The Effect of Limited Proteolysis by Trypsin on the Formation of Soy Protein Isolate Nanofibrils

Nanoﬁbril system constructed by protein self-assembly is widely used in the food industry because of purposive functional properties. Soy protein isolate nanoﬁbrils (SPINs) were reported to form via heating at pH 2.0. In this research, the soy protein isolate (SPI) hydrolysate prepared by trypsin was used as a raw material for the formation of nanoﬁbrils called soy protein isolate hydrolysate nanoﬁbrils (SPIHNs). Microscopic images demonstrated the formation of two nanoﬁbrils. Based on circular dichroism spectroscopy and Thioﬂavin T (ThT) ﬂuorescence spectral, we concluded that β -sheet played an important role in SPIN and SPIHN’s structural composition. At the same time, the α -helix in SPI had not been destroyed, thereby favoring the formation of SPIHN. The surface hydrophobicity of SPIHN continued to increase during the heating process and reached the highest value when heating 8 h. SDS-PAGE analysis showed that peptides produced by enzyme-modiﬁed SPI aﬀected the formation of SPIHN. These results proposed that enzymatic hydrolysis prior to acidic during ﬁbrillation process aﬀected the ﬁbrillation of SPI, and the peptides formed by enzymatic hydrolysis were more eﬃcient for the self-assembly process. This study will provide a theoretical basis for the future research of SPI nanoﬁbril functionality.


Introduction
Self-assembly has been applied as an emerging method for the study of functional properties of proteins. In past studies, many proteins including egg protein [1], whey protein [2,3], soy protein [4,5], and bovine serum albumin [6] had been confirmed to form nanofibrils by self-assembly under acidic conditions (pH 2.0), accompanying the heating process. e nanofibrils are morphologically characterized by a linear fibril structure with a nanometer diameter and a micron length [7]. In general, it has been confirmed that nanofibril generation mainly depended on the interaction of noncovalent bonds, which is the synergistic result of hydrogen bonding, hydrophobic interaction, and electrostatic interaction [7]. Since cysteine residues were protonated during fibrillation, disulfide bonds do not play an important role in fabrication and stabilization of nanofibrils [8]. At the same time, a significant structural change occurs in the self-assembly of proteins and cross-repetitive β-sheet structure provides a unique structure [9]. In food science, researchers focus on wide applications of protein fibrillation such as functional enhancement [10]. e nanofibrils present diverse and reactive functional groups on their surface, which makes them tunable by numerous agents.
Peptides are widely accepted as the building blocks of heat-induced nanofibrils [11]. Hydrolysis occurs before aggregation, and different peptides have a significant effect on the structure of aggregates [12]. erefore, prehydrolyzed proteins rather than intact proteins have accordingly been utilized for fabrication of the nanofibrils. More importantly, the hydrolysis kinetics affect the growth kinetics of nanofibrils the same as temperature, concentration, and time [13]. So enzymatic hydrolysis of proteins could participate in the fibrillation process of nanofibrils and played a role in the growth of nanofibrils.
SPI is a mixture of several proteins that are widely used in industrial production because of their good functional properties, and it plays an important role in plant protein resources. Slender, semiflexible nanofibrils were formed from SPI when heated at 85°C and pH 2.0 [5]. Akkermans et al. found that most properties of soy protein nanofibrils were comparable to whey protein and even better than whey protein in some respects [5]. Long-term research showed that soy protein could be fabricated into various nanostructures.
erefore, nanofibrils which are based on soy protein have widespread application prospects. Enzymatic hydrolysis had been successfully applied to soy protein isolates to produce functional peptides [14]. Enzymatic hydrolysis produces peptides that may provide a new selfassembly strategy for nanofibrils prior to acid hydrolysis. e combination of enzymatic hydrolysis and self-assembly can improve the functional properties of proteins [15]. is study aimed at the hydrolysis of SPI by trypsin and then induced the hydrolysis products to form nanofibrils, comparing the morphological, structural, and functional properties of the SPIHN with SPIN. e objectives of this work were to study the importance of enzymatic hydrolysis in the formation of nanofibrils. e results would reveal the mechanism of enzymatic hydrolysis on the self-assembly process and found new ways to produce nanofibrils. e basic composition and content of SPI are shown in Table 1. Trypsin (with a minimum activity of 250 NFU·mg −1 ), papain (with a minimum activity of 8 × 10 5 U·g −1 ), and neutral protease (with a minimum activity of 5 × 10 4 U·g −1 ) was obtained from Solarbio (Shanghai, China). Bicinchoninic acid (BCA) protein assay kit was purchased from Nanjing Jiancheng Biotechnology Institute Co., Ltd.(Nanjing, China). O-Phthalaldehyde (OPA), 1,4-dithio-DL-threitol (DTT), sodium dodecyl sulfate (SDS), and gel electrophoresis-related reagents were obtained from Solarbio (Shanghai, China). 2-[4-(Dimethylamino)phenyl]-3,6-dimethylbenzothiazolium chloride ( ioflavin T, T) was purchased from Yuanye (Shanghai, China). Anilinonaphthalene-8-sulfonate (ANS) was obtained from Aladdin (Shanghai, China). Other undescribed chemical reagents were of analytical grade.

