A ternary crosslinked nanofiltration membrane, a preparation method and application thereof

Abstract

The application belongs to the technical field of haematococcus pluvialis separation, and specifically discloses a ternary cross-linked nanofiltration membrane, a preparation method and application. The nanofiltration membrane comprises a polyethersulfone ultrafiltration membrane base film, a polydopamine intermediate layer and a ternary cross-linked functional layer sequentially loaded on the surface of the polyethersulfone ultrafiltration membrane base film. The preparation method comprises the following steps: preparing the polydopamine intermediate layer, anchoring oxidized haematococcus pluvialis polysaccharide, covalently cross-linking polyphenylated chitosan, coordination cross-linking iron ions, and post-treatment to obtain the ternary cross-linked nanofiltration membrane. The ternary cross-linked nanofiltration membrane is used for separating haematococcus pluvialis polysaccharide and astaxanthin ester. The ternary cross-linked functional layer simultaneously has three separation abilities of size screening, charge regulation and coordination selection, realizes selective separation of astaxanthin ester and neutral oil with similar molecular weights, and effectively retains macromolecular polysaccharide.

Description

Technical Field

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

[0002] Haematococcus pluvialis is a microalga rich in various bioactive substances. Its intracellular astaxanthin esters possess strong antioxidant activity and are widely used in functional foods, cosmetics, and pharmaceuticals. Meanwhile, Haematococcus pluvialis polysaccharides, another important active ingredient, exhibit immunomodulatory and antioxidant activities and have a wide molecular weight distribution. Extracting and separating these high-value-added components from Haematococcus pluvialis is a key step in realizing the high-value utilization of Haematococcus pluvialis resources.

[0003] Currently, the mainstream processes for obtaining astaxanthin esters and polysaccharides from *Hylocereus pluvialis* typically include cell disruption, solvent extraction, and centrifugation. The extracted *Hylocereus pluvialis* after cell disruption has a complex composition, containing astaxanthin esters, polysaccharides, proteins, neutral lipids, and chlorophyll, among other substances. Astaxanthin esters have a molecular weight of approximately 800-1000 Da, while neutral lipids have a similar molecular weight, making them difficult to distinguish using conventional separation methods. *Hylocereus pluvialis* polysaccharides, on the other hand, have molecular weights ranging from tens of thousands to millions of Da, and polysaccharides in different molecular weight ranges exhibit different biological activities. Therefore, achieving the selective separation of astaxanthin esters and neutral lipids, as well as the effective separation of polysaccharides from other small-molecule lipids, is a core challenge in the deep processing of *Hylocereus pluvialis*.

[0004] Membrane separation technology has been widely used in the field of natural product separation in recent years due to its advantages such as mild operation, no phase change, and ease of scale-up. Nanofiltration and ultrafiltration membranes can separate substances of different molecular weights through size sieving effects. However, there are many problems when using ordinary commercially available membranes directly for the separation of Haematococcus pluvialis extract.

[0005] On the one hand, the extract of *Hylococcus pluvialis* contains a large amount of protein and polysaccharides. These macromolecules are easily adsorbed onto the membrane surface or clog the membrane pores, causing membrane fouling. Flux drops sharply within a short period of operation, and the proportion of irreversible fouling is high, with a low flux recovery rate after membrane cleaning. On the other hand, ordinary polyamide nanofiltration membranes swell severely in organic solvents such as aqueous ethanol, increasing the inter-chain spacing of the polyamide and causing the molecular weight cutoff to drift, resulting in unstable separation performance. Furthermore, astaxanthin esters and neutral oils have similar molecular weights, and the difference in retention rates between the two by ordinary membranes is usually very small, making it difficult to effectively enrich astaxanthin esters.

[0006] To address the aforementioned issues, existing technologies primarily focus on improvements in two areas: membrane surface modification and functional layer design. Regarding membrane surface modification, researchers employ polydopamine coatings to enhance the hydrophilicity of the membrane surface, utilizing the adhesive properties of polydopamine to provide reaction sites for subsequent functionalization. In terms of functional layer construction, chitosan, due to its excellent biocompatibility and abundant amino groups, is frequently used to prepare polyelectrolyte composite membranes, where surface charge and pore size are controlled through electrostatic interactions. Metal polyphenol network membranes have also attracted attention in recent years, utilizing the coordination of polyphenol groups with metal ions to construct selective separation layers, demonstrating certain advantages in dye separation and heavy metal removal.

[0007] However, existing modification technologies still have significant shortcomings. While polydopamine coatings can improve membrane hydrophilicity, their effect on membrane selectivity is limited when used alone. Chitosan-based polyelectrolyte composite membranes are prone to network structure changes under acidic conditions due to amino protonation, and chitosan itself lacks specific interaction sites with astaxanthin ester molecules, failing to solve the problem of selective separation of astaxanthin esters from neutral oils. Although metal polyphenol network membranes introduce coordination crosslinking, the polyphenols are mostly exogenous substances such as tannins, lacking relevance to the Haematococcus pluvialis extraction process. Furthermore, the functional layers of existing metal polyphenol membranes typically only possess single separation capabilities, making it difficult to simultaneously handle complex separation scenarios involving multiple coexisting components with similar properties in Haematococcus pluvialis extracts. In addition, the pore sizes of the functional layers of existing membranes are mostly fixed values, unable to be dynamically adjusted according to the needs of the separation targets, making it difficult for the same membrane to adapt to the fractional separation of polysaccharides of different molecular weights or process optimization under different pH conditions.

[0008] Therefore, there is a need to design a ternary cross-linked nanofiltration membrane, its preparation method, and its application. Summary of the Invention

[0009] To overcome the shortcomings of the existing technology, a ternary crosslinked nanofiltration membrane, its preparation method, and its application are provided.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] A ternary crosslinked nanofiltration membrane, comprising a polyethersulfone ultrafiltration membrane base membrane and a polydopamine intermediate layer and a ternary crosslinked functional layer sequentially loaded on the surface of the polyethersulfone ultrafiltration membrane base membrane;

[0012] The ternary cross-linked functional layer is constructed by covalent and coordination cross-linking of oxidized red algae polysaccharide, polyphenolized chitosan and iron ions. The aldehyde group of oxidized red algae polysaccharide and the amino group of polyphenolized chitosan are covalently cross-linked after Schiff base reaction and reduction. The catechol group of polyphenolized chitosan forms coordination cross-linking with iron ions.

[0013] The oxidized Haematococcus pluvialis polysaccharide is obtained by selective oxidation of Haematococcus pluvialis polysaccharide with sodium periodate after ultrasonic treatment.

[0014] The polyphenolized chitosan was obtained by reducing and purifying chitosan and 3,4-dihydroxybenzaldehyde via a Schiff base reaction.

[0015] The polyethersulfone ultrafiltration membrane base has a molecular weight cutoff of 50 kDa; the polydopamine interlayer has a thickness of 10-20 nm and is loaded on the surface of the polyethersulfone ultrafiltration membrane base; the ternary crosslinked functional layer has a thickness of 20-40 nm and is loaded on the surface of the polydopamine interlayer.

[0016] The preparation method of the oxidized Rhodophyta polysaccharide includes the following steps: Rhodophyta polysaccharide is prepared into an aqueous solution and subjected to ultrasonic treatment. Sodium periodate is added, and the reaction is carried out at 25°C in the dark for 2-4 hours. After the reaction is completed, ethylene glycol is added, with a molar ratio of ethylene glycol to sodium periodate of 1.5-2:1. The reaction is terminated after stirring at room temperature for 30 minutes. The resulting reaction solution is placed into a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed with deionized water for 48 hours, with the water changed every 8 hours, until the conductivity of the dialysate is lower than 10 μS / cm. The oxidized Rhodophyta polysaccharide is obtained by freeze drying.

