Egg white peptide / polyphenol / metal ion ternary synergistic assembly composite particle and application thereof in stable emulsion

Abstract

The invention relates to the technical field of deep processing of egg products, in particular to egg white peptide / polyphenol / metal ion ternary synergistically assembled composite particles and application of the egg white peptide / polyphenol / metal ion ternary synergistically assembled composite particles in stable emulsions.The egg white peptide / polyphenol / metal ion ternary synergistically assembled composite particles are prepared by pre-assembling metal ions and polyphenol to form a metal-phenolic network and synergistically assembling the metal-phenolic network and egg white peptide through an anti-solvent coprecipitation method. The egg white peptide / polyphenol / metal ion ternary synergistic assembly composite particles are formed. Compared with unassembled egg white peptide, the colloidal property of the composite particle is regulated and controlled, the absolute value of surface charge is reduced, the interface hydrophobicity is enhanced, and the egg white peptide molecule conformation is promoted to be converted into an ordered secondary structure mainly comprising beta-folding. The composite particle can be used as a novel stabilizer for constructing an emulsion, and the obtained emulsion is uniform in droplet size, good in dispersity and still stable under the environmental stress conditions of heating, freezing and thawing, different pH values, salt ion strength and the like. The invention provides a new technical approach for the application of the egg white source active peptide in emulsion and functional food.

Description

Technical Field

[0001] This invention belongs to the field of egg product deep processing technology, specifically relating to a ternary synergistic assembly composite particle of egg white peptide / polyphenol / metal ion and its application in stable emulsions. Background Technology

[0002] Emulsions play an irreplaceable role in the modern food industry. Their value lies not only in imparting diverse textural and flavor characteristics to foods such as beverages, sauces, creams, and baked goods, but also in their ability to serve as efficient carriers for the effective encapsulation and delivery of fat-soluble nutrients, bioactive substances, and flavor components. However, emulsions are inherently thermodynamically unstable, primarily due to their high Gibbs free energy resulting from their large oil-water interfacial area. Therefore, during processing, storage, and distribution, emulsions tend to minimize energy by reducing their interfacial area, leading to a series of physical instabilities such as droplet flocculation, irreversible aggregation, and separation or sedimentation due to density differences. Furthermore, the large interfacial area provides favorable conditions for oxygen diffusion and the occurrence and propagation of lipid oxidation reactions. The resulting peroxides and other oxidation products significantly shorten the shelf life of food and adversely affect its nutritional value and sensory quality.

[0003] Peptides, as functional fragments derived from natural proteins, have become a research hotspot for replacing traditional synthetic emulsifiers in food-grade emulsions due to their excellent biocompatibility, tunable molecular structure, and abundant functional groups. Their inherent amphiphilicity allows them to rapidly migrate and adsorb at the oil-water interface, reducing interfacial tension and forming a protective interfacial film, thus inhibiting droplet aggregation and improving the physical and oxidative stability of the emulsion to some extent. Egg white protein has attracted much attention due to its abundant raw material supply, high nutritional value, and outstanding biological activity. Egg white peptides obtained through enzymatic hydrolysis not only retain some nutritional functions but also possess various biological activities such as antioxidant and angiotensin-converting enzyme inhibition, exhibiting certain interfacial adsorption potential. However, limited by their low molecular weight, limited molecular size, and high conformational flexibility, egg white peptides have insufficient adsorption capacity and steric hindrance effect at the oil-water interface, making it difficult to form a dense and mechanically stable interfacial film. This leads to interfacial desorption under environmental disturbances or long-term storage conditions, subsequently causing emulsion aggregation, flocculation, and phase separation, significantly restricting their practical application in food emulsion systems.

[0004] Metal-phenolic networks (MPNs) are supramolecular assembly structures formed by coordination between polyphenol ligands and metal ions. Due to their excellent structural tunability, outstanding interfacial affinity, and the antioxidant activity imparted by polyphenols, they show broad application prospects in interfacial engineering and emulsion stabilization. MPNs can self-assemble in situ at the oil-water interface, significantly improving the physical stability and antioxidant properties of emulsions. However, current research mainly focuses on the interfacial behavior and functional characteristics of MPNs themselves. How to systematically introduce this strategy into small molecule peptide systems to overcome problems such as weak interfacial activity and easy desorption caused by limited molecular size and insufficient steric hindrance remains to be explored. Especially for peptides such as egg white peptides, which have good biocompatibility but limited emulsifying properties, research on using MPNs to mediate their assembly to construct high-performance emulsion systems still faces significant technical obstacles. Therefore, developing a novel ternary synergistic assembly strategy based on metal-phenolic network regulation of peptides / polyphenols / metal ions to synergistically enhance the interfacial adsorption capacity and antioxidant properties of peptides at the molecular level has important theoretical significance and application value for constructing food emulsions with both high stability and functionality. Summary of the Invention

