*3.2. Solubility and Surface Hydrophobicity (H0)*

Solubility is a key functional characteristic that can be used to evaluate the potential use of protein in food and determine its foaming, emulsification, and gel formation ability [37]. As can be seen in Figure 3, the solubility of Co, BL, PPI, and CPI was significantly pH dependent, with solubility exhibiting a U-shaped curve. The minimum solubility was observed at pH 5.0 because the isoelectric point (PI) of globulin is 4.5 and the isoelectric point of myofibrillar protein is 5.3, which is consistent with other studies of PPI [38], CPI [39], and soy–fish mixed protein [14]. When the pH deviates from the isoelectric point, solubility significantly increases because an increase in net charges on the protein surface reinforces the intermolecular electrostatic repulsive force, which promotes ion–dipole interaction between proteins and water molecules [40].

ANS is a fluorescence probe that is sensitive to external polar groups and interacts with hydrophobic groups of protein molecules to generate fluorescence spectra, which can be used to characterize the exposure of hydrophobic groups in proteins. As shown in Figure 4 under different conditions, *H*<sup>0</sup> varied inversely with solubility. Thus, a higher solubility was associated with fewer hydrophobic groups on the protein molecular surface [41].

Comprehensive analysis of the solubility and surface hydrophobicity of the mixed proteins indicated that the solubility of Co was the highest under all pH values except at pH 5.0 (*p* < 0.05), which is similar to the result of our previous study of soy–tilapia Co [14]. Co prepared by ISP was precipitated at pH 5.0. Under this circumstance, pea and CPI molecules carry opposite charges and the intermolecular electrostatic interaction is strengthened to generate soluble Co-aggregates of proteins, triggering the reconstruction of hydrophobic and disulfide bonds [42]. Moreover, some subunits, such as vicilin, will be concentrated "selectively", thus changing the protein properties [43]. BL is formed by mixing PPI and CPI powders, and the molecular interaction is not as strong as that of Co. The molecular interaction of BL involved PPI and CPI at pH 3.0, 7.0, and 9.0, but the solubility (80.77%) at pH 11.0 was a little higher than that of PPI (77.36%) and CPI (79.07%). Under strong alkaline conditions, protein molecules degenerate, and hydrophobic groups are exposed. Protein molecules cluster with each other via hydrophobic bonds, inducing hydrophobic nonlinear superposition [44]. As a result, the *H*<sup>0</sup> (231.53) of BL was lower than that of PPI (302.36) and CPI (234.41). Due to this hydrophobic synergistic effect, the solubility of BL was higher than that of PPI and CPI. Thus, Co has a better synergistic effect on solubility than BL.

**Figure 3.** Solubility of proteins under different pH values. Different letters under the same pH value indicate significant differences (*p* < 0.05).

**Figure 4.** Surface hydrophobicity (*H*0) of proteins under different pH values. Different letters under the same pH value indicate significant differences (*p* < 0.05).

#### *3.3. Foaming Properties*

Foaming plays a critical role in the texture and structure of foods, such as ice cream, cakes, breads, and meringue. Currently, foaming is achieved by replacing a surfactant with protein. Foaming capacity (FC) and foaming stability (FS) are the most common indices used to describe the foaming properties of protein [45]. The effects of pH on the FC and FS of the proteins are shown in Figures 5 and 6. Overall, FC and FS exhibited a U-shaped variation, similar to those observed for solubility. The foaming properties were the weakest at pH 5.0, which is consistent with other studies [46]. When the pH deviated from the PI, the solubility of the protein was improved and the degree of dispersion of the protein molecules in water was increased. Under these circumstances, the surface charges on the protein molecules are increased and the protein structures are opened to expose the hydrophobic amino acid side chains. Hence, the proteins quickly locate at the air–water interface, and the surface tension is decreased, thus improving the FC and FS of the proteins.

**Figure 5.** Foaming capacity of proteins under different pH values. Different letters under the same pH value indicate significant differences (*p* < 0.05).

**Figure 6.** Foaming stability of proteins under different pH values. Different letters under the same pH value indicate significant differences (*p* < 0.05).

Zhao et al. argued that the FC of PPI was similar to that of soy protein isolate, but the FS was slightly higher. PPI is a plant foaming agent with potential application in various food preparations [47]. In this study, the FC and FS of PPI were the highest under all pH values (*p <* 0.05). The maximum value of FC was 103 ± 1.25 at pH 9.0, which is related to the small molecular weights of PPI, small volume, and weaker intermolecular interaction [48]. CPI exhibited the opposite trend. The molecular weights of fibrillin are relatively high and present a linear structure, thus causing significant steric hindrance. The air–water interfacial absorption capacity of fibrillin is significantly lower than that of globulin, and the compactness of the viscous layer is insufficient. Therefore, the FC and FS of fibrillin are relatively low [49]. The foaming performance of the mixed protein was better than that of CPI, but still inferior to that of PPI at all pH values, i.e., there was no synergistic effect, similar to solubility. The FC of Co was lower than that of BL at pH 3.0 and 5.0, and the FS of Co was lower than that of BL under all pH values. This might be because Co contains additional macromolecular soluble aggregates, making it difficult to form a dense viscous layer on the water–air interface. In brief, the addition of PPI is a potential strategy to improve the foaming properties of CPI.

