High-protein milk concentration process based on membrane filtration technology

Abstract

The invention relates to the technical field of membrane separation, and discloses a high-protein milk concentration process based on a membrane filtration technology, the process adopts an electric response membrane grafted with an electric response polymer brush on the surface, and an electric control zymogen modified by genetic engineering is added into milk feed liquid to be treated. The zymogen is modified with an electric field responsive anchoring group and an electric signal activated ferrocene protecting group. In the filtering stage, a first electric field is applied to stretch the polymer brush, so that anti-pollution filtering is realized; and in the cleaning stage, switching to a second electric field to enable the polymer brush to shrink and drive the zymogen to be anchored on the surface of the membrane, and applying an activation pulse to trigger the activity of the zymogen so as to degrade pollutants in situ. On the premise of not interrupting production, not adding chemical cleaning agents and not damaging target product protein, milk concentration is efficiently and continuously carried out, and long-term stable high permeation flux is maintained.

Description

Technical Field

[0001] This invention relates to the field of membrane separation technology, specifically to a high-protein milk concentration process based on membrane filtration technology. Background Technology

[0002] With consumers' growing demand for healthy nutrition, high-protein dairy products, such as concentrated milk, yogurt base, and whey protein products, are experiencing strong market demand. Membrane separation technology, especially ultrafiltration, has become a core technology for the dairy industry in producing high-protein products because it can efficiently separate large protein molecules from small lactose and salt molecules at room temperature, while also being energy-efficient and gentle to operate.

[0003] However, in practical applications, membrane separation technology faces a long-standing and difficult-to-eradicate technical bottleneck: membrane fouling. Milk is a complex colloidal system, and its components, such as casein and whey protein, inevitably adsorb and deposit on the membrane surface and within the pores during filtration, forming a gel layer that clogs the membrane pores. This fouling directly causes a sharp decline in membrane permeate flux, significantly reducing production efficiency and increasing operating costs, making it a key obstacle to the further development of this technology.

[0004] To restore membrane permeability, current processes commonly employ periodic chemical in-place (CIP) cleaning. This method typically requires interrupting production and circulating the membrane modules with strong acids, alkalis, or oxidizing agents. This cleaning method not only results in significant unproductive downtime on the production line, reducing equipment utilization, but the corrosive chemical cleaning agents used also gradually erode the membrane material, shortening the membrane element's lifespan. Furthermore, the large amounts of acidic and alkaline wastewater generated after cleaning also burden subsequent wastewater treatment.

[0005] In search of gentler cleaning strategies, the industry has explored enzymatic cleaning. Utilizing proteases to specifically hydrolyze contaminating proteins seems like an ideal solution. However, this method suffers from a fundamental contradiction: if active proteases are added directly to the milk solution to be treated, their catalytic effect will be indiscriminate. This means that while the proteases degrade contaminants on the membrane surface, they will inevitably also degrade the milk proteins in the solution, the target product, resulting in product loss and quality degradation. Therefore, conventional enzymatic cleaning also cannot be performed simultaneously during production, greatly limiting its application.

[0006] In summary, existing milk concentration processes consistently face a difficult trade-off between production continuity, membrane material lifespan, target product integrity, and cleaning efficiency in addressing membrane fouling issues. Therefore, there is an urgent need in this field for a novel technological solution that can achieve in-situ, efficient control of membrane fouling without interrupting production, using corrosive chemicals, or damaging the target product. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a high-protein milk concentration process based on membrane filtration technology. It aims to solve the technical challenges of membrane damage, production interruption, and chemical residue risks associated with chemical cleaning during downtime after membrane fouling leads to a decline in permeate flux, and the inability of mild enzymatic methods to achieve online application due to their indiscriminate degradation of the target product protein in the feed solution.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution.

[0009] The first aspect of this invention provides a high-protein milk concentration process based on membrane filtration technology, comprising the following steps: Step a) Provide a filtration system containing an electroresponsive membrane and add an electro-controlled enzyme to the milk mixture to be treated.

