A method for separating and extracting astaxanthin from haematococcus pluvialis

Abstract

The present application relates to natural product separation and extraction technical field, specifically disclose a kind of method for separating and extracting astaxanthin with haematococcus pluvialis as raw material, comprising the following steps: (1) haematococcus pluvialis pretreatment preparation haematococcus pluvialis dry powder;(2) using ultrasonic-assisted natural deep eutectic solvent separates and extracts astaxanthin from haematococcus pluvialis dry powder and obtains crude extract;(3) using macroporous resin dynamic adsorption crude extract and purifying and collecting astaxanthin eluate;(4) astaxanthin eluate is concentrated by rotary evaporation under reduced pressure, freeze-dried, and astaxanthin crystal powder is obtained.The method provided by the present application effectively improves the extraction rate of astaxanthin by the ternary synergy of natural deep eutectic solvent, the trans-phase antioxidant synergy of tocopherol and ascorbyl palmitate, and the mild process of ultrasonic-assisted extraction and resin gradient elution, maintains the purity of astaxanthin and the proportion of all-trans astaxanthin, and prolongs the antioxidant retention time of astaxanthin.

Description

Technical Field

[0001] This invention relates to the field of natural product separation and extraction technology, and more specifically, to a method for separating and extracting astaxanthin from Haematococcus pluvialis. Background Technology

[0002] Haematococcus pluvialis is currently the highest known biological source of natural astaxanthin, with astaxanthin content reaching up to approximately 5.0% of cell dry weight under stress conditions such as nitrogen deficiency and high light intensity. However, under stress, Haematococcus pluvialis forms a three-layered, anti-acetylation cell wall approximately 2 μm thick, becoming the strongest barrier to astaxanthin extraction. Existing extraction technologies have several shortcomings: organic solvent extraction carries the risk of excessive solvent residue and poor selectivity; supercritical CO2 extraction faces bottlenecks such as insufficient cell wall disruption efficiency and high equipment costs; high-pressure homogenization can maintain astaxanthin activity, but the disruption rate is only about 70%, requiring pressures above 300 MPa to achieve a disruption rate of over 90%, posing high safety risks and consuming enormous amounts of energy; microwave-assisted extraction suffers from 10%–15% degradation of astaxanthin and cis-trans isomerization due to localized high temperatures. Furthermore, existing technologies generally suffer from fragmented extraction and purification separation steps and low purity of the target product. Therefore, developing a green, efficient, and highly selective integrated method for astaxanthin extraction and purification is of significant industrial importance. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a method for separating and extracting astaxanthin from Haematococcus pluvialis.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] A method for separating and extracting astaxanthin from Haematococcus pluvialis includes the following steps:

[0006] (1) Take Haematococcus pluvialis spores, freeze dry them in a vacuum freeze dryer, grind them at low temperature, pass them through an 80-120 mesh sieve, seal them and store them in a cool, dark place to obtain Haematococcus pluvialis dry powder;

[0007] (2) Weigh out 10-12 parts of Haematococcus pluvialis powder, 120-240 parts of natural eutectic solvent, 0.1-0.3 parts of tocopherol, and 0.08-0.1 parts of ascorbate palmitate by weight.

[0008] (3) Add dried Haematococcus pluvialis, 4 / 5 of the natural eutectic solvent, tocopherol, and ascorbyl palmitate to the ultrasonic reactor in sequence. After purging the oxygen with nitrogen, seal the reactor and extract by ultrasonic stirring. After extraction, cool the reactor and let it stand in the dark to obtain a mixture.

[0009] (4) The mixture was placed in a centrifuge and centrifuged at low temperature to obtain supernatant I and algal residue;

[0010] (5) Add the algal residue to the remaining 1 / 5 of the natural eutectic solvent, mix well and let stand, then place it in a centrifuge to separate it. After centrifugation, obtain supernatant II. Combine supernatant I and supernatant II to obtain crude extract.

[0011] (6) Soak HPD-600 resin in an ethanol-water mixed solvent, then wash it in sequence with deionized water, sodium hydroxide aqueous solution, dilute hydrochloric acid and deionized water until neutral, drain it, pack it into a column with a column diameter-to-height ratio of 1:5~8, and equilibrate it with 3 times the column volume of deionized water to obtain a resin column.

[0012] (7) Pass the crude extract through a resin column and elute with a 30%~40% ethanol aqueous solution at a flow rate of 1~2 BV / h to remove polar impurities. Then elute with an 85%~95% ethanol aqueous solution at a flow rate of 0.5~1 BV / h and collect the astaxanthin eluent. Wash the resin column sequentially with 2~3 BV of 90%~95% ethanol aqueous solution, 2~3 BV of sodium hydroxide aqueous solution, and 3~5 BV of deionized water until neutral and regenerate for reuse.

[0013] (8) Place the collected astaxanthin eluent in a rotary evaporator and evaporate under reduced pressure to 1 / 20 to 1 / 10 of the original volume to obtain astaxanthin concentrate;

[0014] (9) After freezing and solidifying the astaxanthin concentrate at -20~-16℃, place it in a vacuum freeze dryer and freeze dry it at -50~-40℃ and 8~10Pa for 24~36h. Then, seal and store it in a cool, dark place at -6~-5℃ to obtain astaxanthin crystal powder.

[0015] Furthermore, in step (1), the freeze-drying conditions are: temperature -40~-30℃, vacuum degree 10~30Pa, freeze-drying time 24~48h, low-temperature grinding temperature 6~10℃, and sealed light-proof refrigerated storage temperature -8~-4℃.

