JP6283569B2 - Method for producing fat accumulation inhibitor - Google Patents

Description

  The present invention relates to a method for producing a fat accumulation-inhibiting substance using supercritical carbon dioxide from fermented stevia, and a fat accumulation-inhibiting agent comprising the fat accumulation-inhibiting substance as an active ingredient.

  Stevia plants contain terpenoid glycosides such as stevioside and rebaudioside A, and are used as sweeteners because of their sweetness. In addition, the use of steviol glycosides as an antiallergic agent (Patent Document 1) and an antihistamine substance extracted from a fermented yeast ferment of Stevia plant tissue are also known (Patent Document 2).

  On the other hand, as a method for extracting active ingredients from natural agricultural products, there is a method using water and carbon dioxide in a supercritical state or subcritical state (Patent Document 3). Carbon dioxide becomes critical at 31.0 ° C. and pressure 7.4 MPa, and supercritical carbon dioxide has similar properties to nonpolar organic solvents. Moreover, since water is a polar substance, it is said that a hydrophilic substance and a hydrophobic substance can be extracted separately and with high purity by using supercritical carbon dioxide and water. In the embodiment, water is added to coffee beans and stored in an extraction tank, supercritical carbon dioxide is supplied from the lower part of the extraction tank, and the fraction of the carbon oxide layer is discharged supercritically from the upper part of the extraction tank. The water layer is obtained from below.

  Supercritical carbon dioxide extraction is known (Non-Patent Document 1). Non-Patent Document 1 describes that carbon dioxide, which is gaseous at room temperature, becomes a high-density fluid state in a supercritical state and has a property similar to that of a liquid. It is disclosed that a component that can be dissolved in supercritical carbon dioxide can be extracted by contact, and that the dissolved component can be separated and recovered by reducing the pressure of carbon dioxide discharged from the extraction container.

Japanese Patent No. 39591717 Japanese Patent No. 4375758 Japanese Patent No. 4131230

Satoshi Masuda, “Supercritical Carbon Dioxide Utilization Technology in the Ultra High Pressure Region”, [online], Kopelunikus, P11-14, No. 32, Oct. 2007, [Search May 14, 2014], Internet <URL: http: // www. kobelcokaken. co. jp / tech_library / pdf / no32 / d. pdf>

  In recent years, guidance has been made to prevent or improve lifestyle-related diseases by introducing the concept of metabolic syndrome, in which visceral fat obesity is combined with two or more of hyperglycemia, hypertension and dyslipidemia. Dietary therapy for reducing visceral fat includes intake of low-calorie diet, exercise, and others, but a method that can reduce calorie intake safely without difficulty is desired.

  Conventionally, stevia glycosides derived from stevia plants have been used as sweeteners. Stevia glycosides are considered to be effective in preventing obesity because they have lower calories than sucrose. On the other hand, a fermented product obtained by fermenting a stevia plant until steviol glycoside is not detected has no sweetness and cannot be used as a sweetener. However, the extract is a plant-derived component and is highly safe. If there is a component that suppresses fat accumulation in the fermented stevia plant, it can be used as a safe fat accumulation-inhibiting substance.

  In addition, with the increase in the number of adults exhibiting metabolic syndrome, it is preferable that the production of a fat accumulation-inhibiting substance is simple, and development of a fat accumulation-inhibiting agent and a method for producing the same is desired.

  In view of the above-mentioned present situation, an object of the present invention is to provide a method for producing a fat accumulation-inhibiting substance derived from a stevia fermented product.

  Another object of the present invention is to provide a fat accumulation inhibitor comprising the thus produced fat accumulation inhibitor as an active ingredient.

  The present inventors fractionated stevia fermented products by various extraction methods and evaluated the respective medicinal effects.In the hydrophilic polar solvent extract after removing the hydrophobic fraction by supercritical carbon dioxide extraction, The present invention was completed by finding out that an extremely excellent fat accumulation-inhibiting substance was contained.

