阅读说明：本技术 一种酶催化制备茶黄素的方法及其制得的产品 (Method for preparing theaflavin by enzyme catalysis and product prepared by method ) 是由 杨卫国 赵凡伟 闵小华 �金钟 周广勇 管华扬 于 2020-11-23 设计创作，主要内容包括：本发明公开了一种酶催化制备茶黄素的方法及其制得的产品,所述方法包括：步骤S1：将鲜茶叶粉碎,并加入石油醚或乙酸乙酯搅拌均匀,获得混合物；步骤S2：将混合物中的石油醚或乙酸乙酯滤除,获得叶粉；步骤S3：在所述叶粉中,加入茶多酚制品和水,加入乙酸乙酯或乙酸丁酯,获得混合体系；步骤S4：将所述混合体系进行有氧发酵；步骤S5：取发酵液,并进行干燥,获得茶黄素固形物。在鲜茶叶粉加入石油醚或乙酸乙酯脱除咖啡因、茶碱蜡质、叶绿素及非挥发性油脂,以制备含有多酚氧化酶酶系的叶粉,以叶粉为固定化酶催化剂,催化茶多酚制品发酵,制备茶黄素；叶粉中保留了天然固化多酚氧化酶酶系,对酶活性的影响小。(The invention discloses a method for preparing theaflavin by enzyme catalysis and a product prepared by the method, wherein the method comprises the following steps: step S1: crushing fresh tea leaves, adding petroleum ether or ethyl acetate, and uniformly stirring to obtain a mixture; step S2: filtering out petroleum ether or ethyl acetate in the mixture to obtain leaf powder; step S3: adding a tea polyphenol product and water into the leaf powder, and adding ethyl acetate or butyl acetate to obtain a mixed system; step S4: carrying out aerobic fermentation on the mixed system; step S5: taking the fermentation liquor, and drying to obtain theaflavin solid. Adding petroleum ether or ethyl acetate into fresh tea leaf powder to remove caffeine, theophylline wax, chlorophyll and non-volatile oil to prepare leaf powder containing polyphenol oxidase enzyme system, and catalyzing fermentation of tea polyphenol product with the leaf powder as immobilized enzyme catalyst to prepare theaflavin; the leaf powder retains natural immobilized polyphenol oxidase enzyme system, and has small influence on enzyme activity.)

1. A process for the enzymatic preparation of theaflavins, comprising:

step S1: crushing fresh tea leaves, adding petroleum ether or ethyl acetate, and uniformly stirring to obtain a mixture;

step S2: filtering out petroleum ether or ethyl acetate in the mixture to obtain leaf powder;

step S3: adding a tea polyphenol product and water into the leaf powder, and adding ethyl acetate or butyl acetate to obtain a mixed system;

step S4: introducing oxygen into the mixed system for aerobic fermentation;

step S5: taking the fermentation liquor, and drying to obtain theaflavin solid.

2. The enzymatic theaflavin preparation method according to claim 1, wherein in step S1, the ratio of petroleum ether to fresh tea leaves is: 2-30L: 1kg, stirring time 30-90 minutes.

3. The enzymatic theaflavin preparation method according to claim 1, wherein in step S3, the ratio of tea polyphenol product, ethyl acetate, water and fresh tea leaves is: 0.1-1 kg: 2-20L: 0-1.5L: 1 kg.

4. The enzymatic theaflavin preparation process as set forth in claim 1 wherein the fermentation broth is taken and dried by a process comprising:

treating the mixed liquid after aerobic fermentation by a filtering method to obtain fermentation liquid and leaf residues;

washing the leaf residue with ethyl acetate, and filtering to obtain a filtrate solution;

combining the filtrate and the fermentation liquor, and drying under reduced pressure.

5. The enzymatic theaflavin preparation process according to claim 4 further comprising the step of recycling leaf powder: the washed leaf residue is reused as leaf powder for 2-4 times.

6. The enzymatic theaflavin preparation process according to claim 4 further comprising a byproduct extraction process:

mixing the leaf residue with water of 50-70 deg.C, and separating to obtain extractive solution;

drying the extract to obtain black tea extract.

7. The enzymatic theaflavin preparation process of claim 6 further comprising an extract purification process:

distilling the extract under reduced pressure to remove ethyl acetate, filtering, passing through AB-8 macroporous resin column, and collecting effluent liquid on the column;

washing AB-8 macroporous resin column with water, eluting with 20% ethanol water solution, and collecting water eluate and eluate respectively;

and drying the effluent of the upper column to obtain a byproduct tea polysaccharide, drying the water washing liquid to obtain a byproduct caffeine, and drying the eluate to obtain a byproduct theanine.

8. The enzymatic theaflavin production process of claim 1 further comprising a theaflavin solids purification process:

dissolving theaflavin powder with 3% -10% ethanol water solution, filtering, and vacuum drying filtrate to obtain theaflavin finished product.