[0017] The specific steps and parameters of the ultrasonic treatment are as follows: prepare a 10-20 g / L aqueous solution of Haematococcus pluvialis polysaccharide, place it in an ice-water bath and apply ultrasound, with an ultrasonic power of 300-500 W, a frequency of 20-25 kHz, an ultrasonic time of 3-5 s, an interval time of 3-5 s, and a total ultrasonic duration of 30-90 min.

[0018] The molar ratio of sodium periodate to the monosaccharide units with vicinal diol structures in the polysaccharide of *Hylocereus pluvialis* is 0.3-0.6.

[0019] The preparation method of the polyphenolized chitosan includes the following steps: dissolving chitosan in a 0.5%-1% acetic acid aqueous solution to prepare a 5-10 g / L solution, dissolving 3,4-dihydroxybenzaldehyde in ethanol to prepare a 10-20 g / L solution, mixing the two solutions so that the molar ratio of chitosan amino group to 3,4-dihydroxybenzaldehyde in the mixture is 1:0.8-1:1.2, and reacting at 40-60℃ for 2-4 hours to form a Schiff base;

[0020] Sodium borohydride was then added, and the reaction was carried out at 0-4℃ for 2-4 hours; the pH was then adjusted to 5-6 with 0.1 mol / L HCl, and the mixture was stirred at room temperature for 30 minutes.

[0021] The resulting reaction solution was placed into a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with deionized water for 48 hours, with the water changed every 8 hours, until the conductivity of the dialysate was below 10 μS / cm. The solution was then freeze-dried to obtain polyphenolized chitosan.

[0022] The molar ratio of sodium borohydride to 3,4-dihydroxybenzaldehyde is 2:1 to 4:1.

[0023] A method for preparing a ternary crosslinked nanofiltration membrane, the method comprising the following steps:

[0024] Step 1: Preparation of the polydopamine interlayer

[0025] The polyethersulfone ultrafiltration membrane base was immersed in Tris-HCl buffer at pH 8.5, dopamine hydrochloride was added, and self-polymerization was carried out at 25°C for 15-25 min. After removal, it was rinsed with deionized water 3 times, 5 min each time.

[0026] Step 2: Anchoring of Oxidized Haematococcus pluvialis polysaccharides

[0027] Immerse the membrane obtained in step one in an aqueous solution of oxidized red puffball polysaccharide at pH 6-7 and react at room temperature for 30-60 minutes. Remove the membrane and rinse it with deionized water three times for 5 minutes each time.

[0028] Step 3: Covalent cross-linking of polyphenolized chitosan

[0029] The membrane obtained in step two is immersed in a polyphenolized chitosan acetate solution at pH 4.5-5.5 and reacted at room temperature for 30-60 min. The membrane is then removed and rinsed three times with deionized water for 5 min each time. Subsequently, the membrane is immersed in freshly prepared sodium borohydride aqueous solution and reacted in an ice-water bath for 30-60 min. After the reduction is completed, the membrane is removed and rinsed three times with deionized water for 10 min each time.

[0030] Step 4: Coordination and crosslinking of iron ions

[0031] Immerse the membrane obtained in step 3 in ferric chloride solution, adjust the pH to 3.5-4.5 with HCl, and react at room temperature for 10-20 minutes;

[0032] Step 5: Post-processing

[0033] Take out the membrane obtained in step four, rinse it with deionized water until the conductivity of the rinsing solution is less than 5 μS / cm, and air dry it at room temperature to obtain the ternary cross-linked nanofiltration membrane.

[0034] The concentration of dopamine hydrochloride is 2 g / L, the concentration of the oxidized Rhodophyta polysaccharide aqueous solution is 1.5-2.5 g / L, the concentration of the polyphenolized chitosan acetate solution is 1-2 g / L, the concentration of the sodium borohydride solution is 0.5-2 g / L, and the concentration of the ferric chloride solution is 0.3-1 g / L.

[0035] This ternary cross-linked nanofiltration membrane is used for the separation of polysaccharides from Haematococcus pluvialis and astaxanthin esters.

[0036] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0037] 1. This application constructs a polydopamine interlayer on the surface of a polyethersulfone ultrafiltration membrane substrate. The polydopamine interlayer is firmly attached to the substrate surface through the adhesion of catechol groups, transforming the hydrophobic surface into a hydrophilic surface. This reduces the irreversible adsorption of contaminants such as proteins and polysaccharides on the membrane surface, mitigating flux decline caused by membrane fouling. Simultaneously, the amino groups and catechols on the polydopamine interlayer surface provide high-density reaction sites for anchoring oxidized Haematococcus pluvialis polysaccharides, enhancing the interlayer bonding between subsequent functional layers and the substrate membrane from simple physical adsorption to a combination of electrostatic attraction and hydrogen bonding.

[0038] 2. This application employs oxidized Rhodospirulina polysaccharide and polyphenolized chitosan to construct a covalently cross-linked network. The aldehyde groups of oxidized Rhodospirulina polysaccharide and the amino groups of polyphenolized chitosan react with a Schiff base and are then reduced to form stable secondary amine bonds. This covalent cross-linking ensures the functional layer maintains structural integrity under acidic conditions, preventing network collapse caused by imine bond hydrolysis. The amino groups of polyphenolized chitosan impart pH responsiveness to the functional layer. When the pH of the feed solution changes, the polyelectrolyte segments contract or extend, allowing the effective pore size of the functional layer to be dynamically adjusted within a narrow range, adapting to the separation requirements of polysaccharides with different molecular weights. The rigid chain structure of oxidized Rhodospirulina polysaccharide helps form a sieving structure with a narrow pore size distribution in the functional layer, improving the accuracy of size sieving.

[0039] 3. This application introduces iron ions to form coordination crosslinks with the catechol groups of polyphenolized chitosan, constructing a metal polyphenol network. Iron ions, acting as coordination centers, can form weak coordination bonds with the ketone and hydroxyl groups at the ends of astaxanthin ester molecules, increasing the mass transfer resistance of astaxanthin esters on the membrane surface and improving their retention rate. Neutral oil molecules do not contain ketone and hydroxyl groups and cannot form coordination interactions with iron ions; their mass transfer is solely governed by size sieving. Therefore, the ternary crosslinked functional layer simultaneously possesses three separation capabilities: size sieving, charge regulation, and coordination selection, achieving selective separation of astaxanthin esters and neutral oils with similar molecular weights, as well as effective retention of large-molecule polysaccharides.

[0040] 4. In the preparation process of this application, the membrane is removed from the polyphenolized chitosan acetate solution and rinsed before being immersed in freshly prepared sodium borohydride aqueous solution for reduction. This stepwise operation avoids homogeneous cross-linking and gelation of free polyphenolized chitosan in the solution under the action of the reducing agent, ensuring that the reduction reaction only occurs at the Schiff base sites already formed on the membrane surface. Attached Figure Description

[0041] Figure 1 This is a process flow diagram of a method for preparing a ternary cross-linked nanofiltration membrane. Detailed Implementation

[0042] 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.

[0043] In the specific embodiments of this application, the sources of various main raw materials are briefly described as follows:

[0044] Polyethersulfone ultrafiltration membrane base membrane: purchased from Tianjin Membrane Technology Group Co., Ltd., with a molecular weight cutoff of 50 kDa.

[0045] Dopamine hydrochloride: purchased from Hubei Weideli Chemical Technology Co., Ltd., CAS No. 62-31-7.

[0046] Tris(hydroxymethyl)aminomethane: purchased from Meite (Hubei) New Materials Co., Ltd., CAS No. 77-86-1.