That is, the present invention is a method for producing a fat accumulation-inhibiting substance from a stevia fermented product fermented under aerobic conditions using carbon dioxide,
Said stevia fermentation product, a step A of shifting a supercritical state, by contacting the carbon dioxide in a subcritical state or liquid state components dissolved in the carbon dioxide to the carbon dioxide,
A step B in which the stevia fermented product in which the components are transferred to the carbon dioxide side is contacted with a hydrophilic polar solvent,
After the step B, the fat accumulation inhibiting substance contained in the hydrophilic polar solvent is fractionated,
A method for producing a fat accumulation-inhibiting substance is provided.

  Moreover, this invention provides the manufacturing method of the said fat accumulation inhibitory substance characterized by the contact with the said stevia fermented product and the said carbon dioxide being the temperature of 10-130 degreeC, and the pressure of 7-80 MPa. is there.

Further, 30 to 2000 parts by mass of a hydrophilic polar solvent is brought into contact with carbon dioxide in a supercritical state or a subcritical state with respect to 100 parts by mass of the stevia fermented product together with the stevia fermented product,
The present invention provides a method for producing the fat accumulation-suppressing substance, wherein the step A and the step B are performed simultaneously.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the fat accumulation inhibitory substance which can be manufactured simply and the fat accumulation inhibitor which uses the said fat accumulation inhibitory substance as an active ingredient are provided.

It is the schematic of the supercritical carbon dioxide extraction apparatus which can be used by this invention. It is a figure which shows the result observed with the inverted phase-contrast microscope of the mouse | mouth fat precursor cell 3T3-L1 cultured in Example 1. FIG. It is a figure which shows the result of the dose response curve with respect to the fat accumulation inhibitory effect of Example 1. FIG. It is a figure which shows the result of the dose response curve with respect to the fat accumulation inhibitory effect of the comparative example 1. It is a figure which shows the result of the dose response curve with respect to the fat accumulation inhibitory effect of the comparative example 2. It is a figure which shows the result of having evaluated the statistical significant difference of the fat accumulation inhibitory effect of Example 1, the comparative example 1, and the comparative example 2 in a 500 microgram / ml sample.

The first of the present invention is a method for producing a fat accumulation-inhibiting substance from a stevia fermented product using carbon dioxide fermented under aerobic conditions ,
Said stevia fermentation product, a step A of shifting a supercritical state, by contacting the carbon dioxide in a subcritical state or liquid state components dissolved in the carbon dioxide to the carbon dioxide,
A step B in which the stevia fermented product in which the components are transferred to the carbon dioxide side is contacted with a hydrophilic polar solvent,
After the step B, the fat accumulation inhibiting substance contained in the hydrophilic polar solvent is fractionated,
This is a method for producing a fat accumulation-inhibiting substance .

(1) Stevia fermented product The stevia plant (scientific name: Stevia rebaudiana) used in the present invention is a perennial plant belonging to the genus Stevia which is native to South America. Terpenoid glycosides such as stevioside and rebaudioside A extracted from stevia plants are used as sweeteners and are currently cultivated in Asia such as Japan, China and Korea. In the present invention, in particular, Stevia Rebaudiana Bertoni and its related plants can be preferably used. Stevia plants that can be used include Stevia plant stems, leaves, whole plants before buds, mature plant roots and flowers.

  As the stevia plant, a plant harvested at an appropriate time can be used as it is, or a plant that has been dried after harvesting can be used. The dried product is excellent in storage stability and is suitable. Fermentation is started when a dried stevia plant is cut into a predetermined size, pulverized or otherwise chopped or crushed, and yeast is added and stirred. When a stevia plant is fermented with yeast, components that are not present in the stevia plant are produced. As yeast, it is preferable to use Saccharomyces.

  Stevia plants can be fermented by adding yeast to the stevia plants and stirring and leaving them. When using a dry stevia plant, it can carry out by adding water and yeast to a dry stevia plant, stirring, and leaving to stand. The amount of water to be added is sufficient so that the whole is moistened, whereby solid fermentation can be performed under aerobic conditions.