[0016] Furthermore, the preparation method of the natural eutectic solvent in step (2) includes the following steps:

[0017] S1. Weigh out 30-60 parts betaine, 50-100 parts natural organic acid, 20-40 parts D-glucose, and 20-40 parts deionized water by weight.

[0018] S2. Mix betaine, natural organic acid, D-glucose and deionized water in sequence and stir at 200-300 rpm for 40-60 min in a water bath at 50-60℃ to obtain a natural eutectic solvent.

[0019] Furthermore, the natural organic acid in step S1 includes at least one of lactic acid, citric acid, and malic acid.

[0020] Further, in step (3), the flow rate of nitrogen gas is 0.5~1L / min, the conditions for ultrasonic stirring extraction are power 300~600W, frequency 20~25kHz, temperature 45~65℃, rotation speed 150~300rpm, ultrasonic stirring extraction time is 40~80min, cooling temperature is 20~30℃, and the time for standing in the dark is 20~30min.

[0021] Furthermore, in step (4), the temperature of low-temperature centrifugation is 4~8℃, the centrifugation speed is 8000~12000rpm, and the separation time is 15~25min.

[0022] Furthermore, in step (5), the settling time is 10-20 min, the centrifugation speed is 6000-10000 rpm, and the time is 10-20 min.

[0023] Furthermore, in step (6), the mass ratio of ethanol to water in the ethanol-water mixed solvent is 90~95:100, and the soaking time is 20~24h.

[0024] Furthermore, in step (7), the flow rate of the crude extract through the resin column is 0.5~1.5 BV / h, and the sample loading volume is 2~4 BV.

[0025] Furthermore, the conditions for vacuum rotary evaporation in step (8) are a temperature of 45~55℃ and a vacuum degree of -0.09~-0.08MPa.

[0026] In summary, this application includes at least the following beneficial effects:

[0027] (1) The natural eutectic solvent used in this invention is composed of betaine, natural organic acid and D-glucose. The zwitterionic properties of betaine can effectively interfere with the hydrophobic interaction and hydrogen bonding between proteins and astaxanthin in thick-walled spores of Haematococcus pluvialis, promoting the release of astaxanthin from the bound state; the natural organic acid provides abundant carboxyl and hydroxyl groups, forming a stable hydrogen bond network with betaine, providing a suitable polar solvation environment for the released astaxanthin, preventing astaxanthin molecules from π-π stacking and precipitating; D-glucose has 4 hydroxyl groups distributed on its pyranose ring and has a chair configuration, which enables it to form hydrogen bonds with multiple betaine and natural organic acid molecules at the same time, constructing a three-dimensional network with a rigid skeleton and flexible branches. The rigid pyranose ring structure of D-glucose acts as a "molecular wedge" inserted between betaine and organic acid, destroying the dense hydrogen bond network and significantly reducing the viscosity of the system. Meanwhile, the thick-walled spore cell walls of Haematococcus pluvialis are mainly composed of cellulose, hemicellulose, and callose. These polysaccharides form a rigid barrier through dense intramolecular and intermolecular hydrogen bonds. D-glucose, as a monosaccharide, has a much smaller molecular volume than polysaccharides, but it is also rich in hydroxyl groups. When a natural eutectic solvent permeates into the cell wall, the hydroxyl groups on the glucose molecule form competitive hydrogen bonds with the hydroxyl groups on the cell wall polysaccharide chains, vying for hydrogen bond sites originally used to maintain the stability of the cell wall structure. This competitive binding causes local "loosening" or even "breakage" of the hydrogen bond network between the cell wall polysaccharide chains, thereby softening or destroying the cell wall structure, making it easier for astaxanthin to be released. Furthermore, astaxanthin molecules have long conjugated polyene chains, which are highly susceptible to oxidation and cis-trans isomerization. In solution, the polyene chains tend to fold or aggregate through hydrophobic interactions. The axial hydrogen atoms on the pyranose ring of glucose carry a partially positive charge, which can interact with the π electron cloud of the astaxanthin polyene chain through a weak but non-covalent attraction, namely the CH-π interaction. This is a common stabilization mechanism in sugar-carotenoid complexes. This interaction anchors the astaxanthin molecule near the hydrophobic surface of the glucose ring, limiting excessive movement and conformational inversion of its polyene chains, thus significantly inhibiting the formation of the cis isomer. Simultaneously, the hydroxyl environment of glucose provides some antioxidant protection. The three components form a synergistic "cell disruption-dissolution-stabilization" effect at the molecular level, effectively improving the extraction rate and purity of astaxanthin without the addition of any functionalized nanomaterials or chemical modifiers.

[0028] (2) In this invention, tocopherol and ascorbyl palmitate are added during the extraction stage to inhibit the oxidative degradation and cis-isomerization of astaxanthin during extraction and drying. Tocopherol is a lipid-soluble antioxidant that preferentially locates in the hydrophobic region near the conjugated polyene chain of the astaxanthin molecule, directly quenching lipid peroxide free radicals. Ascorbyl palmitate is amphiphilic and can anchor at the natural eutectic solvent-air interface to prevent oxygen diffusion. After quenching free radicals, tocopherol is converted into free radicals and no longer has antioxidant activity. Ascorbyl palmitate can reduce tocopherol and simultaneously convert itself into dehydroascorbic acid. This regeneration cycle allows tocopherol to be reused, significantly extending the antioxidant protection time. The two form a cross-phase antioxidant network of "hydrophobic phase-interface-hydrophilic phase", producing a significant synergistic effect and avoiding the lag problem of adding protective agents only in the drying stage in traditional processes. Detailed Implementation

[0029] It should be noted that, where there is no conflict, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0030] It should be noted that, unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0031] This invention provides a method for separating and extracting astaxanthin from Haematococcus pluvialis, comprising the following steps:

[0032] (1) Weigh out 30-60 parts of betaine, 50-100 parts of natural organic acid, 20-40 parts of D-glucose and 20-40 parts of deionized water by weight.

