Preparation method of high-activity theaflavin

Abstract

The invention discloses a preparation method of high-activity theaflavin. The method sequentially comprises the following steps: preparation and enzyme purification of tea powder, ultrasonic-assisted enzymolysis, oil-water double-liquid phase step type fermentation and online extraction, chromatographic column separation and purification, and membrane concentration and drying. According to the method, an oil-water double-liquid-phase enzymatic oxidation system is constructed, the fermentation process and the extraction process are coupled, the characteristic that the target product theaflavin-3, 3 '-digallate has a higher partition coefficient in an organic phase is utilized, in-situ separation and enrichment in the reaction process are achieved, and product degradation is avoided. In combination with a subsequently adopted specific chromatographic separation technology, the content of the high-activity TF3 monomer in the product is finally successfully increased to 50% or above of the total theaflavin monomer, and the biological activity of the product is improved.

Description

Technical Field

[0001] This invention relates to the field of theaflavin preparation technology, specifically to a method for preparing highly active theaflavin. Background Technology

[0002] Theaflavins are a class of key functional components formed during the fermentation of black tea, first discovered and named by Roberts E.A.H. in the 1950s. They are a class of benzo[a]phenolic ketone compounds formed by the oxidative coupling and condensation reactions of catechins in tea leaves under the catalysis of polyphenol oxidase. They are named "theaflavins" because they are soluble in ethyl acetate and exhibit a bright orange-yellow color. As a core factor determining the "brightness," "intensity," and freshness of black tea liquor, the content and composition of theaflavins directly determine the quality grade of black tea. Their content in dry black tea is typically between 0.3% and 1.5%.

[0003] Theaflavin is not a single substance, but a complex containing more than 12 components. Among them, four monomers dominate in terms of content and activity: theaflavin (TF1), theaflavin-3-gallate (TF2a), theaflavin-3'-gallate (TF2b), and theaflavin-3,3'-digallate (TF3). The main difference in the chemical structure of these four monomers lies in the number and position of the galloyl groups they are attached to, which directly leads to significant differences in their physicochemical properties and biological activities.

[0004] Of all these monomers, TF3 exhibits the strongest biological activity due to the two galloyl groups linked in its molecular structure. Numerous scientific studies have confirmed that TF3 possesses outstanding antioxidant and free radical scavenging capabilities, with its activity order being: TF3 > TF2a ≈ TF2b > TF1. In addition to its excellent antioxidant capacity, TF3 has also been shown to possess various pharmacological potentials, including anti-inflammatory, antiviral, lipid-lowering, blood glucose-lowering, and tumor cell proliferation-inhibiting effects.

[0005] However, the content of TF3 in natural and traditionally processed black tea is relatively low. Currently, the main industrial processes for preparing theaflavins include direct extraction, chemical oxidation, and enzymatic oxidation. Among these, enzymatic oxidation has become the mainstream method due to its mild conditions and good specificity. Even so, in theaflavins products prepared by existing technologies, the proportion of TF3 in the total theaflavins is usually only around 25%, resulting in the actual TF3 content in the final product generally being less than 10%. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and increase the TF3 content in theaflavins.

[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows.

[0008] A method for preparing highly active theaflavins includes the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. After static fermentation, the fresh tea leaves are quick-frozen, pulverized and sieved to obtain tea powder. S002. Add the tea powder to a buffer solution to form a mixed solution, sonicate for 30-60 minutes, then add a potential regulator and polyphenol oxidase to the mixed solution, and perform pre-enzymatic hydrolysis for 30-60 minutes under stirring conditions to obtain an enzymatic hydrolysate. S003. Transfer the enzymatic hydrolysate to the reactor, add organic phase and aqueous phase to the reactor, control the volume ratio of organic phase to aqueous phase to be 2:3~1:4, stir the organic phase and aqueous phase together until uniform, and raise the temperature in the reactor to 25℃~50℃. During the stirring process, continuously introduce sterile air into the reactor, and control the oxygen partial pressure of the sterile air to be 0.10MPa~0.20MPa. S004. Allow the reactor and reaction system to stand until the reaction system separates into layers. Collect the upper organic phase and return the lower aqueous phase to the reactor for a second countercurrent extraction. Combine the organic phases obtained from the two extractions. S005. Load the organic phase onto the paper chromatography column and use a gradient elution with a mixed solvent of distilled water and ethanol as the mobile phase. Collect the absorption peak at 280 nm under the UV detector as the target fraction. S006. The target fraction is pre-concentrated through a nanofiltration membrane, and then the concentrate is transferred to a vacuum belt dryer for continuous drying, and finally pulverized and sieved.

[0009] As a preferred technical solution, in step S001, the quick-freezing treatment is quick-freezing with liquid nitrogen, and the sieving is passing through a 20-mesh to 100-mesh sieve; and the polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, with a specific enzyme activity of 4500 U / mg to 5500 U / mg.

[0010] As a preferred technical solution, in step S002, the buffer solution is a citrate-disodium hydrogen phosphate buffer system with a pH of 5.6 to 6.0; the potential regulator is L-ascorbic acid sodium, and its addition amount is 0.3% to 0.7% of the total mass of the mixed solution; the power of the ultrasonic treatment is 250W to 350W.

[0011] As a preferred technical solution, in step S003, the organic phase is ethyl acetate that has been dehydrated by molecular sieve; the stirring speed is 150 rpm to 250 rpm; and the temperature of the sterile air introduced is 25°C to 35°C, and the relative humidity is 80% to 90%.

[0012] As a preferred technical solution, in step S004, the condition for static stratification is: standing at 20°C to 30°C for 15 to 25 minutes.

[0013] As a preferred technical solution, in step S005, the volume fraction of ethanol in the gradient elution program increases linearly from an initial 5% to a final value of 25%-35%; the column temperature for elution is controlled at 30°C to 40°C.

[0014] As a preferred technical solution, in step S005, the stationary phase of the chromatography column includes a mixture of silica gel and octadecyl bonded silica gel, with a mass mixing ratio of 1:0.5 to 1:1.5.

