A method for multi-product cascade separation of haematococcus based on coupling of low eutectic solvent sequential extraction and enzyme membrane reactor

Abstract

The application belongs to the technical field of natural substance separation and extraction, and particularly relates to a kind of Haematococcus pluvialis multi-product cascade separation method based on eutectic solvent sequential extraction and enzyme membrane reactor coupling, comprising the following steps: (1) cell wall weakening pretreatment step; (2) water-soluble component extraction step; (3) pigment component extraction step; (4) astaxanthin extraction step; (5) enzyme conversion step. Through the synergistic effect of eutectic solvent sequential extraction and enzyme membrane reactor, efficient, green and continuous cascade separation of water-soluble components, pigments, astaxanthin and xylo-oligosaccharides in Haematococcus pluvialis is realized, and product purity and resource utilization are improved.

Description

Technical Field

[0001] This invention belongs to the field of natural substance separation and extraction technology, specifically relating to a multi-product stepwise separation method of Haematococcus pluvialis based on sequential extraction with a low eutectic solvent coupled with an enzyme membrane reactor. Background Technology

[0002] Haematococcus pluvialis is a single-celled microalga, with Haematococcus pluvialis particularly noteworthy for its ability to accumulate high levels of astaxanthin. Astaxanthin is a potent antioxidant, far exceeding the antioxidant capacity of ordinary vitamins, and is widely used in health foods, cosmetics, and pharmaceuticals to enhance immunity, combat aging, and improve skin health. In addition to astaxanthin, Haematococcus pluvialis also contains abundant polysaccharides, proteins, and lipids, which possess various biological activities. For example, polysaccharides can regulate gut microbiota and enhance immunity, while lipids contain essential fatty acids that contribute to cardiovascular health.

[0003] In practical applications, the processing and utilization of *Hydrocotyle vulgaris* faces several drawbacks. First, the cell wall structure of *Hydrocotyle vulgaris* is dense, composed of multiple layers of polysaccharides and glycoproteins. Traditional cell wall disruption methods, such as high-pressure homogenization or mechanical grinding, are energy-intensive and easily lead to the degradation of heat-sensitive components like astaxanthin during processing, affecting product activity and yield. Second, the algae contain complex components, including water-soluble polysaccharides, fat-soluble pigments, and non-polar astaxanthin. Existing extraction technologies often require multiple steps and use large amounts of organic solvents such as acetone and n-hexane. These solvents are highly toxic, pose safety hazards, and can easily cause product residues and environmental pollution. Furthermore, existing methods often focus on the extraction of a single target product, such as astaxanthin, while neglecting the utilization of other high-value components like polysaccharides, leading to resource waste and poor overall economic efficiency. Xylan polysaccharides contained in the cell wall of *Hydrocotyle vulgaris* can be converted into xylooligosaccharides through enzymatic hydrolysis.

[0004] To address these shortcomings, existing technologies have explored various solutions. For example, supercritical carbon dioxide extraction is used to replace organic solvents, avoiding solvent residue. However, this method has high equipment investment and operating costs, poor extraction efficiency for water-soluble components, and difficulty in achieving simultaneous separation of multiple components. Another common method is enzymatic hydrolysis, which uses cellulase or pectinase to disrupt cell walls. However, single enzymatic hydrolysis has limited efficiency and often requires high temperatures or strong acid / alkali conditions, which may damage sensitive components. Furthermore, enzymes are expensive and have low reusability. In recent years, eutectic solvents have been introduced into the field of natural product extraction as a green solvent. They consist of hydrogen bond donors and acceptors, exhibiting low toxicity and biodegradability.

[0005] However, in the extraction of *Haemaphysalis*, the simple application of eutectic solvents still suffers from low selectivity, failing to effectively extract components of different polarities stepwise. Furthermore, solvent recovery is difficult, and the number of times it can be reused is limited. In addition, polysaccharides in *Haemaphysalis* are often treated as byproducts and not converted into higher-value products, such as xylooligosaccharides, a prebiotic traditionally prepared from raw materials like corn cobs. The technology for converting *Haemaphysalis*-derived polysaccharides into xylooligosaccharides is not yet mature, which limits resource utilization efficiency and product added value.

[0006] While existing technologies address the aforementioned shortcomings, several problems remain. For example, combining supercritical fluid extraction and enzymatic hydrolysis results in a complex process, high energy consumption, and inability to achieve continuous production. Eutectic solvents are easily affected by moisture and temperature during extraction, exhibiting poor stability and leading to fluctuations in extraction efficiency. The polysaccharide conversion stage lacks efficient reactors, resulting in incomplete product separation and affecting purity. These problems lead to high processing costs for *Hydrocotyle vulgaris*, limited product variety, and insufficient environmental friendliness. Therefore, there is an urgent need in this field for an innovative method that can achieve efficient and green separation of multiple components from *Hydrocotyle vulgaris*, while simultaneously improving resource utilization through high-value conversion. Thus, a multi-product stepwise separation method for *Hydrocotyle vulgaris* based on sequential extraction with eutectic solvents coupled with an enzyme membrane reactor needs to be designed. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, a multi-product stepwise separation method for Haematococcus pluvialis based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor is provided.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A stepwise separation method for multiple products of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor, comprising the following steps:

[0010] (1) Cell wall weakening pretreatment step: Mix Haematococcus pluvialis powder with weakening agent solution at a mass ratio of 1:15 to 1:25, and treat at a temperature of 35℃ to 45℃ for 25 to 35 minutes to obtain pretreated algal powder;

[0011] In the processing of Haematococcus pluvialis, the first challenge is the extraction process posed by its dense cell walls. Traditional mechanical cell disruption methods generate strong shear forces, easily leading to the degradation of heat-sensitive components such as astaxanthin, while chemical cell disruption methods may introduce impurities or damage active ingredients. This invention employs a cell wall weakening pretreatment step, using a weakening agent solution composed of citric acid, malic acid, and ascorbic acid. Under a mild acidic environment, these organic acids synergistically act on the polysaccharide and glycoprotein structures in the cell wall, loosening them through hydrogen bonding and gentle acid hydrolysis. Simultaneously, the addition of polysorbate 80 acts as a permeabilizer, further promoting the solution's penetration into the cell interior. This pretreatment method effectively weakens the cell wall barrier without using strong mechanical force or high temperatures, creating favorable conditions for the subsequent extraction process.

[0012] (2) Water-soluble component extraction steps: The pretreated algal powder is mixed with a hydrophilic eutectic solvent at a mass ratio of 1:20 to 1:30 and extracted at a temperature of 50℃ to 60℃ for 40 to 60 minutes to separate the water-soluble extract and the first algal residue.

