JP4026723B2 - Method for producing epithea fragalins and method for producing beverages containing epithea fragalins - Google Patents

Description

  The present invention relates to a method for producing epithea fragalins and a method for producing a tea beverage containing epithea fragalins.

  Epithea fragalin-3-O-gallate is, for example, disclosed in Japanese Patent Application Laid-Open No. 2000-226329 (Patent Document 1), Naoto Oku et.al., Biol. Pharm. Bull., 26 (9), 1235-1238 ( 2003) (Non-Patent Document 1) is a useful compound listed as a candidate substance of a matrix metalloproteinase (MMP) inhibitor that is expected to have an antitumor metastasis effect.

  Methods for chemically synthesizing epitheaflagalins include methods using potassium ferricyanide (Gen-ichiro Nonaka, Fumio Hashimoto and Itsuo Nishioka, Chem. Pharm. Bull., 34 (1), 61-65 (1986) (non- Patent document 2)) is known. However, potassium ferricyanide is not approved for use as a food additive.

As an enzymatic synthesis method, a method using peroxidase (Shegmin Sang et. Al., Bioorganic and Medicinal Chemistry, 12 (2), 459-467 (2004) (Non-patent Document 3)) has been reported. In this peroxidase reaction, hydrogen peroxide and pyrogallol are used.
JP 2000-226329 A Naoto Oku et.al., Biol. Pharm. Bull., 26 (9), 1235-1238 (2003) Gen-ichiro Nonaka, Fumio Hashimoto and Itsuo Nishioka, Chem. Pharm. Bull., 34 (1), 61-65 (1986) Shegmin Sang et.al., Bioorganic and Medicinal Chemistry, 12 (2), 459-467 (2004)

  The method using potassium ferricyanide is not applicable to the production of food because potassium ferricyanide is not permitted to be used as a food additive.

Regarding the method using peroxidase, hydrogen peroxide is recognized as a food additive, but since it is supposed to be decomposed or removed before the final food is completed, the step of decomposing or removing it is essential. In particular, hydrogen peroxide itself is stable, but since it quickly generates hydroxy radicals (OH ·) by heavy metal complexes in the living body, such as Fe ^{2+ -complexes} , it oxidizes all organic compounds that make up the living body, It can be a poison. Pyrogallol is not recognized as a food additive.

  The conventional techniques described above have a drawback that they cannot be used directly for food production. Therefore, there has been a demand for a simple method for producing epitheaflagalin and epiteafragalin-3-O-gallate that can be directly used in foods.

  Accordingly, an object of the present invention is to provide a simple method for producing epiteafragalin and epiteafragalin-3-O-gallate that can be directly used in foods.

  Furthermore, the objective of this invention is providing the manufacturing method of the drink containing epitea fragalin and epitea fragalin-3-O-gallate using the said manufacturing method.

  As a result of intensive studies to solve the above problems, the present inventors have made use of various polyphenol oxidases approved as foods and gallic acid recognized as food additives, such as green tea The inventors have found a method for producing epithea fragalins without impairing the flavor, and have completed the present invention.

The present invention is as follows.
[1] To convert epigallocatechin and / or epigallocatechin gallate to epitheafragalin and / or epitheafragalin-3-O-gallate by allowing polyphenol oxidase to act in the presence of gallic acid. The manufacturing method of epitea fragalins containing this.
[2] By adding gallic acid to the tea extract and allowing polyphenol oxidase to act, at least a part of epigallocatechin and / or epigallocatechin gallate contained in the tea extract is converted to epitheafragalin and / or Or the manufacturing method of the drink containing epitea fragalins including converting to epitea fragalin-3-O-gallate.
[3] The production method according to [2], wherein the tea extract is a green tea extract, an oolong tea extract, or a black tea extract.
[4] The production method according to [2] or [3], wherein gallic acid having a molar ratio of 1 to 10 is added to the total amount of epigallocatechin and epigallocatechin gallate contained in the tea extract.
[5] The polyphenol oxidase is selected from the group consisting of laccase (EC1.10.3.2), tyrosinase (EC 1.14.18.1), bilirubin oxidase (EC1.3.3.5), phenol and polyphenol oxidase (EC1.10.3.1) The production method according to any one of [1] to [4], which is at least one kind of enzyme.
[6] The production method according to any one of [1] to [5], wherein the polyphenol oxidase is a free enzyme or an immobilized enzyme.
[7] The polyphenol oxidase is a free enzyme, and further comprises heating the tea extract to inactivate the enzyme after the conversion reaction with the polyphenol oxidase, according to any one of [2] to [5] Production method.
[8] Polyphenol oxidase is an immobilized enzyme, and the conversion reaction with polyphenol oxidase is performed by adding the immobilized enzyme to the tea extract and removing the immobilized enzyme from the tea extract after the conversion reaction. [2] The production method according to any one of [5].
[9] Polyphenol oxidase is an immobilized enzyme, and the conversion reaction with polyphenol oxidase is performed by passing a tea extract to which gallic acid has been added into a container filled with the immobilized enzyme, any of [2] to [5] The production method according to claim 1.
[10] The production method according to any one of [2] to [9], wherein the conversion reaction with polyphenol oxidase is performed while oxygen or air is passed through the tea extract.
[11] The production method according to any one of [2] to [10], wherein the beverage is a green tea beverage, a green tea beverage, a oolong tea beverage, a oolong tea beverage, a tea beverage, or a tea beverage.
[12] The production method according to any one of [2] to [11], wherein the beverage contains 0.0001 to 0.5 mass% of an epitheafragalin.

