Preparation of a self-assembled protein peptide from egg white and its application in emulsions

Abstract

This invention discloses the preparation of egg white self-assembled peptides and their application in emulsions. This technology belongs to the field of food processing technology, specifically focusing on the research of stabilizing high internal phase Pickering emulsions (HIPPEs) with egg white self-assembled peptides. The main raw materials used in this invention include egg white powder, etc. Based on a peptide self-assembly strategy, through enzymatic hydrolysis and pH control techniques, egg white self-assembled peptides with interfacial self-assembly properties, broad pH applicability in emulsion processing applications, good interfacial wetting properties, and biofunctional activity are obtained. Their amino acid sequences include Tyr-Ser-Phe-Ser-Leu (YSFSL), Cys-Phe-Asp-Val (CFDV), and Met-Pro-Phe-Arg (MPFR). The core of this invention lies in the excellent interfacial properties of its egg white self-assembled peptides. This function benefits from its interfacial self-assembly characteristics, effectively simplifying the preparation process of peptide-based emulsions, improving the bioavailability and absorption efficiency of functional active ingredients, and serving as a new strategy for stabilizing peptide-based emulsions, which is beneficial for expanding the application fields of egg white peptides.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a method for preparing egg white self-assembled peptides and their application in constructing Pickering emulsions. Background Technology

[0002] Pickering emulsions are emulsion systems stabilized by solid particles, exhibiting superior stability compared to traditional emulsions stabilized by surfactants (such as Tween-80) or inorganic particles (such as silica). Currently, Pickering emulsions are widely used in chemical synthesis and biomedicine. However, these traditional Pickering stabilizers have limitations in terms of biosafety, biodegradability, and biocompatibility, severely hindering the expanded application of Pickering emulsions in the food industry. Therefore, developing an environmentally friendly, green, and healthy food-grade stabilizer is of profound significance and an urgent need for expanding the application of Pickering emulsions in the food industry.

[0003] Eggs are an important source of protein for the human body. Egg white peptides are rich in multifunctional groups and binding sites, endowing them with a variety of functional activities, such as immune regulation, amino acid supplementation, intestinal repair, and microbiota regulation. Therefore, egg white is an excellent choice for preparing bioactive peptides. However, there is often a significant difference between the nutritional value and self-assembly properties of egg white peptides. Specifically, although low molecular weight peptides obtained from excessive protein hydrolysis possess good biological activity, their short amino acid sequences result in fewer self-assembly sites, making it difficult to form nanoparticles with good interfacial wetting properties. Consequently, their emulsification properties are poor, limiting their application in emulsion processing. Therefore, preparing and regulating the self-assembly characteristics of bioactive peptides based on self-assembly strategies to improve the interfacial wetting properties of egg white peptides could be a new strategy for stabilizing peptide-based emulsions, which would be beneficial for expanding the application areas of egg white peptides.

[0004] To address the aforementioned problems, this invention provides a method for preparing egg white self-assembled peptides, and uses this method to prepare novel peptide-based emulsions. This study utilizes enzymatic hydrolysis technology, employing amino acid sites such as leucine, valine, or arginine as cleavage sites, to significantly enhance the self-assembly properties of egg white peptides. The prepared egg white self-assembled peptides exhibit broad pH applicability in emulsion processing applications. The self-assembly properties of the egg white self-assembled peptides were confirmed by critical micelle concentration (CMC) experiments. Contact angle and Young's modulus indices showed improved interfacial properties. Optical microscopy and laser confocal microscopy revealed the good interfacial structure characteristics of stable high internal phase Pickering emulsions (HIPPEs) of egg white self-assembled peptides. Apparent viscosity and storage / energy dissipation modulus measurements demonstrated the stability of HIPPEs from a macroscopic perspective, and in vitro simulated digestion indicated their potential for functional factor delivery. In summary, this invention simplifies the preparation process of peptide-based emulsions, enhances the self-assembly performance of egg white peptides, and improves the bioavailability and absorption efficiency of functional active ingredients. The present invention aims to develop an egg white self-assembled peptide and provide a method for preparing HIPPEs with good stability, good encapsulation effect of bioactive substances, and simple preparation process and materials. Summary of the Invention

[0005] The present invention aims to develop a method for preparing egg white peptides with self-assembly capability, and to prepare HIPPEs based on this method.

