阅读说明：本技术 一种循环利用多酚氧化酶制备茶黄素的方法 (Method for preparing theaflavin by recycling polyphenol oxidase ) 是由 李定忠 沈宗毕 于 2019-11-06 设计创作，主要内容包括：本发明提供一种循环利用多酚氧化酶制备茶黄素的方法,包括下列各步骤：1)在发酵液中加入多酚氧化酶液、茶液；2)按0.5～2个单位/分钟的量,通入氧气,于20～40℃温度、60～150r/min转速下,发酵40～150min；3)在惰性气体、发酵液温度为20～35℃的环境中过滤；4)透过液经纯化、浓缩、干燥,得茶黄素；多酚氧化酶液返回步骤1)的发酵液中参与发酵,以回收、循环利用多酚氧化酶液。得高纯度茶黄素,回收、循环利用多酚氧化酶液,保持多酚氧化酶的活性,避免多酚氧化酶的浪费,大幅度降低茶黄素的生产成本,便于连续化生产,安全可靠。(The invention provides a method for preparing theaflavin by recycling polyphenol oxidase, which comprises the following steps: 1) adding polyphenol oxidase solution and tea solution into the fermentation liquor; 2) introducing oxygen according to the amount of 0.5-2 units/min, and fermenting for 40-150 min at the temperature of 20-40 ℃ and the rotating speed of 60-150 r/min; 3) filtering in an environment with inert gas and fermentation liquor temperature of 20-35 ℃; 4) purifying, concentrating and drying the permeate to obtain theaflavin; and (3) returning the polyphenol oxidase liquid to the fermentation liquid in the step 1) to participate in fermentation so as to recycle and recycle the polyphenol oxidase liquid. The high-purity theaflavin is obtained, the polyphenol oxidase liquid is recycled, the activity of the polyphenol oxidase is kept, the waste of the polyphenol oxidase is avoided, the production cost of the theaflavin is greatly reduced, the continuous production is convenient, and the method is safe and reliable.)

1. A method for preparing theaflavin by recycling polyphenol oxidase is characterized by comprising the following steps:

1) adding polyphenol oxidase liquid into the fermentation liquor until the mass concentration of fresh tea leaves or plants containing exogenous enzymes is 10-40%, adding tea liquid until the mass concentration of tea polyphenols in the fermentation liquor is 0.5-3.0%, and adjusting the pH value of the fermentation liquor to 3.5-6.5;

2) introducing oxygen into the fermentation liquor obtained in the step 1) according to the amount of 0.5-2 units/min, and fermenting for 40-150 min at the temperature of 20-40 ℃ and the rotating speed of 60-150 r/min to obtain the fermentation liquor;

3) feeding the fermentation liquor obtained in the step 3) into a separation membrane with an interception molecular weight of 5-20 ten thousand Da, filtering in an environment with inert gas and fermentation liquor at the temperature of 20-35 ℃ to obtain a permeate and an interception liquid, and washing the interception liquid for 2-3 times by using a proper amount of buffer solution with the pH value of 3.5-6.5 when the solid content of the interception liquid reaches 8-12% to obtain polyphenol oxidase liquid;

4) purifying, concentrating and drying the permeate liquid obtained in the step 3) by a conventional method to obtain theaflavin;

5) and returning the polyphenol oxidase liquid obtained in the step 3) to the fermentation liquid obtained in the step 1) to participate in fermentation so as to recover and recycle the polyphenol oxidase liquid.

2. The method for preparing theaflavins by recycling polyphenol oxidase as claimed in claim 1, wherein said buffer of step 3) is phosphate buffer.