[0013] (3) Pigment extraction steps: Mix the first algal residue with a medium polar eutectic solvent at a mass ratio of 1:15 to 1:25, and extract at a temperature of 58°C to 65°C for 25 to 40 minutes to obtain the pigment extract and the second algal residue.

[0014] (4) Astaxanthin extraction steps: Mix the second algal residue with a hydrophobic eutectic solvent at a mass ratio of 1:10 to 1:20, and extract at a temperature of 42°C to 50°C for 50 to 70 minutes to separate the astaxanthin extract and protein residue.

[0015] After overcoming the cell wall barrier problem, the simultaneous separation of different polar components in *Haemaphysalis* became a new challenge. Traditional single-solvent systems struggle to handle water-soluble polysaccharides, lipophilic pigments, and strongly nonpolar astaxanthin simultaneously, often requiring multiple solvent systems and complex process switching. This invention designs three types of low-melting-point solvent systems with different polarities for sequential extraction. In the hydrophilic low-melting-point solvent, choline chloride and lactic acid form the basic solvent framework. The specially introduced succinylated trehalose enhances hydrogen bonding with polysaccharides through the introduction of carboxyl groups, improving the extraction selectivity of water-soluble components. Subsequently, the medium-polarity low-melting-point solvent is based on betaine and propylene glycol. The acetylated tea polyphenol ester, through the introduction of acetyl groups, modulates the solvent's lipophilicity, making it more suitable for dissolving pigment components. Finally, the hydrophobic low-melting-point solvent consists of menthol and thymol. Combined with the interfacial activity of polyethylene glycol-modified phytosterol esters, it effectively promotes the transfer of strongly hydrophobic components such as astaxanthin. This sequential extraction strategy based on polarity gradients enables the effective separation of components with different properties.

[0016] (5) Enzyme conversion step: The water-soluble extract and the compound enzyme preparation are introduced into the enzyme membrane reactor. The enzymatic hydrolysis reaction is carried out under the action of the compound enzyme preparation. At the same time, the membrane separation function of the enzyme membrane reactor is used to separate and collect the xylooligosaccharides generated in the reaction in real time to obtain xylooligosaccharide products.

[0017] The weakening agent solution in step (1) is a mixture of citric acid, malic acid and ascorbic acid in a mass ratio of 1:0.5:0.2 to 1:1:0.5, dissolved in deionized water to form a solution with a concentration of 0.8% to 1.2%. The pH value is adjusted to 4.0 to 4.5, and 0.05% to 0.1% of polysorbate 80 is added as a penetrant.

[0018] The water-soluble component extraction step (2) is carried out under ultrasound assistance. The ultrasound assistance adopts a multi-frequency combination with frequencies of 28kHz, 40kHz and 60kHz, a power ratio of 1:0.8:0.5, and a pulse mode. The ratio of ultrasound time to interval time is 1:1 to 1:1.5, and the total ultrasound time is 25 to 40 minutes.

[0019] The pigment extraction step (3) is carried out under microwave assistance. The power of the microwave is controlled in multiple stages, with an initial power of 300W, increasing by 50W every 5 minutes, and not exceeding 450W. Nitrogen gas is introduced for protection at the same time, with a gas flow rate of 0.2L / min to 0.4L / min and an extraction time of 25 to 40 minutes.

[0020] The astaxanthin extraction step (4) was carried out under mechanical stirring and nitrogen protection, with a stirring speed of 280 rpm to 350 rpm and a nitrogen flow rate of 0.1 L / min to 0.3 L / min. An anchor-type stirring paddle was used, and the extraction time was 50 to 70 minutes.

[0021] Although a sequential extraction solvent system has been established, extraction efficiency still needs further improvement, while ensuring that active ingredients are not damaged during extraction. This invention introduces targeted physical field enhancement measures at each extraction stage. In the water-soluble component extraction stage, multi-frequency combined ultrasound technology is employed. The cavitation effect generated by ultrasound waves of different frequencies acts on cell structures at different scales, synergistically enhancing solvent diffusion and component dissolution, while the pulse mode avoids localized overheating caused by continuous ultrasound. In the pigment component extraction stage, multi-segment controlled microwave assistance provides a rapid and uniform heating method, significantly improving mass transfer efficiency. Simultaneously, nitrogen protection effectively prevents the oxidative degradation of photosensitive pigments. In the astaxanthin extraction stage, mechanical stirring ensures thorough mixing of the solvent and materials, while continuous nitrogen protection provides a stable extraction environment for easily oxidized components like astaxanthin. These physical field enhancement measures, combined with the corresponding solvent system, improve the extraction rate while maximizing the preservation of the bioactivity of each component.

[0022] The enzyme membrane reactor in step (5) uses a polyethersulfone hollow fiber membrane with an inner diameter of 1.0 mm to 1.5 mm, a wall thickness of 0.2 mm to 0.3 mm, and a molecular weight cutoff of 7000 Da to 8500 Da; the membrane module packing density is 35% to 45%, the operating pressure is 0.1 MPa to 0.3 MPa, the membrane surface flow rate is 1.0 m / s to 2.0 m / s, and the reaction temperature is 45℃ to 55℃.

[0023] After successfully extracting water-soluble polysaccharides, the challenge lies in efficiently converting them into higher-value products. Traditional enzymatic hydrolysis processes suffer from product inhibition and the inability to reuse enzymes. This invention employs an enzyme membrane reactor system, integrating the enzyme-catalyzed reaction with the membrane separation process. The xylanase, arabinosidase, and polyethylene glycol-modified cellulase in the composite enzyme preparation synergistically act on different sites of the polysaccharide molecule, with the polyethylene glycol-modified cellulase exhibiting better stability and compatibility. During the reaction, the polyethersulfone hollow fiber membrane, with its precise molecular weight cutoff, separates the generated xylooligosaccharides from the enzyme and unreacted substrates in real time. This continuous separation method not only eliminates product feedback inhibition of the enzyme, maintaining a consistently high reaction rate, but also achieves effective enzyme recovery and reuse, significantly improving the process's economic efficiency.

[0024] The hydrophilic eutectic solvent in step (2) is a mixture of choline chloride, lactic acid and succinylated trehalose in a molar ratio of 1:2.5:0.5 to 1:3.5:1, and deionized water is added to adjust the water content to 20% to 30%.

[0025] The preparation method of the succinylated trehalose includes the following steps: dissolving trehalose in a phosphate buffer solution with a pH of 7.5 to 8.5, adding succinic anhydride to carry out the reaction, the reaction temperature is 25°C to 35°C, the reaction time is 2 to 4 hours, the molar ratio of trehalose to succinic anhydride is 1:0.5 to 1:1.2, after the reaction is completed, removing small molecule impurities by dialysis or ultrafiltration, and then freeze-drying to obtain succinylated trehalose.

