*3.1. Effects of Ultrasound Process on the Preparation of ZNP-FSG Complexes* 3.1.1. Emulsifying Properties

The emulsifying activity index (EAI) refers to the ability of proteins absorbed into the oil/water interface. The emulsifying stability index (ESI) is an indicator of the ability of a protein to maintain emulsion stability [35]. The EAI and ESI of the complexes subjected to various ultrasound and homogenization treatments are presented in Figure 2. The results showed that the EAI and ESI of untreated control sample ZNP were 6.09 ± 0.62 m2/g and 12.64 ± 0.43 min, respectively. In comparison with the ZNP, the EAI and ESI of homogenization treated complex (ZNP-FSG)H were significantly increased, up to 10.47 ± 1.20 m2/g and 42.47 ± 4.10 min, respectively. Interestingly, no significant difference was observed between the EAI and ESI of (ZNP-FSG)H and (ZNPU-FSG)H, indicating the emulsification property of the homogenization-treated complex was independent from the ultrasonic treatment of zein. Moreover, (ZNP-FSG)H and (ZNPU-FSG)H exhibited higher EAI and

ESI than that of the (ZNP-FSGU)H, implying that pretreatment of FSG with ultrasonication was not favorable to improve the emulsifying properties of the complex. More importantly, we found that the (ZNP-FSG)HU and (ZNP-FSG)U complex showed significantly lower EAI and ESI than that of (ZNP-FSG)H. This result indicates that further treatment with ultrasonication could dramatically destroy the emulsifying capability of the complex. In fact, the (ZNP-FSG)H and (ZNPU-FSG)H complex had the highest EAI and ESI among all the samples in the current study. Finally, there was no obvious difference among the EAI and ESI between ZNP, (ZNP-FSG)HU, and (ZNP-FSG)U. That is to say, adding FSG did not necessarily improve the emulsification property of ZNP, which also depends on the pretreatment and emulsifying process. The improved emulsifying properties of ZNP-FSG complexes may be correlated with the solubility, structural changes of zein, electrostatic interaction, and hydrophobic interaction between ZNP and FSG [32,36,37].

**Figure 2.** Emulsifying activity index (EAI) and emulsifying stability index (ESI) of emulsions stabilized by ZNP-FSG complexes subjected to various ultrasound and homogenization treatments.

#### 3.1.2. Turbidity Changes

Dispersion turbidity indicates the number and size of dispersed particles to some content [38], which reflects the dispersion state and aggregation degree. Figure 3 illustrates the turbidity of ZNP and ZNP-FSG complexes. Turbidity of most complexes decreased after ultrasound pretreatments, except (ZNP-FSG)HU and (ZNP-FSG)U, which increased. It may be because the stable balances of original complexes were destroyed by extra ultrasound. Contrary to our results, Ma et al. [37] found the ultrasound treatment significantly decreased the turbidity of the soy protein isolate (SPI)—citrus pectin (CP) complex. The type of polysaccharide is probably one of the reasons for the different phenomenon. The turbidity of (ZNPU-FSG) is lowest, and the turbidity of (ZNP-FSG)H is lower than that of (ZNP-FSGU)H. From this, we hypothesized that the particle size changes of proteins induced by ultrasonic treatment decreased the turbidity of the solution most [39], which was related to the smaller protein particle size obtained by ultrasonic treatment. (ZNP-FSGU)H might have had higher turbidity because of the degradation of FSG [36].

**Figure 3.** Turbidity of ZNP-FSG complexes subjected to various ultrasound and homogenization treatments.

#### 3.1.3. Salt Ionic Stability

The stability of the complexes at different ionic strengths (50 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, and 1000 mM) was evaluated by visual observation and turbidity in Figure 4. In Figure 4a, ZNP started to form flocculation and precipitation at a 50 mM NaCl due to electrostatic screening effects [40]. The flocculation extent kept increasing with increased salt concentration. Different from ZNP, it can be seen from the picture that the stability of the ZNP-FSG complex at different salt ion concentrations improvement was attributed to the addition of FSG, even though differences were noticed among complexes with different treatments. The result indicates that FSG is effective in protecting ZNP from salt-induced aggregation, which is probably due to the weakened electrostatic screening effects of ZNP brought by the electrostatic interaction between ZNP and FSG [40]. However, the tolerance of (ZNP-FSG)HU to salt was relatively poor, and flocculation can be clearly observed, as shown in Figure 4a.

The turbidity results, as shown from Figure 4b, is in agreement with the visual observation. The turbidity of (ZNPU-FSG)H and (ZNP-FSG)H decreased as the NaCl concentration increased. The turbidity of (ZNP-FSGU)H, (ZNP-FSG)U, and (ZNP-FSG)HU did not show a great relationship with the increasing salt ion concentration but fluctuated to different degrees. As we can see, the turbidity of (ZNP-FSG)HU and (ZNP-FSG)U showed an obvious upward trend at the salt ion concentration of 1000 mM. This is probably caused by the decreased interaction between ZNP and FSG, which resulted from the electrostatic shielding effect at high ion concentrations. At all the tested ion concentrations, (ZNPU-FSG)H complex showed lower turbidity than the others, which indicated that (ZNPU-FSG)H had the best tolerance of salt. Overall, the complexes with different treatments had a good stability at the salt ion concentration of 1000 mM except for (ZNP-FSG)HU. Increases of (ZNP-FSG)U and (ZNP-FSG)HU in turbidity illustrate the continued aggregation of soluble complexes into insoluble protein/polysaccharide complexes due to charge neutralization.

