Haematococcus multiproduct gradient separation method based on deep eutectic solvent sequential extraction and enzyme membrane reactor coupling

Abstract

The invention belongs to the technical field of natural substance separation and extraction, and particularly relates to a haematococcus multiproduct gradient separation method based on deep eutectic solvent sequential extraction and enzyme membrane reactor coupling, which comprises the following steps: (1) cell wall weakening pretreatment; (2) extracting water-soluble components; (3) extracting pigment components; (4) an astaxanthin extraction step; and (5) enzymatic conversion. Through the synergistic effect of sequential extraction of the eutectic solvent and the enzyme membrane reactor, efficient, green and continuous gradient separation of water-soluble components, pigments, astaxanthin and xylooligosaccharide in haematococcus is realized, and the product purity and the resource utilization rate are improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural substance separation and extraction, and particularly relates to a method for multi-product cascade separation of Haematococcus based on eutectic solvent sequential extraction coupled with enzyme membrane reactor. BACKGROUND

[0002] Haematococcus is a unicellular microalgae, among which Haematococcus pluvialis has attracted much attention due to its ability to accumulate high levels of astaxanthin. Astaxanthin is a powerful antioxidant with antioxidant capacity far exceeding ordinary vitamins, and is widely used in health foods, cosmetics and pharmaceutical products to enhance immunity, anti-aging and improve skin health. In addition to astaxanthin, Haematococcus also contains abundant polysaccharides, proteins and lipids, etc. These components have various biological activities, for example, polysaccharides can regulate intestinal flora and enhance immunity, and lipids contain essential fatty acids for human body, which are helpful for cardiovascular health.

[0003] In practical application scenarios, the processing and utilization of Haematococcus faces many defects. First, the cell wall structure of Haematococcus is dense, composed of multiple layers of polysaccharides and glycoproteins. Traditional methods such as high-pressure homogenization or mechanical grinding have high energy consumption and are prone to cause degradation of heat-sensitive components such as astaxanthin during processing, affecting the activity and yield of the product. Second, the composition of the algae is complex, including water-soluble polysaccharides, fat-soluble pigments and non-polar astaxanthin, etc. Existing extraction techniques often require multiple steps and use a large amount of organic solvents such as acetone and n-hexane, which are highly toxic and pose a safety hazard, easily causing product residue and environmental pollution. In addition, existing methods often focus on the extraction of a single target product, such as astaxanthin, while ignoring the utilization of other high-value components such as polysaccharides, resulting in resource waste and poor overall economic efficiency. Xylan polysaccharides contained in the cell wall of Haematococcus can be converted into xylo-oligosaccharides after enzymatic hydrolysis.

[0004] In order to address these defects, existing technologies have tried various solutions. For example, supercritical carbon dioxide extraction technology is used instead of organic solvents, which avoids solvent residue, but has high equipment investment and operating costs, and has poor extraction effect on water-soluble components, making it difficult to achieve simultaneous separation of multiple components. Another common method is enzymatic hydrolysis, which uses cellulase or pectinase to break down the cell wall, but the efficiency of single enzyme hydrolysis is limited, often requiring high temperature or strong acid and alkali conditions, which can damage sensitive components, and the cost of enzymes is high, with low reuse rate. In recent years, eutectic solvents have been introduced into the field of natural product extraction as a green solvent. They are composed of hydrogen bond donors and acceptors, and are low in toxicity and biodegradable.

[0005] However, in the extraction of red algae, the simple application of a eutectic solvent still has the problem of low selectivity, and different polar components cannot be effectively extracted step by step, and the solvent recovery is difficult, and the number of repeated use is limited. In addition, the polysaccharide component in red algae is often treated as a by-product, and cannot be converted into a higher value product, such as xylo-oligosaccharide, which is traditionally mainly prepared from corn cob and other raw materials. The technology of converting polysaccharide from red algae to xylo-oligosaccharide is not mature, which limits the resource utilization efficiency and product added value.

[0006] The prior art solves the above-mentioned defects, but also has some problems. For example, when supercritical extraction and enzymatic hydrolysis are combined, the process is complex, the energy consumption is high, and continuous production cannot be realized; the eutectic solvent is easily affected by moisture and temperature during extraction, and has poor stability, resulting in fluctuations in extraction efficiency; the polysaccharide conversion link lacks an efficient reactor, and the product separation is not complete, affecting the purity. These problems result in high processing cost of red algae, single product and insufficient environmental friendliness. Therefore, there is an urgent need in the art for an innovative method that can realize efficient and green separation of red algae multi-components, and at the same time improve resource utilization through high-value conversion. Therefore, it is necessary to design a red algae multi-product cascade separation method based on eutectic solvent sequential extraction and enzyme membrane reactor coupling. SUMMARY

[0007] In order to overcome the defects in the prior art, a red algae multi-product cascade separation method based on eutectic solvent sequential extraction and enzyme membrane reactor coupling is provided.

