Protein nanoparticle-nanofiber composite material and preparation method thereof

Abstract

The invention discloses a protein nanoparticle-nanofiber composite material and a preparation method thereof, and belongs to the technical field of food processing and functional protein materials. The method comprises the following steps: (1) taking fresh egg white, diluting and stirring to obtain uniform egg white protein dispersion liquid; (2) performing acylation modification on the egg white protein dispersion liquid to increase surface negative charges and improve conformation flexibility; (3) performing controllable enzymolysis on the acylated egg white protein, so that part of peptide chains of the acylated egg white protein are broken and oligopeptide with self-assembly capability is formed; and (4) carrying out heat treatment on the enzymolysis product, inducing the peptide fragment to be self-assembled to form nano-particles while inactivating the protease through heat treatment, further forming a nano-fiber network dominated by a beta-folding structure, and finally obtaining the protein nano-particle-nano-fiber composite system. Through the synergistic effect of acylation, enzymolysis and heat treatment, accurate conversion of protein from a natural spherical conformation to a functional nanostructure is achieved, and the obtained composite material has the dispersion stability of particles and the network enhancement effect of fibers and has excellent foamability, emulsibility and gelling property.

Description

Technical Field

[0001] This invention belongs to the field of food processing and functional protein materials technology. Specifically, it relates to a protein nanoparticle-nanofiber composite material prepared by an acylation-enzymatic hydrolysis-thermal treatment process and its preparation method, which can be applied to the development of food structure regulation, functional hydrogels, emulsifiers and edible biomaterials. Background Technology

[0002] Proteins, as important natural polymers, are widely used in the food, pharmaceutical, and biomaterials fields due to their excellent biocompatibility, biodegradability, and abundant functional groups. Egg white protein, in particular, as a high-quality animal protein resource, has become an important raw material for the development of functional protein materials due to its outstanding functional properties, wide availability, and low cost. However, natural proteins typically have a compact globular conformation, with hydrophobic groups largely embedded within the molecule, resulting in slow interfacial adsorption rates, weak film-forming ability, and poor dispersion stability. Especially against the backdrop of the increasing demand in the modern food industry for high internal phase emulsions, stable foam systems, and functional gel materials, the performance of natural globular proteins is no longer sufficient to meet the requirements of structural controllability and functional stability.

[0003] To improve the functional properties of proteins, researchers have developed various modification methods. Traditional modification methods include thermal denaturation, chemical cross-linking, and single enzymatic hydrolysis. While these methods improve the processing characteristics of proteins to some extent, they generally suffer from problems such as harsh processing conditions, low precision in structural regulation, and the potential for irreversible aggregation or difficulty in enzyme inactivation. For example, the patent technology with publication number CN111286038A enhances the water solubility of zein through a covalent reaction between polyglyceraldehyde and zein. However, this method relies on strong oxidants and non-selective chemical cross-linking, resulting in limited reaction specificity and difficulty in completely removing potential residues, thus failing to meet the requirements of mild and safe food systems. The patent technology with publication number CN115989866A uses strong acid pretreatment and prolonged thermal induction at 85°C to form soybean protein fibrils. Although it can obtain specific structures, the overall processing conditions are quite harsh, potentially leading to insufficient precision in structural regulation and decreased nutritional and functional retention, and also limiting its extension to green and controllable processing methods.

[0004] In recent years, the nanostructuring of proteins has gradually become an important approach to enhance their functional properties. Studies have shown that nanoparticles can be rapidly adsorbed at gas-liquid or oil-water interfaces to form dense particle layers, thereby enhancing the viscoelasticity and fracture resistance of interfacial films; while nanofibers can form continuous network structures within the system, increasing viscosity and yield stress, providing three-dimensional mechanical support and long-term stability for foams or emulsions. Therefore, how to precisely control protein self-assembly within a system and simultaneously generate these two complementary nanostructures using a mild and green method is a key technological bottleneck currently facing this field. Existing methods are often complex, require stringent conditions, or can only generate nanostructures of a single morphology, failing to achieve synergistic effects between particles and fibers.

[0005] Therefore, there is an urgent need to develop a new method that is simple, mild, cost-effective, and environmentally friendly to achieve the controllable and efficient transformation of proteins from a globular conformation to a "nanoparticle-nanofiber" composite structure, thereby providing a brand-new technical solution for the development of high-performance protein-based functional materials. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of low precision in structural regulation and limited improvement in functional performance in existing protein modification methods, and to provide a green and efficient method for preparing protein nanoparticle-nanofiber composite materials.

