JP2012183010A - Tea beverage and method for producing the same - Google Patents

Abstract

The present invention provides a black tea beverage having a high anti-obesity effect and an effect of suppressing the onset of diabetes.
[MEANS FOR SOLVING PROBLEMS] The present invention is produced from a black tea extract treated with an esterase during and / or after extraction of a black tea extract from black tea leaves, containing polyphenols in an amount of 60 mg / 100 ml or more in terms of tannin, It is a tea beverage having a value lowering effect.
[Selection figure] None

Description

  The present invention relates to a black tea beverage and a method for producing the same.

  In recent years, techniques for treating a tea extract with tannase are known (Patent Documents 1 to 5).

  Patent Document 1 describes that the addition of oxidase and tannase to an aqueous tea extract reduces the occurrence of turbidity during refrigerated storage. Patent Document 2 describes that the turbidity of the tea extract can be significantly decomposed and removed when tannase and chlorogenic acid esterase are used in combination.

  Patent Document 3 describes that by applying tannase treatment in a state containing a high concentration of catechin, the astringency can be reduced without impairing the umami and richness of the tea beverage.

Patent Document 4 describes an antioxidant composition comprising a tea extract with enhanced antioxidant power. Patent Document 4 discloses that tannin-degrading enzyme tannase is allowed to act during and / or after extraction of tea leaves to cleave gallate esters of gallate-type catechins and decompose into non-gallate-type catechins and gallic acid, It is described that epigallocatechin and gallic acid produced by the action of tannase on epigallocatechin gallate have strong antioxidant power.
Patent Document 5 describes that the tea extract or the concentrate thereof is tannase treated to reduce the gallate catechin ratio. In patent document 5, in manufacturing a container-packed tea beverage using a tea extract whose gallate body catechin ratio is adjusted to less than 50% by mass in advance, the content of gallic acid is adjusted to 21 to 150 ppm, and a non-polymer If the gallate catechin ratio in the catechins is adjusted to 0 to 50% by mass and the epimer ratio to 30 to 60% by mass, not only the bitterness is suppressed, but also the long term It is said that the composition change of catechins hardly occurs even when stored.

JP 7-303450 A JP 11-308965 A JP 2005-130809 A JP 2006-83352 A JP 2007-325585 A

  However, the present inventors have newly found that a tannase-treated black tea extract lowers blood glucose level. In addition, this was predicted to be a common action not only for tannase but also for black tea extracts treated with esterases such as chlorogenic acid esterase.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a tea beverage having a high anti-obesity effect.

  According to the present invention, it is produced from a black tea extract treated with an esterase at the time of extracting a black tea extract from black tea leaves and / or after extraction, containing polyphenols in an amount of 60 mg / 100 ml or more in terms of tannin content, and reducing blood sugar levels A black tea beverage having an action is provided.

  Moreover, according to this invention, the container-packed drink formed by filling said drink into the container for tea is provided.

  Moreover, according to this invention, said tea drink used in order to prevent or treat diabetes is provided.

  Furthermore, according to the present invention, the black tea extract is extracted during the step of extracting a black tea extract from black tea leaves, and / or after the step of extracting the black tea extract. A method of producing a black tea beverage having a blood glucose level-lowering effect, comprising a step of treating with esterase, containing 60 mg / 100 ml or more of polyphenol in terms of tannin amount.

  According to this invention, the black tea beverage produced from the black tea extract treated with esterase contains a high concentration polyphenol of 60 mg / 100 ml or more in terms of the amount of tannin. Thereby, a blood glucose level can be reduced by ingesting as a meal. Therefore, it becomes possible to realize a tea beverage that has a high anti-obesity action and an effect of suppressing the onset of diabetes.

  ADVANTAGE OF THE INVENTION According to this invention, the black tea drink from which the suppression effect of diabetes onset is acquired with a high anti-obesity effect is provided.

It is a figure which shows the polyphenol composition ratio of a black tea drink. It is a figure which shows transition of the increase / decrease in the body weight of a mouse | mouth in a mouse | mouth long-term administration test. (A) It is a figure which shows the ratio (%) of the visceral (mesenteric) surrounding fat per body weight of the mouse | mouth in a mouse | mouth long-term administration test. (B) It is a figure which shows the ratio (%) of fat around a kidney per body weight of a mouse | mouth in a mouse | mouth long-term administration test. It is a figure which shows the ratio (%) of the testicular fat per body weight of a mouse | mouth in a mouse | mouth long term administration test. It is a figure which shows transition of the blood glucose level at the time of fasting of the mouse | mouth in a mouse | mouth long-term administration test. It is a figure which shows the ratio of hemoglobin _A1c (what hemoglobin and glucose couple | bonded) in the total hemoglobin in a mouse | mouth long-term administration test. (A) It is a figure which shows the mouse | mouth serum triglyceride value in a mouse | mouth long-term administration test. (B) It is a figure which shows the total cholesterol value in the blood of a mouse | mouth in a mouse | mouth long-term administration test. It is a figure which shows the serum NEFA value of the mouse | mouth in a mouse | mouth long-term administration test. (A) It is a figure which shows the total amount of lipids in a mouse | mouth liver in a mouse | mouth long-term administration test. (B) It is a figure which shows the triglyceride value in the mouse | mouth liver in a mouse | mouth long-term administration test. (A) It is a figure which shows the total cholesterol value in the mouse | mouth liver in a mouse | mouth long-term administration test. (B) It is a figure which shows the NEFA value in a mouse | mouth liver in a mouse | mouth long-term administration test. It is a figure which shows the phospholipid value in the mouse liver in a mouse | mouth long-term administration test. (A) It is a figure which shows the amount of lipid excretion in the mouse | mouth feces of the breeding 89-91 day in a mouse | mouth long-term administration test. (B) It is a figure which shows the triglyceride value in the mouse | mouth feces of the breeding 89-91 day in a mouse | mouth long-term administration test. (A) It is a figure which shows the total cholesterol value in the mouse | mouth feces of the breeding 89-91 day in a mouse | mouth long-term administration test. (B) It is a figure which shows the total bile acid in the mouse | mouth feces of the breeding 89-91 day in a mouse | mouth long-term administration test. (A) It is a figure which shows the fatty acid synthetase activity in the mouse liver in a mouse | mouth long-term administration test. (B) Carnitine palmitoyltransferase activity in mouse liver in mouse long-term administration test. (A) It is a figure which shows the serum leptin value of the mouse | mouth in a mouse | mouth long-term administration test. (B) It is a figure which shows the serum insulin level of the mouse | mouth in a mouse | mouth long-term administration test. It is a figure which shows the mouse | mouth serum adiponectin level in a mouse | mouth long-term administration test. (A) It is a figure which shows the APOA2 expression level of a mouse liver in a mouse | mouth long-term administration test. (B) It is a figure which shows the expression level of FABP1 of a mouse liver in a mouse | mouth long-term administration test. (A) It is a figure which shows the APOC3 expression level of a mouse liver in a mouse | mouth long-term administration test. (B) It is a figure which shows the GCK expression level of a mouse liver in a mouse | mouth long-term administration test. (A) It is a figure which shows the PTEN expression level of a mouse liver in a mouse | mouth long-term administration test. (B) It is a figure which shows the RBP4 expression level of a mouse liver in a mouse | mouth long-term administration test. (A) It is a figure which shows the FBP1 expression level of a mouse liver in a mouse | mouth long-term administration test. (B) It is a figure which shows the NFKBIA expression level of a mouse liver in a mouse | mouth long-term administration test. It is a figure which shows the preparation method of a rat blood sample. It is a figure which shows transition of the increase / decrease in the body weight of a rat in a rat long-term administration test. (A) It is a figure which shows the ratio (%) of the visceral (mesenteric) surrounding fat per body weight of the rat in a rat long-term administration test. (B) It is a figure which shows the ratio (%) of the testicular fat per rat body weight in a rat long-term administration test. It is a figure which shows transition of the fasting blood glucose level of the rat in a rat long-term administration test. It is a figure which shows transition of the blood glucose level of the rat at any time in a rat long-term administration test. (A) It is a figure which shows the ratio of hemoglobin _A1c (what hemoglobin and glucose couple | bonded) in the total hemoglobin of the breeding 4th week in a rat long-term administration test. (B) It is a figure which shows the ratio of hemoglobin _A1c (what hemoglobin and glucose couple | bonded) in the total hemoglobin of the breeding 5th week in a rat long-term administration test. (A) It is a figure which shows the result of the glucose tolerance test of the breeding 4th week in a rat long-term administration test. (B) It is a figure which shows the result of the glucose tolerance test of the breeding 6th week in a rat long-term administration test. (A) It is a figure which shows the serum triglyceride value in a rat long-term administration test. (B) It is a figure which shows the total cholesterol value in rat blood in a rat long-term administration test. It is a figure which shows the result of the rat arteriosclerosis index | exponent in a rat long-term administration test. (A) It is a figure which shows the serum HDL-C value in a rat long-term administration test. (B) It is a figure which shows the serum NEFA value in a rat long-term administration test. (A) It is a figure which shows the total lipid amount in a rat liver in a rat long-term administration test. (B) It is a figure which shows the NEFA value in a rat liver in a rat long-term administration test. (A) It is a figure which shows the triglyceride value in the rat liver in a rat long-term administration test. (B) It is a figure which shows the total cholesterol value in the rat liver in a rat long-term administration test. It is a figure which shows the phospholipid value per rat liver in a rat long-term administration test. (A) It is a figure which shows the serum TBARS value in a rat long-term administration test. (B) It is a figure which shows the TBARS value in a rat liver in a rat long-term administration test. (A) It is a figure which shows the catalase activity of the rat liver in a rat long-term administration test. (B) It is a figure which shows the GSH-Px activity of the rat liver in a rat long-term administration test. (A) It is a figure which shows the serum leptin level of the rat in a rat long-term administration test. (B) It is a figure which shows the serum insulin level of the rat in a rat long-term administration test. (A) It is a figure which shows the serum adiponectin level of a rat in a rat long-term administration test. (B) It is a figure which shows TNF- (alpha) of a rat in a rat long-term administration test. (A) It is a figure which shows the albumin in the rat urine of 44 hours at the 5th week in the rat long-term administration test. (B) It is a figure which shows the creatinine in the rat urine of 44 hours at the 5th week of breeding in the rat long-term administration test. It is a figure which shows 8-OHdG in the rat urine of 44 hours at the 5th week of breeding in the rat long term administration test.