[0010] The electroresponsive membrane is a modified membrane on a membrane substrate grafted with an electroresponsive polymer brush. The electroresponsive polymer brush is a copolymer of 2-(dimethylamino)ethyl methacrylate and acrylic acid. The electrocontrolled proenzyme is a modified inactive protease with two functional groups on its surface: an electric field-induced anchoring group and an electro-signal-activated protecting group, which blocks the active site of the enzyme through steric hindrance.

[0011] Step b) involves the high-efficiency filtration stage. A first electric field, a DC field, is applied across the electroresponsive membrane, with the feed liquid side electrode set as the negative electrode and a voltage ranging from -0.8V to -1.5V. Under this electric field, the electroresponsive polymer brushes grafted onto the membrane surface repel each other due to carrying the same charge, resulting in a fully extended chain conformation. This extended polymer brush forms a dense hydrated three-dimensional barrier on the membrane surface, preventing protein molecules in the milk feed liquid from directly contacting and adsorbing onto the membrane surface, thereby maintaining a high permeate flux. During this stage, the electroreactive enzymes in the feed liquid exist in an inactive, free-flowing state. The transmembrane pressure difference is maintained at 60-120 kPa, and the operating temperature is 48-52°C.

[0012] Step c), an online in-situ self-cleaning stage is performed. The first electric field is switched to a second electric field, which is a DC electric field. The feed liquid side electrode is set as the positive electrode, and the voltage is +1.5V to +2.5V. The reversal of the electric field polarity triggers the following two simultaneous physical processes: First, the electroresponsive polymer brush collapses from an extended state to a contracted state due to the neutralization of charges or the weakening of repulsion, thereby completely exposing the small amount of protein contaminants accumulated in the high-efficiency filtration stage; Second, the anchoring groups induced by the electric field on the surface of the electro-controlled zymogen undergo electrostatic adsorption with the membrane surface or the polymer chain roots under the action of the positive electric field, thereby capturing and anchoring the free electro-controlled zymogen in the feed liquid around the newly exposed contaminants.

[0013] Subsequently, while maintaining the second electric field, an activation pulse is applied. This activation pulse is a square wave high-voltage pulse with a peak voltage of +6.0V to +10.0V. This pulse signal triggers a conformational change or chemical bond breakage of the protective groups on the surface of the electro-zymogen anchored on the membrane surface, releasing the blockage of the enzyme's active site and activating the electro-zymogen in situ, transforming it into a highly active protease. The activated protease efficiently degrades protein contaminants only on the membrane surface, breaking them down into small polypeptide molecules, which are then carried away with the feed liquid.

[0014] In step d), the electric field is restored to the first electric field. The activated enzyme, having lost its anchoring electric field and activation signal, detaches from the membrane surface and reverts to its inactive proenzyme state. Simultaneously, the electroresponsive polymer brush re-extends, restoring the membrane's antifouling capability, and the system enters the next high-efficiency filtration stage.

[0015] A second aspect of the present invention provides an electroresponsive membrane for the above-described process, the preparation method of which is as follows: First, the membrane substrate is pretreated. The membrane substrate is a polyvinylidene fluoride ultrafiltration membrane with an average pore size of 50 nm and a molecular weight cutoff of 100 kDa. The pretreatment steps include: plasma treatment of the membrane substrate, followed by hydroxylation treatment, and finally, 2-bromoisobutyryl bromide is used as an initiator and fixed to the membrane surface by chemical bonds.

[0016] Then, the electroresponsive polymer brush was grafted onto the pretreated membrane substrate using surface-initiated atom transfer radical polymerization. The reactants in the polymerization reaction included monomers 2-(dimethylamino)ethyl methacrylate and acrylic acid in a molar ratio of 4:1 to 6:1; the catalytic system included an initiator, cuprous bromide, and N,N,N',N'',N''-pentamethyldiethylenetriamine in a molar ratio of 1:1.2:1.5 to 1:1.5:2.0. The polymerization reaction was carried out at 65-75°C for 8-12 hours.

