Astaxanthin and cyanidin double-drug multi-layer liposome, preparation method and application thereof

Abstract

This application provides a multilayer liposome with astaxanthin and anthocyanin dual drug loading, its preparation method, and its application, belonging to the field of food processing technology. The multilayer liposome with astaxanthin and anthocyanin dual drug loading comprises the following raw materials: 5-20 parts astaxanthin, 5-20 parts anthocyanin, 20-50 parts soybean lecithin, 5-20 parts hydrogenated soybean lecithin, 3-15 parts phytosterols, 1-8 parts glycerol, 0.5-4 parts chitosan, 0.5-4 parts polygalacturonic acid, 0.5-4 parts vitamin C, 15-60 parts trehalose, 5-40 parts dextrin, and deionized water. This application achieves efficient co-loading and structural stability of fat-soluble astaxanthin and water-soluble anthocyanins by constructing liposomes with a multilayer vesicle structure and utilizing the differences in the physicochemical properties of astaxanthin and anthocyanins, as well as the synergistic protection of the multilayer membrane and the polysaccharide shell.

Description

Technical Field

[0001] This application relates to the field of food processing technology, and in particular to a multilayer liposome with dual drug delivery of astaxanthin and anthocyanins, its preparation method, and its application. Background Technology

[0002] Astaxanthin is a fat-soluble carotenoid with extremely strong antioxidant activity, widely used in functional and health foods. Anthocyanins are water-soluble natural pigments, also possessing excellent antioxidant and anti-inflammatory effects. However, astaxanthin is poorly soluble in water, sensitive to light, heat, and oxygen, and easily degrades in the acidic environment of the stomach; while anthocyanins are soluble in water, they are easily hydrolyzed and lose their biological activity under acidic conditions. Their physicochemical properties are vastly different, making it difficult for traditional delivery systems to simultaneously and efficiently encapsulate and protect both active ingredients, thus limiting their combined application in oral functional foods.

[0003] Currently, liposome technology is widely used for the encapsulation and delivery of active ingredients. However, most existing liposome products have a monolayer structure, which has a high encapsulation efficiency for fat-soluble components but limited capacity for water-soluble components. Furthermore, the monolayer liposome membrane structure is fragile and easily ruptures in gastric acid, leading to leakage of the active ingredient. In addition, existing liposome preparation methods mostly rely on specialized equipment such as high-pressure homogenization and ultrasonic dispersion, which are complex and costly. The resulting products are mostly liquid suspensions, which are susceptible to sedimentation, oxidation, and leakage, making it difficult to meet the long-term storage stability requirements of solid beverages. Therefore, how to simultaneously achieve efficient co-encapsulation of fat-soluble astaxanthin and water-soluble anthocyanins, and improve the structural stability of liposomes in gastric juice and the room-temperature storage stability of the formulation, is a pressing technical problem to be solved in this field. Summary of the Invention

[0004] This application provides a multilayer liposome with dual drug delivery of astaxanthin and anthocyanins, its preparation method, and its application, in order to solve the following technical problem: how to simultaneously achieve efficient co-loading and structural stability of fat-soluble astaxanthin and water-soluble anthocyanins.

[0005] In a first aspect, embodiments of this application provide a multilayer liposome with dual drug-carrying capacity for astaxanthin and anthocyanins. By weight, the multilayer liposome with dual drug-carrying capacity for astaxanthin and anthocyanins comprises the following raw materials: 5-20 parts astaxanthin, 5-20 parts anthocyanins, 20-50 parts soybean lecithin, 5-20 parts hydrogenated soybean lecithin, 3-15 parts phytosterols, 1-8 parts glycerol, 0.5-4 parts chitosan, 0.5-4 parts polygalacturonic acid, 0.5-4 parts vitamin C, 15-60 parts trehalose, 5-40 parts dextrin, and deionized water.

[0006] Optionally, the multilayer liposomes have an average particle size of 300–500 nm, an astaxanthin encapsulation rate of ≥80%, an anthocyanin encapsulation rate of ≥70%, and an absolute value of zeta potential of ≥20 mV.

[0007] Optionally, the total weight of the astaxanthin and the anthocyanin does not exceed 30 parts;

[0008] The weight ratio of the soybean lecithin to the hydrogenated soybean lecithin is (1-3):1.

[0009] Secondly, embodiments of this application provide a method for preparing astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes as described in any one of the first aspects, the method comprising the following steps:

[0010] S1. Dissolve astaxanthin, anthocyanins, soybean lecithin, hydrogenated soybean lecithin, and phytosterols in an ethanol-water solution, and then remove the solvent by vacuum evaporation to form a uniform film on the container wall to obtain mixture A.

[0011] S2. Dissolve vitamin C, trehalose, dextrin, and glycerol in deionized water at 45–55°C to obtain mixture B;

[0012] S3. Quickly pour the mixture B into a container containing the mixture A to completely submerge the film, and stir and hydrate it in a water bath at 45-55°C to form a liposome suspension with a multilayer vesicle structure, thus obtaining mixture C.

[0013] S4. Heat the mixture C to 30-40°C, and add chitosan solution dropwise at a rate of 0.5-2 mL / min. After the addition is complete, continue stirring for 30-60 min to obtain a chitosan-coated liposome suspension.

[0014] S5. Add polygalacturonic acid solution dropwise to the chitosan-coated liposome suspension at a rate of 0.5 to 2 mL / min. After the addition is complete, continue stirring for 30 to 60 min to obtain a chitosan-polygalacturonic acid bilayer coated liposome suspension.

[0015] S6. Spray-dry the chitosan-polygalacturonic acid bilayer-coated liposome suspension to obtain the astaxanthin and anthocyanin dual-drug-loaded multilayer liposomes.

[0016] Optionally, the volume fraction of the ethanol-water solution in S1 is 70-80%, the temperature of the vacuum evaporation is 35-45°C, and the vacuum degree is -0.08 to -0.1 MPa.

[0017] Optionally, the stirring speed for hydration described in S3 is 400–800 rpm, and the time is 45–90 min.

[0018] Optionally, the concentration of the chitosan solution in S4 is 0.5–1.5 wt%, and the pH is 4.5–5.5.