[0008] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions: A red algae multi-product cascade separation method based on eutectic solvent sequential extraction and enzyme membrane reactor coupling, the method comprising the following steps: (1) Cell wall weakening pretreatment step: mixing the Chlorella vulgaris powder with the weakening agent solution at a mass ratio of 1:15 to 1:25, treating at a temperature of 35°C to 45°C for 25 to 35 minutes to obtain pretreated algae powder; The first problem in the process of Haematococcus pluvialis is the extraction of the dense cell wall. Traditional mechanical methods can cause degradation of astaxanthin and other heat-sensitive components due to strong shear force. Chemical methods may introduce impurities or destroy active ingredients. The present application uses a weakener solution composed of citric acid, malic acid and ascorbic acid. These organic acids can synergistically act on the polysaccharide and glycoprotein structures in the cell wall through hydrogen bonding and mild acid hydrolysis to loosen them. The addition of polysorbate 80 as a penetrant further promotes the penetration of the solution into the cell. This pretreatment method effectively weakens the barrier effect of the cell wall without using strong mechanical force or high temperature, creating favorable conditions for subsequent extraction.

[0009] (2) Water-soluble component extraction step: mix the pretreated algae powder with the hydrophilic deep eutectic solvent at a mass ratio of 1:20 to 1:30, extract at a temperature of 50-60°C for 40-60 minutes, and separate to obtain a water-soluble extract and a first algae residue; (3) Pigment component extraction step: mix the first algae residue with the medium-polarity deep eutectic solvent at a mass ratio of 1:15 to 1:25, extract at a temperature of 58-65°C for 25-40 minutes, and separate to obtain a pigment extract and a second algae residue; (4) Astaxanthin extraction step: mix the second algae residue with the hydrophobic deep eutectic solvent at a mass ratio of 1:10 to 1:20, extract at a temperature of 42-50°C for 50-70 minutes, and separate to obtain an astaxanthin extract and a protein residue; After solving the cell wall barrier problem, the simultaneous separation of different polarity components in Haematococcus pluvialis becomes a new challenge. Traditional single solvent systems cannot simultaneously extract water-soluble polysaccharides, lipid-soluble pigments, and strongly non-polar astaxanthin, often requiring multiple solvent systems and complex process switching. The present application designs three types of deep eutectic solvent systems for sequential extraction. The hydrophilic deep eutectic solvent consists of choline chloride and lactic acid, and the specially introduced succinylated trehalose enhances the hydrogen bonding with polysaccharide components through the introduction of carboxyl groups, improving the extraction selectivity of water-soluble components. Subsequently, the medium-polarity deep eutectic solvent uses betaine and propylene glycol as the base, and the acetylated tea polyphenol ester in it adjusts the lipophilicity of the solvent through the introduction of acetyl groups, making it more suitable for the dissolution of pigment components. Finally, the hydrophobic deep eutectic solvent is composed of menthol and thymol, which, combined with the interfacial activity of the polyethylene glycolated phytosterol ester, effectively promotes the transfer of astaxanthin and other strongly hydrophobic components. This sequential extraction strategy based on polarity gradient achieves effective separation of components with different properties.

[0010] (5) Enzymatic conversion step: introducing the water-soluble extract and the complex enzyme preparation into an enzyme membrane reactor, and performing enzymatic reaction under the action of the complex enzyme preparation, while separating and collecting the oligoxylan produced in real time by using the membrane separation function of the enzyme membrane reactor to obtain the oligoxylan product.

[0011] The weakening agent solution in step (1) is prepared by compounding citric acid, malic acid and ascorbic acid in 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, and the ultrasonic assistance uses a multi-frequency combination, with frequencies of 28 kHz, 40 kHz and 60 kHz, a power ratio of 1:0.8:0.5, a pulse mode, a ratio of ultrasonic time to intermittent time of 1:1 to 1:1.5, and a total ultrasonic time of 25 to 40 minutes.

[0012] The pigment component extraction step in step (3) is performed under microwave assistance, and the power of the microwave is controlled in multiple stages, with an initial power of 300 W, an increase of 50 W every 5 minutes, and a maximum of no more than 450 W, while nitrogen gas is introduced for protection, with a gas flow rate of 0.2 L / min to 0.4 L / min, and an extraction time of 25 to 40 minutes.

[0013] The astaxanthin extraction step in step (4) is performed under mechanical stirring and nitrogen protection, with a stirring speed of 280 rpm to 350 rpm, a nitrogen flow rate of 0.1 L / min to 0.3 L / min, an anchor stirring paddle, an extraction time of 50 to 70 minutes; Although the solvent system for sequential extraction is established, the extraction efficiency still needs to be further improved, and at the same time it is necessary to ensure that the active ingredients are not destroyed during the extraction process. The present application introduces targeted physical field strengthening measures at each extraction stage. In the water-soluble component extraction stage, multi-frequency combination ultrasonic technology is used, and the cavitation effects of ultrasonic waves of different frequencies act on cell structures at different scales, synergistically enhancing solvent diffusion and component dissolution, while the pulse mode avoids local overheating caused by continuous ultrasonic waves. In the pigment component extraction stage, multi-stage controlled microwave assistance provides a fast and uniform heating method, significantly improving the mass transfer efficiency, while the coordinated nitrogen protection effectively prevents the oxidative degradation of photosensitive pigments. In the astaxanthin extraction stage, mechanical stirring ensures the thorough mixing of the solvent and the material, and continuous nitrogen protection provides a stable extraction environment for astaxanthin, which is extremely susceptible to oxidation. The combination of these physical field strengthening measures and the corresponding solvent system improves the extraction rate while maximizing the biological activity of each component.