[0047] Haematococcus pluvialis polysaccharide: purchased from Yunnan Aierfa Biotechnology Co., Ltd.

[0048] Sodium periodate: purchased from Jiangsu Zhenri Chemical Co., Ltd., CAS No. 7790-28-5

[0049] Ethylene glycol: purchased from Satellite Chemicals Co., Ltd., CAS No. 107-21-1

[0050] Chitosan: Purchased from Wuhan Kemike Biomedical Technology Co., Ltd., degree of deacetylation 85%.

[0051] 3,4-Dihydroxybenzaldehyde: Purchased from Guangzhou Yuanda New Materials Co., Ltd., CAS No. 139-85-5

[0052] Sodium borohydride: purchased from Ningxia Baister Pharmaceutical Chemical Co., Ltd., CAS No. 16940-66-2

[0053] Ferric chloride: purchased from Hubei Zhongshui Chemical Co., Ltd., CAS No. 7705-08-0

[0054] Hydrochloric acid: purchased from Huanghua Xinsheng Chemical Co., Ltd., CAS No. 7647-01-0

[0055] Acetic acid: purchased from Jinan Chuangshi Chemical Co., Ltd., CAS No. 64-19-7

[0056] The technical solution of this application is as follows:

[0057] A ternary crosslinked nanofiltration membrane, comprising a polyethersulfone ultrafiltration membrane base membrane and a polydopamine intermediate layer and a ternary crosslinked functional layer sequentially loaded on the surface of the polyethersulfone ultrafiltration membrane base membrane;

[0058] The ternary cross-linked functional layer is constructed by covalent and coordination cross-linking of oxidized red algae polysaccharide, polyphenolized chitosan and iron ions. The aldehyde group of oxidized red algae polysaccharide and the amino group of polyphenolized chitosan are covalently cross-linked after Schiff base reaction and reduction. The catechol group of polyphenolized chitosan forms coordination cross-linking with iron ions.

[0059] The oxidized Haematococcus pluvialis polysaccharide is obtained by selective oxidation of Haematococcus pluvialis polysaccharide with sodium periodate after ultrasonic treatment.

[0060] The polyphenolized chitosan was obtained by reducing and purifying chitosan and 3,4-dihydroxybenzaldehyde via a Schiff base reaction.

[0061] Conventional polyethersulfone (PES) ultrafiltration membranes have a highly hydrophobic surface, making them susceptible to adsorption by proteins and polysaccharides through hydrophobic interactions, which can clog pores and cause a significant drop in flux within a short period, along with a high rate of irreversible fouling. This application introduces a polydopamine interlayer onto the surface of the PES ultrafiltration membrane base. Polydopamine is formed by the oxidative polymerization of dopamine hydrochloride in a weakly alkaline Tris-HCl buffer, and its molecular chains are rich in catechol and amino groups. The catechol groups can firmly adhere to the PES ultrafiltration membrane base surface through various non-covalent interactions such as hydrogen bonding, coordination, and π-π stacking, forming a dense coating with a thickness of 10-20 nm. This coating transforms the membrane surface from hydrophobic to hydrophilic, allowing water molecules to form a hydration layer on the membrane surface, creating steric hindrance for contaminants such as proteins and reducing irreversible adsorption. Simultaneously, the amino and catechol groups on the surface of the polydopamine interlayer provide high-density reaction sites for the subsequent anchoring of oxidized Haematococcus pluvialis polysaccharides, enhancing the interlayer binding force from simple physical adsorption to a combination of electrostatic attraction and hydrogen bonding.

[0062] The polyethersulfone ultrafiltration membrane base has a molecular weight cutoff of 50 kDa; the polydopamine interlayer has a thickness of 10-20 nm and is loaded on the surface of the polyethersulfone ultrafiltration membrane base; the ternary crosslinked functional layer has a thickness of 20-40 nm and is loaded on the surface of the polydopamine interlayer.

[0063] The preparation method of the oxidized Rhodophyta polysaccharide includes the following steps: Rhodophyta polysaccharide is prepared into an aqueous solution and subjected to ultrasonic treatment. Sodium periodate is added, and the reaction is carried out at 25°C in the dark for 2-4 hours. After the reaction is completed, ethylene glycol is added, with a molar ratio of ethylene glycol to sodium periodate of 1.5-2:1. The reaction is terminated after stirring at room temperature for 30 minutes. The resulting reaction solution is placed into a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed with deionized water for 48 hours, with the water changed every 8 hours, until the conductivity of the dialysate is lower than 10 μS / cm. The oxidized Rhodophyta polysaccharide is obtained by freeze drying.

[0064] The specific steps and parameters of the ultrasonic treatment are as follows: prepare a 10-20 g / L aqueous solution of Haematococcus pluvialis polysaccharide, place it in an ice-water bath and apply ultrasound, with an ultrasonic power of 300-500 W, a frequency of 20-25 kHz, an ultrasonic time of 3-5 s, an interval time of 3-5 s, and a total ultrasonic duration of 30-90 min.

[0065] The molar ratio of sodium periodate to the monosaccharide units with vicinal diol structures in the polysaccharide of *Hylocereus pluvialis* is 0.3-0.6.

[0066] While the polydopamine interlayer improves hydrophilicity and anchoring, it lacks charge regulation capabilities and a rigid framework. This application anchors oxidized *Rhodotorula purpureus* polysaccharide onto the surface of the polydopamine interlayer. After ultrasonic degradation, the *Rhodotorula purpureus* polysaccharide exposes numerous vicinal diol structures on its molecular chains. Selective oxidation with sodium periodate breaks the C / C bonds of these vicinal diols, generating aldehyde groups, while the carboxyl and sulfate groups are retained. The oxidized *Rhodotorula purpureus* polysaccharide is adsorbed and anchored onto the polydopamine interlayer surface through electrostatic attraction between the carboxyl and sulfate groups and the amino groups on the surface of the polydopamine interlayer, as well as hydrogen bonding between the hydroxyl groups and catechol. As a rigid-chain polysaccharide, its sugar ring framework is not easily bent, forming a relatively regular layered structure when arranged on the membrane surface. This limits the pore size distribution range of the functional layer, improving the selectivity of size sieving. Furthermore, the carboxyl and sulfate groups impart a negative charge to the functional layer, providing a charge basis for subsequent formation of a polyelectrolyte complex with polyphenolized chitosan.

[0067] The preparation method of the polyphenolized chitosan includes the following steps: dissolving chitosan in a 0.5%-1% acetic acid aqueous solution to prepare a 5-10 g / L solution, dissolving 3,4-dihydroxybenzaldehyde in ethanol to prepare a 10-20 g / L solution, mixing the two solutions so that the molar ratio of chitosan amino group to 3,4-dihydroxybenzaldehyde in the mixture is 1:0.8-1:1.2, and reacting at 40-60℃ for 2-4 hours to form a Schiff base;

[0068] Sodium borohydride was then added, and the reaction was carried out at 0-4℃ for 2-4 hours; the pH was then adjusted to 5-6 with 0.1 mol / L HCl, and the mixture was stirred at room temperature for 30 minutes.

[0069] The resulting reaction solution was placed into a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with deionized water for 48 hours, with the water changed every 8 hours, until the conductivity of the dialysate was below 10 μS / cm. The solution was then freeze-dried to obtain polyphenolized chitosan.

[0070] Oxidized Rhodochophora polysaccharide layers anchored solely by electrostatics and hydrogen bonds are not stable enough in acidic environments and cannot provide pH responsiveness.

