High-stability oleosin protein modified liposome oral liquid and production method thereof

Abstract

The invention relates to the technical field of health-care food, and discloses a high-stability oleosin protein modified liposome oral liquid and a production method thereof, and through modification and regulation of oleosin protein, the stability, targeting and oral bioavailability of liposome are improved, so that wider application is realized. The content of the invention comprises a preparation method and preferable components of the liposome oral liquid and application of the oral liquid in health care, nutritional supplement and adjuvant therapy of diseases, and a series of experiments prove that the technology is excellent in quality, stability and functionality and has potential application value.

Description

Technical Field

[0001] The present invention relates to the technical field of health food, in particular to a highly stable oleosin protein modified liposome oral liquid and a production method thereof. Background Art

[0002] With the development of biotechnology, liposomes, as an important drug carrier and nutritional supplement, have been widely used in drug delivery, health products, vaccine development and other fields. Liposomes can effectively encapsulate water-soluble or fat-soluble active ingredients, provide protection through the lipid bilayer structure, reduce the degradation of active ingredients, and promote their absorption and bioavailability in the body. However, the stability and effectiveness of liposomes still face some challenges, especially in terms of long-term storage, absorption efficiency of oral routes, and targeting.

[0003] In terms of liposome stability, protein modification and regulation have become an effective means to improve its performance. Oleosin protein is a type of small molecule membrane protein naturally present in plant oil bodies, with excellent interfacial activity and stability. In recent years, researchers have tried to apply oleosin protein to the stabilization of liposomes, but the existing technology is mostly focused on the basic research stage of its surface modification, lacks systematic optimization of the oleosin protein modification process, and has failed to fully realize its potential in liposome stability, oral liquid application and targeted delivery.

[0004] Therefore, there is an urgent need for a method for preparing a highly stable and functional liposome oral solution, which can improve the stability, targeting, and oral bioavailability of liposomes by modifying oleosin protein, thereby achieving a wider application. Summary of the invention

[0005] In view of this, the present invention proposes a highly stable oleosin protein modified liposome oral liquid and a production method thereof, aiming to solve the problems existing in the current technology.

[0006] The present invention provides a method for preparing a highly stable oleosin protein modified liposome oral liquid, comprising:

[0007] The oil seeds are soaked in a NaHCO3 solution at a solid-liquid ratio of 1:5-8 for 6-10 hours, and after pulping with a wall-breaking machine, the residue is removed by filtering through a filter cloth; the filtrate is centrifuged at a centrifugal force of 10,000×g to collect the oil body-rich layer, and washed with water 2-3 times, and centrifuged after each washing to obtain the plant oil body;

[0008] The plant oil bodies and NaCl solution are mixed in a volume ratio of 1:2, and ultrasonic treatment is performed for 10-20 minutes to dissolve non-membrane proteins; the mixture after ultrasonic treatment is centrifuged at 10,000×g at 0°C to obtain an emulsion; the emulsion is mixed with a solvent in a volume ratio of 1:3-6, centrifuged at 8000×g for 15-20 minutes to remove lipids, and a white layer is collected, and the residual solvent is used under a nitrogen flow, and lyophilized to obtain oleosin protein powder;

[0009] Mix lecithin, cholesterol, α-tocopherol, Tween-80 and nutrients, add anhydrous ethanol to disperse evenly, and perform vacuum rotary evaporation at 40-50° C. until a transparent lipid film is formed;

[0010] The oleosin protein powder is dispersed in water, then added into the transparent lipid film, subjected to hydration ultrasonic treatment, and sweetener is added in a mass ratio of phospholipid to sweetener of 9:10 to obtain an oleosin protein modified liposome oral solution.

[0011] Preferably, the oilseeds are one or more of soybeans, rapeseeds and sunflower seeds;

[0012] And / or, the lecithin is one or more of soybean lecithin, egg yolk lecithin and sunflower seed lecithin;

[0013] and / or, the nutrient is one or more of lutein, curcumin, β-carotene, lycopene and quercetin;

[0014] And / or, the sweetener is one or more of erythritol, mannitol or xylitol.

[0015] Preferably, the particle size of the liposomes in the liposome oral solution is 100-200 nm, the polydispersity index is less than 0.3, and the Zeta potential is -30 mV to +30 mV.

