JP6243270B2 - Koji fermenting composition and koji fruit processing method - Google Patents

Description

  The present invention relates to a koji fermented composition obtained by fermentation of koji fruits and a method for treating koji fruits.

  In general, persimmons are widely cultivated because edible fruits and processed foods using persimmon fruits are edible. When edible berries are used, the fruit portion is used after separating the berries other than fruits. Regarding the use of soot, for example, the following is known.

  In Patent Document 1, a dried fruit of strawberry is soaked in water or any fruit juice to swell, and the pulp of the swelled dried fruit is crushed and sludged, and then liquefied by adding pectin-degrading enzyme. A method for producing grape wine is described in which a sugar source for alcohol fermentation is supplied to the liquefied product and yeast is added to cause alcohol fermentation.

  Patent Document 2 describes that koji is made into a paste and fermented with lactic acid. This lactic acid fermentation is performed using plant-derived lactic acid bacteria. Thereby, it is said that the antioxidant activity function of sputum can be enhanced, and on the other hand, the ability to suppress the decrease in red blood cell deformability due to oxidative stress is not deteriorated.

JP-A-10-191955 JP 2010-252726 A

  In both Patent Documents 1 and 2, pectin-degrading enzyme as a pretreatment for fermentation after crushing or crushing the fruit of persimmon, or cellulase-degrading enzyme simultaneously with fermentation using yeast or lactic acid bacteria as a method of parallel double fermentation The process using was also performed. That is, it is necessary to perform a pretreatment for crushing or crushing fruits before the enzyme treatment, which is complicated.

  Moreover, persimmon contains abundant carbohydrates, vitamins, potassium and the like in fruits, and is known to have antiviral activity, antitumor activity, antiallergic activity and the like in addition to the antioxidant activity described above. Therefore, it is expected to produce a composition having a new function by treating the fruit of persimmon.

  Accordingly, it is an object of the present invention to provide a composition having a new function by simply treating a strawberry fruit and a method for treating the strawberry fruit.

  As a result of intensive studies on the treatment of persimmon fruit, the present inventors have found that the processed product obtained by fermenting persimmon fruit with Aspergillus oryzae has an active ingredient and a fermented persimmon composition having an inhibitory effect on an increase in blood glucose level, an antihypertensive effect, and an anti-obesity effect. It was found to have an action.

That is, in order to achieve the above object, the following inventions [1] to [6] are provided.
[1] persimmon fruit persimmon fermented composition for blood glucose increase inhibitory effect that Yusuke containing as an active ingredient, a treated product obtained by solid fermentation with Aspergillus oryzae.
[2] persimmon fruit persimmon fermented composition for antihypertensive action that Yusuke containing as an active ingredient, a treated product obtained by solid fermentation with Aspergillus oryzae.
[3] The fruit persimmon fermented composition for anti-obesity effect that Yusuke containing as an active ingredient, a treated product obtained by solid fermentation with Aspergillus oryzae persimmon.
[4] The koji fermentation composition according to any one of [1] to [3], wherein the koji mold belongs to the genus Aspergillus.
[5] The koji fermentation composition according to [4], wherein the koji mold is black koji mold.
[6] A method for treating persimmon fruit, comprising: a fermentation process for subjecting persimmon fruit to solid fermentation with koji mold; and an extraction process for extracting an active ingredient by bringing alcohol into contact with the fermented product.

  The koji fermentation composition of the present invention contains a processed product obtained by fermenting koji fruit with koji mold as an active ingredient. In the examples described later, an embodiment using Aspergillus spp. Aspergillus is shown, and in Example 2, it is shown that the koji fermentation composition of the present invention has an α-glucosidase inhibitory activity superior to the treatment with lactic acid bacteria. In Example 3, it is shown that the koji fermentation composition has an angiotensin converting enzyme inhibitory activity superior to the treatment with lactic acid bacteria. In Example 4, the koji fermentation composition has a β-lipase superior to the treatment with lactic acid bacteria. It has been shown to have inhibitory activity.

