JP2011172514A - Method for producing theaflavin cultured using heated fresh tea leaf and tea cell cultured in medium for microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for inexpensively producing theaflavin in a high yield, which is directly useful in a food. <P>SOLUTION: The method for producing theaflavin includes (1) a step for heating a raw material containing tea, (2) a step for pulverizing the raw material heated by the step (1), (3) a step for culturing the tea cells in a medium for microorganism to obtain a tea cell culture and (4) a step for reacting the raw material pulverized by the step (2) with the tea cell culture cultured by the step (3) to form theaflavin. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing theaflavin using heated raw tea leaves and tea cells cultured in a microbial medium, theaflavin produced by the production method, and a food composition containing the theaflavin.

  Theaflavin is a red pigment produced by enzymes in tea leaves in the black tea production process. In recent years, theaflavin has been revealed to have physiological activities such as an antioxidant action, a hypoglycemic action, and an antibacterial action, and is expected to be useful not only as a natural colorant but also as a physiologically active substance.

  Extraction from tea leaves and biosynthesis have been known as methods for producing theaflavin. When extracted from black tea leaves, theaflavins are extracted as theaflavins containing three galloyl esters as follows.

The approximate ratio of the above four compounds contained in black tea is theaflavin: 0.08% by weight, theaflavin-3-O-gallate: 0.3% by weight, theaflavin-3′-O-gallate: 0.2% by weight. %, Theaflavin-3,3′-di-O-gallate: 0.4% by weight. Thus, since the theaflavin content in black tea is small, in the conventional method for producing theaflavin by extraction from black tea leaves, it is possible to obtain theaflavin in a sufficient amount and yield to use theaflavin as a physiologically active substance. could not.

  On the other hand, the biosynthesis methods of theaflavins include methods using potassium ferricyanide, methods using enzyme samples (water-insoluble fraction of Yabukita young leaves), methods using polyphenol oxidase obtained from tea leaves, green tea extract and polyphenol oxidation A method of using a plant extract containing an enzyme, a method of using horseradish peroxidase, a method of contacting processed green tea leaves and polyphenol oxidase, a method of treating a green tea slurry with tannase, and fermenting in an argon or nitrogen atmosphere, A method by fermenting fresh leaf juice is known. However, in any of the methods, as in the extraction method from black tea, it was impossible to obtain theaflavin in a sufficient amount and yield to use theaflavin as a physiologically active substance.

  Patent Documents 1 and 2 report a method for producing theaflavins using hydrolases such as peroxidase (peroxidase) and tannase obtained from cultured tea cells as enzymes for theaflavin conversion reaction (Patent Document 1: “Theaflavins). ”, Patent Document 2:“ Selective production method of theaflavin ”). In these production methods, there are few side reactions, and theaflavins can be synthesized in a high yield as compared with the conventional methods. Unlike the conventional methods, these production methods can selectively produce theaflavin with high yield at a low cost. However, since theaflavins produced by the methods of Patent Documents 1 and 2 use components such as plant hormones and B5 salts that cannot be used as food additives in the production process, it is not possible to add the obtained theaflavins to food compositions. could not.

JP 2007-143461 A Japanese Patent Application No. 2007-182217

  An object of the present invention is to provide a method for producing theaflavin that can be used at a low cost and in a high yield, and the produced theaflavin or a composition containing theaflavin can be directly used in foods.

  The present inventors produce peroxidase or tannase necessary for the theaflavin conversion reaction in the same manner as in the conventional culture in plant media, even when tea cells, which are plant cells, are cultured in microorganism media. Was found unexpectedly. Based on this discovery, we developed a method for producing theaflavins that is inexpensive and easy to apply to foods, using heated fresh tea leaves and tea cells cultured in a microbial medium. In this production method, (1) caffeine, which is a major unnecessary component derived from tea, is decomposed by heating, so that the purity of the target product, theaflavin, is improved, and (2) used as a food additive Since it does not use components such as plant hormones and B5 salts that cannot be used, the theaflavin obtained can be added to food compositions and can be easily applied to foods. It plays.

In certain embodiments, the present invention provides:
(1) a step of heating a raw material containing tea,
(2) a step of pulverizing the raw material heated in the step (1),
(3) a step of culturing tea cells in a microorganism medium to obtain a tea cell culture; and (4) the raw material ground in step (2) and the tea cell culture cultured in step (3). A method for producing theaflavin comprising the step of reacting with a product to produce theaflavin is provided.

  In one aspect, the microorganism medium is a yeast medium containing sugar, a nitrogen source, and inorganic salts.

  In one aspect, the sugar is selected from the group consisting of sucrose and glucose.

  In one aspect, the method for producing theaflavin of the present invention further includes a step of inactivating an enzyme contained in the reaction product in the step (3).

  In one aspect, the deactivation step is performed by acidifying and / or warming the reaction product.

  In one aspect, the method for producing theaflavin of the present invention further includes a step of recovering theaflavin.

  In one aspect, the recovery is performed with an adsorption resin.

  In another embodiment, the present invention provides a substantially pure theaflavin produced by the method for producing theaflavin of the present invention.

  In another embodiment, the present invention provides a food composition comprising theaflavin produced by the method for producing theaflavin of the present invention.

  In another embodiment, the present invention provides a food composition for preventing obesity comprising theaflavin produced by the method for producing theaflavin of the present invention.

  In another embodiment, the present invention provides a food composition for suppressing an increase in blood glucose level, comprising theaflavin produced by the method for producing theaflavin of the present invention.

  In one aspect, the food composition is taken in combination with a high fat diet.

  In one aspect, the food composition further comprises a theaflavin stabilizer.

  According to the present invention, there is provided a method for producing theaflavin that is inexpensive and has a high yield, and that allows easy application of the produced theaflavin to food. In this production method, (1) caffeine, which is a main unnecessary component derived from tea, is decomposed by heating, so that the purity of the target product, theaflavin, is improved accordingly, (2) food addition in the production process Because it does not use components such as plant hormones and B5 salts that cannot be used as foods, the theaflavin obtained can be added to food compositions and has excellent effects such as easy application to foods. It is.

  Therefore, according to the present invention, it is possible to produce theaflavins having various physiological activities at a low cost and in a high yield, and easily apply them to foods to provide food compositions.

FIG. 1 shows the catechins (EC: epicatechin, ECG: epigallocatechin, ECG: epicatechin-3-O-gallate, EGCG: epigallocatechin-3- O-gallate) and caffeine content, and peroxidase activity (POD). FIG. 2 shows the content of catechins and caffeine contained in each of the raw tea leaves with different collection times. FIG. 3 shows the results of a theaflavin (TF) conversion test using raw tea leaves with different collection times. FIG. 4 shows changes in catechin content and peroxidase activity during cryopreservation at −80 ° C. FIG. 5 shows the contents of catechins and caffeine when tea leaves are treated under different heat treatment conditions. FIG. 6 shows the contents of catechins and caffeine when heat treatment is performed with different amounts of heat treatment liquid. FIG. 7A shows the time course of peroxidase activity in cultures in which tea cell culture was performed in media with various amounts of sucrose. FIG. 7B shows the time course of peroxidase activity in a culture in which tea cell culture was performed in a medium containing sucrose or glucose, respectively, as a sugar source. The vertical axis represents peroxidase (POD) activity (U / ml), and the horizontal axis represents culture time (days). FIG. 8 (A) shows the results of peroxidase stability tests with respect to pH. FIG. 8B shows the results of a stability test of peroxidase against heat. FIG. 9 (A) shows the tannase stability test results with respect to pH. FIG. 9 (B) shows the tannase stability test results against heat. FIG. 10 (A) shows the stability test results of theaflavin with respect to pH. FIG. 10B shows the stability test result of theaflavin against heat. FIG. 11 shows the effect of addition of ascorbic acid on the stability of theaflavin. FIG. 11A shows the effect of added concentration of ascorbic acid on the stability of theaflavin. FIG. 11 shows the effect of addition of ascorbic acid on the stability of theaflavin. FIG. 11B shows the thermal stability of the theaflavin solution supplemented with ascorbic acid at a concentration of 0.1%. FIG. 12 shows the stability test results of theaflavin powder obtained by the production method of the present invention, which was pulverized by freeze drying or spray drying. FIG. 13 shows the purification result of theaflavin.

