Trans-ferulic acid bifunctional monomer molecular imprinting polymer and preparation method and application thereof

Abstract

The present application relates to the field of biochemical technology, and discloses a trans-ferulic acid bifunctional monomer molecular imprinting polymer and its preparation method and application. The steps include: preparing a trans-ferulic acid solution; dividing the trans-ferulic acid solution into a first trans-ferulic acid solution and a second trans-ferulic acid solution, adding methacrylic acid to the first trans-ferulic acid solution and stirring and mixing, adding acrylamide to the second trans-ferulic acid solution and stirring and mixing, and then mixing the first trans-ferulic acid solution and the second trans-ferulic acid solution to obtain a mixed solution; adding a cross-linking agent and azobisisobutyronitrile to the mixed solution, passing nitrogen, sealing, heating to the reaction temperature, stirring and reacting to obtain a polymer material; washing the polymer material to remove trans-ferulic acid, washing to neutrality, heating and vacuum drying to obtain a trans-ferulic acid bifunctional monomer molecular imprinting polymer. The imprinting polymer can achieve efficient and specific adsorption, and can be used for large-scale separation and purification of trans-ferulic acid by solid phase extraction.

Description

Technical Field

[0001] The present application relates to the field of biochemical engineering technology, and mainly to a trans-ferulic acid bifunctional monomer molecular imprinting polymer and a preparation method and application thereof. Background Art

[0002] Ferulic Acid is a functional phenolic acid widely found in plants. The content of ferulic acid in food raw materials such as coffee, rice husk, vanilla beans, wheat bran and rice bran is relatively high. It is also the main active ingredient of the traditional Chinese medicine Chuanxiong. The content of ferulic acid in traditional Chinese medicines such as Ferula, Angelica, Chuanxiong, Cimicifuga and Ziziphus jujuba seed is also relatively high. It is one of the active ingredients of these traditional Chinese medicines and is currently used as one of the quality indicators of Chinese patent medicines.

[0003] The chemical name of ferulic acid is 4-hydroxy-3-methoxycinnamic acid, which is cinnamic acid (also known as cinnamic acid, 3-phenyl-2-propenoic acid, molecular structure ). Ferulic acid (generally used as the drug sodium ferulate) has the effects of anti-platelet aggregation, inhibiting the release of 5-hydroxytryptamine from platelets, inhibiting the generation of platelet thromboxane A2 (TXA2), enhancing the activity of prostaglandins, relieving pain, and relieving vasospasm. It is the basic raw material for the production of drugs for the treatment of cardiovascular and cerebrovascular diseases and leukocytopenia. It is mainly used in a variety of products such as Xinxuekang, Limai Capsules and Taitai Oral Liquid. It can also play a role in bodybuilding and skin protection in the human body. Ferulic acid can enter the human body through transdermal absorption, and the permeability coefficient is positively correlated with the concentration, which makes ferulic acid fully applicable as an active ingredient in beauty cosmetics. Ferulic acid has the following three characteristics: 1) Antioxidant effect; it has strong antioxidant properties and can effectively remove various free radicals such as hydrogen peroxide, superoxide radicals, hydroxyl radicals, etc. It is a natural free radical scavenger. It can also remove excess reactive oxygen in the body and inhibit oxidative stress by inhibiting the production of lipid peroxide MDA; 2) Sunscreen effect; ferulic acid can be used as a natural sunscreen, and its maximum ultraviolet absorption peak is at 236nm and 322nm, which can prevent or reduce the damage of ultraviolet rays in this wavelength range to the skin; 3) Whitening effect; the formation of skin melanin is the most fundamental reason for skin darkening, among which tyrosinase is the key enzyme in the formation of melanin, and studies have confirmed that ferulic acid can significantly inhibit the activity of tyrosinase and the proliferation and synthesis of melanocytes, which can reduce the production of melanin and achieve the effect of whitening and removing spots.

[0004] At present, the demand for ferulic acid is gradually increasing, and the market prospects are quite promising. In order to achieve efficient and large-scale separation and purification of ferulic acid, it is necessary to improve the existing purification methods.

[0005] Molecular imprinting technology (MIT) is an interdisciplinary recognition technology that has emerged in recent years. It uses template molecules, functional monomers and cross-linking agents to undergo polymerization reactions under certain conditions to obtain polymers. Such polymers usually have corresponding spatial structures and functional groups of different sizes. The target molecules are separated from the molecularly imprinted polymers in a certain way, and the adsorption sites formed can match the template molecules. In this way, a "memory" hole that matches the template molecules in space and has good selectivity for them is obtained, which can show specific selectivity and discrimination for the target molecules. Because molecular imprinting technology has good molecular recognition ability and stability, it has good application prospects in many aspects such as chromatographic separation, biosensors and solid phase extraction.

[0006] Chinese invention patent CN109400804A proposes a method for preparing a ferulic acid molecular imprinting blended membrane material, which is identified by molecular imprinting technology, but its ferulic acid molecular imprinting blended membrane material only uses one functional monomer, and the recognition site is single. Chinese invention patent CN113087842A also proposes a method for preparing a ferulic acid molecular imprinting polymer, which is prepared by magnetic carbon nanotubes into a molecular imprinting polymer, in which only α-methylacrylic acid is used as a single functional monomer, and the recognition site is also relatively single, which has certain limitations on the specific recognition of ferulic acid and is relatively inefficient. Therefore, the prior art needs to be improved and developed. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a trans-ferulic acid bifunctional monomer molecular imprinting polymer and its preparation method and application, aiming to solve the problem of a single recognition site in the existing molecular imprinting technology for identifying ferulic acid.