[0071] This application introduces polyphenolized chitosan to construct a covalently cross-linked network. The polyphenolized chitosan is obtained by reacting chitosan with 3,4-dihydroxybenzaldehyde via a Schiff base reaction and subsequent reduction, and its molecular chain contains both amino and catechol groups. The aldehyde group of oxidized Haematococcus pluvialis polysaccharide undergoes a condensation reaction with the amino group of the polyphenolized chitosan to form an imine bond, which is then reduced to a secondary amine bond by sodium borohydride. The secondary amine bond remains chemically stable under acidic conditions of pH 3.5-4.5 and will not hydrolyze due to proton catalysis, ensuring the structural integrity of the functional layer during subsequent ferric chloride solution treatment.

[0072] The amino groups of polyphenolized chitosan impart pH responsiveness to the functional layer. When the pH of the feed solution drops to 3-4, the amino groups accept protons to form ammonium ions, increasing the positive charge density. This generates a strong electrostatic attraction with the negative charge of the oxidized Rhodophyta citrate polysaccharide, causing the polyelectrolyte segments to shrink, reducing the layer thickness, and densifying the effective pore size. When the pH of the feed solution rises to 7-8, the carboxyl groups of the oxidized Rhodophyta citrate polysaccharide deprotonate, increasing the negative charge density. The electrostatic repulsion between like charges causes the polyelectrolyte segments to extend, increasing the layer thickness, and swelling the effective pore size. This conformational change allows the pore size of the functional layer to be dynamically adjusted within a narrow range, adapting to the fractional separation requirements of polysaccharides with different molecular weights.

[0073] The molar ratio of sodium borohydride to 3,4-dihydroxybenzaldehyde is 2:1 to 4:1.

[0074] A method for preparing a ternary crosslinked nanofiltration membrane, such as... Figure 1 As shown, the method includes the following steps:

[0075] Step 1: Preparation of the polydopamine interlayer

[0076] The polyethersulfone ultrafiltration membrane base was immersed in Tris-HCl buffer at pH 8.5, dopamine hydrochloride was added, and it was self-polymerized at 25°C for 15-25 min. After removal, it was rinsed with deionized water 3 times, 5 min each time.

[0077] Step 2: Anchoring of Oxidized Haematococcus pluvialis polysaccharides

[0078] Immerse the membrane obtained in step one in an aqueous solution of oxidized red puffball polysaccharide at pH 6-7 and react at room temperature for 30-60 minutes. Remove the membrane and rinse it three times with deionized water for 5 minutes each time.

[0079] Step 3: Covalent cross-linking of polyphenolized chitosan

[0080] The membrane obtained in step two is immersed in a polyphenolized chitosan acetate solution at pH 4.5-5.5 and reacted at room temperature for 30-60 min. The membrane is then removed and rinsed three times with deionized water for 5 min each time. Subsequently, the membrane is immersed in freshly prepared sodium borohydride aqueous solution and reacted in an ice-water bath for 30-60 min. After the reduction is completed, the membrane is removed and rinsed three times with deionized water for 10 min each time.

[0081] Step 4: Coordination and crosslinking of iron ions

[0082] Immerse the membrane obtained in step 3 in ferric chloride solution, adjust the pH to 3.5-4.5 with HCl, and react at room temperature for 10-20 minutes;

[0083] Step 5: Post-processing

[0084] Take out the membrane obtained in step four, rinse it with deionized water until the conductivity of the rinsing solution is less than 5 μS / cm, and air dry it at room temperature to obtain the ternary cross-linked nanofiltration membrane.

[0085] The concentration of dopamine hydrochloride is 2 g / L, the concentration of the oxidized Rhodophyta polysaccharide aqueous solution is 1.5-2.5 g / L, the concentration of the polyphenolized chitosan acetate solution is 1-2 g / L, the concentration of the sodium borohydride solution is 0.5-2 g / L, and the concentration of the ferric chloride solution is 0.3-1 g / L.

[0086] This ternary cross-linked nanofiltration membrane is used for the separation of polysaccharides from Haematococcus pluvialis and astaxanthin esters.

[0087] While the aforementioned covalent crosslinking networks are stable and pH-responsive, they lack selective recognition sites for substances containing ketone and hydroxyl groups. This application introduces iron ions to construct a coordination crosslinking network. The catechol groups on polyphenolized chitosan undergo coordination reactions with iron ions, forming a metal polyphenol network structure. The two hydroxyl oxygens of the catechol form coordination bonds with the empty d orbitals of the iron ion, constructing a three-dimensional crosslinked framework. The iron ion, acting as a coordination center, can accept the lone pair electrons from the terminal ketone and hydroxyl oxygens of the astaxanthin ester molecule through its unfilled d orbitals, forming weak coordination bonds. According to the hard and soft acid-base theory, the iron ion is a boundary acid, while the ketone and hydroxyl oxygens are hard bases, exhibiting a certain coordination tendency. This selective interaction forms affinity sites on the membrane surface. When the astaxanthin ester approaches the membrane surface, it forms transient coordination with the iron ion, increasing its residence time on the membrane surface, reducing the diffusion driving force, and increasing the apparent rejection rate. Neutral fat molecules are mainly triglycerides, containing only ester bonds. The electron cloud density of oxygen atoms is reduced due to their connection with two alkyl groups, and their steric hindrance is relatively large, preventing them from forming effective coordination with iron ions. Their mass transfer behavior is dominated solely by size sieving. As a result, the difference in retention rates between astaxanthin esters and neutral fats widens, achieving selective enrichment.

[0088] Schiff base reduction is often carried out in the same solution as the crosslinking step. Free polyphenolized chitosan in the solution undergoes homogeneous reduction and crosslinking under the action of sodium borohydride, easily forming gels or precipitates, interfering with the construction of the functional layer on the membrane surface. In this application, the membrane is removed from the polyphenolized chitosan acetate solution, rinsed with deionized water to remove unadsorbed free polyphenolized chitosan, and then immersed in freshly prepared sodium borohydride aqueous solution. At this point, only the adsorbed oxidized Rhodophyta globulin-polyphenolized chitosan composite layer remains on the membrane surface. Sodium borohydride only reacts with the Schiff base on the membrane surface; there is no free polyphenolized chitosan in the bulk solution, thus avoiding homogeneous gelation. The reduction reaction is carried out in an ice-water bath. The low temperature reduces the hydrolysis rate of sodium borohydride in the aqueous system, improving the reduction efficiency. After reduction, the membrane is thoroughly rinsed to remove residual sodium borohydride and boric acid byproducts before entering the ferric chloride solution treatment step, ensuring that the coordination crosslinking is not interfered with by the reducing agent.

[0089] Furthermore, this application uses oxidatively modified *Hypoxococcus pluvialis* polysaccharide obtained during the extraction process as a raw material for the membrane functional layer, which is then compounded with polyphenolized chitosan and iron ions to construct a self-derived membrane modification system. The rigid chain structure of *Hypoxococcus pluvialis* polysaccharide and the flexible chain of chitosan interpenetrate to form a semi-interpenetrating network. The carboxyl and sulfate groups of oxidized *Hypoxococcus pluvialis* polysaccharide and the amino groups of polyphenolized chitosan form a polyelectrolyte complex, while iron ions form a coordination crosslink with catechol. The three components are interconnected at the molecular level. The negative charge density of oxidized *Hypoxococcus pluvialis* polysaccharide is higher than that of ordinary sodium alginate, resulting in a denser polyelectrolyte complex layer formed with chitosan. Its rigid framework limits the range of pore size changes during network swelling, making pH-responsive pore size adjustment more precise. This compounding of raw materials is not a simple superposition, but rather, through charge complementarity, structural complementarity, and chemical bonding, the ternary crosslinked functional layer simultaneously possesses three separation capabilities: size sieving, charge regulation, and coordination selection, achieving one-step selective separation of polysaccharides, astaxanthin esters, and neutral oils in *Hypoxococcus pluvialis* extract.

[0090] 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.

