JP2008201683A - Appetite suppressing composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an appetite suppressing composition that obtains a secure appetite suppressing effect, has a high safety and is friendly to the environment. <P>SOLUTION: The appetite suppressing composition comprises the protease treated product of rice saccharification lees and/or spirit fermentation lees, preferably the endopeptidase treated product, more preferably the serine protease treated product, further preferably the serine protease treated product produced by a bacterium of the genus Bacillus and/or Aspergillus as an active ingredient. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an appetite-suppressing composition, and more particularly to an appetite-suppressing composition containing as an active ingredient a protease-treated product of rice saccharified and / or fermented sake lees.

Obesity itself is considered a problem from a cosmetic point of view, but it can also cause adult diseases such as hypertension, diabetes, arteriosclerosis, stroke, angina, hyperlipidemia, Health is also a major social problem. In particular, in modern society, obesity often occurs due to intake of excess energy that exceeds consumption energy due to changes in dietary habits and excessive stress.
Thus, various methods for relieving obesity have been proposed, and in addition to exercising, methods of consuming excess energy by ingesting functional foods that burn body fat, etc. have been performed, for example, Dieting agents containing a specific proportion of amino acid having a body fat reducing effect such as arginine, glutamine, valine, isoleucine and leucine (for example, see Patent Document 1) have been proposed. It was difficult to burn etc. and was not very effective.

  On the other hand, since you will not become obese if you do not consume excessive energy in the body, drugs that have an appetite suppressing action have been developed as a method to keep the intake energy properly, but due to strong side effects, etc. Limited to use only by people with excessive morbid obesity.

In order to solve these safety problems, a safe appetite suppressant using food ingredients taken daily is also being developed, and cholecystokinin (hereinafter sometimes referred to as “CCK”). Has been proposed (for example, see Patent Document 2), and the peptide of the specific amino acid sequence is present in the pepsin degradation product of soybean β-conglycinin peptide. It is disclosed.
CCK is a digestion secreted from duodenal mucosal cells that suppresses gastric emptying of fed foods or has the ability to promote pancreatic enzyme secretion, and has the effect of suppressing satiety by giving a feeling of satiety. It is a tube hormone.
However, the appetite-suppressing action of the peptide having the specific amino acid sequence is not so strong, and the effect is not sufficient, and it has been demanded to develop an appetite-suppressing composition that can provide a more reliable effect. .

  Dietary health supplements containing a large number of amino acids effective for anti-obesity, obtained by subjecting starchy raw materials such as brown rice to fermentation by koji mold, yeast fermentation, and acetic acid fermentation followed by acetic acid removal treatment However, it is generally known that the anti-obesity mechanism of amino acids is a fat burning action, which is different from the appetite suppressing action of the present invention.

Japanese Patent Laid-Open No. 2005-27524 Japanese Patent Application Laid-Open No. 2004-10568 JP 2003-125732 A

As described above, there has not yet been reported an example of succeeding in developing an appetite suppressing composition that has a more reliable effect on appetite suppression and is highly safe.
An object of the present invention is to provide an appetite suppressing composition that provides a reliable appetite suppression effect, is highly safe, and is friendly to the environment.

  As a result of intensive studies to achieve the above object, the present inventors have found that a protease-treated product of rice saccharified koji and / or fermented sake koji has a strong appetite suppressing action, and the present invention is based on such findings. It came to complete.

That is, the present invention according to claim 1 relates to an appetite suppressing composition containing as an active ingredient a protease-treated product of rice saccharified and / or fermented sake lees.
The present invention according to claim 2 relates to an appetite suppressing composition according to claim 1, wherein the protease is an endopeptidase.
The present invention according to claim 3 relates to an appetite suppressing composition according to claim 2, wherein the endopeptidase is a serine protease.
The present invention according to claim 4 relates to an appetite suppressing composition according to claim 3, wherein the serine protease is produced by a microorganism belonging to the genus Bacillus and / or Aspergillus.
The present invention according to claim 5 relates to an appetite suppressing composition containing 0.08 μg to 40 g of the protease-treated product according to any one of claims 1 to 4 per one intake.
The present invention according to claim 6 relates to a method for producing an appetite-suppressing composition, characterized in that rice saccharified and / or fermented sake lees are treated with protease in producing an appetite-suppressing composition.

