A pH-responsive charge-controlled nanofiltration membrane, its preparation method and application

Abstract

This invention belongs to the field of Haematococcus pluvialis separation technology, specifically providing a pH-responsive charge-controlled nanofiltration membrane, its preparation method, and its application. The preparation method includes: preparing OSA, preparing DOPA-OSA, preparing carboxylated chitosan, and preparing the pH-responsive charge-controlled nanofiltration membrane. This nanofiltration membrane is used for the separation of Haematococcus pluvialis polysaccharides and astaxanthin esters. This application constructs a chemically anchored pH-responsive nanofiltration membrane by modifying sodium alginate with catechol, achieving the stepwise separation of Haematococcus pluvialis polysaccharides and astaxanthin esters by charge reversal of the same membrane under different pH conditions. Using DOPA-OSA as an interface modifier, the catechol groups on its molecular chain strongly adhere to the surface of the polyamide thin-layer composite nanofiltration membrane through hydrogen bonding, π-π stacking, and covalent interactions. Simultaneously, the aldehyde groups form Schiff bases with the primary amine of polyethyleneimine, accompanied by catechol self-oxidative crosslinking, constructing a chemically anchored composite primary layer on the base membrane surface.

Description

Technical Field

[0001] This invention belongs to the field of halophyte separation technology, specifically relating to a pH-responsive charge-controlled nanofiltration membrane, its preparation method, and its application. Background Technology

[0002] Nanofiltration membranes are pressure-driven separation membranes that combine size sieving with the Donnan effect, and are widely used in water treatment, the food industry, and natural product extraction. Haematococcus pluvialis is a microalga rich in high-value-added active ingredients, with intracellular astaxanthin ester content reaching 2%-4% of dry weight, along with polysaccharide components with molecular weights of 5-50 kDa. Astaxanthin esters possess strong antioxidant activity and are in high demand in the health supplement, cosmetics, and aquatic feed industries; Haematococcus pluvialis polysaccharides have immunomodulatory functions. The efficient separation of these two components is crucial for enhancing the comprehensive utilization value of Haematococcus pluvialis. Currently, the separation of astaxanthin esters and polysaccharides from Haematococcus pluvialis cell-wall-broken liquid mainly relies on organic solvent extraction combined with column chromatography, or multi-stage membrane filtration processes. The former involves high solvent consumption, cumbersome steps, and the risk of solvent residue, while the latter involves high equipment investment, long operating cycles, and severe membrane fouling.

[0003] Polyamide thin-layer composite nanofiltration membranes are used for the concentration and separation of Haematococcus pluvialis extract due to their moderate retention precision and high flux. However, existing polyamide nanofiltration membranes have a fixed surface charge, making it difficult to reversibly control the charge based on the properties of the feed solution. Haematococcus pluvialis polysaccharide molecules contain uronic acid and sulfate groups, carrying a negative charge in solution, with a molecular weight distribution of 5-50 kDa; astaxanthin ester molecules are electrically neutral, with a molecular weight of approximately 600-900 Da. Because ordinary nanofiltration membranes cannot selectively separate these two molecules based on their charge difference, polysaccharides and astaxanthin esters are simultaneously retained or permeate, resulting in low separation purity and requiring extensive subsequent purification steps. Furthermore, negatively charged polysaccharides readily adsorb onto the membrane surface and within the pores, forming a dense gel layer, causing rapid flux decay, difficulty in cleaning and recovery, shortened service life, and directly increased production costs.

[0004] To improve the selectivity and antifouling properties of membranes, researchers have attempted to coat polyelectrolytes such as polyethyleneimine onto the surface of polyamide-based membranes. This alters the membrane surface charge through electrostatic interactions, utilizing the Donnan effect to trap or allow charged molecules to pass through. Other studies have grafted carboxylated chitosan or zwitterionic polymers onto the membrane surface, using pH changes to regulate the polarity of the membrane surface charge, attempting to achieve reversible separation. These modification methods have improved the membrane's ability to recognize charged molecules to some extent, but significant shortcomings remain. On the one hand, coatings such as polyethyleneimine mainly rely on physical adsorption or simple cross-linking for fixation, resulting in weak interlayer bonding. Under long-term action of transmembrane pressure differentials and feed shear forces, the coating easily peels off from the base membrane surface, causing irreversible degradation of membrane performance, making reuse difficult and economically inefficient. On the other hand, existing pH-responsive membranes typically rely solely on the protonation or deprotonation of a single polyelectrolyte to achieve charge switching, resulting in limited variation in membrane surface charge density. During the reversal from acidic to alkaline conditions, the charge density often changes from weakly positive to near neutral or weakly negative, failing to generate a sufficiently strong electrostatic repulsion force to prevent polysaccharide adsorption. This leads to incomplete polysaccharide release under alkaline conditions, low recovery rates, and the membrane surface remains easily contaminated by polysaccharides. Furthermore, some modified membranes employ complex chemical grafting processes involving multiple reactions and harsh conditions, making industrial scale-up difficult. They also lack chemical anchoring designs for the base membrane, resulting in insufficient interlayer bonding and unreliable long-term operational stability.

[0005] In recent years, inspired by the strong adhesive properties of mussel byssal proteins, compounds containing catechol groups have been used to modify membrane surfaces to enhance the adhesion between coatings and base membranes. However, existing technologies have not yet addressed combining this biomimetic anchoring technology with pH-responsive charge regulation for the stepwise separation of Haematococcus pluvialis polysaccharides and astaxanthin esters. Current technologies often only address adhesion or charge regulation issues in membrane modification, failing to simultaneously achieve separation efficiency and membrane stability.

[0006] Therefore, there is a need to design a pH-responsive charge-controlled nanofiltration membrane, its preparation method, and its application. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, a pH-responsive charge-controlled nanofiltration membrane, its preparation method, and its application are provided.

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

[0009] A method for preparing a pH-responsive charge-controlled nanofiltration membrane, the method comprising the following steps:

[0010] Step 1: Preparation of OSA

[0011] Sodium periodate was added to sodium alginate solution and reacted in the dark. After the reaction was completed, ethylene glycol was added to terminate the reaction. After stirring, the reaction product was dialyzed in deionized water. The resulting retentate was freeze-dried to obtain OSA.

[0012] Step 2: Preparation of DOPA-OSA

[0013] OSA was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer, EDC and NHS were added for activation for 30 min, then DOPA was added and deoxygenated for 30 min. The reaction was carried out at room temperature in the dark for 12-24 h under nitrogen protection. The reaction solution was dialyzed and then freeze-dried to obtain DOPA-OSA.

[0014] Step 3: Preparation of carboxylated chitosan

[0015] Chitosan was dispersed in isopropanol, alkalized, and then reacted with chloroacetic acid. The mixture was then neutralized with glacial acetic acid to a pH of 7.0, filtered, and dried to obtain carboxylated chitosan.

[0016] Step 4: Preparation of pH-responsive charge-controlled nanofiltration membrane

[0017] Polyethyleneimine and DOPA-OSA were prepared into a mixed aqueous solution. The base membrane was immersed in the mixed aqueous solution for reaction, rinsed, and then immersed in a glutaraldehyde aqueous solution. After rinsing, it was immersed in a carboxylated chitosan aqueous solution. After rinsing, a pH-responsive charge-controlled nanofiltration membrane was obtained.

[0018] Step one specifically includes the following steps: dissolve sodium alginate in deionized water to prepare a 1-3 wt% solution, add sodium periodate, react in the dark for 4-6 h at a temperature of 25-35℃, add ethylene glycol to terminate the reaction after the reaction is completed, and continue stirring for 0.5-1 h; put the reaction product into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze it in deionized water, change the water every 4-6 h, dialyze for 48-72 h, take out the retentate in the dialysis bag, and freeze-dry it to obtain OSA.

[0019] The molar ratio of uronic acid structural units in sodium periodate and sodium alginate is 0.3:1-0.6:1, and the molar ratio of ethylene glycol to sodium periodate is 1:1.

