Method for preparing theasinensin a by means of low-temperature enzyme process

Abstract

A method for preparing theasinensin A (TSA) by means of a low-temperature enzyme process. The method comprises using a plant polyphenol oxidase enzyme extract or polyphenol oxidase enzyme formulation as an enzyme source, using epigallocatechin gallate (EGCG) as a substrate, using as a reaction system a buffer solution having a certain pH value, and performing an enzymatic reaction at a low temperature and subsequent steps such as column chromatography, distillation and concentration, liquid phase preparation, and freeze drying, thereby synthesizing TSA in a directional enzymatic manner. The method achieves EGCG enzymatic oxidation at a low temperature and obtains dimeric TSA by means of a conversion reaction, laying a theoretical and practical basis for green, safe, efficient and large-scale industrial production and utilization of TSA.

Description

A method for preparing polyester-type catechin A by low-temperature enzymatic method Technical Field

[0001] The invention belongs to the technical field of deep processing of tea leaves, and particularly relates to a preparation method of polyester-type catechin A. Background Art

[0002] During tea fermentation, epigallocatechin gallate (EGCG) undergoes enzymatic or non-enzymatic oxidation to form o-quinone (EGCGO), which then polymerizes to form unstable dehydroester-catechin A (DTSA). DTSA is then oxidized and reduced to form polyester-catechin A (TSA). TSA has superior antioxidant and anti-inflammatory properties to EGCG. Furthermore, TSA is absorbed significantly faster than EGCG by intestinal epithelial cells.

[0003] In recent years, the Chinese Patent Office has published approximately 20 patents related to polyester catechins. Most involve extraction, separation, purification, and processing of polyester catechin-rich black tea. A small number also cover chemical synthesis methods and exogenous enzymatic synthesis of polyester catechin monomers. Exogenous enzymatic synthesis methods are typically performed at room temperature, resulting in mixtures and relatively low yields. Patent number CN 114015733 B utilizes green tea flakes as raw material, extracts them with hot water, and then cools and centrifuges them to produce a green tea extract. A composite redox enzyme solution and a salt solution are then added to the green tea extract, with precise control of the pH, salt cation concentration, and ratio, as well as the ratio and total amount of polyphenol oxidase and peroxidase solutions. Fermentation is then carried out under optimal temperature conditions and controlled for a controlled period of time to produce a fermentation broth. The fermentation broth is then concentrated by membrane filtration and spray-dried to produce the polyester catechin product. This technology achieves a total polyester catechin content exceeding 30%. Patent CN1097699918 uses loquat pulp as an oxidase source to oxidize a green tea extract at room temperature, resulting in a polyester-catechin mixture with a total content of 18% to 25%. Existing patented technologies often use fresh tea leaves or green tea raw materials for fermentation, using enzymatic oxidation with endogenous tea enzymes or chemical synthesis to produce polyester-catechins or black tea products. Furthermore, pure polyphenol oxidase is expensive. Therefore, a method using plant-derived oxidase to catalyze the conversion of catechin monomers to a high-concentration single polyester-catechin solution improves TSA yield, simplifies TSA separation and purification steps, and minimizes chemical impurities during the process. This provides a theoretical and practical basis for green, safe, and efficient large-scale industrial production and utilization. Summary of the Invention

[0004] The technical problems to be solved by the present invention are: to clarify the characteristics of TSA and the factors affecting the formation of TSA during the enzymatic oxidation of EGCG, to optimize the efficient synthesis parameters for the enzymatic preparation of TSA, to avoid further polymerization and degradation of TSA; and to construct a chromatographic separation technology system for separating and purifying TSA, to improve the purity and yield of TSA.

[0005] The technical solution adopted to solve the above technical problems is to use a plant-derived polyphenol oxidase (PPO) extract or polyphenol oxidase enzyme preparation to aerobically oxidize EGCG to produce TSA under low temperature conditions. The steps of this method are as follows:

[0006] Step 1: placing plant tissue rich in PPO in a phosphate buffer solution with a pH of 6.5 to 7.0, adding polyvinyl pyrrolidone and ascorbic acid, mixing and beating, extracting at room temperature for 10 to 13 hours, centrifuging, and collecting the supernatant to obtain a PPO enzyme solution; or preparing a PPO enzyme solution by using a phosphate buffer solution with a pH of 6.5 to 7.0;

[0007] Step 2: Add EGCG to a phosphate buffer solution with a pH of 6.5 to 7.0, and add the PPO enzyme solution obtained in step 1, then stir the reaction system at 5 to 15° C. for 30 to 60 minutes, then add ascorbic acid and stir at 85 to 95° C. for 8 to 12 minutes to obtain an enzymatic reaction solution;

[0008] Step 3: The enzymatic reaction solution is applied to a macroporous resin chromatography column, eluted with pure water and methanol, and distilled at 50-60°C to obtain a methanol elution concentrate;

[0009] Step 4: Purify the methanol eluted concentrate by high performance preparative liquid chromatography, vacuum concentrate, and freeze-dry to obtain a refined polyester catechin A, wherein the TSA purity is between 91.5% and 97.2%, and the TSA yield is 62.4% to 97.6%.