[0006] 1. This invention provides a method for preparing egg white self-assembled peptides, the method comprising the following steps:

[0007] Step 1: Weigh an appropriate amount of egg white powder and dissolve it in distilled water to prepare a protein solution with a concentration of 1-10% (w / v). Transfer the solution to an enzymatic digestion flask and pretreat the protein solution in an 80-100℃ constant temperature water bath. Then transfer it to a 50-80℃ constant temperature water bath and adjust the pH of the sample to the set value of 6.0-7.0 using 1 mol / L NaOH. Add 5% papain, trypsin, or alkaline protease to the solution, and maintain the pH of the solution using 1 mol / L NaOH until the preset reaction time is reached. After the reaction is complete, quickly place the solution in a 90℃ constant temperature water bath and heat for 10 minutes to inactivate the enzyme, obtaining an egg white self-assembled peptide solution.

[0008] Step 2: After the enzymatic hydrolysate cools to room temperature, it is freeze-dried at low temperature to obtain egg white self-assembled peptides, which are then stored at -20℃ for subsequent detection and analysis. Experimental data show that the degree of hydrolysis of the egg white self-assembled peptides reaches 2%-3%.

[0009] In a preferred manner, the protein solution concentration described in step one tends to be 5%;

[0010] In the preferred embodiment, the pretreatment temperature in the constant temperature water bath described in step one tends to be 90°C;

[0011] In the preferred embodiment, the bio-enzymatic hydrolysis temperature in the constant temperature water bath described in step one tends to be 60°C;

[0012] In a preferred manner, the protease described in step one is preferably papain;

[0013] 2. This invention proposes a method for preparing a Pickering emulsion stabilized by egg white self-assembled peptides, which specifically includes the following steps:

[0014] Step 1: Dissolve the obtained egg white self-assembled peptide lyophilized powder in distilled water and disperse it uniformly under magnetic stirring. At room temperature (25°C), precisely adjust the pH of the egg white self-assembled peptide solution within the range of 3.0–11.0 using 1 mol / L HCl and NaOH to obtain a Pickering emulsion aqueous phase system.

[0015] Step 2: Weigh out corn oil quantitatively as the oil phase of HIPPEs. The ratio of oil phase to water phase is 1:1–4:1.

[0016] Step 3: Mix the aqueous phase prepared in Step 1 with the oil phase prepared in Step 2, and homogenize using a high-speed homogenizer at 10,000–12,000 rpm for 3–5 minutes to obtain the product emulsion.

[0017] Step 4: Accurately weigh the hydrophilic functional factor anthocyanin and dissolve it in the egg white self-assembled peptide solution obtained in Step 1. Then, adjust the pH of the anthocyanin and egg white self-assembled peptide mixed solution at 25°C with 1 mol / L HCl and NaOH to prepare an aqueous phase for later use. Quantitatively weigh the hydrophobic functional factor quercetin and dissolve it in corn oil to prepare an oil phase for later use. Prepare HIPPEs containing functional factors according to the method in Step 3.

[0018] In a preferred embodiment, the aqueous phase in step one is an aqueous phase with a concentration of 0.5%-2.5% for egg white self-assembled peptides.

[0019] In the preferred embodiment, the mixing in step three specifically involves mixing the aqueous phase prepared in step one with the oil phase prepared in step two, with an oil phase addition ratio of 3:1 to the aqueous phase addition, and homogenizing at 12000 r / min for 5 minutes.

