CN101011091A - Fermented tea, extract of fermented tea, composition for inhibiting elevation of blood sugar value, and beverage and foodstuff - Google Patents

Abstract

The present invention provides a new type fermentation tea exhibiting effective medicative effects for maintaining or improving health, fermentation tea extract and alimentary products using same. It means that there are provided fermentation tea which contains leafages of mixed and knead tea and loquat leaf therein and suffers a fermentation course, fermentation tea extract obtained by extracting the tea, and alimentary products containing the fermentation tea and/or the fermentation tea extract.

Description

Fermented tea, fermented tea extract, be used to suppress composition and diet product that blood glucose value rises

Technical field

The present invention relates to mix the leaf that uses tea and loguat leaf and with its fermentation and fermented tea, fermented tea extract that forms and the diet product that use these.In addition, the invention provides and be used to suppress composition that blood glucose value rises and the diet product that contain said composition.

Background technology

Effects (for example, following non-patent literature 1) such as known tea set Wheat Protein, antitumaous effect, pre-anti-cancer, the effect of reduction blood cholesterol, the effect of inhibition increased blood pressure.

In following patent documentation 1, the diet product of the material of the inhibition AMS that contains in the extract of Guava Leaf have been proposed to use.

In addition, now very high to the care of thereby the lifestyle disease that cause former because of irregular dietetic life or hypomotility etc., wherein, diabetes have the tendency that increases considerably.Even do not cause diabetes, high blood glucose value also becomes the signal of various lifestyle diseases, in addition, because obesity is considered to cause easily lifestyle disease, therefore, suppresses the diet product of blood glucose value rising and is attracted attention.

Except the Guava Leaf extract of record in following patent documentation 1, as the food specific for health care of effect with absorption of stably carrying out sugar, known have with indigestible dextrins, little leucosin, L-arabinose, the fermented soya bean extract diet product as active ingredient.

[patent documentation 1] spy opens flat 7-59539 communique

[non-patent literature 1] " chemistry of tea " village pine is respected a youth and compiles (towards storehouse bookstore) p124-191

Summary of the invention

The problem that invention will solve

The raw material or the method for making of tea are varied, still have the part of a lot of the unknowns for effective component.

Because tea is safe food, can habitually absorb, therefore, the expectation exploitation is to keeping or improve the novel tea of the effective drug effect of healthy performance.

In addition, in recent years,, therefore require more effectively to be used to suppress the composition and the diet product of blood glucose value rising to suppressing the raising that focuses on of diet product that blood glucose value rises.

The present invention In view of the foregoing makes, and its purpose is, provides a kind of to keeping or improve novel fermented tea, fermented tea extract that health has effective drug effect and the diet product that use it.

The method of dealing with problems

The invention provides a kind of fermented tea, it is characterized in that, mix, knead the leaf and the loguat leaf of tea, and ferment.

The invention provides a kind of fermented tea, it is characterized in that, the leaf of tea blend and loguat leaf, and disorganize ferment again.

The invention provides a kind of fermented tea, it is characterized in that, the leaf of tea blend and loguat leaf, and grind, ferment again.

The invention provides a kind of fermented tea, it is characterized in that, in the leaf of tea and loguat leaf, add entry and pulverize and mix, ferment again.

The invention provides a kind of fermented tea extract that extracts fermented tea of the present invention and obtain.

The invention provides a kind of diet product that contain fermented tea of the present invention.

The invention provides a kind of diet product that contain fermented tea extract of the present invention.

Diet product of the present invention are suitable as and suppress the diet product that blood glucose value rises.

Diet product of the present invention are suitable as the diet product that are used to prevent or improve diabetes.

Diet product of the present invention are suitable as the diet product that are used to reduce neutral fat.

Diet product of the present invention are suitable as the diet product that are used to reduce cholesterol.

Diet product of the present invention are suitable as the diet product that are used to reduce the serum lipid peroxide.

Diet product of the present invention are suitable as and are used to suppress the diet product that body fat is accumulated.

Diet product of the present invention are suitable as the diet product that are used to suppress increased blood pressure.

In addition; discoveries such as the inventor; the mixed culture fermentation tealeaves of making as raw material with the leaf of tea and loguat leaf as AGH inhibition (maltose inhibition and the invertase inhibition) height that suppresses blood glucose value effect of increasing index; and the effect that the excellent inhibition blood glucose value of performance rises in the experiment of using mouse; to so specific found that of furtheing investigate of bringing the active ingredient of ascending effect on the excellent inhibition blood glucose value; Tea Polyphenols (theasinensin) A; theaflavin derivative with galloyl base; with the epiafzelechin gallate is the OPC of construction unit; and each composition such as polyphenol P of catechin gallic acid ester class oxidative condensation shows high AGH inhibition respectively, and described polyphenol P exists ¹³Show in the C-NMR spectrogram that the molecular weight of its acetylate is 1000～15000 from the signal of the phloroglucin of catechin A ring with from the signal of galloyl base, and 2000 serve as greatly, down to finishing the present invention.

Promptly; the invention provides a kind of composition that blood glucose value rises that is used to suppress; wherein contain be selected from the following substances more than a kind: Tea Polyphenols A, have the theaflavin derivative of galloyl base, be the OPC of construction unit and the polyphenol P of catechin gallic acid ester class oxidative condensation with the epiafzelechin gallate, this polyphenol P exists ¹³Show in the C-NMR spectrogram that the molecular weight of its acetylate is 1000～15000 from the signal of the phloroglucin of catechin A ring with from the signal of galloyl base, and serve as very big with 2000.

Preferably be used for suppressing the composition that blood glucose value rises, also contain the loquat OPC.

In addition; the invention provides a kind of composition that blood glucose value rises that is used to suppress; wherein comprise fermented tea; described fermented tea contain be selected from the following substances more than a kind: Tea Polyphenols A, have the theaflavin derivative of galloyl base, be the OPC of construction unit and the polyphenol P of catechin gallic acid ester class oxidative condensation with the epiafzelechin gallate, this polyphenol P exists ¹³Show in the C-NMR spectrogram that the molecular weight of its acetylate is 1000～15000 from the signal of the phloroglucin of catechin A ring with from the signal of galloyl base, and serve as very big with 2000.

Preferably be used for suppressing the composition that blood glucose value rises, above-mentioned fermented tea also contains the loquat OPC.

In addition; the invention provides a kind of composition that blood glucose value rises that is used to suppress; wherein comprise the fermented tea extract; described fermented tea extract contain be selected from the following substances more than a kind: Tea Polyphenols A, have the theaflavin derivative of galloyl base, be the OPC of construction unit and the polyphenol P of catechin gallic acid ester class oxidative condensation with the epiafzelechin gallate, this polyphenol P exists ¹³Show in the C-NMR spectrogram that the molecular weight of its acetylate is 1000～15000 from the signal of the phloroglucin of catechin A ring with from the signal of galloyl, and serve as very big with 2000.

Preferably be used for suppressing the composition that blood glucose value rises, above-mentioned fermented tea extract also contains the loquat OPC.

In addition, the invention provides a kind of diet product that are used to suppress the composition that blood glucose value rises of the present invention that contain.

The effect of invention

According to the present invention, can obtain having effect, prevention or the effect of improving diabetes, the effect that reduces neutral fat, the effect that reduces cholesterol, the effect that reduces the serum lipid peroxide that suppress blood glucose value and rise, suppress the effect that body fat accumulates or the effect that suppresses increased blood pressure such for novel fermented tea, the fermented tea extract of keeping/improves healthy effective effect and the diet product that use these.

In addition, according to the present invention, can obtain having the composition of good inhibition blood glucose value effect of increasing and the diet product that contain said composition.

Description of drawings

Fig. 1 is the figure that the high-speed liquid phase chromatogram analysis result of the mixed culture fermentation tea extract that the present invention relates to is shown.

Fig. 2 is the flow chart of example that the component separation operation of the mixed culture fermentation tealeaves that the present invention relates to is shown.

Fig. 3 is the figure that the example of the polyphenol that contains in the mixed culture fermentation tealeaves that the present invention relates to is shown.

Fig. 4 illustrates the figure that operates the high-speed liquid phase chromatogram analysis result of the fraction that obtains with the component separation of the mixed culture fermentation tealeaves that the present invention relates to.

Fig. 5 is the figure that the example of the supposition structure of the OPC that contains in the fraction that the component separation operation with the mixed culture fermentation tealeaves that the present invention relates to obtains and mercaptan decomposition thing is shown.

Fig. 6 is high-speed liquid phase chromatogram analysis result's the figure that the water-soluble fraction of the mixed culture fermentation tea extract that the present invention relates to and independent fermented tea extract as a comparative example is shown.

Fig. 7 is high-speed liquid phase chromatogram analysis result's the figure that the solvable fraction of ethyl acetate of the mixed culture fermentation tea extract that the present invention relates to and independent fermented tea extract as a comparative example is shown.

Fig. 8 is the figure that the high-speed liquid phase chromatogram analysis result of the oxidized form high-molecular weight polyphenol fraction of separating from the mixed culture fermentation tealeaves that the present invention relates to and mercaptan decomposition thing thereof is shown.

Fig. 9 illustrates the oxidized form high-molecular weight polyphenol fraction of separating and loquat OPC from the mixed culture fermentation tealeaves that the present invention relates to ¹³The figure of C-NMR spectrogram.

Figure 10 is the curve map that the result of experimental example 2 is shown.

Figure 11 is the curve map that the result of experimental example 3 is shown.

Figure 12 is the curve map that the result of experimental example 4 is shown.

Figure 13 is the curve map that the result of experimental example 8 is shown.

Figure 14 is the curve map that the result of experimental example 13 is shown.

Figure 15 is the curve map that the result of experimental example 13 is shown.

Figure 16 is the curve map that the result of experimental example 13 is shown.

Figure 17 is the curve map that the result of experimental example 13 is shown.

Figure 18 is the curve map that the result of clinical testing example 1 is shown.

Figure 19 is the curve map that the result of clinical testing example 1 is shown.

Figure 20 is the curve map that the result of clinical testing example 1 is shown.

Figure 21 is the curve map that the result of clinical testing example 1 is shown.

Figure 22 is the curve map that the result of clinical testing example 1 is shown.

Figure 23 is the curve map that the result of clinical testing example 1 is shown.

Figure 24 is the curve map that the result of clinical testing example 1 is shown.

Figure 25 is the curve map that the result of clinical testing example 1 is shown.

Figure 26 is the curve map that the result of clinical testing example 1 is shown.

Figure 27 is the curve map that the result of clinical testing example 1 is shown.

Figure 28 is the curve map that the result of clinical testing example 1 is shown.

Figure 29 is the curve map that the result of clinical testing example 1 is shown.

Figure 30 is the curve map that the result of clinical testing example 2 is shown.

The specific embodiment

[I] at first describes fermented tea of the present invention, fermented tea extract and the diet product that contain them.

The raw material of＜fermented tea 〉

In the present invention, the leaf of the tea that uses as raw material is the leaf of the evergreen shrubs Thea sinensis L. of said Theaceae.As this leaf, except that the newly picked and processed tea leaves of plucking at first, can also use the tea that tea, the tea of plucking for the third time, the autumn and winter of plucking for the second time pluck, the tea of harvesting etc., preferably contain the leaf of the tea of the later cheapness of the tea plucked for the second time of many Polyphenols.In addition, these thick tea that cut late are cheap now, and quite a few goes out of use, and can effectively utilize this thick tea.

Particularly, the tealeaves of the China seed that those pestles of east, Nagasaki that use in experimental example described later and analytical test example produce, preferably in the tealeaves that shallow lake overgrown with wild plants north tea tree (ヤ Block キ ) is planted, only to be identified the epiafzelechin-3-O-gallic acid ester that has that exists seldom in natural be the OPC of construction unit owing to contain, so preferably.In addition, have epiafzelechin as containing-the 3-O-gallic acid ester is the tealeaves of the OPC of construction unit, can enumerate the tealeaves that is used for making Darjeeling's black tea that India produces and homemade Qimen (キ one モ Application) black tea.

As hereinafter described, this to have epiafzelechin gallate be that the OPC of construction unit has high AGH inhibition (maltose inhibition and invertase inhibition).

In the present invention, the loguat leaf that uses as raw material is the leaf of the rose family, plant name " loquat ", can use with being not particularly limited.

The manufacture method of＜fermented tea 〉

Fermented tea of the present invention (below, be also referred to as mixed culture fermentation tealeaves) the as described below manufacturing.

At first, as pre-treatment, preferred as required with the leaf drying of loguat leaf and tea, making moisture content is about 50～60 quality %.In addition, preferably be cut into suitable size, for example the size of 1～10mm about square.

Then, with the leaf drying of tea, amount of moisture is reduced, and make it wilting.In this operation, can make with the following method, for example, on one side the leaf that uses rough kneading machine, the pot that directly heats with fire or electricity consumption or tabular utensil to stir tea, Yi Bian be 40～150 ℃ the method that hot-air contacts the leaf of tea that adds with temperature; Tealeaves is put in the container of airtight stirring and the air in the aspiration container, made inside become the state of decompression and stir dry method; The method of using wilting groove and ventilating etc. from the below that is dispersed in online tealeaves.

Wilting by this, if make the moisture content of the leaf of raw material tea reduce to 45～65 quality %, be preferably about 50～60 quality %, owing to be difficult to from the leaf of tea, rub out moisture in the kneading operation below, therefore can prevent the loss of active ingredient, can suppress the reduction of quality simultaneously.In addition, on this aspect of the drying process that can shorten the back, also be preferred.

Then, the leaf limit of tea that to pass through the raw material of wilting operation adds flanging and rubs (kneading), simultaneously, when kneading, add loguat leaf, both are kneaded together, loguat leaf can add when beginning to knead the leaf of tea, perhaps also can add loguat leaf and knead after carry out the leaf that only kneads tea of a period of time.Preferably after beginning time of 0～40% through total kneading time, kneading adds loguat leaf again.Be more preferably from the initial interpolation loguat leaf of kneading operation.

The mass ratio that parches of the leaf of the preferred loguat leaf/tea of addition of loguat leaf is 5/95～35/65 scope, is more preferably 8/92～30/70 scope.If the addition of loguat leaf is in the above-mentioned scope,, can obtain to suppress the raising effect of blood glucose value effect of increasing well by the leaf and the loguat leaf of tea blend.

The known method such as method of the common puy that use uses can be suitably adopted in kneading in the kneading of the leaf of tea.

The kneading time is 15～25 minutes.By being set at this scope, can making and suppress the function well that blood glucose value rises.

In addition, the temperature of the raw material during kneading is 20～40 ℃.Be lower than 20 ℃ of insufficient fermentations, when surpassing 40 ℃, quality reduces significantly.

By this kneading, organizing of the leaf of tea and loguat leaf is destroyed, and the oxidizing ferment such as polyphenol oxidase that contain in the leaf of tea generate oxypolymer with the polyphenol oxidase, the polymerization that contain in the leaf of tea and the loguat leaf.

Then, the mixed material of this state is transferred in the fermentation procedure.That is, the mixed material after the kneading is left standstill under the environment in the fermenting cellar of 20～27 ℃ of temperature, humidity 30～60%RH etc. to be piled into the thick state of several cm.In addition, said in the manufacturing process of teas " fermentation " is meant the meaning of carrying out oxidation reaction by the oxidizing ferment in the leaf.

Fermentation time is 0～4 hour.Here, fermentation time is 0 hour to be owing to begin fermentation in the kneading operation formerly when beginning to knead, and the time that will not knead operation can claim that also fermentation time is 0 hour, even also fermented in 0 hour in fact when being included in the fermentation procedure.When fermentation time surpasses 4 hours, the tendency that the mixed culture fermentation tealeaves that obtains or the taste of its extract, fragrance have reduction.In order to obtain excellent especially AGH inhibition, fermentation time is preferred below 1 hour, the most preferred 0 hour.

Then, behind the fermentation time through regulation, heating raw stops fermentation and carries out drying.For example, raw material is put in the continuous drier, and to the hot blast that wherein is blown into 80～120 ℃ of temperature, making delivery temperature is that 50～60 ℃ of ground are operated.Be to be exactly that thus, can make the moisture content in the raw material is about 5 quality % fully about 10～30 minutes heat time heating time.

In above-mentioned manufacturing process, kneading time, fermentation time are long more, and it is low more that the AGH inhibition becomes.In addition, also step-down of antioxidation (1,1-two picryls-2-phenyl diazanyl (DPPH) cancellation activity).When paying close attention to functional the manufacturing, it is desirable to knead time set is 15～25 minutes, and fermentation time is short as far as possible.

In addition, about taste, the long more bitterness sense of fermentation time is grow more, and fragrance is variation also, and fermentation time is short more, and taste, fragrance are also excellent more.

Like this, can obtain mixed culture fermentation tealeaves.Tealeaves after the heating process is so-called thick tea (waste tea), can also implement fine finishining to it as required, makes fine finishining tea.The same operation of fine finishining in drying, heating, screening, shaping, sorting etc. again and the manufacturing of tealeaves in the past can be suitably adopted in fine finishining.Above-mentioned heating is to heat the operation of seeking to tart up once more.By carrying out fine finishining, can improve qualities such as storage property, fragrance, thereby commodity is improved.In addition, thick tea or fine finishining tea can also be made the Powdered of suitable size.

In addition, as mentioned above, above-mentioned kneading operation can be destroyed the leaf of tea and the tissue of loguat leaf, except the method for using puy, can also use the method for for example rubbing with hand, the method that grinds, add method that entry pulverizes and wait and carry out.

During method that use is rubbed with hand, can suitably use the method for using when rubbing the leaf that mixes tea usually.For example, can adopt what be called as " opening straw mat (ね こ ぶ I) greatly " and rub mixed method etc. with hand, also can use method in addition with having on the concavo-convex place mat fabric of hawser braiding.Particularly, the method for the leaf of the tea by rubbing the raw material that has passed through wilting operation with hand is kneaded, and adds loguat leaf simultaneously when kneading, and both are kneaded together.Preferred 20～30 minutes of the kneading time of this moment, when being lower than 20 minutes or surpassing 30 minutes, the effect step-down that the inhibition blood glucose value of the fermented tea that obtains rises.Other conditions etc. are same as described above.Fermentation procedure can similarly carry out with above-mentioned.