[0010] In the above step 1, the PPO-rich plant tissue includes any one of PPO-rich fruits, vegetables, plant flowers, plant young fruits, etc.

[0011] In the above step 1, the amounts of the PPO-rich plant tissue, polyvinylpyrrolidone, and ascorbic acid added per liter of phosphate buffer having a pH of 6.5 to 7.0 are preferably 900 to 1100 g, 9.0 to 15.0 g, and 1.5 to 2.5 g, respectively.

[0012] In the above step 1, PPO is preferably prepared into a PPO enzyme solution having a concentration of 235,000 to 329,000 U / L using a phosphate buffer solution having a pH of 6.5 to 7.0.

[0013] In the above step 2, the volume ratio of the PPO enzyme solution to the phosphate buffer solution 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 the reaction system is 1.1 to 1.6 g.

[0014] In the above step 2, the amount of ascorbic acid added is preferably 15 to 40 times the mass of EGCG.

[0015] In the above step 3, the filler of the macroporous resin chromatography column is any one of D101, HP-20, etc., and is preferably eluted with 3 to 6 column volumes of pure water and 3 to 6 column volumes of methanol in sequence.

[0016] In the above step 4, the separation conditions of the high performance preparative liquid chromatography are as follows: using a 1010-C18HC preparative chromatography column, using an acetonitrile aqueous solution with a volume concentration of 78% as mobile phase A, pure water as mobile phase B, isocratic elution from 0 to 30 min, mobile phase A is 15%, mobile phase B is 85%, the flow rate is 50 to 75 mL / min, and the detection wavelength is 280 nm.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention utilizes exogenous PPO enzyme to catalyze the production of TSA from EGCG under low temperature conditions, which changes the existing technical status quo of using tea soup extraction or tea endogenous enzyme-catalyzed oxidation liquid to produce multiple dimer products such as theaflavins and polyester-type catechins, which makes separation and purification difficult. It realizes the directed synthesis of TSA from EGCG catalyzed by exogenous PPO enzyme.

[0019] 2. The present invention adopts EGCG to generate TSA in a targeted manner, thereby improving substrate utilization, resulting in a single product, fewer by-products and impurities, and effectively increasing the TSA yield to 62.4% to 97.6%, effectively avoiding the formation of theaflavins and other polyester-type catechins, and reducing the tedious process of separation and purification.

[0020] 3. The present invention uses PPO enzymatically to generate TSA, effectively breaking through the previous preparation method using chemical reagents such as copper chloride, reducing safety issues caused by the use of chemical reagents, and achieving green, environmentally friendly, simple and safe enzymatic reaction preparation of TSA.

[0021] 4. The present invention adopts the method of enzymatically generating TSA by exogenous PPO, which provides a material basis for the biological activity research and functional development of a single polyester-type catechin component, and also provides theoretical and technical support for the subsequent efficient large-scale industrial production and utilization of TSA. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 is a HPLC chromatogram of EGCG.

[0023] FIG2 is an HPLC chromatogram of the TSA preparation solution in Example 5. DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and examples, but the protection scope of the present invention is not limited to these examples. Example 1

[0025] Step 1: Place 500 g of pears in 500 mL of phosphate buffer with a pH of 6.5, add 5 g of polyvinyl pyrrolidone and 1 g of ascorbic acid, mix and pulp, extract at room temperature for 12 hours, centrifuge and collect the supernatant to obtain PPO enzyme solution.

[0026] Step 2: Add 0.1157 g of EGCG to 76 mL of phosphate buffer (pH 6.5), and add 24 mL of the PPO enzyme solution obtained in step 1. Then, stir the reaction system at 10°C for 60 minutes. Then, add 4.5 g of ascorbic acid and stir at 85°C for 10 minutes to obtain an enzymatic reaction solution with a TSA content of 0.89 mg / mL.

[0027] Step 3: The enzymatic reaction solution was loaded onto a D101 macroporous resin chromatography column, 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.

