Immobilized urease catalytic ultrafiltration membrane for degrading urea in yellow rice wine and preparation method of immobilized urease catalytic ultrafiltration membrane
By covalently bonding urease to a polymer ultrafiltration membrane, an integrated catalytic ultrafiltration membrane was constructed, solving the problems of simultaneous degradation of urea and enzyme dissolution in rice wine. This achieved efficient and stable rice wine processing, reduced EC formation, and improved the safety and flavor of rice wine.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-03
- Publication Date
- 2026-03-27
AI Technical Summary
Existing technologies cannot simultaneously, efficiently, and specifically degrade urea within the ultrafiltration clarification unit of rice wine, and there are risks of enzyme leaching contamination and enzyme residue, which affect the safety and flavor of rice wine.
Urease is immobilized on the inner wall of the membrane pores and/or the membrane surface of a polymer ultrafiltration membrane by covalent bonding, and the immobilization of urease is achieved through amidation reaction, thus constructing an integrated catalytic ultrafiltration membrane to achieve urea filtration and degradation.
It achieves stable enzyme fixation with no enzyme residue, improves processing efficiency and enzyme stability, and simultaneously completes clarification and urea degradation, reducing the formation of ethyl carbamate and enhancing the safety and flavor authenticity of rice wine.
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Figure CN121732001A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a kind of immobilized urease catalytic ultrafiltration membrane for degrading urea in yellow rice wine and its preparation method, belong to the cross technical field of polymer separation membrane and enzyme engineering. BACKGROUND
[0002] Yellow rice wine is a traditional Chinese brewing wine, and a potential carcinogen ethyl carbamate (EC) is generated during storage. Studies have shown that the reaction of urea and ethanol in yellow rice wine is one of the main ways to generate EC. Therefore, effectively removing urea in yellow rice wine is the key to controlling the content of EC and improving the safety of yellow rice wine.
[0003] Currently, the methods for reducing urea in yellow rice wine mainly include: 1. Direct addition of urease method: directly adding free urease to degrade urea in the wine body, and then inactivating and removing the enzyme by heating. This method has the risk of enzyme residue, complicated process steps, and the enzyme cannot be reused and may affect the inherent flavor of yellow rice wine.
[0004] 2. Adsorption method: using activated carbon or specific adsorption resin to adsorb urea. This method has poor selectivity, and may simultaneously adsorb effective flavor substances and functional components in yellow rice wine, resulting in changes in wine style.
[0005] 3. Membrane separation method: using nanofiltration or reverse osmosis membrane to remove urea. This method can only achieve physical separation of urea and cannot degrade it. The membrane is easily contaminated, the operating pressure is high, and some beneficial small molecules in yellow rice wine may also be lost.
[0006] Ultrafiltration membranes are widely used for turbidity removal and clarification of yellow rice wine, but their pore size is too large to retain small molecules such as urea. The existing technology lacks a technology that can simultaneously, efficiently and specifically degrade urea in the same processing unit of yellow rice wine ultrafiltration and clarification. Simply physically blending urease into the membrane material can cause enzyme to dissolve out during use, not only contaminating the wine body, but also the enzyme activity cannot be sustained.
[0007] Therefore, it is of great practical significance and innovative value to develop a catalytic ultrafiltration membrane that firmly immobilizes urease and integrates "filtration-degradation" for the yellow rice wine industry. SUMMARY
[0008] To solve the above technical problems, the present application provides a kind of immobilized urease catalytic ultrafiltration membrane for degrading urea in yellow rice wine and its preparation method. The immobilized urease catalytic ultrafiltration membrane integrates and synchronously realizes yellow rice wine ultrafiltration clarification and urea degradation. The preparation method of the present application realizes the firm, high-activity immobilization of urease on the inner wall of membrane pores and / or membrane surface.
[0009] The present application adopts the following technical solutions: In one aspect of the present application, a fixed urease catalytic ultrafiltration membrane for degrading urea in yellow rice wine is provided, in which a polymer ultrafiltration membrane is used as a substrate membrane, and urease is fixed on the inner wall of membrane pores and / or the surface of the substrate membrane through covalent bonds; and the covalent bonds are realized through an amidation reaction.
[0010] Preferably, the amidation reaction is realized by first modifying the substrate membrane with polyethyleneimine (PEI) for amination, and then cross-linking urease with glutaraldehyde as a cross-linking agent.
[0011] Preferably, the material of the substrate membrane is polysulfone.