In the method, the long more AGH inhibition of kneading time, fermentation time becomes low more.In addition, also step-down of antioxidation (DPPH cancellation activity).When paying close attention to functional the manufacturing, it is desirable to knead time set is 20～30 minutes, and fermentation time is short as far as possible.

During method that use grinds, can adopt the method for the common mortar that uses in the grinding of the leaf that uses tea etc., also can use the method that grinds in addition.Particularly, grind the leaf of the tea of the raw material that has passed through wilting operation, grind when sneaking at this simultaneously and add loguat leaf, grind and sneak into two kinds of raw materials.Preferred 15～25 minutes of the time that grinds of this moment, when being lower than 15 minutes or surpassing 25 minutes, the effect step-down that the inhibition blood glucose value of the fermented tea that obtains rises.The temperature of the raw material when grinding is 20～40 ℃.It is insufficient to be lower than 20 ℃ of fermentations, and when surpassing 40 ℃, quality reduces.In addition, add when the interpolation time of loguat leaf can begin at the leaf of tea to grind, perhaps also can after carry out the leaf that only grinds tea of certain hour, add loguat leaf and grind again.Can add loguat leaf again after beginning through 0～40% the time that always grinds the time from grinding.Other conditions etc. are same as described above.

Like this, by grinding, organizing of the leaf of tea and the raw material of loguat leaf is destroyed, and the oxidizing ferment such as polyphenol oxidase that contain in the leaf of tea generate oxypolymer with the polyphenol oxidase, the polymerization that contain in the leaf of tea and the loguat leaf.Fermentation procedure can similarly carry out with above-mentioned.

In the method, grind the long more AGH inhibition of time, fermentation time and become low more.In addition, also step-down of antioxidation (DPPH cancellation activity).When paying close attention to functional the manufacturing, it is desirable to grind time set is 15～25 minutes, and fermentation time is short as far as possible.

In addition, about taste, the long more bitterness sense of fermentation time becomes strong more, and fragrance is variation also, and fermentation time is short more, and taste, fragrance are also excellent more.

When use adds the method that entry pulverizes, can be by in the leaf of tea and loguat leaf, adding entry and pulverizing to stir they are mixed, destroy the tissue of the raw material of the leaf of tea and loguat leaf simultaneously.Pulverize to stir the method that can adopt common blender that use uses etc. in the pulverizing of the leaf of tea is stirred, also can use pulverizing stirring means in addition.Particularly, in the leaf of the tea of the raw material that has passed through wilting operation and loguat leaf, add entry and pulverize stirring.The leaf of tea and the mixed proportion of loguat leaf and above-mentioned same.With respect to total amount 100 mass parts of the leaf and the loguat leaf of tea, preferred 20～200 mass parts of the amount of the water of adding are more preferably 50～150 mass parts.

Pulverizing mixing time is 5～15 minutes, when being lower than 5 minutes or surpassing 15 minutes, and the effect step-down that the inhibition blood glucose value of the fermented tea that obtains rises.The temperature of pulverizing the raw material when stirring is same as described above, is 20～40 ℃.

The interpolation time of loguat leaf can add when beginning to pulverize stirring, perhaps also can add loguat leaf and pulverize stirring again after the leaf of the only pulverizing stirring tea that carries out certain hour.Preferably adding loguat leaf again after beginning through total time of 0～50% of pulverizing mixing time from pulverize stirring.

Like this, by pulverize stirring, the leaf and the loguat leaf of tea are mixed, and simultaneously, these raw tissue is destroyed, and the oxidizing ferment such as polyphenol oxidase that contain in the leaf of tea generate oxypolymer with the polyphenol oxidase, the polymerization that contain in the leaf of tea and the loguat leaf.

Then, in will be, the mixed material of pulverizing after stirring be left standstill under the environment in the fermenting cellar of 20～27 ℃ of temperature, humidity 30～60%RH etc. to be piled into the thick state of several cm by the operation that pulverize to stir the mixed material fermentation that obtain.Preferred 4～12 hours of fermentation time.When fermentation time was lower than 4 hours, owing to do not ferment, the effect that therefore suppresses the blood glucose value rising was low, and the green grass taste is arranged.When fermentation time surpassed 12 hours, the taste of the fermented tea that obtains, fragrance had the tendency of reduction.Under the situation of paying attention to drug effect, preferred 4～12 hours.That is, 4～12 hours AGH inhibition of fermentation time is high especially.In addition, antioxidation (DPPH cancellation activity) also uprises.When paying close attention to functional the manufacturing, it is desirable to pulverize mixing time and remained on 10～15 minutes, fermentation time remained on 4～12 hours.In addition, about taste, if beyond this time, bitterness sense grow then, fragrance is variation also.

Then, behind the fermentation time through regulation, heating raw stops fermentation and carries out drying.For example, the container that has poured into raw material is put in the drying machine, and to be blown into temperature be 80～120 ℃ hot blast that making delivery temperature is that 50～60 ℃ of ground are operated.Preferred about 60～120 minutes of heat time heating time, thus, can make the amount of moisture in the raw material is about 5%.Like this, can obtain fermented tea of the present invention, the fermented tea after the heating process can be ground into Powdered, also can not pulverize and uses as solids.

The composition that contains in＜mixed culture fermentation the tealeaves 〉

As the composition that contains in the mixed culture fermentation tealeaves that obtains like this, can enumerate the composition that in analytical test example described later, separates.

Though whole also indeterminate for the active ingredient of bringing into play by mixed culture fermentation tealeaves of the present invention that helps drug effect, but by experimental example described later and analytical test example as can be known, each composition of following (1)～(4) shows high AGH inhibition (maltose inhibition and invertase inhibition).

(1) Tea Polyphenols A (below, sometimes also brief note for TS-A),

(2) have the galloyl base the theaflavin derivative,

(3) have OPC that epiafzelechin gallate is a construction unit,

(4) the polyphenol P of catechin gallic acid ester class oxidative condensation, this polyphenol P exists ¹³Show in the C-NMR spectrogram that the molecular weight of its acetylate is 1000～15000 from the signal of the phloroglucin of catechin A ring with from the signal of galloyl base, and serve as greatly (below, also brief note is polyphenol P sometimes) with 2000.

＜composition (1)～(4) 〉

(1) Tea Polyphenols A is the compound with structure shown in Figure 3.

(2) particularly, the theaflavin derivative with galloyl base be selected from theaflavin-3-O-gallic acid ester (3-TFG), theaflavin-3 '-O-gallic acid ester (3 '-TFG), theaflavin-3,3 '-two-O-gallic acid ester (in 3,3 '-TFGG) more than a kind.Their structure is shown in Fig. 3.In the middle of these, particularly, theaflavin-3, the AGH inhibition of 3 '-two-O-gallic acid ester is stronger.

(3) having epiafzelechin gallate is that the OPC of construction unit is included among the Fr.7 in the analytical test example 3 described later, by the mercaptan decomposition that adopts mercaptoethanol to carry out, generation is selected from epicatechin (EC), epigallocatechin (EGC), epigallocatechin-3-O-gallic acid ester (EGCg), and epicatechin-3-O-gallic acid ester (ECg (below, sometimes also be designated as ECG)) in more than a kind, be selected from EGC-ME (below, " ME " expression " 4-(2 '-hydroxyethylthio) ether ", for example, EGC-ME represents 4-(2 '-hydroxyethylthio) ether of EGC, below identical), EC-ME, among EGCg-ME and the ECg-ME more than a kind, and epiafzelechin-3-O-gallic acid ester-ME.Structure before the mercaptan decomposition of such OPC is estimated as structure for example shown in Figure 4.In addition, the epiafzelechin gallate in (3) composition is meant epiafzelechin-3-O-gallic acid ester.

This composition (3) comes from the leaf of the tea of raw material, as the leaf of the tea of raw material, is included in to use and contains (3) and have in the mixed culture fermentation tealeaves that epiafzelechin gallate makes as the leaf of the tea of the OPC of construction unit.

Above-mentioned (4) polyphenol P is shown in for example analytical test example 5 described later, obtain by following method: at room temperature use the 60% ethanol (ethanol water of concentration 60 volume %, extract the fermented tea that contains polyphenol P down together), extract is used ether successively, ethyl acetate, n-butanol carries out solvent and distributes, can be dissolved in being partly dissolved in 60% methyl alcohol in the n-butanol, and with the material that obtains with the SephadexLH-20 post with 60% methyl alcohol, 80% methyl alcohol, 100% methyl alcohol, it then is methanol-water-acetone (8: 1: 1) mixed solution, methanol-water-acetone (6: 2: 2) mixed solution, water-acetone (1: 1) mixed solution is wash-out successively, again the eluent that obtains is carried out thin-layer chromatography (toluene-Ethyl formate-formic acid, 1: 7: 1) analyze, only collect the fraction that contains not the material that moves from initial point and concentrate.

＜loquat OPC 〉

In addition, the feature of mixed culture fermentation tealeaves of the present invention is to contain the loquat OPC.Shown in the analytical test example as described later 3, can think that in a plurality of fractions that the extract of the mixed culture fermentation tealeaves high by the AGH inhibition obtains, the loquat OPC is included in the highest fraction (Fr.7) of AGH inhibition, therefore, helps the AGH inhibition.

＜theaflavin class 〉

In addition, the feature of mixed culture fermentation tealeaves of the present invention is, by adding loguat leaf, can promote the theaflavin class (theaflavin (TF), theaflavin-3-O-gallic acid ester (3-TFG), theaflavin-3 '-O-gallic acid ester (3 '-TFG), theaflavin-3, the generation of 3 '-two-O-gallic acid ester (3,3 '-TFGG)).

Promptly, the total content of the theaflavin class that contains in the independent fermented tea of total content of the theaflavin class that contains in the mixed culture fermentation tealeaves than the leaf of the tea of making under the same conditions except not adding loguat leaf is many, for example, is more than 1.5 times, preferred more than 2 times, be more preferably more than 2.5 times.Though the upper limit without limits, inferring can not be more than 4 times.

For example, analytical test example 4 described later and Fig. 7 (high-speed liquid chromatography of the ethyl acetate soluble fraction of fermented tea extract (below, sometimes brief note is HPLC) analysis result) in, mixed culture fermentation tealeaves extract is compared with independent fermented tea extract, the total of the peak area of theaflavin class is increased to about 2.7 times, as can be known by adding the generation that loguat leaf has significantly promoted the theaflavin class.

＜polyphenol P 〉

In addition, the feature of mixed culture fermentation tealeaves of the present invention is, by adding the generation that loguat leaf has promoted above-mentioned (4) composition (polyphenol P).

That is, the content of the polyphenol P that contains in the independent fermented tea of the content of the polyphenol P that contains in the mixed culture fermentation tealeaves than the leaf of the tea of making under the same conditions except not adding loguat leaf is many.For example, be more than 1.2 times, preferred more than 2 times, be more preferably more than 3 times.Though the upper limit without limits, inferring can not be more than 4 times.

For example, in analytical test example 4 described later and Fig. 6 (the HPLC analysis result of the water-soluble fraction of fermented tea extract), mixed culture fermentation tealeaves extract is compared with independent fermented tea extract, the peak area of the material that is detected as the projection of baseline is increased to about 1.59 times (159%), can think that the part of this increase is from promoting the catechuic acid oxidation product that generates by adding loguat leaf.In analytical test example 5, this catechuic acid oxidation product is equivalent to the specific composition as above-mentioned polyphenol P.

＜fragrance component 〉

In addition, mixed culture fermentation tealeaves of the present invention has the fragrance component of following feature and forms: the fragrance component that contains 1-hexanol, anti--the 2-hexenoic aldehyde, acetate blatter alcohol ester, blatter alcohol, linalool, benzaldehyde, gaultherolin, geraniol and nerolidol, in the solid phase micro extraction method, all the peak area than 1-hexanol is big for each peak area of the fragrance component of anti--the 2-hexenoic aldehyde, acetate blatter alcohol ester, blatter alcohol, linalool, benzaldehyde, gaultherolin, geraniol and nerolidol.

In the above-mentioned fragrance component of enumerating, shown in the analytical test example as described later 10, linalool, benzaldehyde, gaultherolin, geraniol and nerolidol contain more in fermented teas such as black tea, are few fragrance components in the loguat leaf of non-fermented teas such as green tea and fermentation.In addition, anti--2-hexenoic aldehyde, acetate blatter alcohol ester and blatter alcohol are the more fragrance components that contains in the loguat leaf of fermentation.Mixed culture fermentation tealeaves of the present invention has these fragrance components with many content, thus, can sample the distinctive fragrance as fermented tea.

＜effect 〉

Mixed culture fermentation tealeaves of the present invention, as described later shown in the experimental example, show high AGH inhibition, by absorbing this mixed culture fermentation tealeaves, the effect that the blood glucose value that can be inhibited rises, and can obtain to prevent or improve the effect of the effect of diabetes, the effect that reduces neutral fat, the effect that reduces cholesterol, reduction serum lipid peroxide, the effect that the inhibition body fat is accumulated or the effect that suppresses increased blood pressure.Preferably obtain more than 2 kinds of these effects simultaneously, can also obtain whole effects simultaneously.

＜fermented tea extract 〉

Fermented tea extract of the present invention (below, be also referred to as the mixed culture fermentation tea extract sometimes) is with extracting that the soluble component of solvent with fermented tea extracts and the extract that obtains.Extracting solvent can be water (comprising warm water or hot water), also can be organic solvent.As the object lesson of organic solvent, can enumerate methyl alcohol, ethanol, acetone etc.As the form that is used to suppress the fermented tea extract of the composition that blood glucose value rises of the present invention, be not particularly limited, can be the solution shape, also can be its concentrate, can also be by dry solids such as freeze dryings.Under the situation of solid, can be block, can also be that it is ground into the Powdered of suitable size.When the fermented tea extract is used for the diet product with the form that contains organic solvent, can use ethanol as organic solvent.

Extraction conditions is not particularly limited, but when water extracts, and preferably fermented tea is immersed in temperature and is in 40～100 ℃ warm water or the hot water to extract in about 3 minutes～60 minutes.The ratio of water and fermented tea is preferably with respect to 100 mass parts water, and fermented tea (moisture content 5 quality %) is about 0.5～20 mass parts, is more preferably about 0.5～5 mass parts.

When extracting with organic solvent, preferably with respect to the fermented tea (moisture content 5 quality %) about 100 mass parts organic solvents use, 0.1～20 mass parts, and at normal pressure or add and depress, under the condition of temperature-20～60 ℃, 1～60 minute time, carry out, but be not limited to this scope, can suitably change.

After extracting processing, remove solid constituent, obtain extract by filtration, centrifugation etc.

The soluble component that contains mixed culture fermentation tealeaves in the mixed culture fermentation tea extract shown in experimental example and the analytical test example, shows high AGH inhibition as described later.In addition, as described later shown in experimental example and the analytical test example, by absorbing this mixed culture fermentation tea extract, effect, the prevention that the blood glucose value that can be inhibited rises or improve diabetes effect, reduce neutral fat effect, reduce cholesterol effect, reduce the serum lipid peroxide effect, suppress effect that body fat accumulates or the effect that suppresses increased blood pressure.Preferably obtain more than 2 kinds of these effects simultaneously, can also obtain whole effects simultaneously.

＜diet product 〉

Diet product of the present invention are the diet product that contain the diet product of mixed culture fermentation tealeaves or contain the mixed culture fermentation tea extract, and so long as diet product that can the orally ingestible form then be not particularly limited.Can also contain mixed culture fermentation tealeaves and mixed culture fermentation tea extract.

Diet product of the present invention are the various beverages that for example contain the mixed culture fermentation tea extract.In addition, the mixed culture fermentation tea extract is that water extracts mixed culture fermentation tealeaves and the extract that obtains, if having the local flavor that can directly drink, this extract can also be made tea beverage.

In addition, be to add mixed culture fermentation tealeaves and/or mixed culture fermentation tea extract in the various food materials food.As food material, be not particularly limited, relate to soy food products such as natto, soya-bean milk, sauce, soy sauce; The paste goods that a flesh of fish sweet potato ball, breaded fish stick, cylindric breaded fish stick etc. are tempered; Meat systems such as ham, sausage processed goods; Numerous food raw material such as snack categories such as maltose, toffee, beans filling glutinous rice dessert, red bean jelly.

The mixed culture fermentation tealeaves in the diet product of the present invention and/or the content of mixed culture fermentation tea extract are arbitrarily, can suitably set according to the degree of the drug effect that goes for and the local flavor and the intake of expectation.

For example, be used to suppress under the situation of the food that blood glucose value rises, can be with separately AGH inhibition of the mixed culture fermentation tealeaves that adds and mixed culture fermentation tea extract (IC for example ₅₀) be benchmark, suitably set according to the local flavor and the intake of ascending effect on the inhibition blood glucose value that goes for and expectation.

When the extract that water is extracted fermented tea and obtain was directly made tea beverage, the ratio of water and fermented tea was preferably with respect to the water of 100 mass parts, and fermented tea (moisture content 5 quality %) is about 0.5～20 mass parts.In addition, preferably to be immersed in temperature be 3 minutes～60 minutes method in 40～100 ℃ warm water or the hot water to extracting method.

Fermented tea is added under the situation in the food material, and for example, with respect to the food material of 100 mass parts, fermented tea (moisture content 5 quality %) suitably determines in the scope of 0.1～200 mass parts.

Diet product of the present invention like this contain mixed culture fermentation tealeaves and/or mixed culture fermentation tea extract, by absorbing this material, effect, the prevention that the blood glucose value that can be inhibited rises or improve diabetes effect, reduce neutral fat effect, reduce cholesterol effect, reduce the serum lipid peroxide effect, suppress effect that body fat accumulates or the effect that suppresses increased blood pressure.Preferably obtain more than 2 kinds of these effects simultaneously, can also obtain whole effects simultaneously.