[0033] (2) After mixing betaine, natural organic acid, D-glucose and deionized water in sequence, stir at 200-300 rpm for 40-60 min in a water bath at 50-60℃ to obtain a natural eutectic solvent.

[0034] (3) Take Haematococcus pluvialis spores, place them in a vacuum freeze dryer, freeze dry them for 24 to 48 hours at a temperature of -40 to -30℃ and a vacuum of 10 to 30 Pa, grind them at a low temperature of 6 to 10℃, pass them through an 80 to 120 mesh sieve, and store them in a sealed, light-proof, cold-stored container at a temperature of -8 to -4℃ to obtain Haematococcus pluvialis dry powder;

[0035] (4) Weigh out 10-12 parts of Haematococcus pluvialis powder, 120-240 parts of natural eutectic solvent, 0.1-0.3 parts of tocopherol, and 0.08-0.1 parts of ascorbate palmitate by weight.

[0036] (5) Add dried Haematococcus pluvialis, 4 / 5 of the natural eutectic solvent, tocopherol, and ascorbyl palmitate to an ultrasonic reactor in sequence. After purging the oxygen with nitrogen at a flow rate of 0.5-1 L / min, seal the reactor and extract the mixture by ultrasonic stirring at a speed of 150-300 rpm under the conditions of power 300-600 W, frequency 20-25 kHz, and temperature 45-65 °C for 40-80 min. After extraction, cool the mixture to 20-30 °C and let it stand in the dark for 20-30 min to obtain the mixture.

[0037] (6) Place the mixture in a centrifuge and centrifuge at 8000-12000 rpm for 15-25 min at a temperature of 4-8℃ to obtain supernatant I and algal residue;

[0038] (7) Add the algal residue to the remaining 1 / 5 of the natural eutectic solvent, mix evenly and let stand for 10-20 minutes, then place it in a centrifuge and centrifuge at 6000-10000 rpm for 10-20 minutes. After centrifugation, obtain supernatant II. Combine supernatant I and supernatant II to obtain crude extract.

[0039] (8) Soak HPD-600 resin in an ethanol-water mixed solvent with a mass ratio of 90~95:100 for 20~24h, then wash it with deionized water, sodium hydroxide aqueous solution, dilute hydrochloric acid and deionized water in sequence until neutral, drain it, pack it into a column with a column diameter-to-height ratio of 1:5~8, and equilibrate it with 3 times the column volume of deionized water to obtain a resin column.

[0040] (9) Pass the crude extract through the resin column at a flow rate of 0.5~1.5 BV / h, with a sample loading of 2~4 BV. Elute with 30%~40% ethanol aqueous solution at a flow rate of 1~2 BV / h to remove polar impurities. Then elute with 85%~95% ethanol aqueous solution at a flow rate of 0.5~1 BV / h and collect the astaxanthin eluent. Wash the resin column sequentially with 2~3 BV of 90%~95% ethanol aqueous solution, 2~3 BV of sodium hydroxide aqueous solution, and 3~5 BV of deionized water until neutral, and then regenerate for reuse.

[0041] (10) The collected astaxanthin eluent was placed in a rotary evaporator and evaporated under reduced pressure at a temperature of 45~55℃ and a vacuum of -0.09~-0.08MPa to 1 / 20~1 / 10 of the original volume to obtain astaxanthin concentrate.

[0042] (11) After freezing and solidifying the astaxanthin concentrate at -20~-16℃, place it in a vacuum freeze dryer and freeze dry it at -50~-40℃ and 8~10Pa for 24~36h. Then, seal and store it in a cool, dark place at -6~-5℃ to obtain astaxanthin crystal powder.

[0043] The natural organic acids include at least one of lactic acid, citric acid, and malic acid.

[0044] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. To ensure the accuracy of the experimental results, the raw materials used for extracting astaxanthin in the embodiments are all Haematococcus pluvialis powder.

[0045] Example 1

[0046] The method for separating and extracting astaxanthin using Haematococcus pluvialis as raw material in this embodiment includes the following steps:

[0047] (1) Weigh out 30 parts betaine, 50 parts lactic acid, 20 parts D-glucose and 20 parts deionized water by weight.

[0048] (2) After mixing betaine, lactic acid, D-glucose and deionized water in sequence, stir at 200 rpm for 40 min in a water bath at 50°C to obtain a natural eutectic solvent.

[0049] (3) Take Haematococcus pluvialis spores, place them in a vacuum freeze dryer, freeze dry them for 24 hours at a temperature of -40℃ and a vacuum of 10Pa, grind them at a low temperature of 6℃, pass them through an 80-mesh sieve, and store them in a sealed, light-proof, cold-stored container at -8℃ to obtain Haematococcus pluvialis dry powder.

[0050] (4) Weigh out 10 parts of Haematococcus pluvialis powder, 120 parts of natural eutectic solvent, 0.1 parts of tocopherol, and 0.08 parts of ascorbyl palmitate by weight.

