Multi-level astaxanthin compound as well as preparation method and application thereof

Abstract

The invention discloses a multi-level astaxanthin compound as well as a preparation method and application thereof, and relates to the field of embedding technologies or astaxanthin preparations. Comprising a first embedding component containing astaxanthin and a second embedding component containing haematococcus pluvialis isolated protein, optionally, the multi-level astaxanthin compound also contains cyclodextrin or a derivative thereof. Through the local micro-embedding operation of the foaming microenvironment, the problems of agglomeration phenomenon, non-uniform embedding and the like caused by the fact that astaxanthin or an astaxanthin solution is usually directly added when the astaxanthin compound is prepared in the prior art and violent stirring is needed to promote the formation of embedded particles are solved.

Description

Technical Field

[0001] The present invention relates to the field of embedding technology or astaxanthin preparations, and more particularly to a multi-level astaxanthin complex, its preparation method and application. Background Art

[0002] Astaxanthin (3,3'-dihydroxy-β-carotene-4,4'-dione, Astaxanthin, abbreviated as ASX) is a fat-soluble carotenoid belonging to the lutein class, which is usually produced by various biological sources such as various marine organisms in nature. It is a powerful antioxidant, and its antioxidant activity is much higher than that of substances such as β-carotene and vitamin E. It can more effectively protect the peroxidation of lipids such as membrane phospholipids, and also has good effects in preventing and treating diseases related to reactive oxygen species. Therefore, it is widely used in the fields of food, health products, cosmetics and medicine. However, due to its strong fat solubility, it is insoluble in water; it is easily decomposed by light, and is also sensitive to heat and oxygen, resulting in poor stability, especially unstable during manufacturing and storage, and its lipophilic and hydrophobic properties lead to very low bioavailability, severely limiting its direct application. In the field of food applications, the small intestine is the main site for the absorption of hydrophobic substances, and the gastric acid environment easily changes the carrier. Therefore, encapsulating hydrophobic substances, including solid dispersions, polymer nanoparticles, lipid particles and microemulsions, is an optional form. Therefore, in the prior art, composite technology is usually used to combine astaxanthin with materials such as stabilizers to improve its stability and solubility, etc.

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

[0004] However, the prior art often focuses on the embedding rate when embedding astaxanthin, and does not improve the size and uniformity of the astaxanthin embedding particle size enough. Uneven and large particle sizes often result in poor embedding effects and serious waste of astaxanthin raw materials during the embedding process, and also affect the bioavailability of astaxanthin. For example, CN119280182A discloses microspheres loaded with astaxanthin and its preparation method, including dissolving zein in an ethanol solution, then adding lecithin and stirring to obtain a zein-lecithin complex; then adding astaxanthin to the zein-lecithin complex and stirring, and then preparing zein-lecithin-astaxanthin microspheres. Directly embedding astaxanthin in this way often brings great waste of raw materials and uneven large particle sizes.

[0005] In the prior art, there is also astaxanthin encapsulated in liposomes. Liposomes are bilayer microcapsules similar to biological membranes, with amphiphilic properties (hydrophilic and hydrophobic), and are commonly used to encapsulate lipophilic substances. Although the liposome technology can appropriately solve the water solubility problem of astaxanthin and obtain liposomes with relatively uniform particle sizes, a large amount of organic solvents are required in the preparation process, and the single liposome itself also has poor encapsulation effect and bioavailability.

[0006] In order to further improve the stability of astaxanthin, the prior art has also proposed various complexes including proteins and polysaccharides and their preparation methods. Although the encapsulates can improve the water solubility and stability of astaxanthin to a certain extent, basically they are directly mechanically encapsulated under vigorous stirring conditions or prepared by using the anti-solvent principle, and it is impossible to overcome the drawbacks of poor particle size and uniformity of astaxanthin encapsulates. This is caused by the following reasons in the existing processes:

[0007] 1) In the existing processes, astaxanthin or astaxanthin solution is usually directly added, and vigorous stirring is required to promote the formation of microspheres or nanoparticles; otherwise, problems such as agglomeration and uneven encapsulation are likely to occur.

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

[0009] 3) In some anti-solvent operations in this field, low-toxic organic solvents such as ethanol cannot fully dissolve astaxanthin; and there are limiting conditions that the organic solvent needs to have poor miscibility with the aqueous solution.