[0014] The enzyme membrane reactor in step (5) adopts a 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, a molecular weight cut-off of 7,000 Da to 8,500 Da, a membrane module filling 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.

[0015] After the water-soluble polysaccharide is successfully extracted, how to efficiently convert it into a higher value product becomes a problem to be solved. The traditional enzymatic hydrolysis process has problems such as product inhibition and enzyme cannot be reused. The present application adopts an enzyme membrane reactor system, which integrates enzyme catalytic reaction and membrane separation process. Xylanase, arabinosidase and polyethylene glycol modified cellulase in the composite enzyme preparation act on different sites of the polysaccharide molecule, and the polyethylene glycol modified cellulase has better stability and compatibility. During the reaction process, the polyether sulfone hollow fiber membrane separates the generated xylo-oligosaccharide from the enzyme and unreacted substrate in real time due to its precise molecular weight cut-off. This continuous separation method not only eliminates the feedback inhibition of the product on the enzyme, but also makes the reaction always maintain at a high rate, and realizes the effective recovery and reuse of the enzyme, thereby significantly improving the economic efficiency of the process.

[0016] The hydrophilic deep eutectic solvent in step (2) is compounded by 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%.

[0017] 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 the trehalose to the succinic anhydride is 1:0.5 to 1:1.2, after the reaction is completed, small molecule impurities are removed by dialysis or ultrafiltration, and then freeze-drying is performed to obtain the succinylated trehalose.

[0018] The medium-polar deep eutectic solvent in step (3) is compounded by 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%.

[0019] 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 the tea polyphenol to the acetic anhydride is 1:1 to 1:2, after the reaction is completed, acetylated tea polyphenol ester is purified by rotary evaporation and ethanol recrystallization.

[0020] The hydrophobic deep eutectic solvent in step (4) is compounded by menthol, thymol and polyethylene glycolated phytosteryl ester at 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 45-55 DEG C for 2-4 hours, and the hydrophobic deep eutectic solvent is obtained.

[0021] The preparation method of the polyethylene glycolated phytosteryl ester comprises dissolving phytosterol and polyethylene glycol monomethyl ether in toluene, adding a catalyst p-toluenesulfonic acid, and reacting at a temperature of 110-130 DEG C for 6-8 hours, with a molar ratio of phytosterol to polyethylene glycol monomethyl ether being 1:1-1:1.5; after the reaction, purification is performed by cooling crystallization and filtration.

[0022] The composite enzyme preparation in step (5) is compounded by xylanase, arabinosidase and polyethylene glycol modified cellulase at 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 0.4-0.7; the preparation process of the composite enzyme preparation is as follows: dissolving the above enzymes in a citrate buffer with a pH value of 5.5-6.0, and the total concentration of the enzymes is 10-20 mg / mL.

[0023] The preparation method of the polyethylene glycol modified cellulase comprises dissolving cellulase in a phosphate buffer with a pH value of 7.0-7.5, and 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; the modification reaction temperature is controlled at 4-8 DEG C, the reaction time is 12-16 hours, the molar ratio of cellulase to polyethylene glycol succinimidyl ester is 1:5-1:10, and after the reaction, purification is performed by using an ultrafiltration membrane with a molecular weight cut-off of 30000 to obtain the polyethylene glycol modified cellulase.

[0024] The present application forms a good synergistic effect between each unit operation from cell wall weakening pretreatment to three-stage sequential extraction, and then to enzyme membrane reactor conversion. The low eutectic solvent used in the whole process system can be recycled and used, avoiding the environmental burden of traditional organic solvents, and realizing the simultaneous obtaining of low oligosaccharides, natural pigments, astaxanthin and feed protein and other high-value products from Haematococcus.

[0025] Compared with the prior art, the present application has the following advantages and beneficial effects: 1. The present application realizes the effective dissociation of Haematococcus pluvialis cell structure and the high-selective separation of target components by the synergistic effect of cell wall weakening pretreatment step and sequential extraction of ternary deep eutectic solvents. Among them, the weakening agent solution preferentially softens the polysaccharide network in the cell wall under mild acidic conditions, creating favorable conditions for subsequent extraction. Subsequently, through the precise design of hydrophilic, moderately polar and hydrophobic deep eutectic solvent systems, water-soluble polysaccharides, pigment components and astaxanthin are extracted step by step. This sequential extraction strategy not only avoids the mutual interference between different polar components, but also significantly improves the extraction efficiency and purity of each target component. In particular, the introduction of components such as succinylated trehalose and acetylated tea polyphenol esters in the modified deep eutectic solvents enhances the affinity between the solvent and the target components through specific intermolecular interactions, thereby achieving more precise separation results.