  Hereinafter, embodiments of the present invention will be described. As described above, the present invention is produced from a black tea extract treated with an esterase when extracting a black tea extract from black tea leaves and / or after extraction, and contains 60 mg / 100 ml or more of polyphenol in terms of tannin amount, It is a tea beverage having a value lowering effect.

<Tea leaves>
The raw material of the black tea extract used in the present invention is not particularly limited as long as it is black tea leaves (totally fermented tea leaves obtained by completely oxidative fermentation of tea leaves of tea tree Camellia sinensis). It may be a leaf or a tea leaf produced by the OTC (non-traditional) manufacturing method. As tea leaves, commercially available ones may be used as they are, but treatments such as pulverization and attrition may be performed.

<Black tea extract>
The tea leaf extract can be obtained, for example, by supplying water or hot water at a constant flow rate to the raw tea packed in the extraction column to obtain a predetermined amount of the extract, or by charging the tea leaves into the extraction kettle and supplying a predetermined amount of water. Alternatively, it can be prepared by appropriately selecting a commonly used method such as a method of immersing in hot water for a certain time and then separating from tea leaves to obtain an extract. The extraction magnification is preferably 3 to 50 times. The extraction magnification refers to the ratio of the volume of the extraction solvent to the tea leaf weight used for extraction. As the extraction condition, for example, the temperature of water or hot water can be 35 ° C. or more and 100 ° C. or less. An extraction aid may be added during extraction. It does not specifically limit as an extraction aid, A conventionally well-known extraction aid can be used. For example, sodium L-ascorbate or L-ascorbic acid can be used as an antioxidant, and sodium bicarbonate or potassium carbonate can be used as a pH adjuster. Moreover, 0.1-10 mass% of these extraction adjuvants can be added and used with respect to the mass of a tea leaf.

  In the present invention, esterase is allowed to act during and / or after extraction of these black tea leaves. The esterase of the present invention is not limited as long as it has an action capable of decomposing condensed tannin. Specifically, the action can decompose gallate-type catechin into non-gallate-type catechin, and / or gallate-type theaflavin is non-gallate-type. What has the effect | action which can be decomposed | disassembled into theaflavins is preferable. For example, pectinase, chlorogenic acid esterase, and tannase can be mentioned, and it is preferable to use tannase or chlorogenic acid esterase or both, and tannase is particularly preferable. Any tannase can be used as long as it has an activity of decomposing tannin. As the chlorogenic acid esterase, any chlorogenic acid esterase can be used as long as it has an activity of decomposing chlorogenic acid ester.

  When esterase is allowed to act after extracting a black tea extract from black tea leaves, the esterase may be added to the black tea extract as it is, or for example, esterase may be added to a black tea extract concentrated about 10 times. When the black tea extract is subjected to esterase treatment, the temperature can be 10 ° C. or higher and 60 ° C. or lower, preferably 20 ° C. or higher and 55 ° C. or lower, more preferably 20 ° C. or higher and 40 ° C. or lower. Further, the pH may be adjusted to 2.0 or more and 8.0 or less, preferably 4.0 or more and 7.0 or less. And esterase can be made to react for 10 minutes or more and 480 minutes or less under such conditions.

  In the present invention, the gallate-type catechin and the gallate-type theaflavin in the black tea extract can be made non-gallate by subjecting the black tea extract to an esterase treatment. By reducing the content of gallate type, astringency is reduced, and by containing non-gallate type polyphenol, an effect of lowering blood glucose level can be obtained. The ratio of the total amount (mg / 100 ml) of non-gallate catechin and non-gallate type theaflavin to the total amount (mg / 100 ml) of gallate-type catechin and gallate-type theaflavin contained in the black tea beverage of the present invention is 2-15. preferable.

<Polyphenol>
The “tea beverage” of the present invention contains a polyphenol of “a degree of bitterness when mixed alone”. Here, “polyphenol” means not only black tea-derived polyphenol obtained from black tea extract, but also polyphenol (added polyphenol) contained in general plants added separately. Here, the "tea leaf-derived polyphenol" is a polyphenol derived from the tea leaf of the main raw material used in the tea beverage of the present invention. In the production of tea beverage, from the tea leaf of the main raw material in water or hot water, etc. It will be extracted. Or separately prepared tea extract.

  Examples of the “tea leaf-derived polyphenol” include epigallocatechin gallate, gallocatechin gallate, epicatechin 3 gallate, and catechin gallate as gallate catechins. Non-gallate catechins include catechin, gallocatechin, epigallocatechin, and epicatechin. Examples of the gallate type theaflavin include theaflavin 3 gallate, theaflavin 3 ′ gallate, and theaflavin 3,3 ′ digallate. Non-gallate type theaflavins include theaflavins.