[0010] Finally, organic solvents that can fully dissolve astaxanthin and have good anti-solvent effects with the aqueous phase often have strong toxicity, which poses great limitations in applications such as food and health products. Moreover, temperature and pH also affect solubility and anti-solvent effects, which makes the control of the particle size of astaxanthin complexes more difficult. Summary of the Invention

[0011] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a multi-level astaxanthin complex, a preparation method and its application. By treating the micellized and dispersed astaxanthin components in a foaming microenvironment, the present invention can achieve excellent uniformity and small particle sizes, and overcome problems such as agglomeration and uneven encapsulation easily formed by the conventional direct encapsulation method.

[0012] In order to achieve the above purpose, the present invention adopts the following technical solutions.

[0013] In a first aspect, the present invention provides a multi - level astaxanthin complex, which includes a first embedded component containing astaxanthin and a second embedded component containing Haematococcus pluvialis isolated protein; optionally, the multi - level astaxanthin complex further contains cyclodextrin or its derivatives.

[0014] Among them, the first embedded component contains microencapsulated astaxanthin microspheres, and the microencapsulated astaxanthin microspheres have been pretreated by nano - micellization dispersion and micro - emulsification treatment mediated by a phospholipid component. Further, the microencapsulated astaxanthin microspheres are obtained through a micro - bubble emulsification treatment step in a foaming micro - environment; and optionally, the multi - level astaxanthin complex is prepared through an embedding treatment step in a foaming micro - environment. Among them, the foaming micro - environment is generated by the contact of two mixtures with a pH difference of not less than 1 or 2; preferably, the acid - base properties of the two mixtures are different, and the alkaline mixture contains a sodium bicarbonate component.

[0015] Further, the astaxanthin complex of the present invention further contains at least one additional component selected from stabilizers and / or encapsulating agents. Among them, the encapsulating agent is preferably a cyclodextrin - type or its derivative, such as hydroxypropyl - β - cyclodextrin or sodium sulfobutyl - β - cyclodextrin.

[0016] Further, the microencapsulated astaxanthin microsphere component of the present invention has been pretreated by nano - micellization dispersion and micro - emulsification treatment mediated by a phospholipid component, among which the phospholipid component is preferably selected from amphiphilic phosphatidyl - type auxiliary phospholipid components, such as phosphatidylethanolamine or phosphatidylcholine - type.

[0017] Specifically, during the preparation of the multi - level astaxanthin complex of the present invention, it undergoes at least one treatment step in a foaming micro - environment without the participation of drastic steps under high - energy physical conditions (such as vigorous stirring or high - speed homogenization). Exemplarily, the microencapsulated astaxanthin microspheres are obtained from a micro - bubble emulsification treatment step in a foaming micro - environment; and the microencapsulated astaxanthin microspheres are prepared into a multi - level astaxanthin complex through an embedding treatment step in a foaming micro - environment.

[0018] Among them, the foaming micro - environment is generated by the contact of two mixtures with a pH difference of not less than 1 or 2.

[0019] Preferably, the foaming micro - environment is generated by the contact of two mixtures with a pH difference of not less than 3.

[0020] More preferably, the acid - base properties of the two mixtures are different.

[0021] In some embodiments, the multi - level astaxanthin complex of the present invention, in addition to containing the first embedded component of astaxanthin, further includes a second embedded component including Haematococcus pluvialis isolated protein and a cyclodextrin - based polymer embedding material. Among them, the protein is preferably Haematococcus pluvialis isolated protein with a molecular weight of 10 - 40 kDa; it has a synergistic effect with the antioxidant property of astaxanthin. Optionally, other proteins can also be used as the embedding material.

[0022] The main preparation steps of the multi - level astaxanthin complex of the present invention are as follows: Dissolve Haematococcus pluvialis isolated protein and an optional cyclodextrin derivative (present or absent) in a sodium bicarbonate solution or its buffer solution according to a certain mass ratio, and stir evenly to obtain an inclusion solution containing Haematococcus pluvialis protein; Drop the solution of the first embedded component containing astaxanthin into the inclusion solution, and perform embedding under the effervescent effect at the mixing interface; After the embedding treatment, adjust the pH to acidic with citric acid, and the multi - level astaxanthin complex is obtained.

[0023] In some embodiments, the following preparation method is adopted: Dissolve the above - mentioned Haematococcus pluvialis isolated protein and cyclodextrin derivative in a sodium bicarbonate solution or its buffer solution (preferably pH 8.0 - 9.0) according to a certain mass ratio, and stir and hydrate until uniform to obtain a Haematococcus pluvialis protein - cyclodextrin - based inclusion complex solution. Drop the solution of the first embedded component containing astaxanthin into the inclusion solution composed of the Haematococcus pluvialis protein / cyclodextrin derivative - sodium bicarbonate buffer solution, and perform embedding treatment under the effervescent effect; After the embedding treatment, adjust the pH to slightly acidic with citric acid and desalt, thereby preparing the multi - level astaxanthin complex.

