Sulfonated GO molecularly imprinted polymer composite membrane as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biochemical engineering, and mainly relates to a sulfonated GO molecularly imprinted polymer composite membrane as well as a preparation method and application thereof. The preparation method of the sulfonated GO molecularly imprinted polymer composite membrane comprises the following steps: dispersing sulfonated graphene oxide in a first solvent, adding a molecularly imprinted polymer for dispersion, then adding PVDF for stirring, and then adding a pore-foaming agent to obtain a membrane casting solution; the membrane casting solution is subjected to membrane scraping, and the sulfonated GO molecularly imprinted polymer composite membrane is obtained through phase conversion. The membrane casting solution is prepared from the molecularly imprinted polymer, the sulfonated graphene oxide, the PVDF and the pore-foaming agent, the composite membrane with good comprehensive performance can be prepared after membrane scraping and phase inversion, the membrane separation effect on a target extract is good, the recognition performance is good, and the application prospect is wide. And the directional filtering effect can be improved, and large-scale separation and purification of trans-ferulic acid can be realized.

Description

Technical Field

[0001] The present application relates to the field of biochemical engineering technology, and mainly to a sulfonated GO molecular imprinting polymer composite membrane and a preparation method and application thereof. Background Art

[0002] Trans-Ferulic acid, also known as (E)-Ferulic acid, is a naturally occurring isomer of ferulic acid. Its chemical name is 4-hydroxy-3-methoxycinnamic acid, an aromatic compound that is widely present in plant cell walls, especially in rye and wheat grains. Trans-Ferulic acid is a yellow powdery crystal with specific physical and chemical properties, such as a melting point of 168-172°C and a boiling point of about 372.3°C. It is soluble in hot water, ethanol and ethyl acetate, slightly soluble in ether, but poorly soluble in benzene and petroleum ether.

[0003] In terms of biological activity, trans-ferulic acid can cause the phosphorylation of β-catenin, thereby promoting proteasome degradation, increasing the expression of the pro-apoptotic factor Bax, and reducing the expression of pro-survival factors. In addition, it has strong antioxidant properties, can effectively remove reactive oxygen species (ROS) and inhibit lipid peroxidation, and has anti-proliferative and anti-migratory effects on the human lung cancer cell line H1299. Trans-ferulic acid is a natural compound with multiple biological activities and wide applications. Its unique chemical structure and biological activity make it have important research value and application potential in many fields. However, there are still many limitations in the large-scale preparation of trans-ferulic acid.

[0004] Molecularly imprinted composite membrane (MICM) is an advanced material that combines molecular imprinting technology and membrane separation technology. Its definition can be summarized as: a composite membrane material obtained by coating or modifying the molecular imprinted polymer (MIP) on the surface of a porous support membrane (such as an ultrafiltration membrane or a microfiltration membrane) to form a cortex with a specific recognition function. Molecularly imprinted composite membranes have the characteristics of specific recognition, high flux, good stability and controllability. The preparation process of molecularly imprinted composite membranes usually includes the following steps: 1) Base membrane selection: Select a suitable commercial porous membrane as a support, such as an ultrafiltration membrane or a microfiltration membrane. 2) Imprinting layer preparation: Mix the raw materials such as template molecules, functional monomers, crosslinkers and initiators evenly to form a precursor solution of the imprinted polymer. Then, the porous support membrane is immersed in the solution, and a molecularly imprinted polymer layer is formed on the surface of the support membrane by light initiation, thermal initiation or chemical initiation. 3) Template elution: The template molecules in the composite membrane are eluted, leaving holes that are complementary to the shape, size and chemical environment of the template molecules. These holes are the recognition sites. 4) Post-treatment: The composite membrane is subjected to necessary post-treatment, such as cleaning and drying, to improve its stability and separation performance.

[0005] The use of molecular imprinting composite membranes has a good application prospect for separating and purifying trans-ferulic acid. Chinese invention patent CN109400804A proposes a method for preparing a ferulic acid molecular imprinting blended membrane material, but only one functional monomer is used, and the recognition site is single. Chinese invention patent CN113087842B also applies molecular imprinting technology, in which α-methyl acrylic acid is used as a functional monomer and magnetic carbon nanotubes are prepared into molecular imprinting polymers. This invention still only uses one functional monomer, and there is a problem of low recognition efficiency for the separation and purification of trans-ferulic acid, which affects the separation and purification efficiency. Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a sulfonated GO molecular imprinting polymer composite membrane and a preparation method and application thereof, aiming to solve the problem of low recognition efficiency when separating and purifying trans-ferulic acid using molecular imprinting technology.

[0007] The technical solution of this application is as follows: In a first aspect, the present application provides a method for preparing a sulfonated GO molecular imprinted polymer composite membrane, which comprises the following steps: Dispersing sulfonated graphene oxide in a first solvent, adding a molecular imprinting polymer to disperse the dispersion, then adding PVDF and stirring, and then adding a porogen to obtain a casting solution; The casting solution is scraped to obtain a sulfonated GO molecular imprinting polymer composite membrane through phase conversion.

[0008] The preparation method provided by the present application can produce a composite membrane with good performance, achieve specific adsorption of the target molecule, and not adsorb other water-soluble impurities, which is conducive to the efficient and highly selective extraction of the target molecule. The membrane separation of the composite membrane can achieve the purpose of efficiently separating and purifying the target product.

[0009] Further, the mass ratio of the sulfonated graphene oxide to the molecular imprinted polymer is 0.9:0.15-0.225; The mass ratio of the molecular imprinting polymer to PVDF is 0.15-0.225:12-18.

[0010] Further, the ratio of the sulfonated graphene oxide to the first solvent is 1:100 (g / ml); The first solvent is N,N-dimethylacetamide; The porogen is one of PEG2000 and PVP, and the mass proportion of the porogen in the casting solution is 2%-12%; The dispersion is carried out by ultrasonic dispersion for 1-2 hours; The PVDF is mechanically stirred for 6-8h after addition; The thickness of the scraping film is 200-300 μm, and the scraping speed is 10-20 mm / s; The phase conversion was carried out by scraping the membrane and then placing it in water for 24 hours for replacement.

[0011] Furthermore, the method for preparing the molecularly imprinted polymer comprises the following steps: dissolving the template molecule in a second solvent; Then add methacrylic acid and acrylamide and mix them, let them stand at 4°C, take the supernatant, add a crosslinking agent and azobisisobutyronitrile, pass nitrogen and seal, heat to the reaction temperature, stir and react, and separate the solid to obtain a polymer material; The template molecules are washed away from the polymer material, and then vacuum dried after washing to obtain the molecular imprinted polymer.

[0012] Further, every 6.5 mmol of the template molecule is added to 10-50 mL of the second solvent for dissolution, and every 6.5 mmol of the template molecule is used with 6-10 mmol of methacrylic acid, 3-8 mmol of acrylamide, 10-20 mmol of a crosslinking agent and 0.1-0.2 mmol of azobisisobutyronitrile; The molecularly imprinted polymer is ground to a particle size of 200-300 meshes.

[0013] Further, the dissolution is carried out by magnetic stirring for 1-2 hours until complete dissolution; the second solvent is one or more of anhydrous ethanol, acetonitrile and methanol; The mixing is carried out by magnetic stirring at room temperature for 2-4 hours; the cross-linking agent is ethylene glycol dimethacrylate; the reaction temperature is 50-70° C.; the stirring reaction is carried out by magnetic stirring for 24-30 hours; The template molecules are washed away by washing the polymer material with anhydrous ethanol using Soxhlet extraction for 36-48 hours until no template molecules are detected in the washing solution; the polymer material is washed with distilled water until it is neutral; and the vacuum drying is performed by maintaining it at 50° C. for 12-15 hours.

[0014] In a second aspect, the present application provides a sulfonated GO molecular imprinted polymer composite membrane, wherein the membrane is prepared by the preparation method of the sulfonated GO molecular imprinted polymer composite membrane as described in the first aspect.

