WO2025201310A1 - 一种低温酶法制备聚酯型儿茶素a的方法 - Google Patents

一种低温酶法制备聚酯型儿茶素a的方法

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Publication number
WO2025201310A1
WO2025201310A1 PCT/CN2025/084664 CN2025084664W WO2025201310A1 WO 2025201310 A1 WO2025201310 A1 WO 2025201310A1 CN 2025084664 W CN2025084664 W CN 2025084664W WO 2025201310 A1 WO2025201310 A1 WO 2025201310A1
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polyphenol oxidase
tsa
low
phosphate buffer
solution
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French (fr)
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付静
赵锦
沈小萌
杨晨曦
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Shaanxi University of Technology
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Shaanxi University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
    • C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • PPO plant-derived polyphenol oxidase
  • the steps of this method are as follows:
  • 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;
  • 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;
  • 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, wherein the TSA purity is between 91.5% and 97.2%, and the TSA yield is 62.4% to 97.6%.
  • the PPO-rich plant tissue includes any one of PPO-rich fruits, vegetables, plant flowers, plant young fruits, etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG1 is a HPLC chromatogram of EGCG.
  • FIG2 is an HPLC chromatogram of the TSA preparation solution in Example 5.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m). 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%.
  • 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.
  • Step 2 Add 0.2068 g of EGCG to 80 mL of phosphate buffer with a pH of 7.0, and add 20 mL of PPO enzyme solution obtained in step 1. Then stir the reaction system at 30°C for 60 minutes, add 4.0 g of ascorbic acid and stir at 90°C for 10 minutes to obtain an enzymatic reaction solution with a TSA content of 0.59 mg/mL.