  Epithea fragalin and epithea fragalin-3-O-gallate which can be directly used for food production can be produced.

  In the present invention, epigallocatechin and / or epigallocatechin gallate is converted to epitheaflagalin and / or epitheafragalin-3-O-gallate by allowing polyphenol oxidase to act in the presence of gallic acid. The present invention relates to a method for producing epiteafragalins.

  Furthermore, the present invention provides at least a part of epigallocatechin and / or epigallocatechin gallate contained in the tea extract by adding gallic acid to the tea extract and allowing polyphenol oxidase to act, respectively. The present invention relates to a method for producing a beverage containing epithea fragalins, comprising converting to phosphorus and / or epithea fragalin-3-O-gallate.

  The manufacturing method of the drink containing the epitea fragalin of this invention is one aspect | mode of the manufacturing method of the epitea fragalin of this invention. Hereinafter, a case where epigallocatechin and / or epigallocatechin gallate is contained in a tea extract, that is, a method for producing a beverage containing epiteafragalins will be described as an example. About the manufacturing method of epitea fragalins, epigallocatechin and / or epigallocatechin gallate is read as a raw material or raw material solution containing epigallocatechin and / or epigallocatechin gallate. be able to.

  In the present invention, the tea extract is not particularly limited as long as it contains epigallocatechin and / or epigallocatechin gallate. Examples of the tea extract include green tea extract, oolong tea extract, and black tea extract.

Green tea extract Green tea is an extract of the leaves of Camellia plants, which are mainly dried from Camellia sinensis and Camellia assamica sprouts. Examples of the green tea extract include SD Green Tea Extract Powder No. 16714 (San-Eigen FFI Co., Ltd.), Sunphenon BG (Taiyo Chemical Co., Ltd.), and the like. As an extraction method, there is a method in which the raw material is extracted with hot water, hydrous alcohol, glycerin aqueous solution, ethyl acetate or the like, purified and concentrated, and spray-dried or freeze-dried.

Oolong tea extract Oolong tea is obtained by fermenting tea leaves similar to green tea extract for a certain period of time and then heating to stop fermentation (semi-fermented tea). As the oolong tea extract, for example, FD oolong tea extract powder No. 16297 (San-Eigen FFI Co., Ltd.). As an extraction method, there is a method in which the raw material is extracted with hot water, hydrous alcohol, glycerin aqueous solution, ethyl acetate or the like, purified and concentrated, and spray-dried or freeze-dried.

Black tea extract Black tea is obtained by strongly fermenting tea leaves similar to the green tea extract and then heating to stop fermentation (strongly fermented tea). Examples of the black tea extract include SD black tea extract powder No. 16691 (Saneigen FFI Co., Ltd.). As an extraction method, there is a method in which the raw material is extracted with hot water, hydrous alcohol, glycerin aqueous solution, ethyl acetate or the like, purified and concentrated, and spray-dried or freeze-dried.

  Examples of tea extract are shown in Table 1 below. However, these tea extracts are examples, and are not limited to these.

EGC and EGCg contents are catalog values of each manufacturer

  In the method of the present invention, it is appropriate to add 1 to 10 gallic acid in a molar ratio with respect to the total amount of epigallocatechin and epigallocatechin gallate contained in the tea extract. Preferably, gallic acid having a molar ratio of 2 to 5 is added to the total amount of epigallocatechin and epigallocatechin gallate.

