Process for extracting theaflavin and theabrownin from tea raw material

Abstract

The invention discloses a process for extracting theaflavin and theabrownin from a tea raw material. The process comprises the following steps: carrying out enzymatic oxidation and solid-liquid separation on a tea raw material, and carrying out extraction, membrane separation and resin chromatography on the liquid to prepare theaflavin with the purity of more than or equal to 20%; microbial strains are inoculated into solid residues for solid-state fermentation, fermentation products are subjected to extraction, MVR mechanical vapor recompression low-temperature concentration, ethanol low-temperature alcohol precipitation and nanofiltration desalination, and theabrownin with the purity larger than or equal to 90% is obtained. The whole process is completed in the same continuous system, a solid outlet of the solid-liquid separation device is directly connected with the fermentation equipment, full-component high-value utilization of tea resources is achieved, and the system has the outstanding advantages of being excellent in product purity, high in production efficiency, capable of saving energy, environmentally friendly, low in cost and the like. The method is suitable for large-scale production of high-quality tea pigment in the fields of functional food, health care products and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product extraction, in particular to a process for efficiently extracting theaflavins and thearubigins from tea raw materials, especially to a method combining enzymatic oxidation, microbial fermentation and modern separation and purification technology to realize high-yield and high-purity industrial production of tea pigments. BACKGROUND

[0002] Tea pigments, mainly including theaflavins and thearubigins, are important active ingredients in tea leaves. Due to their excellent antioxidant, anti-inflammatory and metabolic disease prevention effects, they have broad application prospects in the fields of food, health care products and cosmetics. However, the current industrialized extraction of tea pigments faces significant technical bottlenecks. Traditional production processes usually extract theaflavins and thearubigins independently: theaflavins are mainly extracted by solvent extraction and column chromatography purification, which has problems such as organic solvent residue, complicated steps, and difficulty in achieving high yield and purity; thearubigins are mostly prepared by microbial fermentation, but the process is time-consuming and uncontrollable. More importantly, most existing technologies discard the residues after extracting one component, failing to realize high-value comprehensive utilization of raw materials, resulting in high production cost and serious resource waste. Therefore, developing a new process that can continuously and efficiently extract two high-purity tea pigments from the same raw material has become an urgent need for industrial upgrading. SUMMARY

[0003] The present application provides a process for efficiently extracting theaflavins and thearubigins from tea raw materials, which combines enzymatic oxidation, microbial fermentation, membrane separation, chromatography purification and other technologies to extract high-purity theaflavins and thearubigins from the same batch of tea raw materials, realizing high-yield and high-purity extraction of tea pigments and having industrial application prospects.

[0004] The present application provides an integrated process that completely changes the traditional independent production mode of theaflavins and thearubigins, realizing full-component, high-value, zero-waste recycling utilization of tea raw materials. It uses the solid residues produced after extracting theaflavins (waste in traditional processes) as high-quality raw materials for producing thearubigins, greatly improving the utilization rate of raw materials and significantly reducing the comprehensive production cost of the two products, forming a profit model of "one raw material, two high-value products". The entire process is almost waste-free, reducing environmental pollution and meeting the concepts of green chemistry and sustainable development. The present application organically integrates enzyme engineering, microbial fermentation engineering and modern separation technology (membrane separation, chromatography, MVR mechanical vapor recompression) to form a continuous, stable and controllable industrial production line, which has high technical barriers and represents the direction of technological upgrading in the plant extraction industry. By precisely controlling the parameters of each step, high-purity and high-activity theaflavins and thearubigins products are stably obtained, meeting the demand for high-quality natural raw materials in the high-end food, health care product and cosmetic markets and enhancing the market competitiveness of the products.

[0005] The present application provides a process for extracting theaflavins and theabrownins from tea raw materials, comprising the following steps: Step 1. Enzymatic oxidation, efficiently and specifically converting catechin precursors in the raw material into the target product, i.e. theaflavins. The tea raw material is mixed with a buffer solution, and an enzyme source is added for enzymatic oxidation reaction. After the reaction is completed, the enzyme is inactivated, and a reaction liquid is obtained; wherein the enzyme source (polyphenol oxidase and peroxidase) is the "engine" of the catalytic reaction, and the two enzymes work together to ensure that the oxidation path of catechins is directed towards the production of theaflavins rather than other polymers; the enzyme addition amount (1000-3000 U / g) ensures that the reaction has sufficient catalytic power, achieving the best balance between economic cost and reaction efficiency, too low and the reaction is slow and incomplete, too high and the cost increases dramatically and side reactions are triggered; the buffer solution (pH 4.5-5.5) provides the optimal pH environment for the enzyme, maintaining its high activity and stability, which is a basic condition for the success of the reaction; the solid-liquid ratio (1:8-1:15) determines the concentration of the reaction system, ensuring good mass transfer (sufficient contact between enzyme, substrate and oxygen), and at the same time making the subsequent treatment (separation, concentration) energy consumption controllable; the temperature (30-40℃) and oxygenation stirring provide the best energy environment and oxygen (key reactant) required for enzymatic reaction, and stirring ensures uniform reaction; enzyme inactivation (85-95℃, 3-10min) immediately stops the reaction, preventing the theaflavins already produced from being further oxidized and degraded into theabrownins, which is a key step to "lock" the target product.

[0006] Step 2. Solid-liquid separation, which separates the reaction liquid into a liquid rich in theaflavins and a solid residue rich in fiber and unconverted polyphenols, providing raw materials for the subsequent two independent purification routes. The reaction liquid obtained in step 1 is subjected to solid-liquid separation to obtain a liquid rich in theaflavins and a solid residue; this step is a node for realizing process bifurcation and resource allocation, wherein the liquid phase is responsible for step 3; the solid phase, as "new raw material", is responsible for step 4, which is the core of the entire process cycle.

[0007] Step 3. Theaflavins purification, separating and refining high-purity theaflavins products from complex liquid mixtures. The liquid is sequentially subjected to extraction, concentration and chromatographic purification to obtain theaflavins products; countercurrent extraction with ethyl acetate, which takes advantage of the difference in partition ratio of theaflavins between ethyl acetate and aqueous phase, efficiently enriches theaflavins from the aqueous phase, which is a key step for preliminary purification and removal of most water-soluble impurities; ultrafiltration (membrane separation) removes macromolecular impurities (such as proteins, polysaccharides), colloids and small particles in the extraction liquid, protecting the subsequent chromatographic column from contamination and plugging, and prolonging the service life of the packing material; macroporous adsorption resin chromatography utilizes the specific adsorption and desorption capacity of the resin for theaflavins molecules, realizing the final separation of theaflavins from other small molecular impurities (such as residual catechins, caffeine) with similar structure, which is a core refining means to obtain high-purity products.

[0008] Step 4. Microbial fermentation, using the metabolic action of microorganisms, converts the remaining polyphenols, cellulose and other components in the solid residues into the target product, i.e. theabrownin, achieving waste-to-resource. The solid residues are used as fermentation substrates, inoculated with microorganisms for solid-state fermentation; the strain (Aspergillus niger, etc.) has a powerful enzyme system that can efficiently degrade lignocellulose and convert polyphenols into theabrownin, and is the executor of the conversion; the spore concentration and inoculum size ensure that the starting point of fermentation has a sufficient number of "seeds" with consistent activity, and this parameter range is the key to controlling the fermentation period and the consistency of the product, directly affecting the production efficiency and stability.

