A process for genistein production

Abstract

The application provides a genistein production process and relates to the technical field of plant extraction. The process is characterized in that: first, the sophora fruit is crushed and extracted with hot water to fully dissolve and concentrate the effective components in the sophora fruit; then, MOFs encapsulated naringinase is added to the concentrated solution for directional enzymolysis; after that, a magnetic field is started, and the filter cake is collected by filtration; then, the filter cake is soaked with a solvent and decolorized with activated carbon; finally, genistein is obtained by filtration, heating and concentration, cooling and recrystallization. Compared with the traditional hydrolysis method, the process avoids the damage of strong acid to the product and the application of toxic reagents; compared with the enzyme-assisted extraction method, the naringinase directly acts on the substrate instead of assisting in extraction; compared with the in-situ bioconversion method, the process does not need to match a microbial fermentation enzyme production process, the enzymolysis time is short, and the product directly exists in the liquid phase system, which is beneficial to subsequent separation and purification; the MOFs encapsulated naringinase used in the application can be recycled multiple times, thereby reducing the production cost.

Description

Technical Field

[0001] This application relates to the field of plant extraction technology, specifically to a process for producing genistein. Background Technology

[0002] Genistein is a natural isoflavone compound. It possesses phytoestrogen activity, can bind to estrogen receptors, and exhibits a variety of pharmacological effects, including antioxidant properties, inhibition of tyrosine protein kinase activity, induction of programmed cell death in tumor cells, and inhibition of angiogenesis. It has broad application prospects in the prevention and treatment of osteoporosis, adjuvant therapy for tumors, and cardiovascular protection.

[0003] Sophora japonica fruit is the dried, mature fruit of the Sophora japonica plant, a member of the legume family. The fruit contains over 9% rutin, which, after hydrolysis to remove the glycosyl groups, can be converted into the active aglycone genistein, making it an ideal raw material for genistein preparation. In addition, Sophora japonica fruit also contains various glycosides with genistein as an aglycone, such as genistein glycoside and rutin diglycoside, which can also be converted into genistein after hydrolysis.

[0004] Currently, the processes for preparing genistein from Sophora japonica fruit include acid hydrolysis, enzyme degradation-assisted extraction, and biotransformation. However, all existing technologies have different drawbacks, which restrict their industrial application.

[0005] The traditional acid hydrolysis method involves extracting Sophora japonica fruit with ethanol, then adding hydrochloric acid or sulfuric acid for reflux hydrolysis, followed by extraction and purification with organic solvents such as chloroform, acetone, or ethyl acetate. Chinese patents CN1465571A and CN1465570A both adopt a process route combining acid hydrolysis and organic solvent extraction. This type of process has the following prominent problems: (1) The acid hydrolysis process requires the use of strong acid and is carried out under heating conditions, which places high demands on the corrosion resistance of the reaction equipment, increases equipment investment and maintenance costs, and also poses operational safety hazards; (2) Prolonged heating with strong acid can easily lead to the structural destruction or polymerization of genistein aglycones, affecting the product extraction rate and quality; (3) Subsequent purification requires multiple extractions with toxic reagents such as chloroform and dichloromethane, which not only increases reagent recovery costs but may also lead to excessive organic reagent residues; (4) The separation process generates a large amount of acidic waste liquid and organic waste liquid, increasing production costs.

[0006] Patent CN101760488A employs a strategy of enzymatic hydrolysis followed by extraction. It uses commercial enzymes such as cellulase and β-glucanase to pretreat the Sophora japonica powder, thereby disrupting the cell wall structure, and then extracts it by reflux with ethanol. The process avoids the use of strong acids to a certain extent, but still has the following shortcomings: (1) The cellulase and β-glucanase used in this process are cell wall breaking enzymes. Their target is cellulose and hemicellulose in the plant cell wall. The purpose is to destroy the cell wall structure and help substances such as rutin dissolve, rather than directly hydrolyzing the glycosidic bonds of rutin. If active genistein is to be obtained, additional acid hydrolysis or enzymatic conversion steps are still required; (2) The recrystallization of this process relies solely on ethanol reflux to adjust the pH for crystallization. The steps are simple, and the extract contains a variety of compounds with structures similar to genistein in addition to the target substance, resulting in low product purity; (3) In addition to rutin, sophora japonica contains a variety of glycosidic compounds with genistein as the aglycone, such as genistein and rutin diglycoside. Cellulase and other cell wall breaking enzymes cannot directly act on these compounds, resulting in low raw material utilization.