  In other words, by containing a processed product obtained by fermenting koji fruit with koji mold as an active ingredient, koji fermenting composition has a blood glucose level increase inhibitory action (α-glucosidase inhibitory activity) and a blood pressure increase inhibitory action (angiotensin converting enzyme inhibitory activity). Activity) and anti-obesity action (β-lipase inhibitory activity), so that each koji fermentation composition can be used as a blood glucose level increase inhibitor, blood pressure increase inhibitor and anti-obesity agent.

  A blood sugar level increase inhibitor, for example, has an α-glucosidase inhibitory activity that inhibits the action of α-glucosidase inhibitory activity in the intestine and inhibits the decomposition and absorption of sugar. Ingestion is expected to suppress an increase in blood glucose level.

  An antihypertensive agent, for example, has an angiotensin converting enzyme inhibitory activity that inhibits angiotensin converting enzyme (ACE) involved in an increase in blood pressure. By taking an effective amount of an antihypertensive agent, an increase in blood pressure can be achieved. It is expected to be suppressed.

  The anti-obesity agent is achieved by inhibiting the action of β-lipase in the intestine and having β-lipase inhibitory activity that inhibits the decomposition and absorption of fat.

  The koji used in the present invention is widely cultivated as an edible fruit and is easily available. When using the fruit part of a persimmon for a fermentation process, since it can be used as it is, a fermentation process (fermentation process) can be performed simply.

  In view of using the koji fermentation composition as a pharmaceutical or food, the alcohol used in the extraction step of the present invention is preferably ethanol or the like that is easily available and safe for the human body.

  That is, according to the present invention, an active ingredient having new functionalities such as a blood glucose level increase inhibitory effect, a blood pressure increase inhibitory effect, and an anti-obesity effect is contained only by simply treating the fruit of persimmon and performing simple alcohol extraction. It is possible to easily obtain a koji fermentation composition.

It is a figure which shows the result of the GC-MS analysis of a koji fermentation composition (Black koji mold treatment, 100% ethanol-water extraction (ethanol fraction)). It is a figure which shows the result of the GC-MS analysis of a koji fermentation composition (Black koji mold treatment, 100% ethanol-water extraction (water fraction)). It is a figure which shows the result of the GC-MS analysis of a koji fermentation composition (Black koji mold treatment, 50% ethanol extraction). It is a figure which shows the result of the GC-MS analysis of a koji fermentation composition (lactic acid bacteria treatment, 50% ethanol extraction). It is a figure which shows the result of the GC-MS analysis of a koji fermentation composition (lactic-acid-bacteria treatment, 100% ethanol-water extraction (ethanol fraction)). It is a figure which shows the result of the GC-MS analysis of a koji fermentation composition (lactic-acid-bacteria treatment, 100% ethanol-water extraction (water fraction)).

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
The present invention is a koji fermentation composition containing a processed product obtained by fermentation of koji fruits with koji mold as an active ingredient. The koji fermentation composition can be obtained by a koji fruit processing method having a fermentation process in which koji fruit is fermented with koji mold and an extraction process in which alcohol is brought into contact with the fermented product to extract an active ingredient. .

  The part of the cocoon used in the present invention is a fruit part from which the cocoon part has been removed. As long as the fruit portion is included, it does not matter whether the skin or seeds are present. There are no particular restrictions on the variety of koji used, and any taxonically belonging to Diospyros kaki can be used, but for reasons such as easy availability, it is named, for example, Fuyucho or Jiro It is good. Persimmons are young fruits harvested earlier than the normal harvest time (about 30 to 60 days after fruit set) or mature fruits harvested at the normal harvest time (about 150 to 180 days after fruit set) It is good to use. After removing the cocoon part from the harvested cocoon, it is preferably subjected to washing, removal of fruit skins and seeds, shredding, and sterilization treatment as a raw material for the fermentation process. The sterilization process may be a known process such as boiling, ultraviolet light, or sterilization with chemicals.