[0015] In a third aspect, the present application provides an application of the sulfonated GO molecular imprinted polymer composite membrane as described in the second aspect, wherein the sulfonated GO molecular imprinted polymer composite membrane is prepared using trans-ferulic acid as a template molecule, and the sulfonated GO molecular imprinted polymer composite membrane is used to separate and purify trans-ferulic acid, comprising the following steps: The sulfonated GO molecular imprinted polymer composite membrane is loaded into a membrane sheet of a membrane separation device; The liquid containing trans-ferulic acid is loaded into a membrane separation device, pressure is applied to the membrane to adsorb and filter, and the filtrate is collected; The sulfonated GO molecularly imprinted polymer composite membrane is then eluted with anhydrous ethanol-organic acid water mixtures of gradient concentrations, the eluate is collected, and the filtrate and the eluate are concentrated under reduced pressure to obtain a crude trans-ferulic acid, which is then purified to obtain trans-ferulic acid.

[0016] Further, the liquid containing trans-ferulic acid is a trans-ferulic acid fermentation liquid, and the concentration of trans-ferulic acid in the trans-ferulic acid fermentation liquid is 50-2000 mg / L; The ratio of the flow rate of the trans-ferulic acid fermentation broth to the area of ​​the sulfonated GO molecular imprinted polymer composite membrane is 70-150 L / h:1 m 2 .

[0017] Further, the elution is performed by sequentially using a mixture of anhydrous ethanol-organic acid water with a volume ratio of 10%, 20%, 40% and 60% of anhydrous ethanol for 2 minutes, 2 minutes, 10 minutes and 10 minutes; the organic acid in the mixture of anhydrous ethanol-organic acid water is one of acetic acid or formic acid, and the volume ratio of the organic acid in the organic acid water is one thousandth; The pressure is 0.1 MPa; The pH of the liquid containing trans-ferulic acid is 3-4, and the adsorption temperature is 25-37° C.; The purification step comprises: The crude trans-ferulic acid was dissolved in ultrapure water to a final concentration of 9 g / L, hydrochloric acid was added to adjust the pH to 3, anhydrous ethanol was added in a volume ratio of ultrapure water to anhydrous ethanol of 3:1, and the mixture was stirred at 20° C. for 5 h and then allowed to stand for 24 h.

[0018] Beneficial effects: The present application prepares the casting solution through molecular imprinting polymer, sulfonated graphene oxide, PVDF and porogen. After scraping and phase transformation, a composite membrane with good comprehensive performance can be obtained. It has good membrane separation effect on the target extract and good recognition performance, which is helpful to achieve large-scale separation and purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the application process of the sulfonated GO molecular imprinting polymer composite membrane of the present application.

[0020] Figure 2 This is a physical picture of the sulfonated GO molecular imprinting polymer composite membrane prepared in Example 1 of the present application.

[0021] Figure 3 This is a scanning electron microscope image of the sulfonated GO molecular imprinting polymer composite membrane prepared in Example 1 of the present application.

[0022] Figure 4 This is a scanning electron microscope cross-sectional image of the sulfonated GO molecular imprinting polymer composite membrane prepared in Example 1 of the present application.

[0023] Figure 5 This is a high performance liquid chromatogram of the crystals recovered by recrystallization in Example 4 of the present application.

[0024] Figure 6 This is a time-of-flight mass spectrum (full scan mode) of the crystals recovered by recrystallization in Example 4 of the present application.

[0025] Figure 7 This is the secondary ion pattern of the time-of-flight mass spectrum of the crystals recovered by recrystallization in Example 4 of the present application.

[0026] Figure 8 This is the infrared spectrum of the crystals recovered by recrystallization in Example 4 of the present application.

[0027] Fig. 9 This is the H NMR spectrum of the crystals recovered by recrystallization in Example 4 of the present application.

[0028] Fig.10 This is the NMR carbon spectrum of the crystals recovered by recrystallization in Example 4 of the present application.

[0029] Fig.11 This is the crystal diffraction spectrum of the crystals recovered by recrystallization in Example 4 of the present application.

[0030] Fig.12 This is a scanning electron microscope image of the sulfonated graphene oxide blank film of Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0031] The present application provides a sulfonated GO molecular imprinted polymer composite membrane and its preparation method and application. In order to make the purpose, technical solution and effect of the present application clearer and more specific, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0032] like Figure 1 As shown, the present application provides a method for preparing a sulfonated GO molecular imprinted polymer composite membrane, which comprises the following steps: S1, dispersing sulfonated graphene oxide (sulfonated GO) in a first solvent, adding a molecular imprinting polymer to disperse, then adding PVDF (polyvinylidene fluoride) and stirring, and then adding a porogen to mix to obtain a casting solution; S2. Scrape the casting solution to obtain the sulfonated GO molecular imprinting polymer composite membrane through phase conversion.

[0033] The preparation method provided by the present application can produce a composite membrane with good performance, achieve specific adsorption of the target molecule, and not adsorb other water-soluble impurities, which is conducive to the efficient and highly selective extraction of the target molecule. The membrane separation of the composite membrane can achieve the purpose of efficiently separating and purifying the target product.

[0034] Among them, the dispersion of sulfonated graphene oxide and molecular imprinting polymer can be carried out by ultrasonic dispersion for 1-2 hours; PVDF is mixed by mechanical stirring for 6-8 hours after addition; scraping can be carried out by a scraping machine, the scraping thickness is 200-300μm, and the scraping speed is 10-20mm / s; phase conversion is carried out by placing the scraping film in water for 24 hours, and after the replacement is completed, it can be stored in a mixed solution of water and glycerol with a volume ratio of 1:1 for standby use.

[0035] Furthermore, in step S1, the method for preparing the molecularly imprinted polymer comprises the following steps: S11, dissolving the template molecule in a second solvent; S12, adding methacrylic acid and acrylamide, mixing, letting stand at 4°C, taking the supernatant, adding a crosslinking agent and azobisisobutyronitrile, passing nitrogen, sealing, heating to the reaction temperature, stirring and reacting, separating the solid to obtain a polymer material; S13, washing the polymer material to remove the template molecules, and then vacuum drying after washing to obtain a molecularly imprinted polymer.

[0036] After the molecularly imprinted polymer is prepared, it can be lyophilized for later use.

[0037] Wherein, by introducing nitrogen gas, the subsequent heating reaction process is carried out under a nitrogen atmosphere.

[0038] Furthermore, in step S11, the dissolution can be carried out by magnetic stirring for 1-2 hours until complete dissolution; the second solvent is one or more of anhydrous ethanol, acetonitrile and methanol, and can be specifically selected according to the template molecules that need to be dissolved to ensure that the template molecules can be completely dissolved.

[0039] Further, in step S12, the mixing can be carried out by magnetic stirring at room temperature for 2-4 hours; the cross-linking agent is ethylene glycol dimethacrylate; the reaction temperature is 50-70°C; and the stirring reaction is carried out by magnetic stirring for 24-30 hours.

[0040] The 4° C. standing in step S12 can be performed by placing in a refrigerator. By standing at 4° C., the solubility can be reduced, causing some unwanted polymers or impurities to precipitate or precipitate from the solution. The supernatant can be taken after the solution is stabilized.

[0041] Further, in step S13, the template molecules are washed away by washing the polymer material with anhydrous ethanol using Soxhlet extraction for 36-48 hours until no template molecules are detected in the washing solution; the polymer material is washed with distilled water until it is neutral; and vacuum drying is performed by maintaining it at 50°C for 12-15 hours.

[0042] Further, every 6.5 mmol of the template molecule is added to 10-50 mL of the second solvent for dissolution, and every 6.5 mmol of the template molecule is used with 6-10 mmol of methacrylic acid, 3-8 mmol of acrylamide, 10-20 mmol of a crosslinking agent and 0.1-0.2 mmol of azobisisobutyronitrile.

[0043] Further, after the molecular imprinted polymer is prepared, the molecular imprinted polymer is ground to a particle size of 200-300 mesh. Wherein, the grinding can be performed by agate. Specifically, if the particle size of the molecular imprinted polymer is too large, the pores on the membrane surface will also be of different sizes, and if it is too small, the membrane separation effect will be affected. The present application is beneficial to improving the surface flatness of the composite membrane and stabilizing the water flux by controlling the particle size of the molecular imprinted polymer within a suitable range. The molecular imprinted polymer particles evenly distributed on the membrane surface can also be clearly seen in the electron microscope image, which is beneficial to improving the separation effect.