  The type of “added polyphenol” is not particularly limited as long as it is a polyphenol contained in a plant, but tea polyphenol and fruit polyphenol are preferable.

  “Tea polyphenol” is not particularly limited as long as it is derived from tea, and black tea polyphenol, green tea polyphenol, oolong tea polyphenol and the like can be mentioned. Among these, it is preferable to add black tea polyphenol, which is compatible with sweeteners and does not require the addition of cyclic oligosaccharides. Although the manufacturing method of an addition polyphenol is not specifically limited, For example, what was obtained by extraction from a tea leaf etc. by a conventionally well-known method can be used. Moreover, you may use what is marketed as these addition polyphenols. Further, the added polyphenol may be a concentrate.

  The above-mentioned “tea polyphenol” contains various catechins, such as catechins such as epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, catechin or gallocatechin, which are non-polymers of catechins, etc. In addition, black tea polyphenols include theaflavins, theasinensins, thealvidins, proanthocyanidins and the like.

  The above-mentioned “degree of feeling bitter and astringent when mixed alone” is the degree that it feels sensuously that tea polyphenol is astringent and bitter and cannot be drunk when mixed with black tea. The bitter and astringent taste due to polyphenols is also felt when drinking teas such as black tea, green tea and oolong tea, which are generally extracted with water or hot water. However, when the additive tea polyphenol is mixed into black tea, the bitter taste of black tea becomes difficult to drink as a favorite product. Since bitter and astringent tastes vary depending on the taste of each individual drinking, the degree of bitter and astringent taste is relative, but an example is 60 mg / 100 ml or more in terms of the amount of tannin.

  “Fruit polyphenols” include polyphenols such as apples and grapes. These fruit polyphenols are also known to have an antioxidant action, an antihypertensive action and the like, and the effects of polyphenols are enhanced. Of these, apple polyphenol is particularly preferred because of its good flavor and compatibility with black tea beverages. Apple polyphenol is effective when added in a small amount, and has a low bitter taste when added. Addition of green tea-derived polyphenol to a black tea beverage makes it easier to feel bitterness and astringency.

  Then, as described above, a large amount of polyphenol is ingested by adding the above-mentioned added polyphenol, preferably added black tea polyphenol and / or apple polyphenol, to the black tea-derived polyphenol derived from black tea as the main raw material. can do. That is, many polyphenols can be ingested by one drinking compared with the case of drinking tea extracted only from tea leaves and hot water.

<Polyphenol content>
In the present invention, the proportion of the total amount of the above-described black tea-derived polyphenol and added tea polyphenol in the total polyphenol of the black tea beverage is preferably 1% by mass or more and 100% by mass or less. More preferably, it is 20 mass% or more and 100 mass% or less. Here, the “total polyphenol” means the total amount of the whole polyphenol including the tea polyphenol and the fruit polyphenol added in addition to the tea leaf-derived polyphenol tea.

  The total polyphenol is particularly preferably contained in an amount of 60 mg / 100 ml to 250 mg / 100 ml in terms of the amount of tannin. Thus, by adding the above-mentioned added polyphenol to the esterase-treated black tea extract to contain polyphenol in an amount of 60 mg / 100 ml to 250 mg / 100 ml, antioxidant action, antibacterial action, inhibition of glycolytic enzyme It can be set as the tea drink which has health functions, such as the diet effect by an effect | action. Moreover, it is more preferable that the content of “tea leaf-derived polyphenol” in the black tea beverage is 60 mg / 100 ml or more and 250 mg / 100 ml or less in terms of the tannin amount. This amount of tannin can be measured by the iron tartrate method.

<Tea drink>
The black tea beverage of the present invention contains black tea-derived polyphenols derived from black tea leaves, and added tea polyphenols and / or other polyphenols to a black tea extract treated with esterase, It is preferable to add sugar alcohol.

  The “high-sweetness sweetener” as used herein is a non-sugar sweetener that has a high sweetness and can exhibit sweetness in a very small amount compared to sucrose. The sweetness is a sweetness value compared to a pure sucrose solution when sucrose is set to 1.00. Since it is measured by sensory test, it is difficult to accurately define, but for example, it is said that sucralose is 600 times, acesulfame potassium is 200 times, and stevia is nearly 200 times.

  In addition, “sugar alcohol” is produced (catalytic reduction) by reacting and reducing a saccharide having a reducing group obtained by reducing an aldehyde group of a sugar molecule with hydrogen under high temperature and high pressure. , A general term for chain polyhydric alcohols such as sorbitol, maltitol, and xylitol.

  A sweetener is a seasoning used to sweeten foods, and high-intensity sweeteners and sugar alcohols are included in sweeteners. In the present invention, “high-intensity sweetener and / or sugar alcohol” is any of high-intensity sweeteners such as sucralose, acesulfame potassium, aspartame, stevia, and sugar alcohols such as xylitol, maltitol, and erythritol. Or it may be a sweetener comprising two or more selected from these groups.

  By adding a high-intensity sweetener and / or sugar alcohol, the bitter and astringent taste of tea polyphenols can be alleviated. Slight bitterness is conspicuous with only the high-intensity sweetener, and by combining the sugar alcohol and the high-intensity sweetener, the tea beverage can be suppressed to a lower calorie than adding sweetness by adding sugar.

  Epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, catechin or gallocatechin, tea flavins, theasinensins, thearubidines, proanthocyanidins, etc. contained in tea polyphenols are tannin components, and have astringency as a property. Have. For this reason, if a polyphenol-containing beverage whose amount of polyphenol exceeds the amount that is extracted by drinking with normal hot water etc., it becomes bitter to the extent that it is not suitable for drinking, and the cyclic oligosaccharide is added to mask the bitter taste It is necessary to do.

  On the other hand, according to the above-described tea beverage of the present invention, the gallate catechin is converted into a non-gallate catechin by esterase treatment. Therefore, astringency can be reduced. Further, when astringency remains, it is only necessary to add a high-intensity sweetener and a sugar alcohol, so there is no need to add a cyclic oligosaccharide. Therefore, it is possible to provide a black tea beverage having a blood sugar level lowering effect while suppressing the black tea beverage to a low calorific value.

  By the way, the tea extract subjected to the action of esterase may show a decrease in pH. Therefore, it is desirable to add an alkali to the tea extract after treatment to adjust the pH value before the esterase treatment. Examples of the alkali used here include sodium bicarbonate and potassium carbonate. Specifically, the pH of the black tea beverage is preferably 2.5 to 8.0, more preferably 3.0 to 7.9.

  Moreover, you may use fruit juice, a fragrance | flavor, a sour agent, a nutrient fortifier, a stabilizer, etc. in addition to the above in the tea beverage of this invention.

  In the method for producing a black tea beverage of the present invention, after the above esterase treatment, it is 70 ° C. or higher and 145 ° C. or lower, more preferably 90 ° C. to 142 ° C., 0.02 minutes to 60 minutes, more preferably 0 ° C. A high temperature heating step of heating from 03 minutes to 40 minutes can be performed. By performing such a heating step, a normal tea beverage sterilization step can be performed and at the same time the esterase can be deactivated.

  The tea extract thus obtained is concentrated to a concentration suitable for drinking or diluted with water to obtain a tea beverage. Alternatively, the black tea extract may be powdered and used as an instant powdered tea. The powdered tea obtained here is excellent in solubility in cold water and transparency when the solution is cooled.

