JP4701328B2 - Fermented tea leaves and production method thereof, fermented tea leaf extract and food and drink - Google Patents

Description

  The present invention relates to a fermented tea leaf, a fermented tea leaf extract obtained by mixing and using tea leaves and loquat leaves, and a food and drink using these.

It is known that tea has an antioxidant action, an anticancer action, a cancer prevention, a blood cholesterol lowering action, a blood pressure rise inhibiting effect and the like (for example, Non-Patent Document 1 below).
Patent Document 1 below proposes a diet food using an α-amylase inhibitor contained in an extract of guava leaves.
JP-A-7-59539 “Chemistry of Tea” edited by Keiichiro Muramatsu (Asakura Shoten) p124-191

There are a wide variety of tea ingredients and production methods, and there are many unknown parts regarding medicinal properties.
Since tea is a highly safe food and can be ingested habitually, the development of a new tea that is effective for maintaining or improving health is expected.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel fermented tea leaf, a fermented tea leaf extract having a medicinal effect effective for maintaining or improving health, and a food and drink using the same.

The present invention is a maceration machine, while maceration the leaves of tea, has a maceration mixing and maceration by adding loquat leaf, after該揉twisting step, a step of stopping the fermentation by heating immediately, the maceration step , The total twisting time from the start to the end of the tea leaf twisting is 15 to 25 minutes, and 0-40% of the total twisting time has elapsed, and then the loquat leaves are added. A manufacturing method is provided.
The present invention comprises a step of twisting tea leaves , adding loquat leaves , mixing and twisting, and heating immediately after the twisting step to stop fermentation by twisting tea leaves by the method of manually kneading , In the twisting process, the total twisting time from the start to the end of tea leaf twisting is 20 to 30 minutes, and after the time of 0 to 40% of the total twisting time has elapsed, loquat leaves are added. A method for producing fermented tea leaves is provided.
The present invention, while grinding the tea leaves, the ground step for mixing and sliding grain Mr added loquat leaf, after the grinding step, comprising the step of immediately stopping the heating and fermentation, in the grinding step, Production of fermented tea leaves , characterized in that the total mashing time from the start to the end of mashing of tea leaves is 15 to 25 minutes, and after 0 to 40% of the total mashing time has elapsed, loquat leaves are added. Provide a method .
The present invention includes a pulverization and stirring step in which loquat leaves are added and mixed and pulverized and stirred while adding water to the tea leaves and pulverized and stirred, and after the pulverization and stirring step, the temperature is 20 to 27 ° C. and the humidity is 30 to 60%. After fermentation for 4-12 hours after standing in an RH environment, heating and stopping the fermentation , in the pulverization and stirring step, total pulverization and stirring from the start to the end of tea leaf pulverization and stirring Provided is a method for producing fermented tea leaves , characterized in that the time is 5 to 15 minutes, and after 0 to 50% of the total pulverization and stirring time has elapsed, loquat leaves are added .
The present invention provides fermented tea leaves obtained by the production method of the present invention.
The present invention provides a fermented tea leaf extract obtained by extracting the fermented tea leaf of the present invention.
This invention provides the food-drinks containing the fermented tea leaf of this invention.
This invention provides the food-drinks containing the fermented tea leaf extract of this invention.
The food / beverage products of this invention are suitable as food / beverage products for suppression of an increase in blood glucose level.
The food or drink of the present invention is suitable as a food or drink for preventing or improving diabetes.
The food / beverage products of this invention are suitable as food / beverage products for neutral fat reduction.
The food / beverage products of this invention are suitable as a food / beverage product for cholesterol reduction.
The food / beverage products of this invention are suitable as food / beverage products for serum lipid peroxide reduction.
The food / beverage products of this invention are suitable as food / beverage products for body fat accumulation suppression.
The food / beverage products of this invention are suitable as food / beverage products for blood-pressure rise suppression.

  According to the present invention, health maintenance such as blood glucose level increase inhibitory effect, diabetes prevention or improvement effect, neutral fat reduction effect, cholesterol reduction effect, serum lipid peroxide reduction effect, body fat accumulation suppression effect, or blood pressure increase suppression effect -A novel fermented tea leaf having a medicinal effect effective for improvement, a fermented tea leaf extract, and a food and drink using the same.

<Fermented tea leaves>
In the present invention, the tea leaves used as a raw material are the so-called camellia evergreen shrub Thea sinensis L. Leaves. As this leaf, in addition to the first tea, second tea, third tea, autumn / winter ban tea, banban tea and the like can be used, and cheap tea leaves after the second tea containing a relatively large amount of polyphenols are preferred. The price of these late banchas is currently sluggish and a considerable amount is discarded, but this can be used effectively.
In particular, in the tea leaves of Nagasaki Higashi, which is used in the following experimental examples and analytical test examples, preferably tea leaves of Yabukita seeds, eapiafzeletin-3-O-gallate, which has been confirmed to be little in nature, is used. Proanthocyanidins that are included as constituent units are included, which is preferable. In addition, examples of tea leaves that may contain proanthocyanidins having the epiaphzeletin-3-O-gallate as a constituent unit include tea leaves used in the production of Indian Darjeeling black tea and Chinese tea.
As will be described later, proanthocyanidins having the epiafzeletin gallate as a constituent unit have high AGH inhibitory properties (maltase inhibitory properties and sucrase inhibitory properties).

  In the present invention, loquat leaves used as a raw material are leaves of the family Rosaceae and the plant name “Biwa”, and can be used without particular limitation.

The fermented tea leaves of the present invention (hereinafter sometimes referred to as mixed fermented tea leaves) are produced as follows.
First, as a pretreatment, it is preferable to dry loquat leaves and tea leaves as necessary to keep the water content at about 50 to 60% by mass. Moreover, it is preferable to cut | disconnect to an appropriate dimension, for example, a magnitude | size about 1-10 mm square.
The tea leaves are then dried, the water content is reduced, and the leaves are wilted. In this process, for example, using a roughing machine, a pot or plate-like appliance heated directly or with electricity, stirring the tea leaves and applying heated air at a temperature of 40 to 150 ° C. to the tea leaves, sealing A method in which tea leaves are put into a stirring container, the air in the container is sucked and the inside is decompressed and stirred and dried, and a method in which air is blown from below the tea leaves sprayed on the net using a wilt tank Etc. are used.
If the moisture content of the raw tea leaves is reduced to 45 to 65% by mass, preferably about 50 to 60% by mass due to this wilt, it becomes difficult for water to ooze out from the tea leaves in the next twisting step. The loss of the active ingredient can be prevented and the deterioration of quality can be suppressed. It is also preferable in that the subsequent drying step can be shortened.

Next, the raw tea leaves that have undergone the wilting process are massaged while being pressed (twisting), and loquat leaves are added at the time of twisting, and both are twisted together. Loquat leaves may be added simultaneously with the start of tea leaf twisting, or after only tea leaf twisting for a certain period of time, loquat leaves may be added and further twisted. It is preferable to add loquat leaves after 0 to 40% of the total twisting time has elapsed since the start of twisting. It is more preferable to add loquat leaves from the beginning of the twisting process.
The amount of loquat leaves added is preferably in the range of 5/95 to 35/65, more preferably 8/92 to 30/70. If the addition amount of loquat leaves is within the above range, the effect of improving the blood glucose level increase effect by mixing tea leaves and loquat leaves can be obtained satisfactorily.

For the twisting, a known method such as a method using a normal twisting machine used for twisting tea leaves can be appropriately employed.
The twisting time is 15 to 25 minutes. By setting it within this range, the blood sugar level increase suppressing effect is improved.
Moreover, the temperature of the raw material at the time of twisting shall be 20-40 degreeC. If it is less than 20 ° C, the fermentation is insufficient.

  This twisting destroys the tissue of tea leaves and loquat leaves, and oxidative enzymes such as polyphenol oxidase contained in tea leaves oxidize and polymerize polyphenols contained in tea leaves and loquat leaves, producing an oxidized polymer. To do.

Next, the mixed raw material in this state is transferred to the fermentation process. That is, in a state where the mixed raw material after twisting is deposited to a thickness of several centimeters, the mixture is left in an environment such as a fermentation chamber at a temperature of 20 to 27 ° C. and a humidity of 30 to 60% RH. “Fermentation” in the tea production process means an oxidation reaction by oxidase in leaves.
The fermentation time is 0 to 4 hours. Here, the fermentation time of 0 hours means that in the previous twisting process, fermentation starts simultaneously with the start of twisting, and when the time of the twisting process is not included in the fermentation process, the fermentation time is 0. It can also be called time, and the substance is one that is fermented even at 0 hours. When fermentation time exceeds 4 hours, there exists a tendency for the taste and aroma of the obtained mixed fermented tea leaves or its extract to fall. In order to obtain particularly excellent AGH inhibition, the fermentation time is preferably 1 hour or less, and most preferably 0 hour.

Next, when a predetermined fermentation time has elapsed, the raw material is heated to stop the fermentation and dried. For example, raw materials are put into a continuous dryer, hot air having a temperature of 80 to 120 ° C. is blown into the continuous dryer, and the exhaust temperature is controlled to be 50 to 60 ° C. A heating time of about 10 to 30 minutes is sufficient, so that the water content in the raw material is about 5% by mass.
In the above production process, AGH inhibition becomes lower as the twisting time and fermentation time become longer. Further, the antioxidant action (1,1-dipicryl-2-phenylhydrazyl (DPPH) scavenging activity) is also lowered. If production is performed with attention paid to functionality, it is ideal to set the twisting time to 15 to 25 minutes and to shorten the fermentation time as much as possible.
In addition, the longer the fermentation time, the stronger the bitterness and the worse the aroma, and the shorter the fermentation time, the better the taste and aroma.

  In this way, mixed fermented tea leaves are obtained. What is after a heating process is what is called rough tea, and it is good also as finishing tea by giving finishing processing to this as needed. For the finishing process, steps similar to the finishing process in conventional tea leaf production, such as re-drying, burning, sieving, shaping, and sorting, can be appropriately employed. The above-mentioned burning is a process of reheating to improve the flavor. By performing the finishing process, quality such as storability and fragrance can be improved and merchantability can be improved. Moreover, you may make rough tea or finished tea into the powder form of a suitable magnitude | size.

In addition, as described above, the twisting process only needs to destroy the tissue of tea leaves and loquat leaves. In addition to the method using a twisting machine, for example, a method of squeezing by hand, a method of grinding, a method of pulverizing by adding water Etc. can be used.
In the case of using a method of massaging by hand, a method usually used for massaging tea leaves can be appropriately used. For example, a method of rubbing by hand on an uneven rug knitted with a rough rope called “Nekobuki” can be adopted, and other methods may be used. Specifically, the raw tea leaves that have undergone the wilting process are twisted by hand, and loquat leaves are added at the time of twisting, and both are twisted together. The twisting time in this case is preferably 20 to 30 minutes, and if it is less than 20 minutes or more than 30 minutes, the effect of inhibiting the increase in blood sugar level of the obtained fermented tea becomes low. Other conditions are the same as above. The fermentation process can be performed in the same manner as described above.
In this method, AGH inhibition becomes lower as the twisting time and fermentation time become longer. Further, the antioxidant action (DPPH scavenging activity) is also lowered. If production is performed with attention paid to functionality, it is ideal that the twisting time is 20 to 30 minutes and the fermentation time is as short as possible.

In the case of using the mashing method, a method using a normal mortar used for mashing tea leaves can be employed, and other mashing methods may be used. Specifically, the raw tea leaves that have undergone the wilting process are ground, and loquat leaves are added at the time of this rubbing, and both raw materials are rubbed. In this case, the grinding time is preferably 15 to 25 minutes, and if it is less than 15 minutes or more than 25 minutes, the increase in blood sugar level of the obtained fermented tea becomes low. The temperature of the raw material during the grinding time is 20 to 40 ° C. If it is less than 20 degreeC, it will become inadequate fermentation, and if it exceeds 40 degreeC, it will become a quality fall. Furthermore, the timing of adding loquat leaves may be added simultaneously with the start of grinding of tea leaves, or after grinding of only tea leaves for a certain period of time, loquat leaves may be added and further ground. The loquat leaf can be added after 0 to 40% of the total grinding time has elapsed from the start of grinding. Other conditions are the same as above.
By grinding in this way, the raw tissue of tea leaves and loquat leaves is destroyed, and oxidative enzymes such as polyphenol oxidase contained in tea leaves oxidize and polymerize polyphenols contained in tea leaves and loquat leaves. Things are generated. The fermentation process can be performed in the same manner as described above.
In this method, AGH inhibitory property becomes low, so that grinding time and fermentation time become long. Further, the antioxidant action (DPPH scavenging activity) is also lowered. If production is performed with attention paid to functionality, it is ideal that the grinding time is 15 to 25 minutes and the fermentation time is as short as possible.
In addition, the longer the fermentation time, the stronger the bitterness and the worse the aroma, and the shorter the fermentation time, the better the taste and aroma.