[0019] Optionally, the concentration of the polygalacturonic acid solution in S5 is 0.5–1.5 wt%, and the pH is 6.0–6.5.

[0020] Optionally, the process parameters for spray drying in S6 are: inlet air temperature 85±5℃, outlet air temperature 55±2℃, atomizing disc rotation speed 20000~30000rpm, feed flow rate 5~10 mL / min, and the solid content of the chitosan-polygalacturonic acid bilayer coated liposome suspension before spray drying is 10~20%.

[0021] Thirdly, embodiments of this application provide the application of the astaxanthin and anthocyanin dual-drug multilayer liposomes described in any one of the first aspects in food.

[0022] The technical solutions provided in this application have the following advantages compared with the prior art:

[0023] This application constructs liposomes with multilayer vesicle structures and utilizes the differences in the physicochemical properties of astaxanthin and anthocyanins, as well as the synergistic protection of the multilayer membrane and the polysaccharide shell, to achieve both efficient co-encapsulation and structural stability of fat-soluble astaxanthin and water-soluble anthocyanins.

[0024] In terms of efficient co-encapsulation, astaxanthin, as a lipid-soluble active ingredient, is co-dissolved in an ethanol-water solution with soybean phospholipids, hydrogenated soybean phospholipids, and phytosterols during the organic phase preparation step. Upon vacuum evaporation to form a uniform film, astaxanthin molecules, due to their hydrophobic properties, insert into the hydrophobic regions of the phospholipid bilayer and interact hydrophobically with the phosphatidyl chains, thus anchoring themselves within each lipid membrane layer. Anthocyanins, as a water-soluble active ingredient, enter the liposomes with the aqueous phase during the film hydration step and are retained in the aqueous compartments of the multilayer vesicles. This natural partitioning mechanism based on solubility differences allows the two active ingredients to be enriched in the membrane phase and aqueous phase of the liposomes respectively, achieving partitioned and simultaneous encapsulation without the need for additional separation steps. Furthermore, the multilayer vesicle structure provides multiple concentric bilayers and multiple aqueous compartments, increasing the total encapsulation volume, allowing astaxanthin to be distributed in each membrane layer and anthocyanins to be distributed in each aqueous phase layer, further improving the total encapsulation capacity of the two active ingredients per unit mass of liposome.

[0025] Regarding structural stability, this application achieves protection on three levels: First, soybean phospholipids and hydrogenated soybean phospholipids are compounded in a specific ratio to form a rigid-flexible biphasic membrane structure. Combined with phytosterols embedded in the gaps between phospholipid molecules, this enhances the membrane's mechanical strength and thermal stability. Second, chitosan and polygalacturonic acid are electrostatically adsorbed onto the liposome surface to form a double-layered polysaccharide shell. This shell forms a physical barrier under gastric acid conditions, inhibiting the corrosion of liposomes by gastric juice. Simultaneously, the adhesive properties of chitosan prolong the retention time of liposomes in the intestine. Third, trehalose and dextrin play water-substitution protection and filling support roles respectively during spray drying, fixing the multilayered vesicle structure within an amorphous glassy matrix. This ensures that the liposome powder maintains a relatively unchanged particle size distribution and encapsulation efficiency even after long-term storage at room temperature. Attached Figure Description

[0026] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic flowchart illustrating the preparation method of astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes provided in the embodiments of this application. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] This application provides a dual-drug multilayer liposome for astaxanthin and anthocyanins. By weight, the dual-drug multilayer liposome for astaxanthin and anthocyanins comprises the following raw materials: 5-20 parts astaxanthin, 5-20 parts anthocyanins, 20-50 parts soybean lecithin, 5-20 parts hydrogenated soybean lecithin, 3-15 parts phytosterols, 1-8 parts glycerol, 0.5-4 parts chitosan, 0.5-4 parts polygalacturonic acid, 0.5-4 parts vitamin C, 15-60 parts trehalose, 5-40 parts dextrin, and deionized water.

[0031] In some embodiments, the multilayer liposomes have an average particle size of 300–500 nm, an astaxanthin encapsulation rate of ≥80%, an anthocyanin encapsulation rate of ≥70%, and an absolute zeta potential of ≥20 mV.

[0032] In some embodiments, the total weight of the astaxanthin and the anthocyanin does not exceed 30 parts;

[0033] The weight ratio of the soybean lecithin to the hydrogenated soybean lecithin is (1-3):1.

[0034] It should be noted that in the astaxanthin and anthocyanin dual-drug multilayer liposome of this application, each component, based on its physicochemical properties and functional positioning, jointly constructs a delivery system with a multilayer membrane structure and a polysaccharide shell.

[0035] Astaxanthin, as a fat-soluble active ingredient, not only provides antioxidant function, but can also insert into the hydrophobic region of the phospholipid bilayer to play a role in regulating membrane fluidity and synergistically enhance the structural stability of liposomes with the membrane material. Anthocyanins, as a water-soluble active ingredient, are encapsulated in the aqueous compartments of liposomes, achieving partitioned loading of fat-soluble and water-soluble active ingredients and improving loading efficiency.

[0036] Soybean lecithin and hydrogenated soybean lecithin constitute the main membrane material of liposomes: soybean lecithin is rich in unsaturated fatty acid chains, which endows the membrane material with good flexibility and self-assembly ability; hydrogenated soybean lecithin has a high phase transition temperature, which provides the membrane material with rigidity and thermal stability. When the two are compounded in a specific ratio, they form a rigid-flexible complementary biphasic membrane structure, which provides a basis for the construction of multilayer vesicles.

[0037] Phytosterols can replace traditional cholesterol as membrane structure regulators. Their steroidal ring skeleton can be inserted into the phospholipid bilayer, increasing the membrane's density and mechanical strength. At the same time, phytosterols themselves have the health function of lowering plasma cholesterol, making liposome carriers also functional components.

[0038] Glycerin, as a plasticizer, can regulate the interaction between phospholipid molecules, reduce the membrane phase transition temperature gradient, and promote the formation of a uniform multilayer structure of different phospholipids under the same hydration conditions.

