Preparation method of extract containing theaflavin digallate

Abstract

The invention relates to the field of preparation of theaflavin digallate extracts, in particular to a preparation method of an extract containing theaflavin digallate, which comprises the following steps: step 1, fermenting black tea by adopting an oil-water double-liquid biological fermentation technology; step 2, filtering the fermented black tea in the step 1, namely extracting and separating the fermented product of the black tea by adopting a dispersed state liquid chromatography separation technology; step 3, concentrating the product extracted and separated in the step 2 by adopting a buffer temperature control system concentration technology; step 4, purifying the product in the step 3 again by adopting a dispersed state liquid chromatography separation technology; 5, re-concentrating the re-purified product in the step 4 by adopting a buffer temperature control system concentration technology; and step 6, drying the product in the step 5. The method has the following beneficial effects that the content of TF3 in the final theaflavin product reaches 20% or above, so that the theaflavin product with high TF3 content is produced.

Description

Technical Field

[0001] The invention discloses a method for preparing an extract containing theaflavin digallate, and belongs to the field of preparation of theaflavin digallate extract. Background Art

[0002] Theaflavins are the main components of tea pigments, with a total of 12 components, of which theaflavins, theaflavins-3-gallate, theaflavins-3,3'-digallate and theaflavins-3'-gallate are the four most important theaflavins. TF3 is an important biologically active substance in theaflavins. TF3, a component of theaflavins, has a chemical formula of C 43 H 32 O 20 , orange-yellow, melting point 226-233℃, easily soluble in methanol, ethanol, water, ethyl acetate, n-butanol, acetone. There are four important active monomers in theaflavin, namely TF1\TF2a\TF2b\TF3. Among these four monomers, TF3 has the strongest activity. The order of the antioxidant free radical ability of the four monomers is TF3>TF2a=TF2b>TF1. Therefore, the quality of theaflavin is usually based on the content of TF3. The higher the content of TF3, the better the quality of theaflavin.

[0003] At present, there are three main preparation processes for theaflavins: direct extraction, chemical oxidation and enzymatic oxidation. The method of direct extraction to prepare theaflavins is to extract them directly from black tea with hot water or solvent, and then purify and dry them in the subsequent process. This method is simple and theaflavins are relatively easy to obtain, but theaflavins are relatively easy to be oxidized during the process, and the product content is not high, especially the TF3 content is also low; chemical oxidation mainly uses inorganic oxidants to oxidize catechins to obtain theaflavins. Chemical oxidation can be divided into alkaline oxidation and acidic oxidation according to its oxidation conditions. Although chemical oxidation can also prepare theaflavins products, chemical production will invest more in wastewater treatment system costs than enzymatic production, and The chemical method lacks substrate specificity, has complex byproducts, has certain limitations on product purity, and has serious harmful residues. Currently, the chemical oxidation method has low acceptance. The research on the preparation of theaflavins by enzymatic oxidation mainly uses the oxidation characteristics of polyphenol oxidase and peroxidase in tea to purposefully produce theaflavins. The mechanism is that PPO oxidizes catechins to form quinones, which are then transformed into theaflavins by non-enzymatic secondary oxidation. Enzymatic oxidation to prepare theaflavins is the mainstream process in the industry, but the product content is still not high enough. The content of TF3 is usually only 10%. Therefore, high-quality production technology, improving purification technology, and increasing the content of TF3 in products are the research and development directions of theaflavins, and have also become an important topic in the development of theaflavins products. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for preparing an extract containing theaflavin digallate to solve the problems raised in the above background technology.

[0005] In order to achieve the above object, the present invention is implemented by the following technical scheme: a method for preparing an extract containing theaflavin digallate, comprising the following steps: Step 1: fermenting black tea using oil-water dual liquid biological fermentation technology; Step 2: filtering the black tea fermented in step 1, i.e. extracting and separating the product after black tea fermentation by using dispersed liquid chromatography separation technology; Step 3: Concentrate the product extracted and separated in step 2 using a buffer temperature control system concentration technology; Step 4: Purify the product in step 3 again using dispersed liquid chromatography separation technology; Step 5: re-concentrating the product purified again in step 4 using a buffer temperature control system concentration technology; Step 6: Dry the product in step 5.