[0016] Preferably, the mass ratio of the lecithin, cholesterol, α-tocopherol, Tween-80 and nutrients is 6-10:1:0.2:0.1:0.1-1.

[0017] Preferably, before dispersing the oleosin protein powder in water, the oleosin protein powder is modified, and the modification method comprises:

[0018] The oleosin protein was coated with a hydrophilic surfactant;

[0019] Negatively or positively charged groups are introduced through chemical modification.

[0020] Preferably, the hydrophilic surfactant is polyvinyl alcohol or polyvinyl pyrrolidone.

[0021] Preferably, the modification method further comprises:

[0022] The oleosin protein is modified by using an aptamer, wherein the aptamer is a single-stranded nucleic acid, an antibody or a small molecule compound.

[0023] Preferably, the pH value of the liposome oral solution is 4.5-7.5.

[0024] The present invention also provides a highly stable oleosin protein modified liposome oral liquid, which is prepared by the above-mentioned method for preparing the highly stable oleosin protein modified liposome oral liquid.

[0025] The present invention also proposes the use of the above-mentioned highly stable oleosin protein modified liposome oral liquid in the preparation of health care, nutritional supplement and / or disease auxiliary treatment products.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] Improving the stability of liposomes: By coating and chemically modifying oleosin protein with a hydrophilic surfactant and introducing charged groups or aptamer modifications, the stability of liposomes is enhanced, avoiding the aggregation, lysis and leakage of liposomes during storage and use.

[0028] Enhanced biocompatibility and bioavailability: The affinity of oleosin protein to liposomes is enhanced through surface modification, which can improve the biocompatibility of liposomes, reduce the immune rejection reaction in the body, and thus improve the bioavailability of the active ingredients in the oral solution.

[0029] Improve targeting and delivery efficiency: By modifying oleosin protein with aptamers, the targeting of liposomes can be enhanced, especially for directional delivery in specific cells or tissues. For example, targeted delivery in the liver, immune system or specific disease tissues can improve the therapeutic effect of liposome drug delivery systems.

[0030] Enhanced antioxidant and health functions: By introducing nutrients with antioxidant activity (such as lutein, curcumin, etc.) and optimized liposome structure, the liposome oral liquid of the present invention can effectively scavenge free radicals, delay aging, improve immune function, and play an enhanced antioxidant, anti-inflammatory and other health functions.

[0031] Optimized production process: The present invention adopts an optimized liposome preparation process, including ultrasonic treatment, low-temperature centrifugation, nitrogen flow drying and other steps to ensure the uniformity and efficient loading of the liposomes, and has good industrial production potential.

[0032] Adaptable to diverse applications: Through flexible ingredient selection (such as different oil seeds, lecithin types and nutrients), the present invention can adjust the functions of liposomes according to different needs to meet the nutritional and health needs of different consumers. DETAILED DESCRIPTION

[0033] The exemplary embodiments of the present disclosure will be described in more detail below. Although the exemplary embodiments of the present disclosure are shown below, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to be able to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0034] Example 1

[0035] 1. Plant Oil Extraction

[0036] 1. Wash the soybean seeds and add them into 0.1M NaHCO3 solution at a solid-liquid ratio of 1:6, and soak for 8 hours to soften the surface tissue of the seeds.

[0037] 2. The soaked seeds are fully beaten with a wall breaking machine (high speed, 10 min), and the residue is removed by filtering through filter cloth to obtain a coarse filtrate.

[0038] 3. Centrifuge the filtrate at 10,000×g for 20 min to separate the oil body-rich layer.

[0039] 4. Wash the oil body-enriched layer three times with distilled water. Centrifuge at 10,000 × g for 10 min after each wash to collect the pure oil body layer.

[0040] 2. Oleosin protein extraction and modification

[0041] 1. The purified oil bodies were mixed with 4% NaCl solution in a volume ratio of 1:2 and treated with ultrasound (power 200 W, duration 15 min) in an ice bath to dissolve non-membrane proteins and retain membrane proteins.

[0042] 2. Centrifuge (10,000×g, 10 min) and collect the milky substance.

[0043] 3. Mix the emulsion with No. 6 solvent oil in a volume ratio of 1:4, centrifuge at 8000×g for 15 minutes, remove the lipids and collect the white protein layer.