The appetite-suppressing composition of the present invention has a more reliable appetite-suppressing effect than the conventional one, is highly safe, is environmentally friendly, and can eliminate and prevent obesity without stress. .
That is, according to the appetite suppressing composition of the present invention, it is possible to appropriately maintain the calorie intake without taking excessive energy.
Moreover, since the appetite suppressing composition of the present invention contains a processed product of rice that has been ingested as food as an active ingredient, it is highly safe.
Therefore, according to the present invention, it is possible to provide an appetite-suppressing composition that is very safe and that can surely provide an appetite-suppressing effect.
In addition, rice saccharified rice cake and sake spirit fermented rice cake are usually discarded as industrial waste, and it is assumed that there are not a few negative effects on the environment. In the present invention, from the viewpoint of utilization of industrial waste, an excellent appetite suppressing composition can be obtained from industrial waste that should be discarded.

Hereinafter, the present invention will be described in detail.
First, the present invention is an appetite suppressing composition containing as an active ingredient a protease-treated product of rice saccharified and / or fermented sake lees.

Rice saccharified rice used in the present invention is produced by subjecting rice flour, brown rice flour or the like to starch decomposition treatment, and is by-produced in a vinegar production process, for example.
Moreover, the fermented sake lees used in the present invention are produced by subjecting rice flour or brown rice flour to starch decomposition and then fermented with sake, and examples thereof include so-called sake lees obtained in the sake brewing process.

Since these rice saccharified rice cake and sake spirit fermented rice cake are generated as by-products in the vinegar production process, the sake production process, etc., and are usually discarded, it is not possible to obtain the appetite suppressing composition of the present invention from these. It is also preferable from the viewpoint of waste utilization.
Further, in the present invention, rice saccharified rice bran and sake spirit fermented rice bran can be used singly, but both can be mixed and used at an arbitrary ratio.
Furthermore, because rice saccharified and sake refined rice bran are produced from rice or rice flour, the same appetite suppression effect can be obtained even in protease-treated products such as rice and rice flour. is there.

In the present invention, a target appetite suppressing composition can be prepared by treating these raw materials with a protease. There is no limitation in particular as a protease used in this invention, All things can be utilized.
However, from the viewpoint of the yield of the appetite suppressing composition, it is preferable to use an endopeptidase having the ability to cleave the protein from the inside as a protease to be used, and further, a serine protease whose active center amino acid is serine. More preferably, it is used.
In particular, among serine proteases, it is most preferable to use a serine protease derived from the genus Bacillus or Aspergillus.

Here, examples of serine proteases produced by microorganisms belonging to the genus Bacillus include orientase 22BF (manufactured by HBI) and biolase SP-4FG (manufactured by Nagase Chemtech).
Examples of serine proteases produced by microorganisms belonging to the genus Aspergillus include Sumiteam LP50D (manufactured by Shin Nihon Kagaku Kogyo) and Sumiteam MP (manufactured by Shin Nihon Kagaku Kogyo).

In addition, as the above serine protease, instead of using one in the form of a preparation, even if a culture using a microorganism belonging to the genus Bacillus or Aspergillus that produces these serine proteases is used, it also has an appetite suppressing effect. A composition is obtained.
In addition, the culture using the enzyme agent or the microorganism belonging to the genus Bacillus or Aspergillus may be used alone or in combination of two or more.

Next, the manufacturing method of the appetite suppression composition of this invention is explained in full detail.
The method for producing an appetite suppressing composition of the present invention is characterized in that rice saccharified and / or fermented sake lees are treated with a protease in producing the appetite suppressing composition.
In the method for producing an appetite-suppressing composition of the present invention, first, rice saccharified koji and sake fermented koji are used in an amount of 0.1 to 30 parts by mass, preferably around 5 parts by mass with respect to 100 parts by mass of water as a solvent. This is suspended in water. As the water used for suspension, any water can be used, but it is desirable to use water that does not easily inhibit the enzyme reaction, that is, deionized water, distilled water, ultrapure water, sterilized water, or the like.

  As described above, it is desirable to use by-products in the vinegar production process, the sake production process, and the like for the rice saccharified rice cake and the sake spirit fermented rice cake as the raw materials. When suspending rice saccharified rice cake or sake spirit fermented rice cake in water, the raw material can be suspended as it is, but in order to efficiently carry out the enzyme reaction in the following steps, it is finely divided into powder. It is desirable to use a pulverized one.

In addition, from the viewpoint of further improving the yield, it is more preferable to saccharify the rice saccharified rice cake or the fermented rice cake with amylase before or simultaneously with the “protease treatment”.
The conditions for the saccharification treatment with amylase are not particularly limited. For example, the suspension of rice saccharified koji and / or fermented sake koji obtained as described above is used for the optimum temperature of amylase, for example, 20 The reaction is carried out at a temperature of ˜95 ° C., preferably around 90 ° C. for 10 to 240 minutes, preferably around 120 minutes.

