Electrodriven separation device and separation method for simultaneous separation of lactoferrin and lactoperoxidase in one step

Abstract

The application belongs to the field of membrane separation technology, and proposes an electrically-driven separation device and method for simultaneously separating lactoferrin and lactoperoxidase in one step, which comprises a membrane stack, the membrane stack comprising an anode, a cathode, at least one membrane pair, an anode protection chamber, a cathode protection chamber, a cation exchange membrane and an anion exchange membrane, the membrane pair comprising, in sequence along the direction of the electric field, an anion ultrafiltration membrane, a feed chamber partition, a cation ultrafiltration membrane and a permeate chamber partition, and the cation ultrafiltration membrane and the anion ultrafiltration membrane are both ion-type polymer charged ultrafiltration membranes. The application has a reasonable design, utilizes opposite charge characteristics, and combines an electrically-driven separation device with ion-type polymer charged ultrafiltration membranes, so that the two proteins can be simultaneously separated in opposite directions, and high-purity lactoferrin and lactoperoxidase can be obtained in one step.

Description

Technical Field

[0001] This invention belongs to the field of membrane separation technology, and particularly relates to an electrically driven separation device and method for one-step simultaneous separation of lactoferrin and lactoperoxidase. Background Technology

[0002] Lactoferrin (LF) and lactoperoxidase (LP) are two important functional proteins in whey, possessing various biological activities such as antibacterial, antiviral, and immunomodulatory effects, and have broad application prospects in infant formula, health products, and pharmaceuticals. However, LF (molecular weight approximately 80 kDa, isoelectric point pI≈8.7) and LP (molecular weight approximately 78 kDa, isoelectric point pI≈9.6) have very similar molecular weights, making effective separation by traditional ultrafiltration membranes impossible. Furthermore, both are basic proteins, and while conventional ion-exchange chromatography can separate them, it is costly, has low throughput, and is unsuitable for large-scale production.

[0003] Charged ultrafiltration membranes differ from traditional ultrafiltration membranes in that they are ultrafiltration membranes that combine pore size sieving with electrostatic action. However, existing technologies have the following shortcomings: (1) low integration of membrane modules, requiring multiple independent devices to be connected in series or pH to be adjusted multiple times for multi-stage separation, resulting in large equipment volume and complex operation; (2) unstable performance of charged ultrafiltration membranes, easy detachment and large flux loss in surface grafting, and low ion exchange capacity and easy leaching of additives in blending additives; (3) existing technologies do not fully utilize the opposite charge characteristics of LF and LP under specific pH conditions, and cannot achieve one-step synchronous separation. Summary of the Invention

[0004] This invention addresses the technical problems of ultrafiltration membranes mentioned above by proposing an electro-driven separation device and method for the one-step simultaneous separation of lactoferrin and lactoperoxidase. By utilizing the opposite charge characteristics and combining an electro-driven separation device with an ion-charged polymer ultrafiltration membrane, the two proteins can be separated in opposite directions simultaneously, obtaining high-purity lactoferrin and lactoperoxidase in one step.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The present invention provides an electrically driven separation device with an ion-charged polymer ultrafiltration membrane, comprising a membrane stack, the membrane stack including an anode, a cathode, at least one membrane pair, an anode protection chamber and a cathode protection chamber, an anion exchange membrane disposed on the side of the membrane stack near the anode, and a cation exchange membrane disposed on the side of the membrane stack near the cathode, the membrane pair including an anion ultrafiltration membrane, a feed chamber partition, a cation ultrafiltration membrane and a permeate chamber partition arranged sequentially along the electric field direction, the membrane pair being disposed between the anion exchange membrane and the cation exchange membrane, the cation ultrafiltration membrane comprising a cationic polymer, the main chain of the cationic polymer containing an aromatic ring structure and the side chains containing cation exchange groups, the cation exchange groups being sulfonic acid groups, carboxyl groups or phosphate groups, the anion ultrafiltration membrane comprising an anion polymer, the anion polymer being a polyphenylene ether, polyethersulfone or polysulfone polymer containing quaternary ammonium groups, primary amine groups, secondary amine groups or tertiary amine groups.

[0006] Preferably, the ion exchange capacity of the cationic polymer is 1.0~2.5 mmol / g, and the ion exchange capacity of the anionic polymer is 1.0~2.5 mmol / g.

[0007] Preferably, the anode is a graphite electrode, a titanium-based coated electrode, a platinum electrode, or a stainless steel electrode, and the cathode is a graphite electrode, a titanium-based coated electrode, a platinum electrode, or a stainless steel electrode.