As the koji mold, for example, koji mold belonging to the genus Aspergillus used for the production of shochu is preferably used. The koji mold used for the production of shochu refers to a filamentous fungus that ferments a solid raw material such as boiled cereals such as barley and rice and produces citric acid from sugar. As such koji molds, black koji molds (Aspergillus Awamori) or white koji molds (Aspergillus Kawachii) can be used.
As such a koji mold, a commercially available one can be used, and a strain deposited with a patent depositary institution can also be used. The deposited strain is available, for example, from the Biological Genetic Resource Department (NBRC) of the National Institute for Product Evaluation and Technology, and the black koji mold represented by the deposit number of NBRC4033 can be used. In the present invention, it may be a mutant strain or a derivative strain of the strain described above, and can be used as long as the koji fermentation composition obtained by using such a strain has the action described below.

  The koji mold used in the present invention can be prepared by solid-culturing under suitable conditions using a steamed raw material usually used for solid culture of the koji mold. As raw materials, rice, wheat (barley, wheat, rye and oats) as cereals, soybeans as beans, sweet potatoes as potatoes, etc. Can do. These raw materials may be used alone or in combination of two or more. Alternatively, the koji mold used in the present invention may be prepared using a known fungal medium such as a potato dextrose agar medium.

  After culturing, the obtained koji mold culture may be used as it is, or if necessary, rough purification by centrifugation, solid-liquid separation by filtration, sterilization operation, etc. may be performed. Moreover, you may use a koji mold in combination of 2 or more types as appropriate.

  The fermentation process is performed by bringing the koji fruit part and koji mold prepared as described above into contact with each other as an inoculum and thoroughly stirring and mixing, and repeating blowing, stirring and standing in the range of about 20 to 45 ° C. . The amount of koji mold to be brought into contact with the koji fruit part in the fermentation process is usually preferably about 0.001 to 10% by mass of the dried koji mold powder with respect to the koji fruit part, but is limited to this. Not. What is necessary is just to perform pH, time, etc. at the time of performing a fermentation process on well-known conditions, for example, it is good to carry out over 1-20 days at about pH 3-8. After the fermentation process, the culture is dried at about 40 to 45 ° C. for about 1 day by, for example, ventilation drying.

  After the fermentation process, an extraction process for extracting the active ingredient by bringing alcohol into contact with the fermented product is performed. In the extraction step, an extract can be obtained by immersing, stirring, or refluxing the fermented product in an alcohol that is an extraction solvent such as methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, and isobutanol. As the alcohol, a mixed solution of these alcohols and water, a solution obtained by mixing a plurality of the above-described alcohols and water, or a solution obtained by mixing a plurality of the above-described alcohols may be used. The mixing ratio of alcohol and water is not particularly limited. In this specification, the case where 100% or 50% ethanol is used as alcohol will be described.

As extraction conditions, the extraction temperature can be arbitrarily set as long as it is higher than or equal to the freezing point of the solvent used for extraction and lower than the boiling point. Preferably, the components contained in the fruit of persimmon are not easily denatured or lost due to the reaction. And it is good to set it as the temperature range of the normal temperature-the boiling point of a solvent with comparatively favorable extraction efficiency. Specifically, the extraction pressure is suitably set within the range of 1.0 to 2.5 kg / cm ² . The weight ratio of the solvent with respect to the fermented product can be arbitrarily set within a range of 2: 1 to 1:50, for example, fermented product: solvent, but is not limited thereto. There is no particular limitation on the extraction time, and preferably several hours to several days.

  In an extraction process, it is good to heat-process with respect to the fermentation processed material which added the solvent as needed. The heat treatment is preferably performed under conditions of, for example, 30 minutes to 3 hours and 25 to 35 ° C.

  The koji fermentation composition obtained by the extraction solvent and the extraction conditions described above may be used as it is, but the koji fermentation composition may be further purified and used. As a purification method, specifically, a filtration method using a filter paper or a membrane filter, a liquid separation operation method, a distillation method, a sublimation method, a precipitation method, a recrystallization method, a centrifugal separation method, a vacuum concentration method, a pH adjustment, Although methods, such as salting out, are mentioned and 1 type or 2 types or more of these methods are selected, It is not limited to these methods.

  Further, the obtained koji fermentation composition may be diluted with an appropriate solvent, concentrated and used, or further dried under reduced pressure to prepare a dry powder or a paste.