[0006] Specifically, the oil-water dual liquid biological fermentation technology includes: System construction: First, according to the characteristics of the target product and the selected microorganism, prepare the oil phase and the water phase. The oil phase includes long-chain fatty acids, vegetable oils or other hydrophobic solvents, and the water phase is a culture medium rich in nutrients required for microbial growth; Microbial inoculation: Inoculate the selected microbial strain into the water phase to ensure that the strain can effectively attach and distribute at the oil-water interface, or grow at the interface; Optimization of culture conditions: Adjust and control the temperature, pH value, and oxygen supply parameters of the culture system to create a suitable environment for the growth and product flow of microorganisms; Fermentation process monitoring: Take samples regularly during the fermentation process to monitor microbial growth, product concentration and system stability, and adjust culture conditions when necessary to maintain efficient bioconversion.

[0007] Specifically, the temperature of the culture system is between 60-90° C. and the pH value is between 5-7, wherein the oxygen supply is stirred to increase the solubility, and the stirring rate is 80-120 r / min.

[0008] Specifically, the dispersed liquid chromatography separation technology includes: Solvent selection and ratio: First, two immiscible solvents need to be selected: extraction solvent and dispersant. The volume ratio of the two phases is determined based on the polarity and distribution coefficient of the target analyte. Mixing and dispersion: A small amount of extraction solvent is added to a large amount of dispersant containing the target analyte, and the extraction solvent is quickly dispersed into tiny droplets through violent shaking, ultrasound or other mechanical means to form a dispersed two-phase system. This process greatly increases the contact area between the two phases, which is conducive to the rapid transfer of the analyte; Extraction and equilibrium: Under slight stirring or static conditions, the analyte is transferred from the dispersant to the extraction solvent droplets. In this step, the analyte is distributed according to its distribution coefficient between the two phases to achieve extraction equilibrium; Phase separation: by centrifugation, refrigeration-induced phase separation or the use of magnetic nanoparticles, the droplets are forced to aggregate and separate from the dispersant to form two distinct phases; Solvent removal and enrichment: After collecting the extract phase, most of the extraction solvent and dispersant are removed by heating, nitrogen blowing or rotary evaporation to obtain highly concentrated target analytes; Analysis and determination: The enriched analyte solution is used for subsequent analysis and detection, such as high performance liquid chromatography and gas chromatography-mass spectrometry.

[0009] Specifically, the extraction solvent is an organic phase, and the dispersant is an aqueous phase or a buffer solution.

[0010] Specifically, the buffer temperature control system concentration technology includes: Prepare buffer solution: According to the properties of the material, prepare a suitable buffer solution to maintain the pH stability of the system during the concentration process and protect the activity of the target component; Connect and check the system: introduce the raw material solution and buffer solution into the temperature-controlled concentration system, check whether all pipe connections are sealed, and check whether the temperature sensor in the control system is normal; Start the temperature control system: set the target temperature and start the temperature control device to ensure that the system temperature remains constant during the entire concentration process. The temperature should be selected based on the material properties, usually slightly higher than the freezing point of the material but lower than its thermal denaturation temperature; Start concentration: Start the concentration process by reduced pressure evaporation, reverse osmosis, ultrafiltration or low temperature vacuum concentration. Under temperature control, accelerate the evaporation or permeation rate of the solvent, and monitor the concentration rate and material status at the same time; Monitoring and adjustment: During the entire concentration process, the temperature, pH value, and concentration index of the material are continuously monitored, and the temperature system and concentration rate are fine-tuned as needed to ensure product quality; Termination of concentration and cooling: After reaching the predetermined concentration endpoint, stop heating or vacuuming, and quickly cool the concentrate to prevent subsequent non-enzymatic browning or other adverse reactions; Cleaning and disinfection: After completing all operations, the entire concentration system is thoroughly cleaned and disinfected to prepare for the next batch of production.