  Polyphenol oxidase is not particularly limited as long as it is an enzyme capable of converting epigallocatechin to epitheaflagalin and epigallocatechin gallate to epiteafragalin-3-O-gallate, respectively. There is no. Examples of such polyphenol oxidase include laccase (EC1.10.3.2), tyrosinase (EC 1.14.18.1), bilirubin oxidase (EC1.3.3.5), phenol and polyphenol oxidase (EC1.10.3.1). Mention may be made of at least one enzyme selected from the group. Tyrosinase (EC 1.14.18.1) is also partly classified as 1.10.3.1, but the enzyme classified as 1.10.3.1 is also a part of tyrosinase (EC 1.14.18.1) in the present invention.

  The polyphenol oxidase can be a free enzyme or an immobilized enzyme. When polyphenol oxidase is a free enzyme, a predetermined amount of polyphenol oxidase is added to a tea extract to which gallic acid is added, and a conversion reaction is performed at a predetermined temperature for a predetermined time. The predetermined amount of polyphenol oxidase is, for example, in the range of 10 to 200 U with respect to 1 ml of a solution containing 0.5 (w / v)% to 15 (w / v)% of tea extract.

  Note that how much epigallocatechin and / or epigallocatechin gallate is contained in the tea extract varies depending on the tea extract. For example, in SD green tea extract powder, the sum of epigallocatechin and epigallocatechin gallate is said to be 20 (w / w)% or more, epigallocatechin is about 10 (w / w)%, epigallocatechin gallate is About 13 (w / w)% is included. Thus, for example, a solution containing 1% tea extract has about 1 mg / ml epigallocatechin and about 1.3 mg / ml epigallocatechin gallate.

  The predetermined time for the conversion reaction is, for example, 10 minutes to 15 hours, and the predetermined temperature is in the range of 20 to 60 ° C. The method further comprises heating the tea extract to inactivate the enzyme after the conversion reaction with polyphenol oxidase. The heating condition is suitably 2 to 10 minutes at 70 to 90 ° C.

  Polyphenol oxidase can also be an immobilized enzyme. When using an immobilized enzyme, the conversion reaction with polyphenol oxidase can be performed by adding the immobilized enzyme to a tea extract to which gallic acid has been added. The time and temperature of the conversion reaction are, for example, 10 to 240 minutes, and the predetermined temperature is in the range of 20 to 60 ° C. After the conversion reaction, the immobilized enzyme is removed from the tea extract. The removal can be performed, for example, by filtering the immobilized enzyme.

  When polyphenol oxidase is an immobilized enzyme, the conversion reaction by polyphenol oxidase can also be performed by passing a tea extract to which gallic acid has been added into a container filled with the immobilized enzyme. The circulation conditions of the tea extract can be performed by setting the temperature in the range of 20 to 60 ° C. and the contact time with the immobilized enzyme in the range of 10 to 240 minutes, for example.

  The conversion reaction with polyphenol oxidase can be performed while aeration of oxygen or air through the tea extract. The conversion reaction can be promoted by ventilating oxygen or air. The ventilation condition of oxygen or air can be, for example, 0.2 to 10 L / L / min. Furthermore, in order to enhance the aeration effect, aeration can be performed while stirring the reaction solution.

  The conversion reaction with polyphenol oxidase converts at least a portion of epigallocatechin and / or epigallocatechin gallate contained in the tea extract to epitheaflagalin and / or epitheafragalin-3-O-gallate, respectively. Implement as follows. Depending on the purpose, the amount of gallic acid added and the reaction conditions can be selected so that the total amount of epigallocatechin and / or epigallocatechin gallate is converted to epitheaflagalin and / or epitheafragalin-3-O-gallate. can do.

  The conversion reaction with polyphenol oxidase is performed by adding gallic acid to the tea extract. However, in consideration of the pH dependence of the polyphenol oxidase activity, a buffer may be added to the tea extract for the purpose of pH adjustment. it can. However, considering that the conversion reaction product is used as a beverage as it is, it is preferable to use, for example, phosphoric acid and its salts, citric acid and its salts, etc. as the buffer. Of course, the conversion reaction by polyphenol oxidase can also be performed without adding a buffer.

  The beverage obtained by the production method of the present invention can be used as a beverage as it is. The beverage obtained by the production method of the present invention is specifically a tea beverage, and examples of the tea beverage include a green tea beverage, a green tea beverage, a oolong tea beverage, a oolong tea beverage, a tea beverage, or a tea beverage. Can be mentioned. The beverage obtained by the production method of the present invention can contain, for example, 0.0001 to 0.5% by mass of epithea fragalins.