9. The enzymatic theaflavin preparation process of claim 1 further comprising the step of simultaneously extracting the tea polyphenol preparation:

step S11: withering fresh tea leaves, pulverizing, adding green tea powder, adding petroleum ether and water, stirring at room temperature for 60 min to obtain a mixture,

wherein the weight ratio of the fresh tea leaves to the green tea dust is 0.8-1.2: 1, the weight ratio of petroleum ether, water and green tea dust is 4-10: 0.8-1.2: 1;

step S12: after filtering petroleum ether from the mixture, adding petroleum ether again, stirring at room temperature for 60 minutes, and filtering petroleum ether to obtain a leaf powder mixture;

wherein the weight ratio of the petroleum ether to the green tea dust is 4-10: 1;

step S13: adding ethyl acetate or butyl acetate into the leaf powder mixture to obtain a mixed system;

wherein the weight ratio of the ethyl acetate to the green tea dust is 4-10: 1.

10. The product of the process of any one of claims 1 to 9.

Technical Field

The invention relates to the technical field of tea processing, in particular to a method for preparing theaflavin through enzyme catalysis and a product prepared by the method.

Background

The theaflavin is a main active component in the black tea, and has various excellent pharmacological and health-care effects of diminishing inflammation, inhibiting bacteria, inhibiting viruses, reducing blood fat, losing weight, preventing and treating osteoarthritis, preventing cancers and the like. Because the content of the theaflavin in the black tea is extremely low, the content of the finished product black tea is only 0.5% -2%, and the extraction and purification are difficult, the cost is high, so the black tea is praised as 'tea gold'.

A series of research results show that theaflavins are not only an effective free radical scavenger and antioxidant, but also have various physiological functions of resisting cancer, resisting mutation, resisting bacteria and viruses, improving and treating cardiovascular and cerebrovascular diseases, treating diabetes and the like. Therefore, further research on the mass production and preparation of theaflavins and their biological activities is an important issue in the current research on theaflavins.

The enzymatic oxidation fermentation technology is gradually produced in mass production at home at present, and the technical line mainly comprises: polyphenol Oxidase (PPO) is extracted from natural raw materials such as fresh tea leaves, fruit and vegetable juice and Cladosporium exigucum metabolites, and the substrate tea polyphenol is aerobically fermented to generate theaflavin after proper enrichment, purification and immobilization.

PCT Chinese patent publication No. CN101541184A describes a method for producing a tea product rich in theaflavins: contacting the first material with the second material to form a mixture having a specific ratio of catechins and theaflavins, fermenting the mixture under low temperature oxygenated conditions and supplementing the second material, and after fermentation is complete, recovering and drying from the reaction mixture to obtain a leaf tea product containing a high proportion of theaflavins. Fermenting under the condition of low temperature oxygenation through a mixture of specific catechin proportion and theaflavin proportion.

Chinese patent No. CN1289492C discloses a method for extracting theaflavin: kneading fresh tea leaves, adding water, grinding into slurry, fermenting and oxidizing to obtain a solution containing theaflavin, and carrying out centrifugal filtration, ceramic membrane filtration, solvent extraction or membrane separation on the solution to obtain theaflavin.

A Chinese patent with an authorization publication number of CN102648728B provides an exogenous enzyme method production process of high-theaflavin instant black tea: after tea leaves are extracted by hot water and centrifuged to remove impurities, pear homogenate is added as exogenous enzyme, the mixture is stirred and fermented by slightly acid and temperature control bubbling oxygen, solid-liquid separation of fermentation liquor is carried out, then the fermentation liquor is filtered by a ceramic membrane, and the organic membrane is concentrated and dried to obtain the instant black tea powder with the theaflavin content of 3-4%. However, the theaflavin content of the exogenous enzyme fermentation is low.

The Chinese patent with the publication number of CN101886097B provides a preparation process of high-purity theaflavin: extracting green tea with ethyl acetate, adjusting tea polyphenol substrate composition and concentration, adding enzyme prepared from fresh tea, fermenting under stirring with slightly acidic temperature-controlled bubbling oxygen, separating fermentation liquid, concentrating, washing with water, dissolving, refining with column chromatography resin, concentrating, and drying to obtain high-purity theaflavin. The obtained theaflavin can be used for preparing anti-influenza virus medicine. The process can greatly separate out dark green coke gum generated by copolymerization of chlorophyll caffeine, tea polyphenol and theaflavin during the transfer dissolution, and the yield and the quality of the product are seriously influenced.

Chinese patent with the publication number of CN101565724B introduces a method for the biosynthesis of theaflavin crude extract by using Cladosporium exigua: the method comprises the steps of obtaining a strain of the Mortierella minutissima with high yield of polyphenol oxidase (PPO) by screening a large number of microorganisms, culturing the strain in a culture medium under an optimized fermentation condition to generate the polyphenol oxidase, introducing oxygen into a crude enzyme solution to simulate and oxidize catechin in vitro to prepare theaflavin, and performing membrane filtration and drying to obtain a crude theaflavin extract, wherein the theaflavin content in the crude product is more than 20%.

Chinese patent publication No. CN106957884A discloses a theaflavin product rich in catechins: mixing fully ground tender stem and fresh leaf of tea tree with water saturated ethyl acetate and petroleum ether in a certain proportion, water bath oscillating enzymatic reaction, filtering reaction liquid, concentrating, drying, dissolving and filtering to obtain theaflavin product rich in catechin.

