A multi-level astaxanthin complex, its preparation method and application

Abstract

This invention discloses a multi-level astaxanthin complex, its preparation method, and its applications, relating to the fields of encapsulation technology or astaxanthin formulations. It includes a first encapsulation component containing astaxanthin and a second encapsulation component containing Haematococcus pluvialis isolated protein; optionally, the multi-level astaxanthin complex further contains cyclodextrin or its derivatives. This invention overcomes the problems of aggregation and uneven encapsulation caused by the direct addition of astaxanthin or astaxanthin solution and the need for vigorous stirring to promote the formation of encapsulated particles in existing astaxanthin complex preparation techniques through local microencapsulation in a foaming microenvironment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of embedding technology or astaxanthin preparation, more particularly to a multi-layer astaxanthin compound and a preparation method and application thereof. BACKGROUND

[0002] Astaxanthin (3,3'-dihydroxy-β-carotene-4,4'-dione, Astaxanthin, ASX for short) is a fat-soluble carotenoid, which belongs to the xanthophyll family and is usually produced by various marine organisms and other biological sources in nature. It is a powerful antioxidant, and its antioxidant activity is much higher than that of β-carotene and vitamin e and other substances, and it can more effectively protect the peroxidation of membrane phospholipids and other lipids, and has good effect in preventing and treating diseases related to active oxygen, so it is widely used in food, health care products, cosmetics and pharmaceutical fields. However, due to its strong lipophilicity, it is not soluble in water; and it is easily decomposed by light, and is also sensitive to heat and oxygen, resulting in poor stability, especially during manufacturing and storage. In addition, its lipophilic and hydrophobic properties result in very low bioavailability, which seriously limits its direct application. In the field of food application, the small intestine is the main site for the absorption of hydrophobic substances, and the gastric acid environment can easily change the carrier, so encapsulating hydrophobic substances, including solid dispersions, polymer nanoparticles, lipid particles and microemulsions, is an optional form. Therefore, in the prior art, astaxanthin is usually combined with stabilizers and other materials by complex technology to improve its stability and solubility, etc.

[0003] Currently, the main methods for preparing astaxanthin microemulsion complexes include vigorous stirring method and anti-solvent method. Generally speaking, vigorous mechanical stirring can disperse astaxanthin oil phase in water phase to form an emulsion. The anti-solvent method is to dissolve astaxanthin and alcohol-soluble embedding material in an organic solvent such as ethanol, and then add water, and astaxanthin forms an embedding emulsion during the same precipitation process. These two methods not only have few embedding levels, poor embedding rate, and uneven embedding.

[0004] However, the prior art for embedding astaxanthin often focuses on the embedding rate, and does not improve the size and uniformity of the astaxanthin embedding particle size, and uneven and large particle size often results in poor embedding effect and serious waste of astaxanthin raw materials during the embedding process, and affects the bioavailability of astaxanthin. For example, CN119280182A discloses a microsphere loaded with astaxanthin and a preparation method thereof, which comprises dissolving zein in an ethanol solution, then adding lecithin and stirring to obtain a zein-lecithin complex; then astaxanthin is added to the zein-lecithin complex and stirred, and then a zein-lecithin-astaxanthin microsphere is prepared. Directly embedding astaxanthin in this way often results in great waste of raw materials and uneven large particle size.

[0005] There are also liposome-embedded astaxanthin in the prior art. Liposomes are bilayer microcapsules similar to biological membranes, with amphiphilic (hydrophilic and hydrophobic) properties, which are usually used to embed fat-soluble substances. Although the use of liposome technology can properly solve the water solubility problem of astaxanthin, and obtain liposomes with relatively uniform particle size, a large amount of organic solvent is required in the preparation process, and the single liposome itself also has poor embedding effect and bioavailability.

[0006] In order to further improve the stability of astaxanthin, the prior art also proposes a variety of complexes including proteins and polysaccharides and their preparation methods. Although the embedding material can improve the water solubility and stability of astaxanthin to some extent, it is basically directly mechanically embedded or prepared by using the anti-solvent principle under the condition of intense stirring, which cannot overcome the disadvantages of poor size and uniformity of astaxanthin embedding material particle size. This is due to the following reasons in the existing process:

[0007] 1) In the existing process, astaxanthin or astaxanthin solution is usually directly added, and intense stirring is required to promote the formation of microspheres or nanoparticles; otherwise, problems such as agglomeration and uneven embedding are easily formed.

