3.1.4. Circular Dichroism (CD) Spectroscopy Studies

As shown in Table 1, the second structure compositions of PP were 15.0% α-helix, 28.8% β-sheet, 22.1% β-turn, and 34.2% random coils. After enzymatic hydrolysis with four enzymes, the β-sheet content of enzymatic hydrolysis samples decreased with the increase in *DH*, the α-helix content increased significantly, and there was no significant change in β-turn angle and random coil. When the *DH* was 8%, the β-sheet content of PP treated by flavourzyme, neutrase, alcalase, and trypsin decreased from 28.8% to 21.7%, 20.7%, 18.5%, and 14.1%, respectively. Correspondingly, the α-helix content increased from 15.0% to 22.2%, 24.6%, 26.9%, and 32.1%, respectively. These results indicated that enzymatic hydrolysis caused perturbations of the PP secondary structure and, thus, may have an impact on the function properties of PP. Among them, the structure of β-sheet was more compact and the structure of α-helix was looser, so it can be deduced that the ordered βsheet in the enzymatic hydrolysis products was disrupted to a more flexible and spreading α-helix structure after enzyme treatment [36]. According to the results, under the same *DH*

(2%, 4%, 6%, and 8%), trypsin showed the most obvious effect on the secondary structure of PP, followed by alcalase, neutrase, and flavourzyme. The changes in the secondary structure of PP can correspond to the results of the intrinsic fluorescence and surface hydrophobicity.


**Table 1.** Secondary structure content of PP and PP with different *DH* samples determined by circular dichroism.

#### 3.1.5. Morphology Observation

To gain insight into the effect of enzymatic hydrolysis on the change in the microstructure of PP, SEM micrographs were imaged for PP and PP with different *DH* samples. As shown in Figure 4, the natural PP is irregularly spherical with a smooth surface and a tight structure. After being treated with flavourzyme, neutrase, alcalase, and trypsin, the spherical structure of PP was broken into fragments. In addition, the microstructure of PP showed that the particles gradually became smaller with the increase in *DH*. The smaller particle size of PP made it have more chance to come into contact with water molecules during the dispersion process, thus changing the physicochemical properties of PP [29]. Furthermore, at the same *DH*, the protein particles treated by the four enzymes from small to large were trypsin, alcalase, neutrase, and flavourzyme, respectively. This result can correspond to the above result. The disruption of the protein microstructure may affect the amino acid groups embedded in the molecule and change the surface hydrophobicity, interaction forces, and secondary structure [37].

### 3.1.6. SDS-PAGE

The molecular weight distribution was analyzed to investigate the effect of enzymatic hydrolysis on the PP. Figure 5 shows the SDS-PAGE electrophoresis of PP and PP with different *DH* samples. The electrophoresis pattern of PP showed multiple cleaned bands. The band at 17 kDa, 20–22 kDa, ~40 kDa, ~18 and ~50 kDa, ~60 kDa, ~75 kDa, and ~100 kDa could be attributed to 2S albumin polypeptides and/or γ-vicilin, legumin β, legumin α, vicilin, legumin, convicilin, and lipoxygenase, respectively [5]. After being treated with flavourzyme, neutrase, alcalase, and trypsin, the protein bands became progressively smaller as *DH* increased. Additionally, after enzymatic hydrolysis by flavourzyme and neutrase, the bands were clear at low *DH* (2% and 4%), but with the increase in *DH*, the protein bands were aggregated downward and the bands were gradually indistinguishable. After trypsin and alcalase treatment, the bands gradually moved toward the small molecules with the increase in *DH*, and the color of bands gradually became lighter, indicating that they kept aggregating toward the small molecule fragments after enzymatic hydrolysis. This result was consistent with the results of microstructure. It can also be shown that with the increase in *DH*, the molecular weight of PP gradually becomes smaller, which may affect the functional properties of PP. In addition, the effect of flavourzyme and neutrase on molecular weight of PP was lower than trypsin and alcalase under the same *DH*.

**Figure 4.** Morphology of PP and PP with different *DH* samples.


**Figure 5.** SDS-PAGE profiles of PP and PP with different *DH* samples under nonreducing conditions.

3.1.7. Determination of Total Sulfhydryl Groups

The content of sulfhydryl groups (SH) and disulfide bonds in proteins determines their rigid structure and has a significant impact on their functional properties. Figure 6 shows the variation in the total SH of samples. Compared to PP (3.94 μmol/g), the enzymatic hydrolysis samples resulted in a lower total SH. The decrease in SH content may be due to the destruction of SH within the protein molecule by enzymatic hydrolysis. As known, the spatial structure will unfold after enzymolysis; the more SH is exposed, the SH will be destroyed during enzymatic hydrolysis. As reported by Lepedda et al. [38], the SH in protein can be oxidized by enzymes, resulting the decrease in total SH. When the *DH* was 2% to 6%, the reduced SH content was the same, but when the *DH* was increased to 8%, the total SH of the samples treated by flavourzyme and alcalase further decreased significantly, which may be due to the enhanced oxidation of the SH during the enzymatic hydrolysis process. Moreover, the total SH content was reduced by trypsin > alcalase > neutrase > flavourzyme after treatment with the four enzymes, indicating that the effect of the four enzymes on the SH content and structure of PP was also trypsin > alcalase > neutrase > flavourzyme.

