Method for extracting astaxanthin from haematococcus pluvialis and application thereof

Abstract

This invention provides a method for extracting astaxanthin from Haematococcus pluvialis and its application. The method includes the following steps: S1. Raw material pretreatment; S2. Enzymatic hydrolysis with a complex enzyme; S3. Solubilization with a surfactant; S4. Adsorption by iron(III) oxide-cellulose porous microspheres; S5. Magnetic separation and washing; S6. Astaxanthin elution and microsphere recovery; S7. Concentration and drying. This invention utilizes a complex enzyme consisting of cellulase, alginate lyase, and mannanase to synergistically hydrolyze the multilayer polysaccharide structure of thick-walled spores of Haematococcus pluvialis, thereby fully releasing astaxanthin. Furthermore, iron(III) oxide-cellulose porous microspheres with benzyl and octyl surface modifications are used for highly selective adsorption of astaxanthin, combined with magnetic separation technology, thus improving the astaxanthin yield. The extraction process of this invention is carried out under medium-low temperature conditions, and butylated hydroxytoluene is added to the eluent, effectively avoiding thermal degradation, photolysis, and oxidation of astaxanthin, resulting in a high retention rate of antioxidant activity in the obtained astaxanthin.

Description

Technical Field

[0001] This invention relates to the field of astaxanthin preparation technology and application, specifically to a method for extracting astaxanthin from Haematococcus pluvialis and its application. Background Technology

[0002] Astaxanthin is a carotenoid with strong antioxidant activity, widely used in food, medicine, cosmetics, aquaculture, and agricultural production. Haematococcus pluvialis is the richest natural source of astaxanthin, which accumulates in its thick-walled spores as monoesters, diesters, and small amounts of free astaxanthin. However, the multi-layered, tough cell wall structure of Haematococcus pluvialis spores presents numerous challenges to traditional extraction methods.

[0003] Existing astaxanthin extraction technologies mainly include organic solvent extraction, supercritical CO2 extraction, alkaline cell wall disruption, high-pressure homogenization, and ultrasound-assisted extraction. These methods generally face the following bottlenecks: First, cell wall disruption is difficult. Pure physical methods (such as grinding and high-pressure homogenization) are energy-intensive, require demanding equipment, and are prone to generating localized high temperatures that lead to astaxanthin degradation. Chemical methods (such as strong alkalis and acids) can damage the conjugated double bond structure of astaxanthin, reducing its biological activity. Second, extraction selectivity is poor. Conventional solvent extraction simultaneously dissolves large amounts of impurities such as lipids, chlorophyll, and polysaccharides, making subsequent purification steps cumbersome, consuming large amounts of organic solvents, and causing serious environmental pollution.

[0004] Therefore, developing a mild, efficient, and easily separable astaxanthin extraction method to achieve efficient cell wall disruption of Haematococcus pluvialis thick-walled spores and selective capture of astaxanthin has significant application value and practical significance. Summary of the Invention

[0005] In view of this, the present invention proposes a method for extracting astaxanthin from Haematococcus pluvialis and its application to solve the above problems.

[0006] The technical solution of this invention is implemented as follows: A method for extracting astaxanthin from Haematococcus pluvialis includes the following steps: S1. Raw material pretreatment: Select dried Haematococcus pluvialis powder, add citrate-sodium dihydrogen phosphate buffer at a solid-liquid ratio of 1:15-20 (solid-liquid ratio unit is g / mL), shake in a water bath to obtain a suspension; S2. Complex enzyme hydrolysis: A complex enzyme consisting of cellulase, alginate lyase and mannanase is added to the suspension for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated and then cooled to room temperature to obtain the enzymatic hydrolysate. S3. Surfactant to aid solubilization: Add sodium hydroxide solution to the enzymatic hydrolysate to adjust the pH, then add polysorbate 80, stir well, then centrifuge and take the supernatant; S4. Adsorption of Fe3O4-cellulose porous microspheres: Add Fe3O4-cellulose porous microspheres to the supernatant and shake for adsorption treatment; S5. Magnetic separation and washing: After adsorption, the microspheres are placed in a magnetic field environment, allowed to stand, and the supernatant is removed to obtain microsphere precipitate; then the microsphere precipitate is washed with phosphate buffer. S6. Astaxanthin elution and microsphere recovery: Add eluent to the washed microsphere precipitate, shake and elute, place in a magnetic field environment after elution, let stand, and collect the eluent; repeat elution 2-3 times, combine the eluents; wash the eluted microspheres with purified water and recover them; S7. Concentration and drying: The combined eluent was concentrated under reduced pressure and then freeze-dried to obtain astaxanthin.

[0007] Furthermore, in step S1, the pH of the citrate-sodium dihydrogen phosphate buffer solution is 5.5-6.0, the water bath shaking temperature is 25-30℃, the rotation speed is 120-180 rpm, and the shaking time is 25-35 min.

[0008] Furthermore, in step 2, the mass ratio of cellulase, alginate lyase, and mannanase is 3-5:2.5-3.5:2.5-3.5. Enzymatic hydrolysis is carried out at 38-45℃ for 3.5-4.5 hours, with stirring every 0.5 hours. After the enzymatic hydrolysis is completed, the temperature is raised to 90-95℃ and maintained for 10-15 minutes.

[0009] Furthermore, in step S3, the concentration of sodium hydroxide solution is 1-2 mol / L, adjusted to 6.8-7.2, the amount of polysorbate 80 added is 0.3-0.8% of the volume of the enzymatic hydrolysate, the mixture is stirred at 150-250 rpm for 25-35 min, and the centrifugation speed is 3000-5000 rpm for 10-15 min.

[0010] Furthermore, in step S4, the iron tetroxide-cellulose porous microspheres and the supernatant have a solid-liquid ratio of 1:20-30, with the solid-liquid ratio unit being g / mL, and are adsorbed at 25-30℃ with shaking at 150-200 rpm for 1.8-2.2 h.