[0008] 2) Under the condition of intense stirring, the stirring speed and dropping speed are the key steps, and the speed parameters seriously affect the size and uniformity of the formed particles. Even under the optimized parameters, due to the physical properties of stirring itself, it is difficult to achieve excellent uniformity and small particle size.

[0009] 3) In some anti-solvent operations in the field, low-toxicity organic solvents such as ethanol cannot fully dissolve astaxanthin; and there are limitations that the organic solvent needs to be miscible with the aqueous phase.

[0010] Finally, the organic solvent that can fully dissolve astaxanthin and has good anti-solvent effect with the aqueous phase is often highly toxic, which forms a great limitation in food and health products and other uses. And temperature and pH also affect the solubility and anti-solvent effect, which makes it more difficult to control the particle size of astaxanthin complex. SUMMARY

[0011] In order to solve the problems of the prior art, the purpose of the present application is to provide a multi-level astaxanthin complex, a preparation method and its application. The present application can achieve excellent uniformity and small particle size by treating the micellized dispersed pretreated astaxanthin component in a foaming microenvironment, which overcomes the problems of easy agglomeration and uneven embedding in the conventional direct embedding method.

[0012] In order to achieve the above-mentioned purposes, the present application adopts the following technical solutions.

[0013] In a first aspect, the present invention provides a multi-level astaxanthin complex comprising a first encapsulating component containing astaxanthin and a second encapsulating component containing Haematococcus pluvialis isolated protein; optionally, the multi-level astaxanthin complex further comprises cyclodextrin or a derivative thereof.

[0014] The first encapsulation component contains microencapsulated astaxanthin microspheres, which undergo nano-micelle dispersion pretreatment and phospholipid-mediated microemulsification. Further, the microencapsulated astaxanthin microspheres are obtained via a microbubble emulsification step in a foaming microenvironment; and optionally, the multi-level astaxanthin complex is prepared via an encapsulation step in a foaming microenvironment. The foaming microenvironment is generated by contacting two mixtures with a pH difference of not less than 1 or 2; preferably, the two mixtures have different acidity / alkalinity, and the alkaline mixture contains sodium bicarbonate.

[0015] Furthermore, the astaxanthin complex of the present invention also contains at least one additional component selected from stabilizers and / or inclusion agents. The inclusion agent is preferably a cyclodextrin or a derivative thereof, such as hydroxypropyl-β-cyclodextrin or sodium sulfobutyl-β-cyclodextrin.

[0016] Furthermore, the microencapsulated astaxanthin microspheres of the present invention undergo nano-micelle dispersion pretreatment and phospholipid component-mediated microemulsification treatment, wherein the phospholipid component is preferably derived from amphiphilic phosphatidyl-assisted phospholipid components, such as phosphatidylethanolamine or phosphatidylcholine.

[0017] Specifically, the multi-level astaxanthin complex of the present invention undergoes at least one treatment step in a foaming microenvironment during its preparation, without the need for vigorous steps under high-energy physical conditions (e.g., vigorous stirring or high-speed homogenization). Exemplarily, the microencapsulated astaxanthin microspheres are obtained by a microbubble emulsification treatment step in a foaming microenvironment; and the multi-level astaxanthin complex is prepared by an encapsulation treatment step in a foaming microenvironment of the microencapsulated astaxanthin microspheres.

[0018] The foaming microenvironment is generated by contacting two mixtures with a pH difference of not less than 1 or 2.

[0019] Preferably, the foaming microenvironment is generated by contacting two mixtures with a pH difference of not less than 3.

[0020] More preferably, the two mixtures have different acidity or alkalinity.

[0021] In some embodiments, the multi-level astaxanthin complex of the present invention includes, in addition to a first encapsulating component of astaxanthin, a second encapsulating component comprising Haematococcus pluvialis isolated protein and cyclodextrin-based polymeric encapsulating materials. Preferably, the protein is Haematococcus pluvialis isolated protein with a molecular weight of 10-40 kDa; it also has a synergistic antioxidant effect with astaxanthin. Optionally, other proteins may be used as encapsulating materials.

