CN114807264A - Method for preparing theaflavin by using exogenous enzyme and product prepared by method - Google Patents

Abstract

The invention discloses a method for preparing theaflavin by using exogenous enzyme and a product prepared by the method, wherein the method comprises the following steps: cleaning agricultural products rich in polyphenol oxidase, airing, and freezing to break the wall; crushing the frozen wall-broken agricultural products to obtain exogenous enzymes; adding exogenous enzyme into tea polyphenol product, adding ethyl acetate to form a multiphase fermentation system, and performing aerobic fermentation to obtain fermentation product; and (3) taking the fermentation liquor from the fermentation product, drying to obtain a theaflavin crude product, and separating and purifying the theaflavin crude product by using macroporous resin and an organic roll-type nanofiltration membrane to obtain high-content theaflavin. In the multiphase fermentation system, polyphenol oxidase is soluble in water but not soluble in ester, tea polyphenol and catechin are dissolved out from a solid phase by water, extracted into an ester phase, catalyzed into theaflavin substances on the surface of exogenous enzyme, and extracted into the ester phase to form a relatively stable theaflavin ester phase solution. The agricultural products provide extensive sources of polyphenol oxidase, and exogenous polyphenol oxidase can be obtained all the year round.

Description

Method for preparing theaflavin by using exogenous enzyme and product prepared by method

Technical Field

The invention relates to the technical field of tea processing, in particular to a method for preparing theaflavin by using exogenous enzyme 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 theaflavin in black tea is very low, the content of finished 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'.

Therefore, theaflavin is taken as the key point of new medicine and industrialized research by the national 'nine five' science and technology plan and is listed as the '1035' engineering project which is important in the national science and commission; the research on the theaflavins in foreign countries is earlier, more extensive and deeper, and the research on the human health of the black tea is carried out around, and the research on the human health of the black tea is initiated by the world Food and Agriculture Organization (FAO) in 1995 and is carried out jointly in the United kingdom, the United states and Canada. 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, laccase and Cladosporium exigucum metabolite, 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 controlled 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 with a certain proportion, performing water bath oscillation enzymatic reaction, filtering the reaction solution, 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 an ethyl acetate solution rich in tea polyphenol, crushing fresh tea leaves, mixing the crushed tea leaves with the ethyl acetate solution of the tea polyphenol, ventilating and stirring at normal temperature for fermentation to obtain an ester phase solution rich in theaflavin, filtering, washing with water for removing impurities, concentrating and dissolving the ester phase, centrifuging to remove precipitated coked impurities such as chlorophyll, and performing 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 using immobilized polyphenol oxidase to catalyze high-content tea polyphenol: comprises immobilizing polyphenol oxidase with embedding material, polymerizing high-content tea polyphenols 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 by using exogenous enzyme and a prepared product thereof.

The invention discloses a method for preparing theaflavin by using exogenous enzyme, which comprises the following steps: cleaning agricultural products rich in polyphenol oxidase PPO, airing, and freezing for wall breaking; crushing the frozen agricultural products to obtain exogenous enzyme; adding the exogenous enzyme into a tea polyphenol product, adding ethyl acetate or butyl acetate to form a multiphase fermentation system, and carrying out aerobic stirring fermentation to obtain a fermentation product; and taking ester phase fermentation liquor from the fermentation product, and purifying the fermentation liquor to obtain theaflavin.

Preferably, the agricultural product comprises a fruit, vegetable, rhizome or mushroom having a polyphenol oxidase enzyme.

Preferably, the agricultural product comprises one or a combination of the following: apple, pear, banana, oyster mushroom, needle mushroom, straw mushroom, potato, sweet potato, yam and lotus root.

Preferably, the weight ratio of the exogenous enzyme to the ethyl acetate to the tea polyphenol product is 10-25:100: 1-10.

Preferably, the tea polyphenol product is green tea extract with the tea polyphenol content of 35-99%.

Preferably, the method for taking the fermentation liquid from the fermentation product comprises the following steps: the method for taking fermentation liquor from fermentation products comprises the following steps: filtering the fermentation product to obtain fermentation liquor and filter residue; washing the filter residue with ethyl acetate or butyl acetate to obtain a washing liquid; the wash solution is combined into the fermentation broth.

Preferably, the invention also comprises a method for preparing wine by using filter residue: adding tea polyphenol product and ethyl acetate or butyl acetate into the filter residue, and performing aerobic fermentation again; filtering the secondary fermentation product to obtain secondary filter residue and secondary fermentation liquid; purifying the secondary fermentation liquor to obtain theaflavin; adding water with the volume of 1-2 times into the secondary filter residue, and introducing low-pressure water vapor to remove ethyl acetate by evaporation to obtain a brewing raw material; after the wine brewing raw material is gelatinized, wine making yeast is added for fermentation, and the fruit wine is obtained.

Preferably, the method for purifying the fermentation broth comprises: evaporating the fermentation liquor under reduced pressure to obtain crude theaflavin solid; dissolving the crude theaflavin solid with water, and filtering to obtain filtrate; purifying the filtrate with macroporous resin column, and concentrating with organic rolled nanofiltration membrane; concentrating, and drying at low temperature under reduced pressure to obtain high content theaflavin.