[0044] Specifically, the template molecule can be a target molecule that can react and bind with methacrylic acid or acrylamide. By selecting suitable functional monomers to prepare molecular imprinted polymers, the recognition effect is improved. More preferably, the template molecule is a target molecule that can react with both methacrylic acid and acrylamide, such as trans-ferulic acid with hydroxyl and carboxyl groups. By using bifunctional monomers for preparation, a molecular imprinted polymer with dual recognition sites can be obtained, which is beneficial to improving its specific adsorption performance.

[0045] Furthermore, since the selected methacrylic acid and acrylamide may also react. In the present application, when adding the functional monomer to the second solvent, methacrylic acid may be added first and stirred, so that the template molecule and methacrylic acid are first coordinated to a certain extent, and then acrylamide is added and stirred to reduce unnecessary polymerization. At the same time, methacrylic acid provides a hydrophobic environment, and acrylamide can form hydrogen bonds with the template molecule through its amide group. The combination of the two can form a specific three-dimensional structure in the polymer, better matching the molecular shape of trans-ferulic acid, thereby improving the selective recognition ability.

[0046] The present application prepares molecular imprinting polymers through bifunctional monomers, which can identify the template molecule well, improve the specific recognition efficiency, and help improve the purity and recovery rate. Taking the template molecule as trans-ferulic acid as an example, a molecular imprinting polymer with specific recognition of trans-ferulic acid is formed, and the complex can selectively adsorb trans-ferulic acid without adsorbing other water-soluble impurities, thereby efficiently and highly selectively separating and purifying trans-ferulic acid. Experiments show that the purity of the trans-ferulic acid separated by the present application can reach more than 96%, and the recovery rate can reach more than 85%.

[0047] Furthermore, in step S1, the ratio of sulfonated graphene oxide to the first solvent is 1:100 (g / ml); and the first solvent is N,N-dimethylacetamide.

[0048] Further, in step S1, the mass ratio of molecular imprinting polymer to PVDF is 0.15-0.225:12-18. In the present application, the selected PVDF as the substrate of the composite membrane has excellent chemical stability and mechanical strength, but PVDF has a certain hydrophobicity. If the amount is too much, it will affect the adsorption effect of the molecular imprinting polymer on the extraction solution. By controlling the ratio between the molecular imprinting polymer and PVDF, it is beneficial to maintain the performance of the composite membrane while ensuring the separation and purification effect.

[0049] Furthermore, in step S1, the mass ratio of sulfonated graphene oxide to molecular imprinting polymer is 0.9:0.15-0.225. Sulfonated graphene oxide has high rigidity and strength. If its content in the membrane is too high, it may cause the flexibility of the membrane to decrease. Therefore, it is necessary to control the amount to balance the mechanical strength and flexibility to ensure the stability and operability of the membrane.

[0050] More specifically, the combination of methacrylic acid and acrylamide can provide both hydrophobic and hydrophilic regions. This amphiphilic property helps to capture and release template molecules more effectively, which is particularly important for selective separation in the membrane preparation process and can improve the membrane's permeability selectivity and separation efficiency. Moreover, by controlling the dosage ratio of sulfonated graphene oxide to molecular imprinting polymer, sulfonated graphene oxide has a high surface area and negative charge density, and the sulfonic acid groups on it can match the amino and carboxyl groups in the bifunctional monomer imprinting polymer to form ionic bonds. In this way, the membrane material can not only selectively separate target molecules based on molecular size and shape, but also further improve selectivity through ion exchange mechanisms, which is beneficial to improving the directional filtration effect.

[0051] Microbial synthesis is simple and efficient, and can be prepared on a large scale. The composite membrane made by this application can also be used in the separation and purification of fermentation broth. However, the composition of the fermentation broth is complex, and it is easy to affect the adsorption effect of the composite membrane due to hydrophobic problems. Moreover, due to the hydrophobicity of PVDF and the hydrophilicity of the bifunctional monomer molecular imprinting polymer, the separation and purification effect may be affected due to compatibility issues. This application uses sulfonated graphene oxide to increase the hydrophilicity and dispersion stability of the membrane material. In combination with the use of porogens, compatibility can be improved, and the pore structure formation and stability of the composite membrane can be enhanced. The stock solution required to be extracted can also pass through the composite membrane well. By controlling the amount of each other added, the water flux is more suitable within the provided ratio range. While maintaining a certain extraction flux, the target molecule is extracted, and the target molecule is extracted while being concentrated, reducing energy consumption.

[0052] Furthermore, the porogen is one of PEG2000 or PVP, and the mass proportion of the porogen in the casting solution is 2%-12%. In the present application, the combination of sulfonated graphene oxide and molecular imprinting polymer is good. Without the addition of a porogen, the pores on the membrane surface are relatively small, which is easy to affect the water permeability. In the present application, finger-like pores are formed by adding a certain amount of PEG2000 or PVP. Finger-like pores of different sizes can be seen by observing the cross-section of the membrane through an electron microscope. Among them, the water flux will also increase with the increase of the size of the finger-like pores during membrane separation.

[0053] This application adopts the nonsolvent induced phase separation method to mix the eluted molecular imprinting polymer, sulfonated graphene oxide and PVDF, and then induces the polymer solution to undergo phase separation through a nonsolvent, thereby forming a membrane product with a specific pore structure. The membrane prepared by the NIPS method usually has the characteristics and advantages of an asymmetric membrane with pores in the skin, good flexibility, long-term hydrophilicity and easily regulated pore structure: it has a simple process flow, excellent film-forming performance and easy to regulate pore structure, and has broad application prospects in many fields.

[0054] The present application also provides a sulfonated GO molecular imprinted polymer composite membrane, wherein the composite membrane is prepared by the above-mentioned preparation method of the sulfonated GO molecular imprinted polymer composite membrane. The prepared composite membrane has good specific and selective adsorption performance of the target.

[0055] The present application also provides an application of the sulfonated GO molecular imprinted polymer composite membrane as described above, wherein the sulfonated GO molecular imprinted polymer composite membrane is prepared using trans-ferulic acid as a template molecule, and the sulfonated GO molecular imprinted polymer composite membrane is used to separate and purify trans-ferulic acid, comprising the following steps: The sulfonated GO molecular imprinted polymer composite membrane is loaded into the membrane sheet of the membrane separation device; The liquid containing trans-ferulic acid is loaded into a membrane separation device, pressure is applied to the membrane to adsorb and filter, and the filtrate is collected; The sulfonated GO molecularly imprinted polymer composite membrane is then eluted with a mixture of anhydrous ethanol and organic acid water with gradient concentrations, the eluate is collected, and the filtrate and the eluate are concentrated under reduced pressure to obtain a crude trans-ferulic acid, which is then purified to obtain trans-ferulic acid.

[0056] Specifically, the membrane separation device can be a cross-flow integrated flat membrane separation device or a tubular membrane separation device, etc., as long as the sulfonated GO molecular imprinting polymer composite membrane can be installed for membrane separation. Among them, the cross-flow integrated flat membrane separation device has a long service life and high flux. Through membrane cross-flow filtration, the problem of overproduction or insufficient production of trans-ferulic acid can be reduced, and an efficient balance of membrane separation can be achieved.

[0057] The organic acid in the mixed solution of anhydrous ethanol and organic acid water is one of acetic acid and formic acid, and the volume proportion of the organic acid in the organic acid water is one thousandth.

[0058] Further, elution is carried out by eluting 2 minutes, 2 minutes, 10 minutes and 10 minutes by using the mixed solution of anhydrous ethanol-organic acid water whose volume proportion is 10%, 20%, 40% and 60% respectively. After the present application carries out membrane separation and filtration enrichment to the liquid containing trans-ferulic acid, the industrial cost of trans-ferulic acid adsorbed by the gradient elution recovery membrane is lower, which is conducive to the promotion and application of the large-scale preparation of trans-ferulic acid. Specifically, by using the mixed solution of anhydrous ethanol-organic acid water with a lower concentration of 10% in the elution stage for preliminary elution, the impurities with weaker adsorption strength are mainly removed, and then the impurities with medium adsorption strength are gradually removed by 20% concentration, and some molecules and a small part of trans-ferulic acid that are tightly combined with the composite membrane are eluted by 40% concentration, and finally eluted by 60% concentration for 10 minutes, which helps to elute most of the target molecules. The composite membrane can also be used repeatedly and filtered again after being recovered by elution.