[0026] The medium polar eutectic solvent in step (3) is a mixture of betaine, propylene glycol and acetylated tea polyphenol ester in a molar ratio of 1:1.8:0.3 to 1:2.5:0.8, with deionized water added to adjust the water content to 15% to 25%.

[0027] The preparation method of the acetylated tea polyphenol ester includes: dissolving tea polyphenols in ethyl acetate, adding acetic anhydride and catalyst 4-dimethylaminopyridine to react, the reaction temperature is 40℃ to 50℃, the reaction time is 3 to 5 hours, the molar ratio of tea polyphenols to acetic anhydride is 1:1 to 1:2, and after the reaction is completed, the acetylated tea polyphenol ester is obtained by rotary evaporation and recrystallization with ethanol.

[0028] The hydrophobic eutectic solvent in step (4) is composed of menthol, thymol and polyethylene glycol-modified phytosterol ester in a molar ratio of 1:1.2:0.4 to 1:1.8:0.9; the preparation of the hydrophobic eutectic solvent includes stirring and reacting each component at 45°C to 55°C for 2 to 4 hours to obtain the hydrophobic eutectic solvent.

[0029] The method for preparing the polyethylene glycol-modified phytosterol ester includes: dissolving phytosterol and polyethylene glycol monomethyl ether in toluene, adding a catalyst such as p-toluenesulfonic acid to react, the reaction temperature being 110°C to 130°C, the reaction time being 6 to 8 hours, the molar ratio of phytosterol to polyethylene glycol monomethyl ether being 1:1 to 1:1.5, and purifying by cooling crystallization and filtration after the reaction is completed.

[0030] The complex enzyme preparation in step (5) is composed of xylanase, arabinosidase and polyethylene glycol modified cellulase in a mass ratio of 1:0.3:0.2 to 1:0.6:0.5, wherein the degree of modification of polyethylene glycol modified cellulase is 0.4 to 0.7; the preparation process of the complex enzyme preparation is as follows: dissolve the above enzymes in a citrate buffer with a pH of 5.5 to 6.0, and the total concentration of the enzymes is 10 mg / mL to 20 mg / mL.

[0031] The method for preparing the polyethylene glycol-modified cellulase includes: dissolving cellulase in a phosphate buffer solution with a pH of 7.0-7.5, adding pre-activated polyethylene glycol succinimide ester, wherein the polyethylene glycol succinimide ester is prepared by reacting polyethylene glycol 6000 and N-hydroxysuccinimide under the condition of dicyclohexylcarbodiimide as a condensing agent; controlling the modification reaction temperature to be 4°C to 8°C, the reaction time to be 12 to 16 hours, and the molar ratio of cellulase to polyethylene glycol succinimide ester to be 1:5 to 1:10; after the reaction, purifying the enzyme using an ultrafiltration membrane with a molecular weight cutoff of 30,000 to obtain the polyethylene glycol-modified cellulase.

[0032] This invention achieves excellent synergy between its various unit operations, from cell wall weakening pretreatment to three-stage sequential extraction and then to enzyme membrane reactor conversion. The entire process system uses a recyclable eutectic solvent, avoiding the environmental burden of traditional organic solvents, while simultaneously obtaining multiple high-value products from Haematococcus pluvialis, including xylooligosaccharides, natural pigments, astaxanthin, and feed proteins.

[0033] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0034] 1. This invention achieves effective dissociation of the Haematococcus pluvialis cell structure and highly selective separation of target components through the synergistic effect of a cell wall weakening pretreatment step and a three-stage eutectic solvent sequential extraction. Specifically, the weakening agent solution preferentially softens the polysaccharide network in the cell wall under mild acidic conditions, creating favorable conditions for subsequent extraction. Then, using precisely designed hydrophilic, neutral polar, and hydrophobic eutectic solvent systems, water-soluble polysaccharides, pigments, and astaxanthin are extracted stepwise, respectively. This sequential extraction strategy not only avoids mutual interference between components of different polarities but also significantly improves the extraction efficiency and purity of each target component. In particular, components such as succinylated trehalose and acetylated tea polyphenol esters introduced into the modified eutectic solvent enhance the affinity between the solvent and the target components through specific intermolecular interactions, thereby achieving more precise separation.

[0035] 2. This invention enhances the extraction efficiency at each stage through multi-field coupling enhancement technology. In the hydrophilic component extraction stage, the synergistic cavitation effect generated by multi-frequency ultrasound effectively promotes solvent penetration and mass transfer into the cells. In the neutral polar component extraction stage, multi-segment microwave assistance combined with nitrogen protection achieves rapid and uniform heating while preventing the oxidative degradation of heat-sensitive pigments. In the astaxanthin extraction stage, the combination of mechanical stirring and nitrogen protection ensures the full dissolution of hydrophobic components and maintains their chemical stability. These enhancement measures, combined with the corresponding eutectic solvent system, ensure that the extraction process at each stage maintains a high mass transfer rate while maximizing the protection of the target component's bioactivity.

[0036] 3. This invention achieves efficient conversion and continuous separation of water-soluble polysaccharides into high-value-added xylooligosaccharides through the innovative application of an enzyme membrane reactor. The core advantage of this system lies in the organic integration of enzyme catalysis and membrane separation. The components of the composite enzyme preparation act synergistically on specific sites on the polysaccharide molecular chain, directionally converting it into the target oligosaccharide. Simultaneously, the polyethersulfone hollow fiber membrane, through its precise molecular weight cutoff design, enables real-time separation of large-molecule enzyme preparations and small-molecule products in the reaction system. This design not only eliminates product inhibition effects, allowing the enzymatic reaction to proceed continuously in the positive direction, but also achieves effective recovery and reuse of the enzyme preparation, significantly improving the economic efficiency of the conversion process.