[0017] The present invention also provides an electro-controlled enzyme precursor for the above-mentioned process, the preparation method of which includes: First, the protease gene is subjected to site-directed mutagenesis, introducing a cysteine ​​residue near the enzyme's active site and a sequence encoding 6-10 arginine residues at one end. After expression, this sequence forms the electric field-induced anchoring group.

[0018] Then, the purified mutant protease was chemically modified. A linker arm with a ferrocene group was covalently linked to the newly introduced cysteine ​​residue via a disulfide bond, thereby utilizing the steric hindrance of the ferrocene group to form the electrically activated protecting group. The linker arm with the ferrocene group was attached to the zymogen surface via the reaction of N-(γ-maleimidebutyryloxy)succinimide ester with (6-(ferrocene)hexyl)thiol.

[0019] In the milk solution to be treated, the working concentration of the electro-controlled enzyme proenzyme is 0.02-0.08 mg / mL.

[0020] The total operation time of the online in-situ self-cleaning phase is 45-90 seconds. Specifically, the duration of applying the second electric field is 40-80 seconds, and the duration of applying the activation pulse is 200-800 milliseconds.

[0021] This invention provides a high-protein milk concentration process based on membrane filtration technology. It has the following beneficial effects: 1. This invention utilizes a unified electric field signal to synergistically regulate the physical conformation of the polymer brush on the membrane surface and the bioactivity of electro-enzymes. During the self-cleaning phase, the reversal of the electric field polarity simultaneously causes the polymer brush to contract, exposing contaminants, and captures and anchors inactive zymogens in the feed solution near the contaminants. Subsequent electrical pulses then in situ and instantaneously activate the anchored zymogens. This synergistic mechanism, which couples contaminant exposure, catalyst localization, and activity activation, resolves the technical contradiction of traditional enzyme cleaning's inability to distinguish between contaminants and product proteins. It ensures that the enzyme functions locally and on demand on the membrane surface, thereby guaranteeing the quality and integrity of the target product protein in the circulating feed solution.

[0022] 3. This invention transforms the traditional, lengthy downtime chemical cleaning required in membrane filtration into a seamless, in-situ, online self-cleaning cycle that can be seamlessly integrated into the production process, requiring only tens of seconds. This online cleaning mode does not interrupt the production process and can quickly intervene and restore the process when there is a slight decrease in permeate flux, achieving a high degree of continuity in the filtration production process. This significantly improves the effective operating time of the equipment and overall production efficiency, and reduces the time and material losses caused by downtime cleaning.

[0023] 3. This invention operates entirely under gentle physical conditions, completely avoiding the use of corrosive chemical cleaning agents such as strong acids and alkalis. This not only eliminates irreversible damage to membrane materials caused by chemicals and significantly extends the service life of membrane modules, but also fundamentally eliminates the risk of chemical residues entering the final product. Simultaneously, the absence of chemical waste discharge reduces the environmental impact, and the entire gentle treatment process also helps to maximize the preservation of the natural bioactivity of nutrients in high-protein milk. Detailed Implementation

[0024] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0025] Membrane substrate: Polyvinylidene fluoride (PVDF) hollow fiber ultrafiltration membrane, with an average pore size of 50 nm and a molecular weight cutoff of 100 kDa; Monomer-1: 2-(Dimethylamino)ethylmethacrylate (DMAEMA), chemical formula C8H 15 NO2, CAS No.: 2867-47-2, 99% purity, contains stabilizer; Monomer-2: Acrylic acid (AA), chemical formula C3H4O2, CAS number: 79-10-7, 99% purity, contains stabilizer; Initiator precursor: 2-Bromoisobutyryl bromide (BiBB), chemical formula C4H6Br2O, CAS No.: 20769-85-1, 98% purity; Catalyst: Copper(I)bromide (CuBr), chemical formula CuBr, CAS No.: 7787-70-4, 99.9% purity; Ligand: N,N,N',N'',N''-Pentamethyldiethylenetriamine (PMDETA), chemical formula C9H 23 N3, CAS No.: 3030-47-5, 99% purity; Connector arm component-1: N-(γ-maleimide butyryloxy)succinimide ester (GMBS), chemical formula C 12 H 12N2O6, CAS No.: 80307-21-1, 98% purity; Connecting arm component-2: (6-(ferrocenylhexyl)thiol), chemical formula C 16 H 22 FeS, CAS No.: 163700-11-2, 97% purity; Enzyme source: Subtilisin Carlsberg from Bacillus subtilis, CAS No.: 9014-01-1, enzyme activity ≥2.4U / mg; Genetically engineered host: Escherichia coli BL21 (DE3) competent cells.