[0059] Furthermore, after elution with a mixture of anhydrous ethanol-organic acid water with gradient concentrations, the eluate of the mixture of anhydrous ethanol-organic acid water with a volume ratio of 40% and 60% of anhydrous ethanol is collected. After the eluate is dried, a crude trans-ferulic acid can be obtained with a high purity.

[0060] Further, the liquid containing transferulic acid is preferably transferulic acid fermentation liquid, which can be prepared by transferulic acid production bacteria. Transferulic acid synthesis by microorganisms has the characteristics of simple and efficient, and is convenient for large-scale preparation. The application uses a good composite membrane for membrane separation, which can achieve the purpose of efficient separation and purification of transferulic acid.

[0061] Further, the concentration of trans-ferulic acid in the liquid containing trans-ferulic acid is 50-2000 mg / L. Generally speaking, the amount of trans-ferulic acid in the fermentation liquid containing trans-ferulic acid should not exceed the maximum adsorption capacity of the composite membrane, and controlling the appropriate concentration range is conducive to improving the separation effect.

[0062] Furthermore, the pH of the trans-ferulic acid fermentation broth is 3-4, preferably 3.27, and the adsorption temperature is 25-37° C. By controlling the pH of the sample solution and the adsorption temperature, the separation effect of the sulfonated GO molecular imprinting polymer composite membrane on trans-ferulic acid is improved.

[0063] Furthermore, the ratio of the flow rate of the trans-ferulic acid fermentation broth to the area of ​​the sulfonated GO molecularly imprinted polymer composite membrane is 70-150 L / h:1 m2 Among them, when performing large-scale separation, the area of ​​sulfonated GO molecular imprinting polymer composite membrane can be controlled in the range of 0.5-4m 2 .

[0064] Furthermore, the pressure is 0.1 MPa. If the pressure is too high, it is easy to cause adsorption failure, and if the pressure is too low, the utilization rate is low. Under this pressure, the adsorption effect is better.

[0065] Further, the filtration time is 0.5-1h. Specifically, the sulfonated GO molecular imprinting polymer composite membrane provided by the present application mainly plays a directional filtration function, and the membrane adsorption amount is less than the filtration amount. And by controlling the concentration, flow rate and filtration pressure of the trans-ferulic acid fermentation broth, the filtrate concentration will generally gradually stabilize after filtration for 0.5-1h, and the filtration can also be continued for separation at this time, but the composite membrane is easily affected by the concentration of the concentrate or the accumulation of impurities in the fermentation broth. The membrane separation performance of the composite membrane, therefore, is preferably 0.5-1h, and the membrane separation filtration is repeated after the composite membrane is eluted. It is not easy to block while being conducive to maintaining a high flux, and it is also conducive to the composite membrane being able to maintain the adsorption and directional filtration effect of trans-ferulic acid for a long time.

[0066] Specifically, after the crude trans-ferulic acid is obtained by elution and concentration, it can be purified by recrystallization. The crude trans-ferulic acid is dissolved in ultrapure water to a final concentration of 9 g / L of trans-ferulic acid. Hydrochloric acid is added to adjust the pH to 3. Anhydrous ethanol is added in a volume ratio of ultrapure water to anhydrous ethanol of 3:1. The mixture is stirred at 20°C for 5 hours and then allowed to stand for 24 hours. The purity of the obtained trans-ferulic acid crystals can reach more than 99%.

[0067] The application method provided in the present application has simple process operation and low cost, is convenient for large-scale industrial production, and is of great significance for promoting the industrial development of trans-ferulic acid.

[0068] The invention is further described below by means of specific examples.

[0069] The trans-ferulic acid producing bacteria used in the examples of the present application are from the research group of Xiaolin Shen of Beijing University of Chemical Technology, reference (Targeting cofactors regeneration in methylation and hydroxylation for high level production of Ferulic acid, Xiaolin Shen (Beijing University of Chemical Technology) etc.).

[0070] The fermentation medium and enrichment medium used in the examples of the present application are both M9 medium: 2 mL of 1M MgSO4, 0.1 mL of 1M CaCl2, 200 mL of 5×M9 salt solution (Na2PO4·7H2O: 12.8 g, KH2PO4: 3.0 g, NaCl: 0.5 g, NH4Cl: 1.0 g, dissolved in 200 mL of double distilled water, sterilized at 121°C for 15 min); 20 mL of 20% glucose solution, plus 1000 mL of sterilized double distilled water.

[0071] Example 1 Example 1 The method for separating and purifying trans-ferulic acid fermentation broth using sulfonated GO molecular imprinting polymer composite membrane comprises the following steps: (1) Preparation of trans-ferulic acid fermentation broth: Trans-ferulic acid fermentation broth was prepared using trans-ferulic acid producing bacteria. The specific method was as follows: Escherichia coli was inoculated into a fermentation medium at an inoculation rate of 5%, and the culture was shaken at 37°C for 3 days to obtain a seed solution; then the seed solution was inoculated into an enrichment medium at an inoculation rate of 10%, and a precursor required for trans-ferulic acid fermentation was added, and the culture was shaken at 37°C for 5 days. The culture solution was centrifuged at 5°C and 9000r / min for 15 minutes, and the supernatant was collected to obtain a trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 70 mg / L.

[0072] (2) Preparation of sulfonated GO molecularly imprinted polymer composite membrane: 0.9 g of sulfonated graphene oxide was added to 90 mL of N,N-dimethylacetamide and dispersed by ultrasound for 1 h. 0.225 g of molecular imprinting polymer was added and dispersed by ultrasound for 1 h. Then 12 g of PVDF was added and mechanically stirred for 6 h. Then, 3% of the mass of the porogen PVP in the casting solution was added. The film was scraped by a scraping machine with a depth of 250 μm and a speed of 15 mm / s. The film was then replaced in water for 24 h. After phase inversion, a sulfonated GO molecular imprinting polymer composite membrane was obtained. A label was attached to the front and the membrane was stored in a mixed solution of water and glycerol in a volume ratio of 1:1 for later use.

[0073] Wherein, trans-ferulic acid is used as a template molecule, and the preparation method of the molecularly imprinted polymer is as follows: 6.5 mmol of trans-ferulic acid was added to 10 mL of methanol solution and magnetically stirred for 1 hour until completely dissolved, then 8 mmol of methacrylic acid and 5 mmol of acrylamide were added, magnetically stirred at room temperature for 3 hours, placed in a refrigerator at 4°C and allowed to stand for 3 hours, then the supernatant was taken, and then 15 mmol of ethylene glycol dimethacrylate and 0.122 mmol of azobisisobutyronitrile were added, nitrogen was introduced for 5 minutes, sealed, heated to 60°C, magnetically stirred for reaction for 24 hours, separated, and a polymer material was obtained; The polymer material was washed with ethanol for 36 hours after Soxhlet extraction to remove the template molecule trans-ferulic acid in the polymer material until the template molecule trans-ferulic acid could not be detected in the eluent, and then the eluted polymer material was washed with distilled water until neutral, and vacuum dried at 50°C for 12 hours to obtain molecularly imprinted polymer, which was freeze-dried and ground into 200 mesh on agate for later use.

[0074] The scanning electron microscopy image of the sulfonated GO molecular imprinting polymer composite membrane prepared in Example 1 is as follows: Figure 3 The scanning electron microscope cross-section is shown in Figure 4 shown.

[0075] (3) Membrane adsorption and separation: The membrane extraction adsorption content was measured by applying pressure to the membrane. The concentration of trans-ferulic acid in the trans-ferulic acid fermentation broth was adjusted to 50 mg / L and pH = 3.27. 2 The sulfonated GO molecular imprinting polymer composite membrane was loaded into the membrane sheet of the cross-flow integrated flat membrane separation device (circular membrane sheet with an effective radius of 2 cm). The trans-ferulic acid fermentation broth was loaded into the cross-flow integrated flat membrane separation device. The flow rate was controlled, and the ratio of the flow rate of the trans-ferulic acid fermentation broth to the area of ​​the sulfonated GO molecular imprinting polymer composite membrane was 70L / h:1m 2 At a temperature of 25°C and a humidity of 55%, a pressure of 0.1 MPa was applied to the membrane. After 1 hour of membrane filtration, the filtrate concentration (filtrate is the liquid that permeates the membrane) was 37.97 mg / L, the concentrate concentration (liquid that did not permeate the membrane in time during cross-flow filtration flushed the remaining substances on the membrane surface and returned to the unfiltered tank to be repeatedly concentrated) was 38.33 mg / L, and the membrane flux was 44.86 L / m 2 h, and then the composite membrane was removed and eluted with 50 ml of anhydrous ethanol for 30 min. The maximum adsorption capacity of the membrane performance was determined by elution with anhydrous ethanol, and the trans-ferulic acid concentration was determined and the mass of the eluted trans-ferulic acid was calculated to be 2.52 mg. The mass of the membrane extraction adsorption was calculated to be about 2002.36 mg / m 2 .