[0037] 4. This invention achieves the simultaneous extraction of four high-value products—xylooligosaccharides, natural pigments, astaxanthin, and feed protein—from a single raw material through the systematic integration of unit operations such as cell wall weakening, multi-stage extraction, enzymatic conversion, and membrane separation. The entire process embodies the basic principles of green chemistry, employing a recyclable eutectic solvent that avoids the environmental pollution problems associated with traditional organic solvents, maximizing raw material utilization, and significantly reducing energy consumption and material loss. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] In the specific embodiments of this application, the sources of various main raw materials are briefly described as follows:

[0040] Citric acid: purchased from Sinopharm Chemical Reagent Co., Ltd., CAS number 77-92-9

[0041] Malic acid: purchased from Aladdin Biochemical Technology Co., Ltd., CAS No. 6915-15-7

[0042] Ascorbic acid: purchased from Hebei Liqia Biotechnology Co., Ltd., CAS No.: 23313-12-4

[0043] Polysorbate 80: Purchased from Nanjing Dulai Biotechnology Co., Ltd., CAS No. 9005-65-6

[0044] Choline chloride: purchased from Beijing Bailingwei Technology Co., Ltd., CAS No. 67-48-1

[0045] Lactic acid: purchased from Jinan Renyuan Chemical Co., Ltd., CAS No. 50-21-5

[0046] Trehalose: Purchased from Shandong Fufeng Fermentation Co., Ltd., CAS No. 99-20-7

[0047] Succinic anhydride: purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0048] Betaine: Purchased from Zhejiang Xinhecheng Co., Ltd., CAS No. 107-43-7

[0049] Propylene glycol: purchased from Jiangsu Dena Chemical Co., Ltd., CAS number 57-55-6

[0050] Tea polyphenols: purchased from Xi'an Tianyi Biotechnology Co., Ltd., CAS No. 84650-60-2

[0051] Acetic anhydride: purchased from Jiangsu Zhengdan Chemical Industry Co., Ltd., CAS No. 108-24-7

[0052] 4-Dimethylaminopyridine: purchased from THICA Chemical Industry Development Co., Ltd., CAS No. 1122-58-3

[0053] Menthol: Purchased from Guangzhou Baihua Fragrance Co., Ltd., CAS No. 89-78-1

[0054] Phytosterols: Purchased from Zhejiang Medicine Co., Ltd., Xinchang Pharmaceutical Factory, CAS No. 83-46-5

[0055] Polyethylene glycol monomethyl ether: purchased from Hunan Huateng Pharmaceutical Co., Ltd., CAS No. 9004-74-4

[0056] p-Toluenesulfonic acid: purchased from Tianjin Kemei Chemical Reagent Co., Ltd., CAS No. 6192-52-5

[0057] Xylanase: Purchased from Novozymes China Biotechnology Co., Ltd., product number NS50030, activity ≥5000U / g

[0058] Arabinosidase: Purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS No. 9025-53-2, food grade.

[0059] Cellulase: Purchased from Jiangsu Ruiyang Biotechnology Co., Ltd., CAS No. 9012-54-8, industrial grade.

[0060] Polyethylene glycol 6000: Purchased from Shandong Yousuo Chemical Technology Co., Ltd., CAS No. 25322-68-3

[0061] N-Hydroxysuccinimide: Purchased from Shanghai BIDE Pharmaceutical Technology Co., Ltd., CAS No. 6066-82-6

[0062] The technical solution of this application is as follows:

[0063] A stepwise separation method for multiple products of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor, comprising the following steps:

[0064] (1) Cell wall weakening pretreatment step: Mix Haematococcus pluvialis powder with weakening agent solution at a mass ratio of 1:15 to 1:25, and treat at a temperature of 35℃ to 45℃ for 25 to 35 minutes to obtain pretreated algal powder;

[0065] (2) Water-soluble component extraction steps: The pretreated algal powder is mixed with a hydrophilic eutectic solvent at a mass ratio of 1:20 to 1:30 and extracted at a temperature of 50℃ to 60℃ for 40 to 60 minutes to separate the water-soluble extract and the first algal residue.

[0066] (3) Pigment extraction steps: Mix the first algal residue with a medium polar eutectic solvent at a mass ratio of 1:15 to 1:25, and extract at a temperature of 58°C to 65°C for 25 to 40 minutes to obtain the pigment extract and the second algal residue.

[0067] (4) Astaxanthin extraction steps: Mix the second algal residue with a hydrophobic eutectic solvent at a mass ratio of 1:10 to 1:20, and extract at a temperature of 42°C to 50°C for 50 to 70 minutes to separate the astaxanthin extract and protein residue.

[0068] (5) Enzyme conversion step: The water-soluble extract and the compound enzyme preparation are introduced into the enzyme membrane reactor. The enzymatic hydrolysis reaction is carried out under the action of the compound enzyme preparation. At the same time, the membrane separation function of the enzyme membrane reactor is used to separate and collect the xylooligosaccharides generated in the reaction in real time to obtain xylooligosaccharide products.

[0069] The weakening agent solution in step (1) is a mixture of citric acid, malic acid and ascorbic acid in a mass ratio of 1:0.5:0.2 to 1:1:0.5, dissolved in deionized water to form a solution with a concentration of 0.8% to 1.2%. The pH value is adjusted to 4.0 to 4.5, and 0.05% to 0.1% of polysorbate 80 is added as a penetrant.

[0070] The water-soluble component extraction step (2) is carried out under ultrasound assistance. The ultrasound assistance adopts a multi-frequency combination with frequencies of 28kHz, 40kHz and 60kHz, a power ratio of 1:0.8:0.5, and a pulse mode. The ratio of ultrasound time to interval time is 1:1 to 1:1.5, and the total ultrasound time is 25 to 40 minutes.

[0071] The pigment extraction step (3) is carried out under microwave assistance. The power of the microwave is controlled in multiple stages, with an initial power of 300W, increasing by 50W every 5 minutes, and not exceeding 450W. Nitrogen gas is introduced for protection at the same time, with a gas flow rate of 0.2L / min to 0.4L / min and an extraction time of 25 to 40 minutes.

[0072] The astaxanthin extraction step (4) was carried out under mechanical stirring and nitrogen protection, with a stirring speed of 280 rpm to 350 rpm and a nitrogen flow rate of 0.1 L / min to 0.3 L / min. An anchor-type stirring paddle was used, and the extraction time was 50 to 70 minutes.

[0073] The enzyme membrane reactor in step (5) uses a polyethersulfone hollow fiber membrane with an inner diameter of 1.0 mm to 1.5 mm, a wall thickness of 0.2 mm to 0.3 mm, and a molecular weight cutoff of 7000 Da to 8500 Da; the membrane module packing density is 35% to 45%, the operating pressure is 0.1 MPa to 0.3 MPa, the membrane surface flow rate is 1.0 m / s to 2.0 m / s, and the reaction temperature is 45℃ to 55℃.

[0074] The hydrophilic eutectic solvent in step (2) is a mixture of choline chloride, lactic acid and succinylated trehalose in a molar ratio of 1:2.5:0.5 to 1:3.5:1, and deionized water is added to adjust the water content to 20% to 30%.

[0075] The preparation method of the succinylated trehalose includes the following steps: dissolving trehalose in a phosphate buffer solution with a pH of 7.5 to 8.5, adding succinic anhydride to carry out the reaction, the reaction temperature is 25°C to 35°C, the reaction time is 2 to 4 hours, the molar ratio of trehalose to succinic anhydride is 1:0.5 to 1:1.2, after the reaction is completed, removing small molecule impurities by dialysis or ultrafiltration, and then freeze-drying to obtain succinylated trehalose.