  Stevia plants before fermentation have sweetness due to terpenoid glycosides, but the sweetness disappears as fermentation progresses. In the present invention, the disappearance of sweetness is taken as a measure of the end of fermentation. Such disappearance of sweetness is 1 to 3 weeks at room temperature. In addition to adding yeast at the beginning of fermentation, it may be added during the fermentation.

  In addition to fermenting by adding yeast to Stevia plant, boiling extract obtained by crushing dried Stevia plant and boiling extraction, immersion liquid obtained by soaking in warm water, water from Stevia plant, A predetermined fraction extracted with another solvent may be used as a stevia plant extract, and the yeast may be added thereto for fermentation. Again, the end of fermentation can be confirmed by the disappearance of sweetness.

  Next, the stevia fermented product is dried. Stevia fermented product contains yeast, but the activity of the yeast can be stopped by drying it. Drying is preferably performed at a temperature of 5 to 35 ° C. by air drying, natural drying, or the like. Decomposition, alteration, etc. of the compound contained in the stevia fermented product can be avoided.

(2) Process A
In step A, a stevia fermented product is brought into contact with carbon dioxide in a liquid state, a subcritical state, or a supercritical state (hereinafter also referred to as supercritical carbon dioxide) to convert a component that can be dissolved in the carbon dioxide into the carbon dioxide. This is a process of transferring to carbon. By removing supercritical carbon dioxide and the like after contact out of the system, the dissolved component can be removed from the stevia fermented product. For example, the supercritical carbon dioxide extraction apparatus shown in FIG. 1 can be used. In FIG. 1, 1 is a carbon dioxide cylinder, 2 is a booster, 3 is a preheating pipe, 4 is a thermostatic bath, 5 is a reaction vessel, 6 is a back pressure valve, 7 is a receiver, 8 is a carbon dioxide discharge pipe, and 9 is an extraction. A material take-out tube, 10 is a hydrophilic polar solvent storage unit, 11 is a valve, 12 is a receiver, and 13 is an extract. Inside the reaction vessel 5, wire meshes indicated by symbols a and b are disposed, and ceramic balls c can be placed and filled above the upper wire mesh a and below the lower wire mesh b. A carbon dioxide inlet is formed in the lower part of the reaction vessel 5 and is connected to the preheating pipe 2. A discharge port is provided in the upper part of the reaction vessel 5 and connected to the back pressure valve 6 and the receiver 7. Further, a discharge port for discharging the contents in the reaction vessel 5 is formed in the lower side portion and is connected to the valve 11 and the receiver 12. Note that the preheating tube 3 and the reaction vessel 5 are accommodated in a thermostatic bath 4 filled with water.

  Ceramic balls c are placed in the reaction vessel 5 and the wire mesh b is fixed. Stevia fermented product is charged on the upper part of the wire net b, the wire net a is fixed on the upper part, and the ceramic balls c are placed on the upper part. By charging the stevia fermented product between the metal mesh a and the metal mesh b of the reaction vessel 5, it is possible to prevent floating and clogging. When the hydrophilic polar solvent is charged together with the stevia fermented product, it may be introduced into the reaction vessel 5 from the hydrophilic polar solvent storage unit 10. The thermostat 4 is heated to a temperature of 10 to 130 ° C., the preheating tube 3 and the reaction vessel 5 are heated to a predetermined temperature, and carbon dioxide is supplied from the carbon dioxide cylinder 1 through the booster 2 to the preheating tube 3 and the reaction vessel 5. To introduce. When pressurized to 7-80 MPa with the booster 2, the carbon dioxide is in a liquid, subcritical or supercritical state. Carbon dioxide heated by the preheating tube 3 is introduced at a flow rate of 5 to 4000 ml / min from a carbon dioxide inlet provided in the lower part of the reaction vessel 5. The carbon dioxide introduced into the reaction vessel 5 comes into contact with the stevia fermentation product while moving from the lower portion to the upper portion of the reaction vessel 5. The back pressure valve 6 is controlled to discharge carbon dioxide containing a predetermined component from the carbon dioxide discharge port provided at the top of the reaction vessel 5 to the receiver 7 side. Thereby, the stevia fermented product from which the substance dissolved in supercritical carbon dioxide or the like is removed remains in the reaction vessel 5. After the reaction is completed, the reaction vessel 5 is brought to normal pressure, and then the liquid component remaining in the reaction vessel 5 can be taken out by opening the valve 11 and introducing the contents into the receiver 12. The carbon dioxide introduced into the receiver 7 is discharged out of the system through the carbon dioxide discharge pipe 8, and the extract stored in the receiver 7 can be taken out from the extract take-out pipe 9. By taking out a part of the contents and measuring the amount of the extracted contents, the contact time with carbon dioxide can be determined. Usually, 30 minutes to 20 hours is sufficient.

