A sulfonated GO molecularly imprinted polymer composite membrane, its preparation method and application

Abstract

This application relates to the field of biochemistry and chemical engineering, and mainly relates to a sulfonated GO molecularly imprinted polymer composite membrane, its preparation method and application. Among them, the preparation method of the sulfonated GO molecularly imprinted polymer composite membrane includes the following steps: dispersing sulfonated graphene oxide in a first solvent, adding a molecularly imprinted polymer and dispersing, then adding PVDF and stirring, and then adding a pore-forming agent to obtain a casting solution; scraping the casting solution to obtain a sulfonated GO molecularly imprinted polymer composite membrane through phase inversion. This application prepares a casting solution through a molecularly imprinted polymer, sulfonated graphene oxide, PVDF and a pore-forming agent. After scraping the film and performing phase inversion, a composite membrane with good comprehensive performance can be obtained, which has a good membrane separation effect on the target extract, good recognition performance, can improve the directional filtration effect and helps to achieve the large-scale separation and purification of trans-ferulic acid.

Description

Technical Field

[0001] This application relates to the field of biochemistry and chemical engineering, and mainly relates to a sulfonated GO molecularly imprinted polymer composite membrane, a preparation method thereof, and an 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, which is an aromatic compound and widely exists in plant cell walls, especially abundant in rye and wheat grains. Trans-ferulic acid is a yellow powdery crystal with specific physical and chemical properties. For example, its melting point is 168-172 °C, boiling point is about 372.3 °C, it is soluble in hot water, ethanol and ethyl acetate, slightly soluble in ether, but hardly soluble in benzene and petroleum ether.

[0003] In terms of biological activity, trans-ferulic acid can cause phosphorylation of β-catenin, thereby promoting proteasome degradation, increasing the expression of pro-apoptotic factor Bax, and decreasing the expression of pro-survival factors. In addition, it also has strong antioxidant properties, can effectively remove reactive oxygen species (ROS) and inhibit lipid peroxidation, and has anti-proliferation and anti-migration effects on human lung cancer cell line H1299. Trans-ferulic acid is a natural compound with various 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 at present.

[0004] The molecularly imprinted composite membrane (Molecular Imprinted Composite Membrane, abbreviated as MICM) is an advanced material that combines molecular imprinting technology and membrane separation technology. Its definition can be summarized as follows: A molecularly imprinted polymer (Molecular Imprinted Polymer, MIP) is coated or modified on the surface of a porous support membrane (such as an ultrafiltration membrane or a microfiltration membrane) to form a cortical layer with specific recognition functions, thereby obtaining a composite membrane material. The molecularly imprinted composite membrane has the characteristics of specific recognition, high throughput, good stability, and controllability. The preparation process of the molecularly imprinted composite membrane generally includes the following steps: 1) Selection of the base membrane: Select a suitable commercial porous membrane as the support, such as an ultrafiltration membrane or a microfiltration membrane. 2) Preparation of the imprinted layer: Mix raw materials such as template molecules, functional monomers, cross-linking agents, and initiators evenly to form a precursor solution of the imprinted polymer. Then, immerse the porous support membrane in this solution, and through methods such as photo-initiation, thermal initiation, or chemical initiation, form a layer of molecularly imprinted polymer on the surface of the support membrane. 3) Template elution: Elute the template molecules in the composite membrane, leaving cavities that are complementary to the shape, size, and chemical environment of the template molecules. These cavities are the recognition sites. 4) Post-treatment: Perform necessary post-treatment on the composite membrane, such as cleaning and drying, to improve its stability and separation performance.

[0005] The use of molecularly imprinted composite membranes has good application prospects for the separation and purification of trans-ferulic acid. Chinese invention patent CN109400804A proposed a preparation method of a ferulic acid molecularly imprinted blend membrane material, but only one functional monomer was used therein, and the recognition sites were single. The same molecular imprinting technology was also applied in Chinese invention patent CN113087842B, in which α-methylacrylic acid was used as the functional monomer to prepare a molecularly imprinted polymer with magnetic carbon nanotubes. 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 existing technology still needs to be improved and developed. Summary of the Invention

[0006] In view of the above deficiencies of the prior art, the purpose of this application is to provide a sulfonated GO molecularly imprinted polymer composite membrane, its preparation method, and application, aiming to solve the problem of low recognition efficiency when using molecular imprinting technology for the separation and purification of trans-ferulic acid.

[0007] The technical solution of this application is as follows:

[0008] In the first aspect, this application provides a preparation method of a sulfonated GO molecularly imprinted polymer composite membrane, which includes the following steps:

[0009] Sulfonated graphene oxide is dispersed in a first solvent. After adding a molecularly imprinted polymer and dispersing, PVDF is added and stirred, and then a porogen is added to obtain a casting solution;

[0010] The casting solution is cast into a film and undergoes phase inversion to obtain a sulfonated GO molecularly imprinted polymer composite membrane.

[0011] Through the preparation method provided by this application, a composite membrane with good performance can be prepared, realizing specific adsorption of target molecules and not adsorbing other water-soluble impurities, which is beneficial to achieving efficient and highly selective extraction of target molecules. Through the membrane separation of this composite membrane, the purpose of efficient separation and purification of target products can be achieved.

[0012] Further, the mass ratio of the sulfonated graphene oxide to the molecularly imprinted polymer is 0.9:0.15 - 0.225;

[0013] The mass ratio of the molecularly imprinted polymer to PVDF is 0.15 - 0.225:12 - 18.

[0014] Further, the ratio of the sulfonated graphene oxide to the first solvent is 1:100 (g / ml);

[0015] The first solvent is N,N-dimethylacetamide;

[0016] The porogen is one of PEG2000 or PVP, and the mass proportion of the porogen in the casting solution is 2% - 12%;

[0017] The dispersion is carried out by ultrasonic dispersion for 1 - 2 h;

[0018] After adding PVDF, mechanical stirring is carried out for 6 - 8 h;

[0019] The thickness of the film casting is 200 - 300 μm, and the film casting speed is 10 - 20 mm / s;

[0020] The phase inversion is carried out by placing the cast film in water for 24 h for replacement.

[0021] Further, the preparation method of the molecularly imprinted polymer includes the following steps:

[0022] Dissolve the template molecule in a second solvent;

[0023] Then add methacrylic acid and acrylamide and mix. After standing at 4°C, take the supernatant, then add a crosslinking agent and azobisisobutyronitrile, seal after introducing nitrogen, heat to the reaction temperature and carry out stirring reaction, and then separate the solid to obtain a polymer material;

[0024] Wash the polymer material to remove the template molecule, and then vacuum dry after washing to obtain the molecularly imprinted polymer.

[0025] Further, every 6.5 mmol of the template molecules are dissolved in 10 - 50 mL of the second solvent, and for every 6.5 mmol of the template molecules, 6 - 10 mmol of methacrylic acid, 3 - 8 mmol of acrylamide, 10 - 20 mmol of crosslinking agent, and 0.1 - 0.2 mmol of azobisisobutyronitrile are used;

[0026] The molecularly imprinted polymer is ground to a particle size of 200 - 300 mesh.

[0027] Further, the dissolution is carried out by magnetic stirring for 1 - 2 h until completely dissolved; the second solvent is one or more of anhydrous ethanol, acetonitrile, and methanol;

[0028] The mixing is carried out by magnetic stirring at room temperature for 2 - 4 h; the crosslinking agent is ethylene glycol dimethacrylate; the reaction temperature is 50 - 70 °C; the stirring reaction is carried out by magnetic stirring for 24 - 30 h;

[0029] The washing of the template molecules is carried out by washing the polymeric material with anhydrous ethanol in a Soxhlet extractor for 36 - 48 h until no template molecules can be detected in the washing liquid; the washing is carried out by washing the polymeric material with distilled water until neutral; the vacuum drying is carried out by maintaining at 50 °C for 12 - 15 h.

