WO2007141945A1

WO2007141945A1 - Process for production of epitheaflagallin, and process for production of beverage containing epitheaflagallin

Info

Publication number: WO2007141945A1
Application number: PCT/JP2007/054787
Authority: WO
Inventors: Nobuya Itoh; Yuji Katsube; Keiichi Yamamoto; Noriyuki Nakajima
Original assignee: Kracie Seiyaku, Ltd.; Toyama Prefecture
Priority date: 2006-06-05
Filing date: 2007-03-12
Publication date: 2007-12-13
Also published as: JP2007319140A; JP4026723B2

Abstract

[PROBLEMS] To provide a simple process for production of epitheaflagallin and epitheaflagallin-3-O-gallate which can be used in a food directly, and a process for production of a beverage containing epitheaflagallin and epitheaflagallin-3-O-gallate by utilizing the above-mentioned process. [MEANS FOR SOLVING PROBLEMS] Disclosed is a process for production of an epitheaflagallin compound, which comprises reacting epigallocathechin and/or epigallocathechin gallate with polyphenol oxidase in the presence of gallic acid to convert the epigallocathechin and/or epigallocathechin gallate into epitheaflagallin and/or epitheaflagallin-3-O-gallate, respectively. Also disclosed is a process for production of a beverage containing an epitheaflagallin compound, which comprises adding gallic acid to a tea leaf extract and reacting the mixture with polyphenol oxidase to convert at least a part of epigallocathechin and/or epigallocathechin gallate contained in the tea leaf extract into epitheaflagallin and/or epitheaflagallin-3-O-gallate, respectively.

Description

Specification

EPITEA FLAGALIN MANUFACTURING METHOD AND DRINK PRODUCTION METHOD CONTAINING EPITEA FLAGALIN

Technical field

TECHNICAL FIELD [0001] The present invention relates to a method for producing an epithea fragalin and a method for producing a tea beverage containing the epithela fragalin.

Background art

[0002] Epitea fragalin-3-0-gallate is, for example, disclosed in JP 2000-226329 A (Patent Document 1) and 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 for a matrix meta-oral protease (MMP) inhibitor that is expected to have an antitumor metastatic effect.

[0003] The method of chemically synthesizing the epitheafragalins uses friesian potassium. ), 61-65 (1986) (Non-Patent Document 2)). However, potassium ferricyanide should be approved for use as a food additive.

[0004] In addition, as a synthesis method using an enzyme, a method using peroxidase (Shegmin Sang et. Al., Bioorganic and Medicinal Chemistry, 12 (2), 459-467 (2004) (Non-patent Document 3)) has been reported. Yes. In this peroxidase reaction, hydrogen peroxide and pyrogallol are used.

Patent Document 1: JP 2000-226329 A

Non-patent literature l: Naoto Oku et.al, Biol. Pharm. Bull, 26 (9), 1235-1238 (2003) Non-patent literature 2: Gen-ichiro Nonaka, Fumio Hashimoto and Itsuo Nishioka, Chem. Pharm. Bull , 34 (1), 61-65 (1986)

Non-Patent Document 3: Shegmin Sang et.al., Bioorganic and Medicinal Chemistry, 12 (2), 459-467 (2004)

Disclosure of the invention

Problems to be solved by the invention [0005] The method using ferricyanium potassium cannot be applied to the production of food because ferricyanium potassium is permitted to be used as a food additive.

[0006] Regarding the method using peroxidase, peroxyhydrogen is recognized as a food additive and is required to be decomposed or removed before the final food is finished, so the process of decomposition or removal is essential. It is. In particular, hydrogen peroxide itself is stable, but since a hydroxyl radical (ΟΗ) is rapidly generated by a heavy metal complex in the living body, for example, Fe ²⁺ — complex, all organic compounds that compose the living body are oxidized, Can be a cytotoxin. Pyrogallol is also recognized as a food additive.

[0007] The above conventional technique has a drawback that it cannot be directly used for food production. Therefore, there is a need for an easy process for the production of epitheafragalin and epithelafragalin-3-0-gallate that can be used directly in food!

[0008] Therefore, an object of the present invention is to provide a simple method for producing epitea fragalin and epitea fragrin 3-0-gallate that can be directly used in foods.

