Method for preparing high-interfacial-activity phospholipid-olein nano-composite

Abstract

The invention discloses a method for preparing a high-interfacial-activity phospholipid-olein nano-composite, and belongs to the crossing field of a food technology, a colloid interface chemical technology and a nano-biotechnology. The method comprises two steps of co-dissolution of phospholipid and oleosin and self-assembly of phospholipid and oleosin: dissolving 5mg of phospholipid with the saturation degree of 0-2 in 10mL of distilled water, performing vortex for 3 minutes, then performing ultrasonic treatment for 30-90 seconds under the power of 200-400W to obtain a phospholipid stock solution, then mixing 1mg of oleosin solid powder, 2mL of phospholipid stock solution and 8mL of distilled water, performing vortex for 5 minutes, and performing centrifugal separation to obtain the phospholipid and oleosin self-assembly. The preparation method comprises the following steps: carrying out ultrasonic treatment for 30-90 seconds at the temperature of 25 DEG C, standing for 1-3 hours at the power of 200-400 W, then centrifuging for 5-30 minutes at the speed of 3000-5000 rpm, and collecting supernate obtained after centrifuging, namely the high-interfacial-activity phospholipid-oleosin nano-composite. The particle size of the prepared high-interfacial-activity phospholipid-olein nano-composite ranges from 200 nm to 600 nm, and interfacial tension can be effectively reduced when the high-interfacial-activity phospholipid-olein nano-composite is adsorbed to an oil-water interface.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of food technology, colloidal interface chemistry and nanobiotechnology, and more specifically, relates to a method for preparing highly interfacially active phospholipid-oil body protein nanocomposites. Background Technology

[0002] Phospholipids and proteins are two core components of biological membranes and many natural food systems (such as milk and egg liquid), and their interfacial interactions are crucial for maintaining the structure and function of these systems. In the field of food colloids, mimicking this natural synergistic effect and artificially constructing phospholipid-protein complex systems is considered an important strategy for developing next-generation, efficient, stable, and clean-labeling emulsifiers and delivery systems.

[0003] Oil body proteins are the most abundant intrinsic proteins on plant oil bodies. In nature, they form a monolayer phospholipid membrane with phospholipids, embedded in the phospholipid layer through a hydrophobic central anchor, while their hydrophilic arms extend into the aqueous phase. This unique "pinning" structure endows natural oil bodies with excellent physical and chemical stability. Therefore, oil body proteins are ideal candidates for biomimetic composites with phospholipids. Theoretically, by reconstructing the complexes of oil body proteins and phospholipids, nanoparticles with interfacial properties similar to or even superior to those of natural oil bodies can be obtained, showing great application potential in the food, pharmaceutical, and cosmetic fields.

[0004] However, the controlled and efficient preparation of phospholipid-oil body protein nanocomposites with well-defined structures, uniform sizes, and high interfacial activity in aqueous phase remains a significant technical challenge. Patent CN202110184450.0 describes the preparation of a plant-inspired oil body core-shell liposome using phospholipids and oil body proteins, but this method requires large amounts of organic solvents and suffers from drawbacks such as cumbersome steps, potential impact of residual ethanol on protein activity, and poor process controllability.

[0005] Therefore, this invention aims to develop a highly interfacially active phospholipid-oil body protein complex by leveraging the interaction between the hydrophobic ends of phospholipid molecules and the transmembrane domains of oil body proteins, thereby expanding its application as an emulsifier in Pickering emulsions for food, pharmaceuticals, and cosmetics. Summary of the Invention

[0006] Existing technologies for preparing phospholipid-protein complexes generally suffer from one or more of the following drawbacks: complex processes involving the use and removal of toxic organic solvents, failing to meet green food processing requirements; demanding equipment requirements and stringent process parameter control, making large-scale stable production difficult; resulting in complexes with uneven size, unstable structure, and unsatisfactory interfacial activity; and an inability to accurately simulate the unique spatial structure and interactions between phospholipids and oil proteins in natural oil bodies. To address these shortcomings, the applicant, through extensive preliminary research on the physicochemical properties of oleosin proteins, has creatively discovered that the co-solubility and self-assembly of phospholipids and oil proteins in distilled water can be achieved through the interaction between the hydrophobic ends of phospholipid molecules and the transmembrane domains of oil proteins. The research found that this method not only avoids the use of denaturants and organic solvents but also significantly reduces costs compared to traditional methods.

