Multilayer lutein ester liposome as well as preparation method and application thereof

Abstract

The invention provides a multilayer lutein ester liposome as well as a preparation method and application thereof, and belongs to the field of food processing. The multi-layer lutein ester liposome is prepared from the following raw materials: 30 to 50 parts of lutein ester, 40 to 60 parts of soybean phosphatidylcholine, 5 to 20 parts of cholesterol, 10 to 25 parts of starch sodium octenylsuccinate, 20 to 40 parts of trehalose, 15 to 30 parts of mannitol, 4 to 10 parts of phosphatidyl glycerol, 1 to 2 parts of vitamin E acetate and 1 to 2 parts of ascorbyl palmitate. According to the present invention, through the specific food-grade raw material composition and the accurate ratio relationship, the comprehensive delivery system is constructed for the highly hydrophobic and unstable lutein ester, and the system synchronously achieves the high encapsulation efficiency, the excellent stability, the good water dispersibility and the slow release function.

Description

Technical Field

[0001] This application relates to the field of food processing technology, and in particular to a multilayer lutein ester liposome, its preparation method, and its application. Background Technology

[0002] Lutein esters, as important fat-soluble functional factors, have clear beneficial effects in areas such as visual health. However, their inherent strong hydrophobicity makes them difficult to dissolve and disperse uniformly in water-based food systems, resulting in low bioavailability. Furthermore, the conjugated double bonds in their molecular structure make them extremely sensitive to light, heat, and oxygen, easily degrading and becoming ineffective during processing and storage, leading to poor shelf-life stability. These drawbacks severely limit their application in general food and beverage products.

[0003] To address the aforementioned issues, existing technologies typically employ encapsulation techniques such as liposomes and microcapsules. However, these technologies still have significant limitations for applications in the food industry: First, conventional liposome preparation methods, such as thin-film dispersion and extrusion coating, require specialized equipment, are complex, and costly, making them difficult to meet the food industry's requirements for economic efficiency and large-scale production. Second, the resulting liposomes are mostly monolayer structures, with limited encapsulation efficiency, protection strength of active ingredients, and sustained-release capacity in the gastrointestinal environment. Third, many existing solutions use excipients or processes that do not meet food regulatory requirements, or the final product form (such as suspension) is not easily added to or preserved directly in solid or semi-solid foods. Therefore, how to construct a food-grade delivery system for highly hydrophobic and unstable lutein esters that combines high encapsulation efficiency, excellent stability, good water dispersibility, and sustained-release function is a pressing technical problem that needs to be solved. Summary of the Invention

[0004] This application provides a multilayer lutein ester liposome, its preparation method, and its application to solve the following technical problem: how to construct a food-grade delivery system for highly hydrophobic and unstable lutein esters that combines high encapsulation efficiency, excellent stability, good water dispersibility, and sustained-release function.

[0005] In a first aspect, embodiments of this application provide a multilayer lutein ester liposome, wherein the raw material composition of the multilayer lutein ester liposome, by weight, is as follows: 30-50 parts lutein ester, 40-60 parts soybean phosphatidylcholine, 5-20 parts cholesterol, 10-25 parts sodium octenyl succinate starch, 20-40 parts trehalose, 15-30 parts mannitol, 4-10 parts phosphatidylglycerol, 1-2 parts vitamin E acetate, and 1-2 parts ascorbyl palmitate.

[0006] Optionally, the molar ratio of cholesterol to soybean phosphatidylcholine is 0.2:1 to 0.4:1.

[0007] Optionally, the total mass ratio of trehalose and mannitol to the total mass ratio of soybean phosphatidylcholine and cholesterol is 0.9:1 to 1.5:1.

[0008] Optionally, the mass ratio of vitamin E acetate to ascorbate palmitate is 1:1 to 1:1.5.

[0009] Optionally, the structure of the multilayer lutein ester liposome, from the inside out, comprises:

[0010] The active core is formed by the hydrophobic interaction between lutein esters and cholesterol.

[0011] The phospholipid bilayer surrounding the core of the active ingredient is composed of soybean phosphatidylcholine and cholesterol, and is doped with phosphatidylglycerol.

[0012] An octenyl succinate starch sodium macromolecular space-stabilizing layer attached to the outer surface of the phospholipid bilayer;

[0013] The solid glass matrix that runs through and encapsulates the material is formed by a blend of trehalose and mannitol.

[0014] Optionally, the volume-average hydrated particle size of the structure is 200–400 nm, and the polydispersity index is 0.20–0.35.

[0015] Secondly, embodiments of this application provide a method for preparing multilayer lutein ester liposomes as described in the first aspect, the method comprising the following steps:

[0016] S1. Lutein ester, soybean phosphatidylcholine, cholesterol and vitamin E acetate are dissolved in an organic solvent and stirred and preheated at 60-70°C to obtain a structured organic phase;

[0017] S2. Dissolve water, trehalose, mannitol, ascorbyl palmitate and phosphatidylglycerol by heating to 50-60°C and stirring to obtain a homogeneous aqueous phase;

[0018] S3. Under high-speed shearing, the structured organic phase is injected into the homogeneous aqueous phase for initial emulsification, followed by the addition of sodium octenyl succinate starch, and the induced assembly reaction is continuously carried out under medium-speed stirring to obtain crude liposome emulsion.

[0019] S4. Spray dry the crude liposome emulsion to obtain powdered multilayer lutein ester liposomes.

[0020] Optionally, in step S3, the high-speed shearing speed is 9000-11000 rpm, the medium-speed stirring speed is 1200-1800 rpm, and the duration of the induced assembly reaction is 45-75 min.

[0021] Optionally, in step S4, the inlet air temperature of the spray dryer is 165-175°C, and the outlet air temperature is 75-85°C.

[0022] Thirdly, embodiments of this application provide a food product containing multilayered lutein ester liposomes as described in any one of the first aspects.