[0076] The medium polar eutectic solvent in step (3) is a mixture of betaine, propylene glycol and acetylated tea polyphenol ester in a molar ratio of 1:1.8:0.3 to 1:2.5:0.8, with deionized water added to adjust the water content to 15% to 25%.

[0077] The preparation method of the acetylated tea polyphenol ester includes: dissolving tea polyphenols in ethyl acetate, adding acetic anhydride and catalyst 4-dimethylaminopyridine to react, the reaction temperature is 40℃ to 50℃, the reaction time is 3 to 5 hours, the molar ratio of tea polyphenols to acetic anhydride is 1:1 to 1:2, and after the reaction is completed, the acetylated tea polyphenol ester is obtained by rotary evaporation and recrystallization with ethanol.

[0078] The hydrophobic eutectic solvent in step (4) is composed of menthol, thymol and polyethylene glycol-modified phytosterol ester in a molar ratio of 1:1.2:0.4 to 1:1.8:0.9; the preparation of the hydrophobic eutectic solvent includes stirring and reacting each component at 45°C to 55°C for 2 to 4 hours to obtain the hydrophobic eutectic solvent.

[0079] The method for preparing the polyethylene glycol-modified phytosterol ester includes: dissolving phytosterol and polyethylene glycol monomethyl ether in toluene, adding a catalyst such as p-toluenesulfonic acid to react, the reaction temperature being 110°C to 130°C, the reaction time being 6 to 8 hours, the molar ratio of phytosterol to polyethylene glycol monomethyl ether being 1:1 to 1:1.5, and purifying by cooling crystallization and filtration after the reaction is completed.

[0080] The complex enzyme preparation in step (5) is composed of xylanase, arabinosidase and polyethylene glycol modified cellulase in a mass ratio of 1:0.3:0.2 to 1:0.6:0.5, wherein the degree of modification of polyethylene glycol modified cellulase is 0.4 to 0.7; the preparation process of the complex enzyme preparation is as follows: dissolve the above enzymes in a citrate buffer with a pH of 5.5 to 6.0, and the total concentration of the enzymes is 10 mg / mL to 20 mg / mL.

[0081] The method for preparing the polyethylene glycol-modified cellulase includes: dissolving cellulase in a phosphate buffer solution with a pH of 7.0-7.5, adding pre-activated polyethylene glycol succinimide ester, wherein the polyethylene glycol succinimide ester is prepared by reacting polyethylene glycol 6000 and N-hydroxysuccinimide under the condition of dicyclohexylcarbodiimide as a condensing agent; controlling the modification reaction temperature to be 4°C to 8°C, the reaction time to be 12 to 16 hours, and the molar ratio of cellulase to polyethylene glycol succinimide ester to be 1:5 to 1:10; after the reaction, purifying the enzyme using an ultrafiltration membrane with a molecular weight cutoff of 30,000 to obtain the polyethylene glycol-modified cellulase.

[0082] In subsequent processing, natural pigment products can be obtained from pigment extract using macroporous adsorption resin column purification, astaxanthin products can be obtained from astaxanthin extract using distillation and extraction, and feed protein products can be obtained from protein residue using washing and separation. Other processes in the industry can also be used. All of the above processes are well-known technologies and will not be described in detail here.

[0083] This invention achieves efficient, green, and continuous stepwise separation of water-soluble components, pigments, astaxanthin, and xylooligosaccharides in Haematococcus pluvialis through the synergistic effect of sequential extraction with eutectic solvents and enzyme membrane reactors, thereby improving product purity and resource utilization.

[0084] The present invention will be described in detail below through examples and comparative examples, but the scope of protection of the present invention is not limited to these examples. Unless otherwise specified, the chemical reagents and raw materials used in the following examples and comparative examples are all conventional commercially available products. Example 1

[0085] This embodiment details the implementation process of a multi-product stepwise separation method for Haematococcus pluvialis based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor. First, a cell wall weakening pretreatment is performed. Haematococcus pluvialis powder is mixed with a weakening agent solution at a mass ratio of 1:25 and treated at 45°C for 35 minutes to obtain pretreated powder. The weakening agent solution is a 1.2% solution formed by dissolving a mixture of citric acid, malic acid, and ascorbic acid in deionized water at a mass ratio of 1:1:0.5. The pH is adjusted to 4.0, and 0.1% (w / w) of polysorbate 80 is added as a permeabilizing agent.

[0086] Next, water-soluble components were extracted. The pretreated algal powder was mixed with a hydrophilic eutectic solvent at a mass ratio of 1:30 and extracted at 60℃ for 60 minutes. This step was assisted by multi-frequency ultrasound at frequencies of 28kHz, 40kHz, and 60kHz, with a power ratio of 1:0.8:0.5, using a pulse mode. The ratio of ultrasound time to interval time was 1:1.5, with a total ultrasound time of 40 minutes. The hydrophilic eutectic solvent was a mixture of choline chloride, lactic acid, and succinylated trehalose in a molar ratio of 1:3.5:1, with deionized water added to adjust the water content to 30%. After separation, a water-soluble extract and the first algal residue were obtained.

[0087] Pigment extraction was then performed. The first algal residue was mixed with a moderately polar eutectic solvent at a mass ratio of 1:25 and extracted at 65°C for 40 minutes. This step was microwave-assisted, with an initial power of 300W, increasing by 50W every 5 minutes, up to a maximum power of 450W, while nitrogen gas was introduced for protection at a flow rate of 0.4 L / min. The moderately polar eutectic solvent was a mixture of betaine, propylene glycol, and acetylated tea polyphenol esters in a molar ratio of 1:2.5:0.8, with deionized water added to adjust the water content to 25%. After separation, the pigment extract and the second algal residue were obtained.

[0088] Astaxanthin extraction was then performed. The second algal residue was mixed with a hydrophobic eutectic solvent at a mass ratio of 1:20, and extracted at 50°C for 70 minutes. This step employed mechanical stirring and nitrogen protection, with a stirring speed of 350 rpm and a nitrogen flow rate of 0.3 L / min, using an anchor-type stirrer. The hydrophobic eutectic solvent was a mixture of menthol, thymol, and polyethylene glycol-modified phytosterol esters in a molar ratio of 1:1.8:0.9. After separation, astaxanthin extract and protein residue were obtained.