[0005] The purpose of this invention is to propose a method for preparing a ternary synergistic assembly composite particle of egg white peptide / polyphenol / metal ion, and to utilize the composite particle for the construction and stabilization of emulsions.

[0006] 1. This invention provides a method for preparing egg white peptides, the specific operation steps of which are as follows:

[0007] Step 1: Dissolve egg white protein powder in distilled water to prepare a protein solution with a mass fraction of 1-10% (w / v), and transfer the protein solution to an enzymatic hydrolysis container for pretreatment in a constant temperature water bath at 60-100℃ for 5-10 min; then transfer it to a constant temperature water bath at 50-90℃ and adjust the pH of the solution to 5.0-10.0 using 1 mol / L NaOH or HCl.

[0008] Step 2: Add 2-8% of the protein mass of protease to the solution obtained in Step 1, and carry out the enzymatic hydrolysis reaction in a constant temperature water bath at 50-90℃ for 1-5 h, while maintaining the pH of the system in the range of 5.0-10.0 with 1 mol / L NaOH or HCl; the protease is selected from one or two of acidic protease, neutral protease, alkaline protease, papain, bromelain, flavor protease and Bacillus licheniformis protease;

[0009] Step 3: After the enzymatic hydrolysis reaction is completed, the reaction system is heated in a 90℃ water bath for 10 min to inactivate the enzyme preparation. Then the enzymatic hydrolysate is cooled to room temperature. The cooled enzymatic hydrolysate is then freeze-dried at low temperature to obtain egg white peptide powder. The obtained egg white peptide powder is stored at -20℃ for subsequent detection and analysis.

[0010] 2. This invention provides a method for preparing ternary synergistically assembled composite particles of egg white peptides / polyphenols / metal ions, comprising the preparation of a metal-phenolic network and its induction of egg white peptides to construct ternary synergistically assembled composite particles. The specific operation steps are as follows:

[0011] Step 1: Dissolve the egg white peptides in distilled water to prepare an egg white peptide solution with a concentration of 10-20 mg / mL;

[0012] Step 2: Prepare a metal ion solution with a concentration of 1-6 mol / L and a polyphenol solution with a concentration of 1-6 mol / L. The metal ion solution contains one or more of sodium, potassium, calcium, zinc, magnesium, and iron ions, and the polyphenol solution contains one or more of curcumin, quercetin, resveratrol, astaxanthin, anthocyanins, catechins, and tannic acid. Mix the metal ion solution and the polyphenol solution at a metal to polyphenol molar ratio of 1:3-3:1 to form a metal-polyphenol network pre-assembled solution.

[0013] Step 3: Using an antisolvent co-precipitation method, the metal-polyphenol network pre-assembled solution was slowly added dropwise to the egg white peptide solution obtained in Step 1 at a volume ratio of 1:3-3:1. The pH of the system was adjusted to 5.0-9.0, and after thorough stirring, ternary synergistically assembled composite particles of egg white peptide / polyphenol / metal ions based on a metal-phenolic network were obtained. Subsequently, the assembled powder was prepared by low-temperature freeze-drying and stored at 25°C for subsequent analysis.

[0014] 3. This invention provides a method for preparing a stable emulsion of egg white peptide / polyphenol / metal ion ternary synergistically assembled composite particles based on a metal-phenolic network. The specific operation steps are as follows:

[0015] Step 1: Dissolve the prepared metal-phenolic network-based ternary synergistic composite particle powder of egg white peptide / polyphenol / metal ion in distilled water and disperse it fully under magnetic stirring. At 25°C, use 1 mol / L NaOH or HCl to precisely adjust the pH of the system to 5.0-9.0 to obtain the aqueous phase of Pickering emulsion.

[0016] Step 2: Select one or two of corn oil, coconut oil, olive oil, or soybean oil as the oil phase of the emulsion;

[0017] Step 3: Mix the aqueous phase from Step 1 and the oil phase from Step 2 at a volume ratio of 1:3-3:1, and homogenize using a high-speed homogenizer at 10000-15000 rpm for 3-5 min to obtain an emulsion.