[0008] The technical solution of this application is as follows:

[0009] In a first aspect, the present application provides a method for preparing a trans-ferulic acid bifunctional monomer molecularly imprinted polymer, which comprises the following steps:

[0010] preparing a trans-ferulic acid solution;

[0011] The trans-ferulic acid solution is evenly divided into a first trans-ferulic acid solution and a second trans-ferulic acid solution, methacrylic acid is added to the first trans-ferulic acid solution and the mixture is stirred and mixed, acrylamide is added to the second trans-ferulic acid solution and the mixture is stirred and mixed, and the first trans-ferulic acid solution and the second trans-ferulic acid solution are mixed to obtain a mixed solution;

[0012] Adding a crosslinking agent and azobisisobutyronitrile to the mixed solution, introducing nitrogen, sealing, heating to a reaction temperature, stirring for reaction, and obtaining a polymer material;

[0013] The trans-ferulic acid is washed away from the polymer material, and then the polymer material is washed to neutrality, heated and vacuum dried to obtain the trans-ferulic acid bifunctional monomer molecular imprinting polymer.

[0014] The present application uses methacrylic acid and acrylamide as bifunctional monomers to prepare a trans-ferulic acid bifunctional monomer molecular imprinting polymer, which has good performance in specific and selective adsorption of the trans-ferulic acid target. The target product trans-ferulic acid is subjected to solid phase extraction using the polymer as a solid phase extraction filler, which can achieve the purpose of efficiently separating and purifying trans-ferulic acid.

[0015] Furthermore, the molar ratio of the methacrylic acid to acrylamide in the mixed solution is 8:5.

[0016] In the present application, a specific ratio of bifunctional monomers was screened out through experiments, and the prepared molecularly imprinted polymer had a better elution effect by maintaining the molar ratio of methacrylic acid to acrylamide at 8:5.

[0017] Furthermore, the molar ratio of trans-ferulic acid to acrylamide in the mixed solution is 1.3:1.

[0018] In the present application, by controlling the polymerization of trans-ferulic acid and bifunctional monomers at a suitable ratio, it is beneficial to form a molecular imprinting polymer with good pore position and spatial structure, avoiding excessive closure that makes it difficult to wash out the template molecule and affects the recognition performance, and can also improve the dual recognition effect.

[0019] Further, the reaction temperature is 50-70°C;

[0020] Before heating to the reaction temperature, the temperature was raised to 40°C and maintained for 30 min.

[0021] In the present application, by raising the temperature and keeping it warm for a period of time before cross-linking, trans-ferulic acid and methacrylic acid are dehydrated in an alkaline environment to form a certain degree of intramolecular esterification before cross-linking occurs, so that a good coordination relationship between ferulic acid and the functional monomer can be maintained before cross-linking, which is beneficial to the expression of the dual recognition function of the prepared molecular imprinting polymer.

[0022] Further, every 0.5-7 mmol of trans-ferulic acid is dissolved in 50 mL of solvent, wherein the solvent is one or more of anhydrous ethanol, acetonitrile and methanol;

[0023] The cross-linking agent is ethylene glycol dimethacrylate;

[0024] In the mixed solution, 10-250 mmol of a cross-linking agent and 50-300 mg of azobisisobutyronitrile are used for every 0.5-7 mmol of trans-ferulic acid.

[0025] In a second aspect, the present application further provides a trans-ferulic acid bifunctional monomer molecular imprinted polymer, wherein the polymer is prepared by the preparation method of the trans-ferulic acid bifunctional monomer molecular imprinted polymer as described in the first aspect.

[0026] In a third aspect, the present application also provides an application of the trans-ferulic acid bifunctional monomer molecular imprinting polymer as described in the second aspect, wherein the trans-ferulic acid bifunctional monomer molecular imprinting polymer is applied to separate and purify trans-ferulic acid in a fermentation broth by solid phase extraction, comprising the following steps:

[0027] (1) Preparation: preparing a fermentation broth containing trans-ferulic acid by using a trans-ferulic acid high-producing strain;

[0028] (2) Extraction and separation: adding the fermentation broth containing trans-ferulic acid to a molecular imprinting extraction column filled with the trans-ferulic acid bifunctional monomer molecular imprinting polymer, eluting with anhydrous ethanol and a mixed solution of anhydrous ethanol and organic acid water in sequence, collecting the eluate, and concentrating under reduced pressure to obtain a crude trans-ferulic acid product;

[0029] (3) Purification: The crude trans-ferulic acid is dissolved in ultrapure water, hydrochloric acid is added to adjust the pH to 3-4, anhydrous ethanol is added, and the mixture is stirred at 20-25° C. and allowed to stand to obtain trans-ferulic acid.

[0030] Furthermore, the solid-liquid ratio of the trans-ferulic acid bifunctional monomer molecularly imprinted polymer to the fermentation broth containing trans-ferulic acid is 1 g:30-180 mL.

