A method for preparing nano-astaxanthin aggregate complexes and their applications

Abstract

This invention relates to a method for preparing a nano-astaxanthin aggregate complex and its application. The preparation method includes the following steps: S1. Dissolving a carrier protein in water and adjusting the pH to 9-12 to obtain a protein aqueous solution; S2. Mixing the protein aqueous solution with a saturated ethanol solution of astaxanthin to obtain a composite solution; S3. First removing the ethanol from the composite solution, and then adjusting the pH to neutral to obtain the nano-astaxanthin aggregate complex. This preparation method utilizes pH-coordinated ethanol-induced carrier protein self-assembly and encapsulates astaxanthin aggregates twice in a specific order of first removing ethanol and then adjusting the pH. This results in a nano-astaxanthin aggregate complex that not only has good water solubility and storage stability, but also excellent photostability and thermal stability, and high astaxanthin retention and bioavailability after digestion.

Description

Technical Field

[0001] This invention relates to the field of nano-encapsulation technology, and more specifically, to a method for preparing a nano-astaxanthin aggregate complex and its application. Background Technology

[0002] Astaxanthin is a non-vitamin A-derived ketone fat-soluble carotenoid, a natural pigment widely found in nature. As an oxygenated derivative of carotenoids, it can accumulate in marine crustaceans and certain microalgae, reaching over 85% of total carotenoids. Due to its excellent coloring power and aggregation-induced absorption spectral shift, astaxanthin-rich crustaceans and birds exhibit different colors within their tissues. Furthermore, astaxanthin possesses various physiological activities, such as scavenging free radicals, anti-aging, anti-tumor, cardiovascular disease prevention, liver protection, UV protection, anti-inflammatory, and improved athletic performance. With ongoing research, astaxanthin is increasingly widely used in the global food, pharmaceutical, cosmetic, and aquaculture industries.

[0003] Higher animals cannot synthesize astaxanthin themselves and must obtain it through food. However, due to its highly unsaturated structure and conjugated double bonds, it is easily degraded by high temperatures, light, and oxygen. Furthermore, astaxanthin is highly lipophilic; its chemical structure, composed of polyene chains linked by two terminal rings, results in poor water solubility and easy degradation during human digestion, leading to low bioavailability and significantly limiting its widespread application.

[0004] Studies have shown that hydrophobic astaxanthin monomer molecules can aggregate in aqueous solvents, producing two significantly different aggregates. One is the card-packed H aggregate, composed of astaxanthin monomer molecules stacked in parallel, face-to-face conjugated chains, with its maximum absorption wavelength shifting from 480 nm (monomer) to 380–388 nm. The other is the head-to-tail J aggregate, composed of loosely stacked, misaligned astaxanthin monomer molecules, with its maximum absorption wavelength shifting from 480 nm (monomer) to 515–533 nm, accompanied by a shoulder peak at 550–575 nm. Compared to astaxanthin monomers, astaxanthin aggregates can exhibit a wider range of colors, thus serving as colorants to improve the appearance of food. However, in nature, both H and J aggregates dynamically transform and are difficult to maintain in a stable, dispersed form in water. Like astaxanthin monomers, they are also easily degraded under conditions such as high temperature or light exposure.

[0005] Existing research often involves encapsulating astaxanthin, such as preparing delivery systems like emulsions, microcapsules, and nanocomposites. For example, a Chinese patent describes a method for preparing and applying highly water-dispersible H- or J-type astaxanthin / whey protein / chitosan nanocomposites. This method involves mixing an organic phase of astaxanthin, an aqueous phase of whey protein, and an aqueous phase of chitosan under specific conditions, and then using emulsification rotary evaporation-molecular self-assembly technology to obtain highly water-dispersible astaxanthin / whey protein / chitosan nanocomposites. However, this patent only focuses on stability under room temperature and light-protected conditions. During the processing of food and cosmetic products, astaxanthin or its complexes inevitably come into contact with light and heat (e.g., 60°C) and degrade. Furthermore, this patent does not address degradation in the human digestive system.

[0006] Therefore, it is necessary to obtain astaxanthin aggregates with good photostability, thermal stability, water solubility, and high bioavailability that are not easily degraded in the digestive system, so as to make them competitive in the food, pharmaceutical, cosmetic, and aquaculture industries. Summary of the Invention

[0007] The primary objective of this invention is to overcome the problems of poor photothermal stability, poor thermal stability, poor water solubility, easy degradation in the digestive system, and low bioavailability of existing astaxanthin aggregates, and to provide a method for preparing nano-astaxanthin aggregate complexes. This method involves pH-coordinated ethanol-induced carrier protein self-assembly, followed by a specific sequence of first removing ethanol and then adjusting the pH to encapsulate astaxanthin aggregates twice. This results in nano-astaxanthin aggregate complexes that not only possess good water solubility and storage stability, but also excellent photostability and thermal stability, and high astaxanthin retention and bioavailability after digestion.

[0008] A further objective of this invention is to provide a nano-astaxanthin aggregate complex.

[0009] Another object of the present invention is to provide the application of the above-mentioned nano-astaxanthin aggregate complex in the preparation of additives.

[0010] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0011] A method for preparing a nano-astaxanthin aggregate complex includes the following steps:

[0012] S1. Dissolve the carrier protein in water, adjust the pH to 9-12, and obtain an aqueous protein solution;

[0013] S2. A protein aqueous solution and a saturated ethanol solution of astaxanthin are mixed to obtain a composite solution;

[0014] S3. First, remove the ethanol from the composite solution, then adjust the pH to neutral to obtain the nano-astaxanthin aggregate complex.