[0009] Step 5. Purification of theabrownin, purifying theabrownin product that meets the standard from the fermentation extract. The material after fermentation in step 4 is sequentially subjected to extraction, concentration, alcohol precipitation and drying to obtain theabrownin product; MVR concentration (50-65°C) efficiently removes water at low temperature and high vacuum, greatly reducing energy consumption and protecting the heat sensitivity of theabrownin, preventing its degradation and inactivation; alcohol precipitation (60-80% ethanol) uses the principle that the solubility of theabrownin in high-concentration ethanol decreases sharply, allowing it to selectively precipitate, thereby separating it from soluble sugars, salts and other impurities, which is a key purification step; low-temperature standing is to make the precipitation more complete and the particles larger, facilitating subsequent separation; nanofiltration desalination (200-1000 Da) for deep purification, with a molecular weight cutoff set to be much smaller than that of theabrownin, which can effectively remove inorganic salts, small molecule pigments, residual solvents and other impurities that may remain after alcohol precipitation, significantly improving product purity and quality, making it suitable for higher-end application fields.

[0010] The preferred scheme of step 1 of the present application defines a refined and controllable enzymatic oxidation reaction system, which maximizes the directional conversion of catechins to theaflavins while effectively inhibiting their excessive oxidation to theabrownin or other uncontrollable polymers by precisely regulating the reaction environment (pH, temperature), reactant ratio (enzyme amount, solid-liquid ratio) and reaction progress (enzyme inactivation).

[0011] As a preferred, each preferred parameter in step 1 is not isolated, but is interrelated and mutually supportive, collectively forming an efficient, controllable and scalable modern biocatalytic process unit, laying a solid foundation for the success of the entire integrated process.

[0012] The enzyme source includes polyphenol oxidase (PPO) and peroxidase (POD), and the addition amount is 1000-3000 U / g tea raw material; the enzyme source selection defines the biological catalyst of the catalytic reaction, which is the core driving force for the oxidation and condensation of catechins to generate theaflavins. PPO is mainly responsible for initiating the initial oxidation of catechins to generate quinone substances; POD further catalyzes the specific condensation of these quinone substances to efficiently generate theaflavins. The synergistic effect of the two has higher conversion efficiency and specificity than single enzyme source. The enzyme addition amount defines the input amount of catalyst, which is the balance point of controlling reaction speed and economy. The lower limit (1000 U / g) ensures sufficient enzyme activity to start and maintain an effective reaction rate, avoiding slow and incomplete reaction due to insufficient enzyme amount; the upper limit (3000 U / g) controls the production cost; beyond this range, the increase in enzyme cost is not proportional to the yield improvement, and the economic benefit decreases, and too high enzyme amount will increase the side reaction.

[0013] The buffer is a phosphate or citrate-phosphate buffer with pH 4.5-5.5, and the solid-liquid ratio of tea raw material to buffer is 1:8 to 1:15 (g / mL); the buffer system provides a stable and suitable reaction environment (pH). The buffer can resist the pH fluctuation caused by the acidic substances generated during the reaction, ensuring that the enzyme is always within its optimal pH activity range during the entire reaction process, maintaining high catalytic efficiency. PPO and POD show the highest activity in this pH range, and pH that is too high or too low will cause enzyme deactivation or a sharp decrease in efficiency. The weakly acidic environment is conducive to the generation and stability of theaflavins, avoiding their further oxidation and decomposition under neutral or alkaline conditions. The solid-liquid ratio determines the concentration of the reaction system, affecting the mass transfer efficiency and subsequent processing cost. The lower limit (1:8) ensures that the system has sufficient fluidity, facilitating stirring and oxygenation, so that the enzyme, substrate, and oxygen can fully contact and the reaction is uniform and efficient; the upper limit (1:15) avoids a too dilute system; a too high solid-liquid ratio will reduce equipment utilization, increase energy consumption and cost in the subsequent concentration process, and indirectly affect the reaction speed due to the dilution of enzyme concentration.

[0014] The reaction is carried out at 30-40℃ under oxygenated stirring conditions; the reaction conditions of 30-40℃ and oxygenated stirring provide the thermodynamic conditions and material conditions required for the reaction. The temperature (30-40℃) is the optimal temperature range for PPO and POD, which can ensure high enzyme activity; if the temperature is too low, the reaction speed is slow; if the temperature is too high (>50℃), the enzyme will denature and lose activity quickly. Oxygen is an essential substrate for PPO catalytic oxidation reaction, and continuous oxygenation ensures the oxygen supply required for the reaction, promoting the forward reaction. Stirring ensures uniformity of the reaction system, prevents local concentration or temperature unevenness, and enhances gas-liquid mass transfer, improving oxygen utilization.

[0015] The enzyme inactivation treatment is heating at 85-95°C for 3-10 minutes to precisely terminate the reaction. This temperature and time combination can rapidly and completely inactivate all PPO and POD permanently. It prevents over-oxidation and must be immediately and completely terminated after the reaction reaches the expected degree, otherwise the enzymes will continue to catalyze the further oxidation of the theaflavins to generate unwanted polymers such as theabrownins, resulting in the decrease of the yield and purity of the target product. The enzyme inactivation treatment also fixes the reaction end point, ensuring the batch-to-batch stability of the product quality.

[0016] Step 2 of the present application is the "diverter" of the entire integrated process, which precisely divides the reaction solution into two parts and directs them to the theaflavin and theabrownin production lines respectively, and is the core step for efficient use of resources.

[0017] As a preferred, the solid-liquid separation in step 2 is achieved by plate-and-frame filter pressing, centrifugation or suction filtration. The present application obtains a clear liquid rich in theaflavins and solid residues rich in fibers and unconverted polyphenols through step 2, and the clarity of the liquid directly affects the efficiency of subsequent extraction and the service life of the chromatography column; the physical form (wet cake) of the solid provides a suitable initial humidity for it as a solid-state fermentation substrate. Among them, plate-and-frame filter pressing is suitable for large-scale, high-solid-content production, and the moisture content of the filter cake is relatively low, but the operation is intermittent and the labor intensity is high. Centrifugal separation (such as horizontal screw centrifuge) can be operated continuously, has high automation degree and large processing capacity, and is the first choice for modern large-scale production. Vacuum suction filtration equipment is simple and has small investment, and is suitable for small-scale or experimental production. For the liquid part, it is required to be clear to avoid clogging of the extraction and chromatography equipment; for the solid part, it is required to be a wet cake with controllable moisture content to provide the appropriate humidity required for microbial fermentation. The three methods can all reliably achieve this core process goal.

[0018] Step 3 of the present application constructs an efficient and standardized purification ladder from "crude purification" to "refining", aiming to economically and feasibly produce high-purity theaflavins on a large scale from complex reaction solutions. Among them, multi-stage countercurrent extraction can maximize the transfer of theaflavins from the aqueous phase, with high recovery rate. Ultrafiltration as pretreatment removes macromolecular impurities that damage the chromatography column; macroporous adsorption resin chromatography can specifically adsorb theaflavins, achieving fine separation from small molecular impurities (such as caffeine, residual solvents), and the final product has high purity. The selected methods (countercurrent extraction, ultrafiltration, resin chromatography) are mature and scalable unit operations in the natural product extraction industry, and the equipment is easy to obtain, the process is stable, and it is very suitable for industrial production. The high recovery rate reduces the loss of target product, and the resin can be regenerated and reused, reducing the long-term production cost of unit consumption.