[0022] The main preparation steps of the multi-level astaxanthin complex of the present invention are as follows: Haematococcus pluvialis isolated protein and optional cyclodextrin derivative (present or absent) are dissolved in sodium bicarbonate solution or its buffer solution at a certain mass ratio, and stirred evenly to obtain an inclusion solution containing Haematococcus pluvialis protein; the first encapsulation component solution containing astaxanthin is added dropwise to the inclusion solution, and encapsulation is carried out under the effervescent effect of the mixing interface; after the encapsulation treatment is completed, the pH is adjusted to acidic using citric acid, and the multi-level astaxanthin complex is obtained.

[0023] In some embodiments, the preparation method is as follows: The Haematococcus pluvialis isolated protein and cyclodextrin derivative are dissolved in sodium bicarbonate solution or its buffer solution (preferably pH 8.0-9.0) at a certain mass ratio, and stirred until homogeneous to obtain a Haematococcus pluvialis protein-cyclodextrin inclusion complex solution. A solution containing the first encapsulation component, astaxanthin, is added dropwise to the inclusion solution composed of Haematococcus pluvialis protein / cyclodextrin derivative-sodium bicarbonate buffer solution, and encapsulation is performed under effervescent effect. After encapsulation, the pH is adjusted to slightly acidic using citric acid to remove salts, thereby preparing a multi-layered astaxanthin complex.

[0024] Secondly, the present invention also provides a method for preparing the astaxanthin complex, comprising the following main steps:

[0025] (1) Astaxanthin was pretreated by nano-micelle dispersion;

[0026] (2) Phospholipid-modified astaxanthin microemulsion was prepared by emulsification treatment of astaxanthin nanomicelle dispersion in a foaming microenvironment;

[0027] (3) Optionally, the astaxanthin microemulsion emulsion is subjected to microencapsulation self-assembly treatment;

[0028] (4) Using inclusion materials including Haematococcus pluvialis protein, the astaxanthin complex was encapsulated in a foaming microenvironment to obtain a multi-level astaxanthin complex.

[0029] Specifically, in this invention, the astaxanthin complex is prepared through the following steps:

[0030] S1: Nanomicelle dispersion pretreatment:

[0031] S1-1) Polyoxyethylene oleic acid glyceride (preferably with a degree of polymerization and a melting point below 50°C) is placed in a glass bottle and heated in a water bath until the polyoxyethylene oleic acid glyceride is completely melted. High-purity natural astaxanthin is added under light-protected conditions and stirred initially to mix well. After returning to room temperature, ethanol is added to fully dissolve the mixture. Volatile alcohols are removed using a rotary evaporator to uniformly disperse the astaxanthin.

[0032] S1-2) is then added to a 0.05-0.5M citric acid solution (as a matrix solution), and the mixture is stirred to hydrate and obtain an acidic micelle solution. The solution is then centrifuged or filtered (e.g., through a 0.22 μm microporous membrane) to remove insoluble particles, resulting in an acidic astaxanthin nanomicelle dispersion. Optionally, the hydration treatment can be carried out under ultrasonic conditions or with appropriate stirring.

[0033] The preferred water bath heating temperature is 50-60℃.

[0034] The preferred mass ratio of polyoxyethylene oleic acid glyceride to astaxanthin is 5-15:1.

[0035] In this step, the present invention avoids the operation of dissolving an appropriate amount of astaxanthin in an auxiliary solvent to form an astaxanthin solution, which is often done in the prior art. This is because astaxanthin is not easily soluble in ethanol and has low solubility, which not only leads to the use of a large amount of organic solution, but also affects the subsequent embedding process due to the low solubility of astaxanthin.

[0036] Citric acid is added not only as a stabilizer and antioxidant component, but also as the main acidic component in the mixture, serving as an acidic effervescent agent for subsequent effervescent treatments or foaming microenvironments.

[0037] S2: Microemulsification treatment in a foaming microenvironment to prepare phospholipid-modified astaxanthin microemulsion:

[0038] S2-1) The amphiphilic phosphatidyl-assisted phospholipid component is dissolved in an organic solvent to form a homogeneous phospholipid solution; the astaxanthin nanomicelle dispersion is mixed evenly with the phospholipid solution to obtain an astaxanthin nanomicelle-phospholipid mixture. The organic solvent is preferably ethanol.

[0039] When mixing the astaxanthin nanomicelle dispersion with the phospholipid solution, the mass ratio of the phospholipid component to the astaxanthin is preferably controlled to be (5-20):1.