  A composition obtained by adding water, alcohols, polyhydric alcohols, surfactants, acids, alkalis, thickeners, excipients, preservatives, etc. to the koji fermentation composition thus prepared Is also possible. The shape of the koji fermentation composition may be liquid, solid, powder, granule, pellet, gel, mucous, or the like.

  The koji fermented composition can be administered as a pharmaceutical by oral, enteral, transmucosal, injection or the like. Examples of the administration form include oral administration or intravenous injection such as tablets, capsules, granules, powders, syrups, etc., intramuscular injections, suppositories, inhalants, percutaneous absorption agents, eye drops, nasal drops, etc. Parenteral administration. In order to prepare pharmaceutical preparations of such various dosage forms, the above-described composition is further added to a pharmaceutically acceptable excipient, binder, extender, disintegrant, lubricant, and dispersant. , Buffering agents, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents, and the like can be added in appropriate combinations. Among the administration forms, the preferred form is oral administration, and the content of the koji fermentation composition in the preparation when used as a preparation for oral administration is preferably about 0.1 to 70% by mass, for example.

  When used as a medicine, the daily dose per adult varies depending on various factors such as age, weight, sex, and administration method, but in the case of oral administration, the above plant or extract thereof (in terms of solid content) ), For example, 0.1 to 5000 mg is preferably administered once a day to several times a day.

  Examples of the form of the koji fermentation composition used as a food include the same forms (tablets, capsules, syrups, etc.) as the above-mentioned oral administration preparations. Moreover, you may mix | blend the said koji fermentation composition suitably with various drinks, snacks, dairy products, seasonings, starch processing products, processed meat products, etc.

  To prepare various forms of food products, the above-mentioned koji fermented composition is added to other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, UV absorbers. Agents, antioxidants, humectants, thickeners and the like can be added in appropriate combinations. In general, the content of the koji fermentation composition in the food product is preferably about 0.002 to 5% by mass, for example.

  The koji fermented composition obtained by the method for treating koji fruits of the present invention has a blood glucose rise inhibiting effect, a blood pressure rise inhibiting effect, and an anti-obesity effect.

  The inhibitory action on the increase in blood glucose level is achieved, for example, by having an α-glucosidase inhibitory activity that inhibits the action of α-glucosidase inhibitory activity in the intestine and inhibits the decomposition and absorption of sugar. α-Glucosidase (AGH) breaks down disaccharides such as maltose produced by breaking down starch into glucose. When this glucose enters the blood, the blood sugar level rises. Therefore, it is considered that inhibiting AGH can reduce the amount of glucose produced and suppress an increase in blood glucose level. The α-glucosidase inhibitory activity can be evaluated using, for example, a rat small intestine-derived enzyme.

  The blood pressure increase inhibitory effect is achieved, for example, by having an angiotensin converting enzyme inhibitory activity that inhibits angiotensin converting enzyme (ACE) involved in increasing blood pressure. An increase in blood pressure involves a pressor mechanism (renin / angiotensin system) and an antihypertensive mechanism (kinin / kallikrein system), and angiotensin converting enzyme is an enzyme that acts on both systems. Angiotensin I is converted to angiotensin II having a vasoconstrictive action by an angiotensin converting enzyme. Furthermore, the enzyme also has an action of inactivating bradykinin that lowers blood pressure, and the action of these two systems is involved in the increase of blood pressure. Therefore, it is considered that an increase in blood pressure can be suppressed by inhibiting angiotensin converting enzyme. The angiotensin converting enzyme inhibitory activity can be detected by enzymatic method of 3-Hydroxybutyric acid (3HB) excised from 3-Hydroxybutyryl-Gly-Gly-Gly (3HB-GGG).

  The anti-obesity effect by the anti-obesity action is achieved by having the β-lipase inhibitory activity that inhibits the action of β-lipase in the intestine and inhibits the decomposition and absorption of fat.

  The koji fermentation composition of the present invention is preferably provided as a pharmaceutical composition or functional food containing the koji fermentation composition as an active ingredient. Particularly in the case of functional food, since it can be taken orally on a daily basis, it is thought that it is possible to easily prevent an increase in blood sugar, an increase in blood pressure, and obesity. In addition, the fermented koji composition contained as an active ingredient is suitable for processing foods and the like because it retains the above-described actions even after high-temperature treatment or freeze-drying treatment. Furthermore, even when added, the fermented koji composition has little effect on the taste and flavor of the food itself, so it can be continuously added to various foods.