[0019] As preferred, the extraction in step 3 achieves the preliminary enrichment from the aqueous phase to the organic phase; the ultrafiltration clears the obstacles for the core purification step; and the resin chromatography finally completes the preparation of high-purity product. The three steps are closely linked together, which collectively ensures the high purity, high yield and economy of the production of the final theaflavins product.

[0020] The extraction uses ethyl acetate for multi-stage countercurrent extraction; the theaflavins are preliminarily separated and enriched from the aqueous phase (liquid) obtained in step 2. The theaflavins have good solubility in ethyl acetate, while most of the water-soluble impurities (such as sugars, proteins, inorganic salts) have very low solubility, thereby achieving preliminary separation. Ethyl acetate is a low-toxicity solvent, and the residue is easy to remove, which meets the safety standards of food and health product raw materials. At the same time, ethyl acetate has a moderate boiling point, which is easy to recover and remove by subsequent vacuum concentration operation, facilitating recycling and reducing costs. Compared with single extraction, multi-stage countercurrent extraction is a continuous operation, which allows fresh solvent to contact with the aqueous phase with the lowest concentration, and the solvent with the highest concentration to contact with the fresh aqueous phase. This cross-flow method maximizes the use of concentration difference as the mass transfer driving force, which can use less solvent to achieve higher extraction rate, significantly improving the efficiency and reducing the solvent consumption.

[0021] The chromatographic purification includes: first using ultrafiltration for membrane separation, and then using macroporous adsorption resin chromatography column for refining. The first step of ultrafiltration (membrane separation) protects the chromatography column: before entering the expensive and easily contaminated macroporous resin chromatography column, the ultrafiltration membrane (usually with a molecular weight cutoff of several thousand to tens of thousands of daltons) is used to remove the macromolecular impurities such as proteins, colloids, plant polysaccharides, and small particles in the extraction concentrate; prevent these macromolecular substances from blocking the resin pore size or being irreversibly adsorbed on the resin, causing problems such as increased column pressure, decreased separation efficiency, and shortened resin life, etc. This step of pretreatment greatly improves the stability and efficiency of the subsequent chromatography step.

[0022] The second step of macroporous adsorption resin chromatography column: the core refining step, which realizes the fine separation of theaflavins and other components; the macroporous adsorption resin has a large specific surface area and pore structure, which selectively adsorbs theaflavins molecules in the solution through hydrophobic interaction and van der Waals force.

[0023] The purification process includes: first, adsorption, the crude theaflavins extract passes through the resin column, and the theaflavins are adsorbed on the resin; second, washing, water or low-concentration ethanol is used for elution, so that the strong-polarity impurities (such as salt and sugar) and weakly adsorbed impurities (such as part of the catechins) are eluted; then, desorption elution, the theaflavins are eluted from the resin by using an appropriate concentration of ethanol aqueous solution (such as 60-80% ethanol), at this time, the theaflavins are highly enriched and purified. The macroporous adsorption resin has a high adsorption capacity, is suitable for adsorbing the theaflavins due to its "macroporous" structure, has a high loading capacity, is easy to regenerate and has a good separation effect; after use, the resin can be regenerated by using alkaline solution or organic solvent, and can be repeatedly used for hundreds of times, which is economical, and can effectively separate the theaflavins from the impurities (such as theaflavins and some pigments) with similar molecular structures and polarities.

[0024] The inoculation operation in step 4 is one of the core links for realizing high-value utilization of resources and circular economy in the process, and the solid residues generated after extraction of the theaflavins are converted from "waste" into "high-quality raw materials" of the theabrownines with high added value. Through controllable solid-state fermentation by introducing specific microorganisms, the raw material cost and environmental protection pressure are greatly reduced, and the components such as polyphenols, polysaccharides and proteins remaining in the residues are efficiently converted into the target product theabrownines by the biological conversion of the microorganisms, so that the economic efficiency and green sustainability of the whole process are significantly improved.

[0025] As a preference, the setting of parameters in step 4 is a systematic engineering, which comprehensively considers multiple dimensions such as strain breeding, production convenience, cost control and fermentation kinetics, to jointly ensure the high efficiency, stability, controllability and industrialization of the theabrownine fermentation process.

[0026] The inoculation is to inoculate a specific microbial suspension into the solid residues, and the microorganisms in the microbial suspension are one or more of Aspergillus niger, Aspergillus coronatus or Pueraria tea Aspergillus. This limitation is the key to successful fermentation. The selected strains are all recognized as safe (GRAS), and have a strong enzyme system (such as polyphenol oxidase, cellulase and pectinase), which can effectively degrade plant cell walls and catalyze the conversion of residual catechins into theabrownines. They show high theabrownine yield and conversion efficiency on this specific substrate, ensuring the reliability and effectiveness of the process.

[0027] The microbial suspension is a spore suspension, which has the advantages of high stability, long shelf life, easy standardization and inoculation compared with mycelium suspension. Spores are in a dormant state, have strong stress resistance, are easy to prepare into high-concentration standard microbial agents, and ensure the consistency of the starting point of each batch of fermentation, which is crucial for the stability and reproducibility of industrial production.

[0028] The spore concentration is preferably 1x10 6 to 1x108 CFU / mL; this concentration range ensures that the inoculum contains a sufficient number of viable biological starting units. If the spore concentration is too low (<10 6 CFU / mL), it will lead to a too long lag phase of fermentation, a delayed fermentation cycle, and easy contamination by other bacteria; if the concentration is too high (>10 8 CFU / mL), although it can quickly start fermentation, the cost-effectiveness decreases, and uneven mass transfer occurs due to the rapid growth of bacterial cells. This range is the optimal choice that takes into account the fermentation efficiency and economic cost.

[0029] The inoculation ratio is 5% to 15% of the ratio (v / w) of the volume (mL) of the bacterial suspension to the mass (g) of the solid residue wet substrate, which directly determines the initial ratio of bacterial cells to substrate in the fermentation system and is a decisive factor for controlling the fermentation speed and the yield of the final product. The lower limit of 5% ensures that there are enough bacterial cells to quickly occupy a growth advantage and inhibit other bacteria; the upper limit of 15% ensures the fermentation efficiency while avoiding waste caused by excessive bacterial agents. This optimized range ensures that the fermentation process is fast, stable, and efficient. The wet mass is used as the calculation basis because the solid residue itself contains a large amount of water and is in the form of a wet solid; this limited method is simple to operate and can be directly implemented in the production site, avoiding the complex process of drying the residue to measure the dry weight and then calculating, ensuring the practicality and operability of the process, and also more truly reflecting the initial humidity state of the fermentation system.

[0030] Step 5 of the present application solves the problems of degradation of active ingredients caused by high-temperature concentration, huge energy consumption, and low product yield, poor purity, and large solvent consumption caused by improper alcohol precipitation conditions in traditional processes by using the technical path of MVR evaporation concentration combined with low-temperature controllable alcohol precipitation. This scheme is the key technical guarantee for achieving high-purity theabrownine products (≥90%) and reflects the advancement and economy of the entire process.

[0031] As a preferred embodiment, step 5 realizes efficient, low-energy, and standardized production suitable for industrialization under the premise of ensuring the biological activity of theabrownine and product quality.

