**3. Results and Discussion**

#### *3.1. Protein Composition Analysis*

Non-reducing electrophoresis cannot disrupt the disulphide bonds. Therefore, it reflects the original composition of the various proteins in a mixed protein (Figure 1 and Table 2), and 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), and legumin β (~22 kDa), all aggregates consistent with the literature [25,26]. Compared with legumin α + β, vicilin is more flexible and exhibits higher interfacial activity [27]. Many studies have confirmed that the vicilin/legumin a + B ratio is positively related to a protein's functional properties, including solubility and foaming and emulsifying properties [28,29]. Thus, the ratio of vicilin to legumin a + β is very important. As shown in Table 2, 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 superior to those of BL.

**Figure 1.** Non-reducing electrophoretograms of BL and Co.

**Table 2.** Relative band optical density (%) of BL and Co (non-reducing electrophoretogram).


#### *3.2. Chemical Composition of PPI, CPI, Co and BL*

The chemical composition analysis of PPI, CPI, BL, and Co is shown in Table 3. The protein content of the four protein samples was about 85%, which belongs to high-quality protein powder, indicating that the ISP method is an effective way to concentrate pea protein and grass carp protein. Co was the highest (*p* < 0.05) at 87.91%, indicating that Co has the potential to be used as the raw material of high-protein food. Compared with BL, Co contains less fat. When Co is added to fish sausage, the damage to the gel structure

and nutritional value caused by fat oxidation can be reduced, and the shelf life of the product can be prolonged [30]. We previously speculated that the carbohydrate content of Co should be between PPI and CPI. Interestingly, experimental data showed that the carbohydrate content of Co was only 0.16% lower than CPI (*p* < 0.05), suggesting that co-precipitation enhanced the removal rate of starch or muscle glycogen. It is speculated that this is because the interaction of heterologous proteins reduces protein–carbohydrate (glycoprotein) binding during co-dissolution or precipitation. In addition, Co has low fat and low carbohydrate characteristics, which is in line with the consumption trend. Therefore, Co, as an isolated protein powder, has greater application potential in meat products than BL.


**Table 3.** Chemical composition of PPI, CPI, Co, and BL.

Note: Different letters in the same column indicate a significant difference between samples (*p* < 0.05).

#### *3.3. Cooking Loss*

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The effect of adding different protein pre-emulsified soybean oil on the CL of fish sausage is shown in Figure 2. In this study, due to different emulsifiers, the introduction of soybean oil in the form of protein pre-emulsification will affect CL. CPI fish sausage had the lowest cooking loss (5.24%, *p* < 0.05), indicating that adding fish protein homologous to the fish-sausage matrix in the pre-emulsification emulsion can reduce cooking loss [31]. Compared with the control group, the cooking loss of PPI fish sausage was higher (*p* < 0.05). This is similar to the finding that replacing pig fat with pre-emulsified olive oil stabilised by soy protein isolate increased cooking loss. This is because the plant protein in fish sausage is not tightly bound to the fish matrix, resulting in a loose structure of the sausage that makes it difficult to hold water [32]. However, there was no significant difference in the CL of BL and Co when compared with the control group (*p* > 0.05). It can be seen that the introduction of plant protein in the form of dual proteins in fish sausage can significantly reduce CL.

**Figure 2.** Effects of different protein pre-emulsified soybean oils on the cooking loss of fish sausage. Note: Different letters indicate that there is a significant difference between samples (*p* < 0.05). The error bars represent the standard deviation (*n* = 3).

#### *3.4. Water-Holding Capacity*

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The WHC can reflect the cross-linking degree of the blend gel of pre-emulsified protein and fish meat. The effect of soybean oil pre-emulsified with four kinds of proteins on the water-holding capacity of fish sausage is shown in Figure 3. The results showed that the WHC of the five kinds of fish sausage was greater than 93%, with the control group having the lowest (93.20% *p* < 0.05), indicating that increased protein concentration results in a stronger gel network. This is consistent with the results reported in the literature [33]. The WHC of Co-, PPI-, and CPI fish sausage was higher than 96.00%. Under centrifugation, the water in the capillary of the gel permeates the surface and is removed by filter paper. Therefore, it can be considered that the addition of Co, PPI, and CPI reduces the flow of free water [34]. In addition, the WHC of Co fish sausage was the highest, while that of BL fish sausage was the lowest. Combined with the previous results [18], we speculate that this is due to the antagonistic effect of PPI and CPI in BL on heat-induced gelation, which leads to a loose structure and poor WHC of fish sausage. It can be seen that Co is more beneficial to the water retention of fish-sausage gels than BL.

**Figure 3.** Effect of soybean oil with different protein pre-emulsification on the water retention of fish sausage. Note: Different letters indicate a significant difference between samples (*p* < 0.05). The error bars represent the standard deviation (*n* = 3).

#### *3.5. Folding Test*

The folding test can directly reflect the gel strength of fish sausage and allow researchers to quickly understand its texture characteristics [35]. The folding test results of the five kinds of fish sausages are shown in Figure 4, which confirms the results of CL and WHC. The folding ability of the control group was lower than that of PPI-, CPI-, and Co fish sausage, which was attributed to the weak gel network formed by the lower protein content [36]. However, the lowest folding ability of BL fish sausage was only level 3 (*p* < 0.05). This is due to the antagonism of PPI and CPI in BL, leading to the gel structure loosening.