Preferably, the method for purification of the fermentation broth comprises:

preparing the solid into 3-8% aqueous solution, and filtering to obtain filtrate;

adsorbing and purifying the filtrate by using an AB-8 macroporous resin column, wherein the flow rate of the filtrate on the column is 1 time of the column volume/hour, and the column loading amount of solid matters is as follows: the weight ratio of the AB-8 macroporous resin is 1:10, and the mixture is placed on a column and then is kept stand for 1 to 2 hours;

washing the AB-8 macroporous resin column with water of 4-8 times of column volume;

eluting tea polyphenol on the macroporous resin column by using 30% ethanol, wherein the elution volume of the 30% ethanol is 5-15 times of the column volume to obtain tea polyphenol eluent;

concentrating the tea polyphenol eluate by an organic roll type nanofiltration membrane to obtain 30% ethanol filtrate and trapped fluid, wherein the 30% ethanol filtrate can be reused for tea polyphenol elution, and the trapped fluid is dried under reduced pressure to obtain a tea polyphenol compound product;

eluting theaflavin with 70% ethanol in macroporous resin column with 70% ethanol volume of 3-10 times of column volume to obtain theaflavin eluate;

concentrating the eluent by an organic roll-type nanofiltration membrane until the solid content is 25-50%, and simultaneously obtaining 70% ethanol;

drying the concentrated solution in a continuous vacuum belt dryer at low temperature and low pressure to obtain high-content theaflavin solid.

The invention also provides a product prepared by the method.

Compared with the prior art, the invention has the beneficial effects that: 1. compared with the method for providing polyphenol oxidase by fresh tea leaves, the method has the advantages that the source of agricultural products containing polyphenol oxidase is wider, and cheap exogenous polyphenol oxidase can be obtained all the year round. 2. The ester-water-solid multi-phase fermentation system can more stably maintain the enzyme activity of the polyphenol oxidase PPO and more efficiently promote the generation of theaflavin. 3. The organic roll-type nanofiltration membrane non-phase change concentration technology is combined with the macroporous resin separation technology, so that the theaflavin refining process has greater economic advantages.

Drawings

FIG. 1 is a flow chart of a process for preparing theaflavins with exogenous enzymes in accordance with the present invention;

FIG. 2 is a schematic structural diagram of theaflavins;

FIG. 3 is a process flow diagram of an embodiment;

FIG. 4 product profile of example 1;

FIG. 5 product profile of example 2;

FIG. 6 product profile of example 3;

FIG. 7 product profile of example 4;

FIG. 8 product profile of example 5;

FIG. 9 is a HPLC peak plot of TF standard;

FIG. 10 is a HPLC peak plot of a TF-3-G standard;

FIG. 11 is a HPLC peak plot of TF-3' -G standard;

FIG. 12 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 obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

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

a method for preparing theaflavin by exogenous enzyme, as shown in figure 1, comprises:

step 101: cleaning and airing the agricultural products rich in polyphenol oxidase PPO, and freezing and breaking the walls.

The agricultural products such as fruits, vegetables, rhizomes and mushrooms which are rich in the polyphenol oxidase PPO are rich in the polyphenol oxidase, meanwhile, the edible safety of the agricultural products is high, the enzyme activity of the polyphenol oxidase (PPO) can be further improved by properly airing and withering, and the moisture content of the aired agricultural products is 30-80%. Examples of agricultural products that can be used include, but are not limited to, apples, pears, bananas, oyster mushrooms, needle mushrooms, straw mushrooms, potatoes, sweet potatoes, yams, and lotus roots.

Step 102: and crushing the frozen wall-broken agricultural products to obtain the exogenous enzyme. Freezing to make water in agricultural product cells coalesce into ice crystals to destroy cell walls, increase permeability of the cell walls and release polyphenol oxidase PPO enzyme in the cells;

step 103: and adding the exogenous enzyme into the tea polyphenol product, adding ethyl acetate or butyl acetate to form a multiphase fermentation system, and carrying out aerobic fermentation to obtain a fermentation product. Wherein the tea polyphenol product can be green tea extract with tea polyphenol content of 35-99%, and the tea polyphenol product contains polymerized theaflavin and catechin.

In a multi-phase fermentation system consisting of ester-water-solid, polyphenol oxidase is dissolved in a water phase but not in an ester phase, tea polyphenol and catechin molecules are dissolved out of the solid phase by water, extracted into the ester phase, adsorbed and enriched on the surface of exogenous enzyme and polymerized into theaflavin, and the theaflavin is extracted into the ester phase again and the activity of the enzyme is recovered. The existence of the ester phase reduces the polyphenol concentration in the water phase, so that the polyphenol oxidase can continuously keep high activity; at the same time, the ester phase makes the theaflavins dissolved therein more stable. The theaflavin is dissolved in the ethyl acetate stably even in a low-concentration state, the degradation of the 1.5% theaflavin ethyl acetate solution is not more than 2% after the theaflavin ethyl acetate solution is placed at room temperature in a dark place for 24 hours, and the requirements of various working procedures in the production process on the operation time can be met.

It should be noted that the agricultural products contain abundant water, and after airing, the water still contains part of water, 30-80%, and for the exogenous enzyme with less water content, the water should be supplemented in a multiphase fermentation system.