[0028] Step 4: The methanol elution concentrate was purified by high-performance preparative liquid chromatography using a 1010-C18HC preparative column (50 mm × 250 mm, 10 μm). A 5.0 mL sample volume was loaded, using a 78% by volume acetonitrile-water solution as mobile phase A and pure water as mobile phase B. The elution was isocratic from 0 to 30 min, with mobile phase A at 15% and mobile phase B at 85%, at a flow rate of 60 mL / min, and detection at a wavelength of 280 nm. The resulting TSA preparation was concentrated in vacuo at 55°C and freeze-dried to obtain a purified TSA product with a yield of 56.1% and a purity of 94%.

[0029] Comparative Example 1

[0030] Step 1: Place 300 g of tea in 500 mL of phosphate buffer with a pH of 7.0, add 5 g of polyvinyl pyrrolidone and 1 g of ascorbic acid, mix and slurry, extract at room temperature for 12 hours, centrifuge and collect the supernatant to obtain PPO enzyme solution.

[0031] Step 2: 0.2068 g of EGCG was added to 80 mL of pH 7.0 phosphate buffer, and 20 mL of the PPO enzyme solution obtained in step 1 was added. The reaction system was then stirred at 30°C for 60 minutes. 4.0 g of ascorbic acid was then added and stirred at 90°C for 10 minutes to obtain an enzymatic reaction solution with a TSA content of 0.59 mg / mL.

[0032] Step 3: The enzymatic reaction solution was loaded onto a D101 macroporous resin chromatography column, 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.

[0033] Step 4: The methanol elution concentrate was purified by high-performance preparative liquid chromatography using a 1010-C18HC preparative column (50 mm × 250 mm, 10 μm). A 5.0 mL sample volume was loaded, using a 78% by volume acetonitrile-water solution as mobile phase A and pure water as mobile phase B. Elution was performed isocratically from 0 to 30 min, with mobile phase A at 15% and mobile phase B at 85%, at a flow rate of 60 mL / min, and detection at a wavelength of 280 nm. The resulting TSA preparation was concentrated in vacuo at 55°C and freeze-dried to obtain a refined TSA product with a yield of 27.01% and a purity of 90%. Example 2

[0034] Step 1: Place 500g of apples in 500mL of phosphate buffer with a pH of 7.0, add 5g of polyvinylpyrrolidone and 1g of ascorbic acid, mix and pulp, soak at room temperature for 12 hours, centrifuge and collect the supernatant to obtain PPO enzyme solution.

[0035] Step 2: 0.1604 g of EGCG was added to 68 mL of phosphate buffer (pH 7.0), and 32 mL of the PPO enzyme solution obtained in step 1 was added. The reaction system was then stirred at 10°C for 60 minutes. 4.8 g of ascorbic acid was then added and stirred at 92°C for 11 minutes to obtain an enzymatic reaction solution with a TSA content of 1.23 mg / mL.

[0036] Step 3: The enzymatic reaction solution was loaded onto a D101 macroporous resin chromatography column, eluted with 4 column volumes of pure water and 4 column volumes of methanol, and distilled at 50°C to obtain a methanol elution concentrate.

[0037] Step 4: The methanol elution concentrate was purified by high-performance preparative liquid chromatography using a 1010-C18HC preparative column (50 mm × 250 mm, 10 μm). A 5.0 mL sample volume was loaded, using a 78% by volume acetonitrile-water solution as mobile phase A and pure water as mobile phase B. The elution was isocratic from 0 to 30 min, with mobile phase A at 15% and mobile phase B at 85%, at a flow rate of 60 mL / min, and detection at a wavelength of 280 nm. The resulting TSA preparation was concentrated in vacuo at 55°C and freeze-dried to obtain a refined TSA product with a yield of 48.6% and a purity of 93%. Example 3

[0038] Step 1: Place 200 g of walnut fluff in 500 mL of phosphate buffer with a pH of 6.0, add 5 g of polyvinyl pyrrolidone and 1 g of ascorbic acid, mix and pulp, soak at room temperature for 12 hours, centrifuge and collect the supernatant to obtain PPO enzyme solution.

[0039] Step 2: Add 0.1146 g of EGCG to 35 mL of phosphate buffer (pH 6.5), and add 65 mL of the PPO enzyme solution obtained in step 1. Then, stir the reaction system at 5°C for 60 minutes. Then, add 3.5 g of ascorbic acid and stir at 90°C for 10 minutes to obtain an enzymatic reaction solution with a TSA content of 0.96 mg / mL.