In the case of using a method of pulverizing by adding water, by adding water to the tea leaves and loquat leaves and pulverizing and stirring them, the tea leaves and loquat leaf raw materials can be destroyed. For the pulverization and stirring, a method using an ordinary mixer or the like used for pulverizing and stirring tea leaves can be employed, and other pulverization and stirring methods may be used. Specifically, water is added to the raw tea leaves and loquat leaves that have undergone the wilting step, followed by pulverization and stirring. The mixing ratio of tea leaves and loquat leaves is the same as above. The amount of water added is preferably 20 to 200 parts by weight, more preferably 50 to 150 parts by weight, based on a total of 100 parts by weight of tea leaves and loquat leaves.
The pulverization stirring time is 5 to 15 minutes, and when the pulverization stirring time is less than 5 minutes or exceeds 15 minutes, the increase in blood sugar level of the obtained fermented tea becomes low. The temperature of the raw material at the time of pulverization and stirring is the same as described above, and is set to 20 to 40 ° C.
The timing of adding loquat leaves may be added simultaneously with the start of pulverization stirring, or after pulverizing and stirring only tea leaves for a certain period of time, loquat leaves may be added and further pulverized and stirred. It is preferable to add loquat leaves after 0 to 50% of the total pulverization stirring time has elapsed since the start of pulverization stirring.
By crushing and stirring in this way, tea leaves and loquat leaves are mixed, and the tissue of these raw materials is destroyed, and oxidative enzymes such as polyphenol oxidase contained in tea leaves are polyphenols contained in tea leaves and loquat leaves. Is oxidized and polymerized to produce an oxidized polymer.
Next, in the step of fermenting the mixed raw material obtained by pulverization and stirring, the mixed raw material after pulverization and stirring is deposited to a thickness of several centimeters, such as in a fermentation chamber at a temperature of 20 to 27 ° C. and a humidity of 30 to 60% RH. Leave in the environment. The fermentation time is preferably 4 to 12 hours. If the fermentation time is less than 4 hours, since the fermentation has not progressed, the blood glucose level increase-inhibiting action is low, and the aroma also has a blue odor. When fermentation time exceeds 12 hours, there exists a tendency for the taste and aroma of the obtained fermented tea to fall. In the case where the medicinal effect is emphasized, 4 to 12 hours are preferable. That is, fermentation time of 4 to 12 hours is particularly high in AGH inhibition. In addition, the antioxidant action (DPPH scavenging activity) also increases. If production is performed with attention paid to functionality, it is ideal to keep the pulverization stirring time at 10 to 15 minutes and the fermentation time at 4 to 12 hours. In addition, outside this time, the bitterness and taste become worse with respect to taste.
Then, when a predetermined fermentation time has elapsed, the raw material is heated to stop the fermentation and dried. For example, what poured the raw material into the container is put into a dryer, and hot air with a temperature of 80 to 120 ° C. is blown into the container, and the exhaust temperature is set to 50 to 60 ° C. The heating time is preferably about 60 to 120 minutes, so that the water content in the raw material is about 5%. Thus, the fermented tea of the present invention is obtained, but the product after the heating step may be pulverized or used as a solid without being pulverized.

As a component contained in the mixed fermented tea leaves obtained in this way, the component isolate | separated by the below-mentioned analytical test example is mentioned.
Although the whole picture about the active ingredient which contributes to the medicinal effect played by the mixed fermented tea leaves of the present invention is not yet clear, each component of the following (1) to (4) is based on the following experimental examples and analytical test examples. It has been found to exhibit high AGH inhibition (maltase inhibition and sucrase inhibition).
(1) Theacinensin A (hereinafter sometimes abbreviated as TS-A),
(2) a theaflavin derivative having a galloyl group,
(3) Proanthocyanidins having epiafuzeretin gallate as a constituent unit;
(4) Oxidative condensation of catechin gallates having a catechin A ring-derived phloroglucinol-derived signal and a galloyl group-derived signal in a ¹³ C-NMR spectrum, wherein the molecular weight of the acetylated compound is 1000-15000 with a maximum of 2000. Polyphenol (hereinafter sometimes abbreviated as polyphenol P).

(1) Theasinensin A is a compound having the structure shown in FIG.
(2) Theaflavin derivatives having a galloyl group are specifically theaflavin-3-O-gallate (3-TFG), theaflavin-3′-O-gallate (3′-TFG), theaflavin-3,3 ′. -It is 1 or more types chosen from di-O-gallate (3,3'-TFGG). These structures are shown in FIG. Among these, in particular, theaflavin-3,3′-di-O-gallate has stronger AGH inhibitory properties.
(3) Proanthocyanidins having epiafzeletin gallate as a constituent unit are those described in Fr. 7 and by thiol decomposition with mercaptoethanol, epicatechin (EC), epigallocatechin (EGC), epigallocatechin-3-O-gallate (EGCg), and epicatechin-3-O-gallate ( One or more selected from the group consisting of ECg (hereinafter sometimes referred to as ECG) and EGC-ME (hereinafter “-ME” represents “4- (2′-hydroxyethylthio) ether”). For example, EGC-ME is EGC 4- (2′-hydroxyethylthio) ether, the same applies hereinafter), one selected from the group consisting of EC-ME, EGCg-ME, and ECg-ME. As a result, Epiafzeletin-3-O-gallate-ME is produced. The structure of proanthocyanidins before thiol decomposition is presumed to be as shown in FIG. 4, for example. In addition, the epiafzeletin gallate in (3) component points out an epiafzeletin-3-O-gallate.
This component (3) is derived from the tea leaves of the raw material, and as a raw tea leaf, (3) mixed fermentation produced using tea leaves containing proanthocyanidins having epiafzeletin gallate as a constituent unit Included in tea leaves.

  The above (4) polyphenol P is extracted by, for example, extracting the fermented tea leaves containing this with room temperature extracted with 60% ethanol (60% by volume ethanol aqueous solution, hereinafter the same) as shown in Analytical Test Example 5 described later. The solution was sequentially partitioned with ethyl ether, ethyl acetate, and n-butanol, and the n-butanol soluble part dissolved in 60% methanol was added to a Sephadex LH-20 column using 60% methanol, 80% methanol, 100%. Eluate sequentially eluted with 1% methanol, then methanol-water-acetone (8: 1: 1) mixed solution, methanol-water-acetone (6: 2: 2) mixed solution, water-acetone (1: 1) mixed solution Was analyzed by thin layer chromatography (toluene-ethyl formate-formic acid, 1: 7: 1) and fractions containing material that did not migrate from the origin Only it is obtained collected and concentrated to.

  In addition, the mixed fermented tea leaves of the present invention are characterized by containing loquat proanthocyanidins. As shown in Analytical Test Example 3 described later, biwaproanthocyanidin has the highest AGH-inhibiting fraction (Fr. 7) among a plurality of fractions obtained from the mixed fermented tea leaf extract having a high AGH-inhibiting property. It is considered that it contributes to AGH inhibition.

Moreover, the mixed fermented tea leaves of the present invention can be obtained by adding loquat leaves. TFG) and theaflavin-3,3′-di-O-gallate (3,3′-TFGG)) are also promoted.
That is, the total content of theaflavins contained in the mixed fermented tea leaves is greater than the total content of theaflavins contained in the single fermented tea leaves of tea leaves produced under the same conditions except that no loquat leaves are added, For example, it is 1.5 times or more, preferably 2 times or more, more preferably 2.5 times or more. The upper limit is not limited, but is presumed to be no more than 4 times.
For example, in Analytical Test Example 4 and FIG. 7 (results of high performance liquid chromatography (hereinafter sometimes abbreviated as HPLC) analysis of ethyl acetate soluble fraction of fermented tea leaf extract), the mixed fermented tea leaf extract is used alone. Compared with the fermented tea leaf extract, the total peak area of theaflavins increased by about 2.7 times, indicating that the addition of loquat tea greatly promoted the production of theaflavins.

The mixed fermented tea leaves of the present invention are also characterized in that the production of the component (4) (polyphenol P) is promoted by adding loquat leaves.
That is, the content of polyphenol P contained in the mixed fermented tea leaves is greater than the content of polyphenol P contained in the single fermented tea leaves of tea leaves produced under the same conditions except that no loquat leaves are added. For example, it is 1.2 times or more, preferably 2 times or more, more preferably 3 times or more. The upper limit is not limited, but is presumed to be no more than 4 times.
For example, in Analytical Test Example 4 and FIG. 6 (HPLC analysis result of water-soluble fraction of fermented tea leaf extract) described later, the mixed fermented tea leaf extract is detected as a rise in the baseline as compared with the single fermented tea leaf extract. The peak area of the substance has increased by about 1.59 times (159%), and this increase is considered to be due to the catechin oxidation product whose generation was promoted by the addition of loquat tea. This catechin oxidation product corresponds to the component specified as polyphenol P in Analytical Test Example 5.

The mixed fermented tea leaves of the present invention are composed of 1-hexanol, trans-2-hexenal, 3-hexen-1-ol acetate, 3-hexen-1-ol, linalool, benzaldehyde, methyl salicylate, geraniol, and nerolidol. In the solid-phase microextraction method, trans-2-hexenal, 3-hexen-1-ol acetate, 3-hexen-1-ol, linalool, benzaldehyde, methyl salicylate, geraniol, nerolidol Each peak area has a characteristic fragrance component composition that all are larger than the peak area of 1-hexanol.
Among the aromatic components listed above, as shown in Analytical Test Example 10 described later, linalool, benzaldehyde, methyl salicylate, geraniol, and nerolidol are also relatively contained in fermented teas such as black tea, It is a small aroma component in unfermented tea such as green tea and fermented loquat leaves. Moreover, trans-2-hexenal, 3-hexen-1-ol acetate, and 3-hexen-1-ol are aromatic components that are relatively contained in fermented loquat leaves. The mixed fermented tea leaf of the present invention has a relatively large content of these aroma components, so that a unique scent as a fermented tea can be tasted.

  The mixed fermented tea leaves of the present invention show high AGH inhibitory activity as shown in the experimental examples described later, and by ingesting this, an action to suppress an increase in blood glucose level can be obtained, preventing or improving diabetes, neutrality A fat reduction effect, a cholesterol reduction effect, a serum lipid peroxide reduction effect, a body fat accumulation suppression effect, or a blood pressure increase suppression effect can be obtained. Preferably, two or more of these effects can be obtained simultaneously, and all the effects can be obtained simultaneously.

<Fermented tea leaf extract>
The fermented tea leaf extract of the present invention (hereinafter sometimes referred to as mixed fermented tea leaf extract) is an extract obtained by extracting soluble components of fermented tea leaves with an extraction solvent. The extraction solvent may be water (including warm water or hot water) or an organic solvent. Specific examples of the organic solvent include methanol, ethanol, acetone and the like. The form of the fermented tea leaf extract as the blood glucose level elevation suppressing composition of the present invention is not particularly limited, and may be in the form of a solution, a concentrate thereof, or a solid dried by freeze drying or the like. In the case of a solid, it may be a lump or may be a powder obtained by pulverizing it to an appropriate size. When the fermented tea leaf extract is used in a food or drink in a form containing an organic solvent, ethanol is used as the organic solvent.

Extraction conditions are not particularly limited, but when extracting with water, it is preferable to extract by immersing the fermented tea leaves in warm water or hot water at a temperature of 40 to 100 ° C. for about 3 to 60 minutes. The ratio of water and fermented tea leaves is preferably about 0.5 to 20 parts by mass, more preferably about 0.5 to 5 parts by mass, with respect to 100 parts by mass of water.
In the case of extracting with an organic solvent, about 0.1 to 20 parts by mass of fermented tea leaves (water content 5% by mass) with respect to 100 parts by mass of the organic solvent, at a normal pressure or under pressure, a temperature of -20 to 60 ° C, Although it is preferable to carry out on the conditions for time 1-60 minutes, it is not limited to this range, It can change suitably.
After the extraction treatment, the solid content is removed by filtration, centrifugation, etc. to obtain an extract.

  The mixed fermented tea leaf extract contains soluble components of the mixed fermented tea leaf and exhibits high AGH inhibitory activity as shown in the experimental examples and analytical test examples described below. In addition, as shown in the experimental examples and analytical test examples described later, by ingesting the mixed fermented tea leaf extract, blood glucose level increase inhibitory effect, diabetes prevention or improvement effect, neutral fat reduction effect, cholesterol reduction effect, serum A lipid peroxide reduction effect, a body fat accumulation suppression effect, or a blood pressure increase suppression effect is obtained. Preferably, two or more of these effects can be obtained simultaneously, and all the effects can be obtained simultaneously.

The food or drink of the present invention is not particularly limited as long as it contains mixed fermented tea leaves, or contains mixed fermented tea leaf extracts and can be taken orally. You may contain both mixed fermented tea leaves and mixed fermented tea leaf extracts.
The food / beverage products of this invention are the various drinks containing a mixed fermented tea leaf extract, for example. Further, if the mixed fermented tea leaf extract is obtained by extracting the mixed fermented tea leaf with water and has a flavor that can be drunk as it is, the extract can be used as a tea beverage.
Or it is the foodstuff which added mixed fermented tea leaf and / or mixed fermented tea leaf extract to various food materials. There are no particular restrictions on the food material, soy foods such as natto, soy milk, miso and soy sauce, kneaded products such as hampen, kamaboko and chikuwa, processed meat products such as ham and sausage, rice cakes, caramel, in the middle, and sheep candy A wide variety of confectionery such as.

The content of the mixed fermented tea leaf and / or the mixed fermented tea leaf extract in the food and drink of the present invention is arbitrary, and can be appropriately set according to the degree of medicinal effect to be obtained and the expected flavor and intake. it can.
For example, in the case of foods and drinks for suppressing blood sugar level elevation, the effect of inhibiting blood sugar level increase to be obtained and expected based on the AGH inhibitory properties (for example, IC ₅₀ ) of each of the mixed fermented tea leaves and mixed fermented tea leaf extracts to be added are expected. Can be set as appropriate according to the flavor and intake.