  • 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.
  • 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 ⁇ m). 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%.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m). 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m). 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%.
  • 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.
  • Step 2 Add 0.1146 g EGCG to 90 mL of phosphate buffer (pH 7.0), and add 10 mL of 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.
  • 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.
  • 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 ⁇ m). 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%.
  • 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.
  • 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.

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Abstract

一种低温酶法制备聚酯型儿茶素A的方法，该方法是以植物多酚氧化酶的酶提取物或多酚氧化酶酶制剂为酶源，以表没食子酸儿茶素没食子酸酯（EGCG）为底物，以一定pH值缓冲液作为反应体系，经低温下酶促反应以及后续的柱层析、蒸馏浓缩、液相制备、冷冻干燥等步骤，定向酶促合成聚酯型儿茶素A（TSA）。该方法实现在低温下EGCG酶促氧化并通过转化反应获得二聚体TSA，为TSA绿色、安全、高效大规模工业化生产与利用奠定了理论和实践依据。

Description

一种低温酶法制备聚酯型儿茶素A的方法 技术领域
本发明属于茶叶深加工技术领域，具体涉及一种聚酯型儿茶素A的制备方法。
 背景技术
在茶叶发酵过程中，儿茶素（EGCG）经酶性或非酶性氧化形成邻醌（EGCGO），之后聚合形成不稳定的脱氢聚酯型儿茶素A（DTSA），DTSA再氧化还原形成聚酯型儿茶素A（TSA）。TSA具有优于EGCG的抗氧化、消炎的药理作用。且TSA在肠道上皮细胞被吸收的速率明显高于EGCG。
近年来，由中国专利局公告的关于聚酯型儿茶素的专利约20个，大多数涉及提取、分离纯化和富含聚酯型儿茶素红茶的加工工艺等，也有少量关于聚酯型儿茶素单体化学合成方法和外源酶合成方法。其中的外源酶法合成都是在常温条件下进行，产物多为混合物，而且得率较低。专利号为CN 114015733 B，其特点为以绿茶片末为原料，热水浸提，经冷却离心后得绿茶浸提液；向绿茶浸提液中加入复合氧化还原酶液和盐液，精准调控pH值、盐液阳离子浓度及配比，以及多酚氧化酶液和过氧化物酶液的添加比例和添加总量，在较佳温度条件下控时发酵，得发酵料液；发酵料液经膜滤浓缩、喷雾干燥，得到聚酯型儿茶素产品。该技术获得聚酯型儿茶素类物质总量含量达到30%以上。专利CN1097699918以枇杷果肉为氧化酶酶源，常温氧化绿茶浸提液，得到的聚酯型儿茶素混合物总量大达18%～25%。由于现有专利技术多采用茶鲜叶或绿茶原料进行发酵，茶叶内源酶酶促氧化方法或化学合成法将其制备成聚酯型儿茶素或红茶产品，且纯品多酚氧化酶价格昂贵。因此，通过植物源氧化酶催化儿茶素单体达到高浓度单一聚酯型儿茶素溶液的方法，提高TSA得率，简化TSA分离纯化步骤，最大限度避免过程中的化学杂质，为达到绿色、安全、高效大规模工业化生产与利用奠定了理论和实践依据。
 发明内容
本发明所要解决的技术问题：明确EGCG酶法氧化过程中TSA的特性及影响TSA的生成因素，优化酶法制备TSA的高效合成参数，避免TSA的进一步聚合及降解；构建一种分离纯化TSA的色谱分离技术体系，提高TSA的纯度及得率。