[0020] Step two specifically includes the following steps: dissolving OSA in 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5, adding EDC and NHS, activating for 30 min, then adding DOPA, purging the reaction system with nitrogen gas to remove oxygen for 30 min, and reacting at room temperature in the dark under nitrogen protection for 12-24 h.

[0021] The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water. The water was changed every 4-6 hours, and the dialysis was performed for 48-72 hours. The retentate in the dialysis bag was then removed and freeze-dried to obtain DOPA-OSA.

[0022] The molar ratio of carboxyl groups in EDC to OSA is 1.5:1-2.0:1, the molar ratio of EDC to NHS is 1:0.5-1:0.8, and the mass ratio of DOPA to OSA is 0.2:1-0.5:1.

[0023] Step three specifically includes the following steps: dispersing chitosan with a degree of deacetylation ≥85% in isopropanol, adding a 30%-50% sodium hydroxide solution, and alkalizing at 25-35℃ for 1-2 h; adding chloroacetic acid and reacting at 60-80℃ for 3-5 h, wherein the molar ratio of chloroacetic acid to the repeating unit 2-amino-2-deoxy-D-glucose unit of chitosan is 3:1-5:1; after the reaction, neutralizing to pH 7.0 with glacial acetic acid, filtering, washing the filter cake with a 70%-80% ethanol aqueous solution until neutral, and drying to obtain carboxylated chitosan.

[0024] Step four specifically includes the following steps: dissolving polyethyleneimine and DOPA-OSA together in deionized water, adjusting the pH to 8.0-8.5 with 0.1 M sodium hydroxide solution to obtain a mixed aqueous solution;

[0025] The base membrane was immersed in the mixed aqueous solution and reacted at 25-40℃ for 2-4 h. After removal, it was rinsed with boric acid solution with a pH of 7.4, and then immersed in 0.5-2 wt% glutaraldehyde aqueous solution for crosslinking at 25-35℃ for 1-2 h, and rinsed with deionized water. Then it was immersed in carboxylated chitosan aqueous solution and assembled at 25-35℃ for 1-3 h. After assembly, it was rinsed with citrate-sodium citrate buffer solution and then rinsed thoroughly with deionized water to obtain a pH-responsive charge-controlled nanofiltration membrane.

[0026] The base membrane is a polyamide thin-layer composite nanofiltration membrane;

[0027] The concentration of polyethyleneimine in the mixed aqueous solution is 1-3 wt%, and the concentration of DOPA-OSA is 0.5-1.5 wt%; the boric acid solution is prepared by dissolving boric acid in deionized water and adjusting the pH value to 7.4 with sodium hydroxide.

[0028] The carboxylated chitosan aqueous solution is prepared by dissolving carboxylated chitosan in a phosphate buffer solution with a pH of 7.5-8.0, with a carboxylated chitosan concentration of 0.5-2 wt%.

[0029] The citric acid-sodium citrate buffer solution is obtained by mixing 0.1 M citric acid solution and 0.1 M sodium citrate solution at a volume ratio of 3:1 to 4:1.

[0030] The nanofiltration membrane was prepared by the above method.

[0031] This nanofiltration membrane is used for the separation of polysaccharides from Haematococcus pluvialis and astaxanthin esters.

[0032] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0033] 1. This application uses DOPA-OSA as an interface modifier. The catechol groups on its molecular chain strongly adhere to the surface of the polyamide thin-layer composite nanofiltration membrane through hydrogen bonding, π-π stacking, and covalent interactions. Simultaneously, the aldehyde groups form Schiff bases with the primary amine of polyethyleneimine, accompanied by catechol self-oxidative crosslinking, constructing a chemically anchored composite nascent layer on the base membrane surface. Compared to the existing polyethyleneimine coatings that rely solely on physical adsorption or simple crosslinking for fixation, the modified layer in this application forms multiple chemical bonds with the base membrane, significantly improving interlayer bonding strength. It is less prone to peeling under long-term transmembrane pressure differential and feed shear, maintaining stable membrane separation performance and increasing the number of reusable layers.

[0034] 2. This application establishes a pH-responsive charge-controlled structure by sequentially assembling a polyethyleneimine / DOPA-OSA composite layer and a carboxylated chitosan layer on the surface of a polyamide thin-layer composite nanofiltration membrane. Under acidic conditions, the protonation of the amino groups in the polyethyleneimine / DOPA-OSA layer results in a high-density positive charge on the membrane surface, strongly repelling the negatively charged *Haemaphysalis* polysaccharides, thus trapping the polysaccharides. Meanwhile, the neutral astaxanthin esters are unaffected by electrostatic interactions and permeate through the membrane according to their molecular size. Under alkaline conditions, a large number of carboxyl groups in the carboxylated chitosan and DOPA-OSA ionize, transforming the membrane surface into a high-density negative charge. This generates strong electrostatic repulsion with the negatively charged polysaccharides, releasing them from the membrane surface and allowing them to permeate through the pores. This charge reversal is not a simple neutralization or weakening, but rather a rapid switch from strong positive to strong negative charge, improving separation selectivity and polysaccharide recovery.

[0035] 3. The DOPA-OSA molecular chain of this invention contains both aldehyde and catechol groups, which undergo in-situ self-polymerization and cross-linking within the polyethyleneimine cross-linking network, generating localized hard nodules and forming a microphase separation structure that combines rigidity and flexibility. This structure allows for controlled swelling under acidic conditions, providing more precise molecular channels for astaxanthin esters and reducing polysaccharide blockage within the membrane pores. Simultaneously, the gradient network formed by microphase separation strengthens the hydration layer on the membrane surface, reducing the adhesion tendency of pollutants to the membrane surface, alleviating membrane fouling problems, and enabling the membrane to maintain a stable permeate flux even after multiple pH changes.

[0036] 4. This invention uses the same pH-responsive charge-controlled nanofiltration membrane, achieving the stepwise separation of *Haematococcus pluvialis* polysaccharides and astaxanthin esters simply by adjusting the pH of the feed solution. The first nanofiltration is performed under acidic conditions, where the positive charge on the membrane surface retains the *Haematococcus pluvialis* polysaccharides, and the astaxanthin ester-enriched permeate is collected. The second nanofiltration is performed under alkaline conditions, where the negative charge on the membrane surface releases the polysaccharides, and the polysaccharide recovery solution is collected. The entire process requires no replacement of membrane modules, no organic solvent extraction, and no column chromatography purification, improving the economic efficiency of extracting active components from *Haematococcus pluvialis*. Attached Figure Description

[0037] Figure 1 This is a process flow diagram of a method for preparing a pH-responsive charge-controlled nanofiltration membrane. 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 this application, the Chinese meanings of some English abbreviations are as follows:

[0040] DOPA-OSA: Catechol-modified sodium alginate

[0041] OSA: Sodium Oxygenated Alginate

[0042] EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0043] NHS: N-hydroxysuccinimide

[0044] DOPA: 3,4-Dihydroxyphenylalanine

[0045] In the specific embodiments of this application, the sources of various main raw materials are briefly described as follows:

[0046] Sodium alginate: Purchased from Qingdao Mingyue Seaweed Group Co., Ltd., industrial grade, product code LY1

[0047] Sodium periodate: purchased from Jiangsu Zhenri Chemical Co., Ltd., CAS No. 7790-28-5

[0048] Ethylene glycol: purchased from Satellite Chemicals Co., Ltd., CAS No. 107-21-1

[0049] 2-(N-morpholino)ethanesulfonic acid: purchased from Hubei Jianchu Biomedical Co., Ltd., CAS No. 4432-31-9

[0050] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: purchased from Hubei Jianchu Biopharmaceutical Co., Ltd., CAS No. 25952-53-8

[0051] N-Hydroxysuccinimide: Purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., CAS No. 6066-82-6

[0052] 3,4-Dihydroxyphenylalanine: Purchased from Guangzhou Hewei Pharmaceutical Technology Co., Ltd., CAS No. 59-92-7

[0053] Chitosan: Purchased from Wuhan Kemike Biomedical Technology Co., Ltd., degree of deacetylation 85%.