[0023] The technical solutions provided in this application have the following advantages compared with the prior art:

[0024] This application provides a multilayer lutein ester liposome, which, through its specific food-grade raw material composition and precise ratio, constructs a comprehensive delivery system for highly hydrophobic and unstable lutein esters. This system simultaneously achieves high encapsulation efficiency, excellent stability, good water dispersibility, and sustained-release function. The specific construction logic is as follows:

[0025] First, high encapsulation and sustained-release functions are achieved by constructing a tight structure of the active ingredient core-phospholipid bilayer. Cholesterol (5-20 parts) and soybean phosphatidylcholine (40-60 parts) in the formulation are the core components for achieving this function. During the preparation process, the steroidal ring structure of cholesterol and the hydrophobic long chain of lutein ester (30-50 parts) are pre-complexed through hydrophobic interactions to form the encapsulated active ingredient core. At the same time, sufficient soybean phosphatidylcholine and cholesterol together form a dense phospholipid bilayer, tightly encapsulating the active ingredient core. This not only provides ample space for the hydrophobic lutein ester to achieve a high encapsulation rate, but its multilayer membrane structure itself also constitutes a physical diffusion barrier, requiring the release of lutein ester to pass through multiple phospholipid membranes sequentially, thus structurally laying the foundation for sustained release.

[0026] Second, excellent stability is achieved by establishing a multi-synergistic mechanism of anti-oxidation, electrostatics, steric hindrance, and glassy solidification. This stability system consists of four layers of defense: (1) Endogenous antioxidant network: Vitamin E acetate (1-2 parts) distributed in the hydrophobic region of the phospholipid bilayer and ascorbyl palmitate (1-2 parts) located at the water-oil interface synergistically scavenge free radicals from different spatial sites, providing chemical stability. (2) Electrostatic repulsion: The negative charge carried by phosphatidylglycerol (4-10 parts) makes the surface of liposomes charged, preventing particle aggregation through charge repulsion. (3) Steric hindrance stabilization: Sodium octenyl succinate starch (10-25 parts) inserts into the phospholipid bilayer through its hydrophobic end, and the huge hydrophilic chain forms a physical spatial barrier on the periphery, which, together with electrostatic repulsion, ensures the long-term physical stability of the dispersion system. (4) Solid protection: Trehalose (20-40 parts) and mannitol (15-30 parts) form a solid glass matrix that encapsulates each liposome particle during the final spray drying process. This matrix can isolate moisture and oxygen, solidify the precisely assembled structure in a dry state, and thus provide storage stability.

[0027] Third, excellent water dispersibility is achieved through optimization of particle size and interfacial properties. The direct result of the aforementioned multiple stabilization mechanisms is the formation of liposome particles with uniform particle size and stable structure. The presence of the sodium octenyl succinate starch macromolecular stabilizing layer greatly enhances the hydrophilicity of the particles, enabling them to disperse rapidly in the aqueous phase and form a stable colloidal system. The final product is a powder with good flowability, whose water dispersibility is jointly ensured by the stability of the liposome particles themselves and the rapid solubility of the external sugar alcohol matrix, fully meeting the application requirements for instant food preparation or as an ingredient in aqueous foods.

[0028] In summary, the formulation of this application is a systematic engineering project in which each component performs its specific function and works synergistically: soybean phosphatidylcholine and cholesterol constitute the main structure for encapsulation and sustained release; vitamin E acetate and ascorbate palmitate constitute the internal antioxidant network; phosphatidylglycerol and sodium octenyl succinate starch constitute the external dispersion stabilizing layer; and trehalose and mannitol constitute the solid-state shaping protective layer. These components work synergistically through precise proportioning to ultimately construct a food-grade delivery system that simultaneously solves comprehensive industrial challenges such as hydrophobicity, instability, low absorption rate, and inconvenient application. Attached Figure Description

[0029] 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.

[0030] 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.

[0031] Figure 1 This is a schematic flowchart illustrating the preparation method of multilayer lutein ester liposomes provided in the embodiments of this application. Detailed Implementation

[0032] 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.

[0033] This application provides a multilayer lutein ester liposome, wherein the raw materials of the multilayer lutein ester liposome are, by weight: 30-50 parts lutein ester, 40-60 parts soybean phosphatidylcholine, 5-20 parts cholesterol, 10-25 parts sodium octenyl succinate starch, 20-40 parts trehalose, 15-30 parts mannitol, 4-10 parts phosphatidylglycerol, 1-2 parts vitamin E acetate, and 1-2 parts ascorbyl palmitate.

[0034] In some embodiments, the molar ratio of cholesterol to soybean phosphatidylcholine is 0.2:1 to 0.4:1.

[0035] In some embodiments, the total mass ratio of the trehalose and mannitol to the total mass ratio of soybean phosphatidylcholine and cholesterol is 0.9:1 to 1.5:1.

[0036] In some embodiments, the mass ratio of vitamin E acetate to ascorbate palmitate is 1:1 to 1:1.5.

[0037] It should be noted that the multilayer lutein ester liposomes provided in this application construct a stable delivery system through precise molecular design and functional synergy of each component. The specific functions of each component are as follows:

[0038] (1) Core active ingredient: Lutein ester

[0039] Lutein esters, as the core functional component, are the encapsulation target and the basis of action in this application. Their lipid-soluble molecular structure is encapsulated in the hydrophobic core region of liposomes, thereby isolating them from the external aqueous phase. This directly improves their dispersibility in aqueous systems and provides the structural prerequisite for achieving sustained-release properties.

[0040] (2) Phospholipid bilayer structure components

[0041] These components together constitute the core membrane structure that encapsulates lutein esters.

[0042] Soybean phosphatidylcholine: As a key film-forming material, its amphiphilic molecular structure forms the framework for the phospholipid bilayer of liposomes. The hydrophilic head contacts the water phase outward, while the hydrophobic tail forms a hydrophobic region inward, providing structural space for the encapsulation of lutein esters.