  The invention will now be described, by way of example, with reference to the accompanying drawings, where appropriate. The terms used in this specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  The present inventors have constructed a method for producing a theaflavin-containing powder using a culture medium for microorganisms of tea cells as an enzyme source and a tea leaf extract as a substrate source.

  The production method of the present invention uses fresh tea leaves as a raw material. After the raw tea leaves are heat-treated, they are pulverized to prepare a tea leaf extract. In this extract, catechins contained in the fresh tea leaves are eluted and become a substrate for the theaflavin reaction.

  In order to reduce the amount of caffeine extracted into the tea leaf extract and increase the yield of the target product theaflavin, the production method of the present invention includes a heat treatment step. Since the enzyme in the tea leaf necessary for the theaflavin conversion reaction is inactivated during the heat treatment step, a theaflavin conversion reaction is performed in which the enzyme is separately supplied to oxidize catechin. It is an important feature of the present invention that this enzyme supply is achieved by adding a tea cell culture cultured in a microbial medium rather than a conventional plant medium. Microbial culture media generally do not contain ingredients that cannot be used in food compositions (such as certain vitamins and plant hormones). Therefore, by using the tea cell culture cultured in the microorganism medium of the present invention, theaflavin is produced without using ingredients that cannot be used as food additives such as plant hormones and B5 salts. Can be added to the food composition. This makes it very easy to apply theaflavin to food compositions. This tea cell culture contains the peroxidase and tannase necessary for the theaflavin conversion reaction. A theaflavin conversion reaction is performed using peroxidase and tannase in the tea cell culture. If necessary, peroxidase and tannase can be inactivated to stop the theaflavin conversion reaction after theaflavin production. If necessary, the supernatant can be collected by centrifugation, the supernatant can be concentrated, and theaflavin can be powdered by spray drying or freeze drying.

(Theaflavin)
As used herein, “theaflavin” refers to the following general formula:

The compound which has a benzotropolone ring represented by these. In the production method of the present invention, theaflavin is produced by reacting epicatechin (EC) and epigallocatechin (EGC) contained in tea. This reaction is represented by the following equation:

Epicatechin and epigallocatechin are also produced by the elimination of gallate from epicatechin-3-O-gallate (ECG) and epigallocatechin-3-O-gallate (EGCG) by an enzymatic reaction in tea. obtain. In this specification, these epicatechin, epigallocatechin, epicatechin-3-O-gallate and epigallocatechin-3-O-gallate are collectively referred to as “catechins”.

(Tea heating)
As used herein, “tea” refers to common tea (Camellia sinensis or Camellia assamica). The part of tea used as a raw material in the present invention is not particularly limited, such as seedlings, leaves, cotyledons, stems, and fruits, but is preferably leaves or stems. Examples of tea varieties include Yabukita, Okuhikari, Yamabuki, Sayaka Kaori, Kanaya Midori, Shigarase, etc. The tea cultivar is not particularly important in the present invention. From the results of Example 1 below, tea collected from July to September has a high content of catechins including epicatechin and epigallocatechin, which are starting materials for theaflavin synthesis, and caffeine, which is an impurity. Since the content rate of is less, it is preferable as a raw material in the production method of the present invention. See also FIGS. Similarly, from the results of Example 1, tea within 60 days from collection is preferred as a raw material in the production method of the present invention. See also FIG. However, tea as a raw material in the production method of the present invention is not particularly limited depending on the collection time and the number of days elapsed since collection.

  Caffeine derived from tea is an unnecessary component for theaflavin production. Therefore, in order to increase the purity of theaflavin, the raw tea is first heated to remove caffeine in the production method of the present invention. This heat treatment is performed at about 80 ° C. to about 90 ° C. for about 1 minute or longer, about 3 minutes or longer, about 3 minutes to about 5 minutes, about 3 minutes, about 4 minutes, or about 5 minutes. Preferably, the heat treatment is performed at about 80 ° C. for about 1 minute or longer, about 3 minutes or longer, about 3 minutes to about 5 minutes, about 3 minutes, about 4 minutes, or about 5 minutes. By this heat treatment, about 40% or more, about 50% or more, or about 60% or more of caffeine contained in the raw tea is removed. More preferably, by this heat treatment, about 50% or more of caffeine contained in tea as a raw material, specifically about 51%, about 52%, about 53%, about 54%, about 55%, about 56%. %, About 57%, about 58%, about 59%, or about 60% or more. By this heat treatment, the peroxidase activity in tea is almost completely inactivated.

(Crushing of heated tea)
In the present invention, the tea heated as described above is dehydrated and pulverized, and used for the subsequent reaction. The grinding is performed by a method known to those skilled in the art. For example, in the present invention, an appropriate amount (for example, 100 mL of water per 1 g of tea) is added to heated and dehydrated tea and ground using a mixer.

(Brown cell culture)
The “tea cell” used in the present invention induces callus based on a general callus preparation method well-known to those skilled in the art using a section containing live tea tissue, and transfers it to a medium for culturing. It is prepared by. The tea cell culture of the present invention may be either a cell culture obtained by culturing this tea cell in a solid medium or a culture suspension of cultured cells obtained using a liquid medium. The tea cell culture thus obtained contains peroxidase and tannase produced by the tea cells themselves, and these enzymes act in the subsequent theaflavin conversion reaction.

  Typically, the section is cultured by first sterilizing the section and culturing the section on a solid medium such as an agar medium to induce callus. The induced callus is then fully grown on similar solid or liquid media. The sterilization is performed by ethanol surface sterilization, treatment with hypochlorite, washing with sterilized distilled ion-exchanged water, or the like.

  The present inventors have found that tea cells are normally cultured even when cultured in a microbial medium instead of a plant medium, and produce active peroxidase and tannase. By culturing tea cells not in a plant medium but in a microbial medium, a mixture of theaflavin and medium obtained through the subsequent theaflavin conversion reaction can be directly applied to food. Therefore, in the present invention, tea cells are cultured in a microbial medium instead of a plant medium. Conventionally, it has not been considered that plant tea cells can be cultured in a microbial medium, and it has been extremely unexpected to produce normally active enzymes such as peroxidase and tannase.

  As used herein, the term “microorganism medium” includes any medium that is used in culturing microorganisms including bacteria and fungi and does not contain components that cannot be used as food additives such as plant hormones and B5 salts. Examples of the microbial medium usable in the present invention include, but are not limited to, YPD medium, glucose / yeast extract medium (yeast / mold medium), PGY medium, hyperosmotic medium (yeast medium), and the like. . These media are described in experimental documents such as a biotechnology experiment book (Baifukan) and a microbiology experiment method (Kodansha replay Tyfik). The medium in the present invention may be a solid medium or a liquid medium. The culture medium for microorganisms of the present invention contains, for example, about 10 g / 100 mL or more, more preferably about 15 g / 100 mL or more of sugar. More specifically, the microbial medium of the present invention is about 10 g / 100 mL to about 25 g / 100 mL, about 10 g / 100 mL to about 20 g / 100 mL, about 10 g / 100 mL to about 15 g / 100 mL, about 15 g / 100 mL to about Contains 25 g / 100 mL or about 15 g / 100 mL to about 20 g / 100 mL of sugar. In a more preferred embodiment, the microbial medium of the present invention comprises about 10 g / 100 mL to about 15 g / 100 mL of sugar. In a particularly preferred embodiment, the microbial medium of the present invention comprises about 15 g / 100 mL of sugar.

  Examples of the sugar in the microorganism medium in the present invention include glucose, sucrose, maltose, lactose, sorbitol, fructose, and the like, but not limited thereto, and sugars known in the art can be used. The sugar of the microorganism medium in the present invention is preferably sucrose or glucose, more preferably sucrose.

  The microorganism culture medium of the present invention may contain a nitrogen source or inorganic salts in addition to sugar (preferably sucrose or glucose, more preferably sucrose). Examples of the nitrogen source include, but are not limited to, organic nitrogen sources such as yeast extract, peptone, meat extract, and amino acid solution, or inorganic nitrogen sources such as ammonium sulfate, potassium nitrate, and ammonium chloride. The microbial medium of the present invention may contain one or more nitrogen sources. Examples of inorganic salts include, but are not limited to, magnesium sulfate, magnesium chloride, sodium phosphate, potassium phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, potassium chloride, sodium chloride, or calcium chloride. The microbial medium of the present invention may contain one or more inorganic salts. However, the culture medium for microorganisms of the present invention does not contain components that cannot be used as food additives (for example, plant hormones, nicotinates, pyridoxine hydrochlorides, B5 salts, etc.).

