阅读说明：本技术 一种利用安吉白茶修剪枝叶生产茶黄素的方法 (Method for producing theaflavin by utilizing branches and leaves of Anji white tea pruning ) 是由 田伟 程勇 邵云东 阎卫东 丁建宝 程琨 陈�峰 于 2020-06-01 设计创作，主要内容包括：本发明属于茶黄素提取技术领域,具体公开了一种利用安吉白茶修剪枝叶生产茶黄素的方法,该方法包括缓冲液的制备、酶液的制备、底物溶液的制备、发酵、过滤、萃取以及浓缩干燥步骤,本发明选用安吉白茶修剪枝叶作为酶源,既不引入外源性酶源又实现废弃资源的再利用,有效增加经济效益；并采用双液相发酵方法生产茶黄素,通过对酶液、缓冲液、酶促反应体系等条件的优化,显著提高了酶的转化率,并且反应稳定,能够持续高效进行,茶黄素得率高,因此能够实现产业化。此外,本发明发酵液过滤后的滤饼仍旧具有良好的活性,可作为二次酶源循环利用。(The invention belongs to the technical field of theaflavin extraction, and particularly discloses a method for producing theaflavin by utilizing Anji white tea pruning branches and leaves, which comprises the steps of buffer solution preparation, enzyme solution preparation, substrate solution preparation, fermentation, filtration, extraction, concentration and drying, wherein the Anji white tea pruning branches and leaves are used as an enzyme source, so that the exogenous enzyme source is not introduced, the reutilization of waste resources is realized, and the economic benefit is effectively increased; the method adopts a double-liquid-phase fermentation method to produce theaflavin, obviously improves the conversion rate of enzyme by optimizing the conditions of enzyme solution, buffer solution, enzymatic reaction system and the like, has stable reaction, can be continuously and efficiently carried out, and has high theaflavin yield, thereby realizing industrialization. In addition, the filter cake obtained after the fermentation liquor is filtered still has good activity and can be recycled as a secondary enzyme source.)

1. A method for producing theaflavin by utilizing branches and leaves of Anji white tea pruning is characterized by comprising the following steps: the method comprises the following steps:

s1, preparing a buffer solution: adding salt into water until the mass concentration of the salt is 0.1-10%, and then adding citric acid to adjust the pH value to 4.8-5.2; the salt is (NH)₄)₂SO₄、Na₂HPO₄、K₂CO₃、Na₂SO₄、K₂HPO₄、Na₂CO₃、CaCl₂、KH₂PO₄Or NaCl;

s2, preparation of enzyme solution: collecting fresh Anji white tea, pruning branches and leaves, and adding a buffer solution according to the mass ratio of (45-55) to 1; crushing by a crusher for 2 times, and placing in a refrigeration house for later use;

s3, preparation of a substrate solution: mixing the tea polyphenol solution with a buffer solution to prepare a substrate solution, wherein the substrate concentration is 3-8% by mass percent;

s4, fermentation: mixing the enzyme solution and the substrate solution according to the mass ratio of 1: 5-8, and adding ethyl acetate according to 0.5-1 time of the volume of the substrate solution to form a two-phase reaction system; heating to 30-45 ℃, introducing air or oxygen according to the amount of 0.5-2 units/min, stirring at the rotating speed of 60-150 r/min, and fermenting for 6-12 hours;

s5, filtering: filtering the fermentation liquor in a plate-and-frame filter press; after feeding is finished, compressed air is fed to take away residual feed liquid in the plate frame, and a filter cake is dried; the filter cake can be used as a secondary enzyme source for recycling, and the filtrate is used for the next extraction;

s6, extraction: mixing the filtrate and ethyl acetate according to a volume ratio of 1: (2.5-3.5), extracting, stirring for 20-30 min, standing for 1-2 h for thoroughly layering, and taking an upper organic layer;

s7, concentrating and drying: concentrating the organic layer under reduced pressure, controlling the vacuum degree to be more than 0.06MPa, removing the solvent ethyl acetate, supplementing water for many times, and removing the ethyl acetate residue; and spray drying the extract, and collecting dry powder to obtain the theaflavin product.

2. The method for producing theaflavins by utilizing pruned branches and leaves of Anji white tea as claimed in claim 1, characterized in that: in step S1, the salt is Na₂HPO₄。

3. The method for producing theaflavins by utilizing pruned branches and leaves of Anji white tea as claimed in claim 1, characterized in that: and S2, selecting a 5-10 cm trimmed part from the trimmed branches and leaves of the Anji white tea.