[0026] 2. The present application improves the extraction efficiency of each stage through multi-field coupling enhancement technology. In the hydrophilic component extraction stage, the synergistic cavitation effect produced by multi-frequency ultrasound effectively promotes the penetration and mass transfer of the solvent into the cell interior. In the moderately polar component extraction stage, multi-section microwave assistance combined with nitrogen protection not only realizes rapid and uniform heating, but also prevents the oxidative degradation of heat-sensitive pigments. In the astaxanthin extraction stage, the combination of mechanical stirring and nitrogen protection ensures the sufficient dissolution of hydrophobic components and maintains their chemical stability. These strengthening measures cooperate with the corresponding deep eutectic solvent systems, making the extraction process of each stage maintain a high mass transfer rate while maximizing the protection of the biological activity of the target components.

[0027] 3. The present application realizes the efficient conversion and continuous separation of water-soluble polysaccharides to high-value oligomeric xylose through the innovative application of enzyme membrane reactors. The core advantage of this system is the integration of enzyme-catalyzed reaction and membrane separation process, in which each component of the complex enzyme preparation acts synergistically on specific sites of the polysaccharide molecular chain, directing the conversion of the polysaccharide to the target oligosaccharide. At the same time, the polyether sulfone hollow fiber membrane realizes the real-time separation of macromolecular enzyme preparation and small molecule product in the reaction system through its precise molecular weight cutoff design. This design not only eliminates the product inhibition effect, allowing the enzyme-catalyzed reaction to continue in the positive direction, but also realizes the effective recovery and reuse of the enzyme preparation, significantly improving the economy of the conversion process.

[0028] 4. The present application realizes the simultaneous obtaining of oligomeric xylose, natural pigments, astaxanthin and feed protein from a single raw material through the system integration of cell wall weakening, multi-stage extraction, enzyme conversion and membrane separation. The entire process system embodies the basic principles of green chemistry, the deep eutectic solvents used can be recycled, avoiding the environmental pollution problem of traditional organic solvents, maximizing the utilization rate of raw materials, and significantly reducing energy consumption and material loss. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0030] In the specific embodiments of the present application, the sources of various main raw materials are briefly described as follows: Citric acid: purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd., CAS number 77-92-9 Malic acid: purchased from Aladdin Biochemical Technology Co., Ltd., CAS number 6915-15-7 Ascorbic acid: purchased from Hebei Licha Biological Technology Co., Ltd., CAS number: 23313-12-4 Polysorbate 80: purchased from Nanjunguo Biological Technology Co., Ltd., CAS number 9005-65-6 Choline chloride: purchased from Beijing Bailingwei Technology Co., Ltd., CAS number 67-48-1 Lactic acid: purchased from Jinan Rensheng Chemical Co., Ltd., CAS number 50-21-5 Trehalose: purchased from Shandong Fufeng Fermentation Co., Ltd., CAS number 99-20-7 succinic anhydride: purchased from Shanghai Maikelin Biochemical Technology Co., Ltd. Betaine: purchased from Zhejiang Xinhengcheng Co., Ltd., CAS number 107-43-7 Propylene glycol: purchased from Jiangsu Denka Chemical Co., Ltd., CAS number 57-55-6 Tea polyphenol: purchased from Xi'an Tianyi Biological Technology Co., Ltd., CAS number 84650-60-2 Acetic anhydride: purchased from Jiangsu Zhengdan Chemical Industry Co., Ltd., CAS number 108-24-7 4-dimethylamino pyridine: purchased from TCI Chemical Industry Development Co., Ltd., CAS number 1122-58-3 Menthol: purchased from Guangzhou Baihua Fragrance Co., Ltd., CAS number 89-78-1 Phytosterol: purchased from Zhejiang Pharmaceutical Co., Ltd. Xinchang Pharmaceutical Factory, CAS number 83-46-5 Polyethylene glycol monomethyl ether: purchased from Hunan Huateng Pharmaceutical Co., Ltd., CAS number 9004-74-4 p-toluenesulfonic acid: purchased from Tianjin Kemio Chemical Reagent Co., Ltd., CAS number 6192-52-5 Xylanase: purchased from Novozymes China Biotechnology Co., Ltd., product number NS50030, activity ≥5000 U / g Arabinosidase: purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., CAS number 9025-53-2, food grade Cellulase: purchased from Jiangsu Ruiyang Biotechnology Co., Ltd., CAS number 9012-54-8, industrial grade Polyethylene glycol 6000: purchased from Shandong Yousuo Chemical Technology Co., Ltd., CAS number 25322-68-3 N-hydroxy succinimide: purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd., CAS number 6066-82-6 The technical solution of the present application is: A Haematococcus pluvialis multi-product cascade separation method based on the coupling of eutectic solvent sequential extraction and enzyme membrane reactor, comprising the following steps: (1) Cell wall weakening pretreatment step: mixing Haematococcus pluvialis algae powder and a weakening agent solution at a mass ratio of 1:15 to 1:25, treating at a temperature of 35°C to 45°C for 25 to 35 minutes, to obtain pretreated algae powder; (2) Water-soluble component extraction step: mixing the pretreated algae powder and a hydrophilic eutectic solvent at a mass ratio of 1:20 to 1:30, extracting at a temperature of 50°C to 60°C for 40 to 60 minutes, to separate a water-soluble extract and a first algae residue; (3) Pigment component extraction step: mixing the first algae residue and a medium-polar eutectic solvent at a mass ratio of 1:15 to 1:25, extracting at a temperature of 58°C to 65°C for 25 to 40 minutes, to separate a pigment extract and a second algae residue; (4) Astaxanthin extraction step: mixing the second algae residue and a hydrophobic eutectic solvent at a mass ratio of 1:10 to 1:20, extracting at a temperature of 42°C to 50°C for 50 to 70 minutes, to separate an astaxanthin extract and a protein residue; (5) Enzymatic conversion step: introducing the water-soluble extract and a complex enzyme preparation into an enzyme membrane reactor, and performing enzymatic reaction under the action of the complex enzyme preparation, while using the membrane separation function of the enzyme membrane reactor to separate and collect the oligomeric xylose generated in the reaction in real time, to obtain an oligomeric xylose product.

