Method for preparing theaflavins on basis of substrate immobilization

Abstract

Provided is a method for preparing theaflavins on the basis of substrate immobilization. The method comprises: enriching natural plant monomeric catechin molecular substrates on an inert adsorption carrier in a non-covalent bond manner, performing purification, and then catalyzing the polymerization of the enriched catechin substrates on the carrier into dimeric substances, i.e., theaflavins, by means of a high-selectivity free exogenous enzyme. The purification and enzymatic oxidative condensation of the catechin substrates are integrated and implemented on the same carrier, thus reducing the operational difficulty of the process.

Description

A method for preparing theaflavins by immobilizing a substrate Technical Field

[0001] The invention belongs to the technical field of biosynthesis, and particularly relates to a method for preparing theaflavins by using an immobilized substrate. Background Art

[0002] Theaflavins, dimers of catechins, are a golden pigment found in black tea. Theaflavins are present in only a small fraction of finished tea, at only 0.5-1.5%, making extraction expensive. Known as the "soft gold" of tea, theaflavins are widely used in healthcare. Industrial production of theaflavins typically involves endogenous or exogenous enzymatic fermentation.

[0003] Endogenous enzymatic fermentation utilizes the endogenous polyphenol oxidase (PPO) and peroxidase (POD) in fresh tea leaves, through freezing and temperature-controlled fermentation, to produce tea leaves high in theaflavins, followed by extraction and purification of the high-content theaflavins. Exogenous enzymatic fermentation utilizes an exogenous enzyme source rich in polyphenol oxidase to enzymatically catalyze the synthesis of catechins and produce theaflavins. Existing exogenous enzyme catalysis technologies are divided into two routes: free enzyme catalysis and immobilized enzyme catalysis.

[0004] Immobilized enzyme catalysis involves purifying the extracted crude PPO enzyme and immobilizing it on a suitable carrier through methods such as ionic binding, biospecific interactions, scrap iron-star interactions, hydrophobic interactions, chelation, or affinity binding. The enzyme then contacts a solution containing catechins in the reaction system. Under aerobic conditions and appropriate pH values, the enzyme catalyzes the polymerization of catechin molecules to form theaflavins. This process is costly to purify and immobilize, resulting in limited use of the immobilized enzyme and the generation of significant amounts of industrial waste. During the purification and immobilization process, the PPO enzyme contacts and bonds with multiple active sites on the carrier, solidifying the enzyme's spatial configuration. This alters the enzyme's spatial configuration, structure, and conformation compared to its free state. Once immobilized on the carrier, the enzyme is less likely to change its spatial configuration, which can alter its catalytic selectivity for the substrate catechins and, in particular, interfere with its catalytic ability for ester-type catechins.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing theaflavins by using an immobilized substrate. The method provided by the present invention solves the problems of immobilized enzyme catalysis technology, has low cost and good catalytic effect.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing theaflavins by using an immobilized substrate, comprising the following steps:

[0009] (1) mixing a tea polyphenol solution with an inert adsorption carrier for adsorption, and then washing with water and alcohol in sequence to obtain an immobilized substrate; the inert adsorption carrier comprises a macroporous adsorption resin; the pore size of the macroporous adsorption resin is 10 to 180 angstroms; the concentration of the alcohol solution used for the alcohol washing is 4 to 20 wt%;

[0010] (2) mixing the immobilized substrate with an enzyme solution and conducting an enzymatic reaction under oxygen to obtain an enzyme catalytic carrier; the enzyme in the enzyme solution is polyphenol oxidase PPO;

[0011] (3) Concentrating the eluate obtained by desorption of the enzyme-catalyzed carrier to obtain theaflavins.

[0012] Preferably, in step (1), the concentration of the tea polyphenol solution is 0.5 to 100 g / L.

[0013] Preferably, in step (1), the mass ratio of tea polyphenols to inert adsorption carrier in the tea polyphenol solution is 3 to 15:100.

[0014] Preferably, in step (1), the adsorption temperature is 5 to 35° C., the pH value of the adsorption system is 4.0 to 5.5, and the insulation time is 1 to 5 hours.

[0015] Preferably, in step (1), the macroporous adsorption resin includes one or more of LX-8 resin, LX-5 resin, AB-8 resin, NKA resin and NKA-9 resin;

[0016] The inert adsorption carrier further comprises one or more of silica gel and alumina.