[0018] 4. The beneficial effects of this invention are as follows:

[0019] (1) This invention introduces a metal-phenolic network to regulate the assembly behavior of egg white peptides, thereby constructing a ternary synergistic assembly composite particle based on a metal-phenolic network of egg white peptides / polyphenols / metal ions, which significantly improves the colloidal properties of the composite particle.

[0020] (2) The egg white peptide / polyphenol / metal ion ternary synergistic assembly composite particles can be used as particle stabilizers to construct emulsions. The resulting emulsion droplet particle size is in the micrometer range and is uniformly distributed with good dispersibility. It can remain stable under environmental conditions such as heating, freeze-thaw, different pH values ​​and salt ion strength, and the emulsion separation index is zero.

[0021] (3) The method for preparing egg white peptide / polyphenol / metal ion ternary synergistic assembly composite particles of the present invention adopts a process route that combines anti-solvent co-precipitation and low-temperature freeze drying. It is simple to operate, has good repeatability, and the obtained product has good storage stability, making it suitable for food industry applications.

[0022] (4) This invention provides a new technical approach for the application of egg white peptides in emulsions, which significantly improves the stability of emulsions while giving the system good antioxidant properties, and provides technical support for the development of functional food emulsifiers. Attached Figure Description

[0023] Figure 1 The mass spectra of the amino acid sequences of the egg white peptides prepared in Examples 1 and 2 are shown.

[0024] Figure 2 The images shown are optical microscope images of the composite particle stabilized emulsions of Examples 6-9. E1-E4 correspond to Examples 6-9, respectively. Detailed Implementation

[0025] The present invention will be further described below by way of specific embodiments.

[0026] Example 1: Preparation of egg white peptides

[0027] In this embodiment, egg white protein was treated with different enzymatic hydrolysis conditions to obtain egg white peptides. The specific steps are as follows.

[0028] First preparation method:

[0029] Step 1: Dissolve egg white protein powder in distilled water to prepare a 10% (w / v) protein solution. Transfer the protein solution to an enzymatic hydrolysis container and pretreat it in a 90°C constant temperature water bath for 10 min. Then transfer it to a 56°C constant temperature water bath and adjust the pH of the solution to 10.0 using 1 mol / L NaOH or HCl.

[0030] Step 2: Add one of alkaline protease, papain, or bromelain, accounting for 8% of the protein mass, to the solution obtained in Step 1, and carry out the enzymatic hydrolysis reaction in a constant temperature water bath at 56℃ for 3 hours. During the reaction, maintain the pH of the solution at 10.0 with 1 mol / L NaOH or HCl.

[0031] Step 3: After the enzymatic hydrolysis reaction is completed, the reaction system is heated in a 90℃ constant temperature water bath for 10 min to inactivate the enzyme preparation. The hydrolysate is then cooled to room temperature. The cooled hydrolysate is then freeze-dried at low temperature to obtain egg white peptide powder. The degree of hydrolysis of the obtained egg white peptide was determined to be 25.2%. The obtained egg white peptide powder is stored at -20℃ for subsequent detection and analysis.

[0032] The degree of hydrolysis was determined using the pH-stat method and was defined as the percentage of cleaved peptide bonds out of the total number of peptide bonds. This was achieved by measuring the amount of H+ released during protein hydrolysis. + The resulting change in solution pH, combined with the volume of NaOH added, is used to calculate the degree of hydrolysis:

[0033]

[0034] In the formula:

[0035] h is the number of peptide bonds that were cleaved (mmol / g).

[0036] N is the concentration of NaOH (mol / L);

[0037] B is the volume (mL) of sodium hydroxide.

[0038] m p It refers to the protein content in the substrate;

[0039] h tot It is the gram equivalent of peptide bonds per gram of egg white protein (8.38 meqv / g).

[0040] α represents the average degree of dissociation of the α-amino group, which is taken as 1.105.

[0041] Based on the above preparation method, this embodiment also uses papain and bromelain to enzymatically hydrolyze egg white protein, and the preparation process is as follows.