The Chinese patent with the granted publication number of CN101691591B discloses a method for the industrialized preparation of theaflavin: preparing ethyl acetate solution rich in tea polyphenol, crushing fresh tea leaves, mixing with the ethyl acetate solution of tea polyphenol, ventilating and stirring at normal temperature for fermentation to obtain ester phase solution rich in theaflavin, filtering, washing with water to remove impurities, concentrating and dissolving ester phase, centrifuging to remove coked impurities such as chlorophyll and the like, and spray drying to obtain a theaflavin product. The preparation method can separate out a large amount of pyro-colloidal chlorophyll copolymerization impurities after the ester phase is concentrated and dissolved, thereby seriously reducing the yield and quality of the product.

The Chinese patent with the granted publication number of CN101235025B discloses a preparation method of theaflavin: cleaning fresh tea leaf, pulverizing at low temperature, adding biocatalyst comprising leaf juice of fructus litchi, fructus crataegi, fructus kaki, Aloe, and fructus Myrtilli, stirring at slightly acidic temperature, ventilating and fermenting, separating solid and liquid of fermentation liquid, adding protectant into supernatant, vacuum concentrating and drying, or refining by column chromatography, and vacuum concentrating and drying to obtain the final product.

For example, chinese patent No. CN1155717C, discloses a method for producing high-purity theaflavin by catalyzing high-content tea polyphenol with immobilized polyphenol oxidase: comprises immobilizing polyphenol oxidase with embedding material, polymerizing high-content tea polyphenol with immobilized polyphenol oxidase under certain conditions to obtain theaflavin, extracting with solvent, concentrating, and drying to obtain high-purity theaflavin. The immobilized enzyme has higher cost, and the space phase activity expression of the immobilized enzyme can be changed.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a method for preparing theaflavin through enzyme catalysis and a product prepared by the method.

The invention discloses a method for preparing theaflavin through enzyme catalysis, which comprises the following steps: step S1: crushing fresh tea leaves, adding petroleum ether or ethyl acetate, and uniformly stirring to obtain a mixture; step S2: filtering out petroleum ether or ethyl acetate in the mixture to obtain leaf powder; step S3: adding a tea polyphenol product and water into the leaf powder, and adding ethyl acetate or butyl acetate to obtain a mixed system; step S4: introducing oxygen into the mixed system for aerobic fermentation; step S5: taking the fermentation liquor, and drying to obtain theaflavin solid.

Preferably, in step S1, the ratio of petroleum ether to fresh tea leaves is: 2-30L: 1kg, stirring time 30-90 minutes.

Preferably, in step S3, the ratio of the tea polyphenol product, ethyl acetate, water and fresh tea leaves is: 0.1-1 kg: 2-20L: 0-1.5L: 1 kg.

Preferably, the method for taking and drying the fermentation liquor comprises the following steps:

treating the mixed liquid after aerobic fermentation by a filtering method to obtain fermentation liquid and leaf residues;

washing the leaf residue with ethyl acetate, and filtering to obtain a filtrate solution;

combining the filtrate and the fermentation liquor, and drying under reduced pressure.

Preferably, the method of the present invention further comprises a method of recycling leaf powder: the washed leaf residue is used as leaf powder for repeated use for 2-4 times.

Preferably, the method of the present invention further comprises a method of extracting the by-product:

mixing the leaf residue with water of 50-70 deg.C, and separating to obtain extractive solution;

drying the extract to obtain black tea extract.

Preferably, the method of the present invention further comprises a method of purification of the extract:

distilling the extract under reduced pressure to remove ethyl acetate, filtering, passing through AB-8 macroporous resin column, and collecting effluent liquid on the column;

washing AB-8 macroporous resin column with water, eluting with 20% ethanol water solution, and collecting water eluate and eluate respectively;

and drying the effluent of the upper column to obtain a byproduct tea polysaccharide, concentrating and drying the water washing solution to obtain a byproduct caffeine, and drying the eluate to obtain a byproduct theanine.

Preferably, the process of the invention also comprises a process for the purification of theaflavin solids:

dissolving theaflavin powder with 3% -10% ethanol water solution, filtering, and vacuum drying the filtrate to obtain the final product of theaflavin.

Preferably, the method of the present invention further comprises a method for simultaneously extracting a tea polyphenol product:

step S11: withering fresh tea leaves, pulverizing, adding green tea powder, adding petroleum ether and water, stirring at room temperature for 60 min to obtain a mixture,

wherein the weight ratio of the fresh tea leaves to the green tea dust is 0.8-1.2: 1, the weight ratio of petroleum ether, water and green tea dust is 4-10: 0.8-1.2: 1;

step S12: after filtering petroleum ether from the mixture, adding petroleum ether again, stirring at room temperature for 60 minutes, and filtering petroleum ether to obtain a leaf powder mixture;

wherein the weight ratio of the petroleum ether to the green tea dust is 4-10: 1;

step S13: adding ethyl acetate or butyl acetate into the leaf powder mixture to obtain a mixed system;

wherein the weight ratio of the ethyl acetate to the green tea dust is 4-10: 1.

The invention also provides a product prepared by the method.