[0015] As a preferred technical solution, in step S005, the stationary phase of the chromatography column is a composite packing material of silica gel and adsorption resin, wherein the adsorption resin is a styrene-divinylbenzene copolymer with a specific surface area of ​​500-650 m². 2 / g, with an average pore size of 8-12nm; the dry weight mixing ratio of the silica gel and the adsorption resin is 1:1 to 1:2.

[0016] As a preferred technical solution, in step S005, the stationary phase of the chromatography column comprises, by mass percentage, 40%-60% silica matrix, 15%-25% aniline-pyrrole copolymer microspheres, 10%-20% tea polyphenol-zinc ion complex powder, and 10%-20% aminated mesoporous silica.

[0017] As a preferred technical solution, in step S006, the molecular weight cutoff of the nanofiltration membrane is 800 Da to 1200 Da; the belt surface temperature of the vacuum belt dryer is controlled at 55°C to 65°C, and the system vacuum degree is controlled at -0.090 MPa to -0.098 MPa; and the final pulverization and sieving is through an 80-mesh to 120-mesh sieve.

[0018] The advantages and beneficial effects of this invention are as follows: This invention couples the fermentation and extraction processes by constructing an oil-water two-phase enzymatic oxidation system. This system utilizes the higher solubility of TF3 in the organic phase, achieving in-situ separation and enrichment of the product TF3 simultaneously with the reaction. Timely removal of the product promotes the oxidation reaction to continue in the direction of TF3 generation, improving the conversion rate. Furthermore, it avoids oxidative degradation caused by prolonged residence of TF3 in the aqueous phase, ensuring the stability of the active ingredients. The subsequent liquid chromatography separation technology utilizes the differences in polarity and molecular forces between TF3 and other theaflavin monomers and impurities, achieving highly selective separation and purification of TF3 through a stationary phase and gradient elution program. This invention successfully increases the TF3 content in the final product to over 50% of the total theaflavin monomers. Detailed Implementation

[0019] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below. It is to be understood that the specific embodiments described herein are merely illustrative of this application and not intended to limit it. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0020] The terms “comprising” and “having”, and any variations thereof, used in this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus.

[0021] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly or implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0022] This invention provides a method for preparing highly active theaflavins, comprising the following steps: preparation and enzyme purification of tea powder, ultrasound-assisted enzymatic hydrolysis, oil-water two-phase stepwise fermentation and online extraction, column chromatography separation and purification, and membrane concentration and drying. By constructing an oil-water two-phase enzymatic oxidation system, the fermentation and extraction processes are coupled. Utilizing the higher partition coefficient of the target product theaflavins-3,3'-digallate (TF3) in the organic phase, in-situ separation and enrichment during the reaction process are achieved. Combined with subsequent specific chromatographic separation techniques, the content of highly active TF3 monomers in the product is successfully increased to over 50% of the total theaflavins monomers.

[0023] In step S001, fresh tea leaves and their corresponding polyphenol oxidases are obtained. The fresh tea leaves are subjected to static fermentation, followed by quick-freezing, pulverization, and sieving to obtain tea powder. The purpose of static fermentation is to utilize the polyphenol oxidase system of the fresh leaves under controlled conditions to induce preliminary enzymatic oxidation of precursors such as catechins, forming key intermediate products for theaflavins synthesis. This provides a high content of substrate for subsequent deep conversion and enrichment reactions. Quick-freezing (e.g., liquid nitrogen quick-freezing) instantly lowers the temperature of the fresh leaves, preventing ice crystal formation and damage to cell structure, thus protecting the integrity of polyphenols. Pulverization and sieving increase the specific surface area of ​​the tea powder, facilitating sufficient contact between the substrate and enzyme during subsequent enzymatic hydrolysis and improving reaction efficiency. If the particle size is too large, it will increase mass transfer resistance and lead to incomplete enzymatic hydrolysis; if the particle size is too small, it may cause dust dispersion and loss, affecting the yield. The purification of polyphenol oxidase was carried out by ammonium sulfate fractionation followed by DEAE-cellulose ion exchange chromatography, with the specific enzyme activity controlled within the range of 4500 U / mg to 5500 U / mg. This activity range ensured high catalytic activity and specificity of the enzyme. If the specific enzyme activity was too low, the reaction rate would be slow and the TF3 yield would be low; if it was too high, it might trigger side reactions, leading to a more complex product composition.

[0024] In step S002, the tea powder is added to a buffer solution to form a mixed solution. The mixed solution is then sonicated for 30-60 minutes. Subsequently, a potential regulator and the polyphenol oxidase are added, and pre-enzymatic hydrolysis is performed under stirring for 30-60 minutes to obtain an enzymatic hydrolysate. The buffer solution is a citrate-disodium hydrogen phosphate buffer system with a pH of 5.6 to 6.0. This pH range is close to the optimal pH of polyphenol oxidase, which can maintain the stability of enzyme activity. The potential regulator is L-ascorbic acid sodium, which is added at 0.3% to 0.7% of the total mass of the mixed solution. Its function is to regulate the redox potential, prevent premature oxidation of catechins, and protect enzyme activity. Sonication disrupts the cell walls of the tea powder through cavitation, promotes the release of intracellular contents, and enhances the mass transfer efficiency between the enzyme and the substrate. If the sonication time is too short or the power is too low, the cell wall disruption will be insufficient; if it is too long or too high, it may lead to local overheating and enzyme inactivation. The pre-enzymatic hydrolysis process is carried out under stirring conditions to ensure that the substrate and enzyme are evenly mixed and to initially form theaflavins precursor, laying the foundation for subsequent fermentation.

[0025] In step S003, the enzymatic hydrolysate is transferred to a reactor, and an organic phase and an aqueous phase are added to the reactor. The volume ratio of the organic phase to the aqueous phase is controlled at 2:3 to 1:4. The mixture is stirred until homogeneous, and the temperature of the reaction system is raised to 25°C to 50°C. Simultaneously, sterile air with an oxygen partial pressure of 0.10 MPa to 0.20 MPa is continuously introduced into the reactor during stirring. The organic phase is ethyl acetate that has been dehydrated using molecular sieves. Its high purity avoids interference from impurities, and the high partition coefficient of TF3 in the organic phase is utilized to couple the reaction and extraction. The volume ratio of the aqueous phase to the organic phase is optimized to 2:3 to 1:4. If the organic phase ratio is too high, although it is beneficial for TF3 extraction, it may reduce the efficiency of the enzymatic reaction; if it is too low, the extraction will be insufficient, and the TF3 yield will decrease. The stirring speed is controlled at 150 rpm to 250 rpm to ensure thorough mixing of the two phases while avoiding excessive shear force that could lead to enzyme inactivation. The introduced sterile air provides a suitable oxidative environment, promoting the oxidative coupling of catechins to form TF3. If the oxygen partial pressure is too low, the oxidation reaction will be insufficient; if it is too high, it may lead to over-oxidation and product degradation.