[0011] Furthermore, the iron(III) oxide-cellulose porous microspheres were prepared by the following method: FeCl3·6H2O and FeSO4·7H2O were dissolved in deionized water at a molar ratio of 2:1, under nitrogen protection, and stirred at 30-35℃ until completely dissolved. A 2 mol / L NaOH solution was slowly added dropwise to adjust the pH to 10.0, and the reaction was carried out at 30-35℃ for 0.9-1.1 h. The mixture was then centrifuged, and the Fe3O4 nanoparticles were collected, washed with deionized water until neutral, and then washed once with anhydrous ethanol. Finally, they were vacuum dried at 60-65℃ under a vacuum of -80 to -100 kPa for 1 hour. 8-2.2 h was used to obtain Fe3O4 magnetic cores for later use; microcrystalline cellulose powder was taken and 1-butyl-3-methylimidazolium chloride ionic liquid was added at a solid-liquid ratio of 1:8-12 (solid-liquid ratio unit is g / mL). The mixture was stirred at 75-85℃ until completely dissolved. Then, Fe3O4 magnetic cores were added, with a microcrystalline cellulose to Fe3O4 mass ratio of 9-11:1. The mixture was stirred for 25-35 min to uniformly disperse the magnetic cores, resulting in a dispersion. Then, 4-6% (by weight) of polyethylene glycol 6000 was added as a pore-forming agent, and the mixture was stirred for another 18-22 min to obtain a composite dispersion. The above composite dispersion was slowly... Add the microspheres dropwise to deionized water, and continue stirring for 25-35 minutes after the addition is complete. Allow the mixture to cool to 20-25°C, filter and collect the microspheres, and wash them 5-6 times with deionized water. Then, place the microspheres in anhydrous dimethylformamide and soak for 10-20 minutes. Filter and collect the microspheres, and soak them twice. Add a 7-9% benzyl chloride solution prepared with anhydrous dimethylformamide at a solid-liquid ratio of 1:8-12 (unit: g / mL). Simultaneously, add triethylamine as an acid-binding agent. The molar ratio of benzyl chloride to triethylamine is 1:1.2. The mixture is stirred at a constant temperature of 48-52°C under nitrogen protection for 1.5-2 minutes. After 5 hours of reaction, the microspheres were collected by filtration and washed sequentially with anhydrous dimethylformamide, anhydrous ethanol, and deionized water. The microspheres were then immersed in a 4-6% aqueous solution of octyltriethoxysilane in 95% ethanol at 48-52°C for 1.8-2.2 hours. After the reaction, the microspheres were collected by filtration and washed 2-3 times with anhydrous ethanol, followed by 2-3 times with deionized water. They were then vacuum dried at 60-65°C and a vacuum degree of -80 to -100 kPa for 1.5-2.5 hours to obtain iron(III) oxide-cellulose porous microspheres, which were stored in a sealed container in the dark and in a dry place.

[0012] Furthermore, in step S5, the magnetic field strength is 0.3-0.5T, the mixture is allowed to stand for 5-10 minutes, and the microsphere precipitate is washed 2-3 times with phosphate buffer.

[0013] Furthermore, in step S6, the amount of eluent added is 2.5-3.5 times the volume of the microspheres, and the eluent is composed of 95% ethanol, ethyl acetate and 0.1% butyl hydroxytoluene solution in a volume ratio of 1:1:0.1, and is eluted at 33-37°C with shaking at 150-250 rpm for 25-35 minutes.

[0014] Furthermore, in step S7, the volume is concentrated to 15-25% of the original volume under reduced pressure at 38-42℃ and a vacuum of -80 to -100 kPa, and then freeze-dried to constant weight at -40 to -50℃ and a vacuum of 10-30 Pa.

[0015] Furthermore, the astaxanthin obtained above can be applied to water-soluble fertilizers, aquaculture feed, food, and cosmetics.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention employs a complex enzyme consisting of cellulase, alginate lyase, and mannanase to synergistically hydrolyze the multilayered polysaccharide structure of thick-walled spores of Haematococcus pluvialis, thereby fully releasing astaxanthin. Furthermore, porous iron oxide-cellulose microspheres with surface-modified benzyl groups (π-π stacking) and octyl groups (hydrophobic interaction) are used for highly selective adsorption of astaxanthin, combined with magnetic separation technology, thereby improving the astaxanthin yield.

[0017] 2. The extraction process of this invention is carried out under low or medium temperature conditions, and the antioxidant butylated hydroxytoluene (BHT) is added to the eluent, effectively avoiding thermal degradation, photolysis, and oxidation of astaxanthin. The resulting astaxanthin exhibits significantly lower half-maximal inhibitory concentrations (IC50) against DPPH and ABTS free radicals than commercially available products, and retains high antioxidant activity, making it suitable for food, cosmetics, and pharmaceutical fields where strict activity requirements exist.

[0018] 3. The magnetic microspheres of this invention can be recovered through magnetic separation and reused at least 5 times after washing, with an adsorption capacity decrease of no more than 10%. At the same time, magnetic separation replaces the traditional centrifugation or filtration steps, which is simple to operate, has low energy consumption, and reduces the production cost and environmental burden of astaxanthin extraction. Detailed Implementation

[0019] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0020] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0021] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Example 1

[0022] A method for extracting astaxanthin from Haematococcus pluvialis includes the following steps: S1. Raw material pretreatment: Select dried Haematococcus pluvialis powder, add citrate-sodium dihydrogen phosphate buffer solution with pH 5.5 at a solid-liquid ratio of 1:15 (solid-liquid ratio unit is g / mL), and shake in a water bath at 120 rpm for 35 min at 25℃ to obtain a suspension.

[0023] S2. Enzymatic hydrolysis with a complex enzyme: A complex enzyme consisting of cellulase, alginate lyase, and mannanase was added to the suspension and enzymatically hydrolyzed at 38°C for 4.5 h, with stirring every 0.5 h. The mass ratio of cellulase, alginate lyase, and mannanase was 3:2.5:2.5. After the enzymatic hydrolysis was completed, the temperature was raised to 90°C and held for 15 min to inactivate the enzyme. Then the temperature was cooled to 25°C to obtain the enzymatic hydrolysate.