[0042] The second preparation method:

[0043] Step 1: Dissolve egg white protein powder in distilled water to prepare a 5% (w / v) protein solution. Transfer the protein solution to an enzymatic hydrolysis container and pretreat it in an 80°C constant temperature water bath for 5 min. Then transfer it to a 60°C constant temperature water bath and adjust the pH of the solution to 6.5 using 1 mol / L NaOH or HCl.

[0044] Step 2: Add papain and bromelain, accounting for 2% of the protein mass, to the solution obtained in Step 1, and carry out the enzymatic hydrolysis reaction in a constant temperature water bath at 60℃ for 2 h. During the reaction, maintain the pH of the solution at 6.5 with 1 mol / L NaOH or HCl.

[0045] Step 3: After the enzymatic hydrolysis reaction is completed, the reaction system is heated in a 90℃ constant temperature water bath for 10 min to inactivate the enzyme preparation. The hydrolysate is then cooled to room temperature. The cooled hydrolysate is then freeze-dried at low temperature to obtain egg white peptide powder. The degree of hydrolysis of the obtained egg white peptide was determined (using the same method as in Example 1) to be 1.8%. The obtained egg white peptide powder is stored at -20℃ for subsequent detection and analysis.

[0046] To further identify the sequence of the egg white peptide prepared in this invention, liquid chromatography-tandem mass spectrometry was used for analysis. The specific operation steps are as follows:

[0047] Step 1: Sample dissolution and reduction treatment

[0048] Take 10.0 mg of egg white peptide sample, dissolve it in 100 μL of distilled water, and add 1 μL of 1 mol / L dithiothreitol to make a final concentration of 10 mmol / L. Then, treat the mixture in a 56℃ water bath for 1 h to complete the reduction reaction.

[0049] Step 2: Alkylation reaction and neutralization treatment

[0050] Add 2 μL of a 1 mol / L iodoacetamide solution to the reduced sample obtained in step one to achieve a final concentration of 20 mmol / L. React the mixture at room temperature for 40 min in the dark to complete the alkylation reaction. After the reaction is complete, add 1 μL of a 1 mol / L dithiothreitol solution to neutralize the excess iodoacetamide.

[0051] Step 3: Desalination and Drying

[0052] The sample processed in step two was desalted using a C18 Stage-Tip desalting column. The eluent from the desalted sample was collected and vacuum dried at 45°C to obtain the peptide powder to be analyzed.

[0053] Step 4: Mass spectrometry analysis

[0054] The dried powder sample prepared in step four was reconstituted and then analyzed by mass spectrometry. Chromatographic conditions: pre-column 150 μm inner diameter × 50 mm, analytical column 150 μm inner diameter × 170 mm, both packed with C18; mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 80% acetonitrile containing 0.1% formic acid, flow rate 0.6 mL / min, total analysis time 60 min. Mass spectrometry conditions: first-order resolution 70000, maximum ion implantation time 100 ms, scan range 100-1500; second-order resolution 17,500, maximum implantation time 50 ms.

[0055] Step 5: Data Retrieval and Peptide Screening

[0056] The raw mass spectrometry data were searched using protein databases. Fixed modifications included cysteine ​​alkylation, while variable modifications included methionine oxidation and N-terminal acetylation of peptides. Enzyme digestion was non-specific. Sturgeon and tilapia proteins were selected from the database. The permissible deviation for primary mass spectrometry was 20 ppm, and for secondary mass spectrometry, it was 0.02 Da. Abundances greater than 1.04 × 10⁻⁶ were selected. 7 Furthermore, peptides with an activity score greater than 0.5 were ultimately selected as three candidate peptides, such as... Figure 1 As shown, the amino acid sequences are KGEADAVALDGG, KGEADAVALDGGLVY, and GTEATGSTGAIGNIK.

[0057] Example 2: Preparation of ternary synergistically assembled composite particles of egg white peptide / magnesium ion / curcumin

[0058] This embodiment aims to prepare a ternary synergistically assembled composite particle based on a metal-phenolic network, consisting of egg white peptide / magnesium ion / curcumin.

[0059] Step 1: Take the egg white peptide powder prepared in Example 1, dissolve it in distilled water, and prepare an egg white peptide solution with a concentration of 20 mg / mL.

[0060] Step 2: Weigh curcumin and dissolve it in a 75% ethanol aqueous solution to prepare a 6 mol / L curcumin solution. Separately prepare a 2 mol / L magnesium ion solution. Mix the metal and polyphenol solutions at a molar ratio of 1:3 and stir magnetically to form a homogeneous pre-assembled solution.