[0009] Furthermore, an object of the present invention is to provide a method for producing an epitheta fragalin and a beverage containing an epitheta fragalin-3-0-gallate using the above production method.

Means for solving the problem

[0010] As a result of diligent studies to solve the above problems, the present inventors use various polyphenol oxidases approved as foods and gallic acid recognized as food additives. As a result, the inventors have found a method for producing epitheta fragalins without impairing the flavor of green tea or the like, and have completed the present invention.

[0011] The present invention is as follows.

[1] Conversion to epitheafragalin and / or epithelafragalin-3-0-gallate by the action of polyphenoloxidase in the presence of gallic acid on epigallocatechin and / or epigallocatechin gallate, respectively. A process for producing Epitea fragalins, comprising:

[2] By adding gallic acid to the tea extract and allowing polyphenoloxidase to act, at least a part of the epigallocatechin and / or epigallocatechin gallate contained in the tea extract is added to the epitheafraga. A method for producing a beverage containing Epetica fragalins, comprising converting to phosphorus and / or Epetica fragalin-3-0-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 1 to 10 gallic acid is added in a molar ratio with respect to the total amount of epigallocatechin and epigallocatechin gallate contained in the tea extract.

[5] Polyphenoloxidases are laccase (EC1.10.3.2), tyrosinase (EC 1.14. 18.1), pyrilvinoxidase (EC1.3.3.5), phenol and polyphenoloxidase (EC1.10.3. The production method according to any one of [1] to [4], which is at least one enzyme selected from the group force consisting of 1).

[6] The production method according to any one of [1] to [5], wherein the polyphenol oxidase is a free enzyme or a fixed enzyme.

[7] Polyphenoloxidase is a free enzyme, and further comprises heating the tea extract to inactivate the enzyme after the conversion reaction with polyphenoloxidase [2] to [5] 2. The manufacturing method according to item 1.

[8] Polyphenol oxidase is an immobilized enzyme, and the conversion reaction by polyphenol oxidase is carried out by adding the immobilized enzyme to the tea extract. The production method according to any one of [2] to [5], comprising removing.

[9] Polyphenol oxidase is an immobilized enzyme, and the conversion reaction by polyphenol oxidase is performed by passing a tea extract to which gallic acid has been added into a container filled with immobilized enzyme, [2 ] The manufacturing method of any one of [5].

[10] The production method according to any one of [2] to [9], wherein the conversion reaction with polyphenoloxidase is performed while aeration of oxygen or air to 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 power contains 0.0001 to 0.5% by mass of an epitheafragalin.

The invention's effect

It is possible to produce Epitea Fragarin and Epitea Fragalin-3-0-gallate that can be used directly for food production. BEST MODE FOR CARRYING OUT THE INVENTION

[0013] In the present invention, a polyphenoloxidase is allowed to act on epicarocatechin and / or epicogallocatechin gallate in the presence of gallic acid, respectively, and epitheafragalin and / or epitheafragalin-3- The present invention relates to a method for producing epitheta fragalins, including conversion to 0-gallate.

[0014] Further, in the present invention, at least a part of epigallocatechin and / or epigallocatechin gallate contained in the tea extract is obtained by adding gallic acid to the tea extract and allowing polyphenoloxidase to act. The present invention relates to a method for producing a beverage containing an epithea fragalin, which comprises conversion to epithea fragalin and / or epithela fragalin-3-0-gallate, respectively.

[0015] The method for producing a beverage containing the epitheta fragalin of the present invention is an embodiment of the method for producing the epitheta fragalin of the present invention. In the following, the case where epicarocatechin and / or epicarocatechin gallate is contained in the tea extract, that is, a method for producing a beverage containing an epithea fragalin will be described as an example. As for the method for producing epitheafragalins, epigallocatechin and / or epigallocatechin gallate is replaced with tea extract as a raw material or raw material solution containing epigallocatechin and / or epigallocatechin gallate. can do.

[0016] In the present invention, the tea extract is not particularly limited as long as it contains epicarocatechin and / or epicarocatechin gallate. Examples of the tea extract include green tea extract, oolong tea extract, and black tea extract.