[0051] (5) Add dried Haematococcus pluvialis, 4 / 5 of the natural eutectic solvent, tocopherol, and ascorbyl palmitate to an ultrasonic reactor in sequence. After purging the oxygen with nitrogen at a flow rate of 0.5 L / min, seal the reactor and extract the mixture by ultrasonic stirring at 150 rpm for 40 min at a power of 300 W, a frequency of 20 kHz, and a temperature of 45 °C. After extraction, cool the mixture to 20 °C and let it stand in the dark for 20 min to obtain the mixture.

[0052] (6) Place the mixture in a centrifuge and centrifuge at 8000 rpm for 15 min at 4°C to obtain supernatant I and algal residue;

[0053] (7) Add the algal residue to the remaining 1 / 5 of the natural eutectic solvent, mix evenly and let stand for 10 min, then place it in a centrifuge and centrifuge at 6000 rpm for 10 min. After centrifugation, obtain supernatant II. Combine supernatant I and supernatant II to obtain crude extract.

[0054] (8) Soak HPD-600 resin in an ethanol-water mixed solvent with a mass ratio of 90:100 for 20 hours, then wash it with deionized water, sodium hydroxide aqueous solution, dilute hydrochloric acid and deionized water in sequence until neutral, drain it, pack it into a column with a column diameter-to-height ratio of 1:5, and equilibrate it with 3 times the column volume of deionized water to obtain a resin column.

[0055] (9) Pass the crude extract through the resin column at a flow rate of 0.5 BV / h, with a sample loading of 2 BV. Elute with 30% ethanol aqueous solution at a flow rate of 1 BV / h to remove polar impurities. Then elute with 85% ethanol aqueous solution at a flow rate of 0.5 BV / h. Collect the astaxanthin eluent. Wash the resin column sequentially with 2 BV of 90% ethanol aqueous solution, 2 BV of sodium hydroxide aqueous solution, and 3 BV of deionized water until neutral, and then regenerate for reuse.

[0056] (10) The collected astaxanthin eluent was placed in a rotary evaporator and evaporated under reduced pressure at a temperature of 45°C and a vacuum of -0.09MPa to 1 / 20 of the original volume to obtain astaxanthin concentrate.

[0057] (11) After freezing and solidifying the astaxanthin concentrate at -20°C, it was placed in a vacuum freeze dryer and vacuum freeze-dried at -50°C and 8Pa for 24 hours. It was then sealed and stored in a cool, dark place at -6°C to obtain astaxanthin crystal powder.

[0058] Example 2

[0059] The method for separating and extracting astaxanthin using Haematococcus pluvialis as raw material in this embodiment includes the following steps:

[0060] (1) Weigh out 45 parts betaine, 75 parts citric acid, 30 parts D-glucose and 30 parts deionized water by weight.

[0061] (2) After mixing betaine, citric acid, D-glucose and deionized water in sequence, stir at 250 rpm for 50 min in a water bath at 55°C to obtain a natural eutectic solvent.

[0062] (3) Take Haematococcus pluvialis spores, place them in a vacuum freeze dryer, freeze dry them for 36 hours at a temperature of -35℃ and a vacuum degree of 20Pa, grind them at a low temperature of 8℃, pass them through a 100-mesh sieve, and store them in a sealed, light-proof, cold-stored container at a temperature of -6℃ to obtain Haematococcus pluvialis dry powder.

[0063] (4) Weigh out 11 parts of Haematococcus pluvialis powder, 180 parts of natural eutectic solvent, 0.2 parts of tocopherol, and 0.09 parts of ascorbyl palmitate by weight.

[0064] (5) Add the dried Haematococcus pluvialis powder, 4 / 5 of the natural eutectic solvent, tocopherol, and ascorbyl palmitate to the ultrasonic reactor in sequence. After purging the oxygen with nitrogen at a flow rate of 0.75 L / min, seal the reactor and extract the mixture by ultrasonic stirring at 225 rpm for 60 min at a power of 450 W, a frequency of 22.5 kHz, and a temperature of 55 °C. After extraction, cool the mixture to 25 °C and let it stand in the dark for 25 min to obtain the mixture.

[0065] (6) Place the mixture in a centrifuge and centrifuge at 10,000 rpm for 20 min at 6°C to obtain supernatant I and algal residue;

[0066] (7) Add the algal residue to the remaining 1 / 5 of the natural eutectic solvent, mix well and let stand for 15 minutes, then place it in a centrifuge and centrifuge at 8000 rpm for 15 minutes. After centrifugation, obtain supernatant II. Combine supernatant I and supernatant II to obtain crude extract.

[0067] (8) Soak HPD-600 resin in an ethanol-water mixed solvent with a mass ratio of 92.5:100 for 22 hours, then wash it with deionized water, sodium hydroxide aqueous solution, dilute hydrochloric acid and deionized water in sequence until neutral, drain it, pack it into a column with a column diameter-to-height ratio of 1:7.5, and equilibrate it with 3 times the column volume of deionized water to obtain a resin column.

[0068] (9) Pass the crude extract through the resin column at a flow rate of 1 BV / h, with a sample loading of 3 BV. Elute with 35% ethanol aqueous solution at a flow rate of 1.5 BV / h to remove polar impurities. Then elute with 90% ethanol aqueous solution at a flow rate of 0.75 BV / h. Collect the astaxanthin eluent. Wash the resin column sequentially with 2.5 BV of 92.5% ethanol aqueous solution, 2.5 BV of sodium hydroxide aqueous solution, and 4 BV of deionized water until neutral, and then regenerate for reuse.

[0069] (10) The collected astaxanthin eluent was placed in a rotary evaporator and evaporated under reduced pressure at a temperature of 50°C and a vacuum of -0.085MPa to 1 / 15 of the original volume to obtain astaxanthin concentrate.