[0040] S2-2) Under optional stirring conditions, the astaxanthin nanomicelle-phospholipid mixture is added dropwise to a sodium bicarbonate solution (preferably pH 8.0-8.5). The acidic astaxanthin nanomicelle-phospholipid mixture generates an effervescent effect at the contact interface of the dropwise mixing, thereby constructing a foaming microenvironment, i.e., microbubble emulsification treatment is performed to obtain a phospholipid-treated astaxanthin microemulsion emulsion.

[0041] Optionally, during or after microbubble emulsification, slow stirring or ultrasonic-assisted emulsification can be used, with an ultrasonic power of 50-200 W and a processing time of 0.5-5 min.

[0042] Preferably, the amphiphilic phosphatidyl-associated phospholipid component is selected from phosphatidylethanolamine or phosphatidylcholine.

[0043] S3: Microencapsulation self-assembly process

[0044] The astaxanthin microemulsion obtained by the above microbubble emulsification treatment was transferred to a dialysis bag and dialyzed in a phosphate buffer solution at pH 7.0-7.5 (preferably pH 7.0-7.3) to remove salt impurities and promote the further microencapsulation and self-assembly of astaxanthin micelle molecules in the buffer solution to form a microcapsule structure.

[0045] Optionally, the dialysis time is 12-24 hours, during which the buffer solution is changed 2-5 times.

[0046] After dialysis, the remaining organic solvent (e.g., ethanol) is removed by low-temperature evaporation in a rotary evaporator. Then, citric acid is added to the dialysate to make the solution concentration 0.1-0.5M, and the solution is stirred evenly for acidification. The resulting microencapsulated astaxanthin microsphere dispersion is stored at 4°C for later use.

[0047] S4: Preparation of multi-level astaxanthin complex

[0048] S4-1) Dissolve Haematococcus pluvialis isolated protein in deionized water and dialyze to retain protein components with a molecular weight cutoff of 10-40 kDa to obtain Haematococcus pluvialis isolated protein with a molecular weight of 10-40 kDa.

[0049] The Haematococcus pluvialis isolated protein and cyclodextrin derivative were dissolved in sodium bicarbonate-sodium carbonate buffer (preferably pH 9.0-10.0) at a mass ratio of 2-5:1, and the concentration of Haematococcus pluvialis protein was controlled at 1-20 wt% (preferably 3-10%). The mixture was stirred and hydrated at room temperature or in a water bath at 30-35°C until homogeneous to obtain a Haematococcus pluvialis protein-cyclodextrin inclusion complex solution.

[0050] In some embodiments, the cyclodextrin derivative is selected from HP-β-cyclodextrin.

[0051] For example, the stirring hydration treatment time is 10-30 minutes.

[0052] S4-2) Optionally, under slow stirring (e.g., 50-100 rpm) conditions (with or without stirring), the microencapsulated astaxanthin microsphere dispersion is added dropwise to an inclusion solution composed of Haematococcus pluvialis protein / cyclodextrin derivative-sodium bicarbonate buffer, and the encapsulation is performed under the effervescent effect of the foaming microenvironment; after the encapsulation is completed, the solution is dialyzed to remove salt, and the pH is adjusted to 5.0-7.0 using citric acid to obtain a multi-level astaxanthin complex dispersion; store at 4℃ for later use.

[0053] Optionally, the above-described complex solution can be freeze-dried or spray-dried to obtain solid particles. Freeze-drying is preferred for obtaining solid formulations.

[0054] For example, the freeze-drying conditions are: temperature of -40°C to -50°C, pressure of 10-20 mTorr, and time of 24-48 hours. The spray-drying conditions are: inlet temperature of 100°C to 120°C, outlet temperature of 60°C to 70°C, and flow rate of 10-20 mL / min.

[0055] During the embedding process, slow stirring or ultrasonic-assisted embedding can be used; for example, the ultrasonic power is 50-200 W, and the processing time is 0.5-3 min. Vigorous stirring at medium to high speeds or high-speed homogenization should be avoided.

[0056] This step eliminates the need to remove unencapsulated free astaxanthin. Because the astaxanthin is encapsulated in microencapsulated astaxanthin microspheres, even the very small amount of astaxanthin microspheres not encapsulated by proteins or other macromolecules will not be exposed to oxidation. This avoids the inconvenience of light-protected operation required in existing technologies, as well as the need to remove unencapsulated free astaxanthin (AST) and the resulting waste of raw materials.