[0043] Cholesterol: As a membrane stabilizer, its rigid steroidal ring structure can intercalate into the hydrophobic tail region of soybean phosphatidylcholine. This intercalation can regulate the fluidity of the phospholipid bilayer, increase the compactness and mechanical strength of the membrane, thereby significantly reducing the leakage of active ingredients and improving the tolerance of liposomes to changes in the external environment (such as the gastrointestinal environment).

[0044] Phosphatidylglycerol: As a charge regulator, the negatively charged groups on its molecules give the liposome surface a negative charge. This negative charge effectively prevents liposome particles from approaching each other, aggregating, and fusing during storage through electrostatic repulsion between like charges, thus ensuring the long-term physical stability of the system.

[0045] (3) Outer layer stabilizing and shaping components

[0046] This component forms a physical and state barrier outside the phospholipid bilayer.

[0047] Sodium octenyl succinate starch: As an interfacial steric stabilizer, its hydrophobic group (octenyl) can be anchored into the phospholipid bilayer, while the large hydrophilic starch chain extends fully in water, forming a three-dimensional steric barrier around the liposomes. This steric hindrance effect, combined with the electrostatic repulsion provided by phosphatidylglycerol, constitutes a dual guarantee for the stability of the liposome dispersion system.

[0048] Trehalose and mannitol: As glassy stabilizers, they coexist in the aqueous phase and rapidly dehydrate during spray drying, forming an amorphous glassy solid matrix. This matrix acts like a "solid shell," encapsulating and immobilizing the liposome particles, allowing their structure to be maintained for a long time in the dry state. This prevents phospholipid oxidation and structural collapse before rehydration and greatly improves the flowability and water dispersibility of the final powder product.

[0049] (4) Antioxidant system components

[0050] This component forms a spatially complementary antioxidant network that specifically protects different areas.

[0051] Vitamin E acetate: As a lipid-phase antioxidant, its hydrophobicity allows it to be located in the hydrophobic region of the phospholipid bilayer, directly scavenging free radicals from inside the liposomes and protecting the unsaturated fatty acid chains of soybean phosphatidylcholine and lutein esters from oxidative degradation.

[0052] Ascorbyl palmitate: As an amphiphilic antioxidant, its molecular structure allows it to be positioned at the water-oil interface between the inner and outer layers of the phospholipid bilayer. This position enables it to both assist the antioxidant effect of vitamin E acetate and scavenge free radicals that invade in the aqueous phase, forming a spatial functional synergy and complementarity with vitamin E acetate.

[0053] More importantly, this application is not a simple mixture of components, but a structural design based on their molecular properties. Soybean phosphatidylcholine, cholesterol, and phosphatidylglycerol synergistically construct a dense and charged stable primary membrane. Sodium octenyl succinate starch further enhances dispersion stability by forming an external steric hindrance layer. Trehalose and mannitol solidify and preserve the precisely assembled structure during the drying stage. Throughout, an antioxidant network composed of vitamin E acetate and ascorbate palmitate provides comprehensive protection from different physical sites. Ultimately, all components, centered around lutein ester, work together to achieve a synergistic effect of high encapsulation, sustained release, high stability, and ease of application. The preferred molar ratio of soybean phosphatidylcholine to cholesterol (0.2:1 to 0.4:1) is key to membrane rigidity, while the ratio of the total mass of trehalose and mannitol to the total mass of the phospholipid bilayer components (0.9:1 to 1.5:1) is the basis for forming an effective glassy protective layer.

[0054] In some embodiments, the structure of the multilayer lutein ester liposome, from the inside out, comprises:

[0055] The active core is formed by the hydrophobic interaction between lutein esters and cholesterol.

[0056] The phospholipid bilayer surrounding the core of the active ingredient is composed of soybean phosphatidylcholine and cholesterol, and is doped with phosphatidylglycerol.

[0057] An octenyl succinate starch sodium macromolecular space-stabilizing layer attached to the outer surface of the phospholipid bilayer;

[0058] The solid glass matrix that runs through and encapsulates the material is formed by a blend of trehalose and mannitol.

[0059] In some embodiments, the volume-average hydrated particle size of the structure is 200–400 nm, and the polydispersity index is 0.20–0.35.

[0060] The multilayer lutein ester liposome structure provided in this application, through precisely defined multilayer ordered assembly, exhibits a series of synergistic advantages in stability, functionality, and product performance compared to conventional liposomes.

[0061] First, this structure achieves high encapsulation and active sustained release through the tight composite of the "active ingredient core and phospholipid bilayer." Lutein esters and cholesterol are pre-complexed through hydrophobic interactions, firmly "anchoring" the active ingredient to the innermost layer of the liposome, providing the molecular basis for high encapsulation efficiency. Simultaneously, the outer phospholipid bilayer (composed of soybean phosphatidylcholine and cholesterol, and doped with phosphatidylglycerol) forms a dense physical diffusion barrier. The active ingredient needs to sequentially pass through multiple phospholipid membranes to be released, fundamentally achieving programmed sustained release in the gastrointestinal environment and contributing to improved bioavailability.

[0062] Secondly, this structure achieves excellent physical and chemical stability through a triple external stabilization mechanism of "charge repulsion-steric hindrance-glass solidification". Phosphatidylglycerol in the phospholipid bilayer imparts a negative charge to the liposome surface, preventing particle aggregation through electrostatic repulsion. The sodium octenyl succinate starch macromolecule stabilizing layer attached to its outer surface generates a strong steric hindrance effect through its large hydrophilic chains, synergistically working with electrostatic repulsion to ensure the long-term kinetic stability of the liposomes in aqueous dispersion systems. The outermost solid glass matrix, formed by a blend of trehalose and mannitol, completely solidifies and encapsulates the precisely assembled structure in a dry state, effectively isolating it from moisture and oxygen, preventing phospholipid oxidation and structural collapse, and endowing the final product with excellent storage stability and reconstitution properties.