<Contained beverage>
The container-packed beverage of the present invention is obtained by filling the above-described tea beverage into a beverage container by a conventionally known method.

  As a beverage container, conventionally known glass bottles, metal cans, paper, plastics and the like are used and are not particularly limited. Moreover, although a metal can, a PET bottle, a paper container etc. are used with a container, it is not limited to these. Moreover, the container-packed black tea drink may be filled as it is, and may be sterilized as needed. Of the above, PET bottles are particularly preferred because they are easy to carry, open and close and are lightweight.

  In the case of a container-packed beverage, the aforementioned heating step may be performed on a tea beverage filled in a container such as a can or a PET bottle. In the sterilization step, the heating temperature and the heating time are as described above.

<Blood glucose lowering action>
The blood sugar level lowering action of the black tea beverage of the present invention means an action of lowering at least one of the blood sugar level on an empty stomach or at any time. The fasting blood glucose level lowering effect can be evaluated by, for example, analyzing the blood glucose level after 12 hours of fasting. The blood glucose level lowering effect can be evaluated by analyzing the blood glucose level measured 2 hours after feeding, for example. Furthermore, HbA _1c, may be evaluated by analyzing the blood insulin level. The present invention is particularly excellent in that it has a blood glucose level lowering action as needed.

<Anti-obesity action>
The anti-obesity action obtained by ingesting the black tea beverage of the present invention is not particularly limited as long as it suppresses obesity, but it suppresses body weight gain, suppresses blood sugar level increase, and suppresses serum triglyceride level increase Etc.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1
First, a black tea extract was extracted at an extraction ratio of 20 times from 40 g of black tea leaves (Ceylon black tea from Sri Lanka, manufactured by Mitsui Norin Co., Ltd.). The extraction conditions were 85 ° C. and 7 minutes. Next, the black tea extract obtained by this concentrated extraction was treated with 0.2 g of tannase (Tannase KTFH, manufactured by Kikkoman Corporation) at a temperature of 25 to 30 ° C. for 60 minutes. After this tannase treatment, the pH was adjusted with 1 g of sodium L-ascorbate and 0.3 g of sodium bicarbonate, diluted with pure water to a total volume of 1000 mL, and adjusted to double the expected drinking concentration. Furthermore, ultra-high temperature sterilization by heating at 135 ° C. for 30 seconds was performed to obtain a tea beverage.

(Example 2)
The procedure was the same as in Example 1 except that 20 g of xylitol (manufactured by Danisco Japan) and 0.08 g of sucralose (San-Eigen FFI) were added after tannase treatment.

(Example 3)
The procedure was the same as Example 1 except that 36 g of tea leaves (produced by Kenya, manufactured by Mitsui Norin Co., Ltd.) were used.

(Comparative Example 1)
The procedure was the same as Example 1 except that tannase treatment was not performed.

(Comparative Example 2)
Example 2 was repeated except that tannase treatment was not performed.

(Comparative Example 3)
Example 3 was repeated except that tannase treatment was not performed.

1. Analysis of Black Tea Beverage Example 1 and Comparative Example 1 were analyzed. The analysis items are as follows. The results are shown in Table 1. In Table 1, TF-1 is theaflavin 3-monogallate, TF-1 ′ is theaflavin 3′-monogallate, and TF-2 is theaflavin 3,3′-digallate.
(1) pH
It was measured with a pH meter (product name: pH meter M-13, manufactured by Horiba, Ltd.).
(2) Absorbance The brownness (420 nm) and turbidity (720 nm) were measured with an ultraviolet / visible spectrophotometer (product name: spectrophotometer U-1500, manufactured by Hitachi High-Tech).
(3) Brix value (sugar content)
It was measured with a saccharimeter (product name: digital differential densitometer DD-7, manufactured by Atago Co., Ltd., or digital refractometer RX-5000α, manufactured by Atago Co., Ltd.).
(4) Polyphenol A black tea beverage as a sample was prepared so that the tannin value by the iron tartrate method was 350 mg / 100 ml. About other analysis items, it measured by the Asahi drink company standard method.

  As a result of the analysis, as shown in Table 1 and FIG. 1, in Examples 1 to 3, the proportion of non-gallate catechins in the polyphenol composition increased. Moreover, when Example 1 was compared with Example 3, the difference of the polyphenol composition by the difference in the tea leaf manufacturing method appeared.

2. Mouse long-term administration test After 5 weeks old male C57BL / 6J mice were preliminarily raised for 5 days, fasting blood glucose level and body weight were measured, control group 5 (Con), high fat diet group 6 (HF), comparative example Divided into 6 groups of 1 black tea group (CBT1), 6 black tea groups of Example 1 (CBT2), isoquercitrin group (Iso), 6 theaflavin groups (Thea) It was. The breeding period was 94 days, and the body weight and food intake during the period were measured every day. In addition, blood glucose levels are measured on week 1, 4, 7, 9, 12 (main day 1, day 22, day 22, day 44, day 59, day 81). Measurements of plasma triglycerides and plasma free fatty acids (NEFA) were performed on the eyes (day 81). In addition, Palmus IIIN145 × 195 mm (Natural Materials Exploration Laboratory Co., Ltd.) was placed under a mouse gauge on the 13th week (main breeding day 89-91), and feces were collected for 72 hours.
The mice were bred after 12 hours of fasting after 94 days of main breeding, and blood was collected from the heart. Next, liver, kidney, perirenal fat, testicular fat, and visceral (mesenteric) fat were removed. Excluded organs were frozen in liquid nitrogen and stored frozen (−84 ° C.) except for those prepared immediately for measurement. The blood sample was placed in an Eppendorf tube and allowed to stand for 1 hour, centrifuged (3000 rpm, 15 ° C., 30 minutes), and the resulting supernatant was transferred as serum to a new Eppendorf tube and stored at −84 ° C. until measurement. As for the test results, the mean value is shown by ± standard error, and the significant difference between each group was tested using Fisher's least significant difference method (Fisher-LDS) (hereinafter, all the test results were significant for each group). Is the same).

  The feed composition is shown in Table 2. CBT1 group and CBT2 group were mixed with 80 g of casein at a ratio of 30 ml of black tea, freeze-dried as casein, and fed with the same composition as the high-fat food (black tea casein was corrected to moisture to 20%). The Iso group was supplied with Isoquercitrin powder (manufacturer: EXTRASYNTHESE), and the Thea group with Theaflavin powder (manufactured by Mitsui Norin Co., Ltd.) as a sample and added to a high fat diet.

Fasting blood glucose level was measured using Medisafe Mini GR-102 (Terumo Corporation) after fasting for 12 hours. Hemoglobin A _1c (HbA _1c ) was blood collected from the heart at the time of dissection and was measured using a Micromat II HbA _1c cartridge (Bio-Rad Laboratories).
Plasma triglycerides and plasma NEFA (non-ester type fatty acid) were collected after fasting for 12 hours, the blood was allowed to stand for 30 minutes, centrifuged (3000 rpm, 15 ° C., 20 minutes), and the resulting supernatant was measured as plasma. Plasma triglyceride was measured by enzyme method (GPO / DAOS method) using Triglyceride E-Test Wako (Wako Pure Chemical Industries, Ltd.). The plasma triglyceride concentration was calculated using a standard curve created using a standard solution. Plasma NEFA was measured by enzyme method (ACS / ACOD method) using NEFA C-Test Wako (Wako Pure Chemical Industries, Ltd.). The concentration was calculated using a standard curve created using a standard solution.