[0017] Green tea extract

Green tea is an extract of the leaves of Camellia plants, and is mainly dried from Camellia sinensis, Ca mellia assamica shoots. Examples of the green tea extract include SD green tea extract powder No. 16714 (San-Eigen F'F'I Co., Ltd.), Sanphenon BG (Taiyo Kagaku 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.

[0018] 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). Examples of the oolong tea extract include FD Woolong Tea Extract Powder No. 16297 (San-Eigen F'F'I 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.

[0019] Black tea extract

Black tea is obtained by strongly fermenting the same tea leaves as the green tea extract and then heating to stop the fermentation (strongly fermented tea). Examples of the black tea extract include SD black tea extract powder No. 16691 (San-Eigen F'F'I 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.

[0020] Examples of tea extract are shown in Table 1 below. However, these tea extracts are examples, and are not limited thereto.

[0021] [Table 1]

Product Name Manufacturer No. Polyph I Nord EGC (%) EGCg (%)

Content

SD Green Tea Extract Powder Saneigen 9.4 12.0 No.1 6714 FFI

FD Green Tea Extract Powder Saneigen 2.0 5.5

No.1 6323 F'F 'Eye

Polyph I Non 70A Mitsui Norin 80% or more

(Chiya extract)

TEACALON 90 Tokiwa Phytochemistry 90% or more 35% or more TEACALON 90S Tokiwa Phytochemistry 95% or more 40% or more Green tea extract powder Matsuura Pharmaceutical

Sanphenon EGCg Taiyo Kagaku 90% or more Sanphenon BG Taiyo Kagaku About 40%

FD oolong tea expa

Udaichi No.16297 F'F 'Eye

SD tea extract powder Saneigen (tannin content

(No.1 6600 F'F 'Eye 27.4%)

EGC and EGCg contents are catalog values of each manufacturer

[0022] 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.

[0023] Polyphenol oxidase can convert epigallocatechin to epitheta fragalin, respectively, and epigallocatechin gallate to epitheta fragalin-3-0-gallate. If it is an enzyme, there will be no restriction | limiting in particular. Examples of such polyphenol oxidases include laccase (EC 1.10.3.2), tyrosinase (EC 1.14.18. 1), pyrilvin oxidase (EC 1.3.3.5), phenol and polyphenol oxidase ( Mention may be made of at least one enzyme which is also chosen for group power consisting of EC1.10.3.1). Tyrosinase (EC 1.14.18.1) is partly classified in 1.10.3.1, but enzymes classified as 1.10.3.1 are also part of tyrosinase (EC 1.14.18.1) in the present invention. is there. [0024] The polyphenoloxidase can be a free enzyme or an immobilized enzyme. When polyphenoloxidase is a free enzyme, polyphenoloxidase is added to tea extract to which gallic acid has been added, and a conversion reaction is performed at a predetermined temperature for a predetermined time. The predetermined amount of polyphenoloxidase 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)% tea extract.

[0025] It should be noted that the amount of epicarocatechin and / or epicarocatechin gallate contained in the tea extract differs depending on the tea extract. For example, in the Epigarokatekin the sum of Epigaroka Tekingareto is referred to as a 20 (w / w)% or more! /, Ru SD green tea extract powder, E Pigarokatekin is about 10 _(w / _w)%, the E pin epigallocatechin gallate about About 13 (w / w)% is included. Thus, for example, a solution containing 1% tea extract has about 1 mg / ml of epigallocatechin and about 1.3 mg / ml of epigallocatechin gallate.

[0026] 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 polyphenoloxidase. It is appropriate that the heating condition is 70 to 90 ° C and 2 to LO.

[0027] Polyphenol oxidase can also be an immobilized enzyme. When an immobilized enzyme is used, the conversion reaction with polyphenoloxidase can be performed by adding the immobilized enzyme to a tea extract supplemented with gallic acid. 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.

[0028] When polyphenol oxidase is an immobilized enzyme, the conversion reaction by polyphenol oxidase can also be carried out by passing a tea extract to which gallic acid has been added into a container filled with the immobilized enzyme. The condition for the circulation of the tea extract can be carried out by setting the temperature in the range of 20 to 60 ° C and the contact time with the immobilized enzyme for example in the range of 10 to 240 minutes.