[0024] S3. Surfactant to aid solubilization: Add 1 mol / L sodium hydroxide solution to the enzymatic hydrolysate to adjust the pH to 6.8, then add 0.3% of polysorbate 80 (by volume of the enzymatic hydrolysate), stir at 150 rpm for 35 min until homogeneous, then centrifuge at 3000 rpm for 15 min and collect the supernatant.

[0025] S4. Adsorption of Fe3O4-cellulose porous microspheres: Fe3O4-cellulose porous microspheres were added to the supernatant. The solid-liquid ratio of the Fe3O4-cellulose porous microspheres to the supernatant was 1:20, and the solid-liquid ratio was expressed in g / mL. The adsorption was carried out at 25℃ with shaking at 150 rpm for 2.2 h.

[0026] S5. Magnetic separation and washing: After adsorption, the microspheres were placed in a magnetic field with a strength of 0.3T and allowed to stand for 10 minutes. The supernatant was removed to obtain microsphere precipitate. The microsphere precipitate was then washed twice with phosphate buffer.

[0027] S6. Astaxanthin elution and microsphere recovery: Eluent was added to the washed microsphere precipitate at a volume of 2.5 times the volume of the microspheres. The eluent consisted of a solution of 95% ethanol, ethyl acetate, and 0.1% butylated hydroxytoluene in a volume ratio of 1:1:0.1. The eluent was shaken at 150 rpm for 35 min at 33 °C. After elution, the microspheres were placed in a magnetic field with a strength of 0.3 T and allowed to stand for 10 min. The eluent was collected. The elution was repeated twice, and the eluents were combined. The eluted microspheres were washed with purified water and recovered.

[0028] S7. Concentration and drying: The combined eluent was concentrated to 25% of its original volume at 38°C and a vacuum of -80 to -100 kPa, and then freeze-dried to constant weight at -40°C and a vacuum of 10 Pa to obtain astaxanthin.

[0029] The Fe3O4-cellulose porous microspheres were prepared by the following method: FeCl3·6H2O and FeSO4·7H2O were dissolved in deionized water at a molar ratio of 2:1, and the mixture was protected by nitrogen gas. The mixture was stirred at 30°C until completely dissolved. A 2 mol / L NaOH solution was slowly added dropwise to adjust the pH to 10.0. The mixture was reacted at 30°C for 1.1 h. The Fe3O4 nanoparticles were collected by centrifugation, washed with deionized water until neutral, and then washed once with anhydrous ethanol. Finally, the mixture was vacuum dried at 60°C and -80 kPa for 2.2 h to obtain Fe3O4 magnetic nanoparticles. Core, for later use; Take microcrystalline cellulose powder and add 1-butyl-3-methylimidazolium chloride ionic liquid at a solid-liquid ratio of 1:8 (solid-liquid ratio unit is g / mL). Stir at 75℃ until completely dissolved at a stirring speed of 120 r / min. Add Fe3O4 magnetic cores (microcrystalline cellulose to Fe3O4 mass ratio 9:1). Stir for 25 min to uniformly disperse the magnetic cores to obtain a dispersion. Then add 4% (by weight) of polyethylene glycol 6000 as a pore-forming agent and continue stirring for 18 min to obtain a composite dispersion. Slowly drop the above composite dispersion into deionized water, accelerating the dropping process. The microspheres were added at a rate of 0.5 mL / min, with stirring maintained during the process at a speed of 100 rpm. After the addition was complete, stirring was continued at 100 rpm for 35 min. The mixture was then allowed to cool to 20°C, filtered to collect the microspheres, and washed 5 times with deionized water. Subsequently, the microspheres were placed in anhydrous dimethylformamide and soaked for 10 min. The microspheres were then filtered to collect the microspheres and soaked twice. A 7% benzyl chloride solution prepared with anhydrous dimethylformamide was added at a solid-liquid ratio of 1:8 (solid-liquid ratio in g / mL). Triethylamine was added as an acid-binding agent, with a molar ratio of benzyl chloride to triethylamine of 1:1. 2. The reaction was carried out at 48℃ under nitrogen protection with constant temperature stirring for 2.5 h. After the reaction was completed, the microspheres were collected by filtration and washed successively with anhydrous dimethylformamide, anhydrous ethanol, and deionized water. Then, the microspheres were immersed in a 4% concentration of octyltriethoxysilane in 95% ethanol aqueous solution and the immersion reaction was carried out at 48℃ for 2.2 h. After the reaction was completed, the microspheres were collected by filtration and washed twice with anhydrous ethanol and then twice with deionized water. Then, they were dried under vacuum at 60℃ and -80 kPa for 2.5 h to obtain iron oxide-cellulose porous microspheres, which were placed in a sealed container and stored in a dry place away from light.

[0030] Furthermore, the astaxanthin obtained above can be applied to water-soluble fertilizers, aquaculture feed, food, and cosmetics. Example 2

[0031] A method for extracting astaxanthin from Haematococcus pluvialis includes the following steps: S1. Raw material pretreatment: Select dried Haematococcus pluvialis powder, add citrate-sodium dihydrogen phosphate buffer solution with pH 5.7 at a solid-liquid ratio of 1:18 (solid-liquid ratio unit is g / mL), and shake in a water bath at 28℃ and 150 rpm for 30 min to obtain a suspension.

[0032] S2. Enzymatic hydrolysis with a complex enzyme: A complex enzyme consisting of cellulase, alginate lyase, and mannanase was added to the suspension and enzymatically hydrolyzed at 42°C for 4.0 h, with stirring every 0.5 h during the process. The mass ratio of cellulase, alginate lyase, and mannanase was 4:3:3. After the enzymatic hydrolysis was completed, the temperature was raised to 92°C and held for 12.5 min to inactivate the enzyme. Then the temperature was cooled to 25°C to obtain the enzymatic hydrolysate.

