Method for supercritical separation and extraction of astaxanthin

Abstract

The invention relates to the technical field of separation and extraction of natural products, in particular to a method for supercritical separation and extraction of astaxanthin, which comprises the following steps: (1) weighing raw material components in parts by weight; (2) mixing a biomass raw material rich in astaxanthin with the functionalized deep-eutectic solvent, and performing physical field enhanced extraction to obtain an extracting solution; (3) filtering the extracting solution through a vibrating membrane, and purifying to obtain a trapped fluid containing astaxanthin; and (4) carrying out supercritical CO2 back extraction on the trapped fluid, separating to obtain an astaxanthin product, and recovering the functional eutectic solvent. According to the method, continuous membrane separation and purification under the eutectic solvent system are realized, the purity of astaxanthin and the proportion of all-trans astaxanthin are maintained while the extraction rate of astaxanthin is increased, the solvent can be recycled after being recovered, and the production cost and waste liquid discharge are greatly reduced. All the raw materials are nontoxic and harmless, no organic solvent is used in the technological process, and good application prospects are achieved.

Description

Technical Field

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

[0002] Astaxanthin is a ketocarotenoid with 13 conjugated double bonds and two ketone groups in its molecular structure. It possesses extremely strong antioxidant activity, with its ability to quench singlet oxygen being 550 times that of vitamin E. Astaxanthin has broad application prospects in medicine, health products, cosmetics, and high-end animal feed. Natural astaxanthin mainly comes from biomass resources such as Haematococcus pluvialis and by-products of crustacean aquaculture processing. Haematococcus pluvialis, in particular, can accumulate astaxanthin accounting for 3%–5% of its cell dry weight under stress conditions, making it an ideal raw material for the industrial production of natural astaxanthin.

[0003] The extraction of astaxanthin has long faced the following technical challenges: First, astaxanthin usually exists in organisms in a free or esterified form and is tightly bound to proteins, chitin, or liposomes to form a stable complex structure, which traditional solvents cannot effectively penetrate and release. Second, the long conjugated double bond system in the astaxanthin molecule is extremely sensitive to light, heat, and oxygen, and is prone to isomerization and oxidative degradation during the extraction process, leading to a decrease in product activity. Finally, traditional organic solvent extraction methods pose a risk of solvent residue, and the production of 1 kg of astaxanthin generates 200-300 kg of organic waste liquid with high chemical oxygen demand, resulting in poor environmental compatibility.

[0004] To address these issues, researchers have developed various improved technologies. While supercritical carbon dioxide extraction avoids organic solvent residues, the non-polar nature of CO2 limits its solubility of astaxanthin esters, resulting in an actual extraction rate of only around 75%, and the equipment investment is high. Although ionic liquid extraction can improve solubility by designing specific anions and cations, the bioaccumulation and ecotoxicity of ionic liquids restrict their application in the food industry. Therefore, developing a green astaxanthin extraction process that combines high extraction rate, high purity, high activity retention, continuous operation, and complete non-toxicity remains a pressing technical challenge in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for supercritical separation and extraction of astaxanthin.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for supercritical separation and extraction of astaxanthin includes the following steps:

[0008] (1) Weigh 1-3 parts of biomass raw material rich in astaxanthin, 20-40 parts of functionalized eutectic solvent, 5-15 parts of deionized water, and 10-20 parts of anhydrous ethanol by weight.

[0009] (2) Slowly add deionized water to the functionalized eutectic solvent and stir until homogeneous. Then add biomass raw material rich in astaxanthin and stir until fully mixed to obtain the liquid.

[0010] (3) The material liquid is subjected to ultrasonic-microfluidic synergistic extraction using a series ultrasonic-microfluidic synergistic extraction system, centrifuged, and the supernatant is taken to obtain the extract.

[0011] (4) The extract was purified by filtration through a vibrating membrane to obtain a retentate containing astaxanthin;

[0012] (5) After the retentate is mixed with anhydrous ethanol, it is back-extracted by supercritical CO2, the CO2 is vaporized and recovered, the mixture is collected and the ethanol is removed by vacuum distillation, the functionalized eutectic solvent is recovered, and the precipitated astaxanthin crystals are collected to obtain the astaxanthin product.

[0013] Furthermore, the biomass raw materials rich in astaxanthin in step (1) include at least one of Haematococcus pluvialis powder, shrimp shell powder, and crab shell powder.

[0014] Furthermore, the preparation method of the functionalized eutectic solvent in step (1) includes the following steps:

[0015] S1. Weigh out 20-40 parts of betaine, 40-60 parts of 1,2-propanediol, and 1-3 parts of modified cellulose nanocrystals by weight.

[0016] S2. After mixing betaine with 1,2-propanediol, heat and stir at 300-500 rpm in an oil bath at 75-85°C for 1.5-2.5 hours until a homogeneous, transparent, viscous liquid without solid particles is formed. Cool to 40-50°C to obtain a eutectic solvent.

[0017] S3. Add the modified cellulose nanocrystals to 1 / 5 of the eutectic solvent and stir into a paste. Then add the remaining 4 / 5 of the eutectic solvent and place it in an ultrasonic dispersion device. Disperse the mixture ultrasonically for 30-60 minutes at an ultrasonic power of 200-400W, a frequency of 20-40kHz, and a temperature of 40-50℃. Then raise the temperature to 50-60℃ and magnetically stir at a speed of 400-600rpm for 1-2 hours. Place the mixture in a vacuum dryer and degas it for 20-30 minutes at 40-50℃ and -0.09--0.08MPa to obtain the functionalized eutectic solvent.