[0039] Chitosan and polygalacturonic acid constitute a surface functionalization modification layer: Chitosan, a cationic polysaccharide, becomes positively charged after protonation under acidic conditions, enabling it to electrostatically adsorb onto the negatively charged liposome surface, forming the first protective shell; polygalacturonic acid, an anionic polysaccharide, can further adsorb onto the chitosan layer surface, forming a bilayer polysaccharide shell. This layer-by-layer self-assembled polysaccharide shell forms a physical barrier in the acidic gastric environment, significantly inhibiting the corrosion of liposomes by gastric juice and the leakage of active ingredients. Furthermore, the adhesive properties of chitosan prolong the retention time of liposomes in the intestine, promoting the absorption of active ingredients.

[0040] Vitamin C, as a water-soluble antioxidant, is distributed in the aqueous phase of liposomes; astaxanthin, as a fat-soluble antioxidant, is anchored in the hydrophobic region of the phospholipid bilayer. Together, they form an aqueous-membrane phase partitioned antioxidant network: vitamin C scavenge free radicals in the aqueous phase, while astaxanthin scavenge lipid peroxide free radicals in the membrane phase, exerting their antioxidant functions in different microenvironments and achieving synergistic protection.

[0041] Trehalose and dextrin constitute the freeze-drying protection and matrix formation system. During spray drying, trehalose forms hydrogen bonds with the phospholipid heads through a water substitution mechanism, maintaining the integrity of the liposome membrane and simultaneously forming an amorphous glassy matrix to fix the multilayer structure. Dextrin acts as a filler and drying aid, increasing powder flowability and reducing hygroscopicity. Deionized water serves as the dispersion medium and is an essential continuous phase for liposome formation.

[0042] Figure 1 This is a schematic flowchart illustrating the preparation method of astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes provided in the embodiments of this application.

[0043] Based on a general inventive concept, such as Figure 1 As shown, this application provides a method for preparing astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes as described in any one of the above-mentioned embodiments, the method comprising the following steps:

[0044] S1. Dissolve astaxanthin, anthocyanins, soybean lecithin, hydrogenated soybean lecithin, and phytosterols in an ethanol-water solution, and then remove the solvent by vacuum evaporation to form a uniform film on the container wall to obtain mixture A.

[0045] S2. Dissolve vitamin C, trehalose, dextrin, and glycerol in deionized water at 45–55°C to obtain mixture B;

[0046] S3. Quickly pour the mixture B into a container containing the mixture A to completely submerge the film, and stir and hydrate it in a water bath at 45-55°C to form a liposome suspension with a multilayer vesicle structure, thus obtaining mixture C.

[0047] S4. Heat the mixture C to 30-40°C, and add chitosan solution dropwise at a rate of 0.5-2 mL / min. After the addition is complete, continue stirring for 30-60 min to obtain a chitosan-coated liposome suspension.

[0048] S5. Add polygalacturonic acid solution dropwise to the chitosan-coated liposome suspension at a rate of 0.5 to 2 mL / min. After the addition is complete, continue stirring for 30 to 60 min to obtain a chitosan-polygalacturonic acid bilayer coated liposome suspension.

[0049] S6. Spray-dry the chitosan-polygalacturonic acid bilayer-coated liposome suspension to obtain the astaxanthin and anthocyanin dual-drug-loaded multilayer liposomes.

[0050] In some embodiments, the volume fraction of the ethanol-water solution in S1 is 70-80%, the temperature of the vacuum evaporation is 35-45°C, and the vacuum degree is -0.08 to -0.1 MPa.

[0051] In some embodiments, the stirring speed for hydration in S3 is 400–800 rpm, and the time is 45–90 min.

[0052] In some embodiments, the concentration of the chitosan solution in S4 is 0.5–1.5 wt%, and the pH is 4.5–5.5.

[0053] In some embodiments, the concentration of the polygalacturonic acid solution in S5 is 0.5–1.5 wt%, and the pH is 6.0–6.5.

[0054] In some embodiments, the process parameters for spray drying in S6 are: inlet air temperature 85±5℃, outlet air temperature 55±2℃, atomizing disc rotation speed 20000~30000rpm, feed flow rate 5~10 mL / min, and the solid content of the chitosan-polygalacturonic acid bilayer coated liposome suspension before spray drying is 10~20%.

[0055] It should be noted that S1 represents the organic phase preparation and film formation steps. Astaxanthin, anthocyanins, soybean lecithin, hydrogenated soybean lecithin, and phytosterols are dissolved in a 70-80% (v / v) ethanol aqueous solution. This solvent system can dissolve both the lipid-soluble astaxanthin and phospholipid membrane materials, and accommodate a certain amount of water-soluble anthocyanins, ensuring uniform mixing of the components at the molecular level. The solvent is removed by vacuum evaporation at 35-45°C and a vacuum degree of -0.08 to -0.1 MPa, avoiding damage to the heat-sensitive active ingredients at high temperatures, while simultaneously forming a uniform and dense film on the container wall. In this film, astaxanthin is inserted into the hydrophobic region of the phospholipid bilayer, and phytosterols are embedded in the gaps between phospholipid molecules, forming a phospholipid-astaxanthin-sterol ternary co-assembly structure together with soybean lecithin and hydrogenated soybean lecithin, laying the molecular arrangement foundation for the subsequent formation of multilayer vesicles.

[0056] S2 is the aqueous phase preparation step. Vitamin C, trehalose, dextrin, and glycerol are dissolved in deionized water at 45–55°C. This temperature range is higher than the phase transition temperature of soybean lecithin but close to that of hydrogenated soybean lecithin, which is beneficial for the uniform dispersion of the membrane material during subsequent hydration. Vitamin C is distributed in the aqueous phase, preparing to form a partitioned antioxidant network with astaxanthin in the membrane phase; trehalose and dextrin are dissolved in the aqueous phase as freeze-drying protectants and fillers; glycerol is used as a plasticizer to adjust the flexibility of the subsequent membrane material.