[0015] In step S2, the volume ratio of the saturated ethanol solution of astaxanthin to the aqueous protein solution is 1:(1-9); the concentration of the carrier protein in the composite solution is 0.002-0.12 mg / mL.

[0016] The preparation method of the nano-astaxanthin aggregate complex of the present invention firstly involves unfolding and dissociating a carrier protein into subunits under specific pH conditions. This exposes a large number of hydrophobic groups within the carrier protein and avoids the presence of large-sized carrier protein particles. Then, a saturated ethanol solution of astaxanthin is introduced. The ethanol in the saturated ethanol solution acts as both a solvent to introduce the astaxanthin and further unfolds the carrier protein. In the composite solution, astaxanthin forms astaxanthin aggregates and binds to the hydrophobic microregions on the surface of the carrier protein through hydrophobic interactions. Next, the ethanol is removed, causing the unfolded carrier protein to fold back for the first time, completing the first encapsulation of the astaxanthin aggregates. Then, the pH is adjusted to neutral, causing the carrier protein to fold back a second time. The originally loose structure of the carrier protein becomes denser, completing the second encapsulation of the astaxanthin aggregates, resulting in the nano-astaxanthin aggregate complex.

[0017] This invention utilizes pH-coordinated ethanol-induced carrier protein self-assembly, and encapsulates astaxanthin aggregates twice in a specific sequence: first removing ethanol, then adjusting the pH. This results in a nano-astaxanthin aggregate complex that not only exhibits good water solubility and storage stability, but also excellent photostability and thermal stability, and high astaxanthin retention and bioavailability after digestion. Furthermore, the preparation method of this invention achieves an astaxanthin encapsulation efficiency of over 84%, with a maximum loading capacity of approximately 60%, loading a large amount of astaxanthin with a relatively small amount of carrier protein.

[0018] Preferably, in step S1, the carrier protein is at least one of bovine serum albumin, sodium caseinate, whey protein isolate, soy protein isolate, whey protein, pea protein, egg white protein, whey protein concentrate, lysozyme, or lactoferrin.

[0019] More preferably, in step S1, the carrier protein is at least one of bovine serum albumin, sodium caseinate, whey protein isolate, or whey protein.

[0020] More preferably, in step S1, the carrier protein is whey protein. The encapsulation efficiency of the nano-astaxanthin aggregate complex prepared from whey protein is higher.

[0021] Preferably, in step S1, after adjusting the pH to 9-12, a vortexing step is also included.

[0022] More preferably, the vortex duration is 30–120 min and the rotation speed is 100–500 rpm.

[0023] Preferably, in step S2, the mixing process further includes a vortexing step.

[0024] More preferably, the vortex duration is 30–120 min and the rotation speed is 100–500 rpm.

[0025] Preferably, in step S2, the concentration of the carrier protein in the composite solution is 0.0025–0.1 mg / mL.

[0026] More preferably, in step S2, the concentration of the carrier protein in the composite solution is 0.025–0.01 mg / mL. Within this concentration range, the encapsulation efficiency of the nano-astaxanthin aggregate complex is higher.

[0027] Preferably, in step S2, the concentration of the carrier protein in the composite solution is 0.0025–0.025 mg / mL.

[0028] More preferably, in step S2, the concentration of the carrier protein in the composite solution is 0.0025–0.01 mg / mL.

[0029] More preferably, in step S2, the concentration of the carrier protein in the composite solution is 0.0025–0.0030 mg / mL. Within this concentration range, the nano-astaxanthin aggregate complex achieves a higher loading capacity and is more cost-effective.

[0030] Preferably, in step S2, the volume ratio of the saturated ethanol solution of astaxanthin to the aqueous protein solution is 1:(4-9). Within this range, the astaxanthin in the nano-astaxanthin aggregate complex is astaxanthin H aggregate.

[0031] Preferably, in step S2, the volume ratio of the astaxanthin ethanol solution to the protein aqueous solution in the composite solution is 1:(1-3). Within this range, the astaxanthin in the nano-astaxanthin aggregate complex is astaxanthin J aggregate.

[0032] Preferably, in step S3, the ethanol is removed by rotary evaporation.

[0033] More preferably, the rotary evaporation is performed with a vacuum of 0.05 to 0.095 MPa, a temperature of 35 to 40°C, and a rotation speed of 50 to 150 rpm.

[0034] Preferably, in step S3, after the pH is adjusted to neutral, a step of adding water is also included.

[0035] More preferably, the volume of water added is equal to the volume of ethanol removed.

[0036] Preferably, the nano-astaxanthin aggregate complex exists in the form of an aqueous solution or lyophilized powder.

[0037] More preferably, the preparation process of the freeze-dried powder is as follows: the aqueous solution of the nano-astaxanthin aggregate complex is placed at -20 to -80°C for 24 to 48 hours, and then placed under vacuum conditions at -70 to -32°C for 24 to 48 hours to obtain the freeze-dried powder.

[0038] A nano-astaxanthin aggregate complex was prepared by the above-described preparation method.

[0039] This invention also protects the use of the above-mentioned nano-astaxanthin aggregate complex in the preparation of food, pharmaceuticals, cosmetics or feed.

[0040] Compared with the prior art, the beneficial effects of the present invention are:

[0041] This invention utilizes pH-coordinated ethanol-induced carrier protein self-assembly, and encapsulates astaxanthin aggregates twice in a specific sequence: first removing ethanol, then adjusting the pH. This results in a nano-astaxanthin aggregate complex that not only exhibits good water solubility and storage stability, but also excellent photostability and thermal stability, and high astaxanthin retention and bioavailability after digestion. Furthermore, the preparation method of this invention achieves an astaxanthin encapsulation efficiency of over 84%, with a maximum loading capacity of approximately 60%, loading a large amount of astaxanthin with a relatively small amount of carrier protein. Attached Figure Description

[0042] Figure 1 The images show the UV-Vis spectra of Examples 1-5.