Nanofibril
Formation. SPI was dissolved in 50 mL distilled water at 4% (W/V). e solution was stirred at room temperature and stored at 4°C overnight for full hydration. ree enzymes were added to the solutions, and then the solutions were adjusted to the appropriate reaction conditions of the three enzymes. e fibrillation process followed the method reported by Tang et al. [16]. Next, the hydrolysates were adjusted to pH 2.0 using 6 M HCl and heating at 85°C for fibrillation. In order to terminate the reaction, the heated sample was placed in an ice bath and the sample was preserved at 4°C.

Degree of Hydrolysis (DH).
e DH measurement by the OPA method as reported by Nissen [17]. e 100 mL OPA solution was as follows: 3.81 g sodium tetraborate, 100 mg sodium dodecyl sulfate (SDS), 80 mg OPA (dissolved in 2 mL of anhydrous ethanol), and 88 mg DL-dithiothreitol. 400 μL SPI hydrolysate (100-fold dilution) was added to 3 mL OPA solution and reacted in the dark for 2 min. e absorbance was measured at 340 nm by a UV-visible spectrophotometer (UV2600, Shimadzu, Japan). e results were calculated using a standard curve prepared with serine. e DH was calculated using the following equation: where h 1 and h 0 represent the amino acid concentrations of hydrolyzed and nonhydrolyzed SPI, respectively. h tot is the total amino acid concentration of SPI.

Atomic Force Microscope (AFM).
e microstructure images were analyzed by AFM. e samples were diluted to 0.01 mg·mL −1 with distilled water (pH 2.0). en, 20 μL diluted sample was added to a glass slide and air-dried for 30 min at room temperature. Imaging was performed by Icon (Dimension Icon, Bruker Ltd., US) in the tapping mode, and the scan rate was 1.2 Hz. AFM images were processed by NanoScope Analysis (1.8.0.0).

Particle Size Measurement.
Particle size distribution and light scattering intensity of nanofibrils were measured by Zetasizer Nano (Zetasizer Nano Zs, Malvern Instruments Ltd., UK). e samples' concentration was diluted to 0.1 mg/ mL with distilled water (pH 2.0). e distilled water was filtered by a 0.22 μm filter before being used. e diluted sample was added into the cuvette, and the sample amount according to the reference quantity was provided by the instrument manufacturer. Dynamic light scattering (DLS) analysis was performed with a backscattering angle of 173°a nd a refractive index of 1.45 for protein particles at 25°C.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
. SDS-PAGE in a discontinuous vertical plate system according to Laemmli's method [18]. e gel sheet used in the experiment was 10% resolving gel (pH 8.8) and 5% stacking gel (pH 6.8). e sample was diluted to 2 mg/mL by dilution. e diluted sample was heated in a water bath at 100°C for 4 min, cooled, and centrifuged at 11000 ×g for 5 min. Marker's molecular weight range was from 11 kDa to 180 kDa. 10 μL of sample supernatant and 5 μL of marker were loaded into the lanes. e gel was stained with Coomassie brilliant blue G250 for 30 min, and the gel sheet was then decolorized.

Determination of Surface
Hydrophobicity. Using ANS to measure surface hydrophobicity is the most appropriate method for analyzing the three-dimensional structure of proteins [19]. According to the method of Luo et al. and modifying it [20], the samples were diluted to 0.02, 0.04, 0.08, and 0.16 mg/mL with 0.01 mol/L phosphate buffer (pH 7.0). en, 30 μL ANS solution (8 mmol/L ANS and 0.01 mol/L phosphate buffer at pH 7.0) was added to aliquots of the dilute sample (5 mL), and the mixture was vortexed and reacted in dark for 15 min. e fluorescence spectra were obtained by a fluorescence spectrometer (Hitachi F4500, Hitachi High-Technologies Corp., Japan). e parameter excitation wavelength was 390 nm, emission wavelength was 400-600 nm, and slit width was 10 nm. e initial slope of the regression line in the curve of protein concentration and fluorescence intensity is the surface hydrophobicity of the protein.