[0026] Expression vector: pET-28a(+) plasmid vector; Experimental materials: Commercially available brand-name skim milk.

[0027] Example 1

[0028] Example 1: Preparation of an electroresponsive membrane This embodiment illustrates the specific preparation process of the electroresponsive membrane described in this invention.

[0029] 1. Pretreatment of membrane substrate a) A 20cm x 15cm polyvinylidene fluoride (PVDF) ultrafiltration membrane was placed in a plasma treatment machine (100W power, argon atmosphere, 50Pa pressure) for 180 seconds to introduce peroxy groups on the membrane surface.

[0030] b) Immerse the membrane treated in step a) in 1000 mL of an aqueous solution containing 1 mol / L sodium hydroxide and 10% (w / w) hydrogen peroxide, and react at 70 °C for 2 hours to form hydroxyl groups on the membrane surface. After the reaction, rinse repeatedly with deionized water until the washing solution is neutral, and then dry in a vacuum oven at 60 °C for 4 hours.

[0031] c) Place the dried membrane treated in step b) in 500 mL of anhydrous dichloromethane and add 10 mL of triethylamine. Under nitrogen protection and an ice bath, slowly add 20 mL of anhydrous dichloromethane solution containing 5 mL of 2-bromoisobutyryl bromide (BiBB). After the addition is complete, remove the ice bath and react at room temperature for 24 hours to fix the initiator BiBB on the membrane surface. After the reaction is complete, rinse the membrane sequentially with ethanol and deionized water, and then dry it in a vacuum oven at 60 °C for later use.

[0032] 2. Surface-Initiated Atom Transfer Radical Polymerization (SI-ATRP) a) In a 500 mL three-necked flask, add 16.0 g of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and 2.2 g of acrylic acid (AA) in a molar ratio of approximately 5:1. Then add 5.3 g of N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA) and 200 mL of a methanol / water mixture (volume ratio 1:1), and stir magnetically until completely dissolved.

[0033] b) Continuously pass high-purity nitrogen gas through the solution in step a) for 30 minutes to remove dissolved oxygen.

[0034] c) Under nitrogen protection, 0.85 g of cuprous bromide (CuBr) is added to the solution. At this time, the molar ratio of the initiator (fixed on the membrane surface), CuBr and PMDETA is approximately 1:1.3:1.8.

[0035] d) The PVDF membrane that has been treated in step 1) and has the initiator fixed is completely immersed in the above reaction solution. The reaction system is sealed and placed in a constant temperature water bath at 70°C for 10 hours.

[0036] e) After the reaction is complete, remove the membrane and expose it to air to terminate the polymerization reaction. Immerse the resulting modified membrane in a large volume of deionized water for 48 hours, changing the deionized water every 6 hours to thoroughly remove unreacted monomers, catalysts, and physically adsorbed polymers. Finally, dry the cleaned electroresponsive membrane in a vacuum oven at 60°C for later use.

[0037] Example 2: Preparation of Electrolytically Controlled Enzyme Progeny This embodiment is used to illustrate the specific preparation process of the electro-controlled zymogen described in this invention.

[0038] 1. Gene construction and mutation: The gene encoding Subtilisin Carlsberg was cloned into the pET-28a(+) expression vector. Using overlap extension PCR-mediated site-directed mutagenesis, the codon encoding the serine residue (Ser221) near the active site was mutated to encode cysteine. Simultaneously, a DNA sequence encoding eight arginine residues (Arg8) was inserted downstream of the C-terminus of the protein as an anchoring region. The gene sequence was verified by DNA sequencing.