When the extract obtained by extracting fermented tea leaves with water is used as it is as a tea beverage, the ratio of water and fermented tea leaves is 0.5 to 20 parts by mass of fermented tea leaves (water content 5% by mass) with respect to 100 parts by mass of water. The degree is preferred. The extraction method is preferably a method of immersing in warm water or hot water at a temperature of 40 to 100 ° C. for 3 to 60 minutes.
When adding fermented tea leaves to a food material, for example, it can be appropriately determined within a range of 0.1 to 200 parts by mass of fermented tea (water content 5% by mass) with respect to 100 parts by mass of the food material.
Such a food or drink of the present invention contains mixed fermented tea leaves and / or mixed fermented tea leaf extracts, and by ingesting them, the effect of suppressing blood sugar level elevation, the effect of preventing or improving diabetes, the effect of reducing neutral fat, A cholesterol reduction effect, a serum lipid peroxide reduction effect, a body fat accumulation suppression effect, or a blood pressure increase suppression effect is obtained. Preferably, two or more of these effects can be obtained simultaneously, and all the effects can be obtained simultaneously.

In the following experimental examples and analytical test examples, “%” is “% by mass” unless otherwise specified.
The raw materials “tea leaves” used in the following experimental examples and analytical test examples are the leaves of the third tea of Yabukita cultivated at Nagasaki Prefectural Agricultural and Forestry Experiment Station Higashisonobe Tea Leaf Branch. Further, unless otherwise specified, twisting was performed using a twisting machine.
The evaluation of AGH inhibition in Example 1 and Analytical Test Examples 1 to 5 is based on the method using free AGH by Matsui et al. (J. Agric. Food Chem., 47, 550-553 (1999)). The measurement results are expressed as an inhibition rate (unit:%) of α-glucosidase (AGH) enzyme activity.

[Experimental Example 1]
(Production of mixed fermented tea leaves)
The loquat leaves were put into the tea leaves and swallowed with a twister. The blending ratio of tea leaves and loquat leaves was set to two mass ratios of tea leaf: lowa tea: 90:10 (10% loquat input ratio) and 75:25 (25% loquat input ratio). The time for squeezing was set at 2 minutes for 20 minutes and 40 minutes.
Thereafter, 24 types of mixed fermented tea leaves were obtained through 6 fermentation processes of 0 hour, 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours. In addition, the thing of fermentation process 0 hours is the mixed fermented tea leaves obtained immediately after the end of the mastication.

(Sensory examination)
Three panelists with sensory review experience of green tea examined the flavor and taste of the 24 types of mixed fermented tea leaves obtained above according to the green tea screening method. In the examination, 3 g of mixed fermented tea leaves were poured into the examination tea bowl, 180 ml of hot water was poured into it, and immediately after pouring hot water into the fragrance, the taste was examined after 5 minutes. The results are shown in Table 1.

  As shown in the results of Table 1, regarding the taste and aroma, the twisting time is 20 minutes, the fermentation time is 0 to 4 hours, the aroma is high, and the aftertaste is refreshing and excellent. When the twisting time is 40 minutes, the taste is lowered, and when the fermentation time is 6 hours or more, the deterioration of the fragrance becomes remarkable.

(Measurement of AGH activity)
Of the 24 types of mixed fermented tea leaves obtained above, 16 types excluding those with fermentation times of 6 hours and 24 hours were extracted with hot water at 100 ° C. for 10 minutes (the amount of mixed fermented tea leaves used: 2 0 mg / ml) was measured. The results are shown in Table 2.
In addition, AGH inhibition was measured in the same manner for extracts obtained by similarly extracting commercially available green tea, loquat tea, guava tea, and black tea with hot water. The results are shown in Table 3.

As shown in Table 2, the AGH inhibitory effect is lowered when the blending ratio of loquat leaves is increased with respect to tea leaves, and becomes low even when the twisting time is long. Moreover, there exists a tendency for an inhibition rate to become low as fermentation time becomes long.
In addition, from the results of Tables 2 and 3, in particular, the mixing ratio of loquat leaf 10% by mass, twisting time 20 minutes, fermentation time 0 to 1 hour, and the mixing ratio of loquat leaf 25% by mass, twisting time 20 minutes, fermentation time 0 hour. Under the conditions, AGH inhibition superior to that of green tea was obtained.

[Analysis Test Example 1]
In Table 2, 70% acetone (acetone with a concentration of 70% by volume) mixed fermented tea leaves that showed excellent AGH inhibition (tea leaf to loquat blending ratio 9: 1, twisting time 20 minutes, fermentation time 0 hours) After extraction at room temperature with an aqueous solution, the same shall apply hereinafter), the extract was concentrated to an aqueous solution, ether was added, and the solvent was partitioned to remove fat-soluble components such as chlorophyll. The results of analyzing the obtained aqueous layer by high performance liquid chromatography (HPLC) are shown in FIG.
As shown in FIG. 1, caffeine, epicatechin (EC), epigallocatechin (EGC), epigallocatechin-3-O-gallate (EGCg), epicatechin-3-O-gallate (green tea components) ECa), theasinensin C, theasinensin E, theasinensin A, theasinensin D (overlapping EGCg), theaflavins (theaflavin (TF), theaflavin-) which are dimers formed by oxidation of these catechins (Including 3-O-gallate (3-TFG), theaflavin-3′-O-gallate (3′-TFG), theaflavin-3,3′-di-O-gallate (3,3′-TFGG)) The peak was detected. In addition, the presence of catechin oxidation products detected as a rise in baseline was also observed.

[Analysis Test Example 2]
In Table 2, dry fermented tea leaves obtained by drying mixed fermented tea leaves (tea leaf to loquat blending ratio 9: 1, twisting time 20 minutes, fermentation time 0 hours) that showed excellent AGH inhibitory properties are shown in FIG. Fractionation was performed according to the procedure shown.
<Operation (1)> 125 g of dried fermented tea leaves (water content 5% by mass) were crushed together with 2 L of 70% acetone in a crusher (product name; Waring Blender 7011S type, manufactured by FMI) and filtered. . The residue was extracted with 3 L of 70% acetone to obtain an acetone extract, and then the residue was extracted with 3 L of methanol to obtain a methanol extract. The filtrate obtained by the filtration, the acetone extract, and the methanol extract were combined, concentrated by a rotary evaporator and dried to obtain 59.9 g of extract (dried product).
<Operation (2)> The obtained extract was suspended in water, ether was added and the solvent was partitioned to remove fat-soluble components such as chlorophyll.
<Operation (3)> The aqueous layer obtained in operation (2) is poured into a Sephadex LH-20 column (manufactured by Pharmacia Fine Chemical Co., 4 cm × 28 cm) prepared with water, and water (500 mL), 40% methanol (200 mL). ), 60% methanol (300 mL), 80% methanol (300 mL), methanol (300 mL), and 60% acetone (500 mL).
The eluate was concentrated to fraction Fr. 1 (18.66 g), Fr. 2 (5.79 g), Fr. 3 (3.12 g), Fr. 4 (4.87 g), Fr. 5 (6.01 g), Fr. 6 (3.55 g), Fr. Fractionated to 7 (2.07 g).
When AGH inhibitory activity was measured for each fraction, as shown in Table 4 below, Fr. 6 and 7 showed very high inhibition rates.

Therefore, Fr. 6 and 7 were further separated and purified by the operation shown in FIG.
<Operation (4)> Fr. 6 and Fr. Each of 7 was attached to MCIgel CHP20P (Mitsubishi Chemical Co., Ltd., 3 cm × 28 cm), 20% methanol (100 mL), 30% methanol (100 mL), 40% methanol (100 mL), 50% methanol (100 mL), 60% Elution was successively performed with methanol (100 mL), 70% methanol (100 mL), 80% methanol (100 mL), 90% methanol (100 mL), and methanol (200 mL).
The obtained eluate was concentrated to obtain Fr. 6 to Fr. 6-1 (899 mg), Fr. 6-2 (2.16 g), Fr. A fraction of 6-3 (282 mg) was obtained. In addition, Fr. 7 to Fr. 7-1 (67 mg), Fr. 7-2 (1.17 g), Fr. A fraction of 7-3 (600 mg) was obtained.
Of these, Fr. 6-1 and Fr. 7-1 measured hydrogen nuclear magnetic resonance (< ¹ > H-NMR) spectrum, and compared with the spectrum of the sample, it identified as theasinensin A and theasinensin D, respectively. Fr. 6-3 and Fr. 7-3 was identified as theaflavins from the results of HPLC and thin layer chromatography.

<Operation (5)> Fr. 6-2 and Fr. 7-2 was added to Chromatorex ODS (manufactured by Fuji Silysia Ltd., 3 cm × 30 cm) and 10% methanol (100 mL), 20% methanol (100 mL), 30% methanol (100 mL), 40% methanol ( 100 mL), 50% methanol (100 mL), 60% methanol (100 mL), and methanol (200 mL) were eluted successively.
The obtained eluate was concentrated to obtain Fr. 721 (179 mg), Fr. 722 (1.10 g), Fr. 723 (530 mg), Fr. Fractionated to 724 (1.27 g).
Of these, Fr. 721 measured a ¹ H-NMR spectrum and was identified as a mixture of EGCg and theacinensin A.

<Operation (6)> Fr. A combination of 722 and 723 was poured into a Sephadex LH-20 column (3 cm × 20 cm) and eluted sequentially with ethanol (500 mL), 90% ethanol (200 mL), 20% methanol (300 mL), and 60% acetone (400 mL). .
The obtained eluate was concentrated to obtain Fr. 7221 (3.7 mg), Fr. 7222 (41.3 mg), Fr. 7223 (47.0 mg), Fr. 7224 (166.2 mg), Fr. 7225 (458 mg), Fr. Fractionated to 7226 (657.5 mg).
Of these, Fr. 7221, Fr. 7222, Fr. 7223 and Fr. 7224 was identified as gallic acid, ECg, EGCg, and epigallocatechin-3,3 ′ (4 ′)-O-gallate by measuring the ¹ H-NMR spectrum and comparing it with the spectrum of the sample.

<Operation (7)> Fr. 7225 is attached to Diaion HP20SS (Mitsubishi Chemical Co., Ltd., 2 cm × 20 cm), 20% methanol (100 mL), 25% methanol (100 mL), 30% methanol (100 mL), 35% methanol (100 mL), 40% methanol ( 100 mL), 45% methanol (100 mL), 50% methanol (100 mL), 60% methanol (100 mL), and methanol (200 mL) were sequentially eluted.
The obtained eluate was concentrated to obtain Fr. 72251 (39.6 mg), Fr. 72252 (320 mg), Fr. Fractionated to 72253 (47.7 mg).
Of these, Fr. 72251 was identified as oolong theanine by measuring the ¹ H-NMR spectrum and comparing it with the spectrum of the sample.

<Operation (8)> Fr. Compared to Chromatorex ODS (manufactured by Fuji Silysia, 2 cm × 20 cm), 72252 is 10% methanol (100 mL), 20% methanol (100 mL), 30% methanol (100 mL), 40% methanol (100 mL), 50% methanol. (100 mL), 60% methanol (100 mL), and methanol (200 mL) were eluted successively.
The obtained eluate was concentrated to obtain Fr. 722521 (10.5 mg), Fr. 722522 (38.3 g), Fr. Fractionated to 722523 (60 mg).
Of these, Fr. 722521 measured ¹ H-NMR spectrum and identified as epigallocatechin-3,3 ′ (4 ′)-O-gallate.

<Operation (9)> Fr. 722523 was poured into a Sephadex LH-20 column (1.5 cm × 20 cm) and eluted sequentially with 80% methanol (200 mL), 90% methanol (200 mL), methanol (200 mL), and 60% acetone (200 mL).
The obtained eluate was concentrated to obtain Fr. 7225231 (14.4 mg), Fr. 7225232 (16.1 mg), Fr. Fractionated to 7225233 (15.5 mg).
Of these, Fr. 7225232 was identified as galloyl oolong theanine by measuring the ¹ H-NMR spectrum and comparing it with the spectrum of the sample.

From these results, Fr. The following fractions of 6 and 7 were found to contain the following compounds, respectively. The structure of the following compound is shown in FIG.
Fr. 6-1: Theacinensin A (TS-A),
Fr. 6-3, 7-3: Theaflavin (TF) mixture (including theaflavin, theaflavin-3-O-gallate, theaflavin-3′-O-gallate, theaflavin-3,3′-di-O-gallate),
Fr. 7-1: Teasinensin D (TS-D),
Fr. 7221: gallic acid,
Fr. 7222: Epicatechin-3-O-gallate (ECg),
Fr. 7223: Epigallocatechin-3-O-gallate (EGCg),
Fr. 7224 and Fr. 722521: Epigallocatechin (EGC) -3,3 '(4')-di-O-gallate,
Fr. 72251: Oolong theanine,
Fr. 7225232: Galloyl oolong theanine.

  Moreover, the result of having quantified content of each said compound is shown in FIG. From this result, Fr. The main component of 6 was found to be theacinensin A. In addition, Fr. The main component of 7 was found to be a theaflavin (TF) mixture. The above-mentioned theaflavins contained in the theaflavin mixture were each measured for AGH inhibitory activity, and particularly theaflavin-3,3'-di-O-gallate showed strong enzyme inhibitory activity. This can be seen from the results of analytical test examples 5 and 6 described later.