[0020] In a preferred embodiment, the aqueous phase in step four is an aqueous phase with a pH of 3.0–11.0, consisting of a mixed solution of anthocyanins and egg white self-assembled peptides.

[0021] 3. This invention relates to a method for preparing an egg white self-assembled peptide, and further utilizes this egg white self-assembled peptide to prepare HIPPEs. The detailed steps of the preparation method include: (1) using egg white powder as raw material, and using papain for biological enzymatic hydrolysis to generate egg white self-assembled peptides with self-assembly properties; (2) performing enzyme inactivation treatment on the product obtained after enzymatic hydrolysis to ensure the safety of subsequent steps, and then obtaining peptide powder through vacuum freeze-drying technology for easy storage and transportation; (3) adjusting the pH value of the egg white self-assembled peptide solution to verify its assembly performance under different pH environments, laying the foundation for the construction of the emulsion; (4) dissolving the hydrophilic functional factor anthocyanin in the egg white self-assembled peptide solution to form an aqueous phase system rich in functional factors, enhancing the antioxidant and other properties of the emulsion; (5) dissolving the hydrophilic functional factor anthocyanin in the egg white self-assembled peptide solution to form an aqueous phase system rich in functional factors, enhancing the antioxidant and other properties of the emulsion; (6) finally, through high-speed homogenization, mixing and emulsifying the above aqueous phase and oil phase, and using the interfacial self-assembly properties of the egg white self-assembled peptides to form a stable Pickering emulsion.

[0022] 4. The beneficial effects of the present invention: (1) Egg white self-assembled peptides were prepared by enzymatic hydrolysis technology. The amino acid sequence of the peptides included Tyr-Ser-Phe-Ser-Leu (YSFSL), Cys-Phe-Asp-Val (CFDV), and Met-Pro-Phe-Arg (MPFR), which improved the interfacial wetting properties of the egg white peptides, effectively enhanced the processing performance of the egg white peptides, and expanded the application field of the egg white peptides; (2) Egg white self-assembled peptides stabilized HIPPEs have a wide pH applicability in application. The stable HIPPEs have a stable interfacial structure, high viscosity, and good modulus characteristics, which is conducive to achieving stabilization in the gastrointestinal environment; (3) The preparation process of peptide emulsions was simplified, and the co-delivery of hydrophilic and hydrophobic bioactive substances was successfully achieved, which improved the bioaccessibility and absorption efficiency of functional active ingredients. Attached Figure Description

[0023] Figure 1 The critical micelle value of the egg white self-assembled peptide prepared in Example 3 of this invention; Figure 2 The critical micelle value of the egg white self-assembled peptide prepared in Example 4 of this invention; Figure 3 The critical micelle value of the egg white self-assembled peptide prepared in Example 5 of this invention; Figure 4 The critical micelle value of the egg white self-assembled peptide prepared in Example 6 of this invention; Figure 5 The critical micelle value of the egg white self-assembled peptide prepared in Example 7 of this invention; Figure 6 The three-phase contact angles of the egg white self-assembled peptides prepared in Examples 3-7 of this invention; Figure 7The Young's modulus of the egg white self-assembled peptides prepared in Examples 3-7; Figure 8 Optical micrographs of HIPPEs stabilized by egg white self-assembled peptides prepared in Examples 1-3 of this invention; Figure 9 These are laser confocal microscopy images of HIPPEs stabilized by egg white self-assembled peptides prepared in Examples 3-7 of this invention. Figure 10 The apparent viscosity of the HIPPEs stabilized by the egg white self-assembled peptides prepared in Examples 3-7 of this invention; Figure 11 The energy storage and energy dissipation moduli of the egg white self-assembled peptide-stabilized HIPPEs prepared in Examples 3-7 of this invention; Figure 12 Bioaccessibility of anthocyanins containing functional HIPPEs prepared according to Examples 8-10 of this invention; Figure 13 To ensure the bioavailability of quercetin containing functional HIPPEs as described in Examples 8-10 of this invention; Figure 14 Sequence identification of the egg white self-assembled peptide described in this invention; Figure 15 Sequence identification of the egg white self-assembled peptide described in this invention; Figure 16 Sequence identification of the egg white self-assembled peptide described in this invention; Detailed Implementation