[0030] In a second aspect, the present application provides a sulfonated GO molecularly imprinted polymer composite membrane, which is prepared by the preparation method of the sulfonated GO molecularly imprinted polymer composite membrane as described in the first aspect.

[0031] In a third aspect, the present application provides an application of the sulfonated GO molecularly imprinted polymer composite membrane as described in the second aspect, wherein the sulfonated GO molecularly imprinted polymer composite membrane is prepared using trans - ferulic acid as a template molecule. Using the sulfonated GO molecularly imprinted polymer composite membrane for the separation and purification of trans - ferulic acid includes the following steps:

[0032] Load the sulfonated GO molecularly imprinted polymer composite membrane into the membrane sheet of a membrane separation device;

[0033] Load the liquid containing trans - ferulic acid onto the membrane separation device, apply pressure to the membrane sheet for adsorption and filtration, and collect the filtrate;

[0034] Then, elute the sulfonated GO molecularly imprinted polymer composite membrane successively with a gradient - concentration mixture of anhydrous ethanol - organic acid water, collect the eluate, concentrate the filtrate and the eluate under reduced pressure to obtain a crude product of trans - ferulic acid, and purify it to obtain trans - ferulic acid.

[0035] Further, the liquid containing trans-ferulic acid is a trans-ferulic acid fermentation broth, and the concentration of trans-ferulic acid in the trans-ferulic acid fermentation broth is 50 - 2000 mg / L;

[0036] 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 m 2 。

[0037] Further, the elution is carried out by sequentially using a mixed solution of anhydrous ethanol - organic acid water with the volume ratio of anhydrous ethanol being 10%, 20%, 40% and 60% respectively to elute for 2 minutes, 2 minutes, 10 minutes and 10 minutes; the organic acid in the mixed solution 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;

[0038] The pressure is 0.1 MPa;

[0039] The pH of the liquid containing trans-ferulic acid is 3 - 4, and the adsorption temperature is 25 - 37 °C;

[0040] The purification steps include:

[0041] Dissolve the crude trans-ferulic acid product in ultrapure water to a final concentration of 9 g / L of trans-ferulic acid, add hydrochloric acid to adjust the pH to 3, add anhydrous ethanol according to the volume ratio of ultrapure water to anhydrous ethanol being 3:1, stir at 20 °C for 5 h and then stand for 24 h.

[0042] Beneficial effects: In this application, a casting solution is prepared from a molecularly imprinted polymer, sulfonated graphene oxide, PVDF and a pore-forming agent. After scraping the film and phase inversion, a composite membrane with good comprehensive performance can be obtained, which has a good membrane separation effect on the target extract and good recognition performance, and helps to achieve large-scale separation and purification. Description of the Drawings

[0043] Figure 1 It is a schematic diagram of the application process of the sulfonated GO molecularly imprinted polymer composite membrane of this application.

[0044] Figure 2 It is a physical picture of the sulfonated GO molecularly imprinted polymer composite membrane prepared in Example 1 of this application.

[0045] Figure 3 It is a scanning electron microscope picture of the sulfonated GO molecularly imprinted polymer composite membrane prepared in Example 1 of this application.

[0046] Figure 4 It is a cross-sectional scanning electron microscope picture of the sulfonated GO molecularly imprinted polymer composite membrane prepared in Example 1 of this application.

[0047] Figure 5 This is the high performance liquid chromatography (HPLC) chart of the crystals obtained by recrystallization and recovery in Example 4 of this application.

[0048] Figure 6 This is the time-of-flight mass spectrometry (full scan mode) chart of the crystals obtained by recrystallization and recovery in Example 4 of this application.

[0049] Figure 7 This is the secondary ion time-of-flight mass spectrometry chart of the crystals obtained by recrystallization and recovery in Example 4 of this application.

[0050] Figure 8 This is the infrared spectroscopy chart of the crystals obtained by recrystallization and recovery in Example 4 of this application.

[0051] Figure 9 This is the nuclear magnetic resonance hydrogen spectrum chart of the crystals obtained by recrystallization and recovery in Example 4 of this application.

[0052] Figure 10 This is the nuclear magnetic resonance carbon spectrum chart of the crystals obtained by recrystallization and recovery in Example 4 of this application.

[0053] Figure 11 This is the X-ray diffraction pattern chart of the crystals obtained by recrystallization and recovery in Example 4 of this application.

[0054] Figure 12 This is the scanning electron microscopy image of the sulfonated graphene oxide blank membrane in Comparative Example 1 of this application. Detailed implementation manners

[0055] This application provides a sulfonated GO molecularly imprinted polymer composite membrane, its preparation method and application. To make the purpose, technical solutions and effects of this application clearer and more definite, the following further details this application. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0056] As Figure 1 shown, this application provides a preparation method of a sulfonated GO molecularly imprinted polymer composite membrane, which includes the following steps:

[0057] S1. Disperse sulfonated graphene oxide (sulfonated GO) in a first solvent, add a molecularly imprinted polymer and disperse it, then add PVDF (polyvinylidene fluoride) and stir, and then add a porogen and mix to obtain a casting solution;

[0058] S2. Cast the casting solution and obtain a sulfonated GO molecularly imprinted polymer composite membrane through phase inversion.

[0059] Through the preparation method provided by this application, a composite membrane with good performance can be prepared, realizing specific adsorption of target molecules without adsorbing other water-soluble impurities, which is conducive to achieving efficient and highly selective extraction of target molecules. Through the membrane separation of this composite membrane, the purpose of efficient separation and purification of target products can be achieved.

[0060] Among them, the dispersion of sulfonated graphene oxide and molecularly imprinted polymer can both be carried out by ultrasonic dispersion for 1-2 h; PVDF is mixed by mechanical stirring for 6-8 h after being added; the film scraping can be carried out by a film scraping machine, the thickness of the film scraping is 200-300 μm, and the speed of the film scraping is 10-20 mm / s; the phase inversion is carried out by placing it in water for 24 h for replacement after film scraping, and it can be stored in a mixed solution with a volume ratio of water to glycerol of 1:1 for standby after the replacement is completed.

[0061] Furthermore, in step S1, the preparation method of the molecularly imprinted polymer includes the following steps:

[0062] S11. Dissolve the template molecule in the second solvent;

[0063] S12. Then add methacrylic acid and acrylamide and mix. After standing at 4 °C, take the supernatant, then add the cross-linking agent and azobisisobutyronitrile, seal it after passing nitrogen, heat it to the reaction temperature and carry out stirring reaction, and separate the solid to obtain the polymer material;

[0064] S13. Wash the polymer material to remove the template molecule, and then vacuum dry it after washing to obtain the molecularly imprinted polymer.

[0065] After the preparation of the molecularly imprinted polymer is completed, it can be freeze-dried for standby.

[0066] Among them, by passing nitrogen, the subsequent heating reaction process is carried out in a nitrogen atmosphere.

[0067] Furthermore, in step S11, the dissolution can be carried out by magnetic stirring for 1-2 h until completely dissolved; the second solvent is one or more of anhydrous ethanol, acetonitrile and methanol, and specifically can also be selected according to the template molecule to be dissolved, as long as the template molecule can be completely dissolved.

[0068] Furthermore, in step S12, the mixing can be carried out by magnetic stirring at room temperature for 2-4 h; 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 h.

[0069] Among them, the 4 °C standing in step S12 can be carried out by placing it in a refrigerator. By standing at 4 °C, the solubility can be reduced, promoting some unnecessary polymers or impurities to precipitate or settle out of the solution, and then taking the supernatant after standing stably.

[0070] Further, in step S13, the template molecules are washed away by washing the polymeric material with anhydrous ethanol in a Soxhlet extractor for 36 - 48 h until no template molecules are detected in the washing solution; the washing is carried out by washing the polymeric material with distilled water until neutral; the vacuum drying is carried out by maintaining at 50 °C for 12 - 15 h.