[0031] The weakening agent solution in step (1) is prepared 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 of step (2) is performed under ultrasonic assistance, the ultrasonic assistance uses a combination of multiple frequencies, the frequencies are 28 kHz, 40 kHz and 60 kHz, the power ratio is 1:0.8:0.5, the ultrasonic assistance uses a pulse mode, the ratio of ultrasonic time to intermittent time is 1:1 to 1:1.5, and the total ultrasonic time is 25 to 40 minutes.

[0032] The pigment component extraction step of step (3) is performed under microwave assistance, the power of the microwave uses multi-stage control, the initial power is 300 W, the power is increased by 50 W every 5 minutes, the highest power 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.

[0033] The astaxanthin extraction step of 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 used, and the extraction time is 50 to 70 minutes. The enzyme membrane reactor in step (5) uses a polyether sulfone hollow fiber membrane, the inner diameter of the membrane is 1.0 mm to 1.5 mm, the wall thickness is 0.2 mm to 0.3 mm, the molecular weight cut-off is 7000 Da to 8500 Da, the packing density of the membrane module 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°C to 55°C.

[0034] The hydrophilic deep eutectic solvent in step (2) is compounded by choline chloride, lactic acid and succinylated trehalose at 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%.

[0035] 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 is completed, small molecule impurities are removed by dialysis or ultrafiltration, and then freeze-drying is performed to obtain succinylated trehalose.

[0036] The moderately polar deep eutectic solvent in step (3) is compounded by betaine, propylene glycol and acetylated tea polyphenol ester at 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%.

[0037] 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 to react, the reaction temperature is 40-50 DEG C, the reaction time is 3-5 hours, the molar ratio of the tea polyphenol to the acetic anhydride is 1:1-1:2, and after the reaction is completed, the acetylated tea polyphenol ester is purified by rotary evaporation and ethanol recrystallization.

[0038] The hydrophobic deep eutectic solvent in step (4) is compounded by menthol, thymol and polyethylene glycolated phytosterol ester according to 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 45-55 DEG C for 2-4 hours to obtain the hydrophobic deep eutectic solvent.

[0039] The preparation method of the polyethylene glycolated phytosterol ester comprises the following steps: dissolving phytosterol and polyethylene glycol monomethyl ether in toluene, adding a catalyst p-toluenesulfonic acid to react, the reaction temperature is 110-130 DEG C, the reaction time is 6-8 hours, the molar ratio of the phytosterol to the polyethylene glycol monomethyl ether is 1:1-1:1.5, and after the reaction is completed, the polyethylene glycolated phytosterol ester is purified by cooling crystallization and filtration.

[0040] The composite enzyme preparation in step (5) is compounded by xylanase, arabinosidase and polyethylene glycol modified cellulase according to 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 0.4-0.7; the preparation process of the composite enzyme preparation is as follows: dissolving the above enzymes in a citrate buffer with a pH value of 5.5-6.0, and the total concentration of the enzymes is 10-20 mg / mL.

[0041] The preparation method of the polyethylene glycol modified cellulase comprises the following steps: dissolving cellulase in a phosphate buffer with a pH value of 7.0-7.5, adding pre-activated polyethylene glycol succinimidyl ester, wherein the polyethylene glycol succinimidyl ester is prepared by reacting polyethylene glycol 6000 and 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 is 12-16 hours, the molar ratio of the cellulase to the polyethylene glycol succinimidyl ester is 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.

[0042] In the subsequent processing technology, macroporous adsorption resin column purification process can be used to obtain natural pigment products from the pigment extraction liquid, distillation and extraction can be used to obtain astaxanthin products from the astaxanthin extraction liquid, and washing and separation can be used to obtain feed protein products from the protein residue; other processes in the industry can also be used; the above processes are all known technologies, and will not be described here in detail.

[0043] The application realizes efficient, green and continuous gradient separation of water-soluble components, pigments, astaxanthin and xylo-oligosaccharides in Haematococcus by the synergistic effect of low eutectic solvent sequential extraction and enzyme membrane reactor, and improves product purity and resource utilization.