[0018] Furthermore, the preparation method of modified cellulose nanocrystals in step S1 includes the following steps:

[0019] A1. Weigh out 1-2 parts of cellulose nanocrystals, 0.5-1.5 parts of methoxy polyethylene glycol silane, and 10-20 parts of anhydrous ethanol by weight.

[0020] A2. Add cellulose nanocrystals to anhydrous ethanol, place them in an ultrasonic dispersion device, and ultrasonically disperse them for 10-20 minutes under ultrasonic power of 200-300W and frequency of 20-40kHz to obtain a suspension.

[0021] A3. Add glacial acetic acid dropwise to the suspension to adjust the pH to 4-5, slowly add methoxy polyethylene glycol silane, stir at 150-250 rpm for 30-40 min at room temperature, raise the temperature to 60-80℃, reflux under nitrogen protection for 4-6 h, cool to room temperature after the reaction is completed to obtain the reaction solution;

[0022] A4. Place the reaction solution in a centrifuge and centrifuge at 8000~10000 rpm for 10~12 min. Collect the precipitate, wash the precipitate 3~5 times with anhydrous ethanol, place it in a drying oven and dry it at 40~50℃ for 10~12 h to obtain modified cellulose nanocrystals.

[0023] Furthermore, the series ultrasonic-microjet synergistic extraction system in step (3) includes a stirring tank equipped with an ultrasonic probe, a high-pressure microjet homogenizer, and a circulating cooling coil. The outlet of the stirring tank is connected to the inlet of the high-pressure microjet homogenizer, and the outlet of the high-pressure microjet homogenizer is connected to the inlet of the stirring tank through the circulating cooling coil to form a closed loop.

[0024] Furthermore, the conditions for ultrasonic-microjet synergistic extraction in step (3) are: temperature 45~55℃, rotation speed 50~100rpm, ultrasonic frequency 20~40kHz, power 300~500W, microjet pressure 80~120MPa, and ultrasonic-microjet synergistic extraction time 15~25min.

[0025] Furthermore, in step (3), the centrifugation speed is 8000~10000 rpm and the centrifugation time is 6~10 min.

[0026] Furthermore, in step (4), the membrane type selected for vibrating membrane filtration is a ceramic membrane with a molecular weight cutoff of 20~30kDa, and the conditions for vibrating membrane filtration are a frequency of 20~30Hz, an amplitude of 10~20mm, and a transmembrane pressure of 0.05~0.15MPa.

[0027] Furthermore, the conditions for supercritical CO2 back-extraction in step (5) are a temperature of 40~50℃, a pressure of 25~35MPa, and a CO2 flow rate of 30~60L / h.

[0028] Furthermore, the conditions for vacuum distillation in step (5) are a temperature of 50~60℃ and a pressure of -0.09~-0.08MPa.

[0029] In summary, this application includes at least the following beneficial effects:

[0030] (1) The functionalized eutectic solvent used in this invention is composed of betaine, 1,2-propanediol, and modified cellulose nanocrystals. Betaine, as a zwitterionic hydrogen bond acceptor, contains a positively charged quaternary ammonium group and a negatively charged carboxylate group in its molecular structure, which can simultaneously provide hydrogen bond acceptance sites and electrostatic interaction sites. 1,2-propanediol, as a hydrogen bond donor, contains two hydroxyl groups, which can form a rich hydrogen bond network. The zwitterionic properties of betaine can effectively interfere with the hydrophobic interaction and hydrogen bond binding between proteins and astaxanthin in biomass raw materials, promoting the release of astaxanthin from the bound state; at the same time, a stable hydrogen bond network is formed between the hydroxyl groups of 1,2-propanediol and the carboxylate group of betaine, providing a suitable polar solvation environment for the released astaxanthin and preventing the astaxanthin molecules from π-π stacking and precipitating. When the two are combined, the resulting eutectic solvent has suitable polarity and viscosity, taking into account both extraction efficiency and mass transfer performance. Cellulose nanocrystals are rigid, rod-shaped nanoparticles with a surface rich in hydroxyl groups, exhibiting high specific surface area and good biocompatibility. Polyethylene glycol (PEG) chains are covalently grafted onto the surface of cellulose nanocrystals via silanization, forming a "nanobrush" structure. In eutectic solvents, the PEG chains of the modified cellulose nanocrystals extend and occupy volume, disrupting the original dense hydrogen bond network between betaine and 1,2-propanediol, reducing intermolecular bonding length, and increasing the free volume in the eutectic solvent. The flexibility of the PEG chains provides a "molecular lubrication" effect, reducing internal frictional resistance during shear flow. The nanoscale rigidity of the cellulose nanocrystals can act as "micro-abrasives" during ultrasonic-microfluidic processing, assisting in the physical field breaking down biomass feedstock and further improving extraction efficiency.

[0031] (2) This invention employs a synergistic approach of simultaneous ultrasonic treatment and high-pressure microjets to extract astaxanthin from biomass raw materials. Ultrasonic waves generate cavitation bubbles in the feed solution, which collapse to produce localized high temperatures and pressures, forming microjets and shock waves, effectively disrupting the structure of the biomass raw materials. The high-pressure microjets force the feed solution through the interactive cavity at supersonic speeds, further tearing the pre-broken cells into submicron-sized fragments under the triple action of high-speed shearing, collision, and cavitation. Ultrasonic waves provide cavitation impact, while microjets provide high-speed shearing. The simultaneous cyclic treatment of both significantly reduces the heat exposure time of astaxanthin during the extraction process, lowering the risk of oxidative degradation. After the functionalized eutectic solvent is modified with cellulose nanocrystals to reduce viscosity, the fluid flow is better during the ultrasonic-microjets treatment process, cavitation bubbles are easier to form and collapse, and microjets are more effective at shearing. Under low viscosity conditions, the mass transfer coefficient of astaxanthin molecules diffusing into the solution is significantly improved.