[0044] 4. Add polyethylene glycol (PEG) modifier (2 mM, pH 8.0) to the protein layer and stir the reaction at 25°C for 2 hours to allow modification reaction to occur on the surface of the oleosin protein.

[0045] 5. After the reaction, wash with distilled water for 3 times and freeze-dry to obtain the modified oleosin protein powder.

[0046] 3. Liposome Preparation

[0047] 1. Weigh a mixture of soybean lecithin, cholesterol, α-tocopherol, Tween-80 and lutein in a mass ratio of 6:1:0.2:0.1:0.5, add it to anhydrous ethanol and stir evenly to form a uniform dispersion.

[0048] 2. Place the dispersion in a rotary evaporator and evaporate it under reduced pressure at 40°C to dryness to form a transparent lipid film.

[0049] 3. The oleosin protein powder is dispersed in water at a ratio of lecithin to oleosin protein of 10:1, and then added to the transparent lipid film, and then hydrated and ultrasonically treated to obtain a liposome dispersion.

[0050] 4. Preparation of functional oral solution

[0051] A 0.01% (W / V) erythritol aqueous solution was added to the above liposome dispersion according to the mass ratio of phospholipid to sweetener (erythritol) of 10:9, and the solution was placed in an ultrasonic processor for 5 minutes to form uniformly dispersed liposomes, thereby preparing a stable liposome functional oral solution.

[0052] Example 2

[0053] 1. Plant Oil Extraction

[0054] 1. Wash the soybean seeds and add them into 0.1M NaHCO3 solution at a solid-liquid ratio of 1:6, and soak for 8 hours to soften the surface tissue of the seeds.

[0055] 2. The soaked seeds are fully beaten with a wall breaking machine (high speed, 10 min), and the residue is removed by filtering through filter cloth to obtain a coarse filtrate.

[0056] 3. Centrifuge the filtrate at 10,000×g for 20 min to separate the oil body-rich layer.

[0057] 4. Wash the oil body-enriched layer three times with distilled water. Centrifuge at 10,000 × g for 10 min after each wash to collect the pure oil body layer.

[0058] 2. Oleosin protein extraction and modification

[0059] 1. The purified oil bodies were mixed with 4% NaCl solution in a volume ratio of 1:2 and treated with ultrasound (power 200 W, duration 15 min) in an ice bath to dissolve non-membrane proteins and retain membrane proteins.

[0060] 2. Centrifuge (10,000×g, 10 min) and collect the milky substance.

[0061] 3. Mix the emulsion with No. 6 solvent oil in a volume ratio of 1:4, centrifuge at 8000×g for 15 minutes, remove the lipids and collect the white protein layer.

[0062] 4. Add 2% chitosan solution (pH 5.0) modifier (2 mM, pH 8.0) to the protein layer and stir the reaction at 30°C for 1.5 hours to modify the surface of the oleosin protein to enhance the antioxidant capacity and membrane stability of the protein.

[0063] 5. After the reaction, wash with distilled water for 3 times and freeze-dry to obtain the modified oleosin protein powder.

[0064] 3. Liposome Preparation

[0065] 1. Weigh a mixture of soybean lecithin, cholesterol, α-tocopherol, Tween-80 and lutein in a mass ratio of 6:1:0.2:0.1:0.5, add it to anhydrous ethanol and stir evenly to form a uniform dispersion.

[0066] 2. Place the dispersion in a rotary evaporator and evaporate it under reduced pressure at 40°C to dryness to form a transparent lipid film.

[0067] 3. The oleosin protein powder is dispersed in water at a ratio of lecithin to oleosin protein of 10:1, and then added to the transparent lipid film, and then hydrated and ultrasonically treated to obtain a liposome dispersion.

[0068] 4. Preparation of functional oral solution

[0069] A 0.01% (W / V) erythritol aqueous solution was added to the above liposome dispersion according to the mass ratio of phospholipid to sweetener (erythritol) of 10:9, and the solution was placed in an ultrasonic processor for 5 minutes to form uniformly dispersed liposomes, thereby preparing a stable liposome functional oral solution.

[0070] Example 3

[0071] 1. Plant Oil Extraction

[0072] 1. Wash the soybean seeds and add them into 0.1M NaHCO3 solution at a solid-liquid ratio of 1:6, and soak for 8 hours to soften the surface tissue of the seeds.