[0026] In step S004, the reactor is allowed to stand to allow the reaction system to separate into layers. The upper organic phase is collected, and the lower aqueous phase is refluxed back to the reactor for a second countercurrent extraction. The organic phases obtained from the two extractions are then combined. The standing and separation conditions are 20°C to 30°C for 15 to 25 minutes. This temperature and time range ensures the efficiency and integrity of phase separation. If the standing time is too short, the separation will be incomplete, and the organic phase may contain impurities from the aqueous phase; if it is too long, the production cycle will be prolonged, and energy consumption will increase. The second countercurrent extraction design significantly improves the recovery rate of TF3 and reduces the residue of the product in the aqueous phase.

[0027] In step S005, the combined organic phase is loaded onto the chromatography column, and gradient elution is performed using a mixed solvent of distilled water and ethanol as the mobile phase. The absorption peak at 280 nm wavelength is collected by the UV detector to obtain the target fraction. In the gradient elution program, the volume fraction of ethanol increases linearly from an initial 5% to a final value of 25%-35%, and the column temperature is controlled between 30℃ and 40℃. This elution program is based on the differences in polarity and hydrophobicity between TF3 and other theaflavins (such as TF1, TF2a, and TF2b), achieving highly selective separation by gradually increasing the ethanol concentration. If the initial ethanol concentration is too high or increases too rapidly, TF3 may elute prematurely, resulting in reduced purity; if it is too low or increases too slowly, the separation time will be prolonged, and the efficiency will decrease. The design of the stationary phase of the chromatography column is crucial. The following analysis focuses on three specific stationary phases: In some embodiments, to achieve efficient adsorption and selective elution of TF3, the stationary phase of the chromatography column is a mixture of silica gel and octadecyl-bonded silica gel, wherein the dry basis mass ratio of the silica gel to the octadecyl-bonded silica gel is 1:0.5 to 1:1.5. Silica gel provides a high specific surface area and a rigid framework, while the octadecyl-bonded silica gel introduces hydrophobic interactions, enhancing the retention of TF3.

[0028] In some embodiments, to further improve separation efficiency and stability, the stationary phase of the chromatography column is a composite packing material of silica gel and adsorption resin, wherein the adsorption resin is a styrene-divinylbenzene copolymer with a specific surface area of ​​500-650 m². 2 The silica gel has an average pore size of 8-12 nm per g, and the dry weight mixing ratio of the silica gel to the adsorption resin is 1:1 to 1:2. This resin possesses a high specific surface area and uniform pore size distribution, selectively adsorbing TF3 through π-π interactions and hydrophobic interactions. The addition of silica gel enhances the mechanical strength and chemical stability of the filler, avoiding the compression deformation that may occur with pure resin under high pressure.

[0029] In some embodiments, to achieve precise separation by utilizing multiple interactions, the stationary phase of the chromatography column comprises the following components by mass percentage: 40%-60% silica matrix, 15%-25% aniline-pyrrole copolymer microspheres, 10%-20% tea polyphenol-zinc ion complex powder, and 10%-20% aminated mesoporous silica. The silica matrix provides basic support; the aniline-pyrrole copolymer microspheres enhance their affinity for TF3 through hydrogen bonding and π-π stacking interactions; the tea polyphenol-zinc ion complex powder selectively binds to the ortho-phenolic hydroxyl groups in TF3 through metal coordination; and the aminated mesoporous silica further purifies the TF3 through electrostatic interactions and pore size sieving effects.

[0030] In step S006, the target fraction is pre-concentrated through a nanofiltration membrane to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying, and finally pulverized and sieved to obtain a highly active theaflavins product. The nanofiltration membrane has a molecular weight cutoff of 800 Da to 1200 Da, effectively retaining the target product TF3 (molecular weight approximately 868 Da) while allowing small molecule impurities to pass through. The belt surface temperature of the vacuum belt dryer is controlled at 55°C to 65°C, and the system vacuum is controlled at -0.090 MPa to -0.098 MPa to prevent thermal degradation of TF3.

[0031] The preparation method of aniline-pyrrole copolymer microspheres includes: under argon protection, dissolving aniline and pyrrole monomers in a molar ratio of 1:1 to 1:1.5 in a 1.0 mol / L hydrochloric acid solution; adding ammonium persulfate hydrochloric acid solution as an initiator dropwise under ice-water bath conditions at 0-5℃, with the molar ratio of initiator to monomer being 1:1; after the addition is complete, reacting for 8-12 hours under continuous stirring; after the reaction is completed, filtering and repeatedly washing with deionized water and ethanol until the filtrate is colorless; finally, vacuum drying at 50℃ to constant weight, followed by pulverization and sieving to obtain particle sizes of 45-75 μm.

[0032] The preparation method of tea polyphenol-zinc ion complex powder includes dissolving epigallocatechin gallate in hot water at 60℃ to prepare a 5% (w / v) solution; adding a 0.1 mol / L zinc sulfate aqueous solution under continuous stirring, wherein the molar ratio of zinc ions to epigallocatechin gallate is 1:2; simultaneously maintaining the pH of the system at 7.5-8.0 with a 1.0 mol / L sodium hydroxide solution, and performing the complexation reaction at 60℃ for 2 hours; after cooling the reaction solution, collecting the precipitate by centrifugation, washing it three times with deionized water, freeze-drying it, and pulverizing it through a 400-mesh sieve to obtain a brownish-yellow complex powder with a particle size of less than 38 μm.