[Analysis Test Example 3]
In the above-mentioned analytical test example 2, Fr. The results of HPLC analysis for 7 are shown in FIG. 4A. The analysis conditions are
Column: Cosmosil 5C ₁₈ ARII (manufactured by Nacalai Tesque, 4.6 × 250 mm),
Column temperature: 35 ° C.
Mobile phase: A; 50 mM phosphoric acid, B; CH ₃ CN, B 4% to 30% (39 minutes), 30% to 75% (15 minutes),
Flow rate: 0.8 ml / min,
Detection: Photodiode array detection (Max absorption).
(The HPLC analysis conditions in the following analytical test examples are the same as those in this test example unless otherwise specified.)
From the result of this figure, Fr. In addition to the theaflavin mixture and the above compound found to be present in Analytical Test Example 2 in No. 7, there are many substances that are detected as the origin in thin layer chromatography (TLC) analysis and detected as a bulge in the baseline in HPLC analysis. It became clear that many were included. Such materials are mainly Fr. It was fractionated to 724.
Such a substance is similar to that of EGCg in behavior and UV-visible absorption spectrum in various column chromatograms, and positive for ferric chloride reagent and vanillin hydrochloric acid reagent. It is presumed to be a catechin oxidation product having a molecular weight higher than that of catechins whose existence has been found.
Typically, proanthocyanidins are typical of high-molecular polyphenols contained in tea, and loquat leaves are known to contain proanthocyanidins (biwaproanthocyanidins) containing epicatechin as constituent units, Fr. It is presumed that proanthocyanidins are contained in the “catechin oxidation product having a large molecular weight” in No. 7.

Proanthocyanidins are dimers or polymers having catechins or catechin derivatives as constituent units, and the catechins or catechin derivatives constituting the catechins or catechin derivatives are carbons at the 4-position (C-4) and carbons at the 8-position (C-8). ), Or carbon (C-4) at the 4-position and carbon (C-6) at the 6-position are carbon-carbon bonded.
Therefore, the Fr. For the purpose of clarifying the constituent units of proanthocyanidins contained in Fr. 7 was subjected to thiol decomposition with mercaptoethanol by the following method.
That is, Fr. No. 7 60% ethanol solution (concentration of Fr. 7; 10 mg / mL) mixed with mercaptoethanol test solution (mercaptoethanol 2.5 mL, 0.1% hydrochloric acid 4 mL, ethanol 27.5 mL, water 16 mL) ) 1.8 mL was added and heated at 60 ° C. for 6 hours, and 20 μL of the reaction solution was analyzed by HPLC. The analysis conditions were the same as when obtaining FIG. 4A. The chromatogram after thiol decomposition obtained in this way is shown in FIG. 4B.

  As shown in FIG. 4B, Fr. When 7 is subjected to thiol decomposition, many peaks that are not recognized before decomposition (FIG. 4A) appear. Retention time and UV of the standard (Tanaka, T. et. Al., J. Chem. Soc. Perkin Trans 1, 1994, 3013) for peaks that were not observed before decomposition and peaks that were higher than before decomposition. As a result of comparison with the absorption spectrum, EGC, EC, EGCg, ECg, EGC-ME, EC-ME, EGCg-ME, and ECg-ME were identified.

In FIG. 4B, the peak detected at 39 minutes is Fr. 7 (500 mg) was dissolved in 10 mL of a mercaptoethanol test solution, heated for 6 hours, and then separated by Chromatorex ODS (manufactured by Fuji Silysia) (water-methanol eluent) column chromatography, followed by Sephadex LH-20 (EtOH eluent) column chromatography. As a result of the purification, it was revealed from the analysis result of ¹ H-NMR spectrum that it was epiafzeletin-3-O-gallate-ME. The structural formula of Epiafzeletin-3-O-gallate-ME is shown in FIG.
That is, in the ¹ H-NMR spectrum, the signals of A ring, C ring, galloyl group and hydroxyethylthio group are almost the same as those of EGCg-ME, but the signal of B ring is d7.40 and 6.81. Both were observed as double lines for 2H (J = 8 Hz), indicating that the B ring was p-hydroxyphenol. This was also supported by the fact that TOF (time-of-flight) mass spectrometry showed an [M + H] ⁺ peak at m / z 503.

  Among these identified structural units, EGC, EGCg, and ECg are derived from the terminal unit of the polymer compound, and the thioether compound is derived from the extension unit. Therefore, the structure of the polymer compound before thiol decomposition, that is, the Fr. The structure of proanthocyanidins contained in 7 is estimated to have a structure as shown in FIG. 5, for example.

  So far, only one kind of dianthr obtained from oolong tea has been known as proanthocyanidins having epiafuzeretin gallate as a constitutional unit (Hashimoto, F. et al., Chem. Pharm. Bull.,). 1989, 37, 3255-3263). Usually, the dimer is detected as a clear peak by HPLC, but Fr. In the HPLC of No. 7, no peak corresponding to a dimer that can explain the amount of production of epiafzeletin-3-O-gallate-ME is observed, so Fr. It can be said that Epiafzeletin-3-O-gallate-ME produced by the thiol decomposition of 7 is derived from a trimer or higher proanthocyanidin oligomer or polymer. Such a “proanthocyanidin (oligomer or polymer of trimer or higher) having an epiaphzeletin gallate as a constituent unit” is a novel compound that has not been known so far.

The same analysis test was performed on fermented tea leaves produced in the same manner except that only loaf leaves were used without using loquat leaves, and thioether compounds obtained after thiol decomposition were compared. The peak of O-gallate-ME was detected with almost the same magnitude when loquat leaves were mixed and when they were not mixed. Moreover, even if thiol decomposition was performed on proanthocyanidins extracted from loquat leaves, no epiafzeletin-3-O-gallate-ME was detected. From this, Fr. It can be seen that “Proanthocyanidins having Epiafzeletin gallate as a constituent unit” contained in No. 7 is derived from tea leaves.
Moreover, the peak of EC-ME was clearly larger when the loquat leaves were not mixed than when the loquat leaves were not mixed. Accordingly, “proanthocyanidins having epicatechin as a constituent unit (biwaproanthocyanidins)” contained in loquat leaves are highly effective in inhibiting Fr. 7 is included.

[Analysis Test Example 4]
As shown in FIG. 4B, Fr. Even after thiol degradation of 7, the baseline excitement remains.
This is because Fr. 7. In addition to “Proanthocyanidins with Epiafzeletin gallate as a constituent unit” and “Biwa Proanthocyanidins” described in Analytical Test Example 3 above, there are substances detected as a rise in the baseline in HPLC analysis. It shows that
Therefore, the substances detected as the climax of the baseline in HPLC analysis are mixed fermented tea leaves when tea leaves and loquat leaves are mixed, and fermented tea leaves when they are the same except that loquat leaves are not mixed (hereinafter referred to as single It was compared whether or not the production amount was different in some cases. The production conditions of the mixed fermented tea leaves are the same as those in Analytical Test Examples 1 and 2. The blending ratio of tea leaves and loquat leaves is 9: 1, the twisting time is 20 minutes, and the fermentation time is 0 hours.

About each of mixed fermented tea leaves and individual fermented tea leaves, 3 g of tea leaves (water content 5 mass%) were extracted with 100 mL of 70% acetone at room temperature (extraction time 18 hours) and then filtered. The filtrate was concentrated to an aqueous solution (20 mL), and the solvent was partitioned twice with ether and ethyl acetate.
The remaining aqueous layer was once concentrated, made up to 30 mL with 70% ethanol, and 10 μL thereof was analyzed by HPLC. The HPLC analysis result of the aqueous layer is shown in FIG.
As shown in this figure, the mixed fermented tea leaves clearly have a larger amount of substance detected as a rise in the baseline than the single fermented tea leaves. Comparing the areas of the peaks, the amount of substance detected as a baseline swell in the mixed fermented tea leaves is estimated at 159% of the single fermented tea leaves. This increase includes proanthocyanidins derived from loquat leaves, but the amount of loquat leaves added to mixed fermented tea leaves is only one-tenth that of tea leaves, so only the ingredients originally contained in loquat leaves The 59% increase cannot be explained. Therefore, this increase indicates the presence of catechin oxidation products that are promoted by the addition of loquat leaves and are not produced without the addition of loquat leaves.

On the other hand, after concentrating the ethyl acetate layer obtained above to 30 mL with 80% methanol, 1 mL of that was introduced into ODS short column (TOYOPAK ODS M, TOSO, manufactured by Tosoh Corporation) with 80% methanol and eluted. The liquid was adjusted to 5.0 mL, and 5 μL thereof was analyzed by HPLC. The HPLC analysis result of the ethyl acetate layer is shown in FIG.
As shown in this figure, the peak area of theaflavins in the mixed fermented tea leaves is increased 2.7 times in comparison with the single fermented tea leaves. On the other hand, the peak area of catechins is decreasing, specifically, EGC is 0.24 times, EC is 0.44 times, EGCg is 0.57 times, and ECg is 0.64 times. .
This indicates that in mixed fermented tea leaves, a large amount of high-molecular catechin oxidation products are produced by adding loquat leaves to the tea leaves and squeezing them.

[Analysis Test Example 5]
From the results of the analytical test example 4, in the mixed fermented tea leaves, a large amount of high molecular catechin oxidation products are generated by adding and mixing the loquat leaves into the tea leaves, which is detected as a rise in the baseline in the HPLC analysis. That is, it was found to be detected as the origin in thin layer chromatography (TLC) analysis.
Therefore, for the purpose of specifying a component that specifically increases in such mixed fermented tea leaves, the following method is used to prepare mixed fermented tea leaves (tea leaf to loquat blending ratio 9: 1, twisting time 20 minutes, fermentation time. Among the components contained in (0 hour), the oxidized polymer polyphenol detected as the origin in thin layer chromatography (TLC) analysis was separated.
That is, 228 g of mixed fermented tea leaves (water content 5 mass%) was extracted with 3 L of 60% ethanol three times at room temperature, and the three extracts were combined and concentrated under reduced pressure. The obtained aqueous solution (1.5 L) was sequentially partitioned with ethyl ether, ethyl acetate, and n-butanol, and the ethyl ether soluble part (4.03 g), ethyl acetate soluble part (9.8 g), n- A butanol soluble part (22.1 g) was obtained.
15 g of the n-butanol soluble part was dissolved in 60% methanol, applied to a Sephadex LH-20 column conditioned with 60% methanol, 60% methanol, 80% methanol, 100% methanol, then methanol-water-acetone (8: 1: 1) Elution was performed sequentially with a mixed solution, a methanol-water-acetone (6: 2: 2) mixed solution, and a water-acetone (1: 1) mixed solution. The eluate was analyzed by TLC (toluene-ethyl formate-formic acid, 1: 7: 1), and only fractions containing substances that did not move from the origin were collected and concentrated to obtain a polymer-like substance (oxidized polymer polyphenol fraction) 1 .4 g was obtained. The HPLC analysis result is shown in FIG. 8A.

In FIG. 8A, a small peak around 45 minutes indicates the presence of theaflavins due to its UV absorption, but the peak area was 2% or less of the total rise in the baseline.
The thus obtained polymer-like substance (oxidized polymer polyphenol fraction) was measured for AGH inhibition. The results are shown in Table 5.
In addition, Table 5 also shows the measurement results of AGH inhibitory properties for theaflavin-3,3′-di-O-gallate.

  As shown in Table 5, the oxidized polymer polyphenol fraction separated from the mixed fermented tea leaves by the above method, ie, the polymer polyphenol specifically increased in the mixed fermented tea leaves is theaflavin-3,3′-O—. It showed strong maltase and sucrase inhibitory activity equivalent to digallate. In view of the content, this activity is not due to contaminating theaflavins (peaks near 45 minutes) but is derived from high molecular weight polyphenols.

Furthermore, the oxidized polymer polyphenol fraction obtained above was subjected to thiol decomposition with mercaptoethanol by the following method.
That is, 0.2 mL of a solution obtained by dissolving the oxidized polymer polyphenol obtained above in 60% ethanol (oxidized polymer polyphenol concentration: 10 mg / mL) and 0.8 mL of a mercaptoethanol test solution were mixed. The mixture was reacted at 5 ° C. for 5 hours, and 5 μL thereof was analyzed by HPLC. The result is shown in FIG. 8B.
In the analytical test example 3, Fr. As in the case of HPLC analysis of the 7-thiol-decomposed product, peaks of thioether compounds and catechins including epiafzeletin-3-O-gallate-ME were detected. Further, in FIG. 8B, the bulge of the baseline was observed even after thiol decomposition.
Therefore, the oxidized polymer polyphenol fraction obtained above contains proanthocyanidins having the above-mentioned epiafzeletin gallate as a constituent unit, but the content ratio is small, and there are quite a few substances that are not thiol-degraded. It is suggested that it is included.

Therefore, the ^{13 C-NMR} spectrum of the oxidized polymer polyphenol fraction was measured, it and a ^{13 C-NMR} spectrum of included in loquat leaves "proanthocyanidins configured unit epicatechin (loquat proanthocyanidins)" Compared. The result is shown in FIG. FIG. 9A is a spectrum of biwaproanthocyanidins, and FIG. 9B is a spectrum of the oxidized polymer polyphenol fraction.
From the results of FIG. 9, a signal derived from catechol ring type B ring (Bc) is observed in biwaproanthocyanidin, whereas a signal derived from B ring is present in oxidized high molecular polyphenol separated from mixed fermented tea leaves. Is hardly observed, derived from phloroglucinol (1,3,5-trihydroxybenzene) corresponding to the A ring of catechin, and derived from pyrogallol (1,2,3-trihydroxybenzene) of the galloyl group A small signal was also observed from the C ring, although it was small.
Therefore, the main component of the oxidized polymer polyphenol separated from the mixed fermented tea leaves by the above method is not Biwa proanthocyanidin, but catechin derivatives having various structures in the B ring part are oxidatively condensed and polymerized. It turns out that it is the material which became.
Although the structure of such a high molecular polyphenol is not clear, it is considered that the B rings of catechin gallates are mainly oxidatively condensed and polymerized by analogy with the compound shown in FIG. Various types such as theacinensin, theaflavin, and oolong theanine shown in FIG. 3 are presumed as the binding mode of the B ring, but the details are currently unknown.