**Figure 4.** Visual appearance (**a**) and turbidity (**b**) of various ZNP-FSG complexes prepared in different NaCl concentration, i.e., black for (ZNP-FSG)H, red for (ZNPU-FSG)H, blue for (ZNP-FSGU)H, green for (ZNP-FSG)HU, and purple for (ZNP-FSG)U.

3.1.4. Surface Hydrophobicity

Surface hydrophobicity (H0) is closely related to the emulsifying properties, stability, and function of the complex/particles [41], and a balanced surface hydrophobicity has a beneficial effect on surface activity and interfacial performance in emulsions. The surface hydrophobicity of the complexes is shown in Figure 5. We concluded that the H0 of ZNP could be increased by ultrasonic pretreatment on zein. Ultrasonic cavitation can effectively expose the buried hydrophobic regions of zein from the interior of molecules to the surface [42]. Similar results were obtained by Gao et al. [14], investigating the effect of ultrasound on whey isolated protein. The H0 of (ZNP-FSG)H complexes significantly decreased after adding FSG, which was ascribed to FSG blocking the binding sites between the fluorescent probe and hydrophobic groups of protein. Wang et.al [43] also reported that the hydrophobicity of chitosan-zein complexes decreased due to the addition of chitosan. The surface hydrophobicity of (ZNPU-FSG)H was higher than that of (ZNP-FSG)H, which indicated that the increase in H0 of ZNP after ultrasound improved the H0 of (ZNPU-FSG)H. The H0 value of (ZNP-FSG)HU and (ZNP-FSG)U showed an extreme decrease after sonication, which was independent of whether it had been homogenized or not. This was probably due to the contact between the ultrasound and protein increasing when FSG separates from ZNP after sonication, and hydrophobic groups were reburied. A similar result was reported in the effects of dynamic high-pressure microfluidization treatment on the functional and structural properties of potato protein isolate and its complex with chitosan by Hu et al. [44]. The changing trend of surface hydrophobicity is consistent with that of emulsification capability. Previous research [45] has shown that the adsorption rate was shown to scale with the relative hydrophobicity, which is considered to be helpful to improve emulsifying properties.

**Figure 5.** Surface Hydrophobicity (H0) of ZNP-FSG complexes subjected to various ultrasound and homogenization treatments.

#### 3.1.5. Particle Size

In order to study the effects of the pretreatment methods and operating conditions on different samples, the particle size of complexes was characterized, as shown in Figure 6a. Ultrasound pretreatment was able to significantly decrease the particle size of ZNP from 226.1 nm to 185.4 nm, which was possibly related to the structural changes of protein upon ultrasonication [46]. The particle size of FSG decreased from 767.0 nm to 332.7 nm after ultrasound treatment, presumed to be caused by the degradation of FSG after ultrasound [47]. Similar results were reported for ultrasonication-treated Persian gum and gum tragacanth [48]. The particle size of both (ZNP-FSG)U and (ZNP-FSG)HU was the lowest among all complexes, and there was no significant difference between them. We found out that if the complex eventually requires ultrasonic treatment, there was no difference in particle whether the intermediate process was homogenized or not. There was no significant difference in particle size of (ZNP-FSGU)H and (ZNP-FSG)H, revealing the ultrasound on FSG playing no effect on the particle size of the complex. However, the pre-ultrasonication on zein significantly decreased the particle size of the complex (ZNPU-FSG)H ]. Since the particle size of (ZNP-FSG)HU was lower than that of (ZNP-FSG)H, it can be concluded that the extra ultrasonication is effective to further decrease the particle size of the homogenized complex. In conclusion, ultrasonic treatment of different ingredients has a significant effect on the particle size of the complex prepared. However, homogenization on complexes does not have significant effects on the particle size.

PDI is usually used to represent the particle size distribution of dispersion [37]. As shown in Figure 6b, the PDI values of FSG and FSGU are significantly higher than other samples, indicating a broad particle size distribution [29]. The PDI of (ZNP-FSGU)H, (ZNP-FSG)HU and (ZNP-FSG)U were higher than that of other complexes, which were all around 0.30. The PDI of (ZNPU-FSG)H was the lowest (0.21), showing the uniformity of particle size distribution was increased due to the pre-ultrasonication on zein. The particle size distribution of various ingredients or ZNP -FSG complexes subjected to various ultrasound and homogenization treatments are showing in Figure S1. As is known to all, PDI is not the only reason that describes emulsion stability. We should continue to analyze together with other properties of the complexes.

**Figure 6.** Particle size (**a**), PDI (**b**), and ζ-potential (**c**) of ZNP-FSG complexes subjected to various ultrasound and homogenization treatments. Different lowercase letters (a, b), different capital letters (A, B) and asterisk (\*, \*\*) indicated significant difference (*p* < 0.05).