[0007] The primary objective of this invention is to overcome the aforementioned shortcomings of the prior art and provide a method for preparing highly interfacially active phospholipid-oil body protein nanocomposites that is simple to process, operates under mild conditions, requires no toxic organic solvents, and offers high controllability. The prepared phospholipid-oil body protein nanocomposites exhibit uniform particle size, stable structure, and high interfacial activity, thus broadening the application of oil body proteins in the food, pharmaceutical, and cosmetic fields.

[0008] To achieve the aforementioned objectives, the present invention employs the following technical solutions: This invention provides a technique for preparing highly interfacially active micron-sized oleosin protein particles, comprising the following steps: This invention provides a method for preparing highly interfacially active phospholipid-oil body protein nanocomposites, characterized by comprising the following two core steps: (1) Preparation steps of phospholipid dispersion: Weigh a certain amount of phospholipid and disperse it in distilled water. After vortexing, it is ultrasonically treated to obtain a clear or semi-transparent phospholipid stock solution. (2) Self-assembly step of the complex: The oil protein solid powder, the phospholipid stock solution obtained in step (1) are mixed with distilled water, vortexed and then subjected to ultrasonic treatment; (3) The treated mixture was left to stand and age, then centrifuged and the supernatant was collected, which is the highly interfacially active phospholipid-oil body protein nanocomposite.

[0009] Preferably, in step (1), the saturation of phospholipids is defined as the average number of double bonds in the fatty acid chains of the phospholipid molecule, which ranges from 0 to 2. Phospholipids within this saturation range have suitable membrane fluidity and self-assembly ability at room temperature, and can achieve effective intermolecular intercalation and binding with the hydrophobic regions of oil body proteins.

[0010] Preferably, in step (1), the phospholipid is one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and phosphatidic acid, and the saturation of the hydrophobic fatty acid chain of the phospholipid is between 0 and 2.

[0011] More preferably, in step (1), the phospholipid is one of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine, myristoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, and 1,2-myristoyl-sn-propanetriyl-3-phosphatidylethanolamine.

[0012] Preferably, in step (1), the mass ratio of phospholipid to distilled water is 5 mg: 10 mL. This concentration ensures that the phospholipid can be effectively dispersed and hydrated.

[0013] Preferably, in step (1), the vortex mixing time is 3-5 minutes to ensure that the phospholipid powder is fully wetted and initially dispersed.

[0014] Preferably, in step (1), the actual power output of the ultrasonic treatment is 200-400 W, and the total effective ultrasonic time is 30-90 seconds. This step utilizes the cavitation effect and shear force of ultrasound to break up the phospholipid aggregates and form smaller and more uniformly distributed phospholipid dispersions.

[0015] Preferably, in step (2), the mass-to-volume ratio of the oil-body protein solid powder, phospholipid stock solution, and distilled water is 1 mg: 2 mL: 8 mL. This ratio is derived from extensive experimental optimization, ensuring that the phospholipid and oil-body protein have the optimal molar ratio, thereby driving the formation of a thermodynamically stable nanocomposite.

[0016] Preferably, in step (2), the vortexing time is 5 minutes to ensure that the oil protein powder is fully dispersed and in uniform contact with other components.

[0017] Preferably, in step (2), the actual power output of the ultrasonic treatment is 200-400 W, and the total effective ultrasonic time is 30-90 seconds. This step provides the necessary energy for the interaction between phospholipids and oil body proteins, promotes the reconstruction of the phospholipid bilayer or micelles, and allows the hydrophobic anchoring regions of the oil body proteins to be efficiently "inserted" into them.