  A preferable microorganism medium in the present invention is a yeast medium. The “yeast medium” in the present specification can typically contain a sugar, a nitrogen source and inorganic salts. The yeast medium of the present invention contains about 10 g / 100 mL or more of sugar, more preferably about 15 g / 100 mL or more of sugar. More specifically, the yeast medium of the present invention has about 10 g / 100 mL to about 25 g / 100 mL, about 10 g / 100 mL to about 20 g / 100 mL, about 10 g / 100 mL to about 15 g / 100 mL, about 15 g / 100 mL to about Contains 25 g / 100 mL or about 15 g / 100 mL to about 20 g / 100 mL of sugar. In a more preferred embodiment, the yeast media of the present invention comprises about 10 g / 100 mL to about 15 g / 100 mL of sugar. In a particularly preferred embodiment, the yeast medium of the present invention contains about 15 g / 100 mL of sugar.

  Examples of the sugar for the yeast culture medium in the present invention include glucose, sucrose, maltose, lactose, sorbitol, fructose, and the like. However, sugars known in the art can be used. The sugar of the yeast medium in the present invention is preferably sucrose or glucose, more preferably sucrose.

  Accordingly, the yeast medium of the present invention may contain about 10 g / 100 mL or more, more preferably about 15 g / 100 mL or more of sucrose. More specifically, the yeast medium of the present invention has about 10 g / 100 mL to about 25 g / 100 mL, about 10 g / 100 mL to about 20 g / 100 mL, about 10 g / 100 mL to about 15 g / 100 mL, about 15 g / 100 mL to about 25 g / 100 mL or from about 15 g / 100 mL to about 20 g / 100 mL of sucrose. In a more preferred embodiment, the yeast media of the present invention may comprise about 10 g / 100 mL to about 15 g / 100 mL sucrose. In a particularly preferred embodiment, the yeast medium of the present invention may comprise about 15 g / 100 mL sucrose.

  In one embodiment, the yeast medium of the present invention may contain one or more nitrogen sources. Examples of the nitrogen source used in the yeast medium of the present invention include organic nitrogen sources such as yeast extract, peptone, meat extract and amino acid solution, or inorganic nitrogen sources such as ammonium sulfate, potassium nitrate and ammonium chloride. It is not limited to these. In certain embodiments, the yeast medium of the present invention optionally includes an organic nitrogen source such as yeast extract or peptone. The yeast medium of the present invention can be prepared, for example, from about 0.1 g / 100 mL to about 0.4 g / 100 mL, preferably from about 0.4 g / 100 mL, or from about 0.1 g / 100 mL to about 0.0. 2 g / 100 mL, preferably about 0.2 g / 100 mL of peptone, or a combination thereof. Examples of the yeast extract used in the yeast medium of the present invention include Mist N (Oriental Yeast Industry Co., Ltd.), and examples of peptone include CE90M (Asahi Food and Healthcare Co., Ltd.) It is not limited to these. A person skilled in the art can appropriately determine the type and amount of the organic nitrogen source to be included in the medium to be used based on the common general technical knowledge in the field. In certain embodiments, the yeast media of the present invention comprises about 0.3 g / 100 mL potassium nitrate or about 0.05 g / 100 mL ammonium sulfate, or a combination thereof. Nitrogen sources other than those listed above are well known to those skilled in the art, and such nitrogen sources may also be included in the yeast media of the present invention. A person skilled in the art can appropriately determine the type and amount of the nitrogen source to be included in the medium to be used, based on the common general technical knowledge in the field.

  In one embodiment, the yeast medium of the present invention may contain one or more of these inorganic salts. Examples of the inorganic salts used in the yeast medium of the present invention include magnesium sulfate, magnesium chloride, sodium phosphate, potassium phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, potassium chloride, sodium chloride or calcium chloride. However, it is not limited to these. In certain embodiments, the yeast medium of the present invention comprises about 0.0.3 g / 100 mL potassium dihydrogen phosphate, about 0.0.3 g / 100 mL magnesium sulfate, about 0.0.2 g / 100 mL phosphorus. Contains sodium dihydrogen acid or about 0.00.2 g / 100 mL calcium phosphate, or a combination thereof. Inorganic salts other than those listed above are well known to those skilled in the art, and such inorganic salts can also be included in the yeast culture medium of the present invention. A person skilled in the art can appropriately determine the type and amount of inorganic salts to be included in the medium to be used, based on the common general technical knowledge in the field.

The microorganism medium and / or yeast medium of the present invention does not contain components that cannot be used as food additives (for example, plant hormones, nicotinates, pyridoxine hydrochlorides, B5 salts, etc.). In the present specification, the “component that cannot be used as a food additive” refers to any component such as these plant hormones and B5 salts that cannot be used as a component in a food or food additive. It should be particularly noted that the microorganism medium and / or yeast medium of the present invention does not contain plant hormones. Plant hormones include, but are not limited to, auxin, gibberellin, cytokinin, abscisic acid, ethylene, brassinosteroids, jasmonic acid, florigen, and strigolactone. B5 salts are obtained from Gamborg O. L. Miller R. A. Ojima K .; , Experimental Cell Research, 50, 151-158 (1968), etc., and are well known to those skilled in the art.
In order to cultivate plant cells such as tea cells, it was common knowledge in the art to add the above substances such as plant hormones to the medium. Therefore, even when tea cells are cultured in a medium not containing the above substances, those skilled in the art have not thought that peroxidase and / or tannase in an amount that efficiently causes the theaflavin conversion reaction is secreted by tea cells. However, the present inventors anticipate that even if tea cells are cultured in a medium not containing the above substances, the amount of peroxidase and / or tannase that efficiently causes the theaflavin conversion reaction is sufficiently secreted by the tea cells. I found it outside. Thus, by culturing tea cells using a medium not containing the above substances, the final product, theaflavin, can be obtained without using ingredients that cannot be used as food additives. It can be added to the composition.

(Theaflavin conversion reaction)
In the method for producing theaflavins of the present invention, tea cells were cultured in a tea raw material that had been heated to remove caffeine and then pulverized, and a microbial medium (preferably a yeast medium) that does not contain ingredients that cannot be used as food additives. A theaflavin conversion reaction occurs by reacting with the tea cell culture. Theaflavin is below

It is produced by a conversion reaction according to the formula: Manami Takemoto: Selective production method of theaflavin (PCT / JP2008 / 062579 (2008) and Manami Takemoto: Selective production method of theaflavin See Japanese Patent Application No. 2007-182217 (2007) and the like.

  The theaflavin conversion reaction is typically carried out by rotating at room temperature for about 28 to about 34 hours. Further, it was found that the optimal peroxidase activity: total catechin amount: reaction solution amount for carrying out stable theaflavin conversion reaction was 1 unit: 20 to 35 mg: 100 mL (see Example 4). .

(Deactivation of enzyme)
After completion of the theaflavin conversion reaction, the product of theaflavin is degraded due to the remaining peroxidase and tannase enzyme activities contained in the tea cell culture. Therefore, in one embodiment of the present invention, the enzyme activities of peroxidase and tannase are deactivated after the end of the theaflavin conversion reaction. It should be noted that the inactivation of peroxidase and tannase enzyme activity must be performed under conditions that do not degrade theaflavin. In one embodiment of the invention, the deactivation of peroxidase and tannase enzyme activity is carried out by maintaining at pH 3 for 10 minutes at 70 ° C.