[0008] Preferably, the anion exchange membrane and the cation exchange membrane are homogeneous or heterogeneous ion exchange membranes.

[0009] Preferably, both the cation ultrafiltration membrane and the anion ultrafiltration membrane are ion-charged polymer ultrafiltration membranes, and the preparation method of the ion-charged polymer ultrafiltration membrane includes the following steps:

[0010] P1, Synthetic ionic polymers;

[0011] P2. Preparation of casting solution: Dissolve the ionic polymer obtained in step P1 in a polar organic solvent, add a pore-forming agent, and stir at 40-80°C for 2-6 hours to obtain a homogeneous casting solution; the casting solution contains 15-25 wt% ionic polymer, 60-80 wt% organic solvent, and 0-10 wt% pore-forming agent.

[0012] P3. Degassing: Let the casting solution stand at 40~60℃ for 4~12 hours to degas, or use vacuum degassing for 1~2 hours;

[0013] P4. Film Formation: The degassed casting solution is scraped onto a glass plate or support material to form a film. The wet film thickness is controlled at 150~300 μm. After being kept in an environment with a temperature of 20~30℃ and a relative humidity of 40~70% for 10~60s, it is immersed in a coagulation bath at 20~50℃ to solidify into a film. The coagulation bath is deionized water or a mixed solution of water and organic solvent.

[0014] P5. Post-treatment: Wash the ultrafiltration membrane after membrane formation in deionized water for 24-48 hours to obtain an ion-charged polymer ultrafiltration membrane.

[0015] Preferably, the organic solvent in step P2 includes one or more of N-methylpyrrolidone, N,N-dimethylformamide, and N,N-dimethylacetamide; the porogen includes one or more of polyethylene glycol, polyvinylpyrrolidone, and LiCl.

[0016] A one-step method for simultaneously separating lactoferrin and lactoperoxidase includes the following steps: S1, adjusting the pH of the pretreated feed solution to 9.0~9.3, and introducing it into the feed chamber of an electrically driven separation device equipped with an ion-charged polymer ultrafiltration membrane, while introducing electrode water into the anolyte and catholyte protection chambers; S2, applying a DC voltage between the anode and cathode, with a voltage range of 5 V~30 V and an electric field strength of 0.5 V / cm~5 V / cm; S3, under the drive of the electric field, positively charged lactoperoxidase migrates towards the cathode, passes through the cation ultrafiltration membrane into the cathode-side permeate chamber, and the lactoperoxidase enriched solution is collected from the cathode-side permeate outlet; negatively charged lactoferrin migrates towards the anode, passes through the anion ultrafiltration membrane into the anode-side permeate chamber, and the lactoferrin enriched solution is collected from the anode-side permeate outlet.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0018] 1. This invention starts with polymer molecular design to synthesize cationic and anionic polymers with high ion exchange capacity, and uses these as the main raw materials to prepare charged ultrafiltration membranes via a non-solvent-induced phase separation method. Compared with the surface grafting method, the ion exchange groups are uniformly distributed throughout the entire membrane body (including the inner wall of the membrane pores), eliminating the risk of graft layer detachment and minimizing membrane flux loss. Compared with existing blending methods, the ionic polymer is used as the main membrane-forming material rather than a small amount of additive, achieving an ion exchange capacity as high as 1.0~2.5 mmol / g, without the risk of additive leaching, and with durable and stable performance.

[0019] 2. This invention utilizes the opposite charge characteristics of lactoferrin and lactoperoxidase under pH 9.0~9.3 conditions to achieve synchronous phase-to-phase separation of the two proteins. Compared with traditional methods that require multiple separations, the running time is shortened by more than 40%, energy consumption is reduced by more than 30%, while maintaining high purity (LP≥92%, LF≥91%) and high recovery rate (LP≥87%, LF≥89%). Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 A schematic diagram of an electrically driven separation device for the one-step simultaneous separation of lactoferrin and lactoperoxidase provided in an embodiment;

[0022] Figure 2 The chart shows a comparison of the separation purity and recovery rate of lactoferrin and lactoperoxidase between the present invention and the traditional process (multi-stage ultrafiltration tandem sieving).