[0007] The article (Chen Xiang, Wang Pingping, Wu Xia, et al. In situ biotransformation method to increase the genistein content in Sophora japonica fruit[J]. Chinese Journal of Modern Applied Pharmacy, 2020, 37(18):2201-2205) reported a process for in situ biotransformation of Sophora japonica fruit powder using crude fermentation broth of Rhizopus oryzae, which can increase the genistein content in Sophora japonica fruit by about 14 times. However, the following problems exist: (1) The process uses crude enzyme broth after fermentation of Rhizopus oryzae, which contains a variety of enzymes and microbial metabolites in addition to the target glycosidase. The complex composition makes it difficult to precisely control the reaction process and the industrialization threshold is high; (2) The conversion time of this process is as long as 32 hours, and the yield per unit time is lower than that of the traditional process; (3) This process requires the Rhizopus oryzae to be cultured and fermented first, which increases the number of operation steps.

[0008] Therefore, there is a need in this field to develop a process that is direct in enzymatic hydrolysis, has mild and controllable conditions, and is easy to scale up industrially. Summary of the Invention

[0009] To address the problems of excessive wastewater and waste acid, high toxicity of organic reagents, and low raw material utilization in existing genistein production, this application provides a genistein production process. The process involves first crushing Sophora japonica fruit, extracting and concentrating it with hot water, then adding MOF-encapsulated naringinase to the concentrate for targeted enzymatic hydrolysis. Following this, a magnetic field is activated, the filter cake is collected, and then the cake is soaked in solvent, decolorized with activated carbon, and finally filtered, heated, concentrated, cooled, and recrystallized to obtain genistein. This process features mild reaction conditions, low organic reagent consumption, ease of operation, and high genistein extraction rate. Furthermore, the MOF-encapsulated naringinase can be recycled multiple times, reducing production costs.

[0010] To achieve the above objectives, this application adopts the following technical solution:

[0011] S1. Dry the raw material Sophora japonica fruit, pulverize it, and obtain Sophora japonica fruit powder;

[0012] S2. Place the Sophora japonica powder into an extraction tank, add water, heat up, and extract multiple times at the set temperature to obtain a sophoroside extract.

[0013] S3. Heat and concentrate the rutin extract to obtain rutin concentrate;

[0014] S4. After the naringin concentrate is cooled, MOF-encapsulated naringinase is added. After incubation and stirring for a period of time, the magnetic field is activated to adsorb the MOF-encapsulated naringinase onto the tank wall. The filter cake is then collected by filtration.

[0015] S5. After soaking the filter cake in solvent, add activated carbon for decolorization and filter to obtain a genistein solution.

[0016] S6. After heating and concentrating the genistein solution, let it cool and recrystallize. Filter, wash, crush and dry the crystals to obtain the finished genistein product.

[0017] Furthermore, the mesh size of the Sophora japonica powder in S1 is between 10 and 30 mesh.

[0018] Crushing effectively breaks down the dense structure of cellulose and hemicellulose in the pericarp of Sophora japonica, increasing the contact area between the material and the extraction solvent. This allows hot water to quickly penetrate the particles, accelerating the dissolution and diffusion of rutin and improving extraction efficiency. If the raw material is not crushed or has a too-small mesh size, the effective components inside will have difficulty contacting the solvent, resulting in lower extraction efficiency.

[0019] Setting the upper limit for mesh size at 30 mesh is to prevent dust pollution and equipment blockage caused by excessive mesh size. When the mesh size of the material is too large, a large amount of dust will be generated during the feeding and transfer process, causing a deterioration of the operating environment; excessively fine powder is prone to forming high-viscosity slurry during the extraction and filtration stages, clogging the filter cloth, resulting in increased filtration resistance, decreased filtrate throughput, affecting production continuity and increasing maintenance costs.

[0020] Furthermore, the mass of water added in S2 each time is 3-8 times the mass of the Sophora japonica powder.

[0021] Furthermore, the extraction temperature in S2 is 85-90℃; the extraction time for a single extraction in S2 is 1-3 hours; and the number of extractions in S2 is 3-5 times.

[0022] The hot water extraction temperature is set between 85-90℃. Within this range, hot water effectively softens plant tissues and disrupts cell wall structure, allowing for the full release and dissolution of rutin in the water. Simultaneously, it avoids the oxidative degradation of active ingredients when the temperature is too high, approaching the boiling point. If the temperature is too low, the water's permeability and solubility are insufficient, resulting in incomplete dissolution of rutin and a significant decrease in extraction efficiency. This extraction time and number of extractions ensure that the vast majority of rutin is fully dissolved in the solution under these conditions. Too few extractions result in insufficient extraction and waste of raw materials; too many extractions lead to diminishing marginal returns and increased production costs.