[0063] Third, the precise control of size and uniformity ensures reproducibility of performance and reliability of application. Controlling the volume-average hydrated particle size within 200–400 nm and maintaining a polydispersity index within a narrow range of 0.20–0.35 ensures that the particles have a size suitable for oral absorption and demonstrates that the preparation process can stably and uniformly reproduce the described multilayer structure. Uniform particle size distribution is crucial for consistent batch-to-batch quality and predictable in vivo and in vitro behavior, and is also the foundation for its application as a standardized raw material in food.

[0064] Figure 1 This is a schematic flowchart illustrating the preparation method of multilayer lutein ester liposomes provided in the embodiments of this application.

[0065] like Figure 1 As shown in the embodiments of this application, a method for preparing multilayer lutein ester liposomes as described above is provided, the method comprising the following steps:

[0066] S1. Lutein ester, soybean phosphatidylcholine, cholesterol and vitamin E acetate are dissolved in an organic solvent and stirred and preheated at 60-70°C to obtain a structured organic phase;

[0067] S2. Dissolve water, trehalose, mannitol, ascorbyl palmitate and phosphatidylglycerol by heating to 50-60°C and stirring to obtain a homogeneous aqueous phase;

[0068] S3. Under high-speed shearing, the structured organic phase is injected into the homogeneous aqueous phase for initial emulsification, followed by the addition of sodium octenyl succinate starch, and the induced assembly reaction is continuously carried out under medium-speed stirring to obtain crude liposome emulsion.

[0069] S4. Spray dry the crude liposome emulsion to obtain powdered multilayer lutein ester liposomes.

[0070] In some embodiments, in step S3, the high-speed shearing speed is 9000-11000 rpm, the medium-speed stirring speed is 1200-1800 rpm, and the duration of the induced assembly reaction is 45-75 min.

[0071] In some embodiments, in step S4, the inlet air temperature of the spray dryer is 165-175°C and the outlet air temperature is 75-85°C.

[0072] The preparation method provided in this application is not a simple physical mixing process at each step, but rather aims to precisely drive the directional assembly of molecules from a "solution" to a "multilayer ordered structure." The function of each step is as follows:

[0073] Step S1 (preparation of the structured organic phase) is a crucial pretreatment for forming the "active core" and membrane basis. Lutein ester, soybean phosphatidylcholine, cholesterol, and vitamin E acetate are co-dissolved in an organic solvent at 60–70°C. The core purpose is to promote pre-organization of the components at the molecular level. Specifically, under these thermodynamic conditions, the steroidal ring structure of cholesterol and the hydrophobic long chain of lutein ester can pre-bind through hydrophobic interactions, forming the "prototype of the complex" to be encapsulated. Simultaneously, soybean phosphatidylcholine and cholesterol undergo intermolecular pre-arrangement in this homogeneous solution, while vitamin E acetate is pre-dispersed in this lipid environment. This lays the molecular foundation for the subsequent formation of a well-ordered phospholipid bilayer.

[0074] Step S2 (preparation of a homogeneous aqueous phase) constructs a continuous phase environment with both charge and protective functions. Water, trehalose, mannitol, ascorbyl palmitate, and phosphatidylglycerol are dissolved at 50–60°C. This step achieves multiple functions. On one hand, phosphatidylglycerol is uniformly dispersed in the aqueous phase, and its negatively charged sites await subsequent binding with organic compounds. On the other hand, ascorbyl palmitate, due to its amphiphilicity, is positioned at the water-oil interface to be formed. Most importantly, trehalose and mannitol are completely dissolved, constructing a high-concentration sugar solution environment in the system, preparing for the final formation of the solid glass matrix encapsulating the entire liposome structure in step S4.

[0075] Step S3 (emulsification and induced assembly) is the core step driving the transformation from a single-layer structure to a multilayer structure. This step is divided into two consecutive stages with clearly defined functional purposes. First, the structured organic phase is injected into a homogeneous aqueous phase under high-speed shear at 9000–11000 rpm. The high energy input forces the organic phase to break into extremely small oil droplets, and the lipid molecules (soybean phosphatidylcholine, cholesterol) within it rapidly align at the aqueous phase interface, encapsulating the lutein ester-cholesterol complex to form primary, small-particle-size single-chamber liposomes. Subsequently, sodium octenyl succinate starch is added, and the stirring speed is precisely adjusted to 1200–1800 rpm, entering the "induced assembly reaction" stage. Under this relatively mild shear force, the hydrophobic end of sodium octenyl succinate starch has sufficient time (45–75 min) to insert into the phospholipid bilayer of the primary liposomes. The strong steric hindrance effect generated by its large hydrophilic chain, and the electrostatic repulsion caused by phosphatidylglycerol, work together to make multiple primary liposomes approach each other but not merge during movement. Under the physical induction of continuous stirring, membrane reorganization and nesting eventually occur, thus stably forming a crude liposome emulsion with a few chambers or multiple layers of vesicle structure (i.e., the volume average hydrated particle size increases to 200-400 nm).

[0076] Step S4 (spray drying and curing) is the shaping step that "locks in" the dynamic multilayer structure in the solution into a solid product. The crude liposome emulsion is spray-dried at an inlet air temperature of 165–175°C and an outlet air temperature of 75–85°C, a very rapid dehydration process. Trehalose and mannitol, previously uniformly distributed in the aqueous phase, cannot crystallize upon the instantaneous evaporation of water, but instead form a dense, continuous, amorphous solid glass matrix. This matrix acts like a mold, completely encapsulating and fixing the multilayer liposome vesicle structure assembled in step S3, thus obtaining a powdered product. This step not only removes moisture for better preservation but, more importantly, permanently fixes the multilayered, ordered structure of the liposomes by forming a solid glass matrix, preventing phospholipid molecule rearrangement or structural fusion during storage.