[0012] Preferably, the pore size of the membrane is 10-50 nm; the urease loading of the fixed urease catalytic ultrafiltration membrane is 0.5-5.0 mg / cm 2 ; and the relative activity of the fixed urease is maintained at more than 80% of the initial activity.
[0013] In another aspect of the present application, a preparation method of the fixed urease catalytic ultrafiltration membrane is provided, which comprises the following steps: Step 1. Substrate membrane pretreatment: the ultrafiltration membrane is sequentially immersed in NaOH solution, ethanol solution and ultrapure water for washing, and then is placed in a 4℃ refrigerator for standby; Step 2. Amination modification: the pretreated substrate membrane obtained in step 1 is immersed in a PEI aqueous solution, and is slowly oscillated for reaction; after the reaction is completed, the PEI is removed by washing with ultrapure water to obtain an aminated membrane; Step 3. Covalent immobilization of urease: the aminated membrane obtained in step 2 is immersed in a phosphate buffer solution containing urease, and is subjected to a covalent cross-linking reaction in the presence of a cross-linking agent glutaraldehyde; Step 4. Post-treatment: after the reaction is completed, the membrane is taken out and washed with a large amount of phosphate buffer solution to remove the physically adsorbed urease, thereby obtaining a fixed urease catalytic ultrafiltration membrane.
[0014] Preferably, in step 1, the concentration of the NaOH solution is 0.1 M; and the concentration of the ethanol solution is 30%.
[0015] Preferably, in step 2, the concentration of the polyethyleneimine aqueous solution is 2% (w / v); the oscillation reaction temperature is 25℃; and the oscillation reaction time is 4 hours.
[0016] Preferably, in step 3, the concentration of the urease is 2 mg / mL; the phosphate buffer solution is a 0.1 M phosphate buffer solution with a pH of 7.0; the concentration of the glutaraldehyde is 0.5% (v / v); the cross-linking reaction temperature is 4-25℃; and the cross-linking reaction time is 2-12 hours.
[0017] Yet another aspect of the present application provides an application of the immobilized urease catalytic ultrafiltration membrane described above, which is used in the processing of yellow rice wine, rice wine or other fermented wine drinks, to simultaneously achieve the clarification filtration of the wine body and the degradation of urea, so as to reduce the potential content of ethyl carbamate.
[0018] The urease molecules are firmly anchored in the membrane pore inner wall and / or membrane surface of the ultrafiltration membrane by covalent bonding, to construct a "membrane reactor". When the yellow rice wine flows through the membrane surface and penetrates the membrane pores, the urea therein is in full contact with the immobilized urease and is catalytically decomposed into ammonia and carbon dioxide, so as to complete the degradation of urea while filtering.
[0019] Compared with the prior art, the present application has the following beneficial effects: 1. Functional integration and process simplification: the ultrafiltration separation and biological catalysis are integrated in a single membrane assembly, and the clarification and urea degradation of yellow rice wine are completed in one step, which greatly simplifies the process flow and improves the processing efficiency.
[0020] 2. Stable enzyme immobilization, no residual risk: the urease is fixed by covalent bonding, and the enzyme molecules are firmly connected with the substrate membrane, without the risk of falling off during use and cleaning, which completely solves the safety hazard of free enzyme residue polluting yellow rice wine.
[0021] 3. High enzyme stability and reusability: the immobilized enzyme is in a restricted microenvironment, and its thermal stability and operational stability are significantly higher than those of free enzyme, which realizes the repeated use of the membrane and reduces the processing cost.
[0022] 4. High processing efficiency and strong specificity: urea is rapidly degraded in situ inside the membrane pores, with small mass transfer resistance and high reaction efficiency; at the same time, the enzymatic reaction has high specificity for urea and does not affect other flavor substances in yellow rice wine.
[0023] 5. Control of EC from the source: the present application actively and online removes urea, the precursor of EC, breaks the generation balance of EC, and effectively reduces the generation of EC in the subsequent storage of yellow rice wine. BRIEF DESCRIPTION OF DRAWINGS
[0024] Figure 1 A preparation method diagram of the immobilized urease catalytic ultrafiltration membrane for degrading urea in yellow rice wine is provided for the present application. DETAILED DESCRIPTION
[0025] The present application will be further described below through specific examples, but the scope of protection of the present application is not limited thereto.