As a result of elemental analysis of the oxidized polymer polyphenol fraction, it was 57.06% carbon, 4.51% hydrogen, and 0.35% nitrogen.
Calculated _{EGCg (C 22 H 18 O 11} : carbon 57.65%, hydrogen _{3.96%, C 22 H 18 O} 11 · 2H 2 O: carbon 53.44%, hydrogen 4.49%) compared to Since the hydrogen content is high and the hydrogen content should decrease in normal oxidation, it is suggested that another metabolite may be incorporated during the catechin oxidation process.

Furthermore, after acetylating the obtained oxidized polymer polyphenol and measuring the molecular weight by gel permeation chromatography, the peak top molecular weight was 2000 (number average molecular weight 1400, mass average molecular weight 3200), and the molecular weight distribution was wide. It was about 1000-15000.
The measurement conditions for gel permeation chromatography were as follows.
Column: TSK-gel G 4000H ₆ , diameter 4.6 mm × length 250 mm, manufactured by Tosoh Corporation
Pump: TOSO DP8020, manufactured by Tosoh Corporation
Detector: JASCO UV970 made by JASCO Corporation, 254 nm,
Moving bed: tetrahydrofuran,
Temperature: room temperature,
Flow rate: 1.0 mL / min,
Molecular weight calculation by comparison with standard polystyrene: JASCO 807IT integrator, manufactured by JASCO Corporation.
If the molecular weight increase due to acetylation is 1.5 times by analogy with catechins, the molecular weight is 670 to 10,000 with 1330 as the maximum. Assuming that the catechin gallate is produced only from catechin gallates, it is included from those corresponding to dimers (theaflavins) to those corresponding to 20-mers.

[Analysis Test Example 6]
DMSO (dimethyl sulfoxide) is a pure product (commercial product) of EC, ECG, EGC, EGCg, theaflavin (TF), 3-TFG, 3′-TFG, 3,3′-TFGG, and theacinensin A (TS-A). A sample solution was prepared by dissolving in a mixed solvent consisting of 1: water = 1: 9, and AGH inhibitory activity was measured for each sample solution.
The evaluation of AGH inhibition in this test example and analytical test example 7 was carried out using the pseudo-in vivo method (T. Oki. Et al., Biol. Pharm. Bull., 23) using a carrier on which rat small intestine AGH was immobilized. , 1084-1087 (2000)), and is expressed as a final concentration (IC ₅₀ value, unit: mM) that inhibits enzyme activity by 50%. The results are shown in Table 6.

From the results of Table 6, the strong maltase inhibitory ^{properties, ECG, EGCG, 3-TFG} , 3,3, a -TFGG, the sucrase inhibitory is strong, ECG, ^EGCG, 3,3, in -TFGG It turned out to be. That is, it has been clarified that those having a molecular structure having an ester type for catechin and a polysubstituted OH group of galloyl group for theaflavin are more AGH-inhibiting.

[Analysis Test Example 7]
20 g of mixed fermented tea leaves (tea leaf to loquat blending ratio 9: 1, twisting time 20 minutes, fermentation time 0 hours, moisture content 5 mass%) are extracted with 1000 mL of hot water at 100 ° C. for 10 minutes and centrifuged. The supernatant obtained was freeze-dried and the AGH inhibitory property was examined.
The sample was dissolved in a mixed solvent of DMSO: water = 1: 9 to prepare a sample solution, and AGH inhibitory properties were measured by the pseudo-in vivo method for the sample solution. The contents of catechins (EC, ECG, EGC, EGCg) and theaflavins (TF, 3-TFG, 3′-TFG, 3,3′-TFGG) contained in this sample solution were measured. Then, based on the IC ₅₀ value measured in the analytical test example 6, when the AGH inhibitory effect exhibited by the mixed fermented tea leaves is defined as 100%, the ratio of the AGH inhibitory effect exhibited by each component (activity contribution rate, Unit:%). The results of maltase inhibition are shown in Table 7, and the results of sucrase inhibition are shown in Table 8.

[Analysis Test Example 8]
20 g of the same mixed fermented tea leaves as in Analysis Test Example 7 were extracted with 1 L of hot water at 100 ° C. for 10 minutes. The filtrate obtained by filtering the extract was concentrated with an evaporator and then freeze-dried. 1 mg of water was added to 10 mg of the dried powder, and after stirring, centrifugation (3000 rpm, 10 minutes) was performed to obtain a supernatant, that is, a water-soluble fraction. The AGH inhibitory activity of this water-soluble fraction was measured. The results are shown in Table 9 below.
In addition, the measurement of AGH inhibition in this analysis test example was performed by the in vitro method using the above-mentioned free AGH (Oki. T. et. Al., J. Agric. Food Chem, 47, 550-553 (1999)). .

From the results of Table 9, it can be seen that the water-soluble fraction of the hot water extract of mixed fermented tea leaves exhibits high AGH inhibitory properties.
Therefore, a component separation operation was further performed by the following method on the water-soluble fraction of the hot water extract exhibiting high AGH inhibition.
That is, the water-soluble fraction of the hot water extract was subjected to adsorption chromatography using Sephadex LH-20 (manufactured by Amersham Biosciences). Elution was carried out in the order of water, 50% methanol, 100% methanol, and 70% acetone, and AGH inhibitory measurements were performed in the same manner as described above for the lyophilized fractions obtained from each. The yield and AGH inhibitory properties of each fraction are shown in Table 10 below. Table 10 also shows the measurement results of Table 9.

As shown in Table 10, among the water-soluble fractions of the mixed fermented tea leaf hot water extract, AGH inhibition was particularly high in the 70% acetone-eluted fraction and the 100% methanol-eluted fraction.
Therefore, when the molecular weight of the 70% acetone-eluted fraction and 100% methanol-eluted fraction was measured by gel permeation chromatography, the components contained in both fractions having particularly high AGH inhibitory properties were in the range of molecular weights of 100-1500. Turned out to be.
The measurement conditions of gel permeation chromatography here are as follows.
Column: TSK-gel G2500 PWXL, diameter 7.8 mm x length 250 mm,
Pump: manufactured by Shimadzu Corporation, LC-10Avp,
Detector: SPD-10AV, 254 nm,
Moving layer: 0.1 M, phosphate buffer (pH 7.2, containing 0.1 M sodium chloride),
Temperature: room temperature,
Flow rate: 0.3 mL / min,
Molecular weight calculation by comparison with standard polyethylene glycol.

[Analysis Test Example 9]
Similarly to the analytical test example 8, 20 g of the same mixed fermented tea leaves as in the analytical test example 7 were extracted with 1 L of hot water at 100 ° C. for 10 minutes, and the obtained extract was filtered to obtain a filtrate. The filtrate was applied to a column packed with an adsorbent Porapack Q (product name, manufactured by Waters). Then, the water-soluble component in the column was eluted with 100 ml of water and subsequently with 100 ml of 50% methanol. The water-eluted fraction and 50% methanol-eluted fraction thus obtained were each concentrated and dried, and then AGH inhibitory activity was measured. The results are shown in Table 11 below.
The measurement of AGH inhibition in this analytical test example is performed by the method using free AGH by Matsui et al.

As shown in Table 11, both the water-eluted fraction and 50% methanol-eluted fraction of the mixed fermented tea leaf hot water extract showed high AGH inhibitory properties.
Furthermore, when the molecular weight of the water-eluted fraction and 50% methanol-eluted fraction was measured by gel permeation chromatography under the same conditions as in Analytical Test Example 8, they were included in both fractions having particularly high AGH inhibitory properties. It was found that the component to be obtained is in the range of molecular weight 100-1900.
From the results of Analytical Test Examples 8 and 9, the water-soluble fraction of the hot water extract of mixed fermented tea leaves shows high AGH inhibition, and the molecular weight of the component that greatly contributes to this AGH inhibition is about 100 to 1900. It was found to have a relatively low molecular weight.

[Experiment 2]
Normal rats were fed a hot-water extract (freeze-dried product) of mixed fermented tea leaves and examined the effect on glucose tolerance.
Mixed fermented tea leaves (tea leaf to loquat blending ratio 9: 1, twisting time 20 minutes, fermentation time 0 hours), bancha, loquat leaves dried and ground loquat leaves, and tea leaves without mixing loquat leaves A single fermented tea leaf obtained by twisting only for 20 minutes was prepared as a sample tea leaf. The bancha is bancha (raw tea produced without fermenting tea leaves) using the same tea leaves used as the raw material of the mixed fermented tea leaves and produced according to the normal method for producing green tea.
20 g of each sample tea leaf (water content 5 mass%) was extracted with 1,000 ml of hot water at 100 ° C. for 10 minutes and then filtered. The filtrate was freeze-dried to obtain a powdery extract.
Four weeks old male Sprague were prepared by adding each of the hot water extracts prepared above to a diet based on the AIN-76 composition containing no cholesterol so that the content was 1% by mass (4 species). -Dawley (SD) rats were fed ad libitum.
In addition, as a control, the same rats were allowed to freely ingest a control diet in which no hot water extract was added to the diet.
Rats were housed in an animal breeding room with a light cycle of room temperature 22 ± 1 ° C., humidity 55 ± 5%, and 8:00:00 to 20:00 lighting. After 4 weeks, fasted for 6 hours, 1 g / kg body weight of maltose was orally administered. After administration, blood was collected from the tail vein at 0, 10, 20, 30, and 60 minutes, respectively, and the blood glucose level was measured using a blood glucose test measurement Medisafe chip (manufactured by Terumo).
The result is shown in FIG.

In FIG. 10, the horizontal axis indicates the elapsed time after maltose administration, and the vertical axis indicates the blood glucose level. In addition, the value of a blood glucose level represents the blood glucose level at the time of maltose administration as 0 mg / dl.
As shown in this figure, the blood glucose level of the rat ingesting the hot water extract of the mixed fermented tea leaves is changing at a level lower than that of any sample tea leaves.
From this, it can be seen that an excellent effect of suppressing an increase in blood glucose level can be obtained by ingesting the hot water extract of mixed fermented tea leaves. In addition, the effect is superior to the effect obtained with loquat leaves dried only loquat leaves and tea leaves fermented with tea leaves only. By mixing, twisting and fermenting tea leaves and loquat leaves It turns out that a synergistic effect is acquired.

[Experiment 3]
Rats that developed type 2 diabetes were ingested with mixed fermented tea leaf powder and examined the effect on blood glucose levels.
Mixed fermented tea leaves (tea leaf to loquat blending ratio 9: 1, twisting time 20 minutes, fermentation time 0 hours, moisture content 5% by mass) using a food processor, powder of 60-100 mesh size The powdered mixed fermented tea leaves were obtained by pulverization until it reached a state.
The rats include male Otsuka Long-Evans Tokyo Fatty rats (hereinafter referred to as OLETF rats) that spontaneously develop type 1 diabetes at one month of age, and male Long-Evans Tokyo Otsuka rats (hereinafter referred to as OLETF rats) that do not develop diabetes in the target model animals. LETO rats). Rats were housed in an animal breeding room with a light cycle of room temperature 22 ± 1 ° C., humidity 55 ± 5%, and 8:00:00 to 20:00 lighting.
During the first 3 months, MF chow (manufactured by Oriental Yeast Co., Ltd.) was given to LETO and OLETF rats for preliminary breeding. Since OLETF rats usually develop type 2 diabetes from 5 to 8 months of age, feeding of the test meal was started together with LETO rats from 4 months of age one month before the possibility of onset.
That is, after a 6-hour fast (9: 0 to 15:00) at the age of 4 months, blood was collected from the tail vein and blood glucose levels were measured. Each group was divided into 6 animals so that the body weight and blood glucose levels were equal. The following test meals were fed freely for 5 months.
As the test meal, a purified meal based on AIN-76 was used as a control meal and fed to the LETO rat-control group and the OLETF rat-control group. The composition of the control food (g / kg) is: casein 200, salad oil 100, mineral mixture (AIN-76-MX) 35, vitamin mixture (AIN-76-VX) 10, cellulose 50, choline bitartrate 2, DL-methionine 3. Corn starch 150 and sucrose 450.
In the OLETF rat-mixed fermented tea leaf addition group, 5% of the total feed mass of powdered mixed fermented tea leaf was added to the control food, and the amount of sucrose was reduced by the added amount.
In each group, during the breeding period, food intake was measured every day, and body weight and water intake were measured every other day.

1, 2, 3, 4 and 5 months after the start of test meal administration, blood glucose was measured using a blood glucose test measurement Medisafe chip (manufactured by Terumo) and blood was collected from the tail vein after 6 hours of fasting. FIG. 11 shows the change in blood glucose level of the rat during 5 months after taking the test meal.
In FIG. 11, the horizontal axis indicates the elapsed time (unit: month) from the start of test meal administration, and the vertical axis indicates the blood glucose level.
As shown in this figure, the blood glucose level of LETO rats that did not develop diabetes who took the control diet was low throughout the breeding period. On the other hand, the blood glucose level of OLETF rats that developed diabetes that took the control diet increased over time, and was considered to have developed diabetes.
On the other hand, in a group in which an OLETF rat that develops type 2 diabetes is fed a diet supplemented with powder of mixed fermented tea leaves, the blood glucose level does not increase over time and does not cause diabetes. The level was as low as. From this, it became clear that the powder of mixed fermented tea leaves has an excellent blood glucose rise inhibitory effect.