[0091] Example 1

[0092] The polyethersulfone ultrafiltration membrane base membrane has a molecular weight cutoff of 50 kDa. The polyethersulfone ultrafiltration membrane base membrane was immersed in Tris-HCl buffer at pH 8.5, and dopamine hydrochloride at a concentration of 2 g / L was added. It was self-polymerized at 25°C for 25 min, resulting in a polydopamine interlayer thickness of 20 nm. After removal, it was rinsed three times with deionized water for 5 min each time.

[0093] A 15 g / L aqueous solution of *Hydrocotyle erythrorhizon* polysaccharide was prepared and subjected to sonication in an ice-water bath at a power of 300 W, a frequency of 25 kHz, a sonication time of 4 s, an interval of 3 s, and a total sonication time of 90 min. Sodium periodate was added, with a molar ratio of sodium periodate to monosaccharide units with vicinal diol structures in *Hydrocotyle erythrorhizon* polysaccharide of 0.3. The reaction was carried out at 25 °C in the dark for 2 h. After the reaction was completed, ethylene glycol was added, with a molar ratio of ethylene glycol to sodium periodate of 2:1. The reaction was terminated by stirring at room temperature for 30 min. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed with deionized water for 48 h, changing the water every 8 h, until the conductivity of the dialysate was below 10 μS / cm. The lyophilized solution yielded oxidized *Hydrocotyle erythrorhizon* polysaccharide.

[0094] Chitosan was dissolved in a 0.5% acetic acid aqueous solution to prepare a 7.5 g / L solution, and 3,4-dihydroxybenzaldehyde was dissolved in ethanol to prepare a 10 g / L solution. The two solutions were mixed to achieve a molar ratio of chitosan amino groups to 3,4-dihydroxybenzaldehyde of 1:1.2. The mixture was reacted at 50°C for 2 hours to form a Schiff base. Sodium borohydride was then added, with a molar ratio of sodium borohydride to 3,4-dihydroxybenzaldehyde of 4:1, and the mixture was reacted at 0°C for 2 hours. The pH was then adjusted to 5 with 0.1 mol / L HCl, and the mixture was stirred at room temperature for 30 minutes. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with deionized water for 48 hours, changing the water every 8 hours, until the conductivity of the dialysate was below 10 μS / cm. The resulting solution was then lyophilized to obtain polyphenolized chitosan.

[0095] The polydopamine intermediate layer was immersed in an aqueous solution of oxidized Rhodopsin (2 g / L, pH 6) at room temperature for 60 min. The membrane was then removed and rinsed three times with deionized water for 5 min each time. The membrane was then immersed in a polyphenolized chitosan acetate solution (1 g / L, pH 4.5) at room temperature for 30 min. The membrane was then removed and rinsed three times with deionized water for 5 min each time. Subsequently, the membrane was immersed in a freshly prepared sodium borohydride aqueous solution (2 g / L) and reacted in an ice-water bath for 60 min. After reduction, the membrane was removed and rinsed three times with deionized water for 10 min each time. The membrane was then immersed in a ferric chloride solution (0.65 g / L), and the pH was adjusted to 3.5 with HCl. The reaction was carried out at room temperature for 10 min. The membrane was then removed and rinsed with deionized water until the conductivity of the rinsing solution was below 5 μS / cm. After air-drying at room temperature, the ternary cross-linked nanofiltration membrane with a ternary cross-linked functional layer thickness of 30 nm was obtained.

[0096] Example 2

[0097] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0098] The polyethersulfone ultrafiltration membrane base membrane has a molecular weight cutoff of 50 kDa. The polyethersulfone ultrafiltration membrane base membrane was immersed in Tris-HCl buffer at pH 8.5, and dopamine hydrochloride at a concentration of 2 g / L was added. It was self-polymerized at 25°C for 15 min, resulting in a polydopamine interlayer thickness of 10 nm. After removal, it was rinsed three times with deionized water for 5 min each time.

[0099] A 20 g / L aqueous solution of *Hydrocotyle erythrorhizon* polysaccharide was prepared and ultrasonicated in an ice-water bath at a power of 500 W, a frequency of 20 kHz, a duration of 5 s, an interval of 4 s, and a total ultrasonication time of 30 min. Sodium periodate was added, with a molar ratio of sodium periodate to monosaccharide units with vicinal diol structures in *Hydrocotyle erythrorhizon* polysaccharide of 0.45. The reaction was carried out at 25 °C in the dark for 2 h. After the reaction was completed, ethylene glycol was added, with a molar ratio of ethylene glycol to sodium periodate of 1.5:1. The reaction was terminated by stirring at room temperature for 30 min. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed with deionized water for 48 h, changing the water every 8 h, until the conductivity of the dialysate was below 10 μS / cm. The lyophilized solution yielded oxidized *Hydrocotyle erythrorhizon* polysaccharide.

[0100] Chitosan was dissolved in a 0.5% acetic acid aqueous solution to prepare a 10 g / L solution, and 3,4-dihydroxybenzaldehyde was dissolved in ethanol to prepare a 15 g / L solution. These two solutions were mixed to achieve a molar ratio of chitosan amino groups to 3,4-dihydroxybenzaldehyde of 1:0.8. The mixture was reacted at 60°C for 2 h to form a Schiff base. Sodium borohydride was then added at a molar ratio of 2:1 to 3,4-dihydroxybenzaldehyde, and the reaction was carried out at 2°C for 2 h. The pH was then adjusted to 5.5 with 0.1 mol / L HCl, and the mixture was stirred at room temperature for 30 min. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with deionized water for 48 h, changing the water every 8 h, until the conductivity of the dialysate was below 10 μS / cm. The resulting solution was then lyophilized to obtain polyphenolized chitosan.

[0101] The polydopamine intermediate layer was immersed in an aqueous solution of oxidized Rhodopsin monosodium glutamate (2.5 g / L, pH 6.5) and reacted at room temperature for 30 min. The membrane was then removed and rinsed three times with deionized water for 5 min each time. The membrane was then immersed in a polyphenolized chitosan acetate solution (1.5 g / L, pH 5) and reacted at room temperature for 60 min. The membrane was then removed and rinsed three times with deionized water for 5 min each time. Subsequently, the membrane was immersed in a freshly prepared sodium borohydride aqueous solution (0.5 g / L) and reacted in an ice-water bath for 30 min. After reduction, the membrane was removed and rinsed three times with deionized water for 10 min each time. The membrane was then immersed in a ferric chloride solution (1 g / L), and the pH was adjusted to 4 with HCl. The reaction was carried out at room temperature for 20 min. The membrane was then removed and rinsed with deionized water until the conductivity of the rinsing solution was below 5 μS / cm. After air-drying at room temperature, the ternary cross-linked nanofiltration membrane was obtained, with a ternary cross-linked functional layer thickness of 40 nm.

[0102] Example 3

[0103] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0104] The polyethersulfone ultrafiltration membrane base membrane has a molecular weight cutoff of 50 kDa. The polyethersulfone ultrafiltration membrane base membrane was immersed in Tris-HCl buffer at pH 8.5, and dopamine hydrochloride at a concentration of 2 g / L was added. It was self-polymerized at 25°C for 20 min, resulting in a polydopamine interlayer thickness of 15 nm. After removal, it was rinsed three times with deionized water for 5 min each time.

