3.1.6. ζ-Potential

Figure 6c also shows the ζ-potential of ZNP-FSG complexes subjected to various ultrasound and homogenization processes. The stability of complexes could be studied by measuring the electrostatic stability of the colloidal particles [49]. Particles with ζ-potentials more positive than +30 mV or more negative than −30 mV are normally considered stable [50]. In the absence of FSG, the ζ-potential of the ZNP suspension was around +35 mV, which was due to the fact that the pH was below its isoelectric point (IP) and, therefore, ZNP had a net positive charge. The ζ-potential value of ZNP was increased from +35.2 mV to +45.9 mV after ultrasound pretreatment, suggesting that pretreatment on zein could improve the stability of ZNP. This was consistent with previous results that the ZNP system was stable below pH 6.0 [16]. In fact, ZNP remained stable for several days at 4 ◦C. The ζ-potential value of FSG was increased from −49.7 mV to −47.1 mV after ultrasound treatment, indicating that ultrasonic treatment slightly lowered the stability of FSG. According to Figure 5, the ζ-potential of complexes were negative, which is because the negatively charged FSG molecules interacted with the ZNP through electrostatic force and aggregates on the surface of positively charged ZNP, resulting in charge reversal. Similar results were found in the study on complexes of WPI gel-chitosan and zein-carboxymethyl dextrin [28,51].

The ζ-potential values of ZNP-FSG complexes subjected to various ultrasound and homogenization treatments were obviously different from each other, suggesting that the pretreatment and operating process significantly affected the stability of the complexes. The absolute zeta potentials of complexes ranking by increasing order is (ZNP-FSG)U ≈ (ZNP-FSG)HU < (ZNP-FSGU)H < (ZNP-FSG)H < (ZNPU-FSG)H. First, the absolute zeta potential of (ZNP-FSG)U was equal to that of (ZNP-FSG)HU and was the smallest among all. Thus, it can be seen that homogenization of the complex prior to ultrasonic treatment had no effect on their stability. Second, the absolute ζ-potential of (ZNP-FSG)HU was lower than that of (ZNP-FSG)H, indicating the stability of the complex was significantly decreased by ultrasonic treatment after homogenization, regardless of the ingredients of ultrasound. Based on the above results, we concluded that in order to obtain ZNP-FSG complexes with better stability, ultrasound and homogenization cannot be superimposed on the complex. Maybe the mechanical force and cavitation induced by ultrasound increase the energy and entropy of ZNP-FSG system and break the balance made by homogenization, even the particle size or type of interaction force may be affected as well [14]. Ultrasonic treatment has the capability of decreasing the size of protein [52] and reduces the viscosity of polysaccharides by degrading chains of them [48]. The ζ-potential of (ZNP-FSGU)H reduced in comparison with ZNP, which was ascribed to the decrease of amounts and distributions of charges on the ZNP surface because of the depolymerizing of FSG during the ultrasonic process [30].

However, when applying ultrasonic pretreatment on a single ingredient before homogenization, the complex had better stability. Sample (ZNPU-FSG)H showed the maximum absolute ζ-potential value and the best stability, and indicated its better stability than (ZNP-FSG)H or (ZNP-FSGU)H. We found that the selection of ultrasonic ingredients significantly affected the stability of the complex. When the ultrasonic pretreatment ingredient was protein, the ultrasonic had a positive effect on the stability of the complex, whereas if the ultrasonic pretreatment ingredient was FSG, a negative effect on the stability of the complex was observed. Some possible reasons for the results were speculated from different mechanisms of ultrasonic treatment on different ingredients.

In addition, we tried to discuss the relationship between particle size and the potential of the ZNP-FSG complex. There was no direct correlation between the stability and particle size of the complex nor an obvious correlation between ζ-potential and particle size changes of the ZNP-FSG complex. Although ultrasound is widely used in the pretreatment of various samples in preparing a Pickering emulsion for emulsification and homogenization, its effect on homogeneity was revealed for the first time. Above all, the selection of ingredients for treatment and combination of process technology is very important for

obtaining a good effect on the stability of protein–polysaccharides complex during the emulsification and homogenization.