[0089] Finally, enzymatic conversion was performed. The water-soluble extract and the compound enzyme preparation were introduced into an enzyme membrane reactor, where the enzymatic hydrolysis reaction took place under the action of the compound enzyme preparation. The reaction temperature was 55℃, the operating pressure was 0.3MPa, the membrane flow rate was 2.0m / s, and the reaction time was 5 hours. The compound enzyme preparation was composed of xylanase, arabinosidase, and polyethylene glycol-modified cellulase in a mass ratio of 1:0.6:0.5, with a total enzyme concentration of 20mg / mL. The xylooligosaccharides generated in the reaction were separated and collected in real time using the membrane separation function of the enzyme membrane reactor to obtain the xylooligosaccharide product. Example 2

[0090] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0091] This embodiment employs a multi-product stepwise separation method for *Haemaphysalis* using different process parameters. In the cell wall weakening pretreatment step, algal powder and weakening agent solution are mixed at a mass ratio of 1:15 and treated at 35°C for 25 minutes. The weakening agent solution is a compound of citric acid, malic acid, and ascorbic acid at a mass ratio of 1:0.5:0.2, with a solution concentration of 0.8%, a pH of 4.5, and 0.05% polysorbate 80.

[0092] The extraction of water-soluble components was performed at a mass ratio of 1:20, a temperature of 50℃, and an extraction time of 40 minutes. Ultrasonic assistance was used in pulse mode with an ultrasonic time-to-interval ratio of 1:1, for a total ultrasonic time of 25 minutes. The hydrophilic eutectic solvent was a mixture of choline chloride, lactic acid, and succinylated trehalose in a molar ratio of 1:2.5:0.5, with a water content of 20%.

[0093] The pigment extraction process employed a mass ratio of 1:15, an extraction temperature of 58℃, and an extraction time of 25 minutes. Microwave-assisted extraction was performed with an initial power of 300W and a gas flow rate of 0.2L / min. The moderately polar eutectic solvent was a mixture of betaine, propylene glycol, and acetylated tea polyphenol esters in a molar ratio of 1:1.8:0.3, with a water content of 15%.

[0094] Astaxanthin extraction was performed at a mass ratio of 1:10, a temperature of 42℃, and an extraction time of 50 minutes. Mechanical stirring was maintained at 280 rpm, and nitrogen flow rate was 0.1 L / min. The hydrophobic eutectic solvent was a mixture of menthol, thymol, and polyethylene glycol-modified phytosterol esters at a molar ratio of 1:1.2:0.4.

[0095] The enzymatic conversion step was carried out at a reaction temperature of 45℃, an operating pressure of 0.1MPa, a membrane flow rate of 1.0m / s, and a reaction time of 3 hours. The compound enzyme preparation consisted of xylanase, arabinosidase, and polyethylene glycol-modified cellulase in a mass ratio of 1:0.3:0.2, with a total enzyme concentration of 10mg / mL. Example 3

[0096] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0097] This embodiment provides an alternative parameter combination implementation. In the cell wall weakening pretreatment step, algal powder and weakening agent solution are mixed at a mass ratio of 1:20 and treated at 40°C for 30 minutes. The weakening agent solution is a compound of citric acid, malic acid, and ascorbic acid at a mass ratio of 1:0.8:0.3, with a solution concentration of 1.0%, a pH value of 4.2, and an addition of 0.08% polysorbate 80.

[0098] The extraction of water-soluble components was performed at a mass ratio of 1:25, a temperature of 55℃, and an extraction time of 50 minutes. Ultrasonic assistance was used in pulse mode, with an ultrasonic time-to-interval ratio of 1:1.2 and a total ultrasonic time of 32 minutes. The hydrophilic eutectic solvent was a mixture of choline chloride, lactic acid, and succinylated trehalose in a molar ratio of 1:3.0:0.8, with a water content of 25%.

[0099] The pigment extraction process employed a mass ratio of 1:20, a temperature of 62℃, and an extraction time of 32 minutes. Microwave-assisted extraction was performed with an initial power of 300W and a gas flow rate of 0.3L / min. The moderately polar eutectic solvent was a mixture of betaine, propylene glycol, and acetylated tea polyphenol esters in a molar ratio of 1:2.2:0.5, with a water content of 20%.

[0100] Astaxanthin extraction was performed at a mass ratio of 1:15, a temperature of 46℃, and an extraction time of 60 minutes. Mechanical stirring was maintained at 320 rpm, and nitrogen flow rate was 0.2 L / min. The hydrophobic eutectic solvent was a mixture of menthol, thymol, and polyethylene glycol-modified phytosterol esters at a molar ratio of 1:1.5:0.6.

[0101] The enzymatic conversion step was carried out at a reaction temperature of 50℃, an operating pressure of 0.2MPa, a membrane flow rate of 1.5m / s, and a reaction time of 4 hours. The compound enzyme preparation consisted of xylanase, arabinosidase, and polyethylene glycol-modified cellulase in a mass ratio of 1:0.4:0.3, with a total enzyme concentration of 15mg / mL.

[0102] Comparative Example 1

[0103] In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows:

[0104] The cell wall weakening pretreatment step was omitted, and untreated Haematococcus pluvialis powder was used directly for subsequent extraction.

[0105] Comparative Example 2

[0106] In this comparative example, the similarities with Example 2 will not be repeated, and the differences are as follows:

[0107] In the extraction step of water-soluble components, pure water is used instead of hydrophilic eutectic solvent.

[0108] Comparative Example 3

[0109] In this comparative example, the similarities with Example 3 will not be repeated, and the differences are as follows:

[0110] Ethanol was used instead of a moderately polar eutectic solvent in the pigment extraction step.

[0111] Comparative Example 4

[0112] In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows:

[0113] Nitrogen protection is not used in the astaxanthin extraction process.

[0114] Comparative Example 5

[0115] In this comparative example, the similarities with Example 2 will not be repeated, and the differences are as follows:

[0116] In the enzyme conversion step, a conventional batch enzymatic hydrolysis reaction is used instead of an enzyme membrane reactor.

[0117] Comparative Example 6

[0118] In this comparative example, the similarities with Example 3 will not be repeated, and the differences are as follows:

[0119] Use unmodified common eutectic solvents, i.e., without adding succinylated trehalose, acetylated tea polyphenol esters, and polyethylene glycol-modified phytosterol esters.

[0120] Performance Test Results and Analysis

[0121] The performance of the products obtained in the examples and comparative examples was evaluated using the following test methods: astaxanthin content was determined spectrophotometrically at a wavelength of 478 nm; xylooligosaccharide purity was determined using high-performance liquid chromatography with a differential detector; pigment retention rate was determined by ultraviolet-visible spectrophotometry at the maximum absorption wavelength, with the pigment content of fresh raw materials as the baseline; protein content was determined using the Kjeldahl nitrogen determination method; total extraction efficiency was obtained by calculating the yield of each product to the mass ratio of the raw material; and the antioxidant activity of the products was determined using the DPPH free radical scavenging method. The specific test results are shown in Table 1.