[0033] The preparation method of aminated mesoporous silica includes: using hexadecyltrimethylammonium bromide as a template agent and tetraethyl orthosilicate as a silicon source, preparing a mesoporous silica support by hydrolysis and condensation under alkaline conditions; then dispersing it in toluene, adding 10% of the total mass of the silicon source as an amination modifier of 3-aminopropyltriethoxysilane, and refluxing at 110°C for 6 hours; after the reaction, filtering, washing with ethanol, drying at 80°C, and finally calcining in a muffle furnace at a heating rate of 1°C / min to 550°C for 5 hours to remove the template agent, obtaining aminated mesoporous silica with a pore size of 5-8 nm and a particle size range of 30-50 μm.

[0034] Example 1 This embodiment provides a method for preparing highly active theaflavins, including the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. The fresh tea leaves are subjected to static fermentation, followed by liquid nitrogen quick-freezing treatment, and then crushed through a 20-mesh sieve to obtain tea powder. The polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, and its specific enzyme activity is 5000 U / mg.

[0035] S002. The tea powder is added to a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.8 to form a mixed solution. The mixed solution is ultrasonically treated for 45 minutes with an ultrasonic power of 300W. Then, sodium L-ascorbate is added as a potential regulator at an amount of 0.5% of the total mass of the mixed solution, along with the polyphenol oxidase. The mixture is pre-enzymatically hydrolyzed for 45 minutes under stirring conditions to obtain an enzymatic hydrolysate.

[0036] S003. Transfer the enzymatic hydrolysate to a reactor, add ethyl acetate (dehydrated by molecular sieve) as the organic phase and aqueous phase to the reactor, control the volume ratio of the organic phase to the aqueous phase to be 1:3, stir evenly, raise the temperature of the reaction system to 25°C, and stir at 200 rpm; at the same time, during the stirring process, continuously introduce sterile air with an oxygen partial pressure of 0.15 MPa into the reactor, the temperature of the sterile air is 30°C, and the relative humidity is 85%.

[0037] S004. Allow the reactor to stand to allow the reaction system to separate into layers. Let it stand at 25°C for 20 minutes, collect the upper organic phase, and reflux the lower aqueous phase back to the reactor for a second countercurrent extraction. Combine the two organic phases obtained. S005. Load the combined organic phase onto a chromatography column. The stationary phase of the chromatography column is a mixture of silica gel and octadecyl-bonded silica gel, with a dry basis mass mixing ratio of 1:0.8. Use a mixed solvent of distilled water and ethanol as the mobile phase for gradient elution. The volume fraction of ethanol increases linearly from an initial 5% to a final value of 30%. The column temperature for elution is controlled at 35°C. Collect the absorption peak at 280 nm using a UV detector to obtain the target fraction.

[0038] S006. The target fraction is pre-concentrated through a nanofiltration membrane with a molecular weight cutoff of 1000 Da to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying. The belt surface temperature of the vacuum belt dryer is controlled at 60°C, and the system vacuum degree is controlled at -0.094 MPa. Finally, the product is pulverized through an 80-mesh sieve to obtain a high-activity theaflavins product.

[0039] In the product prepared in this embodiment, the TF3 monomer content accounts for 52% of the total theaflavin monomers.

[0040] Example 2 This embodiment provides a method for preparing highly active theaflavins, including the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. The fresh tea leaves are subjected to static fermentation, then quick-frozen with liquid nitrogen, and pulverized through an 85-mesh sieve to obtain tea powder. The polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, and its specific enzyme activity is 4800 U / mg.

[0041] S002. The tea powder is added to a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.7 to form a mixed solution. The mixed solution is ultrasonically treated for 50 minutes with an ultrasonic power of 280W. Then, sodium L-ascorbate is added as a potential regulator at an amount of 0.4% of the total mass of the mixed solution, along with the polyphenol oxidase. The mixture is pre-enzymatically hydrolyzed for 50 minutes under stirring conditions to obtain an enzymatic hydrolysate.

[0042] S003. Transfer the enzymatic hydrolysate to a reactor, add ethyl acetate (dehydrated by molecular sieve) as the organic phase and aqueous phase to the reactor, control the volume ratio of the organic phase to the aqueous phase to be 1:2, stir evenly, raise the temperature of the reaction system to 30°C, and stir at 180 rpm; at the same time, during the stirring process, continuously introduce sterile air with an oxygen partial pressure of 0.12 MPa into the reactor, the temperature of the sterile air is 28°C, and the relative humidity is 82%.

[0043] S004. Allow the reactor to stand to allow the reaction system to separate into layers. Let it stand at 22°C for 18 minutes, collect the upper organic phase, and reflux the lower aqueous phase back into the reactor for a second countercurrent extraction. Combine the two organic phases obtained. S005. Load the combined organic phase onto a chromatography column. The stationary phase of the chromatography column is a composite packing material of silica gel and adsorption resin, wherein the adsorption resin is a styrene-divinylbenzene copolymer with a specific surface area of ​​550 m². 2 / g, with an average pore size of 10nm; the dry weight mixing ratio of the silica gel and the adsorption resin is 1:1.5; gradient elution is performed using a mixed solvent composed of distilled water and ethanol as the mobile phase, with the volume fraction of ethanol increasing linearly from an initial 5% to a final value of 28%, and the column temperature of the elution is controlled at 32℃; the absorption peak portion at a wavelength of 280nm is collected by the ultraviolet detector to obtain the target fraction.

[0044] S006. The target fraction is pre-concentrated through a nanofiltration membrane with a molecular weight cutoff of 900 Da to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying. The belt surface temperature of the vacuum belt dryer is controlled at 58°C, and the system vacuum degree is controlled at -0.092 MPa. Finally, the product is pulverized through a 105-mesh sieve to obtain a high-activity theaflavins product.

[0045] The performance of the product obtained in this embodiment: In the theaflavins product prepared by this method, the TF3 monomer content accounts for 51% of the total theaflavins monomers.

[0046] Example 3 This embodiment provides a method for preparing highly active theaflavins, including the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. The fresh tea leaves are subjected to static fermentation, then quick-frozen with liquid nitrogen, and pulverized through a 30-mesh sieve to obtain tea powder. The polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, and its specific enzyme activity is 5200 U / mg.

[0047] S002. The tea powder is added to a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.9 to form a mixed solution. The mixed solution is ultrasonically treated for 40 minutes with an ultrasonic power of 320W. Then, sodium L-ascorbate is added as a potential regulator at an amount of 0.6% of the total mass of the mixed solution, along with the polyphenol oxidase. The mixture is pre-enzymatically hydrolyzed for 40 minutes under stirring conditions to obtain an enzymatic hydrolysate.

