Preparation and application of molecularly imprinted resin for extracting soy isoflavone

Abstract

The invention discloses preparation and application of molecularly imprinted resin for extracting soy isoflavone, and belongs to the technical field of natural product separation. The preparation method comprises the following steps: (1) dissolving a functional monomer in acetonitrile, uniformly stirring, and carrying out condensation polymerization at normal temperature to obtain a reaction solution; (2) sequentially adding a pore-foaming agent and a catalyst into the reaction liquid, and uniformly stirring to obtain an organic phase; (3) adding soy isoflavone and a cross-linking agent into water, and uniformly mixing to obtain an oil phase; (4) adding the oil phase into the organic phase, stirring for 20 minutes at normal temperature, standing for 12 hours in a sealed manner, and filtering to obtain solid resin; and (5) washing the solid resin with deionized water, and drying to obtain powder, namely the isoflavone molecularly imprinted resin. The isoflavone molecularly imprinted resin can more effectively and specifically recognize the target molecule isoflavone, the efficiency of selectively extracting the isoflavone is greatly improved, and the separation yield of the isoflavone reaches 90% or above.

Description

Technical Field

[0001] The invention relates to the preparation and application of a molecular imprinting resin for extracting soybean isoflavones, and belongs to the technical field of natural product separation. Background Art

[0002] Soy isoflavones are natural flavonoid compounds synthesized by leguminous plants through the phenylalanine metabolic pathway. Their core structure is a 3-phenylchromone skeleton (C 15 H 10 O2), has the characteristics of weak estrogenic activity, strong antioxidant activity, and dual hormone regulation. The isoflavone content in unprocessed soybean yellow slurry produced by the soybean products industry is usually as high as 30%-50%. Soy isoflavones exist mainly in the form of glycoside conjugates in nature, and it is impossible to directly concentrate and crystallize the isoflavones to purify them. The current extraction method of isoflavones commonly uses resin separation technology, but there is a lack of molecular imprinting resins specifically for extracting isoflavones, resulting in low isoflavone extraction efficiency. The isoflavone product needs to be purified through repeated and complicated steps. It is difficult to obtain an extraction rate of more than 90% for soy isoflavones through simple adsorption in conventional resins.

[0003] Chinese patent CN118684805A discloses the preparation and application of a material for the targeted recovery of isoflavones from soybean yellow slurry. The targeted imprinted material has an extraction efficiency of 60-65% for isoflavones. This patented technology has the following problems: (1) It uses conventional oscillation adsorption and is not designed for the separation of post-reaction materials, which increases the difficulty of post-reaction processing; (2) The targeted imprinted material contains acrylamide and DMF, which is detrimental to food safety; (3) The amount of organic reagent used is large and the polymer polymerization time is too long; (4) The dense pore structure of the polymer is not conducive to the separation and purification of isoflavones, resulting in low extraction efficiency.

[0004] Chinese patent CN118005842A discloses the preparation and application of a molecularly imprinted resin for maltitol purification. Using maltitol as a template molecule, an imprinted resin specifically for maltitol purification was synthesized. Although a specific resin was successfully prepared, the patented technology has the following problems: (1) The resin synthesis adopts a free radical polymerization reaction, which has harsh reaction conditions, slow polymerization, and reagent ratio deviation and insufficient nitrogen deoxygenation are not conducive to polymerization; (2) The toxicity of the intermediate product produced by the decomposition of azobisisobutyronitrile is not suitable for food requirements; (3) The preparation of the resin is complicated, highly technically dependent, and requires a large amount of organic solvent, resulting in high cost.

[0005] Therefore, molecularly imprinted resins capable of specifically extracting isoflavones are crucial for efficient isoflavone extraction. Furthermore, molecularly imprinted resins possess the ability to recognize and selectively adsorb specific molecules. Combining them with shape-memory polymers can imbue the material with shape memory properties that respond to specific molecules, simultaneously enabling molecular recognition and stimuli-responsive shape changes. The combination of molecularly imprinted resins and shape-memory polymers enhances the intelligence and application range of molecularly imprinted resins, broadening their potential applications. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a preparation and application of a molecularly imprinted resin for extracting soybean isoflavones, which solves the problems of low soybean isoflavone extraction efficiency, high preparation cost and safety of molecularly imprinted resins.