[0017] Preferably, in step (1), the method for preparing the tea polyphenol solution includes method 1, method 2 or method 3;

[0018] The method 1 comprises the following steps: stirring and extracting tea leaves with hot water at 65 to 90°C;

[0019] The second method comprises the following steps: mixing commercially available tea polyphenol powder having a content of 30 to 99 wt% with water;

[0020] The third method comprises the following steps: enriching a tea polyphenol solution having a concentration lower than a target concentration.

[0021] Preferably, in method 1, the extraction is performed more than once, and the time for a single extraction is 20 to 60 minutes; and the tea leaves are one or more of large-leaf green tea and small-leaf green tea.

[0022] Preferably, in the second method, the commercially available tea polyphenols powder having a content of 30 to 99 wt% includes one or more commercially available tea polyphenols TP30 to TP99 products.

[0023] Preferably, in step (2), the volume ratio of the immobilized substrate to the enzyme solution is 0.5-1:0.5-1.

[0024] Preferably, in step (2), the temperature of the enzymatic reaction is 5 to 35° C., the pH value of the enzymatic reaction system is 4.5 to 6.5, the oxygen volume concentration is 60 to 90%, and the insulation time is 4 to 8 hours.

[0025] Preferably, the enzymatic reaction adopts A 380 Test the alcohol extract of the inert adsorption carrier to monitor the reaction progress;

[0026] The alcohol extract of the inert adsorption carrier was tested as follows: the immobilized substrate was mixed with ethanol for 30 minutes per reaction, extracted by ultrasonic oscillation at room temperature, and the supernatant was aspirated;

[0027] The absorbance of the supernatant at 380 nm was measured, and the enzymatic reaction was stopped after the absorbance stopped rising.

[0028] Preferably, in step (2), the enzyme solution is polyphenol oxidase from Novozymes or laccase from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., or pear juice obtained by filtering pear homogenate, or crude enzyme solution obtained by filtering fresh tea homogenate.

[0029] Preferably, the pear is a snow pear; the pear homogenate filtration is as follows: the snow pear is frozen and then thawed, then the thawed snow pear is mixed with a pH buffer solution and homogenized, and the pear residue is filtered through four layers of gauze.

[0030] Preferably, the mass ratio of the snow pear to the pH buffer solution is 1:0.2-1; and the pH value of the pH buffer solution is 5.5-6.5.

[0031] Preferably, the pH buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0032] Preferably, in step (3), the desorption eluent is edible ethanol with a concentration of 60 to 80 wt%.

[0033] The invention provides a method for preparing theaflavins by using an immobilized substrate. The method provided by the present invention uses an immobilized substrate to carry out catalytic polymerization. Natural plant monomer catechin molecular substrates are enriched on an inert adsorption carrier in a non-covalent bond manner, achieving non-bonding immobilization to form a multi-molecular aggregate state, which is then purified (washed with water and washed with low-concentration alcohol). A highly selective free exogenous PPO enzyme solution is then added for catalysis. In the aqueous solution, the free PPO enzyme protein chain-shaped molecular framework has a spatially stretched configuration and high catalytic activity for ester-type and non-ester-type catechin substances. At the same time, because most of the substrate is locked on the adsorption carrier, the concentration of the free substrate in the enzyme solution is low, and the substrate's toxic effect on the enzyme and its inhibitory effect on the enzyme activity are greatly reduced. Under the micro-dynamic equilibrium conditions of decomposition-catalytic polymerization-adsorption, the enriched substrate catechins on the catalytic carrier are efficiently polymerized into dimer-type substances, theaflavins. This innovatively combines the purification of the substrate catechins and the enzymatic oxidative condensation on the same carrier, greatly simplifying the operational difficulty of the process and making it easy to operate.

[0034] The present invention innovatively catalyzes the synthesis of theaflavins by immobilizing substrates. Subsequently, the theaflavins-enriched resin can be simply vacuum-dried, and the theaflavins-enriched carrier can be used as a basic template for subsequent molecular functional group grafting modification. This provides a new approach for the enrichment and purification of low-concentration functional small molecules and the subsequent immobilization and targeted catalytic synthesis of functional macromolecules, and has broad development prospects.