(3) Process B
Step B is a step of bringing the stevia fermented product from which components dissolved in supercritical carbon dioxide or the like in Step A have been removed into contact with a hydrophilic polar solvent. Step B can be performed subsequent to step A, or can be performed simultaneously with step A.

  When performing the process B after the process A, the back pressure valve 6 is controlled to discharge carbon dioxide from the carbon dioxide discharge port provided at the upper part of the reaction vessel 5 to the receiver 7 side. Analyze the matter over time to confirm the completion of extraction with supercritical carbon dioxide. Next, a hydrophilic polar solvent is added to the reaction vessel 5 and extracted at a temperature of 10 to 130 ° C., a pressure of 7 to 80 MPa, and a time of 30 minutes to 10 hours. Examples of such hydrophilic polar solvents include water, alcohols such as ethanol and butanol. If the contents of the reaction vessel 5 are taken out after extraction with a hydrophilic polar solvent and subjected to solid-liquid separation, the hydrophilic polar solvent can be separated as a filtrate. This hydrophilic polar solvent contains a hydrophilic polar substance contained in the stevia fermented product.

Step A and step B can be performed simultaneously. In this case, there are a batch method and a continuous method.
The batch method is a method in which the entire amount of a hydrophilic polar solvent is charged in a reaction vessel and extraction is performed with supercritical carbon dioxide or the like. The reaction vessel is charged with 30 to 2000 parts by mass, more preferably 50 to 1000 parts by mass of a hydrophilic polar solvent with respect to 100 parts by mass (dry weight) of the stevia fermentation product together with the stevia fermentation product, and supercritical carbon dioxide or the like is added to the reaction vessel. 5, the back pressure valve 6 is controlled to discharge carbon dioxide from the carbon dioxide outlet provided in the upper part of the reaction vessel 5 to the receiver 7 side. The extract stored in the receiver 7 is analyzed over time to confirm the completion of extraction with supercritical carbon dioxide or the like. Then, if the contents of the reaction vessel 5 are taken out and subjected to solid-liquid separation, the hydrophilic polar solvent charged in the reaction vessel 5 can be collected as a filtrate. This hydrophilic polar solvent contains a hydrophilic polar substance contained in the stevia fermented product.

  The continuous method is a method of performing extraction with supercritical carbon dioxide or the like while continuously supplying a hydrophilic polar solvent together with supercritical carbon dioxide or the like to the reaction vessel 5. When supercritical carbon dioxide or the like is introduced into the reaction vessel 5 from below the reaction vessel and a hydrophilic polar solvent is introduced opposite to the supercritical carbon dioxide, a region containing carbon dioxide as a main component at the top of the reaction vessel 5 And a region mainly composed of a hydrophilic polar solvent is formed in the lower part. Since the dipole moment of carbon dioxide is 0 and the property is similar to that of a nonpolar organic solvent in the supercritical or subcritical state, an organic substance having a low polarity can be extracted. On the other hand, water, methanol, ethanol and the like are polar solvents. When carbon dioxide in a critical state is brought into contact with the stevia fermented product, a hydrophobic substance is extracted in a region containing carbon dioxide as a main component, and a hydrophilic polar substance is extracted in a region containing a hydrophilic polar solvent as a main component. Carbon dioxide can be discharged out of the system from the upper part of the reaction vessel 5 and the hydrophilic polar solvent can be taken out from the lower side of the reaction vessel 5. In addition, a part of hydrophilic polar solvent functions as an entrainer, and can improve the extraction efficiency by a carbon dioxide. Water can be preferably used as the hydrophilic polar solvent. In addition to water, methanol or ethanol may be included.