[0007] In order to solve the above technical problems, the technical solution provided by the present invention is: A preparation method for a molecularly imprinted resin for extracting soybean isoflavones comprises the following steps: (1) Dissolve the functional monomer in acetonitrile and stir evenly, and carry out polycondensation reaction at room temperature to obtain a reaction solution; (2) Add porogen and catalyst to the reaction solution in sequence and stir evenly to obtain an organic phase; (3) Add soybean isoflavones and a cross-linking agent to acetonitrile and mix well to obtain an oil phase; (4) Add the oil phase to the organic phase, stir at room temperature for 20-40 minutes, then seal and let stand for 10-12 hours, and filter to obtain solid resin; (5) Wash the solid resin with deionized water at a pH of 8-10 and dry it at 40-60°C to obtain a powder, which is the isoflavone molecularly imprinted resin.

[0008] Furthermore, the mass ratio of the functional monomer, porogen, catalyst, soy isoflavones and cross-linking agent is (5-20): (10-15): (0.7-1.0): (5-10): (10-20).

[0009] Furthermore, in step (1), the functional monomer is m-aminophenol. In the prior art, conventional molecular imprinting often uses acrylic monomers (e.g., methacrylic acid). The amino and hydroxyl groups of m-aminophenol can provide stronger hydrogen bonding sites, thereby enhancing the specific recognition of isoflavones. Furthermore, compared with isoflavone-MIPs reported in the literature (e.g., dopamine-based MIPs), the multiple bonding sites of m-aminophenol significantly enhance the selectivity factor.

[0010] Furthermore, in step (1), the amount of the functional monomer added is 3-5% of the mass of acetonitrile.

[0011] Furthermore, in step (1), the polycondensation reaction time is 7-10 hours.

[0012] Furthermore, in step (2), the porogen is polyethylene glycol 6000; the catalyst is hydrochloric acid, and the concentration of the hydrochloric acid is 2 mol / L. The hydrophilicity of polyethylene glycol 6000 improves the swelling of the resin in the aqueous phase, reduces the template embedding problem, and achieves an optimal balance between porosity and mechanical strength. In addition, the molecular weight and cross-linking density of polyethylene glycol 6000 can also improve the adsorption capacity of the isoflavone-imprinted resin. Selecting polyethylene glycol 6000 as a porogen can significantly increase the isoflavone adsorption capacity. The present invention uses hydrochloric acid as a catalyst, and by adjusting the hydrochloric acid concentration over a wide range to compensate for the room temperature reaction energy barrier, it avoids the destruction of the template molecular structure that may be caused by traditional polymerization under high temperature conditions.

[0013] Furthermore, in step (3), the cross-linking agent is glutaraldehyde; and the ratio of the cross-linking agent to acetonitrile is (0.2-0.3) g:50 mL. Glutaraldehyde can act as a hydrophilic cross-linking agent to reduce local overpolymerization and form more regular isoflavone-imprinted cavities.

[0014] Furthermore, in step (4), the stirring speed is 50 r / min.

[0015] Furthermore, in step (5), the washing operation is as follows: washing the solid resin with deionized water three times until the washing liquid is clear, then washing it once with ethanol, and washing it once with deionized water to remove residual ethanol. Washing the resin with deionized water and ethanol can remove the soy isoflavones in the solid resin.

[0016] The present invention uses m-aminophenol as a functional monomer, and its amino (-NH2) and hydroxyl (-OH) groups can form non-covalent complexes with phenolic hydroxyl groups, ketone groups and other groups of isoflavones through hydrogen bonds or π-π stacking. Glyoxal is used as a cross-linking agent to undergo a condensation reaction with the phenolic hydroxyl groups of m-aminophenol under acidic conditions. Polyethylene glycol 6000 is used to avoid template embedding caused by excessive local cross-linking, while forming a uniform porous network. Temporary pores are formed during the polymerization process, and a high specific surface area is retained after elution.

[0017] An application of a molecularly imprinted resin for extracting soybean isoflavones comprises the following steps: (1) Powdered isoflavone molecularly imprinted resin is loaded into an SPE extraction cartridge for isoflavone extraction; (2) Deionized water is flowed from the SPE extraction column into the packing area from top to bottom. After the water flows out, the soy isoflavone solution is added. After the solution flows out, it is eluted with deionized water with a pH of 8-9, and the soy isoflavones separated and purified in the packing area are collected.