[0035] Compared with the existing technology, the method provided by the present invention is green and environmentally friendly, does not produce toxic and harmful wastes such as discarded immobilized enzymes, does not use highly toxic, flammable and explosive ethyl acetate and petroleum ether, and does not use toxic and harmful halogenated alkanes in the caffeine removal and impurity removal processes. It can produce high-content tea polyphenols (immobilized substrates) and high-content theaflavins on the same production line, and lays a basic platform for subsequent operations such as modification of active group molecules and molecular modification using theaflavins as substrates.

[0036] Furthermore, in step (1), the adsorption temperature is 5 to 35° C., the pH value of the adsorption system is 4.0 to 5.5, and the holding time is 1 to 5 hours. In step (2), the enzymatic reaction temperature is 5 to 35° C., the pH value of the enzymatic reaction system is 4.5 to 6.5, the oxygen volume concentration is 60 to 90%, and the holding time is 4 to 8 hours. The method provided by the present invention has mild operating conditions, is conducive to safe production, saves energy, and reduces production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] FIG1 is a flow chart of the method for preparing theaflavins using an immobilized substrate provided by the present invention;

[0039] FIG2 is an HPLC detection spectrum of theaflavin prepared in Example 4 of the present invention. DETAILED DESCRIPTION

[0040] The present invention provides a method for preparing theaflavins by using an immobilized substrate, comprising the following steps:

[0041] (1) mixing a tea polyphenol solution with an inert adsorption carrier for adsorption, and then washing with water and alcohol in sequence to obtain an immobilized substrate; the inert adsorption carrier comprises a macroporous adsorption resin; the pore size of the macroporous adsorption resin is 10 to 180 angstroms; the concentration of the alcohol solution used for the alcohol washing is 4 to 20 wt%;

[0042] (2) mixing the immobilized substrate with an enzyme solution and conducting an enzymatic reaction under oxygen to obtain an enzyme catalytic carrier; the enzyme in the enzyme solution is polyphenol oxidase PPO;

[0043] (3) Concentrating the eluate obtained by desorption of the enzyme-catalyzed carrier to obtain theaflavins.

[0044] The present invention mixes the tea polyphenol solution with an inert adsorption carrier (referred to as the first mixing) for adsorption and then sequentially washes with water and alcohol to obtain an immobilized substrate. In the present invention, the concentration of the tea polyphenol solution is preferably 0.5 to 100 g / L.

[0045] In the present invention, the preparation method of the tea polyphenol solution preferably includes method one, method two or method three; the method one preferably includes the following steps: stirring and extracting tea leaves with 65-90°C hot water; the number of extractions is preferably one or more, more preferably two or more; the time for a single extraction is preferably 20-60 minutes, more preferably 30-50 minutes, and further preferably 40 minutes; the tea leaves are preferably one or more of large-leaf green tea and small-leaf green tea; the large-leaf green tea is preferably large-leaf green tea from Fuding, Fujian; the small-leaf green tea is preferably small-leaf green tea from Wuyuan, Jiangxi.

[0046] In the present invention, the second method preferably includes the following steps: mixing commercially available tea polyphenols powder with a content of 30 to 99 wt% and water; the commercially available tea polyphenols powder with a content of 30 to 99 wt% preferably includes one or more commercially available tea polyphenols TP30 products to tea polyphenols TP99 products.

[0047] In the present invention, the third method preferably includes the following steps: enriching the tea polyphenol solution below the target concentration.

[0048] In the present invention, the inert adsorption carrier comprises a macroporous adsorption resin; the pore size of the macroporous adsorption resin is 10 to 180 angstroms; the macroporous adsorption resin preferably comprises one or more of LX-8 resin, LX-5 resin, AB-8 resin, NKA resin, and NKA-9 resin; and the inert adsorption carrier preferably also comprises one or more of silica gel and alumina. The inert adsorption carrier in the present invention can be one or more macroporous adsorption resins, or silica gel and / or alumina, and can be obtained from a wide variety of sources.

[0049] In the present invention, the mass ratio of tea polyphenols to inert adsorption carrier in the tea polyphenol solution is preferably 3 to 15:100, more preferably 5 to 12:100, further preferably 7 to 10:100, and even more preferably 8 to 9:100.

[0050] In the present invention, the first mixing is preferably stirring mixing; the stirring speed is preferably 1 to 200 rpm, more preferably 10 to 100 rpm, and further preferably 30 rpm.