  Such functional foods include, for example, foods for specified health use (foods that include health functional ingredients that affect the physiological functions of the body and have specific effects), nutritional functional foods (for supplementation and supplementation of nutritional ingredients) Food), health supplements, nutritional supplements, and the like. Such functional foods are widely distributed in the market and can be obtained easily and inexpensively. A person skilled in the art may appropriately set the proportion of the koji fermentation composition of the present invention contained in the functional food.

[Example 1]
Examples of the present invention will be described.
The fruit of the persimmon used the fruit of Fuyu persimmon. After removing the skin, straw and seeds, the young fruit was sterilized by boiling in 100 ° C hot water for 10 seconds (fruit raw material). After sterilization, 40.0 g of each sample was transferred to a sterilized 300 mL baffle flask.

  As the koji mold, black koji mold (NBRC4033) was used. The black koji mold was used as an inoculum after culturing at 35 ° C. for 2 days in a potato dextrose slant medium. An appropriate amount of inoculum was suspended in 10 mL of sterilized water, and 2.0 mL was added to the above fruit raw material, followed by fermentation at 35 ° C. for 14 days under aerobic conditions (fermentation step). The fermented processed product was sampled on days 0, 7, 10, and 14 and an extraction process was performed to prepare a koji fermentation composition.

  As a control, lactic acid bacteria (Lactococcus lactis, NBRC12007) were used and added to the fruit material for fermentation. Lactic acid bacteria were preincubated for 2 days at 30 ° C. in MRS medium as seed bacteria. 100 mL of water was added to the above fruit material to make it immersed, and 5.0 mL of the inoculum suspension was added, followed by fermentation at 30 ° C. for 14 days under anaerobic conditions using Aneropac Kenki. The fermented processed product was sampled on days 0, 7, 10, and 14 and an extraction process was performed to prepare a koji fermentation composition.

The extraction process was performed as follows.
-100% ethanol-water extraction The ethanol extract was collected by extracting for 4 days using 30 mL of ethanol, and then extracted for 4 days using 30 mL of water. After the ethanol fraction was dried under reduced pressure, water-soluble and ethanol-insoluble components were precipitated. Therefore, the ethanol-soluble component was used as the ethanol fraction, and the water-soluble and ethanol-insoluble components were combined with the water fraction. Each component was dried under reduced pressure and then redissolved in each solvent to 10% (w / v). In the lactic acid bacteria fermented product, in addition to the above method, 10 mL of sampled immersion water was also integrated into the water fraction.

-50% ethanol extraction Using 50% ethanol, extraction for 4 days was performed twice. In this extraction method, since the extract after drying under reduced pressure was completely dissolved in 50% ethanol, it was dissolved again in 50% ethanol so as to be 10% (w / v) to obtain a sample solution. In the lactic acid bacteria fermented product, in addition to the above method, 10 mL of the sampled immersion water was integrated and the same treatment was performed after drying under reduced pressure.

[Example 2]
The ethanol fraction and water fraction of the koji fermentation composition prepared in Example 1 were examined for α-glucosidase inhibitory activity (inhibition of increasing blood glucose level).

  The enzyme solution was prepared as follows. That is, 1.0 g rat intestinal acetone powder was suspended in 10 mL of 0.1 M sodium phosphate buffer (pH 7.0) and stirred for 30 minutes under ice cooling. Thereafter, centrifugation was performed (12000 rpm × 10 minutes, 4 ° C.), and the supernatant was used as a crude enzyme solution. The prepared enzyme solution was stored at -80 ° C.