[0076] The membrane permeation experiment was carried out on the sulfonated GO molecular imprinting polymer composite membrane. Membrane permeation bottle A and membrane permeation bottle B of the same specifications were set up respectively. The two bottles were connected by a sulfonated GO molecular imprinting polymer composite membrane (effective radius of 2 cm). 120 mL of trans-ferulic acid fermentation broth was placed in membrane permeation bottle A, and the concentration of trans-ferulic acid fermentation broth was 83.3 mg / L and the pH was 3.27; 120 mL of pure water was placed in membrane permeation bottle B. Under the conditions of humidity of 55% and temperature of 37°C, natural permeation was carried out through concentration difference (no pressure was applied to the composite membrane). At this time, a part of trans-ferulic acid not only penetrated into the membrane permeation bottle B filled with pure water, but also adsorbed on the composite membrane. The concentration change of trans-ferulic acid in membrane permeation bottle A and membrane permeation bottle B after 121 hours of permeation was measured. The test results are shown in Table 1: Table 1

[0077] At 37°C, the ferulic acid concentration on both sides of the composite membrane basically reached equilibrium after 121 hours of permeation. After the composite membrane was removed, it was eluted with 50 mL of anhydrous ethanol for 30 minutes. The mass of adsorbed trans-ferulic acid was measured to be 2.08 mg, and the calculated membrane permeation adsorption amount was about 1652.32 mg / m 2 .

[0078] In application, trans-ferulic acid adsorbed on the composite membrane can be extracted and purified by gradient elution; moreover, in the presence of a composite membrane, trans-ferulic acid-producing bacteria and some impurities cannot pass through the membrane, so a preliminary separation is carried out, and trans-ferulic acid can be adsorbed on the composite membrane, and after collection, trans-ferulic acid can be extracted and then purified. Trans-ferulic acid can also pass through the membrane to the membrane permeation bottle B, and the permeate can be collected to extract and purify trans-ferulic acid.

[0079] (4) Recrystallization: After the membrane extraction in step (3), the filtrate is collected, and the sulfonated GO molecular imprinting polymer composite membrane extracted by the membrane is eluted with anhydrous ethanol-organic acid water mixture of gradient concentrations in sequence, and the eluate of the anhydrous ethanol-organic acid water mixture with an anhydrous ethanol volume ratio of 40% and 60% and the filtrate of the membrane extraction are collected, and the collected filtrate and eluate are concentrated under reduced pressure to obtain crude trans-ferulic acid. The crude trans-ferulic acid is dissolved in ultrapure water to a final concentration of trans-ferulic acid of 9 g / L, and hydrochloric acid is added to adjust the pH to 3, and anhydrous ethanol is added in a volume ratio of ultrapure water to anhydrous ethanol of 3:1, and the mixture is stirred at 20° C. for 5 hours and then allowed to stand for 24 hours. The purity of the obtained trans-ferulic acid crystals is above 95.6%, and the overall recovery rate reaches 86.5%.

[0080] The concentration (volume percentage) and elution time of the gradient concentration anhydrous ethanol-organic acid water mixture are shown in Table 2: Table 2

[0081] Example 2 Example 2 The method for separating and purifying trans-ferulic acid fermentation broth using sulfonated GO molecular imprinting polymer composite membrane comprises the following steps: (1) Preparation of trans-ferulic acid fermentation broth: Trans-ferulic acid fermentation broth was prepared using trans-ferulic acid producing bacteria. The specific method was as follows: Escherichia coli was inoculated into a fermentation medium at an inoculation rate of 5%, and the culture was shaken at 37°C for 3 days to obtain a seed solution; then the seed solution was inoculated into an enrichment medium at an inoculation rate of 10%, and a precursor required for trans-ferulic acid fermentation was added, and the culture was shaken at 37°C for 5 days. The culture solution was centrifuged at 5°C and 9000r / min for 15 minutes, and the supernatant was collected to obtain a trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 70 mg / L.

[0082] (2) Preparation of sulfonated GO molecularly imprinted polymer composite membrane: 0.9 g of sulfonated graphene oxide was added to 90 mL of N,N-dimethylacetamide and dispersed by ultrasonication for 1 h. 0.225 g of molecular imprinting polymer was added and dispersed by ultrasonication for 1 h. Then 12 g of PVDF was added and mechanically stirred for 6 h. Then, 10% of the mass of the porogen PVP in the casting solution was added. The film was scraped by a scraping machine with a depth of 250 μm and a speed of 15 mm / s. The film was then replaced in water for 24 h. After phase inversion, a sulfonated GO molecular imprinting polymer composite membrane was obtained. A label was affixed to the front and the membrane was stored in a mixed solution of water and glycerol in a volume ratio of 1:1 for later use.

[0083] Wherein, trans-ferulic acid is used as a template molecule, and the preparation method of the molecularly imprinted polymer is as follows: 6.5 mmol of trans-ferulic acid was added to 10 mL of methanol solution and magnetically stirred for 1 hour until completely dissolved, then 8 mmol of methacrylic acid and 5 mmol of acrylamide were added, magnetically stirred at room temperature for 3 hours, placed in a refrigerator at 4°C and allowed to stand for 3 hours, then the supernatant was taken, and then 15 mmol of ethylene glycol dimethacrylate and 0.122 mmol of azobisisobutyronitrile were added, nitrogen was introduced for 5 minutes, sealed, heated to 60°C, magnetically stirred for reaction for 24 hours, separated, and a polymer material was obtained; The polymer material was washed with ethanol for 36 hours after Soxhlet extraction to remove the template molecule trans-ferulic acid in the polymer material until the template molecule trans-ferulic acid could not be detected in the eluent, and then the eluted polymer material was washed with distilled water until neutral, and vacuum dried at 50°C for 12 hours to obtain molecularly imprinted polymer, which was freeze-dried and ground into 200 mesh on agate for later use.

[0084] A trans-ferulic acid standard solution with a trans-ferulic acid concentration of 1600 mg / L was prepared, and the solvent was ethanol solution. 0.1 g of the sulfonated GO molecular imprinting polymer composite membrane prepared in Example 2 was added to the trans-ferulic acid standard solution and adsorbed for 24 hours. The membrane was taken out and dried, and the overall mass was measured. The adsorption capacity of the sulfonated GO molecular imprinting polymer composite membrane in Example 2 was measured to be 28.86 mg / g.

[0085] (3) Membrane adsorption and separation: The membrane extraction adsorption content was measured by applying pressure to the membrane. The concentration of trans-ferulic acid in the trans-ferulic acid fermentation broth was adjusted to 50 mg / L and pH = 3.51. 2 The sulfonated GO molecular imprinting polymer composite membrane was loaded into the membrane sheet of the cross-flow integrated flat membrane separation device (circular membrane sheet with an effective radius of 2 cm). The trans-ferulic acid fermentation broth was loaded into the cross-flow integrated flat membrane separation device. The flow rate was controlled, and the ratio of the flow rate of the trans-ferulic acid fermentation broth to the area of ​​the sulfonated GO molecular imprinting polymer composite membrane was 70L / h:1m 2 At a temperature of 25°C and a humidity of 55%, a pressure of 0.1 MPa was applied to the membrane. After 1 hour of membrane filtration, the filtrate concentration (filtrate is the liquid that permeates the membrane) was 30.92 mg / L, the concentrate concentration (liquid that did not permeate the membrane in time during cross-flow filtration flushed the remaining substances on the membrane surface and returned to the unfiltered tank to be repeatedly concentrated) was 35.57 mg / L, and the membrane flux was 64.43 L / m 2 After the composite membrane was removed, it was eluted with 50 ml of anhydrous ethanol for 30 min. The concentration of trans-ferulic acid was measured and the mass of trans-ferulic acid eluted was calculated to be 2.66 mg. The mass of membrane extraction adsorption was about 2118.78 mg / m 2 .