[0051] In the present invention, the adsorption temperature is preferably 5 to 35°C, more preferably 10 to 30°C, further preferably 15 to 25°C, and further preferably 20°C. The pH value of the adsorption system is preferably 4.0 to 5.5, more preferably 4.5 to 5.0. The holding time is preferably 1 to 5 hours, more preferably 2 to 4 hours, and further preferably 3 hours. In the present invention, the tea polyphenol solution is stirred and mixed with an inert adsorption carrier to complete the aggregation and adsorption of tea polyphenols.

[0052] In the present invention, the water used for washing is preferably pure water; the end point of the water washing is preferably washing until the washing liquid is substantially colorless. The present invention removes polysaccharide impurities by water washing.

[0053] In the present invention, the concentration of the alcohol solution used for alcohol washing is 4-20 wt%, preferably 8-15 wt%, and more preferably 10-14 wt%. The alcohol solution is preferably edible ethanol. The present invention removes impurities such as caffeine and water-soluble amino acids that interfere with subsequent enzymatic reactions through alcohol washing.

[0054] In the present invention, the alcohol washing preferably further comprises filtering to filter out the inert adsorption carrier for standby use.

[0055] After obtaining the immobilized substrate, the present invention mixes the immobilized substrate with an enzyme solution (referred to as the second mixing) and oxygenates the mixture to carry out an enzymatic reaction to obtain an enzyme catalytic carrier. In the present invention, the enzyme solution is preferably polyphenol oxidase from Novozymes or laccase from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., or pear juice obtained by filtering a pear homogenate, or a crude enzyme solution obtained by filtering a fresh tea homogenate.

[0056] In the present invention, the pear is preferably snow pear; the pear slurry filtration is preferably: the snow pear is frozen and then thawed, and then the thawed snow pear and pH buffer solution are mixed and homogenized, and the pear residue is filtered through four layers of gauze; the thawing is preferably natural thawing; the mass ratio of the snow pear to the pH buffer solution is preferably 1:0.2-1, more preferably 1:0.4-0.8, and further preferably 1:0.6; the pH value of the pH buffer solution is preferably 5.5-6.5, more preferably 5.5-6.0, and further preferably 5.5; the pH buffer solution is preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0057] In the present invention, the concentration of the enzyme solution is preferably 20 to 100 wt %, more preferably 50 to 100 wt %, and even more preferably 80 to 100 wt %.

[0058] In the present invention, the volume ratio of the immobilized substrate to the enzyme solution is preferably 0.5-1:0.5-1, more preferably 0.6-0.9:0.6-0.9, and further preferably 0.7-0.8:0.7-0.8.

[0059] In the present invention, the second mixing is preferably stirring mixing; the stirring speed is preferably 30 to 600 rpm, more preferably 100 to 500 rpm, and further preferably 400 to 500 rpm.

[0060] In the present invention, the speed of oxygen supply is preferably such that the reaction system solution does not foam and overflow.

[0061] In the present invention, the temperature of the enzymatic reaction is preferably 5-35°C, more preferably 10-30°C, further preferably 15-25°C, further preferably 20°C, the pH value of the enzymatic reaction system is preferably 4.5-6.5, more preferably 5.0-6.0, further preferably 5.5, the oxygen volume concentration is preferably 60-90%, more preferably 70-80%, and the insulation time is preferably 4-8 hours, more preferably 5-7 hours, further preferably 6 hours.

[0062] In the present invention, the enzymatic reaction preferably adopts A 380The inert adsorption carrier alcohol extract is tested to monitor the reaction progress; the test inert adsorption carrier alcohol extract is preferably: the test inert adsorption carrier alcohol extract is: take the immobilized substrate (wet state) and ethanol for 30 minutes of reaction, extract with ultrasonic oscillation at room temperature, and draw 10 mL of the supernatant; measure the absorbance of the supernatant at 380 nm, and stop the enzymatic reaction after the absorbance stops rising.

[0063] In the present invention, the mass of the immobilized substrate is preferably 2 g, the volume of the ethanol is preferably 50 mL; the volume concentration of the ethanol is preferably 95%; the time of the ultrasonic oscillation extraction is preferably 5 min; after absorbing 10 mL of the supernatant, the supernatant is preferably added to a 50 mL volumetric flask and the volume is fixed with ethanol with a volume concentration of 95%; the present invention preferably uses ethanol with a volume concentration of 95% as a blank control.