T Fluorescence Assay.
e stock solution (0.8 mg/mL T, 10 mM phosphate, and 150 mM NaCl, pH 7.0) was filtered through a 0.22 μm filter.
T stock solution was sealed with metal foil and stored in the refrigerator at 4°C. e working solution was prepared by diluting the T stock solution 50-fold with the above phosphate-NaCl buffer. 50 μL sample was mixed with 5 mL T working solution, and the mixture was magnetic stirred for 1 min. e fluorescence intensity was obtained by a fluorescence spectrometer (Hitachi F4500, Hitachi High-Technologies Corp., Tokyo, Japan). e excitation wavelength was 460 nm (slit width � 5 nm), and the emission spectra were obtained from 480 to 550 nm with a scanning speed of 240 nm/min.

Circular Dichroism Spectroscopy.
Circular dichroism spectroscopy (CD) can analyze changes in secondary structure of SPIN and SPIHN. Far-UV region (190-260 nm) spectra were measured at room temperature with Jasco spectropolarimeter (model J-815, Jasco, Japan). Different samples were diluted to 0.2 mg/L by distilled water, and the samples were placed in a 1.0 mm path length quartz cells and scanned at 100 nm/min. All data were analyzed by CDNN software.

Statistical Analysis.
All experiences were analyzed in triplicate, and values given in the tables and figures were reported as the mean ± standard deviation (error bars). Statistical analysis was performed using the SPSS (20.0) software. Significance of differences (p < 0.05) between means was determined by Duncan's multiple-range test.

Selection of Enzymes and Determination of Degree of Hydrolysis.
ree common enzymes, including trypsin, papain, and neutral proteases, were selected in this study. Excessive hydrolysis will greatly reduce the production of nanofibril because the nonnucleated regions will be separated from the self-assembled regions of the nanofibril, thus affecting the nanofibril formation process [21], and the degree of hydrolysis of enzyme-modified soy protein was usually controlled between 1% and 25% [22]. According to our research requirement, we controlled the same hydrolysis degree (1%) of three SPI enzymatic hydrolysate (trypsin, 18 min; papain, 5 min; neutral protease 9 min; Figure 1). e three hydrolysates of SPI were formed to nanofibrils via heating at pH 2.0 with different times (1, 2, and 4 h). According to the T fluorescence intensity analysis (Figure 2), it could be concluded that the fluorescence intensity of the trypsin group increased significantly with time, while the fluorescence intensity of the papain and neutral protease groups did not change substantially and the peak values were low. is indicated that less nanofibrils were formed by the papain and neutral protease treatment. e nucleation mechanism indicated that homogeneous nucleation was essential for the fibrillation process, and the wrong nucleation process will lead to the emergence of nonfibril aggregates [23].