[0070] (11) After freezing and solidifying the astaxanthin concentrate at -18°C, it was placed in a vacuum freeze dryer and vacuum freeze-dried for 30 hours at -45°C and 9Pa. It was then sealed and stored in a cool, dark place at -5.5°C to obtain astaxanthin crystal powder.

[0071] Example 3

[0072] The method for separating and extracting astaxanthin using Haematococcus pluvialis as raw material in this embodiment includes the following steps:

[0073] (1) Weigh out 60 parts betaine, 100 parts malic acid, 40 parts D-glucose and 40 parts deionized water by weight.

[0074] (2) After mixing betaine, malic acid, D-glucose and deionized water in sequence, stir at 300 rpm for 60 min in a water bath at 60°C to obtain a natural eutectic solvent.

[0075] (3) Take Haematococcus pluvialis spores, place them in a vacuum freeze dryer, freeze dry them for 48 hours at a temperature of -30℃ and a vacuum of 30Pa, grind them at a low temperature of 10℃, pass them through a 120-mesh sieve, and store them in a sealed, light-proof, cold-stored container at a temperature of -4℃ to obtain Haematococcus pluvialis dry powder.

[0076] (4) Weigh out 12 parts of Haematococcus pluvialis powder, 240 parts of natural eutectic solvent, 0.3 parts of tocopherol, and 0.1 parts of ascorbyl palmitate by weight.

[0077] (5) Add the dried Haematococcus pluvialis powder, 4 / 5 of the natural eutectic solvent, tocopherol, and ascorbyl palmitate to the ultrasonic reactor in sequence. After purging the oxygen with nitrogen at a flow rate of 1 L / min, seal the reactor and extract with ultrasonic stirring at 300 rpm for 80 min at a power of 600 W, a frequency of 25 kHz, and a temperature of 65 °C. After extraction, cool to 30 °C and let stand in the dark for 30 min to obtain the mixture.

[0078] (6) Place the mixture in a centrifuge and centrifuge at 12000 rpm at 8°C for 25 min to obtain supernatant I and algal residue;

[0079] (7) Add the algal residue to the remaining 1 / 5 of the natural eutectic solvent, mix evenly and let stand for 20 minutes, then place it in a centrifuge and centrifuge at 10,000 rpm for 20 minutes. After centrifugation, obtain supernatant II. Combine supernatant I and supernatant II to obtain crude extract.

[0080] (8) Soak HPD-600 resin in an ethanol-water mixed solvent with a mass ratio of 95:100 for 24 hours, then wash it with deionized water, sodium hydroxide aqueous solution, dilute hydrochloric acid and deionized water in sequence until neutral, drain it, pack it into a column with a column diameter-to-height ratio of 1:8, and equilibrate it with 3 times the column volume of deionized water to obtain a resin column.

[0081] (9) Pass the crude extract through the resin column at a flow rate of 1.5 BV / h, with a sample loading of 4 BV. Elute with 40% ethanol aqueous solution at a flow rate of 2 BV / h to remove polar impurities. Then elute with 95% ethanol aqueous solution at a flow rate of 1 BV / h and collect the astaxanthin eluent. Wash the resin column sequentially with 3 BV of 95% ethanol aqueous solution, 3 BV of sodium hydroxide aqueous solution, and 5 BV of deionized water until neutral and regenerate for reuse.

[0082] (10) The collected astaxanthin eluent was placed in a rotary evaporator and evaporated under reduced pressure at a temperature of 55°C and a vacuum of -0.08MPa to 1 / 10 of the original volume to obtain astaxanthin concentrate.

[0083] (11) After freezing and solidifying the astaxanthin concentrate at -16°C, it was placed in a vacuum freeze dryer and vacuum freeze-dried for 36 hours at -40°C and 10Pa. It was then sealed and stored in a cool, dark place at -5°C to obtain astaxanthin crystal powder.

[0084] Comparative Example 1

[0085] The method for separating and extracting astaxanthin using Haematococcus pluvialis as raw material in this comparative example is largely the same as that in Example 1, except that D-glucose was not added to the natural eutectic solvent in this comparative example.

[0086] Comparative Example 2

[0087] The method for separating and extracting astaxanthin using Haematococcus pluvialis as raw material in this comparative example is largely the same as that in Example 1. The difference is that glycerol is used instead of D-glucose as the natural eutectic solvent in this comparative example.

[0088] Comparative Example 3

[0089] The method for separating and extracting astaxanthin using Haematococcus pluvialis as raw material in this comparative example is largely the same as that in Example 1. The difference is that fructose is used instead of D-glucose as the natural eutectic solvent in this comparative example.

[0090] Comparative Example 4

[0091] The method for separating and extracting astaxanthin using Haematococcus pluvialis as raw material in this comparative example is largely the same as that in Example 1. The difference is that no tocopherol was added during the separation and extraction of astaxanthin in this comparative example.

[0092] Comparative Example 5

[0093] The method for separating and extracting astaxanthin using Haematococcus pluvialis as raw material in this comparative example is roughly the same as that in Example 1. The difference is that ascorbyl palmitate was not added when separating and extracting astaxanthin in this comparative example.

[0094] Experimental Example

[0095] Astaxanthin samples were prepared using the methods of Examples 1-3 and Comparative Examples 1-5, respectively, and the following performance tests were performed:

[0096] Viscosity of natural eutectic solvents: The dynamic viscosity of natural eutectic solvents was determined using a rotational viscometer at 25°C with an L2 rotor and a rotation speed of 30 rpm.