[0077] Based on a general inventive concept, embodiments of this application provide a food containing any of the multilayered lutein ester liposomes described above.

[0078] The multilayer lutein ester liposomes of this application can be used as a core ingredient in food because of their comprehensive regulatory compliance, food safety, and the solution to the bottlenecks of traditional application technologies by their unique structure.

[0079] First, regarding regulations and safety, all components of this multilayered lutein ester liposome are food ingredients or food additives permitted for use in China. The core active ingredient, lutein ester, is an approved new food ingredient; soybean phosphatidylcholine, trehalose, and mannitol are all conventional food ingredients; phosphatidylglycerol and sodium octenyl succinate starch are legal food additives; and the two antioxidants (vitamin E acetate and ascorbate palmitate) are also permitted by regulations. The preparation method employs heating, stirring, and spray drying, all standard processes in the food industry, and the organic solvents are completely removed from the final powder product, ensuring the food safety of the final product.

[0080] Secondly, at the technical and functional level, this multilayer structure systematically solves three core challenges in the application of lutein esters in food systems. First, it addresses the challenge of chemical stability: the internal-external active ingredient core – phospholipid bilayer – solid glass matrix structure, combined with a spatially distributed antioxidant network, provides multiple layers of isolation and protection for light-, heat-, and oxygen-sensitive lutein esters, significantly improving their retention rate during processing, storage, and reconstitution, ensuring the effective shelf life of the final product. Second, it addresses the challenge of bioavailability: this structure transforms fat-soluble lutein esters into nanoparticles that are easily and uniformly dispersed in water-based foods. The high affinity between the phospholipid bilayer and biological membranes, along with the sustained-release properties achieved through the multilayer membrane structure, jointly promotes efficient absorption in the intestines. Finally, it addresses the challenge of ease of application: the spray-dried powder form exhibits good flowability and water dispersibility, allowing for convenient and precise addition to various food forms such as solid beverages and dairy products, overcoming the obstacle of oily components not easily mixing homogeneously with water-based food systems.

[0081] Third, in terms of product value, this technology offers a clear competitive advantage. Compared to commonly available lutein ester oleoresins or simple mixed powders, the multilayer lutein ester liposomes of this application represent a leap from providing raw materials to providing functional units with delivery and protection systems. End products developed based on this technology can establish solid and verifiable technical support in terms of efficient absorption, stable sustained release, and ease of use, thereby forming significant market competitiveness.

[0082] 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.

[0083] Example 1

[0084] This embodiment provides a multilayer lutein ester liposome, with the following specific raw material composition by weight: lutein ester: 40 parts, soybean phosphatidylcholine (purity ≥95%): 50 parts, cholesterol: 8 parts, sodium octenyl succinate starch: 18 parts, trehalose: 38 parts, mannitol: 30 parts, phosphatidylglycerol: 7 parts, vitamin E acetate: 1.5 parts, ascorbate palmitate: 1.5 parts.

[0085] Based on the above-mentioned components of multilayer lutein ester liposomes, this embodiment also provides a preparation method, the steps of which are as follows:

[0086] S1. Preparation of structured organic phase: Lutein ester, soybean phosphatidylcholine, cholesterol and vitamin E acetate are placed in anhydrous ethanol and stirred in a 65°C water bath (500 rpm) until completely dissolved to obtain a clear and homogeneous structured organic phase; the amount of anhydrous ethanol used can ensure that the fat-soluble components are fully dissolved and can be completely volatilized by spray drying without residue.

[0087] S2. Preparation of a homogeneous aqueous phase: Deionized water, trehalose, mannitol, ascorbate palmitate and phosphatidylglycerol are mixed and stirred at 400 rpm in a 55°C water bath until completely dissolved to obtain a homogeneous and transparent aqueous phase; wherein the phosphatidylglycerol is dispersed in the aqueous phase with its hydrophilic head facing the aqueous phase, providing a negative charge to the surface of the liposome membrane to be formed subsequently, avoiding liposome aggregation and inducing the formation of a multilayer membrane structure.

[0088] S3. Induction of Liposome Emulsion Formation: The homogeneous aqueous phase was placed in a high-speed shear disperser, and the structured organic phase obtained in step S1 was slowly injected at 10,000 rpm. Primary emulsification was performed by continuous high-speed shearing for 5 minutes to form a uniform O / W emulsion. Subsequently, sodium octenyl succinate starch was added, and the stirring speed was adjusted to 1500 rpm. Induction of assembly was carried out by continuous stirring for 60 minutes to obtain a milky white, non-layered, and highly fluid crude liposome emulsion. Sodium octenyl succinate starch adsorbed onto the liposome surface at this stage, further enhancing the liposome dispersibility and multilayer structure stability.

[0089] S4. Spray Drying and Curing: The above crude liposome emulsion is immediately subjected to spray drying. The process parameters are set as follows: inlet air temperature 170℃, outlet air temperature 80℃, feed pump speed 7mL / min, atomization pressure 0.3MPa, and inlet air volume 30m³ / min. 3 The limits on feed pump speed, atomization pressure, and airflow rate ensure rapid solidification of liposomes, preventing membrane structure damage and guaranteeing complete evaporation of organic solvents. The final product is a free-flowing, light yellow, powdery multilayer lutein ester liposome.

[0090] Example 2

[0091] This embodiment provides a multilayer lutein ester liposome, with the following specific raw material composition by weight: lutein ester: 35 parts, soybean phosphatidylcholine (purity ≥95%): 45 parts, cholesterol: 9 parts, sodium octenyl succinate starch: 12 parts, trehalose: 32 parts, mannitol: 20 parts, phosphatidylglycerol: 5 parts, vitamin E acetate: 1.0 part, ascorbate palmitate: 1.2 parts.