[0031] Furthermore, the concentration of trans-ferulic acid in the fermentation broth containing trans-ferulic acid is 20-2000 mg / L.

[0032] Furthermore, in step (3), the crude trans-ferulic acid is dissolved in ultrapure water to a final concentration of 9-12 g / L;

[0033] In step (3), the volume ratio of ultrapure water to anhydrous ethanol is 3-5:1.

[0034] Beneficial effects: The trans-ferulic acid molecular imprinting polymer prepared by the present application uses methacrylic acid and acrylamide as functional monomers, which can specifically adsorb the target molecule, has good separation and purification effect and high efficiency when applied to solid phase extraction, can facilitate the large-scale production of trans-ferulic acid, and is of great significance to the industrial application of trans-ferulic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the effect curve diagram of the four-factor three-level orthogonal experiment of Examples 1-9 of the present application.

[0036] Figure 2 This is the high performance liquid chromatogram of trans-ferulic acid adsorbed in Example 13 of the present application.

[0037] Figure 3 This is the HPLC chromatogram of trans-ferulic acid standard.

[0038] Figure 4 This is the NMR carbon spectrum of trans-ferulic acid purified in Example 20 of the present application.

[0039] Figure 5 This is the H NMR spectrum of trans-ferulic acid purified in Example 20 of the present application. DETAILED DESCRIPTION

[0040] The present application provides a trans-ferulic acid bifunctional monomer molecular imprinting polymer and its preparation method and application. In order to make the purpose, technical scheme 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.

[0041] The present application provides a method for preparing a trans-ferulic acid bifunctional monomer molecularly imprinted polymer, which comprises the following steps:

[0042] preparing a trans-ferulic acid solution;

[0043] The trans-ferulic acid solution is evenly divided into a first trans-ferulic acid solution and a second trans-ferulic acid solution, methacrylic acid is added to the first trans-ferulic acid solution and the mixture is stirred and mixed, acrylamide is added to the second trans-ferulic acid solution and the mixture is stirred and mixed, and the first trans-ferulic acid solution and the second trans-ferulic acid solution are mixed to obtain a mixed solution;

[0044] Adding a crosslinking agent and azobisisobutyronitrile to the mixed solution, introducing nitrogen, sealing, heating to a reaction temperature, stirring for reaction, and obtaining a polymer material;

[0045] The trans-ferulic acid is washed away from the polymer material, and then the polymer material is washed to neutrality, heated and vacuum dried to obtain a trans-ferulic acid bifunctional monomer molecular imprinting polymer.

[0046] The present application uses trans-ferulic acid as a template molecule, methacrylic acid and acrylamide as bifunctional monomers, and the prepared trans-ferulic acid bifunctional monomer molecular imprinting polymer has more recognition sites and can efficiently and specifically adsorb target molecules, which is beneficial to large-scale separation and purification of trans-ferulic acid, improves production efficiency, and is of great significance to the industrial application of trans-ferulic acid.

[0047] More specifically, the method for preparing the trans-ferulic acid bifunctional monomer molecularly imprinted polymer comprises the following steps:

[0048] S1. Dissolve 0.5-7 mmol of trans-ferulic acid in 50 mL of solvent and stir magnetically until completely dissolved to prepare a trans-ferulic acid solution;

[0049] S2, dividing the trans-ferulic acid solution into a first trans-ferulic acid solution and a second trans-ferulic acid solution, adding methacrylic acid to the first trans-ferulic acid solution, and mixing them under magnetic stirring at room temperature for 2-4 hours, adding acrylamide to the second trans-ferulic acid solution, and mixing them under magnetic stirring at room temperature for 2-4 hours, and then mixing the first trans-ferulic acid solution and the second trans-ferulic acid solution to obtain a mixed solution;

[0050] S3, adding 10-250 mmol ethylene glycol dimethacrylate and 50-300 mg azobisisobutyronitrile to the mixed solution, passing nitrogen for 5 min, sealing, heating to the reaction temperature, stirring and reacting for 8-20 h to obtain a polymer material;

[0051] S4. After washing the polymer material with anhydrous ethanol for 8-10 times by Soxhlet extraction, vacuum drying at 50°C for 10-36h, repeatedly washing the dried polymer material with anhydrous ethanol / acetic acid mixed solution with a volume ratio of 6-10:1 to remove the template molecule trans-ferulic acid in the polymer material until trans-ferulic acid is no longer detected in the washing solution, then washing the polymer material with distilled water until it is neutral, and vacuum drying at 50°C for 12h to obtain a trans-ferulic acid bifunctional monomer molecular imprinting polymer.

[0052] Furthermore, in step S1, the solvent is one or more of anhydrous ethanol, acetonitrile and methanol.

[0053] The preparation method can be used to prepare a bifunctional monomer molecular imprinting polymer with high specific surface activity and good adsorption performance, and can be used to separate and purify trans-ferulic acid.