3. The method for preparing theaflavins by recycling polyphenol oxidase as claimed in claim 1, wherein the permeate of step 2) is purified, concentrated and dried by the following method:

31) feeding the permeation solution into a chromatographic column at a flow rate of 0.5-2.0 BV/h, eluting 3-5 column volumes (BV) with purified water and 15-35% ethanol in sequence at an elution flow rate of 1-3 BV/h, eluting 2-4 column volumes (BV) with 60-80% ethanol in volume fraction and at an elution flow rate of 1-2 BV/h, and collecting 60-80% ethanol eluent to obtain a purified solution;

32) adding a food-grade antioxidant into the purified solution obtained in the step 31) according to the amount of 30-50 mg/Kg, and concentrating by using a separation membrane with the cut-off molecular weight of 100-500 Da, or concentrating under reduced pressure conventionally to obtain a concentrated solution;

33) and (3) carrying out conventional vacuum freeze drying or vacuum drying or spray drying on the concentrated solution obtained in the step 32) to obtain the high-purity theaflavin.

4. The method for preparing theaflavins by recycling polyphenol oxidase as claimed in claim 3, wherein the chromatography column packing of step 31) is MCI resin, or one or more of macroporous resin D130, AB-8, D101, NKA-9, HPD300 and HPD 10; the antioxidant of step 22) is ascorbic acid.

5. The method for preparing theaflavins by recycling polyphenol oxidase as claimed in claim 1, wherein the polyphenol oxidase solution of step 1) is prepared by the following method:

11) crushing fresh tea leaves or plants containing exogenous enzymes into mud, adding phosphate buffer solution with the pH value of 3.5-6.5 which is 3-5 times of the weight of the fresh tea leaves or plants containing exogenous enzymes, and uniformly mixing to obtain a mixture;

12) adding crosslinked polyvinylpyrrolidone (PVPP) into the mixture of step 11) according to the mass ratio of fresh tea leaves or plants containing exogenous enzyme to the crosslinked polyvinylpyrrolidone (PVPP) = 1000: 0.02-0.2, mixing and emulsifying for 5-20 min, and filtering to obtain polyphenol oxidase solution.

6. The method for preparing theaflavins by recycling polyphenol oxidase of claim 5 wherein said phosphate buffer is a citric acid-disodium hydrogen phosphate buffer formulated by the following method: 10-40g of disodium hydrogen phosphate and 11-33g of citric acid monohydrate are added into 100L of purified water, and the mixture is stirred until the solid is completely dissolved.

7. The method for preparing theaflavins by recycling polyphenol oxidase as claimed in claim 5, wherein said phosphate buffer of step 11) is citric acid-disodium hydrogen phosphate buffer containing 20-40% volume fraction ethanol, and is prepared by the following method: adding 10-40g of disodium hydrogen phosphate and 11-33g of citric acid monohydrate into 100L of 20-40% ethanol aqueous solution by volume fraction, and stirring until the solid is completely dissolved.

8. The method for preparing theaflavins by recycling polyphenol oxidase as claimed in claim 5, wherein said exogenous enzyme-containing plant of step 11) is: one or more fruits selected from apple, pear, grape, banana, blueberry, loquat, pineapple, watermelon, orange, peach, watermelon, mango, kiwi fruit and persimmon in any ratio; or one or more vegetables selected from fructus Solani Melongenae, fructus Lycopersici Esculenti, rhizoma Nelumbinis, rhizoma Solani Tuber osi, sweet potato, herba Spinaciae, fructus Cucurbitae Moschatae, rhizoma Dioscoreae, radix Dauci Sativae, and fructus Colocasiae Esculentae at any ratio; or one of kidney bean and soybean; or one or more of semen Setariae, semen Maydis, semen Tritici Aestivi, fructus Hordei vulgaris, and semen Fagopyri Esculenti; or leaf of Forsythia deciduous shrub of Forsythia of Oleaceae.

9. The method for preparing theaflavins by recycling polyphenol oxidase as claimed in claim 1, wherein said tea liquid is green tea extract or commercially available tea polyphenol solution, wherein green tea extract is prepared by the following method:

crushing tea leaves, sieving the crushed tea leaves with a 20-50-mesh sieve, adding purified water according to the feed liquid mass ratio of 1: 10-20, extracting the tea leaves for 30-90 min at the temperature of 70-95 ℃, cooling an extracting solution to 25-45 ℃, clarifying the extracting solution by using a clarification membrane with the molecular weight of 30 ten thousand Da, and then performing reverse osmosis concentration to obtain a green tea extracting solution.