[0023] In the above diagrams: 1. Anode; 2. Cathode; 3. Anode protection chamber; 4. Cathode protection chamber; 5. Anion exchange membrane; 6. Cation exchange membrane; 7. Anion ultrafiltration membrane; 8. Cation ultrafiltration membrane; 9. Feed chamber; 10. Cathode-side permeate chamber; 11. Anode-side permeate chamber. Detailed Implementation

[0024] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0025] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0026] Examples, such as Figure 1As shown, the electro-driven separation device for the one-step simultaneous separation of lactoferrin and lactoperoxidase provided by the present invention includes a membrane stack, which includes an anode 1, a cathode 2, at least one membrane pair, an anode protection chamber, and a cathode protection chamber. An anion exchange membrane 5 is disposed on the side of the membrane stack closest to the anode 1, and a cation exchange membrane 6 is disposed on the side of the membrane stack closest to the cathode 2. The membrane pair includes an anion ultrafiltration membrane 7, a feed chamber partition, a cation ultrafiltration membrane 8, and a permeate chamber partition, sequentially distributed along the electric field direction. The membrane pair is disposed between the anion exchange membrane 5 and the cation exchange membrane 6. The cation ultrafiltration membrane 8 includes a cationic polymer, the main chain of which contains an aromatic ring structure and the side chains contain cation exchange groups, which are sulfonic acid groups, carboxyl groups, or phosphate groups. The ion exchange capacity of the cationic polymer is 1.0~2.5. The anion exchange capacity is 1.0~2.5 mmol / g, and the charge sites are uniformly distributed in the membrane body. The anion ultrafiltration membrane 7 includes anion polymers, which are polyphenylene ether, polyethersulfone, or polysulfone polymers containing quaternary ammonium groups, primary amine groups, secondary amine groups, or tertiary amine groups. The anion polymer has an ion exchange capacity of 1.0~2.5 mmol / g, which matches the charge capacity range of the cation membrane to ensure bidirectional migration equilibrium under an electric field.

[0027] The anion ultrafiltration membrane 7 and cation ultrafiltration membrane 8 provided by this invention are both ion-charged polymer ultrafiltration membranes. These membranes employ a non-solvent-induced phase separation method, which differs from traditional surface grafting and additive blending processes. Using ion-polymers as the main film-forming material, they possess inherent molecular charge, thus solving the problems of membrane charge loss and additive precipitation. The specific preparation method includes the following steps:

[0028] P1. Synthesize ionic polymers, including cationic aromatic ring polymers and anionic amine-modified polymers, which are the source of stable charge on the membrane. Charged groups are grafted onto the side chains of the polymer backbone, penetrating the entire polymer molecule. After membrane formation, the charge is evenly distributed on the membrane surface, the inner wall of the membrane pores, and the membrane support layer.

[0029] P2. Preparation of casting solution: Dissolve the cationic polymer / anionic polymer obtained in step P1 in a polar organic solvent, add a pore-forming agent, and stir at 40-80°C for 2-6 hours to obtain a homogeneous casting solution; the casting solution contains 15-25 wt% cationic polymer / anionic polymer, 60-80 wt% organic solvent, and 0-10 wt% pore-forming agent; wherein the organic solvent is selected from one or more of N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), and N,N-dimethylacetamide (DMAc); the pore-forming agent is selected from one or more of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and LiCl;

[0030] P3. Degassing: Degas the casting solution by standing at 40~60℃ for 4~12 hours, or by vacuum degassing for 1~2 hours; vacuum degassing is preferred for industrial continuous production as it has higher degassing efficiency.

[0031] P4. Film Formation: The degassed casting solution is scraped onto a glass plate or non-woven fabric support material to form a film. The wet film thickness is controlled at 150~300 μm. If it is too thin, it is easy to break down and the flux decays quickly; if it is too thick, the mass transfer resistance is large and the protein migration rate decreases. After being kept in an environment with a temperature of 20~30℃ and a relative humidity of 40~70% for 10~60 seconds, the solvent in the layer evaporates slightly, forming an ultrathin and dense separation layer. Finally, it is immersed in a coagulation bath at 20~50℃ to solidify and form a film. The coagulation bath is deionized water or a mixed solution of water and organic solvent. The solvent precipitates rapidly, and the polymer phase separates and solidifies to form a porous ultrafiltration membrane framework.

[0032] P5. Post-treatment: The ultrafiltration membrane after membrane formation is washed in deionized water for 24-48 hours to completely remove residual organic solvents and free pore-forming agents, avoid leaching of additives from the production process and contamination of whey protein, and stabilize the membrane charge performance, finally obtaining cation ultrafiltration membrane 8 / anion ultrafiltration membrane 7.