[0054] Specifically, since methacrylic acid and acrylamide may also react with each other, if a bifunctional monomer is directly added to a trans-ferulic acid solution, the binding of trans-ferulic acid will be affected. In the present application, the trans-ferulic acid solution is evenly divided into two parts, and then mixed with methacrylic acid and acrylamide respectively, so that it is complexed with trans-ferulic acid, and trans-ferulic acid is firstly coordinated with different functional monomers, and then mixed together for prepolymerization and fixation, so that the template molecule trans-ferulic acid is connected to methacrylic acid and acrylamide, and then polymerized to form a molecular imprinting polymer, which can avoid unnecessary polymerization and affect the performance of the molecular imprinting polymer, and help to ensure the expression of dual recognition sites.

[0055] Further, in step S2, the molar ratio of methacrylic acid to acrylamide in the mixed solution is 8:5. In the present application, trans-ferulic acid has hydroxyl and carboxyl groups, and can react with methacrylic acid and acrylamide as a template molecule to undergo polymerization. However, the performance difference between methacrylic acid and acrylamide will affect the polymerization process, and polymerization may also occur between the two. If the ratio of the two is not controlled, it is easy to affect the expression of the required recognition site. Therefore, the inventors of the present application screened out a specific ratio of bifunctional monomers through multiple rounds of orthogonal experiments, and by maintaining the molar ratio of methacrylic acid to acrylamide at 8:5, the prepared molecularly imprinted polymer has a better elution effect.

[0056] Furthermore, in step S2, the molar ratio of trans-ferulic acid to acrylamide in the mixed solution is 1.3: 1. In the present application, too high or too low an amount of trans-ferulic acid will affect the recognition performance of the prepared molecularly imprinted polymer. The present application controls the polymerization of trans-ferulic acid and bifunctional monomers at a suitable ratio, which is conducive to forming a molecularly imprinted polymer with good pore position and spatial structure, avoids the difficulty of washing out the template molecule due to the high degree of closure and affects the recognition performance, and can also improve the dual recognition effect.

[0057] Further, in step S3, the reaction temperature is 50-70° C., more preferably 55-65° C. In the present application, cross-linking mainly occurs at 55-65° C., and the reaction is accelerated as the temperature increases.

[0058] Specifically, in the process of remixing after the bifunctional monomers are each complexed with trans-ferulic acid, since the hydrogen bonding between acrylamide and trans-ferulic acid is stronger than that between acrylamide and methacrylic acid, direct cross-linking is likely to affect the coordination relationship between them. In this way, certain steric hindrance may be formed during the polymerization process, affecting the performance of the molecular imprinting polymer and easily weakening its dual recognition effect.

[0059] In view of this problem, in the present application, before heating to the reaction temperature, the temperature is first raised to 40°C and maintained for 30 minutes. Specifically, after mixing to obtain a mixed solution, acrylamide can provide an alkaline environment for the mixed solution. By heating and keeping the temperature for a period of time before cross-linking, trans-ferulic acid and methacrylic acid are dehydrated in an alkaline environment to form a certain degree of intramolecular esterification before cross-linking occurs, which can maintain a good coordination relationship between ferulic acid and the functional monomer before cross-linking, which is conducive to the expression of the dual recognition function of the prepared molecular imprinting polymer. Insufficient preheating time will affect the cross-linking effect.

[0060] Through the preparation method provided in the present application, trans-ferulic acid is used as a template molecule and is prepared with a bifunctional monomer. The prepared molecular imprinting polymer can perform dual recognition and has good specific and selective adsorption properties for trans-ferulic acid, which is helpful for purifying trans-ferulic acid.

[0061] The present application also provides a trans-ferulic acid bifunctional monomer molecular imprinted polymer, wherein the trans-ferulic acid bifunctional monomer molecular imprinted polymer is prepared by the preparation method of the trans-ferulic acid bifunctional monomer molecular imprinted polymer as described above. The prepared trans-ferulic acid bifunctional monomer molecular imprinted polymer has high specific surface activity and good adsorption performance, and can be used to separate and purify trans-ferulic acid.

[0062] Specifically, a proper amount of trans-ferulic acid bifunctional monomer molecular imprinting polymer can be loaded into a solid phase extraction column, 0.2-20 mL of a trans-ferulic acid solution (solvent is anhydrous ethanol) with a concentration of 1000-5000 mg / L is loaded, vacuum filtered, and the separation performance of the trans-ferulic acid bifunctional monomer molecular imprinting polymer is verified by solid phase extraction.

[0063] Furthermore, the particle size of the trans-ferulic acid bifunctional monomer molecular imprinting polymer is 80 mesh. In the present application, the molecular imprinting polymer can be ground to a particle size of 80 mesh after preparation, which is convenient for loading into the molecular imprinting extraction column. The particle size is conducive to solid phase extraction, improves the speed of solid phase extraction elution, and improves the recognition degree of trans-ferulic acid.

[0064] The present application also provides an application of the trans-ferulic acid bifunctional monomer molecular imprinting polymer as described above, wherein the trans-ferulic acid bifunctional monomer molecular imprinting polymer is applied to separate and purify trans-ferulic acid in a fermentation broth by solid phase extraction, comprising the following steps:

[0065] (1) Preparation: preparing a fermentation broth containing trans-ferulic acid by using a trans-ferulic acid high-producing strain;

[0066] (2) Extraction and separation: The fermentation broth containing trans-ferulic acid is added to a molecular imprinting extraction column filled with a molecular imprinting polymer of a bifunctional monomer of trans-ferulic acid, and eluted with anhydrous ethanol and a mixture of anhydrous ethanol and organic acid water in sequence, and the eluate is collected and concentrated under reduced pressure to obtain a crude trans-ferulic acid product;

[0067] (3) Purification: Dissolve the crude trans-ferulic acid in ultrapure water, add hydrochloric acid to adjust the pH to 3-4, add anhydrous ethanol, stir at 20-25°C and let stand to obtain trans-ferulic acid.