[0023] Furthermore, the heating and concentration temperature in S3 is 65-75℃.

[0024] If the concentration temperature is too high, the phenolic hydroxyl groups in glycosides such as genistein and sophoridine are prone to oxidation, polymerization, or decomposition at high temperatures, leading to degradation of the active ingredients, decreased product extraction rate, and increased impurities. Simultaneously, localized overheating zones easily form on the container walls at high temperatures, causing organic matter in the feed solution to carbonize and coke on the heated surface, resulting in loss of the target product, reduced extraction rate, and decreased heat transfer efficiency. If the concentration temperature is too low, the solution concentration time is prolonged, reducing equipment utilization and production efficiency.

[0025] Furthermore, the specific gravity of the concentrated sophoroside solution after heating and concentration in S3 is 1.02-1.05.

[0026] Furthermore, the temperature at which MOF-encapsulated naringinase is added in S4 is 43-47℃; the mass of MOF-encapsulated naringinase added in S4 is 1-3% of the mass of the sophoroside concentrate; and the incubation and stirring time in S4 is 15-24h.

[0027] Naringinase plays a key role in the directional hydrolysis and transformation process. Naringinase is not a single enzyme, but a complex enzyme system composed of α-L-rhamnosidase and β-D-glucosidase. Its mechanism of action is as follows: (1) α-L-rhamnosidase first acts on the rhamnosyl group at the end of the sucralose molecule, hydrolyzing and removing it to generate purine and rhamnose; (2) β-D-glucosidase acts on purine, hydrolyzing and removing the remaining glucose group on its molecule, and finally releasing genistein and glucose.

[0028] This hydrolysis process is highly specific, converting water-soluble glycosides into more lipid-soluble aglycones, thereby improving the efficiency of subsequent solvent extraction and significantly enhancing product activity. Compared to traditional acid hydrolysis, naringinase hydrolysis conditions are mild, avoiding the damage to product structure caused by strong acids and reducing the generation of waste acid. Compared to cellulosic disruptors, naringinase acts directly on the glycosidic bonds of the target substrate, achieving a one-step conversion without additional steps. Furthermore, naringinase is widely available, produced through industrial fermentation by fungi such as Aspergillus niger and Aspergillus oryzae, ensuring stable and controllable quality for large-scale application.

[0029] The difference between MOF encapsulation and traditional methods (such as adsorption and covalent bonding) is that the enzyme is embedded in situ inside the MOF crystal, physically immobilizing the enzyme. This effectively solves the problem of enzyme loss compared to traditional methods. At the same time, the MOF framework provides a physical barrier and a mild microenvironment for the enzyme, effectively protecting its activity.

[0030] Furthermore, the preparation of MOF-encapsulated naringinase includes the following steps:

[0031] T1. Mix and stir the naringinase solution, zinc salt solution and magnetic iron oxide at room temperature for 10-30 minutes.

[0032] T2. Add 2-methylimidazole solution to the system and stir at 20-35℃ for 30-60 min;

[0033] T3. Filter, wash and dry the reaction product to obtain MOF-encapsulated naringinase.

[0034] The core strategy of this immobilization method is to use naringinase itself as a nucleation template, utilizing its abundant carboxyl and amino functional groups to preferentially construct zinc ion-rich pre-nucleation clusters. Subsequently, 2-methylimidazole solution is added to trigger coordination self-assembly, building a MOF framework around the naringinase. Compared with traditional adsorption methods, this method firmly locks the enzyme inside the MOF lattice, effectively solving the problem of enzyme leakage.

[0035] The introduction of magnetic iron oxide (Fe3O4) endows the material with convenient magnetic response recovery capabilities. After the reaction, solid-liquid separation can be achieved simply by applying an external magnetic field, greatly simplifying the operation process. This method features mild reaction conditions and simple operation, and is expected to reduce enzyme costs in the production of genistein.

[0036] Furthermore, the concentration of naringinase in T1 is 50-100 g / L; the concentration of zinc salt is 10-20 g / L; the zinc salt includes any one of zinc acetate, zinc sulfate, zinc chloride and zinc citrate; the mass ratio of naringinase, zinc salt and magnetic iron oxide is 60-90:3-10:10-20.

[0037] Furthermore, the concentration of the 2-methylimidazole solution in T2 is 10-20 g / L; the molar ratio of 2-methylimidazole to zinc salt is 1.1-1.3:1.