[II] then describes the diet product that are used to suppress the composition of blood glucose value rising and contain said composition of the present invention.

Of the present inventionly be used to suppress composition that blood glucose value rises and contain and be selected from:

(1) Tea Polyphenols A (below, sometimes also brief note for TS-A),

(2) have the galloyl base the theaflavin derivative,

(3) have OPC that epiafzelechin gallate is a construction unit,

(4) the polyphenol P of catechin gallic acid ester class oxidative condensation, this polyphenol P exists ¹³Show in the C-NMR spectrogram that the molecular weight of its acetylate is 1000～15000 from the signal of the phloroglucin of catechin A ring with from the signal of galloyl base, and serve as the very big composition more than a kind or 2 kinds in (below, also brief note is polyphenol P sometimes) with 2000.Combination when containing more than 2 kinds is arbitrarily, is more preferably to contain whole 4 kinds.

Shown in experimental example and the analytical test example, these (1)～(4) composition is any all to be the composition that shows high AGH inhibition (maltose inhibition and invertase inhibition) as described later.

Of the present invention being used to of containing such composition suppressed the composition that blood glucose value rises, by orally ingestible, and the effect that the blood glucose value that can be inhibited rises.

(1)～(4) explanation of composition and above-mentioned＜composition (1)～(4)〉identical.

It is of the present invention that what be used to suppress composition that blood glucose value rises preferred embodiment is fermented tea more than a kind or the fermented tea extract that contains above-mentioned (1)～(4) composition.

So-called fermented tea is not carry out adopting the inactivating oxidase of heating but the fermented tea that obtains through the operation that will knead as the leaf of raw material, produces in manufacture process because the oxidation of the composition that oxidizing ferment produces.As the form that is used to suppress the fermented tea of the composition that blood glucose value rises of the present invention, be not particularly limited, can be that the tealeaves that will produce fermentation heats and stops fermentation, and carry out dry tealeaves (so-called thick tea), in addition, also can be to have implemented accurately machined fine finishining tea as required.In addition, thick tea or processing tea can also be made the Powdered of suitable size.

So-called fermented tea extract is with extracting that the soluble component of solvent with fermented tea extracts and the extract that obtains.Extracting solvent can be water (comprising warm water or hot water), also can be organic solvent.As the object lesson of organic solvent, can enumerate methyl alcohol, ethanol, acetone etc.As the form that is used to suppress the fermented tea extract of the composition that blood glucose value rises of the present invention, be not particularly limited, can be the solution shape, also can be its concentrate, can also be by dry solids such as freeze dryings.Under the situation of solid, can be block, can also be that it is ground into the Powdered of suitable size.When the fermented tea extract is used for the diet product with the form that contains organic solvent, can use ethanol as organic solvent.

Extraction conditions is not particularly limited, but when water extracts, and preferably fermented tea is immersed in temperature and is in 40～100 ℃ warm water or the hot water to extract in 3 minutes～60 minutes.The ratio of water and fermented tea is preferably with respect to 100 mass parts water, and fermented tea (moisture content 5 quality %) is about 0.5～20 mass parts, is more preferably about 0.5～5 mass parts.

When extracting with organic solvent, preferably with respect to 100 mass parts organic solvents, use the fermented tea (moisture content 5 quality %) about 0.1～20 mass parts, and at normal pressure or add and depress, under the condition of temperature-20～60 ℃, 1～60 minute time, carry out, but be not limited to this scope, can suitably change.

After extracting processing, remove solid constituent, obtain extract by filtration, centrifugation etc.

The leaf and the loguat leaf of the preferred tea blend of fermented tea among the present invention and the mixed culture fermentation tealeaves that kneads, ferments and form.In this mixed culture fermentation tealeaves, contain theaflavin derivative and (4) above-mentioned polyphenol P that above-mentioned (1) Tea Polyphenols A, (2) have the galloyl base.

In addition, as the leaf of the tea of raw material, owing to contain above-mentioned (3) and have the composition that contains all above-mentioned (1)～(4) in the mixed culture fermentation tealeaves of epiafzelechin gallate as the leaf manufacturing of the tea of the OPC of construction unit using, so be more preferably.

Feature at above-mentioned mixed culture fermentation tealeaves is to contain the loquat OPC.

The explanation of loquat OPC and above-mentioned＜loquat OPC〉identical.

In addition, in above-mentioned mixed culture fermentation tealeaves, compare with the independent fermented tea that loguat leaf similarly kneads, ferments and obtain separately respectively with the leaf of tea, the theaflavin class (theaflavin (TF), theaflavin-3-O-gallic acid ester (3-TFG), theaflavin-3 '-O-gallic acid ester (3 '-TFG), theaflavin-3, the total content of 3 '-two-O-gallic acid ester (3,3 '-TFGG)) is more.

In addition, in above-mentioned mixed culture fermentation tealeaves, compare with the fermented tea that loguat leaf similarly kneads, ferments and obtain separately respectively with the leaf of tea, the content of (4) above-mentioned polyphenol P is more.

Above-mentioned mixed culture fermentation tealeaves can be with following method manufacturing.

The raw material of the leaf of the tea that uses as raw material and the explanation of loguat leaf and above-mentioned＜fermented tea〉identical.

Make the method for mixed culture fermentation tealeaves and the manufacture method of above-mentioned＜fermented tea〉identical.

Above-mentioned (1) Tea Polyphenols A that is used for suppressing the composition that blood glucose value rises of the present invention, theaflavin derivative, (3) that (2) have the galloyl base have the content of each composition of OPC that epiafzelechin gallate is a construction unit, (4) polyphenol P, and there is no particular limitation.The AGH inhibition that can have with each composition (IC for example ₅₀) be benchmark, the intake that is used to suppress the composition that blood glucose value rises according to ascending effect on the inhibition blood glucose value that goes for and this is suitably set.

Diet product of the present invention contain the composition that blood glucose value rises that is used to suppress of the present invention, and, so long as the diet product of form that can orally ingestible get final product, be not particularly limited.

For example, contain the various beverages that are used to suppress the fermented tea extract of the composition that blood glucose value rises as of the present invention.In addition, this fermented tea extract is that water extracts fermented tea and the extract that obtains, if having the local flavor that can directly drink, this extract can also be made tea beverage.

In addition, be to be used for suppressing the fermented tea of the composition that blood glucose value rises or the food that the fermented tea extract adds various food materials to as of the present invention.As food material, be not particularly limited, relate to soy food products such as natto, soya-bean milk, beans sauce, soy sauce; The paste goods that a flesh of fish sweet potato ball, breaded fish stick, cylindric breaded fish stick etc. are tempered; Meat systems such as ham, sausage processed goods; Numerous food raw material such as snack categories such as maltose, toffee, beans filling glutinous rice dessert, red bean jelly.

The content that being used in the diet product of the present invention suppressed the composition that blood glucose value rises is arbitrarily, can be to be used to suppress the AGH inhibition that composition itself that blood glucose value rises has (IC for example ₅₀) be benchmark, suitably set according to the effect of the inhibition blood glucose value rising that goes for and the local flavor and the intake of expectation.

For example, water extracted fermented tea and the extract that obtains when directly making tea beverage, the ratio of water and fermented tea is preferably with respect to the water of 100 mass parts, and fermented tea (moisture content 5 quality %) is about 0.5～20 mass parts.In addition, extracting method preferably is immersed in the warm water of 40～100 ℃ of temperature or the hot water 3 minutes～60 minutes method.

In addition, when adding to fermented tea in the food material, for example, with respect to the food material of 100 mass parts, fermented tea (moisture content 5 quality %) can suitably determine in the scope of 0.1～200 mass parts.

Like this contain the diet product that are used to suppress the composition that blood glucose value rises of the present invention, can be used to suppress the effect that composition that blood glucose value rises obtains to suppress the blood glucose value rising by absorbing this.

Embodiment

In following experimental example and analytical test example, unless otherwise specified, " % " is " quality % ".

" leaf of tea " of the raw material that uses in following experimental example and the analytical test example is those pestle tealeaves of east, comprehensive agricultural test site, Nagasaki county and props up a leaf of the tea of plucking for the third time of the ヤ Block キ kind of cultivation.In addition, unless otherwise specified, kneading is to use puy to carry out.

In addition, the evaluation of the AGH inhibition in embodiment 1, the analytical test example 1～5 according to the use that proposes by people such as Song Jing the method for free AGH (J.Agric.Food Chem., 47,550-553, (1999)) carry out, and with the inhibiting rate (unit: %) represent measurement result of alpha-Glucosidase (AGH) enzymatic activity.

[experimental example 1]

(manufacturing of mixed culture fermentation tealeaves)

Loguat leaf is put in the leaf of tea, rubbed mixed with puy.The leaf of tea and the mixed proportion of loguat leaf are the leaf of tea: the mass ratio of loguat leaf is 90: 10 (loquat input ratio is 10%) and 75: 25 (loquat input ratio is 25%) 2 kinds.Rubbing does time is two kinds of 20 minutes and 40 minutes.

After this, 6 kinds of fermentation procedures through 0 hour, 1 hour, 2 hours, 4 hours, 6 hours, 24 hours obtain 24 kinds of mixed culture fermentation tealeaves.In addition, 0 hour tealeaves of fermentation procedure is to rub to mix the mixed culture fermentation tealeaves that obtains when just finishing.

(sense organ examination)

According to the checking method of green tea above-mentioned 24 kinds of mixed culture fermentation tealeaves that obtain are carried out the judge of fragrance and taste by 3 participants of sense organ examination experience with green tea.During examination, be in the examination teacup, to add 3g mixed culture fermentation tealeaves and inject 180ml hot water, after injecting hot water, fragrance passed judgment on, after 5 minutes, taste is examined, the results are shown in table 1.

[table 1]

Loquat is dropped into ratio 10%					Loquat is dropped into ratio 25%
										The kneading time (branch)	Fermentation time (hour)	Fragrance	Taste	Add up to	The kneading time (branch)	Fermentation time (hour)	Fragrance	Taste	Add up to
20	0	3.0	3.0	6.0	20	0	3.0	3.7	6.7
										1	3.0	3.0	6.0	1	3.0	3.0	6.0
	2	2.7	3.0	5.7		2	3.0	3.0	6.0
										4	2.7	3.0	5.7	4	3.3	2.7	6.0
	6	2.0	2.7	4.7		6	2.0	2.0	4.0
										24	1.0	1.3	2.3	24	1.0	1.3	2.3
	40	0	3.0	2.3		5.3	40	0	2.7									2.7	5.4
1					2.3					2.3	4.6	1	2.7	2.3	5.0
		2	2.3	2.7		5.0		2	3.0							2.3	5.3
4					2.7					2.3	5.0	4	2.3	2.7	5.0
		6	3.0	1.7		4.7		6	3.0							2.7	5.7
24					1.0					1.3	2.3	24	1.0	1.0	2.0

Fragrance, taste are divided into full marks with 5 respectively

The tea that in the examination of the sense organ of green tea, the newly picked and processed tea leaves of plucking at first are 6～7 minutes, pluck for the second time for be lower than 5 fens slightly, the tea plucked for the third time is 5 minutes

Shown in the result of table 1, about taste, fragrance, in the time of 0～4 hour, fragrance is dense at 20 minutes kneading time, fermentation time, and aftertaste is salubrious, excellence.The kneading time is when being 40 minutes, and taste reduces, and fermentation time is more than 6 hours the time, and the deterioration of fragrance is obvious.

(mensuration of AGH activity)

In above-mentioned 24 kinds of mixed culture fermentation tealeaves that obtain, to being 16 kinds the mixed culture fermentation tealeaves of 6 hours and 24 hours except fermentation time, be determined at extract (the mixed culture fermentation tealeaves use amount: AGH inhibition 2.0mg/ml) of extracting 10 minutes in 100 ℃ the hot water and obtaining.The results are shown in table 2.

In addition, commercially available green tea, loquat tea, guava tea, black tea are similarly used hot water extracting respectively, and extract is similarly measured the AGH inhibition.The results are shown in table 3.

[table 2]

Loquat is dropped into ratio 10%				Loquat is dropped into ratio 25%
								The kneading time (branch)	Fermentation time (hour)	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)	The kneading time (branch)	Fermentation time (hour)	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)
20	0	81.9	82.7	20	0	70.3	61.9
								1	72.3	67.6	1	66.3	53.6
	2	65.4	60.4		2	62.0	46.9
								4	59.3	45.1	4	58.6	40.6
	40	0	66.9		43.4	40	0							69.6	57.5
1				56.3				36.7	1	58.5	38.9
		2	56.9		32.0		2					55.2	37.9
4				54.1				35.7	4	49.6	28.4

[table 3]

Test portion	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)
			Green tea	61.2	61.3
Loguat leaf	55.4	58.5
			Guava tea	48.3	38.3
Black tea	55.6	52.7

As shown in table 2, the AGH inhibition is, loguat leaf becomes for a long time with respect to the cooperation ratio of the leaf of tea, and even the inhibiting rate step-down is long also step-down of kneading time.In addition, along with fermentation time is elongated, inhibiting rate has the tendency of step-down.

In addition, by table 2,3 result as can be known, be 10 quality %, kneading time to be that 20 minutes, fermentation time are 0～1 hour particularly in the cooperation ratio of loguat leaf, and the cooperation ratio of loguat leaf is 25 quality %, kneading time to be that 20 minutes, fermentation time are under 0 hour the condition, can obtain the AGH inhibition than green tea excellence.

[analytical test example 1]

At room temperature use the 70% acetone (aqueous acetone solution of concentration 70 volume %, down with) extract the mixed culture fermentation tealeaves that is displayed in Table 2 excellent AGH inhibition (the cooperation ratio of the leaf of tea and loguat leaf is that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour), with extract concentrate make the aqueous solution after, add ether and carry out the solvent distribution, remove liposoluble constituents such as chlorophyll.The water layer that obtains is analyzed with high-speed liquid chromatography (HPLC), the results are shown in Fig. 1.

As shown in Figure 1, except caffeine as the green tea composition, epicatechin (EC), epigallocatechin (EGC), epigallocatechin-3-O-gallic acid ester (EGCg), and epicatechin-3-O-gallic acid ester (ECg) in addition, also detected the dimer Tea Polyphenols C that generates by the oxidation of these catechins, Tea Polyphenols E, Tea Polyphenols A, Tea Polyphenols D (overlapping) with EGCg, the theaflavin class (comprises theaflavin (TF), theaflavin-3-O-gallic acid ester (3-TFG), theaflavin-3 '-O-gallic acid ester (3 '-TFG), theaflavin-3, the peak of 3 '-two-O-gallic acid ester (3,3 '-TFGG)).In addition, also found the existence of the catechin oxidation product that the projection as baseline is detected.

[analytical test example 2]

Make the mixed culture fermentation tealeaves that is displayed in Table 2 excellent AGH inhibition (the cooperation ratio of the leaf of tea and loguat leaf is that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour) drying, and should the drying fermented tea according to program classification shown in Figure 2.

＜operation (1)〉with the dry fermented tea of 125g (moisture content 5 quality %) with the acetone of 2L 70% in disintegrating machine (goods name; Rotary blender 7011S type, FMI (エ Off エ system ア イ) company makes) middle broken, filter.Acetone extraction residue with 3L 70% obtains acetone extract, then, uses the 3L methanol extraction residue, obtains methanol extract liquid.Filtrate, above-mentioned acetone extract, above-mentioned methanol extract liquid that above-mentioned filtration is obtained merge, and after concentrating with rotary evaporator, drying obtains 59.9g extract (dry thing).

＜operation (2)〉extract that obtains is suspended in the water, add ether and carry out the solvent distribution, remove liposoluble constituents such as chlorophyll.

＜operation (3)〉will operate Sephadex LH-20 post (the Off ア Le マ シ ア Off ア イ Application ケ ミ カ Le company manufacturing that water layer that (2) obtain is circulated into the water preparation, among the 4cm * 28cm), water (500mL), 40% methyl alcohol (200mL), 60% methyl alcohol (300mL), 80% methyl alcohol (300mL), methyl alcohol (300mL), 60% acetone (500mL) wash-out successively.

Eluent is concentrated, be classified into fraction Fr.1 (18.66g), Fr.2 (5.79g), Fr.3 (3.12g), Fr.4 (4.87g), Fr.5 (6.01g), Fr.6 (3.55g), Fr.7 (2.07g).

When each fraction was measured AGH inhibition activity, shown in following table 4, Fr.6 and Fr.7 showed very high inhibiting rate.

[table 4]

	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)
			Fr.6	94.2	89.1
Fr.7	96.3	91.3

Therefore, with operation shown in Figure 2 Fr.6 and Fr.7 are carried out separation and purification again.

＜operation (4)〉to Fr.6 and Fr.7, (Mitsubishi Chemical Ind makes to be carried in MCIgelCHP20P respectively, 3cm * 28cm), use 20% methyl alcohol (100mL), 30% methyl alcohol (100mL), 40% methyl alcohol (100mL), 50% methyl alcohol (100mL), 60% methyl alcohol (100mL), 70% methyl alcohol (100mL), 80% methyl alcohol (100mL), 90% methyl alcohol (100mL), methyl alcohol (200mL) wash-out successively.

Concentrate the eluent that obtains, obtain the fraction of Fr.6-1 (899mg), Fr.6-2 (2.16g), Fr.6-3 (282mg) by Fr.6.Obtain the fraction of Fr.7-1 (67mg), Fr.7-2 (1.17g), Fr.7-3 (600mg) in addition by Fr.7.

Wherein, Fr.6-1 and Fr.7-1 measure hydrogen nuclear magnetic resonance ( ¹H-NMR) spectrogram compares by the spectrogram with standard items, is accredited as Tea Polyphenols A and Tea Polyphenols D respectively.By the result of HPLC and thin-layer chromatography, Fr.6-3 and Fr.7-3 all are accredited as the theaflavin class.