[0026] The application provides an immobilized urease catalytic ultrafiltration membrane for degrading urea in yellow rice and a preparation method thereof. The urease is stably fixed in the inner wall of the membrane hole and / or the membrane surface of the ultrafiltration membrane by covalent bonding, and an integrated catalytic separation membrane is constructed. The preparation method of the membrane comprises the following steps: the surface of a polysulfone (PSF) matrix membrane is modified by amination, and then the covalent fixation of urease is realized through an amidation reaction. When the catalytic ultrafiltration membrane prepared by the application is used to treat yellow rice, the retention of macromolecular impurities and the enzymatic degradation of small molecular urea can be simultaneously completed, the urea removal rate can reach more than 80%, the enzyme is firmly immobilized and has no risk of dissolution, the process is simple and efficient, and a new solution is provided for controlling the generation of ethyl carbamate (EC) in yellow rice from the source.
[0027] Example 1 The application provides a preparation method of an immobilized urease catalytic ultrafiltration membrane for degrading urea in yellow rice, comprising the following steps: Step 1. Matrix membrane pretreatment: take a polysulfone (PSF) flat plate ultrafiltration membrane with an average pore size of 40 nm, and immerse it in 0.1M NaOH solution, 30% ethanol solution and ultrapure water for 30 minutes, respectively, then wash and clean, and store in a 4℃ refrigerator for standby; Step 2. Amino modification: immerse the pretreated PSF membrane in a 2% (w / v) polyethyleneimine (PEI, molecular weight 70000) aqueous solution, and slowly oscillate at 25℃ for 4 hours; after the reaction is completed, wash with ultrapure water to remove the physically adsorbed PEI, and obtain the aminated PSF membrane; Step 3. Covalent immobilization of urease: immerse the aminated PSF membrane in a 0.1M phosphate buffer (PBS, pH 7.0) containing 2mg / mL urease and 0.5% (v / v) glutaraldehyde, and slowly oscillate at 4℃ for 6 hours; Step 4. Post-treatment: after the reaction is completed, take out the membrane, repeatedly wash with a large amount of PBS (pH 7.0) until there is no ultraviolet absorption at 280nm (indicating no protein dissolution) and the urease activity cannot be detected, obtain the prepared immobilized urease catalytic ultrafiltration membrane (the immobilized amount of urease is 0.5-5.0mg / cm 2 , the relative activity of the immobilized urease remains more than 80% of the initial activity), and store in 4℃ PBS.
[0028] Comparative Example 1 A PSF ultrafiltration membrane physically blended with urease. The traditional phase inversion method is used for membrane preparation: urease powder with a final immobilized amount equivalent to that of Example 1 is directly added to the PSF membrane preparation liquid, stirred uniformly, and then scraped and formed into a solid. The urease in the obtained membrane is only physically embedded.
[0029] Comparative Example 2 An unmodified PSF ultrafiltration membrane. The PSF ultrafiltration membrane is not subjected to any treatment.
[0030] Verification experiment: Enzyme immobilization stability test: The membranes prepared in Example 1 and Comparative Example 1 were respectively placed in flowing PBS (pH 7.0) for 24 hours, and the urease activity and protein content in the washing liquid were detected at intervals. After 24 hours of washing, no significant enzyme activity and protein were detected in the washing liquid of the immobilized urease catalytic ultrafiltration membrane of Example 1, indicating that the enzyme was firmly immobilized by covalent bond; while in the initial stage (first 2 hours) of washing, a large amount of enzyme activity and protein were detected in the washing liquid of the PSF ultrafiltration membrane of Comparative Example 1, and after 24 hours, the enzyme activity in the membrane was lost by more than 60%.
[0031] Yellow rice processing experiment: two stages, namely the circulating reaction stage and the filtrate stage. The circulating reaction stage is that the permeate is pumped into the circulating tank for transmembrane reaction, and the circulation is repeated to make it fully react with the urease on the membrane to degrade urea. This process realizes the enzymatic degradation of small molecular urea, and the time of this stage is controlled at 6 hours, a lower operating pressure is used, and a smaller flow is maintained to make the feed liquid fully react. The filtrate stage is that after the urease reaction is complete, the transmembrane permeate is discharged into the next process. This process realizes the interception of macromolecular impurities. A higher operating pressure is used in this stage to maintain a higher transmembrane flux and improve efficiency, and then the permeate is collected and the urea content is detected. The same batch of traditional semi-dry yellow rice wine is used for yellow rice processing experiments using the membranes prepared in Example 1, Comparative Example 1 and Comparative Example 2, and the urea content of the yellow rice after multiple treatments is detected to obtain the urea removal rate as shown in Table 1. It is worth noting that when the urease activity disappears, the ultrafiltration membrane can be used as a conventional ultrafiltration membrane to intercept macromolecular substances and continue to play its filtering and clarifying role.