4. The method for producing theaflavins by utilizing pruned branches and leaves of Anji white tea as claimed in claim 1, characterized in that: and S2, the grinding mesh number of the trimmed branches and leaves of the Anji white tea is 200-260 meshes.

5. The method for producing theaflavins by utilizing pruned branches and leaves of Anji white tea as claimed in claim 1, characterized in that: the tea polyphenol solution in the step S3 is green tea extract, and the preparation method comprises the following steps: crushing tea leaves, sieving the crushed tea leaves with a 20-50-mesh sieve, adding purified water according to the mass ratio of feed liquid of 1 to (10-20), extracting at the temperature of 70-95 ℃ for 30-90 min, cooling an extracting solution to 25-45 ℃, clarifying with a clarifying membrane with the molecular weight of 30 ten thousand Da, and then performing reverse osmosis concentration to obtain a green tea extracting solution.

6. The method for producing theaflavins by utilizing pruned branches and leaves of Anji white tea as claimed in claim 1, characterized in that: and S4, heating to 30-35 ℃ during fermentation, wherein the fermentation time is 8-12 h.

7. The method for producing theaflavins by utilizing pruned branches and leaves of Anji white tea as claimed in claim 1, characterized in that: feeding the substrate solution in batches at least three times in step S4, wherein the feeding amount of the next batch is not more than that of the previous batch; the batch feeding interval time is not less than 20 min.

8. The method for producing theaflavins by utilizing pruned branches and leaves of Anji white tea as claimed in claim 7, characterized in that: the concentration of the substrate in the step S3 is 5%, and the substrate solution is divided into three batches according to the mass ratio of 2:2:1 in the step S4, and the three batches are fed at an interval of 30 min.

9. The method for producing theaflavins by utilizing pruned branches and leaves of Anji white tea as claimed in claim 1, characterized in that: and S5, adopting filter cloth with the aperture of 280-320 meshes by the plate-and-frame filter press, and controlling the feeding speed to be 200-300L/h.

10. The method for producing theaflavins by utilizing pruned branches and leaves of Anji white tea as claimed in claim 1, characterized in that: in the concentration process of the step S7, the temperature is controlled to be less than 80 ℃, and the relative density is 1.08-1.12 as the concentration end point; adding water in the following amount before collecting paste: adding water into the extract (mass ratio) =1 (2.5-3.5), removing solvent residues, and adding water at least three times.

Technical Field

The invention relates to the technical field of theaflavin extraction, and in particular relates to a method for producing theaflavin by utilizing Anji white tea pruning branches and leaves.

Background

Theaflavin is a golden yellow pigment existing in black tea, is a substance with a polyphenol hydroxyl group having a theapolyphenol ketone structure, is obtained by oxidizing and condensing polyphenols and derivatives thereof, and has been found out to have 12 kinds, wherein Theaflavin (TFs), theaflavin-3-gallate (TF-3-G), theaflavin-3 '-gallate (TF-3' -G) and theaflavin digallate (TFDG) are four most main theaflavins. The content of theaflavins in the black tea is generally 0.3-1.5%, and the tea plays a decisive role in the color, the smell and the quality of the black tea. The theaflavin is not only an important quality component of the black tea, but also has a plurality of potential efficacies 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 a natural colorant of cosmetics and foods, so the theaflavin 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. The extraction method is to directly extract the theaflavin in the black tea, but because the theaflavin is low in content in the black tea, the extraction and purification difficulty is high, and the yield is low. The chemical oxidation method is to convert tea polyphenol into theaflavin by using a chemical reagent, needs to use a large amount of chemical reagents such as potassium ferricyanide, ferric trioxide, magnesium oxide, acid and alkali and the like, has serious environmental pollution and needs to invest more sewage treatment cost, and has the defects of complex by-products and limited product purity and safety due to the lack of substrate specificity of the chemical method. The enzymatic oxidation method is to convert tea polyphenol into theaflavin by utilizing enzyme for fermentation, has mild reaction conditions and strong specificity, can avoid the defects of the extraction method and the chemical oxidation method, and has more advantages.

Disclosure of Invention

In order to solve the problems, the invention provides a method for producing theaflavin by utilizing branches and leaves of Anji white tea pruning, which has the advantages of high enzyme conversion rate, stable reaction rate, high theaflavin yield and capability of realizing industrialization.