[0048] S003. Transfer the enzymatic hydrolysate to a reactor, add ethyl acetate (dehydrated by molecular sieve) as the organic phase and aqueous phase to the reactor, control the volume ratio of the organic phase to the aqueous phase to be 1:4, stir evenly, raise the temperature of the reaction system to 35°C, and stir at 220 rpm; at the same time, during the stirring process, continuously introduce sterile air with an oxygen partial pressure of 0.18 MPa into the reactor, the temperature of the sterile air is 32°C, and the relative humidity is 88%.

[0049] S004. Allow the reactor to stand to allow the reaction system to separate into layers. Let it stand at 28°C for 22 minutes, collect the upper organic phase, and reflux the lower aqueous phase back to the reactor for a second countercurrent extraction. Combine the two organic phases obtained. S005. Load the combined organic phase onto a chromatography column. The stationary phase of the chromatography column consists of aniline-pyrrole copolymer microspheres and silica gel, wherein the mass percentage of the aniline-pyrrole copolymer microspheres is 20%, and the balance is 80% silica gel. A gradient elution is performed using a mixed solvent of distilled water and ethanol as the mobile phase. The volume fraction of ethanol increases linearly from an initial 5% to a final value of 29%. The column temperature for elution is controlled at 38°C. Collect the absorption peak at a wavelength of 280 nm using a UV detector to obtain the target fraction.

[0050] S006. The target fraction is pre-concentrated through a nanofiltration membrane with a molecular weight cutoff of 1100 Da to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying. The belt surface temperature of the vacuum belt dryer is controlled at 62°C, and the system vacuum degree is controlled at -0.096 MPa. Finally, the product is pulverized through a 115-mesh sieve to obtain a high-activity theaflavins product.

[0051] In the product prepared in this embodiment, the TF3 monomer content accounts for 53% of the total theaflavin monomers.

[0052] Example 4 This embodiment provides a method for preparing highly active theaflavins, including the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. The fresh tea leaves are subjected to static fermentation, then quick-frozen with liquid nitrogen, and pulverized through a 40-mesh sieve to obtain tea powder. The polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, and its specific enzyme activity is 4500 U / mg.

[0053] S002. The tea powder is added to a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.6 to form a mixed solution. The mixed solution is ultrasonically treated for 60 minutes with an ultrasonic power of 250W. Then, sodium L-ascorbate is added as a potential regulator at an amount of 0.3% of the total mass of the mixed solution, along with the polyphenol oxidase. The mixture is pre-enzymatically hydrolyzed for 60 minutes under stirring conditions to obtain an enzymatic hydrolysate.

[0054] S003. Transfer the enzymatic hydrolysate to a reactor, add ethyl acetate (dehydrated by molecular sieve) as the organic phase and aqueous phase to the reactor, control the volume ratio of the organic phase to the aqueous phase to be 2:3, stir evenly, raise the temperature of the reaction system to 25°C, and stir at 150 rpm; at the same time, during the stirring process, continuously introduce sterile air with an oxygen partial pressure of 0.10 MPa into the reactor, the temperature of the sterile air being 25°C and the relative humidity being 80%.

[0055] S004. Allow the reactor to stand to allow the reaction system to separate into layers. Let it stand at 20°C for 15 minutes, collect the upper organic phase, and reflux the lower aqueous phase back into the reactor for a second countercurrent extraction. Combine the two organic phases obtained. S005. Load the combined organic phase onto a chromatography column. The stationary phase of the chromatography column consists of tea polyphenol-zinc ion complex powder and silica gel, wherein the mass percentage of tea polyphenol-zinc ion complex powder is 10%, and the balance is 90% silica gel. A gradient elution is performed using a mixed solvent of distilled water and ethanol as the mobile phase. The volume fraction of ethanol increases linearly from an initial 5% to a final value of 25%. The column temperature for elution is controlled at 30°C. Collect the absorption peak at a wavelength of 280 nm using a UV detector to obtain the target fraction.

[0056] S006. The target fraction is pre-concentrated through a nanofiltration membrane with a molecular weight cutoff of 800 Da to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying. The belt surface temperature of the vacuum belt dryer is controlled at 55°C, and the system vacuum degree is controlled at -0.090 MPa. Finally, the product is pulverized through a 120-mesh sieve to obtain a high-activity theaflavins product.

[0057] In the product prepared in this embodiment, the TF3 monomer content accounts for 50% of the total theaflavin monomers.

[0058] Example 5 This embodiment provides a method for preparing highly active theaflavins, including the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. The fresh tea leaves are subjected to static fermentation, then quick-frozen with liquid nitrogen, and pulverized through a 100-mesh sieve to obtain tea powder. The polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, and its specific enzyme activity is 5500 U / mg.

[0059] S002. The tea powder is added to a citrate-disodium hydrogen phosphate buffer solution with a pH of 6.0 to form a mixed solution. The mixed solution is ultrasonically treated for 30 minutes with an ultrasonic power of 350W. Then, sodium L-ascorbate is added as a potential regulator at an amount of 0.7% of the total mass of the mixed solution, along with the polyphenol oxidase. The mixture is pre-enzymatically hydrolyzed for 30 minutes under stirring conditions to obtain an enzymatic hydrolysate.

[0060] S003. Transfer the enzymatic hydrolysate to a reactor, add ethyl acetate (dehydrated by molecular sieve) as the organic phase and aqueous phase to the reactor, control the volume ratio of the organic phase to the aqueous phase to be 1:4, stir evenly, raise the temperature of the reaction system to 30°C, and stir at 250 rpm; at the same time, continuously introduce sterile air with an oxygen partial pressure of 0.20 MPa into the reactor during the stirring process, the temperature of the sterile air is 35°C, and the relative humidity is 90%.