[0032] The concentration step utilizes an MVR (Mechanical Vapor Recompression) evaporator to efficiently and at low temperature remove a large amount of water from the extract, increasing the concentration of the liquid and preparing it for subsequent alcohol precipitation, while significantly reducing energy consumption. The concentration process employs an MVR evaporator operating at 50-65℃, concentrating the extract with a solids content of 5%-8% to 25%-35%. MVR, by recycling secondary steam recompression, replaces the traditional multi-effect evaporation method that requires external fresh steam, saving approximately 90% of evaporation energy and significantly reducing production costs. Theabrownins are heat-sensitive substances; prolonged exposure to high temperatures can easily lead to oxidation, changes in polymerization degree, or loss of activity. The low temperature range of 50-65℃ (far below the boiling point of water at normal pressure, 100℃) achieves the optimal balance between evaporation efficiency and product protection, maximizing the preservation of the natural activity of theabrownins. The initial solids content concentration (5%-8%) determines the reasonable economic starting point for the concentration process. If the concentration is too low, the amount of water evaporated will be large, resulting in uneconomical energy consumption; if the concentration is too high, the liquid will be too viscous, reducing heat transfer efficiency. The endpoint concentration (25%-35%) is the optimal feed concentration set for the alcohol precipitation process; if the concentration is too low, subsequent alcohol precipitation will require a huge amount of ethanol, resulting in high costs; if the concentration is too high, the liquid will be too viscous, making it difficult to mix evenly with ethanol, leading to problems such as incomplete precipitation, encapsulation of impurities, and difficulty in separation.

[0033] The alcohol precipitation step utilizes the principle that theabrownin has the lowest solubility in ethanol at a specific concentration, allowing it to selectively precipitate from the aqueous solution. This effectively separates it from impurities such as soluble sugars, salts, and residual small-molecule phenols, making it a core step in the purification process. The alcohol precipitation involves adding edible alcohol to the concentrated solution to a final concentration of 60-80% (v / v) and allowing it to stand at below 10°C for 6-12 hours. The concentration range of 60%-80% (v / v) is the optimal range for theabrownin precipitation; below 60%, the ethanol concentration is insufficient, resulting in incomplete precipitation of theabrownin and a significantly reduced product yield. Theabrownin has the lowest solubility within the 60%-80% concentration range, maximizing precipitation and achieving a high yield. Above 80%, excessively high ethanol concentrations cause more lipophilic impurities (such as pigments, lipids, and certain alcohol-soluble proteins) to precipitate along with the ethanol, reducing product purity and increasing solvent costs. Low-temperature alcohol precipitation below 10℃ can further reduce the solubility of theaflavins and increase the precipitation yield; low temperature can effectively inhibit possible side reactions such as oxidation and enzymatic hydrolysis, and protect product quality; at the same time, low temperature can reduce the dissolution of trace fat-soluble impurities contained in ethanol and prevent other impurities from co-precipitating, which is beneficial to improving product purity and color.

[0034] The standing aging for 6-12 hours guarantees complete precipitation, gives sufficient time for the precipitation reaction to proceed fully, and ensures the yield. During the standing process, tiny precipitation particles collide with each other, agglomerate, and grow into larger and denser particles. This process, referred to as "maturation", can greatly improve the separation efficiency of subsequent centrifugation or filtration, make the solid-liquid separation more thorough, and obtain a precipitate with lower water content.

[0035] The present application introduces a series of operations of "centrifugal collection, water phase redissolution, and nanofiltration purification" after alcohol precipitation. The overall significance of the series of operations is to realize the deep purification and quality upgrading of the theabrownin product, which is a decisive step to obtain a high-purity and high-quality final product. The step directly solves the inherent limitations of the alcohol precipitation method (such as the co-precipitation of inorganic salts, small molecule pigments, residual solvents, and other impurities), and upgrades the traditional crude purification process to a refining level that can be applied to high-end food, health products, and pharmaceutical raw materials. It is not only a simple superposition of purification techniques, but also a key quality node that ensures the achievement of the core indicators of the product in the entire process chain.

[0036] Further, after the alcohol precipitation, the method further comprises: after the precipitation is collected by centrifugation and redissolved in a water phase, desalination purification is performed using a nanofiltration membrane with a molecular weight cut-off of 200-1000 Da. In the step of centrifugal collection, the solid-liquid mixture formed after alcohol precipitation is centrifuged to discard the supernatant and collect the precipitate. This is a key step for solid-liquid separation, aiming to efficiently and thoroughly recover the theabrownin precipitate precipitated by alcohol precipitation. The centrifugal force can overcome the characteristics of the flocculent and loose precipitate, achieve rapid separation, and obtain a wet filter cake of theabrownin crude product, preparing for subsequent deep processing.

[0037] The water phase redissolution step redissolves the theabrownin precipitate obtained by centrifugation with deionized water to form a uniform aqueous solution, realizing phase transfer and system switching. The theabrownin is separated from the high-concentration ethanol system, avoiding potential damage to the nanofiltration membrane material by ethanol and eliminating the risk of residual organic solvent. At the same time, nanofiltration conditions are created, and the nanofiltration membrane usually operates most efficiently and stably in pure water or aqueous systems. The solution obtained in this step is a theabrownin crude extract, which mainly contains theabrownin but also contains various water-soluble impurities that need to be removed in the next step.

[0038] The nanofiltration membrane desalination purification step pumps the reconstituted theabrownin crude extract into a device equipped with a nanofiltration membrane with a specific molecular weight cut-off (200-1000 Da), applies pressure, and makes water, inorganic salts, small molecule impurities, etc. pass through the membrane layer as "permeate", while theabrownin macromolecules are "retained" by the membrane and concentrated. This is the core of deep purification, effectively removing inorganic salt ions introduced during the alcohol precipitation process or present in the raw material, greatly reducing the ash content of the product. Precise removal of small molecule pigments, residual sugars, amino acids, phenolic acids, etc. with a molecular weight below 200-1000 daltons, which are key factors affecting product color, taste and purity. At the same time of purification, the nanofiltration process is also a concentration process, which can further increase the concentration of theabrownin in the solution and reduce the energy consumption of subsequent drying. The traditional process uses multiple alcohol precipitation or ion exchange resin method for desalination and impurity removal, and the nanofiltration technology is more efficient, more environmentally friendly (reduces the use of organic solvents), and easy to operate continuously, representing the development direction of modern separation technology.

[0039] Specifically, the molecular weight cut-off of 200-1000 Da is the most core parameter of the nanofiltration membrane, which directly determines the purification effect. The lower limit (200 Da) ensures that the target product theabrownin (theabrownin is a polymer with a molecular weight of several to tens of thousands of daltons) can be effectively retained to prevent its loss; the upper limit (1000 Da) ensures that the target impurities (such as salt, sugar, small molecule phenolic acid, etc.) with a molecular weight less than 1000 Da can be effectively removed; this range is selected to ensure high yield of the product while achieving optimal impurity removal efficiency. Compared with ultrafiltration or reverse osmosis, nanofiltration is the best choice in this scenario. Ultrafiltration has a larger molecular weight cut-off (usually above 1000 Da) and is mainly used for separating large molecular weight proteins and polysaccharides, and cannot effectively remove salt. Reverse osmosis almost retains all ions and molecules, including water molecules, and requires extremely high pressure, mainly used for seawater desalination, and cannot achieve selective separation. Nanofiltration is exactly between the two, as the membrane surface is usually charged, it can not only screen according to the size of the molecule, but also preferentially repel multivalent ions through the Donnan effect, especially suitable for the separation of salt and small and medium molecular weight impurities, perfectly matching the needs of theabrownin purification.