[0064] After obtaining the enzyme catalytic support, the present invention concentrates the eluate obtained by desorbing the enzyme catalytic support to obtain theaflavins. In the present invention, the desorption eluent is preferably 60-80 wt% edible ethanol, more preferably 70-75 wt% edible ethanol.

[0065] In the present invention, the enzyme catalytic support is preferably washed with water and then with alcohol before desorption; the water used for the water wash is preferably pure water; and the alcohol used for the alcohol wash is preferably 5-20 wt% edible ethanol, more preferably 10-15 wt% edible ethanol. The present invention removes impurities and purifies the enzyme catalytic support through water washing and alcohol washing.

[0066] In the present invention, the concentration is preferably vacuum concentration; the multiple of the vacuum concentration is preferably 10 to 20 times, more preferably 12 to 18 times, further preferably 14 to 16 times, and the vacuum degree is preferably -0.1 MPa; the target solid content of the concentration is preferably 30 to 40 wt%, more preferably 35 to 37 wt%.

[0067] In the present invention, the concentration preferably further includes drying the obtained concentrate; the drying is preferably freeze drying, spray drying or vacuum drying; the vacuum drying equipment is preferably a vacuum belt dryer.

[0068] The present invention provides a method for preparing theaflavins using an immobilized substrate, and the process flow is shown in Figure 1: In the present invention, a tea polyphenol solution is adsorbed by an inert adsorption carrier and purified, and then oxygen is passed through to carry out an enzymatic reaction. After the reaction, impurities are removed by washing with water and alcohol, and then the enzyme catalytic carrier loaded with the product is desorbed and concentrated to obtain theaflavins.

[0069] In order to further illustrate the present invention, the scheme of the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0070] Example 1

[0071] (1) Take 100 g of Fujian Fuding large-leaf green tea powder and stir extract it twice with 1 liter of 90℃ purified water, each time for 30 minutes. Filter the extract, combine the two filtered extracts, adjust the pH value to 4.0-5.5 with phosphoric acid, and cool the extract to room temperature for use.

[0072] (2) At room temperature, the tea extract was added to 100 g of LX-8 resin, and the mixture was stirred and adsorbed at 30 rpm for 2 hours, and the adsorption bottom liquid was filtered off; the resin was washed with 1 liter of purified water for 4 times, each time for 10 minutes, until the water washing liquid was basically clear and colorless, and the water washing liquid was filtered off; the resin was washed with 1 liter of 6% edible ethanol aqueous solution for 3 times, each time for 10 minutes, and the dilute ethanol washing liquid was filtered off; the resin after adsorption and purification was khaki and was set aside.

[0073] (3) Take 100 g of snow pear, freeze it and then thaw it naturally, add 100 ml of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with a pH value of 5.5, homogenize it, filter out the pear residue through four layers of gauze, and take the filtered pear juice for later use.

[0074] (4) Pour the pear juice into the purification resin of step (2), stir and oxygenate at 30°C, the oxygenation rate is 1 liter / minute, and the stirring speed is 400 rpm. Samples are taken from the resin every 30 minutes during the stirring process. After ethanol extraction, the A380 absorbance change is detected. The absorbance change is linearly increased and then leveled off. Stop the reaction after the absorbance value leveled off. The resin after the reaction changes from khaki to beautiful orange-red.

[0075] (5) The pear juice reaction solution was filtered out (the enzyme activity of the reaction solution was only 10% of the initial value), and the resin was washed 5 times with purified water adjusted to pH 4 with phosphoric acid, until the washing solution was clear and colorless, and the washing solution was filtered out.

[0076] (6) Wash the resin three times with 1 liter of 15% edible ethanol aqueous solution, stirring for 10 minutes each time, filter out the low-alcohol washing liquid, and try to filter out the low-alcohol washing liquid as much as possible in the last time.

[0077] (7) The resin was loaded into a column, and the theaflavins on the resin were eluted with 80% edible ethanol. The eluate was concentrated and dried in a vacuum evaporator to obtain 8.2 g of golden orange powder, with a yield of 8.2% based on the weight of the tea leaves. The total catechin content in the tea leaves was 12.6%, with a yield of 65% based on the total amount of catechins. The product was tested by HPLC, and the theaflavins content was 53.6%.