解决上述技术问题所采用的技术方案是：利用植物源多酚氧化酶（PPO）提取液或多酚氧化酶酶制剂在低温条件下有氧氧化EGCG生成TSA的方法。该方法步骤如下：
步骤1：将富含PPO的植物组织置于pH为6.5～7.0的磷酸缓冲液中，加入聚乙烯吡咯烷酮、抗坏血酸混匀打浆后，常温浸提10～13小时，离心分离，收集上清液，即得PPO酶液；或者取PPO用pH为6.5～7.0的磷酸缓冲液配置成PPO酶液；
步骤2：将EGCG加入pH为6.5～7.0的磷酸缓冲液中，并加入步骤1得到的PPO酶液，然后将反应体系于5～15℃下搅拌30～60分钟，再加入抗坏血酸于85～95℃下搅拌8～12分钟，得到酶促反应液；
步骤3：将酶促反应液上大孔树脂层析柱，经纯水、甲醇洗脱，50～60℃蒸馏得甲醇洗脱浓缩液；
步骤4：将甲醇洗脱浓缩液经高效制备液相色谱纯化后，真空浓缩、冷冻干燥，得到聚酯型儿茶素A精制品，其中TSA纯度在91.5%～97.2%之间，TSA得率为62.4%～97.6%。
上述步骤1中，所述富含PPO的植物组织包括富含PPO的水果、蔬菜、植物的花、植物的幼果等中任意一种。
上述步骤1中，优选每升pH为6.5～7.0磷酸缓冲液中富含PPO的植物组织、聚乙烯吡咯烷酮、抗坏血酸的加入量依次为900～1100g、9.0～15.0g、1.5～2.5g。
上述步骤1中，优选取PPO用pH为6.5～7.0的磷酸缓冲液配置成235000～329000U/L的PPO酶液。
上述步骤2中，优选所述PPO酶液与pH为6.5～7.0的磷酸缓冲液的体积比为10:90～35:65，每升反应体系中EGCG的加入量为1.1～1.6 g。
上述步骤2中，优选所述抗坏血酸的加入量为EGCG质量的15～40倍。
上述步骤3中，所述大孔树脂层析柱的填料为D101、HP-20等中任意一种，优选依次经过3～6倍柱体积纯水、3～6倍柱体积甲醇洗脱。
上述步骤4中，所述高效制备液相色谱的分离条件为：采用1010-C18HC制备色谱层析柱，以体积浓度为78%的乙腈水溶液为流动相A，纯水为流动相B，在0～30 min等梯度洗脱，流动相A为15%、流动相B为85%，流速为50～75mL/min，检测波长为280 nm。
与现有技术相比，本发明的有益效果如下： 
1、本发明在低温条件下利用外源PPO酶促EGCG生成TSA，改变了以往使用茶汤提取或茶叶内源酶酶促氧化液时产生多种二聚体产物如茶黄素组分和聚酯型儿茶素组分导致分离纯化困难的技术现状，实现了利用外源PPO酶催化EGCG定向合成TSA。
2、本发明采用EGCG定向生成TSA，使底物利用率提高，产物单一、副产物和杂质少，TSA得率有效提高至62.4%～97.6%，有效避免了茶黄素与其余聚酯型儿茶素生成，减少了分离纯化的繁琐工序。
3、本发明通过PPO酶促生成TSA，有效突破以往采用氯化铜等化学试剂制备，减少化学试剂使用引起的安全问题等，实现了酶促反应制备TSA的绿色环保、简便安全。
4、本发明采用外源PPO酶促生成TSA的方法，为单一聚酯型儿茶素组分的生物活性研究及其功能性开发提供了物质基础，同时为后续开展TSA高效大规模工业化生产与利用提供了理论与技术支撑。
 附图说明
图1是EGCG的HPLC色谱图。
图2是实施例5中TSA制备液的HPLC色谱图。
 具体实施方式
下面结合附图和实施例对本发明进一步详细说明，但本发明的保护范围不仅限于这些实施例。
实施例1
步骤1：将500g梨置于500mL pH为6.5的磷酸缓冲液中，加入5g聚乙烯吡咯烷酮、1g抗坏血酸混匀打浆后，常温浸提12小时，离心分离，收集上清液，即得PPO酶液。
步骤2：将0.1157g EGCG加入76mL pH为6.5的磷酸缓冲液中，并加入步骤1得到的24mL PPO酶液，然后将反应体系于10℃下搅拌60分钟，再加入4.5g抗坏血酸于85℃下搅拌10分钟，得到酶促反应液，其中TSA含量为0.89mg/mL。
步骤3：将酶促反应液上D101大孔树脂层析柱，依次经过3倍柱体积纯水、3倍柱体积甲醇洗脱，50℃蒸馏得甲醇洗脱浓缩液。
步骤4：将甲醇洗脱浓缩液经高效制备液相色谱纯化，采用1010-C18HC制备色谱层析柱（50mm×250mm，10μm），上样量为5.0mL，以体积浓度为78%的乙腈水溶液为流动相A，纯水为流动相B，在0～30min等梯度洗脱，流动相A为15%、流动相B为85%，流速为60mL/min，检测波长为280nm。所得TSA制备液经55℃真空浓缩后冷冻干燥，得到TSA精制品，得率为56.1%，纯度94%。
对比例1
步骤1：将300g茶置于500mL pH为7.0的磷酸缓冲液中，加入5g聚乙烯吡咯烷酮、1g抗坏血酸混匀打浆后，常温浸提12小时，离心分离，收集上清液，即得PPO酶液。