[0057] Step S3, the membrane hydration and multilayer vesicle formation step, is a crucial step in constructing the core structure of multilayer liposomes. Mixture B at 45–55°C is rapidly poured into a container containing mixture A, ensuring complete immersion of the membrane. Consistent temperature ensures uniform water absorption and expansion during hydration. Hydration is carried out in a 45–55°C water bath at 400–800 rpm for 45–90 minutes with stirring. This temperature, higher than the phase transition temperature of all phospholipid components, keeps the phospholipid molecular chains in a fluid state, facilitating the membrane's detachment from the container wall and spontaneous vesicle formation. A stirring speed of 400–800 rpm is within the range of gentle stirring, avoiding the strong shear forces generated by high-speed stirring that could damage the newly formed vesicle structure. Under these conditions, the phospholipid bilayers absorb water, peel off layer by layer, and curl up to close, forming multilayer vesicles with 3–5 concentric bilayers, i.e., multilayer liposomes. Astaxanthin is anchored in the hydrophobic regions of each bilayer, while anthocyanins are encapsulated in aqueous compartments, achieving partitioned encapsulation of lipid-soluble and water-soluble active ingredients.

[0058] S4 is the chitosan cationic coating step. Mixture C is heated to 30–40°C, and a chitosan solution with a concentration of 0.5–1.5 wt% and a pH of 4.5–5.5 is added dropwise at a rate of 0.5–2 mL / min. After the addition is complete, stirring continues for 30–60 min. Under this pH condition, the amino groups of chitosan are protonated and become positively charged, while the surface of the multilayer liposomes is negatively charged due to the presence of phosphatidyl groups. Adsorption occurs between the two through electrostatic attraction. The slow dropwise addition of 0.5–2 mL / min avoids excessively high local concentrations that could lead to liposome aggregation, and the thorough stirring for 30–60 min ensures that chitosan molecules form a uniform and complete monolayer coating on the liposome surface. After coating, the surface charge of the liposomes changes from negative to positive, providing an electrostatic basis for the subsequent adsorption of the polygalacturonic acid layer.

[0059] S5 is the polygalacturonic acid (PCA) anionic coating step. A PCA solution with a concentration of 0.5–1.5 wt% and a pH of 6.0–6.5 is added dropwise to the chitosan-coated liposome suspension at a rate of 0.5–2 mL / min. After the addition is complete, stirring continues for 30–60 min. Under this pH condition, the carboxyl groups of PCA dissociate and become negatively charged, adsorbing onto the positively charged chitosan layer through electrostatic attraction, forming a chitosan-PCA bilayer polysaccharide shell. Compared to a single chitosan layer, the bilayer structure is denser and can more effectively block gastric acid penetration. Simultaneously, the PCA layer converts the surface charge of the liposomes back to negative, avoiding the non-specific adsorption that might occur in vivo with positively charged liposomes.

[0060] S6 is the spray drying and curing step. The chitosan-polygalacturonic acid double-layer-coated liposome suspension is spray-dried with the following parameters: inlet air temperature 85±5℃, outlet air temperature 55±2℃, atomizing disc rotation speed 20000~30000rpm, and feed flow rate 5~10mL / min. Before spray drying, the solid content of the suspension is controlled to be 10~20%, within which the viscosity is suitable for atomization and can form complete powder particles. The inlet air temperature of 85±5℃ ensures rapid evaporation of moisture while avoiding thermal degradation of astaxanthin and anthocyanins; the outlet air temperature of 55±2℃ ensures that the powder is fully dried and at a suitable temperature when exiting the tower. The high rotation speed of the atomizing disc (20000~30000rpm) disperses the suspension into micron-sized droplets, which are rapidly dried and cured in the hot airflow, fixing the multilayer vesicle structure of the liposomes in an amorphous glassy matrix. Trehalose forms hydrogen bonds with the phospholipid head through a water substitution mechanism, protecting the membrane structure from drying damage; dextrin acts as a filler to increase powder flowability and reduce hygroscopicity.

[0061] Based on a general inventive concept, embodiments of this application provide an application of the astaxanthin and anthocyanin dual-drug multilayer liposomes described in any one of the above-mentioned embodiments in food.

[0062] The astaxanthin and anthocyanin dual-loaded multilayer liposomes provided in this application can be used in food based on the following four technical foundations. First, all raw materials comply with food regulatory requirements. Second, the multilayer liposome structure solves the stability problem of active ingredients during food processing and storage. Astaxanthin and anthocyanins are respectively encapsulated in the hydrophobic region and aqueous compartment of the phospholipid bilayer, achieving partitioned encapsulation of lipid-soluble and water-soluble active ingredients, thus improving loading efficiency. The chitosan-polygalacturonic acid bilayer polysaccharide shell forms a dense protective layer during spray drying, combined with the freeze-drying protection of trehalose and dextrin, ensuring that the liposome powder maintains a basically unchanged particle size distribution and encapsulation efficiency after long-term storage at room temperature, overcoming the defects of liquid liposomes such as easy sedimentation, oxidation, and leakage. Third, the multilayer liposome structure provides functional protection for the product in the gastrointestinal environment. The bilayer polysaccharide shell forms a physical barrier under gastric acid conditions, significantly inhibiting the corrosion of liposomes by gastric juice and the leakage of active ingredients, maintaining a high retention rate of active ingredients in gastric juice. Chitosan's adhesive properties prolong the retention time of liposomes in the intestine, promoting the absorption of active ingredients. Fourth, the preparation process is suitable for large-scale production in the food industry. The integrated process of film hydration-layer self-assembly-spray drying does not require specialized equipment such as high-pressure homogenization or ultrasound; it can be completed using only conventional stirring, rotary evaporation, and spray drying towers. The preparation time is short, the cost is low, and the final product is a free-flowing powder that can be quickly reconstituted into a homogeneous emulsion.

[0063] The present application is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards / industry standards / the disclosure herein; if there are no corresponding national standards / industry standards / the disclosure herein, they are performed according to generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer.

[0064] Example 1

[0065] This embodiment provides a multilayer liposome with dual drug delivery of astaxanthin and anthocyanins. The specific raw material composition by weight is as follows: 12 parts astaxanthin, 12 parts anthocyanins, 30 parts soybean lecithin, 15 parts hydrogenated soybean lecithin (HSPC, CAS No. 92128-87-5), 9 parts phytosterol (β-sitosterol, CAS No. 83-46-5), 3 parts glycerol, 2 parts chitosan, 2 parts polygalacturonic acid (CAS No. 25990-10-7), 2 parts vitamin C, 30 parts trehalose, 20 parts dextrin, and deionized water. It should be noted that all raw materials are existing compounds that comply with food safety standards.