(4) Fat Accumulation Inhibiting Substance The hydrophilic polar solvent obtained in Step B contains a hydrophilic polar substance contained in the stevia fermentation product. If a hydrophilic polar solvent is introduced into the receiver 12 of FIG. 1, the fat accumulation-inhibiting substance 13 can be fractionated. The obtained fat accumulation inhibiting substance may be further purified by silica gel column chromatography, reverse phase column or the like.

  According to the said method, as shown in the Example mentioned later, the fat accumulation inhibitory substance contained in a stevia fermented material can be melt | dissolved and isolate | separated simply and efficiently in a hydrophilic polar solvent. Stevia dried leaves are known to contain more than 80% of glycosides that are purified and have steviol skeleton such as steviol glycosides, but there are “fat-accumulating substances” as components other than sweeteners. It became clear to do.

(5) Fat accumulation inhibitor The fat accumulation inhibitor of the present invention comprises the aforementioned fat accumulation inhibitor as an active ingredient. Examples of the dosage form of the fat accumulation inhibitor include pills, granules, powders, liquids, and sprays.

  In addition to fat accumulation inhibitors, enteric nutrients, health foods, etc. can also be formulated by incorporating fat accumulation inhibitors.

  EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

(Production Example 1)
Preparation of Stevia Fermented Product A dried stevia plant obtained by drying stems and leaves of a stevia plant (scientific name: Stevia rebaudiana) from Hokkaido, Japan was pulverized to about 1 cm or less. When 45 g of yeast (Saccharomyces) and 4,500 g of water were added to 2,000 g of ground stevia plant and stirred under aerobic conditions at a temperature of 25 to 35 ° C., fermentation started. This was left to stand at a temperature of 25 to 35 ° C. for 14 days and fermented aerobically. The sweetness disappeared from the fermented product, and this was regarded as the end of fermentation. The fermented product was dried at a temperature of 25 to 35 ° C. for 10 days to stop yeast fermentation. This is a stevia solid fermented product.

Example 1
The supercritical carbon dioxide extraction apparatus shown in FIG. 1 was used. Twenty ceramic balls having a diameter of 6 mm and a metal wire mesh were fixed to the bottom of a cylindrical reaction vessel having an inner diameter of 30 mm, a length of 200 mm, and an internal volume of about 140 ml. 5 g of stevia solid fermented product obtained in Production Example 1 and 50 ml of water were charged into a reaction vessel, a metal mesh was mounted on the top, and a ceramic ball having a diameter of 6 mm was placed on the top. The preheating tube and the reaction vessel were heated to 60 ° C. in a thermostatic bath. After raising the temperature of carbon dioxide to 80 ° C. with a cylinder pressure, it was press-fitted to 30 MPa and allowed to stand for 30 minutes. Subsequently, carbon dioxide was fed at a predetermined pressure at a flow rate of 7 ml / min for 4 hours. At this time, components released from the back pressure valve were collected in a receiver every hour. After 4 hours, the carbon dioxide feeding was stopped, the pressure was gradually lowered, and the pressure in the reaction vessel was returned to normal pressure.

  The contents in the reaction vessel were taken out and separated into solid and liquid, and the filtrate was lyophilized. Using this lyophilized product as a specimen, the effect of inhibiting fat accumulation was evaluated.