[0018] Preferably, in step (3), the static aging temperature is 25°C and the time is 1-3 hours. This aging process allows the ultrasonically activated composite system to undergo structural adjustment and energy relaxation, making the composite structure more stable and uniform.

[0019] Preferably, in step (3), the centrifugation speed is 3000-5000 rpm and the centrifugation time is 10-30 minutes. This step aims to remove aggregates that have failed to assemble into nanocomposites, or that are too large or too heavy, to ensure the homogeneity and colloidal stability of the composite population in the supernatant.

[0020] The present invention also provides a phospholipid-oil body protein nanocomposite, which is prepared by the above method.

[0021] Preferably, the particle size distribution of the phospholipid-oil body protein nanocomposite is in the range of 200-600 nm.

[0022] Preferably, the phospholipid-oil body protein nanocomposite can be efficiently adsorbed at the oil-water interface and significantly reduce interfacial tension. As determined by the hanging drop method, it can reduce the interfacial tension of the soybean oil-water system from an initial approximately 25 mN / m to below 10 mN / m, exhibiting superior interfacial activity compared to single phospholipids or single oil body proteins.

[0023] This invention also provides the application of the above-mentioned highly interfacially active phospholipid-oil body protein nanocomposite in the preparation of functional foods.

[0024] The present invention has the following beneficial effects: This invention provides a technique for preparing highly interfacially active micron-sized oleosin protein particles. The method involves preparing highly interfacially active micron-sized oily protein particles by combining ethanol-induced self-assembly with ultrasound-membrane filtration technology.

[0025] This method is simple, operates under mild conditions, and is highly controllable. The prepared particles have uniform particle size, stable structure, and high interfacial activity, showing broad application prospects in the fields of functional foods, pharmaceuticals, and cosmetics. Attached Figure Description

[0026] Figure 1 The average particle size diagrams are shown for the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4 and Comparative Example 1.

[0027] Figure 2 The ζ-potential diagrams are for the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4.

[0028] Figure 3 The interfacial tension diagrams are for the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4.

[0029] Figure 4 This is a schematic diagram of a method for preparing highly interfacially active phospholipid-oil body protein nanocomposites. Detailed Implementation

[0030] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0031] Example 1 (1) Preparation of phospholipid dispersion: Accurately weigh 5.0 mg of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine into a 20 mL glass scintillation bottle and add 10 mL of distilled water. Vortex for 3 minutes to fully suspend the phospholipid powder. Then, place the bottle in an ice-water bath for sonication: the total effective sonication time is 90 seconds, and the actual power output is approximately 200 W. The solution changes from turbid to a translucent or slightly opalescent clear solution, which is the phospholipid stock solution.

[0032] (2) Complex self-assembly: Accurately weigh 1.0 mg of oil-body protein solid powder into a 15 mL centrifuge tube. Measure 2.0 mL of the phospholipid stock solution prepared in step (1) and add it to the centrifuge tube, then add 8.0 mL of distilled water. Immediately use a vortex mixer to mix vigorously at maximum speed for 5 minutes to ensure complete dispersion of the protein powder. Subsequently, place the mixture in an ice-water bath for sonication: the total effective sonication time is 90 seconds, and the actual power output is approximately 200 W.

[0033] (3) Transfer the ultrasonically treated mixture to a 25°C constant temperature incubator and let it stand for 1 hour. Finally, centrifuge at 25°C and 3000 rpm for 30 minutes using a benchtop high-speed centrifuge. Carefully aspirate the supernatant with a pipette, avoiding contact with any trace precipitate that may be present at the bottom of the tube. This supernatant is the solution of highly interfacially active phospholipid-oil body protein nanocomposite.

[0034] Example 2 (1) Preparation of phospholipid dispersion: Accurately weigh 5.0 mg of myristoyl phosphatidylcholine into a 20 mL glass scintillation bottle and add 10 mL of distilled water. Vortex for 3 minutes to fully suspend the phospholipid powder. Then, place the bottle in an ice-water bath for ultrasonic treatment: the total effective ultrasonic time is 30 seconds, and the actual power output is about 400 W. The solution changes from turbid to a translucent or slightly opalescent clear solution, which is the phospholipid stock solution.