Compared with the PPO provided by fresh tea leaves, the source of agricultural products containing the PPO is wide, and the exogenous PPO can be obtained all the year round.

The butyl acetate is 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, and can effectively reduce the cost.

Step 104: and obtaining ester phase fermentation liquor from the fermentation product, drying and purifying the ester phase fermentation liquor to obtain high-content theaflavin.

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 103, the weight ratio of the exogenous enzyme, the ethyl acetate and the tea polyphenol product is 10-25:100:1-10, and the preferable ratio is 18-25: 100: 1-5.

The method for obtaining the fermentation liquor from the fermentation product comprises the following steps:

step 201: and filtering the fermentation product to obtain fermentation liquor and filter residue. But not limited thereto, the fermentation broth and the residue may be obtained by centrifugation.

Step 202: washing the filter residue with ethyl acetate or butyl acetate to obtain a washing solution.

Step 203: the washings were combined with the fermentation broth.

The method for purifying the fermentation liquor comprises the following steps:

step 301: and (4) decompressing and evaporating the fermentation liquor to dryness to obtain a theaflavin crude solid.

Step 302: dissolving theaflavin crude solid with water, and filtering to obtain filtrate.

Step 303: after the filtrate is adsorbed by a macroporous resin column, the filtrate is eluted in stages, and the eluates at all stages are concentrated by an organic roll type nanofiltration membrane.

The macroporous resin column contains porous polymer microsphere as separating material combining adsorptivity and screening principle, and the organic compound is separated through elution with certain solvent based on different adsorbability and molecular weight.

The organic roll-type nanofiltration membrane concentration is a novel energy-saving non-phase-change normal-temperature concentration method, the operating temperature is lower than 40 ℃, and the oxidative deterioration of thermosensitive substances and the heating volatilization loss of an organic solvent are effectively prevented. Meanwhile, the concentration treatment cost of membrane concentration is very low, and the membrane has considerable economic benefit advantage compared with vacuum heating evaporation.

Step 304: concentrating, and drying at low temperature under reduced pressure to obtain high content theaflavin.

In one embodiment, the following fermentation broth purification process is employed:

step 401: and preparing the solid into 3-8% aqueous solution, and filtering to obtain filtrate.

Step 402: adsorbing and purifying the filtrate by using an AB-8 macroporous resin column, wherein the flow rate of the filtrate on the column is 1 time of the column volume/hour, and the column loading amount of solid matters is as follows: the weight ratio of the AB-8 macroporous resin is 1:10, and the mixture is placed on a column and stands for 1 to 2 hours.

Step 403: the AB-8 macroporous resin column was washed with 4-8 column volumes of water.

Step 404: eluting tea polyphenol catechin components adsorbed on the macroporous resin column with 30% ethanol, wherein the volume of 30% ethanol eluate is 5-15 times of the column volume to obtain tea polyphenol eluate.

Step 405: filtering and concentrating the tea polyphenol eluate with organic roll-type nanofiltration membrane to obtain 30% ethanol filtrate and retentate, and drying the retentate under reduced pressure to obtain tea polyphenol compound product, wherein the 30% ethanol can be used repeatedly.

In the concentration process of the organic roll-type nanofiltration membrane, ethanol loss cannot be caused, and the filtered ethanol can be reused; and the tea polyphenol or theaflavin in the trapped liquid can not deteriorate due to long-time heating, so that the eluted tea polyphenol can be used as a raw material for secondary fermentation after being concentrated and dried. Thus, the raw materials and the solvent are reused at the same time, and the cost can be optimized.

Step 406: the macroporous resin column uses 70% ethanol to elute theaflavin, and the volume of 70% ethanol elution is 3-10 times of the column volume, so as to obtain theaflavin eluent.

Step 407: concentrating theaflavin eluate to solid content of 25% -50% by organic rolled nanofiltration membrane, and simultaneously obtaining 70% ethanol, wherein the 70% ethanol can be reused. But not limited thereto, the solid holdup can be adjusted according to actual conditions.

Step 408: the concentrated solution obtained by concentration in step 307 is dried at low temperature and low pressure in a continuous vacuum belt dryer to obtain theaflavin solid.

The invention also comprises a method for preparing wine by utilizing filter residues, which comprises the following steps:

step 501: adding tea polyphenols and ethyl acetate or butyl acetate into the residue, and performing aerobic fermentation again.

Step 502: and filtering and washing the secondary fermentation product to obtain secondary filter residue, secondary fermentation liquid and secondary washing liquid.

Step 503: and combining the secondary fermentation liquor and the secondary washing liquid, and purifying to obtain the high-content theaflavin.

Step 504: and adding purified water with the volume of 1-2 times of the secondary filter residue, and introducing low-pressure water vapor to remove ethyl acetate by evaporation to obtain the brewing raw material.

Step 505: after the wine brewing raw material is gelatinized, wine making yeast is added for fermentation, and the fruit wine is obtained.

The filter residue or secondary filter residue is rich in monosaccharide, double pond, and multiple functional polysaccharides such as tea polysaccharide and vitamin mineral, and is used for preparing functional wine by steaming to remove ethyl acetate, and the procedure of steaming to remove ethyl acetate is similar to gelatinization procedure of fermenting wine.