[0122] Table 1 Analysis of Test Results

[0123] As shown in Table 1, the extraction rates of astaxanthin in all three examples exceeded 94%, while Comparative Example 1 only achieved 72.3%. This significant difference directly demonstrates the importance of the cell wall weakening pretreatment step. The mechanism of action lies in the fact that the organic acid components in the weakening agent solution can selectively act on the polysaccharide and glycoprotein structures in the cell wall, disrupting their dense network through gentle acid hydrolysis and hydrogen bonding. Simultaneously, the penetrant polysorbate 80 further enhances the solution's permeability. This pretreatment method effectively weakens the cell wall barrier without using strong mechanical force, creating favorable conditions for subsequent extraction. The astaxanthin extraction rate in Comparative Example 4 decreased to 85.6%, illustrating the necessity of nitrogen protection for astaxanthin, a highly unsaturated compound, which is highly susceptible to oxidative degradation during extraction, leading to reduced extraction rate and product activity.

[0124] Regarding xylooligosaccharide conversion, the example groups all achieved conversion rates exceeding 93%, while Comparative Example 2 only achieved 75.2%, and Comparative Example 5 was even lower at 78.3%. This result reveals the synergistic mechanism between the hydrophilic eutectic solvent and the enzyme membrane reactor. The succinylated trehalose in the hydrophilic eutectic solvent enhances the hydrogen bonding with polysaccharide molecules through the introduced carboxyl groups, not only improving the extraction efficiency of polysaccharides but, more importantly, maintaining the integrity of the polysaccharide molecules and providing a high-quality substrate for subsequent enzymatic hydrolysis. The enzyme membrane reactor, through its unique structural design, achieves simultaneous reaction and separation. On the one hand, the precise retention of the membrane maintains the enzyme concentration in the reaction system; on the other hand, it promptly removes the product xylooligosaccharides, effectively eliminating product inhibition effects and ensuring that the enzymatic reaction remains at a high efficiency.

[0125] Data on pigment retention rates showed that the example groups all maintained above 96%, while Comparative Example 3 decreased to 89.3%. This difference highlights the advantage of moderately polar, eutectic solvents in protecting photosensitive pigments. The introduction of acetylated tea polyphenol esters not only adjusted the solvent's polarity range, making it more suitable for dissolving pigment components, but more importantly, the phenolic hydroxyl groups in its molecular structure provide a certain degree of antioxidant protection. In contrast, the traditional organic solvent ethanol easily leads to the oxidative degradation of pigment molecules under microwave-assisted extraction conditions, which is the main reason for the significant decrease in pigment retention rate in Comparative Example 3.

[0126] The protein yield data showed relatively small differences between the examples and comparative examples, but the example group still exhibited a slight advantage. This indicates that the overall process system had a relatively mild impact on the protein composition, and the three-stage sequential extraction strategy avoided the excessive effects of highly polar solvents, which is beneficial for maintaining the spatial structure and functionality of the protein.

[0127] The DPPH radical scavenging rate test results further validated the product's quality advantages. All example groups maintained high antioxidant activity, especially Example 1, which reached 96.3%, while Comparative Examples 4 and 6 decreased to 90.2% and 92.6%, respectively. This phenomenon can be explained at the molecular level: the antioxidant activity of astaxanthin and pigment components is closely related to the conjugated double bond system in their molecules; any factor that disrupts this structure will directly affect their antioxidant capacity. The lack of nitrogen protection in Comparative Example 4 led to the oxidation of astaxanthin molecules, and the use of an unmodified solvent in Comparative Example 6 may have caused partial degradation of the active ingredient during extraction; both of these directly affect the functional quality of the final product.

[0128] In summary, the technical features of this invention exhibit a significant synergistic effect. Cell wall weakening pretreatment lays the foundation for subsequent extraction; the three-stage eutectic solvent system achieves highly selective extraction of components with different polarities; the physical field-assisted technology improves efficiency while preserving component activity; and the enzyme membrane reactor completes the high-value conversion of water-soluble components. This systematic process design not only significantly improves the yield and quality of each target product, but more importantly, it realizes the high-value utilization of all components of *Haematococcus pluvialis* resources.

[0129] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for the multi-product cascade separation of Haematococcus pluvialis based on coupling of low consolute solvent sequential extraction with enzyme membrane reactor, characterized in that, The method comprises the following steps: (1) a cell wall weakening pretreatment step: mixing Haematococcus pluvialis algae powder with a weakening agent solution at a mass ratio of 1:15 to 1:25, treating at a temperature of 35-45°C for 25-35 minutes to obtain pretreated algae powder; (2) a water-soluble component extraction step: mixing the pretreated algae powder with a hydrophilic deep eutectic solvent at a mass ratio of 1:20 to 1:30, extracting at a temperature of 50-60°C for 40-60 minutes to separate a water-soluble extract and a first algae residue; (3) a pigment component extraction step: mixing the first algae residue with a medium-polarity deep eutectic solvent at a mass ratio of 1:15 to 1:25, extracting at a temperature of 58-65°C for 25-40 minutes to separate a pigment extract and a second algae residue; (4) a astaxanthin extraction step: mixing the second algae residue with a hydrophobic deep eutectic solvent at a mass ratio of 1:10 to 1:20, extracting at a temperature of 42-50°C for 50-70 minutes to separate an astaxanthin extract and a protein residue; (5) an enzyme conversion step: introducing the water-soluble extract and a complex enzyme preparation into an enzyme membrane reactor, performing enzymatic reaction under the action of the complex enzyme preparation, and simultaneously separating and collecting the xylo-oligosaccharides generated in the reaction in real time by using the membrane separation function of the enzyme membrane reactor to obtain a xylo-oligosaccharide product; The hydrophilic deep eutectic solvent is obtained by compounding choline chloride, lactic acid and succinylated trehalose and adding deionized water; The medium-polarity deep eutectic solvent is obtained by compounding betaine, propylene glycol and acetylated tea polyphenol ester and adding deionized water; The hydrophobic deep eutectic solvent is obtained by compounding menthol, thymol and polyethylene glycolated phytosteryl ester and adding deionized water.