Atomic Force Microscope (AFM)
Analysis. AFM as one of the most versatile single-molecule techniques has emerged within the last decade as a powerful tool for the study of the structural properties of nanofibril and the fibrillation process [24,25]. Figure 3 shows the AFM images of SPIN and SPIHN. When unheated, there was no large aggregates in the SPI, and protein exhibited the spherical structure. We tried to study the formation of two kinds of nanofibrils by prolonging the heating time. When heated for 8 h, there was a significant difference in the AFM images of the two nanofibrils. In SPIN (Figure 3(b)), the morphology of the nanofibrils was short and thick with an average length of 302 nm. In SPIHN (Figure 3(c)), nanofibrils with an average length of 194 nm were shorter than SPIN, had almost no branches, and appeared more flexible in morphology. is indicated that enzymatic hydrolysis played a role in the formation mechanism of nanofibrils, which changed the morphology of nanofibrils. Fibril-forming peptides prepared by enzymatic hydrolysis could result in minor differences in binding between peptide units, which were infinitely magnified during nanofibril development, and could lead to morphological differences [26]. Many nonfibril aggregates were observed in SPIN; instead, there was no appearance of aggregates in SPIHN. e appearance of aggregates may indicate that the fibrillation process had entered the later stage, that is, the reverse phase of nanofibril formation, and the nanofibrils were decomposed into different monomers. Whereafter, a portion of the unstable monomers converge to form nonlinear aggregates with different sizes and morphologies [27]. Generally, the nanofibrils entangled and clustered at some domains [2]. But the domains could clearly observe the fibril-like structure, while the aggregates in SPIN appeared as dense spheres. We believed that the formation of aggregates was due to unstable monomers.
It is clear that the unfolding of proteins is a common requirement before self-assembly forms nanofibrils [28]. e conformationally altered monomer or the partially unfolded state has a strong tendency to form protein nanofibrils. Formation of protein aggregates with random shape during the enzymatic hydrolysis of proteins could probably influence the fibrillation extent [29].
3.3. Particle Size Measurement. DLS was very sensitive to the formation of tiny aggregates in protein solutions [30]. Once the monomer particles in the solution were aggregated, the light waves will change greatly. Figure 4 shows that particle size distribution of samples with different heating times. Figure 4(a) shows that SPIN had a small change in particle size distribution between 0 h and 2 h, which may be the preparation stage for fiber formation. Bimodal peaking began to appear when heated for 4 h, four peaks appeared when heated for 8 h, and the peak value of the aggregate had reached 1000 nm. Figure 4(b) shows that the peak of the particle size distribution of SPIHN did not change from 0 h to 2 h, which was consistent with SPIN. When heated for 4 h, the bimodal contribution indicated that the formation of nanofibrils (especially elongation of nanofibrils) was only formed during heating with enough time [31]. Figure 5 shows the average particle size change in SPIN and SPIHN. Enzymatic hydrolysis reduced the average particle size of SPI. e average particle size of SPIN was decreasing from 0 h to 2 h. e phenomenon of SPIHN occurred from 0 h to 4 h. is should be because heating caused the protein to release more peptides, which was known as the preparation phase of the fibrillation process [32]. Akkermans et al. demonstrated the self-assembly fibrillation process was related to the changes in protein conformation and peptide hydrolysis, and peptides were the main building blocks of self-assembly fibrils [11]. e change in the average particle size indicated that the enzymatic hydrolysis did not significantly affect the minimum average particle size, which was consistent with the results of Xia et al [33]. We found a more interesting phenomenon; that is, the minimum average particle size of nanofibrils was between 100 nm and 150 nm. We could boldly guess that the particle size of the system after releasing the peptide also affects the self-assembly process. After heating for 4 h, the average particle size of SPIN and SPIHN increased rapidly. Peptides with excellent self-assembly ability as building blocks rapidly Fluorescence intensity (a.u.) Wavelength (nm)

T Fluorescence Assay.
T is cationic benzothiazoles that combine with the β-sheet structure of nanofibrils to increase the fluorescence intensity significantly [34]. e possible mechanism was that the thiazole nitrogen forms a hydrogen bond with the hydroxyl group of the tissue structure, allowing these dye molecules to specifically bind to the nanofibrils [35]. Vassar demonstrated that T bonded to nanofibrils better than congo red, crystal violet and Van Gieson [36]. T could be used as a fluorescent indicator, which detected the dynamic process of nanofibril formation [37].
Prior to heating, the natural and hydrolyzed SPI fluorescence intensity curves did not change (Figure 6(a)). is was because they contain highly twisted β-sheets structure, but few β-strands cause the binding sites to be short or distorted, resulting in failure to combine with T [38,39]. When the heating time was continuously increased, the fluorescence intensity of SPIN and SPIHN was significantly increased because the β-sheets structure gave rise to a specific sequence of cross-strand ladders during self-assembly, and these specific side chains could allow T to bind [40]. e fluorescence intensity of untreated SPIforming nanofibrils was much lower than the nanofibrils previously reported by Tang et al. because the affinity of T was lower at acidic conditions than at neutral conditions, and this was because the electrostatic repulsion of positive charges hindered the combination of dyes and nanofibrils [41]. However, increasing the ionic strength could shield the charges between dye and nanofibrils to overcome the decrease in affinity at acidic pH [42], but in order to discuss the effect of enzymatic hydrolysis on the formation of nanofibrils, this study did not add salt in system. e fluorescence intensity of SPIN decreased when heating time from 4 h to 8 h. At this time, the average particle size of SPIN reached the maximum value, and the decreased fluorescence intensity may be attributed to the disordered aggregation of nanofibrils into other nanofibrils [43,44], but not all nanofibrils had been transformed, so the fluorescence intensity was not rapidly decreased. e fluorescence intensity of SPIHN reached the maximum at 8 h, which was 2179.46 ± 12.56.
It could be seen from Figure 6(c) that there was no significant difference in the maximum fluorescence intensity of SPIHN when heated for 1 h-4 h, indicating that a large number of nanofibrils were not generated. e maximum fluorescence intensity of SPIN had a significant difference. e process of nanofibrils formation went through the nucleation period [45]. Maximum fluorescence intensity indicated that SPIN generated faster than SPIHN, and  e theory of self-assembly mechanism established by Mishra et al. indicated that nicking will form during the protein selfassembly fibrillation process. When proteins lost peptides, the nicking was formed. ese proteins will form the core of nanofibrils [46]. Enzymatic hydrolysis greatly increased the number of peptides resulting in nicking. erefore, SPIHN had a longer nucleation time, and the maximum fluorescence intensity was significantly increased after heating for 8 h.