[0105] A 10 g / L aqueous solution of *Hydrocotyle erythrorhizon* polysaccharide was prepared and sonicated in an ice-water bath at a power of 400 W, a frequency of 22.5 kHz, a sonication time of 3 s, an interval of 5 s, and a total sonication time of 60 min. Sodium periodate was added, with a molar ratio of sodium periodate to monosaccharide units with vicinal diol structures in *Hydrocotyle erythrorhizon* polysaccharide of 0.6. The reaction was carried out at 25 °C in the dark for 4 h. After the reaction was completed, ethylene glycol was added, with a molar ratio of ethylene glycol to sodium periodate of 1.75:1. The reaction was terminated by stirring at room temperature for 30 min. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed with deionized water for 48 h, changing the water every 8 h, until the conductivity of the dialysate was below 10 μS / cm. The lyophilized solution yielded oxidized *Hydrocotyle erythrorhizon* polysaccharide.

[0106] Chitosan was dissolved in a 0.5% acetic acid aqueous solution to prepare a 5 g / L solution, and 3,4-dihydroxybenzaldehyde was dissolved in ethanol to prepare a 20 g / L solution. The two solutions were mixed to achieve a 1:1 molar ratio of chitosan amino groups to 3,4-dihydroxybenzaldehyde. The mixture was reacted at 40°C for 4 hours to form a Schiff base. Sodium borohydride was then added, with a 3:1 molar ratio of sodium borohydride to 3,4-dihydroxybenzaldehyde, and the mixture was reacted at 4°C for 4 hours. The pH was then adjusted to 6 with 0.1 mol / L HCl, and the mixture was stirred at room temperature for 30 minutes. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with deionized water for 48 hours, changing the water every 8 hours, until the conductivity of the dialysate was below 10 μS / cm. The resulting solution was then lyophilized to obtain polyphenolized chitosan.

[0107] The polydopamine intermediate layer was immersed in an aqueous solution of oxidized Rhodopsin monosodium glutamate (1.5 g / L, pH 7) and reacted at room temperature for 45 min. The membrane was then removed and rinsed three times with deionized water for 5 min each time. The membrane was then immersed in a polyphenolized chitosan acetate solution (2 g / L, pH 5.5) and reacted at room temperature for 45 min. The membrane was then removed and rinsed three times with deionized water for 5 min each time. Subsequently, the membrane was immersed in a freshly prepared sodium borohydride aqueous solution (1.25 g / L) and reacted in an ice-water bath for 45 min. After reduction, the membrane was removed and rinsed three times with deionized water for 10 min each time. The membrane was then immersed in a ferric chloride solution (0.3 g / L), and the pH was adjusted to 4.5 with HCl. The reaction was carried out at room temperature for 15 min. The membrane was then removed and rinsed with deionized water until the conductivity of the rinsing solution was below 5 μS / cm. After air-drying at room temperature, the ternary cross-linked nanofiltration membrane with a ternary cross-linked functional layer thickness of 20 nm was obtained.

[0108] Example 4

[0109] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0110] The polyethersulfone ultrafiltration membrane base membrane has a molecular weight cutoff of 50 kDa. The polyethersulfone ultrafiltration membrane base membrane was immersed in Tris-HCl buffer at pH 8.5, and dopamine hydrochloride at a concentration of 2 g / L was added. It was self-polymerized at 25°C for 20 min, resulting in a polydopamine interlayer thickness of 15 nm. After removal, it was rinsed three times with deionized water for 5 min each time.

[0111] A 15 g / L aqueous solution of *Hydrocotyle erythrorhizon* polysaccharide was prepared and ultrasonicated in an ice-water bath at a power of 400 W, a frequency of 22.5 kHz, a duration of 4 s, an interval of 4 s, and a total ultrasonication time of 60 min. Sodium periodate was added, with a molar ratio of sodium periodate to monosaccharide units with vicinal diol structures in *Hydrocotyle erythrorhizon* polysaccharide of 0.45. The reaction was carried out at 25 °C in the dark for 3 h. After the reaction was completed, ethylene glycol was added, with a molar ratio of ethylene glycol to sodium periodate of 1.75:1. The reaction was terminated by stirring at room temperature for 30 min. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed with deionized water for 48 h, changing the water every 8 h, until the conductivity of the dialysate was below 10 μS / cm. The lyophilized solution yielded oxidized *Hydrocotyle erythrorhizon* polysaccharide.

[0112] Chitosan was dissolved in a 0.5% acetic acid aqueous solution to prepare a 7.5 g / L solution, and 3,4-dihydroxybenzaldehyde was dissolved in ethanol to prepare a 15 g / L solution. The two solutions were mixed to achieve a 1:1 molar ratio of chitosan amino groups to 3,4-dihydroxybenzaldehyde, and reacted at 50°C for 3 h to form a Schiff base. Sodium borohydride was then added, with a 3:1 molar ratio of sodium borohydride to 3,4-dihydroxybenzaldehyde, and the reaction was carried out at 2°C for 3 h. The pH was then adjusted to 5.5 with 0.1 mol / L HCl, and the mixture was stirred at room temperature for 30 min. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with deionized water for 48 h, changing the water every 8 h, until the conductivity of the dialysate was below 10 μS / cm. The resulting solution was then lyophilized to obtain polyphenolized chitosan.

[0113] The polydopamine intermediate layer was immersed in an aqueous solution of oxidized Rhodopsin monosodium glutamate (2 g / L, pH 6.5) and reacted at room temperature for 45 min. The membrane was then removed and rinsed three times with deionized water for 5 min each time. The membrane was then immersed in a polyphenolized chitosan acetate solution (1.5 g / L, pH 5) and reacted at room temperature for 45 min. The membrane was then removed and rinsed three times with deionized water for 5 min each time. Subsequently, the membrane was immersed in a freshly prepared sodium borohydride aqueous solution (1.25 g / L) and reacted in an ice-water bath for 45 min. After reduction, the membrane was removed and rinsed three times with deionized water for 10 min each time. The membrane was then immersed in a ferric chloride solution (0.65 g / L), and the pH was adjusted to 4 with HCl. The reaction was carried out at room temperature for 15 min. The membrane was then removed and rinsed with deionized water until the conductivity of the rinsing solution was below 5 μS / cm. After air-drying at room temperature, the ternary cross-linked nanofiltration membrane was obtained, with a ternary cross-linked functional layer thickness of 30 nm.

[0114] Comparative Example 1

[0115] Compared with Example 1, the difference in this comparative example is that the preparation step of the polydopamine intermediate layer is omitted, and the polyethersulfone ultrafiltration membrane base membrane is directly immersed in the aqueous solution of oxidized red algae polysaccharide for anchoring. The remaining steps and parameters are the same as in Example 1.

[0116] Comparative Example 2

[0117] Compared with Example 1, the difference in this comparative example is that sodium alginate is used instead of oxidized red puffball polysaccharide, that is, sodium alginate aqueous solution is directly used instead of oxidized red puffball polysaccharide aqueous solution for anchoring. The remaining steps and parameters are the same as in Example 1.

[0118] Comparative Example 3

[0119] Compared with Example 1, the difference in this comparative example is that unmodified chitosan is used instead of polyphenolized chitosan. That is, chitosan is directly dissolved in acetic acid and then used to replace the polyphenolized chitosan acetic acid solution for covalent cross-linking. The remaining steps and parameters are the same as in Example 1.

[0120] Comparative Example 4

[0121] Compared with Example 1, the difference in this comparative example is that in step three, the membrane is not removed, and sodium borohydride is directly added to the polyphenolized chitosan acetate solution for reduction. That is, after the membrane is immersed in the polyphenolized chitosan acetate solution for reaction, the membrane is not removed, and sodium borohydride is directly added to the solution. The reaction is carried out in an ice-water bath for 60 minutes. The remaining steps and parameters are the same as in Example 1.

[0122] Comparative Example 5

[0123] Compared with Example 1, the difference in this comparative example is that the coordination crosslinking step of iron ions is omitted. That is, after the reduction in step three, the post-processing is carried out directly without the treatment with ferric chloride solution. The remaining steps and parameters are the same as in Example 1.