[0040] Step 3: The enzymatic reaction solution was loaded onto a D101 macroporous resin chromatography column, eluted with 6 column volumes of pure water and 6 column volumes of methanol, and distilled at 60°C to obtain a methanol elution concentrate.

[0041] Step 4: The methanol elution concentrate was purified by high-performance preparative liquid chromatography using a 1010-C18HC preparative column (50 mm × 250 mm, 10 μm). A 5.0 mL sample volume was loaded, using a 78% by volume acetonitrile-water solution as mobile phase A and pure water as mobile phase B. The elution was isocratic from 0 to 30 min, with mobile phase A at 15% and mobile phase B at 85%, at a flow rate of 60 mL / min, and detection at a wavelength of 280 nm. The resulting TSA preparation was concentrated in vacuo at 55°C and freeze-dried to obtain a refined TSA product with a yield of 53.4% ​​and a purity of 94%. Example 4

[0042] Step 1: Place 300g of potatoes in 500mL of phosphate buffer with a pH of 8.1, add 5g of polyvinylpyrrolidone and 1g of ascorbic acid, mix and beat, then soak at room temperature for 12 hours, centrifuge and collect the supernatant to obtain PPO enzyme solution.

[0043] Step 2: Add 0.1604 g of EGCG to 88 mL of phosphate buffer (pH 8.1), and add 12 mL of the PPO enzyme solution obtained in step 1. Then, stir the reaction system at 5°C for 60 minutes. Then, add 4.5 g of ascorbic acid and stir at 90°C for 10 minutes to obtain an enzymatic reaction solution with a TSA content of 1.27 mg / mL.

[0044] Step 3: The enzymatic reaction solution was loaded onto a D101 macroporous resin chromatography column, eluted with 5 column volumes of pure water and 5 column volumes of methanol, and distilled at 60°C to obtain a methanol elution concentrate.

[0045] Step 4: The methanol elution concentrate was purified by high-performance preparative liquid chromatography using a 1010-C18HC preparative column (50 mm × 250 mm, 10 μm). A 5.0 mL sample volume was loaded, using a 78% by volume acetonitrile-water solution as mobile phase A and pure water as mobile phase B. The elution was isocratic from 0 to 30 min, with mobile phase A at 15% and mobile phase B at 85%, at a flow rate of 60 mL / min, and detection at a wavelength of 280 nm. The resulting TSA preparation was concentrated in vacuo at 55°C and freeze-dried to obtain a purified TSA product with a yield of 54.2% and a purity of 95%. Example 5

[0046] Step 1: Dissolve 7.4 mg of PPO in 100 mL of pH 7.0 phosphate buffer to prepare a 245,000 U / L PPO enzyme solution.

[0047] Step 2: Add 0.1146 g of EGCG to 90 mL of phosphate buffer (pH 7.0), and add 10 mL of the PPO enzyme solution obtained in step 1. Then, stir the reaction system at 10°C for 60 minutes. Then, add 4.3 g of ascorbic acid and stir at 90°C for 10 minutes to obtain an enzymatic reaction solution with a TSA content of 1.02 mg / mL.

[0048] Step 3: The enzymatic reaction solution was loaded onto a D101 macroporous resin chromatography column, eluted with 3 column volumes of pure water and 3 column volumes of methanol, and distilled at 60°C to obtain a methanol elution concentrate.

[0049] Step 4: The methanol elution concentrate was purified by high-performance preparative liquid chromatography using a 1010-C18HC preparative column (50 mm × 250 mm, 10 μm). A 5.0 mL sample volume was loaded, using a 78% by volume acetonitrile-water solution as mobile phase A and pure water as mobile phase B. Elution was performed isocratic from 0 to 30 min, with mobile phase A at 15% and mobile phase B at 85%, at a flow rate of 60 mL / min, and detection at a wavelength of 280 nm. The resulting TSA preparation was concentrated under vacuum at 55°C and then freeze-dried to obtain refined TSA with a yield of 69.6%. As shown in Figures 1 and 2, PPO enzymatically catalyzed EGCG under low-temperature conditions produced highly pure TSA, with a purity of 96%. Example 6

[0050] Step 1: Place 500 g of yam in 500 mL of pH 7.0 phosphate buffer, add 5 g of polyvinyl pyrrolidone and 1 g of ascorbic acid, mix and slurry, soak at room temperature for 12 hours, centrifuge and collect the supernatant to obtain PPO enzyme solution.