[0057] In another aspect, the present invention also provides the microencapsulated astaxanthin microspheres prepared above and their applications or uses, which can be encapsulated by any suitable encapsulating material using conventional techniques to obtain respective astaxanthin complexes.

[0058] This invention presents an astaxanthin complex that combines astaxanthin with different encapsulation components. Utilizing the effervescent effect to assist in microencapsulation and encapsulation, it forms a multilayered complex. This not only overcomes the shortcomings of existing technologies that employ high-energy or violent physical conditions, resulting in insufficient and uneven encapsulation, but also achieves near-complete encapsulation through micro-encapsulation. Furthermore, the addition of beneficial acidic effervescent components endows the complex with additional antioxidant properties and stability. In addition, the use of a specific weakly alkaline buffer solution not only serves as a source of effervescent gas but also helps maintain the protein's native conformation, increasing its stability. It also increases the negative charge on the protein molecule surface, enhancing intermolecular electrostatic repulsion and thus improving the protein's solubility in solution.

[0059] Compared with the prior art, the present invention further includes, but is not limited to, the following beneficial technical effects:

[0060] 1) This invention embeds microencapsulated astaxanthin microspheres in a mixture of Haematococcus pluvialis isolated protein / cyclodextrin derivatives, replacing the direct embedding of astaxanthin in the prior art. Compared with the prior art, the construction of the microenvironment through two foaming processes not only improves the embedding rate of the astaxanthin complex, but also makes the complex particles smaller and more uniform, thereby achieving improved stability and bioavailability. Moreover, it avoids the adverse effects of the vigorous stirring conditions commonly used in the prior art, especially the unstable stirring speed and dropping speed and the uneven local embedding environment, on the size and uniformity of the complex particles.

[0061] Currently, existing technologies for directly encapsulating astaxanthin using pure protein or starch polysaccharide systems exhibit poor bioavailability because these systems lack lipids. Furthermore, formulations obtained through direct astaxanthin encapsulation tend to have larger particle sizes, and the astaxanthin loss rate is high during the encapsulation process. Additionally, while the presence of well-formulated liposomes can improve the bioavailability of the encapsulated lipid-soluble active ingredient astaxanthin, the complexes obtained by encapsulating astaxanthin using liposomes alone exhibit poor stability and are prone to breakage and aggregation.

[0062] 2) The astaxanthin encapsulation complex prepared by this invention achieves an encapsulation rate of over 99.5%, essentially approaching complete encapsulation. Furthermore, the astaxanthin complex, composed of inner and outer encapsulation components, exhibits stable physicochemical properties, with small and uniform particle size within the encapsulated components. This improves the chemical stability and bioavailability of astaxanthin and effectively masks its fishy odor.

[0063] 3) In the preparation process of the astaxanthin microsphere-Haematococcus pluvialis isolated protein / cyclodextrin derivative complex of the present invention, the difference in pH value of different mixture components has an important influence on the morphology and properties of the complex. The difference in pH value constructs the foaming microenvironment in the process of astaxanthin microsphere and final complex formation. This technology, which replaces conventional stirring or antisolvent methods to assist in astaxanthin encapsulation, has not been recorded in the prior art.

[0064] 4) The Haematococcus pluvialis isolated protein used in this invention can be obtained using conventional methods in the art or commercially available. Haematococcus pluvialis isolated protein contains various types of proteins with different molecular weights, typically ranging from tens of thousands to hundreds of thousands of Daltons. The highly stable protein-based embedding material of this invention, by introducing a small amount of cyclodextrin derivative (e.g., 5-50 wt% relative to the protein content), improves the conformational stability and adsorption of the protein. Furthermore, the foaming treatment in the foaming microenvironment overcomes the drawbacks of "high-intensity over-treatment" caused by vigorous stirring and other physical treatments, allowing the protein / cyclodextrin embedding material and the liposome-shaped astaxanthin microspheres to fully contact at the interface of the foaming microenvironment. Small and uniform average particle size complex particles can be prepared under no external energy input or low energy input (e.g., short-time microwave or slow stirring), and the presence of the localized foaming microenvironment significantly inhibits the aggregation tendency caused by vigorous physical conditions (such as rapid stirring), thereby suppressing agglomeration. At the same time, it avoids the drawbacks that often exist in existing technologies, such as the easy destruction of protein conformation during high-energy emulsification.