[0024] Example 1:

[0025] Step 1: Weigh an appropriate amount of egg white powder and dissolve it in distilled water to prepare a 5% (w / v) protein solution. Transfer the solution to an enzymatic digestion flask and pretreat the protein solution in a 90°C water bath. Then transfer it to a 60°C water bath and adjust the pH of the sample to the set value of 6.5 using 1 mol / L NaOH. Add 5% papain to the solution and maintain the pH of the solution using 1 mol / L NaOH until the preset reaction time is reached. After the reaction is complete, quickly place the solution in a 90°C water bath and heat for 10 minutes to inactivate the enzyme, obtaining an egg white self-assembled peptide solution.

[0026] Step 2: After the enzymatic hydrolysate cools to room temperature, it is freeze-dried at low temperature to obtain egg white self-assembled peptides, which are then stored at -20℃ for subsequent detection and analysis.

[0027] Step 3: Dissolve the obtained egg white self-assembled peptide lyophilized powder in distilled water to prepare a 0.5% dispersion, and achieve uniform dispersion by magnetic stirring. At room temperature of 25°C, adjust the pH of the egg white self-assembled peptide solution to 3.0 using 1 mol / L HCl and NaOH to obtain the Pickering emulsion aqueous phase system.

[0028] Step 4: Mix the aqueous phase prepared in Step 3 with corn oil at a ratio of 3:1 (oil phase added: aqueous phase added), and homogenize using a high-speed homogenizer at 12,000 rpm for 5 minutes to obtain the product emulsion.

[0029] Example 2:

[0030] Step 3: Dissolve the obtained egg white self-assembled peptide lyophilized powder in distilled water to prepare a 1.5% dispersion, and achieve uniform dispersion by magnetic stirring. At room temperature of 25°C, adjust the pH of the egg white self-assembled peptide solution to 3.0 using 1 mol / L HCl and NaOH to obtain the Pickering emulsion aqueous phase system.

[0031] Step 4: Mix the aqueous phase prepared in Step 3 with corn oil at a ratio of 3:1 (oil phase added: aqueous phase added), and homogenize using a high-speed homogenizer at 12,000 rpm for 5 minutes to obtain the product emulsion.

[0032] Example 3:

[0033] Step 3: Dissolve the obtained egg white self-assembled peptide lyophilized powder in distilled water to prepare a 2.5% dispersion, and achieve uniform dispersion by magnetic stirring. At room temperature of 25°C, adjust the pH of the egg white self-assembled peptide solution to 3.0 using 1 mol / L HCl and NaOH to obtain the Pickering emulsion aqueous phase system.

[0034] Step 4: Mix the aqueous phase prepared in Step 3 with corn oil at a ratio of 3:1 (oil phase added: aqueous phase added), and homogenize using a high-speed homogenizer at 12,000 rpm for 5 minutes to obtain the product emulsion.

[0035] Example 4:

[0036] Step 3: Dissolve the obtained egg white self-assembled peptide lyophilized powder in distilled water to prepare a 2.5% dispersion, and achieve uniform dispersion by magnetic stirring. At room temperature of 25°C, adjust the pH of the egg white self-assembled peptide solution to 5.0 using 1 mol / L HCl and NaOH to obtain the Pickering emulsion aqueous phase system.

[0037] Step 4: Mix the aqueous phase prepared in Step 3 with corn oil at a ratio of 3:1 (oil phase added: aqueous phase added), and homogenize using a high-speed homogenizer at 12,000 rpm for 5 minutes to obtain the product emulsion.