The invention also comprises a method for detecting the enzyme activity of the exogenous enzyme, which comprises the following steps:

grinding a sample in 2% PVP phosphate buffer solution with the pH value of 5.8 into homogenate in an ice bath environment;

centrifuging the homogenate at 9000r/min for 20min, and collecting supernatant centrifugate (enzyme solution);

adding 0.5mL of enzyme solution into 1.5mL of 0.05mol/L phosphate buffer solution (pH 7.0) and 1.0mL of 0.05mol/L catechol solution to obtain a reaction system;

immediately putting the reaction system into an ultraviolet spectrophotometer, starting timing, measuring and recording the change of absorbance value at the wavelength of 410nm, and calculating the enzymatic activity of the PPO by taking the increase of 0.001 of A410nm every 60 seconds as 1 PPO activity unit:

wherein the unit of the enzyme activity is U/(ml min), and delta A is the change value of absorbance in the reaction time; v is the volume (ml) of the enzyme solution in the reaction system at the time of measurement; t is reaction time (min); d is the dilution factor of the sample. The polyphenol oxidase can catalyze catechol to be oxidized into pyrocatechone, so that the absorbance value at the wavelength of 410nm changes.

Example 1

Aerobic fermentation: cleaning and airing commercially available ample flow pears, freezing for 12 hours, taking out, crushing in a 30 ten thousand-level purification workshop to obtain pomace (exogenous enzyme), measuring the enzyme activity of the pomace to be 617U/(ml _ min), weighing 100 kg of pomace, putting the pomace into a temperature-controlled stainless steel extraction kettle with a sieve plate at the bottom and a side filter outlet, adding 600L of ethyl acetate and 30 kg of green tea water extract with the tea polyphenol content of 35%, blowing oxygen from the bottom of the kettle at room temperature, stirring and fermenting, detecting the absorbance change of fermentation liquor at 380 nm by using an ultraviolet spectrophotometer, stopping fermentation when the A380 absorbance of the fermentation liquor reaches the peak value and starts, and filtering and collecting orange-red fermentation liquor and filter residues. The process flow is shown in figure 3.

And (3) cleaning filter residues: and uniformly stirring and washing the filter residue twice by using 1000 liters of ethyl acetate, filtering and collecting orange-red washing liquid. The fermentation liquor and the washing liquid are combined and decompressed and concentrated to be dry, and 12 kg of brownish red solid is obtained.

And (3) purifying theaflavin: adding 240 kg of water into the solid matter, stirring and dissolving the solid matter to prepare a 3-8% aqueous solution, filtering the solution, feeding the filtrate into an AB-8 macroporous resin column, wherein the resin column is composed of 160 kg of resin, the diameter-height ratio is 1:10, the flow rate of the filtrate on the column is 1 BV/h, BV refers to the column volume of the resin column, and standing the filtrate for 1 h after all the filtrate is fed into the column. Washing the resin with water to remove impurities such as caffeine and small molecular phenolic acid such as gallic acid, wherein the flow rate of water washing is 2 BV/h, and the washing amount is 5 BV; after water washing, the tea polyphenol and catechin components are eluted by 30% ethanol, the cleaning flow rate is 2 BV/h, the cleaning amount is 8BV, the cleaning solution is dealcoholized, dehydrated and concentrated by an organic roll type nanofiltration membrane, the filtered solution is an ethanol aqueous solution, the cleaning solution is concentrated to 25% -50% solid content, and then dried by a continuous vacuum belt type dryer to obtain 8.3 kg of yellow powdery tea polyphenol compound product which can be used for the fermentation process. Eluting the resin with 70% ethanol at an elution flow rate of 1 BV/h and an elution amount of 3BV, concentrating the eluate film to 25-50% of solid content, drying with a continuous vacuum belt drier to obtain 3.0 kg of orange red powder, detecting theaflavin content with High Performance Liquid Chromatography (HPLC) of 61.7%, and detecting the chromatogram as shown in figure 4.

And (3) secondary fermentation: the pomace washed by ethyl acetate can be reused, 500L of butyl acetate, 10 kg of green tea water extract with the tea polyphenol content of 35% and 8.3 kg of tea polyphenol compound product recovered in the previous process are added, oxygen is blown in at room temperature, stirring and fermentation are carried out, an ultraviolet spectrophotometer is used for detecting the absorbance change of the fermentation liquor at 380 nm, the fermentation is stopped when the A380 absorbance of the fermentation liquor reaches the peak value and starts to go away, and orange-red fermentation liquor is filtered and collected. And uniformly stirring and washing the pomace twice by 1000 liters of butyl acetate, filtering and collecting orange-red washing liquid. The fermentation liquor and the washing liquid are combined and dried under reduced pressure to obtain 11.9 kg of brownish red solid. Adding 240 kg water into the solid matter, stirring to fully dissolve the solid matter, filtering, loading the filtrate on AB-8 resin column, washing with water, and washing with 30% ethanol to remove unfermented tea polyphenols and catechin. And eluting theaflavin by using 70% ethanol, concentrating a theaflavin eluent film to 25% -50% solid content, drying by using a continuous vacuum belt type dryer to obtain 2.7 kg of orange red powder, and detecting the theaflavin content by using High Performance Liquid Chromatography (HPLC) to be 58.6%.