  Serum triglyceride and serum NEFA were measured using the previously stored serum. Serum total cholesterol (TC) was measured by an enzyme method (cholesterol oxidase / DAOS method) using cholesterol E-Test Wako (Wako Pure Chemical Industries, Ltd.). The concentration was calculated using a standard curve created using a standard solution.

  Extraction of liver lipids and fecal lipids is described in J. Org. Folch et al. (J. Folch, M. Less, and H. S. Stanley: A simple method for the isolation of total lipids, similar tissues, J. Biol. The lipids were measured by the method described above.

  Liver phospholipid (PL) was measured by a choline oxidase DAOS method using phospholipid C-Test Wako (Wako Pure Chemical Industries, Ltd.).

  Fecal total bile acid was measured by enzyme colorimetric method using total bile acid-Test Wako (Wako Pure Chemical Industries, Ltd.).

  A sample used for measurement of liver lipid metabolism enzyme activity was prepared as follows. 0.5 g is excised from the liver immediately after blood collection, 7 volumes of 0.25M sucrose disruption solution (pH 7.2, containing 1 mM EDTA, 3 mM Tris-HCl) is added, and using a glass homogenizer in ice Homogenized. The homogenate solution was centrifuged (1000 rpm, 4 ° C., 10 minutes), and the resulting supernatant was further centrifuged (9000 rpm, 4 ° C., 10 minutes) to separate the supernatant and the precipitate. The supernatant was further centrifuged (100,000 × g, 4 ° C., 60 minutes), and the supernatant and the precipitate were separated. The obtained supernatant was used as a cytosol fraction for measurement of lipid metabolic enzyme activity in the liver. In order to determine the enzyme activity in mg protein, the protein was quantified by the biuret method using A / GB-Test Wako (Wako Pure Chemical Industries, Ltd.).

For fatty acid synthase activity, 0.5 ml of 0.2 M potassium phosphate buffer (pH 7.0), 0.2 ml of 2.5 mM acetyl-CoA, 0.03 ml of 10 mM NADPH, 0.05 ml of sample, and 0.41 ml of distilled water were sequentially added to the cell. The mixture was put on top and covered with parafilm and the cells were mixed up and down, then mounted on a constant temperature cell holder kept at 30 ° C., and the absorbance was measured over time (339 nm, measurement time 120 seconds). Next, 0.02 ml of 10 mM malonyl-CoA was added and mixed, and then mounted on a constant temperature cell holder kept at 30 ° C., and the absorbance was again measured over time (339 nm, measurement time 120 seconds). The blank used 0.05 ml of 0.2 M potassium phosphate buffer (pH 7.0) to correct the absorbance of the sample. The molecular extinction coefficient was set to 6620 M ⁻¹ cm ⁻¹ , and the enzyme activity was determined by [Formula 1].

[Formula 1]
Fatty acid synthase (mmol / mg) = (Absorbance change / 6620) × 1000 / Amount of protein in sample (mg)

The measurement of carnitine palmitoyltransferase activity was carried out by reacting CoA released from acyl-CoA in a carnitine-dependent manner with dithionitrobenzoic acid (DTNB), and determining the activity value by a reverse reaction in which the resulting yellow pigment was measured over time. Specifically, 0.5 ml of 116 mM Tris-HCl buffer (containing pH 8, 2.5 mM EDTA, 0.2% Triton X-100, 0.005 mM DTNB), 0.02 ml of sample, and 0.45 ml of distilled water were sequentially placed in the cell. The lid was covered with a film, the cells were mixed up and down, and mounted on a thermostatic cell holder kept at 30 ° C., and the absorbance was measured over time (412 nm, Rate Time 120 seconds). Next, 0.02 ml of 2 mM palmitoyl-CoA was added and mixed, and the absorbance was measured over time (412 nm, Rate Time 120 seconds). Further, 0.02 ml of 116 mM Tris-HCl buffer (containing pH 8, 2.5 mM EDTA, 0.2% Triton X-100, 0.005 mM DTNB) was used as a blank, and the absorbance of the sample was corrected. The molecular extinction coefficient was 13600 M ⁻¹ cm ⁻¹ and the enzyme activity was determined by [Formula 2].

[Formula 2]
Carnitine palmitoyltransferase (mmol / mg) = (Absorbance change / 13600) × 1000 / Amount of protein in sample (mg)

  Serum leptin was measured by ELISA using Morinaga leptin measurement kit (Morinaga Biochemical Laboratories). The concentration was calculated using a standard curve created using a standard solution.

  Serum insulin was measured by ELISA using Levis insulin-mouse (S type) (Shibayagi Co., Ltd.). The concentration was calculated using a standard curve created using a standard solution.

  Serum adiponectin was measured by ELISA using a mouse / rat adiponectin ELISA kit (Otsuka Pharmaceutical Co., Ltd.). The concentration was calculated using a standard curve created using a standard solution.

  For gene expression evaluation, RNA extracted from the liver was used to analyze with GeneSQUARE (registered trademark) lifestyle-related disease research mouse (Kurashiki Boseki Co., Ltd.), and DNA microarray analysis was performed. The analysis was performed with GenePix Pro 6.1.0.4 (Axon CNS). After the filter, an expression difference analysis was performed using the HF group as a control. Fold was induced at 2 or more, and less than 0.5 was suppressed. The Thea group was not analyzed.

The results are shown in FIGS.
As shown in FIG. 2, there was a tendency for the body weight to change less in the CBT1 and CBT2 groups than in the HF group. In addition, as shown in FIG. 3A, the visceral (mesenteric) peripheral fat per body weight was significantly decreased in the CBT2 group. As shown in FIG. 3 (b), perirenal fat was also decreased in the CBT2 group, but no significant difference was observed. As shown in FIG. 4, testicular fat was in a decreasing trend in the CBT1 and CBT2 groups compared to the HF group. Regarding fat accumulation, the CBT2 group showed a tendency to decrease overall.

As shown in FIG. 5, the fasting blood glucose level did not differ between the groups until the 44th day of the breeding, but the CBT1 group significantly decreased compared to the HF group on the 59th day. On day 81 of the main breeding, both the CBT1 and CBT2 groups significantly decreased. FIG. 6 is a diagram showing a ratio of hemoglobin A _1c in total hemoglobin.

  FIG. 7 (a) shows the triglyceride (TG) results for serum lipids. As shown in FIG. 7B, no significant difference was observed in the total cholesterol (TC) among the sample groups. As shown in FIG. 8, serum NEFA was significantly decreased in the CBT1 group and decreased in the CBT2 group.

  As shown in FIG. 9 (a), with respect to liver lipids, total lipids were significantly reduced in the CBT1 and CBT2 groups. As shown in FIG. 9 (b), triglyceride (TG) was significantly decreased in the CBT1 group and decreased in the CBT2 group. As shown in FIG. 10 (a), total cholesterol (TC) was significantly decreased in the CBT2 group, and a decreasing tendency was observed in the CBT1 group. As shown in FIG. 10 (b), NEFA significantly decreased in the CBT2 group, and a decreasing tendency was observed in the CBT1 group. As shown in FIG. 11, phospholipid (PL) was significantly reduced in all sample groups.

  As shown in FIG. 12 (a), regarding lipid excretion in feces, total lipids tended to be higher in the CBT2 group than in the HF group. FIG. 12 (b) shows the results of triglyceride (TG). As shown in FIG. 13A, the total cholesterol (TC) tended to be higher in the CBT2 group than in the HF group. As shown in FIG. 13B, the total bile acid tended to be higher in the CBT2 group than in the HF group.