[0024] In the second aspect, the present invention also provides a preparation method of the astaxanthin complex, including the following main steps:

[0025] (1) Perform nano - micellization dispersion pretreatment on astaxanthin;

[0026] (2) Based on the astaxanthin nano - micelle dispersion liquid, perform emulsification treatment in a foaming micro - environment to prepare a phosphatized astaxanthin microemulsion;

[0027] (3) Optionally, perform microencapsulation self - assembly treatment on the astaxanthin microemulsion;

[0028] (4) Use an inclusion material including Haematococcus pluvialis protein to perform embedding treatment in a foaming micro - environment; obtain a multi - level astaxanthin complex.

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

[0030] S1: Nano - micellization dispersion pretreatment:

[0031] S1-1) Place glyceryl polyoxyethylene oleate (preferably with a degree of polymerization having a melting point below 50 °C) in a glass bottle. After heating in a water bath until the glyceryl polyoxyethylene oleate is completely melted, add high-purity natural astaxanthin and stir preliminarily under light-shielded conditions. After restoring to room temperature, add ethanol to fully dissolve the mixture, and use a rotary evaporator to remove volatile alcohols to uniformly disperse astaxanthin;

[0032] S1-2) Then add it to a 0.05 - 0.5 M citric acid solution (as a matrix solution), stir for hydration treatment to obtain an acidic micellar solution, and centrifuge or filter the micellar solution (for example, through a 0.22 μm microporous membrane) to remove insoluble particles, obtaining an acidic astaxanthin nano-micelle dispersion. Optionally, the hydration treatment can be carried out under ultrasonic conditions or appropriate stirring conditions.

[0033] Among them, the water bath heating temperature is preferably 50 - 60 °C.

[0034] Among them, the mass ratio of glyceryl polyoxyethylene oleate to astaxanthin is preferably 5 - 15:1.

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

[0036] Among them, adding citric acid not only serves as a stabilizer and antioxidant component, but also is the main acidic component in the mixture, and is used as an acidic effervescent agent for the subsequent effervescent treatment or the effervescent microenvironment of the foaming microenvironment.

[0037] S2: Microemulsification treatment under a foaming microenvironment to prepare phosphatidylated astaxanthin microemulsion:

[0038] S2-1) Dissolve an amphiphilic phosphatidyl auxiliary phospholipid component in an organic solvent to form a uniform phospholipid solution; mix the astaxanthin nano-micelle dispersion and the phospholipid solution evenly to obtain an astaxanthin nano-micelle - phospholipid mixture. Among them, the organic solvent is preferably ethanol.

[0039] Among them, when mixing the astaxanthin nano-micelle dispersion and the phospholipid solution, it is preferably controlled that the mass ratio of the phospholipid component to astaxanthin is (5 - 20):1.

[0040] S2-2) Under optionally stirring conditions, drop the astaxanthin nano-micelle - phospholipid mixture into a sodium bicarbonate solution (preferably pH 8.0 - 8.5). The acidic astaxanthin nano-micelle - phospholipid mixture generates an effervescent effect at the contact interface of the dropping mixture to construct a foaming microenvironment, that is, carry out microbubble emulsification treatment to obtain a phosphatidylated astaxanthin microemulsion emulsion.

[0041] Optionally, during or after the microbubble emulsification process, slow stirring or ultrasonic-assisted emulsification can be employed. The ultrasonic power is 50 - 200 W, and the treatment time is 0.5 - 5 min.

[0042] Among them, preferably, the amphiphilic phosphatidyl auxiliary phospholipid component is selected from phosphatidylethanolamine or phosphatidylcholine.

[0043] S3: Microencapsulation self-assembly treatment

[0044] Transfer the astaxanthin microemulsion obtained from the above microbubble emulsification treatment to a dialysis bag, and place it in a phosphate buffer solution with a pH of 7.0 - 7.5 (preferably pH 7.0 - 7.3) for dialysis treatment to remove salt impurities, and promote further microencapsulation self-assembly of astaxanthin micelle molecules in the buffer solution to form a microcapsule structure.

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

[0046] After dialysis is completed, evaporate the remaining organic solvent (such as ethanol) at low temperature in a rotary evaporator, and then add citric acid to the dialysis solution again to make its solution concentration 0.1 - 0.5 M, and stir evenly for acidification treatment; namely, obtain a microencapsulated astaxanthin microsphere dispersion, and store it at 4 °C for use.