[0086] At a temperature of 37°C and a pH of 3.51, 120 mL of 83.3 mg / L trans-ferulic acid fermentation broth was used to carry out the same membrane permeation experiment as in Example 1. The test results are shown in Table 3: Table 3

[0087] After the composite membrane was taken out, it was eluted with 50 mL of anhydrous ethanol for 30 min, and the mass of adsorbed trans-ferulic acid was measured to be 2.26 mg. It can be seen that at 37°C, the membrane permeation adsorption capacity of the sulfonated GO molecular imprinting polymer composite membrane of Example 2 was about 1800.56 mg / m 2 .

[0088] (4) Recrystallization: After the membrane extraction in step (3), the filtrate was collected, and the sulfonated GO molecular imprinting polymer composite membrane after membrane extraction was eluted with anhydrous ethanol-organic acid water mixture under the same gradient concentration conditions as in Example 1, and the eluate of the anhydrous ethanol-organic acid water mixture with an anhydrous ethanol volume ratio of 40% and 60% was collected. The collected filtrate and eluate were concentrated under reduced pressure to obtain crude trans-ferulic acid. The crude trans-ferulic acid was dissolved in ultrapure water to a final concentration of 9 g / L of trans-ferulic acid, and hydrochloric acid was added to adjust the pH to 3. Anhydrous ethanol was added in a volume ratio of ultrapure water to anhydrous ethanol of 3:1, and the mixture was stirred at 20°C for 5 hours and then allowed to stand for 24 hours. The purity of the obtained trans-ferulic acid crystals was above 96.2%, and the overall recovery rate reached 86.13%.

[0089] Example 3 Example 3 The method for separating and purifying trans-ferulic acid fermentation broth using sulfonated GO molecular imprinting polymer composite membrane comprises the following steps: (1) Preparation of trans-ferulic acid fermentation broth: Trans-ferulic acid fermentation broth was prepared using trans-ferulic acid producing bacteria. The specific method was as follows: Escherichia coli was inoculated into a fermentation medium at an inoculation rate of 5%, and the culture was shaken at 37°C for 3 days to obtain a seed solution; then the seed solution was inoculated into an enrichment medium at an inoculation rate of 10%, and a precursor required for trans-ferulic acid fermentation was added, and the culture was shaken at 37°C for 5 days. The culture solution was centrifuged at 5°C and 9000r / min for 15 minutes, and the supernatant was collected to obtain a trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 70 mg / L.

[0090] (2) Preparation of sulfonated GO molecularly imprinted polymer composite membrane: 0.9 g of sulfonated graphene oxide was added to 90 mL of N,N-dimethylacetamide and dispersed by ultrasound for 1 h. 0.225 g of molecular imprinting polymer was added and dispersed by ultrasound for 1 h. Then 12 g of PVDF was added and mechanically stirred for 6 h. Then 4.5% of the mass of porogen PEG 2000 in the casting solution was added. The film was scraped by a scraper with a depth of 250 μm and a speed of 15 mm / s. The film was then replaced in water for 24 h. After phase inversion, a sulfonated GO molecular imprinting polymer composite membrane was obtained. A label was affixed to the front and the membrane was stored in a mixed solution of water and glycerol in a volume ratio of 1:1 for later use.

[0091] Wherein, trans-ferulic acid is used as a template molecule, and the preparation method of the molecularly imprinted polymer is as follows: 6.5 mmol of trans-ferulic acid was added to 10 mL of methanol solution and magnetically stirred for 1 hour until completely dissolved, then 8 mmol of methacrylic acid and 5 mmol of acrylamide were added, magnetically stirred at room temperature for 3 hours, placed in a refrigerator at 4°C and allowed to stand for 3 hours, then the supernatant was taken, and then 15 mmol of ethylene glycol dimethacrylate and 0.122 mmol of azobisisobutyronitrile were added, nitrogen was introduced for 5 minutes, sealed, heated to 60°C, magnetically stirred for reaction for 24 hours, separated, and a polymer material was obtained; The polymer material was washed with ethanol for 36 hours after Soxhlet extraction to remove the template molecule trans-ferulic acid in the polymer material until the template molecule trans-ferulic acid could not be detected in the eluent, and then the eluted polymer material was washed with distilled water until neutral, and vacuum dried at 50°C for 12 hours to obtain molecularly imprinted polymer, which was freeze-dried and ground into 200 mesh on agate for later use.

[0092] (3) Membrane adsorption and separation: The membrane extraction adsorption content was measured by applying pressure to the membrane. The concentration of trans-ferulic acid in the trans-ferulic acid fermentation broth was adjusted to 50 mg / L and pH = 3.27. 2 The sulfonated GO molecular imprinting polymer composite membrane was loaded into the membrane sheet of the cross-flow integrated flat membrane separation device (circular membrane sheet with an effective radius of 2 cm). The trans-ferulic acid fermentation broth was loaded into the cross-flow integrated flat membrane separation device. The flow rate was controlled, and the ratio of the flow rate of the trans-ferulic acid fermentation broth to the area of ​​the sulfonated GO molecular imprinting polymer composite membrane was 70L / h:1m 2 At a temperature of 25°C and a humidity of 55%, a pressure of 0.1 MPa was applied to the membrane. After 1 hour of membrane filtration, the filtrate concentration (filtrate is the liquid that permeates the membrane) was 33.83 mg / L, the concentrate concentration (liquid that did not permeate the membrane in time during cross-flow filtration flushed the remaining substances on the membrane surface and returned to the unfiltered tank to be repeatedly concentrated) was 35.81 mg / L, and the membrane flux was 40.57 L / m 2 After the composite membrane was removed, 50 ml of anhydrous ethanol was used for elution for 30 min. The concentration of trans-ferulic acid was determined and the mass of trans-ferulic acid eluted was calculated to be 2.71 mg. The mass of membrane extraction adsorption was about 2152.55 mg / m 2 .

[0093] At a temperature of 37°C and a pH of 3.27, 120 mL of 83.3 mg / L trans-ferulic acid fermentation broth was used to perform the same membrane permeation experiment as in Example 1. The test results are shown in Table 4: Table 4

[0094] After the composite membrane was taken out, it was eluted with 50 mL of anhydrous ethanol for 30 min, and the mass of adsorbed trans-ferulic acid was measured to be 2.12 mg. It can be seen that at 37°C, the membrane permeation adsorption capacity of the sulfonated GO molecularly imprinted polymer composite membrane of Example 3 was about 1683.18 mg / m 2 .

[0095] (4) Recrystallization: After the membrane extraction in step (3), the filtrate was collected, and the sulfonated GO molecular imprinting polymer composite membrane after membrane extraction was eluted with anhydrous ethanol-organic acid water mixture under the same gradient concentration conditions as in Example 1, and the eluate of the anhydrous ethanol-organic acid water mixture with an anhydrous ethanol volume ratio of 40% and 60% was collected. The collected filtrate and eluate were concentrated under reduced pressure to obtain crude trans-ferulic acid. The crude trans-ferulic acid was dissolved in ultrapure water to a final concentration of 9 g / L of trans-ferulic acid, and hydrochloric acid was added to adjust the pH to 3. Anhydrous ethanol was added in a volume ratio of ultrapure water to anhydrous ethanol of 3:1, and the mixture was stirred at 20°C for 5 hours and then allowed to stand for 24 hours. The purity of the obtained trans-ferulic acid crystals was above 95.4%, and the overall recovery rate reached 86.34%.

[0096] Example 4 Example 4 The method for separating and purifying trans-ferulic acid fermentation broth using sulfonated GO molecular imprinting polymer composite membrane comprises the following steps: (1) Preparation of trans-ferulic acid fermentation broth: Trans-ferulic acid fermentation broth was prepared using trans-ferulic acid producing bacteria. The specific method was as follows: Escherichia coli was inoculated into a fermentation medium at an inoculation rate of 5%, and the culture was shaken at 37°C for 3 days to obtain a seed solution; then the seed solution was inoculated into an enrichment medium at an inoculation rate of 10%, and a precursor required for trans-ferulic acid fermentation was added, and the culture was shaken at 37°C for 5 days. The culture solution was centrifuged at 5°C and 9000r / min for 15 minutes, and the supernatant was collected to obtain a trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 70 mg / L.