(Theaflavin stabilizer)
As used herein, “theaflavin stabilizer” refers to any substance that prevents the degradation of theaflavin. Since theaflavin is a substance that is easily oxidatively decomposed, in the present invention, if necessary, an antioxidant is added to theaflavin to stabilize theaflavin. Therefore, in one embodiment of the present invention, the theaflavin stabilizer is an antioxidant. Antioxidants that can be used as stabilizers for theaflavin include, but are not limited to, sulfuric acid, ascorbic acid, citric acid, gallic acid, tocopherol, sodium sulfite, sodium ascorbate, sodium citrate and the like. A preferred theaflavin stabilizer in the present invention is ascorbic acid. The theaflavin produced in the present invention may contain about 0.1% or more ascorbic acid. By including about 0.1% ascorbic acid in theaflavin, about 98% theaflavin remains undegraded after 5 days at 25 ° C. On the other hand, when no ascorbic acid is contained, only about 10% theaflavin remains after 2 days at 25 ° C., and almost all theaflavins are oxidatively decomposed after 3 days.

(Reaction powder)
In order to apply the reaction solution after the theaflavin conversion reaction to health food, it is preferable to pulverize the theaflavin obtained by the reaction in a high yield without substantially decomposing. The reaction solution can be powdered by, for example, freeze drying or spray drying. For example, pulverization in the present invention can be accomplished by lyophilization for about 3 days or longer. Powdering in the present invention can also be accomplished by spray drying until a desired amount of powdering is achieved at an inlet temperature average of 150 ° C and an outlet temperature of 70 ° C to 75 ° C. In pulverization, it is important to carry out without decomposing the theaflavin obtained by the reaction. Therefore, it is preferable to pulverize the reaction solution containing the theaflavin stabilizer.

(Purification of theaflavin)
In the production method of the present invention, theaflavin is purified by recovering theaflavin from the theaflavin conversion reaction solution as necessary to obtain highly pure theaflavin. By this purification, impurities such as caffeine derived from tea leaves contained in the theaflavin conversion reaction solution can be removed. For removal of caffeine, for example, extraction with an organic solvent is generally used, but this method is not suitable for food production and is not used in the present invention. Examples of the purification method used in the present invention include, but are not limited to, purification using an adsorption resin. In a preferred embodiment of the present invention, theaflavin is purified by column chromatography using a column such as a glass column. For example, SEPABEADS (Mitsubishi Chemical) can be used as the resin, and ethanol can be used as the eluent. The target product, theaflavin, elutes in the 30% ethanol fraction. However, the purification of the present invention is not limited to this method.

(Food composition)
Based on theaflavins produced using the method of the present invention, theaflavin-containing food compositions can be produced by methods well known to those skilled in the art.

  The food composition of the present invention can be added to the theaflavin powder or theaflavin-containing culture as it is to a liquid, gel or solid food, or, if necessary, an excipient such as dextrin, lactose or starch, a flavor, a pigment, etc. In addition, it can be produced by processing into pellets, tablets, granules, or the like, or by coating with gelatin or the like and molding into capsules to obtain health foods or dietary supplements. The amount of theaflavin in these food compositions is not uniformly defined, but is 10 to 1,000 mg / day, more preferably 50 to 400 mg / day. The food composition of the present invention can be used for obesity prevention or suppression of increase in blood glucose level.

  “Obesity prevention” in the present invention means prevention of obesity caused by obesity-promoting factors such as intake of a high fat diet. It is understood that theaflavin is a healthy and safe substance in that it prevents only undesired obesity caused by obesity-promoting factors, rather than merely losing weight. In fact, there was no change in body weight in mice fed with a theaflavin-containing food composition produced by the production method of the present invention along with a normal diet (see Example 11).

  In the present invention, “inhibition of blood glucose level increase” refers to suppression of an increase in blood glucose level due to a blood glucose level increase factor such as intake of a high fat diet. As with obesity prevention, it is understood that theaflavin is a healthy and safe substance in that it suppresses only undesired increases in blood glucose levels caused by factors that increase blood glucose levels, rather than merely lowering blood glucose levels. In fact, there was no change in blood glucose level in mice fed with a theaflavin-containing food composition produced by the production method of the present invention together with a normal diet (see Example 11).

  In certain embodiments, the theaflavins of the invention can be taken in admixture with a high fat diet. In another embodiment, the theaflavins of the invention can be taken in combination with a high fat diet. When the theaflavin of the present invention is taken in combination with a high fat diet, theaflavin may be taken before the high fat diet, the theaflavin may be taken simultaneously with the high fat diet, or the high fat diet Theaflavin may be taken after ingestion.

  As used herein, “high fat diet” refers to any diet that can cause obesity and / or increased blood glucose levels in a subject who has consumed it. More specifically, a diet having a fat content exceeding about 3.4 kcal / g and not corresponding to a normal diet is referred to as a “high fat diet”. In embodiments of the present invention, a high fat diet typically refers to a diet that contains about 5 kcal / g or more of fat.

  The theaflavin-containing food composition of the present invention particularly refers to health foods or supplements. The food composition of the present invention refers to any product containing theaflavin. The food / beverage composition of the present invention typically has an effect of preventing obesity and / or suppressing an increase in blood glucose level, as demonstrated in the following examples, and is also marketed with these effects displayed. The theaflavin-containing food composition of the present invention suppresses obesity and / or an increase in blood glucose level that can be caused by those factors when taken in combination with an obesity promoting factor and / or an increase factor in blood glucose level. On the other hand, when there are no undesired factors such as an obesity promoting factor and / or a blood glucose level increasing factor, the theaflavin-containing food composition of the present invention does not have a significant effect on the ingested subject. This is a function of theaflavin that was not known at all. In subjects who are in a healthy state, without causing weight loss or lowering of blood glucose level, obesity caused by the factor in obesity-promoting factors such as a high-fat diet and / or factors that increase blood glucose level, and / or Alternatively, the theaflavin-containing food composition of the present invention has an unexpected and surprising effect in suppressing an increase in blood glucose level.

References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to preferred embodiments for easy understanding. The present invention will now be described based on examples, but it should be understood that the above description and the following examples are provided for illustrative purposes only and are not intended to limit the invention. It is. Accordingly, the scope of the invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

(Examination of tea conditions as Example 1 raw material)
In order to produce theaflavins with high efficiency, it is important to grasp the substrate components for obtaining theaflavins contained in the raw tea. Therefore, in this example, tea leaves were appropriately collected at different times, and changes in the components contained therein were grasped throughout the year. From these results, the most suitable tea procurement time for theaflavin production was determined.

(Test method)
Tea leaves were collected at different times from tea field in Iwata city, and the tea leaves were frozen and stored at -80 ° C. to prevent oxidation. 100 g of well water (4 ° C.) is added to 1 g of frozen tea leaves, pulverized for 1 minute using a mixer (IFM-750G; IWATANI), and then the supernatant liquid is centrifuged (14,000 rpm, 10 minutes, 4 ° C.). It was collected. Caffeine, epicatechin (EC), epigallocatechin (EGC), epicatechin-3-O-gallate (ECG), and epigallocatechin-3-O-gallate (EGCG) contained in the supernatant liquid are UPLC ( Peroxidase activity (POD) was measured by a method well known in the art.

(Results and Discussion)
FIG. 1 shows the results of measurement of the catechin content and peroxidase activity in tea leaves, FIG. 2 shows the content of each component, and FIG. 3 shows the results of the theaflavin reaction test (see Example 4 below for the protocol). As can be seen from FIGS. 1 and 2, the total catechin content and peroxidase activity contained in the tea leaves gradually decreased from the peak in August and increased after one month, as can be seen from FIG. 1 and FIG. There was a trend. On the other hand, the content of caffeine showed a tendency opposite to that of its total catechin content and peroxidase activity. Theaflavin production depends on the concentration of catechins contained in the tea leaves used. As can be seen in FIG. 3, the theaflavin yield was almost constant during the period from June to September, but after that it tended to decrease and increase again from January.

  The examination result of the storage stability of the collected tea leaves is shown in FIG. In this example, tea leaves from Arahataen Co., Ltd. (Makinohara City, Shizuoka Prefecture) were used. As shown in FIG. 4, in the frozen tea leaves, there was almost no change in the components contained therein until the 60th day, but the catechin content gradually decreased after the 60th day. On the other hand, peroxidase activity decreased rapidly after 60 days, and hardly changed after 200 days.

  From the above results, it is considered that tea leaves from July to September are suitable for theaflavin production because they have a low caffeine content and a high content of catechins. Moreover, from the result of the cryopreservation stability of tea leaves shown in FIG. 4, it was found that the number of days that catechins can be stored without decomposition is about 60 days.