[0065] 5) From a microstructural perspective: Haematococcus pluvialis protein has a rough and porous surface, which is beneficial for encapsulation. With increasing pH, the solubility, emulsifying properties, foaming properties, and foam stability of Haematococcus pluvialis protein show a trend of first increasing and then decreasing, while the emulsifying stability shows the opposite trend, which is beneficial for the construction of the foaming microenvironment and promotes microencapsulation in this invention.

[0066] 6) In the preparation process of the complex of this invention, astaxanthin is first encapsulated in microcapsules using phospholipid molecules, forming a phospholipid coating layer, which improves its solubility and stability in the aqueous phase in subsequent processes. The multi-layer protective structure and the use of stabilizers in this invention give the complex higher stability and effectively extend the shelf life of astaxanthin. It also improves the water solubility and bioavailability of astaxanthin. Furthermore, the addition of appropriate stabilizers (such as citric acid) in this invention not only serves as a foaming component in the foaming microenvironment but also helps to inhibit the oxidative degradation of astaxanthin.

[0067] 7) Enhanced antioxidant effect: After storage at 4°C for 30 days, the astaxanthin complex prepared in Example 1 of this invention exhibited a astaxanthin retention rate as high as 95.4±0.6%, indicating its high stability under low-temperature storage conditions. This demonstrates that the multi-level composite system of this invention can effectively protect astaxanthin and prevent its degradation under the influence of external environmental factors. Attached Figure Description

[0068] Figure 1 This is an external structural morphology diagram of the astaxanthin complex prepared in Example 1 of the present invention. Detailed Implementation

[0069] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] The following detailed description of preferred embodiments of the invention, along with the included examples, will make the invention more readily apparent. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of any discrepancy, the definitions in this specification shall prevail.

[0071] Example 1

[0072] 1) Preparation of astaxanthin nanomicelle dispersion

[0073] 11g of polyoxyethylene oleic acid glyceride was placed in a glass bottle and heated in a 55°C water bath until the polyoxyethylene oleic acid glyceride was completely melted. Then, 1g of high-purity natural astaxanthin was added under light-protected conditions and stirred to mix thoroughly. After cooling to room temperature, anhydrous ethanol was added to fully dissolve the mixture, and volatile alcohols were removed using a rotary evaporator to uniformly disperse the astaxanthin. Then, 80mL of 0.1M citric acid solution (instead of deionized water as the aqueous phase) was added, and the mixture was hydrated under 100W ultrasonic conditions or with appropriate stirring to obtain a micelle solution. The micelle solution was filtered through a 0.22μm microporous membrane to obtain an astaxanthin nanomicelle dispersion.

[0074] 2) Preparation of phospholipid-modified astaxanthin microemulsion

[0075] Six g of the auxiliary phospholipid component, dimyristoylphosphatidylethanolamine, was dissolved in ethanol to form a homogeneous phospholipid solution. The astaxanthin nanomicelle dispersion was then mixed uniformly with the phospholipid solution to obtain an astaxanthin nanomicelle-phospholipid mixture. Under slow shaking, the astaxanthin nanomicelle-phospholipid mixture was added dropwise to a sodium bicarbonate solution (pH 8.0-8.2). The dropping rate was controlled to induce an effervescent effect at the interface of the mixture, ensuring thorough microbubble emulsification and yielding a phospholipid-treated astaxanthin microemulsion. The amount of sodium bicarbonate solution added was controlled to ensure the final emulsion was weakly alkaline (approximately pH 7.5-8.0). Slow stirring was used to assist emulsification after the emulsification process was completed.

[0076] 3) Microencapsulated self-assembly

[0077] The astaxanthin emulsion obtained from the above emulsification process was transferred to a dialysis bag and dialyzed in phosphate buffered saline (PBS) solution at pH 7.2-7.3 to remove salt. In the buffer solution, the astaxanthin micelle molecules were further microencapsulated and self-assembled to form microencapsulated structures. The dialysis time was 16 hours, with the buffer solution changed twice during this period. After dialysis, the remaining organic solvent was removed by low-temperature evaporation in a rotary evaporator. Citric acid was added to a molar concentration of 0.12 M, and the mixture was stirred thoroughly for acidification. This yielded a microencapsulated astaxanthin microsphere dispersion, which was stored at 4°C for later use.