The effect of suppressing fat accumulation was evaluated by the following method.
A 5 vol% DMSO aqueous solution was added to the sample and suspended by sonication to prepare a 50 mg / mL test solution stock solution. The test solution stock solution was diluted with a medium to prepare test solutions having specimen concentrations of 2,000, 1,000, and 500 μg / mL.
Three days after seeding mouse adipose precursor cells 3T3-L1 cells (hereinafter simply referred to as 3T3-L1 cells; obtained from Human Science Foundation) for 3 days, penicillin-streptomycin 1% by volume and fetal bovine serum 10 The cells were cultured in a DMEM medium containing volume%. After the culture, the adipogen differentiation assay kit (manufactured by Cayman Chemical Company) was administered to differentiate into adipocytes. In addition, the final concentration of the test solution became 1,000, 500, and 250 μg / mL by administration of the adipocyte differentiation agent. After culturing for 3 days, the culture solution was replaced with a newly prepared test solution-added medium. After further culturing for 4 days, the culture supernatant was removed, the cells were fixed according to the specifications of the adipogenesis assay kit, and then stained with an oil red O solution. The results of observation with an inverted phase contrast microscope are shown in FIG. In addition, cells that do not undergo differentiation induction by an adipogenesis assay kit are used as progenitor cells (undifferentiated), cells that contain only medium are used as untreated controls, and berberine chloride (manufactured by Wako Pure Chemical Industries, Ltd.) is used as the final concentration. FIG. 2 also shows the results of a similar test using a positive control that was added to 1 μg / mL.

  After photographing, a dye extract was added to extract oil red O taken in the fat droplets. Using a microplate reader (Spectra Max M2e, manufactured by Molecular Device Corporation), the absorbance of the extracted oil red O was measured at 520 nm. The average value (n = 3) of the absorbance of progenitor cells (undifferentiated) is BL, the absorbance of the untreated control is CN, the absorbance of each test solution is Sa, and the fat accumulation rate (%) = {(Sa-BL ) / (CN-BL)} × 100. The experimental outline is shown in Table 1, and the fat accumulation rate is shown in Table 2 and FIG.

(Comparative Example 1)
To 80 g of the ground stevia plant used in Example 1, 800 ml of water was added and packed in a retort pack, and the air was extruded and heat sealed. This was maintained at a temperature of 85 to 90 ° C. and normal pressure for 30 minutes, and sterilized. Next, the reaction was performed at a temperature of 35 ° C. and a pressure of 50 MPa for 21 hours. This reaction product had a Brix value of 3.1 indicating sweetness. After the reaction, the extract was subjected to solid-liquid separation, and the filtrate was freeze-dried. Let this be unfermented stevia.
A 5 vol% DMSO aqueous solution was added to unfermented stevia and suspended by sonication to prepare a 50 mg / mL test solution stock solution. Using this test solution stock solution, the fat accumulation inhibitory effect was evaluated in the same manner as in Example 1. The experimental outline is shown in Table 1, and the fat accumulation rate is shown in Table 3 and FIG.

(Comparative Example 2)
To 80 g of the ground stevia plant used in Example 1, 800 ml of water was added and packed in a retort pack, and the air was extruded and heat sealed. This was sterilized at a temperature of 85 to 90 ° C. and normal pressure for 30 minutes. After cooling, the retort pack was opened, 14.4 g of Saccharomyces and 1.6 g of Lactobacillus acidophilus were added, and air was pushed out and heat sealed. The reaction was performed at a temperature of 35 ° C. and a pressure of 50 MPa for 21 hours. This reaction product had a Brix value of 3.4 indicating sweetness.
Next, as in Example 1, the contents of the retort pack were charged into the reaction vessel of the supercritical carbon dioxide extraction apparatus shown in FIG. 1, and carbon dioxide extraction was performed under the conditions of 80 ° C., 30 MPa, and a flow rate of 3.5 L / min. Went for hours. After 4 hours, the carbon dioxide feeding was stopped, the pressure was gradually lowered, and the pressure in the reaction vessel was returned to normal pressure.