[0032] Table 1 Urea removal rate of the membranes prepared in Example 1, Comparative Example 1 and Comparative Example 2
[0033] The present application successfully prepared an immobilized urease catalytic ultrafiltration membrane by covalent bonding technology. The membrane exhibits high enzyme stability, can efficiently and stably degrade urea when processing yellow rice, and the urea removal rate can reach more than 80%, and significantly reduces the generation potential of EC. The membrane can repeatedly process multiple batches of yellow rice, and has high reusability when degrading urea in yellow rice. The "one membrane with double effects" characteristic makes it have great application prospect in the safety quality improvement of yellow rice and other fermented wines.
[0034] The above merely describes preferred embodiments of the present application, and is not intended to limit the present application in any form or in essence. It should be noted that those skilled in the art can make some improvements and supplements without departing from the present application, and these improvements and supplements should also be considered as the protection scope of the present application. For those skilled in the art, some slight changes, modifications and equivalent changes made by using the disclosed technical content without departing from the spirit and scope of the present application are equivalent embodiments of the present application; meanwhile, any equivalent changes, modifications and evolution made according to the essential technology of the present application to the above embodiments are still within the scope of the technical solutions of the present application.
Claims
1. An immobilized urease catalytic ultrafiltration membrane for degrading urea in yellow rice wine, characterized in that: The urease is covalently immobilized on the inner wall of the membrane pores and / or the membrane surface of the high-molecular ultrafiltration membrane by an amide reaction.
2. The immobilized urease catalytic ultrafiltration membrane according to claim 1, characterized by: The amide reaction is realized by first modifying the substrate membrane with polyethyleneimine and then cross-linking the urease with glutaraldehyde as a cross-linking agent.
3. The immobilized urease catalytic ultrafiltration membrane according to claim 1, characterized by: The substrate membrane is made of polysulfone.
4. The immobilized urease catalytic ultrafiltration membrane according to claim 1, characterized by: The pore size of the membrane is 10-50nm; the urease immobilization amount of the immobilized urease catalytic ultrafiltration membrane is 0.5-5.0mg / cm 2 ; the relative activity of the immobilized urease keeps above 80% of the initial activity.
5. A method for the preparation of an immobilized urease catalytic ultrafiltration membrane as claimed in any one of claims 1 to 4, characterized in that, The method comprises the following steps: Step 1. Pretreatment of the substrate membrane: the ultrafiltration membrane is sequentially immersed in a NaOH solution, an ethanol solution and ultrapure water, and then washed with ultrapure water and stored in a refrigerator at 4°C for standby; Step 2. Amino modification: the pretreated substrate membrane obtained in step 1 is immersed in a polyethyleneimine aqueous solution and slowly oscillated for reaction; after the reaction is completed, the polyethyleneimine is removed by washing with ultrapure water, and an amino-modified membrane is obtained; Step 3. Covalent immobilization of urease: the amino-modified membrane obtained in step 2 is immersed in a phosphate buffer solution containing urease, and a covalent cross-linking reaction is performed in the presence of a cross-linking agent glutaraldehyde; Step 4. Post-treatment: after the reaction is completed, the membrane is taken out and washed with a large amount of phosphate buffer solution to remove the physically adsorbed urease, and a urease-immobilized catalytic ultrafiltration membrane is obtained.
6. The method of claim 5, wherein: In step 1, the concentration of the NaOH solution is 0.1M; and the concentration of the ethanol solution is 30%.
7. The method of claim 5, wherein: In step 2, the concentration of the polyethyleneimine aqueous solution is 2% (w / v); the oscillation reaction temperature is 25°C; and the oscillation reaction time is 4 hours.
8. The method of claim 5, wherein: In step 3, the concentration of the urease is 2mg / mL; the phosphate buffer solution is a 0.1M phosphate buffer solution with a pH of 7.0; the concentration of the glutaraldehyde is 0.5% (v / v); the cross-linking reaction temperature is 4-25°C; and the cross-linking reaction time is 2-12 hours.
9. Use of the immobilized urease catalytic ultrafiltration membrane according to any one of claims 1 to 4, characterized in that, The method is used for the treatment of yellow rice wine, rice wine or other fermented wine beverages, simultaneously realizing the clarification and filtration of the wine body and the degradation of urea to reduce the potential content of ethyl carbamate.