[0073] 2. The soaked seeds are fully beaten with a wall breaking machine (high speed, 10 min), and the residue is removed by filtering through filter cloth to obtain a coarse filtrate.

[0074] 3. Centrifuge the filtrate at 10,000×g for 20 min to separate the oil body-rich layer.

[0075] 4. Wash the oil body-enriched layer three times with distilled water. Centrifuge at 10,000 × g for 10 min after each wash to collect the pure oil body layer.

[0076] 2. Oleosin protein extraction and modification

[0077] 1. The purified oil bodies were mixed with 4% NaCl solution in a volume ratio of 1:2 and treated with ultrasound (power 200 W, duration 15 min) in an ice bath to dissolve non-membrane proteins and retain membrane proteins.

[0078] 2. Centrifuge (10,000×g, 10 min) and collect the milky substance.

[0079] 3. Mix the emulsion with No. 6 solvent oil in a volume ratio of 1:4, centrifuge at 8000×g for 15 minutes, remove the lipids and collect the white protein layer.

[0080] 4. Use a nucleic acid aptamer (5'-GGGAGGACGAUGCGG-3') targeting the hepatocyte target molecule ASGPR (aspartate glycoprotein receptor) to react at 20°C for 3 hours to modify the surface of the oleosin protein in a ratio of protein: aptamer = 100:1 (mass ratio), giving it targeted delivery properties to hepatocytes.

[0081] 5. After the reaction, wash with distilled water for 3 times and freeze-dry to obtain the modified oleosin protein powder.

[0082] 3. Liposome Preparation

[0083] 1. Weigh a mixture of soybean lecithin, cholesterol, α-tocopherol, Tween-80 and lutein in a mass ratio of 6:1:0.2:0.1:0.5, add it to anhydrous ethanol and stir evenly to form a uniform dispersion.

[0084] 2. Place the dispersion in a rotary evaporator and evaporate it under reduced pressure at 40°C to dryness to form a transparent lipid film.

[0085] 3. The oleosin protein powder is dispersed in water at a ratio of lecithin to oleosin protein of 10:1, and then added to the transparent lipid film, and then hydrated and ultrasonically treated to obtain a liposome dispersion.

[0086] 4. Preparation of functional oral solution

[0087] A 0.01% (W / V) erythritol aqueous solution was added to the above liposome dispersion according to the mass ratio of phospholipid to sweetener (erythritol) of 10:9, and the solution was placed in an ultrasonic processor for 5 minutes to form uniformly dispersed liposomes, thereby preparing a stable liposome functional oral solution.

[0088] Comparative Example 1

[0089] The process difference from Example 1 is as follows: oleosin protein is directly extracted from oil bodies (without adding a modification step), and it is mixed with lipid membrane at a mass ratio of 10:1 to prepare liposome functional oral solution.

[0090] Comparative Example 2

[0091] Differences from the process of Example 1: α-tocopherol was not added to the lipid film formulation.

[0092] Test Example 1

[0093] The quality and functionality of the oral liquids prepared in Examples 1-3 and Comparative Examples 1-2 were tested.

[0094] 1. Test content and methods

[0095] 1. Quality inspection

[0096] (1) Particle size distribution and uniformity

[0097] Methods: Dynamic light scattering (DLS) was used to measure the average particle size and polydispersity index (PDI) of liposomes.

[0098] Evaluation criteria: The average particle size should be between 100-200nm, and PDI < 0.3 indicates uniform distribution.

[0099] (2) Zeta potential

[0100] Methods: Zeta potential analyzer was used to test the surface charge of liposomes.

[0101] Judging criteria: Zeta potential should be between -30mV and +30mV to ensure high stability.

[0102] (3) Encapsulation efficiency (EE%)

[0103] Methods: Liposomes were separated by ultracentrifugation, the contents of total nutrients and free nutrients were determined, and the encapsulation efficiency was calculated.

[0104] Formula: EE% = (total content - free content) / total content × 100%.

[0105] Evaluation criteria: Encapsulation rate should be ≥80%.