  Subsequently, the contents of the reaction vessel were taken out and subjected to solid-liquid separation, and the filtrate was dried by spray drying. Using this dried product as a sample, a 5 vol% DMSO aqueous solution was added and suspended by sonication to prepare a 50 mg / mL test solution stock solution. Using this test solution stock solution, the fat accumulation inhibitory effect was evaluated in the same manner as in Example 1. The experimental outline is shown in Table 1, and the fat accumulation rate is shown in Table 4 and FIG.

(result)
(1) As is clear by comparing FIGS. 3 to 5, all of Example 1, Comparative Example 1 and Comparative Example 2 have a lower fat accumulation rate than the untreated control at a concentration of 1000 μg / ml, It was found to have a fat accumulation inhibition rate. Thereby, it was suggested that the stevia plant contains the component which has a fat accumulation inhibitory effect.
(2) In Example 1, as shown in FIG. 3, a dose-dependent fat accumulation suppression rate was observed using the amount of oil red O as an index. As shown in the microscopic image of the cultured cells in FIG. 2, there is no difference in the number of cultured cells between 1000 μg / ml administration, 500 μg / ml administration and 250 μg / ml administration. The cytotoxicity of the fat accumulation inhibitory substance was low, and the reduction in the amount of oil red O was estimated to be derived from the fat accumulation inhibitory effect or fat production inhibitory effect of the fat cells.
(3) The result of having evaluated the statistical significance difference of the fat accumulation inhibitory effect of Example 1, the comparative example 1, and the comparative example 2 in a 500 microgram / ml sample is shown in FIG. The samples used in Example 1 were subjected to multiple comparisons by one-way analysis of variance and the Turkey method for Comparative Example 1 and Comparative Example 2, respectively, and a significant difference was detected. The specimens obtained in Example 1, Comparative Example 1, and Comparative Example 2 are common in that they undergo high pressure and high temperature processing conditions. The difference is that Example 1 is fermented under aerobic conditions, but Comparative Example 1 and Comparative Example 2 are subjected to anaerobic and pressurized conditions. Therefore, it was estimated that the difference in the effect of suppressing fat accumulation may be due to the presence or absence of oxygen during the fermentation process.

  According to the present invention, a fat accumulation-inhibiting substance can be easily produced from a stevia fermented product, which is useful.

1 carbon dioxide cylinder,
2 Booster,
3 Preheating tube,
4 constant temperature bath,
5 reaction vessel,
6 Back pressure valve,
7 Receiver,
8 carbon dioxide exhaust pipe,
9 Extract extraction pipe,
10 hydrophilic polar solvent reservoir,
11 Valve,
12 Receiver,
13 Extract

Claims (3)

A method for producing a fat accumulation-inhibiting substance from a stevia fermented product fermented under aerobic conditions using carbon dioxide,
Said stevia fermentation product, a step A of shifting a supercritical state, by contacting the carbon dioxide in a subcritical state or liquid state components dissolved in the carbon dioxide to the carbon dioxide,
A step B in which the stevia fermented product in which the components are transferred to the carbon dioxide side is contacted with a hydrophilic polar solvent,
After the step B, the fat accumulation inhibiting substance contained in the hydrophilic polar solvent is fractionated,
A method for producing a fat accumulation-inhibiting substance.   The method for producing a fat accumulation-inhibiting substance according to claim 1, wherein the stevia fermented product and the carbon dioxide are in contact with each other at a temperature of 10 to 130 ° C and a pressure of 7 to 80 MPa. 30 to 2000 parts by mass of a hydrophilic polar solvent is brought into contact with carbon dioxide in a supercritical state or a subcritical state with respect to 100 parts by mass of the stevia fermented product together with the stevia fermented product,
The process which comprises carrying out A and step B and simultaneously manufacturing method of fat accumulation inhibitor according to claim 1 or 2.