[0054] Isopropanol: Purchased from Jiangsu Yida Chemical Co., Ltd., CAS No. 67-63-0

[0055] Chloroacetic acid: purchased from Hebei Donghua Jiheng Chemical Co., Ltd., CAS No. 79-11-8

[0056] Polyamide thin-layer composite nanofiltration membrane: purchased from Wharton Technology Co., Ltd., with a molecular weight cutoff of 200-500 Da.

[0057] Polyethyleneimine: Purchased from Wuhan Qianglong Chemical New Materials Co., Ltd., CAS No. 106899-94-9

[0058] Boric acid: purchased from Dalian Jinma Boron Industry Technology Group Co., Ltd., CAS No. 10043-35-3

[0059] Glutaraldehyde: Purchased from Hubei Jinghong Biotechnology Co., Ltd., CAS No. 111-30-8

[0060] Phosphate buffer: purchased from Xilong Scientific Co., Ltd.

[0061] Citric acid: purchased from Shandong Yingxuan Industrial Co., Ltd., CAS No. 77-92-9

[0062] Sodium citrate: Purchased from Shandong Yingxuan Industrial Co., Ltd., CAS No. 68-04-2

[0063] Haematococcus pluvialis: Purchased from Yunnan Aierfa Biotechnology Co., Ltd., variety is Haematococcus pluvialis.

[0064] The technical solution of this application is as follows:

[0065] A method for preparing a pH-responsive charge-controlled nanofiltration membrane, such as... Figure 1 As shown, the preparation method includes the following steps:

[0066] Step 1: Preparation of OSA

[0067] Sodium periodate was added to sodium alginate solution and reacted in the dark. After the reaction was completed, ethylene glycol was added to terminate the reaction. After stirring, the reaction product was dialyzed in deionized water. The resulting retentate was freeze-dried to obtain OSA.

[0068] Step 2: Preparation of DOPA-OSA

[0069] OSA was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer, EDC and NHS were added for activation for 30 min, then DOPA was added and deoxygenated for 30 min. The reaction was carried out at room temperature in the dark for 12-24 h under nitrogen protection. The reaction solution was dialyzed and then freeze-dried to obtain DOPA-OSA.

[0070] Step 3: Preparation of carboxylated chitosan

[0071] Chitosan was dispersed in isopropanol, alkalized, and then reacted with chloroacetic acid. The mixture was then neutralized with glacial acetic acid to a pH of 7.0, filtered, and dried to obtain carboxylated chitosan.

[0072] Step 4: Preparation of pH-responsive charge-controlled nanofiltration membrane

[0073] Polyethyleneimine and DOPA-OSA were prepared into a mixed aqueous solution. The base membrane was immersed in the mixed aqueous solution for reaction, rinsed, and then immersed in a glutaraldehyde aqueous solution. After rinsing, it was immersed in a carboxylated chitosan aqueous solution. After rinsing, a pH-responsive charge-controlled nanofiltration membrane was obtained.

[0074] Step one specifically includes the following steps: dissolve sodium alginate in deionized water to prepare a 1-3 wt% solution, add sodium periodate, react in the dark for 4-6 h at a temperature of 25-35℃, add ethylene glycol to terminate the reaction after the reaction is completed, and continue stirring for 0.5-1 h; put the reaction product into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze it in deionized water, change the water every 4-6 h, dialyze for 48-72 h, take out the retentate in the dialysis bag, and freeze-dry it to obtain OSA.

[0075] The molar ratio of uronic acid structural units in sodium periodate and sodium alginate is 0.3:1-0.6:1, and the molar ratio of ethylene glycol to sodium periodate is 1:1.

[0076] Step two specifically includes the following steps: dissolving OSA in 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5, adding EDC and NHS, activating for 30 min, then adding DOPA, purging the reaction system with nitrogen gas to remove oxygen for 30 min, and reacting at room temperature in the dark under nitrogen protection for 12-24 h.

[0077] The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water. The water was changed every 4-6 hours, and the dialysis was performed for 48-72 hours. The retentate in the dialysis bag was then removed and freeze-dried to obtain DOPA-OSA.

[0078] The molar ratio of carboxyl groups in EDC to OSA is 1.5:1-2.0:1, the molar ratio of EDC to NHS is 1:0.5-1:0.8, and the mass ratio of DOPA to OSA is 0.2:1-0.5:1.

[0079] Step three specifically includes the following steps: dispersing chitosan with a degree of deacetylation ≥85% in isopropanol, adding a 30%-50% sodium hydroxide solution, and alkalizing at 25-35℃ for 1-2 h; adding chloroacetic acid and reacting at 60-80℃ for 3-5 h, wherein the molar ratio of chloroacetic acid to the repeating unit 2-amino-2-deoxy-D-glucose unit of chitosan is 3:1-5:1; after the reaction, neutralizing to pH 7.0 with glacial acetic acid, filtering, washing the filter cake with a 70%-80% ethanol aqueous solution until neutral, and drying to obtain carboxylated chitosan.

[0080] Step four specifically includes the following steps: dissolving polyethyleneimine and DOPA-OSA together in deionized water, adjusting the pH to 8.0-8.5 with 0.1 M sodium hydroxide solution to obtain a mixed aqueous solution;

[0081] The base membrane was immersed in the mixed aqueous solution and reacted at 25-40℃ for 2-4 h. After removal, it was rinsed with boric acid solution with a pH of 7.4, and then immersed in 0.5-2 wt% glutaraldehyde aqueous solution for crosslinking at 25-35℃ for 1-2 h, and rinsed with deionized water. Then it was immersed in carboxylated chitosan aqueous solution and assembled at 25-35℃ for 1-3 h. After assembly, it was rinsed with citrate-sodium citrate buffer solution and then rinsed thoroughly with deionized water to obtain a pH-responsive charge-controlled nanofiltration membrane.

[0082] The base membrane is a polyamide thin-layer composite nanofiltration membrane; the base membrane mainly plays a supporting role, the separation is mainly dominated by the charge regulation of the modified layer, and the size sieving is secondary.

[0083] The concentration of polyethyleneimine in the mixed aqueous solution is 1-3 wt%, and the concentration of DOPA-OSA is 0.5-1.5 wt%; the boric acid solution is prepared by dissolving boric acid in deionized water and adjusting the pH value to 7.4 with sodium hydroxide.

[0084] The carboxylated chitosan aqueous solution is prepared by dissolving carboxylated chitosan in a phosphate buffer solution with a pH of 7.5-8.0, with a carboxylated chitosan concentration of 0.5-2 wt%.

[0085] The citric acid-sodium citrate buffer solution is obtained by mixing 0.1 M citric acid solution and 0.1 M sodium citrate solution at a volume ratio of 3:1 to 4:1.

[0086] The nanofiltration membrane was prepared by the above method.

[0087] This nanofiltration membrane is used for the separation of polysaccharides from Haematococcus pluvialis and astaxanthin esters.

[0088] When existing polyamide thin-layer composite nanofiltration membranes are used for separating Haematococcus pluvialis cell-wall-broken liquid, the modified layer and the base membrane are mostly physically adsorbed or simply cross-linked, resulting in weak interlayer bonding. Under long-term transmembrane pressure difference and feed shear, the coating is easily peeled off from the base membrane surface, causing irreversible degradation of membrane performance. Although existing technologies attempt to improve membrane surface charge by coating with polyelectrolytes such as polyethyleneimine, the coating fixation method is singular, the anchoring is not firm, and it is difficult to reuse.