[0035] (2) Complex self-assembly: Accurately weigh 1.0 mg of oil-body protein solid powder into a 15 mL centrifuge tube. Measure 2.0 mL of the phospholipid stock solution prepared in step (1) and add it to the centrifuge tube, then add 8.0 mL of distilled water. Immediately use a vortex mixer to mix vigorously at maximum speed for 5 minutes to ensure complete dispersion of the protein powder. Subsequently, place the mixture in an ice-water bath for sonication: the total effective sonication time is 30 seconds, and the actual power output is approximately 400 W.

[0036] (3) Transfer the ultrasonically treated mixture to a 25°C constant temperature incubator and let it stand for 2 hours. Finally, centrifuge at 25°C and 4000 rpm for 20 minutes using a benchtop high-speed centrifuge. Carefully aspirate the supernatant with a pipette, avoiding contact with any trace precipitate that may be present at the bottom of the tube. This supernatant is the solution of highly interfacially active phospholipid-oil body protein nanocomposite.

[0037] Example 3 (1) Preparation of phospholipid dispersion: Accurately weigh 5.0 mg of 1-palmitoyl-2-oleoylphosphatidylethanolamine into a 20 mL glass scintillation bottle and add 10 mL of distilled water. Vortex for 5 minutes to fully suspend the phospholipid powder. Then, place the bottle in an ice-water bath for ultrasonic treatment: the total effective ultrasonic time is 60 seconds, and the actual power output is about 300 W. The solution changes from turbid to a translucent or slightly opalescent clear solution, which is the phospholipid stock solution.

[0038] (2) Complex self-assembly: Accurately weigh 1.0 mg of oil-body protein solid powder into a 15 mL centrifuge tube. Measure 2.0 mL of the phospholipid stock solution prepared in step (1) and add it to the centrifuge tube, then add 8.0 mL of distilled water. Immediately use a vortex mixer to mix vigorously at maximum speed for 5 minutes to ensure complete dispersion of the protein powder. Subsequently, place the mixture in an ice-water bath for sonication: the total effective sonication time is 60 seconds, and the actual power output is approximately 300 W.

[0039] (3) Transfer the ultrasonically treated mixture to a 25°C constant temperature incubator and let it stand for 3 hours. Finally, centrifuge at 25°C and 5000 rpm for 10 minutes using a benchtop high-speed centrifuge. Carefully aspirate the supernatant with a pipette, avoiding contact with any trace precipitate that may be present at the bottom of the tube. This supernatant is the solution of highly interfacially active phospholipid-oil body protein nanocomposite.

[0040] Example 4 (1) Preparation of phospholipid dispersion: Accurately weigh 5.0 mg into a 20 mL glass scintillation bottle and add 10 mL of distilled water. Vortex for 3 minutes to fully suspend the phospholipid powder. Then, place the bottle in an ice-water bath for ultrasonic treatment: the total effective ultrasonic time is 60 seconds, and the actual power output is about 300W. The solution changes from turbid to a translucent or slightly opalescent clear solution, which is the phospholipid stock solution.

[0041] (2) Complex self-assembly: Accurately weigh 1.0 mg of oil-body protein solid powder into a 15 mL centrifuge tube. Measure 2.0 mL of the phospholipid stock solution prepared in step (1) and add it to the centrifuge tube, then add 8.0 mL of distilled water. Immediately use a vortex mixer to mix vigorously at maximum speed for 5 minutes to ensure complete dispersion of the protein powder. Subsequently, place the mixture in an ice-water bath for sonication: the total effective sonication time is 60 seconds, and the actual power output is approximately 300 W.

[0042] (3) Transfer the ultrasonically treated mixture to a 25°C constant temperature incubator and let it stand for 2 hours. Finally, centrifuge at 25°C and 4000 rpm for 20 minutes using a benchtop high-speed centrifuge. Carefully aspirate the supernatant with a pipette, avoiding contact with any trace precipitate that may be present at the bottom of the tube. This supernatant is the solution of highly interfacially active phospholipid-oil body protein nanocomposite.