[0061] S004. Allow the reactor to stand to allow the reaction system to separate into layers. Let it stand at 30°C for 25 minutes, collect the upper organic phase, and reflux the lower aqueous phase back into the reactor for a second countercurrent extraction. Combine the two organic phases obtained. S005. Load the combined organic phase onto a chromatography column. The stationary phase of the chromatography column consists of aminated mesoporous silica and silica gel, wherein the mass percentage of aminated mesoporous silica is 20% and the mass percentage of silica gel is 80%. A gradient elution is performed using a mixed solvent of distilled water and ethanol as the mobile phase. The volume fraction of ethanol increases linearly from an initial 5% to a final value of 35%. The column temperature for elution is controlled at 40°C. Collect the absorption peak at a wavelength of 280 nm using a UV detector to obtain the target fraction.

[0062] S006. The target fraction is pre-concentrated through a nanofiltration membrane with a molecular weight cutoff of 1200 Da to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying. The belt surface temperature of the vacuum belt dryer is controlled at 65°C, and the system vacuum degree is controlled at -0.098 MPa. Finally, the product is pulverized through a 120-mesh sieve to obtain a high-activity theaflavins product.

[0063] In the product prepared in this embodiment, the TF3 monomer content accounts for 55% of the total theaflavin monomers.

[0064] Example 6 This embodiment provides a method for preparing highly active theaflavins, including the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. The fresh tea leaves are subjected to static fermentation, then quick-frozen with liquid nitrogen, and pulverized through a 50-mesh sieve to obtain tea powder. The polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, and its specific enzyme activity is 4700 U / mg.

[0065] S002. The tea powder is added to a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.7 to form a mixed solution. The mixed solution is ultrasonically treated for 55 minutes with an ultrasonic power of 260W. Then, sodium L-ascorbate is added as a potential regulator at an amount of 0.45% of the total mass of the mixed solution, along with the polyphenol oxidase. The mixture is pre-enzymatically hydrolyzed for 55 minutes under stirring conditions to obtain an enzymatic hydrolysate.

[0066] S003. Transfer the enzymatic hydrolysate to a reactor, add ethyl acetate (dehydrated by molecular sieve) as the organic phase and aqueous phase to the reactor, control the volume ratio of the organic phase to the aqueous phase to be 1:3, stir evenly, raise the temperature of the reaction system to 45°C, and stir at 190 rpm; at the same time, during the stirring process, continuously introduce sterile air with an oxygen partial pressure of 0.14 MPa into the reactor, the temperature of the sterile air is 29°C, and the relative humidity is 83%.

[0067] S004. Allow the reactor to stand to allow the reaction system to separate into layers. Let it stand at 26°C for 19 minutes, collect the upper organic phase, and reflux the lower aqueous phase back to the reactor for a second countercurrent extraction. Combine the two organic phases obtained. S005. Load the combined organic phase onto a chromatography column. The stationary phase of the chromatography column consists of the following components by mass percentage: 50% silica gel matrix, 20% aniline-pyrrole copolymer microspheres, 15% tea polyphenol-zinc ion complex powder, and 15% aminated mesoporous silica. Use a mixed solvent of distilled water and ethanol as the mobile phase for gradient elution. The volume fraction of ethanol increases linearly from an initial 5% to a final value of 30%. The column temperature for elution is controlled at 36°C. Collect the absorption peak at 280 nm using a UV detector to obtain the target fraction.

[0068] S006. The target fraction is pre-concentrated through a nanofiltration membrane with a molecular weight cutoff of 950 Da to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying. The belt surface temperature of the vacuum belt dryer is controlled at 59°C, and the system vacuum degree is controlled at -0.093 MPa. Finally, the product is pulverized through a 120-mesh sieve to obtain a high-activity theaflavins product.

[0069] In the product prepared in this embodiment, the TF3 monomer content accounts for 54% of the total theaflavins monomer content.

[0070] Example 7 This embodiment provides a method for preparing highly active theaflavins, including the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. The fresh tea leaves are subjected to static fermentation, then quick-frozen with liquid nitrogen, and pulverized through an 80-mesh sieve to obtain tea powder. The polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, and its specific enzyme activity is 4900 U / mg.

[0071] S002. The tea powder is added to a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.8 to form a mixed solution. The mixed solution is ultrasonically treated for 35 minutes with an ultrasonic power of 340W. Then, sodium L-ascorbate is added as a potential regulator at an amount of 0.55% of the total mass of the mixed solution, along with the polyphenol oxidase. The mixture is pre-enzymatically hydrolyzed for 35 minutes under stirring conditions to obtain an enzymatic hydrolysate.

[0072] S003. Transfer the enzymatic hydrolysate to a reactor, add ethyl acetate (dehydrated by molecular sieve) as the organic phase and aqueous phase to the reactor, control the volume ratio of the organic phase to the aqueous phase to be 1:2, stir evenly, raise the temperature of the reaction system to 40°C, and stir at 210 rpm; at the same time, during the stirring process, continuously introduce sterile air with an oxygen partial pressure of 0.16 MPa into the reactor, the temperature of the sterile air is 31°C, and the relative humidity is 86%.

[0073] S004. Allow the reactor to stand to allow the reaction system to separate into layers. Let it stand at 24°C for 21 minutes, collect the upper organic phase, and reflux the lower aqueous phase back to the reactor for a second countercurrent extraction. Combine the two organic phases obtained. S005. Load the combined organic phase onto a chromatography column. The stationary phase of the chromatography column consists of the following components by mass percentage: 45% silica gel matrix, 25% aniline-pyrrole copolymer microspheres, 15% tea polyphenol-zinc ion complex powder, and 15% aminated mesoporous silica. Use a mixed solvent of distilled water and ethanol as the mobile phase for gradient elution. The volume fraction of ethanol increases linearly from an initial 5% to a final value of 33%. The column temperature for elution is controlled at 37°C. Collect the absorption peak at 280 nm using a UV detector to obtain the target fraction.

[0074] S006. The target fraction is pre-concentrated through a nanofiltration membrane with a molecular weight cutoff of 1050 Da to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying. The belt surface temperature of the vacuum belt dryer is controlled at 61°C, and the system vacuum degree is controlled at -0.095 MPa. Finally, the product is pulverized through a 110-mesh sieve to obtain a high-activity theaflavins product.

[0075] In the product prepared in this embodiment, the TF3 monomer content accounts for 56% of the total theaflavin monomers.