[0038] Example 5:

[0039] Step 3: Dissolve the obtained egg white self-assembled peptide lyophilized powder in distilled water to prepare a 2.5% dispersion, and achieve uniform dispersion by magnetic stirring. At room temperature of 25°C, adjust the pH of the egg white self-assembled peptide solution to 7.0 using 1 mol / L HCl and NaOH to obtain the Pickering emulsion aqueous phase system.

[0040] Step 4: Mix the aqueous phase prepared in Step 3 with corn oil at a ratio of 3:1 (oil phase added: aqueous phase added), and homogenize using a high-speed homogenizer at 12,000 rpm for 5 minutes to obtain the product emulsion.

[0041] Example 6:

[0042] Step 3: Dissolve the obtained egg white self-assembled peptide lyophilized powder in distilled water to prepare a 2.5% dispersion, and achieve uniform dispersion by magnetic stirring. At room temperature of 25°C, adjust the pH of the egg white self-assembled peptide solution to 9.0 using 1 mol / L HCl and NaOH to obtain the Pickering emulsion aqueous phase system.

[0043] Step 4: Mix the aqueous phase prepared in Step 3 with corn oil at a ratio of 3:1 (oil phase added: aqueous phase added), and homogenize using a high-speed homogenizer at 12,000 rpm for 5 minutes to obtain the product emulsion.

[0044] Example 7:

[0045] Step 3: Dissolve the obtained egg white self-assembled peptide lyophilized powder in distilled water to prepare a 2.5% dispersion, and achieve uniform dispersion by magnetic stirring. At room temperature of 25°C, adjust the pH of the egg white self-assembled peptide solution to 11.0 using 1 mol / L HCl and NaOH to obtain the Pickering emulsion aqueous phase system.

[0046] Step 4: Mix the aqueous phase prepared in Step 3 with corn oil at a ratio of 3:1 (oil phase added: aqueous phase added), and homogenize using a high-speed homogenizer at 12,000 rpm for 5 minutes to obtain the product emulsion.

[0047] Example 8:

[0048] Step 1: Accurately weigh the hydrophilic functional factor anthocyanin and dissolve it in the egg white self-assembled peptide solution. Then, at 25°C, adjust the pH of the anthocyanin and egg white self-assembled peptide mixture to 3.0 using 1 mol / L HCl and NaOH to prepare a Pickering emulsion aqueous phase system containing the functional factor anthocyanin.

[0049] Step 2: Quantitatively weigh the hydrophobic functional factor quercetin and dissolve it in corn oil to prepare a Pickering emulsion oil phase system containing the hydrophobic functional factor quercetin;

[0050] Step 3: Mix the Pickering emulsion aqueous phase system containing the functional factor anthocyanin prepared in Step 1 with the oil phase system containing the hydrophobic functional factor quercetin prepared in Step 2, and homogenize using a high-speed homogenizer at 12,000 rpm for 5 minutes to obtain the product HIPPEs.

[0051] Example 9:

[0052] Step 1: Accurately weigh the hydrophilic functional factor anthocyanin and dissolve it in the egg white self-assembled peptide solution. Then, at 25°C, adjust the pH of the anthocyanin and egg white self-assembled peptide mixture to 7.0 using 1 mol / L HCl and NaOH to prepare a Pickering emulsion aqueous phase system containing the functional factor anthocyanin.

[0053] Step 2: Quantitatively weigh the hydrophobic functional factor quercetin and dissolve it in corn oil to prepare a Pickering emulsion oil phase system containing the hydrophobic functional factor quercetin;

[0054] Step 3: Mix the Pickering emulsion aqueous phase system containing the functional factor anthocyanin prepared in Step 1 with the oil phase system containing the hydrophobic functional factor quercetin prepared in Step 2, and homogenize using a high-speed homogenizer at 12,000 rpm for 5 minutes to obtain the product HIPPEs.