**Figure 6.** Total sulfhydryl content of PP and PP with different *DH* samples. The values reported represent means (*n* = 3) ± standard deviations and different superscript letters indicate significant difference (*p* < 0.05). ((**A**–**D**) are flavourzyme, neutrase, alcalase and trypsin treatment, respectively).

#### 3.1.8. Amino Acid Composition and Average Hydrophobicity Analysis

PP was considered a high-quality protein because its balanced amino acid ratio can fulfil FAO/WHO recommendations [39]. Table 2 shows the results of the amino acid composition of PP with different *DH* samples. Due to the difference in the specific cutting sites of different proteases, different amino acid compositions of peptides can be produced by enzymatic hydrolysis. As summarized in Table 2, 16 amino acids were identified and quantified among all the hydrolysates, and GLU had the highest content (it ranged from 15.70 to 18.99 g/100 g), which was similar to the amino acid composition of PP [3]. It was worth noting that the hydrophobic amino acid (HAA) (including ALA, VAL, MET, ILE, LEU, TYR, PHE, and PRO) content of PP treated by flavourzyme, neutrase, alcalase, and trypsin increased with increasing *DH* (from 2% to 8%). Meanwhile, proteolytic enzyme treatment of food protein can improve the functional and nutritional properties, but also introduce undesirable attributes [40]. Among these, bitterness is considered to be one of the main disadvantages in utilizing protein hydrolysates in food applications. In addition, sensory evaluation showed the color gradually deepened, and the roughness of the taste and the beany flavor gradually decreased with the increase in *DH* after enzymatic hydrolysis with the four enzymes. Meanwhile, the bitterness assessed by tasting was also enhanced with the increase in *DH* (data are displayed in Supplementary Materials Figure S1), and at the same *DH*, the bitterness intensity of PP treated with different enzymes was in the following order: trypsin > alcalase > neutrase > flavourzyme.



It is well known that bitter peptides are spontaneously produced during the proteolytic process and more obvious at high *DH* [41]. The degree of hydrophobicity is considered the most important predictor of peptide bitterness. So far, the Q rule proposed by Ney is still used for the relationship between hydrophobicity and bitterness of polypeptide chains [42]. As expected, the Q values tended to increase with the increase in enzymatic hydrolysis by the four enzymes, which meant that the bitterness gradually increased with the enzymatic hydrolysis. This can be explained by the results of HAA content as mentioned above. However, the Q rule found that the bitter taste was presented when the Q value was greater than 1400 cal/mol, and vice versa [41]. In this study, the Q value of all samples was below the threshold, which was not consistent with the actual bitterness of the products through tasting. Therefore, the hydrophobicity data calculated based on amino acid composition from this study do not support Ney's Q rule as a predictor of bitterness of PP hydrolysates.

In general, the secondary structure of PP changed from ordered β-sheet to α-helix and the molecular weight of PP gradually decreased with the increase in *DH* after treatment with the four enzymes. Furthermore, the total sulfhydryl content and surface hydrophobicity of PP were changed by enzymatic hydrolysis. In addition, the bitterness of PP after enzymatic hydrolysis increased with the increase in *DH*.

#### *3.2. Functional Properties*

#### 3.2.1. Solubility

Solubility plays a crucial role in the functionality of the protein and protein-based systems including gels, emulsions, and foams [43]. Especially for plant protein, boosted properties can benefit from the increase in solubility [44]. The solubility of PP and PP with different *DH* samples is shown in Figure 7. Compared with PP (10.23%), the solubility of PP gradually increased with the increase in *DH*. The solubility of flavourzyme 2%, 4%, 6%, and 7% was 17.54%, 23.68%, 25.46%, and 29.44%, respectively. The solubility of neutrase 2%, 4%, 6%, and 8% was 19.18%, 24.78%, 27.62%, and 30.47%, respectively. The solubility of alcalase 2%, 4%, 6%, and 8% was 40.55%, 41.52%, 46.37%, and 54.97%, respectively. The solubility of trypsin 2%, 4%, 6%, and 8% was 41.91%, 43.49%, 54.22%, and 58.14%, respectively. The increased solubility may be due to the decreased molecular size of PP and smaller peptides when the PP was hydrolyzed. This was consistent with the results of SDS-PAGE. In addition, the effect of different enzymes on PP at the same *DH* was different. From the experimental results, the degree of change induced by the four enzymes on PP solubility was: trypsin > alcalase > neutrase > flavourzyme. This meant that trypsin had the best enzymatic hydrolysis effect on PP of all the enzymes.