[0044] The application will be described in detail below by examples and comparative examples, but the protection scope of the application is not limited to these examples. The chemical reagents and raw materials used in the following examples and comparative examples are all conventional commercially available products unless otherwise specified. Example 1

[0045] This example describes in detail the implementation process of the Haematococcus multi-product gradient separation method based on low eutectic solvent sequential extraction and enzyme membrane reactor coupling. First, the cell wall is weakened by pretreatment, and Haematococcus pluvialis powder is mixed with a weakening agent solution at a mass ratio of 1:25, treated at a temperature of 45℃ for 35 minutes, and the pretreated algae powder is obtained. The weakening agent solution is prepared by mixing citric acid, malic acid and ascorbic acid at a mass ratio of 1:1:0.5, dissolving in deionized water to form a solution with a concentration of 1.2%, adjusting the pH value to 4.0, and adding 0.1% (mass fraction) polysorbate 80 as a penetrant.

[0046] Then, the water-soluble components are extracted by mixing the pretreated algae powder with a hydrophilic eutectic solvent at a mass ratio of 1:30, and extracting at a temperature of 60℃ for 60 minutes. This step uses multi-frequency ultrasonic assistance, with frequencies of 28kHz, 40kHz and 60kHz, power ratios of 1:0.8:0.5, pulse mode, ultrasonic time to intermittent time ratio of 1:1.5, and total ultrasonic time of 40 minutes. The hydrophilic eutectic solvent is prepared by mixing choline chloride, lactic acid and succinylated trehalose at a molar ratio of 1:3.5:1 and adding deionized water to adjust the water content to 30%. After separation, the water-soluble extract and the first algae residue are obtained.

[0047] Subsequently, the pigment components are extracted by mixing the first algae residue with a medium-polarity eutectic solvent at a mass ratio of 1:25, and extracting at a temperature of 65℃ for 40 minutes. This step uses microwave assistance, with an initial power of 300W, increasing by 50W every 5 minutes, and a maximum power of 450W. Nitrogen gas is introduced for protection at a flow rate of 0.4L / min. The medium-polarity eutectic solvent is prepared by mixing betaine, propylene glycol and acetylated tea polyphenol ester at a molar ratio of 1:2.5:0.8 and adding deionized water to adjust the water content to 25%. After separation, the pigment extract and the second algae residue are obtained.

[0048] Then astaxanthin extraction was performed by mixing the second algal residue with a hydrophobic deep eutectic solvent at a mass ratio of 1:20 at a temperature of 50℃ for 70 minutes. Mechanical stirring and nitrogen protection were used in this step, with a stirring speed of 350 rpm, a nitrogen flow rate of 0.3 L / min, and an anchor stirring paddle. The hydrophobic deep eutectic solvent was composed of menthol, thymol, and polyethylene glycolated phytosteryl ester at a molar ratio of 1:1.8:0.9. After separation, astaxanthin extract and protein residue were obtained.

[0049] Finally, enzymatic conversion was performed by introducing the water-soluble extract and a complex enzyme preparation into an enzyme membrane reactor for enzymatic reaction under the action of the complex enzyme preparation. The reaction temperature was 55℃, the operating pressure was 0.3 MPa, the membrane surface flow rate was 2.0 m / s, and the reaction time was 5 hours. The complex enzyme preparation was composed of xylanase, arabinosidase, and polyethylene glycol-modified cellulase at a mass ratio of 1:0.6:0.5, and the total enzyme concentration was 20 mg / mL. Through the membrane separation function of the enzyme membrane reactor, the reaction-generated xylooligosaccharides were separated and collected in real time to obtain the xylooligosaccharide product. Example 2

[0050] In this example, the same as in Example 1 will not be repeated, and the differences are as follows: In this example, a different process parameter was used to implement the Haematococcus pluvialis multi-product cascade separation method. In the cell wall weakening pretreatment step, the algal powder was mixed with a weakening agent solution at a mass ratio of 1:15 at a temperature of 35℃ for 25 minutes. The weakening agent solution was composed 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 value of 4.5, and a polysorbate 80 addition amount of 0.05%.

[0051] The water-soluble component extraction step used a mass ratio of 1:20, a temperature of 50℃, and an extraction time of 40 minutes. The ultrasonic assistance parameters were pulse mode, a ratio of ultrasonic time to intermittent time of 1:1, and a total ultrasonic time of 25 minutes. The hydrophilic deep eutectic solvent was composed of choline chloride, lactic acid, and succinylated trehalose at a molar ratio of 1:2.5:0.5, with a water content of 20%.

[0052] The pigment component extraction step used a mass ratio of 1:15, a temperature of 58℃, and an extraction time of 25 minutes. The microwave assistance started with a power of 300 W, and the gas flow rate was 0.2 L / min. The medium-polarity deep eutectic solvent was composed of betaine, propylene glycol, and acetylated tea polyphenol ester at a molar ratio of 1:1.8:0.3, with a water content of 15%.