[0018] Furthermore, in step (1), the SPE extraction column has a capacity of 10-20 mL and a height of 10-20 cm. The SPE extraction column includes three scale areas: 0 mL area, 5 mL area, and 10 mL area. Each scale area can accommodate 2-3 g of isoflavone molecularly imprinted resin. The SPE extraction column is equipped with two sieve plates, which are respectively located on the upper and lower surfaces of the isoflavone molecularly imprinted resin after the isoflavone molecularly imprinted resin is loaded, forming an upper and lower fixed structure. The remaining capacity of each SPE extraction column after the isoflavone molecularly imprinted resin is filled is 5-10 mL.

[0019] Furthermore, in step (1), the specific steps of filling the isoflavone molecularly imprinted resin are as follows: fixing the lower sieve plate to the bottom layer of the SPE extraction column and compacting it, slowly placing the dry powdered isoflavone molecularly imprinted resin on the lower sieve plate in the SPE extraction column through a medicine spoon and flattening it as a water outlet, until the isoflavone molecularly imprinted resin is filled to the 3mL mark, stopping the feeding, and then installing the upper sieve plate at the top of the SPE extraction column and compacting it as an inlet, with the filler area between the upper and lower sieve plates. The upper sieve plate separates large particle impurity components, the filler area between the upper and lower sieve plates specifically adsorbs soybean isoflavones, and the lower sieve plate prevents the resin in the filler area from mixing with the water sample and losing it, so that the molecularly imprinted resin remains in the filler area. The upper sieve plate, the filler area, and the lower sieve plate are connected in sequence to form a closed system, thereby achieving efficient separation of soybean isoflavones and impurity components.

[0020] Furthermore, in step (2), the pH value of the deionized water is 3-5, and the pH value is adjusted to 3-5 using 2 mol / L hydrochloric acid. Under acidic conditions, the phenolic hydroxyl groups of the imprinted resin are protonated, thereby enhancing the hydrogen bonding interaction with the amino or hydroxyl groups of the isoflavones, forming a more stable complex, which is beneficial for the subsequent adsorption of the isoflavones.

[0021] Furthermore, in step (2), the volume ratio of the deionized water to the soy isoflavone solution is 1:4.

[0022] Furthermore, in step (2), after the soy isoflavone solution is added, there is no feeding and taking out operation during the running of the soy isoflavone solution in the isoflavone molecularly imprinted resin.

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) The isoflavone molecularly imprinted resin prepared by the present invention can specifically adsorb more target molecules of isoflavones, has high selectivity for adsorption of isoflavones, and has no adsorption effect on other miscellaneous proteins in soybean yellow slurry, such as genistein. It can easily achieve the separation of isoflavones and other miscellaneous proteins, and the extraction yield of isoflavones reaches more than 95%.

[0024] (2) The isoflavone molecularly imprinted resin prepared by the present invention can be used for the extraction of soybean isoflavones in various fields. The isoflavone molecularly imprinted resin is used to specifically adsorb the isoflavone components in yellow slurry water, and the extraction effect is obvious. The extraction efficiency of the soybean isoflavone components is high, which is more than 15% higher than that of normal phenolic resin or molecularly imprinted polymer process.

[0025] (3) The present invention only uses the SPE extraction cartridge extraction mode during separation, which reduces the dependence and cost of high-energy consumption equipment and can reduce costs. In addition, the recycling of the SPE extraction cartridge extraction realizes the separation and enrichment of multiple components, reducing the generation of waste energy consumption during the separation process.

[0026] (4) The preparation method of the isoflavone molecularly imprinted resin of the present invention is simple, and the reaction is carried out at room temperature, which reduces energy consumption and equipment requirements. The isoflavone molecularly imprinted resin has a high yield and is easy to use. Compared with the preparation method of molecular imprinting in the prior art, the amount of organic reagents and toxic reagents used is greatly reduced, which significantly improves safety and reduces costs.