[0076] Example 8 This embodiment provides a method for preparing highly active theaflavins, including the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. The fresh tea leaves are subjected to static fermentation, then quick-frozen with liquid nitrogen, and pulverized through a 20-mesh sieve to obtain tea powder. The polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, and its specific enzyme activity is 5300 U / mg.

[0077] S002. The tea powder is added to a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.9 to form a mixed solution. The mixed solution is ultrasonically treated for 42 minutes with an ultrasonic power of 270W. Then, sodium L-ascorbate is added as a potential regulator at an amount of 0.65% of the total mass of the mixed solution, along with the polyphenol oxidase. The mixture is pre-enzymatically hydrolyzed for 42 minutes under stirring conditions to obtain an enzymatic hydrolysate.

[0078] S003. Transfer the enzymatic hydrolysate to a reactor, add ethyl acetate (dehydrated by molecular sieve) as the organic phase and aqueous phase to the reactor, control the volume ratio of the organic phase to the aqueous phase to be 1:4, stir evenly, raise the temperature of the reaction system to 50°C, and stir at 230 rpm; at the same time, continuously introduce sterile air with an oxygen partial pressure of 0.19 MPa into the reactor during the stirring process, the temperature of the sterile air is 33°C, and the relative humidity is 89%.

[0079] S004. Allow the reactor to stand to allow the reaction system to separate into layers. Let it stand at 29°C for 23 minutes, collect the upper organic phase, and reflux the lower aqueous phase back to the reactor for a second countercurrent extraction. Combine the two organic phases obtained. S005. Load the combined organic phase onto a chromatography column. The stationary phase of the chromatography column consists of the following components by mass percentage: 60% silica gel matrix, 15% aniline-pyrrole copolymer microspheres, 10% tea polyphenol-zinc ion complex powder, and 15% aminated mesoporous silica. Use a mixed solvent of distilled water and ethanol as the mobile phase for gradient elution. The volume fraction of ethanol increases linearly from an initial 5% to a final value of 34%. The column temperature for elution is controlled at 39°C. Collect the absorption peak at 280 nm using a UV detector to obtain the target fraction.

[0080] S006. The target fraction is pre-concentrated through a nanofiltration membrane with a molecular weight cutoff of 1150 Da to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying. The belt surface temperature of the vacuum belt dryer is controlled at 63°C, and the system vacuum degree is controlled at -0.097 MPa. Finally, the product is pulverized through a 120-mesh sieve to obtain a high-activity theaflavins product.

[0081] In the product prepared in this embodiment, the TF3 monomer content accounts for 57% of the total theaflavin monomers.

[0082] Example 9 This embodiment provides a method for preparing highly active theaflavins, including the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. The fresh tea leaves are subjected to static fermentation, then quick-frozen with liquid nitrogen, and pulverized through a 40-mesh sieve to obtain tea powder. The polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, and its specific enzyme activity is 4600 U / mg.

[0083] S002. The tea powder is added to a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.6 to form a mixed solution. The mixed solution is ultrasonically treated for 58 minutes with an ultrasonic power of 255W. Then, sodium L-ascorbate is added as a potential regulator at an amount of 0.35% of the total mass of the mixed solution, along with the polyphenol oxidase. Pre-enzymatic hydrolysis is carried out under stirring conditions for 58 minutes to obtain an enzymatic hydrolysate.

[0084] S003. Transfer the enzymatic hydrolysate to a reactor, add ethyl acetate (dehydrated by molecular sieve) as the organic phase and aqueous phase to the reactor, control the volume ratio of the organic phase to the aqueous phase to be 2:3, stir evenly, raise the temperature of the reaction system to 28°C, and stir at 160 rpm; at the same time, continuously introduce sterile air with an oxygen partial pressure of 0.11 MPa into the reactor during the stirring process, the temperature of the sterile air is 26°C, and the relative humidity is 81%.

[0085] S004. Allow the reactor to stand to allow the reaction system to separate into layers. Let it stand at 21°C for 16 minutes, collect the upper organic phase, and reflux the lower aqueous phase back to the reactor for a second countercurrent extraction. Combine the two organic phases obtained. S005. Load the combined organic phase onto a chromatography column. The stationary phase of the chromatography column consists of the following components by mass percentage: 40% silica gel matrix, 20% aniline-pyrrole copolymer microspheres, 20% tea polyphenol-zinc ion complex powder, and 20% aminated mesoporous silica. Use a mixed solvent of distilled water and ethanol as the mobile phase for gradient elution. The volume fraction of ethanol increases linearly from an initial 5% to a final value of 26%. The column temperature for elution is controlled at 31°C. Collect the absorption peak at 280 nm using a UV detector to obtain the target fraction.

[0086] S006. The target fraction is pre-concentrated through a nanofiltration membrane with a molecular weight cutoff of 850 Da to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying. The belt surface temperature of the vacuum belt dryer is controlled at 56°C, and the system vacuum degree is controlled at -0.091 MPa. Finally, the product is pulverized through a 100-mesh sieve to obtain a high-activity theaflavins product.

[0087] In the product prepared in this embodiment, the TF3 monomer content accounts for 51% of the total theaflavins monomers.

[0088] Example 10 This embodiment provides a method for preparing highly active theaflavins, including the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. The fresh tea leaves are subjected to static fermentation, followed by liquid nitrogen quick-freezing treatment, and then crushed through a 70-mesh sieve to obtain tea powder. The polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, and its specific enzyme activity is 5400 U / mg.

[0089] S002. The tea powder is added to a citrate-disodium hydrogen phosphate buffer solution with a pH of 6.0 to form a mixed solution. The mixed solution is ultrasonically treated for 38 minutes with an ultrasonic power of 330W. Then, sodium L-ascorbate is added as a potential regulator at an amount of 0.65% of the total mass of the mixed solution, along with the polyphenol oxidase. Pre-enzymatic hydrolysis is carried out under stirring conditions for 38 minutes to obtain the enzymatic hydrolysate.

[0090] S003. Transfer the enzymatic hydrolysate to a reactor, add ethyl acetate (dehydrated by molecular sieve) as the organic phase and aqueous phase to the reactor, control the volume ratio of the organic phase to the aqueous phase to be 1:3, stir evenly, raise the temperature of the reaction system to 32°C, and stir at 240 rpm; at the same time, during the stirring process, continuously introduce sterile air with an oxygen partial pressure of 0.17 MPa into the reactor, the temperature of the sterile air is 34°C, and the relative humidity is 87%.