[0007] To achieve the above-mentioned technical objectives, the inventors introduced a synergistic process of acylation-enzymatic hydrolysis-thermal treatment to realize the controllable transformation of egg white protein molecules from a globular conformation to a nanostructure, thereby significantly improving its interfacial activity, gelation properties, and dispersion stability.

[0008] Specifically, the objective of this invention is achieved as follows: a method for preparing a protein nanoparticle-nanofiber composite material, the method comprising the following steps:

[0009] (1) Dispersing egg white: Wash fresh eggs, dry the surface moisture, and use an egg separator to obtain the egg white part; mix the egg white with distilled water, gently stir with a magnetic stirrer, and sieve to obtain a uniform egg white protein dispersion.

[0010] (2) Acylation modification: Add the acylation agent to the egg white protein dispersion, and use sodium hydroxide to adjust the pH of the system to stabilize at 8.0-8.2 to carry out acylation modification, so as to increase the negative charge on its surface and improve the conformational flexibility; after stabilizing for 0.5-1 h, adjust the pH of the system to 7.0-7.2 with hydrochloric acid to end the reaction and obtain the acylated egg white protein dispersion;

[0011] (3) Controllable enzymatic hydrolysis: Add protease to the acylated egg white protein dispersion and enzymatically hydrolyze it for 10-20 min under mild conditions of 35-45℃ water bath, so that some peptide chains break down and form oligopeptides with self-assembly ability, to obtain acylated-enzymatically hydrolyzed egg white protein dispersion.

[0012] (4) Heat treatment-induced self-assembly: The acylated-enzymatically hydrolyzed egg white protein dispersion was heat-treated in a water bath at 80-100℃ for 10-20 min. This heat treatment inactivates the protease, and the protein fragments in the system self-assemble into nanoparticles and induce the formation of β-sheet-dominated nanofiber structures, thereby obtaining egg white protein nanoparticle-nanofiber composite material.

[0013] More preferably, in the preparation method of the protein nanoparticle-nanofiber composite material as described above, the volume mixing ratio of egg white and distilled water in step (1) is 1:2 to 1:4 (v / v).

[0014] More preferably, in the preparation method of the protein nanoparticle-nanofiber composite material as described above, the stirring time in step (1) is 1 to 3 hours, and the sieve mesh size for sieving is 60 to 80 mesh.

[0015] More preferably, in the preparation method of the protein nanoparticle-nanofiber composite material as described above, the acylating agent is added to the egg white protein dispersion in step (2) by a small amount and multiple times.

[0016] More preferably, in the preparation method of the protein nanoparticle-nanofiber composite material as described above, the acylating agent in step (2) is selected from one or more of succinic anhydride, maleic anhydride and acetic anhydride.

[0017] More preferably, in the preparation method of the protein nanoparticle-nanofiber composite material as described above, in step (2), the acylating agent is added to the egg white protein dispersion at a ratio of 1:100 to 1:200 (w / v).

[0018] More preferably, in the preparation method of the protein nanoparticle-nanofiber composite material as described above, the method of adding protease to the acylated egg white protein dispersion in step (3) is a small-volume, multiple-time method.

[0019] More preferably, in the preparation method of the protein nanoparticle-nanofiber composite material as described above, the protease in step (3) is selected from one or more of papain, alkaline protease and trypsin.

[0020] More preferably, in the preparation method of the protein nanoparticle-nanofiber composite material as described above, the enzyme activity of the protease in step (3) is 500-1000 U / mg, and it is added to the acylated egg white protein dispersion at a ratio of 0.01-0.1% (w / v).

[0021] In addition, the present invention also provides a protein nanoparticle-nanofiber composite material prepared according to the above method.

[0022] Compared with the prior art, the protein nanoparticle-nanofiber composite material and its preparation method disclosed in this invention have the following advantages and advancements:

[0023] (1) Precise structural regulation: This invention achieves the controllable transformation of egg white protein from a natural spherical structure to a composite structure of nanoparticles and nanofibers through a synergistic process of acylation-enzymatic hydrolysis-thermal treatment, overcoming the problems of uncontrollable structure and limited performance improvement of traditional modification methods.

[0024] (2) Significantly improved performance: The composite material obtained by the present invention has both the high dispersibility of nanoparticles and the network reinforcement effect of nanofibers, which significantly improves the foaming, emulsifying and gelling properties of the system.