[0039] 2. Protein expression and purification: The correctly constructed recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells. The bacterial culture was incubated in LB medium containing kanamycin at 37°C to the logarithmic growth phase (OD600 ≈ 0.6–0.8), and expression was induced at 20°C for 16 hours after the addition of 0.5 mM IPTG. The bacterial cells were collected by centrifugation, and the supernatant was collected after sonication and centrifugation. The His-tagged recombinant protein was purified by Ni-NTA affinity chromatography. The purified protein was dialyzed in phosphate-buffered saline (PBS, pH 7.4) to obtain high-purity mutant protease.

[0040] 3. Covalent linkage of ferrocene protecting groups: 10 mg of purified mutant protease was dissolved in 10 mL of PBS buffer (pH 7.2). Separately, 15 mg of N-(γ-maleimide butyryloxy)succinimide ester (GMBS) and 18 mg of (6-(ferrocene)hexyl)thiol were reacted in dimethyl sulfoxide to prepare a ferrocene linker with a maleimide functional group. This linker was slowly added to the protein solution at a 10-fold molar excess and reacted at room temperature in the dark for 4 hours. During the reaction, the maleimide group of the linker underwent a specific addition reaction with the thiol group of cysteine ​​residues on the surface of the mutant protease. After the reaction, the mixture was dialyzed to remove unreacted ferrocene linkers, finally yielding an electro-enzyme progenitor solution with surface-modified anchoring groups and electro-activation protecting groups, which was stored at 4 °C for later use.

[0041] Example 3: The high-protein milk concentration process of the present invention This embodiment illustrates the specific operation process of the high-protein milk concentration process based on membrane filtration technology described in this invention.

[0042] 1. System construction and feed preparation: The electroresponsive membrane prepared in Example 1 was assembled into a cross-flow membrane filtration assembly, and a peristaltic pump, a pressure sensor, and a programmable DC power supply were connected. Commercially available skim milk was used as the milk feed solution to be treated, and the electro-controlled proenzyme prepared in Example 2 was added to it, so that its final working concentration in the feed solution was 0.05 mg / mL.

[0043] 2. Continuous filtration and online self-cleaning circulation operation: The process is set to perform a total of 5 "filtration-cleaning" cycles, as follows: a) First high-efficiency filtration stage: Start the peristaltic pump, control the transmembrane pressure difference (TMP) to 80 kPa, and maintain the feed temperature at 50°C. Simultaneously, apply the first electric field (DC electric field, feed side electrode is negative, voltage is -1.0V) across the electroresponsive membrane. This stage continues for 60 minutes, and the permeate flux is recorded.

[0044] b) First online in-situ self-cleaning stage: After 60 minutes of filtration, without stopping the liquid circulation, the electric field is directly switched to the second electric field (DC electric field, with the liquid side electrode as the positive electrode and a voltage of +2.0V), and this electric field is maintained for 59.5 seconds. In the last 500 milliseconds of this stage, a square wave high-voltage pulse with a peak voltage of +8.0V and a pulse width of 500 milliseconds is applied. The total duration of the entire self-cleaning stage is 60 seconds.

[0045] c) Subsequent cycles: After the first self-cleaning stage ends, immediately restore the electric field to the setting of the first electric field (-1.0V) and begin the second high-efficiency filtration stage. Repeat steps a) and b) for a total of 5 cycles.

[0046] Comparative example: Comparative Example 1: The difference from Example 3 is that milk concentration was performed using a pristine PVDF ultrafiltration membrane that had not been modified as in Example 1, and no electric field was applied throughout the operation. All other operating conditions, such as transmembrane pressure difference, temperature, and cycle time, were the same as in Example 3.