[0078] 4) Preparation of multi-level astaxanthin complex

[0079] Haematococcus pluvialis isolated protein was dissolved in deionized water, and the protein fraction with a cutoff of 10-40 kDa was retained by dialyzing to obtain Haematococcus pluvialis isolated protein. The obtained Haematococcus pluvialis isolated protein and HP-β-cyclodextrin were dissolved in sodium bicarbonate-sodium carbonate buffer (pH 9.5) at a mass ratio of 3:1, controlling the Haematococcus pluvialis protein concentration at 4.5 wt%. The mixture was stirred and hydrated at 35°C for 15 min until homogeneous, yielding an inclusion complex solution. Under slow stirring, 20 mL of the acidified microencapsulated astaxanthin microsphere dispersion was added dropwise to 100 mL of the above inclusion complex solution composed of Haematococcus pluvialis protein / cyclodextrin derivative-sodium bicarbonate buffer, allowing for self-encapsulation under the effervescent effect of the foaming microenvironment. After encapsulation, the mixture was dialyzed to remove salt, and the pH was adjusted to 6.0 using citric acid to obtain a multi-layered astaxanthin complex dispersion. The morphology of the obtained astaxanthin complex particles is shown in [reference needed]. Figure 1 Its particles are relatively uniform, with a particle size distribution between 0.10 and 0.11 micrometers.

[0080] Alternatively, the above-mentioned complex solution can be freeze-dried to obtain solid particles.

[0081] Comparative Example 1

[0082] Steps 1)-3) are the same as in Example 1. The difference is that in step 4), deionized water is used instead of sodium bicarbonate buffer solution, and stirring is performed. The specific operation is as follows:

[0083] Haematococcus pluvialis isolated protein and HP-β-cyclodextrin were dissolved in deionized water at a mass ratio of 3:1, controlling the Haematococcus pluvialis protein concentration at 4.5 wt%. The mixture was stirred and hydrated at 35°C for 15 min until homogeneous, yielding an inclusion complex solution. Under slow stirring, 20 mL of the acidified microencapsulated astaxanthin microsphere dispersion was added dropwise to 100 mL of the above Haematococcus pluvialis protein / cyclodextrin derivative-deionized water inclusion complex solution, followed by rapid stirring at 200 rpm for encapsulation; this resulted in a multi-layered astaxanthin complex dispersion. The resulting astaxanthin complex had a particle size distribution between 140-160 nm and an encapsulation efficiency of approximately 97%.

[0084] Comparative Example 2

[0085] The method was implemented according to Comparative Example 1, except that deionized water was used instead of sodium bicarbonate solution in step 2), meaning that no micro-encapsulation operation under a local effervescent environment was performed in any of the steps. The resulting astaxanthin complex particles had a size distribution between 155-175 nm and an encapsulation rate of approximately 92%.

[0086] Comparative Example 3

[0087] 1) Weigh 1g of astaxanthin and dissolve it in 80ml of anhydrous ethanol. Stir well at room temperature in the dark to obtain an astaxanthin ethanol solution. Dissolve Haematococcus pluvialis isolated protein and corn starch in deionized water at a mass ratio of 3:1, control the Haematococcus pluvialis protein concentration to 4.5wt%, and hydrate by stirring at 50℃ for 15min until homogeneous to obtain an inclusion complex solution.

[0088] 2) Under rapid stirring at 300 rpm, 20 mL of astaxanthin ethanol solution was added dropwise to 100 mL of the above inclusion complex solution. The mixture was stirred continuously in a 55°C water bath for 3 hours for encapsulation. Then, the ethanol was removed by vacuum rotary evaporation at 25°C, and the unencapsulated free astaxanthin was removed by centrifugation at 3000 rpm for 15 minutes, yielding an astaxanthin complex dispersion. The average particle size of the obtained astaxanthin complex exceeded 0.23 micrometers. After storage at 4°C under sterile conditions for 30 days, the astaxanthin retention rate was only about 78%, indicating poor stability under low-temperature storage conditions.

[0089] The results for each experimental group are shown in Table 1 below. The encapsulation efficiency was determined by conventional UV detection methods in this field, the particle size was determined by a Zetasizer nanoparticle size analyzer, the morphology was observed by electron microscopy, and the stability was determined by UV spectrophotometry (characterized by the retention rate of the complex after storage at 4°C for 30 days).