[0025] (3) Green and controllable process: The entire process of this invention is mild and does not require the use of organic solvents or toxic crosslinking agents. The reaction process is easy to monitor and adjust, the product is highly safe, and it meets the requirements of green chemistry and sustainable development.

[0026] (4) Broad application prospects: The protein nanoparticle-nanofiber composite material obtained by this invention can be used as a functional structural factor in food emulsifiers, foaming agents, edible hydrogels and biocompatible carrier materials, providing a new technical path for the efficient utilization of high-value-added protein resources. Attached Figure Description

[0027] Figure 1 SDS-PAGE of the egg white protein dispersion systems prepared in Examples 1-5.

[0028] Figure 2 Transmission electron microscopy images of the egg white protein dispersion systems prepared in Examples 1-5.

[0029] Figure 3 The particle size distribution diagrams are for the egg white protein dispersion systems prepared in Examples 1-5.

[0030] Figure 4 The content of secondary structures in the egg white protein dispersion systems prepared in Examples 1-5.

[0031] Figure 5The results show the surface hydrophobicity of the egg white protein dispersion systems prepared in Examples 1-5.

[0032] Figure 6 The foaming properties of the egg white protein dispersion systems prepared in Examples 1-5 are shown. Detailed Implementation

[0033] To better explain the present invention, the main contents of the present invention are further illustrated below with reference to specific embodiments, but the scope of protection of this application is not limited to the following embodiments.

[0034] Example 1 (Conventional Dispersion)

[0035] (1) Dilute fresh egg whites with distilled water at a ratio of 1:2 (mL / mL) and gently stir with a magnetic force for 2 h.

[0036] (2) Pass the egg white through a 60-mesh sieve to ensure even dispersion.

[0037] (3) Adjust the pH to 7.0 with hydrochloric acid to obtain a fresh egg white protein dispersion.

[0038] Example 2 (Enzymatic hydrolysis after dispersion)

[0039] (1) Dilute fresh egg whites with distilled water at a ratio of 1:2 (mL / mL) and gently stir with a magnetic force for 2 h.

[0040] (2) Pass the egg white through a 60-mesh sieve to ensure even dispersion.

[0041] (3) Adjust the pH to 7.0 with hydrochloric acid to obtain a fresh egg white protein dispersion.

[0042] (4) Add 0.05% (g / mL) of papain (800 U / mg) to it, and hydrolyze it in a water bath at 45℃ for 10 min to obtain the enzymatically hydrolyzed egg white protein dispersion.

[0043] Example 3 (Acylation after Dispersion)

[0044] (1) Dilute fresh egg whites with distilled water at a ratio of 1:2 (mL / mL) and gently stir with a magnetic force for 2 h.

[0045] (2) Pass the egg white through a 60-mesh sieve to ensure even dispersion.

[0046] (3) Add succinic anhydride (1:150, g / mL) in small amounts several times, adjust the pH to above 8.0 with sodium hydroxide, and after the pH stabilizes at around 8.0 for 1 h, adjust the pH to 7.0 with hydrochloric acid to obtain succinylated egg white protein dispersion.

[0047] Example 4 (Acylation and Enzymatic Hydrolysis after Dispersion)

[0048] (1) Dilute fresh egg whites with distilled water at a ratio of 1:2 (mL / mL) and gently stir with a magnetic force for 2 h.

[0049] (2) Pass the egg white through a 60-mesh sieve to ensure even dispersion.

[0050] (3) Add succinic anhydride (1:150, g / mL) in small amounts several times, adjust the pH to above 8.0 with sodium hydroxide, and after the pH stabilizes at around 8.0 for 1 h, adjust the pH to 7.0 with hydrochloric acid to obtain succinylated egg white protein dispersion.

[0051] (4) Add 0.05% (g / mL) of papain (800 U / mg) to it, and enzymatically hydrolyze in a water bath at 45℃ for 10 min to obtain succinylated-enzymatically hydrolyzed egg white protein dispersion.

[0052] Example 5 (Acylation after dispersion, enzymatic hydrolysis, and heat treatment)

[0053] (1) Dilute fresh egg whites with distilled water at a ratio of 1:2 (mL / mL) and gently stir with a magnetic force for 2 h.

[0054] (2) Pass the egg white through a 60-mesh sieve to ensure even dispersion.