2. The method according to claim 1, wherein the method is a method for the multi-product gradient separation of Haematococcus pluvialis based on the coupling of sequential deep eutectic solvent extraction and enzyme membrane reactor, characterized in that, The weakening agent solution in step (1) is obtained by compounding citric acid, malic acid and ascorbic acid at a mass ratio of 1:0.5:0.2 to 1:1:0.5, dissolving in deionized water to form a solution with a concentration of 0.8% to 1.2%, adjusting the pH value to 4.0 to 4.5, and adding 0.05% to 0.1% of polysorbate 80 as a penetrant; The water-soluble component extraction step in step (2) is performed under ultrasonic assistance, the ultrasonic assistance adopts a multi-frequency combination, the frequencies are 28 kHz, 40 kHz and 60 kHz, the power ratio is 1:0.8:0.5, the ultrasonic time and the intermittent time ratio is 1:1 to 1:1.5, and the total ultrasonic time is 25 to 40 minutes; The pigment component extraction step in step (3) is performed under microwave assistance, the microwave power adopts multi-stage control, the initial power is 300 W, it is increased by 50 W every 5 minutes, the highest does not exceed 450 W, nitrogen gas is introduced for protection at the same time, the gas flow is 0.2 L / min to 0.4 L / min, and the extraction time is 25 to 40 minutes; The astaxanthin extraction step in step (4) is performed under mechanical stirring and nitrogen protection, the stirring speed is 280 rpm to 350 rpm, the nitrogen flow is 0.1 L / min to 0.3 L / min, an anchor stirring paddle is adopted, and the extraction time is 50 to 70 minutes; The enzyme membrane reactor in step (5) adopts polyether sulfone hollow fiber membrane, with an inner diameter of 1.0 mm to 1.5 mm, a wall thickness of 0.2 mm to 0.3 mm, and a molecular weight cut-off of 7000 Da to 8500 Da; the membrane module has a packing density of 35% to 45%, an operating pressure of 0.1 MPa to 0.3 MPa, a membrane surface flow rate of 1.0 m / s to 2.0 m / s, and a reaction temperature of 45°C to 55°C.

3. The method according to claim 1, wherein the method is a method for the multi-product gradient separation of Haematococcus pluvialis based on the coupling of deep eutectic solvent sequential extraction and enzyme membrane reactor, characterized in that, The hydrophilic deep eutectic solvent in step (2) is composed of choline chloride, lactic acid and succinylated trehalose in a molar ratio of 1:2.5:0.5 to 1:3.5:1, and deionized water is added to adjust the water content to 20% to 30%.

4. The method according to claim 3, wherein the method is a method for the multi-product gradient separation of Haematococcus pluvialis based on the coupling of sequential deep eutectic solvent extraction and enzyme membrane reactor, characterized in that, The preparation method of the succinylated trehalose comprises the following steps: dissolving trehalose in a phosphate buffer with a pH value of 7.5 to 8.5, adding succinic anhydride for reaction, the reaction temperature is 25°C to 35°C, the reaction time is 2 to 4 hours, the molar ratio of trehalose to succinic anhydride is 1:0.5 to 1:1.2, after the reaction, small molecular impurities are removed by dialysis or ultrafiltration, and then freeze-drying is performed to obtain succinylated trehalose.

5. The method according to claim 1, wherein the method is a method for the multi-product gradient separation of Haematococcus pluvialis based on the coupling of deep eutectic solvent sequential extraction and enzyme membrane reactor, characterized in that, The medium-polar deep eutectic solvent in step (3) is composed of betaine, propylene glycol and acetylated tea polyphenol ester in a molar ratio of 1:1.8:0.3 to 1:2.5:0.8, and deionized water is added to adjust the water content to 15% to 25%.

6. The method according to claim 5, wherein the method is a method for the multi-product gradient separation of Haematococcus pluvialis based on the coupling of sequential deep eutectic solvent extraction and enzyme membrane reactor, characterized in that, The preparation method of the acetylated tea polyphenol ester comprises the following steps: dissolving tea polyphenol in ethyl acetate, adding acetic anhydride and a catalyst 4-dimethylaminopyridine for reaction, the reaction temperature is 40°C to 50°C, the reaction time is 3 to 5 hours, the molar ratio of tea polyphenol to acetic anhydride is 1:1 to 1:2, after the reaction, acetylated tea polyphenol ester is purified by rotary evaporation and ethanol recrystallization.

7. The method according to claim 1, wherein the method is a method for the multi-product gradient separation of Haematococcus pluvialis based on the coupling of deep eutectic solvent sequential extraction and enzyme membrane reactor, characterized in that, The hydrophobic deep eutectic solvent in step (4) is composed of menthol, thymol and polyglycolized phytosterol ester in a molar ratio of 1:1.2:0.4 to 1:1.8:0.9; the preparation of the hydrophobic deep eutectic solvent comprises stirring and reacting the components at 45°C to 55°C for 2 to 4 hours to obtain the hydrophobic deep eutectic solvent.

8. The method according to claim 7, wherein the method is a method for the multi-product gradient separation of Haematococcus pluvialis based on the coupling of sequential deep eutectic solvent extraction and enzyme membrane reactor, characterized in that, The preparation method of the polyglycolized phytosterol ester comprises the following steps: dissolving phytosterol and polyethylene glycol monomethyl ether in toluene, adding a catalyst p-toluenesulfonic acid for reaction, the reaction temperature is 110°C to 130°C, the reaction time is 6 to 8 hours, the molar ratio of phytosterol to polyethylene glycol monomethyl ether is 1:1 to 1:1.5, and after the reaction, purification is performed by cooling crystallization and filtration.

9. The method according to claim 1, wherein the method is a method for the multi-product gradient separation of Haematococcus pluvialis based on the coupling of deep eutectic solvent sequential extraction and enzyme membrane reactor, characterized in that, The complex enzyme preparation in step (5) is composed of xylanase, arabinosidase and polyethylene glycol modified cellulase in a mass ratio of 1:0.3:0.2 to 1:0.6:0.5, wherein the modification degree of the polyethylene glycol modified cellulase is 0.4 to 0.7; the preparation process of the complex enzyme preparation is as follows: dissolving the above-mentioned enzymes in a citrate buffer with a pH value of 5.5 to 6.0, and the total concentration of the enzymes is 10 mg / mL to 20 mg / mL.

10. The method according to claim 9, wherein the method is a method for the multi-product gradient separation of Haematococcus pluvialis based on the coupling of sequential deep eutectic solvent extraction and enzyme membrane reactor, characterized in that, The preparation method of the polyethylene glycol modified cellulase comprises the following steps: dissolving the cellulase in a phosphate buffer with pH 7.0-7.5, adding pre-activated polyethylene glycol succinimidyl ester, wherein the polyethylene glycol succinimidyl ester is prepared by reacting polyethylene glycol 6000 with N-hydroxysuccinimide under the condition of dicyclohexyl carbodiimide as a condensing agent; controlling the modification reaction temperature to be 4-8 DEG C, the reaction time to be 12-16 hours, and the molar ratio of the cellulase to the polyethylene glycol succinimidyl ester to be 1:5-1:10; and after the reaction is completed, the polyethylene glycol modified cellulase is purified by using an ultrafiltration membrane with a molecular weight cut-off of 30000.

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