**Figure 4.** Effects of different protein pre-emulsified soybean oil on the folding capacity of fish sausage.

#### *3.6. TPA*

The TPA results of the five sausage samples are shown in Table 4. The hardness, cohesiveness, and chewiness of fish sausage added with Co were significantly higher than those of PPI and CPI (*p* < 0.05), which indicated the synergistic effect of Co. According to previous studies [18], it is speculated that disulphide bonds account for the highest proportion of intermolecular forces of Co gel, which makes the hardness and cohesion of Co fish sausage stronger and thus necessitating greater energy consumption during chewing. However, the thermal denaturation temperature of PPI (82 ◦C) [37] and CPI (56 ◦C) [38] contained in BL is obviously different, which leads to the loose structure of heat-induced gel formed by BL, which leads to the lowest hardness, cohesion, and chewiness of BL fish sausage (*p* < 0.05), showing an obvious antagonistic effect. The hardness, cohesion, and chewiness of the blank control group were the lowest, but the springiness was the highest (*p* < 0.05). This is similar to the research results of improving the texture characteristics of fish sausage by adding pea protein [39]. Adding protein can enhance the interaction between protein molecules in surimi gel, thus improving the texture characteristics of the gel.


**Table 4.** Effects of different protein pre-emulsified soybean oil on the fish-sausage texture.

Note: Different letters in the same column indicate a significant difference between samples (*p* < 0.05).

#### *3.7. Colour*

Colour impacts its application in food, and colour tests can objectively evaluate the colour of protein samples. L\* represents sample brightness; the positive value of a\* denotes redness, and the negative value represents greenness. A positive value of b\* is yellowness, and a negative value is blueness. The effect of different protein pre-emulsified soybean oil on the colour of fish sausage is shown in Figure 5 and Table 5. The brightness (L\*) and whiteness of PPI fish sausage were lower than those of the other groups (*p* < 0.05), and yellowness (b\*) was higher than those in other groups (*p* < 0.05). This is related to the yellowish colour of PPI, which is attributed to the natural yellow plant pigments in peas

that are thermally stable. The brightness (L\*) of CPI fish sausage was the highest, reaching 86.37 (*p* < 0.05), and the yellowness (b\*) was the lowest (*p* < 0.05), but the whiteness was significantly higher than that of other groups (*p* < 0.05) [40]. The redness/greenness (a\*) of the fish sausage was lower in the five groups, which had little influence on the colour. The four colour indices of the fish sausage in the control group, i.e., BL and Co, were all between PPI and CPI, indicating that the relative white of CPI had a masking effect on the relative yellow of PPI [41]. Therefore, adding plant protein in the form of dual proteins can reduce the adverse effect of yellow pigments in plants on fish sausage, and the colour quality of BL is superior than that of Co.

**Figure 5.** Appearance of soybean oil fish sausage pre-emulsified with different proteins.


**Table 5.** Effect of soybean oil with different protein pre-emulsification on the colour of fish sausage.

Note: Different letters in the same column indicate a significant difference between samples (*p* < 0.05).

#### *3.8. Water Distribution Analysis*

Low-field nuclear magnetic resonance (LF-NMR) is a useful method to study the content, state, distribution, migration, diffusion, and the interaction of water molecules with other molecules. These properties of water are the key factors in determining the quality of fish-sausage gel [42]. Figure 6 shows the effect of soybean oil pre-emulsified with different proteins on water distribution in fish sausage. The relaxation time T of hydrogen protons in fish sausage reflects their degree of binding or freedom. The higher the T, the lower the binding force of hydrogen protons, leading to decreased stability and increased fluidity. The peak area of each stage represents water content with a different binding degree in fish sausage. T1 represents the bound water tightly adsorbed with macromolecules such as protein; T2 represents the water locked in the gel network of fish sausage, which has a certain fluidity and is classified as not easily allowing the flow of water; and T3 represents the water outside the gel network structure of fish sausage: free water with high fluidity [43]. It can be seen from Figure 6 that the T1 peak area of fish sausage containing pea protein components, such as PPI-, BL-, and Co fish sausage, is more significant. This may be due to the existence of pea protein in the gel in the form of filling after emulsification, and the degree of interaction with the gel matrix is weak. In contrast, the adsorption capacity of plant protein (PPI) to water is considerably stronger than that of fish protein, resulting in a higher volume of bound water (T1) [44]. Only the BL fish sausage had a pronounced T3 peak, indicating that it had a high free-flowing water content and a low gel water-holding capacity. However, the difference between T1 and T2 peaks of

CPI fish sausage was not noticeable, and the peak distribution was similar to that of the control group, indicating that the CPI bonded well with the fish gel matrix during gelation, leaving appropriate gaps to lock the adsorbed water in the gel network [45]. Generally speaking, the water distribution of Co fish sausage was between that of CPI- and PPI fish sausage, with no discernible T3 peak. Again, BL fish sausage showed an antagonistic effect.

**Figure 6.** Effects of different protein pre-emulsified soybean oil on water distribution of fish sausage.