[0038] Furthermore, the mass of solvent added in S5 is 20-30 times the mass of the filter cake; the mass of activated carbon added in S5 is 3-10% of the mass of the filter cake; and the decolorization time is 30-90 min.

[0039] Furthermore, the solvent added to S5 includes either ethanol or n-propanol.

[0040] Alcohols are selected as the dissolving medium for the filter cake. Utilizing the excellent solubility of genistein in alcohol solvents, the target product is efficiently transferred from the filter cake to the liquid phase system, while a large amount of water-soluble impurities remain in the solid or aqueous phase, achieving preliminary separation of the target product from the main impurities. Compared with existing technologies, this application uses alcohol reagents to replace organic solvents such as chloroform and dichloromethane in traditional acid hydrolysis methods, avoiding the quality and safety hazards caused by toxic solvent residues.

[0041] After extraction by soaking in alcohol solvents, the extract is further decolorized using activated carbon. Activated carbon's rich porous structure adsorbs and removes residual plant pigments, colloids, and some small molecule impurities from the extract, effectively eliminating the interference of impurities on crystallization. This ensures that genistein is selectively precipitated in the form of regular crystals, significantly improving the purity and appearance quality of the product.

[0042] Furthermore, the mass of the genistein solution after heating and concentration in S6 is 0.2-0.4 times that before heating.

[0043] The concentration factor of alcohol solvents is key to achieving efficient crystallization separation. Choosing this ratio ensures that the active ingredients and impurities remain completely dissolved in the hot solution and do not precipitate prematurely, while creating suitable conditions for subsequent recrystallization.

[0044] If the concentration factor is too low, the supersaturation of the solution increases rapidly. Not only will the target product precipitate quickly, but pigments, colloids, and other impurities in the solution will also precipitate along with the genistein due to solubility saturation, forming co-precipitates or mixed crystals. This results in a large number of impurities being encapsulated in the crystalline product, which cannot be effectively removed even with subsequent recrystallization. If the concentration factor is too high, the supersaturation of genistein in the solution is insufficient, the crystallization driving force weakens, and a large amount of the target product remains in the mother liquor and cannot precipitate. At the same time, additional energy consumption for alcohol solvent recovery and mother liquor treatment costs are required, leading to an increase in production costs.

[0045] Beneficial technical effects:

[0046] This application provides a process for extracting genistein from Sophora japonica fruit. The process involves first crushing the Sophora japonica fruit and then extracting it with hot water to fully dissolve and concentrate the effective components, such as genistein. Then, naringinase is added to the concentrate for targeted enzymatic hydrolysis. Compared to traditional acid hydrolysis methods, this application avoids the damage to the product caused by strong acids and the use of toxic reagents. Compared to enzymatic-assisted extraction methods, naringinase directly acts on the glycosidic bonds of the substrate to achieve precise molecular structure transformation, rather than assisting extraction. Compared to in-situ biotransformation methods, this application does not require an additional microbial fermentation enzyme production step, has a short enzymatic hydrolysis time, and the product exists directly in the liquid phase system, facilitating subsequent separation and purification.

[0047] This application employs MOFs to encapsulate the enzyme, effectively improving its reusability, stability, and catalytic activity. MOFs provide a rigid protective shell for the enzyme, enhancing its tolerance to the external environment. Simultaneously, their porous structure facilitates rapid diffusion of substrate molecules to the enzyme's active site, reducing mass transfer resistance and accelerating the reaction rate. The introduction of magnetic components endows the material with magnetic responsive properties, enabling rapid solid-liquid separation via an applied magnetic field. Furthermore, the pore-confining properties of MOFs effectively prevent enzyme molecule aggregation and conformational changes, effectively solving the problem of enzyme detachment in traditional adsorption methods.

[0048] In this application, alcohol solvent extraction effectively extracts genistein from the filter cake into the organic phase while removing a large amount of water-soluble impurities. Activated carbon decolorization effectively removes pigments and macromolecular impurities, providing a pure mother liquor for subsequent recrystallization. Recrystallization yields a high-purity product. The entire process uses only two solvents: water and alcohol reagents, avoiding the use of toxic solvents such as chloroform and dichloromethane. It is environmentally friendly, requires minimal equipment, and is easy to operate, showing promising prospects for industrial application.

[0049] In summary, this application effectively overcomes the shortcomings of existing technologies, such as acid hydrolysis destroying products, enzymatic hydrolysis-assisted extraction not directly converting products, and difficulty in separating in-situ bioconversion products, and realizes efficient and green industrial preparation of high-purity genistein from Sophora japonica pods. Attached Figure Description

[0050] Figure 1 This is a flow chart of a dye lignin production process. Detailed Implementation

[0051] The technical solution of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments and specific features in the embodiments are detailed descriptions of the technical solution of this application, rather than limitations thereof.