[0061] Step 3: Under magnetic stirring, the pre-assembled solution prepared in Step 2 is slowly added dropwise to the egg white peptide solution in Step 1 at a volume ratio of 3:1. After the addition is complete, the pH of the system is adjusted to 7.0 using 1 mol / L NaOH or HCl, and stirring is continued for 30 min. The resulting solution is then freeze-dried at low temperature to obtain composite particle powder, which is stored at 25℃.

[0062] Example 3: Preparation of ternary synergistically assembled composite particles of egg white peptide / zinc ion / catechin-anthocyanin

[0063] This embodiment aims to prepare a ternary synergistically assembled composite particle based on a metal-phenolic network, consisting of egg white peptides, zinc ions, catechins, and anthocyanins.

[0064] Step 1: Take the egg white peptide powder prepared in Example 1, dissolve it in distilled water, and prepare an egg white peptide solution with a concentration of 20 mg / mL.

[0065] Step 2: Weigh out catechins and anthocyanins, and dissolve them together in a 75% ethanol aqueous solution to prepare a 2 mol / L polyphenol solution. Separately prepare a 6 mol / L zinc ion solution. Mix the metal and polyphenol solutions at a molar ratio of 3:1 and stir magnetically to form a homogeneous pre-assembled solution.

[0066] Step 3: Under magnetic stirring, the pre-assembled solution prepared in Step 2 is slowly added dropwise to the egg white peptide solution in Step 1 at a volume ratio of 1:3. After the addition is complete, the pH of the system is adjusted to 6.0 using 1 mol / L NaOH or HCl, and stirring is continued for 30 min. The resulting solution is then freeze-dried at low temperature to obtain composite particle powder, which is stored at 25℃.

[0067] Example 4: Preparation of ternary synergistically assembled composite particles of egg white peptide / calcium ion-magnesium ion / quercetin

[0068] This embodiment aims to prepare a ternary synergistically assembled composite particle based on a metal-phenolic network, consisting of egg white peptide / calcium ion-magnesium ion / quercetin.

[0069] Step 1: Take the egg white peptide powder prepared in Example 1, dissolve it in distilled water, and prepare an egg white peptide solution with a concentration of 10 mg / mL.

[0070] Step 2: Weigh quercetin and dissolve it in a 75% ethanol aqueous solution to prepare a quercetin solution with a concentration of 4 mol / L. Separately prepare a metal ion solution with a concentration of 2 mol / L, containing 2 mol / L calcium ions and 2 mol / L magnesium ions. Mix the metal and polyphenol solutions at a molar ratio of 1:2 and form a homogeneous pre-assembled solution under magnetic stirring.

[0071] Step 3: Under magnetic stirring, the pre-assembled solution prepared in Step 2 is slowly added dropwise to the egg white peptide solution in Step 1 at a volume ratio of 2:1. After the addition is complete, the pH of the system is adjusted to 8.0 using 1 mol / L NaOH or HCl, and stirring is continued for 30 min. The resulting solution is then freeze-dried at low temperature to obtain composite particle powder, which is stored at 25℃.

[0072] Example 5: Preparation of ternary synergistically assembled composite particles of egg white peptide / zinc ion / quercetin

[0073] This embodiment focuses on preparing a ternary synergistically assembled composite particle based on a metal-phenolic network, consisting of egg white peptides, zinc ions, and quercetin.

[0074] Step 1: Take the egg white peptide powder prepared in Example 1, dissolve it in distilled water, and prepare an egg white peptide solution with a concentration of 10 mg / mL.

[0075] Step 2: Weigh quercetin and dissolve it in a 75% ethanol aqueous solution to prepare a quercetin solution with a concentration of 4 mol / L. Separately prepare a zinc ion solution with a concentration of 4 mol / L. Mix the metal and polyphenol solutions at a molar ratio of 1:1 and form a homogeneous pre-assembled solution under magnetic stirring.

[0076] Step 3: Under magnetic stirring, the pre-assembled solution prepared in Step 2 is slowly added dropwise to the egg white peptide solution in Step 1 at a 1:1 volume ratio. After the addition is complete, the pH of the system is adjusted to 7.0 using 1 mol / L NaOH or HCl, and stirring is continued for 30 min. The resulting solution is then freeze-dried at low temperature to obtain composite particle powder, which is stored at 25℃.