(Example 2 Removal of caffeine by heating)
In order to increase the theaflavin purity, it is important to remove components unnecessary for the production of tea-derived theaflavin, which is a raw material. Therefore, in this example, a method for removing caffeine, which is an unnecessary component derived from tea, was examined.

(Test method)
100 g of well water was added to 1 g of tea leaves, which were heat-treated at temperatures of 70 ° C., 80 ° C. and 90 ° C. 100 mL of well water was again added to the dehydrated tea leaves, and pulverized for 1 minute using a mixer (IFM-750G; IWATANI). Various components including caffeine in the supernatant obtained by centrifugation (14,000 rpm, 10 minutes, 4 ° C.) were analyzed.

(result)
Catechins (epicatechin (EC), epigallocatechin (EGC), epicatechin-3-O-gallate (ECG), epigallocatechin contained in tea leaves after heating at temperatures of 70 ° C., 80 ° C. and 90 ° C. -3-O-gallate (EGCG)) and caffeine content and peroxidase activity (POD) are shown in FIG. As can be seen from FIG. 5, the caffeine removal could not be confirmed by the heat treatment for 30 minutes in the 70 ° C. treatment. However, the peroxidase activity required for the theaflavin conversion reaction was completely inactivated. When heat treatment was carried out at 80 ° C. for 3 minutes, about 58% of caffeine could be removed, but no further removal was observed even if the treatment time was longer. The 90 ° C. heat treatment was almost the same as the 80 ° C. heat treatment, and the caffeine removal rate by the heat treatment was about 60%. In addition, catechins which are substrates for the theaflavin reaction were not affected, but the peroxidase activity was almost completely inactivated. Therefore, it was clarified that peroxidase supply from tea leaves to the theaflavin conversion reaction cannot be expected by removing caffeine.

  FIG. 6 shows the contents of catechins and caffeine when heat treatment is performed with different amounts of heat treatment liquid. As can be seen from FIG. 6, the caffeine removal rate was about 57% up to 4 g of tea leaves with respect to 100 mL of well water, but when the amount of tea leaves was further increased, a decrease in the caffeine removal rate was observed. From the above results, it was found that in order to remove caffeine from tea leaves, it is desirable to carry out at a concentration of at most about 4 g / 100 mL of tea leaves.

(Example 3 Composition of tea cell culture medium)
(the purpose)
In order to efficiently perform the theaflavin conversion reaction, it is necessary to provide a sufficient amount of peroxidase. For this purpose, it is important to culture tea cells that serve as a source of enzymes. A conventional medium used for tea cell culture always contains components that cannot be used as food additives (for example, plant hormones and specific vitamins). Then, the composition for the novel culture medium for manufacturing theaflavin without using such a component was examined.

(Test method)
The medium composition used is shown in Table 1 below.

  (Table 1 Composition of various media used in this example [g / 100 mL])

The components of the medium compositions RUN1 to RUN5 shown in Table 1 were dissolved in 100 mL of well water, placed in a 200 mL Erlenmeyer flask, and sterilized by autoclaving (121 ° C., 20 minutes). Inoculate 10 mL each of tea cells (in this experiment, we used tea cells from Dr. Takemoto, Shizuoka Prefectural University, but any other tea cells can be used) and rotate at 25 ° C and 120 rpm. Shaking culture was performed. Samples were collected over time as appropriate during the culture, and peroxidase activity, pH, and residual sugar concentration were measured.

(result)
FIG. 7A shows time-dependent changes in peroxidase activity in tea cell cultures cultured using the media shown in Table 1. As shown in FIG. 7A, peroxidase was produced even when cultured in a yeast medium that did not contain ingredients that could not be used as food additives such as vitamins, B5 salts, and plant hormones. In addition, it was found that the peroxidase activity of the culture cultured in the yeast medium having an amount of sucrose of 5 g / 100 mL was considerably low, and it was preferable that the yeast medium contained 10 g / 100 mL or more of sucrose. It has also been found that the most preferred amount of sucrose is 15 g / 100 mL.

  Based on the medium composition of RUN-3 in Table 1, the time-dependent change in peroxidase activity in tea cell cultures cultured using a medium in which the peptone and yeast extract concentrations were changed was determined. As a result, 0.2 g / 100 mL of peptone was obtained. It was revealed that the peroxidase activity was highest in tea cell cultures cultured in a medium having the following medium composition including 0.4 g / 100 mL of yeast extract (detailed data not shown). Therefore, the medium composition shown in Table 2 below was determined as the optimum medium composition in the present invention.

  (Table 2 Optimal medium composition [g / 100 mL])

In addition, changes in peroxidase activity when glucose was used instead of sucrose were also examined. The components of the sucrose medium and glucose medium shown in Table 3 below were dissolved in 100 mL of well water, placed in a 200 mL Erlenmeyer flask, and sterilized with an autoclave (121 ° C., 20 minutes). 10 mL of tea cells were inoculated at a time, and cultured with rotary shaking at 25 ° C. and 120 rpm. Samples were taken over time as appropriate, and peroxidase activity, pH, and residual sugar concentration were measured.

  (Table 3 Composition of sucrose medium and glucose medium)

FIG. 7B shows the peroxidase activity in tea cell cultures cultured using the medium shown in Table 3 immediately after the start of culture (0 day), 7 days, 15 days, 20 days, 28 days, 34 days, and 41 days. The change with time is shown. Sufficient peroxidase activity was detected in both the sucrose medium and the glucose medium. However, in the vicinity of 28 to 34 days after the start of the culture showing a high value of peroxidase activity, the peroxidase activity of the culture cultured in the sucrose medium was observed in the glucose medium. It was about 1.2 times the peroxidase activity of the cultured culture. From this, the sugar source in the microorganism medium in the present invention may be sucrose, glucose or other sugars, but sucrose is considered particularly preferable.

(Examination of Example 4 theaflavin conversion reaction conditions)
(the purpose)
In order to construct a highly efficient theaflavin production process, it is important to convert raw material catechins to theaflavins in a short time with a minimum amount of enzyme. Therefore, the optimum enzyme concentration and the optimum reaction solution amount with respect to the catechin amount were examined.

(Test method)
100 mL of well water was added to 10 g of dried tea leaves to extract catechins as substrates for 5 hours (25 ° C.), and the supernatant obtained by centrifugation (3,000 rpm, 10 minutes, room temperature) was used as a substrate solution. The theaflavin conversion test was performed using a 200 mL Erlenmeyer flask, and the culture supernatant of tea cells was added to a catechin-containing solution adjusted to the concentration shown in Table 4 using well water, and the mixture was shaken at 25 ° C. and 120 rpm. It was done. The reaction volume is 100 mL. In this specification, “Total Cat.” Indicates the total of catechins that are substrates for the reaction.

  (Table 4 Yield and yield of theaflavins relative to peroxidase activity and catechin composition)

In addition, as an examination of the optimal amount of reaction solution / charged concentration, the total amount of catechins (Total Cat.) Was 44.8 mg, the peroxidase activity was 1.3 units, and the reaction solution amount was changed to 25 to 200 mL as shown in Table 5 below. The same conversion reaction was performed.

  (Table 5 Composition for studying reaction volume)

(Results and discussion)
From the result of Table 4, when the catechin density | concentration with respect to peroxidase activity (Total Cat.) Increased, the tendency for the recovery rate of theaflavins also to fall was seen. At the same time, the theaflavin reaction time also increased (data not shown). On the other hand, when the substrate concentration was lowered with respect to the enzyme activity, by-product formation was observed due to overreaction (data not shown).

  From the results of theaflavin yield and yield in Table 5, Total Cat. /Peroxidase=44.8/1.3=34.5 It was found that 100 mL or more was optimal as the amount of the reaction solution. When the amount of the reaction solution was less than 100 mL, a significant decrease in the theaflavin yield was observed. As the cause of this, the generation of by-products due to overreaction or the decomposition of the generated theaflavin can be considered as in the case where the catechin concentration is lowered with respect to the peroxidase activity as described above.

  As described above, it was found that peroxidase activity: total catechin amount: reaction liquid amount = 1 unit: 20 to 35 mg: 100 mL is preferable as a reaction composition for performing an efficient theaflavin conversion reaction.