步骤2：将0.2068g EGCG加入80mL pH为7.0的磷酸缓冲液中，并加入步骤1得到的20mL PPO酶液，然后将反应体系于30℃下搅拌60分钟，再加入4.0g抗坏血酸于90℃下搅拌10分钟，得到酶促反应液， 其中TSA含量为0.59mg/mL。
步骤3：将酶促反应液上D101大孔树脂层析柱，依次经过3倍柱体积纯水、3倍柱体积甲醇洗脱，50℃蒸馏得甲醇洗脱浓缩液。
步骤4：将甲醇洗脱浓缩液经高效制备液相色谱纯化，采用1010-C18HC制备色谱层析柱（50mm×250mm，10μm），上样量为5.0mL，以体积浓度为78%的乙腈水溶液为流动相A，纯水为流动相B，在0～30min等梯度洗脱，流动相A为15%、流动相B为85%，流速为60mL/min，检测波长为280nm。所得TSA制备液经55℃真空浓缩后冷冻干燥，得到TSA精制品，得率为27.01%，纯度90%。
实施例2
步骤1：将500g苹果置于500mL pH为7.0的磷酸缓冲液中，加入5g聚乙烯吡咯烷酮、1g抗坏血酸混匀打浆后，常温浸提12小时，离心分离，收集上清液，即得PPO酶液。
步骤2：将0.1604g EGCG加入68mL pH为7.0的磷酸缓冲液中，并加入步骤1得到的32mL PPO酶液，然后将反应体系于10℃下搅拌60分钟，再加入4.8g抗坏血酸于92℃下搅拌11分钟，得到酶促反应液，其中TSA含量为1.23mg/mL。
步骤3：将酶促反应液上D101大孔树脂层析柱，依次经过4倍柱体积纯水、4倍柱体积甲醇洗脱，50℃蒸馏得甲醇洗脱浓缩液。
步骤4：将甲醇洗脱浓缩液经高效制备液相色谱纯化，采用1010-C18HC制备色谱层析柱（50mm×250mm，10μm），上样量为5.0mL，以体积浓度为78%的乙腈水溶液为流动相A，纯水为流动相B，在0～30min等梯度洗脱，流动相A为15%、流动相B为85%，流速为60mL/min，检测波长为280nm。所得TSA制备液经55℃真空浓缩后冷冻干燥，得到TSA精制品，得率为48.6%，纯度93%。
实施例3
步骤1：将200g核桃花絮置于500mL pH为6.0的磷酸缓冲液中，加入5g聚乙烯吡咯烷酮、1g抗坏血酸混匀打浆后，常温浸提12小时，离心分离，收集上清液，即得PPO酶液。
步骤2：将0.1146g EGCG加入35mL pH为6.5的磷酸缓冲液中，并加入步骤1得到的65mL PPO酶液，然后将反应体系于5℃下搅拌60分钟，再加入3.5g抗坏血酸于90℃下搅拌10分钟，得到酶促反应液，TSA含量为0.96mg/mL。
步骤3：将酶促反应液上D101大孔树脂层析柱，依次经过6倍柱体积纯水、6倍柱体积甲醇洗脱，60℃蒸馏得甲醇洗脱浓缩液。
步骤4：将甲醇洗脱浓缩液经高效制备液相色谱纯化，采用1010-C18HC制备色谱层析柱（50mm×250mm，10μm），上样量为5.0mL，以体积浓度为78%的乙腈水溶液为流动相A，纯水为流动相B，在0～30min等梯度洗脱，流动相A为15%、流动相B为85%，流速为60mL/min，检测波长为280nm。所得TSA制备液经55℃真空浓缩后冷冻干燥，得到TSA精制品，得率为53.4%，纯度94%。
实施例4
步骤1：将300g土豆置于500mL pH为8.1的磷酸缓冲液中，加入5g聚乙烯吡咯烷酮、1g抗坏血酸混匀打浆后，常温浸提12小时，离心分离，收集上清液，即得PPO酶液。
步骤2：将0.1604g EGCG加入88mL pH为8.1的磷酸缓冲液中，并加入步骤1得到的12mL PPO酶液，然后将反应体系于5℃下搅拌60分钟，再加入4.5g抗坏血酸于90℃下搅拌10分钟，得到酶促反应液，TSA含量为1.27mg/mL。
步骤3：将酶促反应液上D101大孔树脂层析柱，依次经过5倍柱体积纯水、5倍柱体积甲醇洗脱，60℃蒸馏得甲醇洗脱浓缩液。
步骤4：将甲醇洗脱浓缩液经高效制备液相色谱纯化，采用1010-C18HC制备色谱层析柱（50mm×250mm，10μm），上样量为5.0mL，以体积浓度为78%的乙腈水溶液为流动相A，纯水为流动相B，在0～30min等梯度洗脱，流动相A为15%、流动相B为85%，流速为60mL/min，检测波长为280nm。所得TSA制备液经55℃真空浓缩后冷冻干燥，得到TSA精制品，得率为54.2%，纯度95%。
实施例5
步骤1：将7.4mg PPO置于100mL pH为7.0的磷酸缓冲液中，配置成245000U/L的PPO酶液。
步骤2：将0.1146g EGCG加入90mL pH为7.0的磷酸缓冲液中，并加入步骤1得到的10mL PPO酶液，然后将反应体系于10℃下搅拌60分钟，再加入4.3g抗坏血酸于90℃下搅拌10分钟，得到酶促反应液，TSA含量为1.02mg/mL。