[0071] Further, every 6.5 mmol of template molecules are dissolved in 10 - 50 mL of a second solvent, and for every 6.5 mmol of template molecules, 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 are used.

[0072] Further, after the molecularly imprinted polymer is prepared, the molecularly imprinted polymer is ground to a particle size of 200 - 300 mesh. Among them, the grinding can be carried out by agate. Specifically, too large a particle size of the molecularly imprinted polymer will result in different pore sizes on the membrane surface, and too small a particle size will also affect the membrane separation effect. By controlling the particle size of the molecularly imprinted polymer within a suitable range in this application, it is beneficial to improve the surface flatness of the composite membrane, stabilize the water flux, and it can also be clearly seen in the electron microscope images that the molecularly imprinted polymer particles are evenly distributed on the membrane surface, which is beneficial to improving the separation effect.

[0073] Specifically, the template molecule can be a target molecule that can react and bind with methacrylic acid or acrylamide. By selecting a suitable functional monomer to prepare the molecularly imprinted polymer, the recognition effect can be 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 a bifunctional monomer for preparation, a molecularly imprinted polymer with dual recognition sites can be prepared, which is beneficial to improving its specific adsorption performance.

[0074] Further, since the selected methacrylic acid and acrylamide may also react with each other. In this application, when adding the functional monomers to the second solvent, methacrylic acid can be added first and stirred to allow the template molecules to first undergo a certain coordination with methacrylic acid, and then acrylamide is added and stirred to reduce unnecessary polymerization. At the same time, methacrylic acid provides a hydrophobic environment, while acrylamide can form hydrogen bonds with the template molecules through its amide groups. 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.

[0075] This application prepares a molecularly imprinted polymer through a bifunctional monomer, which can achieve good recognition of the template molecule, improve the specific recognition efficiency, and is beneficial to improving the purity and recovery rate. Taking the template molecule trans-ferulic acid as an example, a molecularly imprinted polymer with specific recognition of trans-ferulic acid is formed. This complex can selectively adsorb trans-ferulic acid without adsorbing other water-soluble impurities, thereby separating and purifying trans-ferulic acid efficiently and with high selectivity. Experiments show that the purity of the trans-ferulic acid separated by this application can reach over 96%, and the recovery rate can reach over 85%.

[0076] Further, in step S1, the ratio of sulfonated graphene oxide to the first solvent is 1:100 (g / ml); the first solvent is N,N-dimethylacetamide.

[0077] Further, in step S1, the mass ratio of the molecularly imprinted polymer to PVDF is 0.15 - 0.225:12 - 18. In this application, the selected PVDF as the substrate of the composite membrane has excellent chemical stability and mechanical strength. However, PVDF has a certain degree of hydrophobicity, and if the dosage is too high, it will affect the adsorption effect of the molecularly imprinted polymer on the extraction stock solution. By controlling the ratio between the molecularly imprinted polymer and PVDF, it is beneficial to maintain the performance of the composite membrane while ensuring the separation and purification effect.

[0078] Further, in step S1, the mass ratio of sulfonated graphene oxide to the molecularly imprinted 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 lead to a decrease in the flexibility of the membrane. Therefore, it is necessary to control the dosage to balance the mechanical strength and flexibility to ensure the stability and operability of the membrane.

[0079] More specifically, the combination of methacrylic acid and acrylamide can provide both hydrophobic and hydrophilic regions simultaneously. This amphiphilic property helps to capture and release the template molecule more effectively, which is particularly important for selective separation during the membrane preparation process and can improve the permeation selectivity and separation efficiency of the membrane. Moreover, by controlling the dosage ratio of sulfonated graphene oxide to the molecularly imprinted polymer, sulfonated graphene oxide has a high surface area and negative charge density, and the sulfonic acid groups on it can match with the amino and carboxyl groups in the bifunctional monomer imprinted polymer to form ionic bonds. In this way, the membrane material can not only selectively separate the target molecule depending on the molecular size and shape, but also further improve the selectivity through the ion exchange mechanism, which is beneficial to improving the directional filtration effect.

[0080] Microbial synthesis is characterized by simplicity, high efficiency, and large-scale integrated preparation. The composite membrane prepared in this application can also be applied to the separation and purification of fermentation broth. However, the components of the fermentation broth are complex, and the adsorption effect of the composite membrane is easily affected by hydrophobic problems. Moreover, the separation and purification effect may be affected by the compatibility between the hydrophobicity of PVDF and the hydrophilicity of the bifunctional monomer molecularly imprinted polymer. By using sulfonated graphene oxide in this application, the hydrophilicity and dispersion stability of the membrane material can be increased. By combining with a pore-forming agent, the compatibility can be improved, the pore structure formation and stability of the composite membrane can be enhanced, and the original solution to be extracted can also pass through the composite membrane well. By controlling the addition amounts of each other, the water flux is more appropriate within the provided proportion range. While maintaining a certain extraction flux, the target molecule is extracted, realizing the extraction and concentration of the target molecule at the same time, and reducing energy consumption.

[0081] Furthermore, the pore-forming agent is one of PEG2000 or PVP, and the mass ratio of the pore-forming agent in the casting solution is 2%-12%. In this application, the combination performance of sulfonated graphene oxide and the molecularly imprinted polymer is good. Without adding a pore-forming agent, the pore channels on the membrane surface are relatively small, which is likely to affect the water permeability. By adding a certain amount of PEG2000 or PVP in this application, finger-like pores will be formed. By observing the cross-section of the membrane through an electron microscope, finger-like pores with different sizes can be seen. Among them, when performing membrane separation, the water flux will also increase as the size of the finger-like pores increases.

[0082] This application uses the nonsolvent-induced phase separation (NIPS) method to mix the eluted molecularly imprinted polymer, sulfonated graphene oxide, and PVDF, and then induces phase separation of the polymer solution through a nonsolvent, thereby forming a membrane product with a specific pore structure. The membranes prepared by the NIPS method usually have the characteristics and advantages of asymmetric membranes with porous skins, good flexibility, long-term hydrophilicity, and easily adjustable pore structures: its process flow is simple, the film-forming performance is excellent, and the pore structure is easily adjustable, and it has a wide range of application prospects in many fields.

[0083] This application also provides a sulfonated GO molecularly imprinted polymer composite membrane, which is prepared by the preparation method of the sulfonated GO molecularly imprinted polymer composite membrane as described above. The prepared composite membrane has good specific and selective adsorption performance for the target substance.

[0084] This application also provides an application of the sulfonated GO molecularly imprinted polymer composite membrane as described above. Among them, the sulfonated GO molecularly imprinted polymer composite membrane is prepared using trans-ferulic acid as a template molecule. Using the sulfonated GO molecularly imprinted polymer composite membrane for the separation and purification of trans-ferulic acid includes the following steps:

[0085] Load the sulfonated GO molecularly imprinted polymer composite membrane into the membrane sheet of the membrane separation device;

[0086] Load the liquid containing trans-ferulic acid into the membrane separation device, apply pressure to the membrane sheet for adsorption and filtration, and collect the filtrate;

[0087] Then, elute the sulfonated GO molecularly imprinted polymer composite membrane successively with a mixed solution of anhydrous ethanol - organic acid water with gradient concentrations, collect the eluate, concentrate the filtrate and the eluate under reduced pressure to obtain the crude trans-ferulic acid, and obtain trans-ferulic acid after purification.

[0088] 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 it can install the sulfonated GO molecularly imprinted polymer composite membrane for membrane separation. Among them, the cross-flow integrated flat membrane separation device has a longer service life and higher flux. By performing membrane cross-flow filtration, the problems of overproduction or insufficient production of trans-ferulic acid can be reduced, and the efficient balance of membrane separation can be achieved.

[0089] Among them, the organic acid in the mixed solution 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.