[0097] Astaxanthin extraction rate (%): The mass of astaxanthin in the extract was determined by high performance liquid chromatography (HPLC, C18 column, methanol-acetonitrile mobile phase, detection wavelength 476 nm), and the ratio of the mass of astaxanthin in the extract to the total mass of astaxanthin in the raw material.

[0098] Astaxanthin purity (%): The percentage of astaxanthin in the total extract of the final product was determined by HPLC.

[0099] All-trans astaxanthin ratio (%): The proportion of all-trans configuration in total astaxanthin was determined by HPLC (C30 column, normal phase system).

[0100] Antioxidant retention rate (%): The astaxanthin product was sealed and placed in a 60℃ constant temperature chamber for accelerated oxidation for 30 days. The astaxanthin content was determined by periodic sampling using high performance liquid chromatography (HPLC). The mass percentage of astaxanthin before and after acceleration was calculated as the antioxidant retention rate.

[0101] The specific test results are shown in Table 1:

[0102] Table 1: Sample test results of Examples 1-3 and Comparative Examples 1-5

[0103] project Solvent viscosity (mPa·s) Extraction rate (%) purity(%) All-trans astaxanthin percentage (%) Antioxidant retention rate (%) Example 1 190 94.8 98.3 93.5 94.2 Example 2 220 96.1 98.6 96.7 95.8 Example 3 200 95.9 98.4 94.9 94.5 Comparative Example 1 420 78.5 91.5 85.2 86.5 Comparative Example 2 310 85.7 94.2 88.1 90.3 Comparative Example 3 260 89.5 96.1 90.2 91.8 Comparative Example 4 190 93.2 97.5 88.9 86.7 Comparative Example 5 190 93.8 97.9 91.2 90.5

[0104] As shown in Table 1, in Examples 1-3, astaxanthin was isolated and extracted from Haematococcus pluvialis using the method of the present invention. The extraction rate of astaxanthin exceeded 94%, the purity of astaxanthin was greater than 98%, the proportion of all-trans astaxanthin was greater than 93%, and the antioxidant retention rate was greater than 94%. This is because the natural eutectic solvent used in Examples 1-3 consisted of betaine, natural organic acids, and D-glucose. The zwitterionic properties of betaine effectively interfered with the hydrophobic interactions and hydrogen bonding between proteins and astaxanthin in the thick-walled spores of Haematococcus pluvialis, promoting the release of astaxanthin from its bound state. The natural organic acids provided abundant carboxyl and hydroxyl groups, forming a stable hydrogen bond network with betaine, providing a suitable polar solvation environment for the released astaxanthin and preventing the astaxanthin molecules from π-π stacking and precipitating. The D-glucose has four hydroxyl groups distributed on its pyranose ring, and its spatial configuration is chair-shaped. This allows it to simultaneously form hydrogen bonds with multiple betaine and natural organic acid molecules, constructing a three-dimensional network with a rigid framework and flexible branches. The rigid pyran ring structure of D-glucose acts as a "molecular wedge" inserted between betaine and organic acids, disrupting the dense hydrogen bond network and significantly reducing the system's viscosity. Meanwhile, the thick-walled spore cell walls of Haematococcus pluvialis are mainly composed of cellulose, hemicellulose, and callose. These polysaccharides form a rigid barrier through dense intramolecular and intermolecular hydrogen bonds. D-glucose, as a monosaccharide, has a much smaller molecular volume than polysaccharides, but is also rich in hydroxyl groups. When natural eutectic solvents penetrate the cell wall, the hydroxyl groups on the glucose molecules form competitive hydrogen bonds with the hydroxyl groups on the cell wall polysaccharide chains, vying for hydrogen bond sites originally used to maintain cell wall structural stability. This competitive binding causes local "loosening" or even "breakage" of the hydrogen bond network between cell wall polysaccharide chains, thereby softening or destroying the cell wall structure and making it easier for astaxanthin to be released. Furthermore, astaxanthin molecules possess long conjugated polyene chains, making them highly susceptible to oxidation and cis-trans isomerism. In solution, the polyene chains tend to fold or aggregate through hydrophobic interactions. The axial hydrogen atoms on the pyranose ring of glucose carry a partially positive charge, which can interact with the π-electron cloud of the astaxanthin polyene chain through a weak but non-covalent attraction, namely the CH-π interaction. This is a common stabilization mechanism in sugar-carotenoid complexes. This interaction "anchors" the astaxanthin molecule near the hydrophobic surface of the glucose ring, limiting excessive movement and conformational inversion of its polyene chain, thereby significantly inhibiting the formation of the cis isomer. Simultaneously, the hydroxyl environment of glucose also provides some antioxidant protection. The three components form a synergistic effect of "wall disruption-dissolution-stabilization" at the molecular level, effectively improving the extraction rate and purity of astaxanthin without the addition of any functionalized nanomaterials or chemical modifiers.

[0105] Meanwhile, in Examples 1-3, tocopherol and ascorbyl palmitate were added during the extraction stage to inhibit the oxidative degradation and cis-isomerization of astaxanthin during extraction and drying. Tocopherol, a lipid-soluble antioxidant, preferentially locates in the hydrophobic region near the conjugated polyene chain of the astaxanthin molecule, directly quenching lipid peroxide free radicals. Ascorbyl palmitate is amphiphilic and can anchor at the natural eutectic solvent-air interface to prevent oxygen diffusion. After quenching free radicals, tocopherol is converted into free radicals and no longer has antioxidant activity. Ascorbyl palmitate can reduce tocopherol and simultaneously convert itself into dehydroascorbic acid. This regeneration cycle allows tocopherol to be reused, significantly extending the antioxidant protection time. The two form a cross-phase antioxidant network of "hydrophobic phase-interface-hydrophilic phase," producing a significant synergistic effect, avoiding the lag problem of adding protective agents only in the drying stage in traditional processes, and effectively improving the proportion of all-trans astaxanthin and the antioxidant retention rate of astaxanthin.