[0032] (3) This invention applies vibrating membrane filtration technology to the purification of functionalized eutectic solvent extracts. In traditional membrane separation, the high viscosity of the eutectic solvent leads to rapid thickening of the concentration polarization layer and blockage of the membrane pores, causing the flux to decay to an unusable level within minutes. This invention uses vibrating membrane filtration technology, where the membrane module undergoes periodic reciprocating vibration. The resulting shear force continuously "throws" contaminants deposited on the membrane surface, maintaining the membrane flux at a stable level. In this invention, vibrating membrane separation directly retains astaxanthin, and the permeated functionalized eutectic solvent can be recycled back to the extraction step without any eluent, achieving zero organic waste discharge in the purification process. At the same time, supercritical CO2 back-extraction is used, which is oxygen-free and light-free throughout the process, avoiding the thermal isomerization caused by traditional spray drying. Ethanol is added as an entrainer to the functionalized eutectic solvent containing astaxanthin. Ethanol and CO2 form a supercritical mixed fluid, enhancing the solubility of astaxanthin. At the same time, the addition of ethanol further reduces the viscosity of the functionalized eutectic solvent, promoting mass transfer during back-extraction. After supercritical back-extraction, the separated and collected mixture is subjected to vacuum distillation to remove ethanol, and the functionalized eutectic solvent can be restored to its initial viscosity and extraction performance, which significantly reduces material consumption and production costs. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] This invention provides a method for supercritical separation and extraction of astaxanthin, comprising the following steps:

[0036] (1) Weigh 1-2 parts of cellulose nanocrystals, 0.5-1.5 parts of methoxy polyethylene glycol silane, and 10-20 parts of anhydrous ethanol by weight.

[0037] (2) Add cellulose nanocrystals to anhydrous ethanol, place them in an ultrasonic dispersion device, and ultrasonically disperse them for 10-20 minutes under ultrasonic power of 200-300W and frequency of 20-40kHz to obtain a suspension.

[0038] (3) Add glacial acetic acid to the suspension to adjust the pH to 4-5, slowly add methoxy polyethylene glycol silane, stir at 150-250 rpm for 30-40 min at room temperature, raise the temperature to 60-80℃, reflux under nitrogen protection for 4-6 h, and cool to room temperature after the reaction to obtain the reaction solution.

[0039] (4) Place the reaction solution in a centrifuge and centrifuge at 8000~10000rpm for 10~12min. Collect the precipitate, wash the precipitate with anhydrous ethanol 3~5 times, place it in a drying oven and dry it at 40~50℃ for 10~12h to obtain modified cellulose nanocrystals.

[0040] (5) Weigh 20-40 parts of betaine, 40-60 parts of 1,2-propanediol, and 1-3 parts of modified cellulose nanocrystals by weight.

[0041] (6) After mixing betaine with 1,2-propanediol, heat and stir at 300-500 rpm for 1.5-2.5 h in an oil bath at 75-85 °C until a homogeneous, transparent, viscous liquid without solid particles is formed. Cool to 40-50 °C to obtain a eutectic solvent.

[0042] (7) Add the modified cellulose nanocrystals to 1 / 5 of the eutectic solvent and stir into a paste. Then add the remaining 4 / 5 of the eutectic solvent and place it in an ultrasonic dispersion device. Under the conditions of ultrasonic power of 200~400W, frequency of 20~40kHz and temperature of 40~50℃, ultrasonic dispersion is performed for 30~60min. Then the temperature is raised to 50~60℃ and magnetically stirred at a speed of 400~600rpm for 1~2h. Place it in a vacuum dryer and degas it for 20~30min under the conditions of 40~50℃ and -0.09~-0.08MPa to obtain the functionalized eutectic solvent.

[0043] (8) Weigh out 1-3 parts of biomass raw material rich in astaxanthin, 20-40 parts of functionalized eutectic solvent, 5-15 parts of deionized water, and 10-20 parts of anhydrous ethanol by weight.

[0044] (9) Slowly add deionized water to the functionalized eutectic solvent and stir until homogeneous. Then add biomass raw material rich in astaxanthin and stir until fully mixed to obtain the liquid.

[0045] (10) The material liquid is subjected to ultrasonic-microjet synergistic extraction for 15-25 min in a series ultrasonic-microjet synergistic extraction system at a temperature of 45-55℃, a rotation speed of 50-100 rpm, an ultrasonic frequency of 20-40 kHz, a power of 300-500 W, and a microjet pressure of 80-120 MPa. The mixture is then centrifuged at a speed of 8000-10000 rpm for 6-10 min. The supernatant is collected to obtain the extract.

[0046] (11) Select a ceramic membrane with a molecular weight cutoff of 20~30kDa as a vibrating membrane, and purify the extract by filtration through the vibrating membrane under the conditions of frequency 20~30Hz, amplitude 10~20mm and transmembrane pressure 0.05~0.15MPa to obtain a cutoff solution containing astaxanthin.