[0078] Example 2

[0079] (1) Take 100 g of Jiangxi Wuyuan small-leaf green tea powder and stir-extract it twice with 1 liter of 90°C purified water, each time for 30 minutes. Filter the extract, combine the two filtered extracts, adjust the pH value to 4.0-5.5 with phosphoric acid, and cool the extract to room temperature for use.

[0080] (2) At room temperature, the tea extract was added to 100 g of LX-5 resin, stirred and adsorbed at a speed of 30 rpm for 2.5 hours, and the adsorption bottom liquid was filtered off; the resin was washed with 1 liter of purified water for 4 times, each time for 10 minutes, until the water washing liquid was basically colorless, and the water washing liquid was filtered off; the resin was washed with 1 liter of 5% edible ethanol aqueous solution for 3 times, each time for 10 minutes, and the dilute ethanol washing liquid was filtered off; the resin after adsorption and purification was khaki and was set aside.

[0081] (3) Take 100 g of snow pear, freeze it and then thaw it naturally, add 50 ml of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with a pH value of 5.5, homogenize it, filter out the pear residue through four layers of gauze, and take the filtered pear juice for later use.

[0082] (4) Pour the pear juice into the purification resin of step (2), stir and oxygenate at 30°C, the oxygenation rate is 1 liter / minute, and the stirring speed is 400 rpm. Samples are taken from the resin every 30 minutes during the stirring process. After ethanol extraction, the A380 absorbance change is detected. The absorbance change is linearly increased and then leveled off. Stop the reaction after the absorbance value leveled off. The resin after the reaction exhibits a beautiful orange-red color.

[0083] (5) The pear juice reaction solution was filtered out (the enzyme activity of the reaction solution was only 11% of the initial value), and the resin was washed 5 times with purified water adjusted to pH 4 with phosphoric acid, until the washing solution was clear and colorless, and the washing solution was filtered out.

[0084] (6) Wash the resin three times with 1 liter of 12% edible ethanol aqueous solution, stirring for 10 minutes each time, filter out the low-alcohol washing liquid, and try to filter out the low-alcohol washing liquid as much as possible in the last time.

[0085] (7) The resin was loaded into a column, and the theaflavins on the resin were eluted with 80% edible ethanol. The eluate was concentrated and dried in a vacuum evaporator to obtain 7.6 g of golden orange powder, with a yield of 7.6% based on the weight of the tea leaves. The total catechin content in the tea leaves was 11.3%, with a yield of 67% based on the total amount of catechins. The product was tested by HPLC, and the theaflavins content was 56.7%.

[0086] Example 3

[0087] (1) Weigh 10 g of a commercially available low-caffeine tea polyphenol TP90 product and dissolve it in 200 ml of purified water. The TP90 product has EGCG ≥ 40%, EGC ≥ 10%, a total amount of catechins 71%, and caffeine ≤ 1%.

[0088] (2) At room temperature, add the above solution to 100 g of AB-8 resin, adjust the pH value of the solution to 4 with phosphoric acid, stir and adsorb at a speed of 30 rpm for 2 hours, and filter out the adsorption bottom liquid; wash the resin with 1 liter of pure water twice, stirring for 5 minutes each time, until the washing liquid is basically colorless, and filter out the washing liquid; the resin after adsorption and purification is brown-yellow and is ready for use.

[0089] (3) Take 100 g of snow pear, freeze it and then thaw it naturally, add 50 ml of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with a pH value of 5.5, homogenize it, filter out the pear residue through four layers of gauze, and take the filtered pear juice for later use.

[0090] (4) Pour the pear juice into the purification resin of step (2), stir and oxygenate at 30°C, the oxygenation rate is 1 liter / minute, and the stirring speed is 500 rpm. Samples are taken from the resin every 30 minutes during the stirring process. After ethanol extraction, the A380 absorbance change is detected. The absorbance change is linearly increased and then leveled off. Stop the reaction after the absorbance value leveled off. The resin after the reaction exhibits a beautiful deep orange-red color.

[0091] (5) The pear juice reaction solution was filtered out (the enzyme activity of the reaction solution was only 4% of the initial value), and the resin was washed four times with purified water adjusted to pH 4 with phosphoric acid until the washing solution was clear and colorless, and the washing solution was filtered out.