[0077] Experimental results show that the ternary synergistic composite particles based on a metal-phenolic network prepared in this invention exhibit good colloidal stability, antioxidant activity, and emulsifying properties. Taking Example 5 as an example, as shown in Table 1, the average particle size of the prepared egg white peptide / zinc ion / quercetin ternary synergistic composite particles is 268 nm, and the PDI is 0.19, indicating good dispersibility. Its potential is -26.1 mV, indicating that the composite particles have a negative charge on their surface, which is beneficial for their dispersion stability in the aqueous phase. The composite particles achieved 74.2% and 64.3% scavenging rates of DPPH and ABTS free radicals, respectively, confirming their excellent antioxidant activity. Its emulsifying activity index is 5.85 m. 2 / g, with an emulsification stability index of 28 min. Furthermore, taking Examples 2-5 as examples, after 6 months of storage at 25°C, the stability of the egg white peptides increased by more than 26%, and the particle redispersibility reached 95% or higher, indicating that the composite particles have excellent long-term storage stability.

[0078] Table 1. Physicochemical properties characterization of egg white peptide / polyphenol / metal ion ternary synergistic composite particles

[0079] Implementation Cases Particle size (d.nm) PDI Potential (mV) DPPH clearance rate (%) ABTS clearance rate (%) <![CDATA[Emulsifying activity index (m 2 / g)]]> Emulsification stability index (min) Example 2 158 0.12 -34.3 32.1 21.4 3.44 15 Example 3 174 0.17 -12.6 43.6 35.7 3.89 19 Example 4 224 0.23 -31.8 56.9 50.1 4.53 17 Example 5 268 0.19 -26.1 74.2 64.3 5.85 28

[0080] Example 6: Emulsion stabilized by ternary synergistic assembly of egg white peptide / magnesium ion / curcumin composite particles

[0081] In this embodiment, the composite particles prepared in Example 2 are used as a stabilizer to prepare an emulsion.

[0082] Step 1: Dissolve the lyophilized composite particle powder prepared in Example 2 in distilled water to prepare a dispersion with a concentration of 10 mg / mL. Disperse thoroughly under magnetic stirring to obtain a homogeneous aqueous phase.

[0083] Step 2: Select corn oil as the oil phase of the emulsion.

[0084] Step 3: Mix the aqueous phase and oil phase at a volume ratio of 3:1, and homogenize using a high-speed homogenizer at 10,000 rpm for 3 min to obtain the emulsion of the composite particle stabilizer.

[0085] Example 7: Emulsion stabilized by ternary synergistic assembly of egg white peptides / zinc ions / catechins-anthocyanins into a composite particle

[0086] In this embodiment, the composite particles prepared in Example 3 are used as a stabilizer to prepare an emulsion.

[0087] Step 1: Take the lyophilized composite particle powder prepared in Example 3, dissolve it in distilled water, and prepare a dispersion with a concentration of 10 mg / mL. Disperse it thoroughly under magnetic stirring to obtain a homogeneous aqueous phase.

[0088] Step 2: Select soybean oil as the oil phase of the emulsion.

[0089] Step 3: Mix the aqueous phase and oil phase at a volume ratio of 2:1, and homogenize using a high-speed homogenizer at 11,000 rpm for 4 min to obtain the emulsion of the composite particle stabilizer.

[0090] Example 8: Emulsion stabilized by ternary synergistic assembly of egg white peptide / calcium ion-magnesium ion / quercetin composite particles

[0091] In this embodiment, the composite particles prepared in Example 4 are used as a stabilizer to prepare an emulsion.

[0092] Step 1: Dissolve the lyophilized composite particle powder prepared in Example 4 in distilled water to prepare a dispersion with a concentration of 10 mg / mL. Disperse thoroughly under magnetic stirring to obtain a homogeneous aqueous phase.

[0093] Step 2: Select coconut oil as the oil phase of the emulsion.

[0094] Step 3: Mix the aqueous phase and oil phase at a volume ratio of 1:1, and homogenize using a high-speed homogenizer at 12000 rpm for 5 min to obtain the emulsion of the composite particle stabilizer.

[0095] Example 9: Emulsion stabilized by ternary synergistic assembly of egg white peptide / zinc ion / quercetin composite particles

[0096] In this embodiment, the composite particles prepared in Example 5 are used as a stabilizer to prepare the emulsion.