[0027] (5) The isoflavone molecularly imprinted resin of the present invention can be used as an isoflavone carrier and combined with a shape memory polymer to achieve intelligent release. By introducing a shape memory polymer structure and combining it with a dynamic cross-linking or environmental variable response mechanism, the intelligence and application range of the molecularly imprinted resin can be improved, thereby broadening the application field of the molecularly imprinted resin. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The absorbance changes of isoflavones after the resins prepared under different conditions in Experimental Example 5 adsorbed soy isoflavones water samples. DETAILED DESCRIPTION

[0029] Example 1 A preparation method for a molecularly imprinted resin for extracting soybean isoflavones comprises the following steps: (1) Dissolve 0.22 g of m-aminophenol in 1 mL of acetonitrile and stir evenly. Carry out polycondensation reaction at room temperature for 8 h to obtain a reaction solution. (2) Add 0.24 g of polyethylene glycol 6000 and 150 μL of 2 mol / L hydrochloric acid to the reaction solution and stir evenly to obtain an organic phase; (3) Add 0.1 g of soy isoflavones and 0.2902 g of glutaraldehyde to 50 mL of acetonitrile and mix well to obtain the oil phase; (4) Add the oil phase to the organic phase, stir at 50 r / min at room temperature for 20 min, then seal and let stand for 11 h, and filter to obtain solid resin; (5) The solid resin was washed three times with deionized water until the washing liquid was clear, then washed once with 2 mL of ethanol, and washed once with deionized water to remove the residual ethanol. After drying at 50 °C, a powder was obtained, which was the isoflavone molecularly imprinted resin.

[0030] Example 2 A preparation method for a molecularly imprinted resin for extracting soybean isoflavones comprises the following steps: (1) Dissolve 0.22 g of m-aminophenol in 1 mL of acetonitrile and stir evenly. Carry out polycondensation reaction at room temperature for 7 h to obtain a reaction solution. (2) Add 0.24 g of polyethylene glycol 6000 and 150 μL of 2 mol / L hydrochloric acid to the reaction solution and stir evenly to obtain an organic phase; (3) Add 0.1 g of soy isoflavones and 0.2902 g of glutaraldehyde to 50 mL of acetonitrile and mix well to obtain the oil phase; (4) Add the oil phase to the organic phase, stir at 50 r / min for 40 min at room temperature, then seal and let stand for 10 h, and filter to obtain solid resin; (5) The solid resin was washed three times with deionized water until the washing liquid was clear, then washed once with 2 mL of ethanol, and washed once with deionized water to remove the residual ethanol. After drying at 40 °C, a powder was obtained, which was the isoflavone molecularly imprinted resin.

[0031] Example 3 A preparation method for a molecularly imprinted resin for extracting soybean isoflavones comprises the following steps: (1) Dissolve 0.22 g of m-aminophenol in 1 mL of acetonitrile and stir evenly. Carry out polycondensation reaction at room temperature for 10 h to obtain a reaction solution. (2) Add 0.24 g of polyethylene glycol 6000 and 150 μL of 2 mol / L hydrochloric acid to the reaction solution and stir evenly to obtain an organic phase; (3) Add 0.1 g of soy isoflavones and 0.2902 g of glutaraldehyde to 50 mL of acetonitrile and mix well to obtain the oil phase; (4) Add the oil phase to the organic phase, stir at 50 r / min for 30 min at room temperature, then seal and let stand for 12 h, and filter to obtain solid resin; (5) The solid resin was washed three times with deionized water until the washing liquid was clear, then washed once with 2 mL of ethanol, and washed once with deionized water to remove the residual ethanol. After drying at 60 °C, a powder was obtained, which was the isoflavone molecularly imprinted resin.

[0032] Example 4 An application of a molecularly imprinted resin for extracting soybean isoflavones comprises the following steps: (1) 0.1 g of powdered isoflavone molecularly imprinted resin was loaded into the SPE extraction cartridge for isoflavone extraction; (2) 2 mL of deionized water with a pH of 3-5 was flowed from the SPE extraction column into the packing area from top to bottom. After the water flowed out, 8 mL of soy isoflavones water sample with a temperature of room temperature and a pH of 6.2 was added. At this time, the pH of the soy isoflavones water sample was not adjusted. After the solution flowed out, it was eluted with deionized water with a pH of 8-9, and the soy isoflavones separated and purified in the packing area were collected.