[0029] The conversion reaction with polyphenol oxidase can be performed while aeration of oxygen or air to the tea extract. The conversion reaction is accelerated by ventilating oxygen or air. You can. The oxygen or air ventilation conditions can be, for example, 0.2 to: LO LZLZmin. Furthermore, in order to increase the effect of aeration, it is necessary to aerate while stirring the reaction solution.

[0030] The conversion reaction with polyphenoloxidase is carried out by converting at least a part of epigallocatechin and / or epigallocatechin gallate contained in tea extract to epitheafragaline and / or epitheafragalin-3-0-gallate, respectively. To convert to Depending on the purpose, the amount of gallic acid added and the reaction conditions should be adjusted so that the total amount of epigallocatechin and / or epigallocatechin gallate is converted to epitheafragalin and / or epithelafragalin-3-0-gallate. You can choose.

[0031] The conversion reaction with polyphenoloxidase is performed by adding gallic acid to the tea extract, but the purpose of the tea extract is to adjust the pH in consideration of the pH dependence of the polyphenoloxidase activity. A buffering agent can also be added. However, considering that the conversion reaction product can be used as a beverage as it is, it is preferable to use, for example, phosphoric acid and its salts, quenoic acid and its salts, etc. as the buffer. Of course, the conversion reaction with polyphenoloxidase can also be performed without adding a buffer.

[0032] The beverage obtained by the production method of the present invention can be directly used as a beverage. 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 style beverage, an oolong tea beverage, an oolong tea style beverage, a tea beverage, or a tea style. Beverages 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.

[0033] Furthermore, the beverage obtained by the production method of the present invention can be used as a beverage as it is. A solution obtained by treating a tea extract with polyphenoloxidase in the presence of gallic acid is extracted and purified or concentrated. After spray drying or freeze-drying, cocoon powder is prepared by sizing. Such concentrated solutions or extract powders can also be used as raw materials for various forms of foods and health care products.

[0034] Examples of foods to which the concentrated solution or extract powder can be applied include gums, confectionery, candy, and supplements. Concentrated solution or extract powder can be applied Examples of health care products include oral cleaning solutions and toothpastes.

Example

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

[0036] Example 1: Separation and purification of epitea fragalin, epitea fragalin 3-0-gallate and determination of structure in green tea extract treated with laccase

Green tea extract 250g, gallic acid monohydrate 20g, phosphate buffer solution (pH 2.4) 600mL, laccase Y1.66g (Daiwa Kasei Co., Ltd., 200.000U) with water added to a total volume of 2L at 55 ° C The reaction was performed 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: epitetea fragalin) and an increased peak (peak 2: (Epithea fragalin-3-0-gallate) was detected (Figure 1).

[0037] Detector: UV absorptiometer (measurement wavelength: 320 nm)

Column: YMC- Pack ODS-A A-312 (6.0 φ X 150mm)

Column temperature: 40 ° C

Mobile phase: 0.05mol / L Sodium dihydrogen phosphate sample solution Z-acetonitrile (10: 3) Flow rate: 1.0mL / min

[0038] 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 lOOmL was added twice, washed with alkali, washed with water (100mL x 2 times), and then 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 440mm), mobile phase: 0.05mo 1 / L sodium dihydrogen phosphate test solution Z-acetonitrile mixture (17: 3) → (4: 1)), fractions containing a large amount of component 1 and component 2 were collected, concentrated on a rotary evaporator, extracted with ethyl acetate, and the ethyl acetate layer was dried over anhydrous sodium sulfate. After filtration with a cotton plug, the filtrate was dried with a rotary evaporator to obtain a residue 770 mg (Fr.l) containing a large amount of component 1 and a residue 730 mg (Fr.2) containing a large amount of component 2. Each oxalic acid-treated silica gel column chromatography (packed with 10 g of oxalic acid-treated silica gel (column diameter 1.5 cm)) Elution (eluent: ethyl acetate Z-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 (Figure 2).

In addition, the same oxalic acid-treated silica gel column chromatography was performed on Fr.2, and recrystallization was performed with ethanol (99.5) Z water to obtain 14 mg of orange crystals (FIG. 3).