0.4 mL of 250 mM maltose aqueous solution was added as a substrate to 0.48 mL of distilled water. To this reaction solution, 0.1 mL of each extract (ethanol fraction and water fraction, final concentration: 1 to 20 mg / mL) was added as an inhibitor, followed by preincubation at 37 ° C. for 5 minutes after stirring. Thereafter, 0.02 mL of α-glucosidase crude enzyme solution was added, and the reaction was performed at 37 ° C. for 40 minutes. After completion of the reaction, 1.0 mL of 0.2 M sodium carbonate aqueous solution was added to stop the reaction. Glucose produced in the same reaction was measured using Glucose CII-Test Wako. After determining the residual activity, the 50% inhibitory concentration (IC ₅₀ , μg / mL) was calculated. The results are shown in Tables 1 to 3 (the smaller the value, the higher the activity).

  Table 1 shows the α-glucosidase inhibitory activity of the 100% ethanol-water extract of the koji fermentation composition by black koji mold, and Table 2 shows the 50% ethanol extract of the koji fermentation composition by black koji mold and lactic acid bacteria. The results of examining the α-glucosidase inhibitory activity are shown respectively.

In the treatment with Aspergillus niger, α-glucosidase inhibitory activity was confirmed only in the water fraction from Table 1 (100% ethanol-water extract). In addition, the activity was enhanced after the 7th day, and the highest activity (IC ₅₀ = 8016.7 μg / mL) was found on the 10th day. In addition, the treatment with black koji mold has enhanced activity on the 7th and 14th days from Table 2 (50% ethanol extract), and the activity of the lactic acid bacteria 100% ethanol-water extract on the 14th day (IC ₅₀ = 3800.0 μg / mL) was found to show higher activity (IC ₅₀ = 1187.9 μg / mL).

From this, it was recognized that α-glucosidase inhibitory activity was enhanced by fermentation in any koji fermentation composition treated with black koji mold or lactic acid bacteria. α-Glucosidase inhibitory activity varies depending on the extraction method. In the treatment with Aspergillus niger, 100% ethanol-water extraction shows high activity in 7-10 days fermentation treatment, 50% ethanol extraction in 7-14 days fermentation treatment High activity was shown.
In other words, it is considered that the treatment with black koji mold has an enhanced α-glucosidase inhibitory activity enhancing effect superior to the treatment with lactic acid bacteria. It was recognized as having activity. The ratio of the inhibitory activity of the black koji mold treatment and the inhibitory activity of the lactic acid bacteria treatment at this time was about 3.2 times (1187.9 / 3800.0).

Example 3
About the koji fermentation composition prepared in Example 1, the angiotensin converting enzyme inhibitory activity (blood pressure rise inhibitory effect) was investigated.
In this test, the inhibitory activity against angiotensin converting enzyme (ACE) involved in blood pressure elevation was measured using Ace Kit-Double Estee (Angiotensin converting enzyme inhibitory activity measuring kit) (Dojindo Laboratories Co., Ltd.). The rate (IC ₅₀ ) was determined. In this example, the inhibitory activity of 100% ethanol-water extract (water fraction) and 50% ethanol extract was examined.

  The test was conducted according to the method of using the kit. That is, 20 μL of an angiotensin converting enzyme and aminoacylase mixed solution prepared in ultrapure water, 20 μL of a substrate solution (3-Hydroxybutyryl-Gly-Gly-Gly (3HB-GGG)), and 20 μL of a sample solution are mixed and incubated at 37 ° C. for 1 hour. Reacted. To this, 200 μL of a mixture of 3-hydroxybutyrate dehydrogenase and coenzyme was added and allowed to react at room temperature for 10 minutes. After the reaction, absorbance at 450 nm was measured, and the inhibition rate was calculated based on the obtained data. The results are shown in Table 3 (100% ethanol-water extract) and Table 4 (50% ethanol extract) (the smaller the value, the higher the activity).

From Table 3 (100% ethanol-water extract), in the treatment with Aspergillus niger, the activity is enhanced after the seventh day, and the highest activity (IC ₅₀ = 182.9 μg / mL) is exhibited on the seventh day. It has been found.

From Table 4 (50% ethanol extract), the treatment with Aspergillus niger increases the activity after the 7th day, and shows the highest activity (IC ₅₀ = 13.5 μg / mL) on the 7th day. found. Moreover, in the treatment with lactic acid bacteria, the activity was enhanced after the seventh day, and the highest activity (IC ₅₀ = 45.6 μg / mL) was found on the seventh day.