#### *3.2. Effects of Ultrasound Process on the Emulsion of ZNP-FSG Complexes*

#### 3.2.1. Storage Stability of Emulsions

Effects of ultrasonic pretreatment on the physicochemical changes of ZNP-FSG complex and the characteristics of subsequent oil-in-water emulsions were investigated. The creaming index is the ratio of the height of the serum phase to the total height of the emulsion. The creaming index of the emulsions stored at 4 ◦C for 14 days is shown in Table 2. The creaming index was used as a measure of the stability of Pickering emulsions. The occurrence of the phase separation phenomenon proves that the emulsion has poor storage stability [53]. The appearance of the emulsions prepared with ZNP showed slight creaming as early as 30 min after the emulsions were prepared. The creaming index value of ZNP-E exhibited the highest of 25% after 30 min and then increased to 30%, indicating that ZNP-E possessed the worst stability among all the emulsions. The creaming index of (ZNP-FSGU)-E, (ZNP-FSG)HU-E, and (ZNP-FSG)U-E in turn increase. In other words, their stability decreases in turn. However, (ZNP-FSG)-E and (ZNPU-FSG)-E remained physically stable for 14 days at 4 ◦C. This was consistent with the result that their corresponding complexes had the highest EAI and ESI values.

**Table 2.** The creaming index (%) of emulsions stabilized by ZNP-FSG complexes subjected to various ultrasound and homogenization treatments.


#### 3.2.2. Freeze-Thaw Stability of Emulsions

The emulsions might undergo the freeze-thaw process. Many emulsions were extremely unstable after three cycles of freeze-thaw treatment, some phenomena such as creaming, flocculation, and coalescence occurred [54]. The freeze-thaw stability of all emulsions stabilized by complexes subjected to various ultrasound and homogenization treatments were evaluated by the creaming index and oiling off and are shown in Figure 7.

It could be seen from Figure 7 that both the oiling off and creaming index of emulsions increased with the number of freeze-thaw treatment cycles. Obviously, ZNP-E had the highest oiling off and creaming index. Compared with ZNP-E, the oiling off and creaming index of ZNP-FSG complexes subjected to various ultrasound and homogenization treatments all decreased sharply, indicating that the addition of FSG improved the freeze-thaw stability of the emulsion. However, different treatments play important roles in the process. As expected, the oiling off and the creaming index of (ZNP-FSG)U-E and (ZNP-FSG)HU-E were higher than other emulsions except for ZNP-E. Surprisingly, (ZNP-FSGU)H-E showed the lowest oiling off, followed by (ZNPU-FSG)H-E, and then the (ZNP-FSG)H-E, the sequence of which was different from the order of the creaming index. The creaming index of (ZNPU-FSG)-E was the lowest after three freeze-thaw cycles, followed by (ZNP-FSGU)H-E and (ZNP-FSG)H-E.

**Figure 7.** The oiling off and creaming index of the Pickering emulsions stabilized by ZNP-FSG complexes subjected to various ultrasound and homogenization treatments. 1, 2, 3 represent freezethaw cycles, respectively.

After the emulsion is frozen, the droplets can undergo coalescence because of the oil phase and water phase crystallization-induced destruction of the emulsions interface film [55]. Then, the addition of FSG could increase the thickness of the interfacial film and provide enough repulsion to prevent droplet aggregation [56]. The improvement of the freeze-thaw stability of the emulsion is closely related to the emulsification and surface hydrophobicity of the ZNP-FSG complexes [57]. As discussed above, ZNPU-FSG)H exhibited superior emulsifying properties mainly attributed to the great ability of decreasing the interfacial tension. Thus, a decrease in freeze-thawing stability of (ZNP-FSG)U-E and (ZNP-FSG)HU-E would be directly associated with a combination of reduced emulsification ability and surface hydrophobicity of complexes. Next, the structure of emulsions was further studied.