[0097] Step 1: Take the lyophilized composite particle powder prepared in Example 5, dissolve it in distilled water, and prepare a dispersion with a concentration of 10 mg / mL. Disperse it thoroughly under magnetic stirring to obtain a homogeneous aqueous phase.

[0098] Step 2: Select olive oil as the oil phase of the emulsion.

[0099] Step 3: Mix the aqueous phase and oil phase at a volume ratio of 1:3, and homogenize using a high-speed homogenizer at 13000 rpm for 3 min to obtain the emulsion of the composite particle stabilizer.

[0100] To evaluate the performance of the prepared emulsion, its microstructure and physical stability were characterized. For example... Figure 2 The optical microscopy observations shown indicate that the emulsions stabilized by the composite particles described in Examples 6-9 exhibit small and uniformly distributed droplets, with no obvious droplet aggregation or flocculation observed. This suggests that the construction of the metal-phenolic network enhances the interfacial adsorption capacity of the egg white peptide composite particles and forms a dense and highly cross-linked protective layer at the oil-water interface through the multidentate coordination between metal ions, polyphenols, and egg white peptides, thereby effectively inhibiting droplet aggregation and Ostwald ripening. Further stability test results (as shown in Table 2) show that the stability of emulsions stabilized by different composite particles varies. Among them, the emulsion stabilized by the egg white peptide / zinc ion / quercetin composite particles (Example 9) performed best. Its emulsion exudation index was 0% under 70°C heating treatment, repeated freeze-thaw cycles, and at a salt ionic strength of 150 mM NaCl; the emulsion exudation index was only 2.6% under pH 6.0-9.0 conditions. These results confirm that the emulsion constructed in this invention possesses excellent physical stability and tolerance to environmental stress.

[0101] Table 2 Emulsion stability evaluation

[0102] Implementation Cases Storage emulsification index (%) High-temperature emulsification index (%) Freeze-thaw emulsion separation index (%) pH-treated emulsification index (%) Salt-treated emulsion separation index (%) Example 6 66.7 4.7 30 65.2 55.6 Example 7 55.6 5 6.7 52.4 60 Example 8 47.6 0 20 32.4 30.7 Example 9 2.5 0 0 2.6 0

[0103] It should be noted that the freeze-drying, centrifugation, homogenization and mass spectrometry analysis methods mentioned in this invention are all conventional processes that are commonly known and used by those skilled in the art, and their corresponding operation procedures can fully characterize the relevant technical features. Therefore, the above processes will not be described again in this specification.

[0104] The raw materials used in the implementation of this invention include, but are not limited to, curcumin, quercetin, catechin, anthocyanins, zinc chloride, calcium chloride, magnesium chloride, and dithiothreitol, all of which are commercially available reagents under existing technical conditions and can be obtained by those skilled in the art through conventional procurement methods. Unless otherwise specified, other raw materials, equipment, and reagents involved in this invention may also be selected from commercially available products commonly used in the field or obtained through conventional methods.

[0105] It should also be understood that the foregoing embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the scope of protection of the present invention. Equivalent changes or reasonable substitutions made by those skilled in the art to the relevant technical solutions without departing from the technical concept and substance of the present invention should all be considered to fall within the scope of protection of the present invention.