  Furthermore, the beverage obtained by the production method of the present invention can be used as it is as a beverage, but a solution obtained by treating a tea extract with polyphenol oxidase in the presence of gallic acid is extracted, purified or concentrated, and spray dried. Or freeze-dry and prepare extract powder by sizing. Such concentrated solutions or extract powders can also be used as raw materials for various forms of food and health care products.

  Examples of foods to which the concentrated solution or extract powder can be applied include gums, confectionery, candy, and supplements. Examples of the health care product to which the concentrated solution or the extract powder can be applied include an oral cleaning liquid and a toothpaste.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 : Detection, separation, purification and structure determination of epitheafragalin, epitheaflavalin-3-O-gallate in laccase-treated green tea extract, 250 g of green tea extract, 20 g of gallic acid monohydrate, phosphate buffer ( Water (pH 2.4) 600 mL, laccase Y 1.66 g (Daiwa Kasei Co., Ltd., 200,000 U) was added to a total volume of 2 L, and the reaction was carried out at 55 ° C. for 10 hours. When this enzyme reaction solution was measured by liquid chromatography under the following analysis conditions, a peak that was not observed with laccase untreated (peak 1: epitheafragalin) and an increased peak (peak 2: epithea) (Flagalin-3-O-gallate) was detected (Fig. 1).

Detector: UV absorptiometer (measurement wavelength: 320nm)
Column: YMC-Pack ODS-A A-312 (6.0φ × 150mm)
Column temperature: 40 ° C
Mobile phase: 0.05mol / L sodium dihydrogen phosphate sample / acetonitrile mixture (10: 3)
Flow rate: 1.0mL / min

Next, these two components were isolated and purified.
2 L of the reaction solution after laccase treatment was extracted twice with 2 L of ethyl acetate. To the ethyl acetate layer, 5% aqueous sodium hydrogen carbonate solution (100 mL) was added twice, washed with alkali, washed with water (100 mL × 2 times), and dried over anhydrous sodium sulfate. This solution was filtered with a cotton plug, and the filtrate was dried with a rotary evaporator to obtain 90 g of a residue. Dissolved in mobile phase, reversed-phase column chromatography (column: Lobar Column LiChroprep RP-18 (37 x 440 mm), mobile phase: 0.05 mol / L sodium dihydrogen phosphate sample / acetonitrile mixture (17: 3) → ( 4: 1)), each fraction containing a large amount of component 1 and component 2 is collected, concentrated on a rotary evaporator, extracted with ethyl acetate, the ethyl acetate layer is dried over anhydrous sodium sulfate, and cotton plug After filtration, the filtrate was dried on a rotary evaporator to obtain a residue 770 mg (Fr.1) rich in component 1 and a residue 730 mg (Fr.2) rich in component 2. Each was subjected to oxalic acid-treated silica gel column chromatography (packed with 10 g of oxalic acid-treated silica gel (column diameter: 1.5 cm) and eluted (eluent: ethyl acetate / hexane mixture (1: 1) → (2: 1)). Furthermore, it was dissolved in a small amount of ethyl acetate, and a small amount of hexane was added to obtain 10 mg of almost single orange crystals (Fig. 2).
In Fr.2, the same oxalic acid-treated silica gel column chromatography was performed, and recrystallization was performed with ethanol (99.5) / water to obtain 14 mg of orange crystals (FIG. 3).

[Analysis conditions]
Column: TSK gel ODS-80Ts (4.6φ × 150mm)
Column temperature: 40 ° C
Mobile phase: Water / acetonitrile / phosphoric acid mixture (500: 150: 1)
Flow rate: 1.0mL / min
Detection wavelength: 254 nm (each lower side), 210 nm (each upper side)

3) Structural analysis of two components The obtained two components were subjected to instrumental analysis (NMR, MALDI-TOFMS, ESI-precision MS, ultraviolet absorption spectrum). The analysis conditions for each instrumental analysis are as follows.
NMR: ¹³ C-NMR: 100 MHz, solvent CD ₃ OD. ¹ H-NMR: 400 MHz, solvent CD ₃ OD.
・ MALDI-TOFMS: AXIMA-CFR plus (Shimadzu / KRATO, ver2.4.0, reflectron mode), matrix solution: 2,5-dihydroxybenzoic acid (2,5-DHB), cationizing agent: NaCl
・ ESI-Precision MS: AgilentLC1100 / ABI QSTARXL
-UV-visible absorption spectrum: UV-visible spectrophotometer (Shimadzu Corporation, UV250). The sample solution was a 50% ethanol solution of about 0.01 mg / mL.