[0055] Example 10:

[0056] Step 1: Accurately weigh the hydrophilic functional factor anthocyanin and dissolve it in the egg white self-assembled peptide solution. Then, at 25°C, adjust the pH of the anthocyanin and egg white self-assembled peptide mixture to 11.0 using 1 mol / L HCl and NaOH to prepare a Pickering emulsion aqueous phase system containing the functional factor anthocyanin.

[0057] Step 2: Quantitatively weigh the hydrophobic functional factor quercetin and dissolve it in corn oil to prepare a Pickering emulsion oil phase system containing the hydrophobic functional factor quercetin;

[0058] Step 3: Mix the Pickering emulsion aqueous phase system containing the functional factor anthocyanin prepared in Step 1 with the oil phase system containing the hydrophobic functional factor quercetin prepared in Step 2, and homogenize using a high-speed homogenizer at 12,000 rpm for 5 minutes to obtain the product HIPPEs.

[0059] The critical micelle concentration (CMC) of the egg white self-assembled peptides prepared in this invention was determined: Appropriate amounts of the egg white self-assembled peptide solutions prepared in Examples 3-7 were taken and diluted to suitable concentrations. Using Nile red as a fluorescent probe, the self-assembly characteristics of the egg white self-assembled peptides were studied, and the fluorescence intensity was measured using a fluorescence spectrometer. Specific analysis and theoretical basis include:

[0060] (1) Nile red is used as a fluorescent probe to determine the critical micelle concentration (CMC) of egg white self-assembled peptides, which is the concentration corresponding to the inflection point of fluorescence intensity mutation.

[0061] (2) At concentrations below the CMC, fluorescence intensity hardly changes with concentration.

[0062] (3) When the concentration of self-assembled peptides in egg white exceeds the CMC, the fluorescence intensity is positively correlated with the protein concentration. The abrupt change in fluorescence intensity proves the formation of nanoparticles.

[0063] Test results as follows Figures 1-5 As shown.

[0064] The comparison of the test data shows that pH affects the charge change and hydrophobic interaction of different functional groups in Examples 3 to 7. In Examples 3 and 4, the CMC value is lower, which is conducive to promoting the formation of nanoparticle structure and promoting self-assembly behavior.

[0065] The three-phase contact angle of the egg white self-assembled peptides prepared in this invention was measured: Appropriate amounts of the egg white self-assembled peptides prepared in Examples 3 to 7 were taken, and the three-phase contact angle was evaluated using a Dataphysics OCA20 contact angle meter. The Young-Laplace equation was used to fit the contact angle values. Specific analysis and theoretical basis include:

[0066] (1) The wettability of a substance between the oil phase and the water phase can be described by the three-phase contact angle (θ), which is closely related to the interfacial properties.

[0067] (2) A contact angle close to 90° is of great significance for enhancing the stability of the emulsion.

[0068] Test results as follows Figure 6 As shown in the figure, different letters in the data graph represent significant differences (p<0.05).

[0069] Comparison of test data shows that, except for Example 4, the θ values ​​of the egg white self-assembled peptides in Examples 3 to 7 are consistently below 90°, indicating that they tend to stabilize oil-in-water emulsions. The θ value of the powder in Example 4 is closest to 90°, indicating that the weakly acidic pH significantly optimizes the interfacial wetting properties of the egg white self-assembled peptides.

[0070] The Young's modulus of the egg white self-assembled peptide nanoparticles prepared in this invention was determined: the egg white self-assembled peptide sample solution was deposited onto a cut mica sheet and dried at room temperature. The microstructure of the sample was observed using an atomic force microscope. The images were analyzed using Nanoscope software version 3.00, and the average Young's modulus value was calculated. Specific analysis and theoretical basis include:

[0071] (1) Young's modulus is an important indicator for evaluating the stiffness of materials.

[0072] (2) Materials with higher stiffness are less likely to deform at the oil-water interface, which helps stabilize the interface.

[0073] Test results as follows Figure 7 As shown in the figure, different letters in the data graph represent significant differences (p<0.05).