[0090] Furthermore, the elution is carried out by successively using mixed solutions of anhydrous ethanol - organic acid water with the volume ratios of anhydrous ethanol being 10%, 20%, 40% and 60% to elute for 2 minutes, 2 minutes, 10 minutes and 10 minutes respectively. After the membrane separation and filtration enrichment of the liquid containing trans-ferulic acid in this application, the industrial cost of recovering the trans-ferulic acid adsorbed on the membrane by gradient elution is lower, which is beneficial to the popularization and application of the large-scale preparation of trans-ferulic acid. Specifically, by using a mixed solution of anhydrous ethanol - organic acid water with a lower concentration of 10% for preliminary elution in the elution stage, mainly removing impurities with weak adsorption strength, then gradually removing impurities with medium adsorption strength through 20% concentration, eluting some molecules and a small part of trans-ferulic acid that bind tightly to the composite membrane through 40% concentration, and finally eluting for 10 minutes through 60% concentration, which helps to elute most of the target molecules. After the composite membrane is eluted and recovered, it can also be reused and filtered again.

[0091] Furthermore, after eluting with a mixed solution of anhydrous ethanol - organic acid water with gradient concentrations, collect the eluates of the mixed solutions of anhydrous ethanol - organic acid water with the volume ratios of anhydrous ethanol being 40% and 60%. After drying the eluates, crude trans-ferulic acid can be obtained, and the purity is also relatively high.

[0092] Furthermore, the liquid containing trans-ferulic acid is preferably a trans-ferulic acid fermentation broth, which can be prepared by trans-ferulic acid-producing bacteria. Synthesizing trans-ferulic acid by microorganisms has the characteristics of simplicity and high efficiency, facilitating large-scale preparation. In this application, by using a composite membrane with good performance for membrane separation, the purpose of efficient separation and purification of trans-ferulic acid can be achieved.

[0093] Furthermore, 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 broth containing trans-ferulic acid should preferably not exceed the maximum adsorption capacity of the composite membrane. Controlling the appropriate concentration range is beneficial to improving the separation effect.

[0094] Furthermore, the pH of the trans-ferulic acid fermentation broth is 3 - 4, more preferably 3.27; the adsorption temperature is 25 - 37 °C. By controlling the pH of the sample loading solution and the adsorption temperature, it is beneficial to improve the separation effect of the sulfonated GO molecularly imprinted polymer composite membrane on trans-ferulic acid.

[0095] 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 m 2 . Among them, when performing large-scale separation, the area of the sulfonated GO molecularly imprinted polymer composite membrane can be controlled at 0.5 - 4 m 2 .

[0096] Furthermore, the pressure is 0.1 MPa. If the pressure is too high, it is easy to cause adsorption failure; if the pressure is too low, the utilization rate is low. The adsorption effect is better at this pressure.

[0097] Furthermore, the filtration time is 0.5 - 1 h. Specifically, the sulfonated GO molecularly imprinted polymer composite membrane provided in this application mainly plays a directional filtration function, and the membrane adsorption amount is less than the filtrate amount. By controlling the concentration, flow rate, and filtration pressure of the trans-ferulic acid fermentation broth, generally, after filtration for 0.5 - 1 h, the concentration of the filtrate will gradually tend to be stable. At this time, filtration can continue for separation. However, the membrane separation performance of the composite membrane is easily affected by the increase in the concentration of the concentrated solution or the accumulation of impurities in the fermentation broth. Therefore, the preferred filtration time is 0.5 - 1 h, and after eluting the composite membrane, membrane separation filtration is repeated. This is beneficial to maintaining a high flux and not being easily blocked, and is also beneficial for the composite membrane to maintain the adsorption and directional filtration effect on trans-ferulic acid for a long time.

[0098] Specifically, after obtaining the crude trans-ferulic acid through elution and concentration, it can be purified by recrystallization. The crude trans-ferulic acid is dissolved in ultrapure water until the final concentration of trans-ferulic acid is 9 g / L. Hydrochloric acid is added to adjust the pH to 3, and absolute ethanol is added according to the volume ratio of ultrapure water to absolute ethanol of 3:1. After stirring at 20 °C for 5 h and then standing for 24 h, the purity of the obtained trans-ferulic acid crystals can reach over 99%.

[0099] The application method provided by this application has simple process operation, low cost, and is convenient for large-scale industrial production, which is of great significance for promoting the industrial development of trans-ferulic acid.

[0100] The following is further illustrated by specific examples.

[0101] The trans-ferulic acid-producing bacteria used in the examples of this application are from the research group of Xiaolin Shen of Beijing University of Chemical Technology, and the reference is (Targeting cofactors regeneration in methylation and hydroxylation for high level production of Ferulic acid, Xiaolin Shen (Xiaolin Shen of Beijing University of Chemical Technology), etc.).

[0102] The fermentation medium and enrichment medium used in the examples of this application are both M9 medium: 2 mL of 1 M MgSO4, 0.1 mL of 1 M 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, added with 1000 mL of sterilized double-distilled water.

[0103] Example 1

[0104] The method for separating and purifying the trans-ferulic acid fermentation broth using the sulfonated GO molecularly imprinted polymer composite membrane in Example 1 includes the following steps:

[0105] (1) Preparation of trans-ferulic acid fermentation broth:

[0106] The method for preparing trans-ferulic acid fermentation broth using a trans-ferulic acid-producing bacterium is as follows: Escherichia coli is inoculated into a fermentation medium at an inoculation amount of 5% and shaken at 37 °C for 3 days to obtain a seed solution; then the seed solution is inoculated into an enrichment medium at an inoculation amount of 10%, and a precursor required for trans-ferulic acid fermentation is added, and shaken at 37 °C for 5 days. The culture solution is centrifuged at 5 °C and 9000 r / min for 15 min, and the supernatant is collected to obtain a trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 70 mg / L.

[0107] (2) Preparation of sulfonated GO molecularly imprinted polymer composite membrane:

[0108] 0.9 g of sulfonated graphene oxide is added to 90 mL of N,N-dimethylacetamide and dispersed by ultrasonic treatment for 1 h. Then 0.225 g of molecularly imprinted polymer is added and dispersed by ultrasonic treatment for 1 h. Then 12 g of PVDF is added and mechanically stirred for 6 h for mixing. Then a porogen PVP with a mass ratio of 3% in the casting solution is added, and the film is cast with a film thickness of 250 μm at a casting speed of 15 mm / s. Then it is replaced in water for 24 h, and a sulfonated GO molecularly imprinted polymer composite membrane is obtained through phase inversion. A label is attached to the front side, and then it is stored in a mixed solution with a volume ratio of water to glycerol of 1:1 for standby.

[0109] Among them, using trans-ferulic acid as the template molecule, the preparation method of the molecularly imprinted polymer is as follows:

[0110] 6.5 mmol of trans-ferulic acid is added to 10 mL of methanol solution and magnetically stirred for 1 h until completely dissolved. Then 8 mmol of methacrylic acid and 5 mmol of acrylamide are added, and magnetically stirred at room temperature for 3 h. After standing in a 4 °C refrigerator, the supernatant is taken. Then 15 mmol of ethylene glycol dimethacrylate and 0.122 mmol of azobisisobutyronitrile are added, nitrogen is introduced for 5 min, sealed, heated to 60 °C, and magnetically stirred and reacted for 24 h, and then separated to obtain a polymer material;

[0111] The polymer material is washed with ethanol by Soxhlet extraction for 36 h to remove the template molecule trans-ferulic acid in the polymer material until the template molecule trans-ferulic acid cannot be detected in the eluate. Then the washed polymer material is washed with distilled water until neutral, vacuum dried at 50 °C for 12 h to obtain a molecularly imprinted polymer, freeze-dried, and ground to 200 meshes with an agate for standby.

[0112] The scanning electron microscope image of the sulfonated GO molecularly imprinted polymer composite membrane prepared in Example 1 is as Figure 3 shown, and the scanning electron microscope cross-sectional image is as Figure 4 shown.