(1) Structural analysis of component 1 In the ¹ H-NMR spectrum of component 1, 5.98 ppm (1H, d, J = 2.4 Hz) and 5.97 ppm (1H, d, J derived from the A ring and C ring of epicatechin = 2.4Hz), 4.81 ppm (1H, s), 4.27 ppm (1H, m), 2.93 ppm, 2.82 ppm (2H, m). Further, the structural formula shown in FIG. 4 was obtained by analysis using ¹³ C-NMR spectrum and HMQC and HMBC.
When the UV-visible absorption spectrum of component 1 (50% ethanol solution of about 0.01 mg / mL) was measured, it showed maximum absorption at 306 nm and 281 nm (FIG. 5). In addition, in the measurement of MALDI-TOFMS, m / z 423 was strongly detected by addition of NaCl as a cationizing agent, and m / z 401 disappeared. Therefore, [M + H] ⁺ was 401 and the molecular weight was 400. The structure was supported (Figs. 6 and 7).

  This component 1 is in agreement with the spectrum data of non-patent literature (Gen-ichiro Nonaka, Fumio Hashimoto and Itsuo Nishioka, Chem. Pharm. Bull., 34 (1), 61-65 (1986). And a compound designated as pyrogallin.

(2) Structural analysis of component 2 In the ¹ H-NMR spectrum of component 2, the hydrogen signal bound to C-2 and C-3 in the epicatechin part is shifted to the lower magnetic field side, so C-3 The hydroxyl group is considered to be an ester, and the structural formula in FIG. 8 is substituted with a galloyl group. When the UV-visible absorption spectrum of Component 2 (50% ethanol solution of about 0.01 mg / mL) was measured, it was similar to Component 1 and showed maximum absorption at 302 nm and 280 nm (FIG. 9). In the measurement of MALDI-TOFMS (Figs. 10 and 11), m / z 575 was strongly detected and m / z 553 disappeared due to the addition of NaCl as the cationizing agent, and [M + H] ⁺ was 553. The molecular weight was 552, and the structure of component 2 was supported because it was larger than component 1 by 152 corresponding to the molecular weight of the galloyl group.

  This component 2 is described in Non-Patent Document 2 (Gen-ichiro Nonaka, Fumio Hashimoto and Itsuo Nishioka, Chem. Pharm. Bull., 34 (1), 61-65 (1986)), and Shegmin Sang et.al., The compound was consistent with the spectral data of Bioorganic and Medicinal Chemistry, 12 (2), 459-467 (2004) and was named epitheafragalin-3-O-gallate.

  The results of the two-component instrumental analysis are summarized in Table 2.

Example 2: Production of epithea fragalin and epithea fragalin-3-O-gallate from tea extract by laccase (Part 1)
Dissolve 250 g of SD Green Tea Extract Powder (San-Ei Gen FF Eye) in 1000 mL of 10% ethanol solution, 600 mL of phosphate buffer (pH 2.4), 100 mL of 20% gallic acid ethanol solution, enzyme solution (Daiwalaccase Y120, 200,000 U, 1.66 g, white rot fungus-derived Trametes sp., EC 1.10.3.2) 200 mL, water 100 mL was added to make a total of 2000 mL, and the enzyme reaction was started while stirring at 55 ° C. 0.83 g / 50 mL of enzyme solution was added 4 hours and 6 hours after the start of the reaction. In this case, the production amounts of epiteafragalin and epiteafragalin 3-O-gallate were analyzed by liquid chromatography under the following analytical conditions. The results are shown in Table 3.

[Analysis conditions]
Detector: UV absorptiometer (measurement wavelength: 280nm)
Column: YMC-Pack ODS-A A-312 (6.0φ × 150mm)
Column temperature: 30 ° C
Mobile phase: 0.05mol / L sodium dihydrogen phosphate sample / acetonitrile mixture (10: 3)
Flow rate: 1.0mL / min

Example 3: Production of epithea fragalin and epithea fragalin-3-O-gallate from tea extract with laccase (Part 2)
To 100 ml of water, 1 g of SD green tea extract powder (Saneigen F.F.I), 0.48 g of gallic acid monohydrate, and Daiwa Laccase Y120 2500U-10000U were added, and an enzyme reaction was performed at 50 ° C. At the start of the treatment, about 4.4 gallic acid was present in a molar ratio with respect to the total amount of epigallocatechin and epigallocatechin gallate. FIG. 12 and FIG. 13 show changes in the production amounts of epitheaflagalin and epiteafragalin-3-O-gallate at this time. The maximum production was 2.78 mg / g (50 ° C, 30 minutes, laccase 5000U) for epitheaflavalin and 15.41 mg / g (50 ° C, 30 minutes, laccase 10000U) for epitheaflavalin-3-gallate. It was.

