*2.13. Statistical Analysis*

All experimental measurements were performed at least three times. SPSS 25.0 was used for all statistical analyses. Statistical differences between two groups were determined with one-way analysis of variance (ANOVA) and Duncan's multiple range test (*p* < 0.05). The results are expressed as the mean ± standard deviation (SD).

### **3. Results and Discussion**

#### *3.1. Protein Composition Analysis*

The protein compositions of BL and Co were analyzed via reducing and non-reducing SDS-PAGE. As can be seen in Figure 2 and Table 1, both BL and Co contained myosin heavy chain (MHC, ~200 kDa), convicilin (~70 kDa), legumin (~62 kDa), vicilin (~50 kDa), actins (AC, ~42 kDa), legumin α (~38 kDa), tropomyosin (TM, ~34 kDa), legumin β (~22 kDa), and aggregates consistent with the literature [28,29]. To sum up, both BL and Co effectively combined pea and CPI, and thus, both are suitable for further testing.

**Figure 2.** Reducing (**a**) and non-reducing (**b**) electrophoretograms of protein blends (BL) and protein co-precipitates (Co).

**Table 1.** Relative band optical density (%) of BL and Co (non-reducing electrophoretogram).


Reducing SDS-PAGE reflects the specific subunit composition of proteins. BL and Co were both composed of pea and carp (1:1). However, the MHC, legumin α, and legumin β protein bands differed significantly in reducing SDS-PAGE, indicating that Co and BL showed different degrees of crosslinking or degeneration with subunits of CPI. Similar studies have suggested that such differences might be attributed to different electrostatic and hydrophobic interactions and the effects of disulfide bond in proteins during preparation of Co by the ISP method [10]. Co contains a higher number of MHCs, indicating that its functional properties might have some advantages [30]. The reduced MHC retention of Co in non-reducing electrophoresis indicates that some of the MHC components of Co entered soluble aggregates. Legumin is composed of six pairs of subunits and each pair contains an α-polypeptide (acid subunit) and a β-polypeptide chain

(alkaline subunit) linked via a disulfide bond. The disulfide bond can only be opened under reducing conditions, thus dissociating the protein subunits [31]. SDS can decompose hexamer legumin to 60 kDa dimer legumin α + β under heating conditions. However, DTT destroys the non-covalent bonds of the legumin hexamer as well as the disulfide bonds between the α- and β-polypeptide chains. Hence, no legumin α + β band is detected at 60 kDa in the reducing SDS-PAGE. In contrast, the disulfide bond between the α- and β-polypeptide chains remains intact in the absence of DTT [32]. Therefore, legumin α + β bands appeared at the position of 60 kDa following non-reducing electrophoresis. The optical density of Co was significantly less than that of BL, indicating that legumin α + β components of Co also partially entered the aggregate.

Non-reducing electrophoresis cannot disrupt the disulfide bonds. Therefore, it reflects the original composition of the various proteins in a mixed protein. Compared with proteins separated via reducing SDS-PAGE, many soluble aggregates were blocked at the top of the separated gel and the concentration of Co (19.62%) was 1.69-fold higher than that of BL (11.61%) (Table 1), indicating the presence of additional bonds in Co. Simultaneously, as mentioned above, some of pea and grass carp proteins entered the aggregates, indicating that co-precipitation leads to the formation of disulfide bonds between pea protein and grass carp protein, resulting in altered subunit composition. Current studies indicate that the formation of disulfide bonds between globulin molecules enhances the flexibility of the protein structure and improves its foaming and emulsifying capacity as well as its stability. When proteins are adsorbed onto the O/W interface, the stability of the emulsion is improved [33]. Compared with legumin α + β, vicilin is more flexible and exhibits better interfacial activity [34]. Many studies have confirmed that the vicilin/legumin α + β ratio is positively related to a protein's functional properties, including solubility, and foaming and emulsifying properties [35,36]. Thus, the ratio of vicilin to legumin α + β is very important. As shown in Table 1, the proportion of Co (203.99%) is 2.82-fold larger than that of BL (72.36%). Therefore, the analysis of protein composition indicates that the functional properties of Co might be better than those of BL.