From this, it was recognized that the angiotensin-converting enzyme inhibitory activity is enhanced by fermentation in any koji fermentation composition treated with black koji mold or lactic acid bacteria. In the treatment with Aspergillus niger, both 100% ethanol-water extraction and 50% ethanol extraction showed high activity in the fermentation treatment for 7 to 14 days.
That is, it is considered that the treatment with black koji mold has an angiotensin converting enzyme inhibitory activity enhancement effect superior to the treatment with lactic acid bacteria. It was recognized as having At this time, the ratio of the inhibitory activity of the black koji mold treatment and the inhibitory activity of the lactic acid bacteria treatment was about 3.3 times (13.5 / 45.6).

Example 4
The ethanol fraction and water fraction of the koji fermentation composition prepared in Example 1 were examined for β-lipase inhibitory activity (anti-obesity effect).

To 50 μL of the fluorescent methylumbelliferone oleate solution, 25 μL of the sample solution (final concentration: 2.5 to 2500 mg / mL) was added and preincubated at 37 ° C. for 5 minutes. Thereafter, 25 μL of β-lipase was added, and an enzyme reaction was performed at 37 ° C. for 30 minutes. After completion of the reaction, 50 μL of 100 mM citrate buffer (pH 4.2) was added to stop the reaction, and the fluorescence of released 4-methylumbelliferone was measured at an excitation wavelength of 355 nm and a fluorescence wavelength of 460 nm. After determining the residual activity, the 50% inhibitory concentration (IC ₅₀ , μg / mL) was calculated. The results are shown in Tables 5 to 7 (the smaller the value, the higher the activity).

Table 5 shows the β-lipase inhibitory activity of the 100% ethanol-water extract of the koji fermentation composition by Aspergillus niger, and Table 6 shows the β of the 100% ethanol-water extract of the koji fermentation composition by lactic acid bacteria. As a result of examining the -lipase inhibitory activity, Table 7 shows the results of examining the β-lipase inhibitory activity of the 50% ethanol extract of the koji fermentation composition by black koji mold and lactic acid bacteria, respectively.

In the treatment with Aspergillus niger, β-lipase inhibitory activity was confirmed in both the ethanol fraction and the water fraction from Table 5 (100% ethanol-water extract). In particular, it was found that the water fraction showed high activity on day 7 (IC ₅₀ = 10.2 μg / mL) and day 14 (IC ₅₀ = 2.0 μg / mL). These were found to exhibit higher activity than the activity of 100% ethanol-water extract of lactic acid bacteria (day 7: IC ₅₀ = 110.7 μg / mL, day 14: IC ₅₀ = 21.6 μg / mL). .
Further, from Table 7 (50% ethanol extract), it was found that the activity was enhanced after the 7th day and showed the highest activity on the 14th day (IC ₅₀ = 1.43 μg / mL). The activity at this time was found to be higher than the activity of the 50% ethanol extract of lactic acid bacteria on the 14th day (IC ₅₀ = 1891.3 μg / mL).

On the other hand, in the treatment with lactic acid bacteria, β-lipase inhibitory activity was confirmed in both the ethanol fraction and the water fraction from Table 6 (100% ethanol-water extract). The ethanol fraction was found to be highly active on day 7 (IC ₅₀ = 12.0 μg / mL) and day 10 (IC ₅₀ = 15.3 μg / mL). The water fraction was found to be highly active on day 10 (IC ₅₀ = 63.7 μg / mL) and day 14 (IC ₅₀ = 21.6 μg / mL).

From this, it was recognized that any fermented koji fermented composition by treatment with black koji mold and lactic acid bacteria enhances β-lipase inhibitory activity by fermentation.
In the treatment with black koji mold, 100% ethanol-water extraction showed high activity in the fermentation treatment on the 7th and 14th days, and 50% ethanol extraction showed high activity in the fermentation treatment on the 7th to 14th days.
That is, it was considered that the treatment with Aspergillus niger has a better α-glucosidase inhibitory activity enhancing effect than the treatment with lactic acid bacteria. In particular, in the treatment with Aspergillus niger, the inhibitory activity at the time of 100% ethanol-water extraction after 14-day fermentation was 10.8 times (2.0 / 21.6) the inhibitory activity at the time of lactic acid bacteria treatment.
In addition, in the treatment with Aspergillus niger, the inhibitory activity at the time of 50% ethanol extraction after 14-day fermentation was about 1323 times (1.43 / 1891.3) of the inhibitory activity at the time of lactic acid bacteria treatment.