步骤3：将酶促反应液上D101大孔树脂层析柱，依次经过3倍柱体积纯水、3倍柱体积甲醇洗脱，60℃蒸馏得甲醇洗脱浓缩液。
步骤4：将甲醇洗脱浓缩液经高效制备液相色谱纯化，采用1010-C18HC制备色谱层析柱（50mm×250mm，10μm），上样量为5.0mL，以体积浓度为78%的乙腈水溶液为流动相A，纯水为流动相B，在0～30min等梯度洗脱，流动相A为15%、流动相B为85%，流速为60mL/min，检测波长为280nm。所得TSA制备液经55℃真空浓缩后冷冻干燥，得到TSA精制品，得率为69.6%。由图1和图2可见，在低温条件下PPO酶促EGCG生成了高纯度的TSA，TSA纯度为96%。
实施例6
步骤1：将500g山药置于500mL pH为7.0磷酸缓冲液中，加入5g聚乙烯吡咯烷酮、1g抗坏血酸混匀打浆后，常温浸提12小时，离心分离，收集上清液，即得PPO酶酶液。
步骤2：将0.1146g EGCG加入85mL pH为6.5的磷酸缓冲液中，并加入步骤1得到的15mL PPO酶液，然后将反应体系于5℃下搅拌60分钟，再加入4.3g抗坏血酸于92℃下搅拌10分钟，得到酶促反应液，TSA含量为1.59mg/mL。
步骤3：将酶促反应液上D101大孔树脂层析柱，依次经过3倍柱体积纯水、3倍柱体积甲醇洗脱，50℃蒸馏得甲醇洗脱浓缩液。
步骤4：将甲醇洗脱浓缩液经高效制备液相色谱纯化，采用1010-C18HC制备色谱层析柱（50mm×250mm，10μm），上样量为5.0mL，以体积浓度为78%的乙腈水溶液为流动相A，纯水为流动相B，在0～30min等梯度洗脱，流动相A为15%、流动相B为85%，流速为60mL/min，检测波长为280nm。所得TSA制备液经55℃真空浓缩后冷冻干燥，得到TSA精制品，得率为97.6%，纯度96%。

Claims (8)

  1. 一种低温酶法制备聚酯型儿茶素A的方法，其特征在于包括以下步骤：
    步骤1：将富含多酚氧化酶的植物组织置于pH为6.5～7.0的磷酸缓冲液中，加入聚乙烯吡咯烷酮、抗坏血酸混匀打浆后，常温浸提10～13小时，离心分离，收集上清液，即得多酚氧化酶酶液；或者取多酚氧化酶用pH为6.5～7.0的磷酸缓冲液配置成多酚氧化酶酶液；
    步骤2：将表没食子酸儿茶素没食子酸酯加入pH为6.5～7.0的磷酸缓冲液中，并加入步骤1得到的多酚氧化酶酶液，然后将反应体系于5～15℃下搅拌30～60分钟，再加入抗坏血酸于85～95℃下搅拌8～12分钟，得到酶促反应液；
    步骤3：将酶促反应液上大孔树脂层析柱，经纯水、甲醇洗脱，50～60℃蒸馏得甲醇洗脱浓缩液；
    步骤4：将甲醇洗脱浓缩液经高效制备液相色谱纯化后，真空浓缩、冷冻干燥，得到聚酯型儿茶素A精制品。
  2. 根据权利要求1所述的低温酶法制备聚酯型儿茶素A的方法，其特征在于：步骤1中，所述富含多酚氧化酶的植物组织包括富含多酚氧化酶的水果、蔬菜、植物的花、植物的幼果中任意一种。
  3. 根据权利要求1所述的低温酶法制备聚酯型儿茶素A的方法，其特征在于：步骤1中，每升pH为6.5～7.0磷酸缓冲液中富含多酚氧化酶的植物组织、聚乙烯吡咯烷酮、抗坏血酸的加入量依次为900～1100g、9.0～15.0g、1.5～2.5g。
  4. 根据权利要求1所述的低温酶法制备聚酯型儿茶素A的方法，其特征在于：步骤1中，取多酚氧化酶用pH为6.5～7.0的磷酸缓冲液配置成235000～329000U/L的多酚氧化酶酶液。
  5. 根据权利要求1所述的低温酶法制备聚酯型儿茶素A的方法，其特征在于：步骤2中，所述多酚氧化酶酶液与pH为6.5～7.0的磷酸缓冲液的体积比为10:90～35:65，每升反应体系中表没食子酸儿茶素没食子酸酯的加入量为1.1～1.6 g。
  6.  根据权利要求1所述的低温酶法制备聚酯型儿茶素A的方法，其特征在于：步骤2中，所述抗坏血酸的加入量为表没食子酸儿茶素没食子酸酯质量的15～40倍。
  7. 根据权利要求1所述的低温酶法制备聚酯型儿茶素A的方法，其特征在于：步骤3中，所述大孔树脂层析柱的填料为D101或HP-20，依次经过3～6倍柱体积纯水、3～6倍柱体积甲醇洗脱。
  8. 根据权利要求1所述的低温酶法制备聚酯型儿茶素A的方法，其特征在于：步骤4中，所述高效制备液相色谱的分离条件为：采用1010-C18HC制备色谱层析柱，以体积浓度为78%的乙腈水溶液为流动相A，纯水为流动相B，在0～30 min等梯度洗脱，流动相A为15%、流动相B为85%，流速为50～75mL/min，检测波长为280 nm。
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