[0047] Comparative Example 2: Compared to Example 3, the difference is that the electro-enzyme precursor prepared in Example 2 is not added to the milk mixture to be treated. The online in-situ self-cleaning stage only involves switching the electric field and does not include the anchoring and activation steps of the electro-enzyme precursor. Everything else is the same.

[0048] Comparative Example 3: The difference from Example 3 is that, instead of the electro-enzymogen prepared in Example 2, unmodified subtilisin with equivalent protein activity was added to the milk mixture to be treated. All other aspects are the same.

[0049] Comparative Example 4: Compared to Example 3, the difference lies in the absence of an online in-situ self-cleaning stage regulated by an electric field. Instead, after each high-efficiency filtration stage has run for 60 minutes, the feed solution circulation is paused, and a traditional chemical online cleaning method is used: 0.1 mol / L sodium hydroxide solution is circulated and cleaned at 50°C for 15 minutes, followed by rinsing with deionized water until neutral, before starting the next filtration stage. Everything else remains the same.

[0050] Test example: Test Example 1: Evaluation of Membrane Permeation Flux and Flux Recovery Rate Test method: Initial flux determination: Before the milk concentration experiment, all membrane modules to be tested (used in Example 3 and Comparative Examples 1, 2, and 4) were subjected to pure water filtration at 25°C and 60 kPa operating pressure until the permeate flux stabilized. This stable flux was recorded as the initial pure water flux (J). w ).

[0051] Operational flux monitoring: During the continuous operation of Example 3 and Comparative Examples 1, 2, and 4, the permeate volume and time were recorded at 5 minutes after the start of each high-efficiency filtration stage and 5 minutes before the end (i.e., 55 minutes). The permeate flux (J) at the beginning and end of each stage was calculated. p ).

[0052] Data normalization: To eliminate the influence of individual membrane variability, all measured permeation fluxes (J) are normalized. p Divide by its corresponding initial pure water flux (J) w ), to obtain the normalized flux (J p / J w ).

[0053] Flux recovery rate (FRR) calculation: After completing all 5 cycles, the membrane module was thoroughly cleaned with deionized water, and then its pure water flux (J) was measured again under the same conditions. w-final Flux recovery rate (FRR) is calculated using the following formula: ; The test results of Example 3 and Comparative Examples 1, 2, and 4 during continuous operation, including normalized flux and final flux recovery rate, are recorded in the table below.

[0054] Table 1. Normalized flux and flux recovery rate of Example 3 and Comparative Examples 1, 2, and 4 during continuous operation.

[0055] Results analysis: Table 1 shows that the process in Example 3 maintained a high normalized flux and minimal flux decay throughout five consecutive filtration-cleaning cycles. Its final flux recovery rate (FRR) reached 97.2%. This result indicates that during the high-efficiency filtration stage, the applied first electric field causes the electroresponsive polymer brushes on the membrane surface to extend, forming a physical barrier that effectively slows down the initial adsorption and deposition of proteins on the membrane surface. When switching to the second electric field for online self-cleaning, the contracted state of the polymer brushes exposes the deposited contaminants. Simultaneously, the electro-enzyme anchored therein, triggered by an activation pulse, degrades the protein contaminants in situ, thereby achieving a high degree of recovery of membrane filtration performance.

[0056] Comparative Example 1, using an unmodified membrane, showed a sharp decline in normalized flux within the first cycle, which continued to decrease to extremely low levels in subsequent cycles, ultimately resulting in an FRR of only 13.5%, indicating severe irreversible membrane fouling. In Comparative Example 2, relying solely on electric field switching to drive conformational changes in the polymer brush, while mitigating some fouling compared to Comparative Example 1, still resulted in significant cumulative fouling due to the lack of a degradation mechanism for stubbornly adsorbed proteins, leading to a continuous decline in flux and an FRR of less than 50%. These two comparative examples, conversely, demonstrate the necessity of the synergistic effect between electroresponsive polymer brushes and electro-controlled zymogens for maintaining high flux and achieving efficient online cleaning.