＜operation (5)〉material that Fr.6-2 and Fr.7-2 are merged is carried in ChromatorexODS (シ リ シ ア company of Fuji makes, 3cm * 30cm), use 10% methyl alcohol (100mL), 20% methyl alcohol (100mL), 30% methyl alcohol (100mL), 40% methyl alcohol (100mL), 50% methyl alcohol (100mL), 60% methyl alcohol (100mL), methyl alcohol (200mL) wash-out successively.

The eluent that obtains is concentrated, be classified into Fr.721 (179mg), Fr.722 (1.10g), Fr.723 (530mg), Fr.724 (1.27g).

Wherein, Fr.721 measures ¹The H-NMR spectrogram is accredited as the mixture of EGCg and Tea Polyphenols A.

＜operation (6)〉materials that Fr.722 and 723 is merged are circulated into Sephadex LH-20 post and (among the 3cm * 20cm), use ethanol (500mL), 90% ethanol (200mL), 20% methyl alcohol (300mL), 60% acetone (400mL) wash-out successively.

The eluent that obtains is concentrated, be classified into Fr.7221 (3.7mg), Fr.7222 (41.3mg), Fr.7223 (47.0mg), Fr.7224 (166.2mg), Fr.7225 (458mg), Fr.7226 (657.5mg).

Wherein, Fr.7221, Fr.7222, Fr.7223, Fr.7224 measure ¹The H-NMR spectrogram compares by the spectrogram with standard items, is accredited as gallic acid, ECg, EGCg, epigallocatechin-3 respectively, 3 ' (4 ')-O-gallic acid ester.

＜operation (7)〉Fr.7225 is carried in DiaionHP20SS, and (Mitsubishi Chemical Ind makes, 2cm * 20cm), use 20% methyl alcohol (100mL), 25% methyl alcohol (100mL), 30% methyl alcohol (100mL), 35% methyl alcohol (100mL), 40% methyl alcohol (100mL), 45% methyl alcohol (100mL), 50% methyl alcohol (100mL), 60% methyl alcohol (100mL), methyl alcohol (200mL) wash-out successively.

The eluent that obtains is concentrated, be classified into Fr.72251 (39.6mg), Fr.72252 (320mg), Fr.72253 (47.7mg).

Wherein, Fr.72251 measures ¹The H-NMR spectrogram compares by the spectrogram with standard items, is accredited as oolong tea propylhomoserin (ウ one ロ Application テ ア ニ Application).

＜operation (8)〉Fr.72252 is carried in ChromatorexODS, and (シ リ シ ア company of Fuji makes, 2cm * 20cm), use 10% methyl alcohol (100mL), 20% methyl alcohol (100mL), 30% methyl alcohol (100mL), 40% methyl alcohol (100mL), 50% methyl alcohol (100mL), 60% methyl alcohol (100mL), methyl alcohol (200mL) wash-out successively.

The eluent that obtains is concentrated, be classified into Fr.722521 (10.5mg), Fr.722522 (38.3mg), Fr.722523 (60mg).

Wherein, Fr.722521 measures ¹The H-NMR spectrogram is accredited as epigallocatechin-3,3 ' (4 ')-O-gallic acid ester.

＜operation (9)〉Fr.722523 is circulated into Sephadex LH-20 post (among the 1.5cm * 20cm), uses 80% methyl alcohol (200mL), 90% methyl alcohol (200mL), methyl alcohol (200mL), 60% acetone (200mL) wash-out successively.

The eluent that obtains is concentrated, be classified into Fr.7225231 (14.4mg), Fr.7225232 (16.1mg), Fr.7225233 (15.5mg).

Wherein, Fr.7225232 measures ¹The H-NMR spectrogram compares by the spectrogram with standard items, is accredited as galloyl base oolong tea propylhomoserin.

By these results as can be known, AGH suppresses to contain following compound respectively in the following fraction of active high Fr.6 and 7.The structure of following compound is shown in Fig. 3.

Fr.6-1: Tea Polyphenols A (TS-A),

Fr.6-3,7-3: theaflavin (TF) mixture (comprising theaflavin, theaflavin-3-O-gallic acid ester, theaflavin-3 '-O-gallic acid ester, theaflavin-3,3 '-two-O-gallic acid ester),

Fr.7-1: Tea Polyphenols D (TS-D),

Fr.7221: gallic acid,

Fr.7222: epicatechin-3-O-gallic acid ester (ECg),

Fr.7223: epigallocatechin-3-O-gallic acid ester (EGCg),

Fr.7224 and Fr.722521: epigallocatechin (EGC)-3,3 ' (4 ')-two-O-gallic acid ester,

Fr.72251: the oolong tea propylhomoserin,

Fr.7225232: galloyl base oolong tea propylhomoserin.

In addition, the results are shown among Fig. 2 the content quantitative of above-mentioned each compound.By this result as can be known, the main component of the Fr.6 that AGH inhibition activity is high is Tea Polyphenols A.In addition we know, the main component of the Fr.7 that AGH inhibition activity is high is theaflavin (TF) mixture.When measuring the AGH inhibition activity of the above-mentioned theaflavin class that contains in this theaflavin mixture respectively, particularly, theaflavin-3,3 '-O-gallic acid ester shows strong enzyme inhibition activity.This can be known by the result of analytical test example 5,6 described later.

[analytical test example 3]

To showing that in above-mentioned analytical test example 2 very high AGH suppresses active Fr.7 and carries out the HPLC analysis, the results are shown in Fig. 4 A.Analysis condition is:

Post: Cosmosil 5C ₁₈ARII (Na カ ラ イ テ ス Network company makes, and 4.6 * 250mm)

Column temperature: 35 ℃

Mobile phase: A; 50mM phosphoric acid, B; CH ₃CN, B 4% to 30% (39 minutes), 30% to 75% (15 minutes)

Flow velocity: 0.8ml/min

Detect: adopt photodiode array to detect (Max absorbance).

(in addition, the HPLC analysis condition in following analytical test example, unless otherwise specified, identical with this test example).

Result by this figure can be clear and definite: in Fr.7, except the theaflavin mixture and above-claimed cpd that in above-mentioned analytical test example 2, determine to exist, also contain considerable in thin-layer chromatography (TLC) is analyzed as initial point detected and in HPLC analyzes as the protruding detected material of baseline.These materials are mainly by among the Fr.724 of classification in Fig. 2.

Because behavior and ultraviolet-visible absorption spectroscopy and the EGCg in various column chromatographys is similar for these materials, and it is positive to iron chloride reagent and vanillic aldehyde hydrochloric acid reagent, therefore, be speculated as the big catechin oxidation product of catechin of determining existence in the above-mentioned analytical test example 2 of molecular weight ratio.

Usually known, in the typical material of the high-molecular weight polyphenol that tea contained, there is OPC, containing in loguat leaf with the epicatechin is the OPC (loquat OPC) of construction unit, therefore, contain OPC in " the catechin oxidation product that molecular weight is big " of supposition in above-mentioned Fr.7.

OPC is to be the dimer or the polymer of construction unit with catechin or catechin-derived thing, constitutes its catechin or 4 carbon (C-4) of catechin-derived thing and 8 carbon (C-8) or 4 carbon (C-4) and 6 carbon (C-6) and carries out the carbon-to-carbon combination.

Therefore, for the unit of the formation OPC that contains among the clear and definite above-mentioned Fr.7, Fr.7 has been carried out adopting the mercaptan decomposition of mercaptoethanol according to following method.

Promptly, in 60% ethanolic solution of the Fr.7 of the 0.2mL (concentration of Fr.7: add 1.8mL mercaptoethanol test solution (having mixed 0.1% hydrochloric acid, the 27.5mL ethanol of 2.5mL mercaptoethanol, 4mL, the solution of 16mL water) 10mg/mL), heated 6 hours down at 60 ℃, analyze the reactant liquor of 20 μ L with HPLC.Analysis condition is identical when obtaining Fig. 4 A.Spectrogram after the mercaptan decomposition that obtains like this is shown in Fig. 4 B.

Shown in this Fig. 4 B, when Fr.7 is carried out mercaptan decomposition, the peak of discovery has appearred much not having in (Fig. 4 A) before decomposition.For undiscovered peak before decomposition and than the peak that increases before decomposing, carry out and standard items (Tanaka, T.et.al., J.Chem.Soc.Perkin Trans 1,1994, the result of the retention time 3013) and the comparison of ultraviolet absorption spectrum has identified EGC, EC, EGCg, ECg, EGC-ME, EC-ME, EGCg-ME, ECg-ME.

In addition, in Fig. 4 B, Fr.7 (500mg) is dissolved in the 10mL mercaptoethanol test solution, heat after 6 hours, with Chromatorex ODS (manufacturing of シ リ シ ア company of Fuji) (water-methanol eluent) column chromatography, then with Sephadex LH-20 (EtOH eluent) column chromatography separating purification, by ¹The analysis result of H-NMR spectrogram can be clear and definite, is epiafzelechin-3-O-gallic acid ester-ME at 39 minutes detected peaks.The structural formula of epiafzelechin-3-O-gallic acid ester-ME is shown in Fig. 5.

That is, exist ¹In the H-NMR spectrogram; though the signal of A ring, C ring, galloyl base and ethoxy sulfenyl and the signal of EGCg-ME are much at one; but the signal of observing the B ring is for to be the doublet (J=8Hz) that integral area is equivalent to 2H at d7.40 and 6.81 places, and this expression B ring is para hydroxybenzene phenol.In addition, in TOF (flight time type) mass spectral analysis, show at m/z 503 places [M+H] ⁺This conclusion is also supported at the peak.

In the construction unit of these evaluations, EGC, EGCg, ECg come from the terminal units of macromolecular compound, and sulfide compound comes from extension unit.Therefore, the structure of the macromolecular compound before the mercaptan decomposition, the structure of promptly inferring the OPC that contains among the above-mentioned Fr.7 is to have structure for example shown in Figure 5.

Up to now, with the epiafzelechin gallate be the OPC of construction unit only know a kind of dimer that obtains from oolong tea (Hashimoto, F.et.al., Chem.Pharm.Bull., 1989,37,3255-3263).Usually, though dimer is detected as clear and definite peak in HPLC, but in the HPLC of Fr.7, owing to find to be equivalent to illustrate the dimeric peak of degree of the growing amount of epiafzelechin-3-O-gallic acid ester-ME, therefore, epiafzelechin-3-O-gallic acid ester-ME of generating by mercaptan decomposition of Fr.7 can be described as cyanine oligomer or the polymer that comes from more than the tripolymer.Should " is the OPC (oligomer or polymer that tripolymer is above) of construction unit with the epiafzelechin gallate " be unknown up to now novel compound.

In addition, carry out same analytical test for the fermented tea of similarly making the leaf that only uses tea except not adding loguat leaf, when comparing the sulfide compound that obtains after the mercaptan decomposition, mix the situation of loguat leaf, the situation of not mixing loguat leaf, go out the peak of epiafzelechin-3-O-gallic acid ester-ME with almost same size detection.In addition, even the OPC that will extract carries out mercaptan decomposition, do not detect epiafzelechin-3-O-gallic acid ester-ME from loguat leaf yet.Hence one can see that, and " is the OPC of construction unit with the epiafzelechin gallate " contained among the Fr.7 comes from the leaf of tea.

In addition, compare with the situation of not mixing loguat leaf, the peak of the EC-ME when mixing loguat leaf is obviously big.Hence one can see that, and " is the OPC (loquat OPC) of construction unit with the epicatechin " contained in the loguat leaf is included among the high Fr.7 of AGH inhibition.

[analytical test example 4]

Shown in Fig. 4 B, after the Fr.7 that the AGH inhibition is high carries out mercaptan decomposition, the projection of residual baseline still.

This demonstrates, in Fr.7, and except above-mentioned analytical test example 3 described " is the OPC of construction unit with the epiafzelechin gallate " and " loquat OPC ", the material that also exists HPLC to be detected as the projection of baseline in analyzing.

Therefore, compared the mixed culture fermentation tealeaves under the situation of leaf that has mixed tea and loguat leaf and except do not mix loguat leaf with the fermented tea under the external kindred circumstances (below, be sometimes referred to as independent fermented tea) in, whether the growing amount of the material that is detected as the projection of baseline in HPLC analyzes is identical.In addition, creating conditions of mixed culture fermentation tealeaves is identical with analytical test example 1,2, and the leaf of tea and the mixed proportion of loguat leaf are that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour.

For mixed culture fermentation tealeaves and independent fermented tea, at room temperature use the acetone extraction 3g tealeaves (moisture content 5 quality %) (extraction time is 18 hours) of 100mL 70% respectively, filter then.Filtrate concentrating made the aqueous solution (20mL), then, respectively carry out 2 solvents with ether, ethyl acetate and distribute.

After residual water layer concentrated, make 30mL, its 10 μ L is analyzed with HPLC with 70% ethanol.The HPLC analysis result of this water layer is shown in Fig. 6.

As shown in the drawing, to compare with independent fermented tea, the amount of substance that is detected as the baseline projection of mixed culture fermentation tealeaves is obviously many.During the comparison peak area, the amount of substance that is detected as the baseline projection in mixed culture fermentation tealeaves is estimated as 159% of independent fermented tea.Though this increase part also comprises the OPC from loguat leaf, but, can not illustrate that therefore the composition that just is included in the loguat leaf because of script just increases by 59% because the addition of loguat leaf in mixed culture fermentation tealeaves only be 1/10th a amount of the leaf of tea.Therefore, this increase part demonstrates owing to add loguat leaf and promote to generate, when not adding loguat leaf with regard to the existence of the catechin oxidation product that can not generate.

On the other hand, after the above-mentioned ethyl acetate layer that obtains concentrated, make 30mL with 80% methyl alcohol after, wherein 1mL in ODS short column (TOYOPAK ODS M, TOSO, the manufacturing of East ソ one company) with 80% methyl alcohol conducting, eluent is made 5.0mL, and wherein 5 μ L analyze with HPLC.The HPLC analysis result of this ethyl acetate layer is shown in Fig. 7.

As shown in the drawing, to compare with independent fermented tea, the peak area total of the theaflavin class of mixed culture fermentation tealeaves is increased to 2.7 times.On the other hand, the peak area of catechin reduces, and particularly, EGC reduces to 0.24 times, EC and reduces to 0.44 times, EGCg and reduce to 0.57 times, ECg and reduce to 0.64 times.

This demonstrates, and in mixed culture fermentation tealeaves, rubs mixed loguat leaf by adding in tealeaves, generates a large amount of high molecular catechin oxidation products.

[analytical test example 5]

By the result of analytical test example 4 as can be known, in mixed culture fermentation tealeaves, rub mixed loguat leaf by in tealeaves, adding, the high molecular catechin oxidation product of a large amount of generations, it is detected as the baseline projection in HPLC analyzes, that is, be detected as initial point in thin-layer chromatography (TLC) is analyzed.

Therefore, in order to identify the composition that in such mixed culture fermentation tealeaves, increases specifically, according to following method, in the composition that in mixed culture fermentation tealeaves (leaf of tea and the mixed proportion of loguat leaf are that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour), contains, be separated in thin-layer chromatography (TLC) and be detected the oxidized form high-molecular weight polyphenol as initial point in analyzing.

That is, at room temperature use 3L 60% alcohol extract 228g mixed culture fermentation tealeaves (moisture content is 5 quality %) 3 times, 3 times extracts are merged decompression concentrate.The aqueous solution (1.5L) that obtains is carried out solvent with ether, ethyl acetate, n-butanol successively distribute, obtain ether soluble fraction (4.03g), ethyl acetate soluble fraction (9.8g), n-butanol soluble fraction (22.1g).

15g n-butanol soluble fraction is dissolved in 60% methyl alcohol, and be carried on the Sephadex LH-20 post with 60% ethanol damping, successively with 60% methyl alcohol, 80% methyl alcohol, 100% methyl alcohol, then be that methanol-water-acetone (8: 1: 1) mixed solution, methanol-water-acetone (6: 2: 2) mixed solution, water-acetone (1: 1) mixed solution carry out wash-out.Eluent is carried out TLC (toluene-Ethyl formate-formic acid, 1: 7: 1) analyze, only collect and contain the fraction of the material that does not leave initial point and concentrate, obtain the material (oxidized form high-molecular weight polyphenol fraction) of 1.4g polymer shape.Its HPLC analysis result is shown among Fig. 8 A.

In Fig. 8 A, absorb from its UV, near the small peak 45 minutes is represented to mix and is had the theaflavin class, but peak area to be the baseline projection whole below 2%.

The polymer shape material (oxidized form high-molecular weight polyphenol fraction) that obtains is like this measured the AGH inhibition.It the results are shown in table 5.

In addition, for theaflavin-3, the measurement result of the AGH inhibition of 3 '-two-O-gallic acid ester also merges and is shown in Table 5.

[table 5]

	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)
			The oxidized form high-molecular weight polyphenol	49.5	66.5
Theaflavin-3,3 '-two-O-gallic acid ester	53.8	93.0

As shown in table 5, the oxidized form high-molecular weight polyphenol fraction of from mixed culture fermentation tealeaves, separating with said method, that is, the high-molecular weight polyphenol that increases specifically in mixed culture fermentation tealeaves shows and theaflavin-3, the maltose of 3 '-two-O-gallic acid ester equality strength and sucrose enzyme inhibition activity.Consider that from content this activity is not to come from the theaflavin class (near the peaks 45 minutes) of sneaking into, but comes from high-molecular weight polyphenol.

In addition, for the above-mentioned oxidized form high-molecular weight polyphenol fraction that obtains, adopt the mercaptan decomposition of mercaptoethanol according to following method.

That is, with 0.2mL make the above-mentioned oxidized form high-molecular weight polyphenol that obtains be dissolved in solution in 60% ethanol (concentration of oxidized form high-molecular weight polyphenol: 10mg/mL) and 0.8mL mercaptoethanol test solution mix, 60 ℃ of reactions 5 hours down, analyze wherein 5 μ L with HPLC.It the results are shown in Fig. 8 B.