[0089] To address the aforementioned shortcomings, we prepared DOPA-OSA and co-assembled it with polyethyleneimine onto the surface of a polyamide thin-layer composite nanofiltration membrane. The catechol groups on the DOPA-OSA molecular chain possess strong reducing and multidentate coordination capabilities, enabling them to adhere to multiple functional groups such as amide and carboxyl groups on the polyamide membrane surface through hydrogen bonding, π-π stacking, and covalent interactions. Simultaneously, its aldehyde groups undergo a Schiff base reaction with the primary amine of polyethyleneimine, forming dynamic covalent bonds. Under alkaline conditions, the catechol further undergoes auto-oxidation to generate quinone structures, which then undergo Michael addition with amino groups, constructing a three-dimensional cross-linked network. This multi-layer chemical bonding firmly anchors the polyethyleneimine layer to the base membrane, rather than relying solely on physical adsorption or single cross-linking. This significantly enhances the interlayer bonding force, allowing the modified layer to remain intact even after multiple pH changes and long-term operation, resulting in stable membrane separation performance.

[0090] After solving the anchoring problem, existing pH-responsive membranes typically rely solely on the protonation or deprotonation of a single polyelectrolyte to achieve charge switching. The range of change in membrane surface charge density is limited. During the reversal from acidic to alkaline conditions, the charge density often changes from weakly positive to near neutral or weakly negative, which cannot generate a sufficiently strong electrostatic repulsion force to prevent polysaccharide adsorption, resulting in incomplete polysaccharide release under alkaline conditions.

[0091] To address this issue, we sequentially assembled a polyethyleneimine / DOPA-OSA composite layer and a carboxylated chitosan layer on the surface of a polyamide thin-layer composite nanofiltration membrane, forming a pH-responsive charge-controlled structure. From the perspective of charge control principles, in the polyethyleneimine / DOPA-OSA layer, polyethyleneimine contains a large amount of primary and secondary amines. Under acidic conditions, the amino groups protonate and become positively charged, exhibiting Donan repulsion against the negatively charged *Aureobasidium pluvialis* polysaccharide. In the carboxylated chitosan layer, the carboxyl groups are fully ionized and become negatively charged under alkaline conditions, while the free carboxyl groups on the DOPA-OSA molecular chains further increase the negative charge density. The strength of the Donan effect is directly related to the membrane surface charge density; the higher the charge density, the stronger the repulsion force against like-charged ions. Therefore, a high-density positive charge can effectively retain the negatively charged *Aureobasidium pluvialis* polysaccharide, while a high-density negative charge can release the polysaccharide from the membrane surface and allow it to permeate through the membrane pores through strong electrostatic repulsion, achieving a rapid switch from strong positive to strong negative charge, thus improving separation selectivity and polysaccharide recovery rate.

[0092] Even after addressing anchoring and charge control issues, mass transfer resistance within the membrane pores and membrane fouling remain key factors affecting separation efficiency. Existing modified membranes often lack precise control over the pore structure, allowing large polysaccharides to accumulate and clog the pores, leading to flux reduction.

[0093] To this end, we utilized the aldehyde and catechol groups simultaneously present in the DOPA-OSA molecular chain to induce in-situ self-polymerization and cross-linking within the polyethyleneimine cross-linked network. From the perspective of structural regulation, catechol undergoes auto-oxidation under alkaline conditions to generate catechol quinone. The quinone structure can undergo Michael addition and Schiff base reactions with the amino groups of polyethyleneimine, forming hard nodular regions with high local cross-linking density. These hard nodules are dispersed within the flexible polyethyleneimine network, forming a microphase-separated structure. The hard nodular regions limit the overall swelling degree of the network, maintaining a suitable pore size under acidic conditions and preventing a decrease in retention rate due to excessive pore swelling. Simultaneously, the flexible regions maintain the membrane's hydration capacity, forming a dense hydration layer on the membrane surface, reducing the tendency of pollutants to adhere to the membrane surface. The gradient network formed by microphase separation also promotes turbulent disturbance of the feed solution on the membrane surface, reduces the thickness of the concentration polarization layer, provides more precise molecular channels for astaxanthin esters, reduces the blockage of polysaccharides in the membrane pores, alleviates membrane fouling problems, and enables the membrane to maintain a stable permeate flux after multiple pH changes.

[0094] 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.

[0095] Example 1

[0096] Sodium alginate was dissolved in deionized water to prepare a 3 wt% solution. Sodium periodate was added, with a molar ratio of uronic acid structural units in sodium periodate to sodium alginate of 0.45:1. Based on the average molecular weight of repeating units in sodium alginate of 198 g / mol, the reaction was carried out in the dark for 6 h at 25 °C. After the reaction was completed, ethylene glycol of the same amount as sodium periodate was added to terminate the reaction, and stirring was continued for 0.5 h. The reaction product was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water. The water was changed every 5 hours, and the dialysis was repeated for 60 hours. The retentate in the dialysis bag was removed and placed in a lyophilization tray with a thickness not exceeding 1 cm. The tray was pre-frozen at -60°C for 3 hours and then transferred to a freeze dryer. The cold trap temperature was -50°C and the vacuum degree was 30 Pa. The first drying temperature was -30°C and held for 18 hours. The second drying temperature was 25°C and held for 6 hours. The vacuum degree was 20 Pa and the product was dried to constant weight. The freeze-dried product yielded OSA with a viscosity-average molecular weight of 18 kDa and an aldehyde content of 280 μmol / g.

[0097] OSA was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5. EDC (hereinafter referred to as EDC) and NHS were added to activate the carboxyl groups in OSA. The molar ratio of EDC to the carboxyl groups in OSA was 1.75:1, and the molar ratio of EDC to NHS was 1:0.5. After activation for 30 min, DOPA was added. The mass ratio of DOPA to OSA was 0.5:1. Nitrogen gas was introduced into the reaction system to remove oxygen for 30 min. The reaction was carried out at room temperature in the dark for 18 h under nitrogen protection. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water. The water was changed every 5 hours, and the dialysis was repeated for 60 hours. The retentate in the dialysis bag was removed and placed in a lyophilization tray with a thickness not exceeding 1 cm. The tray was pre-frozen at -60°C for 3 hours and then transferred to a freeze dryer. The cold trap temperature was -50°C and the vacuum degree was 30 Pa. The first drying temperature was -30°C and held for 18 hours. The second drying temperature was 25°C and held for 6 hours. The vacuum degree was 20 Pa and the tray was dried to constant weight. The lyophilized product was DOPA-OSA, hereinafter referred to as DOPA-OSA, with a DOPA grafting rate of 22%.

[0098] Chitosan with a degree of deacetylation ≥85% was dispersed in isopropanol, with the repeating unit of chitosan being 2-amino-2-deoxy-D-glucose units. A 30% (w / w) sodium hydroxide solution was added, and the mixture was alkalized at 30°C for 2 h. Chloroacetic acid was then added, with a molar ratio of chloroacetic acid to chitosan repeating units of 4:1, and the reaction was carried out at 60°C for 5 h. After the reaction, the mixture was neutralized to pH 7.0 with glacial acetic acid, filtered, and the filter cake was washed with a 75% (v / v) ethanol aqueous solution until neutral. The mixture was then dried to obtain carboxylated chitosan with a degree of carboxylation of 60%, a molecular weight of 90,000 Da, and good water solubility.