[0106] Compared to Example 1, the solvent viscosity of Comparative Example 1 increased significantly. This is because glucose acts as a "molecular wedge," disrupting the dense hydrogen bond network between betaine and natural organic acids. Without glucose, excessive cross-linking of hydrogen bonds reduces free volume, decreases fluidity, and significantly increases mass transfer resistance. The hydroxyl groups of glucose compete with algal cell wall polysaccharides for hydrogen bonds, aiding in cell wall softening. Without glucose, cell wall disruption primarily relies on the acidity of natural organic acids, weakening the disruption ability and reducing efficiency, leading to a significant decrease in extraction rate. The absence of glucose also results in the loss of CH-π interactions between glucose and astaxanthin, making the astaxanthin polyene chain more susceptible to cis-trans isomerization, decreasing configurational stability, and lowering the all-trans ratio. The hydroxyl environment of glucose has a certain stabilizing effect on free radicals; its absence slightly increases oxidative degradation and reduces antioxidant retention.

[0107] Comparative Example 2 used glycerol instead of glucose in Example 1. Glycerol is a linear trihydroxy molecule, which cannot effectively expand the hydrogen bond network like the rigid pyran ring of glucose, resulting in poor viscosity reduction and the system still maintaining a high viscosity, affecting mass transfer. Although the hydroxyl groups of glycerol can form hydrogen bonds, it lacks the "competitive insertion" effect of glucose on polysaccharide chains, resulting in weak cell wall disruption assistance and incomplete cell wall destruction, leading to a lower extraction rate. Furthermore, the glycerol system is more polar, which will co-extract more polar impurities, leading to a decrease in purity. Glycerol molecules do not have a hydrophobic ring structure and cannot form a CH-π stabilizing effect with the conjugated double bond of astaxanthin. The lack of CH-π interaction results in a significantly lower all-trans ratio than in Example 1.

[0108] Comparative Example 3 uses fructose instead of glucose in Example 1. Fructose has a five-membered furan ring, while glucose has a six-membered pyran ring. The chair configuration of the pyran ring has a more stable spatial orientation and more axial hydrogens, forming a better hydrogen bond network with betaine and natural organic acids, resulting in better viscosity reduction. Furthermore, fructose is more hygroscopic, which can affect water activity in natural eutectic solvents, indirectly affecting extraction efficiency and leading to a decrease in astaxanthin extraction rate. The furan ring of fructose is less hydrophobic, resulting in a slightly weaker ability to stabilize the astaxanthin configuration, while the pyran ring of glucose has a larger hydrophobic surface and stronger CH-π interaction. Therefore, the proportion of all-trans astaxanthin in Comparative Example 3 is significantly lower than that in Example 1.

[0109] In Comparative Example 4, no tocopherol was added to the natural eutectic solvent, only ascorbate palmitate was retained. Tocopherol primarily targets the hydrophobic regions of astaxanthin molecules, while ascorbate palmitate acts at the water-DES interface. Together, they form a "transphase antioxidant network." Without tocopherol, the hydrophobic regions of astaxanthin are not effectively protected, leading to increased oxidative degradation and isomerization, and a significant decrease in the proportion of all-trans astaxanthin. Furthermore, tocopherol can directly quench lipid peroxide free radicals, while ascorbate palmitate regenerates tocopherol and scavenge aqueous free radicals. Without tocopherol, using ascorbate palmitate alone induces cis-isomerization under oxidative stress, significantly reducing the antioxidant retention rate, indicating that long-term stability is heavily dependent on tocopherol. In Comparative Example 5, no ascorbate palmitate was added to the natural eutectic solvent, only tocopherol was retained. Ascorbate palmitate is amphiphilic and can anchor at the natural eutectic solvent-air interface, preventing oxygen diffusion into the solution. Without ascorbyl palmitate, oxygen more readily interacts with astaxanthin, leading to oxidation and a significant decrease in the proportion of all-trans astaxanthin. Ascorbyl palmitate can reduce and regenerate oxidized tocopherol. When tocopherol is used alone, it cannot be recovered after consumption, interrupting the regeneration cycle, reducing protective durability, and significantly decreasing oxidation retention. Therefore, tocopherol and ascorbyl palmitate form a complementary synergy in their action space, free radical scavenging spectrum, and regeneration cycle. This synergistic mechanism achieves a high all-trans proportion and excellent long-term stability. Adding either one alone cannot simultaneously protect the hydrophobic conjugated core of astaxanthin, block interfacial oxygen diffusion, and achieve the cyclic regeneration of antioxidants.

[0110] This invention provides a method for separating and extracting astaxanthin from Haematococcus pluvialis. Through the synergistic effect of a natural eutectic solvent (ternary process), the cross-phase antioxidant synergy of tocopherol and ascorbate palmitate, combined with a gentle process of ultrasound-assisted extraction and resin gradient elution, the extraction rate of astaxanthin is effectively improved, while maintaining its purity and the proportion of all-trans astaxanthin. This also prolongs the antioxidant retention time of astaxanthin and significantly reduces production costs and wastewater discharge. All raw materials are non-toxic and harmless, and no organic solvents are used in the process, demonstrating promising application prospects.