[0051] Step 2: Add 0.1146 g of EGCG to 85 mL of phosphate buffer (pH 6.5), and add 15 mL of the PPO enzyme solution obtained in step 1. Then, stir the reaction system at 5°C for 60 minutes. Then, add 4.3 g of ascorbic acid and stir at 92°C for 10 minutes to obtain an enzymatic reaction solution with a TSA content of 1.59 mg / mL.

[0052] Step 3: The enzymatic reaction solution was loaded onto a D101 macroporous resin chromatography column, 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.

[0053] Step 4: The methanol elution concentrate was purified by high-performance preparative liquid chromatography using a 1010-C18HC preparative column (50 mm × 250 mm, 10 μm). A 5.0 mL sample volume was loaded, using a 78% by volume acetonitrile-water solution as mobile phase A and pure water as mobile phase B. The elution was isocratic from 0 to 30 min, with mobile phase A at 15% and mobile phase B at 85%, at a flow rate of 60 mL / min, and detection at a wavelength of 280 nm. The resulting TSA preparation was concentrated in vacuo at 55°C and freeze-dried to obtain a purified TSA product with a yield of 97.6% and a purity of 96%.

Claims

1. A method for preparing polyester-type catechin A by low-temperature enzymatic method, characterized in that The following steps are involved: Step 1: placing plant tissue rich in polyphenol oxidase in a phosphate buffer solution with a pH of 6.5 to 7.0, adding polyvinyl pyrrolidone and ascorbic acid, mixing and beating, extracting at room temperature for 10 to 13 hours, centrifuging, and collecting the supernatant to obtain a polyphenol oxidase enzyme solution; or preparing a polyphenol oxidase enzyme solution by using a phosphate buffer solution with a pH of 6.5 to 7.0; Step 2: adding epigallocatechin gallate to a phosphate buffer solution having a pH of 6.5 to 7.0, and adding the polyphenol oxidase enzyme solution obtained in Step 1, then stirring the reaction system at 5 to 15° C. for 30 to 60 minutes, then adding ascorbic acid and stirring at 85 to 95° C. for 8 to 12 minutes to obtain an enzymatic reaction solution; Step 3: The enzymatic reaction solution is applied to a macroporous resin chromatography column, eluted with pure water and methanol, and distilled at 50-60°C to obtain a methanol elution concentrate; Step 4: Purify the methanol eluted concentrate by high performance preparative liquid chromatography, vacuum concentrate, and freeze-dry to obtain a refined polyester catechin A.

2. The method for preparing polyester-type catechin A by low-temperature enzymatic method according to claim 1, characterized in that: In step 1, the plant tissue rich in polyphenol oxidase includes any one of fruits, vegetables, plant flowers and young fruits of plants rich in polyphenol oxidase.

3. The method for preparing polyester-type catechin A by low-temperature enzymatic method according to claim 1, characterized in that: In step 1, the amounts of plant tissue rich in polyphenol oxidase, polyvinyl pyrrolidone, and ascorbic acid added per liter of phosphate buffer with a pH of 6.5-7.0 are 900-1100 g, 9.0-15.0 g, and 1.5-2.5 g, respectively.

4. The method for preparing polyester-type catechin A by low-temperature enzymatic method according to claim 1, characterized in that: In step 1, polyphenol oxidase is prepared into a polyphenol oxidase solution with a pH of 6.5 to 7.0 phosphate buffer solution at a concentration of 235,000 to 329,000 U / L.

5. The method for preparing polyester-type catechin A by low-temperature enzymatic method according to claim 1, characterized in that: In step 2, the volume ratio of the polyphenol oxidase solution to the phosphate buffer solution 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.

6. The method for preparing polyester-type catechin A by low-temperature enzymatic method according to claim 1, characterized in that: In step 2, the amount of ascorbic acid added is 15 to 40 times the mass of epigallocatechin gallate.

7. The method for preparing polyester-type catechin A by low-temperature enzymatic method according to claim 1, characterized in that: In step 3, the filler of the macroporous resin chromatography column is D101 or HP-20, and eluted with 3 to 6 column volumes of pure water and 3 to 6 column volumes of methanol in sequence.

8. The method for preparing polyester-type catechin A by low-temperature enzymatic method according to claim 1, characterized in that: In step 4, the separation conditions of the high performance preparative liquid chromatography are as follows: using a 1010-C18HC preparative chromatography column, using an acetonitrile aqueous solution with a volume concentration of 78% as mobile phase A, pure water as mobile phase B, isocratic elution in 0-30 min, mobile phase A is 15%, mobile phase B is 85%, the flow rate is 50-75 mL / min, and the detection wavelength is 280 nm.

Images