[0039] [Analysis conditions]

Column: TSK gel ODS-80Ts (4.6 φ X 150mm)

Column temperature: 40 ° C

Mobile phase: Water Z-acetonitrile Z phosphoric acid mixture (500: 150: 1)

Flow rate: 1.0mL / min

Detection wavelength: 2 ⁵ 4 (each lower), 210 (each upper)

[0040] 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 0D _o NMR: 400 MHz, solvent CD OD.

3 3

• MALDHTOFMS: AXIMA- CFR plus (Shimadzu / KRATO, ver2.4.0, reflectron mode), matrix solution: 2,5-dihydroxybenzoic acid (2,5-DHB), cationizing agent: NaCl • ES precision MS : AgilentLC 1100 / ABI QSTARXL

• UV-visible absorption spectrum: UV-visible spectrophotometer (Shimadzu Corporation, UV250) _{o The} sample solution was approximately 0.01 mg / mL of 50% ethanol solution.

[0041] (1) Structural analysis of component 1

In component 1 ¹ ! "I-NMR ^ vector, 5.98 ppm (lH, d, J = 2.4 Hz), 5.97 ppm (lH, d, J = 2.4 Hz) ゝ 4.81 derived from epicatechin A ring and C ring Signals of ppm (lH, s) ゝ 4.27 ppm (lH, m), 2.93 ppm, 2.82 ppm (2H, m) were observed, and ¹³ C-NMR ^ vector and further analysis by HMQC and HMBC The structural formula is 4.

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 (Figure 5). In addition, in the measurement of MALDI-TOFMS, m / z 423 was strongly detected and m / z 401 disappeared due to the addition of NaCl as a cationizing agent. Thus, [M + H] + was 401, the molecular weight was 400, and the structure was supported (FIGS. 6 and 7).

[0042] This component 1 is consistent with the spectrum data of non-patent literature (Gen-ichiro Nonaka, Fumio Hashimoto and Itsuo Nishioka, Chem. Pharm. Bull, 34 (1), 61-65 (1986). These compounds were named peatafragalin and pyrogallin.

[0043] (2) Structural analysis of component 2

In the ¹ ! ”I-NMR ^ vector of component 2, the hydrogen signal bound to C-2 and C-3 of the epicatechin moiety is shifted to the lower magnetic field side! /, So the hydroxyl group of C-3 is The structural formula shown in Fig. 8 is considered to be an ester and substituted with a galloyl group, and when the UV-visible absorption spectrum of component 2 (approximately 0.01 mg / mL of 50% ethanol solution) was measured, Similar to 1 and showed maximum absorption at 302 nm and 280 nm (Fig. 9) In the measurement of MALDI-TOFMS (Figs. 10 and 11), the addition of NaCl as the catalyzing agent resulted in m / z 575 From the fact that it was detected strongly and m / z 553 disappeared, [M + H] + was 553, the molecular weight was 552, and the component was also increased by 152 corresponding to the molecular weight of the galloyl group. Supported the structure.

[0044] It should be noted that 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 , Bioorganic and Medicinal Chemistry, 12 (2), 459-467 (2004), and was named as Epitea Fragalin-3-0-gallate.

[0045] Table 2 summarizes the results of instrumental analysis of the two components.

[0046] [Table 2]

About ingredient and ingredient 2

Example 2: Tea Extract Strength by Laccase Epitea Fragarin, Epitea Fragalin-3- 0-Gallate Making (Part 1)

Dissolve 250 g of SD Green Tea Extract Powder (San-Ei Gen F F Eye) in 10% ethanol solution lOOOmL, phosphate buffer (pH 2.4) 600mL, 20% gallic acid ethanol solution 100mL, enzyme solution (Daisen Laccase Y120, 200.000U, 1.66g, white rot fungus-derived Trametes sp., ECI.10.3.2) 200mL and lOOmL water were added to make a total of 2000mL, and the enzyme reaction was started with 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 amount of epitheafragalin and epithelafragalin 3-0-gallate produced was analyzed by liquid chromatography under the following analytical conditions. The results are shown in Table 3. [0048] [Analysis conditions]

Detector: UV absorptiometer (measurement wavelength: 280nm)

Column: YMC- Pack ODS-A A-312 (6.0 φ X 150mm)

Column temperature: 30 ° C

Mobile phase: 0.05mol / L Sodium dihydrogen phosphate sample solution Z-acetonitrile mixture (10: 3) Flow rate: 丄. OmL / min