Technical Field

The invention relates to a method for preparing theaflavin, in particular to a method for preparing theaflavin by recycling polyphenol oxidase, belonging to the technical field of biology.

Technical Field

Theaflavins (TFs), originally discovered by Roberts e.a.h (1957), are a generic name for compounds that are soluble in ethyl acetate and have an orange yellow color and a benzophenone structure, formed by oxidation of polyphenols. The theaflavin components with the clear structures comprise 25 components, wherein the main components comprise: theaflavin (TF), theaflavin-3-monogallate (theaflavin-3-gallate, TF-3-G), theaflavin-3 '-monogallate (theaflavin-3' -gallate, TF-3 '-G), and theaflavin-3, 3' -digallate (theaflavin-3, 3 '-gallate, TF-3, 3' -G), and epigallocatechin-3-gallate (TF 4), among others. The theaflavin content in the black tea is generally 0.3% -1.5%, the theaflavin is a main component in the black tea, plays a decisive role in the color, aroma and quality of the black tea, can make the tea color "bright", has strong taste and freshness, is known as "soft gold" in the tea, and has high safety. The theaflavin is not only an important quality component of the black tea, but also has various potential effects related to human health, such as antioxidation, cancer prevention and resistance, blood fat reduction, cardiovascular disease prevention, diabetes resistance, antibiosis, antivirus and the like, and in addition, the theaflavin is also a natural colorant of cosmetics and foods. Therefore, the method is widely concerned at home and abroad and becomes a research hotspot.

Currently, there are roughly three methods for preparing theaflavins: extraction, chemical oxidation, enzymatic oxidation. An extraction method comprises the following steps: because the theaflavin content in the black tea is low, the difficulty of directly extracting and purifying the theaflavin from the black tea is high, and the yield is low; chemical oxidation method: a large amount of potassium ferricyanide, ferric trioxide, magnesium oxide, acid-base and other chemical reagents are needed, the environmental pollution is serious, more sewage treatment cost is needed, and the purity and safety of the product are limited due to the fact that a chemical method lacks substrate specificity, byproducts are complex; the enzymatic oxidation method has mild reaction conditions and strong controllability, can avoid the defects of the two methods, and is a more suitable method for in vitro enzymatic oxidation. However, in the industrial production of theaflavins by enzymatic oxidation, because polyphenol oxidase is not recovered and reused, after a single fermentation is completed, in order to prevent theaflavins from being oxidized continuously in the subsequent purification, concentration and drying processes, enzyme inactivation and inactivation are only carried out by stopping the reaction, thus leading to the waste of polyphenol oxidase, leading to the increase of production cost by 30-50%, and the enzyme inactivation and inactivation are usually carried out by heat inactivation, but theaflavins have the problem of poor heat stability, thus leading to the oxidative degradation of theaflavins with low extraction rate.

In the prior art of producing theaflavin by enzymatic oxidation, chinese patents such as CN1155717C and CN02071232A have better enzyme stability after immobilization, but the technology of fermenting theaflavin by immobilized polyphenol oxidase still has the following disadvantages:

in the stage of extracting and purifying polyphenol oxidase and immobilizing the polyphenol oxidase, the activity loss of the polyphenol oxidase is serious, and particularly in the stage of immobilizing the polyphenol oxidase, the activity of the immobilized polyphenol oxidase is reduced to about 50 percent of that before immobilization;most of solvents used for extracting polyphenol oxidase are toxic acetone, so that the environment is polluted, and toxic solvents are contained in the product to influence the application of the product, thereby limiting the industrial development of theaflavin.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a method for preparing theaflavin by recycling polyphenol oxidase, and provide products such as theaflavin powder, liquid beverage, tablets, capsules and the like which are convenient to carry and drink for people.