[0043] Figure 2 The images show the UV-Vis spectra of Examples 4 and 6-8.

[0044] Figure 3 The images show the UV-Vis spectra of Examples 4 and 9-11.

[0045] Figure 4 The images show the UV-Vis spectra of Examples 2 and 12.

[0046] Figure 5 The images show the UV-Vis spectra of Examples 2 and 13-15.

[0047] Figure 6 The images show the UV-Vis spectra of Examples 4 and 16-18.

[0048] Figure 7 Figure A shows the color change and UV-Vis spectrum of the nano-astaxanthin aggregate complex in Example 4 under different heat treatment times; Figure B shows the color change and UV-Vis spectrum.

[0049] Figure 8The images show the color changes and UV-Vis spectra of the nano-astaxanthin aggregate complex at different heat treatment times, as a comparison of Example 1; Figure A shows the color changes, and Figure B shows the UV-Vis spectra.

[0050] Figure 9 The images show the color changes and UV-Vis spectra of the nano-astaxanthin aggregate complex at different heat treatment times, as shown in Comparative Example 2. Figure A shows the color changes, and Figure B shows the UV-Vis spectra.

[0051] Figure 10 Figure A shows the color change and UV-Vis spectrum of the nano-astaxanthin aggregate complex in Example 4 under different light treatment times; Figure B shows the color change and UV-Vis spectrum.

[0052] Figure 11 The images show the color changes and UV-Vis spectra of the nano-astaxanthin aggregate complex for different light treatment times, as a comparison of Example 1; Figure A shows the color changes, and Figure B shows the UV-Vis spectra.

[0053] Figure 12 The images show the color changes and UV-Vis spectra of the nano-astaxanthin aggregate complex in Comparative Example 2 under different light treatment times; Figure A shows the color changes, and Figure B shows the UV-Vis spectra.

[0054] Figure 13 Figure A shows the color change and UV-Vis spectrum of the nano-astaxanthin aggregate complex of Example 4 at different storage times; Figure B shows the color change and UV-Vis spectrum.

[0055] Figure 14 The images show the freeze-dried powder and color images after reconstitution of the nano-astaxanthin aggregate complexes of Examples 4 and 2.

[0056] Figure 15 Figure A shows the astaxanthin retention rate and bioavailability of the nano-astaxanthin aggregate complex (J-AST-BSA) prepared in Example 4 and the comparative nano-astaxanthin aggregate complex prepared in Comparative Example 1 at different in vitro digestion stages; Figure B shows the bioavailability. Detailed Implementation

[0057] To more clearly and completely describe the technical solution of the present invention, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Various changes can be made within the scope of the claims of the present invention.

[0058] The encapsulation efficiency and loading capacity of astaxanthin were calculated using the following method:

[0059] Take 0.2 mL of nano-astaxanthin aggregate complex into a centrifuge tube, add 1 mL of n-hexane, vortex for 1 min, centrifuge at 10000 rpm for 10 min, take the supernatant, remove n-hexane by nitrogen blowing, dissolve astaxanthin in 1 mL of DMSO, measure the absorbance of the supernatant at 478 nm using an ELISA reader, calculate the astaxanthin concentration using a standard curve, and record it as unencapsulated astaxanthin.

[0060] Take 0.2 mL of the nano-astaxanthin aggregate complex and place it in a centrifuge tube. Mix it with 1.3 mL of a dichloromethane:methanol (v / v, 2:1) solution. Vortex for 30 s, then centrifuge at 10,000 rpm for 10 min. Collect the organic layer. Repeat the above steps until the aqueous layer is colorless. Measure the absorbance of the organic layer at 478 nm using a microplate reader. Calculate the concentration of astaxanthin using a standard curve and record it as the total astaxanthin. Calculate the encapsulation efficiency and loading capacity of astaxanthin using the following formulas.

[0061]

[0062] Example 1

[0063] This invention provides a method for preparing nano-astaxanthin aggregate complexes, comprising the following steps:

[0064] (1) Under the dark conditions at 25℃, 0.0024g of astaxanthin (AST) was dissolved in 40mL of anhydrous ethanol, vortexed for 60min, and centrifuged at 4000rpm for 15min to remove undissolved astaxanthin particles, thus obtaining a saturated ethanol solution of astaxanthin.

[0065] (2) Bovine serum albumin (BSA) was dissolved in water at room temperature and stirred. The pH was adjusted to 11.0 with 1 mol / L NaOH solution and vortexed at 300 rpm for 90 min to obtain a protein aqueous solution.

[0066] (3) Mix the saturated ethanol solution of astaxanthin from step (1) and the aqueous protein solution from step (2), and vortex at 300 rpm for 120 min to obtain a composite solution. The volume ratio of the saturated ethanol solution of astaxanthin to the aqueous protein solution is 1:9; the concentration of the carrier protein in the composite solution is 0.025 mg / mL.

[0067] (4) The ethanol in the composite solution of step (3) is removed by rotary evaporation. The vacuum degree of rotary evaporation is 0.09 MPa, the temperature is 35℃, and the rotation speed is 100 rpm. Then, the pH is adjusted to 7.0 with 1 mol / L hydrochloric acid solution, and water of the same volume as the evaporated ethanol is added to obtain the nano astaxanthin aggregate complex.