[0049] [Table 3]

Production amount of epitheaflavalins per gram of green tea extract (mg / g)

Example 3 Production of Epitea Fragarin and Epitea Fragalin-3-0-Galate with Tea Extraction Power Using Laccase (Part 2)

SD green tea extract powder (Saneigen F “F” eye) lg, 0.48 g of gallic acid monohydrate, Daiwa Laccase Y120 2500U-10000U was added to lOOmL of water, and the enzyme reaction was carried out at 50 ° C. At the start of the treatment, approximately 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 epitheafragalin and epithelafragalin-3-0-gallate at this time. The maximum yield is 2.78 mg / g for epitheafragalin (50 ° C, 30 minutes, laccase 5000U), and 15.41 mg / g for epitheafragalin-3-gallate (50 ° C, 30 minutes, laccase 10000U). there were

[0051] Example 4: Production of Epithea fragalin, Epetica fragalin-3-0-gallate from tea extract by tyrosinase or pyrilbinoxydase

Epigalocatechin gallate 6.9 mg, gallic acid in 20 mmol / L potassium phosphate buffer Add 2.8mg of monohydrate and polyphenoloxidase (tyrosinase 20U or pyrilvin oxidase 0.4U) in a total of 1.5mL of solution at 25 ° C for 3 hours for tyrosinase, and for pyrilvin 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.

[0052] [Analysis conditions]

Detector: UV absorptiometer (measurement wavelength: 280nm)

Column: YMC- Pack ODS-A A- 312 (6.0 φ X 150mm) (Epitea fragalin-3-gallate analysis)

Cadenza CD-C18 (4.6 X 75mm) (Epitea fragalin analysis)

Mobile phase: 0.05mol / L Sodium dihydrogen phosphate sample solution Z-acetonitrile mixture (10: 3) Flow rate: 1.0mL / min

[0053] 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 pyrilvinoxydase (EC.1.3.3.5) is derived from Myrothesium sp. From Wako Pure Chemicals Ichinano Enzym. I used something.

[0054] [Table 4] Amount of epitheaflavalins produced (mg / mL reaction solution)

[0055] Example 5: Production of Epepitealagalin-3-0-Galate from Epigalocatechin Gallate (Control of Anti)

Epigalocatechin gallate 4.6 mg, gallic acid 1.9 mg, laccase 20U (added once or twice) in sodium phosphate buffer at each pH, and 50 mL at 50 ° C in total for 1 mL The reaction was performed for 3 hours. The reaction solution was measured by the liquid chromatography method under the analysis conditions shown in Example 2. Epitetea fragalin-3-0-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 enzyme increased when the enzyme was added in two batches rather than at once.

[0056] [Table 5] Epitea fragalin-3- Amount of O-galle (mg / mL reaction solution)

[0057] Example 6: Production of Epithea Fragarin-3-0-Galate from Epigalocatechin Gallate

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, Epigalocatechin-3-0-gallate: gallic acid = 1: 2 produced more than 1: 1 (Example 5).

[0058] [Table 6]

Epitea Fragalin-3- ί? ~ Galate production (mg / mL reaction solution)

[0059] Example 7: Production of Epitea Fragallin-3-0-Galate from Epigalocatechin Gallate (垂 ¼ droop)

In the production of theaflavalins by laccase, 10 L / L / min of air was aerated, and the amount of epiteafragalin-3-gallate produced was measured. 20 mmol / L potassium phosphate buffer solution (pH 6.5) / ethanol mixture (20: l) 50 mL of epigallocatechin gallate 0.23 g, gallic acid monohydrate 0.19 g, Daiwa laccase 1000 U (9.3 mg) Then, at 50 ° C for 30 minutes, the following method (1) or (2) was performed in a lOOmL Erlenmeyer flask. (1) Shaking only, (2) Reaction while aeration and aeration. The reaction solution was measured by liquid chromatography under the analysis conditions shown in Example 2. It was confirmed that the amount of epiteafragalin-3-0-gallate produced increased by aeration.