[0047] (12) After the retentate is mixed with anhydrous ethanol, supercritical CO2 back-extraction is carried out at a temperature of 40~50℃, a pressure of 25~35MPa and a CO2 flow rate of 30~60L / h. CO2 is vaporized and recovered. The mixture is collected and the ethanol is removed by vacuum distillation at a temperature of 50~60℃ and a pressure of -0.09~-0.08MPa. The functionalized eutectic solvent is recovered and the precipitated astaxanthin crystals are collected to obtain the astaxanthin product.

[0048] The astaxanthin-rich biomass raw material includes at least one of Haematococcus pluvialis powder, shrimp shell powder, and crab shell powder; the series-connected ultrasonic-microjet synergistic extraction system includes a stirring tank equipped with an ultrasonic probe, a high-pressure microjet homogenizer, and a circulating cooling coil. The outlet of the stirring tank is connected to the inlet of the high-pressure microjet homogenizer, and the outlet of the high-pressure microjet homogenizer is connected to the inlet of the stirring tank through the circulating cooling coil, forming a closed loop.

[0049] 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.

[0050] Example 1

[0051] The supercritical separation and extraction method for astaxanthin in this embodiment includes the following steps:

[0052] (1) Weigh 1 part of cellulose nanocrystals, 0.5 parts of methoxy polyethylene glycol silane, and 10 parts of anhydrous ethanol by weight.

[0053] (2) Add cellulose nanocrystals to anhydrous ethanol, place them in an ultrasonic dispersion device, and ultrasonically disperse them for 10 min under ultrasonic power of 200W and frequency of 20kHz to obtain a suspension.

[0054] (3) Add glacial acetic acid to the suspension to adjust the pH to 4, slowly add methoxy polyethylene glycol silane, stir at 150 rpm for 30 min at room temperature, raise the temperature to 60℃, reflux under nitrogen protection for 4 h, and cool to room temperature after the reaction to obtain the reaction solution.

[0055] (4) Place the reaction solution in a centrifuge and centrifuge at 8000 rpm for 10 min. Collect the precipitate, wash the precipitate three times with anhydrous ethanol, place it in a drying oven and dry it at 40°C for 10 h to obtain modified cellulose nanocrystals.

[0056] (5) Weigh 20 parts of betaine, 40 parts of 1,2-propanediol and 1 part of modified cellulose nanocrystals by weight respectively;

[0057] (6) After mixing betaine with 1,2-propanediol, heat and stir at 300 rpm for 1.5 h in an oil bath at 75 °C until a homogeneous, transparent, viscous liquid without solid particles is formed. Cool to 40 °C to obtain a eutectic solvent.

[0058] (7) Add the modified cellulose nanocrystals to 1 / 5 of the eutectic solvent and stir into a paste. Then add the remaining 4 / 5 of the eutectic solvent and place it in an ultrasonic dispersion device. Under the conditions of ultrasonic power 200W, frequency 20kHz and temperature 40℃, ultrasonic dispersion is performed for 30min. Then the temperature is raised to 50℃ and magnetically stirred at a speed of 400rpm for 1h. Place it in a vacuum dryer and degas for 20min at 40℃ and -0.09MPa to obtain the functionalized eutectic solvent.

[0059] (8) Weigh out 1 part of Haematococcus pluvialis dry powder, 20 parts of functionalized eutectic solvent, 5 parts of deionized water and 10 parts of anhydrous ethanol by weight.

[0060] (9) Slowly add deionized water to the functionalized eutectic solvent and stir until homogeneous. Then add Haematococcus pluvialis powder and stir until fully mixed to obtain the solution.

[0061] (10) Place the liquid in a series ultrasonic-microjets synergistic extraction system and perform ultrasonic-microjets synergistic extraction for 15 min at a temperature of 45℃, a rotation speed of 50 rpm, an ultrasonic frequency of 20 kHz, a power of 300 W, and a microjets pressure of 80 MPa. Then centrifuge at 8000 rpm for 6 min and take the supernatant to obtain the extract.

[0062] (11) A ceramic membrane with a molecular weight cutoff of 20 kDa was selected as the vibrating membrane. The extract was purified by filtration through the vibrating membrane under the conditions of a frequency of 20 Hz, an amplitude of 10 mm, and a transmembrane pressure of 0.05 MPa to obtain a retentate containing astaxanthin.

[0063] (12) After the retentate is mixed with anhydrous ethanol, supercritical CO2 back-extraction is carried out at a temperature of 40℃, a pressure of 25MPa and a CO2 flow rate of 30L / h. CO2 is vaporized and recovered. The mixture is collected and the ethanol is removed by vacuum distillation at a temperature of 50℃ and a pressure of -0.09MPa. The functionalized eutectic solvent is recovered and the precipitated astaxanthin crystals are collected to obtain the astaxanthin product.

[0064] Example 2

[0065] The supercritical separation and extraction method for astaxanthin in this embodiment includes the following steps:

[0066] (1) Weigh 1.5 parts of cellulose nanocrystals, 1 part of methoxy polyethylene glycol silane, and 15 parts of anhydrous ethanol by weight.

[0067] (2) Add cellulose nanocrystals to anhydrous ethanol, place them in an ultrasonic dispersion device, and ultrasonically disperse them for 15 minutes under ultrasonic power of 250W and frequency of 30kHz to obtain a suspension.