In above-mentioned analytical test example 3, similarly, detect when carrying out the HPLC analysis with the sulfide compound headed by epiafzelechin-3-O-gallic acid ester-ME and the peak of catechin with the material that the Fr.7 mercaptan decomposition is obtained.In addition, in Fig. 8 B, after mercaptan decomposition, also observe the projection of baseline.

Has hinted from this situation, in the above-mentioned oxidized form high-molecular weight polyphenol fraction that obtains, contained the above-mentioned OPC that epiafzelechin gallate is a construction unit that has, but that it contains is proportional few, contain a great deal of not by the material of mercaptan decomposition.

Therefore, measure this oxidized form high-molecular weight polyphenol fraction ¹³The C-NMR spectrogram, and with itself and " is the OPC (loquat OPC) of construction unit with the epicatechin " in loguat leaf, contained ¹³The C-NMR spectrogram compares.It the results are shown in Fig. 9.Fig. 9 A is the spectrogram of loquat OPC, and Fig. 9 B is the spectrogram of above-mentioned oxidized form high-molecular weight polyphenol fraction.

By the result of Fig. 9 as can be known; in the loquat OPC, observe signal from the ring-like B ring of catechol (Bc); in contrast; in the oxidized form high-molecular weight polyphenol that from mixed culture fermentation tealeaves, separates, almost do not observe signal, but observe the phloroglucin (1,3 that encircles from A corresponding to catechin from the B ring; the 5-trihydroxy benzene) signal and the pyrogallol 1 that comes from the galloyl base; 2,3-trihydroxy benzene) though signal is the little signal that also confirms part from the C ring.

Hence one can see that, and the main component of the oxidized form high-molecular weight polyphenol that separates from mixed culture fermentation tealeaves with said method is not the loquat OPC, but have the catechin-derived thing oxidative condensation of various structures and the material of producing high-molecular at the B loop section.

Though the structure of such high-molecular weight polyphenol is also indeterminate, analogize by compound shown in Figure 3, can think it mainly is that the B of catechin gallic acid ester class encircles each other oxidative condensation and the structure of producing high-molecular.As the combination of B ring, can infer Tea Polyphenols shown in Figure 3 is arranged, all kinds such as theaflavin, oolong tea propylhomoserin, but its concrete condition is also indeterminate now.

In addition, with the result that this oxidized form high-molecular weight polyphenol carries out elementary analysis, carbon is 57.06%, hydrogen is 4.51%, nitrogen is 0.35%.

Calculated value (C with EGCg ₂₂H ₁₈O ₁₁: carbon is 57.65%, hydrogen is 3.96%; C ₂₂H ₁₈O ₁₁2H ₂O: carbon is 53.44%, hydrogen is 4.49%) compare, hydrogeneous rate height, because hydrogeneous rate should reduce in common oxidation, therefore, hint has the possibility of the metabolite of sneaking into other in the process of catechin oxidation.

In addition, the oxidized form high-molecular weight polyphenol that obtains is carried out after the acetylation, during by the gel permeation chromatography molecular weight; peak top molecular weight is that 2000 (number-average molecular weight is 1400; the quality mean molecule quantity is 3200), molecular weight distribution is wide, is about 1000～15000.

The condition determination of gel permeation chromatography is as follows.

Post: TSK-gel G 4000H ₆, diameter 4.6mm * length 250mm, East ソ one company makes

Pump: TOSO DP8020, East ソ one company makes

Detector: JASCO UV970 Japan beam split company makes 254nm

Mobile layer: oxolane

Temperature: room temperature

Flow velocity: 1.0mL/min

Calculate by the molecular weight with the comparison of polystyrene standard: JASCO 807IT integrator, Japanese beam split company makes.

In addition, analogize from catechin, because the molecular weight that causes of acetylation increases part when being 1.5 times, molecular weight is for being 670～10000 of maximum with 1330.If supposition is only generated by the catechin gallic acid ester, then contain and be equivalent to dimeric material (theaflavin) to the material that is equivalent to 20 aggressiveness.

[analytical test example 6]

With EC, ECG, EGC, EGCg, theaflavin (TF), 3-TFG, 3 '-TFG, 3, the pure product (commercially available product) of 3 '-TFGG and Tea Polyphenols A (TS-A) are dissolved in by DMSO (methyl-sulfoxide): in the mixed solvent of water=make at 1: 9, the preparation sample solution carries out AGH to each sample solution and suppresses determination of activity.

The evaluation of the AGH inhibition in this test example and the analytical test example 7 is according to the pseudo-in vivo method (T.Oki.et.al. that has used the immobilized carrier of rat small intestine AGH, Biol.Pharm.Bull., 23,1084-1087 (2000)) to carry out, enzymatic activity is with the 50% final concentration (IC that suppresses ₅₀Value, unit: mM) expression.It the results are shown in table 6.

[table 6]

The maltose inhibition		The invertase inhibition
				IC 50(mM)	IC 50(mM)
	EC ECG EGC EGCg		1.010 0.053 1.260 0.040			EC ECG EGC EGCg	1.080 0.172 0.921 0.169
TF 3-TFG 3’-TFG 3，3’-TFGG		0.500 0.010 0.136 0.058		TF 3-TFG 3’-TFG 3，3’-TFGG	＞10 1.024 0.573 0.159
	TS-A		0.142			TS-A	0.286

Result by table 6 can be clear and definite, the maltose strong inhibition capability be ECG, EGCG, 3-TFG, 3,3 '-TFGG, the invertase strong inhibition capability be ECG, EGCG, 3,3 '-TFGG.That is it is stronger, to have the AGH inhibition of material of molecular structure of the polysubstituted OH base that comprises the galloyl base in the ester type in the catechin, the theaflavin.

[analytical test example 7]

(leaf of tea and the mixed proportion of loguat leaf are that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour with 20g mixed culture fermentation tealeaves with the hot water of 100 ℃ of 1000mL, moisture content is 5 quality %) extracted 10 minutes, the supernatant freeze drying that centrifugation is obtained, and the material that freeze drying is obtained is as test portion, investigation AGH inhibition.

Test portion is dissolved in DMSO: in the mixed solvent of water=1: 9, the preparation sample solution about this sample solution, adopts above-mentioned pseudo-in vivo method mensuration AGH inhibition.In addition, measure catechin (EC, ECG, EGC, EGCg) and theaflavin class (TF, 3-TFG, 3 '-TFG, 3,3 '-TFGG) the content that contains in this sample solution.Then, based on the IC that in above-mentioned analytical test example 6, measures ₅₀Value, obtaining will be by the AGH inhibition of mixed culture fermentation tealeaves performance ratio (active contribution rate, the unit: %) by the AGH inhibition of each composition performance as 100% o'clock.The maltose inhibition the results are shown in table 7, the invertase inhibition the results are shown in table 8.

[table 7]

(1) maltose inhibition

[table 8]

(2) invertase inhibition

[analytical test example 8]

With the hot water of 100 ℃ of 1L 20g and analytical test example 7 same mixed culture fermentation tealeaves were extracted 10 minutes.With extracting liquid filtering, after with evaporimeter the filtrate that obtains being concentrated, freeze drying.In dried powder 10mg, add 1ml water, after the stirring, carry out centrifugal treating (3000rpm, 10 minutes), obtain supernatant, be water-soluble fraction.Measure the AGH inhibition of this water-soluble fraction.It the results are shown in table 9.

In addition, the mensuration of the AGH inhibition in this analysis test example adopts and has used the in vitro method (Oki.T.et.al., J.Agric.Food Chem, 47,550-553 (1999)) of above-mentioned free AGH to carry out.

[table 9]

	Maltose inhibition IC 50(mg/ml)	Invertase inhibition IC 50(mg/ml)
			Hot water extract's water-soluble fraction	0.05	0.29

As shown in Table 9, the hot water extract's of mixed culture fermentation tealeaves water-soluble fraction shows high AGH inhibition.

Therefore, with following method the hot water extract's of the high AGH inhibition of this demonstration water-soluble fraction is carried out the composition lock out operation again.

That is, the water-soluble fraction with this hot water extract supplies in the adsorption charomatography of having used SephadexLH-20 (Amersham Biosciences manufacturing).Wash-out carries out with the order of water, 50% methyl alcohol, 100% methyl alcohol, 70% acetone, carries out the material that freeze drying obtains for the various fractions that will obtain, and measures with the above-mentioned AGH inhibition that similarly carries out.The yield of each fraction and AGH inhibition are shown in following table 10.In addition, in table 10, also merge the measurement result that table 9 is shown.

[table 10]

	Yield (%)	Maltose inhibition IC 50(mg/ml)	Invertase inhibition IC 50(mg/ml)
				(hot water extract's water-soluble fraction)	(100)	(0.05)	(0.29)
Water-soluble fraction	53.7	Unrestraint	Unrestraint
				50% methanol-eluted fractions fraction	9.6	0.32	1.03
100% methanol-eluted fractions fraction	17.9	0.21	0.26
				70% acetone elutriated fraction	18.8	0.05	0.12

As shown in table 10, in mixed culture fermentation tealeaves hot water extract's the water-soluble fraction, the AGH inhibition height of 70% acetone elutriated fraction and 100% methanol-eluted fractions fraction particularly.

Therefore, by gel permeation chromatography during to this 70% acetone elutriated fraction and 100% methanol-eluted fractions fraction determining molecular weight, the clear and definite composition that contains in extra high two fractions of these AGH inhibitions is in the scope of molecular weight 100～1500.

The condition determination of the gel permeation chromatography here is as follows.

Post: TSK-gel G2500 PWXL, diameter 7.8mm * length 250mm

Pump: Shimadzu Seisakusho Ltd. makes, LC-10Avp

Detector: SPD-10AV, 254nm

Mobile layer: 0.1M, phosphate buffer (pH7.2 contains 0.1M sodium chloride)

Temperature: room temperature

Flow velocity: 0.3mL/min

Calculate by the molecular weight that relatively carries out with the standard polyethylene glycol

[analytical test example 9]

With above-mentioned analytical test example 8 similarly, with the hot water of 100 ℃ of 1L 20g and analytical test example 7 same mixed culture fermentation tealeaves were extracted 10 minutes, the extract that filtration obtains obtains filtrate.This filtrate supplying to has been filled in the post of Porapack Q (goods name, Waters company makes).Then, with 100ml water, then with the water soluble ingredient in the 100ml 50% methanol-eluted fractions post.After the water elution fraction and the concentrated respectively also drying of 50% methanol-eluted fractions fraction that obtain like this, measure AGH and suppress active.It the results are shown in following table 11.

The mensuration of the AGH inhibition in this analysis test example according to the use that proposes by people such as Song Jing the method for free AGH carry out, with inhibiting rate (unit: value representation %).

[table 11]

	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)
			The water elution fraction	71.3	41.9
50% methanol-eluted fractions fraction	97.2	93.9

As shown in table 11, mixed culture fermentation tealeaves hot water extract's water elution fraction and 50% methanol-eluted fractions fraction any all shows high AGH inhibition.

In addition, to this water elution fraction and 50% methanol-eluted fractions fraction with the same condition of above-mentioned analytical test example 8 under during by the gel permeation chromatography molecular weight, the clear and definite composition that contains in extra high two fractions of these AGH inhibitions is in the scope of molecular weight 100～1900.

By the result of analytical test example 8,9 as can be known, the hot water extract's of mixed culture fermentation tealeaves water-soluble fraction shows high AGH inhibition, and the molecular weight of the big composition of the contribution of this AGH inhibition is about 100～1900 lower molecular weight.

[experimental example 2]

The hot water extract's (freeze drying product) who allows normal rat absorb mixed culture fermentation tealeaves, the influence that research brings anti-sugared ability.

The independent fermented tea of preparing mixed culture fermentation tealeaves (leaf of tea and the mixed proportion of loguat leaf are that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour), thick tea, make the dry and loguat leaf pulverized of loguat leaf and not mixing loguat leaf and only the leaf kneading of tea was obtained in 20 minutes is as test portion tealeaves.In addition, above-mentioned thick tea uses the leaf with the identical tea that uses as the raw material of mixed culture fermentation tealeaves, is the thick tea made according to the autofrettage of common green tea (not making the leaf fermentation of tea and the thick tea of manufacturing).

Allow various test portion tealeaves (moisture content is 5 quality %) 20g after 10 minutes, filter with 100 ℃ of hot water extracting of 1000ml.With the filtrate drying, obtain pulverous extract.

Make freely ingest the respectively feed (4 kinds) of following acquisition of male Sprague-Dawley (SD) rat in 4 ages in week: the AIN-76 that does not contain cholesterol being consisted of the hot water extract of the above-mentioned preparation of interpolation in the bait of basic composition and making containing ratio is 1 quality %.

In addition, in contrast, allow same rat freely absorb the control Food of in bait, not adding the hot water extract.

The indoor raising of animal feeding that the light that rat is lit a lamp at 22 ± 1 ℃ of room temperatures, humidity 55 ± 5%, 8:00～20:00 circulates.After 4 weeks, in fasting after 6 hours, the maltose of oral administration 1g/kg body weight.After the administration, after 0,10,20,30 and 60 minute,, use blood sugar test to measure medical safe pipe (メ デ イ セ one Off チ Star プ) (manufacturing of テ Le モ company) and measure blood glucose value respectively from tail vein blood.

It the results are shown in Figure 10.

In Figure 10, transverse axis is represented the elapsed time after the maltose administration, and the longitudinal axis is represented the value of blood glucose value.In addition, the value of blood glucose value is that blood glucose value during with the maltose administration is represented as 0mg/dl.

As shown in the drawing, absorbed the hot water extract's of mixed culture fermentation tealeaves the blood glucose value of rat and on than all low level of any one test portion tealeaves, passed.

Hence one can see that, and the hot water extract by picked-up mixed culture fermentation tealeaves can obtain the effect that excellent inhibition blood glucose value rises.In addition we know, the loguat leaf that this effect obtains than dry loguat leaf only, only make the dry and resulting effect excellence of tealeaves that obtains of the leaf of tea, by leaf and the loguat leaf that makes tea mix, kneading, fermentation, can obtain synergy.

[experimental example 3]

Allow the powder of the rat picked-up mixed culture fermentation tealeaves of suffering from diabetes B, the influence that research brings blood glucose value.

(leaf of tea and the mixed proportion of loguat leaf are that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour with mixed culture fermentation tealeaves to use food processor, moisture content 5 quality %) pulverize processing, up to the pulverulence that becomes 60～100 order sizes, obtain the powder-mixed fermented tea.

The male Otuka Long-EvansTokushima Fatty rat of the natural occurrence diabetes B at rat 1 monthly age of use (below, be called the OLETF rat) and as the male Long-Evans Tokushima Otsuka rat that does not suffer from diabetes of its object model animal (below, be called the LETO rat).The indoor raising of animal feeding that the light that rat is turned on light at 22 ± 1 ℃ of room temperatures, humidity 55 ± 5%, 8:00～20:00 circulates.

During initial 3 months LETO and the solid-state feed of OLETF rat administration MF (オ リ エ Application Le yeast industry (strain) manufacturing) are prepared raising.Because the OLETF rat is usually at monthly age at 5 monthly ages to 8 morbidity diabetes B,, begin ingestion experiment food with the LETO rat from having 4 monthly ages before 1 month of initiation potential.

That is, fasting 6 hours when 4 monthly ages (behind the 9:00～15:00), from tail vein blood, is measured blood glucose value, is equally divided into groups with 6 every group according to body weight and blood glucose value, make its following test food 5 months of freely ingesting.

Test food is with based on the purifying food of AIN-76 food in contrast, allows LETO rat-control group and OLETF rat-control group ingest.The composition of control Food (g/kg) is casein 200, salad oil 100, mineral mixing (AIN-76-MX) 35, vitamin mixing (AIN-76-VX) 10, cellulose 50, lipotropin 2, DL-methionine 3, cornstarch 150 and sucrose 450.

In OLETF rat-mixed culture fermentation tealeaves interpolation group, in above-mentioned control Food, add the powder-mixed fermented tea of feed gross mass 5%, and deduct the sucrose amount of its addition part.

Any one group during feeding in, food ration every day is measured, body weight and water uptake were measured every 1 day.

From medicine-feeding test food, 1,2,3,4 and 5 months after, fasting after 6 hours from tail vein blood and use blood sugar test to measure medical safe pipe (manufacturings of テ モ Le company) mensuration blood glucose value.The variation of the rat blood sugar value between picked-up test food 5 months is shown in Figure 11.

In Figure 11, the elapsed time after transverse axis is represented to begin from medicine-feeding test food (unit: month), the longitudinal axis is represented the value of blood glucose value.

As shown in the drawing, the blood glucose value that has absorbed the LETO rat that does not suffer from diabetes of control Food is low value during whole nursing.On the other hand, the blood glucose value that has absorbed the OLETF rat that suffers from diabetes of control Food rises in time, is counted as onset diabetes.

In contrast, the OLETF rat that suffers from diabetes B having been absorbed in the group of feed of the powder that has added mixed culture fermentation tealeaves, even also do not rise through long-time blood glucose value, is low-level with the LETO rat equal extent of the diabetes of not falling ill.Hence one can see that, and the powder of mixed culture fermentation tealeaves has the effect that excellent inhibition blood sugar rises.

[experimental example 4]

Allow hot water extract's (freeze drying product) of the rat picked-up mixed culture fermentation tealeaves of suffering from diabetes B, study the influence that blood glucose value is brought.

(leaf of tea and the mixed proportion of loguat leaf are that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour with 20g mixed culture fermentation tealeaves, moisture content 5 quality %) with the hot water extracting of 100 ℃ of 1000mL 10 minutes, and the filtrate that filtration obtains carried out freeze drying, obtain pulverous mixed culture fermentation tea extract.