[0092] (6) Wash the resin three times with 1 liter of 10% edible ethanol aqueous solution, stirring for 10 minutes each time, filter out the low-alcohol washing liquid, and try to filter out the low-alcohol washing liquid as much as possible in the last time.

[0093] (7) The resin was loaded into a column and the theaflavins on the resin were eluted with 65% edible ethanol. The eluate was concentrated and dried in a vacuum evaporator to obtain 7.5 g of golden orange powder. The yield was 75% based on the raw material tea polyphenols. The product was tested by HPLC and the theaflavins content was 62.7%.

[0094] Example 4

[0095] (1) Weigh 10 g of commercially available low-caffeine tea polyphenols TP95 product and dissolve it in 200 ml of purified water. The TP95 product has EGCG ≥ 45%, EGC ≥ 10%, a total amount of catechins 75.6%, and caffeine ≤ 1%.

[0096] (2) At room temperature, the above solution was added to 100 g of NKA-9 resin, and the pH value of the solution was adjusted to 4 with phosphoric acid. The mixture was stirred and adsorbed at a speed of 30 rpm for 2 hours, and the adsorption bottom liquid was filtered out. The resin was washed with 1 liter of pure water for 5 minutes each time, and the washing liquid was basically colorless. The washing liquid was filtered out. The resin after adsorption and purification turned brown and was set aside.

[0097] (3) Take 100 g of snow pear, freeze it and then thaw it naturally, add 30 ml of disodium hydrogen phosphate-sodium phosphate buffer with a pH value of 5.5, homogenize it, filter out the pear residue through four layers of gauze, and take the filtered pear juice for later use.

[0098] (4) Pour the pear juice into the purification resin prepared in step (2), stir and aerate at 30°C at a rate of 1 L / min and a stirring speed of 500 rpm. Sample the resin every 30 minutes during the stirring process. After ethanol extraction, measure the A380 absorbance change. The absorbance change is linearly increasing and then leveling off. Stop the reaction when the absorbance value leveling off. The resin after the reaction is a beautiful deep orange-red color.

[0099] (5) The pear juice reaction solution was filtered out (the enzyme activity of the reaction solution was only 4% of the initial value), and the resin was washed four times with purified water adjusted to pH 4 with phosphoric acid until the washing solution was clear and colorless, and the washing solution was filtered out.

[0100] (6) Wash the resin three times with 1 liter of 15% edible ethanol aqueous solution, stirring for 10 minutes each time, filter out the low-alcohol washing liquid, and try to filter out the low-alcohol washing liquid as much as possible in the last time.

[0101] (7) The resin was loaded into a column and the theaflavins on the resin were eluted with 75% edible ethanol. The eluate was concentrated and dried in a vacuum evaporator to obtain 7.3 g of golden yellow powder. The yield was 73% based on the raw material tea polyphenols. The product was tested by HPLC and the theaflavins content was 63.9%.

[0102] The theaflavins prepared in this example were subjected to HPLC analysis using the following high performance liquid chromatograph: Shimadzu LC-10ATVP system controller, LC-10ATVP binary pump, SPD-M20A diode array detector, Shimadzu LC-Solution chromatography workstation; chromatographic analysis parameters: chromatographic column: Hypersil BDS (C18, 5μ, 4.6×250 mm); mobile phase A: 2% acetic acid; mobile phase B: acetonitrile-ethyl acetate (volume ratio: 21:3); gradient elution: mobile phase B: linear gradient from 18% to 30% over 30 min; flow rate: 0.9 mL / min; column temperature: 40°C; detection wavelength: 280 nm; injection volume: 10 μL; standards of the four monomer components of theaflavins were purchased externally, and a standard calibration curve was constructed using the external standard method. The resulting detection spectrum is shown in FIG2 . As can be seen from FIG2 , the present invention adopts the immobilized substrate free enzyme catalysis technology to catalyze and obtain balanced four groups of theaflavins: TF, TF-3-G, TF-3'-G, and TFDG. This shows that the method provided by the present invention has high enzyme catalysis efficiency.

[0103] As can be seen from the above examples, the method provided by the present invention has low cost, good catalytic effect, simple steps, and convenient operation, which greatly simplifies the operational difficulty of the process and is green and environmentally friendly.

