*3.1. Structure Properties*

#### 3.1.1. Degree of Enzymolysis (*DH*)

In this study, pea protein (PP) was hydrolyzed by four enzymes (flavourzyme, neutrase, alcalase, and trypsin) for 5 h. As shown in Figure 1, the enzymolysis degree (*DH*) of PP increased firstly and then tended to be flat with the increase in enzymolysis time. The *DH* of PP was most intense during the first 1 h, its *DH* increased rapidly, and the *DH* of PP increased slowly after 1 h. This was consistent with Avramenko et al. [26], who pointed out that the *DH* of the lentil isolate hydrolyzed by trypsin was most rapid in the first 40 min, and then the *DH* increased slowly. Figure 1 indicated that the *DH* of PP treated by four enzymes reached 19.88% (trypsin), 16.78% (alcalase), 8.97% (neutrase), and 7% (flavourzyme) at 5 h. In the same enzymatic hydrolysis time, the enzymatic hydrolysis degree after four kinds of enzyme treatment from large to small is trypsin, alcalase, neutrase, and flavourzyme. This was closely related to the activity unit and specific restriction site of the enzyme. A similar result had been reported for pea protein isolate treated with 11 proteolytic enzymes at different enzymatic hydrolysis times [12]. Compared to previous studies, it was found that excessive enzymatic hydrolysis (*DH* ≥ 10%) showed adverse effects on the properties of PP. In addition, it was worth noting that the enzymatic hydrolysis limit of flavourzyme was 7%. Therefore, in this study, the enzymatic hydrolysates with the *DH* of 2%, 4%, 6%, and 8% (flavourzyme is 7%) of the four enzymes were selected for subsequent studies in order to clarify the effects of different enzymes on the physical and chemical properties of PP at the same *DH*.

**Figure 1.** The *DH* of PP enzymolyzed by four enzymes (alcalase, neutrase, flavourzyme, and trypsin) using pH-stat method for 5 h.

#### 3.1.2. Intrinsic Fluorescence Spectroscopy

The intrinsic fluorescence of aromatic amino acid residues (Trp, Tyr, and Phe) is very sensitive to the microenvironment, and the emission fluorescence spectra of proteins are used to investigate the changes in their tertiary structure [27]. Therefore, the maximum fluorescence intensity and the wavelength (λmax) at the maximum fluorescence intensity are effective indicators to observe the structure and conformational changes in proteins [28]. Figure 2A–D show the fluorescence intensity of PP treated with flavourzyme, neutrase, alcalase, and trypsin, respectively. The intensities of the emission fluorescence of PP exhibited a maximum absorption at 337 nm. The λmax was affected by enzymatic treatment, and a shift in λmax to longer wavelengths (bathochromic shift) was observed for PP treated by flavourzyme enzymolysis (345.8, 345.4, 350.4, and 350.4 nm for 2%, 4%, 6%, and 7%, respectively), neutrase enzymolysis (346.8, 346.4, 350.2, and 350.4 nm for 2%, 4%, 6%, and 8%, respectively), alcalase enzymolysis (349, 349.6, 350.2, and 349.2 nm for 2%, 4%, 6%, and 8%, respectively), and trypsin enzymolysis (349.2, 350.4, 350.6, and 350.4 nm for 2%, 4%, 6%, and 8%, respectively). These results suggested that the microenvironment of chromophores in PP became more polar and hydrophilic after enzymatic treatment owing to the increased contact between the fluorophore and the aqueous medium [29]. In addition, the fluorescence intensity of PP after enzymatic hydrolysis increased significantly; the fluorescence intensity of all the four enzymes changed most significantly when *DH* increased from 0 to 2%. This indicated that the enzymatic hydrolysis process made more aromatic groups be exposed to solvent and more available to emit fluorescence [30,31]. With the increase in the *DH*, this process gradually slowed down. Among the four enzymes, trypsin-enzymolyzed PP had the highest fluorescence absorption peak. This indicated that trypsin changed the structure of PP most obviously.

**Figure 2.** Intrinsic fluorescence of PP and PP with different *DH* samples ((**A**–**D**) are flavourzyme, neutrase, alcalase and trypsin treatment, respectively).

#### 3.1.3. Surface Hydrophobicity

The surface hydrophobicity largely determines the protein structure and properties [32]. The changes in surface hydrophobicity of proteins after enzymatic hydrolysis were related to the type of enzyme and protein and time of enzymatic hydrolysis [33]. The surface hydrophobicity of PP after enzymatic hydrolysis by four enzymes is shown in Figure 3. Compared with PP (16,102 ± 1136), flavourzyme and neutrase showed a gradual decrease in H0 with increasing enzymatic hydrolysis. The H0 of flavourzyme 2%, 4%, 6%, and 7% was 10,418 ± 784, 9010 ± 527, 8804 ± 485, and 8693 ± 153, respectively. The H0 of neutrase 2%, 4%, 6%, and 8% was 19,191 ± 1234, 17,037 ± 478, 14,353 ± 222, and 11,761 ± 563, respectively. Speculatively, the above two enzymes caused the PP molecules

to stretch and be partially hydrolyzed into smaller fragments, and at the same time, these small fragments would reassemble under the action of hydrophobic and disulfide bonds, resulting in a decrease in surface hydrophobic groups [34]. The H0 of alcalase 2%, 4%, 6%, and 8% was 19,191 ± 1234, 17,037 ± 478, 14,353 ± 222, and 11,761 ± 564, respectively. The H0 of trypsin 2%, 4%, 6%, and 8% was 21,274 ± 554, 18,869 ± 937, 14,387 ± 78, and 13,219 ± 987, respectively. The surface hydrophobicity of the PP treated by alcalase and trypsin tended to increase and then decrease with the increase in the *DH*, and the surface hydrophobicity of PP was greatest at a *DH* of 2%. A similar result has been reported for rice glutelin treated with trypsin [18]. The increase in hydrophobicity was due to the partial enzymatic hydrolysis that fully stretched the protein structure, thus exposing the hydrophobic sites wrapped inside the protein molecules [35]. With further increases in enzymatic hydrolysis, the hydrophobicity of the protein decreased. Enzymatic hydrolysis broke down hydrophobic regions or led to protein–protein aggregation, thereby reducing the number or surface area of hydrophobic groups exposed to water [26,34]. In general, trypsin enzymatic hydrolysis has the greatest effect on the surface hydrophobicity of PP.