[0053] The astaxanthin extraction step used a mass ratio of 1:10, a temperature of 42℃, and an extraction time of 50 minutes. The mechanical stirring speed was 280 rpm, and the nitrogen flow rate was 0.1 L / min. The hydrophobic deep eutectic solvent was composed of menthol, thymol, and polyethylene glycolated phytosteryl ester at a molar ratio of 1:1.2:0.4.

[0054] The enzymatic conversion step was carried out at a reaction temperature of 45°C, an operating pressure of 0.1 MPa, a membrane surface flow rate of 1.0 m / s, and a reaction time of 3 hours. The complex enzyme preparation was compounded by xylanase, arabinosidase, and polyethylene glycol modified cellulase at a mass ratio of 1:0.3:0.2, and the total enzyme concentration was 10 mg / mL. Example 3

[0055] In this example, the same as example 1 will not be repeated, the difference is described as follows: This example provides another embodiment of the parameter combination. In the cell wall weakening pretreatment step, the algal powder is mixed with the weakening agent solution at a mass ratio of 1:20, and treated at a temperature of 40°C for 30 minutes. The weakening agent solution is compounded by citric acid, malic acid and ascorbic acid at a mass ratio of 1:0.8:0.3, the solution concentration is 1.0%, the pH value is 4.2, and the addition amount of polysorbate 80 is 0.08%.

[0056] The water-soluble ingredient extraction step was carried out at a mass ratio of 1:25, a temperature of 55°C, and an extraction time of 50 minutes. The ultrasonic assistance parameters were pulse mode, a ratio of ultrasonic time to intermittent time of 1:1.2, and a total ultrasonic time of 32 minutes. The hydrophilic deep eutectic solvent was compounded by choline chloride, lactic acid, and succinylated trehalose at a molar ratio of 1:3.0:0.8, and the water content was 25%.

[0057] The pigment ingredient extraction step was carried out at a mass ratio of 1:20, a temperature of 62°C, and an extraction time of 32 minutes. The microwave assistance started at a power of 300 W, and the gas flow was 0.3 L / min. The medium-polar deep eutectic solvent was compounded by betaine, propylene glycol, and acetylated tea polyphenol ester at a molar ratio of 1:2.2:0.5, and the water content was 20%.

[0058] The astaxanthin extraction step was carried out at a mass ratio of 1:15, a temperature of 46°C, and an extraction time of 60 minutes. The mechanical stirring speed was 320 rpm, and the nitrogen flow was 0.2 L / min. The hydrophobic deep eutectic solvent was compounded by menthol, thymol, and polyethylene glycolated phytosteryl ester at a molar ratio of 1:1.5:0.6.

[0059] The enzymatic conversion step was carried out at a reaction temperature of 50°C, an operating pressure of 0.2 MPa, a membrane surface flow rate of 1.5 m / s, and a reaction time of 4 hours. The complex enzyme preparation was compounded by xylanase, arabinosidase, and polyethylene glycol modified cellulase at a mass ratio of 1:0.4:0.3, and the total enzyme concentration was 15 mg / mL.

[0060] Comparative Example 1 In this comparative example, the same as example 1 will not be repeated, the difference is described as follows: The cell wall weakening pretreatment step was omitted, and the subsequent extraction was directly carried out using untreated Haematococcus pluvialis algal powder.

[0061] Comparative Example 2 In the present comparative example, the same as in Example 2 will not be repeated, and the differences are described as follows: Pure water was used instead of the hydrophilic eutectic solvent in the water-soluble ingredient extraction step.

[0062] Comparative Example 3 In the present comparative example, the same as in Example 3 will not be repeated, and the differences are described as follows: Ethanol was used instead of the medium-polar eutectic solvent in the pigment ingredient extraction step.

[0063] Comparative Example 4 In the present comparative example, the same as in Example 1 will not be repeated, and the differences are described as follows: Nitrogen protection was not used in the astaxanthin extraction step.

[0064] Comparative Example 5 In the present comparative example, the same as in Example 2 will not be repeated, and the differences are described as follows: Traditional batch enzymatic reaction was used instead of the enzyme membrane reactor in the enzyme conversion step.

[0065] Comparative Example 6 In the present comparative example, the same as in Example 3 will not be repeated, and the differences are described as follows: Unmodified ordinary eutectic solvent was used, i.e., without adding succinylated trehalose, acetylated tea polyphenol ester, and polyethylene glycolated phytosterol ester.

[0066] Performance test results and analysis The following test methods were used to evaluate the performance of the products obtained in the examples and comparative examples: astaxanthin content was determined by spectrophotometry at a wavelength of 478 nm; xylo-oligosaccharide purity was determined by 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 reference; protein content was determined by the Kjeldahl method; total extraction efficiency was calculated by the ratio of product yield to raw material mass; and antioxidant activity of the product was determined by the DPPH free radical scavenging method. The test results are shown in Table 1.