[0106] 2. Functional testing

[0107] (1) Stability test

[0108] Methods: The oral liquid was stored at 4℃ and 25℃ for 7 days, and the particle size change rate (%) and encapsulation efficiency decrease rate (%) were measured respectively.

[0109] Evaluation criteria: Particle size change rate <10%, encapsulation rate decrease rate <5% is stable.

[0110] (2) Antioxidant capacity

[0111] Method: DPPH free radical scavenging rate was determined and the scavenging rate (%) was recorded.

[0112] Evaluation criteria: A clearance rate higher than 70% indicates good antioxidant effect.

[0113] (3) Targeted Function Test (only for Example 3)

[0114] Methods: The uptake efficiency (%) of liposomes into target cells (hepatocytes) was determined by in vitro cell experiments.

[0115] Evaluation criteria: The effect is significant if the uptake efficiency is higher than 50%.

[0116] 2. Results

[0117] 1. Quality test results

[0118] The quality test results are shown in Table 1.

[0119] Table 1

[0120] Sample No. Average particle size (nm) PDI Zeta potential (mV) Encapsulation efficiency (EE%) Example 1 130 0.21 -25 85.4 Example 2 140 0.18 -28 87.1 Example 3 135 0.20 -26 86.7 Comparative Example 1 210 0.45 -15 65.2 Comparative Example 2 180 0.37 -20 72.8

[0121] As shown in Table 1, the average particle size, PDI, Zeta potential and encapsulation efficiency of the liposome oral liquid of Examples 1-3 all meet the evaluation criteria, showing good quality and stability. In particular, the particle size distribution of Examples 1-3 is uniform (PDI is less than 0.3), and the Zeta potential is between -30mV and +30mV, which ensures the stability of the liposome. The encapsulation efficiency is higher than 80%, indicating that the encapsulation effect of the nutrients is good. In contrast, the oral liquid of Comparative Examples 1-2 performs poorly in terms of particle size distribution, Zeta potential and encapsulation efficiency, especially Comparative Example 1 has a larger particle size, a higher PDI, a Zeta potential closer to zero, and an encapsulation efficiency significantly lower than that of the embodiment, indicating that its stability is poor.

[0122] 2. Stability test results

[0123] The stability test results are shown in Table 2.

[0124] Table 2

[0125]

[0126]

[0127] As can be seen from Table 2, the particle size change rate and encapsulation rate reduction rate of the liposome oral liquid of Examples 1-3 at 4°C and 25°C are lower than those of Comparative Examples 1-2, showing good stability. In particular, the particle size change rate of Examples 1-3 is less than 10%, and the encapsulation rate reduction rate is less than 5%, which meets the evaluation criteria, indicating that it can maintain good stability at different temperatures. In contrast, the particle size change rate and encapsulation rate reduction rate of the oral liquid of Comparative Examples 1-2 at 4°C and 25°C are both high, especially the particle size change rate and encapsulation rate reduction rate of Comparative Example 1 at 25°C reached 25.3% and 30.2%, respectively, indicating that its stability is poor.

[0128] 3. Functional test results

[0129] The functional test results are shown in Table 2.

[0130] Table 3

[0131] Sample No. DPPH clearance rate (%) Targeted uptake efficiency (%) Example 1 78.4 - Example 2 82.1 - Example 3 81.5 62.3 Comparative Example 1 45.3 - Comparative Example 2 55.7 30.8

[0132] As shown in Table 3, the DPPH free radical scavenging rates of the liposome oral liquids of Examples 1-3 are all higher than 70%, showing good antioxidant capacity. In particular, the scavenging rate of Example 2 is the highest, reaching 82.1%, indicating that it has the best antioxidant effect. In addition to having antioxidant capacity, the liposome oral liquid of Example 3 also has a targeting function, and its targeted uptake efficiency is 62.3%, which is significantly higher than 30.8% of Comparative Example 2, indicating that the introduction of the oleosin protein modified with the targeting function can effectively improve the targeted delivery characteristics of liposomes to hepatocytes.

[0133] In summary, the liposome oral liquids of Examples 1-3 performed well in terms of quality, stability and functionality. In particular, Example 3 not only maintained good antioxidant capacity by introducing oleosin protein with targeted functional modification, but also endowed liposomes with targeted delivery properties to specific cells, and thus had potential application value.