  As shown in FIG. 14 (a), the results of fatty acid synthase activity are shown for liver lipid metabolism enzyme activity. As shown in FIG. 14 (b), carnitine palmitoyltransferase activity tended to be high in the CBT2 group.

  Regarding hormones and cytokines, as shown in FIG. 15 (a), serum leptin tended to decrease in the CBT2 group. As shown in FIG. 15 (b), insulin also tended to decrease in the CBT2 group. As shown in FIG. 16, adiponectin was not different between groups.

  Regarding gene expression analysis, as a lipid metabolism-related gene, expression of APOA2 (apolipoprotein A-II) and FABP1 (fatty acid binding protein 1, liver) was suppressed in the black tea group (FIGS. 17A and 17B). , APOC3 (apolipoprotein C-III) showed a tendency to suppress expression (FIG. 18 (a)). Moreover, GCK (glucokinase) expression is suppressed in the black tea group as a carbohydrate metabolism-related gene (FIG. 18 (b)), and expression is suppressed in PTEN (phosphate and tenthin homolog) and RBP4 (retinol binding protein 4, plasma). (Fig. 19 (a), (b)). In addition, representatively, in the black tea group, FBP1 (fructosbiphosphatase 1) was induced to induce expression, and NFKBIA (nuclear factor of kappa light polypeptide gene enhancer in B-cells was suppressed). ), (B)).

3. Rat long-term administration test After 7-week-old male GK rats were preliminarily raised for 5 days, fasting blood glucose levels and body weights were measured, and each group consisted of 5 animals in the control group (Con) and Comparative Example 1 black tea group (CBT1). Divided into 3 groups of black tea group (CBT2) of Example 1. As a control group of GK rats, 5 7-week-old male Wistar rats (Wistar) were used. The breeding period was 39 days, and the body weight and food intake during the period were measured every day. In addition, the fasting blood glucose level was measured once a week, and the blood glucose level was measured at any time during the 4th and 6th weeks of the breeding (26th and 38th days of the breeding). Further, HbA _1c was measured on the 4th and 5th weeks of the breeding (day 22 and 34 of the breeding). In addition, a glucose tolerance test was performed after fasting for 10 hours on the 4th and 6th week (main breeding days 27 and 38). Furthermore, urine was collected for 44 hours using a metabolic cage on the fifth week of the breeding (days 32 and 33 of the breeding).
The rats were bred after 12 hours of fasting after 39 days of this breeding, and blood was collected from the heart. Next, the liver, kidney, testicular fat, and visceral (mesenteric) fat were removed. Excluded organs were frozen in liquid nitrogen and stored frozen (−84 ° C.) except for those prepared immediately for measurement. The method for preparing the blood sample is shown in FIG. For TBARS, add 0.1 ml of the collected blood to 1.9 ml of 0.9% (w / v) sodium chloride solution, stir gently, and then centrifuge (3000 rpm, 15 ° C., 30 minutes) Obtained. 0.5 ml of this supernatant was transferred to a brown test tube with a stopper and stored frozen at −84 ° C. until measurement. In addition, after adding 1.9 ml of 0.9% (w / v) sodium chloride solution to the erythrocyte fraction thus obtained and stirring, it is centrifuged (3000 rpm, 15 ° C., 15 minutes) and again. An erythrocyte fraction was obtained. The same operation was repeated three times, and 2.9 ml of distilled water was added to the washed erythrocyte fraction and stirred to obtain hemolyzed erythrocytes. The hemolyzed erythrocytes were transferred to an Eppendorf tube and stored frozen at −84 ° C. until measurement.

  The feed composition is shown in Table 3. CBT1 group and CBT2 group were mixed with 80 g of casein at a ratio of 30 ml of black tea, freeze-dried casein, and fed the same composition as high fat food (black tea casein was adjusted to moisture and made 20% by weight) .

  Fasting blood glucose levels were measured on day 1, 8, 15, 22, 37, and 40 after a 12-hour fast. In the glucose tolerance test, a 40% (w / v) D-glucose solution was orally administered using a sonde so that the glucose concentration was 2 g per kg of body weight after fasting for 10 hours. The blood glucose level was measured at 15, 30, 60, and 120 minutes after administration, with 0 minute immediately before oral administration. The blood glucose level was measured using Medisafe Mini GR-102 (manufactured by Terumo Corporation).

Hemoglobin A _1c (HbA _1c ) was blood collected at 4th and 5th week (main day 22 and day 34) of this breeding, and was measured using a Micromat IIHbA _1c cartridge (Bio-Rad Laboratories). Serum triglyceride, serum NEFA and serum HDL cholesterol (HDL-C) were collected after fasting for 12 hours after meal and processed in the same manner as in “2. Mouse long-term administration test”. Serum triglyceride and serum NEDA were measured by the same method as in “2. Mouse long-term administration test”. The concentration was calculated using a standard curve created using a standard solution. Serum HDL cholesterol (HDL-C) was measured by cholesterol oxidase / DAOS method using cholesterol E-Test Wako (Wako Pure Chemical Industries, Ltd.). The arteriosclerosis index was determined by [Formula 3].

[Formula 3]
Atherosclerosis index = (TC-HDL-C) / HDL-C

  Extraction of liver lipids is described in J. Org. Folch et al. (J. Folch, M. Less, and H. S. Stanley: A simple method for the isolation of total lipids, similar tissues, J. Biol. The lipids were measured by the method described above.

  Serum lipid peroxide (serum TBARS) is obtained by following the fluorescence method (TBA method) of Yagi et al. (Kunio Yagi: Vitamin, 49, 403 (1975)) using blood collected from the heart at the time of dissection. The resulting thiobarbituric acid reactant (TBARS) was measured. That is, to 0.5 ml of the above collected blood that had been cryopreserved, 4.0 ml of 1 / 12N sulfuric acid was added and mixed, and then 0.5 ml of 10% (w / v) phosphotungstic acid was added and stirred. . After standing at room temperature for 5 minutes, the mixture was centrifuged (3000 rpm, 15 ° C., 10 minutes), the supernatant was discarded, and the remaining supernatant was blotted off with a filter paper and removed. To this precipitate, 2.0 ml of 1 / 12N sulfuric acid and 0.3 ml of 10% (w / v) phosphotungstic acid were added to sufficiently suspend the white precipitate. Further, the mixture was centrifuged (3000 rpm, 15 ° C., 10 minutes), the supernatant was removed by the method described above, 4.0 ml of distilled water was added and suspended sufficiently, and used for the subsequent operations. The standard contains 5 nmol / ml (saline solution: 0.9% (w / v) sodium chloride solution) 1,1,3,3 tetraethoxypropane solution 0.1 ml with distilled water 3.9 ml. Was used in the same manner as the sample, using 4.0 ml of distilled water as a blank. Next, 1.0 ml of TBA reagent (0.67% (w / v) 2-thiobarbituric acid solution) was added and mixed well. Then, it heated for 60 minutes in the boiling water bath (100 degreeC), and cooled for 5 minutes under running water. Subsequently, 5.0 ml of n-butanol was added and extraction was performed by shaking. This was centrifuged (2000 rpm, 15 ° C., 10 minutes), and the fluorescence of the obtained supernatant (n-butanol layer) was measured at an excitation wavelength (Ex) of 515 nm and a fluorescence wavelength (Em) of 553 nm. The TBARS value was obtained from [Formula 4].