[0091] S004. Allow the reactor to stand to allow the reaction system to separate into layers. Let it stand at 27°C for 24 minutes, collect the upper organic phase, and reflux the lower aqueous phase back to the reactor for a second countercurrent extraction. Combine the two organic phases obtained. S005. Load the combined organic phase onto a chromatography column. The stationary phase of the chromatography column consists of the following components by mass percentage: 55% silica gel matrix, 18% aniline-pyrrole copolymer microspheres, 12% tea polyphenol-zinc ion complex powder, and 15% aminated mesoporous silica. Use a mixed solvent of distilled water and ethanol as the mobile phase for gradient elution. The volume fraction of ethanol increases linearly from an initial 5% to a final value of 29%. The column temperature for elution is controlled at 34°C. Collect the absorption peak at 280 nm using a UV detector to obtain the target fraction.

[0092] S006. The target fraction is pre-concentrated through a nanofiltration membrane with a molecular weight cutoff of 1000 Da to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying. The belt surface temperature of the vacuum belt dryer is controlled at 64°C, and the system vacuum degree is controlled at -0.096 MPa. Finally, the product is pulverized through an 80-mesh sieve to obtain a high-activity theaflavins product.

[0093] In the product prepared in this embodiment, the TF3 monomer content accounts for 58% of the total theaflavin monomers.

[0094] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing highly active theaflavins, characterized in that, Includes the following steps: S001. Obtain fresh tea leaves and their corresponding polyphenol oxidase. The fresh tea leaves are subjected to static fermentation, then quick-frozen, crushed and sieved to obtain tea powder. S002. Add the tea powder to a buffer solution to form a mixed solution, sonicate the mixed solution for 30 min-60 min, then add a potential regulator and the polyphenol oxidase to it, and perform pre-enzymatic hydrolysis for 30 min-60 min under stirring conditions to obtain an enzymatic hydrolysate. S003. Transfer the enzymatic hydrolysate to a reactor, add an organic phase and an aqueous phase to the reactor, control the volume ratio of the organic phase to the aqueous phase to be 2:3 to 1:4, stir evenly and raise the temperature of the reaction system to 25℃~50℃, and at the same time, continuously introduce sterile air with an oxygen partial pressure of 0.10MPa~0.20MPa into the reactor during the stirring process; S004. Allow the reactor to stand to allow the reaction system to separate into layers, collect the upper organic phase, and return the lower aqueous phase to the reactor for a second countercurrent extraction. Combine the organic phases obtained from the two extractions. S005. Load the combined organic phase onto the chromatography column and use a mixed solvent of distilled water and ethanol as the mobile phase for gradient elution. Collect the absorption peak at 280 nm wavelength detected by the ultraviolet detector to obtain the target fraction. S006. The target fraction is pre-concentrated through a nanofiltration membrane to obtain a concentrate. The concentrate is then transferred to a vacuum belt dryer for continuous drying. Finally, it is pulverized and sieved to obtain a high-activity theaflavin product.

2. The method for preparing highly active theaflavins according to claim 1, characterized in that, In step S001, the quick-freezing treatment is liquid nitrogen quick-freezing, and the sieving is passing through a 20-mesh to 100-mesh sieve; and the polyphenol oxidase is purified by ammonium sulfate fractionation precipitation and DEAE-cellulose ion exchange chromatography, with a specific enzyme activity of 4500 U / mg to 5500 U / mg.

3. The method for preparing highly active theaflavins according to claim 1, characterized in that, In step S002, the buffer solution is a citrate-disodium hydrogen phosphate buffer system with a pH of 5.6 to 6.0; the potential regulator is L-ascorbic acid sodium, and its addition amount is 0.3% to 0.7% of the total mass of the mixed solution; the ultrasonic treatment power is 250W to 350W.

4. The method for preparing highly active theaflavins according to claim 1, characterized in that, In step S003, the organic phase is ethyl acetate that has been dehydrated by molecular sieves; the stirring speed is 150 rpm to 250 rpm; and the temperature of the sterile air introduced is 25°C to 35°C, and the relative humidity is 80% to 90%.

5. The method for preparing highly active theaflavins according to claim 1, characterized in that, In step S004, the condition for allowing the reaction system to separate into layers is: to allow it to stand at 20°C to 30°C for 15 to 25 minutes.

6. The method for preparing highly active theaflavins according to claim 1, characterized in that, In step S005, during the gradient elution process, the volume fraction of ethanol increases linearly from an initial 5% to a final value of 25%-35%; the column temperature for elution is controlled between 30°C and 40°C.

7. The method for preparing highly active theaflavins according to claim 1 or 6, characterized in that, In step S005, the stationary phase of the chromatography column is a mixture of silica gel and octadecyl bonded silica gel, and the dry basis mass mixing ratio of the silica gel and the octadecyl bonded silica gel is 1:0.5 to 1:1.

5.

8. The method for preparing highly active theaflavins according to claim 1 or 6, characterized in that, In step S005, the stationary phase of the chromatography column is a composite packing material of silica gel and adsorption resin, wherein the adsorption resin is a styrene-divinylbenzene copolymer with a specific surface area of ​​500-650 m². 2 / g, with an average pore size of 8-12nm; the dry weight mixing ratio of the silica gel and the adsorption resin is 1:1 to 1:

2.

9. The method for preparing highly active theaflavins according to claim 1 or 6, characterized in that, In step S005, the stationary phase of the chromatography column is composed of the following components in mass percentage: 40%-60% silica matrix, 15%-25% aniline-pyrrole copolymer microspheres, 10%-20% tea polyphenol-zinc ion complex powder, and 10%-20% aminated mesoporous silica.

10. The method for preparing highly active theaflavins according to claim 1, characterized in that, In step S006, the molecular weight cutoff of the nanofiltration membrane is 800 Da to 1200 Da; the belt surface temperature of the vacuum belt dryer is controlled at 55°C to 65°C, and the system vacuum degree is controlled at -0.090 MPa to -0.098 MPa; and the final pulverization and sieving is through an 80-mesh to 120-mesh sieve.