Compared with the prior art, the invention has the beneficial effects that: adding petroleum ether or ethyl acetate into fresh tea leaf powder to remove caffeine, theophylline wax, chlorophyll and non-volatile oil in the fresh tea leaf to prepare leaf powder containing polyphenol oxidase enzyme system, and catalyzing a tea polyphenol product to ferment and polymerize in a mixed phase of water, ethyl acetate or butyl acetate by using the leaf powder as an immobilized enzyme catalyst to prepare theaflavin; during the preparation process of the leaf powder, a natural solidified polyphenol oxidase enzyme system is reserved, and the influence on the enzyme activity is small; the fermentation post-treatment is simple, the fermentation liquor is directly separated, concentrated, filtered and dried; the product does not contain water-soluble enzyme, so that the theaflavin activity and content in the product are high.

Drawings

FIG. 1 is a flow chart of the enzymatic theaflavin preparation process of the present invention;

FIG. 2 is a schematic structural diagram of theaflavins;

FIG. 3 is a high performance liquid chromatography assay of the product prepared in example 1;

FIG. 4 is a high performance liquid chromatography assay of the product prepared in comparative example 1;

FIG. 5 is a high performance liquid chromatography assay of the product prepared in example 2;

FIG. 6 is a flow chart of a process for simultaneous extraction of a tea polyphenol product;

FIG. 7 is a HPLC peak plot of TF standard;

FIG. 8 is a HPLC peak plot of a TF-3-G standard;

FIG. 9 is a HPLC peak plot of TF-3' -G standard;

FIG. 10 is a HPLC peak profile of a TFDG standard.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention is described in further detail below with reference to the attached drawing figures:

an enzymatic theaflavin preparation process, as shown in figure 1, comprising:

step S1: pulverizing fresh folium Camelliae sinensis, adding petroleum ether or ethyl acetate, and stirring to obtain mixture.

Fresh tea leaves are used for the production of enzymes, petroleum ether is a light petroleum product, a mixture of hydrocarbons of low relative molecular mass (the main components are pentane and hexane), and is insoluble in water. The petroleum ether and ethyl acetate are used for removing caffeine, theophylline wax, chlorophyll and non-volatile oil in fresh tea.

Step S2: the petroleum ether or ethyl acetate in the mixture was filtered off to obtain leaf powder.

Wherein the leaf powder contains a naturally immobilized polyphenol oxidase enzyme system.

Step S3: adding tea polyphenol product and water into the leaf powder, and adding ethyl acetate or butyl acetate to obtain a multiphase mixed system.

The water phase is used for dissolving tea polyphenol and catechin substances, then the tea polyphenol and the catechin substances are diffused into the ethyl acetate phase and are polymerized and catalyzed to be theaflavin substances on the enzyme surface of the leaf powder, and the theaflavin substances are dissolved again and transferred into the ethyl acetate phase and the activity of the enzyme is recovered. Meanwhile, water forms a water film on the surface of the leaf powder, and polyphenol oxidase is adsorbed on the active groups of the wood structure of the leaf powder and is fully developed in the water film. The wet leaf powder is in a fully dispersed state in ethyl acetate or butyl acetate phase, and the specific surface area is obtained as large as possible; the theaflavin is dissolved in the ethyl acetate and is relatively stable even in a low-concentration state, the theaflavin ethyl acetate solution with the concentration of 1.5% can not be degraded by more than 2% after being placed in a dark place for 24 hours at room temperature, and the requirements of various working procedures on the operation time in the production process can be met.

The butyl acetate is an edible spice which is allowed to be used in the national standard GB2760, is widely used for preparing essences such as apricot, banana, pineapple, pear and the like, and has low toxicity; the butyl acetate has a higher boiling point of 126.5 ℃, has little loss during aerobic fermentation by introducing oxygen, is far lower than the loss of ethyl acetate, can effectively reduce the cost, has larger difference between the boiling points of the butyl acetate and the petroleum ether, and is easy to fractionate the mixed solvent.

Step S4: and carrying out aerobic fermentation on the mixed system. Oxygen can be blown into the mixture system, and the enzymatic oxidative fermentation can be stirred at room temperature or at the low temperature of 0-20 ℃.

Step S5: taking the fermentation liquor, and drying to obtain theaflavin solid. The theaflavin solid is brownish red.

Adding petroleum ether or ethyl acetate into fresh tea leaf powder to remove caffeine, theophylline wax, chlorophyll and non-volatile oil in the fresh tea leaf to prepare leaf powder containing polyphenol oxidase enzyme system, and catalyzing tea polyphenol fermentation polymerization in a mixed phase of water phase, ethyl acetate or butyl acetate by using the leaf powder as an immobilized enzyme catalyst to prepare theaflavin; in the preparation process of the leaf powder, a natural solidified polyphenol oxidase enzyme system is reserved, the influence on the enzyme activity is small, and the composition of theaflavin substances in a fermentation product is similar to that in natural black tea. The fermentation post-treatment is simple, and fermentation liquor is directly separated, concentrated, filtered and dried; the product does not contain water-soluble enzyme, so that the theaflavin activity and content in the product are high.

Wherein the structure of theaflavin is shown in figure 2, the theaflavin component comprises Theaflavin (TF), theaflavin monoester (TF-3-G or TF-3' -G), and theaflavin diester (TFDG), and the original reactants of theaflavin are shown in the following table:

theaflavin component name	R1	R2	Starting reactants
				TF	H	H	EGC+EC
TF-3-G	gallate	H	EGCG+EC
				TF-3'-G	H	gallate	EGC+ECG
TFDG	gallate	gallate	EGCG+ECG

Wherein EGC is epigallocatechin, EGCG is epigallocatechin gallate, EC is epicatechin, ECG is epicatechin gallate, gallote is gallate, and H is hydrogen.