[0057] Comparative Example 4, employing a traditional chemical cleaning method, also achieved a high FRR value (89.4%), but its flux still exhibited a stepwise decline after each cycle, indicating that chemical cleaning causes some irreversible damage. More importantly, this method requires suspending production and introducing chemical reagents for cleaning for up to 15 minutes. In contrast, the online in-situ self-cleaning process used in Example 3 can be completed in just 60 seconds without interrupting feed circulation or introducing any external chemicals. This confirms that the technical solution provided by this invention can effectively control membrane fouling and restore performance without interrupting production or using chemicals, ensuring the continuity and gentleness of the process.

[0058] Test Example 2: Evaluation of the integrity of the target product protein Test method: Sample collection: For the experimental procedures of Example 3 and Comparative Example 3, 2 mL samples were collected from the feed circulation tank on the concentration side at the initial state (0 minutes) and after each "filtration-cleaning" cycle.

[0059] Protein concentration determination: The total protein concentration in the samples was determined using the Bradford method. The specific steps were as follows: An appropriately diluted sample solution was reacted with Bradford's reagent for 5 minutes, and then the absorbance was measured at 595 nm using a UV-Vis spectrophotometer. The precise protein concentration (unit: mg / mL) of each sample was calculated by comparing it with a standard curve prepared using bovine serum albumin (BSA).

[0060] Relative protein concentration calculation: To evaluate the concentration stability of the target product protein in the concentrated feed solution during continuous operation, the protein concentration measured at each time point was calculated. (C) n The relative protein concentration is calculated by dividing the initial protein concentration (C0) of the experimental group by the relative protein concentration. The calculation formula is as follows: relative protein concentration ; Test Results The relative protein concentration changes of the concentrated side feed solution during continuous operation in Example 3 and Comparative Example 3 are recorded in the table below.

[0061] Table 2. Relative protein concentrations of the concentrated side feed solution in Example 3 and Comparative Example 3 during continuous operation.

[0062] Results analysis: The data in Table 2 show that, under the process conditions of Example 3, after five complete "filtration-cleaning" cycles, the relative protein concentration in the concentrated side feed solution remained at 99.2%, demonstrating extremely high stability. This result confirms that the electro-controlled proenzyme used in this invention exists in an inactive state in the feed solution during the high-efficiency filtration stage and does not degrade the target product protein in the circulating feed solution. Only under a specific electric field and pulse signal during the self-cleaning stage is the proenzyme instantaneously and in situ anchored to the membrane surface and activated, and its catalytic effect is strictly limited to the membrane-liquid interface, thereby ensuring the integrity of the product protein in the bulk feed solution.

[0063] In contrast, in Comparative Example 3, due to the addition of an unmodified, continuously active protease, the relative protein concentration of the concentrated side feed solution continuously decreased with the extension of the operating time, with only 78.5% remaining after five cycles. This indicates that the unregulated protease, while degrading contaminants on the membrane surface, also indiscriminately degraded milk proteins in the circulating feed solution, leading to a significant loss of the target product. This phenomenon is the fundamental reason why traditional enzyme cleaning methods cannot be directly applied to continuous filtration processes.

[0064] By directly comparing the data from Example 3 and Comparative Example 3, it can be confirmed that the technical solution provided by this invention, namely, modifying the zymogen through electric field-induced anchoring groups and electrically activated protecting groups, and combining this with spatiotemporal synchronous regulation using electric field signals, successfully solves the technical problem of uncontrollable catalyst activity in online enzyme cleaning, which leads to the degradation of the target product. This solution ensures that the enzyme's catalytic activity is expressed only at the required time (self-cleaning stage) and location (membrane surface), achieving targeted removal of membrane contaminants while effectively protecting the quality of the target product in the feed solution.