The invention is realized by the following technical scheme: a method for preparing theaflavin by recycling polyphenol oxidase is characterized by comprising the following steps:

1) adding polyphenol oxidase liquid into the fermentation liquor until the mass concentration of fresh tea leaves or plants containing exogenous enzymes is 10-40%, adding tea liquid until the mass concentration of tea polyphenols in the fermentation liquor is 0.5-3.0%, and adjusting the pH value of the fermentation liquor to 3.5-6.5;

2) introducing oxygen into the fermentation liquor obtained in the step 1) according to the amount of 0.5-2 units/min, and fermenting for 40-150 min at the temperature of 20-40 ℃ and the rotating speed of 60-150 r/min to obtain the fermentation liquor;

3) feeding the fermentation liquor obtained in the step 2) into a separation membrane with an interception molecular weight of 5-20 ten thousand Da, filtering in an environment with inert gas and fermentation liquor temperature of 20-35 ℃ to obtain a permeate and an interception liquid, and when filtering is carried out until the solid content of the interception liquid reaches 8-12%, washing the interception liquid for 2-3 times by using a proper amount of buffer solution with a pH value of 3.5-6.5 to obtain polyphenol oxidase liquid;

4) purifying, concentrating and drying the permeate liquid obtained in the step 3) by a conventional method to obtain theaflavin;

5) and returning the polyphenol oxidase liquid obtained in the step 3) to the fermentation liquid obtained in the step 1) to participate in fermentation so as to recover and recycle the polyphenol oxidase liquid.

The buffer solution in the step 3) is phosphate buffer solution.

Purifying, concentrating and drying the permeate liquid obtained in the step 2) by the following method:

21) feeding the permeation solution into a chromatographic column at a flow rate of 0.5-2.0 BV/h, eluting 3-5 column volumes (BV) with purified water and 15-35% ethanol in sequence at an elution flow rate of 1-3 BV/h, eluting 2-4 column volumes (BV) with 60-80% ethanol in volume fraction and at an elution flow rate of 1-2 BV/h, and collecting 60-80% ethanol eluent to obtain a purified solution;

22) adding a food-grade antioxidant into the purified solution obtained in the step 21) according to the amount of 30-50 mg/Kg, and concentrating by using a separation membrane with the cut-off molecular weight of 100-500 Da, or concentrating under reduced pressure conventionally to obtain a concentrated solution;

23) and (3) carrying out conventional vacuum freeze drying or vacuum drying or spray drying on the concentrated solution obtained in the step (22) to obtain the high-purity theaflavin.

The chromatography column of the step 21) is filled with MCI resin or one or more of macroporous resin D130, AB-8, D101, NKA-9, HPD300 and HPD 10.

The antioxidant of step 22) is ascorbic acid.

The polyphenol oxidase solution of step 1) is prepared by the following method:

11) crushing fresh tea leaves or plants containing exogenous enzymes into mud, adding phosphate buffer solution with the pH value of 3.5-6.5 which is 3-5 times of the weight of the fresh tea leaves or plants containing exogenous enzymes, and uniformly mixing to obtain a mixture;

12) adding crosslinked polyvinylpyrrolidone (PVPP) into the mixture of step 11) according to the mass ratio of fresh tea leaves or plants containing exogenous enzyme to the crosslinked polyvinylpyrrolidone (PVPP) = 1000: 0.02-0.2, mixing and emulsifying for 5-20 min, and filtering to obtain polyphenol oxidase solution.

The phosphate buffer solution is a citric acid-disodium hydrogen phosphate buffer solution and is prepared by the following method: 10-40g of disodium hydrogen phosphate and 11-33g of citric acid monohydrate are added into 100L of purified water, and the mixture is stirred until the solid is completely dissolved.