As a comparative example, extract the thick tea of making according to the manufacture method of common green tea (leaf of tea is fermented and the thick tea of manufacturing) under same condition, filtration, drying obtain pulverous thick tea extraction.In addition, extract the loguat leaf that is dried to same moisture content under same condition, filtration, drying obtain pulverous loquat-leaf extract.

Rat is used the rat same with above-mentioned experimental example 3, and is also identical up to the feeding conditions at 4 monthly ages.

Fasting 6 hours when 4 monthly ages (behind the 9:00～15:00), from tail vein blood, is measured blood glucose value, is equally divided into groups with 6 every group according to body weight and blood glucose value, make its following test food 5 months of freely ingesting.

For the control Food group of LETO rat and OLETF rat, the control Food same of ingesting with experimental example 3.

In OLETF rat-mixed culture fermentation tealeaves interpolation group, in above-mentioned control Food, add the powder-mixed fermented tea extract of feed gross mass 5%, and deduct the sucrose amount of its addition part.

In OLETF rat-thick tea interpolation group, in above-mentioned control Food, add the pulverous thick tea extraction of feed gross mass 5%, and deduct the sucrose amount of its addition part.

In OLETF rat-loguat leaf interpolation group, in above-mentioned control Food, add the Powdered loquat-leaf extract of feed gross mass 5%, and deduct the sucrose amount of its addition part.

During feeding, food ration is measured every day, and body weight and water uptake were measured every 1 day.

From medicine-feeding test food, 1,2,3,4 and 5 months after, similarly measure blood glucose value with experimental example 3.The variation of 5 months rat blood sugar value of picked-up test food is shown in Figure 12.

The transverse axis among Figure 12 and the longitudinal axis and Figure 11 are same.As shown in the drawing, the blood glucose value that has absorbed the LETO rat that does not suffer from diabetes of control Food is low value during whole nursing.On the other hand, the blood glucose value that has absorbed the OLETF rat that suffers from diabetes of control Food rises in time, is counted as onset diabetes.

In contrast, allow the OLETF rat that suffers from diabetes B absorb in the hot water extract's's (freeze drying product) who is added with mixed culture fermentation tealeaves the group of feed, even also do not rise through long-time blood glucose value, be low-level with the LETO rat equal extent of the diabetes of not falling ill.Hence one can see that, and the hot water extract of mixed culture fermentation tealeaves has the effect that excellent inhibition blood sugar rises.

[experimental example 5]

Other effect when research allows the powder of the rat picked-up mixed culture fermentation tealeaves of suffering from diabetes B.

With the above-mentioned experimental example 3 shape mixed culture fermentation tealeaves that similarly makes powder.

As a comparative example, make to use the thick tea that the leaf of the same tea of the leaf of the tea that uses with raw material as mixed culture fermentation tealeaves makes (do not make the leaf fermentation of tea and make thick tea), similarly pulverize processing, obtain pulverous thick tea.In addition, the loguat leaf that is dried to same moisture content is pulverized processing handle, obtain pulverous loguat leaf.

Rat is used the rat with above-mentioned experimental example 3 similarity conditions, and is also identical up to the feeding conditions at 4 monthly ages.

Fasting 6 hours when 4 monthly ages (behind the 9:00～15:00), from tail vein blood, is measured blood glucose value, is equally divided into groups with 6 every group according to body weight and blood glucose value, make its following test food 5 months of freely ingesting.

For the control Food group of LETO rat and OLETF rat, the control Food same of ingesting with experimental example 3.

In OLETF rat-mixed culture fermentation tealeaves interpolation group, in above-mentioned control Food, add the powder-mixed fermented tea of feed gross mass 5%, and deduct the sucrose amount of its addition part.

In OLETF rat-thick tea interpolation group, in above-mentioned control Food, add the pulverous thick tea of feed gross mass 5%, and deduct the sucrose amount of its addition part.

In OLETF rat-loguat leaf interpolation group, in above-mentioned control Food, add the Powdered loguat leaf of feed gross mass 5%, and deduct the sucrose amount of its addition part.

During feeding, food ration is measured every day, and body weight and water uptake were measured every 1 day.

After medicine-feeding test food began 5 months, the adipose tissue of serum, liver and kidney and testis periphery was slaughtered and taked to fasting after 6 hours.

In following table, show the measurement result of serum insulin concentration, serum lipid peroxide concentration, adipose tissue mass, serum cholesterol, S-TG, liver cholesterol, liver triglyceride respectively.

[table 12]

Test portion	Insulin concentration (ng/ml)
		The LETO-control Food	7.30
The OLETF-control Food	3.68
		The thick cakes and sweetmeats thing of OLETF-powder	8.18
OLETF-powder loguat leaf food	3.12
		OLETF-powder fermented tea food	7.75

By the result of table 12, make the reduction of the group discovery insulin of OLETF rat picked-up control Food or loguat leaf.In addition, by blood-sugar level measuring as can be known, these groups are in the state of onset diabetes.In contrast, even suffer from the OLETF rat of diabetes, allow its insulin concentration that absorbs the group of powder-mixed fermented tea rise to the level of the LETO rat of the diabetes of not falling ill.Can confirm that thus mixed culture fermentation tealeaves has the effect of improving the insulin secretion reduction that causes owing to onset diabetes.

[table 13]

Test portion	Serum lipid peroxide concentration (nmol/ml)
		The LETO-control Food	11.86
The OLETF-control Food	63.78
		The thick cakes and sweetmeats thing of OLETF-powder	92.83
OLETF-powder loguat leaf food	78.57
		OLETF-powder fermented tea food	35.23

By the result of table 13,, allow its serum lipid peroxide concentration of absorbing the group of powder-mixed fermented tea compare also low with other OLETF rat group even suffer from the OLETF rat of diabetes.Can confirm that thus mixed culture fermentation tealeaves has the effect that reduces serum lipid peroxide concentration.

[table 14]

Test portion	Adipose tissue mass (g)
		The LETO-control Food	38.2
The OLETF-control Food	131.1
		The thick cakes and sweetmeats thing of OLETF-powder	77.1
OLETF-powder loguat leaf food	80.1
		OLETF-powder fermented tea food	76.8

By the result of table 14, even suffer from the OLETF rat of diabetes, allow the kidney of group of its picked-up powder-mixed fermented tea and the adipose tissue mass of testis periphery, it is low also to have a mind to the free burial ground for the destitute than the OLETF rat group of picked-up control Food.Thus can be clear and definite, mixed culture fermentation tealeaves has the effect that the inhibition body fat is accumulated.

[table 15]

Test portion	Serum cholesterol (mg/dl)	S-TG (mg/dl)	Liver cholesterol (mg/g)	Liver triglyceride (mg/g)
					The LETO-control Food	147	55.6	3.28	9.78
The OLETF-control Food	253	234	5.98	49.5
					The thick cakes and sweetmeats thing of OLETF-powder	183	127	3.43	16.9
OLETF-powder loguat leaf food	238	237	4.90	34.1
					OLETF-powder fermented tea food	177	139	3.08	12.6

By the result of table 15, even suffer from the OLETF rat of diabetes, the serum of the group of picked-up powder-mixed fermented tea and the cholesterol concentration and the neutral fat concentration of liver, also the OLETF rat group than the picked-up control Food reduces.Thus can be clear and definite, mixed culture fermentation tealeaves has effect that reduces cholesterol and the effect that reduces neutral fat.

[experimental example 6]

Other effect when research makes hot water extract's (freeze drying thing) of rat picked-up mixed culture fermentation tealeaves of morbidity diabetes B.

With above-mentioned experimental example 4 similarly, make the free ingestion experiment food of LETO rat and OLETF rat 5 months.

After the test food of throwing something and feeding began 5 months, the adipose tissue of serum, liver and kidney and testis periphery was slaughtered and taked to fasting after 6 hours.

In following table, show the measurement result of serum insulin concentration, adipose tissue mass, S-TG, liver triglyceride respectively.

[table 16]

Test portion	Insulin concentration (ng/ml)
		The LETO-control Food	7.82
The OLETF-control Food	3.10
		The thick tea extraction food of OLETF-	2.75
OLETF-loquat-leaf extract food	2.55
		OLETF-mixed culture fermentation tea extract food	8.33

By the result of table 16, the group that allows the OLETF rat absorb control Food, thick tea extraction or loquat-leaf extract is found the reduction of insulin.In addition, by blood-sugar level measuring as can be known, these groups are in the state of onset diabetes.In contrast, even suffer from the OLETF rat of diabetes, the insulin concentration of the group of picked-up mixed culture fermentation tea extract rises to the level of the LETO rat of the diabetes of not falling ill.Can confirm that thus the hot water extract of mixed culture fermentation tealeaves has the effect of improving the insulin secretion reduction that causes owing to onset diabetes.

[table 17]

Test portion	Adipose tissue mass (g)
		The LETO-control Food	37.0
The OLETF-control Food	125.0
		The thick tea extraction food of OLETF-	115.8
OLETF-loquat-leaf extract food	110.2
		OLETF-mixed culture fermentation tea extract food	94.7

By the result of table 17, even suffer from the OLETF rat of diabetes, the kidney of the group of picked-up mixed culture fermentation tea extract and the adipose tissue mass of testis periphery, also the OLETF rat group than the picked-up control Food is low.Thus can be clear and definite, the hot water extract of mixed culture fermentation tealeaves has the effect that the inhibition body fat is accumulated.

[table 18]

Test portion	S-TG (mg/dl)	Liver triglyceride (mg/g)
			The LETO-control Food	70.1	12.4
The OLETF-control Food	290	88.8
			The thick tea extraction food of OLETF-	278	105.5
OLETF-loquat-leaf extract food	312	74.3
			OLETF-mixed culture fermentation tea extract food	170	43.9

By the result of table 18, even suffer from the OLETF rat of diabetes, the serum of the group of picked-up mixed culture fermentation tea extract and the neutral fat concentration of liver, also the OLETF rat group than the picked-up control Food reduces.Thus can be clear and definite, the hot water extract of mixed culture fermentation tealeaves has the effect that reduces neutral fat.

[experimental example 7]

Other effect when studying the hot water extract's (freeze drying product) who allows normal rat absorb mixed culture fermentation tealeaves.

Promptly, with the same condition of experimental example 2 under, freely the ingest 4 kinds of feeds and the control Food of similarly having added material that hot water extract's freeze drying of test portion tealeaves (thick tea, fermented tea, loquat tea, mixed culture fermentation tealeaves) is separately obtained respectively with above-mentioned experimental example 2 of Sprague-Dawley (SD) rat that allowed for 4 ages in week fed.

After 4 weeks, serum, liver and adipose tissue were slaughtered and taked to fasting after 6 hours.

In following table, show the measurement result of serum lipid peroxide concentration, adipose tissue mass, serum neutral fat (triglyceride), liver neutral fat (triglyceride) respectively.

[table 19]

Test portion	Serum lipid peroxide concentration (nmol/ml)
		Contrast	61.7
Thick tea extraction	30.5
		Independent fermented tea extract	38.2
The loquat tea extraction	64.1
		The mixed culture fermentation tea extract	36.5

By the result of table 19, in normal rat,, to compare with the situation of picked-up control Food by the hot water extract of picked-up mixed culture fermentation tealeaves, serum lipid peroxide concentration reduces.Can confirm thus, even the hot water extract of mixed culture fermentation tealeaves at the normal healthy state, also brings into play the effect that reduces the serum lipid peroxide.

[table 20]

Test portion	Adipose tissue mass (g)
		Contrast	12.7
Thick tea extraction	6.87
		Independent fermented tea extract	8.71
The loquat tea extraction	10.8
		The mixed culture fermentation tea extract	8.20

By the result of table 20, in normal rat,, to compare with the situation of picked-up control Food by the hot water extract of picked-up mixed culture fermentation tealeaves, adipose tissue mass is low.Can confirm thus, even the hot water extract of mixed culture fermentation tealeaves at the normal healthy state, also brings into play and suppresses the effect that body fat is accumulated.

[table 21]

Test portion	Serum neutral fat concentration (mg/dL)	Liver neutral fat concentration (mg/g)
			Contrast	87.7	37.0
Thick tea extraction	75.8	19.7
			Independent fermented tea extract	77.8	26.1
The loquat tea extraction	62.9	31.0
			The mixed culture fermentation tea extract	55.2	18.6

By the result of table 21, in normal rat,, to compare with the situation of picked-up control Food by the hot water extract of picked-up mixed culture fermentation tealeaves, the neutral fat concentration of serum and liver is also low.Can confirm thus, even the hot water extract of mixed culture fermentation tealeaves at the normal healthy state, also brings into play the effect that reduces neutral fat.

[experimental example 8]

The influence that research brings type 1 diabetes by the hot water extract who absorbs mixed culture fermentation tealeaves.

Promptly, that in the bait that consists of basic composition with the AIN-76 that does not contain cholesterol, add 1 quality % and hot water extract's (freeze drying product) the same mixed culture fermentation tealeaves of experimental example 2, and allow male Sprague-Dawley (SD) rat in 4 all ages freely ingest.As a comparative example, the SD rat is thrown something and fed in same bait, do not add the control Food of mixed culture fermentation tea extract.Feeding conditions is the condition same with experimental example 2.

Ingesting beginning day and ingest beginning after 14 days, fasting is after 6 hours, by tail vein blood, uses blood sugar test to measure medical safe pipe (manufacturings of テ モ Le company) mensuration blood glucose value.

In addition, ingest after the above-mentioned blood sampling of beginning after 14 days, continue fasting again, after 9 hours (fasting total time: 15 hours), the streptozotocin (STZ) of administration 30mg/kg body weight.This STZ is the medicament with following effects: destroy the pancreas beta Cell of islet, cause the insulin secretion infringement, thereby cause type 1 diabetes.Behind the administration STZ, the food of freely ingesting again.

From the beginning after 7 days and after 14 days of ingesting again (from initial ingest 3 week of beginning the back and 4 week the back), fasting by tail vein blood, used blood sugar test to measure medical safe pipe (manufacturing of テ モ Le company) mensuration blood glucose value after 6 hours.

In addition, from the beginning 2 week backs of ingesting again (from initial 4 week of the beginning back of ingesting), fasting after 6 hours (after the above-mentioned blood sampling) slaughter, take serum and pancreas pancreas islet.Giving glucose to the pancreas pancreas islet that takes out stimulates, and measures secretion of insulin.

The variation of blood glucose value of ingesting after the beginning is shown in Figure 13.In addition, in the measurement result of insulin secretion shown in the following table and the measurement result of serum cholesterol concentration and serum neutral fat concentration.

In Figure 13, transverse axis is represented the elapsed time, and the longitudinal axis is represented the value of blood glucose value.In addition, the value of blood glucose value is that the blood glucose value that begins day of ingesting is expressed as 100mg/dL.

As shown in the drawing, the blood glucose value of the rat of picked-up control Food rose after administration STZ14 days in time, was counted as the type 1 diabetes morbidity.In contrast, absorbed the hot water extract's of mixed culture fermentation tealeaves rat, the rising of 14 days later blood glucose values is suppressed very for a short time.

[table 22]

	Insulin secretion (μ U/mL/islet) from pancreas islet
		Contrast	3.48
The mixed culture fermentation tea extract	5.96

By the result of table 22, from the beginning 4 week backs (after administration STZ2 weeks) of ingesting because the insulin secretion that the stimulation of glucose causes from the pancreas pancreas islet, hot water extract's the rat that has absorbed mixed culture fermentation tealeaves is than the rat height that has absorbed control Food.The hot water extract of clear and definite thus mixed culture fermentation tealeaves is because protection pancreas pancreas islet and can keep insulin secretion function, and hinted that the hot water extract of mixed culture fermentation tealeaves is effective to the prevention type 1 diabetes.

[table 23]

	Serum cholesterol concentration (mg/dL)	Serum neutral fat concentration (mg/dL)
			Contrast	124	336
The mixed culture fermentation tea extract	89	138

By the result of table 23, from the serum cholesterol concentration and the serum neutral fat concentration of the beginning 4 week backs (after administration STZ2 week) of ingesting, the rat that has absorbed the hot water extract of mixed culture fermentation tealeaves shows the value lower than the rat that has absorbed control Food.The hot water extract of clear and definite thus mixed culture fermentation tealeaves has the effect that reduces serum lipids concentration in type 1 diabetes.

[experimental example 9]

In order to confirm the security of fermented tea, carry out zoopery.

Promptly, use food processor that mixed culture fermentation tealeaves (leaf of tea and the mixed proportion of loguat leaf are that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour) is pulverized processing, up to the pulverulence that becomes 60～100 order sizes, obtain the powder-mixed fermented tea, with this mixed culture fermentation tealeaves with every day the 5000mg/kg body weight consumption to be that male rat is oral throw something and feed to the male Sprague-Dawley in 4 ages in week.In 22 ± 1 ℃ of temperature, humidity 55 ± 5%, freely absorb when feeding for 4 weeks under the condition of feed and water, to find deadly, yet no abnormal changes of weight is not observed the unusual of internal organs yet in the dissection inspection of feeding after finishing.

Therefore, infer the lethal dose (LD of fermented tea for rat ₅₀) more than the 5000mg/kg body weight, it is quite high to be judged as security.

[experimental example 10]

(manufacturing of mixed culture fermentation tealeaves-with the method for hand kneading)

Loguat leaf is put in the leaf of tea, adopted with the method for hand kneading and knead.The leaf of tea and the mixed proportion of loguat leaf are the leaf of tea: the mass ratio of loguat leaf is 90: 10 (loquat input ratio is 10%) and 75: 25 (loquat input ratio is 25%) 2 kinds.Rub the angle of incidence (with the time of hand kneading) and be two kinds of 25 minutes and 50 minutes.

After this, through 0 hour, 1 hour, 2 hours, 4 hours 4 kinds of fermentation procedures, obtain 16 kinds of mixed culture fermentation tealeaves.In addition, 0 hour tealeaves of fermentation procedure is the mixed culture fermentation tealeaves that obtains when rubbing into firm the end.