[0033] S3. Surfactant to aid solubilization: Add 1.5 mol / L sodium hydroxide solution to the enzymatic hydrolysate to adjust the pH to 7.0, then add 0.5% of polysorbate 80 (by volume of the enzymatic hydrolysate), stir at 200 rpm for 30 min until homogeneous, then centrifuge at 4000 rpm for 12.5 min and collect the supernatant.

[0034] S4. Adsorption of Fe3O4-cellulose porous microspheres: Fe3O4-cellulose porous microspheres were added to the supernatant. The solid-liquid ratio of the Fe3O4-cellulose porous microspheres to the supernatant was 1:25, and the unit of solid-liquid ratio was g / mL. The adsorption was carried out at 28℃ with shaking at 180 rpm for 2 h.

[0035] S5. Magnetic separation and washing: After adsorption, the microspheres were placed in a magnetic field with a strength of 0.4T and allowed to stand for 8 minutes. The supernatant was removed to obtain microsphere precipitate. The microsphere precipitate was then washed twice with phosphate buffer.

[0036] S6. Astaxanthin elution and microsphere recovery: Eluent was added to the washed microsphere precipitate at a volume of 3 times the volume of the microspheres. The eluent consisted of a solution of 95% ethanol, ethyl acetate, and 0.1% butylated hydroxytoluene in a volume ratio of 1:1:0.1. The mixture was eluted at 35°C and 200 rpm for 30 min. After elution, the mixture was placed in a magnetic field with a strength of 0.4T and allowed to stand for 8 min. The eluent was collected. The elution was repeated 3 times, and the eluents were combined. The eluted microspheres were washed with purified water and recovered.

[0037] S7. Concentration and drying: The combined eluent was concentrated to 20% of its original volume under reduced pressure at 40°C and -90 kPa, and then freeze-dried to constant weight at -45°C and 20 Pa to obtain astaxanthin.

[0038] The iron(III) oxide-cellulose porous microspheres were prepared by the following method: FeCl3·6H2O and FeSO4·7H2O were dissolved in deionized water at a molar ratio of 2:1, under nitrogen protection, and stirred at 33°C until completely dissolved. A 2 mol / L NaOH solution was slowly added dropwise to adjust the pH to 10.0, and the reaction was maintained at 33°C for 1 hour. The Fe3O4 nanoparticles were collected by centrifugation, washed with deionized water until neutral, and then washed once with anhydrous ethanol. Subsequently, they were vacuum dried at 62°C and -90 kPa for 2 hours to obtain Fe3O4 magnetic cores. Take microcrystalline cellulose powder and add 1-butyl-3-methylimidazolium chloride ionic liquid at a solid-liquid ratio of 1:10 (solid-liquid ratio unit is g / mL). Stir at 80℃ until completely dissolved at a stirring speed of 150 r / min. Add Fe3O4 magnetic cores (microcrystalline cellulose to Fe3O4 mass ratio of 10:1) and stir for 30 min to uniformly disperse the magnetic cores to obtain a dispersion. Then add 5% (by mass) of polyethylene glycol 6000 as a pore-forming agent and continue stirring for 20 min to obtain a composite dispersion. Slowly drop the above composite dispersion into deionized water, accelerating the dropping process. The addition rate was 0.8 mL / min, with stirring maintained during the process at a speed of 120 rpm. After the addition was complete, stirring continued at 120 rpm for 30 min. The mixture was allowed to cool to 22°C, and the microspheres were collected by filtration and washed 6 times with deionized water. Subsequently, the microspheres were placed in anhydrous dimethylformamide and soaked for 15 min. The microspheres were collected by filtration and soaked twice. An 8% benzyl chloride solution prepared with anhydrous dimethylformamide was added at a solid-liquid ratio of 1:10 (solid-liquid ratio in g / mL). Triethylamine was added as an acid-binding agent, with a molar ratio of benzyl chloride to triethylamine of 1:10. 1.2 The reaction was carried out at 50℃ under nitrogen protection with constant temperature stirring for 2 hours. After the reaction was completed, the microspheres were collected by filtration and washed successively with anhydrous dimethylformamide, anhydrous ethanol, and deionized water. Then, the microspheres were immersed in a 5% concentration of octyltriethoxysilane in 95% ethanol aqueous solution and the immersion reaction was carried out at 50℃ for 2 hours. After the reaction was completed, the microspheres were collected by filtration and washed 3 times with anhydrous ethanol and then 2 times with deionized water. Then, they were vacuum dried at 62℃ and vacuum degree -90kPa for 2 hours to obtain iron oxide-cellulose porous microspheres, which were placed in a sealed container and stored in a dry place away from light.

[0039] Furthermore, the astaxanthin obtained above can be applied to water-soluble fertilizers, aquaculture feed, food, and cosmetics. Example 3

[0040] A method for extracting astaxanthin from Haematococcus pluvialis includes the following steps: S1. Raw material pretreatment: Select dried Haematococcus pluvialis powder, add citrate-sodium dihydrogen phosphate buffer solution with pH 6.0 at a solid-liquid ratio of 1:20 (solid-liquid ratio unit is g / mL), and shake in a water bath at 30℃ and 180 rpm for 25 min to obtain a suspension.

[0041] S2. Enzymatic hydrolysis with a complex enzyme: A complex enzyme consisting of cellulase, alginate lyase, and mannanase was added to the suspension and enzymatically hydrolyzed at 45°C for 3.5 h, with stirring every 0.5 h during the process. The mass ratio of cellulase, alginate lyase, and mannanase was 5:3.5:3.5. After the enzymatic hydrolysis was completed, the temperature was raised to 95°C and held for 10 min to inactivate the enzyme. Then the temperature was cooled to 25°C to obtain the enzymatic hydrolysate.

[0042] S3. Surfactant to aid solubilization: Add 2 mol / L sodium hydroxide solution to the enzymatic hydrolysate to adjust the pH to 7.2, then add 0.8% of polysorbate 80 (by volume of the enzymatic hydrolysate), stir at 250 rpm for 25 min until homogeneous, then centrifuge at 5000 rpm for 10 min and collect the supernatant.