[0043] Comparative Example 1 This comparative example is used to illustrate the effect of a single phospholipid system.

[0044] Accurately weigh 5.0 mg of 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine into a 20 mL glass scintillation bottle and add 10 mL of distilled water. Vortex for 3 minutes to fully suspend the phospholipid powder. Then, place the bottle in an ice-water bath and sonicate: the total effective sonication time is 60 seconds, and the actual power output is approximately 300 W. The solution changes from turbid to a translucent or slightly opalescent clear solution, which is the phospholipid stock solution. Take 2.0 mL of the phospholipid stock solution prepared in step (1) and add it to the centrifuge tube, then add 8.0 mL of distilled water. Immediately use a vortex mixer to mix vigorously at maximum speed for 5 minutes. Then, place the mixture in an ice-water bath and sonicate: the total effective sonication time is 60 seconds, and the actual power output is approximately 300 W. Transfer the sonicated mixture to a 25°C constant temperature incubator and let it stand for 2 hours. Finally, using a benchtop high-speed centrifuge, centrifuge at 25°C and 4000 rpm for 20 minutes, and carefully use a pipette to aspirate the supernatant for comparative analysis.

[0045] Example Quality Inspection and Result Analysis (1) Determination of the average particle size of the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4 and Comparative Example 1: The average particle size of the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4 and Comparative Example 1 was determined using a micron particle size analyzer.

[0046] Figure 1 The figures show the average particle size of the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4 and Comparative Example 1. The particle size of Examples 1-4 is less than 600 nm. The addition of phospholipids can effectively reduce the degree of self-aggregation of oil body proteins, thereby reducing the particle size. The main factor causing this phenomenon is that the hydrophobic fatty acid chains at the hydrophobic end of the phospholipids can bind to the transmembrane domains of oil body proteins through hydrophobic interactions, reducing the tendency of hydrophobic interactions between protein transmembrane domains while increasing their solubility. Among the four examples, Example 1 has the largest particle size, while Example 3 has the smallest particle size. The reason for this difference is mainly inferred to be due to differences in phospholipid composition, phospholipid molecular weight, and the amount bound to the transmembrane domains of oil body proteins.

[0047] (2) Measurement of the ζ-potential of the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4: A potentiometer was used to measure the ζ-potential changes of the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4.

[0048] Figure 2 The results are the absolute values ​​of the ζ-potential of the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4. The results show that the absolute values ​​of the ζ-potential in all four examples are greater than 20 mV; the absolute value of the ζ-potential in Example 1 is higher than that in Example 2; the absolute values ​​of the potentials in Examples 3 and 4 are similar. Unsaturated phosphatidylcholine contains a C=C bond compared to saturated phosphatidylcholine. Unlike C=C, which consists of only one σ bond, unsaturated phosphatidylcholine also contains a π bond formed by the stacking of unhybridized p orbitals. The π bond has characteristics such as low bond energy, high electron density, and spatial exposure, making it easier to participate in covalent or non-covalent interactions. Therefore, the interaction between unsaturated phosphatidylcholine and saturated phosphatidylethanolamine in binding with oil body proteins differs, resulting in significant differences in particle size and absolute ζ-potential. The absolute ζ-potential of unsaturated phosphatidylethanolamine and saturated phosphatidylethanolamine is not significantly different. This phenomenon is mainly due to the small surface potential caused by the small molecule ethanolamine forming the hydrophilic group at the head.

[0049] (3) Measurement of interfacial tension of the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4: An interfacial tension meter was used to measure and determine the changes in interfacial tension of the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4.

[0050] like Figure 3 As shown, in the initial stage, the interfacial tension values ​​of Examples 1-4 gradually decreased with increasing time. This is because the phospholipid-protein complex gradually adsorbed from the aqueous phase to the interface, resulting in a decrease in interfacial tension. Among them, Example 1 had the fastest adsorption rate. The main reason for this difference is that Example 1 has a stronger attraction with the oil phase, thus reducing the time required for adsorption. Compared with Example 1, the adsorption rate of Example 2 was slower and the interfacial tension increased. This was due to the difference in the saturation of the fatty acid chains at the hydrophobic ends of the phospholipids. Phospholipids with a high degree of unsaturation have a weaker molecular structure and stronger interaction with oil proteins. They are also more prone to molecular rearrangement at the interface, resulting in a faster adsorption rate and a stronger ability to reduce interfacial tension.