[0055] (3) Add succinic anhydride (1:150, g / mL) in small amounts several times, adjust the pH to above 8.0 with sodium hydroxide, and after the pH stabilizes at around 8.0 for 1 h, adjust the pH to 7.0 with hydrochloric acid to obtain succinylated egg white protein dispersion.

[0056] (4) Add 0.05% (g / mL) of papain (800 U / mg) to it and hydrolyze it in a water bath at 45℃ for 10 min.

[0057] (5) After 10 min in a 90℃ water bath, a succinylated-enzymatically hydrolyzed-heat-treated egg white protein dispersion (nanoparticle-nanofiber composite material) was obtained.

[0058] Example 6 Method Identification

[0059] (1) SDS-PAGE analysis

[0060] To verify the effect of the method of the present invention on the regulation of protein structure, samples were prepared according to the steps of Examples 1 to 5, and their molecular weight distribution and structural changes were analyzed by SDS-PAGE.

[0061] Dilute the sample dispersion to 2 mg / mL, mix with loading buffer at a 4:1 ratio, and heat in a boiling water bath for 10 min. After cooling, add the protein sample and marker to the pre-prepared gel. Set the initial voltage to 80 V, and adjust the voltage to 120 V after half an hour. Stain the gel surface with Coomassie Brilliant Blue for 60 min, and shake thoroughly to ensure uniform staining. After staining, perform diffusion destaining in destaining solution (237.5 mL of 95% ethanol and 50 mL of glacial acetic acid mixed with water to a final volume of 1000 mL) for 4–8 h until the background is clear.

[0062] Figure 1 SDS-PAGE results showed that the sample prepared by the method in Example 1 exhibited a distinct main band at approximately 45 kDa, representing the main protein structure. The main band in the sample of Example 2 was significantly weakened, with the appearance of 10-25 kDa fragments, indicating partial enzymatic hydrolysis. The migration position of the sample in Example 3 shifted upwards compared to Example 1, indicating successful succinylation modification and resulting in an increase in apparent molecular weight. The high molecular weight band in the sample of Example 4 was further weakened, while the number of low molecular weight fragments increased, indicating more thorough enzymatic hydrolysis. The EI-SEW band remained weak, and no obvious high molecular weight aggregation bands were observed, suggesting that succinylation effectively inhibited heat-induced aggregation, maintaining good sample dispersibility. Under non-reducing conditions, high molecular weight aggregation bands were present in all samples, while under reducing conditions, these bands were significantly weakened, indicating that aggregation mainly relied on disulfide bonds. The results indicate that enzymatic hydrolysis and acylation successfully altered the original structure of egg white protein, causing partial molecular breakage and the formation of low molecular weight peptides, providing a foundation for the subsequent formation of nanostructures.

[0063] (2) Transmission electron microscopy analysis

[0064] The sample was diluted to 1 mg / mL, then dropped onto a dry copper grid, and 2% phosphotungstic acid was added for negative staining. After it dried naturally, it was observed by transmission electron microscopy.

[0065] Figure 2Transmission electron microscopy (TEM) results showed that the sample prepared by the method in Example 1 mainly exhibited irregular macromolecular aggregates with blurred particle boundaries and severe aggregation, indicating that proteins in their native state are prone to forming disordered aggregates. In Example 2, the sample showed the coexistence of heterogeneous small particles and flocculent aggregates, suggesting that although enzymatic hydrolysis destroyed some macromolecular structures, the exposed hydrophobic groups easily led to secondary association. Example 3 produced near-spherical nanoparticles with regular morphology and uniform dispersion, proving that acylation effectively inhibited non-specific aggregation between proteins. In Example 4, the particles were well dispersed, with short fibers or protofibrillary structures visible in some areas, suggesting that the fragments generated by enzymatic hydrolysis promoted localized ordered assembly. Example 5 exhibited a typical "nanoparticle + nanofiber" composite morphology, with uniformly distributed particles interconnected by slender fibers, forming a sparse network structure. Overall, after synergistic treatment by acylation and enzymatic hydrolysis, the structure of the protein gradually transformed from disordered aggregates into a stable composite nanosystem.