[0066] Based on the components of the above-mentioned astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes, this embodiment also provides a preparation method, the steps of which are as follows:

[0067] S1. Preparation of the organic phase and film formation: Astaxanthin, anthocyanins, soybean lecithin, hydrogenated soybean lecithin, and phytosterols were dissolved in an 85% (v / v) aqueous ethanol solution with a solid-to-solvent mass ratio of 1:8. The solution was stirred at 25°C and 1500 rpm until completely dissolved. The solvent was then removed by vacuum evaporation at 40°C and -0.09 MPa. After evaporation, vacuum was continued for 15 minutes to completely remove residual ethanol, forming a uniform and transparent film on the container wall, thus obtaining mixture A.

[0068] S2. Preparation of aqueous phase: Heat deionized water to 50°C, purge with nitrogen for protection, add vitamin C, trehalose, dextrin, and glycerol, stir at 700 rpm for 20 min until completely dissolved, the mass ratio of solids to deionized water is 1:5, keep warm at 50°C to obtain mixture B.

[0069] S3. Preparation of polysaccharide solution: Dissolve chitosan in 1% acetic acid solution to prepare a 1.0 wt% chitosan solution, and adjust the pH to 5.0; dissolve polygalacturonic acid in deionized water to prepare a 1.0 wt% polygalacturonic acid solution, and adjust the pH to 6.3.

[0070] S4. Membrane hydration to form multilayer vesicles: Quickly pour mixture B at 50°C into a container containing mixture A to completely immerse the membrane. Hydrate in a 50°C water bath at 600 rpm for 60 minutes to form a liposome suspension with a multilayer vesicle structure, thus obtaining mixture C.

[0071] S5. Chitosan coating: Heat mixture C to 35°C, and add chitosan solution dropwise at a rate of 1.0 mL / min while stirring at 400 rpm. After the addition is complete, continue stirring for 45 min to obtain a chitosan-coated liposome suspension.

[0072] S6. Polygalacturonic acid layer coating: Polygalacturonic acid solution was added dropwise to the chitosan-coated liposome suspension at a rate of 1.0 mL / min. After the addition was completed, the mixture was stirred for 45 min to obtain a chitosan-polygalacturonic acid bilayer coated liposome suspension.

[0073] S7. Spray Drying and Curing: The chitosan-polygalacturonic acid double-layer coated liposome suspension was spray dried, with the solid content of the suspension controlled at 15% before spray drying. Process parameters were set as follows: inlet air temperature 90℃, outlet air temperature 55℃, atomizing disc speed 25000rpm, and feed flow rate 8mL / min. The powder at the bottom of the column was collected and cooled to room temperature to obtain a free-flowing powdered astaxanthin and anthocyanin dual-drug-loaded multilayer liposome powder.

[0074] Example 2

[0075] This embodiment provides a multilayer liposome with dual drug delivery of astaxanthin and anthocyanins. The specific raw material composition by weight is as follows: 8 parts astaxanthin, 16 parts anthocyanins, 25 parts soybean lecithin, 12 parts hydrogenated soybean lecithin, 6 parts phytosterols, 2 parts glycerol, 1.5 parts chitosan, 1.5 parts polygalacturonic acid, 1.5 parts vitamin C, 25 parts trehalose, 15 parts dextrin, and deionized water.

[0076] Based on the components of the above-mentioned astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes, this embodiment also provides a preparation method, the steps of which are as follows:

[0077] S1. Preparation of the organic phase and film formation: Astaxanthin, anthocyanins, soybean lecithin, hydrogenated soybean lecithin, and phytosterols were dissolved in a 90% (v / v) aqueous ethanol solution with a solid-to-solvent mass ratio of 1:10. The solution was stirred at 25°C and 1500 rpm until completely dissolved. The solvent was then removed by vacuum evaporation at 40°C and -0.09 MPa. After evaporation, vacuum was continued for 15 minutes to completely remove residual ethanol, forming a uniform and transparent film on the container wall, thus obtaining mixture A.

[0078] S2. Preparation of the aqueous phase: Heat deionized water to 50°C, purge with nitrogen for protection, add vitamin C, trehalose, dextrin, and glycerol, and stir at 700 rpm for 20 minutes until completely dissolved. The mass ratio of solids to deionized water is 1:6. Maintain the temperature at 50°C to obtain mixture B. The solids in the aqueous phase are the sum of vitamin C, trehalose, dextrin, and glycerol. Calculate the amount of deionized water needed based on this ratio.

[0079] S3. Preparation of polysaccharide solution: Dissolve chitosan in 1% acetic acid solution to prepare a 1.2 wt% chitosan solution, and adjust the pH to 5.0; dissolve polygalacturonic acid in deionized water to prepare a 1.2 wt% polygalacturonic acid solution, and adjust the pH to 6.3.

[0080] S4. Membrane hydration to form multilayer vesicles: Quickly pour mixture B at 50°C into a container containing mixture A to completely immerse the membrane. Hydrate in a 50°C water bath at 500 rpm for 75 minutes to form a liposome suspension with a multilayer vesicle structure, thus obtaining mixture C.

[0081] S5. Chitosan coating: Heat mixture C to 35°C, and add chitosan solution dropwise at a rate of 1.0 mL / min while stirring at 400 rpm. After the addition is complete, continue stirring for 45 min to obtain a chitosan-coated liposome suspension.

[0082] S6. Polygalacturonic acid layer coating: Polygalacturonic acid solution was added dropwise to the chitosan-coated liposome suspension at a rate of 1.0 mL / min. After the addition was completed, the mixture was stirred for 45 min to obtain a chitosan-polygalacturonic acid bilayer coated liposome suspension.