[0092] Based on the above-mentioned components of multilayer lutein ester liposomes, this embodiment also provides a preparation method, the steps of which are as follows:

[0093] S1. Preparation of structured organic phase: Lutein ester, soybean phosphatidylcholine, cholesterol and vitamin E acetate are placed in anhydrous ethanol and stirred in a water bath at 62°C (500 rpm) until completely dissolved to obtain a clear and homogeneous structured organic phase; the amount of anhydrous ethanol used can ensure that the fat-soluble components are fully dissolved and can be completely volatilized by spray drying without residue.

[0094] S2. Preparation of a homogeneous aqueous phase: Deionized water, trehalose, mannitol, ascorbyl palmitate and phosphatidylglycerol are mixed and stirred at 400 rpm in a 52°C water bath until completely dissolved to obtain a homogeneous and transparent aqueous phase.

[0095] S3. Induction of Liposome Emulsion Formation: The homogeneous aqueous phase was placed in a high-speed shear disperser, and the structured organic phase obtained in step S1 was slowly injected at 9500 rpm. Primary emulsification was performed by continuous high-speed shearing for 5 minutes to form a uniform O / W emulsion. Subsequently, sodium octenyl succinate starch was added, and the stirring speed was adjusted to 1300 rpm. Induction of assembly was carried out by continuous stirring for 50 minutes to obtain a milky white, non-layered, and highly fluid crude liposome emulsion. Sodium octenyl succinate starch adsorbed onto the liposome surface at this stage, further enhancing the liposome dispersibility and multilayer structure stability.

[0096] S4. Spray Drying and Curing: The above-mentioned crude liposome emulsion was immediately spray-dried. The process parameters were set as follows: inlet air temperature 168℃, outlet air temperature 78℃, and the feed pump speed was adjusted according to the material viscosity to ensure stable outlet air temperature. The limited process parameters ensured rapid curing of the liposomes, avoided membrane structure damage, and ensured complete evaporation of organic solvents. The final product was a free-flowing, light yellow, powdery multilayer lutein ester liposome.

[0097] Example 3

[0098] This embodiment provides a multilayer lutein ester liposome, with the following specific raw material composition by weight: lutein ester: 48 parts, soybean phosphatidylcholine (purity ≥95%): 55 parts, cholesterol: 7 parts, sodium octenyl succinate starch: 22 parts, trehalose: 40 parts, mannitol: 28 parts, phosphatidylglycerol: 9 parts, vitamin E acetate: 1.3 parts, ascorbate palmitate: 1.5 parts.

[0099] Based on the above-mentioned components of multilayer lutein ester liposomes, this embodiment also provides a preparation method, the steps of which are as follows:

[0100] S1. Preparation of structured organic phase: Lutein ester, soybean phosphatidylcholine, cholesterol and vitamin E acetate are placed in anhydrous ethanol and stirred in a water bath at 68°C (500 rpm) until completely dissolved to obtain a clear and homogeneous structured organic phase; the amount of anhydrous ethanol used can ensure that the fat-soluble components are fully dissolved and can be completely volatilized by spray drying without residue.

[0101] S2. Preparation of a homogeneous aqueous phase: Deionized water, trehalose, mannitol, ascorbate palmitate and phosphatidylglycerol are mixed and stirred at 400 rpm in a 58°C water bath until completely dissolved to obtain a homogeneous and transparent aqueous phase.

[0102] S3. Induction of Liposome Emulsion Formation: The homogeneous aqueous phase was placed in a high-speed shear disperser, and the structured organic phase obtained in step S1 was slowly injected at 10500 rpm. Primary emulsification was performed by continuous high-speed shearing for 5 minutes to form a uniform O / W emulsion. Subsequently, sodium octenyl succinate starch was added, and the stirring speed was adjusted to 1700 rpm. Induction of assembly was carried out by continuous stirring for 70 minutes to obtain a milky white, non-layered, and highly fluid crude liposome emulsion. Sodium octenyl succinate starch adsorbed onto the liposome surface at this stage, further enhancing the liposome dispersibility and multilayer structure stability.

[0103] S4. Spray Drying and Curing: The above-mentioned crude liposome emulsion was immediately spray-dried. The process parameters were set as follows: inlet air temperature 172℃, outlet air temperature 82℃, and the feed pump speed was adjusted according to the material viscosity to ensure stable outlet air temperature. The limited process parameters ensured rapid curing of the liposomes, avoided membrane structure damage, and ensured complete evaporation of organic solvents. The final product was a free-flowing, light yellow, powdery multilayer lutein ester liposome.

[0104] Comparative Example 1

[0105] This comparative example is modified from the one disclosed in Example 1 as follows:

[0106] The cholesterol component is omitted. The types, amounts, and preparation steps of the remaining components are exactly the same as in Example 1.

[0107] Comparative Example 2

[0108] This comparative example is modified from the one disclosed in Example 1 as follows:

[0109] The soybean phosphatidylcholine with a purity ≥95% was replaced with an equal mass of ordinary soybean phosphatidylcholine (phosphatidylcholine content of approximately 30%). The types, amounts, and preparation steps of the remaining components were exactly the same as in Example 1.

[0110] Comparative Example 3

[0111] This comparative example is modified from the one disclosed in Example 1 as follows:

[0112] Vitamin E acetate and ascorbate palmitate are omitted. The types, amounts, and preparation steps of the remaining components are exactly the same as in Example 1.

[0113] Comparative Example 4

[0114] This comparative example is modified from the one disclosed in Example 1 as follows:

[0115] Maltodextrin was used in place of sodium octenyl succinate. The types, amounts, and preparation steps of the remaining components were exactly the same as in Example 1.