Surface Hydrophobicity Determination.
Surface hydrophobicity can reflect the number of hydrophobic groups on the surface of protein molecules and also reflects change in the molecular structure of proteins [47]. e change in surface hydrophobicity can well reflect the effect of hydrophobic interaction on the formation of protein aggregates [48]. Table 2 shows the surface hydrophobicity of SPIN and SPIHN at pH 2.0 for different heating times (0, 1, 2, 4, and 8 h). e surface hydrophobicity of untreated SPI was 865.16 ± 4.90. When heating began, the surface hydrophobicity of SPIN decreased drastically. e previously described nicking theory could explain this phenomenon, and the special region of nicking with high hydrophobicity and low charge will form the core of nanofibrils [46]. Nucleation process resulted in aggregation of hydrophobic groups exposed to heat. In the initial stage of heating (from 1 h to 2 h), the surface hydrophobicity had a significant increase. Nanofibril formation process leaded to peptides with a higher surface hydrophobicity self-assembled to fibrillar structures during the heating at pH 2.0 [49]. When heated for 4 h, the surface hydrophobicity had a significant decrease. After reaching a maximum, the surface hydrophobicity of SPIN tended to decrease because of aggregation, during which the exposed hydrophobic groups were buried in the nanofibrils [50].
In SPIHN, the surface hydrophobicity of SPI modified by trypsin significantly increased. Trypsin-modified SPI will produce hydrophobic amino acids, such as arginine and lysine [51]. e surface hydrophobicity of SPIHN continued to increase at different heating times, which was much greater than SPIN. e result of SPIHN was consistent with that reported by Gao et al [8], and Gao found that nanofibrils formed by the four enzyme-modified proteins all showed an increase in surface hydrophobicity. Partial unfolding of proteins during fibrillation will expose more hydrophobic groups, which may promote the formation of nanofibril. In addition, surface hydrophobicity was a structure-related function, depending on the size and shape of protein molecule, amino acid composition, and sequence, as well as any intramolecular or intermolecular cross-links [52,53]. erefore, it was necessary to choose more reasonable means to modify the protein and improve the surface hydrophobicity. Xu et al. reported that the surface hydrophobicity of the high concentration SPI had a high value during heating,   [32]. Changing protein concentration was probably the easiest way. Figure 7 shows SDS-PAGE images of SPIN and SPIHN. Untreated SPI and trypsinmodified SPI have two high-molecular-weight major bands (86 kDa and 74 kDa). As the heating time increased, both of the main bands disappeared. After heating for 2 h, neither of SPIN nor SPIHN observed these two bands. In addition to the two major bands, many low-molecular-weight bands had emerged, which might be peptide fragments released by heating at acidic pH. ere were persistent bands of between 25 and 63 kDa that remained dominant at 1, 2, and 4 h. When heated to 8 h, there were almost no bands in the lane A5, the molecular weight was concentrated at 19 kDa in the lane B5, and a part of the molecular weight was <17 kDa. SPIHN had stronger bands than SPIN at 19 kDa at different times, which indicated that the prehydrolyzed sample was heated at acidic conditions producing a heterogeneous mixture of the peptides affecting the formation of SPIHN. e AFM image showed that SPIHN had better fibrillation outcome than SPIN when heated for 8 h. Combined with electrophoresis bands, we concluded that the low-molecular-weight peptide (about 19 kDa) assisted SPIHN with formation of more nanofibrils. e low-molecular-weight peptide produced by enzymatic hydrolysis provided many building blocks for the formation of nanofibrils. Mohammadian et al. reported that the heterogeneous compounds formed by prehydrolysis affected the self-assembly process of WPI nanofibrils [15].