In step S1, the ratio of petroleum ether to fresh tea leaves is: 2-30L: 1kg, stirring time 30-90 minutes.

In step S3, the ratio of the tea polyphenol product, ethyl acetate, water and fresh tea leaves is: 0.1-1 kg: 2-20L: 0-1.5L: 1 kg.

In step S5, the method of taking and drying the fermentation broth includes:

step S101: the mixed liquid after aerobic fermentation is treated by a filtering method to obtain fermentation liquid and leaf residues.

Step S102: the leaf residue was washed with ethyl acetate and filtered to obtain a filtrate.

Step S103: combining the filtrate and the fermentation liquor, and drying under reduced pressure.

The filtrate liquid is obtained by washing the leaf residue and filtering, so that the recovery efficiency of theaflavin is improved.

The method of the present invention may further comprise a method of recycling leaf powder: the washed leaf residue is reused as leaf powder for 2-4 times.

The process of the invention may also include a process for extracting by-products:

mixing the leaf residue with water of 50-70 deg.C, and separating to obtain extractive solution; drying the extract to obtain black tea extract.

The substances contained in the leaf residue are extracted by hot water to extract a byproduct black tea extract which is rich in theanine, caffeine and tea polysaccharide.

Wherein, in extracting the by-product, the extract can be further purified:

step S111: and distilling the extract under reduced pressure to remove ethyl acetate, filtering, passing through an AB-8 macroporous resin column, and collecting effluent liquid on the column.

Step S112: washing AB-8 macroporous resin column with water, eluting with 20% ethanol water solution, and collecting water eluate and eluate respectively.

Step S114: and drying the effluent of the upper column to obtain a byproduct tea polysaccharide, drying the water washing liquid to obtain a byproduct caffeine, and drying the eluate to obtain a byproduct theanine.

The method of the invention also comprises a method for purifying theaflavin solid:

step S6: dissolving theaflavin powder with 3% -10% ethanol water solution, filtering, and vacuum drying filtrate to obtain theaflavin finished product.

In one embodiment, the finished theaflavin product is prepared to have a theaflavin content of greater than 40% as a powdered finished product.

The tea polyphenol product is used as main substrate for theaflavin fermentation, and can be purchased from market, or separated from green tea, such as green tea obtained by water extraction. As shown in fig. 6, the present invention also includes a method for simultaneously extracting a tea polyphenol product:

step S11: withering fresh tea leaves, crushing, adding green tea dust, adding petroleum ether and water, and stirring at room temperature for 60 minutes to obtain a mixture, wherein the weight ratio of the fresh tea leaves to the green tea dust is 0.8-1.2: 1, the weight ratio of petroleum ether, water and green tea dust is 4-10: 0.8-1.2: 1.

the green tea dust contains rich tea polyphenols, and petroleum ether and water are added simultaneously with fresh tea powder for removing caffeine, theophylline wax, chlorophyll and non-volatile oil in fresh tea powder and green tea dust.

Step S12: after filtering petroleum ether from the mixture, adding petroleum ether again, stirring at room temperature for 60 minutes, and filtering petroleum ether to obtain a leaf powder mixture; wherein the weight ratio of the petroleum ether to the green tea dust is 4-10: 1. Adding petroleum ether again to further remove caffeine, theophylline wax, chlorophyll and non-volatile oil.

Step S13: adding ethyl acetate or butyl acetate into the leaf powder mixture to obtain a mixed system; wherein the weight ratio of the ethyl acetate to the green tea dust is 4-10: 1.

The mixed system is subjected to aerobic fermentation in step S4.

Example 1

Preparing leaf powder: 100 kg of fresh tea leaves (picked in 8 months in Jiangsu Wuxi Mashan) picked freshly are withered, then are cut and crushed, are put into a temperature-controlled stainless steel extraction kettle with a sieve plate at the bottom and a side filter outlet, 800 liters of petroleum ether are added, and are stirred for 60 minutes at room temperature, a dark green petroleum ether solution is filtered, and petroleum ether is recovered.

Aerobic fermentation: adding 700L ethyl acetate, 20 kg green tea water extract with tea polyphenol content of 35% and 15L water into extraction kettle, blowing oxygen at room temperature or 0-20 deg.C, stirring, and fermenting. And detecting the absorbance change of the fermentation liquor at 380 nm by using an ultraviolet spectrophotometer, stopping fermentation when the absorbance of A380 of the fermentation liquor reaches a peak value and begins to go flat, and filtering to obtain the fermentation liquor of leaf residues and orange red.

Preparing theaflavin solid: the leaf residues are evenly stirred and washed twice by 1000 liters of ethyl acetate, filtered and collected with orange-red washing liquid. And combining the fermentation liquor and the washing liquid, and concentrating under reduced pressure until the fermentation liquor and the washing liquid are dried to obtain a brownish red theaflavin solid.