[0113] (3) Membrane adsorption and separation:

[0114] The membrane extraction adsorption content was measured under the condition of 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 the pH was 3.27. A sulfonated GO molecularly imprinted polymer composite membrane with an area of 12.57 cm 2 was packed into the membrane sheet of a cross-flow integrated flat membrane separation device (a circular membrane sheet with an effective radius of 2 cm). The trans-ferulic acid fermentation broth was loaded onto 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 molecularly imprinted polymer composite 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 sheet. After 1 hour of membrane filtration, the filtrate concentration (the filtrate is the liquid that permeates through the membrane) was 37.97 mg / L, and the concentrate concentration (the liquid that did not permeate through the membrane in time during the cross-flow filtration process scours the substances remaining on the membrane surface and flows back to the unfiltered pool for repeated concentration) was 38.33 mg / L. The membrane flux was 44.86 L / m 2 ·h. After removing the composite membrane, it was eluted with 50 ml of absolute ethanol for 30 min. The maximum adsorption capacity in the membrane performance was measured by elution with absolute ethanol. The trans-ferulic acid concentration was measured, and the mass of trans-ferulic acid eluted was calculated to be 2.52 mg. The mass of membrane extraction adsorption calculated was approximately 2002.36 mg / m 2 .

[0115] A membrane permeation experiment was carried out on the sulfonated GO molecularly imprinted polymer composite membrane. A membrane permeation bottle A and a membrane permeation bottle B with the same specifications were set up. The two bottles were connected by a sulfonated GO molecularly imprinted polymer composite membrane (effective radius of 2 cm). 120 mL of trans-ferulic acid fermentation broth with a concentration of 83.3 mg / L and a pH of 3.27 was loaded into the membrane permeation bottle A; 120 mL of pure water was loaded into the membrane permeation bottle B. Under the conditions of a humidity of 55% and a temperature of 37 °C, natural permeation was carried out through the concentration difference (no pressure was applied to the composite membrane). At this time, in addition to a part of trans-ferulic acid penetrating into the membrane permeation bottle B filled with pure water, a part was also adsorbed on the composite membrane. The changes in the trans-ferulic acid concentration after 121 h of permeation in the membrane permeation bottle A and the membrane permeation bottle B were measured. The test results are shown in Table 1:

[0116] Table 1

[0117]

[0118] At 37 °C, after 121 h of permeation, the ferulic acid concentrations on both sides of the composite membrane basically reached equilibrium. After removing the composite membrane, it was eluted with 50 mL of absolute ethanol for 30 min. The mass of adsorbed trans-ferulic acid was measured to be 2.08 mg. The calculated membrane permeation adsorption capacity was approximately 1652.32 mg / m 2 .

[0119] In terms of application, the trans-ferulic acid adsorbed on the composite membrane can be extracted and purified by gradient elution; moreover, in the presence of the composite membrane, the trans-ferulic acid-producing bacteria and some impurities cannot pass through the membrane, and a preliminary separation is carried out. In addition, the trans-ferulic acid can be adsorbed on the composite membrane. After collection, the trans-ferulic acid can be extracted and then purified. The trans-ferulic acid can also pass through the membrane to the membrane permeation B bottle, and the permeate collected can also be used to extract and purify the trans-ferulic acid.

[0120] (4) Recrystallization:

[0121] After the membrane extraction in step (3), the filtrate is collected, and then the sulfonated GO molecularly imprinted polymer composite membrane after membrane extraction is eluted successively with anhydrous ethanol-organic acid water mixed solutions with gradient concentrations. The eluates of the anhydrous ethanol-organic acid water mixed solutions with 40% and 60% by volume of anhydrous ethanol and the filtrate after membrane extraction are collected. The collected filtrate and eluate are concentrated under reduced pressure to obtain the crude trans-ferulic acid. 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 according to the volume ratio of ultrapure water to anhydrous ethanol of 3:1, and after stirring at 20 °C for 5 h and then standing for 24 h, the purity of the obtained trans-ferulic acid crystals is above 95.6%, and the overall recovery rate reaches 86.5%.

[0122] Among them, the concentration (volume ratio) of the anhydrous ethanol-organic acid water mixed solution with gradient concentration and the elution time are shown in Table 2:

[0123] Table 2

[0124]

[0125] Example 2

[0126] The method for separating and purifying the trans-ferulic acid fermentation broth using the sulfonated GO molecularly imprinted polymer composite membrane in Example 2 includes the following steps:

[0127] (1) Preparation of trans-ferulic acid fermentation broth:

[0128] The trans-ferulic acid fermentation broth is prepared using the trans-ferulic acid-producing bacteria. The specific method is as follows: Escherichia coli is inoculated into the fermentation medium at an inoculation amount of 5%, and shaken at 37 °C for 3 d to obtain the seed solution; then the seed solution is inoculated into the enrichment medium at an inoculation amount of 10%, the precursor required for trans-ferulic acid fermentation is added, and shaken at 37 °C for 5 d. The culture solution is centrifuged at 5 °C and 9000 r / min for 15 min, and the supernatant is collected to obtain the trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 70 mg / L.

[0129] (2) Preparation of sulfonated GO molecularly imprinted polymer composite membrane:

[0130] Add 0.9 g of sulfonated graphene oxide to 90 mL of N,N-dimethylacetamide, disperse it by ultrasonic wave for 1 h, add 0.225 g of molecularly imprinted polymer, disperse it by ultrasonic wave for 1 h, then add 12 g of PVDF and stir mechanically for 6 h to mix. Then add porogen PVP with a mass ratio of 10% in the casting solution, scrape the film with a film scraper at 250 μm and a scraping speed of 15 mm / s, then replace it in water for 24 h, and obtain the sulfonated GO molecularly imprinted polymer composite membrane through phase inversion. Stick a label on the front, and then store it in a mixed solution with a volume ratio of 1:1 of water and glycerol for standby.

[0131] Among them, using trans-ferulic acid as the template molecule, the preparation method of the molecularly imprinted polymer is as follows:

[0132] Add 6.5 mmol of trans-ferulic acid to 10 mL of methanol solution and stir magnetically for 1 h until completely dissolved. Then add 8 mmol of methacrylic acid and 5 mmol of acrylamide, stir magnetically at room temperature for 3 h, place it in a refrigerator at 4 °C and let it stand, then take the supernatant. Then add 15 mmol of ethylene glycol dimethacrylate and 0.122 mmol of azobisisobutyronitrile, introduce nitrogen for 5 min, seal it, heat it to 60 °C, and stir magnetically for 24 h to separate and obtain the polymer material;

[0133] Wash the polymer material with ethanol in a Soxhlet extractor for 36 h to remove the template molecule trans-ferulic acid in the polymer material until the template molecule trans-ferulic acid cannot be detected in the eluate. Then wash the eluted polymer material with distilled water until neutral, dry it in vacuum at 50 °C for 12 h to obtain the molecularly imprinted polymer, freeze-dry it, and grind it with an agate to 200 mesh for standby.

[0134] Prepare a trans-ferulic acid standard solution with a concentration of 1600 mg / L of trans-ferulic acid, and the solvent is ethanol solution. Add 0.1 g of the sulfonated GO molecularly imprinted polymer composite membrane prepared in Example 2 to the trans-ferulic acid standard solution and adsorb for 24 h. Then take it out, dry it, and measure the overall mass. The adsorption capacity of the sulfonated GO molecularly imprinted polymer composite membrane in Example 2 is measured to be 28.86 mg / g.