[0104] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing theaflavins using an immobilized substrate, comprising the following steps: (1) Mix a tea polyphenol solution with an inert adsorption carrier for adsorption, followed by washing with water and then with alcohol successively to obtain an immobilized substrate; the inert adsorption carrier includes macroporous adsorption resin; the pore size of the macroporous adsorption resin is 10 - 180 Å; the concentration of the alcohol solution used for alcohol washing is 4 - 20 wt%; (2) Mix the immobilized substrate with an enzyme solution and conduct an enzymatic reaction while passing oxygen to obtain an enzyme - catalyzed carrier; the enzyme in the enzyme solution is polyphenol oxidase PPO; (3) Concentrate the eluate obtained by desorbing the enzyme - catalyzed carrier to obtain theaflavins.

2. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the tea polyphenol solution is 0.5 - 100 g / L.

3. The preparation method according to claim 1 or 2, characterized in that, In step (1), the mass ratio of tea polyphenols in the tea polyphenol solution to the inert adsorption carrier is 3 - 15:

100.

4. The preparation method according to claim 1, characterized in that, In step (1), the temperature of adsorption is 5 - 35 °C, the pH value of the adsorption system is 4.0 - 5.5, and the heat - preservation time is 1 - 5 h.

5. The preparation method according to claim 1, characterized in that, In step (1), the macroporous adsorption resin includes one or more of LX - 8 resin, LX - 5 resin, AB - 8 resin, NKA resin, and NKA - 9 resin; The inert adsorption carrier also includes one or more of silica gel and alumina.

6. The preparation method according to claim 1 or 2, characterized in that, In step (1), the preparation method of the tea polyphenol solution includes Method 1, Method 2, or Method 3; Method 1 includes the following steps: Stir - extract tea leaves with hot water at 65 - 90 °C; Method 2 includes the following steps: Mix commercially available tea polyphenol powder with a content of 30 - 99 wt% and water; Method 3 includes the following steps: Enrich a tea polyphenol solution with a concentration lower than the target concentration.

7. The preparation method according to claim 6, characterized in that, In Method 1, the number of extraction times is more than 1, and the single - extraction time is 20 - 60 min; the tea leaves are one or more of large - leaf green tea and small - leaf green tea.

8. The preparation method according to claim 6, characterized in that, In Method 2, the commercially available tea polyphenol powder with a content of 30 - 99 wt% includes one or more of tea polyphenol TP30 product - tea polyphenol TP99 product.

9. The preparation method according to claim 1, characterized in that, In step (2), the volume ratio of the immobilized substrate to the enzyme solution is 0.5 - 1:0.5 - 1.

10. The preparation method according to claim 1 or 9, characterized in that, In step (2), the temperature of the enzymatic reaction is 5 - 35 °C, the pH value of the enzymatic reaction system is 4.5 - 6.5, the volume concentration of oxygen is 60 - 90%, and the heat - preservation time is 4 - 8 hours.

11. The preparation method according to claim 1 or 10, characterized in that, The enzymatic reaction uses A 380 Test the alcohol extract of the inert adsorption carrier and monitor the progress of the reaction; The test inert adsorption carrier alcohol - extract is: Mix the immobilized substrate and ethanol every 30 min of the reaction, ultrasonically oscillate and extract at room temperature, and aspirate the supernatant; Measure the absorbance value of the supernatant at 380 nm, and stop the enzymatic reaction after the absorbance value stops rising.

12. The preparation method according to claim 1 or 9, characterized in that, In step (2), the enzyme solution is polyphenol oxidase from Novozymes or laccase from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., or pear juice obtained by filtering pear homogenate, or crude enzyme solution obtained by filtering fresh tea leaf homogenate.

13. The preparation method according to claim 12, characterized in that, The pear is Sydney pear; the filtering of pear homogenate is: Freeze and then thaw the Sydney pear, then mix the thawed Sydney pear and pH buffer and homogenize, and filter out pear residues with four - layer gauze.

14. The preparation method according to claim 13, wherein The mass ratio of the Sydney pear to the pH buffer is 1:0.2 - 1; the pH value of the pH buffer is 5.5 - 6.

5.

15. The preparation method according to claim 13 or 14, characterized in that, The pH buffer solution is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution.

16. The preparation method according to claim 1, characterized in that, In step (3), the eluent for desorption is edible ethanol with a concentration of 60-80 wt%.

Images