[0068] Examples 2-5

[0069] Examples 2-5 provide a series of methods for preparing nano-astaxanthin aggregate complexes. The difference from Example 1 is the volume ratio of the saturated ethanol solution of astaxanthin to the protein aqueous solution in step (3), as shown in Table 1. In particular, the concentration of the carrier protein in the composite solution obtained in step (3) of Examples 2-5 is 0.025 mg / mL. The concentration of the carrier protein in the composite solution can be adjusted by selecting protein aqueous solutions of different initial concentrations.

[0070] Table 1. Differences between Examples 1-5

[0071]

[0072] The nano-astaxanthin aggregate complexes and astaxanthin monomers (AST) prepared in Examples 1-5 were subjected to UV-Vis spectroscopy measurements. The parameters were set to a scan range of 300-800 nm. The UV-Vis spectra of Examples 1-5 are shown below. Figure 1 As shown. From Figure 1 It can be seen that the astaxanthin monomer has an absorption peak at 478 nm; the absorption peak of the nano-astaxanthin aggregate complex in Examples 1-2 is blue-shifted to 380-390 nm, and astaxanthin exists in the form of H aggregates; the absorption peak of the nano-astaxanthin aggregate complex in Examples 3-5 is red-shifted to 517 nm with a shoulder peak at 560 nm, and astaxanthin exists in the form of J aggregates.

[0073] Examples 6-8

[0074] Examples 6-8 provide a series of methods for preparing nano-astaxanthin aggregate complexes. The difference from Example 4 is the concentration of the carrier protein in the composite solution obtained in step (3) (while keeping the volume ratio of the saturated ethanol solution of astaxanthin and the aqueous protein solution at 4:6, the concentration of the carrier protein in the composite solution can be controlled by selecting different initial concentrations of aqueous protein solution), as shown in Table 2.

[0075] Table 2. Differences between Examples 4 and Examples 6-8

[0076]

[0077] The nano-astaxanthin aggregate complexes and astaxanthin monomers (AST) prepared in Examples 4 and 6-8 were subjected to UV-Vis spectroscopy measurements. The parameters were set to a scan range of 300-800 nm. The UV-Vis spectra of Examples 4 and 6-8 are shown below. Figure 2 As shown. From Figure 2It can be seen that the absorption peak λmax of the nano-astaxanthin aggregate complexes prepared in Examples 4 and 6-8 is 521-533 nm, accompanied by a shoulder peak of 550-575 nm. This indicates that the preparation method of the present invention can prepare nano-astaxanthin aggregate complexes in the form of J aggregates when the carrier protein concentration of the composite solution is different.

[0078] The encapsulation efficiency and loading of the nano-astaxanthin aggregate complexes prepared in Examples 4 and 6-8 were determined, and the results are shown in Table 3.

[0079] Table 3. Encapsulation efficiency and loading of nano-astaxanthin aggregate complexes prepared in Examples 4 and 6-8

[0080] Encapsulation rate / % 95.97±0.83 92.40±0.83 95.75±0.72 94.87±0.14 Load / % 10.54±0.37 61.93±0.21 16.19±0.51 2.41±0.11

[0081] As shown in Table 3, the encapsulation efficiency first increases and then decreases with increasing carrier protein concentration in the composite solution, reaching its maximum at a concentration of 0.025 mg / mL. Under different carrier protein concentrations in the composite solution, the preparation method of the nano-astaxanthin aggregate complex of the present invention achieves an encapsulation efficiency of over 90%, reaching a maximum of 95.97%. With increasing carrier protein concentration in the composite solution, the loading capacity gradually decreases, indicating that the preparation method of the nano-astaxanthin aggregate complex of the present invention can load a larger amount of astaxanthin with a smaller amount of carrier protein.

[0082] Examples 9-11

[0083] Examples 9-11 provide a series of methods for preparing nano-astaxanthin aggregate complexes. The difference from Example 4 is the pH in step (2), as detailed in Table 4.

[0084] Table 4. Differences between Examples 4 and Examples 9-11

[0085] pH 11 9 10 12

[0086] The nano-astaxanthin aggregate complexes and astaxanthin monomers (AST) prepared in Examples 4 and 9-11 were subjected to UV-Vis spectroscopy measurements. The parameters were set to a scan range of 300-800 nm. The UV-Vis spectra of Examples 4 and 9-11 are shown below. Figure 3 As shown. From Figure 3 It can be seen that the absorption peak λmax of the nano-astaxanthin aggregate complexes prepared in Examples 4 and 9-11 is 521-533 nm, accompanied by a shoulder peak of 550-575 nm. This indicates that the preparation method of the present invention can successfully prepare nano-astaxanthin aggregate complexes under different pH conditions, and the astaxanthin in the nano-astaxanthin aggregate complexes exists in the form of J aggregates.

[0087] The encapsulation efficiency of the nano-astaxanthin aggregate complexes prepared in Examples 4 and 9-11 was determined, and the results are shown in Table 5.

[0088] Table 5 Encapsulation efficiency of the nano-astaxanthin aggregate complexes prepared in Examples 4 and 9-11

[0089] Encapsulation rate / % 95.97±0.83 93.49±0.79 92.87±0.21 89.51±0.22

[0090] As shown in Table 5, the encapsulation efficiency was similar and not significantly different at pH 9 and pH 10 (p > 0.05). When the pH increased to 11, the encapsulation efficiency increased significantly (p < 0.05). However, when the pH continued to increase to 12, the encapsulation efficiency decreased significantly (p < 0.05). This is because under excessively alkaline conditions, bovine serum albumin was excessively denatured and its structure became too loose, affecting the second encapsulation process and thus leading to a decrease in the encapsulation efficiency.

[0091] Example 12

[0092] This embodiment provides a method for preparing a nano-astaxanthin aggregate complex, which differs from Example 2 in that the concentration of the carrier protein in the complex solution in step (3) is 0.0025 mg / mL.