[0135] (3) Membrane adsorption and separation:

[0136] By applying pressure to the membrane, measure the membrane extraction adsorption content. Adjust the concentration of trans-ferulic acid in the trans-ferulic acid fermentation broth to 50 mg / L and the pH = 3.51. The area is 12.57 cm 2The sulfonated GO molecularly imprinted polymer composite membrane is loaded into the membrane sheet of a cross-flow integrated flat membrane separation device (a circular membrane sheet with an effective radius of 2 cm). The trans-ferulic acid fermentation broth is loaded onto the cross-flow integrated flat membrane separation device. Control the flow rate, and 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 L / h:1 m 2 , at a temperature of 25 °C and a humidity of 55%, apply a pressure of 0.1 mpa to the membrane sheet. After 1 hour of membrane filtration, the filtrate concentration (the filtrate is the liquid passing through the membrane) is 30.92 mg / L, and the concentrate concentration (the liquid that does not pass through the membrane in time during cross-flow filtration and flushes the substances remaining on the membrane surface and flows back to the unfiltered pool for repeated concentration) is 35.57 mg / L. The membrane flux is 64.43 L / m 2 ·h. After removing the composite membrane, elute it with 50 ml of absolute ethanol for 30 min, measure the trans-ferulic acid concentration, and calculate that the mass of the eluted trans-ferulic acid is 2.66 mg, and the mass adsorbed by the membrane extraction is about 2118.78 mg / m 2 .

[0137] At a temperature of 37 °C and a pH of 3.51, use 120 mL of a trans-ferulic acid fermentation broth with a concentration of 83.3 mg / L to perform the same membrane permeation experiment as in Example 1. The test results are shown in Table 3:

[0138] Table 3

[0139]

[0140] After removing the composite membrane, elute it with 50 mL of absolute ethanol for 30 min, and measure that the mass of the adsorbed trans-ferulic acid is 2.26 mg. It can be seen that at 37 °C, the membrane permeation adsorption capacity of the sulfonated GO molecularly imprinted polymer composite membrane in Example 2 is about 1800.56 mg / m 2 .

[0141] (4) Recrystallization:

[0142] After the membrane extraction in step (3), collect the filtrate, and then elute the sulfonated GO molecularly imprinted polymer composite membrane after membrane extraction with an absolute ethanol-organic acid water mixture with the same gradient concentration conditions as in Example 1 in sequence. Collect the eluates of the absolute ethanol-organic acid water mixture with the volume ratio of absolute ethanol being 40% and 60%. Concentrate the collected filtrate and eluate under reduced pressure to obtain the crude trans-ferulic acid. Dissolve the crude trans-ferulic acid in ultrapure water until the final concentration of trans-ferulic acid is 9 g / L. Add hydrochloric acid to adjust the pH to 3, add absolute ethanol according to the volume ratio of ultrapure water to absolute ethanol being 3:1, stir at 20 °C for 5 h, and then stand for 24 h. The purity of the obtained trans-ferulic acid crystals is above 96.2%, and the overall recovery rate reaches 86.13%.

[0143] Example 3

[0144] The method for separating and purifying the trans-ferulic acid fermentation broth using the sulfonated GO molecularly imprinted polymer composite membrane in Example 3 includes the following steps:

[0145] (1) Preparation of trans-ferulic acid fermentation broth:

[0146] Use the trans-ferulic acid-producing bacteria to prepare the trans-ferulic acid fermentation broth. The specific method is as follows: Inoculate Escherichia coli into the fermentation medium at an inoculation amount of 5%, and shake culture at 37°C for 3 days to obtain the seed liquid; then inoculate the seed liquid into the enrichment medium at an inoculation amount of 10%, add the precursors required for trans-ferulic acid fermentation, shake culture at 37°C for 5 days, centrifuge the culture solution at 5°C and 9000 r / min for 15 min, collect the supernatant, and obtain the trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 70 mg / L.

[0147] (2) Preparation of sulfonated GO molecularly imprinted polymer composite membrane:

[0148] Add 0.9 g of sulfonated graphene oxide to 90 mL of N,N-dimethylacetamide, ultrasonically disperse for 1 h, add 0.225 g of the molecularly imprinted polymer, ultrasonically disperse for 1 h, then add 12 g of PVDF and mechanically stir for 6 h to mix. Then add the porogen PEG 2000 with a mass ratio of 4.5% in the casting solution, use a film scraper to scrape the film to a thickness of 250 μm at a scraping speed of 15 mm / s, then displace it in water for 24 h, and obtain the sulfonated GO molecularly imprinted polymer composite membrane through phase inversion. Stick a label on the front, and then store it in a mixed solution with a volume ratio of 1:1 of water and glycerol for standby.

[0149] Among them, using trans-ferulic acid as the template molecule, the preparation method of the molecularly imprinted polymer is as follows:

[0150] Add 6.5 mmol of trans-ferulic acid to 10 mL of methanol solution and stir magnetically for 1 h until completely dissolved. Then add 8 mmol of methacrylic acid and 5 mmol of acrylamide, stir magnetically at room temperature for 3 h, place it in a 4°C refrigerator and let it stand. Take the supernatant, then add 15 mmol of ethylene glycol dimethacrylate and 0.122 mmol of azobisisobutyronitrile, introduce nitrogen for 5 min, seal, heat to 60°C, and stir magnetically for 24 h to separate and obtain the polymeric material;

[0151] Wash the polymeric material with ethanol in a Soxhlet extractor for 36 h to remove the template molecule trans-ferulic acid in the polymeric material until the template molecule trans-ferulic acid cannot be detected in the eluate. Then wash the eluted polymeric material with distilled water until it is neutral, dry it in vacuo at 50°C for 12 h to obtain the molecularly imprinted polymer, freeze-dry it, and grind it with an agate mortar to 200 mesh for standby.

[0152] (3) Membrane adsorption and separation:

[0153] By applying pressure to the membrane, the membrane extraction adsorption content is measured. Adjust the concentration of trans-ferulic acid in the trans-ferulic acid fermentation broth to 50 mg / L, and the pH = 3.27. Place the sulfonated GO molecularly imprinted polymer composite membrane with an area of 12.57 cm 2 in the membrane sheet of the cross-flow integrated flat membrane separation device (a circular membrane sheet with an effective radius of 2 cm). Load the trans-ferulic acid fermentation broth onto the cross-flow integrated flat membrane separation device. Control the flow rate, and 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 L / h:1 m 2 , at a temperature of 25 °C and a humidity of 55%, apply a pressure of 0.1 mpa to the membrane sheet. After 1 hour of membrane filtration, the filtrate concentration (the filtrate is the liquid passing through the membrane) is 33.83 mg / L, and the concentrate concentration (the liquid that does not pass through the membrane in time during cross-flow filtration flushes the substances remaining on the membrane surface and returns to the unfiltered pool for repeated concentration) is 35.81 mg / L. The membrane flux is 40.57 L / m 2 ·h. After removing the composite membrane, elute it with 50 ml of anhydrous ethanol for 30 min, measure the concentration of trans-ferulic acid, and calculate that the mass of trans-ferulic acid obtained by elution is 2.71 mg, and the mass of membrane extraction adsorption is about 2152.55 mg / m 2 .

[0154] At a temperature of 37 °C and a pH of 3.27, perform the same membrane permeation experiment as in Example 1 using 120 mL of trans-ferulic acid fermentation broth with a concentration of 83.3 mg / L. The test results are shown in Table 4:

[0155] Table 4

[0156]

[0157] After removing the composite membrane, elute it with 50 mL of anhydrous ethanol for 30 min, and the measured mass of adsorbed trans-ferulic acid is 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 in Example 3 is about 1683.18 mg / m 2 .

[0158] (4) Recrystallization:

[0159] After the membrane extraction in step (3), the filtrate was collected, and the sulfonated GO molecularly imprinted polymer composite membrane after membrane extraction was eluted successively with an ethanol-organic acid water mixture with the same gradient concentration conditions as in Example 1. The eluates of the ethanol-organic acid water mixture with 40% and 60% by volume of ethanol were collected. The collected filtrate and eluate were concentrated under reduced pressure to obtain a crude product of trans-ferulic acid. The crude product of 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. Ethanol was added according to a volume ratio of ultrapure water to ethanol of 3:1. After stirring at 20 °C for 5 h and then standing for 24 h, the purity of the obtained trans-ferulic acid crystals was above 95.4%, and the overall recovery rate reached 86.34%.