[0068] (3) Add glacial acetic acid to the suspension to adjust the pH to 4.5, slowly add methoxy polyethylene glycol silane, stir at 200 rpm for 35 min at room temperature, raise the temperature to 70℃, reflux under nitrogen protection for 5 h, and cool to room temperature after the reaction to obtain the reaction solution;

[0069] (4) Place the reaction solution in a centrifuge and centrifuge at 9000 rpm for 11 min. Collect the precipitate, wash the precipitate 4 times with anhydrous ethanol, place it in a drying oven and dry it at 45°C for 11 h to obtain modified cellulose nanocrystals.

[0070] (5) Weigh out 30 parts of betaine, 50 parts of 1,2-propanediol, and 2 parts of modified cellulose nanocrystals by weight.

[0071] (6) After mixing betaine with 1,2-propanediol, heat and stir at 300~500 rpm for 2 hours in an oil bath at 80°C until a homogeneous, transparent, viscous liquid without solid particles is formed. Cool to 45°C to obtain a eutectic solvent.

[0072] (7) Add the modified cellulose nanocrystals to 1 / 5 of the eutectic solvent and stir into a paste. Then add the remaining 4 / 5 of the eutectic solvent and place it in an ultrasonic dispersion device. Under the conditions of ultrasonic power 300W, frequency 30kHz and temperature 45℃, ultrasonic dispersion is performed for 45min. Then the temperature is raised to 55℃ and magnetically stirred at 500rpm for 1.5h. Place it in a vacuum dryer and degas for 25min at 45℃ and -0.085MPa to obtain the functionalized eutectic solvent.

[0073] (8) Weigh out 2 parts of Haematococcus pluvialis dry powder, 30 parts of functionalized eutectic solvent, 10 parts of deionized water and 15 parts of anhydrous ethanol by weight.

[0074] (9) Slowly add deionized water to the functionalized eutectic solvent and stir until homogeneous. Then add Haematococcus pluvialis powder and stir until fully mixed to obtain the solution.

[0075] (10) Place the liquid in a series ultrasonic-microjets synergistic extraction system and perform ultrasonic-microjets synergistic extraction for 20 min at a temperature of 50℃, a rotation speed of 75 rpm, an ultrasonic frequency of 30 kHz, a power of 400 W, and a microjets pressure of 100 MPa. Then centrifuge at 9000 rpm for 8 min and take the supernatant to obtain the extract.

[0076] (11) A ceramic membrane with a molecular weight cutoff of 25 kDa was selected as the vibrating membrane. The extract was purified by filtration through the vibrating membrane under the conditions of frequency 25 Hz, amplitude 15 mm and transmembrane pressure 0.01 MPa to obtain the astaxanthin-containing retentate.

[0077] (12) After the retentate is mixed with anhydrous ethanol, supercritical CO2 back-extraction is carried out at a temperature of 45℃, a pressure of 30MPa and a CO2 flow rate of 45L / h. CO2 is vaporized and recovered. The mixture is collected and the ethanol is removed by vacuum distillation at a temperature of 55℃ and a pressure of -0.085MPa. The functionalized eutectic solvent is recovered and the precipitated astaxanthin crystals are collected to obtain the astaxanthin product.

[0078] Example 3

[0079] The supercritical separation and extraction method for astaxanthin in this embodiment includes the following steps:

[0080] (1) Weigh 2 parts of cellulose nanocrystals, 1.5 parts of methoxy polyethylene glycol silane, and 20 parts of anhydrous ethanol by weight.

[0081] (2) Add cellulose nanocrystals to anhydrous ethanol, place them in an ultrasonic dispersion device, and ultrasonically disperse them for 20 min under ultrasonic power of 300W and frequency of 40kHz to obtain a suspension.

[0082] (3) Add glacial acetic acid to the suspension to adjust the pH to 5, slowly add methoxy polyethylene glycol silane, stir at 250 rpm for 40 min at room temperature, raise the temperature to 80℃, reflux under nitrogen protection for 6 h, and cool to room temperature after the reaction to obtain the reaction solution.

[0083] (4) Place the reaction solution in a centrifuge and centrifuge at 10,000 rpm for 12 min. Collect the precipitate, wash the precipitate 5 times with anhydrous ethanol, place it in a drying oven and dry it at 50°C for 12 h to obtain modified cellulose nanocrystals.

[0084] (5) Weigh out 40 parts of betaine, 60 parts of 1,2-propanediol, and 3 parts of modified cellulose nanocrystals by weight.

[0085] (6) After mixing betaine with 1,2-propanediol, heat and stir at 500 rpm for 2.5 h in an oil bath at 85 °C until a homogeneous, transparent, viscous liquid without solid particles is formed. Cool to 50 °C to obtain a eutectic solvent.

[0086] (7) Add the modified cellulose nanocrystals to 1 / 5 of the eutectic solvent and stir into a paste. Then add the remaining 4 / 5 of the eutectic solvent and place it in an ultrasonic dispersion device. Under the conditions of ultrasonic power 400W, frequency 40kHz and temperature 50℃, ultrasonic dispersion is performed for 60min. Then the temperature is raised to 60℃ and magnetically stirred at a speed of 600rpm for 2h. Place it in a vacuum dryer and degas for 30min under the conditions of 50℃ and -0.08MPa to obtain the functionalized eutectic solvent.

[0087] (8) Weigh out 1-3 parts of Haematococcus pluvialis powder, 40 parts of functionalized eutectic solvent, 5 parts of deionized water, and 20 parts of anhydrous ethanol by weight.

[0088] (9) Slowly add deionized water to the functionalized eutectic solvent, stir until uniform, then add Haematococcus pluvialis powder, stir until fully mixed, and obtain the liquid.