[0033] Comparative Example 1 The difference from Example 1 is that 0.1 g of soy isoflavones is not added in step (3).

[0034] Comparative Example 2 The difference from Example 1 is that in step (3), 50 mL of acetonitrile and 0.2902 g of glutaraldehyde are replaced with 50 mL of water and 0.2902 g of glyoxal.

[0035] Comparative Example 3 The difference from Example 1 is that: in step (3), 0.1 g of soy isoflavones, 0.25 g of 4-vinylpyridine, 0.226 g of N-isopropylacrylamide, 0.17 g of hydrogen peroxide, and 0.2902 g of glyoxal are added to 50 mL of acetonitrile and mixed uniformly to obtain an oil phase; Comparative Example 4 The difference from Example 1 is that in step (3), 0.1 g of soy isoflavones, 0.25 g of 4-vinylpyridine, 0.226 g of N-isopropylacrylamide, 0.17 g of hydrogen peroxide, and 0.2902 g of glutaraldehyde are added to 50 mL of acetonitrile and mixed uniformly to obtain an oil phase; Comparative Example 5 The difference from Example 1 is that in step (3), 50 mL of acetonitrile is replaced by 50 mL of water.

[0036] Comparative Example 6 The difference from Example 1 is that: in step (3), 0.1 g of soy isoflavones, 0.25 g of 4-vinylpyridine, 0.226 g of N-isopropylacrylamide, 0.17 g of hydrogen peroxide, and 0.2902 g of glutaraldehyde are added to 50 mL of water and mixed uniformly to obtain an oil phase; Experimental Example 1 Soybean isoflavone water samples with soy isoflavone contents of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, and 2.0% were prepared, respectively, and were labeled as samples 1-7. The isoflavone molecularly imprinted resin of Example 1 was used to perform experiments according to the method of Example 4. The soy isoflavone solution separated and purified in the filler area (labeled as component A) and the filtrate after adsorption (labeled as component B) were collected, their absorbances were detected, and the extraction rate of soy isoflavones was calculated.

[0037] The calculation formula of extraction rate is X= is the isoflavone concentration of the original water sample; is the absorbance of isoflavones in water sample after original adsorption; is the absorbance of isoflavones in the original water sample; X is the adsorption rate of isoflavones in the water sample by isoflavone-imprinted resin.

[0038] The results are listed in Table 1.

[0039] Table 1. Adsorption properties of isoflavone molecularly imprinted resin As shown in Table 1, Component A obtained from samples 1-7 all contained significant amounts of soy isoflavones, with absorbances close to those of aqueous isoflavone samples. Component B, the filtrate after adsorption, contained virtually no residual soy isoflavones and exhibited a significantly lower absorbance. Calculations indicate that the resins of the present invention achieved extraction rates exceeding 88% for soy isoflavones. Observation during the experiments revealed that samples 1-7 were yellowish-brown in color, while the filtrates after adsorption were clear, transparent, and colorless. This demonstrates that the isoflavone molecularly imprinted resins of the present invention effectively adsorb soy isoflavones, resulting in a high soy isoflavone extraction rate.

[0040] Experimental Example 2 The resins prepared in Example 1 and Comparative Examples 1-6 were used according to the method of Example 4 to adsorb a 0.2% soy isoflavone aqueous sample for different adsorption times, followed by separation. The absorbance of the adsorbed filtrate was then measured. The results are listed in Table 2.

[0041] Table 2. Absorbance of the filtrate after adsorption As shown in Table 2, the absorbance of the filtrates obtained by adsorbing soy isoflavones in aqueous samples for different adsorption times using the isoflavone molecularly imprinted resin of Example 1 was low, all below 0.1. Furthermore, the absorbance of the filtrates decreased with increasing adsorption time. Compared with the resins of Comparative Examples 1-6, the absorbance of the filtrates after adsorption in Example 1 was significantly lower. This indicates that the resin of the present invention exhibits excellent specific adsorption of soy isoflavones and high extraction efficiency even with the same adsorption time. Therefore, the isoflavone molecularly imprinted resin of the present invention exhibits highly selective adsorption of soy isoflavones and a high extraction rate.

[0042] Experimental Example 3 The resins prepared in Example 1 and Comparative Examples 1-6 were used according to the method of Example 4 to adsorb 0.2% soy isoflavones in water at different pH values and then separated. The absorbance of the adsorbed filtrate was measured. The results are listed in Table 3.