[Experimental Example 4]
Rats that develop type 2 diabetes were ingested with hot water extract (freeze-dried product) of mixed fermented tea leaves and examined the effect on blood glucose levels.
20 g of mixed fermented tea leaves (tea leaf and loquat blending ratio 9: 1, twisting time 20 minutes, fermentation time 0 hours, water content 5 mass%) are extracted with hot water 1000 mL at 100 ° C. for 10 minutes and filtered. The obtained filtrate was freeze-dried to obtain a powdered mixed fermented tea leaf extract.
As a comparative example, bancha (produced without fermenting tea leaves) produced according to a normal green tea production method was extracted, filtered and dried under the same conditions to obtain a powdered bancha extract. In addition, loquat leaves dried to have the same water content were extracted, filtered and dried under the same conditions to obtain powdered loquat leaf extract.
Rats were used under the same conditions as in Experimental Example 3 above, and the breeding conditions up to 4 months of age were the same.
After being fasted for 6 hours at the age of 4 months (9: 00-15: 00), blood was collected from the tail vein and the blood glucose level was measured. The following test meals were fed freely for 5 months.
The control diet group of LETO rats and OLETF rats were fed the same control diet as in Experimental Example 3.
In the OLETF rat-mixed fermented tea leaf addition group, 5% of the total feed mass of the powdered mixed fermented tea leaf extract was added to the control food, and the sucrose amount corresponding to the added amount was reduced.
In the OLETF rat-bancha addition group, the powdery bancha extract was added to the control diet at 5% of the total feed mass, and the amount of sucrose was reduced by the added amount.
In the OLETF rat-loquat leaf addition group, powdered loquat leaf extract was added to the control diet at 5% of the total feed mass, and the sucrose amount corresponding to the added amount was reduced.
During the breeding period, food intake was measured every day, and body weight and water intake were measured every other day.

Blood glucose levels were measured in the same manner as in Experimental Example 3 after 1, 2, 3, 4 and 5 months from the start of test meal administration. FIG. 12 shows the change in the blood glucose level of the rat during the 5 months after taking the test meal.
The vertical and horizontal axes in FIG. 12 are the same as those in FIG. As shown in this figure, the blood glucose level of LETO rats that did not develop diabetes who took the control diet was low throughout the breeding period. On the other hand, the blood glucose level of OLETF rats that developed diabetes that took the control diet increased over time, and was considered to have developed diabetes.
On the other hand, in the group in which OLETF rats that develop type 2 diabetes were fed with a mixed fermented tea leaf hot-water extract (freeze-dried product), blood glucose levels increased over time The level was as low as in LETO rats that did not develop diabetes. From this, it was clarified that the hot water extract of mixed fermented tea leaves has an excellent blood glucose rise inhibitory effect.

[Experimental Example 5]
Other effects were examined when rats with type 2 diabetes were fed mixed fermented tea leaf powder.
Powdered mixed fermented tea leaves were produced in the same manner as in Experimental Example 3 above.
As a comparative example, bancha (raw tea produced without fermenting tea leaves) produced using the same tea leaves used as the raw material for mixed fermented tea leaves was produced and pulverized in the same manner to obtain powdered bancha tea. Got. Moreover, the powdered loquat leaf was obtained by pulverizing the dried loquat leaf so as to have the same water content.
Rats were used under the same conditions as in Experimental Example 3 above, and the breeding conditions up to 4 months of age were the same.
After being fasted for 6 hours at the age of 4 months (9: 00-15: 00), blood was collected from the tail vein and the blood glucose level was measured. The following test meals were fed freely for 5 months.
The control diet group of LETO rats and OLETF rats were fed the same control diet as in Experimental Example 3.
In the OLETF rat-mixed fermented tea leaf addition group, 5% of the total feed mass of powdered mixed fermented tea leaf was added to the control food, and the amount of sucrose was reduced by the added amount.
In the OLETF rat-bancha addition group, powdered bancha was added to the control diet at 5% of the total feed mass, and the amount of sucrose was reduced by the amount added.
In the OLETF rat-louse leaf added group, powdered loquat leaves were added to the control diet at 5% of the total feed mass, and the sucrose amount corresponding to the added amount was reduced.
During the breeding period, food intake was measured every day, and body weight and water intake were measured every other day.

Five months after the start of administration of the test meal, after 6 hours of fasting, the mice were sacrificed and serum, liver, and adipose tissue around the kidney and testicles were collected.
The following table shows the measurement results of serum insulin concentration, serum lipid peroxide concentration, adipose tissue mass, serum cholesterol, serum triglyceride, liver cholesterol, and liver triglyceride, respectively.

  From the results shown in Table 12, the group in which the OLETF rats were fed the control diet or loquat leaves showed a decrease in insulin. These groups were found to have developed diabetes by measuring blood glucose levels. On the other hand, even in the OLETF rat that develops diabetes, in the group fed with the powdered mixed fermented tea leaves, the insulin concentration increased to the level of the LETO rat that does not develop diabetes. From this, it was recognized that mixed fermented tea leaves have the effect of improving the decrease in insulin secretion due to the onset of diabetes.

  From the results of Table 13, even in OLETF rats that develop diabetes, the group fed with powdered mixed fermented tea leaves had a lower serum lipid peroxide concentration than other OLETF rat groups. From this, it was recognized that mixed fermented tea leaves have the effect of reducing serum lipid peroxide concentration.

  From the results shown in Table 14, even in the OLETF rats that develop diabetes, the group fed with the powdered mixed fermented tea leaves had significantly more adipose tissue mass around the kidney and testicles than the OLETF rats group fed with the control diet. It was low. From this, it was confirmed that mixed fermented tea leaves have an effect of suppressing body fat accumulation.

  From the results of Table 15, even in the OLETF rat that develops diabetes, the group fed with the powdered mixed fermented tea leaves was higher in serum and liver cholesterol and triglyceride concentrations than the OLETF rat group ingested the control diet. Had fallen. From this, it was confirmed that mixed fermented tea leaves have a cholesterol-reducing effect and a neutral fat-reducing effect.

[Experimental Example 6]
Other effects were examined when rats with onset of type 2 diabetes were fed with hot water extract (freeze-dried product) of mixed fermented tea leaves.
In the same manner as in Experimental Example 4, LETO rats and OLETF rats were allowed to eat freely for 5 months.
Five months after the start of administration of the test meal, after 6 hours of fasting, the mice were sacrificed and serum, liver, and adipose tissue around the kidney and testicles were collected.
The following table shows the measurement results of serum insulin concentration, adipose tissue mass, serum triglyceride, and liver triglyceride, respectively.

  From the results shown in Table 16, a decrease in insulin was observed in the group in which the OLETF rats were fed with the control diet, the bancha extract, or the loquat leaf extract. These groups were found to have developed diabetes by measuring blood glucose levels. On the other hand, even in the OLETF rats that develop diabetes, the group fed with the mixed fermented tea leaf extract had the insulin concentration increased to the level of LETO rats that did not develop diabetes. From this, it was recognized that the hot water extract of mixed fermented tea leaves has the effect of improving the decrease in insulin secretion due to the onset of diabetes.

  From the results of Table 17, even in the OLETF rats that develop diabetes, the group fed with the mixed fermented tea leaf extract had a lower adipose tissue mass around the kidney and testicles than the OLETF rats group fed with the control diet . From this, it was recognized that the hot water extract of mixed fermented tea leaves has an effect of suppressing body fat accumulation.

  From the results of Table 18, even in OLETF rats that develop diabetes, serum and liver triglyceride concentrations in the group fed with the mixed fermented tea leaf extract were lower than those in the OLETF rat group fed with the control diet. It was. From this, it was confirmed that the hot water extract of mixed fermented tea leaves has a neutral fat reducing effect.

[Experimental Example 7]
Other effects were examined when normal rats were fed with hot water extract (freeze-dried product) of mixed fermented tea leaves.
That is, four kinds of feeds and control foods to which the freeze-dried hot water extracts of the same sample tea leaves (bancha, single fermented tea leaves, loquat teas, mixed fermented tea leaves) as in Experimental Example 2 were tested. Under the same conditions as in Example 2, 4-week-old male Sprague-Dawley (SD) rats were freely fed and bred.
After 4 weeks, fasted for 6 hours and then sacrificed to collect serum, liver and adipose tissue.
The following table shows the measurement results of serum lipid peroxide concentration, adipose tissue mass, serum neutral fat (triglyceride), and liver neutral fat (triglyceride).

  From the results in Table 19, the serum lipid peroxide concentration decreased in the normal rats by ingesting the hot water extract of the mixed fermented tea leaves as compared with the case of ingesting the control diet. From this, it was recognized that the hot water extract of mixed fermented tea leaves exerted a serum lipid peroxide reducing effect even under normal health conditions.

  From the results of Table 20, even in normal rats, the adipose tissue mass was lower by ingesting the hot water extract of mixed fermented tea leaves than in the case of ingesting the control diet. From this, it was recognized that the hot water extract of mixed fermented tea leaves exerts an effect of suppressing body fat accumulation even in a normal health state.

  From the results shown in Table 21, serum and liver neutral fat concentrations were lower in normal rats by ingesting the hot water extract of mixed fermented tea leaves than in the case of ingesting the control diet. From this, it was recognized that the hot water extract of mixed fermented tea leaves exerted a neutral fat reducing effect even in a normal health state.

[Experimental Example 8]
The effect of mixed fermented tea leaves on the intake of hot water extract on type 1 diabetes was investigated.
That is, the hot water extract (freeze-dried product) of the same mixed fermented tea leaves as in Experimental Example 2 was added to a diet based on the AIN-76 composition containing no cholesterol and added to a diet of 4 weeks old male Sprague- Dawley (SD) rats were fed ad libitum. As a comparative example, SD rats were given a control diet in which no mixed fermented tea leaf extract was added to the same diet. The breeding conditions were the same as in Experimental Example 2.
After fasting for 6 hours after the start of feeding and 14 days after the start of feeding, blood was collected from the tail vein, and blood glucose was measured using a blood glucose test measurement Medisafe chip (manufactured by Terumo).
In addition, after the blood collection 14 days after the start of feeding, further fasting was continued for 9 hours (total fasting time: 15 hours), and 30 mg / kg body weight of streptozocin (STZ) was administered. This STZ is a drug having an action of destroying pancreatic islets of Langerhans and causing insulin secretion injury to develop type 1 diabetes. After STZ administration, food was freely fed again.
7 and 14 days after the start of refeeding (3 weeks and 4 weeks after the start of the first feeding), fasted for 6 hours, blood was collected from the tail vein, and blood glucose test measurement Medisafe chip (manufactured by Terumo) Was used to measure blood glucose levels.
In addition, two weeks after the start of refeeding (four weeks after the start of the first feeding), 6 hours after fasting (after the above blood collection), the mice were killed to collect serum and pancreatic islets of Langerhans. Glucose stimulation was given to the pancreatic islets of Langerhans, and insulin secretion was measured.
The change in blood glucose level after the start of feeding is shown in FIG. The following table shows the measurement results of insulin secretion and the measurement results of serum cholesterol concentration and serum triglyceride concentration.

In FIG. 13, the horizontal axis indicates the elapsed time, and the vertical axis indicates the blood glucose level. In addition, the value of the blood sugar level is expressed as 100 mg / dL of the blood sugar level on the feeding start date.
As shown in this figure, the blood glucose level of the rat that took the control diet increased over time from day 14 after administration of STZ, and was considered to have developed type 1 diabetes. On the other hand, in the rats ingesting the hot water extract of mixed fermented tea leaves, the increase in blood glucose level after 14th was suppressed to a small level.

  From the results shown in Table 22, insulin secretion induced by glucose stimulation from the pancreatic islets of Langerhans 4 weeks after the start of feeding (2 weeks after STZ administration) Higher than ingested rats. From this, it became clear that the hot water extract of mixed fermented tea leaves maintains the insulin secretion function by protecting pancreatic islets of Langerhans, and the hot water extract of mixed fermented tea leaves is effective in preventing type 1 diabetes. It has been suggested.

  From the results shown in Table 23, the serum cholesterol concentration and serum triglyceride concentration after 4 weeks from the start of feeding (2 weeks after STZ administration) were ingested with the control diet in rats fed with the hot water extract of mixed fermented tea leaves. The value was lower than that of the rat. From this, it became clear that the hot-water extract of mixed fermented tea leaves has a serum lipid concentration lowering action in type 1 diabetes.

[Experimental Example 9]
Animal experiments were conducted to confirm the safety of fermented tea leaves.
That is, mixed fermented tea leaves (tea leaf to loquat blending ratio 9: 1, twisting time 20 minutes, fermentation time 0 hours) until a powder state of 60-100 mesh size using a food processor The powdered mixed fermented tea leaves obtained by pulverization were orally administered to 4-week-old Sprague-Dawley male rats at a dose of 5000 mg / kg body weight per day. When bred for 4 weeks under conditions of temperature 22 ± 1 ° C, humidity 55 ± 5%, feed and water free feeding, no death was observed, no abnormal weight change was observed, and also at autopsy after the breeding No organ abnormalities were observed.
Therefore, the lethal dose (LD ₅₀ ) of fermented tea to rats was estimated to be more than 5000 mg / kg body weight, and the safety was judged to be quite high.

[Experimental Example 10]
(Production of mixed fermented tea leaves-hand brewing method)
The loquat leaves were put into the tea leaves and twisted by the method of squeezing them by hand. The blending ratio of tea leaves and loquat leaves was set to two mass ratios of tea leaf: lowa tea: 90:10 (10% loquat input ratio) and 75:25 (25% loquat input ratio). The time for squeezing (hand squeezing time) was 25 minutes and 50 minutes.
Then, 16 types of mixed fermented tea leaves were obtained through four fermentation processes of 0 hours, 1 hour, 2 hours, and 4 hours. In addition, the thing of fermentation process 0 hours is the mixed fermented tea leaves obtained immediately after the end of the mastication.
The obtained mixed fermented tea leaves were extracted with hot water at 100 ° C. for 10 minutes in the same manner as in Experimental Example 1 (the amount of mixed fermented tea leaves used: 2.0 mg / ml). In the same manner as above, AGH inhibition (maltase inhibition and sucrase inhibition) was measured, and DPPH (1,1-dipicryl-2-phenylhydrazyl) scavenging activity was measured by the following method. The respective results are shown in Tables 24 and 25. In addition, it shows that an antioxidant effect is so high that the measured value of DPPH elimination activity is large. Further, sensory examination was conducted in the same manner as in Experimental Example 1. The results are shown in Table 26.