[0011] Beneficial effects of the present invention: On the basis of the traditional enzymatic chlorination process for preparing theaflavins, the key points affecting the biotransformation of TF3 were found out by in-depth analysis of the molecular physical and chemical properties of TF3. On the basis of screening special substrate raw materials, the oil-water dual-liquid biofermentation technology was developed. In the process of biofermentation and transformation, the oil-soluble theaflavins monomer TF3 was selectively extracted and enriched, so that the conversion of the theaflavins monomer TF3 was increased by more than 25%. In the process of product purification, the dispersed liquid chromatography separation technology was developed by taking advantage of the oily nature of theaflavins TF3, which effectively removed the water-based impurities and enriched the oily component TF3, thereby increasing the content of the theaflavins monomer TF3 to more than 20%. In the process of product concentration, the buffer temperature control system concentration technology was adopted, that is, the pH buffer system was used to enhance the stability of theaflavins during the concentration process, and the theaflavins were concentrated at a certain temperature, so that the overall degradation rate of theaflavins was controlled within 5%, and the content of TF3 in the final theaflavins product reached more than 20%, thereby producing a high TF3 theaflavins product. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 The present invention is a flowchart of a method for preparing an extract containing theaflavin digallate; DETAILED DESCRIPTION

[0013] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0014] See also Figure 1 The present invention provides a technical solution: a method for preparing an extract containing theaflavin digallate, comprising the following steps: Step 1: fermenting black tea using oil-water dual liquid biological fermentation technology; Oil-water dual liquid biological fermentation technology includes: System construction: First, according to the characteristics of the target product and the selected microorganism, prepare the oil phase and the water phase. The oil phase includes long-chain fatty acids, vegetable oils or other hydrophobic solvents, and the water phase is a culture medium rich in nutrients required for microbial growth; Microbial inoculation: Inoculate the selected microbial strain into the water phase to ensure that the strain can effectively attach and distribute on the oil-water interface, or grow on the interface; Optimize culture conditions: Adjust and control the temperature, pH value, and oxygen supply parameters of the culture system to create a suitable environment for microbial growth and product flow; Fermentation process monitoring: Take samples regularly during the fermentation process to monitor the growth of microorganisms, product concentration, and system stability, and adjust the culture conditions when necessary to maintain efficient bioconversion. The temperature of the culture system is 60-90°C and the pH value is between 5-7. The oxygen supply is stirred to increase solubility, and the stirring rate is 80-120r / min.

[0015] Step 2: filtering the black tea fermented in step 1, i.e. extracting and separating the product after black tea fermentation by using dispersed liquid chromatography separation technology; Dispersed liquid chromatography separation techniques include: Solvent selection and proportioning: First, two immiscible solvents need to be selected: extraction solvent and dispersant. The volume ratio of the two phases is determined according to the polarity and distribution coefficient of the target analyte. Mixing and dispersion: Add a small amount of extraction solvent to a large amount of dispersant containing the target analyte, and quickly disperse the extraction solvent into tiny droplets through violent oscillation, ultrasound or other mechanical means to form a dispersed two-phase system. This process greatly increases the contact area between the two phases, which is conducive to the rapid transfer of the analyte. Extraction and balance: Transfer the analyte from the dispersant to the target analyte under slight stirring or static conditions. Into the extraction solvent droplets, in this step, the analyte is distributed according to its distribution coefficient between the two phases to achieve extraction equilibrium; phase separation: through centrifugation, cold storage induced phase separation or the use of magnetic nanoparticles and other methods, the droplets are caused to aggregate and separate from the dispersant to form clear two phases; solvent removal and enrichment: after collecting the extraction phase, most of the extraction solvent and dispersant are removed by heating, nitrogen blowing or rotary evaporation to obtain highly concentrated target analytes; analysis and determination: the enriched analyte solution is used for subsequent analysis and detection, such as high performance liquid chromatography and gas chromatography-mass spectrometry. The extraction solvent is an organic phase and the dispersant is an aqueous phase or a buffer.