[0124] Performance Test Results and Analysis

[0125] The ternary crosslinked nanofiltration membranes prepared in each embodiment and comparative example were evaluated using the following test methods, and the results are shown in Table 1.

[0126] The ternary crosslinked nanofiltration membranes prepared in each embodiment and comparative example were cut to have an effective filtration area of ​​50 cm². 2 The membrane was installed in a cross-flow filtration device. A 70% ethanol-water extract of *Hylocereus pluvialis* was used as the feed solution. This extract was obtained by homogenizing *Hylocereus pluvialis* under high pressure, extracting with a 70% ethanol-water mixture, and centrifuging to remove denatured proteins and cell wall fragments. The extract contained approximately 3 g / L of polysaccharide, 0.5 g / L of astaxanthin ester, and 0.8 g / L of neutral lipids. The test was conducted under the conditions of an operating pressure of 0.8 MPa, a temperature of 30 °C, and a cross-flow velocity of 1.5 m / s. The pure water flux was calculated by measuring the permeate volume per unit time. The polysaccharide rejection rate was calculated by determining the polysaccharide concentration in the feed solution and permeate using the phenol-sulfuric acid method. The astaxanthin ester rejection rate was calculated by determining the astaxanthin ester concentration in the feed solution and permeate using high-performance liquid chromatography. The neutral lipid rejection rate was calculated by determining the lipid content in the feed solution and permeate using Soxhlet extraction. Membrane fouling performance was evaluated by measuring flux decline rate after 2 hours of continuous operation, and flux recovery rate was measured after rinsing with deionized water for 30 minutes. Operational stability was evaluated by measuring flux retention rate after 8 hours of continuous operation.

[0127] As shown in Table 1, the polysaccharide rejection rate of *Haemaphysalis* in Examples 1 to 4 all reached over 95%, the astaxanthin ester rejection rate reached over 90%, the neutral oil rejection rate was controlled between 38% and 45%, the flux decline rate after 2 hours of operation was less than 15%, the flux recovery rate after water rinsing was higher than 89%, and the flux retention rate after 8 hours of operation was higher than 86%. These data indicate that the ternary crosslinked nanofiltration membrane of this application exhibits excellent performance in both separation selectivity and antifouling properties.

[0128] Comparative Example 1 omitted the polydopamine interlayer, and the oxidized Rhodophyta citrate polysaccharide was directly anchored on the surface of the polyethersulfone ultrafiltration membrane substrate. Due to the strong hydrophobicity and lack of reactive sites on the polyethersulfone ultrafiltration membrane substrate surface, the oxidized Rhodophyta citrate polysaccharide adhered via weak physical adsorption, resulting in poor interlayer bonding and easy detachment of the functional layer during operation. Simultaneously, the hydrophobic surface of the substrate led to severe irreversible adsorption of contaminants such as proteins and polysaccharides, exacerbating membrane fouling.

[0129] Test results showed that the pure water flux of Comparative Example 1 was only 32 L·m -2 ·h -1 ·bar -1 The polysaccharide rejection rate of *Haemaphysalis* was 85%, the astaxanthin ester rejection rate was 78%, the flux decay rate after 2 hours of operation was 32%, the flux recovery rate after water rinsing was 68%, and the flux retention rate after 8 hours of operation was 60%. All indicators were significantly lower than those in Example 1. This indicates that the polydopamine interlayer plays an important role in enhancing interlayer bonding and improving membrane hydrophilicity.

[0130] Table 1 Analysis of Test Results

[0131] Example 1 45 96 92 42 12 91 88 Example 2 48 95 90 45 14 89 86 Example 3 42 97 93 40 11 92 89 Example 4 50 98 95 38 10 94 92 Comparative Example 1 32 85 78 58 32 68 60 Comparative Example 2 38 88 83 52 25 76 70 Comparative Example 3 36 87 72 60 28 74 68 Comparative Example 4 28 82 70 62 35 65 55 Comparative Example 5 52 92 62 55 18 84 80

[0132] Comparative Example 2 used sodium alginate instead of oxidized Rhodophytaecos polysaccharide. Sodium alginate is a flexible-chain polysaccharide with easily bent and entangled molecular chains, resulting in a wider pore size distribution in the functional layer formed on the membrane surface, thus reducing the size sieving accuracy. Simultaneously, sodium alginate lacks sulfate groups, resulting in a lower negative charge density than oxidized Rhodophytaecos polysaccharide, leading to insufficient density in the polyelectrolyte composite layer formed with polyphenolized chitosan. Test results showed that Comparative Example 2 exhibited a Rhodophytaecos polysaccharide rejection rate of 88%, an astaxanthin ester rejection rate of 83%, a neutral oil rejection rate of 52%, a flux decay rate of 25% after 2 hours of operation, a flux recovery rate of 76% after water rinsing, and a flux retention rate of 70% after 8 hours of operation, all inferior to Example 1. This indicates that the rigid chain structure and high charge density of oxidized Rhodophytaecos polysaccharide are crucial for improving separation selectivity and functional layer stability.

[0133] Comparative Example 3 used unmodified chitosan instead of polyphenolized chitosan. Ordinary chitosan lacks catechol groups and cannot form coordination crosslinks with iron ions, resulting in a lack of a metal polyphenol network structure in the functional layer. Therefore, the functional layer lacks selective coordination ability for astaxanthin esters, and the separation factor between astaxanthin esters and neutral oils decreases significantly. Furthermore, after amino protonation under acidic conditions, the Schiff base crosslinking network of ordinary chitosan is prone to hydrolysis, leading to poor structural stability of the functional layer. Test results showed that the astaxanthin ester rejection rate in Comparative Example 3 was only 72%, and the neutral oil rejection rate was 60%, a difference of only 12 percentage points, far lower than the 50 percentage points in Example 1. The flux decay rate after 2 hours of operation was 28%, the flux recovery rate after water rinsing was 74%, and the flux retention rate after 8 hours of operation was 68%. The antifouling performance and operational stability were also inferior to Example 1, indicating that the catechol groups in polyphenolized chitosan are indispensable for constructing the coordination crosslinking network and achieving selective separation.

[0134] In Comparative Example 4, sodium borohydride was added directly for reduction without removing the membrane in step three. A large amount of free polyphenolized chitosan in the solution underwent homogeneous cross-linking under the action of the reducing agent, forming gels or precipitates that blocked the membrane pores and covered the membrane surface, resulting in uneven construction of the functional layer. Test results showed that the pure water flux of Comparative Example 4 was only 28 L·m⁻¹. -2 ·h -1 ·bar -1 The sample exhibited the worst performance among all samples, with a polysaccharide rejection rate of 82%, an astaxanthin ester rejection rate of 70%, a neutral lipid rejection rate of 62%, a flux decay rate of 35% after 2 hours of operation, a flux recovery rate of 65% after water rinsing, and a flux retention rate of 55% after 8 hours of operation. This confirms the necessity of stepwise reduction operation for avoiding homogeneous gelation and ensuring the uniform construction of the functional layer on the membrane surface.

[0135] Comparative Example 5 omitted the iron ion coordination crosslinking step. Only the covalent crosslinking between oxidized Haematococcus pluvialis polysaccharide and polyphenolized chitosan existed in the functional layer, lacking a metal polyphenol network. Due to the absence of iron ion coordination centers, the ketone and hydroxyl groups at the ends of the astaxanthin ester molecules could not form weak coordination bonds with the membrane surface, resulting in the loss of selective affinity. Test results showed that Comparative Example 5 had an astaxanthin ester rejection rate of only 62% and a neutral oil rejection rate of 55%, a difference of only 7 percentage points, indicating a near loss of selective separation ability. Although Comparative Example 5 had a higher pure water flux, reaching 52 L·m⁻¹, -2 ·h -1 ·bar -1 This is because the crosslinking density of the functional layer is reduced and the pore size is slightly larger after the lack of coordination crosslinking. However, the retention rate of Haematococcus pluvialis polysaccharide also drops to 92%. The flux decay rate is 18% after 2 hours of operation, the flux recovery rate is 84% ​​after water rinsing, and the flux retention rate is 80% after 8 hours of operation. The overall performance is still inferior to that of Example 1. This fully demonstrates that iron ion coordination crosslinking plays a key role in the selective separation of astaxanthin esters and neutral oils.