[0074] Comparison of test data shows that the stiffness of the egg white self-assembled peptides increased in Examples 3 to 7. Egg white self-assembled peptides with higher stiffness are less prone to deformation at the oil-water interface, thus preventing interfacial film rupture and enhancing interfacial wettability. Optimized wettability facilitates more efficient and convenient adsorption of egg white self-assembled peptides at the oil-water interface, contributing to the maintenance of HIPPE stability.

[0075] The HIPPEs prepared according to this invention were photographed using an optical microscope: 10 μL of freshly prepared HIPPEs from Examples 1 to 3 were dropped onto a glass slide, covered with a coverslip, and placed on the stage of an optical microscope to observe the droplet size distribution. Specific analysis and theoretical basis include:

[0076] (1) The less droplets aggregate to form large droplets, the more stable the system is.

[0077] (2) The smaller the droplet size and the more uniform the distribution, the more stable the system.

[0078] Test results as follows Figure 8 As shown.

[0079] The comparison of test data shows that in Examples 1 to 3, the size of the emulsion droplets decreased sequentially, and the droplets became more uniform. This indicates that the egg white self-assembled peptides can act as emulsifiers to cover a larger interface area, thereby better binding the droplets, preventing the migration between droplets, and inhibiting the occurrence of droplet aggregation.

[0080] Laser confocal microscopy was used to determine the HIPPEs prepared according to this invention: Nile red (0.1 wt%) and Nile blue (0.1 wt%) were dissolved in isopropanol and used to stain the oil and aqueous phases of Examples 3 to 7, respectively. The microstructure of the HIPPEs was observed. Specific analysis and theoretical basis include:

[0081] (1) The denser the interface network structure, the better it can bind droplets, and the higher the stability of HIPPEs.

[0082] (2) The more uniform the droplet distribution and the smaller the size, the higher the stability of HIPPEs.

[0083] Test results as follows Figure 9 As shown.

[0084] Comparison of test data shows that in Examples 3 to 7, acidic pH conditions help the emulsion form a denser interfacial network structure, which can better bind droplets, prevent droplet migration, and inhibit droplet aggregation. The egg white self-assembled peptide-stabilized HIPPEs prepared by this invention have good stability.

[0085] The apparent viscosity and storage and dissipation moduli of the HIPPEs prepared in this invention were determined: Examples 3 to 7 were tested using a hybrid rheometer with a stainless steel parallel plate clamp. The shear rate in the scanning mode was recorded to assess the change in apparent viscosity of the samples, and the oscillation frequency under strain conditions was recorded to determine the storage modulus (G') and dissipation modulus (G"). Specific analysis and theoretical basis include:

[0086] (1) The increase in viscosity of HIPPEs indicates the formation of a dense interfacial network in HIPPEs.

[0087] (2) The greater the difference between the energy storage modulus and the energy dissipation modulus, the more the rigid interface network structure can be enhanced to better confine the oil droplet migration, thus making the HIPPEs droplet distribution more uniform and the size smaller.

[0088] Test results as follows Figure 10 , 11 As shown.

[0089] The comparison of test data shows that, in Examples 3 to 7, the friction between HIPPE droplets can be increased under acidic pH, thereby giving HIPPE higher viscosity, obtaining a larger energy storage modulus value, and enhancing the rigid interface network structure.

[0090] The bioavailability of the HIPPEs prepared in this invention was determined: HIPPEs containing functional factors prepared in Examples 8 to 10 were used to study the in vitro gastrointestinal digestion behavior of HIPPEs according to the INFOOGEST 2.0 digestion protocol. After simulating gastrointestinal digestion, the bioavailability of the functional factors was determined using a UV-Vis spectrophotometer. Specific analysis and theoretical basis include:

[0091] (1) Higher biological accessibility indicates better stability of functional factors in the gastrointestinal tract.

[0092] (2) The higher the biological accessibility, the higher the absorption and utilization rate of functional factors.