[Formula 4]
Serum TBARS (nmol / ml of blood) = 0.5 × (f / F) × (1 / 0.1) × (2.0 / 0.5)
0.5: Standard (nmol / ml)
f / F: fluorescence value of sample / fluorescence value of standard (2.0 / 0.5): (saline solution + blood) / supernatant

  Liver peroxidized lipid (liver TBARS) was obtained by analyzing the liver extracted at the time of dissection by the method of Uchiyama and Mihara et al. -278 (1978)), and the thiobarbituric acid reactant (TBARS) produced with lipid peroxidation was measured according to the TBA method described above. That is, 0.5 g of the liver that had been cryopreserved was added 5 times as much as 0.1 M potassium phosphate buffer (containing pH 7.4, 1 mM EDTA), and homogenized in ice using a Teflon (registered trademark) homogenizer. did. Further, 2 times the amount of 2.3% (w / v) KCl was added and homogenized. This homogenate solution was used as a liver lipid peroxide measurement sample. 0.5 ml of this sample is put into a brown tube with a stopper, 1 ml (w / v) phosphoric acid 0.3 ml, TBA reagent (0.67% (w / v) 2-thiobarbituric acid solution) 0 ml was added and mixed well. Then, it heated for 45 minutes in the boiling water bath (100 degreeC), and cooled for 5 minutes under running water. Next, 4.0 ml of n-butanol was added, and the mixture was vigorously stirred and extracted. This was centrifuged (2000 rpm, 15 ° C., 10 minutes), and fluorescence of the obtained supernatant (n-butanol layer) was measured at an excitation wavelength (Ex) of 515 nm and a fluorescence wavelength (Em) of 553 nm. In addition, 10 nmol / ml (physiological saline) 1,1,3,3 tetraethoxypropane solution was used for the standard, and physiological saline was used instead of each sample for the blank, and the same operation was used. The TBARS value was obtained from [Formula 5].

[Formula 5]
Liver TBARS (nmol / ml of liver) = (f / F) × 10 × (9.0 / 0.5) × (1 / liver weight used)
f / F: fluorescence value of sample / fluorescence value of standard 10: standard substance nmol
(9.0 / 0.5): total amount of homogenate / amount of homogenate used for measurement

For the antioxidant enzyme activity in the liver, a sample was prepared using the homogenate solution prepared in the above-described liver TBARS measurement. Centrifugation (1000 rpm (150 × g), 4 ° C., 3 minutes) was performed to remove fibrous substances from the homogenate solution. The supernatant was centrifuged (3000 rpm (1400 × g), 4 ° C., 10 minutes) to remove cell debris and the like. This supernatant was sonicated in ice (50 W, 2 minutes), further centrifuged (9500 rpm (14000 × g), 4 ° C., 20 minutes), and the resulting supernatant was used for the antioxidant enzyme activity in the liver. A measurement sample was obtained. Furthermore, in order to determine enzyme activity in mg of protein, protein was quantified by the biuret method using A / GB-Test Wako (Wako Pure Chemical Industries, Ltd.).
Catalase activity is a decrease in absorbance at 240 nm based on the decomposition of H ₂ O ₂ , and ultraviolet absorption method (Naoshi Kaneda, Nobuo Ueda, “Lipid peroxide experimental method”, Medical Drug Publishing, B. Chance and A. C. Maehly: Assay of catalase and peroxidase, Vol. 2,764-775, Academic Press (1955)). Specifically, 1.0 ml of 50 mM sodium phosphate buffer (pH 7.0), 0.5 ml of 30 mM H ₂ O ₂ and 20 μl of sample were placed in the cell, covered with a parafilm, mixed up and down, and spectral photo Absorbance was measured over time with a meter (240 nm, Rag Time: 1 second, Rate Time: 15 seconds). A 50 mM sodium phosphate buffer (pH 7.0) and 30 mM H ₂ O ₂ preincubated at 25 ° C. were used. Note that 20 μl of 50 mM sodium phosphate buffer was used instead of the sample as a blank, and the absorbance of the sample was corrected. Catalase activity was expressed as enzyme activity per mg protein, expressed as 1 U of the amount of enzyme that decomposes 1 μmol of H ₂ O ₂ per minute. Catalase activity was determined by [Formula 6].

[Formula 6]
Catalase activity (U)
= Absorbance change per minute / absorbance corresponding to 1 μmol of H ₂ O ₂ Catalase activity (U / mg of protein)
= Catalase activity (U) / (Amount of protein in sample used for measurement (mg)

  Glutathione peroxidase (GSH-Px) activity was measured by measuring the activity of GSH-Px coupled with the action of GSSG-R using the reduction of NADPH when glutathione reductase (GSSG-R) acts (RA). Lawrence and R. F. Burk: Glutatione peroxidase activity in selenium defensive rat liver, Biochem. Biophys. Res. Commum., 71, 952-958 (1990) Changes in glutathione peroxidase activity and oxidized protein level, Journal of Japanese Society of Nutrition and Food, Vol. 43, 2, 121-125 (1990)). That is, 0.1 M sodium phosphate buffer (containing pH 7.0, 4 mM EDTA) 1.0 ml, 0.01 M sodium azide 0.2 ml, 0.01 M reduced glutathione (GSH) 0 preincubated in the cell at 25 ° C. .2 ml, 1.5 mM NADPH 0.2 ml, and distilled water 1.0 ml were put in order, covered with parafilm, and mixed by moving the cell up and down. Next, 0.2 ml of 0.3% (w / v) GSSG-R (yeast 200 U / mg), 20 μl of sample, and 20 μl of 70% (w / v) tert-butyl hydroperoxide were added and mixed in the same manner. The absorbance was measured over time with a spectral photometer (340 nm, Rag Time: 5 seconds, Rate Time: 20 seconds). In addition, since the decrease in NADPH due to tert-butyl hydroperoxide itself is not negligible, 20 μl of 0.1 M sodium phosphate buffer (pH 7.0, containing 4 mM EDTA) was used instead of the sample as a blank, and the absorbance of the sample was corrected. . The activity of GSH-Px is expressed as the enzyme activity per mg of protein expressed as 1 U of the amount of enzyme that decomposes 1 μmol of NADPH per minute. In addition, GSH-Px activity was calculated | required by [Formula 7].

[Formula 7]
GSH-Px activity (U) = 0.482 × Amount of change in absorbance per minute
= 0.482 x (change in absorbance of sample-absorbance of blank)
0.482: 1 μmol NADPH decrease constant = (absorbance corresponding to 1 / NADPH 1 μmol)
GSH-Px activity (U / mg of protein)
= GSH-Px activity (U) / (Amount of protein in sample used for measurement (mg)

  Serum leptin was measured by ELISA using rat leptin ELISA kit Wako (Wako Pure Chemical Industries, Ltd.). The concentration was calculated using a standard curve created using a standard solution.

  Serum insulin was measured by ELISA using Levis Insulin Kit (for rat-T) (Shiba Goat Co., Ltd.). The concentration was calculated using a standard curve created using a standard solution.

  Serum adiponectin was measured by ELISA using a mouse / rat adiponectin ELISA kit (Otsuka Pharmaceutical Co., Ltd.). The concentration was calculated using a standard curve created using a standard solution.

  Serum TNF-α was measured by ELISA using Tumor necrosis factor α rat (RPN2744) (GE Healthcare Biosciences). The concentration was calculated using a standard curve created using a standard solution.