Claims

1. A high-protein milk concentration process based on membrane filtration technology, characterized in that, Includes the following steps: a) Provides a filtration system comprising an electroresponsive membrane, and adds an electro-controlled enzyme progeny to the milk mixture to be treated; wherein the electroresponsive membrane is a modified membrane grafted with an electroresponsive polymer brush on a membrane substrate, the electroresponsive polymer brush being a copolymer of 2-(dimethylamino)ethyl methacrylate and acrylic acid; the surface of the electro-controlled enzyme progeny is modified with an electric field-induced anchoring group and an electric signal-activated protecting group; b) High-efficiency filtration stage: A first electric field is applied to both sides of the electroresponsive membrane to cause the electroresponsive polymer brush to be in an extended state for milk concentration; the first electric field is a DC electric field, the feed liquid side electrode is the negative electrode, and the voltage is -0.8V to -1.5V; c) Online in-situ self-cleaning stage: The first electric field is switched to the second electric field, causing the electroresponsive polymer brush to be in a contracted state and the electro-controlled zymogen to be anchored to the membrane surface; then, while maintaining the second electric field, an activation pulse is applied to activate the anchored electro-controlled zymogen to degrade contaminants on the membrane surface. The second electric field is a DC electric field, with the feed liquid side electrode being the positive electrode and a voltage of +1.5V to +2.5V; the activation pulse is a square wave high-voltage pulse with a peak voltage of +6.0V to +10.0V. d) Restore the electric field to the first electric field and proceed to the next high-efficiency filtration stage.

2. The high-protein milk concentration process based on membrane filtration technology according to claim 1, characterized in that, The electroresponsive membrane is prepared by grafting the electroresponsive polymer brush onto the membrane substrate using a surface-initiated atom transfer radical polymerization method. In the polymerization reaction, the molar ratio of 2-(dimethylamino)ethyl methacrylate to acrylic acid is 4:1 to 6:1, and the molar ratio of initiator, cuprous bromide, and N,N,N',N'',N''-pentamethyldiethylenetriamine is 1:1.2:1.5 to 1:1.5:2.

0. The reaction is carried out at 65-75°C for 8-12 hours.

3. The high-protein milk concentration process based on membrane filtration technology according to claim 1, characterized in that, The method for preparing the electro-controlled zymogen includes: performing site-directed mutagenesis on the protease gene, introducing a cysteine ​​residue near the enzyme active site, and introducing a sequence encoding 6-10 arginine residues as the anchoring group; then using disulfide bonds to covalently link the linker arm with a ferrocene group to the cysteine ​​residue to form the electro-activated protecting group.

4. The high-protein milk concentration process based on membrane filtration technology according to claim 1, characterized in that, During the high-efficiency filtration stage, the transmembrane pressure difference is maintained at 60-120 kPa, and the operating temperature is 48-52℃.

5. The high-protein milk concentration process based on membrane filtration technology according to claim 1, characterized in that, The total operation time of the online in-situ self-cleaning stage is 45-90 seconds.

6. The high-protein milk concentration process based on membrane filtration technology according to claim 5, characterized in that, The online in-situ self-cleaning stage specifically includes: applying the second electric field for 40-80 seconds and applying the activation pulse for 200-800 milliseconds.

7. The high-protein milk concentration process based on membrane filtration technology according to claim 1, characterized in that, In the milk solution to be treated, the working concentration of the electro-controlled enzyme proenzyme is 0.02-0.08 mg / mL.

8. The high-protein milk concentration process based on membrane filtration technology according to claim 1, characterized in that, The membrane substrate is a polyvinylidene fluoride ultrafiltration membrane with an average pore size of 50 nm and a molecular weight cutoff of 100 kDa.

9. A high-protein milk concentration process based on membrane filtration technology according to claim 2, characterized in that, Before surface-initiated atom transfer radical polymerization, the method for preparing the electroresponsive membrane further includes: plasma treatment of the membrane substrate, followed by hydroxylation treatment, and finally fixing 2-bromoisobutyryl bromide as an initiator on the membrane surface.

10. A high-protein milk concentration process based on membrane filtration technology according to claim 3, characterized in that, The linker arm with the ferrocene group is attached to the surface of the zymogen by reacting N-(γ-maleimide butyryloxy)succinimide ester with (6-(ferrocene)hexyl)thiol.

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