[0097] (2) Preparation of sulfonated GO molecularly imprinted polymer composite membrane: 0.9 g of sulfonated graphene oxide was added to 90 mL of N,N-dimethylacetamide and dispersed by ultrasonication for 1 h. 0.225 g of molecular imprinting polymer was added and dispersed by ultrasonication for 1 h. Then 12 g of PVDF was added and mechanically stirred for 6 h. Then 6% of the mass of porogen PEG 2000 in the casting solution was added. The film was scraped by a scraper with a depth of 250 μm and a speed of 15 mm / s. The film was then replaced in water for 24 h. After phase inversion, a sulfonated GO molecular imprinting polymer composite membrane was obtained. A label was affixed to the front and the membrane was stored in a mixed solution of water and glycerol in a volume ratio of 1:1 for later use.

[0098] Wherein, trans-ferulic acid is used as a template molecule, and the preparation method of the molecularly imprinted polymer is as follows: 6.5 mmol of trans-ferulic acid was added to 10 mL of methanol solution and magnetically stirred for 1 hour until completely dissolved, then 8 mmol of methacrylic acid and 5 mmol of acrylamide were added, magnetically stirred at room temperature for 3 hours, placed in a refrigerator at 4°C and allowed to stand for 3 hours, then the supernatant was taken, and then 15 mmol of ethylene glycol dimethacrylate and 0.122 mmol of azobisisobutyronitrile were added, nitrogen was introduced for 5 minutes, sealed, heated to 60°C, magnetically stirred for reaction for 24 hours, separated, and a polymer material was obtained; The polymer material was washed with ethanol for 36 hours after Soxhlet extraction to remove the template molecule trans-ferulic acid in the polymer material until the template molecule trans-ferulic acid could not be detected in the eluent, and then the eluted polymer material was washed with distilled water until neutral, and vacuum dried at 50°C for 12 hours to obtain molecularly imprinted polymer, which was freeze-dried and ground into 200 mesh on agate for later use.

[0099] (3) Membrane adsorption and separation: The membrane extraction adsorption content was measured by applying pressure to the membrane. The concentration of trans-ferulic acid in the trans-ferulic acid fermentation broth was adjusted to 50 mg / L and pH = 3.27. 2 The sulfonated GO molecular imprinting polymer composite membrane was loaded into the membrane sheet of the cross-flow integrated flat membrane separation device (circular membrane sheet with an effective radius of 2 cm). The trans-ferulic acid fermentation broth was loaded into the cross-flow integrated flat membrane separation device. The flow rate was controlled, and the ratio of the flow rate of the trans-ferulic acid fermentation broth to the area of ​​the sulfonated GO molecular imprinting polymer composite membrane was 70L / h:1m 2 At a temperature of 25°C and a humidity of 55%, a pressure of 0.1 MPa was applied to the membrane. After 1 hour of membrane filtration, the filtrate concentration (filtrate is the liquid that permeates the membrane) was 37.63 mg / L, the concentrate concentration (liquid that did not permeate the membrane in time during cross-flow filtration flushed the remaining substances on the membrane surface and returned to the unfiltered tank to be repeatedly concentrated) was 37.209 mg / L, and the membrane flux was 53.94 L / m 2 After the composite membrane was removed, it was eluted with 50 ml of anhydrous ethanol for 30 min. The concentration of trans-ferulic acid was determined and the mass of trans-ferulic acid eluted was calculated to be 2.83 mg. The mass of membrane extraction adsorption was about 2249.31 mg / m 2 .

[0100] At a temperature of 37°C and a pH of 3.27, 120 mL of 83.3 mg / L trans-ferulic acid fermentation broth was used to perform the same membrane permeation experiment as in Example 1. The test results are shown in Table 5: Table 5

[0101] After the composite membrane was taken out, it was eluted with 50 mL of anhydrous ethanol for 30 min, and the mass of adsorbed trans-ferulic acid was measured to be 2.44 mg. It can be seen that at 37°C, the membrane permeation adsorption capacity of the sulfonated GO molecularly imprinted polymer composite membrane of Example 4 was about 1939.04 mg / m 2 .

[0102] (4) Recrystallization: After the membrane extraction in step (3), the filtrate was collected, and the sulfonated GO molecular imprinting polymer composite membrane after membrane extraction was eluted with anhydrous ethanol-organic acid water mixture under the same gradient concentration conditions as in Example 1, and the eluate of the anhydrous ethanol-organic acid water mixture with an anhydrous ethanol volume ratio of 40% and 60% was collected. The collected filtrate and eluate were concentrated under reduced pressure to obtain crude trans-ferulic acid. The crude trans-ferulic acid was dissolved in ultrapure water to a final concentration of 9 g / L of trans-ferulic acid, and hydrochloric acid was added to adjust the pH to 3. Anhydrous ethanol was added in a volume ratio of ultrapure water to anhydrous ethanol of 3:1, and the mixture was stirred at 20°C for 5 hours and then allowed to stand for 24 hours. The purity of the obtained trans-ferulic acid crystals was above 96.4%, and the overall recovery rate reached 87.17%.

[0103] The crystals recovered by recrystallization in Example 4 were characterized, and their HPLC, flight time mass spectrum, infrared spectrum, nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and crystal diffraction spectrum were as follows: Figure 5 , Figure 6-Figure 7 , Figure 8 , Fig. 9 , Fig.10 and Fig.11 As shown ( Fig.11 This indicates that the main crystalline substance contained in the sample is trans-ferulic acid). This proves that the composite membrane of the present application can separate and recover high-purity trans-ferulic acid crystals.

[0104] Comparative Example 1 The method for separating and purifying trans-ferulic acid in the fermentation broth using the sulfonated graphene oxide blank membrane in Comparative Example 1 is substantially the same as that in Example 1, except that the method for preparing the sulfonated graphene oxide blank membrane in Comparative Example 1 comprises the following steps: 0.9 g of sulfonated graphene oxide was added to 90 mL of N,N-dimethylacetamide and ultrasonically dispersed for 1 h. 12 g of PVDF was added and mechanically stirred for 6 h. PVP, a porogen accounting for 0.3% by mass in the casting solution, was added. The film was scraped with a scraping machine at a speed of 250 μm and 15 mm / s. The film was then replaced in water for 24 h. After phase inversion, a sulfonated graphene oxide blank film was obtained. A label was attached to the front side and the film was stored in a mixed solution of water and glycerol in a volume ratio of 1:1 for later use.

[0105] The scanning electron microscope image of the sulfonated graphene oxide blank film prepared in Comparative Example 1 is as follows: Fig.12 shown.

[0106] The membrane extraction adsorption content was measured by applying pressure to the membrane. The concentration of trans-ferulic acid in the trans-ferulic acid fermentation broth was adjusted to 45.92 mg / L, pH = 3.27, and the area of ​​12.57 cm 2 The sulfonated graphene oxide blank membrane was loaded into the membrane of the cross-flow integrated flat membrane separation device, and the flow rate was controlled. The ratio of the flow rate of the trans-ferulic acid fermentation liquid to the area of ​​the sulfonated graphene oxide blank membrane was 70 L / h:1 m 2 At a temperature of 25°C and a humidity of 55%, a pressure of 0.1 MPa was applied to the membrane. After 1 hour of membrane filtration, the filtrate concentration (filtrate is the liquid that permeates the membrane) was 32.150 mg / L, the concentrate concentration (liquid that did not permeate the membrane in time during cross-flow filtration flushed the remaining substances on the membrane surface and returned to the unfiltered tank to be repeatedly concentrated) was 32.26 mg / L, and the membrane flux was 42.48 L / m 2 After the composite membrane was removed, it was eluted with 50 ml of anhydrous ethanol for 30 min. The concentration of trans-ferulic acid was determined and the mass of trans-ferulic acid eluted was calculated to be 1.51 mg. The mass of membrane extraction adsorption was about 1204.76 mg / m 2 .