[0089] (10) Place the liquid in a series ultrasonic-microjets synergistic extraction system and perform ultrasonic-microjets synergistic extraction for 25 min at a temperature of 55℃, a rotation speed of 100 rpm, an ultrasonic frequency of 40 kHz, a power of 500 W, and a microjets pressure of 120 MPa. Then centrifuge at 10000 rpm for 10 min and take the supernatant to obtain the extract.

[0090] (11) Select a ceramic membrane with a molecular weight cutoff of 20~30kDa as a vibrating membrane, and purify the extract by filtration through the vibrating membrane under the conditions of frequency 30Hz, amplitude 20mm and transmembrane pressure 0.15MPa to obtain a retentate containing astaxanthin.

[0091] (12) After the retentate is mixed with anhydrous ethanol, supercritical CO2 back-extraction is carried out at a temperature of 50℃, a pressure of 35MPa and a CO2 flow rate of 60L / h. CO2 is vaporized and recovered. The mixture is collected and then the ethanol is removed by vacuum distillation at a temperature of 60℃ and a pressure of -0.08MPa. The functionalized eutectic solvent is recovered and the precipitated astaxanthin crystals are collected to obtain the astaxanthin product.

[0092] Comparative Example 1

[0093] The supercritical separation and extraction method for astaxanthin in this comparative example is largely the same as that in Example 1, except that modified cellulose nanocrystals were not added to the eutectic solvent in this comparative example.

[0094] Comparative Example 2

[0095] The supercritical separation and extraction method for astaxanthin in this comparative example is largely the same as that in Example 1, except that the cellulose nanocrystals added to the eutectic solvent in this comparative example were not modified.

[0096] Comparative Example 3

[0097] The supercritical separation and extraction method for astaxanthin in this comparative example is largely the same as that in Example 1. The difference is that this comparative example does not use ultrasound-microfluidic synergistic extraction when separating and extracting astaxanthin, but instead uses ultrasound alone.

[0098] Comparative Example 4

[0099] The supercritical separation and extraction method for astaxanthin in this comparative example is largely the same as that in Example 1. The difference is that this comparative example does not use ultrasonic-microfluidic synergistic extraction when separating and extracting astaxanthin, but instead uses a single microfluidic extraction method.

[0100] Comparative Example 5

[0101] The supercritical separation method for extracting astaxanthin in this comparative example is largely the same as that in Example 1. The difference is that this comparative example does not use vibrating membrane technology for filtration during membrane separation, but instead uses a static membrane for filtration separation.

[0102] Experimental Example

[0103] Astaxanthin samples were prepared using the methods of Examples 1-3 and Comparative Examples 1-5, respectively, and the following performance tests were performed:

[0104] Solvent viscosity: The dynamic viscosity of the eutectic solvent was determined using a rotational viscometer at 25°C with an L2 rotor and a rotation speed of 30 rpm.

[0105] 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.

[0106] Astaxanthin purity (%): The percentage of astaxanthin in the total extract of the final product was determined by HPLC.

[0107] Steady-state membrane flux (L·m⁻²·h⁻¹): Permeation flux measured after the membrane separation has stabilized (30 min).

[0108] All-trans astaxanthin ratio (%): The proportion of all-trans configuration in total astaxanthin was determined by HPLC (C30 column, normal phase system).

[0109] Extraction rate decrease after solvent recycling 5 times (%): The percentage decrease in extraction rate after 5 extractions with the same solvent compared to the 1st extraction.

[0110] The specific test results are shown in Table 1:

[0111] Table 1. Detection results of samples from Examples 1-3 and Comparative Examples 1-4

[0112] project Solvent viscosity (mPa·s, 25℃) Extraction rate (%) purity(%) All-trans astaxanthin percentage (%) Membrane steady-state flux (LMH) The extraction rate decreased by (%) after 5 solvent cycles. Example 1 190 97.1 98.2 94.4 67 4.5 Example 2 190 97.3 98.5 94.7 68 4.4 Example 3 190 97.0 98.3 94.3 66 4.6 Comparative Example 1 420 78.5 91.2 91.5 12 12.3 Comparative Example 2 360 85.3 93.5 92.3 25 8.9 Comparative Example 3 190 84.6 96.8 90.8 64 5.1 Comparative Example 4 190 82.1 95.9 91.2 65 4.9 Comparative Example 5 190 96.8 97.8 93.5 8 4.6

[0113] As shown in Table 1, Examples 1-3, using the method of this invention, successfully separated and extracted astaxanthin from Haematococcus pluvialis powder. The extraction rate of astaxanthin exceeded 97%, and the purity of astaxanthin was greater than 98%, with the proportion of all-trans astaxanthin exceeding 94%. This is because the functionalized eutectic solvent used in Examples 1-3 was a compound of betaine, 1,2-propanediol, and modified cellulose nanocrystals. The zwitterionic properties of betaine effectively interfered with the hydrophobic interactions and hydrogen bonding between proteins and astaxanthin in the biomass raw materials, promoting the release of astaxanthin from its bound state. Simultaneously, a stable hydrogen bond network was formed between the hydroxyl groups of 1,2-propanediol and the carboxyl groups of betaine, providing a suitable polar solvation environment for the released astaxanthin and preventing π-π aggregation and precipitation of astaxanthin molecules. Polyethylene glycol chains were covalently grafted onto the surface of cellulose nanocrystals via silanization, forming a "nanobrush" structure. The polyethylene glycol chains of modified cellulose nanocrystals extend and occupy volume in eutectic solvents, disrupting the original dense hydrogen bond network between betaine and 1,2-propanediol, reducing intermolecular bonding length, and increasing the free volume of the eutectic solvent. The flexibility of the polyethylene glycol chains provides a "molecular lubrication" effect, reducing internal frictional resistance during shear flow. The nanoscale rigidity of cellulose nanocrystals can act as "micro-abrasives" during ultrasonic-microfluidic processing, assisting in the physical field breaking down biomass feedstocks. After viscosity reduction by modified cellulose nanocrystals, the functionalized eutectic solvent exhibits better fluid flow during ultrasonic-microfluidic processing, with easier formation and collapse of cavitation bubbles, resulting in more effective microfluidic shearing. Under low viscosity conditions, the mass transfer coefficient of astaxanthin molecules diffusing into the solution is significantly improved, further enhancing extraction efficiency.