[0043] Table 3. Absorbance of the filtrate after adsorption As shown in Table 3, when an unadjusted aqueous soy isoflavone sample was adsorbed at a pH of 6.5 and a temperature of 25°C using the resins of Example 1 and Comparative Examples 1-6, the absorbance of the filtrate after adsorption by the resin of Example 1 was significantly lower than that of the filtrate after adsorption by the resins of Comparative Examples 1-6. When the pH of the aqueous soy isoflavone sample was adjusted to 3-5 and then adsorbed using the resins of Example 1 and Comparative Examples 1-6, the absorbance of the filtrate after adsorption by the resin of Example 1 was significantly lower than that of the filtrate after adsorption by the resins of Comparative Examples 1-6. Furthermore, the change in absorbance of the filtrate after adsorption by the resin of Example 1 was minimal before and after pH adjustment, indicating that the resin of Example 1 is less affected by pH and its adsorption efficiency remains unaffected under pH changes. This demonstrates that the isoflavone molecularly imprinted resin prepared by the present invention still exhibits excellent adsorption efficiency for soy isoflavones, exhibits stable performance unaffected by pH, and exhibits a simple and adaptable preparation method.

[0044] Experimental Example 4 The resins prepared in Example 1 and Comparative Examples 1-6 were used according to the method of Example 4 to adsorb a 0.2% soy isoflavone aqueous sample at different temperatures and then separated. The absorbance of the filtrate after adsorption was measured. The results are listed in Table 4.

[0045] Table 4. Absorbance of the filtrate after adsorption As shown in Table 4, when an unadjusted aqueous soy isoflavone sample with a pH of 6.5 and a temperature of 25°C was adsorbed using the resins of Example 1 and Comparative Examples 1-6, the absorbance of the filtrate after adsorption by the resin of Example 1 was significantly lower than that of the filtrate after adsorption by the resins of Comparative Examples 1-6. When the temperature of the aqueous soy isoflavone sample was adjusted to 50°C and then adsorbed using the resins of Example 1 and Comparative Examples 1-6, the absorbance of the filtrate after adsorption by the resin of Example 1 was significantly lower than that of the filtrate after adsorption by the resins of Comparative Examples 1-6. Furthermore, the change in absorbance of the filtrate after adsorption by the resin of Example 1 was minimal before and after temperature adjustment, indicating that the resin of Example 1 is less affected by temperature and its adsorption effect remains unaffected under temperature changes. This demonstrates that the isoflavone molecularly imprinted resin prepared by the present invention still exhibits excellent adsorption efficiency for soy isoflavones, exhibits stable performance unaffected by temperature, and exhibits a simple and adaptable preparation method.

[0046] Experimental Example 5 An isoflavone extraction experiment was conducted on a soy isoflavone water sample at 25°C, containing 0.2% soy isoflavone and at a pH of 6.5. An equal amount of 0.1 g of resin was added to the filler area of an SPE extraction column to extract the isoflavones. The resins were prepared in Example 1 and Comparative Examples 1-6. The absorbance of the isoflavones in the solution was measured over time. The results are shown in the attached figure. Figure 1 .

[0047] from Figure 1 It can be seen that the absorbance of the original soybean isoflavone water sample is 3.5. The absorbance of the filtrate after adsorption in Example 1 changes the fastest, the amount of change is the largest, and the absorbance reduction is significantly higher than the absorbance reduction of the filtrate after adsorption by the resin prepared under the conditions of Comparative Examples 1-6, indicating that the resin in Example 1 is not only friendly to the preparation conditions, but also has good isoflavone-specific adsorption performance. Comparative Example 1 lacks specific binding sites for isoflavones and exhibits the worst isoflavone absorbance, indicating that using soybean isoflavones as a template is a necessary condition for the specific adsorption of soybean isoflavones. After the resin in Comparative Example 5 is prepared using water as a solvent to adsorb the soybean isoflavone water sample, its absorbance decrease is lower than that in Example 1, indicating that the adsorption performance of the resin prepared using an organic solvent will be improved. Moreover, by comparing Comparative Example 3 with Comparative Example 4, it can be seen that the resin prepared using glutaraldehyde as a cross-linking agent has better adsorption performance for isoflavones than the resin prepared using glyoxal as a cross-linking agent. It is shown that the present invention optimizes the preparation conditions of the isoflavone molecularly imprinted resin, so that the obtained isoflavone molecularly imprinted resin has good performance and high adsorption efficiency for soybean isoflavones.