(Example 5: Examination of inactivation conditions of peroxidase and tannase)
(the purpose)
If the peroxidase activity and tannase activity remain after completion of the theaflavin conversion reaction, theaflavin is degraded. Therefore, it is preferable to deactivate these enzyme activities after completion of the reaction.

5-1. Inactivation of peroxidase and tannase (Test method)
The supernatant recovered from the tea cell culture by centrifugation (14,000 rpm, 5 minutes) is used as the enzyme solution. To examine pH stability, adjust the pH to 3-9 using 78% sulfuric acid and 48% NaOH, leave it at 25 ° C. for 1 hour, and then measure the enzyme activity of peroxidase and tannase by a method well known in the art did. The thermostability was verified by adjusting the pH of the supernatant to 5 and then maintaining at 30-70 ° C. for 1 hour, and then measuring the enzyme activity of peroxidase and tannase.

(result)
Peroxidase activity was very stable at pH 4-7. Only about 40% inactivation was observed at pH 3 (see FIG. 8A). Also, it was hardly inactivated up to 50 ° C. with respect to heat (see FIG. 8B). Further, the pH was adjusted to 3 and maintained at 70 ° C. As a result, it was found that the peroxidase activity was almost inactivated in 10 minutes (data not shown).

  The tannase activity was stable in the neutral region (pH 4-8), but it tended to be rapidly deactivated in the acidic region (<pH 4) (see FIG. 9A). Moreover, thermal stability was comparatively bad, and it was shown that it deactivates rapidly at 40 degreeC or more (refer FIG. 9B). As with peroxidase, the pH was adjusted to 3 and maintained at 70 ° C. As a result, it was almost inactivated in 10 minutes.

5-2. Stability of theaflavin It was verified whether the target product, theaflavin, remained stable without being decomposed under the peroxidase and tannase inactivation conditions.

(Test method)
In order to examine the pH stability of the theaflavin solution, the pH was adjusted to 3-9 using 78% sulfuric acid and 48% NaOH, and left at 25 ° C. for 1 hour, and then the theaflavin residual rate was measured. The thermal stability was verified by adjusting the pH to 3 and maintaining at 30-70 ° C. for 1 hour and measuring the theaflavin residual rate.

(result)
Theaflavin was relatively stable in the acidic range (pH 3-5) but extremely unstable in the neutral to basic range (pH 6-) (see FIG. 10A). Moreover, even if it adjusted to pH 3 and heat-processed at 70 degreeC for 1 hour, it was hardly decomposed | disassembled (refer FIG. 10B).

(Examination of Example 6 Theaflavin Stabilizer)
(the purpose)
In order to obtain the stability of theaflavin from the completion of the theaflavin conversion reaction to commercialization, it is important to prevent theaflavin that is easily oxidatively degraded. In the present invention, theaflavin is stabilized by adding an antioxidant to theaflavin as necessary to prevent oxidative degradation of theaflavin. In this example, the types of antioxidants that effectively prevent oxidative degradation of theaflavin were examined.

(Test method)
In order to prevent the oxidative degradation of theaflavin, each antioxidant was added to the theaflavin solution to a concentration of 0.05%, and then maintained at 70 ° C. for 1 hour, and the theaflavin residual rate was measured.

(Results and discussion)
The effects of various antioxidants are shown in Table 6 below. It has been demonstrated that the addition of each antioxidant improves the stability of theaflavin. High stability effects were observed especially in sulfuric acid and ascorbic acid.

  (Table 6 Effects of various antioxidants)

The influence of the concentration of ascorbic acid added is shown in FIG. It was found that the stability of theaflavin improved with increasing ascorbic acid addition concentration. In particular, at a concentration of 0.1% or higher, theaflavin was hardly degraded even when stored at 25 ° C. for 5 days (see FIG. 11A). When ascorbic acid was added at a concentration of 0.1%, theaflavin hardly decomposed even after treatment at 70 ° C. for 1 hour (see FIG. 11B).

  Next, the effects of pH adjustment with sulfuric acid and addition of ascorbic acid were examined. The results are shown in Table 7 below. When the pH was adjusted to 3.0 with sulfuric acid and when ascorbic acid was added at a concentration of 0.1% (pH 4.3), almost the same stability effect was obtained.

  (Table 7 Effect of sulfuric acid + ascorbic acid)

From the above results, it is possible to keep the generated theaflavin stable by adding ascorbic acid at a concentration of 0.1 (g / L)% as a stabilizer after the reaction is completed and heating to 70 ° C. It was concluded.

(Example 7: Examination of theaflavin powdering conditions)
(the purpose)
In mass production of theaflavins by the method for producing theaflavins of the present invention, it is preferable that a process for stably pulverizing the theaflavin conversion reaction liquid is established. Therefore, the powdering process was examined using the theaflavin solution obtained by the theaflavin conversion reaction.

7.1 Powdering of the theaflavin conversion reaction solution (Test method)
100 mL of well water was added to 1 g of tea leaves and heat-treated at 80 ° C. 100 mL of well water was again added to the dehydrated tea leaves and pulverized for 1 minute using a mixer (IFM-750G; IWATANI) to obtain a tea leaf pulverized liquid. Sucrose 15 g / 100 mL, yeast extract 0.4 g / 100 mL, peptone 0.2 g / 100 mL, KNO ₃ 0.3 g / 100 mL, (NH ₄ ) ₂ SO ₄ 0.05 g / 100 mL, KH ₂ PO ₄ 0.0.3 g / 100 mL, MgSO ₄ · 7H ₂ O 0.03 g / 100 mL, NaH ₂ PO ₄ · H ₂ O 0.00.2 g / 100 mL, CaCl ₂ · 2H ₂ O 0.00.2 g / 100 mL (100 mL ) Was inoculated with 10 mL of tea cells, and cultured with shaking at 25 ° C. and 120 rpm for about 35 hours to obtain a tea cell culture solution. The tea leaf crushed liquid and the tea cell culture liquid were placed in a 200 mL Erlenmeyer flask so that the peroxidase activity: total catechin amount: reaction liquid amount = 1 unit: 20-35 mg: 100 mL, and about 25 ° C., 120 rpm rotation shaking. Theaflavin conversion reaction was performed for 3 to 5 hours.

The pH of this reaction solution was adjusted to 3 using 78% H ₂ SO ₄ and then heat-treated at 70 ° C. for 10 minutes to deactivate peroxidase and tannase. Next, the crushed tea leaves were removed by centrifugation (8,000 rpm, 10 minutes), and the concentrate was adjusted using a evaporator with a target of Brix (solid content concentration in the solution) of 20%. The theaflavin-containing powder was prepared by freeze drying (FD) or spray drying (SD) using the obtained concentrated liquid.

(result)
The composition of the conversion reaction solution obtained by the theaflavin conversion reaction is shown in Table 8 below. The theaflavin yield of the conversion reaction solution was 47%, and Brix was 0.5%.

  (Table 8 Composition of conversion reaction liquid)

It was confirmed that both peroxidase and tannase were inactivated by heat treatment after pH adjustment, and concentration was performed using an evaporator. When 600 mL of the conversion reaction solution was charged into the evaporator and operated at 60 ° C. or lower, the time required for Brix to be 20% or more (40-fold concentration) was about 1 hour. Decomposition of each component including theaflavin in this concentration step was hardly observed, and there was almost no change in the composition of the conversion reaction solution (see Table 9 below).

  (Table 9 Composition of conversion reaction liquid after concentration)

Next, freeze drying and spray drying were performed using the concentrated solution, and a powdering test was performed. The FD operated for 3 days. SD operated with an average inlet temperature of 150 ° C. (maximum 160 ° C.) and an outlet temperature of 70-75 ° C. The test results are shown in Table 10. In both SD and FD, decomposition of each component including theaflavin during the powdering process was hardly observed. There was almost no difference in the content of each component in the produced powder. The process tested this time was shown to produce a powder with 36% by weight theaflavin, 25% by weight caffeine, 17% by weight ascorbic acid and 6% by weight gallic acid.

  (Table 10 Results of powdering test)

7.2 Powder stability test (Purpose)
In order to supply products of a certain quality, product stability is important. Therefore, a stability test was examined using the theaflavin-containing powder produced by freeze-drying or spray-drying obtained in 7.1 above.