[0093] Test results as follows Figure 12 , 13 As shown in the figure, different letters in the data graph represent significant differences (p<0.05).

[0094] Comparison of test data shows that, as demonstrated in Examples 8-10, compared to free functional factors, HIPPEs stabilized by egg white self-assembled peptides have relatively higher bioavailability. The interfacial network structure formed by egg white self-assembled peptides can protect the encapsulated functional factors from degradation by gastric acid and enzymes, thereby improving the absorption and utilization rate of the functional factors.

[0095] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing egg white self-assembled peptides, characterized in that, The preparation steps include the following: Step 1: Weigh an appropriate amount of egg white powder and dissolve it in distilled water to prepare a protein solution with a concentration of 1% - 10% (w / v); transfer the above solution to an enzymatic digestion flask and place it in an 80 - 100℃ constant temperature water bath for pretreatment of the protein solution; then transfer it to a 50 - 80℃ constant temperature water bath and adjust the sample to the set pH value of 6.0 - 7.0 using 1mol / L NaOH; add 5% papain to the solution and maintain the pH of the solution using 1mol / L NaOH until the preset reaction time is reached; after the reaction is completed, quickly place the solution in a 90℃ constant temperature water bath and heat for 10 minutes to inactivate the enzyme, obtaining an egg white self-assembled peptide solution; Step 2: After the enzymatic hydrolysate cools to room temperature, the egg white self-assembled peptides obtained by low-temperature freeze drying are stored at -20℃ for subsequent detection and analysis. Experimental data show that the degree of hydrolysis of the egg white self-assembled peptides reaches 2%-3%. The amino acid sequence of the egg white self-assembled peptides includes Tyr-Ser-Phe-Ser-Leu (YSFSL), Cys-Phe-Asp-Val (CFDV), and Met-Pro-Phe-Arg (MPFR).

2. The application of the egg white self-assembled peptide prepared by the method of claim 1, characterized in that, Preparation of Pickering emulsions for stabilizing self-assembled peptides in egg white.

3. A method for preparing a Pickering emulsion stabilized by egg white self-assembled peptides, characterized in that, Includes the following steps: Step 1: Dissolve the lyophilized egg white self-assembled peptide obtained in claim 1 in distilled water and achieve uniform dispersion by stirring with a magnetic stirrer; at room temperature of 25°C, precisely adjust the pH of the egg white self-assembled peptide solution within the range of 3.0 – 11.0 using 1 mol / L HCl and NaOH to obtain the Pickering emulsion aqueous phase system; Step 2: Weigh out corn oil quantitatively as the oil phase of high internal phase Pickering emulsions (HIPPEs); Step 3: Mix the aqueous phase system prepared in Step 1 with the oil phase system prepared in Step 2. The ratio of oil phase to water phase is 1:1 to 4:

1. Use a high-speed homogenizer to homogenize at 10,000 to 12,000 rpm for 3 to 5 minutes to obtain the product emulsion. Step 4: Accurately weigh the hydrophilic functional factor anthocyanin and dissolve it in the egg white self-assembled peptide solution obtained in Step 1. Then, adjust the pH of the anthocyanin and egg white self-assembled peptide mixed solution at 25 °C with 1 mol / L HCl and NaOH to prepare an aqueous phase for later use. Quantitatively weigh the hydrophobic functional factor quercetin and dissolve it in corn oil to prepare an oil phase for later use. Prepare HIPPEs containing functional factors according to the method in Step 3.

4. The method for preparing the egg white self-assembled peptide-stabilized Pickering emulsion according to claim 3, characterized in that, The concentration of the egg white self-assembled peptide solution is 0.5% - 2.5%.

5. The application of the egg white self-assembled peptide prepared by the method of claim 1 in the food industry.

6. The application of the egg white self-assembled peptide-stabilized Pickering emulsion prepared by the method of any one of claims 3 or 4 in the food industry.

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