[0114] The steady-state membrane flux in Examples 1-3 was greater than 65 LMH. This is because applying vibrating membrane filtration technology to the purification of functionalized eutectic solvent extracts, the membrane module undergoes periodic reciprocating vibration. The resulting shear force continuously "throws" contaminants deposited on the membrane surface, maintaining the membrane flux at a stable level. Furthermore, the extraction rate decreased by less than 5% after five cycles of solvent recycling in Examples 1-3, demonstrating that the functionalized eutectic solvent of this invention, after ethanol removal by vacuum distillation, can recover to its initial viscosity and extraction performance and be recycled, significantly reducing material consumption and production costs.

[0115] In Comparative Example 1, the eutectic solvent did not contain modified cellulose nanocrystals, resulting in a significantly higher solvent viscosity than in Example 1. This high viscosity led to a substantial increase in mass transfer resistance, reducing the extraction rate to 78.5%, far lower than in Example 1. Furthermore, the steady-state membrane flux of Comparative Example 1 was only 12 LMH, insufficient for continuous production. In contrast, Example 1, by using modified cellulose nanocrystals to reduce the viscosity of the eutectic solvent, increased the flux to 67 LMH. After five cycles, the extraction rate of Comparative Example 1 decreased by 12.3% because the unmodified cellulose nanocrystal-functionalized eutectic solvent exhibited disrupted hydrogen bond networks and rapid performance degradation after repeated use, making it unsuitable for recycling.

[0116] In Comparative Example 2, the cellulose nanocrystals added to the eutectic solvent were not grafted with polyethylene glycol. Although the solvent viscosity was lower than that of Comparative Example 1, it was still much higher than that of Example 1. This is because, although the original cellulose nanocrystals can break some hydrogen bonds, they lack the "molecular lubrication" effect of polyethylene glycol chains, and the cellulose nanocrystals themselves are prone to aggregation, resulting in limited viscosity reduction. This leads to a significantly higher steady-state membrane flux in Comparative Example 2 compared to Example 1. Furthermore, the aggregation of cellulose nanocrystals reduces the effective functionalized area, resulting in insufficient selective recognition of astaxanthin. Consequently, the extraction rate and purity of astaxanthin are lower than those in Example 1.

[0117] Comparative Example 3 used ultrasound extraction alone, achieving an extraction rate of 84.6%, while Comparative Example 4 used microfluidic extraction alone, achieving an extraction rate of 82.1%. Both were significantly lower than in Example 1. This is because ultrasound alone did not completely break down the thick-walled spores, and microfluidic extraction alone did not achieve sufficient initial penetration of the cell wall. In Example 1, ultrasound was responsible for cavitation impact and pre-breakdown on the "surface," while microfluidic extraction was responsible for high-speed shear tearing on the "line." The effect of simultaneous cyclic treatment by both was far superior to that of a single physical field, and their synergy was necessary to further improve the cell disruption rate, thereby effectively increasing the astaxanthin extraction rate.

[0118] Comparative Example 5 used a static membrane for filtration. Even though the solvent viscosity of Comparative Example 5 was the same as that of Example 1, the steady-state flux of Comparative Example 5 was only 8 LMH, which could not meet the requirements of continuous production. This is because concentration polarization forms rapidly at high viscosity, and the gel layer on the membrane surface causes a severe flux decay in a short time. Example 1, on the other hand, used a vibrating membrane to generate shear on the membrane surface, effectively removing contaminants and maintaining the flux at 68 LMH. The purity of Comparative Example 5 was slightly lower than that of Example 1 because the static membrane is more prone to the "penetration" effect, allowing some small molecule impurities to pass through the membrane pores, resulting in a decrease in purity.

[0119] This invention provides a supercritical separation and extraction method for astaxanthin. By functionalizing a eutectic solvent with modified cellulose nanocrystals, and employing ultrasonic-microfluidic synergistic extraction combined with vibrating membrane technology, the steady-state membrane flux is improved, achieving continuous membrane separation and purification in a eutectic solvent system. Combined with supercritical CO2 back-extraction, this effectively increases the astaxanthin extraction rate while maintaining its purity and the proportion of all-trans-astaxanthin. The solvent can be recycled after recovery, significantly reducing production costs and wastewater discharge. All raw materials are non-toxic and harmless, and the process uses no organic solvents, demonstrating promising application prospects.

[0120] 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.