[0134] 3. Principle and Mechanism

[0135] 1. Microscopic mechanism of Oleosin protein modification

[0136] (1) Structural characteristics of oleosin protein

[0137] Natural structure: Oleosin is an oil body-specific membrane protein with amphipathic properties, consisting of a hydrophobic region and two hydrophilic regions: the hydrophobic region can be embedded in the liposome bilayer to form a stable hydrophobic interaction with phospholipids. The hydrophilic region is exposed to the outside and can specifically interact with molecules in aqueous solution (such as antioxidants or aptamers).

[0138] Protein modification effect: By modifying (such as aldehyde or sulfhydrylation), the external chemical properties of the protein are changed to provide specific binding sites. The interaction strength between the protein and phospholipid molecules is increased, and the mechanical stability of the liposome is enhanced.

[0139] (2) Contribution of protein to stability

[0140] Interface stability: The amphiphilic structure of Oleosin forms a stable layer at the interface between the aqueous phase and the lipid phase, which reduces fluidity and increases rigidity, thereby reducing the possibility of liposome disintegration.

[0141] Antioxidant protection: The modified protein can bind to antioxidants, blocking free radicals from attacking the phospholipid membrane and improving the antioxidant stability of liposomes at the molecular level.

[0142] 2. Liposome microstructure optimization mechanism

[0143] (1) Stability of lipid bilayer

[0144] Function of cholesterol: Cholesterol is inserted into the gaps between the phospholipid bilayers, filling the gaps, increasing the tightness and fluidity of the membrane, and preventing the liposomes from rupturing under external forces.

[0145] Choice of lecithin: Soy or egg yolk lecithin, due to its high phosphatidylcholine content, can form a dense lipid bilayer structure, providing good stability and encapsulation ability.

[0146] Distribution of antioxidants: Antioxidants such as α-tocopherol and curcumin are distributed in the bilayer and protect phospholipids from oxidative degradation by trapping free radicals.

[0147] (2) Introduction of aptamers

[0148] Aptamer binding to target: Aptamers, as nucleic acid sequences, bind to target molecules (such as cell surface receptors or inflammatory factors) through their specific spatial structures, thereby enhancing the targeting of liposomes.

[0149] Effect of liposome surface modification: Aptamer-modified liposomes enhance the retention time of liposomes at the target site through weak interactions (hydrogen bonds or electrostatic interactions), thereby improving their bioavailability.

[0150] 3. Synergistic mechanism

[0151] (1) Synergy between antioxidants and oleosin protein

[0152] Distribution and transfer of antioxidants: Antioxidants are distributed at the interface within the liposome membrane, forming a dynamic antioxidant protective layer.

[0153] Free radical scavenging: α-tocopherol captures free radicals and protects the membrane structure; curcumin participates in the termination of free radical chain reactions within the membrane.

[0154] (2) Synergy between liposomes and aptamers

[0155] Dual targeting mechanism: Oleosin protein modification improves the structural stability of liposomes, while nucleic acid aptamers provide molecular-level targeting, allowing liposomes to be precisely positioned in target cells or tissues.

[0156] Improved loading efficiency: The modified liposomes have higher encapsulation efficiency, ensuring that antioxidants and nutrients are efficiently delivered.

[0157] 4. Microscopic experimental verification

[0158] (1) Phospholipid membrane imaging

[0159] Technique: The thickness and uniformity of the liposome membrane were observed by transmission electron microscopy (TEM).

[0160] The results show that the liposome membrane thickness in the example is 4-5 nm and is evenly distributed; the membrane thickness in the comparative example is uneven and defects are prone to occur.

[0161] (2) Raman spectroscopy analysis of protein binding

[0162] Technology: Analysis of protein binding sites on phospholipid membranes by Raman spectroscopy.

[0163] The results showed that the Raman shift in liposomes was significant after oleosin protein modification, indicating that the interaction between the protein and the membrane was enhanced.

[0164] (3) Real-time detection of free radical capture

[0165] Technique: Dynamic trapping of free radicals using electron spin resonance (ESR) analysis.

[0166] The experiment showed that the free radical scavenging rate in the example was twice that in the comparative example, proving that the synergistic effect of the antioxidant and oleosin protein was significant.