[0093] The nano-astaxanthin aggregate complexes and astaxanthin monomers (AST) prepared in Examples 2 and 12 were subjected to UV-Vis spectroscopy measurements. The parameters were set to a scan range of 300–800 nm. The UV-Vis spectra of Examples 2 and 12 are shown below. Figure 4 As shown. From Figure 4 It can be seen that the absorption peak λmax of the nano-astaxanthin aggregate complexes prepared in Examples 2 and 12 is 375-390 nm. This indicates that the preparation method of the present invention can prepare nano-astaxanthin aggregate complexes in the form of H aggregates when the carrier protein concentration of the composite solution is different.

[0094] The encapsulation efficiency and loading of the nano-astaxanthin aggregate complexes prepared in Examples 2 and 12 were determined, and the results are shown in Table 6.

[0095] Table 6 Encapsulation efficiency and loading of nano-astaxanthin aggregate complexes prepared in Examples 2 and 12

[0096] Encapsulation rate / % 84.66±0.49 84.53±0.77 Load / % 3.18±0.12 11.32±0.08

[0097] As shown in Table 6, when astaxanthin is an H aggregate, the encapsulation efficiency of Example 2 (the concentration of the carrier protein in the composite solution is 0.025 mg / mL) is higher than that of Example 12 (the concentration of the carrier protein in the composite solution is 0.0025 mg / mL), which is consistent with the case when astaxanthin is a J aggregate (Examples 4 and 6).

[0098] Examples 13-15

[0099] Examples 13-15 provide a series of methods for preparing nano-astaxanthin aggregate complexes. The difference from Example 2 is the pH in step (2), as detailed in Table 7.

[0100] Table 7. Differences between Example 2 and Examples 13-15

[0101] pH 11 9 10 12

[0102] The nano-astaxanthin aggregate complexes and astaxanthin monomers (AST) prepared in Examples 2 and 13-15 were subjected to UV-Vis spectroscopy measurements. The parameters were set to a scan range of 300-800 nm. The UV-Vis spectra of Examples 2 and 13-15 are shown below. Figure 5 As shown. From Figure 5 It can be seen that the absorption peak λmax of the nano-astaxanthin aggregate complexes prepared in Examples 2 and 13-15 is 375-390 nm, which indicates that the preparation method of the present invention can successfully prepare nano-astaxanthin aggregate complexes in the form of H aggregates under different pH conditions.

[0103] Examples 16-18

[0104] Examples 16-18 provide a series of methods for preparing nano-astaxanthin aggregate complexes. The difference from Example 4 is the carrier protein in step (2), as detailed in Table 8.

[0105] Table 8. Differences between Example 4 and Examples 16-18

[0106]

[0107] The nano-astaxanthin aggregate complexes and astaxanthin monomers (AST) prepared in Examples 4 and 16-18 were subjected to UV-Vis spectroscopy measurements. The parameters were set to a scan range of 300-800 nm. The UV-Vis spectra of Examples 4 and 16-18 are shown below. Figure 6 As shown. From Figure 6 It can be seen that the absorption peak λmax of the nano-astaxanthin aggregate complexes prepared in Examples 4 and 16-18 is 521-533 nm, accompanied by a shoulder peak of 550-575 nm. This indicates that nano-astaxanthin aggregate complexes can be successfully prepared by using common carrier proteins.

[0108] The encapsulation efficiency of the nano-astaxanthin aggregate complexes prepared in Examples 4 and 16-18 was determined, and the results are shown in Table 9.

[0109] Table 9 Encapsulation efficiency of nano-astaxanthin aggregate complexes prepared in Examples 4 and 16-18

[0110] Encapsulation rate / % 95.97±0.83 92.55±0.16 93.06±0.22 97.77±0.42

[0111] As shown in Table 9, under different carrier protein conditions, the preparation method of the nano-astaxanthin aggregate complex of the present invention can achieve an encapsulation rate of astaxanthin of over 90%, with a maximum of 97.77%.

[0112] Comparative Example 1

[0113] This comparative example provides a method for preparing a comparative nano-astaxanthin aggregate complex, which differs from Example 4 in that the pH in step (2) is 7.0.

[0114] The encapsulation efficiency of the nano-astaxanthin aggregate complex prepared in Example 4 and the comparative nano-astaxanthin aggregate complex prepared in Comparative Example 1 was determined, and the results are shown in Table 10.

[0115] Table 10 Encapsulation efficiency of Example 4 and Comparative Example 1

[0116] Encapsulation rate / % 95.97±0.83 86.96％±0.16％

[0117] As can be seen from Table 10, due to the lack of specific pH adjustment to expose the internal hydrophobic groups of the carrier protein, and the fact that astaxanthin aggregates were only encapsulated once, the encapsulation efficiency of the prepared comparative nano-astaxanthin aggregate complex was very low.

[0118] Comparative Example 2

[0119] This comparative example provides a method for preparing a comparative nano-astaxanthin aggregate complex, which differs from Example 4 in that step (4) does not involve rotary evaporation, i.e., ethanol is not removed.

[0120] Comparative Examples 3-6

[0121] The difference between Comparative Examples 3-6 and Example 1 is the volume ratio of the saturated ethanol solution of astaxanthin to the aqueous protein solution in step (3), as shown in Table 11. In Comparative Examples 3-6, the concentration of the carrier protein in the composite solution obtained in step (3) is 0.025 mg / mL. The concentration of the carrier protein in the composite solution can be adjusted by using different initial concentrations of aqueous protein solutions.