Example 4: Production of epitheaflagalin and epiteafragalin-3-O-gallate from tea extract with tyrosinase or bilirubin oxidase
6.9 mg epigallocatechin gallate, 2.8 mg gallic acid monohydrate and polyphenol oxidase (tyrosinase 20U or bilirubin oxidase 0.4U) in 20 mmol / L potassium phosphate buffer, in a total of 1.5 mL solution, In the case of tyrosinase, the reaction was performed at 25 ° C. for 3 hours, and in the case of bilirubin oxidase, the reaction was performed at 45 ° C. for 2 hours.
The reaction solution was measured by the liquid chromatography method under the following analysis conditions.

[Analysis conditions]
Detector: UV absorptiometer (measurement wavelength: 280nm)
Column: YMC-Pack ODS-A A-312 (6.0φ × 150mm) (when analyzing epitheafragalin-3-gallate)
Cadenza CD-C18 (4.6φ × 75mm) (at the time of epitheafragalin analysis)
Mobile phase: 0.05mol / L sodium dihydrogen phosphate sample / acetonitrile mixture (10: 3)
Flow rate: 1.0mL / min

Table 4 shows the total amount of epitheaflavalins produced at this time.
Tyrosinase (EC1.14.18.1) is derived from Mushroom from Funakoshi-Worthington Biochemical Corp., and bilirubin oxidase (EC.1.3.3.5) is derived from Mycothesium sp. From Wako Pure Chemicals-Amano Enzyme. used.

Example 5: Production of epitheaflavalin-3-O-gallate from epigallocatechin gallate (control of reaction pH))
Epigallocatechin gallate 4.6 mg, gallic acid 1.9 mg, laccase 20U (added once or twice) in sodium phosphate buffer at each pH, total 1 mL in a test tube at 50 ° C for 3 hours Reaction was performed. The reaction solution was measured by the liquid chromatography method under the analysis conditions shown in Example 2. Epitheaflavalin-3-O-gallate was produced at pH 4.5 to pH 8, and the maximum production was observed at pH 6.0 to 6.5. In addition, the amount of the enzyme increased when the enzyme was added in two batches rather than at one time.

Example 6: Production of epitheafragalin-3-O-gallate from epigallocatechin gallate (control of the amount of gallic acid added 1)
The reaction was carried out in the same manner as in Example 5 except that the amount of gallic acid was doubled to 3.8 mg. The reaction solution was measured by the liquid chromatography method under the analysis conditions shown in Example 2. In terms of molar ratio, epigallocatechin-3-O-gallate: gallic acid = 1: 2 produced more than 1: 1 (Example 5).

Example 7: Production of epiteafragalin-3-O-gallate from epigallocatechin gallate (control of air flow)
In the production of theaflavalins by laccase, 10 L / L / min of air was aerated, and the amount of epitheaflavalin-3-gallate produced was measured. 20 mmol / L potassium phosphate buffer (pH 6.5) / ethanol mixture (20: 1) to 50 mL of epigallocatechin gallate 0.23 g, gallic acid monohydrate 0.19 g, Daiwa Laccase 1000 U (9.3 mg), The reaction of either of the following methods (1) and (2) was carried out in a 100 mL Erlenmeyer flask at 50 ° C. for 30 minutes. (1) Shaking only, (2) Aeration, aeration, and reaction. The reaction solution was measured by the liquid chromatography method under the analysis conditions shown in Example 2. It was confirmed that the amount of epitheaflavalin-3-O-gallate produced increased by aeration.

Example 8: Production of epitheaflagalin from epigallocatechin (control of reaction pH)
Epigallocatechin 3.1 mg, gallic acid 1.9 mg, laccase 20U (added once or added in two portions) in potassium phosphate buffer at each pH, 50 mL in a test tube for a total of 1 mL The reaction was performed for 2 hours. The reaction solution was measured by liquid chromatography under the analysis conditions shown in Example 4. Epiteaflavalin was produced at pH 4.7 to pH 7.5, and the production amount was highest at pH 6.5.