The phosphate buffer solution in the step 11) and the step 3) is a citric acid-disodium hydrogen phosphate buffer solution containing 20-40% of ethanol by volume fraction, and is prepared by the following method: adding 10-40g of disodium hydrogen phosphate and 11-33g of citric acid monohydrate into 100L of 20-40% ethanol aqueous solution by volume fraction, and stirring until the solid is completely dissolved.

The plant containing the exogenous enzyme in the step 11) is as follows: one or more fruits selected from apple, pear, grape, banana, blueberry, loquat, pineapple, watermelon, orange, peach, mango, kiwi fruit and persimmon in any ratio; or one or more vegetables selected from fructus Solani Melongenae, fructus Lycopersici Esculenti, rhizoma Nelumbinis, rhizoma Solani Tuber osi, sweet potato, herba Spinaciae, fructus Cucurbitae Moschatae, rhizoma Dioscoreae, radix Dauci Sativae, and fructus Colocasiae Esculentae at any ratio; or one of kidney bean and soybean; or one or more of semen Setariae, semen Maydis, semen Tritici Aestivi, fructus Hordei vulgaris, and semen Fagopyri Esculenti; or leaf of Forsythia deciduous shrub of Forsythia of Oleaceae.

The tea liquid is green tea extract or tea polyphenol solution purchased from the market, wherein the green tea extract is prepared by the following method:

crushing tea leaves, sieving the crushed tea leaves with a 20-50-mesh sieve, adding purified water according to the feed liquid mass ratio of 1: 10-20, extracting the tea leaves for 30-90 min at the temperature of 70-95 ℃, cooling an extracting solution to 25-45 ℃, clarifying the extracting solution by using a clarification membrane with the molecular weight of 30 ten thousand Da, and then performing reverse osmosis concentration to obtain a green tea extracting solution.

For convenience in carrying and drinking, the theaflavin product can be made into powder, liquid, tablet, capsule, pill, suspension, etc.; wherein the theaflavin content in the liquid is 0.3-1.2 g/L, 0.40-0.8 g/L is selected, the concentration of theaflavin is too high, the astringency is too heavy, and most people cannot accept the theaflavin; too low to achieve health care effect; the solution of the liquid is purified water, or appropriate concentration of fruit juice, cow milk or sheep milk, or carbonated beverage containing carbon dioxide gas.

A proper amount of conventional dietetic auxiliary materials or/and pharmaceutic auxiliary materials can be added into the theaflavin prepared by the invention, and the theaflavin-containing food and health care products or powder, solution, tablets, capsules and pills which are convenient to carry and drink are prepared by a conventional method; the auxiliary materials are maltodextrin, sucrose, lactose, galactose, corn starch, gelatin, microcrystalline cellulose, superfine silica gel powder, carboxymethyl cellulose and the like which are used as carriers or excipients.

In order to stabilize theaflavin in the preparation, a proper amount of food grade antioxidant can be added.

Appropriate amount of food-grade sodium sulfite, sodium bisulfite, vitamin C, etc. can be added into theaflavin.

The invention has the following advantages and effects: by adopting the scheme, high-purity theaflavin can be prepared, polyphenol oxidase liquid can be recovered and recycled to the greatest extent, the activity and safety of polyphenol oxidase are kept, the waste of polyphenol oxidase is avoided, the production cost of theaflavin is greatly reduced, continuous production is facilitated, a thermal inactivation passivation process is omitted by recovering polyphenol oxidase in fermentation liquor, toxic solvents are not used, safety and reliability are realized, the process is simplified, the thermal degradation of theaflavin caused by the thermal inactivation passivation process is reduced, the polyphenol oxidase can be continuously recovered and utilized for at least 10 times, the enzyme activity still keeps more than 85% of the original level, and reliable technical support is provided for the industrial production process of theaflavin.

Drawings

FIG. 1 is a process flow diagram of the present invention.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following examples which are intended to illustrate the invention. The following examples are merely illustrative of the present invention and the scope of the present invention is not limited to the following examples. All the technologies realized based on the above-mentioned summary of the invention belong to the scope of the present invention.