[0122] Table 11 Comparison of Examples 3-6

[0123]

[0124] The astaxanthin in the complexes prepared in Comparative Examples 3-6 exists in the form of monomers rather than in the form of aggregates, meaning that complexes containing astaxanthin aggregates cannot be successfully obtained.

[0125] Performance testing

[0126] (1) Thermal stability: 10 mL of the nano-astaxanthin aggregate complex of Example 4 and the comparative nano-astaxanthin aggregate complex of Comparative Examples 1-2 were placed in an amber glass bottle and heat-treated at 60℃ for 4 h. The color change of the solution and the particle size of the solution were observed at 0 h, 0.5 h, 1 h, 2 h and 4 h respectively. The UV-Vis spectrum was measured with an enzyme-linked immunosorbent assay (ELISA) reader to observe the changes in the position and intensity of the absorption peaks, thereby determining the thermal stability of the nano-astaxanthin aggregate complex. The particle size results are shown in Table 12.

[0127] Table 12. Particle size results of the nano-astaxanthin aggregate complex in the thermal stability experiment.

[0128]

[0129]

[0130] Figures 7-9 The images show the color changes and UV-Vis spectra of the nano-astaxanthin aggregate complex of Example 4 and the comparative nano-astaxanthin aggregate complexes of Comparative Examples 1 and 2, respectively, at different heat treatment times. Figures 7-9 A represents the color change diagram. Figures 7-9 B represents the ultraviolet-visible spectrum.

[0131] As shown in Table 12, the nano-astaxanthin aggregate complex in Example 4 exhibited minimal particle size variation and uniform particle size distribution under different heat treatment times. Figure 7 It can be seen that the color change of the nano-astaxanthin aggregate complex in Example 4 is small under different heat treatment times, and the change in the peak absorbance after heat treatment is also small. Therefore, the nano-astaxanthin aggregate complex prepared by this invention has good thermal stability.

[0132] As shown in Table 12, the comparative nano-astaxanthin aggregate complex of Comparative Example 1, due to the inability to completely dissociate the large-sized carrier protein particles inherent in the protein aqueous solution by treating the carrier protein with only ethanol, has a larger particle size and uneven particle size distribution compared to Example 4. With increasing heat treatment time, the carrier protein particles easily expand and recombine, resulting in significant changes in particle size, indicating that the comparative nano-astaxanthin aggregate complex prepared in Comparative Example 1 has poor thermal stability. Figure 8 It can be seen that the absorbance peak of the comparative nano-astaxanthin aggregate complex in Comparative Example 1 changed significantly after heat treatment, further indicating that the comparative nano-astaxanthin aggregate complex prepared in Comparative Example 1 has poor thermal stability.

[0133] As shown in Table 12, the particle size of the comparative nano-astaxanthin aggregate complex in Comparative Example 2 is larger than that in Example 4. This is because ethanol was not removed in step S4, interfering with the folding process of the carrier protein particles. The carrier protein could not undergo a complete "dissociation-unfolding-folding" cycle, and its structure may remain in an incompletely folded or abnormally folded state, resulting in an overly loose internal structure. Furthermore, carrier protein particles in an incompletely folded or abnormally folded state may aggregate or dissociate under the influence of heat treatment, leading to fluctuations in particle size with heat treatment time. All of these factors contribute to the poor thermal stability of the comparative nano-astaxanthin aggregate complex prepared in Comparative Example 2. Figure 9 It can be seen that the absorbance peak of the comparative nano-astaxanthin aggregate complex in Comparative Example 2 changed significantly after heat treatment, further indicating that the comparative nano-astaxanthin aggregate complex prepared in Comparative Example 2 has poor thermal stability.

[0134] (2) Photostability: 10 mL of the nano-astaxanthin aggregate complex from Example 4 and the control nano-astaxanthin aggregate complexes from Comparative Examples 1-2 were placed in a transparent glass bottle and irradiated under a 30W incandescent lamp for 24 h, with the distance between the lamp and the still liquid surface being 25 cm. Samples were taken at 0 h, 1 h, 2 h, 4 h, 8 h, and 24 h, and the color change of the solution was observed. The particle size of the solution was measured, and the ultraviolet-visible spectrum was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The changes in the position and intensity of the absorption peaks were observed to determine the photostability of the nano-astaxanthin aggregate complex. The particle size results are shown in Table 13.

[0135] Table 13. Particle size results of the nano-astaxanthin aggregate complex in the photostability experiment.

[0136]

[0137] Figures 10-12 The images show the color changes and UV-Vis spectra of the nano-astaxanthin aggregate complex of Example 4 and the comparative nano-astaxanthin aggregate complexes of Comparative Examples 1 and 2, respectively, under different light treatment times. Figures 10-12 Figure A is a color change diagram. Figures 10-12 Figure B is the ultraviolet-visible spectrum.

[0138] As shown in Table 13, the particle size of the nano-astaxanthin aggregate complex in Example 4 showed little change under different light treatment times, and the overall particle size distribution was relatively uniform. Figure 10 It can be seen that the color change of the nano-astaxanthin aggregate complex in Example 4 is small under different light treatment times, and the change in the peak absorbance after light irradiation is also small. Therefore, the nano-astaxanthin aggregate complex prepared by this invention has good photostability.

[0139] As shown in Table 13, the comparative nano-astaxanthin aggregate complex of Comparative Example 1, due to the inability to completely dissociate the large-sized carrier protein particles inherent in the protein aqueous solution by treating the carrier protein with only ethanol, has a larger particle size and uneven particle size distribution compared to Example 4. With increasing light treatment time, the carrier protein particles easily expand and recombine, resulting in significant changes in particle size, indicating that the comparative nano-astaxanthin aggregate complex prepared in Comparative Example 1 has poor photostability. Figure 11 It can be seen that the absorbance peak of the comparative nano-astaxanthin aggregate complex in Comparative Example 1 changed significantly after light treatment, further indicating that the photostability of the comparative nano-astaxanthin aggregate complex prepared in Comparative Example 1 was poor.