In order to achieve the purpose, the invention adopts the following specific technical scheme:

a method for producing theaflavin by utilizing branches and leaves of Anji white tea pruning comprises the following steps:

s1, preparing a buffer solution: adding salt into water until the mass concentration of the salt is 0.1-10%, and then adding citric acid to adjust the pH value to 4.8-5.2; the salt is (NH)₄)₂SO₄、Na₂HPO₄、K₂CO₃、Na₂SO₄、K₂HPO₄、Na₂CO₃、CaCl₂、KH₂PO₄Or NaCl;

s2, preparation of enzyme solution: collecting fresh Anji white tea, pruning branches and leaves, and adding a buffer solution according to the mass ratio of (45-55) to 1; pulverizing for 2 times by pulverizer, and placing in cold storage for use.

S3, preparation of a substrate solution: mixing the tea polyphenol solution with a buffer solution to prepare a substrate solution, wherein the substrate concentration is 3-8% by mass percent;

s4, fermentation: mixing the enzyme solution and the substrate solution according to the mass ratio of 1: 5-8, and adding ethyl acetate according to 0.5-1 time of the volume of the substrate solution to form a two-phase reaction system; heating to 30-45 ℃, introducing air or oxygen according to the amount of 0.5-2 units/min, stirring at the rotating speed of 60-150 r/min, and fermenting for 6-12 hours;

s5, filtering: filtering the fermentation liquor in a plate-and-frame filter press; after feeding is finished, compressed air is fed to take away residual feed liquid in the plate frame, and a filter cake is dried; the filter cake can be used as a secondary enzyme source for recycling, and the filtrate is used for the next extraction;

s6, extraction: mixing the filtrate and ethyl acetate according to a volume ratio of 1: (2.5-3.5), extracting, stirring for 20-30 min, standing for 1-2 h for thoroughly layering, and taking an upper organic layer;

s7, concentrating and drying: concentrating the organic layer under reduced pressure, controlling the vacuum degree to be more than 0.06MPa, removing the solvent ethyl acetate, supplementing water for many times, and removing the ethyl acetate residue; and spray drying the extract, and collecting dry powder to obtain the theaflavin product.

Preferably, the salt in step S1 is Na₂HPO₄。

Preferably, in the step S2, the trimmed parts of 5-10 cm are selected from the trimmed branches and leaves of the Anji white tea.

Preferably, the grinding mesh number of the trimmed branches and leaves of the Anji white tea in the step S2 is 200-260 meshes.

Preferably, the tea polyphenol solution in step S3 is green tea extract, and the preparation method is as follows: crushing tea leaves, sieving the crushed tea leaves with a 20-50-mesh sieve, adding purified water according to the mass ratio of feed liquid of 1 to (10-20), extracting at the temperature of 70-95 ℃ for 30-90 min, cooling an extracting solution to 25-45 ℃, clarifying with a clarifying membrane with the molecular weight of 30 ten thousand Da, and then performing reverse osmosis concentration to obtain a green tea extracting solution.

Preferably, the temperature is increased to 30-35 ℃ during the fermentation in the step S4, and the fermentation time is 8-12 h.

Preferably, the substrate solution is fed in batches in step S4, at least three batches are carried out, and the feeding amount of the next batch does not exceed the feeding amount of the previous batch; the batch feeding interval time is not less than 20 min.

Further, in step S3, the substrate concentration is 5%, and in step S4, the substrate solution is divided into three batches according to the mass ratio of 2:2:1, and the batches are fed at intervals of 30 min.

Preferably, the plate-and-frame filter press in step S5 adopts filter cloth with aperture of 280 meshes and 320 meshes, and the feeding speed is controlled to be 200-300L/h.

Preferably, in the concentration process in the step S7, the temperature is controlled to be less than 80 ℃, and the relative density is 1.08-1.12 as the concentration end point; adding water in the following amount before collecting paste: adding water into the extract (mass ratio) =1 (2.5-3.5), removing solvent residues, and adding water at least three times.

The invention has the following beneficial effects:

1. the tea branches and leaves are selected as enzyme sources, so that no exogenous enzyme source is introduced in the fermentation process, the characteristic of taking the local conditions into full play, the selected branches and leaves are tea branches abandoned by tea growers when the tea growers trim the tea trees, the characteristic of recycling waste resources is reflected, the cost is reduced, and extra income is increased for the local tea growers.