[0048] In summary, the isoflavone molecularly imprinted resin prepared by the present invention has excellent adsorption properties and highly selective adsorption of isoflavones. It can meet the isoflavone extraction needs in various fields and is suitable for the extraction of isoflavones from soybean slurry at different temperature stages in the soy product industry, with high extraction efficiency. It still has excellent adsorption effect on soybean isoflavones under a wide range of extraction conditions, its performance is stable, and it is not affected by pH and temperature. The extraction process is more economical and energy-saving, and has wide application in various fields.

Claims

1. Preparation of a molecularly imprinted resin for extracting soybean isoflavones, characterized by: The following steps are involved: (1) Dissolve the functional monomer in acetonitrile and stir evenly, and carry out polycondensation reaction at room temperature to obtain a reaction solution; (2) Add porogen and catalyst to the reaction solution in sequence and stir evenly to obtain an organic phase; (3) Add soybean isoflavones and a cross-linking agent to acetonitrile and mix well to obtain an oil phase; (4) Add the oil phase to the organic phase, stir at room temperature for 20-40 minutes, then seal and let stand for 10-12 hours, and filter to obtain solid resin; (5) Wash the solid resin with deionized water at a pH of 8-10 and dry it at 40-60°C to obtain a powder, which is the isoflavone molecularly imprinted resin.

2. The preparation of the molecularly imprinted resin for extracting soybean isoflavones according to claim 1, characterized in that: The mass ratio of the functional monomer, the porogen, the catalyst, the soybean isoflavones and the cross-linking agent is (5-20): (10-15): (0.7-1.0): (5-10): (10-20).

3. The preparation of the molecularly imprinted resin for extracting soybean isoflavones according to claim 1, characterized in that: In step (1), the functional monomer is m-aminophenol.

4. The preparation of the molecularly imprinted resin for extracting soybean isoflavones according to claim 1, characterized in that: In step (1), the amount of the functional monomer added is 3-5% of the mass of acetonitrile.

5. The preparation of the molecularly imprinted resin for extracting soybean isoflavones according to claim 1, characterized in that: In step (2), the porogen is polyethylene glycol 6000; and the catalyst is hydrochloric acid.

6. The preparation of the molecularly imprinted resin for extracting soybean isoflavones according to claim 1, characterized in that: In step (3), the cross-linking agent is glutaraldehyde.

7. A use of the molecularly imprinted resin for extracting soybean isoflavones according to claim 1, characterized in that: The following steps are involved: (1) Powdered isoflavone molecularly imprinted resin is loaded into an SPE extraction cartridge for isoflavone extraction; (2) Deionized water is flowed from the SPE extraction column into the packing area from top to bottom. After the water flows out, the soy isoflavone solution is added. After the solution flows out, it is eluted with deionized water with a pH of 8-9, and the soy isoflavones separated and purified in the packing area are collected.

8. The use of the molecularly imprinted resin for extracting soybean isoflavones according to claim 7, characterized in that: In step (1), the SPE extraction column has a capacity of 10-20 mL and a height of 10-20 cm. The SPE extraction column includes three scale areas: a 0 mL area, a 5 mL area, and a 10 mL area. Each scale area can accommodate 2-3 g of isoflavone molecularly imprinted resin. The SPE extraction column is equipped with two sieve plates, which are respectively located on the upper and lower surfaces of the isoflavone molecularly imprinted resin after the isoflavone molecularly imprinted resin is loaded, forming an upper and lower fixation. The remaining capacity of each SPE extraction column after the isoflavone molecularly imprinted resin is filled is 5-10 mL.

9. The use of the molecularly imprinted resin for extracting soybean isoflavones according to claim 7, characterized in that: In step (2), the pH value of the deionized water is 3-5.

10. The use of the molecularly imprinted resin for extracting soybean isoflavones according to claim 7, characterized in that: In step (2), the volume ratio of the deionized water to the soy isoflavone solution is 1:4.

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