[0043] S4. Adsorption of Fe3O4-cellulose porous microspheres: Fe3O4-cellulose porous microspheres were added to the supernatant. The solid-liquid ratio of the Fe3O4-cellulose porous microspheres to the supernatant was 1:30, and the solid-liquid ratio was expressed in g / mL. The adsorption was carried out at 30℃ with shaking at 200 rpm for 1.8 h.

[0044] S5. Magnetic separation and washing: After adsorption, the microspheres were placed in a magnetic field with a strength of 0.5T and allowed to stand for 5 minutes. The supernatant was removed to obtain microsphere precipitate. The microsphere precipitate was then washed three times with phosphate buffer.

[0045] S6. Astaxanthin elution and microsphere recovery: Eluent was added to the washed microsphere precipitate at a volume of 3.5 times the volume of the microspheres. The eluent consisted of a solution of 95% ethanol, ethyl acetate, and 0.1% butylated hydroxytoluene in a volume ratio of 1:1:0.1. The eluent was shaken at 250 rpm for 25 min at 37 °C. After elution, the microspheres were placed in a magnetic field with a strength of 0.5 T and allowed to stand for 5 min. The eluent was collected. The elution was repeated 3 times, and the eluents were combined. The eluted microspheres were washed with purified water and recovered.

[0046] S7. Concentration and drying: The combined eluent was concentrated to 15% of its original volume at 42°C and a vacuum of -100 kPa, and then freeze-dried to constant weight at -50°C and a vacuum of 30 Pa to obtain astaxanthin.

[0047] The Fe3O4-cellulose porous microspheres were prepared by the following method: FeCl3·6H2O and FeSO4·7H2O were dissolved in deionized water at a molar ratio of 2:1, and the mixture was protected with nitrogen gas and stirred at 35°C until completely dissolved. A 2 mol / L NaOH solution was slowly added dropwise to adjust the pH to 10.0, and the reaction was carried out at 35°C for 1.1 h. The mixture was then centrifuged, and the Fe3O4 nanoparticles were collected, washed with deionized water until neutral, and then washed once with anhydrous ethanol. Finally, the nanoparticles were vacuum dried at 65°C and -100 kPa for 1.8 h to obtain Fe3O4 magnetic nanoparticles. Core, for later use; Take microcrystalline cellulose powder and add 1-butyl-3-methylimidazolium chloride ionic liquid at a solid-liquid ratio of 1:12 (solid-liquid ratio unit is g / mL). Stir at 85℃ until completely dissolved, stirring speed 180 r / min. Add Fe3O4 magnetic core, with a microcrystalline cellulose to Fe3O4 mass ratio of 11:1. Stir for 25 min to uniformly disperse the magnetic core to obtain a dispersion. Then add 6% (by mass) of polyethylene glycol 6000 as a pore-forming agent to the dispersion and continue stirring for 22 min to obtain a composite dispersion. Slowly drop the above composite dispersion into deionized water, accelerating the dropping process. The microspheres were added at a rate of 1 mL / min, with stirring maintained during the process at a speed of 150 rpm. After the addition was complete, stirring was continued at 150 rpm for 25 min. The mixture was then allowed to cool to 25°C, filtered to collect the microspheres, and washed 6 times with deionized water. Subsequently, the microspheres were placed in anhydrous dimethylformamide and soaked for 20 min. The microspheres were then filtered to collect the microspheres, and the soaking was repeated twice. A 9% benzyl chloride solution prepared with anhydrous dimethylformamide was added at a solid-liquid ratio of 1:12 (solid-liquid ratio in g / mL). Triethylamine was added as an acid-binding agent, with a molar ratio of benzyl chloride to triethylamine of 1:1.2. The reaction was carried out at 52℃ under nitrogen protection with constant temperature stirring for 1.5 h. After the reaction was completed, the microspheres were collected by filtration and washed successively with anhydrous dimethylformamide, anhydrous ethanol, and deionized water. Then, the microspheres were immersed in a 6% concentration of octyltriethoxysilane in 95% ethanol aqueous solution and reacted at 52℃ for 1.8 h. After the reaction was completed, the microspheres were collected by filtration and washed three times with anhydrous ethanol and then three times with deionized water. Then, they were vacuum dried at 65℃ and vacuum degree -100 kPa for 1.5 h to obtain iron oxide-cellulose porous microspheres, which were stored in a sealed container in the dark and in a dry place.

[0048] Furthermore, the astaxanthin obtained above can be applied to water-soluble fertilizers, aquaculture feed, food, and cosmetics. Comparative Example 1

[0049] Compared with Example 2, the difference in this comparative example is that only an equal mass of cellulase was used instead of the complex enzyme in step S2. Comparative Example 2

[0050] Compared with Example 2, this comparative example differs in that it does not use iron tetroxide-cellulose porous microspheres during the extraction process. The method for extracting astaxanthin from Haematococcus pluvialis includes the following steps: S1. Raw material pretreatment: Select dried Haematococcus pluvialis powder, add citrate-sodium dihydrogen phosphate buffer solution with pH 5.7 at a solid-liquid ratio of 1:18 (solid-liquid ratio unit is g / mL), and shake in a water bath at 28℃ and 150 rpm for 30 min to obtain a suspension.

[0051] S2. Enzymatic hydrolysis with a complex enzyme: A complex enzyme consisting of cellulase, alginate lyase, and mannanase was added to the suspension and enzymatically hydrolyzed at 42°C for 4.0 h, with stirring every 0.5 h during the process. The mass ratio of cellulase, alginate lyase, and mannanase was 4:3:3. After the enzymatic hydrolysis was completed, the temperature was raised to 92°C and held for 12.5 min to inactivate the enzyme. Then the temperature was cooled to 25°C to obtain the enzymatic hydrolysate.