Claims

1. A method for supercritical separation and extraction of astaxanthin, characterized in that, Includes the following steps: (1) Weigh 1-3 parts of biomass raw material rich in astaxanthin, 20-40 parts of functionalized eutectic solvent, 5-15 parts of deionized water, and 10-20 parts of anhydrous ethanol by weight. (2) Slowly add deionized water to the functionalized eutectic solvent and stir until homogeneous. Then add biomass raw material rich in astaxanthin and stir until fully mixed to obtain the liquid. (3) The material liquid is subjected to ultrasonic-microfluidic synergistic extraction using a series ultrasonic-microfluidic synergistic extraction system, centrifuged, and the supernatant is taken to obtain the extract. (4) The extract was purified by filtration through a vibrating membrane to obtain a retentate containing astaxanthin; (5) After the retentate is mixed with anhydrous ethanol, it is back-extracted by supercritical CO2, the CO2 is vaporized and recovered, the mixture is collected and the ethanol is removed by vacuum distillation, the functionalized eutectic solvent is recovered, and the precipitated astaxanthin crystals are collected to obtain the astaxanthin product.

2. The method for supercritical separation and extraction of astaxanthin according to claim 1, characterized in that, The biomass raw materials rich in astaxanthin in step (1) include at least one of Haematococcus pluvialis powder, shrimp shell powder, and crab shell powder.

3. The method for supercritical separation and extraction of astaxanthin according to claim 1, characterized in that, The preparation method of the functionalized eutectic solvent in step (1) includes the following steps: S1. Weigh out 20-40 parts of betaine, 40-60 parts of 1,2-propanediol, and 1-3 parts of modified cellulose nanocrystals by weight. S2. After mixing betaine with 1,2-propanediol, heat and stir at 300-500 rpm in an oil bath at 75-85°C for 1.5-2.5 hours until a homogeneous, transparent, viscous liquid without solid particles is formed. Cool to 40-50°C to obtain a eutectic solvent. S3. Add the modified cellulose nanocrystals to 1 / 5 of the eutectic solvent and stir into a paste. Then add the remaining 4 / 5 of the eutectic solvent and place it in an ultrasonic dispersion device. Disperse the mixture ultrasonically for 30-60 minutes at an ultrasonic power of 200-400W, a frequency of 20-40kHz, and a temperature of 40-50℃. Then raise the temperature to 50-60℃ and magnetically stir at a speed of 400-600rpm for 1-2 hours. Place the mixture in a vacuum dryer and degas it for 20-30 minutes at 40-50℃ and -0.09--0.08MPa to obtain the functionalized eutectic solvent.

4. The method for supercritical separation and extraction of astaxanthin according to claim 3, characterized in that, The preparation method of modified cellulose nanocrystals in step S1 includes the following steps: A1. Weigh out 1-2 parts of cellulose nanocrystals, 0.5-1.5 parts of methoxy polyethylene glycol silane, and 10-20 parts of anhydrous ethanol by weight. A2. Add cellulose nanocrystals to anhydrous ethanol, place them in an ultrasonic dispersion device, and ultrasonically disperse them for 10-20 minutes under ultrasonic power of 200-300W and frequency of 20-40kHz to obtain a suspension. A3. Add glacial acetic acid dropwise to the suspension to adjust the pH to 4-5, slowly add methoxy polyethylene glycol silane, stir at 150-250 rpm for 30-40 min at room temperature, raise the temperature to 60-80℃, reflux under nitrogen protection for 4-6 h, cool to room temperature after the reaction is completed to obtain the reaction solution; A4. Place the reaction solution in a centrifuge and centrifuge at 8000~10000 rpm for 10~12 min. Collect the precipitate, wash the precipitate 3~5 times with anhydrous ethanol, place it in a drying oven and dry it at 40~50℃ for 10~12 h to obtain modified cellulose nanocrystals.

5. The method for supercritical separation and extraction of astaxanthin according to claim 1, characterized in that, The series ultrasonic-microjet synergistic extraction system in step (3) includes a stirring tank equipped with an ultrasonic probe, a high-pressure microjet homogenizer, and a circulating cooling coil. The outlet of the stirring tank is connected to the inlet of the high-pressure microjet homogenizer, and the outlet of the high-pressure microjet homogenizer is connected to the inlet of the stirring tank through the circulating cooling coil to form a closed loop.

6. The method for supercritical separation and extraction of astaxanthin according to claim 1, characterized in that, The conditions for ultrasonic-microjet synergistic extraction in step (3) are: temperature 45~55℃, rotation speed 50~100rpm, ultrasonic frequency 20~40kHz, power 300~500W, microjet pressure 80~120MPa, and ultrasonic-microjet synergistic extraction time 15~25min.

7. The method for supercritical separation and extraction of astaxanthin according to claim 1, characterized in that, In step (3), the centrifugation speed is 8000~10000 rpm and the centrifugation time is 6~10 min.

8. The method for supercritical separation and extraction of astaxanthin according to claim 1, characterized in that, In step (4), the membrane type selected for vibrating membrane filtration is a ceramic membrane with a molecular weight cutoff of 20~30kDa. The conditions for vibrating membrane filtration are a frequency of 20~30Hz, an amplitude of 10~20mm, and a transmembrane pressure of 0.05~0.15MPa.

9. The method for supercritical separation and extraction of astaxanthin according to claim 1, characterized in that, The conditions for supercritical CO2 back-extraction in step (5) are: temperature 40~50℃, pressure 25~35MPa, and CO2 flow rate 30~60L / h.

10. The method for supercritical separation and extraction of astaxanthin according to claim 1, characterized in that, The conditions for vacuum distillation in step (5) are a temperature of 50~60℃ and a pressure of -0.09~-0.08MPa.