Example 9: Production of epitheaflagalin from epigallocatechin (control of the amount of gallic acid added)
The reaction was carried out in the same manner as in Example 8 except that the amount of gallic acid was doubled to 3.8 mg. The reaction solution was measured by liquid chromatography under the analysis conditions shown in Example 4. In terms of molar ratio, epigallocatechin: gallic acid = 1: 2 produced more than 1: 1 (Example 8).

Example 10: Production of epitheaflagalin from epigallocatechin (control of aeration rate)
In the production of theaflavalins by laccase, air was aerated and the production amount of epitheaflagalin was measured. To 50 mL of 20 mmol / L potassium phosphate buffer (pH 7.0) / ethanol mixture (20: 1), add 0.16 g of epigallocatechin, 0.19 g of gallic acid monohydrate, and 1000 U (9.3 mg) of Daiwalacase, 50 The reaction was carried out in a 100 mL Erlenmeyer flask by either of the following methods (1) and (2) at 30 ° C. for 30 minutes. (1) Only shaking, (2) While shaking, 10 L / L / min air was bubbled and reacted. The reaction solution was measured by liquid chromatography under the analysis conditions shown in Example 4. It was confirmed that the production rate of epitheaflavalin was increased by aeration.

Example 11: Comparison of the known method and the method for producing epitheaflagalin according to the method of the present invention The enzymatic reaction with laccase was carried out as follows. Add 0.1 mg of epigallocatechin, 1.9 mg of gallic acid monohydrate and 0.5 U of laccase in 0.1 mol / L potassium phosphate buffer (pH 4.5), and carry out the enzyme reaction at 50 ° C for 8 hours in a total volume of 5 mL. It was. Moreover, the enzyme reaction using peroxidase by the known method (Shegmin Sang et.al., Bioorganic and Medicinal Chemistry, 12 (2), 459-467 (2004)) was performed as follows. In 0.1mol / L potassium phosphate buffer (pH 6.0), add epigallocatechin 3.1mg, pyrogallol 1.3mg, 30% hydrogen peroxide water 0.02mL, peroxidase (from horseradish, Toyobo) 0.5U, The enzyme reaction was carried out at 45 ° C. for 8 hours with a total volume of 5 mL. The reaction solution was measured by the liquid chromatograph method under the analysis conditions shown in Example 2.
In the known production method using peroxidase, purpurogallin was produced in addition to epitheaflavalin (FIG. 14), but this was not observed in the method using the laccase of the present invention (FIG. 15). Purpurogallin is a highly irritating compound and is not preferred for food production.

Example 12: Comparison of the known method and the method for producing epitheaflavalin-3-O-gallate by the method of the present invention, part 1
The enzymatic reaction with laccase was performed as follows. In 0.1 mol / L potassium phosphate buffer (pH 4.5), epigallocatechin-3-O-gallate 4.6 mg, gallic acid monohydrate 1.9 mg, laccase 0.5 U was added, and the total volume was 5 mL at 50 ° C. The enzyme reaction was performed for 8 hours.
Moreover, the enzyme reaction using peroxidase by the known method (Shegmin Sang et.al., Bioorganic and Medicinal Chemistry, 12 (2), 459-467 (2004)) was performed as follows. In 0.1 mol / L potassium phosphate buffer (pH 6.0), epigallocatechin-3-O-gallate 4.6 mg, pyrogallol 1.3 mg, 30% hydrogen peroxide solution 0.02 mL, peroxidase (from horseradish, Toyobo) 0.5 U was added, and the enzyme reaction was carried out at 45 ° C. for 8 hours with a total volume of 5 mL.
The reaction solution was measured under the analysis conditions of Example 3 by liquid chromatography.
In the method using peroxidase, purpurogallin was produced in addition to epitheaflavalin-3-O-gallate (FIG. 16), but in the case of the method using the laccase of the present invention, no purprogalin was produced (FIG. 17). ).