[0068] By applying the prepared trans-ferulic acid bifunctional monomer molecular imprinting polymer to solid phase extraction, the provided separation and purification method is simple and efficient and can be simply adjusted and applied to large-scale production.

[0069] Furthermore, in step (1), the concentration of trans-ferulic acid in the fermentation broth containing trans-ferulic acid is 20-2000 mg / L.

[0070] Specifically, the anhydrous ethanol may be analytical grade anhydrous ethanol, which has good purification and elution effects, while the use of an ethanol solution will reduce the elution effect.

[0071] Further, in step (2), the organic acid in the mixture of anhydrous ethanol and organic acid water is one of formic acid, acetic acid or trifluoroacetic acid, wherein the volume ratio of anhydrous ethanol to organic acid water is 6-10:1-4, wherein the volume proportion of organic acid in organic acid water is 1 / 1000 to 3 / 1000. In the present application, the mixture of anhydrous ethanol and organic acid water can be specifically prepared by anhydrous ethanol and formic acid aqueous solution, acetic acid aqueous solution or trifluoroacetic acid aqueous solution, and respectively prepared into a mixture of anhydrous ethanol and formic acid water, a mixture of anhydrous ethanol and acetic acid water or a mixture of anhydrous ethanol and trifluoroacetic acid water for elution.

[0072] Furthermore, in step (2), the solid-liquid ratio of the trans-ferulic acid bifunctional monomer molecular imprinting polymer to the fermentation broth containing trans-ferulic acid is 1 g:30-180 mL.

[0073] Furthermore, in step (3), the crude trans-ferulic acid is dissolved in ultrapure water to a final concentration of 9-12 g / L;

[0074] In step (3), the volume ratio of ultrapure water to anhydrous ethanol is 3-5:1.

[0075] More specifically, the separation and purification method of trans-ferulic acid by solid phase extraction comprises the following steps:

[0076] (1) Preparation: A fermentation broth containing trans-ferulic acid is prepared using a trans-ferulic acid high-yielding strain, wherein the specific method is as follows: Escherichia coli is inoculated at a rate of 5% into a fermentation medium, and cultured at 37°C for 3 days with shaking to obtain a seed solution; then, the seed solution is inoculated at a rate of 10% into an enrichment medium, and cultured at 37°C for 5 days with shaking, the culture solution is centrifuged at 5°C and 9000 r / min for 15 minutes, and the supernatant is collected to obtain a fermentation broth rich in trans-ferulic acid having a trans-ferulic acid concentration of 20-2000 mg / L;

[0077] (2) Extraction and separation: The fermentation broth containing trans-ferulic acid is treated by molecular imprinting solid phase extraction method, and the fermentation broth containing trans-ferulic acid is added to a molecular imprinting extraction column filled with trans-ferulic acid bifunctional monomer molecular imprinting polymer, the solid-liquid ratio of trans-ferulic acid bifunctional monomer molecular imprinting polymer to the fermentation broth containing trans-ferulic acid is 1g:30-180mL, and then eluted with 10 column volumes of anhydrous ethanol and 10 column volumes of a mixed solution of anhydrous ethanol and organic acid water, respectively, the eluate is collected, and concentrated under reduced pressure to obtain a crude trans-ferulic acid; the vacuum degree of the reduced pressure concentration is -0.9MPa, and the rotation speed is 150-180 rpm;

[0078] (3) Purification: Dissolve the crude trans-ferulic acid in ultrapure water until the final concentration of trans-ferulic acid is 9-12 g / L, add hydrochloric acid to adjust the pH to 3-4, add anhydrous ethanol, and the volume ratio of ultrapure water to anhydrous ethanol is 3-5:1. Stir at 20-25°C for 5-6 hours and then let stand for 12-24 hours to obtain crystals of trans-ferulic acid.

[0079] The trans-ferulic acid bifunctional monomer molecular imprinting polymer provided in the present application has good separation and purification effect and high efficiency when applied to solid phase extraction. The solid phase extraction method can facilitate the large-scale production of trans-ferulic acid, which is of great significance to the industrial application of trans-ferulic acid.

[0080] The invention is further described below by means of specific examples.

[0081] The fermentation medium and enrichment medium used in the examples of the present application are both M9 medium, and the preparation method is as follows: 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, and 1000 mL of sterilized double distilled water.

[0082] In order to determine the optimal amount of methacrylic acid and acrylamide, the effects of different amounts of methacrylic acid and molar ratio of acrylamide to trans-ferulic acid on molecular imprinting polymer factor (IF) were investigated using anhydrous ethanol as solvent. The following design uses methacrylic acid, acrylamide, crosslinking agent, and temperature as the investigation factors to design a four-factor three-level orthogonal experiment.