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

[0048] S4-1) Dissolve Haematococcus pluvialis isolate protein in deionized water, and dialyze the protein component with a cut-off molecular weight of 10 - 40 kDa to obtain Haematococcus pluvialis isolate protein with a molecular weight of 10 - 40 kDa.

[0049] Dissolve the above-mentioned Haematococcus pluvialis isolate protein and cyclodextrin derivative in a sodium bicarbonate - sodium carbonate buffer solution (preferably pH 9.0 - 10.0) at a mass ratio of 2 - 5:1, control the concentration of Haematococcus pluvialis protein to be 1 - 20 wt% (preferably 3 - 10%), and stir and hydrate at room temperature or a water bath temperature of 30 - 35 °C until uniform to obtain a Haematococcus pluvialis protein - cyclodextrin inclusion complex solution.

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

[0051] Among them, exemplarily, the stirring and hydration treatment time is 10 - 30 min.

[0052] S4-2) Optionally, with or without stirring under slow stirring conditions (e.g., 50 - 100 rpm), add the microencapsulated astaxanthin microsphere dispersion dropwise to an inclusion solution composed of 2 - 10 times the volume of Haematococcus pluvialis protein / cyclodextrin derivative - sodium bicarbonate buffer, and perform the embedding treatment under the effervescent effect of the foaming microenvironment; after the embedding treatment, dialyze to remove salts, and adjust the pH to 5.0 - 7.0 with citric acid to obtain a multi - level astaxanthin complex dispersion; store it at 4°C for later use.

[0053] Optionally, the above complex solution can be lyophilized or spray - dried to obtain solid particles. Preferably, lyophilization is used to obtain solid preparations.

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

[0055] During the embedding treatment, slow stirring or ultrasonic assistance can be used for the embedding treatment; exemplarily, the ultrasonic power is 50 - 200 W, and the treatment time is 0.5 - 3 min. Avoid violent stirring at medium - high speeds or high - speed homogenization.

[0056] In this step, there is no need to remove un - embedded free astaxanthin; because astaxanthin has been encapsulated by microencapsulated astaxanthin microspheres, and very little of the astaxanthin microsphere components that are not embedded by macromolecules such as proteins will not be exposed and oxidized. Thus, it avoids the inconvenience of the need for light - avoidance operation in the prior art for this step, as well as the operation of removing un - embedded free astaxanthin (AST) and raw material waste.

[0057] In the third aspect, the present invention also provides the astaxanthin complex prepared above, as well as its applications or uses in the fields of food, cosmetics, pharmaceuticals, or health products.

[0058] In the food field, encapsulated astaxanthin can improve water - solubility and stability, making it easier to be absorbed by the human body, and thus better exert its health - care effects, such as enhancing immunity and protecting the eyes, etc.

[0059] In addition, it can be used as an auxiliary additive component in food, which can inhibit the oxidation of oils and fats in food, and improve the flavor and additional nutritional value of food.

[0060] The astaxanthin complex of the present invention can also be used in health products or pharmaceuticals, for example, for improving diseases such as eye diseases and cardiovascular diseases, thereby improving the stability of the drug component astaxanthin.

[0061] In another aspect, the present invention also provides the microencapsulated astaxanthin microspheres prepared as described above and their applications or uses, which can be encapsulated by any suitable embedding material using conventional techniques to obtain respective astaxanthin complexes.

[0062] The astaxanthin complex of the present invention combines astaxanthin with different embedding components, and adopts the effervescent effect to assist the microencapsulation and embedding processes to form a multi-layer complex morphology. It not only overcomes the disadvantages of insufficient and uneven embedding caused by high-energy or drastic physical conditions in the prior art, and can basically achieve complete embedding by means of microencapsulation operation; but also makes the complex have additional antioxidant properties and stability through the addition of beneficial acidic effervescent components. In addition, the use of a specific weak alkaline buffer not only serves as a source of effervescent gas components, but also helps to maintain the native conformation of proteins, increase their stability, and make the protein molecules carry more negative charges on the surface, increasing the electrostatic repulsion between molecules, thereby enhancing the solubility of proteins in solution.

[0063] Compared with the prior art, the present invention further includes but is not limited to the following beneficial technical effects:

[0064] 1) The present invention embeds the microencapsulated astaxanthin microspheres in a mixture of Haematococcus pluvialis isolated protein / cyclodextrin derivative, instead of directly embedding astaxanthin in the prior art; compared with the prior art, through the construction of a two-step foaming microenvironment, not only the embedding rate of the astaxanthin complex is improved, but also the particle size of the complex is smaller and more uniform, thereby obtaining improved stability and bioavailability; and it avoids the adverse effects of the drastic stirring operation conditions commonly used in the prior art, especially the instability of the stirring speed and dropping speed and the uneven local embedding environment on the size and uniformity of the complex particles.