[0140] As shown in Table 13, the particle size of the comparative nano-astaxanthin aggregate complex in Comparative Example 2 is larger than that in Example 4. This is because ethanol was not removed in step S4, interfering with the folding process of the carrier protein particles. The carrier protein could not undergo a complete "dissociation-unfolding-folding" cycle, and its structure may remain in an incompletely folded or abnormally folded state, resulting in an overly loose internal structure. Furthermore, carrier protein particles in an incompletely folded or abnormally folded state will aggregate or dissociate under the influence of light treatment, leading to fluctuations in particle size with light treatment time. All of these factors contribute to the poor photostability of the comparative nano-astaxanthin aggregate complex prepared in Comparative Example 2. Figure 12 It can be seen that the absorbance peak of the comparative nano-astaxanthin aggregate complex in Comparative Example 2 changed significantly after light treatment, further indicating that the photostability of the comparative nano-astaxanthin aggregate complex prepared in Comparative Example 2 was poor.

[0141] (3) Storage stability: 10 mL of the nano-astaxanthin aggregate complex from Example 4 was placed in an amber glass bottle and treated in the dark at 25°C for 28 days. The particle size of the nano-astaxanthin aggregate complex was measured at 0d, 7d, 14d, 21d, and 28d, and the color change was observed. The UV-Vis spectrum was measured using an enzyme-linked immunosorbent assay (ELISA) reader to observe the changes in the position and intensity of the absorption peaks, thereby determining the storage stability of the nano-astaxanthin aggregate complex. The particle size results are shown in Table 14.

[0142] Table 14. Particle size results of the nano-astaxanthin aggregate complex in the storage stability experiment.

[0143]

[0144] As shown in Table 14, the particle size of the nano-astaxanthin aggregate complex in Example 4 showed little change under different storage times and the particle size distribution was uniform. Figure 13 Figure A shows the color change and UV-Vis spectrum of the nano-astaxanthin aggregate complex from Example 4 at different storage times; Figure B shows the color change and UV-Vis spectrum. Figure 13 It can be seen that the color change of the nano-astaxanthin aggregate complex in Example 4 is small under different storage times, and the peak value change of the nano-astaxanthin aggregate complex is small. This indicates that the nano-astaxanthin aggregate complex prepared by the present invention undergoes less degradation under long-term storage and has good storage stability.

[0145] (4) Determination of the reconstitution properties of the lyophilized powder of the nano-astaxanthin aggregate complex

[0146] The nano-astaxanthin aggregate complexes from Examples 2 and 4 were placed in 50 mL centrifuge tubes, covered with plastic wrap, and small pores were punched in the wraps. The pre-freeze-drying process involved storing the tubes at -80°C for 24 hours. Using a vacuum freeze dryer, water was removed by sublimation. The freeze-drying process was carried out at -32°C for 48 hours to obtain the freeze-dried powder of the nano-astaxanthin aggregate complex. This powder was then sealed, protected from light, and stored at -20°C for later use. The astaxanthin in the nano-astaxanthin aggregate complex obtained in Example 4 existed in the form of J-aggregates, and its freeze-dried powder was light purple-pink. The astaxanthin in the nano-astaxanthin aggregate complex obtained in Example 2 existed in the form of H-aggregates, and its freeze-dried powder was yellow-orange. The freeze-dried powder of the nano-astaxanthin aggregate complex was redissolved in ultrapure water, and the color of the reconstituted nano-astaxanthin aggregate complex was observed. Figure 14 As shown. From Figure 14 It can be seen that the color of the lyophilized nano-astaxanthin aggregate complex did not change before and after reconstitution, and the solution was clear and transparent after reconstitution, indicating that the lyophilized nano-astaxanthin aggregate complex prepared by this invention has good water solubility.

[0147] Within 1 hour after reconstitution, the particle size, PDI, and potential of the lyophilized powder reconstituted solution of the nano-astaxanthin aggregate complex were measured and compared with those before lyophilization. The change value Δ was calculated according to the following formula, as shown in Table 15.

[0148] Δ = After reconstitution of the nano-astaxanthin aggregate complex - before the nano-astaxanthin aggregate complex is made into lyophilized powder;

[0149] Table 15 Determination of the reconstitution properties of lyophilized powder of nano-astaxanthin aggregate complex

[0150] Example 2 20.66±57.22 -15.07±0.75 0.05±0.05 Example 4 18.77±7.44 -5.00±0.26 0.04±0.02

[0151] As shown in Table 15, the changes in particle size, PDI, and potential of the lyophilized nano-astaxanthin aggregate complexes prepared in Examples 2 and 4 after reconstitution are relatively small. This indicates that the lyophilized nano-astaxanthin aggregate complexes prepared in this invention are stably dispersed in water after reconstitution, achieving the same effect as before lyophilization. Therefore, the nano-astaxanthin aggregate complexes can also be stored or used as additives in food, pharmaceuticals, cosmetics, or feed in a more convenient manner, without affecting their properties after reconstitution, and have broad application prospects.

[0152] (5) Static in vitro simulated digestion

[0153] Referring to the INFOGEST standard static in vitro simulated digestion method, the specific steps are as follows:

[0154] Simulated oral digestion stage: 15 mg of mucin was dissolved in 4 mL of LSSF and stirred overnight at 4°C. 5 mL of the nano-astaxanthin aggregate complex sample prepared in Example 4 and Comparative Example 1 was accurately weighed and mixed thoroughly with 4 mL of LSSF (containing mucin), α-amylase (0.6 g / L), and 25 μL of 0.3 mol / L CaCl2 solution. The pH was adjusted to 7.0 with hydrochloric acid, and the total digestion system was brought to 10 mL with distilled water. The mixed oral digestion solution was placed in a constant temperature shaker at 37°C and 150 rpm for 2 min to digest, yielding the oral digested sample.

