increase at 30 min during the SGF incubation. The different influence of polysaccharide type (CMC or ALG) on gastrointestinal fate of colloidal carriers can be attributed to different molecular characteristics. FE-SEM microscopic observation further confirmed that exposure to simulated gastrointestinal conditions had a significant effect on the morphology of the 7,8- DHF-loaded nano-complexes (Figure 9C). For DHF-S/Z, irregular shapes have developed after they were added into the SGF and SIF, similar to an anomalous sheet structure. This shape acquisition was due to irregular aggregates appearing after SGF digestion. Furthermore, DHF-CMC/S/Z and DHF-ALG/S/Z exhibited a spherical shape. After gastrointestinal digestion, the nanoparticles possessed a relatively spherical morphology, resembling the cross-linked structure of large nanoparticles. Overall, CMC and ALG effectively protected the stability of DHF-S/Z nanoparticles through the GIT. Particularly, ALG performed extremely well.

**Figure 9.** Influence of in vitro digestion time on the particle size (**A**) and bio-accessibility of DHF-S/Z, DHF-CMC/S/Z, and DHF-ALG/S/Z (**B**), FE-SEM images of DHF-S/Z, DHF-CMC/S/Z, and DHF-ALG/S/Z, 15,000× magnification times (**C**).

After being exposed to simulated gastrointestinal conditions, 7,8-DHF bioaccessibility was measured after centrifugation and collection of micelle phases. As shown in Figure 9C, the S/Z was broken down when exposed to SGF digestion, and the core of 7,8-DHF was released, resulting in a low bioaccessibility. The bioaccessibility of DHF-S/Z gradually increased in SIF digestion, primarily due to the emulsibility of bile salts and sophorolipids. Another reason for this increase was the binding of hydrolyzed peptides of zein protein [33]. In the presence of CMC, low levels of bioaccessibility were exhibited in SGF digestion. This behavior was introduced by the vulnerability of CMC to acid,

pepsin enzymes, and the alkali ions of SGF juices [53]. However, the bioaccessibility of DHF-CMC/S/Z was higher than that of DHF-S/Z, indicating a synergistic effect existed among CMC, sophorolipid and zein on controlling the release of 7,8-DHF. Furthermore, the presence of ALG further increased bioaccessibility to 75.46% (Figure 9B), showing the highest bioaccessibility of 7,8-DHF after GIT digestion compared to DHF-CMC/S/Z and DHF-S/Z. These results indicated that ALG increased the solubilization capacity of mixed micelles in small intestine fluids. Collectively, our results demonstrated that encapsulating 7,8-DHF in polysaccharide coated S/Z nanoparticles can promote an appreciable increase in its in vitro bioaccessibility.