[0033] Furthermore, the anode 1 is a graphite electrode, a titanium-based coated electrode, a platinum electrode, or a stainless steel electrode, and the cathode 2 is a graphite electrode, a titanium-based coated electrode, a platinum electrode, or a stainless steel electrode. Titanium-based coated electrodes are preferred in industrial applications due to their corrosion resistance, low electrolysis byproducts, and moderate cost.

[0034] The anion exchange membrane 5 and cation exchange membrane 6 are commercially available homogeneous or heterogeneous ion exchange membranes that block protein and inorganic salt ions from crossing the membrane into the electrode chamber, prevent the electrode water from contaminating the protein enrichment solution, and simultaneously facilitate the electrode water circuit.

[0035] To improve the efficiency of automated production, the electrically driven separation device provided by the present invention further includes a pH online monitoring module, a conductivity online monitoring module, and a programmable logic controller. The programmable logic controller adjusts the applied voltage and feed flow rate according to the pH and conductivity monitoring values. By collecting the pH of the feed chamber and the conductivity of the feed liquid in real time, it automatically adjusts the DC output voltage and the feed pump flow rate in a closed loop to maintain the optimal electric field environment for protein migration and realize continuous automated production.

[0036] A one-step method for simultaneously separating lactoferrin and lactoperoxidase includes the following steps: S1, whey raw material pretreatment: defatting, casein removal, and coarse filtration to remove macromolecular impurities; adjusting the pH of the pretreated solution to 9.0~9.3, and passing it into the feed chamber 9 of an electrically driven separation device equipped with an ion-charged polymer ultrafiltration membrane. Lactoperoxidase LP carries a positive charge, and lactoferrin LF carries a negative charge; simultaneously, electrode water is introduced into the anolyte and catholyte protection chambers to stabilize the electrode electric field, neutralize the hydrogen ions and hydroxide ions generated during electrolysis, and avoid drastic pH fluctuations in the electrode chambers; S2, applying a DC voltage between anode 1 and cathode 2, with a voltage range of 5 V~30 V and an electric field strength of 0.5 V / cm~5. V / cm; S3. Under the drive of the electric field, positively charged lactoperoxidase migrates to cathode 2, passes through cation ultrafiltration membrane 8 and enters cathode-side permeate chamber 10. Lactoperoxidase LP enriched solution is collected from the outlet of cathode-side permeate chamber 10. Negatively charged lactoferrin migrates to anode 1, passes through anion ultrafiltration membrane 7 and enters anode-side permeate chamber 11. Lactoferrin LF enriched solution is collected from the outlet of anode-side permeate chamber 11. The remaining waste liquid in feed chamber 9 is waste whey after removing LF and LP, which can be discharged or recycled to recover other whey proteins.

[0037] This invention starts with polymer molecular design to synthesize cationic and anionic polymers with high ion exchange capacity, and uses these as the main raw materials to prepare charged ultrafiltration membranes via a non-solvent-induced phase separation method. Compared with the surface grafting method, the ion exchange groups are uniformly distributed throughout the entire membrane body (including the inner wall of the membrane pores), eliminating the risk of graft layer detachment and minimizing membrane flux loss. Compared with existing blending methods, the ionic polymer is used as the main membrane-forming material rather than a small amount of additive, achieving an ion exchange capacity as high as 1.0~2.5 mmol / g, without the risk of additive leaching, and with long-lasting and stable performance.

[0038] This invention utilizes the opposite charge properties of lactoferrin and lactoperoxidase under pH 9.0-9.3 conditions to achieve simultaneous phase-to-phase separation of the two proteins. Compared with traditional methods that require multiple separations, the operating time is reduced by more than 40%, equipment investment by more than 40%, and energy consumption by more than 30%, while maintaining high purity (LP≥92%, LF≥91%) and high recovery rate (LP≥87%, LF≥89%).