[0065] At present, the prior art uses pure protein or polysaccharide systems such as starch to directly embed astaxanthin, which has poor bioavailability because pure protein or polysaccharide systems do not contain lipids. Moreover, the particle size of the preparation obtained by directly embedding astaxanthin is large, and the loss rate of astaxanthin is high during the embedding process. In addition, although the presence of excellent morphological liposomes can improve the bioavailability of the embedded lipophilic active substance astaxanthin. However, the complex obtained by simply embedding astaxanthin in liposomes has poor stability and is prone to rupture and aggregation.

[0066] 2) The embedding rate of the astaxanthin embedding complex prepared by the present invention can basically reach more than 99.5%, which is basically close to complete embedding, and the physicochemical properties of the astaxanthin complex composed of internal and external embedding components are stable, and the internal embedding particle size is small and uniform; it improves the chemical stability and bioavailability of astaxanthin. It can well cover up the fishy smell of astaxanthin.

[0067] 3) During the preparation of the astaxanthin microsphere-Haematococcus pluvialis isolated protein / cyclodextrin derivative complex of the present invention, the difference in the pH value of different mixture components has an important impact on the morphology formation and properties of the complex. The difference in pH value constructs a foaming microenvironment during the formation of astaxanthin microspheres and the final complex. This technology that assists in the encapsulation of astaxanthin by replacing the conventional stirring or anti-solvent method has not been recorded in the prior art.

[0068] 4) The Haematococcus pluvialis isolated protein used in the present invention can be obtained by conventional means in the art or purchased commercially. The Haematococcus pluvialis isolated protein contains various different types of proteins with different molecular weights, usually ranging from tens of thousands to hundreds of thousands of daltons. The highly stable protein-based encapsulant of the present invention improves the conformational stability and adsorption of the protein by introducing a small amount of cyclodextrin derivative (for example, 5-50 wt% relative to the protein amount). Additionally, under the foaming treatment of the foaming microenvironment, it overcomes the drawbacks brought by "high-intensity over-treatment" such as intense stirring. This enables the protein / cyclodextrin encapsulating material and the astaxanthin microspheres in the form of liposomes to come into full contact at the interface of the foaming microenvironment. Complex particles with small and uniform average particle sizes can be prepared with no external energy input or low energy input (such as short-time microwave or slow stirring). The existence of the local foaming microenvironment can significantly inhibit the aggregation tendency brought by intense physical conditions (such as rapid stirring), thereby suppressing the agglomeration phenomenon. At the same time, it also avoids the drawbacks that often exist in the prior art, namely, the protein conformation is easily damaged during the high-energy emulsification process.

[0069] In addition, the Haematococcus pluvialis protein is also a natural antioxidant, forming an antioxidant synergistic effect with astaxanthin and enhancing the antioxidant ability. The synergistic effect of the Haematococcus pluvialis protein and other antioxidant synergists with astaxanthin enhances the antioxidant effect of the complex. It can be widely applied in the fields of food, health products, cosmetics, and medicine.

[0070] 5) From a microscopic structure perspective: The surface of the Haematococcus pluvialis protein is rough and porous at the microscopic level, which is conducive to encapsulation. As the pH increases, the solubility, emulsifying property, foaming property, and foam stability of the Haematococcus pluvialis protein first increase and then decrease, while the emulsification stability shows the opposite trend, which is beneficial to the construction of the foaming microenvironment of the present invention and the promotion of microencapsulation.

[0071] 6) During the preparation of the complex of the present invention, phospholipid molecules are first used to form microcapsules to encapsulate astaxanthin, forming a phospholipid coating layer, which improves its solubility and stability in the aqueous phase in subsequent processes. The use of the multi-layer protection structure and stabilizer in the present invention makes the complex have higher stability, effectively extending the shelf life of astaxanthin. It also improves the water solubility and bioavailability of astaxanthin. Moreover, the addition of an appropriate amount of stabilizer (such as citric acid) in the present invention not only serves as a foaming component of the foaming microenvironment but also helps to inhibit the oxidative degradation of astaxanthin.

[0072] 7) With enhanced antioxidant effect: After the astaxanthin complex prepared in Example 1 of the present invention was stored at 4°C for 30 days, the retention rate of astaxanthin was as high as 95.4 ± 0.6%, indicating its high stability under low-temperature storage conditions. It is proved that the multi-level composite system of the present invention can effectively protect astaxanthin and prevent its degradation under the influence of external environmental factors. Description of the Drawings

[0073] Figure 1 It is a topographical diagram of the external structure of the astaxanthin complex prepared in Example 1 of the present invention. Detailed Description of the Specific Embodiment

[0074] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0075] The following detailed description of the preferred implementation methods of the present invention and the included embodiments can more easily understand the content of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. When there is a contradiction, the definition in this specification shall prevail.