#### 3.2.3. Emulsion Droplets Size Distribution

The appearance and droplets size of emulsions (micron scale) stabilized by complexes subjected to various ultrasound and homogenization treatments were presented in Figure 8. The Pickering emulsion of ZNP-FSG can be infinitely diluted in water, indicating they were oil/water emulsions. As shown in Figure 8, all the emulsions exhibited unimodal distributions. It can be observed that emulsion stabilized by individual ZNP tended to separate into two layers. The droplet size of the emulsion decreased from 60.03 ± 5.72 μm to 48.43 ± 3.34 μm with the addition of FSG, and no serum phase was observed. The droplets size of (ZNP-FSG)HU-E and (ZNP-FSG)U-E were 60.03 ± 1.35 μm and 59.97 ± 3.16 μm, respectively. However, the emulsions broke after 30 min at room temperature.

Emulsion droplets size is one of the key parameters to characterize the properties of the emulsion. The droplets size distribution of emulsion depends on the breakup and coalescence of droplets. The high shear force (ultrasound and homogenization) can cause breakup of droplets, and the droplets coalescence may be attributed to surfactants [35]. The presence of FSG decreased the interfacial wettability and promoted complexes to absorb irreversibly on the oil/water interface, which is beneficial to the stability of the emulsion. However, the changing trend of particle size of emulsion was similar to that of complexes, (ZNPU-FSG)H-E obtained the lowest droplets size of emulsions (46.9 ± 0.30 μm) because

of the small particle size of the complex. According to the results of storage stability and freeze-thaw stability above, (ZNPU-FSG)H-E showed relatively good storage stability and freeze-thaw stability compared with other groups of emulsions.

**Figure 8.** The particle size distribution of emulsions stabilized by ZNP-FSG complexes subjected to various ultrasound and homogenization treatments. Insets are photographs of fresh emulsions (0. ZNP-E, 1. (ZNP-FSG)H-E, 2. (ZNPU-FSG)H-E, 3. (ZNP-FSGU)H-E, 4. (ZNP-FSG)HU-E, 5. (ZNP-FSG)U-E). The emulsion droplets sizes are shown in the table.

#### 3.2.4. Microstructure of Emulsions

Figure 9 shows the optical microscope photographs and CLSM of Pickering emulsions prepared by complexes subjected to various ultrasound and homogenization treatments. The droplets size distribution is consistent with the result of the droplets microstructure of the emulsion. The droplets of emulsion stabilized by ZNP alone has large droplets size and irregular shape. The creaming index of ZNP-E reached 25% in 30 min and eventually reached the maximum of 30% in 4 days. Pickering emulsions stabilized by ZNP-FSG had no aqueous phase, manifesting that the addition of FSG increased the stability of Pickering emulsions. (ZNPU-FSG)H-E showed significantly more droplets and more regular shapes compared to other emulsions, which is consistent with the observations for emulsions stabilized by pea protein with ultrasound [52]. Compared with (ZNPU-FSG)H-E, the droplets size of (ZNP-FSGU)H-E showed obviously thicker walls and irregular shapes. In the course of observation, we found that the droplet size of (ZNP-FSG)U-E and (ZNP-FSG)HU-E increased gradually over time, and the phase separation phenomenon was observed in 30 min, suggesting their stability were poor. In addition, the presence of oil droplets can be observed clearly on the emulsion droplet surface, which could be explained by two reasons. Firstly, ultrasound increased the frequency of contact collisions between droplets, leading to droplet coalescence. Secondly, over-processing of complexes was caused by ultrasonic and high-speed homogenization.

Figure 9b shows CLSM images of Pickering emulsions after 24 h storage at 4 ◦C. Corn oil was dyed with Nile red. All the above results showed that homogenization had no significant effect on the complexes and emulsion, thus (ZNP-FSG)U-E was not shown in the CLSM diagram. (ZNPU-FSG)-E can prevent collisions and coalescence of droplets and contained a fairly uniform dispersion of spherical oil droplets. The bigger oil droplet size was observed in (ZNP-FSGU)-E and (ZNP-FSG)HU-E. The oil droplet size variation observed by CLSM was in agreement with the storage stability.

**Figure 9.** Optical micrographics (**a**) and Confocal laser scanning microscopy (CLSM) (**b**) of emulsions stabilized by ZNP and FSG complexes subjected to various ultrasound and homogenization treatments.