Filtering residues and preparing wine: after the fruit residues are steamed to remove ethyl acetate or butyl acetate, because bitter micromolecule polyphenols are removed and the fruit residues are rich in fructose, glucose, functional tea polysaccharide, vitamins, mineral substances and other nutrients, wine-making yeast is added for fermentation to obtain the fruit wine.

Example 2

Aerobic fermentation: cleaning and airing red ripe apples purchased in the market, freezing for 12 hours, taking out, crushing in a 30 ten thousand-level purification workshop to obtain pomace (exogenous enzyme), measuring the enzyme activity of the pomace to be 530U/(ml × min), weighing 60 kg of pomace, putting the pomace into a temperature-controlled stainless steel extraction kettle with a sieve plate at the bottom and a side filter outlet, adding 600L of ethyl acetate and 20 kg of green tea water extract with the tea polyphenol content of 35%, blowing oxygen from the bottom of the kettle at room temperature, stirring and fermenting, detecting the absorbance change of fermentation liquor at 380 nm by using an ultraviolet spectrophotometer, stopping fermentation when the A380 absorbance of the fermentation liquor reaches the peak value and starts, and filtering and collecting orange-red fermentation liquor and filter residues.

And (3) cleaning filter residues: and uniformly stirring and washing the filter residue twice by using 1000 liters of ethyl acetate, filtering and collecting orange-red washing liquid. The fermentation liquor and the washing liquid are combined and decompressed and concentrated to be dry, and 12 kg of brownish red solid is obtained.

And (3) purifying theaflavin: adding 240 kg of water into the solid matter, stirring and dissolving the solid matter to prepare a 3-8% aqueous solution, filtering the solution, feeding the filtrate into an AB-8 macroporous resin column, wherein the resin column is composed of 160 kg of resin, the diameter-height ratio is 1:10, the flow rate of the filtrate on the column is 1 BV/h, BV refers to the column volume of the resin column, and standing the filtrate for 1 h after all the filtrate is fed into the column. Washing the resin with water to remove impurities such as caffeine and small molecular phenolic acid such as gallic acid, wherein the flow rate of the water washing is 2 BV/h, and the washing amount is 5 BV; after washing, 30% ethanol is used for eluting tea polyphenol and catechin components, the washing flow rate is 2 BV/h, the washing amount is 8BV, the washing liquid is dealcoholized, dehydrated and concentrated by an organic roll type nanofiltration membrane, the filtered liquid is ethanol aqueous solution, the washing liquid is concentrated to 25% -50% solid content, and then the drying liquid is dried by a continuous vacuum belt type dryer, 5.8 kg of yellow powdery tea polyphenol compound product is obtained, and the yellow powdery tea polyphenol compound product can be used for the fermentation process. Eluting the resin with 70% ethanol at an elution flow rate of 1 BV/h and an elution amount of 3BV, concentrating the eluate film to 25% -50% solid content, drying with a continuous vacuum belt drier to obtain 1.8 kg of orange red powder, detecting theaflavin content with High Performance Liquid Chromatography (HPLC) of 59.8%, and detecting the chromatogram as shown in figure 5.

And (3) secondary fermentation: the pomace washed by ethyl acetate can be reused, 500L of butyl acetate, 10 kg of green tea water extract with 35% of tea polyphenol content and 5.8 kg of tea polyphenol compound product recovered in the purification process are added, oxygen is blown in and stirred for fermentation at room temperature, an ultraviolet spectrophotometer is used for detecting the absorbance change of the fermentation liquor at 380 nm, the fermentation is stopped when the A380 absorbance of the fermentation liquor reaches the peak value and begins to go away, and orange-red fermentation liquor is filtered and collected. The fruit 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 drying under reduced pressure to obtain 7.8 kg of a brownish red solid. Adding 240 kg of water into the solid matter, stirring to fully dissolve the solid matter, filtering, putting the filtrate on an AB-8 resin column, washing with water, and washing with 30% ethanol to remove unfermented tea polyphenol and catechin. And eluting theaflavin by using 70% ethanol, concentrating a theaflavin eluent film to 25% -50% solid content, drying by using a continuous vacuum belt type dryer to obtain 1.5 kg of orange red powder, and detecting the theaflavin content by using High Performance Liquid Chromatography (HPLC) to be 57.3%.

Example 3

Aerobic fermentation: cleaning and airing needle mushrooms purchased in the market, freezing for 12 hours, taking out, crushing in a 30 ten thousand-level purification workshop to obtain fruit residues (exogenous enzyme), measuring the enzyme activity of the fruit residues to be 390U/(ml × min), weighing 110 kg of the fruit residues, putting the fruit residues into a temperature-controlled stainless steel extraction kettle with a sieve plate at the bottom and a side filter outlet, adding 600L of ethyl acetate and 40 kg of green tea water extract with the tea polyphenol content of 35%, blowing oxygen from the bottom of the kettle at room temperature, stirring and fermenting, detecting the absorbance change of fermentation liquor at 380 nm by using an ultraviolet spectrophotometer, stopping fermentation when the A380 absorbance of the fermentation liquor reaches the peak value and starts, and filtering and collecting orange-red fermentation liquor and filter residues.