#### 3.2.2. Foaming Performance

The foaming capacity and foaming stability of PP and PP with different *DH* samples are shown in Figure 8. The foaming ability of the enzymatically digested PP was significantly higher than the native PP, which was consistent with the solubility results. Solubility is a prerequisite for protein-foaming ability, and the protein should first dissolve in the aqueous phase and then rapidly stretch to form a dense layer of protein molecules around the air and foaming [45]. The foaming ability and foaming stability of protein was also related to the *DH*. After the four enzymes' treatment, the foaming ability of PP treated with flavourzyme, alcalase, and trypsin showed an increasing trend and then decreased, reaching the best levels at 6% (101.48%), 6% (164.44%), and 4% (168.88%) of *DH*, respectively. The foaming ability of PP treated by neutrase increased gradually. Speculatively, limited enzymatic hydrolysis produced peptides with balanced hydrophilic/hydrophobic groups, and the amphiphilic parts of proteins and peptides have a stronger capacity to decrease the surface tension at the air–water interface. However, high *DH* usually had an adverse effect on foaming properties. On the one hand, the increase in *DH* increased the protein solubility, but the best structure of protein spheres was destroyed, thus resulting in a reduction in the foaming ability. On the other hand, excessive enzymolysis made the hydrolysates more hydrophilic, disturbing the hydrophilic/hydrophobic balance, which led to the decreased

foam formation ability [46]. In addition, after the four enzymes (flavourzyme, neutrase, alcalase, and trypsin) treatment, the foaming stability of PP was decreased compared with native PP.

**Figure 7.** Solubility of PP and PP with different *DH* samples. Different lowercase letters are used to indicate the significant differences (*p* < 0.05) of the same enzyme under different *DH*, while different uppercase letters are used to indicate the significant differences (*p* < 0.05) of different enzymes under the same *DH*.

**Figure 8.** The foamability and foam stability of PP and PP with different *DH* samples ((**A**–**D**) are flavourzyme, neutrase, alcalase, and trypsin treatment, respectively). The values reported represent means (*n* = 3) ± SDs and different superscript letters indicate significant difference (*p* < 0.05).

#### 3.2.3. Emulsifying Performance

Proteins are a kind of natural emulsifier that are widely used in food emulsion preparation due to their amphiphilic nature. The emulsifying property of protein represents the ability of protein molecules in an emulsion to adsorb to the oil–water interface and is usually characterized by emulsifying activity and emulsifying stability [47]. Usually, the stronger the emulsification, the more stable the emulsion which forms and the less likely it is to form an aggregation. Figure 9 shows the changes in the emulsifying properties and emulsion stability of PP and PP with different *DH* samples. As shown in Figure 9, the emulsification ability and emulsion stability of PP treated with the four enzymes were significantly improved, which may be related to the increased solubility and peptide chain flexibility of the PP enzymatic hydrolysis products. All four enzymes showed an increasing trend and then a decrease in emulsifying properties as the *DH* increased, and all of them were highest at a *DH* of 6%. When the *DH* was 6%, the *EAI* of PP treated by flavourzyme, neutrase, alcalase, and trypsin increased from 24.55 to 42.36, 52.22, 49.55, and 52.16 m2/g, respectively, while the *ESI* increased from 30.02 to 87.54, 89.23, 77.93, and 84.88%, respectively. This may be due to the fact that excessive enzymolysis further reduces the molecular weight of the protein and reduces the amphiphilicity of the peptide, thus inhibiting the interaction between protein molecules at the oil–water interface and reducing the viscoelasticity of the interface membrane [47,48]. In addition, it may be due to the reduced charge repulsion between low-molecular-weight peptides preventing the proteins from either stretching or rearranging at the interface [49].

**Figure 9.** The *EAI* and *ESI* of PP and PP with different *DH* samples ((**A**–**D**) are flavourzyme, neutrase, alcalase, and trypsin treatment, respectively). The different superscript letters of same index indicate significant difference (*p* < 0.05).

Based on the above results and analysis, the solubility of PP gradually increased with the increase in *DH* after treatment with the four enzymes. Furthermore, the foaming capacity of PP treated with four enzymes was significantly improved, and the best foaming capacity was observed for flavourzyme, alcalase, trypsin, and neutrase with *DH*s of 6%, 6%, 4%, and 8%, respectively. In addition, the emulsification ability and emulsion stability of PP treated with the four enzymes were significantly improved, and all of them were the highest at a *DH* of 6%. Overall, the functional properties of PP treated with the four enzymes were the best when the *DH* was 6%.