[0016] Step 3: Concentrate the product extracted and separated in step 2 using a buffer temperature control system concentration technology; Buffer temperature control system concentration technology includes: Prepare buffer solution: Prepare appropriate buffer solution according to the material properties to maintain the pH stability of the system during the concentration process and protect the activity of the target component; Connect and check the system: Introduce the raw material liquid and buffer solution into the temperature-controlled concentration system, check whether all pipe connections are sealed, and check whether the temperature sensor in the control system is normal; Start the temperature control system: Set the target temperature and start the temperature control device to ensure that the system temperature remains constant during the entire concentration process. The temperature selection needs to be based on the material properties, usually slightly higher than the freezing point of the material but lower than its thermal denaturation temperature; Start concentration: Start the concentration process by reduced pressure evaporation, reverse osmosis, ultrafiltration or low-temperature vacuum concentration. Under temperature control conditions, accelerate the evaporation or permeation rate of the solvent, and monitor the concentration rate and material status at the same time; Monitor and adjust: During the entire concentration process, continuously monitor the temperature, pH value, and concentration indicators of the material, and fine-tune the temperature system and concentration rate as needed to ensure product quality; Stop concentration and cooling: After reaching the predetermined concentration end point, stop heating or vacuuming, and quickly cool the concentrate to prevent subsequent non-enzymatic browning or other adverse reactions; Cleaning and disinfection: After completing all operations, thoroughly clean and disinfect the entire concentration system to prepare for the next batch of production.

[0017] Step 4: Purify the product in step 3 again using dispersed liquid chromatography separation technology; Step 5: re-concentrating the product purified again in step 4 using a buffer temperature control system concentration technology; Step 6: Dry the product in step 5.

[0018] The oil-water dual-liquid bio-fermentation technology is used to selectively extract and enrich the oil-soluble theaflavins monomer TF3 during the bio-fermentation conversion process, which increases the conversion of theaflavins monomer TF3 by more than 25%; During the product purification process, the dispersed liquid chromatography separation technology was developed by taking advantage of the oily nature of theaflavins TF3 to effectively remove water-based impurities and enrich the oily component TF3, thereby increasing the content of theaflavins monomer TF3 to more than 20%; During the product concentration process, a buffer temperature control system concentration technology is adopted, that is, a pH buffer system is used to enhance the stability of theaflavins during the concentration process. At the same time, the concentration of theaflavins is carried out at a certain temperature, so that the overall degradation rate of theaflavins is controlled within 5%, and the TF3 content in the final theaflavins product reaches more than 20%, thereby producing a high TF3 theaflavins product.

[0019] The high-content theaflavins product enriched in TF3 produced through the above steps has a TF3 content of more than 20%, and its quality is significantly better than that of traditional theaflavins products. In the process of theaflavin bio-fermentation conversion, the oil-water dual-liquid bio-fermentation technology is used to increase the conversion of theaflavin monomer TF3 by more than 25%. In the process of product purification, the dispersed liquid chromatography separation technology is used to increase the content of theaflavin monomer TF3 to more than 20%.

[0020] Although this specification is described according to implementation modes, not every implementation mode includes only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing an extract containing theaflavin digallate, characterized in that The following steps are involved: Step 1: fermenting black tea using oil-water dual liquid biological fermentation technology; Step 2: filtering the black tea fermented in step 1, i.e. extracting and separating the product after black tea fermentation by using dispersed liquid chromatography separation technology; Step 3: Concentrate the product extracted and separated in step 2 using a buffer temperature control system concentration technology; Step 4: Purify the product in step 3 again using dispersed liquid chromatography separation technology; Step 5: re-concentrating the product purified again in step 4 using a buffer temperature control system concentration technology; Step 6: Dry the product in step 5.

2. The method for preparing an extract containing theaflavin digallate according to claim 1, characterized in that: The oil-water dual liquid biological fermentation technology comprises: System construction: First, according to the characteristics of the target product and the selected microorganism, prepare the oil phase and the water phase. The oil phase includes long-chain fatty acids, vegetable oils or other hydrophobic solvents, and the water phase is a culture medium rich in nutrients required for microbial growth; Microbial inoculation: Inoculate the selected microbial strain into the water phase to ensure that the strain can effectively attach and distribute at the oil-water interface, or grow at the interface; Optimization of culture conditions: Adjust and control the temperature, pH value, and oxygen supply parameters of the culture system to create a suitable environment for the growth and product flow of microorganisms; Fermentation process monitoring: Take samples regularly during the fermentation process to monitor microbial growth, product concentration and system stability, and adjust culture conditions when necessary to maintain efficient bioconversion.

3. The method for preparing an extract containing theaflavin digallate according to claim 2, characterized in that: The temperature of the culture system is 60-90° C., the pH value is between 5-7, and the oxygen supply is stirred to increase the solubility, and the stirring rate is 80-120 r / min.

4. The method for preparing an extract containing theaflavin digallate according to claim 1, characterized in that: The dispersed liquid chromatography separation technology comprises: Solvent selection and ratio: First, two immiscible solvents need to be selected: extraction solvent and dispersant. The volume ratio of the two phases is determined based on the polarity and distribution coefficient of the target analyte. Mixing and dispersion: A small amount of extraction solvent is added to a large amount of dispersant containing the target analyte, and the extraction solvent is quickly dispersed into tiny droplets through violent shaking, ultrasound or other mechanical means to form a dispersed two-phase system. This process greatly increases the contact area between the two phases, which is conducive to the rapid transfer of the analyte; Extraction and equilibrium: Under slight stirring or static conditions, the analyte is transferred from the dispersant to the extraction solvent droplets. In this step, the analyte is distributed according to its distribution coefficient between the two phases to achieve extraction equilibrium; Phase separation: by centrifugation, refrigeration-induced phase separation or the use of magnetic nanoparticles, the droplets are forced to aggregate and separate from the dispersant to form two distinct phases; Solvent removal and enrichment: After collecting the extract phase, most of the extraction solvent and dispersant are removed by heating, nitrogen blowing or rotary evaporation to obtain highly concentrated target analytes; Analysis and determination: The enriched analyte solution is used for subsequent analysis and detection, such as high performance liquid chromatography and gas chromatography-mass spectrometry.

5. The method for preparing an extract containing theaflavin digallate according to claim 4, characterized in that: The extraction solvent is an organic phase, and the dispersant is an aqueous phase or a buffer.

6. The method for preparing an extract containing theaflavin digallate according to claim 1, characterized in that: The buffer temperature control system concentration technology includes: Prepare buffer solution: According to the properties of the material, prepare a suitable buffer solution to maintain the pH stability of the system during the concentration process and protect the activity of the target component; Connect and check the system: introduce the raw material solution and buffer solution into the temperature-controlled concentration system, check whether all pipe connections are sealed, and check whether the temperature sensor in the control system is normal; Start the temperature control system: set the target temperature and start the temperature control device to ensure that the system temperature remains constant during the entire concentration process. The temperature should be selected based on the material properties, usually slightly higher than the freezing point of the material but lower than its thermal denaturation temperature; Start concentration: Start the concentration process by reduced pressure evaporation, reverse osmosis, ultrafiltration or low temperature vacuum concentration, etc., accelerate the evaporation or permeation rate of the solvent under temperature control conditions, and monitor the concentration rate and material status at the same time; Monitoring and adjustment: During the entire concentration process, the temperature, pH value, and concentration index of the material are continuously monitored, and the temperature system and concentration rate are fine-tuned as needed to ensure product quality; Termination of concentration and cooling: After reaching the predetermined concentration endpoint, stop heating or vacuuming, and quickly cool the concentrate to prevent subsequent non-enzymatic browning or other adverse reactions; Cleaning and disinfection: After completing all operations, the entire concentration system is thoroughly cleaned and disinfected to prepare for the next batch of production.

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