And (3) cleaning filter residues: and uniformly stirring and washing the filter residue twice by using 1000 liters of ethyl acetate, filtering and collecting orange-red washing liquid. The fermentation liquor and the washing liquid are combined and decompressed and concentrated to be dry, and 16 kg of brownish red solid is obtained.

And (3) purifying theaflavin: adding 240 kg of water into the solid matter, stirring and dissolving the solid matter to prepare a 3-8% aqueous solution, filtering the solution, feeding the filtrate into an AB-8 macroporous resin column, wherein the resin column is composed of 160 kg of resin, the diameter-height ratio is 1:10, the flow rate of the filtrate on the column is 1 BV/h, BV refers to the column volume of the resin column, and standing the filtrate for 1 h after all the filtrate is fed into the column. Washing the resin with water to remove impurities such as caffeine and small molecular phenolic acid such as gallic acid, wherein the flow rate of the water washing is 2 BV/h, and the washing amount is 5 BV; after washing, using 30% ethanol to elute tea polyphenol and catechin components, washing at the flow rate of 2 BV/h and the washing amount of 8BV, dealcoholizing, dehydrating and concentrating the cleaning solution by using an organic roll type nanofiltration membrane, wherein the filtering solution is an ethanol aqueous solution, concentrating the cleaning solution to the solid content of 25-50%, and drying by using a continuous vacuum belt type dryer to obtain 9 kg of yellow powdery tea polyphenol compound product which can be used for the fermentation process. Eluting the resin with 70% ethanol at an elution flow rate of 1 BV/h and an elution amount of 3BV, concentrating the eluate film to 25% -50% solid content, drying with a continuous vacuum belt drier to obtain 2.2 kg of orange red powder, detecting theaflavin content with High Performance Liquid Chromatography (HPLC) of 59.5%, and detecting the chromatogram as shown in FIG. 6.

And (3) secondary fermentation: and (3) reusing the pomace washed by ethyl acetate, adding 500L of butyl acetate, 10 kg of green tea water extract with the tea polyphenol content of 35% and 9 kg of tea polyphenol compound product, blowing oxygen at room temperature, stirring and fermenting, detecting the absorbance change of the fermentation liquor at 380 nm by using an ultraviolet spectrophotometer, stopping fermentation when the A380 absorbance of the fermentation liquor reaches the peak value and begins to go around, filtering and collecting orange-red fermentation liquor. The fruit 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 drying under reduced pressure to obtain 14 kg of a brownish red solid. Adding 240 kg water into the solid matter, stirring to fully dissolve the solid matter, filtering, loading the filtrate on AB-8 resin column, washing with water, and washing with 30% ethanol to remove unfermented tea polyphenols and catechin. And eluting theaflavin by using 70% ethanol, concentrating a theaflavin eluent film to 25% -50% solid content, drying by using a continuous vacuum belt type dryer to obtain 2 kg of orange red powder, and detecting the theaflavin content by using High Performance Liquid Chromatography (HPLC) to be 57.5%.

Example 4

Aerobic fermentation: washing and airing commercially available potatoes, freezing for 12 hours, taking out, crushing in a 30 ten thousand-level purification workshop to obtain pomace (exogenous enzyme), measuring the enzyme activity of the pomace to be 410U/(ml × min), weighing 115 kg of pomace, putting the pomace into a temperature-controlled stainless steel extraction kettle with a sieve plate at the bottom and a side filter outlet, adding 600L of ethyl acetate and 50 kg of green tea water extract with the tea polyphenol content of 35%, blowing oxygen from the bottom of the kettle at room temperature, stirring and fermenting, detecting the absorbance change of fermentation liquor at 380 nm by using an ultraviolet spectrophotometer, stopping fermentation when the A380 absorbance of the fermentation liquor reaches the peak value and begins to go on, and filtering and collecting orange-red fermentation liquor and filter residues.

And (3) cleaning filter residues: and uniformly stirring and washing the filter residue twice by using 1000 liters of ethyl acetate, filtering and collecting orange-red washing liquid. The fermentation liquor and the washing liquid are combined and decompressed and concentrated to be dry, and 19 kg of brownish red solid is obtained.

And (3) purifying theaflavin: adding 240 kg of water into the solid matter, stirring and dissolving the solid matter to prepare a 3-8% aqueous solution, filtering the solution, feeding the filtrate into an AB-8 macroporous resin column, wherein the resin column is composed of 160 kg of resin, the diameter-height ratio is 1:10, the flow rate of the filtrate on the column is 1 BV/h, BV refers to the column volume of the resin column, and standing the filtrate for 1 h after all the filtrate is fed into the column. Washing the resin with water to remove impurities such as caffeine and small molecular phenolic acid such as gallic acid, wherein the flow rate of the water washing is 2 BV/h, and the washing amount is 5 BV; after washing, using 30% ethanol to elute tea polyphenol and catechin components, washing at a flow rate of 2 BV/h and a washing amount of 8BV, dealcoholizing, dehydrating and concentrating the cleaning solution by using an organic roll type nanofiltration membrane, concentrating the filtered solution into an ethanol aqueous solution, concentrating the cleaning solution to a solid content of 25-50%, and drying by using a continuous vacuum belt type dryer to obtain 15 kg of yellow powdery tea polyphenol compound product which can be used for a fermentation process. Eluting the resin with 70% ethanol at an elution flow rate of 1 BV/h and an elution amount of 3BV, concentrating the eluate film to 25% -50% solid content, drying with a continuous vacuum belt drier to obtain 2.7 kg of orange red powder, detecting theaflavin content with High Performance Liquid Chromatography (HPLC) of 60.1%, and detecting the chromatogram as shown in FIG. 7.