[0039] like Figure 2 As shown, a conventional ultrafiltration membrane sieving experiment was conducted using ordinary ultrafiltration membranes with molecular weight cutoffs of 30 kDa and 100 kDa connected in series. Because the molecular weights of the two membranes were similar (LF approximately 80 kDa, LP approximately 78 kDa), the separation effect was poor. Testing revealed that the purity of LP was only 58.3%, with a recovery rate of 71%; the purity of LF was 62.5%, with a recovery rate of 73%.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An electro-driven separation device for the one-step simultaneous separation of lactoferrin and lactoperoxidase, comprising a membrane stack, the membrane stack including an anode, a cathode, at least one membrane pair, an anode protection chamber, and a cathode protection chamber, wherein an anion exchange membrane is disposed on the side of the membrane stack near the anode, and a cation exchange membrane is disposed on the side of the membrane stack near the cathode, characterized in that, The membrane pair includes an anion exchange membrane, a feed chamber partition, a cation exchange membrane, and a permeate chamber partition, which are sequentially distributed along the direction of the electric field. The membrane pair is disposed between the anion exchange membrane and the cation exchange membrane. The cation exchange membrane includes a cation polymer, the main chain of which contains an aromatic ring structure and the side chain contains cation exchange groups, which are sulfonic acid groups, carboxyl groups, or phosphate groups. The anion exchange membrane includes an anion polymer, which is a polyphenylene ether, polyethersulfone, or polysulfone polymer containing quaternary ammonium groups, primary amine groups, secondary amine groups, or tertiary amine groups.

2. The electrically driven separation device for one-step simultaneous separation of lactoferrin and lactoperoxidase according to claim 1, characterized in that, The ion exchange capacity of the cationic polymer is 1.0~2.5 mmol / g, and the ion exchange capacity of the anionic polymer is 1.0~2.5 mmol / g.

3. The electrically driven separation device for one-step simultaneous separation of lactoferrin and lactoperoxidase according to claim 2, characterized in that, The anode is a graphite electrode, a titanium-based coated electrode, a platinum electrode, or a stainless steel electrode, and the cathode is a graphite electrode, a titanium-based coated electrode, a platinum electrode, or a stainless steel electrode.

4. The electrically driven separation device for one-step simultaneous separation of lactoferrin and lactoperoxidase according to claim 3, characterized in that, The anion exchange membrane and cation exchange membrane are homogeneous or heterogeneous ion exchange membranes.

5. The electrically driven separation device for one-step simultaneous separation of lactoferrin and lactoperoxidase according to claim 1, characterized in that, Both the cation and anion ultrafiltration membranes are ion-charged polymer ultrafiltration membranes, and the preparation method of the ion-charged polymer ultrafiltration membrane includes the following steps: P1, Synthetic ionic polymers; P2. Preparation of casting solution: Dissolve the ionic polymer obtained in step P1 in a polar organic solvent, add a pore-forming agent, and stir at 40-80°C for 2-6 hours to obtain a homogeneous casting solution; the casting solution contains 15-25 wt% ionic polymer, 60-80 wt% organic solvent, and 0-10 wt% pore-forming agent. P3. Degassing: Let the casting solution stand at 40~60℃ for 4~12 hours to degas, or use vacuum degassing for 1~2 hours; P4. Film Formation: The degassed casting solution is scraped onto a glass plate or support material to form a film. The wet film thickness is controlled to be 150~300μm. After being kept in an environment with a temperature of 20~30℃ and a relative humidity of 40~70% for 10~60s, it is immersed in a coagulation bath at 20~50℃ to solidify into a film. The coagulation bath is deionized water or a mixed solution of water and organic solvent. P5. Post-treatment: Wash the ultrafiltration membrane after membrane formation in deionized water for 24-48 hours to obtain an ion-charged polymer ultrafiltration membrane.

6. The electrically driven separation device for one-step simultaneous separation of lactoferrin and lactoperoxidase according to claim 5, characterized in that, The organic solvent in step P2 includes one or more of N-methylpyrrolidone, N,N-dimethylformamide, and N,N-dimethylacetamide; the porogen includes one or more of polyethylene glycol, polyvinylpyrrolidone, and LiCl.

7. A one-step method for simultaneously separating lactoferrin and lactoperoxidase, characterized in that, The electro-driven separation device for simultaneous one-step separation of lactoferrin and lactoperoxidase according to any one of claims 1 to 6 includes the following steps: S1. Adjust the pH of the pretreated solution to 9.0~9.3, introduce it into the feed chamber, and introduce electrode water into the anode protection chamber and the cathode protection chamber; S2. Apply a DC voltage between the anode and cathode, with a voltage range of 5 V to 30 V and an electric field strength of 0.5 V / cm to 5 V / cm; S3. Under the drive of the electric field, positively charged lactoperoxidase migrates to the cathode, passes through the cation ultrafiltration membrane and enters the permeate chamber on the cathode side. The lactoperoxidase enrichment is collected from the permeate outlet on the cathode side. Negatively charged lactoferrin migrates to the anode, passes through the anion ultrafiltration membrane and enters the permeate chamber on the anode side. The lactoferrin enrichment is collected from the permeate outlet on the anode side.

Images