[0052] Example 1

[0053] like Figure 1 As shown, this embodiment provides a process for producing genistein, including the following steps:

[0054] S1. Dry the raw material Sophora japonica fruit and grind it to 10 mesh to obtain Sophora japonica fruit powder;

[0055] S2. Place the Sophora japonica powder into an extraction tank, add 3 times the mass of water, heat to 85℃, and extract 3 times at this temperature, with each extraction time being 1 hour, to obtain the sophoroside extract.

[0056] S3. The rutin extract was heated and concentrated at 65°C to a specific gravity of 1.02 to obtain the rutin concentrate.

[0057] S4. After the sophoroside concentrate is cooled to 43°C, 1% of its mass of MOF-encapsulated naringin is added to the sophoroside concentrate. Then, the mixture is kept warm and stirred for 15 hours. The magnetic field is activated to adsorb the MOF-encapsulated naringin onto the tank wall. The filter cake is then collected by filtration.

[0058] S5. Soak the filter cake in 20 times its weight of ethanol, then add 3% of the filter cake's weight of activated carbon and stir to decolorize for 30 minutes. Filter to obtain a genistein solution.

[0059] S6. Heat and concentrate the genistein solution to 0.2 times its original mass, let it cool and recrystallize, filter, wash, crush and dry the crystals to obtain the finished genistein product.

[0060] The preparation of naringinase encapsulated in MOFs in S4 includes the following steps:

[0061] T1. Mix naringinase solution, zinc acetate solution and magnetic iron oxide at room temperature for 10 min; the mass ratio of naringinase, zinc acetate and magnetic iron oxide is 60:10:20; the concentration of naringinase solution is 50 g / L; the concentration of zinc acetate solution is 10 g / L.

[0062] T2. Add a 10 g / L 2-methylimidazole solution to the system and stir at 20 °C for 30 min. The molar ratio of 2-methylimidazole to zinc acetate is 1.1:1.

[0063] T3. Filter, wash and dry the reaction product to obtain MOF-encapsulated naringinase.

[0064] Example 2

[0065] like Figure 1 As shown, this embodiment provides a process for producing genistein, including the following steps:

[0066] S1. Dry the raw material Sophora japonica fruit and grind it to 30 mesh to obtain Sophora japonica fruit powder;

[0067] S2. Place the Sophora japonica powder into an extraction tank, add 8 times the mass of water, heat to 87°C, and extract 5 times at this temperature, with each extraction time being 3 hours, to obtain the sophoroside extract.

[0068] S3. The rutin extract was heated and concentrated at 75°C to a specific gravity of 1.05 to obtain the rutin concentrate.

[0069] S4. After the sophoroside concentrate is cooled to 47°C, 3% of its mass of MOFs-encapsulated naringinase is added to the sophoroside concentrate. Then, the mixture is kept warm and stirred for 24 hours. The magnetic field is activated to adsorb the MOFs-encapsulated naringinase onto the tank wall. The filter cake is then collected by filtration.

[0070] S5. Soak the filter cake in 30 times its weight of n-propanol, then add 10% of the weight of activated carbon (based on the filter cake weight) and stir to decolorize for 90 minutes. Filter to obtain a genistein solution.

[0071] S6. Heat and concentrate the genistein solution to 0.4 times its original mass, let it cool and recrystallize, filter, wash, crush and dry the crystals to obtain the finished genistein product.

[0072] The preparation of naringinase encapsulated in MOFs in S4 includes the following steps:

[0073] T1. Mix naringinase solution, zinc chloride solution and magnetic iron oxide at room temperature for 10 min; the mass ratio of naringinase, zinc chloride and magnetic iron oxide is 90:3:10; the concentration of naringinase solution is 100 g / L; the concentration of zinc chloride solution is 20 g / L.

[0074] T2. Add a 20 g / L 2-methylimidazole solution to the system and stir at 20 °C for 60 min. The molar ratio of 2-methylimidazole to zinc chloride is 1.3:1.

[0075] T3. Filter, wash and dry the reaction product to obtain MOF-encapsulated naringinase.

[0076] Example 3

[0077] like Figure 1 As shown, this embodiment provides a process for producing genistein, including the following steps:

[0078] S1. Dry the raw material Sophora japonica fruit and grind it to 20 mesh to obtain Sophora japonica fruit powder;

[0079] S2. Place the Sophora japonica powder into an extraction tank, add 6 times the mass of water, heat to 86℃, and extract 4 times at this temperature, with each extraction time being 2 hours, to obtain the sophoroside extract.