[0116] Comparative Example 5

[0117] This comparative example is modified from the one disclosed in Example 1 as follows:

[0118] In preparation step S3, after adding sodium octenyl succinate starch, the step of "continuous stirring at 1500 rpm to induce assembly for 60 minutes" is omitted, and the process is directly proceeded to the spray drying step. The remaining components and amounts, as well as other preparation steps, are exactly the same as in Example 1.

[0119] Comparative Example 6

[0120] This comparative example is modified from the one disclosed in Example 1 as follows:

[0121] Instead of a stepwise preparation method, the components are directly physically mixed. The specific operation is as follows: all raw materials are placed in a dry powder mixer and mixed at room temperature at a conventional speed for 30 minutes to obtain a uniform light yellow mixed powder.

[0122] The performance of lutein ester liposomes obtained in Examples 1-3 and Comparative Examples 1-6 was measured, and the results are shown in Table 1. The performance measurement methods are as follows:

[0123] Encapsulation efficiency and drug loading: High-speed centrifugation-high-performance liquid chromatography (HPLC) was used. An appropriate amount of sample was dispersed in water, and free lutein esters were separated by high-speed centrifugation. After demulsification with an organic solvent, the lutein ester content in the supernatant (free) and the precipitate (encapsulated) was determined by HPLC. The encapsulation efficiency (%) was calculated as follows: (encapsulation amount / total feed amount) × 100%; drug loading (%) = (encapsulation amount / total weight of liposomes) × 100%.

[0124] Particle size and polydispersity index: Dynamic light scattering method was used. After appropriate dilution with deionized water, the sample was placed in a nanoparticle size and Zeta potential analyzer, and its volume average hydrated particle size and polydispersity index were determined at 25℃.

[0125] 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.

[0126] Accelerated stability testing: A high-temperature and high-humidity accelerated testing method was used. Powder samples were sealed in vials and placed in a constant temperature and humidity chamber at 40℃ and 75% relative humidity. Samples were taken periodically to determine the lutein ester retention rate: Retention rate (%) = (content after storage / initial content) × 100%.

[0127] In vitro sustained-release performance: Dynamic dialysis was used. The gastrointestinal environment was simulated by incubation in simulated gastric fluid at pH 1.2 for 2 hours, followed by transfer to simulated intestinal fluid at pH 6.8 for further incubation. Samples were taken periodically, and an equal volume of release medium was added to determine the cumulative release rate of lutein esters.

[0128] Table 1. Physicochemical properties of lutein ester liposomes in Examples 1-3 and Comparative Examples 1-6

[0129]

[0130] According to the data in Table 1, the multilayer lutein ester liposomes prepared in Examples 1-3 of this application exhibit excellent and stable core physicochemical properties: encapsulation efficiency of 92.8%-94.5% and drug loading of 17.1%-18.8%; average hydrated particle size controlled at the nanoscale of 305-332 nm, with extremely uniform distribution (polydispersity index PDI of 0.21-0.23); excellent system stability, characterized by a high absolute Zeta potential (-36.2 to -39.8 mV) and a lutein ester retention rate of 90.5%-91.8% after 30 days of accelerated storage at 40℃ / 75% humidity; and significant in vitro sustained-release characteristics, with a release rate of only 22.5%-25.3% in simulated gastric juice (pH 1.2) after 2 hours, while continuous release in simulated intestinal juice, with a cumulative release rate of 85.9%-88.1% after 4 hours.

[0131] Comparative Example 1 (cholesterol-free) showed a sharp drop in encapsulation efficiency to 68.3%, with increased average particle size and wider distribution, a gastric juice release rate of 45.6%, and an accelerated retention rate of 75.4%. This directly demonstrates that cholesterol is indispensable for building a dense phospholipid bilayer, and its absence leads to the inability of the liposome membrane to effectively encapsulate active ingredients, resulting in a loose and unstable structure.

[0132] Although Comparative Example 2 (using ordinary soybean phospholipids) was better than Comparative Example 1 in all aspects, it was significantly inferior to the Example. This indicates that the non-phosphatidylcholine components in ordinary phospholipids can interfere with the formation of regular membrane structures, highlighting the necessity of using high-purity soybean phosphatidylcholine to ensure the high performance and consistency of the product.

[0133] Comparative Example 3 (without antioxidant) had similar physical encapsulation parameters (encapsulation efficiency, particle size) to the Example, but its accelerated retention rate plummeted to 72.6%. This highlights that the antioxidant network composed of vitamin E acetate and ascorbate palmitate mainly plays a chemical protection role and is crucial for maintaining the chemical stability of the active ingredient during storage.

[0134] Comparative Example 4 (maltodextrin replacing sodium octenyl succinate starch) showed a significantly increased particle size of 485 nm and uneven distribution, with a sharp drop in the absolute value of the Zeta potential, indicating a severe decrease in physical stability. This demonstrates that the synergistic effect of macromolecular steric hindrance and electrostatic stability provided by sodium octenyl succinate starch is the core of maintaining the dispersion stability of nanoparticles and preventing aggregation, and its function cannot be replaced by ordinary sugars.

[0135] Although the particles in Comparative Example 5 (without the induced assembly step) were small, the gastric juice release rate was as high as 58.7%, resulting in the loss of sustained-release function. This confirms that the "induced assembly" process step is the key to driving the formation of multilayer / few-compartment structures with gastric corrosion resistance, rather than a simple emulsification process.

[0136] The performance of Comparative Example 6 (direct physical mixing) was completely ineffective, proving that the entire step-by-step preparation method of this application is the fundamental prerequisite for realizing the nano-encapsulation of active ingredients and obtaining any functional liposome structure.

[0137] To evaluate the performance of the multilayer lutein ester liposomes of this application in the final product application, the following key performance determination methods and data were designed for solid beverage or instant beverage applications. The performance results are shown in Table 2, and the performance determination methods are as follows:

[0138] 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 (in seconds) required for the powder to completely dissolve and the solution to become a homogeneous transparent or homogeneous emulsion.