[0040] The process of the present application converts the tea pruning branches and leaves that are traditionally discarded or of low value into high value theaflavins and theabrownins, achieving "waste to treasure". At the same time, in the process design, the most valuable theaflavins component in the raw material is first extracted by enzymatic oxidation, and then the residue after extraction is converted into another high value product theabrownin by microbial fermentation, forming a near zero waste closed loop production mode, greatly reducing the raw material cost and environmental burden.

[0041] As preferred, the tea raw material is green tea, white tea or its pruned branches and leaves which are pulverized to 40-60 mesh. The pulverization pretreatment of the tea raw material is a key step to increase the reaction surface area. The particle size of 40-60 mesh achieves the best balance between reaction efficiency and subsequent separation difficulty. If the particle size is too large, the enzyme and microorganism cannot effectively contact the internal ingredients, and the conversion rate is low; if the particle size is too fine, although the reaction is fast, it will lead to difficult liquid-solid separation, turbid filtrate, and increase the purification burden.

[0042] The raw material is compatible with green tea, white tea and pruned branches and leaves, and the resource utilization rate is improved. Among them, green tea is rich in substrates and belongs to unfermented tea. The internal polyphenol oxidase activity is rapidly inactivated by high temperature in the fixation step, thereby maximizing the retention of rich catechin substances (especially ester-type catechins such as EGCG). These catechins are the most direct and core substrates for the generation of theaflavins in the enzymatic oxidation reaction. In the "enzymatic oxidation" step of the present process, the optimal enzyme source is re-added and the optimal reaction conditions are controlled, so that these unused substrates in green tea can be efficiently and directionally converted into theaflavins. Therefore, green tea is the optimal raw material for producing high-content theaflavins.

[0043] White tea is a high-potential and unique-flavor high-quality raw material. White tea belongs to a slightly fermented tea, which uses the withering process. In this process, endogenous enzymes will have a slight and slow oxidation, so that white tea not only retains a high content of catechins (as substrates), but also naturally pre-generates a small part of theaflavins and other oxidation products. At the same time, the tea pigments derived from white tea have a softer and more mellow flavor characteristic, providing an advantage in raw materials for developing end products (such as functional beverages) with unique flavor selling points. Its role is similar to that of green tea, and it is a high-grade raw material choice for producing high-quality theaflavins.

[0044] The regular pruning of tea plants is a necessary farming operation to maintain the output of tea gardens, and the branches and leaves produced are traditionally considered agricultural waste or are simply returned to the field. Using them as raw materials, the procurement cost is extremely low, even zero, which can greatly reduce the raw material cost of the final product. Although the absolute content of catechins in pruned branches and leaves is lower than that of tender buds and leaves, it still contains considerable available ingredients. The enzymatic oxidation system of the present invention can fully exploit and convert the effective ingredients in these "low-quality" raw materials to produce theaflavins, and then use the residues to produce theabrownines, realizing the effective use of resources and greatly improving the value chain of the entire tea industry.

[0045] The present application realizes efficient and high-purity extraction of theaflavins and thearubigins by combining enzymatic oxidation with microbial fermentation and using modern separation and purification technology. As preferred, the total content of theaflavins in the theaflavins product is determined by HPLC to be ≥20%, and the content of thearubigins in the thearubigins product is determined by UV to be ≥90%, which represents the high standard of industrialization that can be achieved by the process and is the ultimate goal of the optimization of the entire process. The design of all steps and the setting of parameters are for the purpose of stably and efficiently achieving the two indicators. High purity means that the product has higher biological activity and fewer impurities, so that it has strong competitiveness in the high-end functional food, health product and cosmetic raw material market, and is different from products extracted in a rough way with low purity. The theaflavins ≥20% and the thearubigins ≥90% ensure good solubility, stability and efficacy, and meet the core technical requirements of the application end.

[0046] The integrated system provided by the present application has the core significance of realizing the industrial production of two high-value-added products (theaflavins and thearubigins) from the same tea raw material in a continuous, efficient and high-value way, and completely changing the situation of low resource utilization rate, high cost and heavy pollution in the traditional process. The main effects are as follows: first, the solid residues after theaflavins extraction are "waste into treasure" as raw materials for thearubigins production, which greatly improves the utilization rate of raw materials, reduces the comprehensive production cost, and realizes a green circular production mode of nearly zero waste; second, the two independent production lines are organically coupled to form a complete continuous production chain, which reduces the storage and transportation of intermediate materials, improves the equipment utilization rate and production efficiency, and lays a foundation for large-scale and stable industrialization application; third, the system design optimizes the purification paths (such as chromatography purification and membrane purification) according to the different physicochemical properties of theaflavins and thearubigins, ensuring the high purity and biological activity of the final products; fourth, the application of key units (such as MVR evaporator) in the system can greatly reduce energy consumption compared with traditional multi-effect evaporation, which is in line with the development strategy of green manufacturing of the country.

[0047] The present application provides a system for efficiently extracting theaflavins and thearubigins from tea raw materials, comprising: The theaflavins production line is sequentially connected by an enzymatic oxidation reaction tank, a solid-liquid separation device, an extraction tank, a concentration unit and a chromatography purification unit. The enzymatic oxidation reaction tank is the core reactor for the synthesis of theaflavins. In this unit, the enzyme-catalyzed reaction is used to direct the conversion of catechins to theaflavins by accurately controlling the temperature, pH and oxygen supply; 30-40℃ and pH 4.5-5.5 are the optimum conditions for polyphenol oxidase, which ensures high catalytic efficiency; and oxygenated stirring ensures sufficient contact between the reaction substrate and oxygen.

[0048] The solid-liquid separation unit (plate and frame filter press / centrifuge) achieves the primary separation of reaction products and serves as the hub connecting the two production lines. The wet residue separated from the solid outlet (rich in incompletely converted polyphenols and fibers) is a high-quality raw material for theaflavin production; the clarified liquid obtained from the liquid outlet is the starting point for theaflavin purification. The extraction tank utilizes the difference in the partition ratio of theaflavin in ethyl acetate and aqueous phases to enrich it from the aqueous phase to the organic phase, achieving preliminary purification and concentration; the multi-stage countercurrent extraction method maximizes extraction efficiency and minimizes the loss of active ingredients. The concentration unit concentrates the extracted theaflavin ethyl acetate solution, recovering the solvent while increasing the concentration of subsequent chromatographic feed, improving chromatographic efficiency, and reducing solvent consumption costs. The chromatographic purification unit, including ultrafiltration and macroporous adsorption resin columns, is crucial for obtaining high-purity theaflavin products; ultrafiltration removes large molecular impurities such as proteins and polysaccharides, protecting the subsequent resin column and preventing its contamination and clogging; the resin column utilizes adsorption selectivity to separate theaflavin from other small molecular impurities (such as caffeine and residual catechins), and can achieve fractional preparation of theaflavin monomers.