(Measurement method of DPPH elimination activity)
DPPH scavenging activity (unit: μmol-Trolox / mg) was measured by the following method (hereinafter the same).
The pulverized mixed fermented tea leaves were dissolved in 80% ethanol solution and diluted to 0.5 mg / ml using the same 80% ethanol solution. A mixed solution of 12 ml of 400 μM DPPH, 12 ml of 200 mM MES buffer (pH 6.0) and 12 ml of 20% ethanol is prepared, and 0.9 ml each of the mixed solution is dispensed into each of the test tubes (a) to (f) using an Eppendorf multipipette. After the injection, (300-x) μl of 80% ethanol was added to each of the test tubes (a) to (f) (x is the amount of analysis sample added, specifically, the test tubes (a) to (f) X in 0) is 0, 30, 60, 120, 180, 240 in this order). Furthermore, the analytical sample (x) μl was added to each of the test tubes (a) to (f) and stirred, and the absorbance at a wavelength of 520 nm was measured 20 minutes after the analytical sample was added.
Specifically, 30 μl of the analysis sample is added to the test tube (b) 30 seconds after the start of time measurement, 60 μl of the analysis sample is added to the test tube (c) 60 seconds later, and the test tube (d ), 120 μl was added to the test tube (e) after 180 seconds, and 240 μl was added to the test tube (f) after 240 seconds. The absorbance of the test tube (a) is measured 20 minutes after the start of time measurement, the absorbance of the test tube (b) is measured 20 minutes and 30 seconds later, and the absorbance of the test tube (c) is measured 21 minutes later. The absorbance was measured every 30 seconds.
On the other hand, a calibration curve was created using Trolox as a standard sample. The horizontal axis of the calibration curve is the amount of sample added (unit: μmol), and the vertical axis is the absorbance at 520 nm.
The measured value for the analytical sample is plotted with the sample addition amount (unit: μl) on the horizontal axis and the absorbance at 520 nm on the vertical axis, with respect to the addition amount (x ′ μl) within a range where linear absorbance decrease continues. The absorbance (ΔA520) value is determined. Next, the Trolox addition amount (unit: μmol) when the same (ΔA520) is obtained in the calibration curve is obtained. From the Trolox equivalent amount thus obtained (unit: μmol), the value of the erasing activity per 1 mg of mixed fermented tea leaves (unit: μmol-Trolox / mg) is determined.

[Experimental Example 11]
(Production of mixed fermented tea leaves-grinding method)
Put tea leaves and loquat leaves in a mortar and grind them. The blending ratio of tea leaves and loquat leaves was set to two mass ratios of tea leaf: lowa tea: 90:10 (10% loquat input ratio) and 75:25 (25% loquat input ratio). The grinding time was set to two types of 20 minutes and 40 minutes.
Then, 16 types of mixed fermented tea leaves were obtained through four fermentation processes of 0 hours, 1 hour, 2 hours, and 4 hours. In addition, the thing of fermentation process 0 hours is the mixed fermented tea leaves obtained immediately after grinding.
About the obtained mixed fermented tea leaves, AGH inhibitory activity was measured in the same manner as in Experimental Example 1, and DPPH elimination activity was also measured. The respective results are shown in Tables 27 and 28. Further, sensory examination was conducted in the same manner as in Experimental Example 1. The results are shown in Table 29.

[Experimental example 12]
(Production of mixed fermented tea leaves-crushing and stirring method)
Tea leaves, loquat leaves and water were put into a mixer (product name: Mill and Mixer, manufactured by Tescom) and pulverized and stirred. The blending ratio of tea leaves and loquat leaves was set to two mass ratios of tea leaf: lowa tea: 90:10 (10% loquat input ratio) and 75:25 (25% loquat input ratio). The amount of water added was 100 parts by mass with respect to 100 parts by mass in total of tea leaves and loquat leaves. The pulverization stirring time was set to two types of 10 minutes and 20 minutes.
Thereafter, 20 types of mixed fermented tea leaves were obtained through five fermentation processes of 1 hour, 4 hours, 8 hours, 12 hours, and 16 hours.
About the obtained mixed fermented tea leaves, AGH inhibitory activity was measured in the same manner as in Experimental Example 1, and DPPH elimination activity was also measured. The respective results are shown in Tables 30 and 31. Further, sensory examination was conducted in the same manner as in Experimental Example 1. The results are shown in Table 32.

[Analysis Test Example 10]
(Aroma component analysis)
Using dried loquat leaves only, fermented loquat leaves only, commercially available black tea (Darjeeling black tea and Assam black tea), tea leaves cultivated at Nagasaki Prefectural Agricultural and Forestry Experiment Station Higashisonobe Tea Industry Branch Fermented black tea, green tea produced without fermentation using the same tea leaves (rough tea), and mixed fermented tea leaves (tea leaf to loquat blending ratio 9: 1, twisting time 20 minutes, fermentation time 0 hours ) Was used as a sample, and the fragrance component was quantitatively analyzed.
In general, pretreatment of tea-based aroma components is performed by a column concentration method or the like. Here, solid-phase microextraction was used as a simple and rapid sample extraction, concentration, and chromatographic introduction method. For quantification, cyclohexanol was added as an internal standard to the sample to be measured, and the peak area of each aroma component was determined as a relative peak area ratio with respect to the internal standard.

  Specifically, the following method was used. The sample was prepared in a 200 mL Erlenmeyer flask, 20 g of the sample, 1 mL of 30% sodium chloride aqueous solution and 50 μL of 1% cyclohexanol as the internal standard were added and sealed, then heated at 80 ° C. for 5 minutes to stabilize the Erlenmeyer flask. Then, the collection tube was inserted for 20 minutes to trap the generated aroma component. The collection tube trapping the aroma component was inserted into the inlet of the gas chromatograph heated to 250 ° C., and the aroma component was introduced into the gas chromatograph column for 3 minutes for analysis. The collection tube used was polydimethylcyclohexane / carboxen / divinylbenzene manufactured by SUPELCO. As for the analysis conditions of the gas chromatograph, the column uses a Stabil-WAX (polyethylene glycol system) 60 m × 0.25 mm and a film thickness of 0.25 μ manufactured by Reapect. The temperature was raised at 10 ° C./min. The inlet temperature was 250 ° C. and the helium pressure was 120 kPa. Gas chromatograph mass spectrometry for component identification was performed at an I / F temperature of 250 ° C., an ionization voltage of 70 eV, and an ionization current of 60 μA. Relative peak area with respect to internal standard for 9 components: 1-hexanol, trans-2-hexenal, 3-hexen-1-ol acetate, 3-hexen-1-ol, linalool, benzaldehyde, methyl salicylate, geraniol, nerolidol The ratio was measured. The results are shown in Table 33.

From the results in the table, the mixed fermented tea leaves contain all 9 components listed in the table. Among these, linalool, benzaldehyde, methyl salicylate, geraniol, and nerolidol are also relatively abundant in black tea fermented with tea leaves in the laboratory, and are also included in two types of commercial black tea. However, the content in green tea and fermented loquat leaves is relatively low. Moreover, trans-2-hexenal, 3-hexen-1-ol acetate, and 3-hexen-1-ol are characteristically contained in fermented loquat leaves. Comparing the peak area of 8 components other than 1-hexanol (value of relative area with respect to internal standard) with the peak area of 1-hexanol (value of relative area with respect to internal standard), each peak area of all 8 components is Only the mixed fermented tea leaves are larger than the peak area of hexanol.
In addition, 1-hexanol is always detected in mixed fermented tea leaves even if the production conditions such as blending ratio of tea leaves and loquat leaves, twisting time, fermentation time, etc. are changed, and always more than the other 8 ingredients listed in the table. It is an aroma component detected in a small amount.

[Experimental Example 13]
KK-A ^y mice that spontaneously develop type 2 diabetes have a hot water extract of mixed fermented tea leaves (freeze-dried product), a fraction rich in theacinensin (hereinafter referred to as TS fraction), and a fraction high in theaflavin (hereinafter referred to as TS). TF fraction) was ingested, and the effect on blood glucose level was examined.
The hot water extract of mixed fermented tea leaves is obtained by mixing 20 g of mixed fermented tea leaves (tea leaf to loquat blending ratio 9: 1, twisting time 20 minutes, fermentation time 0 hours, water content 5 mass%) at 100 ° C. Extraction was performed with 1,000 ml for 10 minutes, followed by filtration. The filtrate was freeze-dried and used as a powder.
As the TS fraction, the Fr. 6 was freeze-dried and powdered.
As the TF fraction, the Fr. 7 was freeze-dried and powdered.
Mice were raised in an animal breeding room with a light cycle of room temperature 22 ± 1 ° C., humidity 55 ± 5%, and 8:00:00 to 20:00 lighting.
A 5-week-old KK-A ^y mouse was used, and MF solid feed (produced by Oriental Yeast Co., Ltd.) was given for the first two weeks for preliminary breeding. Since KK-A ^y mice spontaneously develop diabetes around 7-8 weeks of age, feeding of the test meal was started at the age of 7 weeks, which is likely to develop. That is, after 7 hours of fasting for 6 hours (9: 00-15: 00), blood was collected from the tail vein and blood glucose level was measured. Separately, the following test meals were allowed to eat freely for 6 weeks

The test meals are as follows.
Control food: A purified food based on the AIN-76 composition was used as a control food. The mass composition (g / kg) of the control food is 200 g / kg of casein, 100 g / kg of corn oil, 35 g / kg of the mineral mixture (AIN-76-MX), 10 g / kg of the vitamin mixture (AIN-76-VX), cellulose. 50 g / kg, choline bitartrate 2 g / kg, DL-methionine 3 g / kg, corn starch 150 g / kg and sucrose 450 g / kg.
Extract-added diet: 1 part by mass of the powdered hot water extract (lyophilized product) was added to 100 parts by mass of the control diet, and the amount of sucrose added was subtracted from the control diet.
-TS-added food: Since the hot water extract (freeze-dried product) contains 5.4% by mass of theacinensin, the above-mentioned extract-added test meal and the control food are adjusted so that the content of theacinensin is the same. TS fraction was added and the amount of sucrose added was reduced from the control diet.
-TS double-added food: The TS fraction was added to the control food so that the content of theasinensin was double that of the extract-added test food, and the amount of sucrose added was reduced from the control food.
-TF-added food: Since the hot water extract (lyophilized product) contains 4.8% by mass of theaflavin, the above-described extract-added test meal has the same content of theaflavin as the control food. The TF fraction was added, and the amount of sucrose added was reduced from the control diet.
-TF double addition food: The TF fraction was added to the control food so that the theaflavin content was twice that of the extract-added test food, and the amount of sucrose added was reduced from the control food.
TS + TF-added diet: The TS fraction and TF fraction are added to the control diet so that the theasinensin content and theaflavin content are equal to the extract-added test diet, respectively. Reduced from the control diet.

During the breeding period, food intake was measured every day, and body weight and water intake were measured every other day.
Two, four and six weeks after the start of feeding the test meal, blood glucose was measured using a Medisafe chip (manufactured by Terumo Corp.) after 6 hours of fasting and blood was collected from the tail vein. FIGS. 14 to 17 show changes in blood glucose levels in mice for 6 weeks after taking the test meal.
As shown in FIG. 14, the blood glucose level of the mice that took the control diet increased with time and exceeded 300 mg / dl after 6 weeks. Also, since the amount of water intake increased in the same manner, mice that received the control diet are considered to have developed diabetes. On the other hand, the blood glucose level of the mice that received the extract-added diet tended to decrease over time, and the value after 6 weeks decreased to about half that of the mice that received the control diet.
As shown in FIG. 15, the blood glucose level of the mice ingesting the TS-added diet decreased after 6 weeks, and when the TS2-fold added diet was ingested, the blood glucose level was further effectively decreased.
As shown in FIG. 16, the blood glucose level of the mice that received the TF-added diet decreased after 6 weeks, and when the TF double-added diet was consumed, the blood glucose level decreased after 4 weeks.
As shown in FIG. 17, the blood glucose level of the mice that received the TS + TF-added diet was almost the same level as that of the mice that received the extract-added diet after 4 weeks.
From the above results, it was clarified that the blood glucose rise inhibitory action of the hot water extract of mixed fermented tea leaves was mainly exerted by theacinensin and theaflavin.

[Clinical trial example 1]
We investigated the effects of ingestion of beverages containing mixed fermented tea leaf extract on blood glucose levels, serum neutral fat, serum cholesterol, body fat percentage and blood pressure in adults. This study was approved by the Prefectural Nagasaki Siebold University Research Ethics Committee and was conducted in compliance with the spirit of the Declaration of Helsinki.
The subject is a Nagasaki prefectural government official who applied for recruitment within the Nagasaki prefectural office, and has a daily life similar to that of a healthy person. The 28 persons who were judged and received sufficient explanation about the contents of this study and submitted their consent form by their own free will.
The test beverage was prepared by roasting mixed fermented tea leaves against 100 parts by mass of hot water at 90 ° C. (tea leaf to loquat blending ratio 9: 1, twisting time 20 minutes, fermentation time 0 hours, moisture content (1% by mass) was added for 0.8 minutes and extracted for 4 minutes, and the filtrate was filtered and filled in a 200 ml pack and sealed.
The test drinks were allowed to be drunk for 3 months at breakfast or lunch and dinner, each with a total of 2 drinks per day. Blood intake, body weight measurement, body fat percentage measurement, and blood pressure (systolic blood pressure and diastolic blood pressure) were measured on an early morning fasting, 1 month, 2 months, and 3 months after ingestion of the test beverage. In addition, eating and drinking after 9:00 pm the day before blood collection was prohibited. There were no special restrictions on meals during the three-month test period, and guidance was given to live a life that was not different from everyday life.
As measurement items for blood components, total protein, A / G ratio, total cholesterol, HDL-cholesterol, neutral fat, free fatty acid, uric acid, urea nitrogen, creatinine, Na, Cl, K, GOT, GPT, γ-GTP Phospholipid, Ca, inorganic phosphorus, cholinesterase, LDH, ALP, albumin, direct bilirubin, indirect bilirubin, insulin, blood glucose, hemoglobin A1c, white blood cell count, red blood cell count, hemoglobin content, hematocrit, MCV, MCH, MCHC, platelet count It was measured.