[0083] The preparation method of the trans-ferulic acid bifunctional monomer molecularly imprinted polymer of Examples 1-9 of the present application is:

[0084] Dissolve 5 mmol of trans-ferulic acid in 50 mL of anhydrous ethanol and stir magnetically until completely dissolved to prepare a trans-ferulic acid solution;

[0085] The trans-ferulic acid solution is evenly divided into a first trans-ferulic acid solution and a second trans-ferulic acid solution, methacrylic acid is added to the first trans-ferulic acid solution, and the mixture is magnetically stirred at room temperature for 3 hours, acrylamide is added to the second trans-ferulic acid solution, and the mixture is magnetically stirred at room temperature for 3 hours, and the first trans-ferulic acid solution and the second trans-ferulic acid solution are mixed to obtain a mixed solution;

[0086] Ethylene glycol dimethacrylate and 50 mg of azobisisobutyronitrile were added to the mixed solution, nitrogen was introduced for 5 minutes, the mixture was sealed, heated to 40° C. and maintained for 30 minutes, then heated to the reaction temperature, and subjected to magnetic stirring for polymerization for 20 hours, and solid-liquid separation was performed and the solid was retained to obtain a polymer material;

[0087] The polymer material was washed with anhydrous ethanol for 10 times by Soxhlet extraction, and then vacuum dried at 50° C. for 12 h. The dried polymer material was repeatedly washed with an anhydrous ethanol / acetic acid mixed solution with a volume ratio of 8:1 to remove the template molecule trans-ferulic acid in the polymer material until no trans-ferulic acid was detected in the washing solution. The polymer material was then washed with distilled water until neutral, and vacuum dried at 50° C. for 12 h to obtain the trans-ferulic acid bifunctional monomer molecular imprinting polymer of Example 1-9. The trans-ferulic acid bifunctional monomer molecular imprinting polymer of Example 1-9 was ground into 80 mesh for later use.

[0088] After synthesis, the template molecule ferulic acid will be washed away to form a hole that can specifically adsorb ferulic acid. 0.15 g of each of the trans-ferulic acid bifunctional monomer molecular imprinting polymers of Examples 1-9 was loaded on a solid phase extraction column, and 2 mL of a trans-ferulic acid solution (solvent is anhydrous ethanol) with a concentration of 1000 mg / L was loaded. The eluent was a mixture of methanol and formic acid water, wherein the volume ratio of methanol to formic acid water (formic acid volume ratio is 1 / 1000) was 7:3. The collected eluent was vacuum filtered at a vacuum degree of -0.9 MPa, and the separation performance of the trans-ferulic acid bifunctional monomer molecular imprinting polymers of Examples 1-9 was verified by solid phase extraction. The experimental results of trans-ferulic acid recovery are shown in Table 1:

[0089] Table 1

[0090] factor Methacrylic acid (mmol) Acrylamide (mmol) Cross-linking agent (mmol) Reaction temperature (℃) Experimental results (mg) Example 1 1 3 20 60 0.234 Example 2 8 0 25 60 0.237 Example 3 5 3 25 55 0.233 Example 4 5 5 15 60 0.230 Example 5 5 0 20 65 0.152 Example 6 8 3 15 65 0.334 Example 7 8 5 20 55 0.285 Example 8 1 0 15 55 0.299 Example 9 1 5 25 65 0.268

[0091] The effect curve of the orthogonal experiment is as follows Figure 1 As shown, it can be seen from the orthogonal experimental results that the molar ratio of methacrylic acid to acrylamide in the bifunctional monomer is 8:5, and the orthogonal results show that the temperature is better at 55°C, but considering the reaction speed and cross-linking degree, the reaction temperature is more preferably 65°C.

[0092] While maintaining the molar ratio of methacrylic acid to acrylamide at 8:5, the molar ratio of the template molecule trans-ferulic acid is optimized. The preparation method of the trans-ferulic acid bifunctional monomer molecular imprinting polymer of Examples 10-15 is substantially the same as that of Example 1, except that the trans-ferulic acid solutions of Examples 10-13 are prepared by adding 1 mmol, 2 mmol, 4 mmol and 6.5 mmol trans-ferulic acid to 50 mL of anhydrous ethanol, respectively, the trans-ferulic acid solution of Example 14 is prepared by adding 6.5 mmol trans-ferulic acid to 50 mL of acetonitrile, and Example 15 uses 50 mL of methanol as the solvent without adding the template molecule trans-ferulic acid. The raw material formula of the trans-ferulic acid bifunctional monomer molecular imprinting polymer of Examples 10-15 is shown in Table 2:

[0093] Table 2

[0094]

[0095] According to the raw material ratios shown in Table 2, the trans-ferulic acid bifunctional monomer molecularly imprinted polymers of Examples 10-15 were prepared, and the adsorption performance on the fermentation broth containing trans-ferulic acid was tested.

[0096] In the examples of the present application, the trans-ferulic acid high-producing strain used is from the research group of Shen Xiaolin of Beijing University of Chemical Technology, and the trans-ferulic acid high-producing strain is used to produce the fermentation broth of trans-ferulic acid.