Example 5
The fermented koji composition prepared in Example 1 was subjected to GC-MS analysis.
For GC-MS analysis, Agilent Technologies 1260 infinity (manufactured by Agilent Technologies) is used as the HPLC apparatus, Scherzo SS-C18 (150 mm × φ2 mm) (manufactured by Intact Corporation) as the column, and Agilent Technologies 6430 Triple Quad as the MS detector. LC / MS (manufactured by Agilent Technologies) was used.
The mobile phase was A: water (0.2% acetic acid + 0.2% formic acid added), B: water-methanol = 1: 1 (100 mM ammonium acetate added), flow rate 0.3 mL / min, column temperature 40 ° C. Ion mode: The conditions were positive.

  The results are shown in FIGS. FIG. 1 shows the result of black koji mold treatment (100% ethanol-water extraction (ethanol fraction)), and FIG. 2 shows the result of black koji mold treatment (100% ethanol-water extraction (water fraction)). Shows the results of black koji mold treatment (50% ethanol extraction), FIG. 4 shows the results of lactic acid bacteria treatment (50% ethanol extraction), and FIG. 5 shows the results of lactic acid bacteria treatment (100% ethanol-water extraction (ethanol fraction)). The results are shown, and FIG. 6 shows the results of lactic acid bacteria treatment (100% ethanol-water extraction (water fraction)).

  As a result, it was recognized that the fermentation products in each composition differed between the koji fermentation composition treated with black koji mold and the koji fermentation composition treated with lactic acid bacteria. Specifically, in the koji fermentation composition treated with black koji mold, a characteristic peak considered to be a fermentation product was observed in 19 to 25 minutes (FIGS. 1 to 3). On the other hand, such a peak could not be confirmed in the fermented koji composition treated with lactic acid bacteria. In FIG. 2, on the 14th day of the black koji mold treatment, a plurality of peaks (arrows) that could not be confirmed so far were confirmed.

In other words, in the fermented composition of black koji mold and the koji fermented composition of lactic acid bacteria treatment, such fermentation products are different, and therefore the fermentation mechanism of black koji mold and lactic acid bacteria on the koji fruit is different. It was speculated.
In addition, high α-glucosidase inhibitory activity, angiotensin-converting enzyme inhibitory activity, and β-lipase inhibitory activity are observed by the 14-day fermentation after treatment with Aspergillus niger. Fermentation having a characteristic peak as shown in FIG. It was speculated that the product was a factor.

  INDUSTRIAL APPLICABILITY The present invention can be used for fermentation treatment of persimmon fruit, and the persimmon fermentation composition obtained by fermentation can be used as a pharmaceutical and a food having a blood glucose level increase inhibitory effect, a blood pressure increase inhibitory effect and an anti-obesity effect.

Claims (6)

Fruit persimmon fermented composition for blood glucose increase inhibitory effect that Yusuke containing as an active ingredient, a treated product obtained by solid fermentation with Aspergillus oryzae persimmon. Fruit persimmon fermented composition for antihypertensive action that Yusuke containing as an active ingredient, a treated product obtained by solid fermentation with Aspergillus oryzae persimmon. Fruit persimmon fermented composition for anti-obesity effect that Yusuke containing as an active ingredient, a treated product obtained by solid fermentation with Aspergillus oryzae persimmon.   The koji-fermenting composition according to any one of claims 1 to 3, wherein the koji mold is an Aspergillus genus.   The koji fermentation composition according to claim 4, wherein the koji mold is black koji mold. The processing method of the strawberry fruit which has a fermentation process which solid- processes the fruit of an strawberry with an aspergillus, and the extraction process which makes an alcohol contact a fermentation processed material and extracts an active ingredient.

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