[0052] S3. Surfactant to aid solubilization: Add 1.5 mol / L sodium hydroxide solution to the enzymatic hydrolysate to adjust the pH to 7.0, then add 0.5% of polysorbate 80 (by volume of the enzymatic hydrolysate), stir at 200 rpm for 30 min until homogeneous, then centrifuge at 4000 rpm for 12.5 min and collect the supernatant.

[0053] S4. Extraction: Transfer the supernatant to a separatory funnel, add an equal volume of ethyl acetate, shake at 250 rpm for 5 min, allow to stand for separation, and collect the upper organic phase. Repeat the extraction twice with an equal volume of ethyl acetate, and combine the organic phases. Wash the combined organic phase once with an equal volume of saturated brine, then dry with anhydrous sodium sulfate, filter to remove the desiccant, and obtain an astaxanthin organic solution.

[0054] S5. Concentration and drying: The astaxanthin organic solution was concentrated to 20% of its original volume at 40℃ and a vacuum of -90kPa, and then freeze-dried to constant weight at -45℃ and a vacuum of 20Pa to obtain astaxanthin. Comparative Example 3

[0055] Compared with Example 2, the difference in this comparative example is that the iron(III) oxide-cellulose porous microspheres were prepared by the following method: FeCl3·6H2O and FeSO4·7H2O were dissolved in deionized water at a molar ratio of 2:1, under nitrogen protection, and stirred at 33°C until completely dissolved. A 2 mol / L NaOH solution was slowly added dropwise to adjust the pH to 10.0, and the reaction was carried out at 33°C for 1 hour. The Fe3O4 nanoparticles were collected by centrifugation, washed with deionized water until neutral, and then washed once with anhydrous ethanol. Subsequently, they were vacuum dried at 62°C and -90 kPa for 2 hours to obtain Fe3O4 magnetic cores for later use. Microcrystalline cellulose powder was taken and 1-butyl-3-methylimidazolium chloride ionic liquid was added at a solid-liquid ratio of 1:10 (solid-liquid ratio in g / mL). The mixture was stirred at 80°C until... Completely dissolve the microcrystalline cellulose (MCC) and add Fe3O4 magnetic cores at a stirring speed of 150 r / min. The mass ratio of MCC to Fe3O4 is 10:1. Stir for 30 min to ensure uniform dispersion of the magnetic cores and obtain a dispersion. Then, add 5% (by weight) of polyethylene glycol 6000 as a pore-forming agent to the dispersion and continue stirring for 20 min to obtain a composite dispersion. Slowly drop the above composite dispersion into deionized water at a dropping rate of 0.8 mL / min, while maintaining stirring at a stirring speed of 120 r / min. After the addition is complete, continue stirring at 120 r / min for 30 min. Allow the mixture to stand and cool to 22 °C, filter and collect the microspheres, wash them 6 times with deionized water, and then vacuum dry them at 62 °C and a vacuum degree of -90 kPa for 2 h to obtain iron oxide-cellulose porous microspheres. Store them in a sealed container in the dark and dry place. Astaxanthin yield detection

[0056] Using the same batch of Haematococcus pluvialis dried algae powder, extraction was performed according to the extraction methods of Examples 1-3 and Comparative Examples 1-3, with three parallel samples in each group, and 5.00 g of dried algae powder was taken from each parallel sample. The astaxanthin extracted from each group was accurately weighed, and 5.0 mg was dissolved in 1 mL of 95% ethanol aqueous solution containing 0.01% BHT. After ultrasonic filtration, the astaxanthin content was determined by HPLC external standard method (chromatographic column C18, mobile phase methanol:acetonitrile:water = 85:10:5, detection wavelength 474 nm). The mass of astaxanthin in the sample was calculated according to the standard curve, and the yield was calculated using the following formula: Astaxanthin yield (mg / g) = (mass of astaxanthin) / (mass of initial dried algae powder). The average value of each group was recorded in Table 1.

[0057] Table 1

[0058] Comparing Example 2 with Comparative Example 1, Example 2 used a composite membrane composed of cellulase, alginate lyase, and mannanase in a specific ratio. These three enzymes acted on cellulose, alginate, and mannan respectively, forming a synergistic hydrolysis effect. Cellulase cleaved β-1,4 glycosidic bonds, disintegrating the cellulose backbone; alginate lyase degraded alginate through a β-elimination reaction, eliminating the colloidal barrier; and mannanase hydrolyzed mannan, further loosening the inner cell wall. When the three enzymes acted simultaneously, various structural polysaccharides of the cell wall were decomposed synchronously, exposing more astaxanthin-rich plasma membranes and lipid droplets, allowing for efficient release of astaxanthin into the liquid phase. In contrast, Comparative Example 1 used only an equal mass of a single cellulase, which could not effectively remove alginate and mannan. The cell wall was not completely damaged, and a large amount of astaxanthin remained encapsulated in the residual cell wall, resulting in a low release rate and a significantly reduced yield.

[0059] Comparing Example 2 with Comparative Examples 2-3, Comparative Example 2 used traditional extraction. Although ethyl acetate can dissolve astaxanthin, a large number of lipophilic impurities (chlorophyll, fatty acids, etc.) simultaneously entered the organic phase during extraction, resulting in low product purity. During subsequent concentration and drying, impurities may encapsulate astaxanthin to form a viscous paste, and some astaxanthin may degrade due to oxidation or mechanical loss, resulting in a low actual yield of active ingredients. Comparative Example 3 used magnetic microspheres without benzyl and silane modification. Their surface only had cellulose hydroxyl groups, and astaxanthin was only adsorbed through weak hydrogen bonds and physical adsorption, resulting in low adsorption capacity and poor selectivity, thus the yield was significantly lower than that of this method. The iron oxide-cellulose porous microspheres used in Example 2 underwent two-step functionalization modification: first, benzyl was grafted to provide π-π stacking sites, and then octyltriethoxysilane was bonded to provide a hydrophobic surface. Astaxanthin molecules possess long conjugated polyene chains and aromatic cyclohexenone rings at both ends, enabling them to undergo π-π stacking interactions with the benzene ring of benzyl groups. Simultaneously, their hydrophobic long chains interact hydrophobically with the alkyl chains of octyltriethoxysilanes. This dual non-covalent interaction gives the microspheres high affinity and selectivity for astaxanthin, allowing them to actively adsorb astaxanthin from complex enzymatic hydrolysis supernatants (containing impurities such as polysaccharides, proteins, and chlorophyll). The use of an ethanol / ethyl acetate / BHT system during the elution stage effectively disrupts the π-π stacking and hydrophobic interactions, resulting in complete elution of astaxanthin. Therefore, Example 2 shows a significantly higher astaxanthin yield compared to Comparative Examples 2-3. Product activity Comparative Example 4

[0060] The difference between this comparative example and Example 2 is that the astaxanthin used is commercially available astaxanthin.