[0083] S7. Spray Drying and Curing: The chitosan-polygalacturonic acid double-layer coated liposome suspension was spray dried, with the solid content of the suspension controlled at 12% before spray drying. Process parameters were set as follows: inlet air temperature 85℃, outlet air temperature 55℃, atomizing disc speed 25000rpm, and feed flow rate 8mL / min. The powder at the bottom of the column was collected and cooled to room temperature to obtain a free-flowing powdered astaxanthin and anthocyanin dual-drug multilayer liposome powder.

[0084] Example 3

[0085] This embodiment provides a multilayer liposome with dual drug delivery of astaxanthin and anthocyanins. The specific raw material composition by weight is as follows: 15 parts astaxanthin, 10 parts anthocyanins, 35 parts soybean lecithin, 18 parts hydrogenated soybean lecithin, 10 parts phytosterols, 4 parts glycerol, 2.5 parts chitosan, 2.5 parts polygalacturonic acid, 2.5 parts vitamin C, 35 parts trehalose, 25 parts dextrin, and deionized water.

[0086] Based on the components of the above-mentioned astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes, this embodiment also provides a preparation method, the steps of which are as follows:

[0087] S1. Preparation of the organic phase and film formation: Astaxanthin, anthocyanins, soybean lecithin, hydrogenated soybean lecithin, and phytosterols were dissolved in an 80% (v / v) aqueous ethanol solution with a solid-to-solvent mass ratio of 1:7. The solution was stirred at 25°C and 1500 rpm until completely dissolved. The solvent was then removed by vacuum evaporation at 40°C and -0.09 MPa. After evaporation, vacuum was continued for 15 minutes to completely remove residual ethanol, forming a uniform and transparent film on the container wall, thus obtaining mixture A.

[0088] S2. Preparation of the aqueous phase: Heat deionized water to 50°C, purge with nitrogen for protection, add vitamin C, trehalose, dextrin, and glycerol, and stir at 700 rpm for 20 minutes until completely dissolved. The mass ratio of solids to deionized water is 1:4.5. Maintain the temperature at 50°C to obtain mixture B. The solids in the aqueous phase are the sum of vitamin C, trehalose, dextrin, and glycerol. Calculate the amount of deionized water needed based on this ratio.

[0089] S3. Preparation of polysaccharide solution: Dissolve chitosan in 1% acetic acid solution to prepare a 0.8 wt% chitosan solution, and adjust the pH to 5.0; dissolve polygalacturonic acid in deionized water to prepare a 0.8 wt% polygalacturonic acid solution, and adjust the pH to 6.3.

[0090] S4. Membrane hydration to form multilayer vesicles: Quickly pour mixture B at 50°C into a container containing mixture A to completely immerse the membrane. Hydrate in a 50°C water bath at 700 rpm for 50 minutes to form a liposome suspension with a multilayer vesicle structure, thus obtaining mixture C.

[0091] S5. Chitosan coating: Heat mixture C to 35°C, and add chitosan solution dropwise at a rate of 1.5 mL / min while stirring at 400 rpm. After the addition is complete, continue stirring for 35 min to obtain a chitosan-coated liposome suspension.

[0092] S6. Polygalacturonic acid layer coating: Polygalacturonic acid solution was added dropwise to the chitosan-coated liposome suspension at a rate of 1.5 mL / min. After the addition was completed, the mixture was stirred for 35 min to obtain a chitosan-polygalacturonic acid bilayer coated liposome suspension.

[0093] S7. Spray Drying and Curing: The chitosan-polygalacturonic acid bilayer-coated liposome suspension was spray-dried, with the solid content of the suspension controlled at 18% before spray drying. Process parameters were set as follows: inlet air temperature 95℃, outlet air temperature 57℃, atomizing disc speed 25000rpm, and feed flow rate 8mL / min. The powder at the bottom of the column was collected and cooled to room temperature to obtain a free-flowing powdered astaxanthin and anthocyanin dual-drug-loaded multilayer liposome powder.

[0094] Comparative Example 1

[0095] This comparative example is modified from the one disclosed in Example 1 as follows:

[0096] Chitosan and polygalacturonic acid are omitted, meaning the polysaccharide layer-by-layer self-assembly modification steps S5 and S6 are not performed. The remaining raw material composition and preparation method are the same as in Example 1. Specifically, after hydration of the S4 film, mixture C is directly obtained. This mixture C is then spray-dried and cured without undergoing the chitosan layer coating and polygalacturonic acid layer coating steps.

[0097] Comparative Example 2

[0098] Based on the disclosure in Example 1, this comparative example is modified as follows: hydrogenated soybean lecithin is omitted, and the amount of soybean lecithin is adjusted to 45 parts (the same as the total amount of 30 parts of soybean lecithin and 15 parts of hydrogenated soybean lecithin in Example 1). The composition of other raw materials and preparation methods are the same as in Example 1.

[0099] Comparative Example 3

[0100] Based on the disclosure in Example 1, this comparative example is modified as follows: phytosterols are replaced with an equal weight of cholesterol, that is, 9 parts of phytosterols are replaced with 9 parts of cholesterol. The composition of the remaining raw materials and the preparation method are the same as in Example 1.

[0101] The physicochemical and application properties of the astaxanthin and anthocyanin dual-drug multilayer liposomes from Examples 1-3 and Comparative Examples 1-3 were determined, and the results are shown in Tables 1 and 2. The performance determination methods are as follows:

[0102] Encapsulation efficiency and drug loading: High-speed centrifugation-high-performance liquid chromatography (HPLC) was used. The encapsulation efficiency and drug loading of astaxanthin and anthocyanins were determined separately. Appropriate sample amounts were dispersed in water, and the free active substances were separated by high-speed centrifugation. After demulsification with an organic solvent, the contents of astaxanthin and anthocyanins in the supernatant (free) and precipitate (encapsulated) were determined by HPLC. Encapsulation efficiency (%) = (encapsulated amount / total feed amount) × 100%; Drug loading (%) = (encapsulated amount / total weight of liposomes) × 100%.

[0103] Particle size and polydispersity index: Dynamic light scattering method was used. After appropriate dilution with deionized water, the samples were placed in a nanoparticle size and zeta potential analyzer, and their volume average hydrated particle size and polydispersity index (PDI) were determined at 25℃.