[0107] At a temperature of 37°C and a pH of 3.27, 120 mL of 83.3 mg / L trans-ferulic acid fermentation broth was used to perform the same membrane permeation experiment as in Example 1. The test results are shown in Table 6: Table 6

[0108] After the composite membrane was taken out, it was eluted with 50 mL of anhydrous ethanol for 30 min, and the mass of adsorbed trans-ferulic acid was measured to be 1.01 mg. It can be seen that at 37°C, the membrane permeation adsorption amount of the sulfonated graphene oxide blank membrane of Comparative Example 1 was about 806.99 mg / m 2 .

[0109] (4) Recrystallization: After the membrane extraction in step (3), the filtrate was collected, and the sulfonated GO molecular imprinting polymer composite membrane after membrane extraction was eluted with anhydrous ethanol-organic acid water mixture under the same gradient concentration conditions as in Example 1, and the eluates of the anhydrous ethanol-organic acid water mixture with an anhydrous ethanol volume ratio of 40% and 60% were collected. The collected filtrate and eluate were concentrated under reduced pressure to obtain crude trans-ferulic acid. The crude trans-ferulic acid was dissolved in ultrapure water to a final concentration of 9 g / L of trans-ferulic acid, and hydrochloric acid was added to adjust the pH to 3. Anhydrous ethanol was added in a volume ratio of ultrapure water to anhydrous ethanol of 3:1, and the mixture was stirred at 20°C for 5 hours and then allowed to stand for 24 hours. The crystals obtained by standing were subjected to the above recrystallization operation three times. The purity of the obtained trans-ferulic acid crystals was above 95.8%, and the overall recovery rate reached 85.1%.

[0110] The recovery rate of Comparative Example 1 is also at a high level, but it needs to be repeated recrystallization three times to achieve a relatively good purity and recovery rate, indicating that the recognition effect of the sulfonated graphene oxide blank membrane of Comparative Example 1 is poor, resulting in low purity of the filtrate, which is difficult to meet the performance requirements of directional filtration of trans-ferulic acid.

[0111] The sulfonated GO molecular imprinting polymer composite membrane provided in the embodiment of the present application has good separation effect and recognition performance, can well adsorb trans-ferulic acid and easily elute it, which is helpful to achieve large-scale separation and purification of trans-ferulic acid.

[0112] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made according to the above description, and all these improvements and changes should fall within the scope of protection of the present application.

Claims

1. A method for preparing a sulfonated GO molecularly imprinted polymer composite membrane, characterized in that: The following steps are involved: Dispersing sulfonated graphene oxide in a first solvent, adding a molecular imprinting polymer to disperse the dispersion, then adding PVDF and stirring, and then adding a porogen to obtain a casting solution; The casting solution is scraped to obtain a sulfonated GO molecular imprinting polymer composite membrane through phase conversion.

2. The method for preparing the sulfonated GO molecularly imprinted polymer composite membrane according to claim 1, characterized in that: The mass ratio of the sulfonated graphene oxide to the molecular imprinting polymer is 0.9:0.15-0.225; The mass ratio of the molecular imprinting polymer to PVDF is 0.15-0.225:12-18.

3. The method for preparing the sulfonated GO molecularly imprinted polymer composite membrane according to claim 1, characterized in that: The ratio of the sulfonated graphene oxide to the first solvent is 1:100 (g / ml); The first solvent is N,N-dimethylacetamide; The porogen is one of PEG2000 and PVP, and the mass proportion of the porogen in the casting solution is 2%-12%; The dispersion is carried out by ultrasonic dispersion for 1-2 hours; The PVDF is mechanically stirred for 6-8h after addition; The thickness of the scraping film is 200-300 μm, and the scraping speed is 10-20 mm / s; The phase conversion was carried out by scraping the membrane and then placing it in water for 24 hours for replacement.

4. The method for preparing the sulfonated GO molecularly imprinted polymer composite membrane according to claim 1, characterized in that: The preparation method of the molecularly imprinted polymer comprises the following steps: dissolving the template molecule in a second solvent; Then add methacrylic acid and acrylamide and mix them, let them stand at 4°C, take the supernatant, add a crosslinking agent and azobisisobutyronitrile, pass nitrogen and seal, heat to the reaction temperature, stir and react, and separate the solid to obtain a polymer material; The template molecules are washed away from the polymer material, and then vacuum dried after washing to obtain the molecular imprinted polymer.

5. The method for preparing the sulfonated GO molecularly imprinted polymer composite membrane according to claim 4, characterized in that: Every 6.5 mmol of the template molecule is added into 10-50 mL of the second solvent for dissolution, and every 6.5 mmol of the template molecule is used with 6-10 mmol of methacrylic acid, 3-8 mmol of acrylamide, 10-20 mmol of a crosslinking agent and 0.1-0.2 mmol of azobisisobutyronitrile; The molecularly imprinted polymer is ground to a particle size of 200-300 meshes.

6. The method for preparing the sulfonated GO molecularly imprinted polymer composite membrane according to claim 4, characterized in that: The dissolution is carried out by magnetic stirring for 1-2 hours until complete dissolution; the second solvent is one or more of anhydrous ethanol, acetonitrile and methanol; The mixing is carried out by magnetic stirring at room temperature for 2-4 hours; the cross-linking agent is ethylene glycol dimethacrylate; the reaction temperature is 50-70° C.; the stirring reaction is carried out by magnetic stirring for 24-30 hours; The template molecules are washed away by washing the polymer material with anhydrous ethanol using Soxhlet extraction for 36-48 hours until no template molecules are detected in the washing solution; the polymer material is washed with distilled water until it is neutral; and the vacuum drying is performed by maintaining it at 50° C. for 12-15 hours.

7. A sulfonated GO molecular imprinted polymer composite membrane, characterized in that: The composite membrane is prepared by the method for preparing the sulfonated GO molecular imprinting polymer composite membrane as described in any one of claims 1 to 6.

8. An application of the sulfonated GO molecular imprinted polymer composite membrane as claimed in claim 7, characterized in that: The sulfonated GO molecular imprinted polymer composite membrane is prepared by using trans-ferulic acid as a template molecule, and the sulfonated GO molecular imprinted polymer composite membrane is used to separate and purify trans-ferulic acid, comprising the following steps: The sulfonated GO molecular imprinted polymer composite membrane is loaded into a membrane sheet of a membrane separation device; The liquid containing trans-ferulic acid is loaded into a membrane separation device, pressure is applied to the membrane to adsorb and filter, and the filtrate is collected; The sulfonated GO molecularly imprinted polymer composite membrane is then eluted with anhydrous ethanol-organic acid water mixtures of gradient concentrations, the eluate is collected, and the filtrate and the eluate are concentrated under reduced pressure to obtain a crude trans-ferulic acid, which is then purified to obtain trans-ferulic acid.

9. The use of the sulfonated GO molecular imprinted polymer composite membrane according to claim 8, characterized in that: The liquid containing trans-ferulic acid is a trans-ferulic acid fermentation liquid, and the concentration of trans-ferulic acid in the trans-ferulic acid fermentation liquid is 50-2000 mg / L; The ratio of the flow rate of the trans-ferulic acid fermentation broth to the area of ​​the sulfonated GO molecular imprinted polymer composite membrane is 70-150 L / h:1 m 2 .

10. The use of the sulfonated GO molecular imprinted polymer composite membrane according to claim 8, characterized in that: The elution is performed by sequentially using a mixture of anhydrous ethanol-organic acid water with an absolute ethanol volume ratio of 10%, 20%, 40% and 60% for 2 minutes, 2 minutes, 10 minutes and 10 minutes; the organic acid in the absolute ethanol-organic acid water mixture is one of acetic acid or formic acid, and the volume ratio of the organic acid in the organic acid water is one thousandth; The pressure is 0.1 MPa; The pH of the liquid containing trans-ferulic acid is 3-4, and the adsorption temperature is 25-37° C.; The purification step comprises: The crude trans-ferulic acid was dissolved in ultrapure water to a final concentration of 9 g / L, hydrochloric acid was added to adjust the pH to 3, anhydrous ethanol was added in a volume ratio of ultrapure water to anhydrous ethanol of 3:1, and the mixture was stirred at 20° C. for 5 h and then allowed to stand for 24 h.

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