[0060] [Table 7]

Epitea Fragalin-3- 0 Galate production (mg / mL reaction solution)

[0061] Example 8: Production of Epigalocatechin Cathetea Fragarin (Reverse ¾) Control of H)

Epigalocatechin 3. lmg, gallic acid 1.9mg, lath in potassium phosphate buffer at each pH Case 20U (added once or added in two portions) and reacted in a test tube at 50 ° C for 2 hours for a total of 1 mL. The reaction solution was measured by the liquid chromatographic method under the analysis conditions shown in Example 4. Epitea fragalin was produced at pH 4.7 to pH 7.5, and the production amount was most numerous at pH 6.5.

[Table 8]

Production amount of epitheaflavalin (mg / mL reaction solution)

[0063] Example 9: Production of Epigalocatechinca Epitea Fragalin (Control of Addition of Gallic Acid

1

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 the liquid chromatographic method under the analysis conditions shown in Example 4. In terms of molar ratio, Epigalocatechin: gallic acid = 1: 2 produced more power than 1: 1 (Example 8).

[Table 9]

Production amount of epitheaflavalin ( _mg / mL reaction solution)

Example 10: Production of Epigalocatechin Cepitea Fragarin (Control of Air Volume)

In the production of theaflavalins by laccase, air was aerated and the amount of epitheaflavalin produced was measured. 50 mmol of 20 mmol / L potassium phosphate buffer (pH 7.0) / ethanol mixture (20: l) was added with 0.16 g of epigallocatechin, 0.19 g of gallic acid monohydrate, and 100 U (9.3 mg) of dairy laccase, 50 One of the following methods (1) and (2) was performed in a lOOmL Erlenmeyer flask at 30 ° C for 30 minutes. (1) Only with shaking, (2) While reacting, air was reacted with lOL / L / min air. The reaction solution was measured by the liquid chromatography method under the analysis conditions shown in Example 4. It was confirmed that the production rate of Epitea fragallin was increased by aeration.

[0066] [Table 10] Production amount of Ephiaflagalin (mg / mL reaction solution)

[0067] Example 11: Known Method Comparison of Epitea Fragaline Making Method by the Method of the Present Invention

The enzymatic reaction with laccase was performed as follows. In 0.1 mol / L potassium phosphate buffer (pH 4.5), add 3.1 mg of epicarocatechin, 1.9 mg of gallic acid monohydrate, 0.5 U of laccase, and carry out enzymatic reaction at 50 ° C for 8 hours in a total volume of 5 mL It was. In addition, an enzymatic reaction using peroxidase by a 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 Epigalocatechin 3.1mg, Pyrogallol 1.3mg, 30% hydrogen peroxide solution 0.02mL, Peroxidase (from horseradish rust, 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 under the analysis conditions shown in Example 2 by liquid chromatography.

In the known production method with peroxidase, purpurogallin was produced in addition to the epitheaflavalin (Fig. 14), but it was not recognized 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.

[0068] [Table 11]

Production amount of epitheaflavalin ( _mg / mL reaction solution)

[0069] Example 12: Comparison of known methods and the manufacturing method of Epitea fragalin-3-0-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), add epicarocatechin-3-0-gallate 4.6 mg, gallic acid monohydrate 1.9 mg, laccase 0.5 U, total volume 5 mL 50 ° C, enzyme reaction was performed for 8 hours.

3; The enzyme reaction using peroxidase by a known method (Shegmin bang 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), Epigalocatechin-3-0-gallate 4.6 mg, Pyrogallol 1.3 mg, 30% hydrogen peroxide solution 0.02 mL, Peroxidase (from horseradish rust, Toyobo Co., Ltd. Enzyme reaction was carried out at 45 ° C for 8 hours in a total volume of 5 mL with 0.5 U.

The reaction solution was measured under the analysis conditions of Example 3 by liquid chromatography. In the peroxidase production method, purprogalin was produced in addition to epitea fragalin-3-0-gallate (Fig. 16) force. In the method using the laccase of the present invention, no production of pull-up gallin was observed. (Figure 17).