[0080] S3. The rutin extract was heated and concentrated at 70°C to a specific gravity of 1.03 to obtain the rutin concentrate.

[0081] S4. After the sophoroside concentrate is cooled to 45°C, 2% of its mass of MOF-encapsulated naringin is added to the sophoroside concentrate. Then, the mixture is kept warm and stirred for 17 hours. The magnetic field is activated to adsorb the MOF-encapsulated naringin onto the tank wall. The filter cake is then collected by filtration.

[0082] S5. Soak the filter cake in ethanol at 25 times the weight of the filter cake, then add 6% activated carbon (by weight of the filter cake) and stir to decolorize for 60 minutes. Filter to obtain a genistein solution.

[0083] S6. Heat and concentrate the genistein solution to 0.3 times its original mass, let it cool and recrystallize, filter, wash, crush and dry the crystals to obtain the finished genistein product.

[0084] The preparation of naringinase encapsulated in MOFs in S4 includes the following steps:

[0085] T1. Mix and stir the naringinase solution, zinc sulfate solution, and magnetic iron oxide at room temperature for 30 minutes; the mass ratio of naringinase, zinc sulfate, and magnetic iron oxide is 70:5:15; the concentration of naringinase solution is 60 g / L; the concentration of zinc sulfate solution is 15 g / L.

[0086] T2. Add a 15 g / L 2-methylimidazole solution to the system and stir at 35 °C for 40 min. The molar ratio of 2-methylimidazole to zinc sulfate is 1.2:1.

[0087] T3. Filter, wash and dry the reaction product to obtain MOF-encapsulated naringinase.

[0088] Example 4

[0089] like Figure 1 As shown, this embodiment provides a process for producing genistein, including the following steps:

[0090] S1. Dry the raw material Sophora japonica fruit and grind it to 25 mesh to obtain Sophora japonica fruit powder;

[0091] S2. Place the Sophora japonica powder into an extraction tank, add 5 times the mass of water, heat to 90℃, and extract 3 times at this temperature, with each extraction lasting 1.5 hours, to obtain the sophoroside extract.

[0092] S3. The rutin extract was heated and concentrated at 70°C to a specific gravity of 1.04 to obtain the rutin concentrate.

[0093] S4. After the sophoroside concentrate is cooled to 45°C, 2% of its mass of MOFs-encapsulated naringin is added to the sophoroside concentrate. Then, the mixture is kept warm and stirred for 20 hours. The magnetic field is activated to adsorb the MOFs-encapsulated naringin onto the tank wall. The filter cake is then collected by filtration.

[0094] S5. Soak the filter cake in 22 times its weight of n-propanol, then add 6% of the filter cake weight of activated carbon and stir to decolorize for 50 minutes. Filter to obtain a genistein solution.

[0095] S6. Heat and concentrate the genistein solution to 0.2 times its original mass, let it cool and recrystallize, filter, wash, crush and dry the crystals to obtain the finished genistein product.

[0096] The preparation of naringinase encapsulated in MOFs in S4 includes the following steps:

[0097] T1. Mix naringinase solution, zinc chloride solution and magnetic iron oxide at room temperature for 15 min; the mass ratio of naringinase, zinc chloride and magnetic iron oxide is 90:3:10; the concentration of naringinase solution is 70 g / L; the concentration of zinc chloride solution is 13 g / L.

[0098] T2. Add a 16 g / L 2-methylimidazole solution to the system and stir at 30 °C for 50 min. The molar ratio of 2-methylimidazole to zinc chloride is 1.1:1.

[0099] T3. Filter, wash and dry the reaction product to obtain MOF-encapsulated naringinase.

[0100] Example 5

[0101] This embodiment is a test of the recovery activity of MOF-encapsulated naringinase. The test method is as follows: after the magnetic field is started in S4 in Example 2, the MOF-encapsulated naringinase is adsorbed onto the tank wall. Then, the magnetic field is removed and the next batch of sophoridine concentrate is added directly to the reaction system for repeated catalysis. The other process parameters remain unchanged.

[0102] Comparative Example 1

[0103] The difference between this comparative example and Example 3 is that the water extraction step in S2 is omitted, and Sophora japonica powder is directly used for subsequent naringinase hydrolysis treatment, while the other process parameters remain unchanged.