And (3) purifying theaflavin: adding 60 kg of 5% ethanol aqueous solution into the solid, stirring to fully dissolve the solid, carrying out suction filtration or filter pressing or centrifugal filtration on the solution, concentrating the filtrate to 25-50% of solid content, drying by using a continuous vacuum belt dryer to obtain 6.9 kg of orange red powder, and detecting the theaflavin content by High Performance Liquid Chromatography (HPLC) to be 45.2% as shown in figure 3.

Recycling leaf residues: the leaf residue washed by ethyl acetate can be reused, 600 liters of butyl acetate, 20 kilograms of green tea aqueous extract with 35 percent of tea polyphenol content and 15 liters of water are added, oxygen is blown in at room temperature or low temperature and stirred for fermentation, an ultraviolet spectrophotometer is used for detecting the absorbance change of the fermentation liquor at 380 nanometers, the fermentation is stopped when the A380 absorbance of the fermentation liquor reaches the peak value and begins to go around, and the leaf residue and orange-red fermentation liquor are obtained by filtering. And uniformly mixing and washing the leaf residues twice by using 1000 liters of butyl acetate, filtering and collecting orange-red washing liquid. And combining the fermentation liquor and the washing liquid, and concentrating under reduced pressure until the fermentation liquor and the washing liquid are dried to obtain the brownish red theaflavin solid. Adding 60 kg of 5% ethanol aqueous solution into the theaflavin solid, stirring for full dissolution, performing suction filtration or filter pressing or centrifugal filtration, concentrating the filtrate to 25% -50% solid content, and drying by using a continuous vacuum belt dryer to obtain 6.8 kg of orange red powder, wherein the theaflavin content is 43.7% by High Performance Liquid Chromatography (HPLC).

And (3) extracting by-products: adding 800 liters of hot water with the temperature of 70 ℃ into the leaf residues, stirring and extracting for 30 minutes, separating to obtain an extracting solution, evaporating the extracting solution under reduced pressure to remove ethyl acetate or butyl acetate, filtering, then putting the extracting solution on an AB-8 macroporous resin column, collecting effluent liquid on the column, washing the filtrate on the column with water in an amount which is 5 times the column volume, eluting the filtrate with 20 percent ethanol in an amount which is 3 times the column volume, and respectively collecting water washing liquid and eluent passing through the AB-8 macroporous resin column. Concentrating and drying the effluent to obtain a byproduct tea polysaccharide, concentrating and drying the byproduct caffeine in the water washing solution, concentrating the eluate, standing for crystallization, filtering, and drying to obtain the byproduct theanine.

Comparative example 1

Preparing fresh tea powder: 100 kg of fresh tea leaves (picked in 8 months in Jiangsu Wuxi Mashan mountain) which are picked freshly are withered, cut into pieces and crushed, and then put into a stainless steel extraction kettle with a sieve plate at the bottom and a side filter outlet.

Aerobic fermentation: adding 600 liters of ethyl acetate, 20 kilograms of green tea aqueous extract with the tea polyphenol content of 35 percent and 15 kilograms of water into an extraction kettle, blowing oxygen into the extraction kettle at room temperature or low temperature, stirring and fermenting, detecting the absorbance change of fermentation liquor at 380 nanometers by using an ultraviolet spectrophotometer, stopping fermentation when the absorbance of A380 of the fermentation liquor reaches the peak value and begins to go around, filtering and collecting dark green fermentation liquor.

Preparing theaflavin solid: the leaf residues are evenly stirred and washed twice by 1000 liters of ethyl acetate, and green washing liquid is collected after filtration. And combining the fermentation liquor and the washing liquid, and concentrating under reduced pressure to dry to obtain dark green theaflavin solid.

And (3) purifying theaflavin: adding 80 kg of 5% ethanol aqueous solution into theaflavin solid, stirring to fully dissolve the solid to obtain a yellow green turbid solution containing a large amount of suspended colloidal chlorophyll, performing sedimentation type centrifugation, collecting supernatant, concentrating the supernatant to a solid content of 25% -50%, drying or spray drying by using a continuous vacuum belt drier to obtain 3.1 kg of orange red powder, detecting the theaflavin content by High Performance Liquid Chromatography (HPLC) to 36.7%, and detecting the HPLC to be shown in figure 4.

Example 2

Preparing leaf powder: 100 kg of fresh tea leaves (picked in 8 months in Jiangsu Wuxi Mashan) picked freshly are withered, cut into pieces, crushed, put into a stainless steel extraction kettle with a sieve plate at the bottom and a side filter outlet, added with 800 liters of petroleum ether, stirred for 60 minutes at room temperature, filtered to remove dark green petroleum ether solution and recovered with petroleum ether.

Aerobic fermentation: adding ethyl acetate 600L, 10 kg of low-caffeine green tea extract (caffeine content is less than 1%) containing 85% of tea polyphenols and water 8L into the extraction kettle, blowing oxygen gas at room temperature or low temperature, stirring, fermenting, detecting absorbance change of the fermentation liquor at 380 nm with an ultraviolet spectrophotometer, stopping fermentation when A380 absorbance of the fermentation liquor reaches the peak value and starts to go, filtering, and collecting orange red fermentation liquor.

Preparing theaflavin solid: the leaf residues are evenly stirred and washed twice by 800 liters of ethyl acetate, and orange-red washing liquid is collected after filtration. And combining the fermentation liquor and the washing liquid, and concentrating under reduced pressure to dryness to obtain a brownish red theaflavin solid.