[0076] Example 1

[0077] 1) Preparation of astaxanthin nanomicelle dispersion

[0078] Place 11 g of glyceryl oleate polyethylene glycol in a glass bottle. After heating in a water bath at 55°C until the glyceryl oleate polyethylene glycol is completely melted, add 1 g of high-purity natural astaxanthin under light-shielded conditions and stir evenly initially. After cooling to room temperature, add anhydrous ethanol to fully dissolve the mixture, and use a rotary evaporator to remove the volatile alcohols to uniformly disperse the astaxanthin; then add 80 mL of 0.1 M citric acid solution (instead of deionized water as the aqueous phase), and perform hydration treatment under 100 W ultrasonic conditions or with appropriate stirring to obtain a micelle solution. Filter the micelle solution through a 0.22 μm microporous membrane to obtain the astaxanthin nanomicelle dispersion.

[0079] 2) Preparation of phosphatized astaxanthin microemulsion

[0080] Dissolve 6 g of the auxiliary phospholipid component dimyristoyl phosphatidylethanolamine in an ethanol solvent to form a uniform phospholipid solution; mix the above-mentioned astaxanthin nanomicelle dispersion with the phospholipid solution evenly to obtain an astaxanthin nanomicelle-phospholipid mixture; under the condition of slow shaking, drop the astaxanthin nanomicelle-phospholipid mixture into a sodium bicarbonate solution (pH 8.0 - 8.2), control the dropping speed so that the solution produces an effervescent effect at the contact interface of the dropping and mixing, and fully carry out microbubble emulsification to obtain a phosphatidylated astaxanthin microemulsion. Control the addition amount of the sodium bicarbonate solution so that the finally formed emulsion is weakly alkaline (about pH 7.5 - 8.0). Among them, after the emulsification is completed, slow stirring is used to assist emulsification.

[0081] 3) Microencapsulation self-assembly

[0082] Transfer the astaxanthin emulsion obtained by the above emulsification treatment to a dialysis bag, place it in a phosphate buffer solution (PBS solution) with a pH of 7.2 - 7.3 for dialysis to remove salts, and promote the further microencapsulation self-assembly of astaxanthin micelle molecules in the buffer solution to form a microencapsulated structure. The dialysis time is 16 hours, and the buffer solution is changed 2 times during this period. After dialysis is completed, evaporate the remaining organic solvents at low temperature in a rotary evaporator, add citric acid to a molar concentration of 0.12 M, and stir evenly for acidification treatment; that is, obtain a microencapsulated astaxanthin microsphere dispersion, and store it at 4 °C for later use.

[0083] 4) Preparation of multi-level astaxanthin complex

[0084] Dissolve the Haematococcus pluvialis isolated protein in deionized water, dialyze and retain the protein components with a molecular weight cut-off of 10 - 40 kDa to obtain the Haematococcus pluvialis isolated protein; dissolve the obtained Haematococcus pluvialis isolated protein and HP-β-cyclodextrin in a sodium bicarbonate-sodium carbonate buffer solution (pH 9.5) at a mass ratio of 3:1, control the Haematococcus pluvialis protein concentration to be 4.5 wt%, and stir and hydrate at a water bath temperature of 35 °C for 15 min until uniform to obtain an inclusion complex solution. Under the condition of slow stirring, take 20 ml of the acidified microencapsulated astaxanthin microsphere dispersion and drop it into 100 mL of the above-mentioned inclusion complex solution composed of Haematococcus pluvialis protein / cyclodextrin derivative-sodium bicarbonate buffer solution, and carry out self-embedding treatment under the effervescent effect of the foaming microenvironment; after the embedding treatment is completed, dialyze to remove salts, and adjust the pH to 6.0 with citric acid to obtain a multi-level astaxanthin complex dispersion. The particle morphology of the obtained astaxanthin complex is shown in Figure 1 and its particles have better uniformity, and the particle size distribution is between 0.10 - 0.11 microns.

[0085] Optionally, the above complex solution can also be freeze-dried to obtain a solid particle form.