[0099] Using a polyamide thin-layer composite nanofiltration membrane as the base membrane, polyethyleneimine and DOPA-OSA were dissolved together in deionized water to prepare a mixed aqueous solution. The concentration of polyethyleneimine was 2 wt% (molecular weight 6000 Da), and the concentration of DOPA-OSA was 0.5 wt%. The pH was adjusted to 8.5 with 0.1 M sodium hydroxide solution. The base membrane was immersed in this mixed aqueous solution at 32°C for 3 h. After removal, it was rinsed with a boric acid solution at pH 7.4 to remove unreacted substances. This boric acid solution was prepared by dissolving boric acid in deionized water and adjusting the pH to 7.4 with sodium hydroxide, with a concentration of 0.08 M. The membrane was then immersed in a 0.5 wt% glutaraldehyde aqueous solution and crosslinked at 30°C for 2 h, followed by thorough rinsing with deionized water. The primary layer modified membrane was immersed in an aqueous solution of carboxylated chitosan prepared with phosphate buffer (PBS) at pH 7.6 (1.25 wt%) as the solvent. The assembly time was 30°C and 1 h. After assembly, the membrane was rinsed with a citrate-sodium citrate buffer to re-protonate the amino groups and re-protonate the carboxyl groups, forming a stable hydrogen bond network. The citrate-sodium citrate buffer was prepared by mixing 0.1 M citric acid solution and 0.1 M sodium citrate solution at a volume ratio of 3:1. The membrane was then thoroughly rinsed with deionized water to obtain a pH-responsive charge-controlled nanofiltration membrane.

[0100] Example 2

[0101] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0102] Sodium alginate was dissolved in deionized water to prepare a 1 wt% solution. Sodium periodate was added, with a molar ratio of uronic acid structural units in sodium periodate to sodium alginate of 0.6:1. The reaction was carried out in the dark for 4 h at 30 °C. After the reaction was completed, ethylene glycol of an equal amount to sodium periodate was added to terminate the reaction, and stirring was continued for 1 h. The reaction product was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water, with the water changed every 5 h. Dialysis was carried out for 60 h. The retentate in the dialysis bag was removed and placed in a lyophilization tray with a thickness not exceeding 1 cm. It was pre-frozen at -60 °C for 3 h, and then transferred to a freeze dryer. The cold trap temperature was -50 °C, and the vacuum degree was 30 Pa. The first drying temperature was -30 °C for 18 h, and the second drying temperature was 25 °C for 6 h, with a vacuum degree of 20 Pa. The product was dried to constant weight and lyophilized to obtain OSA (hereinafter referred to as OSA), with a viscosity-average molecular weight of 15 kDa and an aldehyde content of 380 μmol / g.

[0103] OSA was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5. EDC and NHS were added to activate the carboxyl groups in OSA. The molar ratio of EDC to carboxyl groups in OSA was 2.0:1, and the molar ratio of EDC to NHS was 1:0.65. After activation for 30 min, DOPA was added. The mass ratio of DOPA to OSA was 0.2:1. Nitrogen gas was purged into the reaction system to remove oxygen for 30 min. The reaction was carried out at room temperature in the dark for 24 h under nitrogen protection. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water. The water was changed every 5 hours, and the dialysis was repeated for 60 hours. The retentate in the dialysis bag was removed and placed in a lyophilization tray with a thickness not exceeding 1 cm. The tray was pre-frozen at -60°C for 3 hours and then transferred to a freeze dryer. The cold trap temperature was -50°C and the vacuum degree was 30 Pa. The first drying temperature was -30°C and held for 18 hours. The second drying temperature was 25°C and held for 6 hours. The vacuum degree was 20 Pa and the tray was dried to constant weight. DOPA-OSA was obtained by lyophilization with a DOPA grafting rate of 15%.

[0104] Chitosan with a degree of deacetylation ≥85% was dispersed in isopropanol, with the repeating unit of chitosan being 2-amino-2-deoxy-D-glucose units. A 40% (w / w) sodium hydroxide solution was added, and the mixture was alkalized at 30°C for 1 h. Chloroacetic acid was then added, with a molar ratio of chloroacetic acid to chitosan repeating units of 5:1, and the reaction was carried out at 70°C for 3 h. After the reaction, the mixture was neutralized to pH 7.0 with glacial acetic acid, filtered, and the filter cake was washed with a 75% (v / v) ethanol aqueous solution until neutral. The mixture was then dried to obtain carboxylated chitosan with a degree of carboxylation of 72%, a molecular weight of 85,000 Da, and good water solubility.

[0105] Using a polyamide thin-layer composite nanofiltration membrane as the base membrane, polyethyleneimine and DOPA-OSA were dissolved together in deionized water to prepare a mixed aqueous solution, wherein the concentration of polyethyleneimine was 3 wt% (molecular weight 6000 Da) and the concentration of DOPA-OSA was 1.0 wt%. The pH was adjusted to 8.0 with 0.1 M sodium hydroxide solution to obtain the mixed aqueous solution. The base membrane was immersed in this mixed aqueous solution at 40℃ for 3 h. After removal, it was rinsed with boric acid solution at pH 7.4 to remove unreacted substances. Then it was immersed in 1.25 wt% glutaraldehyde aqueous solution and crosslinked at 30℃ for 1 h, followed by thorough rinsing with deionized water. The above primary layer modified membrane was immersed in a carboxylated chitosan aqueous solution prepared with PBS buffer at pH 7.6 as the solvent, with a carboxylated chitosan concentration of 2 wt%, at 30℃ for 2 h. After assembly, the membrane was rinsed with a citrate-sodium citrate buffer solution and then thoroughly rinsed with deionized water. The citrate-sodium citrate buffer solution was prepared by mixing 0.1 M citric acid solution and 0.1 M sodium citrate solution at a volume ratio of 3.5:1 to obtain a pH-responsive charge-controlled nanofiltration membrane.

[0106] Example 3

[0107] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0108] Sodium alginate was dissolved in deionized water to prepare a 2 wt% solution. Sodium periodate was added, with a molar ratio of uronic acid structural units in sodium periodate to sodium alginate of 0.3:1. The reaction was carried out in the dark for 5 h at 35℃. After the reaction was completed, ethylene glycol of an equal amount to sodium periodate was added to terminate the reaction, and stirring was continued for 0.8 h. The reaction product was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water, with the water changed every 5 h. Dialysis was carried out for 60 h. The retentate in the dialysis bag was removed and placed in a lyophilization tray with a thickness not exceeding 1 cm. It was pre-frozen at -60℃ for 3 h, and then transferred to a freeze dryer. The cold trap temperature was -50℃, and the vacuum degree was 30 Pa. The first drying temperature was -30℃ for 18 h, and the second drying temperature was 25℃ for 6 h, with a vacuum degree of 20 Pa. The product was dried to constant weight and lyophilized to obtain OSA (hereinafter referred to as OSA), with a viscosity-average molecular weight of 22 kDa and an aldehyde content of 220 μmol / g.

[0109] OSA was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5. EDC and NHS were added to activate the carboxyl groups in OSA. The molar ratio of EDC to carboxyl groups in OSA was 1.5:1, and the molar ratio of EDC to NHS was 1:0.8. After activation for 30 min, DOPA was added. The mass ratio of DOPA to OSA was 0.35:1. Nitrogen gas was purged into the reaction system for 30 min to remove oxygen. The reaction was carried out at room temperature in the dark for 12 h under nitrogen protection. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water. The water was changed every 5 hours, and the dialysis was repeated for 60 hours. The retentate in the dialysis bag was removed and placed in a lyophilization tray with a thickness not exceeding 1 cm. The tray was pre-frozen at -60°C for 3 hours and then transferred to a freeze dryer. The cold trap temperature was -50°C and the vacuum degree was 30 Pa. The first drying temperature was -30°C and held for 18 hours. The second drying temperature was 25°C and held for 6 hours. The vacuum degree was 20 Pa and the tray was dried to constant weight. DOPA-OSA was obtained by freeze drying with a DOPA grafting rate of 20%.

[0110] Chitosan with a degree of deacetylation ≥85% was dispersed in isopropanol, with the repeating unit of chitosan being 2-amino-2-deoxy-D-glucose units. A 50% (w / w) sodium hydroxide solution was added, and the mixture was alkalized at 30°C for 1.5 h. Chloroacetic acid was then added, with a molar ratio of chloroacetic acid to chitosan repeating units of 3:1, and the reaction was carried out at 80°C for 5 h. After the reaction, the mixture was neutralized to pH 7.0 with glacial acetic acid, filtered, and the filter cake was washed with a 75% (v / v) ethanol aqueous solution until neutral. The mixture was then dried to obtain carboxylated chitosan with a degree of carboxylation of 58%, a molecular weight of 110,000 Da, and good water solubility.