And (3) purifying theaflavin: adding 100 liters of 5 percent ethanol aqueous solution into the solid, stirring to fully dissolve the solid, carrying out suction filtration or filter pressing or centrifugal filtration, concentrating the filtrate to 25 to 50 percent of solid content, drying or spray drying by using a continuous vacuum belt dryer to obtain 10.2 kilograms of orange red powder, detecting the theaflavin content by High Performance Liquid Chromatography (HPLC) to 51.6 percent, and the HPLC detection chart is shown in figure 5.

Recycling leaf residues: the leaf residue after ethyl acetate washing can be reused, 600 liters of ethyl acetate, 10 kilograms of low-caffeine green tea extract with 85 percent of tea polyphenol content (the caffeine content is less than 1 percent) and 6 liters of water are added, oxygen is blown in at room temperature or low temperature and stirred for fermentation, an ultraviolet spectrophotometer is used for detecting the absorbance change of the fermentation solution at 380 nm, the fermentation is stopped when the A380 absorbance of the fermentation solution reaches the peak value and starts to go away, and orange-red fermentation solution is filtered and collected. The leaf residues are evenly stirred and washed twice by 800 liters of ethyl acetate, filtered and collected with orange-red washing liquid. And combining the fermentation liquor and the washing liquid, and concentrating under reduced pressure until the fermentation liquor and the washing liquid are dried to obtain a brownish red theaflavin solid. Adding 100 liters of 5 percent ethanol aqueous solution into the solid matter, stirring to fully dissolve the solid matter, carrying out suction filtration or filter pressing or centrifugal filtration, concentrating the filtrate to 25 to 50 percent of solid content, drying or spray drying by using a continuous vacuum belt dryer to obtain 9.2 kilograms of orange red powder, and detecting the theaflavin content by High Performance Liquid Chromatography (HPLC) to be 48.5 percent.

The leaf residue after ethyl acetate washing can be reused, 600 liters of ethyl acetate, 9 kilograms of low-caffeine green tea extract (the caffeine content is less than 1 percent) with the tea polyphenol content of 85 percent and 5 liters of water are added, oxygen is blown in at room temperature or low temperature, stirring and fermentation are carried out, an ultraviolet spectrophotometer is used for detecting the absorbance change of the fermentation liquor at 380 nanometers, the fermentation is stopped when the A380 absorbance of the fermentation liquor reaches the peak value and begins to go, and orange-red fermentation liquor is filtered and collected. The leaf residues are stirred and washed twice with 900 liters of ethyl acetate, filtered and collected with orange-red washing liquid. Combining the fermentation liquor and the washing liquid, and concentrating under reduced pressure to dryness to obtain a red-brown solid. Adding 90 liters of 5 percent ethanol aqueous solution into the solid matter, stirring to fully dissolve the solid matter, carrying out suction filtration or filter pressing or centrifugal filtration, concentrating the filtrate to 25 to 50 percent of solid content, drying or spray drying by using a continuous vacuum belt dryer to obtain 8.7 kilograms of orange red powder, and detecting the theaflavin content by using a High Performance Liquid Chromatography (HPLC) to be 47.3 percent.

And (3) extracting by-products: adding 800 liters of hot water with the temperature of 70 ℃ into the leaf residues, stirring and extracting for 30 minutes, separating to obtain an extracting solution, evaporating ethyl acetate from the extracting solution under reduced pressure, filtering, then putting the extracting solution on an AB-8 macroporous resin column, collecting effluent liquid on the column, washing the filtrate on the column with water in 5 times of the column volume, eluting the filtrate with 20 percent ethanol in 3 times of the column volume, and respectively collecting water washing liquid and eluent passing through the AB-8 macroporous resin column. Concentrating and drying the effluent liquid on the column to obtain tea polysaccharide, concentrating and drying the water washing liquid to be rich in caffeine, concentrating the eluate, standing for crystallization, filtering, and drying to obtain theanine.

Comparative example 2

Preparing fresh tea powder: 100 kg of fresh tea leaves (picked in 8 months in Jiangsu Wuxi Mashan mountain) which are picked freshly are withered, cut into pieces and crushed, and then put into a stainless steel extraction kettle with a sieve plate at the bottom and a side filter outlet.

Aerobic fermentation: adding ethyl acetate 600L, low-caffeine green tea extract (caffeine content is less than 1%) containing tea polyphenols 10 kg, and water 8 kg into the extraction kettle, blowing oxygen at room temperature or low temperature, stirring, fermenting, detecting absorbance change of fermentation liquor at 380 nm with ultraviolet spectrophotometer, stopping fermentation when A380 absorbance of the fermentation liquor reaches peak value and begins to go, filtering, and collecting dark green fermentation liquor.

Preparing theaflavin solid: the leaf residues are evenly stirred and washed twice by 800 liters of ethyl acetate, and green washing liquid is collected after filtration. And combining the fermentation liquor and the washing liquid, and concentrating under reduced pressure to dry to obtain dark green theaflavin solid.