[0167] 5. Summary of Microscopic Principles

[0168] At the structural level: Oleosin protein enhances the interfacial stability of liposomes through physical and chemical modifications; cholesterol and phospholipids work synergistically to improve the tightness of the membrane.

[0169] Functional level: Antioxidants and aptamers are distributed in key positions of liposomes, providing antioxidant protection and targeted recognition functions, respectively.

[0170] Synergistic effect: The combination of protein modification and aptamer enhances the stability and targeting of liposomes, achieving multifunctional synergistic effects at the nanometer scale.

[0171] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a highly stable oleosin protein modified liposome oral solution, characterized in that: include: The oil seeds are soaked in a NaHCO3 solution at a solid-liquid ratio of 1:5-8 for 6-10 hours, and after pulping with a wall-breaking machine, the residue is removed by filtering through a filter cloth; the filtrate is centrifuged at a centrifugal force of 10,000×g to collect the oil body-rich layer, and washed with water 2-3 times, and centrifuged after each washing to obtain the plant oil body; The plant oil bodies and NaCl solution are mixed in a volume ratio of 1:2, and ultrasonic treatment is performed for 10-20 minutes to dissolve non-membrane proteins; the mixture after ultrasonic treatment is centrifuged at 10,000×g at 0°C to obtain an emulsion; the emulsion is mixed with a solvent in a volume ratio of 1:3-6, centrifuged at 8000×g for 15-20 minutes to remove lipids, and a white layer is collected, and the residual solvent is used under a nitrogen flow, and lyophilized to obtain oleosin protein powder; Mix lecithin, cholesterol, α-tocopherol, Tween-80 and nutrients, add anhydrous ethanol to disperse evenly, and perform vacuum rotary evaporation at 40-50° C. until a transparent lipid film is formed; The oleosin protein powder is dispersed in water, then added into the transparent lipid film, subjected to hydration ultrasonic treatment, and sweetener is added in a mass ratio of phospholipid to sweetener of 9:10 to obtain an oleosin protein modified liposome oral solution.

2. The method for preparing the highly stable oleosin protein modified liposome oral solution according to claim 1, characterized in that: The oilseeds are one or more of soybeans, rapeseeds and sunflower seeds; And / or, the lecithin is one or more of soybean lecithin, egg yolk lecithin and sunflower seed lecithin; and / or, the nutrient is one or more of lutein, curcumin, β-carotene, lycopene and quercetin; And / or, the sweetener is one or more of erythritol, mannitol or xylitol.

3. The method for preparing the highly stable oleosin protein modified liposome oral solution according to claim 1, characterized in that: The particle size of the liposome in the liposome oral liquid is 100-200 nm, the polydispersity index is less than 0.3, and the Zeta potential is -30 mV to +30 mV.

4. The method for preparing the highly stable oleosin protein modified liposome oral liquid according to claim 1, characterized in that: The mass ratio of the lecithin, cholesterol, α-tocopherol, Tween-80 and nutrients is 6-10:1:0.2:0.1:0.1-1.

5. The method for preparing the highly stable oleosin protein modified liposome oral solution according to claim 1, characterized in that: Before dispersing the oleosin protein powder in water, the oleosin protein powder is modified, and the modification method comprises: The oleosin protein was coated with a hydrophilic surfactant; Negatively or positively charged groups are introduced through chemical modification.

6. The method for preparing the highly stable oleosin protein modified liposome oral liquid according to claim 5, characterized in that: The hydrophilic surfactant is polyvinyl alcohol or polyvinyl pyrrolidone.

7. The method for preparing the highly stable oleosin protein modified liposome oral liquid according to claim 5, characterized in that: The modification method also includes: The oleosin protein is modified by using an aptamer, wherein the aptamer is a single-stranded nucleic acid, an antibody or a small molecule compound.

8. The method for preparing the highly stable oleosin protein modified liposome oral solution according to claim 1, characterized in that: The pH value of the liposome oral solution is 4.5-7.

5.

9. A highly stable oleosin protein modified liposome oral solution, characterized in that: The liposome oral liquid is prepared by the method for preparing the highly stable oleosin protein modified liposome oral liquid according to any one of claims 1 to 8.

10. Use of the highly stable oleosin protein modified liposome oral liquid according to claim 9 in the preparation of products for health care, nutritional supplementation and / or disease adjuvant treatment.

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