[0160] Example 4

[0161] The method for separating and purifying the trans-ferulic acid fermentation broth using the sulfonated GO molecularly imprinted polymer composite membrane in Example 4 includes the following steps:

[0162] (1) Preparation of trans-ferulic acid fermentation broth:

[0163] The trans-ferulic acid fermentation broth was prepared using the trans-ferulic acid-producing bacterium. The specific method was as follows: Escherichia coli was inoculated into the fermentation medium at an inoculation amount of 5%, and shaken at 37 °C for 3 d to obtain a seed solution; then the seed solution was inoculated into the enrichment medium at an inoculation amount of 10%, and the precursor required for trans-ferulic acid fermentation was added, and shaken at 37 °C for 5 d. The culture solution was centrifuged at 5 °C and 9000 r / min for 15 min, and the supernatant was collected to obtain a trans-ferulic acid fermentation broth with a trans-ferulic acid concentration of 70 mg / L.

[0164] (2) Preparation of sulfonated GO molecularly imprinted polymer composite membrane:

[0165] 0.9 g of sulfonated graphene oxide was added to 90 mL of N,N-dimethylacetamide, sonicated for 1 h for dispersion, 0.225 g of the molecularly imprinted polymer was added, sonicated for 1 h for dispersion, then 12 g of PVDF was added and mechanically stirred for 6 h for mixing. Then, 6% by mass of the pore-forming agent PEG 2000 was added to the casting solution, and the film was cast with a film thickness of 250 μm at a casting speed of 15 mm / s. Then, it was replaced in water for 24 h, and the sulfonated GO molecularly imprinted polymer composite membrane was obtained by phase inversion. A label was attached to the front, and then it was stored in a mixed solution of water and glycerol with a volume ratio of 1:1 for standby.

[0166] Among them, using trans-ferulic acid as the template molecule, the preparation method of the molecularly imprinted polymer was as follows:

[0167] 6.5 mmol of trans-ferulic acid was added to 10 mL of methanol solution and stirred magnetically for 1 h until completely dissolved. Then, 8 mmol of methacrylic acid and 5 mmol of acrylamide were added, and the mixture was stirred magnetically at room temperature for 3 h. After standing in a refrigerator at 4 °C, the supernatant was taken. Then, 15 mmol of ethylene glycol dimethacrylate and 0.122 mmol of azobisisobutyronitrile were added. Nitrogen was introduced for 5 min, and then the mixture was sealed and heated to 60 °C and stirred magnetically for 24 h. After separation, a polymeric material was obtained.

[0168] The polymeric material was washed with ethanol in a Soxhlet extractor for 36 h to remove the template molecule trans-ferulic acid in the polymeric material until the template molecule trans-ferulic acid could not be detected in the eluate. Then, the washed polymeric material was washed with distilled water until neutral, dried in vacuo at 50 °C for 12 h to obtain a molecularly imprinted polymer, which was freeze-dried and ground with agate to 200 mesh for standby.

[0169] (3)Membrane adsorption and separation:

[0170] By applying pressure to the membrane, the membrane extraction adsorption content was measured. The concentration of trans-ferulic acid in the trans-ferulic acid fermentation broth was adjusted to 50 mg / L, and the pH = 3.27. A sulfonated GO molecularly imprinted polymer composite membrane with an area of 12.57 cm 2 was loaded into the membrane sheet of a cross-flow integrated flat membrane separation device (a circular membrane sheet with an effective radius of 2 cm). The trans-ferulic acid fermentation broth was loaded onto 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 molecularly imprinted polymer composite 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 sheet. After 1 hour of membrane filtration, the filtrate concentration (the filtrate is the liquid passing through the membrane) was 37.63 mg / L, and the concentrate concentration (the liquid that did not pass through the membrane in time during cross-flow filtration and washed the residual substances on the membrane surface and flowed back to the unfiltered pool for repeated concentration) was 37.209 mg / L. The membrane flux was 53.94 L / m 2 ·h. After removing the composite membrane, it was eluted with 50 ml of absolute ethanol for 30 min. The concentration of trans-ferulic acid was measured, and the mass of trans-ferulic acid eluted was calculated to be 2.83 mg. The mass of membrane extraction adsorption was approximately 2249.31 mg / m 2 .

[0171] At a temperature of 37 °C and a pH of 3.27, the same membrane permeation experiment as in Example 1 was carried out using 120 mL of a trans-ferulic acid fermentation broth with a concentration of 83.3 mg / L. The test results are shown in Table 5:

[0172] Table 5

[0173]

[0174] After removing the composite membrane, 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 .

[0175] (4) Recrystallization:

[0176] After membrane extraction in step (3), the filtrate was collected, and the sulfonated GO molecularly imprinted polymer composite membrane after membrane extraction was eluted successively with an anhydrous ethanol-organic acid water mixture under the same gradient concentration conditions as in Example 1. 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 a crude product of trans-ferulic acid. The crude product of trans-ferulic acid was dissolved in ultrapure water until the final concentration of trans-ferulic acid was 9 g / L. Hydrochloric acid was added to adjust the pH to 3, and anhydrous ethanol was added according to the volume ratio of ultrapure water to anhydrous ethanol of 3:1. After stirring at 20 °C for 5 h and then standing for 24 h, the purity of the obtained trans-ferulic acid crystals was above 96.4%, and the overall recovery rate reached 87.17%.

[0177] The crystals recovered by recrystallization of Example 4 were characterized. Their high-performance liquid chromatography, time-of-flight mass spectrometry, infrared spectroscopy, nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, and crystallization diffraction spectrum were respectively as Figure 5 、 Figures 6 - 7 、 Figure 8 、 Figure 9 、 Figure 10 and Figure 11 shown ( Figure 11 indicating that the main crystalline substance contained in the sample is trans-ferulic acid). It is proved that high-purity trans-ferulic acid crystals can be separated and recovered through the composite membrane of the present application.

[0178] Comparative Example 1

[0179] The method for separating and purifying trans-ferulic acid from fermentation broth using a sulfonated graphene oxide blank membrane in Comparative Example 1 was basically the same as that in Example 1, except that the preparation method of the sulfonated graphene oxide blank membrane in Comparative Example 1 included the following steps:

[0180] Add 0.9 g of sulfonated graphene oxide to 90 mL of N,N-dimethylacetamide, ultrasonically disperse for 1 h, add 12 g of PVDF, mechanically stir for 6 h, add PVP as a pore-forming agent with a mass ratio of 0.3% in the casting solution, use a film scraper to scrape the film to a thickness of 250 μm at a scraping speed of 15 mm / s, then displace it in water for 24 h, and obtain a sulfonated graphene oxide blank membrane through phase inversion. Stick a label on the front, and then store it in a mixed solution with a volume ratio of water to glycerol of 1:1 for standby.

[0181] The scanning electron micrograph of the sulfonated graphene oxide blank membrane prepared in Comparative Example 1 is as Figure 12 shown.

[0182] Under the condition of applying pressure to the membrane, measure the membrane extraction and adsorption content. Adjust the concentration of trans-ferulic acid in the trans-ferulic acid fermentation broth to 45.92 mg / L, with a pH of about 3.27. Place a sulfonated graphene oxide blank membrane with an area of 12.57 cm 2 in the membrane sheet of a cross-flow integrated flat membrane separation device, control the flow rate, and the ratio of the flow rate of the trans-ferulic acid fermentation broth to the area of the sulfonated graphene oxide blank membrane is 70 L / h:1 m 2 . At a temperature of 25 °C and a humidity of 55%, apply a pressure of 0.1 mpa to the membrane sheet. After 1 hour of membrane filtration, the filtered concentration (the filtrate is the liquid passing through the membrane) is 32.150 mg / L, and the concentrate concentration (the liquid that has not passed through the membrane in time during cross-flow filtration flushes the substances remaining on the membrane surface and returns to the unfiltered pool for repeated concentration) is 32.26 mg / L. The membrane flux is 42.48 L / m 2 ·h. After removing the composite membrane, elute it with 50 ml of absolute ethanol for 30 min, measure the concentration of trans-ferulic acid and calculate that the mass of trans-ferulic acid obtained by elution is 1.51 mg, and the mass of membrane extraction and adsorption is about 1204.76 mg / m 2 .