[0111] Using a polyamide thin-layer composite nanofiltration membrane as the base membrane, polyethyleneimine and DOPA-OSA were dissolved together in deionized water to prepare a mixed aqueous solution, wherein the concentration of polyethyleneimine was 1 wt% (molecular weight 6000 Da) and the concentration of DOPA-OSA was 1.5 wt%. The pH was adjusted to 8.25 with 0.1 M sodium hydroxide solution to obtain the mixed aqueous solution. The base membrane was immersed in this mixed aqueous solution at 25°C for 3 h. After removal, it was rinsed with boric acid solution at pH 7.4 to remove unreacted substances. Then it was immersed in 2 wt% glutaraldehyde aqueous solution and crosslinked at 30°C for 1.5 h, followed by thorough rinsing with deionized water. The above primary layer modified membrane was immersed in a carboxylated chitosan aqueous solution prepared with PBS buffer at pH 7.6 as the solvent, with a carboxylated chitosan concentration of 0.5 wt%, at 30°C for 3 h. After assembly, the membrane was rinsed with a citrate-sodium citrate buffer solution, which was prepared by mixing 0.1 M citric acid solution and 0.1 M sodium citrate solution at a volume ratio of 4:1. The membrane was then thoroughly rinsed with deionized water to obtain a pH-responsive charge-controlled nanofiltration membrane.

[0112] Example 4

[0113] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0114] Sodium alginate was dissolved in deionized water to prepare a 2 wt% solution. Sodium periodate was added, with a molar ratio of uronic acid structural units in sodium periodate to sodium alginate of 0.5:1. The reaction was carried out in the dark for 5 h at 30 °C. After the reaction was completed, ethylene glycol of an equal amount to sodium periodate was added to terminate the reaction, and stirring was continued for another 0.5 h. The reaction product was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water, with the water changed every 5 h. Dialysis was carried out for 60 h. The retentate in the dialysis bag was removed and placed in a lyophilization tray with a thickness not exceeding 1 cm. The tray was pre-frozen at -60 °C for 3 h and then transferred to a freeze dryer. The cold trap temperature was -50 °C and the vacuum degree was 30 Pa. The first drying temperature was -30 °C for 18 h, and the second drying temperature was 25 °C for 6 h at a vacuum degree of 20 Pa. The product was dried to constant weight and lyophilized to obtain OSA (hereinafter referred to as OSA), which has a viscosity-average molecular weight of 20 kDa and an aldehyde content of 320 μmol / g.

[0115] OSA was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5. EDC and NHS were added to activate the carboxyl groups in OSA. The molar ratio of EDC to carboxyl groups in OSA was 1.8:1, and the molar ratio of EDC to NHS was 1:0.65. After activation for 30 min, DOPA was added. The mass ratio of DOPA to OSA was 0.4:1. Nitrogen gas was purged into the reaction system for 30 min to remove oxygen. The reaction was carried out at room temperature in the dark for 18 h under nitrogen protection. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water. The water was changed every 5 hours, and the dialysis was repeated for 60 hours. The retentate in the dialysis bag was removed and placed in a lyophilization tray with a thickness not exceeding 1 cm. The tray was pre-frozen at -60°C for 3 hours and then transferred to a freeze dryer. The cold trap temperature was -50°C and the vacuum degree was 30 Pa. The first drying temperature was -30°C and held for 18 hours. The second drying temperature was 25°C and held for 6 hours. The vacuum degree was 20 Pa and the tray was dried to constant weight. DOPA-OSA was obtained by lyophilization with a DOPA grafting rate of 25%.

[0116] Chitosan with a degree of deacetylation ≥85% was dispersed in isopropanol, with the repeating unit of chitosan being 2-amino-2-deoxy-D-glucose units. A 40% (w / w) sodium hydroxide solution was added, and the mixture was alkalized at 30°C for 1.5 h. Chloroacetic acid was then added, with a molar ratio of chloroacetic acid to chitosan repeating units of 4:1, and the reaction was carried out at 70°C for 4 h. After the reaction, the mixture was neutralized to pH 7.0 with glacial acetic acid, filtered, and the filter cake was washed with a 75% (v / v) ethanol aqueous solution until neutral. The mixture was then dried to obtain carboxylated chitosan with a degree of carboxylation of 65%, a molecular weight of 100,000 Da, and good water solubility.

[0117] Using a polyamide thin-layer composite nanofiltration membrane as the base membrane, polyethyleneimine and DOPA-OSA were dissolved together in deionized water to prepare a mixed aqueous solution, wherein the concentration of polyethyleneimine was 2 wt% (molecular weight 6000 Da) and the concentration of DOPA-OSA was 1.0 wt%. The pH was adjusted to 8.2 with 0.1 M sodium hydroxide solution to obtain the mixed aqueous solution. The base membrane was immersed in this mixed aqueous solution at 30°C for 3 h. After removal, it was rinsed with boric acid solution at pH 7.4 to remove unreacted substances. Then it was immersed in a 1.0 wt% glutaraldehyde aqueous solution and crosslinked at 30°C for 1.5 h, followed by thorough rinsing with deionized water. The above primary layer modified membrane was immersed in a carboxylated chitosan aqueous solution prepared with PBS buffer at pH 7.6 as the solvent, with a carboxylated chitosan concentration of 1.0 wt%, at 30°C for 2 h. After assembly, the membrane was rinsed with a citrate-sodium citrate buffer solution, which was prepared by mixing 0.1 M citric acid solution and 0.1 M sodium citrate solution at a volume ratio of 3.5:1. The membrane was then thoroughly rinsed with deionized water to obtain a pH-responsive charge-controlled nanofiltration membrane.

[0118] Comparative Example 1

[0119] Compared to Example 4, step four did not use DOPA-OSA. Instead, polyethyleneimine was dissolved in deionized water to prepare a 2 wt% solution. The pH was adjusted to 8.2 with 0.1 M sodium hydroxide solution. The base membrane was immersed in this solution at 30°C for 3 hours. After removal, it was rinsed with deionized water and then immersed in a 1.0 wt% glutaraldehyde aqueous solution for crosslinking at 30°C for 1.5 hours. It was then thoroughly rinsed with deionized water. Next, it was immersed in a carboxylated chitosan aqueous solution prepared with PBS buffer at pH 7.6 (1.0 wt% carboxylated chitosan concentration), at 30°C for 2 hours. After assembly, it was rinsed with citrate-sodium citrate buffer and then thoroughly rinsed with deionized water to obtain the control membrane. The remaining steps and separation conditions were exactly the same as in Example 4.

[0120] Comparative Example 2

[0121] Compared to Example 4, step four does not involve the assembly of the carboxylated chitosan layer. Instead, after preparing the polyethyleneimine / DOPA-OSA composite primary layer and crosslinking it with glutaraldehyde, the control membrane is thoroughly rinsed with deionized water, without further carboxylated chitosan impregnation and rinsing steps. The remaining steps and separation conditions are exactly the same as in Example 4.

[0122] Comparative Example 3

[0123] Compared to Example 4, sodium periodate was not used in step one. Instead, unoxidized sodium alginate was used directly as the raw material. Following the conditions in step two of Example 4, sodium alginate was dissolved in a 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5, activated with EDC and NHS, and then reacted with DOPA to prepare an aldehyde-free sodium alginate-DOPA graft, hereinafter referred to as DOPA-SA, which replaced DOPA-OSA for membrane preparation in step four. The remaining steps and separation conditions were exactly the same as in Example 4.