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis.
T fluorescence spectroscopy showed that the fluorescence intensity of SPIHN from 0 h to 4 h did not increase significantly compared with SPIN, which may be related to the formation of heterogeneous compounds. e surge at fluorescence intensity of SPIHN at 8 h may indicate that the peptide produced by enzymatic hydrolysis self-assembled to form more nanofibrils.
According to some reports, after long heating of β-lactoglobulin and pea protein, the protein was completely hydrolyzed into peptides smaller than 20 kDa, and no new bands appeared, which was consistent with the results of our study [31,54]. e process of self-assembly of different proteins continuously generated low molecular peptides. ese results may indicate that self-assembly was the process of transferring macromolecular proteins to small molecular peptides.

Circular Dichroism (CD) Spectroscopy Analysis.
e formation of protein nanofibrils is accompanied by changes in the secondary structure, and we can better understand the self-assembly process according to the changes in the secondary structure. When not heated, the α-helix and β-sheet contents of SPI were 16.37 ± 0.55% and 31.70 ± 0.44%, respectively. When heated for 1, 2, and 4 h, the α-helix of SPIN gradually decreased, while the β-sheet increased continuously (Tables 3 and 4). is indicated that changes in secondary structure were very important for the formation of nanofibrils. Tang et al. demonstrated that the linear aggregates formed by the soy protein self-assembly process were accompanied by the destruction and reorganization of the secondary structure [16], which was consistent with the results of our study.
is was because the α-helix was converted to the β-chains, and then, the structural basis was increased for the formation of nanofibrils [55]. Fandrich et al. found that the molecular structure within the nanofibrils was different from the β-sheet in the native proteins [56]. When heated for 8 h, the β-sheet structure, which was one of the indexes for evaluating the formation of nanofibrils [34], decreased to 22.23 ± 0.12%, and the β-turn increased to 24.13 ± 0.21%. e previous analysis indicated that the nanofibrils had entered the reverse reaction phase after heating for 8 h. Aggregation resulted in the destruction of the β-sheet structure of the nanofibril.
Enzymatic hydrolysis affected the secondary structure of SPI, resulting in the decrease in α-helix (7.23% vs. 16.37%) and the increase in β-sheet (41.60% vs. 31.70%). In SPIHN, the β-sheet continued to increase and reached 62.13 ± 0.15% after heating for 8 h. With the increase in heating time, the content of β-sheet structure in SPIHN increased significantly, which was consistent with the surface hydrophobicity variation trend. e β-sheet formation occurred concurrently with the formation of aggregates with hydrophobic domains [57]. We were not talking too much about the dominance of β-sheets in nanofibrils. Because we found an interesting phenomenon, enzymatic hydrolysis did not lead to the destruction of the α-helix structure in SPIHN, which provided a good basis for the formation of nanofibrils. Gao et al. found the same conclusion with the nanofibrils formed by WPC [8]. e α-helix was used as components of coiled coils, which is the basic folding pattern found in native proteins [58]. It was found that the structural sequence that forms the α-helix requires the participation of hydrophobic residues and polar residues [59]. Dipeptides kinetic studies had found that peptide bonds with C-Asp terminal residues exhibited instability under thermal and acid conditions [60]. e appearance of peptides with N-terminal-Asp was confirmed by hydrolysis of proteins [29,61]. Trypsin cleaves the carboxyl sides of the lysine and arginine residues in the polypeptide chain. Aspartic acid, arginine, and lysine are all polar amino acids. More polar amino acids could increase the content of α-helix structure.

Conclusion
Hydrolysates produced by papain and neutral protease modification of SPI could not be heated to form nanofibrils at pH 2.0. e nanofibrils formation of SPI and SPI enzymatic hydrolysate were multistage under heating at pH 2.0. SPIHN fibrillated to a more extent and had a more flexible structure. Enzymatic hydrolysis provided more building blocks for the formation of nanofibrils, which improved the properties of nanofibers. In addition, the particle size distribution, T fluorescence spectrum, and surface hydrophobicity reflected the dynamic process of self-assembly, and SPIN entered the reverse reaction stage earlier. e formation of SPIN and SPIHN was a process that breaks down high-molecular-weight proteins into low-molecularweight peptides. e α-helix of the trypsin-modified SPI was not destroyed, which provided a structural basis for the better formation of nanofibrils.
is study showed that SPIHN formed by trypsin modification could be readily applied to the food industry, and the next research will focus on the preparation of hydrogels using SPIHN as a delivery system.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest.