Method for producing theasinensin A by low-temperature enzymatic method

Abstract

A method for producing theasinensin A by a low-temperature enzymatic method is disclosed. Using a plant polyphenol oxidase enzyme extract or polyphenol oxidase enzyme preparation as the enzyme source, epigallocatechin gallate (EGCG) as the substrate, and a buffer solution with a specific pH value as the reaction system, theasinensin A (TSA) is synthesized by a directed enzymatic reaction through steps such as low-temperature enzymatic reaction and subsequent column chromatography, distillation concentration, liquid phase preparation, and freeze-drying. This method enables enzymatic oxidation of EGCG at low temperatures and yields dimerized TSA via a conversion reaction, establishing a theoretical and practical foundation for environmentally friendly, safe, efficient, and large-scale industrial production and utilization of TSA.

Description

Technical Field

[0001] The present invention belongs to the field of advanced tea processing technology, and particularly relates to a method for producing theacinesin A.

Background Art

[0002] In the fermentation process of tea leaves, catechin (EGCG) is oxidized enzymatically or non-enzymatically to form o-quinone (EGCGO), and then polymerizes to form unstable dehydrotheacinesin A (DTSA), and DTSA is further redoxed to form theacinesin A (TSA). TSA has better antioxidant and anti-inflammatory pharmacological effects than EGCG. And the rate at which TSA is absorbed by intestinal epithelial cells is significantly faster than that of EGCG.

[0003] In recent years, the Chinese National Intellectual Property Office has published approximately 20 patents related to theasinensin. Most of these patents relate to extraction, separation and purification, and processing processes for black tea rich in theasinensin. A small number of patents also exist for chemical synthesis and exogenous enzymatic synthesis methods of theasinensin monomer. The exogenous enzymatic synthesis methods are all carried out at room temperature, and the products are mostly mixtures with relatively low yields. Patent document 1: CN114015733 B is characterized by the steps of obtaining a green tea extract by using green tea powder as a raw material, extracting it with hot water, cooling and centrifuging it; adding a complex oxidoreductase solution and saline solution to the green tea extract, precisely controlling the pH value, the cation concentration and mixing ratio of the saline solution, and the addition ratio and total amount of polyphenol oxidase solution and peroxidase solution, and fermenting it under relatively good temperature conditions with controlled time to obtain a fermented liquid; and concentrating the fermented liquid by membrane filtration and spray drying to obtain a theasinensin product. The total content of theasinensin-like substances obtained by this technology reaches 30% or more. Patent Document 2: CN1097699918 uses loquat pulp as an oxidizing enzyme source to oxidize green tea extract at room temperature, and the total content of the resulting theasinensin mixture reaches 18% to 25%. Conventional patented technologies mainly use fresh tea leaves or green tea raw materials for fermentation, and thus produce theasinensin or black tea products by enzymatic oxidation using endogenous enzymes in tea leaves or by chemical synthesis, and pure polyphenol oxidase is expensive. Therefore, a method that catalyzes catechin monomers with plant-derived oxidizing enzymes to achieve a high-concentration single theasinensin solution improves TSA yield, simplifies TSA separation and purification steps, minimizes the avoidance of chemical impurities in the process, and lays the theoretical and practical foundation for environmentally friendly, safe, and efficient large-scale industrial production and use. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Chinese patent 114015733B [Patent Document 2] Chinese Patent No. 1097699918 [Overview of the project] [Problems that the invention aims to solve]

[0005] The technical problems that this invention aims to solve are to clarify the properties of TSA and the factors that affect TSA production during the enzymatic oxidation process of EGCG, to optimize efficient synthesis parameters for TSA preparation by enzymatic method, to avoid further polymerization and degradation of TSA, to construct a chromatographic separation technology system for separating and purifying TSA, and to improve the purity and yield of TSA. [Means for solving the problem]

[0006] The technical solution employed to address the above technical challenges is a method for producing TSA by aerobic oxidation of EGCG under low temperature conditions using a plant-derived polyphenol oxidase (PPO) extract or polyphenol oxidase enzyme preparation. The steps of this method are as follows: In Step 1, plant tissue rich in PPO is placed in a phosphate buffer with a pH of 6.5-7.0, polyvinylpyrrolidone and ascorbic acid are added and mixed uniformly to form a slurry, which is then extracted at room temperature for 10-13 hours, centrifuged, and the supernatant is collected to obtain a PPO enzyme solution. Alternatively, PPO can be taken and a PPO enzyme solution prepared using a phosphate buffer with a pH of 6.5-7.0. In step 2, EGCG is added to a phosphate buffer with a pH of 6.5-7.0, and the PPO enzyme solution obtained in step 1 is added. The reaction system is then stirred at 5-15°C for 30-60 minutes, and ascorbic acid is added. The mixture is then stirred at 85-95°C for 8-12 minutes to obtain the enzyme reaction solution. In step 3, the enzyme reaction solution is subjected to a macroporous resin chromatography column, eluted with pure water and methanol, and distilled at 50-60°C to obtain a methanol-eluted concentrate. In step 4, the methanol eluate concentrate was purified by high-performance preparative liquid chromatography, followed by vacuum concentration and freeze-drying to obtain a purified theasinensin A product. The purity of the TSA was 91.5% to 97.2%, and the yield of the TSA was 62.4% to 97.6%.

[0007] In step 1 above, the plant tissue rich in PPO includes one of the following: a fruit, vegetable, plant flower, or young fruit of a plant, all of which are rich in PPO.

[0008] In step 1 above, the amounts of plant tissue rich in PPO, polyvinylpyrrolidone, and ascorbic acid added to a phosphate buffer solution with a pH of 6.5 to 7.0 per liter are preferably 900 to 1100 g, 9.0 to 15.0 g, and 1.5 to 2.5 g, respectively.

[0009] In step 1 above, it is preferable to take the PPO and prepare a PPO enzyme solution of 235,000 to 329,000 U / L using a phosphate buffer with a pH of 6.5 to 7.0.

[0010] In step 2 above, the volume ratio of the PPO enzyme solution to the phosphate buffer with a pH of 6.5 to 7.0 is preferably 10:90 to 35:65, and the amount of EGCG added per liter of reaction system is preferably 1.1 to 1.6 g.

[0011] In step 2 above, the amount of ascorbic acid added is preferably 15 to 40 times the mass of EGCG.

[0012] In step 3 above, the packing material for the macroporous resin chromatography column is one of D101, HP-20, etc., and it is preferable to sequentially elute with 3 to 6 times the column volume of pure water and 3 to 6 times the column volume of methanol.

[0013] In step 4 above, the separation conditions for the high-speed preparative liquid chromatography are as follows: a 1010-C18HC preparative chromatography column is used, with a 78% volume concentration acetonitrile aqueous solution as mobile phase A and pure water as mobile phase B, and isogradient elution is performed for 0 to 30 minutes, with mobile phase A at 15% and mobile phase B at 85%, a flow rate of 50 to 75 mL / min, and a detection wavelength of 280 nm. [Effects of the Invention]

[0014] Compared to the conventional technology, the beneficial effects of the present invention are as follows: 1. The present invention improves upon the technical challenges of conventional methods, such as the difficulty in separating and purifying TSA from EGCG due to the various dimeric products, including theaflavin and theasinensin components, generated when using tea liquor extraction or oxidation solutions with endogenous tea leaf enzymes. By utilizing an exogenous PPO enzyme reaction to produce TSA from EGCG under low-temperature conditions, the invention achieves targeted synthesis of TSA from EGCG via an exogenous PPO enzyme reaction. 2. The present invention improves substrate utilization by selectively generating TSA using EGCG, resulting in a single product with fewer by-products and impurities, effectively increasing the TSA yield to 62.4% to 97.6%, effectively avoiding the formation of theaflavins and other theasinensins, and reducing the complexity of the separation and purification process. 3. The present invention effectively overcomes the use of chemical reagents such as copper chloride in conventional production by generating TSA by a PPO enzyme reaction, thereby reducing safety issues associated with the use of chemical reagents and realizing an environmentally friendly, simple, and safe TSA preparation by an enzymatic reaction. 4. The present invention employs a method for producing TSA by an exogenous PPO enzyme reaction, providing a material basis for the study of the bioactivity of the single component theasinensin and its functional development, as well as providing theoretical and technical support for the subsequent development of efficient large-scale industrial production and utilization of TSA. [Brief explanation of the drawing]

[0015] [Figure 1] This is an HPLC chromatogram of EGCG. [Figure 2] It is the HPLC chromatogram of the TSA preparation solution in Example 5.

Embodiments for Carrying out the Invention

[0016] Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and examples, but the protection scope of the present invention is not limited to these examples.

[0017] Example 1 In Step 1, 500 g of pears are put into 500 mL of a phosphate buffer solution with a pH of 6.5, 5 g of polyvinylpyrrolidone and 1 g of ascorbic acid are added and mixed uniformly to form a slurry, then extracted at room temperature for 12 hours, centrifuged, and the supernatant is recovered to obtain a PPO enzyme solution.

[0018] In Step 2, 0.1157 g of EGCG is added to 76 mL of a phosphate buffer solution with a pH of 6.5, and 24 mL of the PPO enzyme solution obtained in Step 1 is added. Then, the reaction system is stirred at 10 °C for 60 minutes, and further 4.5 g of ascorbic acid is added, and stirred at 85 °C for 10 minutes to obtain an enzyme reaction solution with a TSA content of 0.89 mg / mL.

[0019] In Step 3, the enzyme reaction solution is applied to a D101 macroporous resin chromatography column, eluted sequentially with 3 times the column volume of pure water and 3 times the column volume of methanol, and distilled at 50 °C to obtain a methanol elution concentrate.

[0020] In Step 4, the methanol elution concentrate is purified by high-speed preparative liquid chromatography. An XBridge C18 preparative chromatography column (50 mm×250 mm, 10 μm) is adopted. With a sample injection volume of 5.0 mL, an aqueous acetonitrile solution with a volume concentration of 78% is used as mobile phase A, pure water is used as mobile phase B, and isocratic elution is performed in 0 - 30 minutes. Mobile phase A is 15%, mobile phase B is 85%, the flow rate is 60 mL / min, and the detection wavelength is 280 nm. The obtained TSA preparation solution is vacuum concentrated at 55 °C and then freeze-dried to obtain a TSA purified product with a yield of 56.1% and a purity of 94%.

[0021] Comparative Example 1 In Step 1, 300 g of tea is put into 500 mL of phosphate buffer solution with a pH of 7.0, 5 g of polyvinylpyrrolidone and 1 g of ascorbic acid are added and mixed uniformly to form a slurry, which is extracted at room temperature for 12 hours, centrifuged, and the supernatant is recovered to obtain a PPO enzyme solution.

[0022] In Step 2, 0.2068 g of EGCG is added to 80 mL of phosphate buffer solution with a pH of 7.0, and 20 mL of the PPO enzyme solution obtained in Step 1 is added. After the reaction system is stirred at 30 °C for 60 minutes, 4.0 g of ascorbic acid is further added and stirred at 90 °C for 10 minutes to obtain an enzyme reaction solution with a TSA content of 0.59 mg / mL.

[0023] In Step 3, the enzyme reaction solution is applied to a D101 macroporous resin chromatography column and sequentially eluted with 3 column volumes of pure water and 3 column volumes of methanol, and distilled at 50 °C to obtain a methanol elution concentrate.

[0024] In step 4, the methanol eluate concentrate was purified by high-performance preparative liquid chromatography. A 1010-C18HC preparative chromatography column (50 mm × 250 mm, 10 μm) was used, with a sample injection volume of 5.0 mL. An aqueous acetonitrile solution with a volume concentration of 78% was used as mobile phase A, and pure water as mobile phase B. Elution was performed with an isogradient for 0 to 30 minutes, resulting in 15% mobile phase A and 85% mobile phase B, a flow rate of 60 mL / min, and a detection wavelength of 280 nm. The resulting TSA preparation solution was vacuum concentrated at 55°C and then freeze-dried to obtain a purified TSA product with a yield of 27.01% and a purity of 90%.

[0025] Example 2 In Step 1, 500g of apples are placed in 500mL of phosphate buffer with a pH of 7.0, 5g of polyvinylpyrrolidone and 1g of ascorbic acid are added and mixed uniformly to form a slurry. This slurry is then extracted at room temperature for 12 hours, and the supernatant is collected by centrifugation to obtain a PPO enzyme solution.

[0026] In step 2, 0.1604 g of EGCG is added to 68 mL of phosphate buffer with a pH of 7.0, and then 32 mL of the PPO enzyme solution obtained in step 1 is added. The reaction system is then stirred at 10°C for 60 minutes, and then 4.8 g of ascorbic acid is added. The mixture is then stirred at 92°C for 11 minutes to obtain an enzyme reaction solution with a TSA content of 1.23 mg / mL.

[0027] In step 3, the enzyme reaction solution is passed through a D101 macroporous resin chromatography column, and eluted sequentially with four times the column volume of pure water and four times the column volume of methanol. The methanol eluate concentrate is then obtained by distillation at 50°C.

[0028] In step 4, the methanol eluate concentrate was purified by high-performance preparative liquid chromatography. A 1010-C18HC preparative chromatography column (50 mm × 250 mm, 10 μm) was used, with a sample injection volume of 5.0 mL. An aqueous acetonitrile solution with a volume concentration of 78% was used as mobile phase A, and pure water as mobile phase B. Elution was performed with an isogradient for 0 to 30 minutes, resulting in 15% mobile phase A and 85% mobile phase B, a flow rate of 60 mL / min, and a detection wavelength of 280 nm. The resulting TSA preparation solution was vacuum concentrated at 55°C and then freeze-dried to obtain a purified TSA product with a yield of 48.6% and a purity of 93%.

[0029] Example 3 In Step 1, 200g of walnut blossoms are placed in 500mL of phosphate buffer with a pH of 6.0, 5g of polyvinylpyrrolidone and 1g of ascorbic acid are added and mixed uniformly to form a slurry. This slurry is then extracted at room temperature for 12 hours, centrifuged, and the supernatant is collected to obtain a PPO enzyme solution.

[0030] In step 2, 0.1146 g of EGCG is added to 35 mL of phosphate buffer with a pH of 6.5, and then 65 mL of the PPO enzyme solution obtained in step 1 is added. The reaction system is then stirred at 5°C for 60 minutes, and then 3.5 g of ascorbic acid is added. The system is then stirred at 90°C for 10 minutes to obtain an enzyme reaction solution with a TSA content of 0.96 mg / mL.

[0031] In step 3, the enzyme reaction solution is subjected to a D101 macroporous resin chromatography column, and eluted sequentially with six times the column volume of pure water and then six times the column volume of methanol. The methanol eluate concentrate is obtained by distillation at 60°C.

[0032] In step 4, the methanol eluate concentrate was purified by high-performance preparative liquid chromatography. A 1010-C18HC preparative chromatography column (50 mm × 250 mm, 10 μm) was used, with a sample injection volume of 5.0 mL. An aqueous acetonitrile solution with a volume concentration of 78% was used as mobile phase A, and pure water as mobile phase B. Elution was performed with an isogradient for 0 to 30 minutes, resulting in 15% mobile phase A and 85% mobile phase B, a flow rate of 60 mL / min, and a detection wavelength of 280 nm. The resulting TSA preparation solution was vacuum concentrated at 55°C and then freeze-dried to obtain a purified TSA product with a yield of 53.4% ​​and a purity of 94%.

[0033] Example 4 In Step 1, 300g of potatoes are placed in 500mL of phosphate buffer with a pH of 8.1, 5g of polyvinylpyrrolidone and 1g of ascorbic acid are added and mixed uniformly to form a slurry. This slurry is then extracted at room temperature for 12 hours, and the supernatant is collected by centrifugation to obtain a PPO enzyme solution.

[0034] In step 2, 0.1604 g of EGCG is added to 88 mL of phosphate buffer with a pH of 8.1, and then 12 mL of the PPO enzyme solution obtained in step 1 is added. The reaction system is then stirred at 5°C for 60 minutes, and then 4.5 g of ascorbic acid is added. The system is then stirred at 90°C for 10 minutes to obtain an enzyme reaction solution with a TSA content of 1.27 mg / mL.

[0035] In step 3, the enzyme reaction solution is passed through a D101 macroporous resin chromatography column, and eluted sequentially with five times the column volume of pure water and then five times the column volume of methanol. The methanol eluate concentrate is then obtained by distillation at 60°C.

[0036] In step 4, the methanol eluate concentrate was purified by high-performance preparative liquid chromatography. A 1010-C18HC preparative chromatography column (50 mm × 250 mm, 10 μm) was used, with a sample injection volume of 5.0 mL. An aqueous acetonitrile solution with a volume concentration of 78% was used as mobile phase A, and pure water as mobile phase B. Elution was performed with an isogradient for 0 to 30 minutes, resulting in 15% mobile phase A and 85% mobile phase B, a flow rate of 60 mL / min, and a detection wavelength of 280 nm. The resulting TSA preparation solution was vacuum concentrated at 55°C and then freeze-dried to obtain a purified TSA product with a yield of 54.2% and a purity of 95%.

[0037] Example 5 In Step 1, 7.4 mg of PPO is added to 100 mL of phosphate buffer at pH 7.0 to prepare a PPO enzyme solution of 245,000 U / L.

[0038] In step 2, 0.1146 g of EGCG is added to 90 mL of phosphate buffer with a pH of 7.0, and then 10 mL of the PPO enzyme solution obtained in step 1 is added. The reaction system is then stirred at 10°C for 60 minutes, and then 4.3 g of ascorbic acid is added. The system is then stirred at 90°C for 10 minutes to obtain an enzyme reaction solution with a TSA content of 1.02 mg / mL.

[0039] In step 3, the enzyme reaction solution is passed through a D101 macroporous resin chromatography column, and eluted sequentially with three times the column volume of pure water and three times the column volume of methanol. The methanol eluate concentrate is then obtained by distillation at 60°C.

[0040] In step 4, the methanol eluate concentrate was purified by high-performance preparative liquid chromatography. A 1010-C18HC preparative chromatography column (50 mm × 250 mm, 10 μm) was used, with a sample injection volume of 5.0 mL. An aqueous acetonitrile solution with a volume concentration of 78% was used as mobile phase A, and pure water as mobile phase B. Elution was performed with an isogradient for 0 to 30 minutes, resulting in 15% mobile phase A and 85% mobile phase B. The flow rate was 60 mL / min, and the detection wavelength was 280 nm. The resulting TSA preparation solution was vacuum concentrated at 55°C and then freeze-dried to obtain a purified TSA product with a yield of 69.6%. As can be seen from Figures 1 and 2, under low-temperature conditions, high-purity TSA was produced from EGCG by the PPO enzyme reaction, with a TSA purity of 96%.

[0041] Example 6 In Step 1, 500g of yam is placed in 500mL of phosphate buffer with a pH of 7.0, 5g of polyvinylpyrrolidone and 1g of ascorbic acid are added and mixed uniformly to form a slurry. This slurry is then extracted at room temperature for 12 hours, and the supernatant is collected by centrifugation to obtain a PPO enzyme solution.

[0042] In step 2, 0.1146 g of EGCG is added to 85 mL of phosphate buffer with a pH of 6.5, and then 15 mL of the PPO enzyme solution obtained in step 1 is added. The reaction system is then stirred at 5°C for 60 minutes, and then 4.3 g of ascorbic acid is added. The mixture is then stirred at 92°C for 10 minutes to obtain an enzyme reaction solution with a TSA content of 1.59 mg / mL.

[0043] In step 3, the enzyme reaction solution is passed through a D101 macroporous resin chromatography column, and eluted sequentially with three times the column volume of pure water and three times the column volume of methanol. The methanol eluate concentrate is then obtained by distillation at 50°C.

[0044] In step 4, the methanol eluate concentrate was purified by high-performance preparative liquid chromatography. A 1010-C18HC preparative chromatography column (50 mm × 250 mm, 10 μm) was used, with a sample injection volume of 5.0 mL. An aqueous acetonitrile solution with a volume concentration of 78% was used as mobile phase A, and pure water as mobile phase B. Elution was performed with an isogradient for 0 to 30 minutes, resulting in 15% mobile phase A and 85% mobile phase B, a flow rate of 60 mL / min, and a detection wavelength of 280 nm. The resulting TSA preparation solution was vacuum concentrated at 55°C and then freeze-dried to obtain a purified TSA product with a yield of 97.6% and a purity of 96%.

Claims

1. A method for producing theasinensin A by low-temperature enzymatic method, Step 1 involves placing plant tissue rich in polyphenol oxidase into a phosphate buffer with a pH of 6.5–7.0, adding polyvinylpyrrolidone and ascorbic acid, mixing uniformly to form a slurry, extracting at room temperature for 10–13 hours, centrifuging, and collecting the supernatant to obtain a polyphenol oxidase enzyme solution, or taking polyphenol oxidase and preparing a polyphenol oxidase enzyme solution using a phosphate buffer with a pH of 6.5–7.

0. Step 2 involves adding epigallocatechin gallate to a phosphate buffer with a pH of 6.5–7.0, then adding the polyphenol oxidase enzyme solution obtained in Step 1, stirring the reaction system at 5–15°C for 30–60 minutes, and finally adding ascorbic acid and stirring at 85–95°C for 8–12 minutes to obtain the enzyme reaction solution. Step 3 involves applying the enzyme reaction solution to a macroporous resin chromatography column, eluting it with pure water and methanol, and distilling it at 50-60°C to obtain a methanol-eluted concentrate. The process includes step 4, in which the methanol eluate concentrate is purified by high-performance preparative liquid chromatography, followed by vacuum concentration and freeze-drying to obtain a purified theasinensin A product. A method for producing theasinensin A by low-temperature enzymatic method, characterized by the following:

2. In step 1, the plant tissue rich in polyphenol oxidase includes one of the following: a fruit, a vegetable, a plant flower, or a young fruit of a plant, all of which are rich in polyphenol oxidase. A method for producing theasinensin A by the low-temperature enzymatic method according to claim 1.

3. In Step 1, the amounts of polyphenol oxidase-rich plant tissue, polyvinylpyrrolidone, and ascorbic acid added to a phosphate buffer solution with a pH of 6.5–7.0 per liter are 900–1100 g, 9.0–15.0 g, and 1.5–2.5 g, respectively. A method for producing theasinensin A by the low-temperature enzymatic method according to claim 1.

4. In step 1, polyphenol oxidase is taken and a polyphenol oxidase enzyme solution of 235,000 to 329,000 U / L is prepared using a phosphate buffer with a pH of 6.5 to 7.

0. A method for producing theasinensin A by the low-temperature enzymatic method according to claim 1.

5. In step 2, the volume ratio of the polyphenol oxidase enzyme solution to the phosphate buffer with a pH of 6.5 to 7.0 is 10:90 to 35:65, and the amount of epigallocatechin gallate added per liter of reaction system is 1.1 to 1.6 g. A method for producing theasinensin A by the low-temperature enzymatic method according to claim 1.

6. In step 2, the amount of ascorbic acid added is 15 to 40 times the mass of epigallocatechin gallate. A method for producing theasinensin A by the low-temperature enzymatic method according to claim 1.

7. In step 3, the packing material of the macroporous resin chromatography column is D101 or HP-20, and elution is performed sequentially with 3 to 6 times the column volume of pure water and 3 to 6 times the column volume of methanol. A method for producing theasinensin A by the low-temperature enzymatic method according to claim 1.

8. In step 4, the separation conditions for the high-performance preparative liquid chromatography are as follows: a 1010-C18HC preparative chromatography column is used, a 78% volume aqueous acetonitrile solution is used as mobile phase A, and pure water is used as mobile phase B. Eclectic elution is performed with equal gradient for 0 to 30 minutes, with mobile phase A at 15% and mobile phase B at 85%, a flow rate of 50 to 75 mL / min, and a detection wavelength of 280 nm. A method for producing theasinensin A by the low-temperature enzymatic method according to claim 1.

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