(Test method)
The theaflavin-containing powder was stored in an aluminum pouch, and then stored at 4 ° C. and 25 ° C. using an acceleration tester (40 ° C., humidity 75%). Sampled appropriately and analyzed components (Results)
The stability test results are shown in FIG. Theaflavin powder pulverized by freeze-drying and theaflavin powder pulverized by spray-drying showed similar behavior. It was shown that the accelerated tester started to decompose at the same time as storage. Although the addition of ascorbic acid prevents oxidation, it is thought that the effect of heat is large. In the samples stored at 4 ° C. and 25 ° C., almost no decomposition was observed until around the 50th day of storage, but after that a slight decomposition of TF began to be observed.

(Example 8 Purification of theaflavin reaction solution)
(the purpose)
In order to produce a theaflavin product having a higher theaflavin content, it is preferable to remove impurities such as caffeine derived from tea leaves contained in the theaflavin conversion reaction solution. Extraction removal with an organic solvent is a common caffeine removal method, but this method is not suitable for food production. Therefore, caffeine removal methods that can be used as food were examined.

(Test method)
100 mL of well water was added to 1 g of tea leaves and heat-treated at 80 ° C. 100 mL of well water was again added to the dehydrated tea leaves and pulverized for 1 minute using a mixer (IFM-750G; IWATANI) to obtain a tea leaf pulverized liquid. Sucrose 15 g / 100 mL, yeast extract 0.4 g / 100 mL, peptone 0.2 g / 100 mL, KNO ₃ 0.3 g / 100 mL, (NH ₄ ) ₂ SO ₄ 0.05 g / 100 mL, KH ₂ PO ₄ 0.0.3 g / 100 mL, MgSO ₄ · 7H ₂ O 0.03 g / 100 mL, NaH ₂ PO ₄ · H ₂ O 0.00.2 g / 100 mL, CaCl ₂ · 2H ₂ O 0.00.2 g / 100 mL (100 mL ) Was inoculated with 10 mL of tea cells, and cultured with shaking at 25 ° C. and 120 rpm for about 35 hours to obtain a tea cell culture solution. The tea leaf crushed liquid and the tea cell culture liquid were placed in a 200 mL Erlenmeyer flask so that the peroxidase activity: total catechin amount: reaction liquid amount = 1 unit: 20-35 mg: 100 mL, and about 25 ° C., 120 rpm rotation shaking. Theaflavin conversion reaction was performed for 3 to 5 hours.

  Purification by column chromatography was examined using this theaflavin conversion reaction solution. As the column, a glass column (3.0 mm ID × 40 cm) packed with a synthetic adsorption resin SEPABEADS (Mitsubishi Chemical) was used. Elution was performed using an ethanol solution as the eluent while changing the ethanol concentration stepwise.

(result)
The test results are shown in FIG. When the ethanol concentration of the eluate was 10% (elution volume 0 to 400 mL), elution of ascorbic acid was confirmed, but elution of theaflavin, caffeine, gallic acid, and catechins was hardly observed. When the ethanol concentration was increased to 20% (elution volume: 400 to 1000 mL), elution of caffeine, gallic acid, and catechins could be confirmed, and elution of theaflavin was not observed. When the ethanol concentration was increased to 30% (elution volume 1000-1700 mL), elution of theaflavin could be confirmed. This fraction with an ethanol concentration of 30% contained almost only theaflavin, and a highly pure theaflavin could be obtained. The composition of each fraction is shown in Table 11 below.

  This test showed that TF and caffeine, gallic acid, and catechins can be separated by the gradient of ethanol concentration. However, the recovery rate of theaflavin by the column chromatogram was 37%. High purity TF could be prepared, but showed recovery issues. In the future, it will be necessary to study the conditions in more detail and increase the recovery rate.

  (Table 11 Components contained in each fraction)

(Example 9: Implementation of the production method of the present invention using a 5 kL fermenter)
The product manufacturing process using a 5 kL fermenter using the manufacturing process constructed this time is shown below.

(1) Caffeine removal by heat treatment of tea leaves and pulverization of tea leaves The raw tea leaves used as a raw material is 59.7 kg. Caffeine is obtained by infiltrating 59.7 kg of frozen tea leaves (-20 ° C) into 1,500 L of well-heated water at 80 ° C (optimal ratio is 100 mL of well water for 4 g of tea leaves) for 3 minutes. Remove. After the treatment, the tea leaves are collected using a dehydrator.

  The heat-treated tea leaves are pulverized to extract catechin as a substrate. 25 kg of well water is added to 1.0 kg of heat-treated tea leaves and pulverized. When a multi-mill grinder (GM4-25; Glow Engineering) is used, tea leaves can be pulverized with a processing capacity of 18.0-25.2 kg · tea leaves / hour. The resulting tea leaf grind is about 1,550 kL. The total amount of catechin contained in this tea leaf pulverized liquid is about 1,400 g.

(2) Tea cell culture Tea cells are cultured using a 200 L fermentor (amount charged is 120 L). The culture period is about 30 days, and the peroxidase activity at the time of culture is about 500 mU / mL.

(3) Theaflavin conversion reaction The optimal reaction composition for performing a stable theaflavin conversion reaction is peroxidase activity: total catechin amount: reaction solution amount = 1 unit: 20 to 35 mg: 100 mL (see Example 4 above) ) Since the amount of the reaction solution in the 5 kL fermenter is 4 kL, the peroxidase activity: total catechin amount: reaction solution amount = 40 k units: 1,400 g: 4 kL. The required amount of tea leaf crushed liquid, well water, and tea cell culture solution are added to the 5 kL fermentor to start the reaction. The reaction is carried out at 25 ° C. The reaction time is 3-5 hours. Collect the reaction solution as appropriate and conduct component analysis. The reaction is stopped when the theaflavin content no longer increases. Table 12 shows the composition of the reaction solution. The theaflavin conversion efficiency will be around 45%.

  (Table 12 Composition of reaction solution)

(5) Inactivation of peroxidase and tannase After the pH of the above reaction solution was adjusted to 3 using 78% H ₂ SO ₄ , this reaction solution was heated to 70 ° C. in a 5 kL fermentor, and at 70 ° C. for 10 minutes. Maintain and inactivate peroxidase and tannase. Subsequently, the ground tea leaves are removed by centrifugation (8,000 rpm, 10 minutes), and the supernatant is recovered.

(6) Concentration Concentrate the collected supernatant using an evaporator with a target of Brix of 20%. When the evaporator is operated at 60 ° C. or lower, the time required to bring 600 mL of Brix to 20% or more (about 40-fold concentration) is about 1 hour. Ascorbic acid is added to the concentrate as a stabilizer so as to be 0.1% by weight.

(7) Production of product by pulverization About 100 L of the reaction liquid concentrated by spray drying is pulverized. Spray drying was operated at an inlet temperature of 150 ° C. By production with a 5 kL fermentor, 1.14 kg of powder with 39.6% by weight of theaflavin, 30.3% by weight of caffeine, 14.67% by weight of ascorbic acid, and 7.1% by weight of gallic acid can be produced. (See Table 13 below). The production efficiency of theaflavin according to the present invention is comparable to the efficiency of conventional methods using components that cannot be used as food additives (for example, plant hormones and specific vitamins), and can sufficiently be put into practical use. It became clear that there was.

  (Table 13 Composition of theaflavin-containing powder)

(Example 10 Production of high purity product)
In order to produce a highly pure theaflavin product, it is necessary to perform purification by column chromatography. The process for producing a high-purity product using a 5 kL fermenter is shown below.

(1) Caffeine removal by heat treatment of tea leaves and pulverization of tea leaves The raw tea leaves used as a raw material is 59.7 kg. Caffeine is obtained by infiltrating 59.7 kg of frozen tea leaves (-20 ° C) into 1,500 L of well-heated water at 80 ° C (optimal ratio is 100 mL of well water for 4 g of tea leaves) for 3 minutes. Remove. After the treatment, the tea leaves are collected using a dehydrator.

  The heat-treated tea leaves are pulverized to extract catechin as a substrate. 25 kg of well water is added to 1.0 kg of heat-treated tea leaves and pulverized. When a multi-mill grinder (GM4-25; Glow Engineering) is used, tea leaves can be pulverized with a processing capacity of 18.0-25.2 kg · tea leaves / hour. The resulting tea leaf grind is about 1,550 kL. The total amount of catechin contained in this tea leaf pulverized liquid is about 1,400 g.