[0086] Comparative Example 1

[0087] Steps 1)-3) are the same as those in Example 1. The difference is that in step 4), sodium bicarbonate buffer solution is not used, but deionized water is used instead, and stirring is carried out. The specific operation is as follows:

[0088] Haematococcus pluvialis protein isolate and HP-β-cyclodextrin were dissolved in deionized water at a mass ratio of 3:1, and the concentration of Haematococcus pluvialis protein was controlled at 4.5 wt%. The mixture was stirred and hydrated at 35 °C for 15 min until homogeneous to obtain an inclusion complex solution. Under the condition of 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-deionized water, and the embedding treatment was carried out under the condition of rapid stirring at 200 rpm; thus, a multi-level astaxanthin complex dispersion was obtained. The particle size distribution of the obtained astaxanthin complex was between 140-160 nm, and the embedding rate was about 97%.

[0089] Comparative Example 2

[0090] The method of Comparative Example 1 was implemented, except that in step 2), deionized water was used instead of sodium bicarbonate solution, that is, there was no microencapsulation operation in a local effervescent environment in all steps. The particle size distribution of the obtained astaxanthin complex was between 155-175 nm, and the embedding rate was about 92%.

[0091] Comparative Example 3

[0092] 1) Weigh 1 g of astaxanthin and dissolve it in 80 ml of absolute ethanol. Stir evenly in the dark at room temperature to obtain an astaxanthin ethanol solution. Haematococcus pluvialis protein isolate and corn starch were dissolved in deionized water at a mass ratio of 3:1, and the concentration of Haematococcus pluvialis protein was controlled at 4.5 wt%. The mixture was stirred and hydrated at 50 °C for 15 min until homogeneous to obtain an inclusion complex solution.

[0093] 2) Under the condition of rapid stirring at 300 rpm, 20 ml of the astaxanthin ethanol solution was added dropwise to 100 mL of the above inclusion complex solution, and the embedding treatment was carried out by continuous stirring in a water bath at 55 °C for 3 h. Then, ethanol was removed by vacuum rotary evaporation at 25 °C, and unembedded free astaxanthin was removed by centrifugation at 3000 rpm for 15 min to obtain an astaxanthin complex dispersion. The average particle size of the obtained astaxanthin complex exceeded 0.23 microns. After storing the prepared astaxanthin complex in a sterile environment at 4 °C for 30 days, the retention rate of astaxanthin was only about 78%, indicating that it had poor stability under low-temperature storage conditions.

[0094] The results of each experimental group are shown in Table 1 below. Among them, the entrapment efficiency was determined by the conventional ultraviolet detection method in the art, the particle size was determined by a Zetasizer nanoparticle size analyzer; the morphology was observed by an electron microscope, and the stability was determined by the ultraviolet spectrophotometry method (characterized by the retention rate of the complex after storage at 4°C for 30 days).

[0095] Table 1 Particle size distribution and retention rate of the products of each experimental group

[0096]

[0097]

[0098] The above table shows that, compared with Example 1, under the same operating conditions, Comparative Examples 1-2, which respectively lack the micro-encapsulation treatment step in the foaming microenvironment, have significantly decreased entrapment efficiency and retention rate of the complex. At the same time, Comparative Example 3, which is used as a blank control and adopts the conventional direct encapsulation method in the art, has significantly lower entrapment efficiency and retention rate than the examples and Comparative Examples 1-2 of the present invention.

[0099] Although the present invention has been described in detail by way of reference to the embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and all such modifications or substitutions should be within the scope of the present invention / Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A multi-level astaxanthin complex, characterized in that, It includes a first embedded component containing astaxanthin, and a second embedded component containing Haematococcus pluvialis isolated protein; optionally, the multi-level astaxanthin complex further contains a cyclodextrin or its derivative component.

2. The multi-level astaxanthin complex according to claim 1, wherein The first embedded component contains microencapsulated astaxanthin microspheres, and the microencapsulated astaxanthin microspheres are prepared through nano-micellization dispersion pretreatment and phospholipid component-mediated microemulsification treatment.

3. The multi-level astaxanthin complex according to any one of claims 1-2, wherein The microencapsulated astaxanthin microspheres are obtained through a microbubble emulsification treatment step in a foaming microenvironment; and optionally, the multi-level astaxanthin complex is prepared through an embedding treatment step in a foaming microenvironment; wherein, the foaming microenvironment is generated by contacting two mixtures with a pH difference of not less than 1 or 2; Preferably, the acid-base properties of the two mixtures are different, and the alkaline mixture contains a sodium bicarbonate component.