[0097] The fermentation broth containing trans-ferulic acid is prepared by a trans-ferulic acid high-producing strain, and the specific method is as follows: Escherichia coli (trans-ferulic acid high-producing strain) is inoculated into a fermentation medium at an inoculation rate of 5%, and shake-cultured at 37°C for 3 days to obtain a seed liquid; then the seed liquid is inoculated into an enrichment medium at an inoculation rate of 10%, and shake-cultured at 37°C for 5 days, the culture liquid is centrifuged at 5°C and 9000r / min for 15 minutes, and the supernatant is collected to obtain a fermentation broth rich in trans-ferulic acid with a trans-ferulic acid concentration of 69 mg / L.

[0098] 0.15 g of the molecular imprinted polymers of Examples 10-15 were respectively loaded into the solid phase extraction column, the eluent was 4 mL of a mixed solution of anhydrous ethanol / water with a volume ratio of 4:6, the loading liquid was 20 mL of the fermentation liquid containing trans-ferulic acid, and the eluent was 10 mL of anhydrous ethanol. Under a vacuum degree of -0.9 MPa, the eluent was first used for elution, then the fermentation liquid was loaded, and finally eluted, and the eluent was loaded on a high performance liquid chromatography for determination of the adsorption amount of trans-ferulic acid. The test results are shown in Table 3:

[0099] Table 3

[0100] Group Liquid chromatography peak area data (μV*sec) Concentration of trans-ferulic acid in the eluate (μg / mL) Example 10 2742706 73.504 Embodiment 11 1801071 47.460 Example 12 2122453 56.349 Example 13 2949721 79.230 Embodiment 14 2605434 69.707 Embodiment 15 1477980 38.524

[0101] The HPLC chromatogram of the adsorbed trans-ferulic acid of Example 13 is as follows Figure 2 As shown, the HPLC chromatogram of trans-ferulic acid standard is as follows Figure 3 As shown, by comparing the test results, it can be found that the trans-ferulic acid bifunctional monomer molecular imprinting polymer prepared in Example 13 has a relatively good adsorption effect on trans-ferulic acid, and the molar ratio of trans-ferulic acid to acrylamide during preparation is preferably 1.3:1.

[0102] The dynamic adsorption of the trans-ferulic acid bifunctional monomer molecular imprinting polymer prepared in Example 13 was tested under different injection volumes. The solid phase extraction columns of Examples 16-20 were all filled with 0.15 g of the trans-ferulic acid bifunctional monomer molecular imprinting polymer prepared in Example 13. Different volumes of fermentation broth containing trans-ferulic acid (concentration of 69.405 mg / L) were used to separate and purify trans-ferulic acid by solid phase extraction, respectively, comprising the following steps:

[0103] (1) Preparation: A fermentation broth containing trans-ferulic acid is prepared by a trans-ferulic acid high-producing strain, wherein the specific method is as follows: Escherichia coli (trans-ferulic acid high-producing strain) is inoculated into a fermentation medium at an inoculum amount of 5%, and cultured at 37°C for 3 days with shaking to obtain a seed solution; then, the seed solution is inoculated into an enrichment medium at an inoculum amount of 10%, and cultured at 37°C for 5 days with shaking, and the culture solution is centrifuged at 5°C and 9000 r / min for 15 minutes, and the supernatant is collected to obtain a fermentation broth rich in trans-ferulic acid with a trans-ferulic acid concentration of 69.405 mg / L;

[0104] (2) Extraction and separation: The fermentation broth containing trans-ferulic acid was treated by molecular imprinting solid phase extraction method. 5 mL, 10 mL, 15 mL, 20 mL and 25 mL of the fermentation broth containing trans-ferulic acid were added to a molecular imprinting extraction column filled with 0.15 g of the trans-ferulic acid bifunctional monomer molecular imprinting polymer of Example 13, and then eluted with 10 column volumes of anhydrous ethanol and 10 column volumes of a mixture of anhydrous ethanol and formic acid water in sequence. The eluate was collected and concentrated under reduced pressure to obtain a crude trans-ferulic acid product; the vacuum degree of the reduced pressure concentration was -0.9 MPa and the rotation speed was 170 rpm;

[0105] The volume ratio of anhydrous ethanol to formic acid water in the mixture of anhydrous ethanol and formic acid water is 6:4, and the volume proportion of formic acid in formic acid water is one thousandth;

[0106] (3) Purification: 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 anhydrous ethanol, and the volume ratio of ultrapure water to anhydrous ethanol is 3:1. Stir at 20°C for 5 h and then let stand for 24 h to obtain crystals of trans-ferulic acid.

[0107] The concentration of trans-ferulic acid in the eluent in step (2) was determined by high performance liquid chromatography. The test results are shown in Table 4:

[0108] Table 4

[0109] Group Example 16 Embodiment 17 Embodiment 18 Embodiment 19 Embodiment 20 Fermentation broth volume (mL) 5 10 15 20 25 Liquid chromatography peak area data (μV*sec) 2784061 5260185 8153887 10313578 13177662 Trans-ferulic acid content in fermentation broth (μg) 347.025 694.05 1041.075 1388.1 1735.125 Concentration of trans-ferulic acid in the eluent (μg / mL) 75.537 138.815 212.764 267.955 341.147 Eluted trans-ferulic acid / μg 302.147 555.259 851.055 1071.820 1364.589 Enrichment factor EF 1.08 2.00 3.06 3.86 4.91