[0070] Example 13: Comparison of Epepiafragarin-3-0-gallate production method by known method and method of the present invention Part 2

In the case of peroxidase enzyme reaction by a known method, add 4.6 mg of epicarocatechin gallate, 1.3 mg of pyrogallol, and 20 U of peroxidase 20 U (10 U divided into 2 portions) in potassium phosphate buffer (pH 5.5) The enzyme reaction was performed at 45 ° C for 3 hours. In addition, in the case of the enzymatic reaction according to the present invention, 4.6 mg of epigallocatechin gallate, 1.9 mg of gallic acid monohydrate, and 20 U of laccase (10 U divided into two portions) are added in potassium phosphate buffer (pH 6.5). The total volume 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 the liquid chromatography method.

In the case of peroxidase, 1.46 mg of epitheaflavalin-3-0-gallate was produced and 0.31 mg of pulprogalin was produced (Fig. 18). In the case of the method using the laccase of the present invention, 0.19 mg of epitheaflavalin-3-0-gallate was produced, and the production of by-product purprogalin was not observed (FIG. 19).

Industrial applicability

[0071] The present invention is useful in the field of production of beverages containing Epitea fragallins.

Brief Description of Drawings

[0072] [Fig. 1] Chromatogram of green tea extract treated with laccase (top: laccase treated, bottom: laccase untreated)

[Figure 2] Chromatogram of isolated component 1

[Figure 3] Chromatogram of isolated component 2

[Figure 4] Structural formula of component 1

[Figure 5] UV-visible absorption spectrum of component 1 (UV-250)

[Figure 6] Mass spectrum of component 1 (matrix: 2,5-DHB) [Fig.7] Mass spectrum of component 1 (matrix: 2,5-DHB + NaCl)

[Figure 8] Structural formula of component 2

[Figure 9] UV-Vis absorption spectrum of component 2 (Shimadzu UV-250)

[Figure 10] Mass spectrum of component 2 (matrix: 2,5-DHB)

[Figure 11] Mass spectrum of component 2 (Matrix: 2,5-DHB + NaCl)

圆 12] Changes in the amount of epitea fragallin produced (relationship with the amount of added enzyme)

[Fig.13] Change in the amount of Epitea fragalin-3-gallate produced

[Fig.14] Comparison results of the method for producing epitheta fragalin (in the case of peroxidase)

[Fig.15] Comparison results of the Epepitealagalin production method (in the case of laccase)

[Fig.16] Comparison results of the method for producing Epitea fragalin-3-0-gallate

[Fig. 17] Comparison results of Epepitea flagaline-3-0-gallate production method (in the case of laccase) [Fig. 18] Comparison results of Epepitea flagarin-3-0-gallate production method (in the case of peroxidase) )

[Fig. 19] Comparison results for Epitetea fragalin-3-0-gallate production method (for laccase)

Claims

The scope of the claims

[2] By adding gallic acid to the tea extract and allowing polyphenoloxidase to act, at least a part of the epigallocatechin and / or epigallocatechin gallate contained in the tea extract is separated into epithelia. A method for producing a beverage containing epithela fragalins, comprising conversion to fragalin and / or epithela fragalin-3-0-gallate.

[3] 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.

[4] The production method according to claim 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] Polyphenol oxidase is laccase (EC1.10.3.2), tyrosinase (EC 1.14.1)

8.1), pyrilbinoxidase (EC1.3.3.5), phenol and polyphenol oxidase (EC1.10.3.1) The manufacturing method according to 1.

6. The production method according to any one of claims 1 to 5, wherein the polyphenol oxidase is a free enzyme or a fixed enzyme.

[7] The polyphenoloxidase is a free enzyme, and further comprises inactivating the enzyme by heating the tea extract after the conversion reaction with the polyphenoloxidase.

5. The manufacturing method according to item 1, wherein the deviation is 1.

[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 after the conversion reaction, the immobilized enzyme is immobilized from the tea extract. The production method according to any one of claims 2 to 5, comprising removing an enzyme.

[9] 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. The production method according to any one of 2 to 5.

[10] The production method according to any one of [2] to [9], wherein the conversion reaction with polyphenol oxidase is performed while aeration of oxygen or air to the tea extract.

[11] The production method according to any one of claims 2 to 10, wherein the beverage is a green tea beverage, a green tea beverage, an oolong tea beverage, an oolong tea beverage, a tea beverage, or a tea beverage.

[12] The production method according to any one of [2] to [2] above, wherein the beverage contains 0.0001 to 0.5 mass% of the epitheaflavalins.