[0124] Performance Test Results and Analysis

[0125] The membranes prepared in Examples 1-4 and Comparative Examples 1-3 were cut into 1 cm × 1 cm samples and placed in electrolyte solutions of different pH values. The zeta potential of the membrane surface was measured using an electrophoretic light scattering instrument, with a test pH range of 3.0-8.0. Using the seat drop method, 2 μL of deionized water was dropped onto the membrane surface, and the static water contact angle was recorded using a contact angle meter.

[0126] After high-pressure homogenization and cell wall disruption, *Hydrocotyle erythrorhizon* was diluted with water to a solid content of 7.5 wt%. Cell debris was removed by centrifugation, and the supernatant contained 1.25 g / L of *Hydrocotyle erythrorhizon* polysaccharide and 0.3 g / L of astaxanthin ester. The supernatant was adjusted to pH 4.0 with citrate-sodium hydroxide buffer, and nanofiltration was performed using the membranes prepared in Examples 1-4 and Comparative Examples 1-3 at 27°C and a transmembrane pressure difference of 0.75 MPa. The permeate was collected from the permeate side to obtain the astaxanthin ester-enriched permeate; the retentate was collected from the retentate side to obtain the polysaccharide-enriched retentate. The polysaccharide-enriched retentate was diluted 3.5 times with deionized water and adjusted to pH 7.0 with sodium hydroxide solution. This retentate was then used as fresh feed for a second nanofiltration separation on the same nanofiltration membrane at 27°C and a transmembrane pressure difference of 0.75 MPa. The permeate from the second nanofiltration was collected from the permeate side to obtain the polysaccharide recovery solution.

[0127] Permeate and retentate were collected. The concentration of polysaccharides from *Haemaphysalis* was determined using the phenol-sulfuric acid method, and the concentration of astaxanthin esters was determined using high-performance liquid chromatography (HPLC). The polysaccharide rejection rate, astaxanthin ester permeate rate, and polysaccharide permeate rate were calculated. Under a constant transmembrane pressure difference, the volume of permeate per unit time per unit membrane area was recorded, and the membrane flux was calculated. The same membrane was repeatedly subjected to acidic nanofiltration and alkaline nanofiltration five times each, and the flux was measured each time. The flux retention rate after five cycles was calculated. The test results are shown in Tables 1-3.

[0128] Table 1 shows the results of membrane surface charge and hydrophilicity tests. As can be seen from Table 1, for membrane surface charge data, the zeta potential of Examples 1-4 was +28 to +35 mV under acidic conditions and -35 to -42 mV under alkaline conditions, with a charge reversal amplitude of 63-77 mV, indicating that the membrane surface achieved a rapid switch from strongly positive to strongly negative charge. Example 4 showed the largest charge reversal amplitude, reaching 77 mV. This is because its DOPA-OSA concentration and DOPA grafting rate were at optimal levels, resulting in sufficient charge density formed by catechol and carboxyl groups on the membrane surface.

[0129] Comparative Example 1, without DOPA-OSA and relying solely on the physical adsorption of polyethyleneimine, exhibited a zeta potential of only +18 mV under acidic conditions and -12 mV under alkaline conditions, with a charge reversal amplitude of only 30 mV. This is because the lack of catechol anchoring and carboxyl amplification from DOPA-OSA resulted in a loose bond between the polyethyleneimine layer and the base film, leading to partial detachment and insufficient charge density. Furthermore, the absence of additional carboxyl sources to enhance the negative charge further exacerbated the problem. Comparative Example 2, without carboxylated chitosan assembly, showed a zeta potential of only -25 mV and a reversal amplitude of 47 mV under alkaline conditions, lower than the examples. This indicates that the lack of charge contribution from the carboxylated chitosan layer meant that the carboxyl groups of DOPA-OSA alone could not achieve sufficient negative charge density. Comparative Example 3, using aldehyde-free DOPA-SA, exhibited weak anchoring ability, resulting in partial detachment of the modified layer, reduced charge density, and a reversal amplitude of only 35 mV.

[0130] Based on the hydrophilicity data of the membrane surface, the water contact angles of Examples 1-4 were 35-42°, indicating that the membrane surface had good hydrophilicity. This is because DOPA-OSA and carboxylated chitosan introduced abundant hydrophilic groups such as hydroxyl, carboxyl, and amino groups, forming a dense hydration layer on the membrane surface, reducing the tendency of pollutants to adhere to the membrane surface. Example 4 had the smallest water contact angle of 35°, exhibiting the best hydrophilicity. This may be related to its optimized parameters, high DOPA grafting rate, and high degree of assembly of carboxylated chitosan, resulting in a high density of hydrophilic groups on the membrane surface and a denser hydration layer.

[0131] Comparative Example 1 has a water contact angle of 55°, and its hydrophobicity is significantly stronger than that of the Example. This is because it lacks the hydrophilic groups of catechol in DOPA-OSA. The amino groups of polyethyleneimine alone are insufficient to form a sufficient hydration layer, and the hydrophobic areas on the membrane surface are exposed, making it easy for pollutants to adhere.

[0132] Comparative Example 2 had a water contact angle of 48°, higher than the example. Although DOPA-OSA provided some hydrophilic groups, it lacked further hydrophilic modification of the carboxylated chitosan layer, resulting in insufficient hydration layer thickness. Comparative Example 3 had a water contact angle of 52°. DOPA-SA lacked aldehyde groups, had low grafting efficiency, few hydrophilic groups on the membrane surface, and the shedding of the modified layer led to the exposure of hydrophobic regions of the base membrane.

[0133] Table 1 Results of membrane surface charge and hydrophilicity tests

[0134] Example 1 +28 -35 63 42 Example 2 +32 -38 70 38 Example 3 +30 -36 66 40 Example 4 +35 -42 77 35 Comparative Example 1 +18 -12 30 55 Comparative Example 2 +22 -25 47 48 Comparative Example 3 +15 -20 35 52

[0135] From Table 2, the polysaccharide rejection rate of Examples 1-4 was 78%-88%, the astaxanthin ester permeation rate was 86%-93%, and the membrane flux was 38-48 L / m²·h. Example 4 showed the highest polysaccharide rejection rate at 88%, an astaxanthin ester permeation rate of 93%, and a flux of 48 L / m²·h. This was because its membrane surface had a high positive charge density, which generated sufficient dominance against the negatively charged Haematococcus pluvialis polysaccharide. At the same time, the microphase separation structure provided a smooth molecular channel for astaxanthin ester, reducing mass transfer resistance.

[0136] In Comparative Example 1, the polysaccharide rejection rate was only 52%, the astaxanthin ester permeation rate was 72%, and the flux was 28 L / m²·h. This was because the polyethyleneimine layer was not firmly anchored, and some of it detached, resulting in insufficient positive charge density and an inability to effectively repel polysaccharides. At the same time, the detached polymer blocked the membrane pores, causing a decrease in flux.

[0137] Comparative Example 2 showed a polysaccharide rejection rate of 58%, an astaxanthin ester permeation rate of 75%, and a flux of 30 L / m²·h. Although DOPA-OSA provided some anchoring, the lack of a carboxylated chitosan layer resulted in limited negative charge density on the membrane surface under alkaline conditions, incomplete electrostatic release of polysaccharides, and easy polysaccharide fouling of the membrane pores. Comparative Example 3 showed a polysaccharide rejection rate of 48%, an astaxanthin ester permeation rate of 68%, and a flux of 25 L / m²·h. DOPA-SA lacked aldehyde groups and could not form a Schiff base with polyethyleneimine, leading to significant detachment of the modified layer and the membrane surface almost reverting to the performance of the original polyamide nanofiltration membrane.