2. The polyphenol oxidase in the pruned branches and leaves of the Anji white tea has good catalytic activity, and in order to make the polyphenol oxidase fully play a role, the pruned branches and leaves are mixed with the buffer solution, then the pruned branches and leaves and the buffer solution are crushed together, the membrane structure of cells is damaged by crushing, the enzyme is released or exposed, the enzyme obtains good protection effect due to the existence of the buffer solution, and the enzyme solution is convenient to store and is not easy to inactivate after being prepared. More optimally, 5-10 cm of Anji white tea is selected to prune branches and leaves to obtain polyphenol oxidase with high content and strong activity; the buffer solution is a citric acid-disodium hydrogen phosphate system with the pH value of 4.8-5.2, not only has a protection effect on enzyme, but also provides proper reaction conditions for subsequent fermentation; the crushing mesh number of the pruned branches and leaves is controlled to be 200-260 meshes, so that on one hand, the enzyme and the substrate have sufficient contact area, on the other hand, the oxygen diffusion rate in the fermentation process is increased, the oxygen absorption level is improved, and the reaction efficiency is improved.

3. The two-phase fermentation is adopted, ethyl acetate is added to form a two-phase reaction system, and by utilizing the characteristic that the product theaflavin is easy to dissolve in an organic solvent and not easy to dissolve in a water phase, the theaflavin is enriched from the water phase to the organic phase, so that the concentration of the theaflavin in the water phase is reduced, the oxidation reaction is carried out in the forward direction, the reaction rate is increased, and the fermentation effect is improved. Specifically, the following conditions need to be strictly controlled to obtain an optimal enzymatic reaction system: the method comprises the following steps of (1) controlling the concentration of a substrate to be 3-8% (mass percentage), and mixing enzyme liquid and the substrate solution according to the mass ratio of 1: 5-8 so as to obtain the proper concentration of the substrate and the proportion of the substrate and the enzyme, so that the reaction can be carried out fully and rapidly; secondly, adding ethyl acetate according to 0.5-1 time of the volume of the substrate solution, wherein the reaction speed is reduced when the ethyl acetate dosage is lower than or higher than the interval, theaflavin transfer is influenced when the ethyl acetate dosage is too low, the possibility of enzyme inactivation caused by the increase of the opportunity of contacting with the enzyme when the ethyl acetate dosage is too high is increased, and the residual quantity is also increased, so that the secondary utilization of the enzyme is influenced. More optimally, the substrate solution is fed in batches, the feeding is carried out in at least three batches, the feeding amount of the next batch is not more than that of the previous batch, and the batch feeding interval time is not less than 20min, so that the substrate concentration is linearly controlled, the phenomenon that the reaction speed is too slow due to the fact that the initial substrate concentration is too high to inhibit the activity of enzyme is avoided, the continuous and efficient operation of the reaction is ensured, and the stability of the fermentation process is improved.

4. During reduced pressure concentration, ethyl acetate residue is removed through multiple water replenishing before collection, and the quality and the safety of the theaflavin product are improved.

5. The filter cake after the fermentation liquor filtration can be recycled as a secondary enzyme source, and in order to ensure that the secondary enzyme still has good activity, the invention protects the enzyme from the following two aspects in the process: firstly, enzyme is always in a buffer solution environment, and the temperature and the pH value of the buffer solution environment are also strictly controlled; secondly, the contact between the ethyl acetate and the enzyme in the reaction process is reduced and the residue of the ethyl acetate in the filter cake is reduced by controlling the amount of the ethyl acetate, and finally the filter cake is dried to remove the residual ethyl acetate, so that the harm of the ethyl acetate is reduced to the minimum, and the great reduction and even inactivation of the enzyme activity are effectively avoided.

6. Compared with single liquid phase fermentation, the method improves the theaflavin content by about 30 percent, improves the net yield by about 10 percent and improves the conversion rate by about 30 percent. Thus, the two liquid phase reaction system of the present invention has significant advantages.

In conclusion, the method adopts double-liquid-phase fermentation, remarkably improves the conversion rate of enzyme by optimizing conditions such as enzyme solution, buffer solution, an enzymatic reaction system and the like, has stable reaction, can be continuously and efficiently carried out, and has high theaflavin yield, thereby realizing industrialization.

Detailed Description

The present invention will be further described with reference to the following specific examples.