Example 13: Comparison of known methods and epitheafragalin-3-O-gallate production method according to the present invention Part 2
In the case of peroxidase enzyme reaction by a known method, 4.6 mg of epigallocatechin gallate, 1.3 mg of pyrogallol and 20 U of peroxidase (10 U divided into 2 portions) are added in potassium phosphate buffer (pH 5.5), and the total volume is 1 mL. The enzyme reaction was performed at 45 ° C. for 3 hours. In the case of the enzyme reaction according to the present invention, 4.6 g of epigallocatechin gallate, 1.9 mg of gallic acid monohydrate, and 20 U of laccase (10 U divided into two portions) in potassium phosphate buffer (pH 6.5) The total amount was 1 mL, and the enzyme reaction was performed at 45 ° C. for 3 hours. The reaction solution was measured under the analysis conditions of Example 3 by liquid chromatography.
In the case of peroxidase, 1.46 mg of epitheaflavalin-3-O-gallate was produced and 0.31 mg of purpurogallin was produced (FIG. 18). In the case of the method using the laccase of the present invention, 0.19 mg of epitheaflavalin-3-O-gallate was produced, and the production of by-product purprogalin was not observed (FIG. 19).

  The present invention is useful in the field of producing beverages containing epitea fragallins.

Chromatogram of green tea extract treated with laccase (top: laccase treated, bottom: laccase untreated) Chromatogram of isolated component 1 Chromatogram of isolated component 2 Structural formula of component 1 UV-visible absorption spectrum of component 1 (UV-250) Mass spectrum of component 1 (matrix: 2,5-DHB) Mass spectrum of component 1 (matrix: 2,5-DHB + NaCl) Structural formula of component 2 UV-visible absorption spectrum of component 2 (Shimadzu UV-250) Mass spectrum of component 2 (matrix: 2,5-DHB) Mass spectrum of component 2 (matrix: 2,5-DHB + NaCl) Changes in the amount of epitheaflagalin produced (in relation to the amount of added enzyme) Changes in the amount of epiteafragallin-3-gallate produced (Relationship with the amount of added enzyme) Comparison results of epitea fragalin production method (in the case of peroxidase) Comparison results of epitea fragalin production method (in the case of laccase) Comparison results of epiteafragalin-3-O-gallate production method (in the case of peroxidase) Comparison results of epiteafragalin-3-O-gallate production method (in the case of laccase) Comparison results of epiteafragalin-3-O-gallate production method (in the case of peroxidase) Comparison results of epiteafragalin-3-O-gallate production method (in the case of laccase)

Claims (12)

  Epigallocatechin and / or epigallocatechin gallate, by allowing polyphenol oxidase to act in the presence of gallic acid to convert to epitheaflagalin and / or epiteafragalin-3-O-gallate, respectively. A method for producing epitea fragalins.   By adding gallic acid to the tea extract and allowing polyphenol oxidase to act, at least a portion of epigallocatechin and / or epigallocatechin gallate contained in the tea extract is converted to epiteafragalin and / or epitea, respectively. The manufacturing method of the drink containing epitea flagarins including converting into furagarin-3-O-gallate.   The production method according to claim 2, wherein the tea extract is a green tea extract, an oolong tea extract, or a black tea extract.   The manufacturing method of Claim 2 or 3 which adds 1-10 gallic acid by molar ratio with respect to the total amount of epigallocatechin and epigallocatechin gallate contained in a tea extract.   The polyphenol oxidase is at least one selected from the group consisting of laccase (EC1.10.3.2), tyrosinase (EC 1.14.18.1), bilirubin oxidase (EC1.3.3.5), phenol and polyphenol oxidase (EC1.10.3.1) It is a seed enzyme, The manufacturing method of any one of Claims 1-4.   The production method according to any one of claims 1 to 5, wherein the polyphenol oxidase is a free enzyme or an immobilized enzyme.   The production method according to any one of claims 2 to 5, wherein polyphenol oxidase is a free enzyme, and further comprises inactivating the enzyme by heating the tea extract after the conversion reaction with polyphenol oxidase.   The polyphenol oxidase is an immobilized enzyme, and the conversion reaction by the polyphenol oxidase is performed by adding the immobilized enzyme to the tea extract and removing the immobilized enzyme from the tea extract after the conversion reaction. 6. The production method according to any one of 5 above.   The polyphenol oxidase is an immobilized enzyme, and the conversion reaction by the polyphenol oxidase is performed by passing a tea extract to which gallic acid is added into a container filled with the immobilized enzyme. Manufacturing method.   The production method according to any one of claims 2 to 9, wherein the conversion reaction with polyphenol oxidase is performed while aeration of oxygen or air to the tea extract.   The manufacturing method according to any one of claims 2 to 10, wherein the beverage is a green tea beverage, a green tea beverage, a oolong tea beverage, a oolong tea beverage, a tea beverage, or a tea beverage.   The manufacturing method of any one of Claims 2-11 in which a drink contains 0.0001-0.5 mass% of epitea flagarins.