#### *3.4. Emulsifying Properties*

#### 3.4.1. EAI and ESI

EAI refers to the oil–water interface area for stabilization of the unit mass of the emulsifier; it reflects the resistance of a protein to emulsion stratification caused by flocculation and aggregation. Thus, the EAI can be used to evaluate the emulsifying properties of

proteins. ESI refers to the ability of proteins to remain in a stable state within a certain period, without phase layering or separation [25]. EAI and ESI are key indices that reflect the functional properties of proteins. As shown in Figures 7 and 8, the EAI and ESI of all proteins first decreased and then increased under all pH values. This phenomenon is similar to the trend of the solubility and foaming properties, but contrary to the *H*<sup>0</sup> value. The emulsifying properties and emulsifying stability near PI were the lowest because, at PI, the protein surface carries few charges and lacks electrostatic repulsion. Accordingly, proteins aggregate, and flocculation occurs [50]. Increasing pH can lead to the degeneration of proteins. The protein molecular conformation in the "melting state" is soft and relaxed. The residual groups of the exposed hydrophobic amino acids point to the oil phase, while the hydrophilic groups point to the water phase, thus enhancing the interaction with oil phases [51]. As a result, the emulsifying properties are enhanced. It can be seen that the pH changes the ability of proteins to stabilize oil droplets via expansion and adsorption on interfaces by regulating electrostatic interactions and *H*<sup>0</sup> for stability.

**Figure 7.** Emulsifying activity index of proteins under different pH values. Different letters under the same pH value indicate significant differences (*p* < 0.05).

**Figure 8.** Emulsifying stability index of proteins under different pH values. Different letters under the same pH value indicate significant differences (*p* < 0.05).

The ESI values of BL and Co did not exhibit any clear pattern. At pH 3.0, the ESI of BL was higher than that of Co (*p* < 0.05). At pH 7.0, the ESI of BL was lower than that of Co (*p* < 0.05). There were no significant differences at pH 5.0, 9.0, and 11.0 (*p* > 0.05). However, testing the particle size of the emulsions and analysis under CLSM revealed that Co stabilized smaller oil droplets (discussed later), indicating improved stability. The testing time of ESI was only 10 min, which was not sufficient to complete the maturation process and allow adsorption of the protein on the interface.

### 3.4.2. Particle Size and Zeta-Potential Analysis

Particle size is an important index used to measure the stability of an emulsion [52]. The particle size distributions of protein emulsions were evaluated using the volumeweighted mean diameter (*d*4,3) (Figure 9), supplemented with the surface-area-weighted mean diameter (*d*3,2), and *d*- <sup>43</sup> and *d*- <sup>32</sup> values of the emulsion with 1% SDS (Table 2). The effect of the pH value on the particle size was analyzed. The results indicated that the *d*4,3 values of the emulsions at pH 3.0 and 5.0 were relatively high (3.29 ± 0.27 μm and 22.79 ± 2.75 μm for Co) because the electrostatic repulsion among proteins is insufficient to overcome attractions (Figure 10), thus resulting in particle clusters and increasing the particle size. According to Laplace theory, the droplet size of an emulsion decreases with a reduction in two-phase interface tension under constant homogeneity [53]. The *d*4,3 values of the emulsions at pH 9.0 and 11.0 were significantly lower than at other pH values (0.94 ± 0.05 μm and 0.54 ± 0.01 μm for Co), which is consistent with a previous study reporting a significant decrease in the *d*4,3 of soy protein isolate under alkaline pH (pH 9.0–12.0) [54]. Small particle size is conducive to the formation of a stable emulsion [55]. Therefore, the most stable emulsion systems were achieved at pH 11.0. Moreover, as shown in Figures 9 and 10, all proteins exhibited single-peak particle size distributions at pH 3.0 and 11.0, which is related to the high net charges. At pH 5.0 and 7.0, relatively large particle size peaks (10–100 μm) were observed. Under these conditions, oil droplets develop flocculence, which is attributed to bridging protein aggregates between the droplets. Alternatively, it is difficult to achieve ripening and saturation on the oil–water interface due to the low protein–oil ratio, thus leading to bridging flocculation [56]. The particle size of CPI under all pH values was smaller than that of PPI, which is consistent with the results of EAI, because the molecular flexibility of myofibrillar protein (MP) is higher than that of globulin and, thus, leads to directional expansion on the oil–water interface. Therefore, CPI can stabilize smaller oil droplets. The particle size of Co was smaller than that of BL under all pH values, except for pH 5.0 (*p* < 0.05), which demonstrates the higher emulsifying capacity of Co. The trends in *d*3,2, *d*- 4,3, and *d*- 3,2 variation were similar to that of *d*4,3.