[0104] Comparative Example 2

[0105] The difference between this comparative example and Example 3 is that the S5 activated carbon decolorization step is omitted, while the other process parameters remain unchanged.

[0106] Comparative Example 3

[0107] The difference between this comparative example and Example 3 is that naringinase in S4 is replaced with cellulase, while the other process parameters remain unchanged.

[0108] Comparative Example 4

[0109] The difference between this comparative example and Example 3 is that the naringinase is not encapsulated with MOFs and is directly added to the reaction system in a free state for enzymatic hydrolysis, while the other process parameters remain unchanged.

[0110] The products obtained from the above embodiments and comparative examples were analyzed, and the results are as follows. The extraction rate calculation formula is shown in the table: .

[0111] Table 1 Product Analysis of Examples 1-4 and Comparative Examples 1-3

[0112]

[0113] Table 2. Example 5: Detection of naringinase recovery activity encapsulated in MOFs.

[0114]

[0115] The extraction rates and purity of the products in Examples 1-4 were all in a high range, proving that the process has good feasibility in extracting genistein.

[0116] This application uses hot water as the extraction solvent to fully dissolve and concentrate the effective components such as rutin in Sophora japonica fruit in the concentrated solution, providing a homogeneous reaction system for subsequent enzymatic hydrolysis and significantly improving reaction efficiency. Simultaneously, it removes a large number of lipophilic impurities, preventing contamination and ensuring product purity. Naringinase hydrolysis exhibits high substrate specificity, precisely hydrolyzing the glycosidic bonds of rutin under mild conditions to generate genistein without side reactions. Furthermore, Sophora japonica fruit also contains various glycosides with genistein as an aglycone, such as genistein and rutin diglycoside. Naringinase can simultaneously hydrolyze these precursors, further improving the total extraction rate of the target product and achieving full conversion and utilization of raw materials. After enzymatic hydrolysis, soaking in an alcohol solvent combined with activated carbon decolorization effectively removes pigments and some small molecule impurities, providing a pure mother liquor for subsequent recrystallization and ensuring product purity meets standards.

[0117] In Comparative Example 1, the absence of a water extraction step resulted in a significant decrease in both product extraction rate and purity. The active ingredients, such as rutin, are encapsulated within the plant cell walls. Without water extraction, they cannot be fully released into the liquid phase. Naringinase can only act on a small amount of substrate on the surface of the powder particles. In the solid-state system, the limited contact between the enzyme and substrate, coupled with high mass transfer resistance, significantly reduces reaction efficiency, and a large amount of rutin fails to be effectively hydrolyzed and transformed. Secondly, the sophora japonica powder is rich in various substances such as oils, waxes, proteins, and pectin. These substances can form physical barriers on the powder surface, hindering enzyme-substrate contact, and may also non-specifically bind to the enzyme, reducing its effective activity and further inhibiting the enzymatic hydrolysis reaction. More importantly, without water extraction, a large amount of hydrophobic impurities will enter the alcohol solvent soaking system along with the enzymatic hydrolysis process, mixing with the target product. Some of these impurities co-crystallize with genistein during crystallization, and some pigments are difficult to completely remove by activated carbon decolorization, severely interfering with the crystallization process and leading to a significant decrease in product purity.

[0118] In Comparative Example 2, without activated carbon decolorization, the product purity decreased significantly, while the extraction rate remained relatively unchanged. This is because when alcohol solvents soak and dissolve the target product, they also introduce small amounts of residual plant pigments, colloids, and some small-molecule impurities from the filter cake into the solution. Without activated carbon decolorization, these impurities would directly enter the crystallization system. Pigments would adsorb onto the crystal surface or embed into the crystal lattice, leading to a darker product color and a decline in appearance quality. Some small-molecule impurities would co-precipitate with genistein during crystallization or interfere with the orderly arrangement of crystals, significantly reducing product purity. Activated carbon, with its abundant porous structure, can effectively remove pigments and some small-molecule impurities from the extract through physical adsorption, providing a pure mother liquor for subsequent crystallization. The lack of significant change in extraction rate is because activated carbon adsorbs only extremely low amounts of pigments and trace impurities, not genistein itself, thus having no significant impact on the extraction rate. However, the decrease in purity indicates that without decolorization, pigments and small-molecule impurities would mix into the product, severely affecting product quality.

[0119] In Comparative Example 3, replacing naringinase with cellulase resulted in no product formation. This is because without naringinase, the glycosidic bonds of rutin cannot be hydrolyzed, thus preventing the formation of genistein. Cellulase, on the other hand, only acts on the cellulose components of plant cell walls and cannot hydrolyze the glycosidic bonds of rutin to generate genistein, fully demonstrating the irreplaceable role of naringinase in this process.