And (3) secondary fermentation: and adding 500L of butyl acetate, 10 kg of green tea water extract with the tea polyphenol content of 35% and 15 kg of tea polyphenol compound product recovered in the purification process into the pomace washed by ethyl acetate for reuse, blowing oxygen at room temperature, stirring and fermenting, detecting the absorbance change of the fermentation liquor at 380 nm by using an ultraviolet spectrophotometer, stopping fermentation when the A380 absorbance of the fermentation liquor reaches the peak value and starts to go, filtering and collecting orange-red fermentation liquor. The fruit 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 drying under reduced pressure to obtain 20 kg of brownish red solid. Adding 240 kg water into the solid matter, stirring to fully dissolve the solid matter, filtering, loading the filtrate on AB-8 resin column, washing with water, and washing with 30% ethanol to remove unfermented tea polyphenols and catechin. And eluting theaflavin by using 70% ethanol, concentrating a theaflavin eluent film to 25% -50% solid content, drying by using a continuous vacuum belt type dryer to obtain 2.2 kg of orange red powder, and detecting the theaflavin content by using High Performance Liquid Chromatography (HPLC) to be 56.9%.

Example 5

Aerobic fermentation: cleaning and airing commercially available sweet potatoes, freezing for 12 hours, taking out, crushing in a 30 ten thousand-level purification workshop to obtain pomace (exogenous enzyme), measuring the enzyme activity of the pomace to be 490U/(ml × min), weighing 120 kg of pomace, putting the pomace into a temperature-controlled stainless steel extraction kettle with a bottom sieve plate and a side filter outlet, adding 600 liters of ethyl acetate and 35 kg of green tea water extract with the tea polyphenol content of 35%, blowing oxygen from the bottom of the kettle at room temperature, stirring and fermenting, detecting the absorbance change of fermentation liquor at 380 nm by using an ultraviolet spectrophotometer, stopping fermentation when the A380 absorbance of the fermentation liquor reaches the peak value and starts to go away, and filtering and collecting orange-red fermentation liquor and filter residues.

And (3) cleaning filter residues: and uniformly stirring and washing the filter residue twice by using 1000 liters of ethyl acetate, filtering and collecting orange-red washing liquid. The fermentation liquor and the washing liquid are combined and decompressed and concentrated to be dry, and 14 kg of brownish red solid is obtained.

And (3) purifying theaflavin: adding 240 kg of water into the solid matter, stirring and dissolving the solid matter to prepare a 3-8% aqueous solution, filtering the solution, feeding the filtrate into an AB-8 macroporous resin column, wherein the resin column is composed of 160 kg of resin, the diameter-height ratio is 1:10, the flow rate of the filtrate on the column is 1 BV/h, BV refers to the column volume of the resin column, and standing the filtrate for 1 h after all the filtrate is fed into the column. Washing the resin with water to remove impurities such as caffeine and small molecular phenolic acid such as gallic acid, wherein the flow rate of the water washing is 2 BV/h, and the washing amount is 5 BV; after washing, using 30% ethanol to elute tea polyphenol and catechin components, wherein the washing flow rate is 2 BV/h, the washing amount is 8BV, the washing liquid is dealcoholized, dehydrated and concentrated by using an organic roll type nanofiltration membrane, the filtered liquid is ethanol aqueous solution, the washing liquid is concentrated to 25% -50% solid content, and then, using a continuous vacuum belt type drier to dry, obtaining 10.6 kg of yellow powdery tea polyphenol multiplex product, which can be used for the fermentation process. Eluting the resin with 70% ethanol at an elution flow rate of 1 BV/h and an elution amount of 3BV, concentrating the eluate film to 25% -50% solid content, drying with a continuous vacuum belt drier to obtain 2.6 kg of orange red powder, detecting theaflavin content with High Performance Liquid Chromatography (HPLC) of 60.4%, and detecting the chromatogram as shown in figure 8.

And (3) secondary fermentation: the pomace washed by ethyl acetate can be reused, 500L of butyl acetate, 10 kg of green tea water extract with 35% of tea polyphenol content and 10.6 kg of tea polyphenol compound product recovered in the purification process are added, oxygen is blown in and stirred for fermentation at room temperature, an ultraviolet spectrophotometer is used for detecting the absorbance change of the fermentation liquor at 380 nm, the fermentation is stopped when the A380 absorbance of the fermentation liquor reaches the peak value and begins to go away, and orange-red fermentation liquor is filtered and collected. The fruit 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 drying under reduced pressure to obtain 16 kg of brownish red solid. Adding 240 kg water into the solid matter, stirring to fully dissolve the solid matter, filtering, loading the filtrate on AB-8 resin column, washing with water, and washing with 30% ethanol to remove unfermented tea polyphenols and catechin. And eluting theaflavin by using 70% ethanol, concentrating a theaflavin eluent film to 25% -50% solid content, drying by using a continuous vacuum belt type dryer to obtain 2.3 kg of orange red powder, and detecting the theaflavin content by using High Performance Liquid Chromatography (HPLC) to be 57.2%.