[0139] 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.

[0140] Centrifugal sedimentation rate: To quantify stability, 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%.

[0141] 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).

[0142] Table 2. Application performance of lutein ester liposomes in Examples 1-3 and Comparative Examples 1-6

[0143]

[0144] 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 (33-38 seconds), maintaining uniformity without precipitation after standing for 24 hours, extremely low centrifugal sedimentation rate (0.5%-0.7%), and outstanding sensory experience (color and taste scores ≥4.6 points, no off-odor). This proves that the multilayer lutein ester liposome powder prepared in this application has ideal instant solubility and colloidal stability.

[0145] Comparative Example 1 (cholesterol-free) dissolved more slowly and produced flocculent precipitate (precipitation rate 3.2%), resulting in a significant decrease in taste score. This indicates that the absence of cholesterol leads to a less dense liposome membrane structure, making the particles more prone to aggregation and sedimentation, and may also result in an increased grainy texture due to incomplete encapsulation.

[0146] Comparative Example 2 (using ordinary soybean phospholipids) showed observable deterioration in all indicators, proving that impurities in ordinary phospholipids can interfere with the interfacial properties of liposomes, affecting their hydration and dispersion efficiency and long-term dispersion stability.

[0147] Although Comparative Example 3 (without antioxidants) had acceptable physicochemical stability, it exhibited a "slight oily taste," which directly demonstrates the decisive role of the antioxidant network composed of vitamin E acetate and ascorbate palmitate in maintaining the pure flavor of the product in its prepared state and preventing off-flavors caused by oil oxidation.

[0148] Comparative Example 4 (maltodextrin replacing sodium octenyl succinate starch) showed severely deteriorated performance, exhibiting difficulties in dissolution, severe stratification and precipitation (precipitation rate 15.5%), and poor taste. This irrefutably demonstrates that the steric stabilization provided by the unique amphiphilic structure of sodium octenyl succinate starch is the most crucial factor ensuring the formation of a stable emulsion and achieving application feasibility.

[0149] Comparative Example 5 (without induced assembly step) showed "slight floating oil droplets", indicating that the absence of this step prevented the formation of a complete multilayer structure that could effectively encapsulate the lipid phase, resulting in some oils not being stably encapsulated and precipitating out.

[0150] Comparative Example 6 (direct physical mixing) was completely insoluble and ineffective, and all indicators failed. This demonstrates from the opposite extreme that the step-by-step preparation method of this application is the only way to transform lutein esters from "hydrophobic powder" into "water-dispersible functional units".

[0151] 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.

[0152] 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 multilayered lutein ester liposome, characterized in that, The raw material composition of the multilayer lutein ester liposome, by weight, is as follows: 30-50 parts lutein ester, 40-60 parts soybean phosphatidylcholine, 5-20 parts cholesterol, 10-25 parts sodium octenyl succinate starch, 20-40 parts trehalose, 15-30 parts mannitol, 4-10 parts phosphatidylglycerol, 1-2 parts vitamin E acetate, and 1-2 parts ascorbyl palmitate.

2. The multilayer lutein ester liposome according to claim 1, characterized in that, The molar ratio of cholesterol to soybean phosphatidylcholine is 0.2:1 to 0.4:

1.

3. The multilayer lutein ester liposome according to claim 1, characterized in that, The total mass ratio of trehalose and mannitol to the total mass ratio of soybean phosphatidylcholine and cholesterol is 0.9:1 to 1.5:

1.

4. The multilayer lutein ester liposome according to claim 1, characterized in that, The mass ratio of vitamin E acetate to ascorbate palmitate is 1:1 to 1:1.

5.

5. The multilayer lutein ester liposome according to claim 1, characterized in that, The structure of the multilayered lutein ester liposome, from the inside out, comprises: The active core is formed by the hydrophobic interaction between lutein esters and cholesterol. The phospholipid bilayer surrounding the core of the active ingredient is composed of soybean phosphatidylcholine and cholesterol, and is doped with phosphatidylglycerol. An octenyl succinate starch sodium macromolecular space-stabilizing layer attached to the outer surface of the phospholipid bilayer; The solid glass matrix that runs through and encapsulates the material is formed by a blend of trehalose and mannitol.

6. The multilayer lutein ester liposome according to claim 5, characterized in that, The structure has a volume-average hydrated particle size of 200–400 nm and a polydispersity index of 0.20–0.

35.

7. A method for preparing multilayered lutein ester liposomes according to any one of claims 1 to 6, characterized in that, The method includes the following steps: S1. Lutein ester, soybean phosphatidylcholine, cholesterol and vitamin E acetate are dissolved in an organic solvent and stirred and preheated at 60-70°C to obtain a structured organic phase; S2. Dissolve water, trehalose, mannitol, ascorbyl palmitate and phosphatidylglycerol by heating to 50-60°C and stirring to obtain a homogeneous aqueous phase; S3. Under high-speed shearing, the structured organic phase is injected into the homogeneous aqueous phase for initial emulsification, followed by the addition of sodium octenyl succinate starch, and the induced assembly reaction is continuously carried out under medium-speed stirring to obtain crude liposome emulsion. S4. Spray dry the crude liposome emulsion to obtain powdered multilayer lutein ester liposomes.

8. The method for preparing multilayer lutein ester liposomes according to claim 7, characterized in that, In step S3, the high-speed shearing speed is 9000-11000 rpm, the medium-speed stirring speed is 1200-1800 rpm, and the duration of the induced assembly reaction is 45-75 min.

9. The method for preparing multilayer lutein ester liposomes according to claim 7, characterized in that, In step S4, the inlet air temperature of the spray dryer is 165-175°C, and the outlet air temperature is 75-85°C.

10. A food product, characterized in that, Multilayer lutein ester liposomes containing any one of claims 1 to 6.

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