**Figure 9.** Droplet size distribution of proteins under different pH values. (**a**) pH = 3.0; (**b**) pH = 5.0; (**c**) pH = 7.0; (**d**) pH = 9.0; (**e**) pH = 11.0.


**Table 2.** Average particle size of proteins at different pH values.

Note: Each value represents the mean ± SD (*n* = 3). Different letters under the same pH indicate significant differences (*p* < 0.05).

**Figure 10.** Zeta potential of proteins under different pH values. Different letters under the same pH value indicate significant differences (*p* < 0.05).

Zeta potential is an index of particle repulsion or attraction intensity. The higher the absolute zeta-potential value, the higher the stability of an emulsion [57]. The zeta potentials of droplets under different pH values were evaluated (Figure 10). For all samples, the zeta potential changed from positive to negative when the pH increased from 3.0 to 11.0. This trend can be attributed to the PI values of PPI and CPI, which were 4.5 and 5.2, respectively. At pH 5.0, the zeta potentials of the emulsions were the lowest (2.32 ± 0.31 mV for Co), and the net charges were the lowest, which is related to the negatively charged aggregates on the interfaces. The zeta potentials of the emulsions increased significantly when the pH increased from 7.0 to 11.0 because when the pH is higher than the PI of a protein, electrostatic repulsion increases, and flocculation of droplets is inhibited. Consistent with the particle size results, the zeta potential of CPI was higher than that of PPI (*p <* 0.05). The zeta potential of Co was higher than that of BL under all pH values, except at pH 5.0, which is attributed to the better dispersing capacity of Co and the higher sensitivity of charging properties to pH. This is related to AP and Γ.

#### 3.4.3. Interface Protein Adsorption (AP) and Interface Protein Content (Γ)

AP refers to the percentage ratio between the protein content adsorbed on the oil droplet surface and the protein content in the continuous phase. Γ refers to the protein content on the unit area of a droplet [43]. The AP (%) and Γ values of PPI, CPI, BL, and Co under different pH values are listed in Table 3. At pH 5.0, the AP (%) of all four protein emulsions was the lowest (2.96–11.42%), indicating the presence of abundant protein isolates in the emulsions. The protein solubility decreased due to the electrostatic shielding effect at pH 5.0. When the pH deviated from the PI, the AP increased gradually, which is consistent with the study of Liang and Tang [43]. The Γ value of an emulsion reflects the thickness of the stable oil–droplet interface membrane. Although it is not directly correlated with the particle size of emulsion droplets or AP, it is still influenced by solubility. The overall trends of variation were also similar to those of AP. Nevertheless, it is interesting that the AP of BL at pH 5.0 was higher than at pH 7.0, and the AP of Co at pH 5.0 was higher than at pH 3.0 and 7.0. This indicates that, during emulsification via high-speed shearing, insoluble proteins can also adsorb onto the interface and an emulsion with a similar stable Pickering effect is formed [58]. The results indicated that the AP% and Γ of CPI were higher than those of PPI, and the AP% and Γ of Co were higher than those of BL. These results further demonstrate that surface hydrophobicity is a major and decisive factor influencing the emulsifying properties of a protein under high solubility.

**Table 3.** Interface protein adsorption and interface protein content of proteins at different pH values.


Note: Each value represents the mean ± SD (*n* = 3). Different letters under the same pH value indicate significant differences (*p* < 0.05).

#### 3.4.4. Confocal Laser Scanning Microscopy (CLSM)

CLSM is often used to analyze the microstructure of an emulsion; it reflects the original particle size distribution, dispersion, and stability of emulsion particles. The red and green zones represent oil (Ar laser 559 nm) and protein (He/Ne laser 633 nm), respectively. The microstructures of the protein emulsions under different pH values are shown in Figure 11. The overall results were consistent with the observed particle size results. Almost all oil droplets were spherical and wrapped in an outer protein layer. At pH 5.0, all four protein emulsions showed serious flocculation. The emulsion particles were large and irregular and were mostly distributed in clusters. Although the oil droplets stabilized by Co were small, evident protein aggregation was observed. A pH value approaching PI is the main cause for the bridging flocculation of oil droplets and proteins [6]. When the pH was far from the PI, electrostatic repulsion increased with the increase in the solubility of the protein, while the particle size of the emulsion droplets decreased rapidly. Similarly, the emulsion particle sizes stabilized by BL and Co were smaller and distributed more uniformly under the same pH (e.g., pH 9.0 and 11.0), which is consistent with the results obtained with soy–whey mixed protein at pH 2.0–11.0 [6]. The variation trend of Co was more obvious. CLSM directly validated the EAI/ESI, AP/Γ, and the results of particle size distribution, indicating that Co has a better emulsifying effect than BL.

**Figure 11.** Micromorphology of protein emulsions under different pH values.