[0120] In Comparative Example 4, naringinase without MOF encapsulation showed a slight decrease in extraction rate, but the free enzyme was difficult to recover and reuse, increasing enzyme costs. This fully demonstrates that MOF encapsulation of naringinase not only does not reduce enzyme catalytic activity but also enhances it. This is because the porous structure of MOFs provides a stable catalytic environment for the enzyme, reducing interference from external impurities on enzyme activity. Furthermore, MOFs can be repeatedly recycled, reducing production costs.

[0121] In Example 5, the activity of the MOF-encapsulated naringinase showed no significant change after 8 cycles. This fully demonstrates that the MOF-encapsulated naringinase has good reusability and stability. Furthermore, its recovery method is magnetic field recovery, eliminating the need for cumbersome operations such as centrifugation and filtration, resulting in high recovery efficiency, simple operation, and suitability for continuous industrial production.

[0122] The above description is merely a preferred embodiment of this application. The scope of protection of this application is not limited to the above embodiments. All technical solutions falling within the scope of this application's concept are within the scope of protection of this application. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of this application should also be considered within the scope of protection of this application.

Claims

1. A process for producing lignin dyes, characterized in that, The production process includes the following steps: S1. Dry the raw material Sophora japonica fruit, pulverize it, and obtain Sophora japonica fruit powder; S2. Place the Sophora japonica powder into an extraction tank, add water, heat up, and extract multiple times at the set temperature to obtain a sophoroside extract. S3. Heat and concentrate the rutin extract to obtain rutin concentrate; S4. After the naringin concentrate is cooled, MOF-encapsulated naringinase is added. After incubation and stirring for a period of time, the magnetic field is activated to adsorb the MOF-encapsulated naringinase onto the tank wall. The filter cake is then collected by filtration. S5. After soaking the filter cake in solvent, add activated carbon for decolorization and filter to obtain a genistein solution. S6. After heating and concentrating the genistein solution, let it cool and recrystallize. Filter, wash, crush and dry the crystals to obtain the finished genistein product.

2. The genistein production process as described in claim 1, characterized in that, The powder from Sophora japonica fruit in S1 has a mesh size of 10-30.

3. The genistein production process as described in claim 1, characterized in that, In S2, the mass of water added each time is 3-8 times the mass of the Sophora japonica powder; the extraction temperature is 85-90℃; the extraction time for a single extraction in S2 is 1-3 hours; and the number of extractions in S2 is 3-5 times.

4. The genistein production process as described in claim 1, characterized in that, The heating and concentration temperature in S3 is 65-75℃; the specific gravity of the concentrated sophoroside solution after heating and concentration is 1.02-1.

05.

5. The genistein production process as described in claim 1, characterized in that, The temperature at which MOF-encapsulated naringinase is added in S4 is 43-47℃; the mass of MOF-encapsulated naringinase added in S4 is 1-3% of the mass of the naringin concentrate; and the incubation and stirring time in S4 is 15-24h.

6. The genistein production process as described in claim 1, characterized in that, The preparation method of naringinase encapsulated in MOFs in S4 includes the following steps: T1. Mix and stir the naringinase solution, zinc salt solution and magnetic iron oxide at room temperature for 10-30 minutes. T2. Add 2-methylimidazole solution to the system and stir at 20-35℃ for 30-60 min; T3. Filter, wash and dry the reaction product to obtain MOF-encapsulated naringinase.

7. The genistein production process as described in claim 6, characterized in that, The concentration of naringinase in T1 is 50-100 g / L; the concentration of zinc salt is 10-20 g / L; the zinc salt includes any one of zinc acetate, zinc sulfate, zinc chloride and zinc citrate; the mass ratio of naringinase, zinc salt and magnetic iron oxide is 60-90:3-10:10-20.

8. The genistein production process as described in claim 6, characterized in that, The concentration of 2-methylimidazole solution in T2 is 10-20 g / L; the molar ratio of 2-methylimidazole to zinc salt is 1.1-1.3:

1.

9. The genistein production process as described in claim 1, characterized in that, The mass of solvent added in S5 is 20-30 times the mass of the filter cake; the mass of activated carbon added in S5 is 3-10% of the mass of the filter cake; the decolorization time is 30-90 min; the solvent added in S5 includes either ethanol or n-propanol.

10. The genistein production process as described in claim 1, characterized in that, The mass of the genistein solution after heating and concentration in S6 is 0.2-0.4 times that before heating.

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