[0066] (3) Particle size analysis

[0067] The sample dispersion was diluted to 2 mg / mL, and the particle size distribution of different samples was determined by a nanoparticle size analyzer. Figure 3 The results showed that the sample prepared by the method in Example 1 exhibited a distinct multimodal distribution with a PDI of approximately 0.5, indicating the presence of numerous heterogeneous aggregates in the system. The particle size distribution was wide and unstable, consistent with transmission electron microscopy observations. The sample in Example 2 showed a further widening of the particle size distribution, indicating that while enzymatic hydrolysis can destroy some macromolecular structures, exposed hydrophobic groups and reaction sites are prone to secondary association, leading to increased polydispersity. The sample in Example 3 showed a significant decrease in average particle size and PDI, indicating that acylation can effectively inhibit non-specific aggregation between proteins and promote the formation of well-dispersed nanoparticles. The sample in Example 4 showed a slight increase in PDI, suggesting that enzymatic hydrolysis, while maintaining nanoparticle size, introduced some fragment diversity, resulting in a slightly wider particle size distribution. The sample in Example 5 had the smallest average particle size and a slightly higher PDI than Example 3, but still maintained a low level. Combined with microscopic results, this indicates that the system formed a composite structure composed of nanoparticles and nanofibers, with the non-spherical morphology leading to a relatively higher PDI.

[0068] (4) Secondary structure analysis

[0069] The sample dispersion was diluted to 0.1 mg / mL and measured using circular dichroism spectroscopy. Specific parameters were: optical length of the cuvette 0.1 cm, scanning wavelength 190-240 nm, resolution 0.1 nm, and scan speed 100 nm / min. To reduce noise, each spectrum was the average of three scans. The protein secondary structure ratio was then predicted using Young's equations using the protein secondary structure calculation software, ProteinSecondary Structure Estimation.

[0070] Depend on Figure 4 As can be seen from the circular dichroism chromatogram results, the sample prepared by the method in Example 1 mainly exhibits α-helices, β-sheets, and random coils, maintaining the ordered nature of the protein's native conformation. In Example 2, the proportion of α-helices increased while the proportion of β-sheets decreased, indicating that enzymatic hydrolysis partially unfolded the protein and exposed the helical regions, reducing intermolecular aggregation. In Example 3, acylation introduced a negative charge, weakening the stability of the helical structure and causing the protein to transform from a tightly ordered helical conformation to a looser folded state, enhancing dispersibility and providing a structural basis for subsequent self-assembly. The structural characteristics of the Example 4 sample were similar to the former, but the α-helices were slightly restored, indicating that enzymatic hydrolysis introduced a certain degree of structural diversity while maintaining dispersibility. The Example 5 sample showed significant enrichment of β-sheets and a reduction in α-helices, with further rearrangement of the protein structure forming an ordered network dominated by β-sheets, consistent with the nanoparticle and nanofiber composite morphology observed by transmission electron microscopy. Overall, this indicates that the protein underwent a transformation from a helical to a folded structure under the synergistic effect of acylation-enzymatic hydrolysis-thermal treatment, providing a structural basis for the formation of composite materials.

[0071] (5) Surface hydrophobicity analysis

[0072] The sample dispersion was diluted to obtain concentration gradients of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1 mg / mL. Then, 20 μL of ANS (8 mM) was added to 4 mL of the diluted solution. The fluorescence spectrum of the sample mixture was measured at 390 / 470 nm using a fluorescence spectrophotometer with a slit width of 5 nm. A linear equation was plotted with protein concentration on the x-axis and fluorescence intensity on the y-axis; the slope of the resulting equation can be defined as the surface hydrophobicity of the protein.

[0073] Figure 5The results showed that the samples prepared by the methods in Examples 1 and 2 maintained high surface hydrophobicity, indicating that unmodified or only enzymatically hydrolyzed proteins still exposed a large number of hydrophobic groups, making them prone to intermolecular aggregation. The surface hydrophobicity of the samples in Examples 3 and 4 decreased significantly, indicating that acylation, by introducing carboxyl groups onto the protein molecule surface, weakened hydrophobic interactions, thereby enhancing the dispersibility and solubility of the system. The surface hydrophobicity of the sample in Example 5 significantly increased compared to Examples 3 and 4, indicating that during enzyme inactivation and heat treatment, the protein underwent conformational rearrangement and formed an ordered structure dominated by β-sheets, with some hydrophobic side chains being re-exposed on the fiber surface. This trend is consistent with the results of circular dichroism spectroscopy and transmission electron microscopy, indicating that the protein underwent a dynamic regulatory process of "hydrophobic exposure-hydrophilization-re-exposure" under the synergistic effect of acylation-enzymatic hydrolysis-heat treatment, providing a molecular basis for the stability and self-assembly of the composite material.