Claims

1. A method for preparing a ternary synergistically assembled composite particle of egg white peptide / polyphenol / metal ion, characterized in that, Includes the following steps: (1) Preparation of egg white peptides Step 1: Dissolve egg white protein powder in distilled water to prepare a protein solution with a mass fraction of 1-10% (w / v), and transfer the protein solution to an enzymatic hydrolysis container for pretreatment in a constant temperature water bath at 60-100℃ for 5-10 minutes. Then transfer it to a constant temperature water bath at 50-90℃, and adjust the pH of the solution to 5.0-10.0 using 1 mol / L NaOH or HCl; Step 2: Add 2-8% of the protein mass of protease to the solution obtained in Step 1, and carry out the enzymatic hydrolysis reaction in a constant temperature water bath at 50-90℃ for 1-5 h, while maintaining the pH of the system between 5.0 and 10.0 with 1 mol / L NaOH or HCl; the protease is selected from one or two of acidic protease, neutral protease, alkaline protease, papain, bromelain, flavor protease and Bacillus licheniformis protease; Step 3: After the enzymatic hydrolysis reaction is completed, the reaction system is heated in a 90℃ constant temperature water bath for 10 min to inactivate the enzyme preparation. Then the enzymatic hydrolysate is cooled to room temperature. The cooled enzymatic hydrolysate is then freeze-dried at low temperature to obtain egg white peptide powder. The obtained egg white peptide powder is stored at -20℃ for subsequent detection and analysis. (2) Preparation of egg white peptide / polyphenol / metal ion ternary synergistic assembly composite particles Step 1: Dissolve the egg white peptide described in (1) in distilled water to prepare an egg white peptide solution with a concentration of 10-20 mg / mL; Step 2: Prepare a metal ion solution with a concentration of 1-6 mol / L and a polyphenol solution with a concentration of 1-6 mol / L; the metal ion in the metal ion solution is selected from one or more of sodium ions, potassium ions, calcium ions, zinc ions, magnesium ions, and iron ions, and the polyphenol in the polyphenol solution is selected from one or more of curcumin, quercetin, resveratrol, astaxanthin, anthocyanins, catechins, and tannic acid; mix the metal ion solution and the polyphenol solution at a metal to polyphenol molar ratio of 1:3-3:1 to form a metal-polyphenol network pre-assembled solution; Step 3: Using the antisolvent coprecipitation method, the metal-polyphenol network pre-assembled solution was slowly added dropwise to the egg white peptide solution obtained in Step 1 at a volume ratio of 1:3-3:1, and the pH of the system was adjusted to 5.0-9.

0. After thorough stirring, egg white peptide / polyphenol / metal ion ternary synergistic composite particles based on the metal-phenolic network were obtained. Subsequently, the composite particle powder was obtained by low-temperature freeze drying and stored at 25°C for subsequent analysis.

2. The method for preparing a ternary synergistically assembled composite particle of egg white peptide / polyphenol / metal ion according to claim 1, characterized in that, The egg white peptide contains the following amino acid sequence: GTEATGSTGAIGNIK, KGEADAVALDGGLVY, and KGEADAVALDGG.

3. A ternary synergistically assembled composite particle of egg white peptide / polyphenol / metal ion, characterized in that: The composite particles were prepared according to the method described in any one of claims 1-2, which are ternary synergistic assembly of egg white peptide / polyphenol / metal ion.

4. The egg white peptide / polyphenol / metal ion ternary synergistic assembly composite particle according to claim 3, characterized in that, After co-assembly, the colloidal properties of the composite particles are improved, which is manifested by a decrease in the absolute value of surface charge, the enrichment of hydrophobic groups on the particle surface, which enhances the hydrophobicity of the interface, and promotes the transformation of the protein peptide molecule conformation to an ordered β-sheet secondary structure dominated by sheet structure. After storage at 25℃ for 6 months, the stability of protein peptide is improved by more than 26%, and the redispersibility of the particles reaches 95% or more.

5. A method for preparing a stable emulsion of egg white peptide / polyphenol / metal ion ternary synergistically assembled composite particles, characterized in that, Includes the following steps: Step 1: Dissolve the egg white peptide / polyphenol / metal ion ternary synergistic composite particle powder based on metal-phenolic network obtained according to claim 1 or 2 in distilled water, and fully disperse it under magnetic stirring. At 25°C, use 1 mol / L NaOH or HCl to precisely adjust the pH of the system to 5.0-9.0 to obtain the aqueous phase of Pickering emulsion. Step 2: Select one or two of corn oil, coconut oil, olive oil, or soybean oil as the oil phase of the emulsion; Step 3: Mix the aqueous phase from Step 1 and the oil phase from Step 2 at a volume ratio of 1:3-3:1, and homogenize using a high-speed homogenizer at 10000-15000 rpm for 3-5 min to obtain an emulsion.

6. A stable emulsion composed of egg white peptide / polyphenol / metal ion ternary synergistically assembled composite particles, characterized in that, The emulsion prepared by the method according to claim 5 has droplet size in the micrometer range and uniform distribution, with no obvious droplet aggregation under an optical microscope; and its emulsion exfoliation index is zero under environmental stress conditions of heating at 70°C for 60 min, freeze-thaw cycles, pH 6.0-9.0, and 0-150 mM NaCl ion strength.

7. A functional product, characterized in that: Its components include the egg white peptide / polyphenol / metal ion ternary synergistic assembly composite particles as described in claim 3, or the egg white peptide / polyphenol / metal ion ternary synergistic assembly composite particles stabilized emulsion as described in claim 6.

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