[0061] Astaxanthin from Example 2 and Comparative Example 4 were weighed separately to determine their DPPH and ABTS free radical scavenging abilities. Three parallel samples were set up for each group. 5.0 mg of each parallel sample was weighed and dissolved in 10 mL of anhydrous ethanol containing 0.01% BHT. The solutions were sonicated to prepare a 0.5 mg / mL stock solution, which was then serially diluted with anhydrous ethanol to a series of concentrations of 2.5, 5, 10, 20, 40, and 80 μg / mL for IC50 determination.

[0062] DPPH free radical scavenging capacity determination: Mix 2 mL of each concentration sample solution with 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution, incubate at room temperature in the dark for 30 min, and measure the absorbance at 517 nm (sample A). A blank control was set up: 2 mL sample solution + 2 mL anhydrous ethanol (background A), and a negative control was set up: 2 mL anhydrous ethanol + 2 mL DPPH solution (control A). The DPPH free radical scavenging rate was calculated using the following formula: DPPH free radical scavenging rate (%) = [1 - (Sample A - Background A) / Control A] × 100% Plot a curve with sample concentration on the x-axis (μg / mL) and clearance rate on the y-axis, and calculate the half-maximal inhibitory concentration (IC50, μg / mL).

[0063] ABTS free radical scavenging capacity determination: Prepare 7 mmol / L ABTS aqueous solution and 2.45 mmol / L potassium persulfate aqueous solution, mix equal volumes, and react at 25°C in the dark for 14 h to obtain ABTS⁺ stock solution.

[0064] Dilute the stock solution with PBS (pH 7.4) to achieve an absorbance of 0.70 at 734 nm.

[0065] Take 0.2 mL of sample solutions of different concentrations, add 3.8 mL of ABTS⁺ working solution, mix well, and react at 25°C in the dark for 6 min.

[0066] Absorbance was measured at 734 nm (sample B). Blank control: 0.2 mL sample solution + 3.8 mL PBS (background B). Negative control: 0.2 mL anhydrous ethanol + 3.8 mL ABTS⁺ working solution (control B). ABTS radical scavenging rate was calculated using the following formula: ABTS free radical scavenging rate (%) = [1 - (Sample B - Background B) / Control B] × 100% Plot a curve with sample concentration on the x-axis (μg / mL) and clearance rate on the y-axis, and calculate the half-maximal inhibitory concentration (IC50, μg / mL).

[0067] IC50 (half-maximal inhibitory concentration) refers to the concentration of astaxanthin required to scavenge 50% of free radicals, usually expressed in μg / mL. A lower IC50 value indicates a lower concentration of astaxanthin required to achieve the same scavenging effect, meaning stronger antioxidant activity. The average value of the IC50 calculated by DPPH and ABTS is recorded in Table 2.

[0068] Table 2

[0069] As shown in Table 2, the IC50 values ​​of Example 2 are all lower than those of commercially available products, indicating that the astaxanthin extracted by this invention has higher antioxidant activity. Because the extraction process of this invention is gentler, it effectively protects the natural structure of astaxanthin. Astaxanthin molecules contain multiple conjugated double bonds, making them sensitive to light, heat, and oxygen, and prone to cis-trans isomerization or oxidative degradation, thus reducing their antioxidant activity. This invention employs medium-to-low temperature operations in the enzymatic hydrolysis, adsorption, elution, and concentration steps, and adds 0.1% butylated hydroxytoluene (BHT) as an antioxidant to the eluent, avoiding high-temperature oxidation and photolysis. Commercially available astaxanthin is usually extracted using traditional organic solvents (which may involve high-temperature reflux or prolonged exposure to air), leading to the oxidation or isomerization of some astaxanthin and a decrease in activity. Therefore, the product obtained by this invention retains a more complete conjugated double bond structure and natural configuration, exhibiting stronger free radical scavenging ability.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting astaxanthin from Haematococcus pluvialis, characterized in that, Includes the following steps: S1. Raw material pretreatment: Select dried Haematococcus pluvialis powder, add citrate-sodium dihydrogen phosphate buffer at a solid-liquid ratio of 1:15-20 (solid-liquid ratio unit is g / mL), shake in a water bath to obtain a suspension; S2. Complex enzyme hydrolysis: A complex enzyme consisting of cellulase, alginate lyase and mannanase is added to the suspension for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated and then cooled to room temperature to obtain the enzymatic hydrolysate. S3. Surfactant to aid solubilization: Add sodium hydroxide solution to the enzymatic hydrolysate to adjust the pH, then add polysorbate 80, stir well, then centrifuge and take the supernatant; S4. Adsorption of Fe3O4-cellulose porous microspheres: Add Fe3O4-cellulose porous microspheres to the supernatant and shake for adsorption treatment; S5. Magnetic separation and washing: After adsorption, the microspheres are placed in a magnetic field environment, allowed to stand, and the supernatant is removed to obtain microsphere precipitate; then the microsphere precipitate is washed with phosphate buffer. S6. Astaxanthin elution and microsphere recovery: Add eluent to the washed microsphere precipitate, shake and elute, place in a magnetic field environment after elution, let stand, and collect the eluent; repeat elution 2-3 times, combine the eluents; wash the eluted microspheres with purified water and recover them; S7. Concentration and drying: The combined eluent was concentrated under reduced pressure and then freeze-dried to obtain astaxanthin.