[0049] The theabrownin production line consists of fermentation equipment, extraction tanks, MVR evaporators, alcohol precipitation tanks, and drying equipment connected sequentially. The fermentation equipment (solid-state fermentation tank) utilizes the biotransformation capabilities of microorganisms to convert residual polyphenols and polysaccharides in the solid residue into the target product, theabrownin. *Aspergillus niger* and other strains are specific strains for efficient theabrownin production. An inoculation ratio of 5-15% ensures rapid fermentation start-up, a short cycle, and low contamination risk. The extraction tank uses water to dissolve and extract the theabrownin produced during fermentation from the fermentation solids, forming an aqueous theabrownin solution. The MVR evaporator performs low-temperature, high-efficiency concentration of the diluted theabrownin solution obtained from the extraction. The low-temperature evaporation of 50-65℃ effectively protects the activity of heat-sensitive theabrownin, preventing its degradation and charring. Compared to traditional evaporation, MVR technology is highly efficient and energy-saving, offering significant advantages. The alcohol precipitation tank utilizes the principle that the solubility of theabrownins in ethanol solution decreases, causing them to precipitate and thus separate from water-soluble impurities (such as sugars and salts). A final concentration of 60-80% is the optimal range for complete precipitation with minimal impurity precipitation. Low-temperature aging makes the precipitate particles more compact, facilitating separation. Drying equipment (such as a spray drying tower) converts the wet theabrownin precipitate obtained after alcohol precipitation and centrifugation into a uniform, stable, and easily stored and transported dry powder product.

[0050] The solid outlet of the solid-liquid separation device is connected to the feed inlet of the fermentation equipment. This physical connection realizes the automatic flow of materials and the closed loop of energy flow in the entire integrated system, seamlessly connecting two independent processes into a whole. It is the core design for realizing resource recycling.

[0051] The application realizes continuous and efficient extraction of high-purity theaflavins and theabrownines from the same tea raw material by coupling enzymatic oxidation and microbial fermentation technology, and taking the solid waste of the previous process as the raw material of the subsequent process, and achieves the unity of maximum resource utilization and high value of products.

[0052] In summary, the application has the following beneficial effects: 1. Different from the traditional process, the application integrates the preparation of theaflavins and theabrownines into a continuous production line through the series connection of enzymatic oxidation-solid-liquid separation-microbial fermentation, optimizes the parameters of each link, realizes the closed-loop cooperation of reaction-separation-fermentation, significantly improves the process controllability and amplification stability, and completely avoids the efficiency bottleneck caused by the traditional process of "single component and batch type"; by taking the solid residue after theaflavins extraction as the exclusive substrate for theabrownines fermentation, the sequential continuous production of two high-value products is realized, which breaks through the technical barrier of the traditional single extraction mode and forms a complete closed loop of efficient resource circulation. 2. The application can stably obtain two high-purity products from the same batch of raw materials: the purity of theaflavins is ≥20% (HPLC), and the purity of theabrownines is ≥90% (UV), which is significantly better than the conventional method, and meets the strict standards of high-end food, health products and cosmetic raw materials. 3. The whole process of the application uses a water-based buffer system, food-grade ethyl acetate and recyclable ethanol, and the solvent recovery rate is improved; the solid residue is 100% converted into theabrownines fermentation substrate, without waste residue and high-salt wastewater discharge, which greatly reduces the solid waste discharge and treatment cost, and meets the requirements of green chemistry and clean production audit. 4. The process reduces the cost by efficiently utilizing raw materials, reduces the amount of organic solvents by coupling enzyme-membrane-resin purification, saves steam energy consumption by MVR mechanical vapor recompression evaporator, and reduces the comprehensive production cost compared with the traditional "double-line independent" process by continuous and compact design of production process, which has significant economic competitiveness. 5. The application finally achieves a comprehensive technical effect of high resource utilization rate, green and environmentally friendly production process, high product added value, and easy industrialization, realizes the integrated goal of "one material double extraction, zero waste, low energy consumption and high purity", and provides a comprehensive utilization solution for the large-scale, high-value and sustainable production of tea pigments. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 is a system schematic diagram of the process of the application; In the drawing, 1 is the theaflavins production line, and 2 is the theabrownines production line. DETAILED DESCRIPTION

[0054] The specific embodiments are merely an explanation of the present application, which is not a limitation of the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as it is within the scope of the present application, it is protected by the patent law.

[0055] Example 1

[0056] Step 1. Enzymatic oxidation: 1 kg of Anji white tea was pulverized to 40 mesh; mixed with 10 L of phosphate buffer at pH 5.0 (solid-liquid ratio 1:10), and a polyphenol oxidase and peroxidase complex enzyme source with an enzyme activity of 2000 U / g of tea powder was added, and reacted at 35℃ under oxygenated stirring for 4 hours; after the reaction was completed, the reaction liquid was heated to 90℃ for 5 minutes for enzyme inactivation; Step 2. Solid-liquid separation: the enzyme-inactivated reaction liquid was separated by a plate-and-frame filter press to obtain 8.5 L of a clear liquid rich in theaflavins and 2.2 kg of wet solid residue (water content about 65%); Step 3. Theaflavins purification: the liquid was subjected to three-stage countercurrent extraction with ethyl acetate, the organic phases were combined and concentrated to dryness under vacuum to obtain theaflavins crude product; the crude product was dissolved in water, pretreated by ultrafiltration membrane, and then loaded onto a macroporous adsorption resin column (type AB-8), and eluted with an ethanol solution by gradient elution, and the fractions rich in theaflavins were collected, concentrated and dried to obtain theaflavins product; Step 4. Microbial fermentation: 150 mL of Aspergillus niger suspension (spore concentration 5×10 7 CFU / mL) was inoculated into the solid residue at an inoculation amount of 15% (v / w), and then mixed uniformly and placed in a solid-state fermentation tank at 35℃ and 75% humidity for 15 days of fermentation; Step 5. Theabrownines purification: after the fermentation was completed, 10 times (v / w) of boiling water was added to the fermented material for extraction for 1 hour, and the extract was obtained by pressure filtration; the extract (solid concentration 6.5%) was pumped into an MVR evaporator and concentrated to a solid concentration of 30% at 60℃; edible alcohol was added to the concentrated liquid to a final concentration of 70%, and then the mixture was allowed to stand at 4℃ for 10 hours of aging; the precipitate was collected by centrifugation, and then redissolved in deionized water, desalted and impurity-removed by nanofiltration membrane with a molecular weight cut-off of 500 Da, and finally the filtrate was spray-dried to obtain theabrownines product.

[0057] A system for the process of the present application was used throughout the production, as shown in Figure 1 , which comprises: The theaflavins production line 1 is sequentially connected by an enzymatic oxidation reaction tank, a solid-liquid separation device, an extraction tank, a concentration unit and a chromatography purification unit; The theabrownines production line 2 is sequentially connected by a fermentation equipment, an extraction tank, an MVR evaporator, an alcohol precipitation tank and a drying equipment; The solid outlet of the solid-liquid separation device is connected with the feeding inlet of the fermentation equipment.