Wherein, the following high performance liquid chromatograph is adopted in the detection of theaflavin: 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 min at a flow rate of 0.9ml/min at a column temperature of 40 ℃ and a detection wavelength of 280nm, and the amount of sample was 10. mu.l.

As shown in FIGS. 9-12, the HPLC peak profiles of the theaflavin component standard are shown, which are the HPLC peak profiles of the TF standard, the TF-3-G standard, the TF-3'-G standard and the TFDG standard respectively, and it can be found from the HPLC peak profiles that the TF standard peak time is 19.477min, the TF-3-G standard peak time is 23.193min, the TF-3' -G standard peak time is 25.204min and the TFDG standard peak time is 26.272 min. As shown in FIGS. 4 to 8, catechins were eluted in the form of broad and unresolved peaks at the start of the chromatography, while theaflavins were eluted within 15 to 30 minutes. According to the method, a standard calibration curve is constructed through 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.

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.

Claims (10)

1. A method for preparing theaflavins by exogenous enzymes, comprising:

cleaning agricultural products, airing, and freezing to break the walls;

crushing the frozen wall-broken agricultural products to obtain exogenous enzymes;

adding the exogenous enzyme into a tea polyphenol product, adding ethyl acetate or butyl acetate to form a multiphase fermentation system, and carrying out aerobic stirring fermentation to obtain a fermentation product;

and obtaining ester phase fermentation liquor from the fermentation product, drying the ester phase fermentation liquor, and purifying to obtain high-content theaflavin.

2. The method of claim 1, wherein the agricultural product comprises a fruit, vegetable, rhizome or mushroom having the polyphenol oxidase PPO.

3. The method of claim 1, wherein the agricultural product comprises one or a combination of: apple, pear, banana, oyster mushroom, needle mushroom, straw mushroom, potato, sweet potato, yam and lotus root.

4. The method of claim 1, wherein the weight ratio of the exogenous enzyme to the ethyl acetate to the tea polyphenol product is 10-25:100: 1-10.

5. The method of claim 1, wherein the tea polyphenol product comprises from 35% to 99% tea polyphenol.

6. The method of claim 1, wherein the step of removing the fermentation broth from the fermentation product comprises:

filtering the fermentation product to obtain ester phase fermentation liquor and filter residue;

washing the filter residue with ethyl acetate or butyl acetate to obtain a washing liquid;

the wash solution is combined into the fermentation broth.

7. The method of claim 6, further comprising the step of using the filter residue to produce wine:

adding tea polyphenol product and ethyl acetate or butyl acetate into the filter residue, and performing aerobic fermentation again;

filtering and washing the secondary fermentation product to obtain secondary filter residue, secondary fermentation liquid and washing liquid;

mixing the secondary fermentation liquor and the washing liquor, and drying and purifying to obtain theaflavin;

adding water with the volume of 1-2 times into the secondary filter residue, and introducing low-pressure water vapor to remove ethyl acetate by evaporation to obtain a brewing raw material;

after the wine brewing raw material is gelatinized, wine making yeast is added for fermentation, and the fruit wine is obtained.

8. The method of claim 1, wherein the method of purifying the fermentation broth comprises:

the fermentation liquor is decompressed and evaporated to dryness to obtain a theaflavin crude product solid;

dissolving the solid with water, and filtering to obtain filtrate;

purifying the filtrate with macroporous resin column, and concentrating with organic rolled nanofiltration membrane;

concentrating, and drying at low temperature under reduced pressure to obtain theaflavin.

9. The method of claim 8, wherein the method of broth purification comprises:

preparing the solid into 3-8% aqueous solution, and filtering to obtain filtrate;

purifying the filtrate by using an AB-8 macroporous resin column, wherein the flow rate of the filtrate on the column is 1 time of the column volume/hour, and the column loading amount of solid matters is as follows: the weight ratio of the AB-8 macroporous resin is 1:10, and the mixture is placed on a column and then is kept stand for 1 to 2 hours;

washing the AB-8 macroporous resin column with water of 4-8 times of column volume;

eluting tea polyphenols and catechin components on macroporous resin column with 30% ethanol, wherein the elution volume of 30% ethanol is 5-15 times of column volume to obtain tea polyphenols eluate;

concentrating the tea polyphenol eluent by an organic roll type nanofiltration membrane to obtain 30% ethanol filtrate and trapped fluid, and reusing the 30% ethanol filtrate; concentrating the trapped liquid membrane to solid content of 25% -50%, and drying under reduced pressure to obtain tea polyphenol compound product;

eluting theaflavin with 70% ethanol in a macroporous resin column, wherein the volume of 70% ethanol elution is 3-10 times of the column volume to obtain theaflavin eluate;

concentrating theaflavin eluent by an organic roll type nanofiltration membrane until the solid content is 25% -50%, and simultaneously obtaining 70% ethanol for reuse;

drying the concentrated solution in a continuous vacuum belt dryer at low temperature and low pressure to obtain high-content theaflavin solid.

10. A product obtainable by the process of any one of claims 1 to 9.

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