2. The method for extracting astaxanthin from Haematococcus pluvialis as described in claim 1, characterized in that, In step S1, the pH of the citrate-sodium dihydrogen phosphate buffer solution is 5.5-6.0, the water bath shaking temperature is 25-30℃, the rotation speed is 120-180 rpm, and the shaking time is 25-35 min.

3. The method for extracting astaxanthin from Haematococcus pluvialis as described in claim 1, characterized in that, In step S2, the mass ratio of cellulase, alginate lyase, and mannanase is 3-5:2.5-3.5:2.5-3.

5. Enzymatic hydrolysis is carried out at 38-45℃ for 3.5-4.5 hours, with stirring every 0.5 hours. After the enzymatic hydrolysis is completed, the temperature is raised to 90-95℃ and maintained for 10-15 minutes.

4. The method for extracting astaxanthin from Haematococcus pluvialis as described in claim 1, characterized in that, In step S3, the concentration of sodium hydroxide solution is 1-2 mol / L, adjusted to 6.8-7.2, the amount of polysorbate 80 added is 0.3-0.8% of the volume of the enzymatic hydrolysate, the mixture is stirred at 150-250 rpm for 25-35 min, and the centrifugation speed is 3000-5000 rpm for 10-15 min.

5. The method for extracting astaxanthin from Haematococcus pluvialis as described in claim 1, characterized in that, In step S4, the iron tetroxide-cellulose porous microspheres and the supernatant have a solid-liquid ratio of 1:20-30, with the solid-liquid ratio unit being g / mL. The adsorption is carried out at 25-30℃ with shaking at 150-200 rpm for 1.8-2.2 h.

6. The method for extracting astaxanthin from Haematococcus pluvialis as described in claim 5, characterized in that, The iron(III) oxide-cellulose porous microspheres were prepared by the following method: FeCl3·6H2O and FeSO4·7H2O were dissolved in deionized water at a molar ratio of 2:1, under nitrogen protection, and stirred at 30-35℃ until completely dissolved. A 2 mol / L NaOH solution was slowly added dropwise to adjust the pH to 10.0, and the reaction was carried out at 30-35℃ for 0.9-1.1 h. The mixture was then centrifuged, and the Fe3O4 nanoparticles were collected, washed with deionized water until neutral, and then washed once with anhydrous ethanol. Subsequently, they were vacuum dried at 60-65℃ and a vacuum degree of -80 to -100 kPa for 1.8 hours. -2.2h, Fe3O4 magnetic cores were obtained and set aside. Microcrystalline cellulose powder was taken and 1-butyl-3-methylimidazolium chloride ionic liquid was added at a solid-liquid ratio of 1:8-12 (solid-liquid ratio in g / mL). The mixture was stirred at 75-85℃ until completely dissolved. Then, Fe3O4 magnetic cores were added at a mass ratio of 9-11:1 (microcrystalline cellulose to Fe3O4). The mixture was stirred for 25-35min to ensure uniform dispersion of the magnetic cores, resulting in a dispersion. Then, 4-6% (by weight) of polyethylene glycol 6000 was added as a pore-forming agent, and the mixture was stirred for another 18-22min to obtain a composite dispersion. The above composite dispersion was then slowly dripped... Add the microspheres to deionized water, and after the addition is complete, continue stirring for 25-35 minutes. Allow to cool to 20-25℃, filter and collect the microspheres, and wash with deionized water 5-6 times. Then, place the microspheres in anhydrous dimethylformamide and soak for 10-20 minutes, filter and collect the microspheres, and soak twice. Add a 7-9% benzyl chloride solution prepared with anhydrous dimethylformamide at a solid-liquid ratio of 1:8-12 (solid-liquid ratio unit is g / mL), and simultaneously add triethylamine as an acid-binding agent. The molar ratio of benzyl chloride to triethylamine is 1:1.

2. The reaction is carried out at 48-52℃ under nitrogen protection with constant temperature stirring for 1.5-2.5 minutes. After the reaction was completed, the microspheres were collected by filtration and washed successively with anhydrous dimethylformamide, anhydrous ethanol, and deionized water. Then, the microspheres were immersed in a 4-6% aqueous solution of octyltriethoxysilane in 95% ethanol at 48-52℃ for 1.8-2.2 h. After the reaction was completed, the microspheres were collected by filtration and washed 2-3 times with anhydrous ethanol, and then washed 2-3 times with deionized water. Then, they were vacuum dried at 60-65℃ and a vacuum degree of -80 to -100 kPa for 1.5-2.5 h to obtain iron oxide-cellulose porous microspheres, which were stored in a sealed container in the dark and in a dry place.

7. The method for extracting astaxanthin from Haematococcus pluvialis as described in claim 1, characterized in that, In step S5, the magnetic field strength is 0.3-0.5T, the mixture is left to stand for 5-10 minutes, and the microsphere precipitate is washed 2-3 times with phosphate buffer.

8. The method for extracting astaxanthin from Haematococcus pluvialis as described in claim 1, characterized in that, In step S6, the amount of eluent added is 2.5-3.5 times the volume of the microspheres. The eluent is composed of 95% ethanol, ethyl acetate and 0.1% butyl hydroxytoluene solution in a volume ratio of 1:1:0.

1. The eluent is eluted at 33-37℃ with shaking at 150-250 rpm for 25-35 minutes.

9. The method for extracting astaxanthin from Haematococcus pluvialis as described in claim 1, characterized in that, In step S7, the volume is concentrated to 15-25% of the original volume under reduced pressure at 38-42℃ and a vacuum of -80 to -100 kPa, and then freeze-dried to constant weight at -40 to -50℃ and a vacuum of 10-30 Pa.

10. The application of astaxanthin extracted from Haematococcus pluvialis using the method for extracting astaxanthin according to any one of claims 1-9, characterized in that, The astaxanthin is used in water-soluble fertilizers, aquaculture feed, food, and cosmetics.

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