4. The multi-level astaxanthin complex according to any one of claims 1-3, which is prepared by the following steps: dissolving Haematococcus pluvialis isolated protein and optionally a cyclodextrin derivative in a sodium bicarbonate solution or its buffer solution according to a certain mass ratio, and stirring evenly to obtain an inclusion solution containing Haematococcus pluvialis protein; dropping a solution of the first embedded component containing astaxanthin into the inclusion solution, and performing embedding under the effervescence effect at the mixing interface; after the embedding treatment is completed, adjusting the pH to acidic with citric acid to obtain the multi-level astaxanthin complex.

5. A preparation method of a multi-level astaxanthin complex, characterized in that, It includes the following steps: (1) Perform nano-micellization dispersion pretreatment on astaxanthin; (2) Based on the astaxanthin nano-micelle dispersion, perform emulsification treatment in a foaming microenvironment to prepare a phospholipidized astaxanthin microemulsion; (3) Optionally, perform microencapsulation self-assembly treatment on the astaxanthin microemulsion; (4) Use an inclusion material including Haematococcus pluvialis protein to perform micro-embedding treatment in a foaming microenvironment to obtain a multi-level astaxanthin complex.

6. The preparation method according to claim 5, characterized in that, The preparation method includes the following specific steps: S1: Nano-micellization dispersion pretreatment: Place glyceryl oleate polyoxyethylene in a glass bottle, heat it in a water bath until glyceryl oleate polyoxyethylene is completely melted, add high-purity natural astaxanthin and stir evenly preliminarily under light-shielded conditions, add ethanol to dissolve the mixture fully after restoring to room temperature, and remove volatile alcohols using a rotary evaporator to disperse astaxanthin evenly; Then, add it to a 0.05-0.5M citric acid solution, stir for hydration treatment to obtain an acidic micelle solution, and filter the micelle solution to remove insoluble particles to obtain an acidic astaxanthin nano-micelle dispersion; wherein, the mass ratio of glyceryl oleate polyoxyethylene to astaxanthin is 5-15:1; S2: Microemulsification treatment in a foaming microenvironment: Dissolve an amphiphilic phosphatidyl auxiliary phospholipid component in an organic solvent to form a uniform phospholipid solution; mix the astaxanthin nano-micelle dispersion and the phospholipid solution evenly to obtain an astaxanthin nano-micelle-phospholipid mixture; under optionally stirring conditions, drop the astaxanthin nano-micelle-phospholipid mixture into a sodium bicarbonate solution, and the acidic astaxanthin nano-micelle-phospholipid mixture generates an effervescence effect at the contact interface of the dropping mixture for microbubble emulsification treatment to obtain a phospholipidized astaxanthin microemulsion. Preferably, the organic solvent is ethanol; Among them, when the astaxanthin nanomicelle dispersion is mixed with the phospholipid solution, the mass ratio of phospholipid to astaxanthin is controlled to be (5 - 20):1; S3: Microencapsulation self-assembly treatment: Transfer the astaxanthin microemulsion obtained by the above microbubble emulsification treatment into a dialysis bag, place it in a phosphate buffer solution for dialysis to remove salts, and further perform microencapsulation self-assembly in the buffer solution; after dialysis is completed, evaporate the remaining organic solvent at low temperature in a rotary evaporator, add citric acid to the dialysis solution to make its solution concentration 0.1 - 0.5 M, and stir evenly for acidification treatment; namely, obtain the microencapsulated astaxanthin microsphere dispersion; S4: Prepare multi-level 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 - 40 kDa; dissolve the Haematococcus pluvialis protein and the cyclodextrin derivative in a sodium bicarbonate - sodium carbonate buffer solution at a mass ratio of 2 - 5:1, control the Haematococcus pluvialis protein concentration to be 1 - 20 wt%, and stir for hydration treatment until uniform to obtain the Haematococcus pluvialis protein - cyclodextrin inclusion complex solution; 2) Optionally, under slow stirring conditions, add the microencapsulated astaxanthin microsphere dispersion dropwise to an inclusion complex solution composed of 2 - 10 times the volume of the Haematococcus pluvialis protein / cyclodextrin derivative - sodium bicarbonate buffer solution, and perform embedding under the effervescent effect of the foaming microenvironment; after the embedding treatment is completed, dialyze to remove salts, and adjust the pH to 5.0 - 7.0 with citric acid to obtain the multi-level astaxanthin complex.

7. The astaxanthin complex obtained by the preparation method according to any one of claims 5 - 6.

8. The microencapsulated astaxanthin microspheres obtained by the preparation method according to any one of claims 5 - 6.

9. Use of the astaxanthin complex according to claim 7 in the fields of food, cosmetics, pharmaceuticals or health products.

10. Use of the microencapsulated astaxanthin microspheres according to claim 8 in the preparation of astaxanthin complex.

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