For the mixed culture fermentation tealeaves that obtains, with experimental example 1 similarly, with 100 ℃ hot water extracting 10 minutes, obtain the extract (use amount of mixed culture fermentation tealeaves: 2.0mg/ml), for this extract, measure AGH inhibition (maltose inhibition and invertase inhibition) similarly to Example 1, in addition, measure DPPH (1,1-two picryls-2-phenyl diazanyl) cancellation activity with following method.Separately the results are shown in table 24,25.In addition, the measured value of DPPH cancellation activity is big more, and the expression antioxidation is high more.In addition, similarly carry out the sense organ examination with experimental example 1.The results are shown in table 26.

(assay method of DPPH cancellation activity)

(unit: mensuration μ mol-Trolox/mg) is carried out (down together) according to following method to DPPH cancellation activity.

Mixed culture fermentation tealeaves is pulverized, and the tealeaves after will pulverizing is dissolved in 80% ethanolic solution, uses 80% same ethanolic solution to be diluted to 0.5mg/ml.The mixed solution of the 200mM MES buffer solution (pH6.0) of 400 μ M DPPH, the 12ml of making 12ml and 20% ethanol of 12ml, use エ Star ペ Application De Le Off マ Le チ ピ ペ Star ト that this mixed solution is injected into each 0.9ml of test tube (a)～(f) respectively, (x is the addition of analysis specimen to add 80% ethanol of (300-x) μ l in each of test tube (a)～(f), particularly, the x in test tube (a)～(f) is followed successively by 0,30,60,120,180,240).In addition, in test tube (a)～(f), add (x) μ l analysis specimen respectively and stir, add after 20 minutes, be determined at the absorbance under the 520nm.

Particularly, mensuration from the outset is after 30 seconds, in test tube (b), add 30 μ l analysis specimens, in test tube (c), add 60 μ l analysis specimens after 60 seconds, in test tube (d), add 120 μ l analysis specimens after 120 seconds, in test tube (e), add 180 μ l analysis specimens after 180 seconds, in test tube (f), add 240 μ l analysis specimens after 240 seconds.Then, the mensuration between was measured the absorbance of test tube (a) after 20 minutes from the outset, measures the absorbance of test tube (b) after 20 minutes 30 seconds, measures the absorbance of test tube (c) after 21 minutes, carried out the mensuration of absorbance like this every 30 seconds.

On the other hand, use Trolox as standard test portion production standard curve.The transverse axis of calibration curve is that (unit: μ mol), the longitudinal axis is the absorbance under the 520nm to the test portion addition.

With transverse axis as the test portion addition (unit: μ l), with the longitudinal axis as the absorbance under the 520nm, depict the measured value of analysis specimen, in absorbance continues scope that straight line reduces, obtain value with respect to the absorbance (Δ A520) of addition (x ' μ l).Then, obtain Trolox addition (unit: μ mol) when in calibration curve, obtaining identical (Δ A520).From the Trolox a great deal of that obtains like this (unit: μ mol) obtain the value (unit: μ mol-Trolox/mg) of the cancellation activity of every 1mg mixed culture fermentation tealeaves.

[table 24]

Loguat leaf drops into ratio 10%				Loguat leaf drops into ratio 25%
								Knead the time (branch) with hand	Fermentation time (hour)	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)	Knead the time (branch) with hand	Fermentation time (hour)	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)
25	0	82.3	85.4	25	0	75.9	62.9
								1	74.5	76.8	1	68.2	55.2
	2	62.9	63.1		2	63.5	50.0
								4	55.0	49.9	4	52.4	41.1
	50	0	63.3		52.7	50	0							70.2	61.4
1				54.8				36.7	1	62.6	45.6
		2	52.7		32.8		2					52.8	40.2
4				50.0				30.1	4	45.3	37.7

[table 25]

Loguat leaf drops into ratio 10%			Loguat leaf drops into ratio 25%
						Knead the time (branch) with hand	Fermentation time (hour)	Cancellation activity (μ mol-Trolox/mg)	Knead the time (branch) with hand	Fermentation time (hour)	Cancellation activity (μ mol-Trolox/mg)
	0	1.5		0	1.2

25	1	1.3	25	1	1.0
						2	1.0	2	0.8
	4	0.8		4	0.6
						50	0	1.2	50	0	1.0
1	1.1	1	0.9
				2	0.9		2	0.9
4	0.8	4	0.5

[table 26]

Loguat leaf drops into ratio 10%					Loguat leaf drops into ratio 25%
										Knead the time (branch) with hand	Fermentation time (hour)	Fragrance	Taste	Add up to	Knead the time (branch) with hand	Fermentation time (hour)	Fragrance	Taste	Add up to
25	0	3.0	3.5	6.5	25	0	3.5	3.0	6.5
										1	3.0	3.5	6.5	1	3.0	3.0	6.0
	2	2.5	3.0	5.5		2	2.0	3.0	5.0
										4	2.0	2.0	4.0	4	2.0	2.0	4.0
	50	0	2.5	2.5		5.0	50	0	3.0									3.0	6.0
1					2.0					2.0	4.0	1	2.5	2.5	5.0
		2	2.0	2.0		4.0		2	2.5							2.5	5.0
4					1.5					1.5	3.0	4	2.0	1.5	3.5

[experimental example 11]

(manufacturing of mixed culture fermentation tealeaves-grind method)

In mortar, add leaf and the loguat leaf of the bitter edible plant and grind.The leaf of tea and the mixed proportion of loguat leaf are the leaf of tea: the mass ratio of loguat leaf is 90: 10 (loquat input ratio is 10%) and 75: 25 (loquat input ratio is 25%) 2 kinds.The time of grinding is two kinds of 20 minutes and 40 minutes.

After this, through 0 hour, 1 hour, 2 hours, 4 hours 4 kinds of fermentation procedures, obtain 16 kinds of mixed culture fermentation tealeaves.In addition, 0 hour tealeaves of fermentation procedure is the mixed culture fermentation tealeaves that obtains when grinding firm the end.

For the mixed culture fermentation tealeaves that obtains, similarly measure the AGH inhibition with experimental example 1, in addition, measure DPPH cancellation activity.Separately the results are shown in table 27,28.In addition, similarly carry out the sense organ examination with experimental example 1.The results are shown in table 29.

[table 27]

Loguat leaf drops into ratio 10%				Loguat leaf drops into ratio 25%
								Grind the time (branch)	Fermentation time (hour)	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)	Grind the time (branch)	Fermentation time (hour)	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)
20	0	81.5	87.1	20	0	79.5	68.1
								1	76.9	77.0	1	73.4	59.3
	2	65.8	61.8		2	68.6	54.3
								4	61.2	60.0	4	54.9	45.0
	40	0	69.8		70.9	40	0							75.3	62.8
1				62.5				63.4	1	65.9	49.9
		2	54.8		58.6		2					56.4	43.5
4				48.6				50.5	4	50.6	40.0

[table 28]

Loguat leaf drops into ratio 10%			Loguat leaf drops into ratio 25%
						Grind the time (branch)	Fermentation time (hour)	Cancellation activity (μ mol-Trolox/mg)	Grind the time (branch)	Fermentation time (hour)	Cancellation activity (μ mol-Trolox/mg)
20	0	1.5	20	0	1.3
						1	1.3	1	1.1
	2	1.1		2	1.1
						4	0.9	4	1.0
	40	0		1.2	40					0	1.1
1			1.1			1	1.0
		2		0.9				2	0.7
4			0.7			4	0.6

[table 29]

Loguat leaf drops into ratio 10%					Loguat leaf drops into ratio 25%
										Grind the time (branch)	Fermentation time (hour)	Fragrance	Taste	Add up to	Grind the time (branch)	Fermentation time (hour)	Fragrance	Taste	Add up to
20	0	3.0	3.0	6.0	20	0	3.0	2.5	5.5
										1	2.5	3.0	5.5	1	3.0	2.0	5.0
	2	2.5	2.5	5.0		2	2.5	2.0	4.5
										4	2.0	2.0	4.0	4	2.0	1.5	3.5
	40	0	2.5	2.5		5.0	40	0	2.5									2.5	5.0
1					2.5					2.5	5.0	1	2.5	2.5	5.0
		2	2.0	2.5		4.5		2	2.5							2.5	5.0
4					1.5					1.5	3.0	4	1.5	1.5	3.0

[experimental example 12]

(manufacturing of mixed culture fermentation tealeaves-pulverizing stirring method)

In blender (goods name: ミ Le ア Application De ミ キ サ one, テ ス コ system company makes), add the leaf and the loguat leaf of tea and pulverize stirring.The leaf of tea and the mixed proportion of loguat leaf are the leaf of tea: the mass ratio of loguat leaf is 90: 10 (loquat input ratio is 10%) and 75: 25 (loquat input ratio is 25%) 2 kinds.Leaf and loguat leaf with respect to tea amount to 100 mass parts, and the addition of water is 100 mass parts.Pulverize mixing time and be two kinds of 10 minutes and 20 minutes.

After this, through 1 hour, 4 hours, 8 hours, 12 hours, 16 hours 5 kinds of fermentation procedures, obtain 20 kinds of mixed culture fermentation tealeaves.

For the mixed culture fermentation tealeaves that obtains, similarly measure the AGH inhibition with experimental example 1, in addition, measure DPPH cancellation activity.Separately the results are shown in table 30,31.In addition, similarly carry out the sense organ examination with experimental example 1.The results are shown in table 32.

[table 30]

Loguat leaf drops into ratio 10%				Loguat leaf drops into ratio 25%
								Pulverize mixing time (branch)	Fermentation time (hour)	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)	Pulverize mixing time (branch)	Fermentation time (hour)	Maltose inhibiting rate (%)	Invertase inhibiting rate (%)
10	1	50.2	42.3	10	1	45.2	40.0
								4	75.4	49.8	4	72.8	69.5
	8	76.3	56.3		8	73.9	70.1
								12	72.3	56.3	12	72.4	69.9
	16	55.2	50.0		16	61.0	58.2
								20	1	42.3	39.6	20	1	45.6	41.2
4	56.8	49.7	4	59.8	55.0
						8	65.2		50.0	8	65.6		61.0
12	61.9	50.0	12	66.6	59.6
						16	45.5		45.2	16	51.8		49.2

[table 31]

Loguat leaf drops into ratio 10%			Loguat leaf drops into ratio 25%
						Pulverize mixing time (branch)	Fermentation time (hour)	Cancellation activity (μ mol-Trolox/mg)	Pulverize mixing time (branch)	Fermentation time (hour)	Cancellation activity (μ mol-Trolox/mg)
10	1	1.0	10	1	0.8
						4	1.2	4	1.0
	8	1.3		8	1.0
						12	1.3	12	0.9
	16	0.7		16	0.6
						20	1	0.8	20	1	0.6
4	1.1	4	0.9
				8	1.1		8	0.9
12	1.0	12	0.8
				16	0.7		16	0.5

[table 32]

Loguat leaf drops into ratio 10%					Loguat leaf drops into ratio 25%
										Pulverize mixing time (branch)	Fermentation time (hour)	Fragrance	Taste	Add up to	Pulverize mixing time (branch)	Fermentation time (hour)	Fragrance	Taste	Add up to
10	1	1.5	1.5	3.0	10	1	2.0	1.5	3.5
										4	2.5	3.0	5.5	4	3.0	3.0	6.0
	8	3.0	3.5	6.5		8	3.5	3.0	6.5
										12	3.0	3.0	6.0	12	3.5	3.0	6.5
	16	1.5	2.5	4.0		16	2.0	2.0	4.0
										20	1	1.0	2.0	3.0	20	1	1.5	2.0	3.5
4	2.0	2.5	4.5	4	2.5	2.5	5.0
								8	2.5		2.5	5.0	8	2.5		3.0	5.5
12	3.0	2.5	5.5	12	3.0	2.0	5.0
								16	2.0		2.0	4.0	16	2.5		1.5	4.0

[analytical test example 10]

(analysis of fragrance component)

The material that dry loguat leaf is only obtained, only ferment material that loguat leaf obtains, commercially available black tea (Darjeeling's black tea and Assam tea), use at those pestle tealeaves of east, comprehensive agricultural test site, Nagasaki county and prop up a black tea that the leaf of the tea of cultivation ferments, use the leaf of identical tea but do not ferment and the green tea (thick tea) made and mixed culture fermentation tealeaves (leaf of tea and the mixed proportion of loguat leaf are that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour) etc. are various as sample, carry out the quantitative analysis of fragrance component.

The pre-treatment of the fragrance component of teas is adopted the post concentration method to wait to carry out usually.Here, as simple and easy/rapidly test portion extraction, concentrate, the chromatogram introductory technique, adopt the solid phase micro extraction method to carry out.Quantitatively obtain by the following method: in as the test portion of determination object, add cyclohexanol, obtain the relative peak area ratio of the peak area of a fragrance component with respect to internal standard compound as internal standard compound.

Particularly, carry out according to following method.The adjustment of test portion is following to be carried out: add 30% sodium-chloride water solution, 50 μ L of 20g sample, 1mL 1% cyclohexanol as internal standard compound in the conical flask of 200mL, heated 5 minutes down at 80 ℃ the sealing back, make in the conical flask stable after, insert collecting pipe 20 minutes, and collected the fragrance component that produces.The collecting pipe of having collected fragrance component is inserted into the inlet of the gas chromatograph that is heated to 250 ℃, fragrance component was imported in the post of gas chromatograph 3 minutes, analyze.Poly dimethyl cyclohexane/carboxen/ divinylbenzene that collecting pipe uses SUPELCO company to make.For the analysis condition of gas-chromatography, stabil-WAX (polyethylene glycols) 60m * 0.25mm that post is made for Reapect company, thickness uses 0.25 μ m, and column temperature after 3 minutes, is warmed up to 250 ℃ with 10 ℃/minute 70 ℃ of maintenances.The inlet temperature is 250 ℃, and helium pressure is set at 120kPa.The gaschromatographic mass spectrometric analysis that is used for the composition evaluation carries out under 250 ℃ of I/F temperature, ionization voltage 70eV, ionization electric current 60 μ A.For 1-hexanol, anti--the 2-hexenoic aldehyde, acetate blatter alcohol ester, blatter alcohol, linalool, benzaldehyde, gaultherolin, geraniol, 9 kinds of compositions of nerolidol, measure relative peak area ratio with respect to internal standard compound.It the results are shown in table 33.

[table 33]

	Loguat leaf (drying)	Loguat leaf (fermentation)	Commercially available black tea (Ah Sa grand-mother)	Commercially available black tea (Darjeeling)	Black tea (test site)	Mixed culture fermentation tealeaves	Green tea
								The 1-hexanol	1.16	0.47	-	-	0.06	0.14	0.02
Instead-the 2-hexenoic aldehyde	3.13	1.81	1.10	1.05	1.14	5.09	0.01
								Acetate blatter alcohol ester	tr.	1.33	-	-	tr.	0.41	tr.
Blatter alcohol	2.91	2.51	0.12	0.00	0.39	0.49	0.12
								Linalool	0.22	0.08	1.67	0.97	2.27	1.84	0.13
Benzaldehyde	0.12	0.69	1.05	1.04	1.05	4.26	0.02
								Gaultherolin	0.20	0.03	1.73	1.83	2.67	1.52	0.01
Geraniol	0.00	0.00	0.87	0.97	3.01	3.37	0.04
								Nerolidol	0.06	0.12	0.21	0.17	3.68	2.20	0.06

Annotate) with respect to the relative area of internal standard compound

Tr.:, be below the minimum area though can detect

Result by table contains all 9 kinds of compositions of enumerating in the table in mixed culture fermentation tealeaves.In the middle of these, linalool, benzaldehyde, gaultherolin, geraniol and nerolidol contain many in the black tea of the leaf fermentation of the tea that makes the test site, in 2 kinds of commercially available black tea, also contain, but the content in the loguat leaf of green tea and fermentation is fewer.In addition, anti--2-hexenoic aldehyde, acetate blatter alcohol ester, blatter alcohol contain many especially in the loguat leaf of fermentation.With the peak area of the peak area of 8 kinds of compositions beyond the 1-hexanol (with respect to the value of the relative area of internal standard compound) and 1-hexanol (with respect to the value of the relative area of internal standard compound) when comparing, all the peak area than 1-hexanol is big to have only in the mixed culture fermentation tealeaves 8 kinds of each all peak areas of composition.

In addition, in mixed culture fermentation tealeaves, even the leaf of change tea and the mixed proportion of loguat leaf, kneading time, fermentation time etc. are created conditions, also one detect the 1-hexanol surely, and be detected amount other 8 kinds of fragrance components that composition lacks than table 1 record.

[experimental example 13]

Allow the KK-A of natural occurrence diabetes B ^yMouse absorb respectively mixed culture fermentation tealeaves hot water extract's (freeze drying product), contain a large amount of Tea Polyphenols fraction (below, be called the TS fraction) and contain a large amount of theaflavin fraction (below, be called the TF fraction), and study the influence that blood glucose value is brought.

The hot water extract of mixed culture fermentation tealeaves uses the following extract that obtains: (leaf of tea and the mixed proportion of loguat leaf are that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour with the hot water extracting mixed culture fermentation tealeaves of 100 ℃ of 1000ml, moisture content 5 quality %) after 10 minutes, filter, filtrate freeze drying disease is made pulverous material.

As the TS fraction, use pulverous material is made in the Fr.6 freeze drying that obtains in the above-mentioned analytical test example 2.

As the TF fraction, use pulverous material is made in the Fr.7 freeze drying that obtains in the above-mentioned analytical test example 2.

The indoor raising of animal feeding that the light that mouse is turned on light at 22 ± 1 ℃ of room temperatures, humidity 55 ± 5%, 8:00～20:00 circulates.