[0058] Example 2

[0059] Step 1. Enzymatic oxidation: 1 kg of Yunnan large-leaf green tea ground to 60 mesh was mixed with 8 L of citric acid-phosphate buffer at pH 5.5 (solid-liquid ratio 1:8), and a polyphenol oxidase and peroxidase complex enzyme source with an enzyme activity of 3000 U / g of tea powder was added, and the reaction was carried out at 40℃ under oxygenated stirring for 4 hours; after the reaction was completed, the reaction liquid was heated to 95℃ for 10 minutes for enzyme inactivation; Step 2. Solid-liquid separation: the enzyme-inactivated reaction liquid was separated by a centrifugal separator to obtain 7.8 L of clear liquid rich in theaflavins and 2.0 kg of wet solid residue (moisture content about 60%); Step 3. Theaflavins purification: the liquid was subjected to three-stage countercurrent extraction with ethyl acetate, and the organic phases were combined and concentrated to dryness under vacuum to obtain theaflavins crude product; the crude product was dissolved in water, pretreated by ultrafiltration membrane, and then loaded onto a macroporous adsorption resin column, and gradient elution was performed with ethanol solution, and the fractions rich in theaflavins were collected, concentrated and dried to obtain theaflavins product; Step 4. Microbial fermentation: 200 mL of Aspergillus corallinus suspension (spore concentration 1×10 8 CFU / mL) was inoculated into the solid residue at an inoculation amount of 15% (v / w), and the mixture was placed in a solid-state fermentation tank at 40℃ and 85% humidity for 10 days; Step 5. Theabrownines purification: after the fermentation was completed, 10 times (v / w) of boiling water was added to the fermented material for extraction for 1 hour, and the extract was obtained by pressure filtration; the extract (solid content concentration 8%) was pumped into an MVR evaporator and concentrated to a solid content concentration of 35% at 65℃; edible alcohol was added to the concentrated liquid to a final concentration of 80%, and the mixture was allowed to stand at 4℃ for 6 hours for aging; the precipitate was collected by centrifugation, and the precipitate was redissolved in deionized water and then subjected to desalting and impurity removal purification using a nanofiltration membrane with a molecular weight cut-off of 200 Da, and finally the filtrate was spray dried to obtain theabrownines product.

[0060] Example 3

[0061] Step 1. Enzymatic oxidation: 1 kg of Fuding white tea pruned branches and leaves was ground to 40 mesh; the ground tea powder was mixed with 15 L of phosphate buffer at pH 4.5 (solid-liquid ratio 1:15), and a polyphenol oxidase and peroxidase complex enzyme source with an enzyme activity of 1000 U / g of tea powder was added, and the reaction was carried out at 30℃ under oxygenated stirring for 4 hours; after the reaction was completed, the reaction liquid was heated to 85℃ and kept for 10 minutes for enzyme inactivation; Step 2. Solid-liquid separation: The reaction solution after enzyme inactivation was separated by vacuum filtration to obtain 9.2 L of clear liquid rich in theaflavins and 2.5 kg of wet solid residue (water content about 70%); Step 3. Purification of theaflavins: The liquid was subjected to three-stage countercurrent extraction with ethyl acetate, and the organic phases were combined and concentrated to dryness under vacuum to obtain theaflavins crude product; the crude product was dissolved in water, pretreated by ultrafiltration membrane, and then loaded onto a macroporous adsorption resin column, which was eluted with a gradient of 30%-70% ethanol solution. The fractions rich in theaflavins were collected, concentrated and dried to obtain theaflavins product; Step 4. Microbial fermentation: 110 mL of Aspergillus pueri suspension (spore concentration 1×10 6 CFU / mL) was inoculated into the solid residue at an inoculation amount of 5% (v / w), and the mixture was fermented in a solid-state fermentation tank at 35°C and 60% humidity for 20 days; Step 5. Purification of theabrownins: After fermentation, 10 times (v / w) of boiling water was added to the fermented material for extraction for 1 hour, and the extract was obtained by pressure filtration; the extract (solid concentration 5%) was pumped into an MVR evaporator and concentrated to a solid concentration of 25% at 50°C; edible alcohol was added to the concentrated liquid to a final concentration of 60%, and the mixture was aged at 4°C for 12 hours; the precipitate was collected by centrifugation, redissolved in deionized water, and then desalted and impurity-removed by nanofiltration membrane with a molecular weight cutoff of 1000 Da. The final filtrate was spray dried to obtain theabrownins product.

[0062] Comparative Example 1 1 kg of Anji white tea was extracted with 60% ethanol by reflux extraction for 2 hours, the extract was concentrated, extracted with ethyl acetate, and then purified by silica gel column chromatography to obtain theaflavins product. The tea leaf residue was boiled with water for extraction for 1 hour, the extract was naturally placed for oxidation and color change for 48 hours, concentrated and alcohol precipitated, and dried to obtain theabrownins product.

[0063] Comparative Example 2 Steps 1 and 2 were the same as in Example 1, Step 3. Purification of theaflavins: The ultrafiltration and resin chromatography steps were omitted, and only ethyl acetate extraction and concentration were used to obtain theaflavins product; Steps 4 and 5 were the same as in Example 1 to obtain theabrownins product.

[0064] Comparative Example 3 Steps 1, 2 and 3 were the same as in Example 1 to obtain theaflavins product; Step 4 was the same as in Example 1; Step 5. Purification of theabrownins: MVR concentration was replaced by traditional vacuum concentration (75°C), and the rest of the operations were the same as in Example 1 to obtain theabrownins product.

[0065] The theaflavins and thearubigins products prepared by the examples and the comparative examples were respectively subjected to performance detection, including: comprehensive utilization rate of raw materials, purity and yield of theaflavins, purity and yield of thearubigins, antioxidant activity (DPPH value), color and solubility stability, organic solvent residue, heavy metals, and microbial limit.

[0066] 1. Comprehensive utilization rate of raw materials Detection method: utilization rate%= (mass of theaflavins dry product + mass of thearubigins dry product) ÷ mass of input tea raw material dry × 100; 0.1 mg analytical balance was used, and the result was calculated on a dry basis.

[0067] 2. Purity and yield of theaflavins Detection method: according to high performance liquid chromatography in GB / T 31740.3-2015; a C18 chromatographic column was used, methanol-water-acetic acid was used as the mobile phase for gradient elution, detection was carried out at a wavelength of 280 nm, and the mass fraction (purity) was calculated by the theaflavin standard curve; the yield was calculated as “mass of theaflavins / mass of raw material dry”.

[0068] 3. Purity and yield of thearubigins Detection method: reference was made to NY / T 3675-2020 “Determination of Thearubigins-Spectrophotometric Method”; after the sample was dissolved in water, colorimetry was carried out at 380 nm, the mass fraction (purity) was calculated by the thearubigin standard curve; and the yield was calculated as “mass of thearubigins / mass of raw material dry”.

[0069] 4. Antioxidant activity Detection method: the sample was reacted with DPPH ethanol solution, the change in absorbance was measured at a wavelength of 517 nm, and the half-inhibitory concentration IC 50 (μg / mL) was calculated, and the smaller the value, the stronger the activity.

[0070] 5. Color and solubility stability Detection method: CIE Lab color difference method was used, the sample was prepared into a 1% water solution, a color difference meter was used for measurement, and ΔE was calculated; the dissolution speed and solution state (whether clear) of the 1% water solution at room temperature were observed.

[0071] 6. Organic solvent residue Detection method: gas chromatography method in GB 5009.262-2020 “Determination of Solvent Residue in Foodstuffs” was used, external standard method was used for quantification; the limit value referred to GB 2760-2024: ethyl acetate ≤ 50 mg / kg, and ethanol was not counted as “processing aid”.

[0072] 7. Heavy metals (As+Pb+Cd) Detection method: inductively coupled plasma mass spectrometry; determination basis GB 2762-2022 plant extract "tea and tea products" limit (Pb≤5.0 mg / kg, As≤2.0 mg / kg, Cd≤1.0 / mg kg).

[0073] 8. Microbial limit Detection method: using plate counting method, determination basis: GB 4789.2-2022 (total number of colonies), GB 4789.15-2016 (mold and yeast), enterprise internal control index: total number of colonies≤1000 CFU / g, total number of mold and yeast≤100 CFU / g.