[0090] Table 1. Particle size distribution and retention rate of products from each experimental group.

[0091] Class Average particle size (pm) Retention rate (30 days) Embedding rate Example 1 0.11 95.4% 99.7% Comparative Example 1 0.15 87.7% 95.2% Comparative Example 2 0.17 85.8% 94.4% Comparative Example 3 0.25 78.4% 81.3%

[0092] The table above shows that, compared to Example 1, under the same operating conditions, Comparative Examples 1-2, which lacked the micro-embedding treatment step in a foaming microenvironment, exhibited significantly lower encapsulation rates and retention rates of their composites. Meanwhile, Comparative Example 3, serving as a blank control, employed a conventional direct encapsulation method, and its encapsulation rate and retention rate were significantly lower than those of the Examples of the Present Invention and Comparative Examples 1-2.

[0093] Although the present invention has been described in detail by way of reference to embodiments, it is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a multilayered astaxanthin complex, characterized in that, The preparation method comprises the following specific steps: S1: nanomicellization dispersion pretreatment: Put polyoxyethylene glyceryl oleate in a glass bottle, heat in a water bath until the polyoxyethylene glyceryl oleate is completely melted, then add high-purity natural astaxanthin under light-proof conditions, stir and mix uniformly, restore room temperature, add ethanol to fully dissolve the mixture, and remove volatile alcohol by using a rotary evaporator to uniformly disperse astaxanthin; Then, add a 0.05-0.5M citric acid solution, stir and hydrate to obtain an acidic micellar solution, filter the micellar solution to remove insoluble particles, and obtain an acidic astaxanthin nanomicellar dispersion; The mass ratio of polyoxyethylene glyceryl oleate to astaxanthin is 5-15:1; S2: microemulsion treatment in a foaming microenvironment: Dissolve the amphiphilic phosphatidyl auxiliary phospholipid component in an organic solvent to form a uniform phospholipid solution; mix the astaxanthin nanomicellar dispersion and the phospholipid solution uniformly to obtain an astaxanthin nanomicellar-phospholipid mixture; under optional stirring conditions, add the astaxanthin nanomicellar-phospholipid mixture dropwise into a sodium bicarbonate solution, and perform microbubble emulsification treatment on the acidic astaxanthin nanomicellar-phospholipid mixture at the contact interface of the dropwise mixing to obtain a phosphatidylated astaxanthin microemulsion emulsion; The organic solvent is ethanol; When the astaxanthin nanomicellar dispersion and the phospholipid solution are mixed, the mass ratio of phospholipid to astaxanthin is controlled to be (5-20):1; S3: microencapsulated self-assembly treatment: Transfer the astaxanthin microemulsion emulsion obtained by the above microbubble emulsification treatment to a dialysis bag, dialyze to remove salt in a phosphate buffer, and further microencapsulate and self-assemble in the buffer; after dialysis is completed, remove the remaining organic solvent by low-temperature evaporation in a rotary evaporator, add citric acid to the dialysate to make the solution concentration 0.1-0.5M, and stir uniformly to perform acidification treatment; thus, a microencapsulated astaxanthin microsphere dispersion is obtained; S4: preparation of a multilevel astaxanthin complex 1) Dissolve the Haematococcus pluvialis protein in deionized water, and dialyze the Haematococcus pluvialis protein with a molecular weight cut-off of 10-40kDa; dissolve the Haematococcus pluvialis protein and the cyclodextrin derivative in a sodium bicarbonate-sodium carbonate buffer at a mass ratio of 2-5:1, control the Haematococcus pluvialis protein concentration to be 1-20wt%, and stir and hydrate until uniform to obtain a Haematococcus pluvialis protein-cyclodextrin inclusion complex solution; 2) Under slow stirring conditions, add the microencapsulated astaxanthin microsphere dispersion to 2-10 times the volume of the Haematococcus pluvialis protein / cyclodextrin derivative-sodium bicarbonate buffer to form a complexing solution, and perform embedding under the foaming microenvironment of the effervescence effect; after the embedding treatment is completed, dialyze to remove salt, and adjust the pH to 5.0-7.0 using citric acid to obtain a multilevel astaxanthin complex. The slow stirring conditions are 50-100rpm.

2. The astaxanthin complex obtained by the preparation method of claim 1.

3. Use of the astaxanthin complex of claim 2 in the field of cosmetics.

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