[0111] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for separating and extracting astaxanthin from Haematococcus pluvialis, characterized in that, Includes the following steps: (1) Take Haematococcus pluvialis spores, freeze dry them in a vacuum freeze dryer, grind them at low temperature, pass them through an 80-120 mesh sieve, seal them and store them in a cool, dark place to obtain Haematococcus pluvialis dry powder; (2) Weigh out 10-12 parts of Haematococcus pluvialis powder, 120-240 parts of natural eutectic solvent, 0.1-0.3 parts of tocopherol, and 0.08-0.1 parts of ascorbate palmitate by weight. (3) Add the dried Haematococcus pluvialis powder, 4 / 5 of the natural eutectic solvent, tocopherol, and ascorbyl palmitate to the ultrasonic reactor in sequence. After purging the oxygen with nitrogen, seal the reactor and extract by ultrasonic stirring. After extraction, cool the reactor and let it stand in the dark to obtain a mixture. (4) The mixture was centrifuged at low temperature to obtain supernatant I and algal residue; (5) Add the algal residue to the remaining 1 / 5 of the natural eutectic solvent, mix well and let stand, then place it in a centrifuge to separate it. After centrifugation, obtain supernatant II. Combine supernatant I and supernatant II to obtain crude extract. (6) Soak HPD-600 resin in an ethanol-water mixed solvent, then wash it in sequence with deionized water, sodium hydroxide aqueous solution, dilute hydrochloric acid and deionized water until neutral, drain it, pack it into a column with a column diameter-to-height ratio of 1:5~8, and equilibrate it with 3 times the column volume of deionized water to obtain a resin column. (7) Pass the crude extract through a resin column and elute with a 30%~40% ethanol aqueous solution at a flow rate of 1~2 BV / h to remove polar impurities. Then elute with an 85%~95% ethanol aqueous solution at a flow rate of 0.5~1 BV / h and collect the astaxanthin eluent. Wash the resin column sequentially with 2~3 BV of 90%~95% ethanol aqueous solution, 2~3 BV of sodium hydroxide aqueous solution, and 3~5 BV of deionized water until neutral and regenerate for reuse. (8) Place the collected astaxanthin eluent in a rotary evaporator and evaporate it under reduced pressure to 1 / 20 to 1 / 10 of the original volume to obtain astaxanthin concentrate. (9) After freezing and solidifying the astaxanthin concentrate at -20~-16℃, place it in a vacuum freeze dryer and freeze dry it at -50~-40℃ and 8~10Pa for 24~36h. Then, seal and store it in a cool, dark place at -6~-5℃ to obtain astaxanthin crystal powder.

2. The method for separating and extracting astaxanthin from Haematococcus pluvialis as raw material according to claim 1, characterized in that, The freeze-drying conditions in step (1) are: temperature -40~-30℃, vacuum degree 10~30Pa, freeze-drying time 24~48h, low-temperature grinding temperature 6~10℃, and sealed and protected from light refrigeration temperature -8~-4℃.

3. The method for separating and extracting astaxanthin from Haematococcus pluvialis as raw material according to claim 1, characterized in that, The preparation method of the natural eutectic solvent in step (2) includes the following steps: S1. Weigh out 30-60 parts betaine, 50-100 parts natural organic acid, 20-40 parts D-glucose, and 20-40 parts deionized water by weight. S2. Mix betaine, natural organic acid, D-glucose and deionized water in sequence and stir at 200-300 rpm for 40-60 min in a water bath at 50-60℃ to obtain a natural eutectic solvent.

4. The method for separating and extracting astaxanthin from Haematococcus pluvialis as raw material according to claim 3, characterized in that, The natural organic acids in step S1 include at least one of lactic acid, citric acid, and malic acid.

5. The method for separating and extracting astaxanthin from Haematococcus pluvialis as raw material according to claim 1, characterized in that, In step (3), the flow rate of nitrogen gas is 0.5~1L / min, the conditions for ultrasonic stirring extraction are power 300~600W, frequency 20~25kHz, temperature 45~65℃, rotation speed 150~300rpm, ultrasonic stirring extraction time is 40~80min, cooling temperature is 20~30℃, and the time for standing in the dark is 20~30min.

6. The method for separating and extracting astaxanthin from Haematococcus pluvialis as raw material according to claim 1, characterized in that, In step (4), the temperature for low-temperature centrifugation is 4~8℃, the centrifugation speed is 8000~12000rpm, and the separation time is 15~25min.

7. The method for separating and extracting astaxanthin from Haematococcus pluvialis as raw material according to claim 1, characterized in that, In step (5), the settling time is 10-20 min, the centrifugation speed is 6000-10000 rpm, and the time is 10-20 min.

8. The method for separating and extracting astaxanthin from Haematococcus pluvialis as raw material according to claim 1, characterized in that, In step (6), the mass ratio of ethanol to water in the ethanol-water mixed solvent is 90~95:100, and the soaking time is 20~24h.

9. The method for separating and extracting astaxanthin from Haematococcus pluvialis as raw material according to claim 1, characterized in that, In step (7), the flow rate of the crude extract through the resin column is 0.5~1.5 BV / h, and the loading volume is 2~4 BV.

10. The method for separating and extracting astaxanthin from Haematococcus pluvialis as raw material according to claim 1, characterized in that, The conditions for vacuum rotary evaporation in step (8) are a temperature of 45~55℃ and a vacuum degree of -0.09~-0.08MPa.