The total amount of urine was measured with a graduated cylinder and filtered (TOYO NO. 2). Furthermore, urine subjected to ultrafiltration (8000 rpm, 4 ° C., 20 minutes) with a 1 ml weighed Amincon Ultra was used as a specimen.
Creatinine in urine was measured by the Jeff method using Creatinine-Test Wako (Wako Pure Chemical Industries, Ltd.). The concentration was calculated using a standard curve created using a standard solution.
Albumin in urine was measured by BCG method using A / GB-Test Wako (Wako Pure Chemical Industries, Ltd.). The concentration was calculated using a standard curve created using a standard solution.
Urine 8-OHdG was measured by ELISA using New 8-OHdG Check (Japan Aging Control Research Laboratories). The concentration was calculated using a standard curve created using a standard solution. Moreover, 8-OHdG per creatinine was calculated | required by [Formula 8] from 8-OHdG density | concentration.

[Formula 8]
8-OHdG (ng / creatinine) = (8-OHdG concentration (ng / ml) × urine output excreted in 44 hours (ml)) / creatinine concentration (mg / 44 hours)

  For gene expression evaluation, RNA extracted from the liver was used to analyze with a 44,000 probe Whole Rat Genome chip (Agilent Technologies), and DNA microarray analysis was performed. The analysis was performed by Gene Spring (Agilent Technologies). The differential expression analysis was performed for each of the 44000 probes and the 24452 probes after filtering, and the Con group as a control. Expression difference analysis is performed (i) statistical analysis (T-Test, p <0.01), (ii) Fold Change analysis (magnification comparison, 2 times or more) for those with significant difference in (i), The sample extracted in (ii) was determined to have an expression difference. For each probe set determined to have an expression difference, Gene Ontology analysis using Fisher's Exact Test was performed.

  The results are shown in FIGS. As shown in FIG. 22, there was a tendency for the increase and decrease in body weight to be less increased in the CBT1 and CBT2 groups than in the control group. As shown in FIG. 23 (a), there was no significant difference in the visceral (mesenteric) surrounding fat per body weight. As shown in FIG. 23 (b), the testicular fat was not different between the control group and the CBT1 and CBT2 groups, and the Wistar group showed a high value.

  As shown in FIG. 24, the fasting blood glucose level significantly decreased in the CBT2 group compared to the Con group on the 22nd day of the breeding. On the 27th and 40th day of this breeding, a low tendency was observed in the CBT1 group and the CBT2 group. On the other hand, the blood glucose level at any time significantly decreased in the CBT2 group in both measurements (FIG. 25). HbA1c showed a low value in the Wistar group at 4 and 5 weeks of breeding, but no difference was found between the CBT1 group and the CBT2 group and the Con group (FIGS. 26A and 26B). )).

  As shown in FIG. 27, in the glucose tolerance test, no difference was observed between the black tea group (CBT1 group and CBT2 group) and the Con group at the 4th and 6th week of this breeding.

  As shown in FIG. 28 (a), with regard to serum lipids, triglycerides (TG) tended to decrease compared to the CBT1 group and the CBT2 group and the Con group. As shown in FIG. 28 (b), the total cholesterol (TC) was significantly decreased in the CBT1 group and the CBT2 group as compared to the Con group, and a significant decrease was observed at the 1% level in the CBT1 group. The arteriosclerosis index was significantly higher in the Wistar group, and the CBT1 group and the CBT2 group showed a decreasing tendency compared to the Con group (FIG. 29). No significant difference was observed for HDL-C and NEFA (FIGS. 30 (a) and (b)).

  As shown in FIGS. 31 (a) and 31 (b), no significant difference was observed for liver lipids and total lipids and NEFA. As shown in FIG. 32 (a), triglyceride (TG) was significantly higher in the Wistar group, and no difference was observed between the CBT1 group and the CBT2 group and the Con group. Total cholesterol (TC) was significantly reduced in the CBT1 group compared to the Con group (FIG. 32 (b)). PL was significantly lower in the Wistar group, and there was no difference between the black tea group and the Con group (FIG. 33).

  Regarding lipid peroxide, serum TBARS was significantly reduced in the CBT2 group compared to the Con group (FIG. 34 (a)). Liver TBARS showed a downward trend in the CBT2 group compared to the Con group. (Fig. 34 (b))

  Regarding the liver antioxidant enzyme activity, as shown in FIG. 35 (a), the catalase activity tended to be higher in the CBT2 group than in the Con group. As shown in FIG. 35 (b), GSH-Px activity also showed a higher tendency in the CBT2 group than in the Con group.

  Regarding hormones and cytokines, as shown in FIG. 36 (a), serum leptin showed a significantly lower value in the Wistar group, but there was no difference between the CBT1 group and the CBT2 group and the Con group. It was. As shown in FIG. 36 (b), insulin showed a tendency to decrease in the CBT1 group and the CBT2 group as compared to the Con group. As shown in FIG. 37 (a), adiponectin tended to be higher in the CBT2 group than in the Con group. As shown in FIG. 37 (b), no difference was observed for TNF-α.

  Regarding urine protein, as shown in FIG. 38 (a), albumin was low in the Wistar group, and no difference was found between the CBT1 group and the CBT2 group and the Con group. No difference between groups was observed for creatinine and 8-OHdG (FIG. 38 (b), FIG. 39).

  Regarding gene expression analysis, 44 probes of CBT1 group showed an expression difference, and the top 6 GO terms had angiotensin type I receptor activity, bradykinin receptor activity, feeding behavior, and feeding behaviour. . For the CBT2 group, 26 probes showed differential expression, and the top 6 GO terms included extracellular ligand-gate activity, serotonin-activated activity, selective channel activity, and ligand-gate activity. , Neurotransmitter receptor activity, and postsynthetic membrane. For the Wistar group, there was a difference in the expression of the 623 probe, and the top six GO terms were retinoid metabolite, HC, diterpendoid metaproscess, antigen processing and presentation, and hormones. . There was an expression difference between the CBT1 group and the CBT2 group, and there were three overlapping ones, two of which were ESTs. The remaining one was up against the Con group and was NADH Dehydrogenase.

  From the above results, it is suggested that black tea polyphenol beverage intake suppresses the absorption of sugar when taken together with sugar, and that the effect is related to the black tea polyphenol degradation product degraded by esterase. Inferred. Therefore, it was suggested that diabetes caused by a high-fat diet can be prevented or treated by taking black tea polyphenol beverage.

Claims (9)

  A black tea beverage which is produced from a black tea extract treated with an esterase and / or after extraction from black tea leaves and contains 60 mg / 100 ml or more of polyphenols in terms of the amount of tannin, and has a blood glucose lowering effect.   The black tea beverage according to claim 1, wherein the esterase contains one or both of tannase and chlorogenic acid esterase.   The tea beverage according to claim 1 or 2, wherein the ratio of the total amount of non-gallate catechin and non-gallate type theaflavin to the total amount of gallate-type catechin and gallate-type theaflavin is 2 or more and 15 or less.   The black tea beverage according to any one of claims 1 to 3, comprising either or both of a high-intensity sweetener and a sugar alcohol.   The black tea beverage according to any one of claims 1 to 4, wherein the total black content of the black tea beverage is 60 mg / 100 ml or more and 250 mg / 100 ml or less in terms of tannin amount.   A packaged beverage obtained by filling the beverage container with the tea beverage according to any one of claims 1 to 5.   The black tea drink according to any one of claims 1 to 5, which is used for preventing or treating diabetes. Extracting a black tea extract from black tea leaves;
Treating the black tea extract with an esterase during the step of extracting the black tea extract and / or after the step of extracting the black tea extract;
Including
A method for producing a black tea beverage having a blood sugar level-lowering effect, containing polyphenol in an amount of 60 mg / 100 ml or more in terms of tannin amount.   The method for producing a black tea beverage according to claim 8, wherein the esterase contains one or both of tannase and chlorogenic acid esterase.