[0183] At a temperature of 37 °C and a pH of 3.27, use 120 mL of a trans-ferulic acid fermentation broth with a concentration of 83.3 mg / L to conduct the same membrane permeation experiment as in Example 1. The test results are shown in Table 6:

[0184] Table 6

[0185]

[0186] After removing the composite membrane, elute it with 50 mL of absolute ethanol for 30 min, and measure that the mass of adsorbed trans-ferulic acid is 1.01 mg. It can be seen that at 37 °C, the membrane permeation and adsorption amount of the sulfonated graphene oxide blank membrane in Comparative Example 1 is about 806.99 mg / m 2 .

[0187] (4) Recrystallization:

[0188] After the membrane extraction in step (3), the filtrate was collected, and the sulfonated GO molecularly imprinted polymer composite membrane after membrane extraction was eluted successively with an ethanol-organic acid water mixed solution under the same gradient concentration conditions as in Example 1. The eluates of the ethanol-organic acid water mixed solution with the volume fraction of ethanol being 40% and 60% were collected. The collected filtrate and eluate were concentrated under reduced pressure to obtain a crude product of trans-ferulic acid. The crude product of trans-ferulic acid was dissolved in ultrapure water until the final concentration of trans-ferulic acid was 9 g / L. Hydrochloric acid was added to adjust the pH to 3, and ethanol was added according to the volume ratio of ultrapure water to ethanol being 3:1. After stirring at 20 °C for 5 h, it was left to stand for 24 h. 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%.

[0189] The recovery rate of Comparative Example 1 was also at a relatively high level, but it was necessary to perform repeated recrystallization three times to achieve relatively good purity and recovery rate, indicating that the recognition effect of the sulfonated graphene oxide blank membrane in Comparative Example 1 was poor, resulting in low purity of the filtrate and difficulty in meeting the performance requirements for the directional filtration of trans-ferulic acid.

[0190] The separation effect and recognition performance of the sulfonated GO molecularly imprinted polymer composite membrane provided in the embodiment of the present application are good. It can adsorb trans-ferulic acid well and is easy to elute, which helps to realize the large-scale separation and purification of trans-ferulic acid.

[0191] It should be understood that the application of the present application is not limited to the above examples. For those of ordinary skill in the art, it can be improved or transformed according to the above description. All such improvements and transformations should fall within the protection scope of the present application.

Claims

1. A preparation method of a sulfonated GO molecularly imprinted polymer composite membrane, characterized in that, It includes the following steps: Disperse sulfonated graphene oxide in the first solvent, add the molecularly imprinted polymer and disperse, then add PVDF and stir, and then add the porogen to obtain a casting solution; Cast the casting solution, and obtain a sulfonated GO molecularly imprinted polymer composite membrane through phase inversion; The mass ratio of the sulfonated graphene oxide to the molecularly imprinted polymer is 0.9:0.15 - 0.225; The mass ratio of the molecularly imprinted polymer to PVDF is 0.15 - 0.225:12 - 18; The preparation method of the molecularly imprinted polymer includes the following steps: Dissolve the template molecule in the second solvent; Then add methacrylic acid and acrylamide and mix. After standing at 4°C, take the supernatant, then add the crosslinking agent and azobisisobutyronitrile, seal after introducing nitrogen, heat to the reaction temperature and stir for reaction, and then separate the solid to obtain the polymeric material; Wash the polymeric material to remove the template molecule, and then vacuum dry after washing to obtain the molecularly imprinted polymer; The template molecule is trans-ferulic acid.

2. The preparation method of 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 or 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 h; After adding PVDF, mechanical stirring is carried out for 6 - 8 h; The thickness of the casting is 200 - 300 μm, and the casting speed is 10 - 20 mm / s; The phase inversion is carried out by placing the cast film in water for 24 h for replacement; 3. The preparation method of the sulfonated GO molecularly imprinted polymer composite membrane according to claim 1, characterized in that, Dissolve every 6.5 mmol of the template molecule in 10 - 50 mL of the second solvent. For every 6.5 mmol of the template molecule, 6 - 10 mmol of methacrylic acid, 3 - 8 mmol of acrylamide, 10 - 20 mmol of the crosslinking agent and 0.1 - 0.2 mmol of azobisisobutyronitrile are used; The molecularly imprinted polymer is ground to a particle size of 200 - 300 mesh.

4. The preparation method of the sulfonated GO molecularly imprinted polymer composite membrane according to claim 1, characterized in that, The dissolution is carried out by magnetic stirring for 1 - 2 h until completely dissolved; 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 h; the crosslinking agent is ethylene glycol dimethacrylate; the reaction temperature is 50 - 70°C; the stirring reaction is carried out by magnetic stirring for 24 - 30 h; The washing of the template molecule is carried out by washing the polymeric material with anhydrous ethanol in a Soxhlet extractor for 36 - 48 h until the template molecule cannot be detected in the washing liquid; the washing is carried out by washing the polymeric material with distilled water until it is neutral; the vacuum drying is carried out by maintaining at 50°C for 12 - 15 h.

5. A sulfonated GO molecularly imprinted polymer composite membrane, characterized in that, It is prepared by the preparation method of the sulfonated GO molecularly imprinted polymer composite membrane according to any one of claims 1 - 4.

6. Use of the sulfonated GO molecularly imprinted polymer composite membrane as described in claim 5, characterized in that, The sulfonated GO molecularly imprinted polymer composite membrane is prepared with trans-ferulic acid as the template molecule. Using the sulfonated GO molecularly imprinted polymer composite membrane for the separation and purification of trans-ferulic acid includes the following steps: Load the sulfonated GO molecularly imprinted polymer composite membrane into the membrane sheet of the membrane separation device; Load the liquid containing trans-ferulic acid into the membrane separation device, apply pressure to the membrane sheet for adsorption and filtration, and collect the filtrate; Then, elute the sulfonated GO molecularly imprinted polymer composite membrane successively with gradient concentration of anhydrous ethanol-organic acid water mixture, collect the eluate, concentrate the filtrate and eluate under reduced pressure to obtain the crude trans-ferulic acid, and purify it to obtain trans-ferulic acid.

7. Use of the sulfonated GO molecularly imprinted polymer composite membrane according to claim 6, characterized in that, The liquid containing trans-ferulic acid is the trans-ferulic acid fermentation broth, and the concentration of trans-ferulic acid in the trans-ferulic acid fermentation broth 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 molecularly imprinted polymer composite membrane is 70 - 150 L / h:1 m 2 .

8. Use of the sulfonated GO molecularly imprinted polymer composite membrane according to claim 6, characterized in that, The elution is carried out by successively using the mixture of anhydrous ethanol-organic acid water with the volume ratio of anhydrous ethanol being 10%, 20%, 40% and 60% to elute for 2 minutes, 2 minutes, 10 minutes and 10 minutes respectively; the organic acid in the anhydrous 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 temperature of the adsorption is 25-37 °C; The steps of the purification include: Dissolve the crude trans-ferulic acid in ultrapure water to a final concentration of 9 g / L of trans-ferulic acid, add hydrochloric acid to adjust the pH to 3, add anhydrous ethanol according to the volume ratio of ultrapure water to anhydrous ethanol being 3:1, stir at 20 °C for 5 h and then stand for 24 h.

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