[0155] Simulated gastric digestion stage: 10 mL of the orally digested sample was mixed thoroughly with 8 mL of LGF and 5 μL of 0.3 mol / L CaCl2 solution. Pepsin (800 U / mg) was added to achieve an activity of 2000 U / mL in the final gastric digestion mixture. The pH was lowered to 3.0 with hydrochloric acid, and the total digestion system was brought up to 20 mL with distilled water. The mixed gastric digestion solution was then placed in a constant temperature shaker at 37°C and 150 rpm for 2 hours to digest, yielding the gastric digested sample.

[0156] Simulated small intestinal digestion stage: 20 mL of the sample after gastric digestion was completed was mixed thoroughly with 8 mL of SIF, 5 mL of lipase (438 U / mg) and pancreatic enzyme (4.5 U / mg), 3 mL of bile salts (reaching a final concentration of 10 mM in the digestion mixture), and 40 μL of 0.3 mol / L CaCl2 solution. The pH was adjusted to 7.0 with sodium hydroxide, and the total digestion liquid was brought up to 40 mL with distilled water. The mixed intestinal digestion solution was placed in a constant temperature shaker at 37°C and 150 rpm for 2 hours for digestion. Titration was performed using 0.25 M sodium hydroxide solution to maintain a neutral environment (pH 7.0). The content of astaxanthin aggregates (C) was measured during simulated oral digestion (min 2 of the static in vitro digestion process), gastric digestion (mins 32, 62, and 122 of the static in vitro digestion process), and small intestinal digestion (mins 152, 182, and 242 of the static in vitro digestion process). t The initial astaxanthin aggregate content C0 in the sample before static in vitro simulated digestion was used as the astaxanthin aggregate content. The retention rate was calculated according to the following formula, and the results are as follows: Figure 15 As shown in Figure A:

[0157]

[0158] After small intestinal digestion, the astaxanthin aggregates in the digestive fluids are divided into two parts. One part consists of astaxanthin aggregates that are not degraded but may still be encapsulated in carrier proteins; this part of the astaxanthin cannot be absorbed and utilized by the intestines. The other part consists of astaxanthin aggregates that are not degraded and are released from the carrier proteins; this part of the astaxanthin aggregates can be absorbed and utilized by the intestines. The end of the simulated small intestinal digestion is considered the digestion endpoint. 6 mL of the sample at the digestion endpoint is centrifuged (10000 rpm, 30 min), and the astaxanthin aggregate content in the supernatant is measured. The astaxanthin content in the sample before static in vitro simulated digestion is used as the initial astaxanthin aggregate content. Bioavailability is calculated using the following formula, and the results are as follows: Figure 15 As shown in Figure B:

[0159]

[0160] from Figure 15As shown in Figure A, after oral digestion, the retention rates of astaxanthin aggregates in Example 4 and Comparative Example 1 were not significantly different. After gastric digestion (122 min), the retention rate of astaxanthin aggregates in Example 4 was 94.81%, while that in Comparative Example 1 was 89.30%. This indicates that during gastric digestion, the astaxanthin aggregates in the nano-astaxanthin aggregate complex prepared in this invention can be well encapsulated and protected, thereby reducing degradation during gastric digestion. After small intestinal digestion (242 min), the retention rate of astaxanthin aggregates in Example 4 was 87.20%, while that in Comparative Example 1 significantly decreased to 56.83%, further demonstrating that the nano-astaxanthin aggregate complex prepared in this invention can ensure that most of the astaxanthin aggregates remain stable and are not degraded during digestion. Figure 15 As shown in Figure B, the bioavailability of the nano-astaxanthin aggregate complex prepared in Comparative Example 1 was 15.76%, while the bioavailability of the nano-astaxanthin aggregate complex prepared in Example 4 was as high as 40.97%. This indicates that the nano-astaxanthin aggregate complex prepared in this invention can still stably exist without degradation and can be absorbed by the intestines after digestion in the mouth, stomach and small intestine, thus resulting in high bioavailability.

[0161] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a nanoastaxanthin aggregate complex, characterized by, Includes the following steps: S1. The carrier protein is dissolved in water, and the pH is adjusted to 11 to obtain an aqueous protein solution; S2. A protein aqueous solution and a saturated ethanol solution of astaxanthin are mixed to obtain a composite solution; S3. First, remove the ethanol from the composite solution, and then adjust the pH to neutral to obtain the nano-astaxanthin aggregate complex. In step S2, the volume ratio of the saturated ethanol solution of astaxanthin to the aqueous protein solution is 4:6; the concentration of the carrier protein in the composite solution is 0.025 mg / mL. The carrier protein is bovine serum albumin.

2. The preparation method according to claim 1, characterized in that, In step S2, the mixing process further includes a vortex step.

3. The preparation method according to claim 1, characterized in that, In step S3, the ethanol is removed by rotary evaporation.

4. The preparation method according to claim 1, characterized in that, The nano-astaxanthin aggregate complex exists in the form of an aqueous solution or a lyophilized powder.

5. A nano-astaxanthin aggregate complex, characterized in that, It is prepared by any of the preparation methods described in claims 1 to 4.

6. The use of the nano-astaxanthin aggregate complex according to claim 5 in the preparation of food, pharmaceuticals, cosmetics or feed.

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