(2) Culture of tea cells Tea cells were cultured using a 200 L fermentor (amount charged was 120 L). The culture period is about 30 days, and the peroxidase activity at the time of culture is about 500 mU / mL.

(3) Theaflavin conversion reaction The optimal reaction composition for performing a stable theaflavin conversion reaction is peroxidase activity: total catechin amount: reaction solution amount = 1 unit: 20 to 35 mg: 100 mL (see Example 4 above) ) Since the amount of the reaction solution in the 5 kL fermenter was 4 kL, the peroxidase activity: total catechin amount: reaction solution amount = 40 k units: 1,400 g: 4 kL. The required amount of tea leaf crushed liquid, well water, and tea cell culture solution are added to the 5 kL fermentor to start the reaction. The reaction is carried out at 25 ° C. The reaction time is 3-5 hours. Collect the reaction solution as appropriate and conduct component analysis. The reaction is stopped when the theaflavin content no longer increases. The composition of the reaction solution is the same as that shown in Table 12 above. Theaflavin conversion efficiency is around 45%.

(5) Inactivation of peroxidase and tannase After the pH of the above reaction solution was adjusted to 3 using 78% H ₂ SO ₄ , this reaction solution was heated to 70 ° C. in a 5 kL fermentor, and at 70 ° C. for 10 minutes. Maintain and inactivate peroxidase and tannase. Subsequently, the ground tea leaves are removed by centrifugation (8,000 rpm, 10 minutes), and the supernatant is recovered.

(6) Purification A resin tower packed with 37.9 L of SEPABEADS (Mitsubishi Chemical) is required to process 4 kL of the reaction solution from the 5 kL fermenter. The purification operation is performed at 4 ° C. An ethanol (EtOH) solution is used as the eluent, and the solution is passed at a flow rate SV = 1.8.

  The ethanol concentration is changed stepwise from 10% to 40% to elute each component. At 10% EtOH, there will be little elution of each component. At 20% EtOH, elution of caffeine and catechins is observed. The eluate is appropriately analyzed, and when elution of caffeine and catechins is completed, 30% EtOH is passed through. TF is eluted in the 30% EtOH fraction. The eluate is appropriately analyzed, and when elution of theaflavin is completed, 40% EtOH is passed through. The yield and composition of theaflavin by purification are shown in Table 14 below. By performing the resin purification process from the 5 kL fermenter production, 195.6 g of high-purity product with 98.3% by weight theaflavin could be produced.

  (Table 14 Yield of purified theaflavin powder)

(Example 11 Functional evaluation of theaflavin mixture)
(11-1 Production of theaflavin mixture)
The theaflavin used in this example was prepared by adding 10 mg of commercially available caffeine (Wako Pure Chemical Industries, Ltd.) to the purified theaflavin solution prepared in Example 8.

(11-2 Anti-obesity effect, blood glucose lowering effect of theaflavin mixture)
Using C57BL6 male mice at 4 weeks of age, the anti-obesity effect and blood glucose lowering effect of theaflavin were examined.

  For mice fed with a normal diet (CE2, manufactured by Nippon Claire Co., Ltd.) and a high-fat diet diet (High Fat Diet 32, manufactured by Nippon Claire Co., Ltd.) shown in Table 15 below, water or theaflavin mixture ( Any beverage containing 2 mg of theaflavin per 100 ml of water and 10 mg of caffeine) was given, and changes in body weight, blood glucose level during tail vein hunger and fasting of the tail vein were verified for 9 months.

(1) Rearing Experiment After purchasing a 4-week-old male mouse, C57BL6, a high-fat diet-induced obese model mouse, pre-bred for 7 days to use the healthy animal for the experiment. The environment of the breeding room was a constant temperature of 23 ± 1 ° C., a humidity of 55 ± 5%, and a light / dark period of 12 hours (between 8:00 and 20:00). The experimental animals were housed in an animal breeding room with 5 mice per cage using a plastic cage.

  (Table 15: amount of ingredients in 100 g of normal diet (CE2) and high fat diet (HFD32))

(2) Experimental groups and experimental conditions The experimental groups were 8 groups, and the number of each group was n = 10. The food and drink given to each group are shown in Table 16 below. Feeding and water supply were free intake.

  (Table 16 Feed and drink given to each group)

Table 17 shows the change in weight gain during the 9 months after the start of the experiment. The weight gain rate was calculated according to the following formula based on the body weight one week after the start of breeding.
Weight increase rate = (weight of measurement month−weight 1 week after start of experiment) ÷ weight 1 week after start of experiment × 100
(Table 17 Weight gain of each group)

In the normal diet-theaflavin group (group II), there was no significant difference in weight gain compared to the normal diet-water group (group I). On the other hand, in the high fat diet-theaflavin group (VI group), an average decrease in body weight gain rate of about 6% was observed compared to the high fat diet-water group (Group III).

  The blood glucose level was measured at 1 pm. Blood was collected from the tail vein, and the blood glucose level was measured with a simple blood glucose measurement system (manufactured by Terumo). The measurement results are shown in Table 18 below. In the normal diet-theaflavin group (Group I), the blood glucose level slightly decreased as compared with the normal diet-water group (Group II) as a control, and remained at about 150 units. In the high fat diet-theaflavin group (Group VI), the blood glucose level significantly decreased as needed compared to the high fat diet-water group (Group III), and it remained at about 170 to 180 units.

  (Table 18 Blood glucose levels at any time in each group)

In addition, fasting blood glucose levels were measured for each group after 14 hours of fasting without plastic bedding. The measurement results are shown in Table 19 below. The normal diet-water group (group I) and the normal diet-theaflavin group (group II) showed comparable blood glucose levels. In the high fat diet-theaflavin group (Group VI), the blood glucose level was lower than in the high fat diet-water group (Group III).

  (Table 19 Fasting blood glucose levels for each group)

From the above results, there was no significant difference between group I and group II in terms of body weight, but when comparing group III and group IV, the weight gain in group IV was 8% compared to group III. Declined. Therefore, it became clear that the theaflavin mixture manufactured by the manufacturing method of this invention has an obesity prevention effect.

  Regarding blood glucose level, no significant difference was found between Group I and Group II, but when Group III and Group IV were compared, blood glucose level decreased by 20% in Group IV compared to Group III. Therefore, it became clear that the theaflavin mixture manufactured by the manufacturing method of this invention has a blood glucose level raise inhibitory effect.

  In addition, since the same measurement results were obtained for blood glucose level and body weight in Group I and Group II, the theaflavin mixture was found to have the effect of lowering blood glucose level and weight in the normal state of ingesting a normal diet. It was confirmed that it was safe.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention.

Claims (13)

(1) a step of heating a raw material containing tea,
(2) a step of pulverizing the raw material heated in the step (1),
(3) a step of culturing tea cells in a microorganism medium to obtain a tea cell culture; and (4) the raw material crushed in step (2) and the tea cell culture cultured in step (3). A method for producing theaflavin comprising a step of reacting a product to produce theaflavin. The production method according to claim 1, wherein the microorganism medium is a yeast medium containing sugar, a nitrogen source, and inorganic salts. The production method according to claim 2, wherein the sugar is selected from the group consisting of sucrose and glucose. The manufacturing method of any one of Claims 1-3 which further includes the process of deactivating the enzyme contained in the reaction material in the said (3) process. The production method according to claim 4, wherein the deactivation step is performed by acidifying and / or heating the reaction product. The manufacturing method of any one of Claims 1-5 which further includes the process of collect | recovering the theaflavins. The manufacturing method according to claim 6, wherein the recovery is performed using an adsorption resin. A substantially pure theaflavin produced by the production method according to claim 7. The food composition containing theaflavin manufactured by the manufacturing method of any one of Claims 1-7. The food composition for obesity prevention containing theaflavin manufactured by the manufacturing method of any one of Claims 1-7. A food composition for suppressing blood sugar level elevation, comprising theaflavin produced by the production method according to any one of claims 1 to 7. The food composition according to claim 10, wherein the food composition is taken in combination with a high fat diet. The food composition according to any one of claims 9 to 12, further comprising a theaflavin stabilizer.