[0067] Table 1 Analysis test results

[0068] As can be seen from Table 1, the extraction rates of astaxanthin in the three examples are all more than 94%, while that of Comparative Example 1 is only 72.3%, which directly proves the importance of the cell wall weakening pretreatment step. The mechanism is that the organic acid components in the weakening agent solution can selectively act on the polysaccharide and glycoprotein structures in the cell wall, destroying its dense network through mild acid hydrolysis and hydrogen bonding, while the penetration agent polysorbate 80 further enhances the penetration ability of the solution. This pretreatment method effectively weakens the cell wall barrier without using strong mechanical force, creating favorable conditions for subsequent extraction. The astaxanthin extraction rate of Comparative Example 4 drops to 85.6%, which shows the necessity of nitrogen protection for astaxanthin, a highly unsaturated compound, which is easily oxidized and degraded during extraction, resulting in a decrease in extraction rate and product activity.

[0069] In terms of xylo-oligosaccharide conversion, the conversion rates of the example group are all above 93%, while that of Comparative Example 2 is only 75.2%, and that of Comparative Example 5 is as low as 78.3%. This result reveals the synergistic mechanism of hydrophilic deep eutectic solvent and enzyme membrane reactor. The succinylated trehalose in the hydrophilic deep eutectic solvent enhances the hydrogen bonding with polysaccharide molecules through the introduced carboxyl group, not only improving the extraction efficiency of polysaccharide, but more importantly, maintaining the integrity of polysaccharide molecules, providing high-quality substrates for subsequent enzymatic reactions. The enzyme membrane reactor, through its unique structural design, realizes the simultaneous reaction and separation, on the one hand, maintains the concentration of enzymes in the reaction system through the precise cutoff of the membrane, on the other hand, removes the product xylo-oligosaccharide in time, effectively eliminating the product inhibition effect, so that the enzymatic reaction always maintains a high efficiency.

[0070] The data of pigment retention rate shows that the example group maintains above 96%, while that of Comparative Example 3 drops to 89.3%. This difference reflects the advantage of medium-polar deep eutectic solvent in protecting light-sensitive pigments. The introduction of acetylated tea polyphenol ester not only adjusts the polarity range of the solvent, making it more suitable for the dissolution of pigment components, but more importantly, the phenolic hydroxyl group in its molecular structure can provide certain antioxidant protection. In contrast, the traditional organic solvent ethanol is easy to cause oxidative degradation of pigment molecules under microwave-assisted extraction conditions, which is the main reason for the significant decrease in pigment retention rate of Comparative Example 3.

[0071] From the protein yield data, the difference between the examples and the comparative examples is relatively small, but it can still be observed that the example group is slightly superior. This shows that the entire process system has a relatively mild effect on protein components, and the strategy of three-stage sequential extraction avoids the excessive use of strong polar solvents, which is conducive to maintaining the spatial structure and functionality of proteins.

[0072] The test results of DPPH free radical scavenging rate further verify the quality advantage of the product. The example groups all maintain high antioxidant activity, especially Example 1 reaches 96.3%, while Comparative Examples 4 and 6 decrease to 90.2% and 92.6% respectively. This phenomenon can be explained from the molecular level: the antioxidant activity of astaxanthin and pigment components is closely related to the conjugated double bond system in their molecules, and any factor that causes the destruction of this structure will directly affect its antioxidant capacity. Comparative Example 4 lacks nitrogen protection, which leads to oxidation of astaxanthin molecules, and Comparative Example 6 uses unmodified solvents, which may cause partial degradation of active ingredients during extraction, all of which directly affect the functional quality of the final product.

[0073] In summary, the technical features of the present application show obvious synergistic effects. Cell wall weakening pretreatment lays the foundation for subsequent extraction, three-level eutectic solvent system realizes high selectivity extraction of different polar components, physical field assisted technology improves efficiency while protecting the activity of components, and 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, realizes the full component high value utilization of Haematococcus resources.

[0074] The above describes the preferred embodiments of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A method for multi-product stepwise separation of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor, characterized in that, The method includes the following steps: (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; (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. (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. (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. (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.

2. The method for multi-product stepwise separation of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor, as described in claim 1, is characterized in that... 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. 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. 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. At the same time, nitrogen gas is introduced for protection, with a gas flow rate of 0.2L / min to 0.4L / min, and the extraction time is 25 to 40 minutes. 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. 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℃.

3. The method for multi-product stepwise separation of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor, as described in claim 1, is characterized in that... 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%.

4. The method for multi-product stepwise separation of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor, as described in claim 3, is characterized in that... 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.

5. The method for multi-product stepwise separation of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor according to claim 1, characterized in that, 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%.

6. The method for multi-product stepwise separation of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor, as described in claim 5, is characterized in that... 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.

7. The method for multi-product stepwise separation of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor according to claim 1, characterized in that, 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.

8. The method for multi-product stepwise separation of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor according to claim 7, characterized in that, 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.

9. The method for multi-product stepwise separation of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor according to claim 1, 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 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.

10. The method for multi-product stepwise separation of *Rhodotorula pulmonata* based on sequential extraction with a eutectic solvent coupled with an enzyme membrane reactor according to claim 9, characterized in that, 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, the molar ratio of cellulase to polyethylene glycol succinimide ester to be 1:5 to 1:10, and purifying the enzyme using an ultrafiltration membrane with a molecular weight cutoff of 30,000 after the reaction to obtain the polyethylene glycol-modified cellulase.

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