[0051] (4) Determination of the interaction between phospholipids and oil bodies in the highly interfacially active phospholipid-oil body protein nanocomposites prepared in Examples 1-4: After generating the three-dimensional structures using AlphaFold 2 constraints, the three-dimensional structures of oleosin and glycinin were used to generate pdbqt files using Chimera v1.16. Subsequently, after generating the docking parameter files using AutoDockFR, the docking box was defined using the ligand expansion method for flexible docking. As shown in Table 1, the binding free energies between the phospholipids and target proteins in Examples 1-4 were -9.3904 kcal / mol, -9.2362 kcal / mol, -8.9553 kcal / mol, and -8.2634 kcal / mol, respectively. Example 1 had the highest binding free energy, while Example 4 had the lowest. The main reason for this difference is the difference in the interaction between the hydrophilic end groups and the hydrophobic end fatty acid chain saturation and the oil body protein.

[0052] Table 1

[0053] Figure 4 The present invention illustrates a process for preparing highly interfacially active phospholipid-oil body protein nanocomposites.

[0054] In summary, the present invention has the following beneficial effects: This invention provides a technique for preparing highly interfacially active micron-sized oleosin protein particles. The method involves ethanol-induced self-assembly combined with ultrasound-membrane filtration to prepare highly interfacially active micron-sized oil-body protein particles. This method is simple, operates under mild conditions, and is highly controllable. The prepared particles have uniform particle size, stable structure, and high interfacial activity, showing broad application prospects in functional foods, pharmaceuticals, and cosmetics.

[0055] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A method for preparing highly interfacially active phospholipid-oil body protein nanocomposites, characterized in that, Includes the following steps: (1) Weigh a certain amount of phospholipids and dissolve them in distilled water. After vortexing and ultrasonic treatment, a phospholipid stock solution is obtained. (2) Mix the oil protein solid powder, phospholipid stock solution and distilled water, vortex and then sonicate. (3) The treated mixture is left to stand and age, then centrifuged and the supernatant is collected, which is the highly interfacially active phospholipid-oil body protein nanocomposite.

2. The method according to claim 1, characterized in that, In step (1), the phospholipid is one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and phosphatidic acid, and the saturation of the hydrophobic fatty acid chain of the phospholipid is between 0 and 2.

3. The method according to claim 1, characterized in that, In step (1), the vortex oscillation time is 3-5 minutes, the actual power output of the ultrasonic treatment is 200-400 W, and the total effective ultrasonic time of the ultrasonic treatment is 30-90 seconds.

4. The method according to claim 1, characterized in that, In step (2), the mass-volume ratio of oil protein solid powder, phospholipid stock solution and distilled water is 1 mg: 2 mL: 8 mL.

5. The method according to claim 1, characterized in that, In step (2), the actual power output of the ultrasonic treatment is 200-400 W, and the total effective ultrasonic time of the ultrasonic treatment is 30-90 seconds.

6. The method according to claim 1, characterized in that, In step (3), the settling and aging time is 1-3 hours.

7. The method according to claim 1, characterized in that, In step (3), the centrifugation speed is 3000-5000 rpm and the centrifugation time is 10-30 minutes.

8. A highly interfacially active phospholipid-oil body protein nanocomposite, characterized in that, Prepared by the method described in any one of claims 1 to 7.

9. The highly interfacially active phospholipid-oil body protein nanocomposite according to claim 8, characterized in that, The particle size range of the highly interfacially active phospholipid-oil body protein nanocomposite is 200-600 nm.

10. The application of the highly interfacially active phospholipid-oil body protein nanocomposite according to claims 8-9 in the preparation of functional foods.

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