[0074] (6) Foaming property analysis

[0075] Whisk the egg whites for 30 seconds to obtain foam. The ratio of the initial volume of egg whites to the volume of the foam after whisking is defined as the foaming property.

[0076] Figure 6 The foaming experiment results showed that the sample prepared by the method in Example 1 had the lowest foaming performance, indicating that the natural protein molecules have a compact structure and a slow interfacial migration rate, making it difficult to form a stable film at the gas-liquid interface. The foaming performance of Example 2 was significantly improved, indicating that enzymatic hydrolysis partially unfolded the protein and reduced intramolecular binding, thus facilitating bubble formation. Compared with Example 1, the foaming performance of the samples in Examples 3 and 4 was further improved, indicating that acylation enhanced the interfacial activity of the protein after regulating the charge distribution, making it easier to adsorb and stabilize bubbles. The sample in Example 5 had the highest foaming performance, showing a significant advantage, indicating that the nanoparticle-nanofiber composite material formed under the synergistic effect of acylation-enzymatic hydrolysis-thermal treatment has higher interfacial adsorption capacity and film formation strength. This result is consistent with the aforementioned surface hydrophobicity and secondary structure analysis, indicating that the β-sheet-dominated ordered structure and moderate hydrophobic exposure jointly promote the construction of the interfacial film, thereby significantly improving the foaming performance of the protein system.

Claims

1. A method for preparing a protein nanoparticle-nanofiber composite material, characterized in that, The method includes the following steps: (1) Dispersing egg white: Wash fresh eggs, dry the surface moisture, and use an egg separator to obtain the egg white part; mix the egg white with distilled water, gently stir with a magnetic stirrer, and sieve to obtain a uniform egg white protein dispersion. (2) Acylation modification: Add the acylation agent to the egg white protein dispersion, adjust the pH of the system with sodium hydroxide to stabilize it at 8.0-8.2, and after stabilizing for 0.5-1 h, adjust the pH of the system to 7.0-7.2 with hydrochloric acid to end the reaction, and obtain the acylated egg white protein dispersion; (3) Controllable enzymatic hydrolysis: Add protease to the acylated egg white protein dispersion and hydrolyze in a water bath at 35-45℃ for 10-20 min to obtain the acylated-enzymatically hydrolyzed egg white protein dispersion; (4) Heat treatment-induced self-assembly: The acylated-enzymatically hydrolyzed egg white protein dispersion was heat-treated in a water bath at 80-100℃ for 10-20 min to obtain egg white protein nanoparticle-nanofiber composite material.

2. The method for preparing the protein nanoparticle-nanofiber composite material according to claim 1, characterized in that, The volume ratio of egg white to distilled water in step (1) is 1:2 to 1:

4.

3. The method for preparing the protein nanoparticle-nanofiber composite material according to claim 1, characterized in that, The stirring time in step (1) is 1 to 3 hours, and the sieve mesh size for sieving is 60 to 80 mesh.

4. The method for preparing the protein nanoparticle-nanofiber composite material according to claim 1, characterized in that, In step (2), the acylating agent is added to the egg white protein dispersion in small amounts multiple times.

5. The method for preparing the protein nanoparticle-nanofiber composite material according to claim 1, characterized in that, The acylating agent in step (2) is selected from one or more of succinic anhydride, maleic anhydride and acetic anhydride.

6. The method for preparing the protein nanoparticle-nanofiber composite material according to claim 5, characterized in that, In step (2), the acylating agent is added to the egg white protein dispersion at a ratio of 1:100 to 1:200 (w / v).

7. The method for preparing the protein nanoparticle-nanofiber composite material according to claim 1, characterized in that, In step (3), the protease is added to the acylated egg white protein dispersion in small amounts and multiple times.

8. The method for preparing the protein nanoparticle-nanofiber composite material according to claim 1, characterized in that, The protease mentioned in step (3) is selected from one or more of papain, alkaline protease and trypsin.

9. The method for preparing the protein nanoparticle-nanofiber composite material according to claim 8, characterized in that, The enzyme activity of the protease mentioned in step (3) is 500-1000 U / mg, and it is added to the acylated egg white protein dispersion at a ratio of 0.01-0.1% (w / v).

10. A protein nanoparticle-nanofiber composite material prepared by the method according to any one of claims 1-9.

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