[0138] Table 2 Results of the first nanofiltration separation performance test

[0139] Example 1 78 86 38 Example 2 82 89 42 Example 3 80 87 40 Example 4 88 93 48 Comparative Example 1 52 72 28 Comparative Example 2 58 75 30 Comparative Example 3 48 68 25

[0140] From the second nanofiltration separation performance and cycle stability in Table 3, the polysaccharide permeation rate of Examples 1-4 was 80%-90%, and the flux retention rate after 5 cycles was 85%-95%. Example 4 had the highest polysaccharide permeation rate, reaching 90%, and a flux retention rate of 95%. This is because under alkaline conditions, the high-density negative charge on the membrane surface generates strong electrostatic repulsion with the polysaccharide, inhibiting concentration polarization and membrane fouling. At the same time, the chemical anchoring of DOPA-OSA ensured that the modified layer remained firmly attached after multiple pH changes.

[0141] Comparative Example 1 showed a polysaccharide permeability of only 32% and a flux retention rate of 55%. This was due to the low negative charge density on the membrane surface, which failed to effectively repel the polysaccharide, and the shedding of the modified layer leading to membrane pore blockage and performance degradation. Comparative Example 2 showed a polysaccharide permeability of 42% and a flux retention rate of 68%. The lack of a carboxylated chitosan layer amplified the negative charge, resulting in insufficient electrostatic repulsion on the membrane surface and incomplete polysaccharide release.

[0142] Comparative Example 3 showed a polysaccharide permeability of 28%, a flux retention rate of 50%, a large amount of modified layer peeling off, almost no charge response capability on the membrane surface, polysaccharide retention, and severe membrane fouling.

[0143] Test results show that this application constructs a chemically anchored pH-responsive nanofiltration membrane by modifying sodium alginate with catechol, and achieves the stepwise separation of Haematococcus pluvialis polysaccharide and astaxanthin ester by charge reversal of the same membrane under different pH conditions.

[0144] Table 3 Results of the second nanofiltration separation performance and cycle stability test

[0145] Example 1 80 85 Example 2 84 88 Example 3 82 86 Example 4 90 95 Comparative Example 1 32 55 Comparative Example 2 42 68 Comparative Example 3 28 50

[0146] 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 preparing a pH-responsive charge-controlled nanofiltration membrane, characterized in that, The preparation method includes the following steps: Step 1: Preparation of OSA Sodium periodate was added to sodium alginate solution and reacted in the dark. After the reaction was completed, ethylene glycol was added to terminate the reaction. After stirring, the reaction product was dialyzed in deionized water. The resulting retentate was freeze-dried to obtain OSA. Step 2: Preparation of DOPA-OSA OSA was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer, EDC and NHS were added for activation for 30 min, then DOPA was added and deoxygenated for 30 min. The reaction was carried out at room temperature in the dark for 12-24 h under nitrogen protection. The reaction solution was dialyzed and then freeze-dried to obtain DOPA-OSA. Step 3: Preparation of carboxylated chitosan Chitosan was dispersed in isopropanol, alkalized, and then reacted with chloroacetic acid. The mixture was then neutralized with glacial acetic acid to a pH of 7.0, filtered, and dried to obtain carboxylated chitosan. Step 4: Preparation of pH-responsive charge-controlled nanofiltration membrane Polyethyleneimine and DOPA-OSA were prepared into a mixed aqueous solution. A polyamide thin-layer composite nanofiltration membrane was used as the base membrane and immersed in the mixed aqueous solution for reaction. After rinsing, it was immersed in a glutaraldehyde aqueous solution, rinsed, and then immersed in a carboxylated chitosan aqueous solution. After rinsing, a pH-responsive charge-controlled nanofiltration membrane was obtained.

2. The method for preparing a pH-responsive charge-controlled nanofiltration membrane according to claim 1, characterized in that, Step one specifically includes the following steps: dissolve sodium alginate in deionized water to prepare a 1-3 wt% solution, add sodium periodate, react in the dark for 4-6 h at a temperature of 25-35℃, add ethylene glycol to terminate the reaction after the reaction is completed, and continue stirring for 0.5-1 h; put the reaction product into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze it in deionized water, change the water every 4-6 h, dialyze for 48-72 h, take out the retentate in the dialysis bag, and freeze-dry it to obtain OSA.

3. The method for preparing a pH-responsive charge-controlled nanofiltration membrane according to claim 2, characterized in that, The molar ratio of uronic acid structural units in sodium periodate and sodium alginate is 0.3:1-0.6:1, and the molar ratio of ethylene glycol to sodium periodate is 1:

1.

4. The method for preparing a pH-responsive charge-controlled nanofiltration membrane according to claim 1, characterized in that, Step two specifically includes the following steps: dissolving OSA in 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5, adding EDC and NHS, activating for 30 min, then adding DOPA, purging the reaction system with nitrogen gas to remove oxygen for 30 min, and reacting at room temperature in the dark under nitrogen protection for 12-24 h. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water. The water was changed every 4-6 hours, and the dialysis was performed for 48-72 hours. The retentate in the dialysis bag was then removed and freeze-dried to obtain DOPA-OSA.

5. The method for preparing a pH-responsive charge-controlled nanofiltration membrane according to claim 4, characterized in that, The molar ratio of carboxyl groups in EDC to OSA is 1.5:1-2.0:1, the molar ratio of EDC to NHS is 1:0.5-1:0.8, and the mass ratio of DOPA to OSA is 0.2:1-0.5:

1.

6. The method for preparing a pH-responsive charge-controlled nanofiltration membrane according to claim 1, characterized in that, Step three specifically includes the following steps: dispersing chitosan with a degree of deacetylation ≥85% in isopropanol, adding a 30%-50% sodium hydroxide solution, and alkalizing at 25-35℃ for 1-2 h; adding chloroacetic acid and reacting at 60-80℃ for 3-5 h, wherein the molar ratio of chloroacetic acid to the repeating unit 2-amino-2-deoxy-D-glucose unit of chitosan is 3:1-5:1; after the reaction, neutralizing to pH 7.0 with glacial acetic acid, filtering, washing the filter cake with a 70%-80% ethanol aqueous solution until neutral, and drying to obtain carboxylated chitosan.

7. The method for preparing a pH-responsive charge-controlled nanofiltration membrane according to claim 1, characterized in that, Step four specifically includes the following steps: dissolving polyethyleneimine and DOPA-OSA together in deionized water, adjusting the pH to 8.0-8.5 with 0.1 M sodium hydroxide solution to obtain a mixed aqueous solution; The base membrane was immersed in the mixed aqueous solution and reacted at 25-40℃ for 2-4 h. After removal, it was rinsed with boric acid solution with a pH of 7.4, and then immersed in 0.5-2 wt% glutaraldehyde aqueous solution for crosslinking at 25-35℃ for 1-2 h, and rinsed with deionized water. Then it was immersed in carboxylated chitosan aqueous solution and assembled at 25-35℃ for 1-3 h. After assembly, it was rinsed with citrate-sodium citrate buffer solution and then rinsed thoroughly with deionized water to obtain a pH-responsive charge-controlled nanofiltration membrane.

8. The method for preparing a pH-responsive charge-controlled nanofiltration membrane according to claim 7, characterized in that, The concentration of polyethyleneimine in the mixed aqueous solution is 1-3 wt%, and the concentration of DOPA-OSA is 0.5-1.5 wt%; the boric acid solution is prepared by dissolving boric acid in deionized water and adjusting the pH to 7.4 with sodium hydroxide. The carboxylated chitosan aqueous solution is prepared by dissolving carboxylated chitosan in a phosphate buffer solution with a pH of 7.5-8.0, with a carboxylated chitosan concentration of 0.5-2 wt%. The citric acid-sodium citrate buffer solution is obtained by mixing 0.1 M citric acid solution and 0.1 M sodium citrate solution at a volume ratio of 3:1 to 4:

1.

9. A pH-responsive charge-controlled nanofiltration membrane, characterized in that, The nanofiltration membrane is prepared by the method described in any one of claims 1-8.

10. An application of the pH-responsive charge-controlled nanofiltration membrane as described in claim 9, characterized in that, This nanofiltration membrane is used for the separation of polysaccharides from Haematococcus pluvialis and astaxanthin esters.

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