[0104] Zeta potential: determined using electrophoretic light scattering. The diluted sample was placed in a dedicated electrode cell, and its Zeta potential was measured using the same instrument.

[0105] Accelerated stability testing: A high-temperature and high-humidity accelerated testing method was used. Powder samples were sealed in vials and stored in a constant temperature and humidity chamber at 40℃ and 75% relative humidity for 30 days. Samples were taken periodically to determine the retention rates of astaxanthin and anthocyanins. Retention rate (%) = (content after storage / initial content) × 100%. The table presents the average retention rates of the two active ingredients.

[0106] In vitro sustained-release performance: Dynamic dialysis was used. The gastrointestinal environment was simulated; the mixture was incubated in simulated gastric fluid (pH 1.2) for 2 hours, then transferred to simulated intestinal fluid (pH 6.8) for another 4 hours. Samples were taken periodically, and equal volumes of release medium were added to determine the cumulative release rates of astaxanthin and anthocyanins. The 2-hour release rate in gastric fluid and the 4-hour cumulative release rate in intestinal fluid are the average of the release rates of the two active ingredients.

[0107] Solubility and dispersibility: Take 1.0g of sample powder, add it to 200mL of pure water at 25℃, stir in the same direction at a uniform speed (2 revolutions per second), and record the time (seconds) required for the powder to completely dissolve and the solution to become a homogeneous emulsion.

[0108] Solution stability (static observation): Place the freshly prepared solution in a 25°C environment and observe and record the solution state at 0.5 hours, 2 hours, and 24 hours respectively, noting whether there is visible layering, flocculation, or precipitation.

[0109] Centrifugal sedimentation rate: Take 10 mL of freshly prepared solution and place it in a centrifuge tube. Centrifuge at 3000 rpm for 15 minutes. Carefully remove the supernatant, weigh and calculate the mass of the wet precipitate. Centrifugal sedimentation rate (%) = (mass of wet precipitate / total mass of solution) × 100%.

[0110] Sensory indicators: Freshly prepared solutions will be scored by trained sensory evaluators (at least 5 people). Scoring criteria: Color uniformity (1-5 points, 1 point for severe unevenness, 5 points for highly uniformity), smoothness of texture (1-5 points, 1 point for obvious gritty or grainy texture, 5 points for smooth texture without grains), presence of unpleasant odors (record whether there is a noticeable oxidized oil smell or other off-odors).

[0111] Table 1. Physicochemical properties of astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes in Examples 1-3 and Comparative Examples 1-3

[0112]

[0113] Note: Total drug loading = (Astaxanthin encapsulation amount + Anthocyanin encapsulation amount) / Total liposome weight × 100%. Accelerated 30-day average retention rate is the arithmetic mean of astaxanthin and anthocyanin retention rates. The average release rate in gastric juice over 2 hours and the cumulative average release rate in intestinal juice over 4 hours are the average values ​​of the two active ingredients.

[0114] Table 2. Application performance of astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes in Examples 1-3 and Comparative Examples 1-3

[0115]

[0116] Based on the physicochemical performance data in Table 1, the astaxanthin and anthocyanin dual-drug-loaded multilayer liposomes prepared in Examples 1-3 of this application exhibit excellent and stable core physicochemical performance indicators: astaxanthin encapsulation efficiency is as high as 89.8%–92.5%, anthocyanin encapsulation efficiency is 82.9%–85.1%, and total drug loading reaches 16.2%–17.3%; the average hydrated particle size is controlled at the nanoscale of 318–348 nm, and the distribution is uniform (PDI is 0.22–0.24). The system exhibits excellent stability, characterized by a high absolute Zeta potential (-30.8 to -33.6 mV) and an average retention rate of 89.3% to 91.2% of the active ingredient after 30 days of accelerated storage at 40°C and 75% humidity. It also demonstrates significant in vitro sustained-release properties, with an average release rate of only 17.2% to 20.1% in simulated gastric fluid (pH 1.2) after 2 hours, while it can continuously release in simulated intestinal fluid, with a cumulative average release rate of 84.5% to 87.5% after 4 hours.

[0117] Comparative Example 1 (omitting chitosan and polygalacturonic acid, without polysaccharide layer-by-layer self-assembly modification) showed a sharp drop in the accelerated 30-day average retention rate to 71.5%, while the average 2-hour release rate from gastric juice increased to 42.3%. This indicates that the chitosan-polygalacturonic acid bilayer polysaccharide shell plays a crucial role in inhibiting gastric acid permeation, preventing active ingredient leakage, and maintaining the long-term storage stability of the formulation. Liposomes lacking the polysaccharide shell rapidly release the active ingredient into gastric juice and cannot achieve the gastric protective function.

[0118] Comparative Example 2 (omitting hydrogenated soybean lecithin and using only ordinary soybean lecithin) showed lower encapsulation efficiency (85.6% for astaxanthin and 76.3% for anthocyanins) and total drug loading (15.1%) compared to the Example. The average particle size increased to 395 nm and the distribution broadened (PDI 0.31). The accelerated retention rate (75.8%) and gastric juice release rate (35.6%) were also significantly inferior to the Example. This demonstrates that the rigid-flexible complementary biphasic membrane structure formed by the combination of hydrogenated soybean lecithin and soybean lecithin is indispensable for constructing dense and stable multilayer vesicles, and that ordinary lecithin alone cannot provide sufficient membrane rigidity and thermal stability.

[0119] Comparative Example 3 (with phytosterols replaced by cholesterol) showed similar physical encapsulation parameters such as encapsulation efficiency, particle size, and zeta potential to the examples, but the average retention rate after 30 days decreased to 74.2%, and a slight oxidative odor was observed in its application performance. This indicates that phytosterols can not only replace cholesterol as a membrane structure regulator to maintain membrane stability, but also possess antioxidant synergistic functions that cholesterol lacks. This allows for better protection of the chemical stability of the active ingredient during storage, while avoiding the potential oxidative odor risk associated with cholesterol.