Use the KK-A in 5 ages in week ^yMouse, the solid-state feed of MF of throwing something and feeding in 2 initial weeks (オ リ エ Application Le yeast industry (strain) manufacturing) prepares raising.Because KK-A ^yMouse is the natural occurrence diabetes about 7～8 ages in week, therefore, and from having 7 ages in week of initiation potential, beginning ingestion experiment food.That is, (behind the 9:00～15:00),, measure blood glucose value, equally divide into groups, allow it freely ingest following 6 weeks of test food with 7 every group according to body weight and blood glucose value from tail vein blood in the fasting 6 hours during ages of 7 weeks.

Test food is following 7 kinds.

Control Food: the purifying food that will form based on AIN-76 is food in contrast.The quality of control Food is formed (g/kg): casein 200g/kg, corn oil 100g/kg, mineral intermixture (AIN-76-MX) 35g/kg, vitamin mixtures (AIN-76-VX) 10g/kg, cellulose 50g/kg, lipotropin 2g/kg, DL-methionine 3g/kg, cornstarch 150g/kg and sucrose 450g/kg.

Extract adds food: add the above-mentioned pulverous hot water extract of 1 mass parts (freeze drying product) in the above-mentioned control Food of 100 mass parts, and deduct the sucrose of its addition part from control Food.

TS adds food: owing to contain the Tea Polyphenols of 5.4 quality % among the above-mentioned hot water extract (freeze drying product), therefore, in control Food, add above-mentioned TS fraction, and the content that makes it to add with the said extracted thing Tea Polyphenols in the test food equates, deducts its addition sucrose partly again from control Food.

2 times of TS add foods: add above-mentioned TS fraction in control Food, and the content that makes Tea Polyphenols deducts the sucrose of its addition part again for the said extracted thing adds 2 times of test food from control Food.

TF adds food: owing to contain the theaflavin of 4.8 quality % among the above-mentioned hot water extract (freeze drying product), therefore, in control Food, add above-mentioned TF fraction, and the content that makes it to add with the said extracted thing theaflavin in the test food equates, deducts its addition sucrose partly again from control Food.

2 times of TF add foods: add above-mentioned TF fraction in control Food, and the content that makes theaflavin deducts the sucrose of its addition part again for the said extracted thing adds 2 times of test food from control Food.

TS+TF adds food: add above-mentioned TS fraction and above-mentioned TF fraction in control Food, and make it to equate with polyphenol content and the theaflavin content that the said extracted thing adds in the test food respectively, from control Food, deduct the total sucrose partly of their addition again.

Between feeding period, food ration is measured every day, and body weight and water uptake are measured every other day.

, after 6 hours,, and use blood sugar test to measure medical safe pipe (manufacturing of テ Le モ company) and measure blood glucose value after 2,4,6 weeks at beginning ingestion experiment food from tail vein blood in fasting.The variation of the blood glucose value of the mouse in 6 weeks of picked-up test food is shown in Figure 14 to Figure 17.

As shown in figure 14, the blood glucose value that has absorbed the mouse of control Food rises in time, surpasses 300mg/dl after 6 weeks.In addition, because water uptake increases too, the mouse that has absorbed control Food is counted as onset diabetes.On the other hand, absorbed the blood glucose value that extract adds the mouse of food and had the tendency that reduces in time, the value after 6 weeks is reduced to the only about half of of the mouse that absorbed control Food.

As shown in figure 15, absorbed TS and added blood glucose value reduction after 6 weeks of the mouse of food, absorbed 2 times of TS and added under the situation of foods, reduced more effectively.

As shown in figure 16, absorbed TF and added blood glucose value reduction after 6 weeks of the mouse of food, absorbed 2 times of TF and added under the situation of foods, from the back reduction of 4 weeks.

As shown in figure 17, absorbed the blood glucose value of mouse that TS+TF adds food after 4 weeks with absorbed the mouse that extract adds food and be almost same level.

Can be clear and definite by above result, the effect that the hot water extract's of mixed culture fermentation tealeaves inhibition blood glucose value rises is mainly by Tea Polyphenols and theaflavin performance.

[clinical trial example 1]

Research picked-up contains the influence that the beverage of the extract of mixed culture fermentation tealeaves brings adult's blood glucose value, serum neutral fat, serum cholesterol, body fat rate and blood pressure.In addition, this test obtains admitting of the upright Nagasaki Seybolt university research Ethics Committee in county, and implements in accordance with the spirit of Declaration of Helsinki.

The testee is Nagasaki county Room office worker who recruits in the Room, Nagasaki county, carry out same daily life with the people of health, do not accept doctor's patient compliance instruction, be not conceived person and be that the doctor is judged as suitable people, content for this test, accept explanation fully, and propose letter of consent by me, totally 28 people according to my free wish.

The test beverage uses the beverage of following making: with respect to the hot water of 90 ℃ of 100 mass parts, (leaf of tea and the mixed proportion of loguat leaf are that 9: 1, kneading time are that 20 minutes, fermentation time are 0 hour to the material that adds 0.83 mass parts that mixed culture fermentation tealeaves roasting is fried in shallow oil and obtain, moisture content 1 quality %), extract after 4 minutes and filter, in each wrapper, respectively fill 200ml filtrate, sealing.

Make the subject when breakfast or Chinese meal and dinner, drink 1 bottle respectively, drank 2 bottles, and drank in 1 day with 3 months.Before picked-up test beverage, the picked-up beginning is after 1 month, after 2 months and take a blood sample on an empty stomach the morning after 3 months, carries out body weight determination, the body fat rate is measured and blood pressure (systolic pressure and diastolic pressure) is measured.In addition, blood sampling 9 fasting later on evening before that day.3 months diet for duration of test is not particularly limited, and instructs it according to carrying out with daily identical life.

As mensuration project, gross protein, A/G ratio, T-CHOL, HDL-cholesterol, neutral fat, free fatty, uric acid, urea nitrogen, creatinine, Na, Cl, K, GOT, GPT, γ-GTP, phosphatide, Ca, Phos, cholinesterase, LDH, ALP, albumin, bilirubin direct, indirect bilirubin, insulin, blood sugar, hemoglobin A lc, white blood cell count, erythrocyte number, hemochrome amount, hematocrit, MCV, MCH, MCHC, platelet count have been measured for blood constituent.

During 3 months of picked-up test beverage, 28 experimenters' mean value is in the normal range (NR) in all mensuration projects.

Figure 18, the 19th illustrates the curve map of the measurement result of blood glucose value, Figure 18 represents all 28 people's mean value, and the blood glucose value (initial value) that Figure 19 represents to absorb before the test beverage begins is the rate of change of the above people's (10) of 110mg/dL mean value with respect to initial value.This rate of change is that (unit: the value of %) calculating, " * " expression among the figure has significant poor (following identical) with respect to initial value by (mean value-initial value of measured value)/initial value * 100.

Figure 20, the 21st illustrates the curve map of the measurement result of serum neutral fat concentration, Figure 20 represents all 28 people's mean value, and the serum neutral fat concentration (initial value) that Figure 21 represents to absorb before the test beverage begins is the rate of change of the above people's (9) of 150mg/dL mean value with respect to initial value.

Figure 22, the 23rd illustrates the curve map of the measurement result of serum cholesterol concentration, Figure 22 represents all 28 people's mean value, and the serum cholesterol concentration (initial value) that Figure 23 represents to absorb before the test beverage begins is the rate of change of the above people's (11) of 220mg/dL mean value with respect to initial value.

Figure 24, the 25th illustrates the curve map of the measurement result of body fat rate, Figure 24 represents all 28 people's mean value, and the body fat rate (initial value) that Figure 25 represents to absorb before the test beverage begins is the rate of change of the mean value of the people (15) more than 25% with respect to initial value.

Figure 26, the 27th illustrates the curve map of the measurement result of maximal blood pressure, Figure 26 represents all 28 people's mean value, and the maximal blood pressure (initial value) that Figure 27 represents to absorb before the test beverage begins is the rate of change of the above people's (14) of 140mmHg mean value with respect to initial value.

Figure 28, the 29th illustrates the curve map of the measurement result of minimal blood pressure, Figure 28 represents all 28 people's mean value, and the minimal blood pressure (initial value) that Figure 29 represents to absorb before the test beverage begins is the rate of change of the above people's (10) of 90mmHg mean value with respect to initial value.

By these results as can be known, for blood glucose value, though the change in duration of test of proprietary mean value, the high people of initial value is in the picked-up beginning after 2 months and after 3 months, and compares before the picked-up, has a mind to the free burial ground for the destitute reduce.

For serum neutral fat concentration, though the change in duration of test of proprietary mean value, the high people of initial value is in the picked-up beginning after 2 months and after 3 months, and compares before the picked-up, reduces about 40%.

For serum cholesterol concentration, though the change in duration of test of proprietary mean value, the high people of initial value is in the picked-up beginning after 2 months and after 3 months, and compares before the picked-up, has a mind to the free burial ground for the destitute reduce.

For the body fat rate, though the change in duration of test of proprietary mean value, high and the people that lipophilia arranged slightly of initial value is in the picked-up beginning after 3 months, and compares before the picked-up, has a mind to the free burial ground for the destitute reduce.

For maximal blood pressure, though the change in duration of test of proprietary mean value, the high people of initial value is in the picked-up beginning after 1 month, after 2 months and after 3 months, and compares before the picked-up, has a mind to the free burial ground for the destitute reduce.

For minimal blood pressure, though the change in duration of test of proprietary mean value, the high people of initial value is in the picked-up beginning after 2 months and after 3 months, and compares before the picked-up, has a mind to the free burial ground for the destitute reduce.

Can be clear and definite by these results, contain the beverage of the extract of mixed culture fermentation tealeaves by picked-up, the effect of these values that can improve is arranged respectively among the people of high tendency in blood glucose value, serum neutral fat, serum cholesterol, maximal blood pressure, minimal blood pressure.In addition, can also be clear and definite, the effect that can obtain making the people's that lipophilia is arranged a little body fat rate to reduce.In addition, because above-mentioned each measured value of normal people is reduced, therefore be evaluated as safe beverage.

[clinical testing example 2]

Research picked-up contains the influence that the beverage of the extract of mixed culture fermentation tealeaves brings the body fat rate of ragazza.In addition, this test obtains admitting of the upright Nagasaki Seybolt university research Ethics Committee in county, and implements in accordance with the spirit of Declaration of Helsinki.

The testee is this school schoolgirl who recruits in the upright Nagasaki Seybolt university in county, carry out same daily life with the people of health, do not accept doctor's patient compliance instruction, be not conceived person and be that the doctor is judged as suitable people, content for this test, accept explanation fully, and propose letter of consent by me, totally 54 adult schoolgirls according to my free wish.

The test beverage uses the beverage same with above-mentioned clinical testing example 1.In addition, beverage in contrast uses the green tea beverage of commercially available 200ml wrapper.

Make 27 subjects drink test beverage, 27 subjects and drink the contrast beverage, when breakfast or Chinese meal and dinner, drink 1 bottle respectively, drank 2 bottles, and drank in 1 day with 3 months.

Before beginning to absorb, measure proprietary blood glucose value and body fat rate, drink group and contrast beverage at the test beverage and drink group and divide into groups according to mean value respectively almost equally.The test beverage is identical with the outward appearance of contrast beverage, from not discerning in appearance.Before picked-up test beverage, the picked-up beginning is after 1 month, after 2 months and take a blood sample on an empty stomach the morning after 3 months, carries out body weight determination, body fat is measured and blood pressure determination.In addition, blood sampling 9 fasting later on evening before that day.3 months diet for duration of test is not particularly limited, and instructs it according to carrying out with daily identical life.

The mensuration project of blood constituent is identical with above-mentioned clinical testing example 1.

During 3 months of picked-up test beverage, 54 experimenters' mean value is in the normal range (NR) in all mensuration projects.In addition, during these 3 months,, all do not observe difference for all mensuration projects absorbing 27 testees that test beverage and absorbing between 27 testees of contrast beverage.

Figure 30 represents that the body fat rate (initial value) before on-test is the rate of change of the body fat rate of the people (11 of picked-up test beverage persons, 13 of picked-up contrast beverage persons) more than 25% with respect to initial value.

As shown in the drawing, the initial value of fat percentage is the philtrum that lipophilia is arranged a little more than 25% in vivo, has absorbed the group of test beverage and has compared with the group of picked-up contrast beverage, and the body fat rate after 3 months is had a mind to the free burial ground for the destitute and reduced.

Can be clear and definite by these results, contain the beverage of the extract of mixed culture fermentation tealeaves by picked-up, the effect that can obtain making the body fat rate of the ragazza that lipophilia is arranged a little to reduce.Can confirm in addition, even normal ragazza picked-up also is safe.

Industrial applicibility

According to the present invention, can obtain for prevention or improve fermented tea, fermented tea extract and the diet product that above-mentioned diabetes or hyperlipemia have effective drug effect.

And fermented tea of the present invention has following advantage: can effectively utilize the loguat leaf that is not used effectively up to now, as the leaf of the tea of raw material, can also use and still be sliced off the tealeaves that abandons now.

Claims (31)

1. a fermented tea wherein, mixes, kneads the leaf and the loguat leaf of tea, and ferments.

2. fermented tea, wherein, the leaf of tea blend and loguat leaf, and disorganize ferment again.

3. fermented tea, wherein, the leaf of tea blend and loguat leaf, and grind, ferment again.

4. fermented tea wherein, adds entry and pulverizes in the leaf of tea and loguat leaf and mix, and ferments again.

5. any one described fermented tea in the claim 1～4, wherein, the content of the theaflavin class that contains in the above-mentioned fermented tea is than the fermentation and the content of the theaflavin class that contains in the independent fermented tea that obtains is many separately of the leaf of above-mentioned tea.

6. any one described fermented tea in the claim 1～4, wherein, above-mentioned fermented tea contains the polyphenol P of catechin gallic acid ester class oxidative condensation, and this polyphenol P exists ¹³Show in the C-NMR spectrogram from the signal of the phloroglucin of catechin A ring with from the signal of galloyl base; the molecular weight of its acetylate is 1000～15000; and 2000 serving as greatly, and the content of this polyphenol P in the above-mentioned fermented tea is than the fermentation and the content of this polyphenol P of containing in the independent fermented tea that obtains is many separately of the leaf of above-mentioned tea.

7. any one described fermented tea in the claim 1～4, wherein, above-mentioned fermented tea contains the fragrance component of 1-hexanol, anti--the 2-hexenoic aldehyde, acetate blatter alcohol ester, blatter alcohol, linalool, benzaldehyde, gaultherolin, geraniol and nerolidol, in the solid phase micro extraction method, all the peak area than 1-hexanol is big for each peak area of the fragrance component of anti--the 2-hexenoic aldehyde, acetate blatter alcohol ester, blatter alcohol, linalool, benzaldehyde, gaultherolin, geraniol and nerolidol.

8. fermented tea extract, it extracts any one described fermented tea in claim 1～4 and obtains.

9. diet product, it contains any one described fermented tea in the claim 1～4.

10. diet product, it contains the described fermented tea extract of claim 8.

11. the described diet product of claim 9, it is used to suppress blood glucose value and rises.

12. the described diet product of claim 10, it is used to suppress blood glucose value and rises.

13. the described diet product of claim 9, it is used for prevention or improves diabetes.

14. the described diet product of claim 10, it is used for prevention or improves diabetes.

15. the described diet product of claim 9, it is used to reduce neutral fat.

16. the described diet product of claim 10, it is used to reduce neutral fat.

17. the described diet product of claim 9, it is used to reduce cholesterol.

18. the described diet product of claim 10, it is used to reduce cholesterol.

19. the described diet product of claim 9, it is used to reduce the serum lipid peroxide.

20. the described diet product of claim 10, it is used to reduce the serum lipid peroxide.

21. the described diet product of claim 9, it is used to suppress body fat and accumulates.

22. the described diet product of claim 10, it is used to suppress body fat and accumulates.

23. the described diet product of claim 9, it is used to suppress increased blood pressure.

24. the described diet product of claim 10, it is used to suppress increased blood pressure.

25. one kind is used to suppress the composition that blood glucose value rises; wherein contain be selected from the following substances more than a kind: Tea Polyphenols A, have the galloyl base the theaflavin derivative, to have with the epiafzelechin gallate be the OPC of construction unit and the polyphenol P of catechin gallic acid ester class oxidative condensation, this polyphenol P exists ¹³Show in the C-NMR spectrogram that the molecular weight of its acetylate is 1000～15000 from the signal of the phloroglucin of catechin A ring with from the signal of galloyl base, and serve as very big with 2000.

26. the described composition that is used to suppress the blood glucose value rising of claim 25 wherein also contains the loquat OPC.

27. one kind is used to suppress the composition that blood glucose value rises; wherein comprise fermented tea; described fermented tea contain be selected from the following substances more than a kind: Tea Polyphenols A, have the galloyl base the theaflavin derivative, to have with the epiafzelechin gallate be the OPC of construction unit and the polyphenol P of catechin gallic acid ester class oxidative condensation, this polyphenol P exists ¹³Show in the C-NMR spectrogram that the molecular weight of its acetylate is 1000～15000 from the signal of the phloroglucin of catechin A ring with from the signal of galloyl base, and serve as very big with 2000.

28. the described composition that is used to suppress the blood glucose value rising of claim 27, wherein, above-mentioned fermented tea also contains the loquat OPC.

29. one kind is used to suppress the composition that blood glucose value rises; wherein comprise the fermented tea extract; described fermented tea extract contain be selected from the following substances more than a kind: Tea Polyphenols A, have the galloyl base the theaflavin derivative, to have with the epiafzelechin gallate be the OPC of construction unit and the polyphenol P of catechin gallic acid ester class oxidative condensation, this polyphenol P exists ¹³Show in the C-NMR spectrogram that the molecular weight of its acetylate is 1000～15000 from the signal of the phloroglucin of catechin A ring with from the signal of galloyl base, and serve as very big with 2000.

30. the described composition that is used to suppress the blood glucose value rising of claim 29, wherein, above-mentioned fermented tea extract also contains the loquat OPC.

31. diet product wherein contain any one described composition that is used to suppress the blood glucose value rising in the claim 25～30.

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