[0136] Test results show that this application constructs a ternary cross-linked functional layer by oxidizing Haematococcus pluvialis polysaccharide, polyphenolized chitosan and iron ions, enabling the nanofiltration membrane to simultaneously possess size sieving, charge regulation and coordination selection capabilities, achieving one-step selective separation of polysaccharides, astaxanthin esters and neutral oils in Haematococcus pluvialis extract, and the pore size of the functional layer can be dynamically adjusted according to the pH of the feed solution.

[0137] 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 ternary cross-linked nanofiltration membrane, characterized in that, The nanofiltration membrane comprises a polyethersulfone ultrafiltration membrane base membrane and a polydopamine intermediate layer and a ternary crosslinked functional layer sequentially loaded on the surface of the polyethersulfone ultrafiltration membrane base membrane. The ternary cross-linked functional layer is constructed by covalent and coordination cross-linking of oxidized red algae polysaccharide, polyphenolized chitosan and iron ions. The aldehyde group of oxidized red algae polysaccharide and the amino group of polyphenolized chitosan are covalently cross-linked after Schiff base reaction and reduction. The catechol group of polyphenolized chitosan forms coordination cross-linking with iron ions. The oxidized Haematococcus pluvialis polysaccharide is obtained by selective oxidation of Haematococcus pluvialis polysaccharide with sodium periodate after ultrasonic treatment. The polyphenolized chitosan was obtained by reducing and purifying chitosan and 3,4-dihydroxybenzaldehyde via a Schiff base reaction.

2. The ternary crosslinked nanofiltration membrane according to claim 1, characterized in that, The polyethersulfone ultrafiltration membrane base has a molecular weight cutoff of 50 kDa; the polydopamine interlayer has a thickness of 10-20 nm and is loaded on the surface of the polyethersulfone ultrafiltration membrane base; the ternary crosslinked functional layer has a thickness of 20-40 nm and is loaded on the surface of the polydopamine interlayer.

3. The ternary crosslinked nanofiltration membrane according to claim 1, characterized in that, The preparation method of the oxidized Rhodophyta polysaccharide includes the following steps: Rhodophyta polysaccharide is prepared into an aqueous solution and subjected to ultrasonic treatment. Sodium periodate is added, and the reaction is carried out at 25°C in the dark for 2-4 hours. After the reaction is completed, ethylene glycol is added, with a molar ratio of ethylene glycol to sodium periodate of 1.5-2:

1. The reaction is terminated after stirring at room temperature for 30 minutes. The resulting reaction solution is placed into a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed with deionized water for 48 hours, with the water changed every 8 hours, until the conductivity of the dialysate is lower than 10 μS / cm. The oxidized Rhodophyta polysaccharide is obtained by freeze drying.

4. The ternary crosslinked nanofiltration membrane according to claim 3, characterized in that, The specific steps and parameters of the ultrasonic treatment are as follows: prepare a 10-20 g / L aqueous solution of Haematococcus pluvialis polysaccharide, place it in an ice-water bath and apply ultrasound, with an ultrasonic power of 300-500 W, a frequency of 20-25 kHz, an ultrasonic time of 3-5 s, an interval time of 3-5 s, and a total ultrasonic duration of 30-90 min.

5. The ternary crosslinked nanofiltration membrane according to claim 3, characterized in that, The molar ratio of sodium periodate to the monosaccharide units with vicinal diol structures in the polysaccharide of *Hylocereus pluvialis* is 0.3-0.

6.

6. The ternary crosslinked nanofiltration membrane according to claim 1, characterized in that, The preparation method of the polyphenolized chitosan includes the following steps: dissolving chitosan in a 0.5%-1% acetic acid aqueous solution to prepare a 5-10 g / L solution, dissolving 3,4-dihydroxybenzaldehyde in ethanol to prepare a 10-20 g / L solution, mixing the two solutions so that the molar ratio of chitosan amino group to 3,4-dihydroxybenzaldehyde in the mixture is 1:0.8-1:1.2, and reacting at 40-60℃ for 2-4 hours to form a Schiff base; Sodium borohydride was then added, and the reaction was carried out at 0-4℃ for 2-4 hours; the pH was then adjusted to 5-6 with 0.1 mol / L HCl, and the mixture was stirred at room temperature for 30 minutes. The resulting reaction solution was placed into a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with deionized water for 48 hours, with the water changed every 8 hours, until the conductivity of the dialysate was below 10 μS / cm. The solution was then freeze-dried to obtain polyphenolized chitosan.

7. A ternary crosslinked nanofiltration membrane according to claim 6, characterized in that, The molar ratio of sodium borohydride to 3,4-dihydroxybenzaldehyde is 2:1 to 4:

1.

8. A method for preparing a ternary crosslinked nanofiltration membrane as described in any one of claims 1-7, characterized in that, The method includes the following steps: Step 1: Preparation of the polydopamine interlayer The polyethersulfone ultrafiltration membrane base was immersed in Tris-HCl buffer at pH 8.5, dopamine hydrochloride was added, and it was self-polymerized at 25°C for 15-25 min. After removal, it was rinsed with deionized water 3 times, 5 min each time. Step 2: Anchoring of Oxidized Haematococcus pluvialis polysaccharides Immerse the membrane obtained in step one in an aqueous solution of oxidized red puffball polysaccharide at pH 6-7 and react at room temperature for 30-60 minutes. Remove the membrane and rinse it three times with deionized water for 5 minutes each time. Step 3: Covalent cross-linking of polyphenolized chitosan The membrane obtained in step two is immersed in a polyphenolized chitosan acetate solution at pH 4.5-5.5 and reacted at room temperature for 30-60 min. The membrane is then removed and rinsed three times with deionized water for 5 min each time. Subsequently, the membrane is immersed in freshly prepared sodium borohydride aqueous solution and reacted in an ice-water bath for 30-60 min. After the reduction is completed, the membrane is removed and rinsed three times with deionized water for 10 min each time. Step 4: Coordination and crosslinking of iron ions Immerse the membrane obtained in step 3 in ferric chloride solution, adjust the pH to 3.5-4.5 with HCl, and react at room temperature for 10-20 minutes; Step 5: Post-processing Take out the membrane obtained in step four, rinse it with deionized water until the conductivity of the rinsing solution is less than 5 μS / cm, and air dry it at room temperature to obtain the ternary cross-linked nanofiltration membrane.

9. The method for preparing a ternary crosslinked nanofiltration membrane according to claim 8, characterized in that, The concentration of dopamine hydrochloride is 2 g / L, the concentration of the oxidized Rhodophyta polysaccharide aqueous solution is 1.5-2.5 g / L, the concentration of the polyphenolized chitosan acetate solution is 1-2 g / L, the concentration of the sodium borohydride aqueous solution is 0.5-2 g / L, and the concentration of the ferric chloride solution is 0.3-1 g / L.

10. An application of a ternary crosslinked nanofiltration membrane as described in any one of claims 1-7, characterized in that, This ternary cross-linked nanofiltration membrane is used for the separation of polysaccharides from Haematococcus pluvialis and astaxanthin esters.

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