[0120] According to the application performance data in Table 2, the products of Examples 1-3 of this application exhibit excellent reconstitution characteristics: rapid dissolution and dispersion (36-42 seconds), maintaining uniformity without precipitation after standing for 24 hours, extremely low centrifugal sedimentation rate (0.5%-0.8%), and outstanding sensory experience (color and taste scores ≥4.6 points, no off-odor). This proves that the multilayer liposome powder prepared in this application has ideal instant solubility and colloidal stability.

[0121] Comparative Example 1 (without the polysaccharide shell) dissolved more slowly (58 seconds), produced flocculent precipitate (precipitation rate 3.5%), and had a significantly lower taste score. This indicates that the absence of the polysaccharide shell makes the liposome particles more prone to aggregation and sedimentation, and may also lead to an increased grainy texture due to incomplete encapsulation.

[0122] Comparative Example 2 (unhydrogenated soybean phospholipids) showed observable deterioration in all application indicators, proving that the lack of a rigid-flexible complementary membrane structure interferes with the interfacial properties of liposomes, affecting their hydration and dispersion efficiency and long-term dispersion stability.

[0123] Although Comparative Example 3 (cholesterol replaced with phytosterol) had acceptable physicochemical stability, it showed a slight oxidized taste, which directly proves the advantages of phytosterol in maintaining the pure flavor of the product in the prepared state and preventing oxidized off-flavors.

[0124] The range descriptions used herein, such as numerical ranges and proportional ranges, include all possible sub-ranges and single numerical values ​​within that range. For example, the range descriptions of "1 to 6" or "1 to 6" cover all sub-ranges (such as 1 to 3, 2 to 5, etc.) and single numbers (such as 1, 2, 3, 4, 5, 6) between 1 and 6. Unless otherwise specified, the terms "including" and "contains" as used herein mean "including but not limited to"; relational terms such as "first" and "second" are used only to distinguish different entities or operations and do not imply an actual order or relationship; "and / or" indicates that multiple situations can exist individually or simultaneously; expressions such as "at least one," "multiple," and "at least one" refer to any combination of the corresponding objects, including combinations of single or multiple objects. The proportional relationships mentioned herein, such as mass ratios and molar ratios, should be understood as the correspondence between the first and second terms of a proportional formula, according to the order of description. The raw materials, reagents, instruments, and equipment used herein can all be obtained through commercial purchase or prepared using existing methods.

[0125] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A multilayer liposome with dual drug delivery of astaxanthin and anthocyanins, characterized in that, By weight, the astaxanthin and anthocyanin dual-drug-carrying multilayer liposome comprises the following raw materials: 5-20 parts astaxanthin, 5-20 parts anthocyanin, 20-50 parts soybean lecithin, 5-20 parts hydrogenated soybean lecithin, 3-15 parts phytosterols, 1-8 parts glycerol, 0.5-4 parts chitosan, 0.5-4 parts polygalacturonic acid, 0.5-4 parts vitamin C, 15-60 parts trehalose, 5-40 parts dextrin, and deionized water.

2. The astaxanthin and anthocyanin dual-drug-carrying multilayer liposome according to claim 1, characterized in that, The average particle size of the multilayer liposomes is 300-500 nm, the astaxanthin encapsulation rate is ≥80%, the anthocyanin encapsulation rate is ≥70%, and the absolute value of the zeta potential is ≥20 mV.

3. The astaxanthin and anthocyanin dual-drug-carrying multilayer liposome according to claim 1, characterized in that, The total weight of the astaxanthin and the anthocyanin shall not exceed 30 parts; The weight ratio of the soybean lecithin to the hydrogenated soybean lecithin is (1-3):

1.

4. A method for preparing astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes according to any one of claims 1 to 3, characterized in that, The method includes the following steps: S1. Dissolve astaxanthin, anthocyanins, soybean lecithin, hydrogenated soybean lecithin, and phytosterols in an ethanol-water solution, and then remove the solvent by vacuum evaporation to form a uniform film on the container wall to obtain mixture A. S2. Dissolve vitamin C, trehalose, dextrin, and glycerol in deionized water at 45–55°C to obtain mixture B; S3. Quickly pour the mixture B into a container containing the mixture A to completely submerge the film, and stir and hydrate it in a water bath at 45-55°C to form a liposome suspension with a multilayer vesicle structure, thus obtaining mixture C. S4. Heat the mixture C to 30-40°C, and add chitosan solution dropwise at a rate of 0.5-2 mL / min. After the addition is complete, continue stirring for 30-60 min to obtain a chitosan-coated liposome suspension. S5. Add polygalacturonic acid solution dropwise to the chitosan-coated liposome suspension at a rate of 0.5 to 2 mL / min. After the addition is complete, continue stirring for 30 to 60 min to obtain a chitosan-polygalacturonic acid bilayer coated liposome suspension. S6. Spray-dry the chitosan-polygalacturonic acid bilayer-coated liposome suspension to obtain the astaxanthin and anthocyanin dual-drug-loaded multilayer liposomes.

5. The method for preparing astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes according to claim 4, characterized in that, The volume fraction of the ethanol-water solution in S1 is 70-80%, the temperature of the vacuum evaporation is 35-45℃, and the vacuum degree is -0.08 to -0.1 MPa.

6. The method for preparing astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes according to claim 4, characterized in that, The stirring speed for hydration described in S3 is 400–800 rpm, and the time is 45–90 min.

7. The method for preparing astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes according to claim 4, characterized in that, The concentration of the chitosan solution in S4 is 0.5–1.5 wt%, and the pH is 4.5–5.

5.

8. The method for preparing astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes according to claim 4, characterized in that, The polygalacturonic acid solution in S5 has a concentration of 0.5–1.5 wt% and a pH of 6.0–6.

5.

9. The method for preparing astaxanthin and anthocyanin dual-drug-carrying multilayer liposomes according to claim 4, characterized in that, The process parameters for spray drying described in S6 are: inlet air temperature 85±5℃, outlet air temperature 55±2℃, atomizing disc rotation speed 20000~30000rpm, feed flow rate 5~10 mL / min, and the solid content of the chitosan-polygalacturonic acid double-layer coated liposome suspension before spray drying is 10~20%.

10. The application of a multilayer liposome containing astaxanthin and anthocyanins as described in any one of claims 1 to 3 in food.

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