During the three months of taking the test beverage, the average value of 28 subjects was within the normal range in all measurement items.
18 and 19 are graphs showing blood glucose level measurement results. FIG. 18 is an average value of all 28 persons, and FIG. 19 is a human whose blood glucose level (initial value) before the start of test beverage intake is 110 mg / dL or more. The average value of (10 persons) is represented by the fluctuation rate with respect to the initial value. The fluctuation rate is a value calculated by (average value of measured values−initial value) / initial value × 100 (unit:%), and “*” in the figure is significantly different from the initial value. (Hereinafter the same).
20 and 21 are graphs showing the measurement results of serum triglyceride concentration. FIG. 20 shows the average value of all 28 persons, and FIG. 21 shows the serum triglyceride concentration (initial value) before the start of test beverage intake. The average value of humans (9 persons) having a dose of 150 mg / dL or more is expressed as a variation rate with respect to the initial value.
22 and 23 are graphs showing the measurement results of serum cholesterol concentration, FIG. 22 is the average value of all 28 persons, and FIG. 23 is the serum cholesterol concentration (initial value) before starting the test beverage intake is 220 mg / dL or more. The average value of humans (11 persons) is expressed as a variation rate with respect to the initial value.
24 and 25 are graphs showing the measurement results of the body fat percentage, FIG. 24 is an average value of all 28 persons, and FIG. 25 is a body fat percentage (initial value) before starting the test beverage intake of 25% or more. The average value of humans (15 persons) is represented by the rate of change with respect to the initial value.
FIGS. 26 and 27 are graphs showing the measurement results of systolic blood pressure. FIG. 26 shows an average value of all 28 persons, and FIG. 27 shows a human having a maximum blood pressure (initial value) before starting the test beverage intake of 140 mmHg or more (14 The average value of the name) is expressed as the rate of change with respect to the initial value.
28 and 29 are graphs showing the measurement results of the diastolic blood pressure, FIG. 28 is an average value of all 28 persons, and FIG. 29 is a human (10) whose diastolic blood pressure (initial value) before starting the test beverage intake is 90 mmHg or more. The average value of the name) is expressed as the rate of change with respect to the initial value.
From these results, the average value of the blood sugar level did not change during the test period, but in humans with a high initial value, it significantly decreased 2 months after the start of intake and 3 months later than before the intake.
Regarding the serum triglyceride concentration, the average value of all members did not change during the test period, but in humans with a high initial value, it decreased by about 40% after 2 months and 3 months after ingestion compared to before intake.
Regarding the serum cholesterol concentration, the average value of all the members did not change during the test period, but in humans with a high initial value, it significantly decreased after 2 months and 3 months after ingestion compared to before intake.
Regarding the body fat percentage, the average value of all the members did not change during the test period, but the initial value was slightly high in humans with a slightly high tendency toward obesity after 3 months from the start of intake.
Regarding the systolic blood pressure, the average value of all the members did not change during the test period, but in the human with a high initial value, it significantly decreased compared to before intake after 1 month, 2 months and 3 months after the start of intake.
As for diastolic blood pressure, the average value of all the members did not change during the study period, but in humans with a high initial value, it significantly decreased compared to before 2 months after ingestion and after 3 months.
From these results, by ingesting a beverage containing the mixed fermented tea leaf extract, in humans who tend to have high blood sugar levels, serum triglycerides, serum cholesterol, systolic blood pressure and diastolic blood pressure, these It became clear that the effect which improves a value is acquired. In addition, it has been clarified that an effect of reducing the body fat percentage of a human who is somewhat obese tends to be obtained. Furthermore, since it does not reduce the above measured values in normal humans, it is evaluated as a highly safe beverage.

[Clinical trial example 2]
We investigated the effects of ingestion of beverages containing extracts of mixed fermented tea leaves on body fat percentage in young girls. This study was approved by the Prefectural Nagasaki Siebold University Research Ethics Committee and was conducted in compliance with the spirit of the Declaration of Helsinki.
The subject is a female student of this university who applied for recruitment at the prefectural Nagasaki Siebold University. 54 adult female students who have been judged appropriate and who have received sufficient explanations about the content of the exam and who submitted their consent form on their own free will.
The test beverage used was the same as in the above clinical test example 1. As a control beverage, a commercially available green tea beverage with a 200 ml paper pack was used.
The test drink was given to 27 subjects and the control drink was given to 27 subjects for a total of 2 drinks each day at breakfast or lunch and dinner for 3 months.
Prior to the start of ingestion, the blood sugar level and body fat percentage of all the members were measured, and the test drink intake group and the control drink intake group were divided into groups so that the average values were almost equal. The appearance of the test beverage and the control beverage was the same, so that they could not be distinguished from the appearance. Blood intake, body weight measurement, body fat measurement, and blood pressure measurement were performed on an early morning fasting, 1 month, 2 months, and 3 months after ingestion of the test beverage. In addition, eating and drinking after 9:00 pm the day before blood collection was prohibited. There were no special restrictions on meals for the three months of the exam, and guidance was given to live as usual.
The measurement items of blood components were the same as in the above clinical test example 1.
During the three months of ingesting the test beverage, the average value of 54 subjects in all measurement items was within the normal range. In addition, during the three months, no difference was observed in all measurement items between 27 subjects who took the test beverage and 27 subjects who took the control beverage.
FIG. 30 shows the body fat percentage of humans (11 in the test drink ingestor and 13 in the control drink ingestor) whose body fat percentage (initial value) before the start of the test is 25% or more, as a percentage of the initial value. ing.
As shown in this figure, the body fat percentage after 3 months in the test fat-taken group in subjects with a slightly obese tendency whose initial body fat percentage is 25% or more is higher than that in the group that took the control drink. Significantly decreased.
From these results, it was clarified that the effect of lowering the body fat percentage of young women who are slightly obese tends to be obtained by ingesting a beverage containing an extract of mixed fermented tea leaves. It was also confirmed that it was safe for normal young girls to take.

According to the present invention, fermented tea leaves, fermented tea leaf extracts, and foods and drinks having medicinal effects effective for the prevention or improvement of diabetes and hyperlipidemia as described above can be obtained.
Moreover, the fermented tea leaves of the present invention can effectively use loquat leaves that have not been used effectively so far, and there is also an advantage that tea leaves that have been cut up until now can be used as raw tea leaves. Have.

It is a figure which shows the high performance liquid chromatography analysis result of the mixed fermented tea leaf extract which concerns on this invention. It is a flowchart which shows the example of the component separation operation of the mixed fermented tea leaves which concern on this invention. It is a figure which shows the example of the polyphenol contained in the mixed fermented tea leaf which concerns on this invention. It is a figure which shows the high-speed liquid-romatography analysis result of the fraction obtained by the component separation operation of the mixed fermented tea leaf which concerns on invention. It is a figure which shows the presumed structure of the proanthocyanidin contained in the fraction obtained by the component separation operation of the mixed fermented tea leaf which concerns on invention, and the example of the thiol decomposition product. It is a figure which shows the high performance liquid chromatography analysis result of the water-soluble fraction of the mixed fermented tea leaf extract which concerns on this invention, and the independent fermented tea leaf extract as a comparative example. It is a figure which shows the high performance liquid chromatography analysis result of the ethyl acetate soluble fraction of the mixed fermented tea leaf extract which concerns on this invention, and the independent fermented tea leaf extract as a comparative example. It is a figure which shows the high performance liquid chromatographic analysis result of the oxidation type high molecular polyphenol fraction isolate | separated from the mixed fermented tea leaf which concerns on this invention, and its thiol decomposition product. It is a figure which shows the ¹³ C-NMR spectrum of the oxidation-type high molecular weight polyphenol fraction isolate | separated from the mixed fermented tea leaf which concerns on this invention, and Biwa proanthocyanidin. 10 is a graph showing the results of Experimental Example 2. 10 is a graph showing the results of Experimental Example 3. 10 is a graph showing the results of Experimental Example 4. 10 is a graph showing the results of Experimental Example 8. 14 is a graph showing the results of Experimental Example 13. 14 is a graph showing the results of Experimental Example 13. 14 is a graph showing the results of Experimental Example 13. 14 is a graph showing the results of Experimental Example 13. 2 is a graph showing the results of Clinical Test Example 1. 2 is a graph showing the results of Clinical Test Example 1. 2 is a graph showing the results of Clinical Test Example 1. 2 is a graph showing the results of Clinical Test Example 1. 2 is a graph showing the results of Clinical Test Example 1. 2 is a graph showing the results of Clinical Test Example 1. 2 is a graph showing the results of Clinical Test Example 1. 2 is a graph showing the results of Clinical Test Example 1. 2 is a graph showing the results of Clinical Test Example 1. 2 is a graph showing the results of Clinical Test Example 1. 2 is a graph showing the results of Clinical Test Example 1. 2 is a graph showing the results of Clinical Test Example 1. 6 is a graph showing the results of Clinical Test Example 2.

Claims (18)

While twisting tea leaves with a twisting machine, adding a loquat leaf , mixing and twisting, and after the twisting process, immediately heating and stopping the fermentation,
In the twisting step, the total twisting time from the start to the end of twisting tea leaves is 15 to 25 minutes, and after the time of 0 to 40% of the total twisting time has elapsed, loquat leaves are added. method of manufacturing a fermented tea leaves to be. In method massaged by hand, while maceration the leaves of tea, has a maceration mixing and maceration by adding loquat leaf, after該揉twisting step, a step of stopping the fermentation by heating immediately,
In the twisting step, the total twisting time from the start to the end of tea leaf twisting is set to 20 to 30 minutes, and 0-40% of the total twisting time has elapsed, and then loquat leaves are added. method of manufacturing a fermented tea leaves to be. While ground leaves of tea, has a grinding step for mixing and sliding grain Mr added loquat leaf, after the grinding step, a step of stopping the fermentation by heating immediately,
In the mashing step, the total mashing time from the start to the end of mashing of tea leaves is 15 to 25 minutes, and after the time of 0 to 40% of the total mashing time has elapsed, loquat leaves are added. method of manufacturing a fermented tea leaves to be. A crushing and stirring step in which water is added to tea leaves and pulverized and stirred while adding loquat leaves and mixed and pulverized and stirred, and after the pulverizing and stirring step, in an environment of temperature 20 to 27 ° C. and humidity 30 to 60% RH And then fermenting for 4 to 12 hours, followed by heating to stop the fermentation ,
In the pulverization and stirring step, the total pulverization and stirring time from the start to the end of the pulverization and stirring of tea leaves is set to 5 to 15 minutes, and after 50 to 50% of the total pulverization and stirring time has elapsed, loquat leaves are added. A method for producing fermented tea leaves. Fermented tea leaves obtained by the production method according to claim 1. 6. The fermented tea leaf according to claim 5 , wherein the content of theaflavins contained in the fermented tea leaf is greater than the content of theaflavins contained in a single fermented tea leaf obtained by fermenting the tea leaf alone. Oxidation of catechin gallates in which the fermented tea leaves show a signal derived from phloroglucinol and galloyl group of catechin A ring in ¹³ C-NMR spectrum, and the molecular weight of the acetylated compound is 1000 to 15000 with a maximum of 2000 Condensed polyphenol P is contained, and the content of the polyphenol P in the fermented tea leaf is larger than the content of the polyphenol P contained in the single fermented tea leaf obtained by fermenting the tea leaf alone. The fermented tea leaves according to claim 5 . The fermented tea leaves comprise aroma components of 1-hexanol, trans-2-hexenal, 3-hexen-1-ol acetate, 3-hexen-1-ol, linalool, benzaldehyde, methyl salicylate, geraniol, and nerolidol; In the solid phase microextraction method, the peak areas of the aromatic components of trans-2-hexenal, 3-hexen-1-ol acetate, 3-hexen-1-ol, linalool, benzaldehyde, methyl salicylate, geraniol, and nerolidol are Both are larger than the peak area of 1-hexanol, The fermented tea leaves of Claim 5 characterized by the above-mentioned. A fermented tea leaf extract obtained by extracting the fermented tea leaf according to claim 5 . A food or drink containing the fermented tea leaves according to claim 5 . Food / beverage products containing the fermented tea leaf extract of Claim 9 . The food or drink according to claim 10 or 11, which is used for suppressing an increase in blood glucose level. The food or drink according to claim 10 or 11, which is used for prevention or improvement of diabetes. The food or drink according to claim 10 or 11 , which is used for reducing neutral fat. The food or drink according to claim 10 or 11 , which is for cholesterol reduction. The food or drink according to claim 10 or 11, which is used for reducing serum lipid peroxide. The food or drink according to claim 10 or 11, which is used for suppressing body fat accumulation. The food or drink according to claim 10 or 11 , which is used for suppressing blood pressure elevation.

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