And (3) purifying theaflavin: adding 100 kg of 5% ethanol aqueous solution into the solid, stirring to fully dissolve the solid to obtain a yellow-green turbid solution containing a large amount of suspended colloidal chlorophyll, performing sedimentation type centrifugation, concentrating the supernatant to 25-50% of solid content, and drying or spray drying by using a continuous vacuum belt drier to obtain 4.8 kg of orange red powder, wherein the content of theaflavin is detected by High Performance Liquid Chromatography (HPLC) to be 40.1%.

Example 3

Preparing leaf powder: 100 kg of fresh tea leaves (picked in 8 months of Jiangsu Wuxi Mashan) picked freshly are withered, cut into pieces and crushed, put into a stainless steel extraction kettle with a sieve plate at the bottom and a side filter outlet, then 100 kg of raw material green tea dust for extracting tea polyphenol is added, 800 kg of petroleum ether and 100 kg of water are added, the mixture is stirred for 60 minutes at room temperature, and a dark green petroleum ether solution is filtered and recovered. Adding 800L petroleum ether into the extraction kettle, stirring at room temperature for 60 min, filtering to remove light green petroleum ether solution, and recovering petroleum ether.

Aerobic fermentation: adding 800 kg ethyl acetate into the extraction kettle, blowing oxygen at room temperature or low temperature of 0-20 ℃, stirring and fermenting, detecting the absorbance change of the fermentation liquor at 380 nm by using an ultraviolet spectrophotometer, and stopping fermentation when the absorbance of A380 of the fermentation liquor reaches the peak value and begins to go flat.

Preparing theaflavin solid: filtering and collecting orange red fermentation liquor, uniformly stirring and washing the leaf residues twice by using 1500 kilograms of ethyl acetate, filtering and collecting orange red washing liquor, combining the fermentation liquor and the washing liquor, and concentrating under reduced pressure until the orange red theaflavin solid is dried.

And (3) purifying theaflavin: adding 100 kilograms of 5 percent ethanol aqueous solution into the solid matter, stirring to fully dissolve the solid matter, carrying out suction filtration or filter pressing or centrifugal filtration, concentrating the filtrate to 25 to 50 percent of solid content, drying by using a continuous vacuum belt type drying machine set to obtain 10.2 kilograms of orange red powder, and detecting the content of theaflavin by using High Performance Liquid Chromatography (HPLC) to be 45.1 percent.

And (3) extracting by-products: adding 800 liters of hot water with the temperature of 70 ℃ into the leaf residues, stirring and extracting for 30 minutes, separating to obtain an extracting solution, evaporating ethyl acetate from the extracting solution under reduced pressure, filtering, then putting the extracting solution on an AB-8 macroporous resin column, collecting effluent liquid on the column, washing the filtrate on the column with water in 5 times of the column volume, eluting the filtrate with 20 percent ethanol in 3 times of the column volume, and respectively collecting water washing liquid and eluent passing through the AB-8 macroporous resin column. Concentrating and drying the effluent liquid on the column to obtain tea polysaccharide, concentrating and drying the water washing liquid to be rich in caffeine, concentrating the eluate, standing for crystallization, filtering, and drying to obtain theanine.

	Enzyme	Tea polyphenol product	Fermentation yield	Theaflavin content
					Example 1	Leaf powder	Green tea water extract	6.9 kg	45.20％
Comparative example 1	Fresh tea powder	Green tea water extract	3.1 kg	36.70％
					Example 2	Leaf powder	Low caffeine green tea extract	10.2 kg	51.60％
Comparative example 2	Fresh tea powder	Low caffeine green tea extract	4.8 kg	40.10％
					Example 3	Leaf powder	Mixing green tea powder and fresh tea powder to prepare leaf powder	10.2 kg	45.10％

As shown in the table, the fermentation yield of examples 1-3 is significantly improved and the theaflavin content is significantly improved compared with comparative examples 1 and 2; compared with the example 1, the fermentation yield is obviously improved, and the theaflavin content is obviously improved in the example 2.

Wherein, in the detection, the following high performance liquid chromatograph is adopted: shimadzu LC-10ATVP system controller, LC-10ATVP binary pump, SPD-M20A diode array detector, Shimadzu LC-Solution chromatography workstation; chromatographic analysis parameters: the chromatographic column was Hypersil BDS (C18,5u,4.6X250mm), mobile phase A was 2% acetic acid, mobile phase B was acetonitrile-ethyl acetate (volume ratio 21:3), gradient elution was performed, mobile phase B was changed from 18% linear gradient to 30% in 30 minutes, flow rate was 0.9ml/min, column temperature was 40 ℃, detection wavelength was 280nm, and sample injection was 10. mu.l.

As shown in FIGS. 7-10, they are HPLC peak profiles of theaflavin component standard, and are respectively HPLC peak profiles of TF standard, TF-3-G standard, TF-3' -G standard and TFDG standard. As shown in FIGS. 3-5, catechins eluted in the form of broad and unresolved peaks at the beginning of the chromatography, while theaflavins eluted in 15-30 minutes. According to the invention, a standard calibration curve is constructed by a theaflavin standard substance, a theaflavin peak is obtained by taking the calibration curve as reference, the peak area is calculated, and the content of theaflavin is calculated according to the peak area, and as can be seen from fig. 3-5, the components of the theaflavin of the products prepared in example 1 and example 2 are similar to those of the natural fermentation comparative example 1.

The invention also provides a product prepared by the method, which comprises theaflavin and byproducts.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.