[0110] Among them, when the trans-ferulic acid fermentation broth is loaded with a small volume, the trans-ferulic acid bifunctional monomer molecular imprinted polymer in the extraction column can absorb most of the trans-ferulic acid, and as the volume of the fermentation broth increases, the amount of trans-ferulic acid adsorbed by the imprinted polymer increases, but the amount of trans-ferulic acid that penetrates out also increases. Therefore, it is necessary to test the appropriate enrichment multiple, in which the amount of trans-ferulic acid that penetrates out is as small as possible when the volume of the adsorbed fermentation broth is as large as possible. Taking 25 mL of the fermentation broth containing trans-ferulic acid in Example 20 as an example, the NMR carbon spectrum and NMR hydrogen spectrum of the trans-ferulic acid purified in Example 20 are as follows: Figure 4 and Figure 5 As shown, the maximum adsorption capacity of the molecularly imprinted polymer prepared in Example 13 is 9.097 mg / g, the purity of the separated and purified trans-ferulic acid is high, the recovery rate of the fermentation broth can reach 78.64%, and the enrichment factor also reaches 4.91.

[0111] The trans-ferulic acid molecularly imprinted polymer prepared by the bifunctional monomer in the embodiment of the present application can efficiently and specifically adsorb the target molecule, and can achieve large-scale separation and purification of trans-ferulic acid through solid phase extraction, which is of great significance.

[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 trans-ferulic acid bifunctional monomer molecularly imprinted polymer, characterized in that: The following steps are involved: preparing a trans-ferulic acid solution; The trans-ferulic acid solution is evenly divided into a first trans-ferulic acid solution and a second trans-ferulic acid solution, methacrylic acid is added to the first trans-ferulic acid solution and the mixture is stirred and mixed, acrylamide is added to the second trans-ferulic acid solution and the mixture is stirred and mixed, and the first trans-ferulic acid solution and the second trans-ferulic acid solution are mixed to obtain a mixed solution; Adding a crosslinking agent and azobisisobutyronitrile to the mixed solution, introducing nitrogen, sealing, heating to a reaction temperature, stirring for reaction, and obtaining a polymer material; Washing the polymer material to remove trans-ferulic acid, then washing the polymer material to neutrality, heating and vacuum drying to obtain the trans-ferulic acid bifunctional monomer molecular imprinting polymer; The reaction temperature is 50-70°C; Before heating to the reaction temperature, the temperature was first raised to 40°C and maintained for 30 min; The molar ratio of the methacrylic acid to acrylamide in the mixed solution is 8:5; The molar ratio of trans-ferulic acid to acrylamide in the mixed solution is 1.3:

1.

2. The method for preparing the trans-ferulic acid bifunctional monomer molecularly imprinted polymer according to claim 1, characterized in that: Each 0.5-7 mmol of trans-ferulic acid is dissolved in 50 mL of solvent, wherein the solvent is one or more of anhydrous ethanol, acetonitrile and methanol; The cross-linking agent is ethylene glycol dimethacrylate; In the mixed solution, 10-250 mmol of a cross-linking agent and 50-300 mg of azobisisobutyronitrile are used for every 0.5-7 mmol of trans-ferulic acid.

3. A trans-ferulic acid bifunctional monomer molecular imprinting polymer, characterized in that: The invention is prepared by the method for preparing the trans-ferulic acid bifunctional monomer molecular imprinting polymer as described in any one of claims 1 to 2.

4. An application of the trans-ferulic acid bifunctional monomer molecularly imprinted polymer as claimed in claim 3, characterized in that: The trans-ferulic acid bifunctional monomer molecular imprinting polymer is applied to separate and purify trans-ferulic acid in fermentation broth by solid phase extraction, comprising the following steps: (1) Preparation: preparing a fermentation broth containing trans-ferulic acid by using a trans-ferulic acid high-producing strain; (2) Extraction and separation: adding the fermentation broth containing trans-ferulic acid to a molecular imprinting extraction column filled with the trans-ferulic acid bifunctional monomer molecular imprinting polymer, eluting with anhydrous ethanol and a mixed solution of anhydrous ethanol and organic acid water in sequence, collecting the eluate, and concentrating under reduced pressure to obtain a crude trans-ferulic acid product; (3) Purification: The crude trans-ferulic acid is dissolved in ultrapure water, hydrochloric acid is added to adjust the pH to 3-4, anhydrous ethanol is added, and the mixture is stirred at 20-25° C. and allowed to stand to obtain trans-ferulic acid.

5. The use of the trans-ferulic acid bifunctional monomer molecularly imprinted polymer according to claim 4, characterized in that: The solid-liquid ratio of the trans-ferulic acid bifunctional monomer molecular imprinting polymer to the fermentation liquid containing trans-ferulic acid is 1 g:30-180 mL.

6. The use of the trans-ferulic acid bifunctional monomer molecularly imprinted polymer according to claim 4, characterized in that: The concentration of trans-ferulic acid in the fermentation broth containing trans-ferulic acid is 20-2000 mg / L.

7. The use of the trans-ferulic acid bifunctional monomer molecularly imprinted polymer according to claim 4, characterized in that: In step (3), the crude trans-ferulic acid is dissolved in ultrapure water to a final concentration of 9-12 g / L; In step (3), the volume ratio of ultrapure water to anhydrous ethanol is 3-5:1.

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