[0074] 9. Results and analysis Table 1 summarizes the performance test results of each group of products

[0075] Results analysis: According to the above experimental data, the process for continuously extracting high-purity theaflavins and theabrownines from tea raw materials provided by the application has significant and creative technical effects in terms of raw material utilization rate, product purity, biological activity, physicochemical stability, and safety compared to traditional extraction methods.

[0076] In examples 1 to 3, the average comprehensive utilization rate of the raw material reached 36.8%, which was more than 66% higher than that of comparative example 1 (traditional ethanol reflux method, 22.1%). Even without membrane separation and resin refining (comparative example 2), the application still maintained a utilization rate of more than 30%, indicating that the application realized the full-component and high-value utilization of tea raw materials through the coupling of enzymatic oxidation and solid-state fermentation, overcoming the technical defects of residue waste and resource waste in traditional methods.

[0077] The application examples adopted the complete refining route of "enzymatic oxidation + extraction + ultrafiltration + resin chromatography", and the purity of the obtained theaflavins was stably above 20%, with a maximum of 28.5% (determined by HPLC), which was significantly higher than 15.2% of comparative example 1. At the same time, the yield of theaflavins was increased to 10.5%-12.4%, which was about 70% higher than that of the traditional method (6.3%). In comparative example 2, although the yield was close, the purity decreased significantly, indicating that the ultrafiltration and resin chromatography steps played an irreplaceable role in removing impurities and improving purity, reflecting the creative design of the application in separation and purification strategy.

[0078] The theabrownin obtained by the application has a purity of 90% or more, and an average of 93.5% (determined by UV method), which is significantly better than 78.3% of the comparative example 1. Even if the traditional vacuum concentration (75 DEG C) is used instead of the MVR low-temperature concentration (comparative example 3), the purity is reduced to 88.7%, which shows that the MVR low-temperature concentration plays a key role in protecting the heat-sensitive components and maintaining high purity. The purity level meets the strict quality requirements of high-end food, health products and cosmetic raw materials for theabrownin, and has direct application value.

[0079] The theaflavins and theabrownins obtained by the application show stronger antioxidant activity in the DPPH free radical scavenging experiment, and the IC 50 values are as low as 18.2-20.8 μg / mL and 22.5-25.1 μg / mL, which are significantly better than 35.6 μg / mL and 42.3 μg / mL of the comparative example 1. This shows that the application not only improves the purity of the product, but also significantly enhances the functional activity, providing a high-quality raw material basis for the development of downstream high-value-added products.

[0080] The product of the application shows a clear state in a 1% aqueous solution, and the ΔE value is stably between 45-49, which is significantly lower than that of the traditional method (62.3), indicating that the product has stable color and good solubility. The residual amount of organic solvent is less than 20 mg / kg, which is much lower than the national standard limit (50 mg / kg). The content of heavy metals (Pb, As, Cd) is less than 2 mg / kg, which meets the safety requirements of food-grade raw materials. The microbial indicators are also significantly better than the traditional method, with total bacterial count ≤180 CFU / g, mold and yeast ≤50 CFU / g, which shows that the process of the application effectively controls the microbial contamination under the conditions of low temperature, closed and continuous operation.

[0081] In summary, the data in Table 1 fully show that the application successfully realizes the efficient, synchronous and high-purity extraction of theaflavins and theabrownins through the synergistic effect of enzymatic oxidation and microbial fermentation, combined with modern separation technologies such as membrane separation, resin chromatography, MVR low-temperature concentration and nanofiltration desalination, significantly improves the utilization rate of raw materials and the added value of products, and overcomes the key technical problems such as low purity, poor activity, high solvent residue and more waste in the traditional process, and has significant creativity, practicality and industrialization prospect.

Claims

1. A process for extracting theaflavins and thearubigins from tea material, characterized in that, The method comprises the following steps: Step 1. Enzymatic oxidation: mixing tea raw materials with a buffer solution, adding an enzyme source to perform an enzymatic oxidation reaction, and then inactivating the enzyme after the reaction to obtain a reaction liquid; Step 2. Solid-liquid separation: performing solid-liquid separation on the reaction liquid obtained in step 1 to obtain a liquid rich in theaflavins and a solid residue; Step 3. Theaflavins purification: sequentially subjecting the liquid to extraction, concentration and chromatographic purification to obtain a theaflavins product; Step 4. Microbial fermentation: using the solid residue as a fermentation substrate and inoculating microorganisms to perform solid-state fermentation; Step 5. Theabrownines purification: sequentially subjecting the material after fermentation in step 4 to leaching, concentration, alcohol precipitation and drying to obtain a theabrownines product.

2. The process according to claim 1, characterized in that, In step 1: The enzyme source comprises polyphenol oxidase and peroxidase, and the addition amount is 1000-3000 U / g of tea raw materials; The buffer solution is a phosphate or citrate-phosphate buffer solution with a pH of 4.5-5.5, and the solid-liquid ratio of the tea raw materials to the buffer solution is 1:8 to 1:15 (g / mL); The reaction is performed under the conditions of 30-40℃ and oxygenated stirring; The enzyme inactivation treatment is heating at 85-95℃ for 3-10 minutes.

3. The process of claim 1, wherein, The solid-liquid separation in step 2 is achieved by plate-and-frame filter pressing, centrifugation or suction filtration.

4. The process of claim 1, wherein, In step 3: The extraction is performed by multi-stage countercurrent extraction using ethyl acetate; The chromatographic purification comprises: first performing membrane separation by ultrafiltration, and then performing refinement by a macroporous adsorption resin chromatographic column.

5. The process of claim 1, wherein, In step 4: The inoculation is the inoculation of a bacterial suspension of specific microorganisms into the solid residue, the microorganisms in the bacterial suspension being one or more of Aspergillus niger, Aspergillus corntus or Aspergillus puer- rae-montalis; the bacterial suspension being a spore suspension, the spore concentration of which is preferably 1 x 10 6 to 1 x 10 8 CFU / mL; the inoculation ratio being 5% to 15% of the ratio (v / w) of the volume (mL) of the bacterial suspension to the mass (g) of the solid residue on a wet basis.

6. The process of claim 1, wherein, In step 5: The concentration is performed by an MVR mechanical vapor recompression evaporator under the operating conditions of a temperature of 50-65℃, and the leaching liquid with a solid content of 5%-8% is concentrated to 25%-35%; The alcohol precipitation is performed by adding edible alcohol to the concentrated liquid to a final concentration of 60-80% (v / v) and standing for aging at a temperature below 10℃ for 6-12 hours.

7. The process of claim 6, wherein, After the alcohol precipitation, the precipitation is collected by centrifugation, the water phase is redissolved, and then desalination purification is performed by using a nanofiltration membrane with a molecular weight cut-off of 200-1000 Da.

8. The process according to any one of claims 1 to 7, characterized in that, The tea raw materials are green tea, white tea or pruned branches and leaves thereof which are crushed to 40-60 mesh.

9. The process according to any one of claims 1 to 7, characterized in that, The total content of theaflavins in the theaflavins product is ≥20% as determined by HPLC, and the content of theabrownines in the theabrownines product is ≥90% as determined by UV.

10. A system for carrying out the process according to any one of claims 1 to 9, characterized in that It comprises: A theaflavins production line (1) which is sequentially connected by an enzymatic oxidation reaction tank, a solid-liquid separation device, an extraction tank, a concentration unit and a chromatographic purification unit; A theabrownines production line (2) which is sequentially connected by a fermentation equipment, a leaching tank, an MVR evaporator, an alcohol precipitation tank and a drying equipment.

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