  As the saccharification reaction solution with amylase used for the saccharification treatment, amylase may be added as it is to the suspension of rice saccharified and / or fermented sake lees, but preferably the pH value is 4.0 to 9.0. Preferably, it is desirable to adjust to around 6.4. Any pH adjusting substance can be used for adjusting the pH value as long as the enzyme reaction is not inhibited. Specifically, for example, calcium hydroxide and hydrochloric acid can be used.

Examples of amylase that can be used for the saccharification treatment include α-amylase, β-amylase, isoamylase, and pullulanase. Specifically, Christase (manufactured by Yamato Kasei Co., Ltd.), liquefying enzyme T (manufactured by HBI), liquefying enzyme 6T (manufactured by HBI), Kokugen T (manufactured by Yamato Kasei), spitase XT404 (manufactured by Nagase Chemtech) Etc. can be used.
The addition amount of each amylase used in the saccharification treatment is 0.001 to 10 parts by mass, preferably about 0.1 parts by mass with respect to 100 parts by mass of the solvent used in the reaction solution. The amount of amylase added to the saccharification treatment is not the total amount of amylase used in the reaction, but the amount added for each amylase, that is, the amount that can be added to each of the five types when, for example, five types of amylase are used. .

After the saccharification reaction treatment, amylase is inactivated by heat treatment at 95 to 100 ° C. for 10 to 120 minutes, preferably at 100 ° C. for about 30 minutes.
Furthermore, after cooling this enzyme-reaction liquid to 0-10 degreeC, it is centrifuged at 100-20000g for 1-60 minutes, and a deposit can be used for protease treatment by removing a supernatant liquid.

The appetite suppressing composition in the present invention is obtained by performing “protease treatment” on the above-described rice saccharified and / or fermented sake lees.
That is, the appetite suppressing composition of the present invention can be manufactured by treating rice saccharified and / or sake fermented koji with a protease in manufacturing the appetite suppressing composition.

As described above, any protease can be used as the protease treatment in the present invention, but endopeptidase is preferred, serine protease is more preferred, and among serine proteases, Bacillus and Aspergillus are also included. Most preferably, a serine protease derived from is used.
As described above, examples of serine proteases produced by microorganisms belonging to the genus Bacillus include orientase 22BF (manufactured by HBI) and biolase SP-4FG (manufactured by Nagase Chemtech).
Examples of serine proteases produced by microorganisms belonging to the genus Aspergillus include Sumiteam LP50D (manufactured by Shin Nihon Kagaku Kogyo) and Sumiteam MP (manufactured by Shin Nihon Kagaku Kogyo).

In addition, as the above serine protease, instead of using one in the form of a preparation, even if a culture using a microorganism belonging to the genus Bacillus or Aspergillus that produces these serine proteases is used, it also has an appetite suppressing effect. A composition is obtained.
In addition, the culture using the enzyme agent or the microorganism belonging to the genus Bacillus or Aspergillus may be used alone or in combination of two or more.

As the solution used for the protease treatment in the present invention, any solution can be used as long as it does not inhibit the protease reaction. However, it is desirable to use a buffer solution having the optimum pH and optimum salt concentration of the protease to be used.
For example, when using the above serine protease, water prepared with sodium hydroxide at a pH around 7.2 can be used, but preferably contains 1 to 0.1 mM Tris and 0.1 to 10 mM calcium chloride. It is desirable to use a pH 4.0-9.0 buffer, more preferably a pH 7.2 buffer containing 10 mM Tris and 2 mM calcium chloride.
In addition, when a buffer solution is not used as the solution, it is desirable to adjust the pH to 6.5 to 7.0 again after completion of the enzyme reaction.

In the protease treatment according to the present invention, the “saccharified precipitate” obtained in the above step is 0.1 to 10% (w / v), preferably 1% (w / v) with respect to the solution used for the protease treatment. v) Resuspend in the solution used for the protease treatment so that it is contained before and after.
In addition, when the above saccharification treatment is not performed, the raw material rice saccharified rice cake and sake spirit fermented rice cake are 0.1 to 10% (w / v), preferably 1%, with respect to the solution used for the protease treatment. (W / v) Prepare to contain before and after.

The amount of protease used in the protease treatment in the present invention is the saccharification-treated rice bran and sake refined fermented rice cake, which are raw materials when the saccharification treatment is not performed on the “saccharification-treated precipitate” obtained in the above step. In contrast, 0.01 to 10% (w / w), preferably 0.1 to 2% (w / w), more preferably about 0.5% (w / w), respectively.
In addition, when the titer of the protease to be used is inferior, or when using a culture using microorganisms of the genus Bacillus or Aspergillus, it is possible to increase the production efficiency by adding an amount exceeding the above range. .

  The reaction conditions for the protease treatment in the present invention are the optimum temperature of the protease to be used, for example, when using the above serine protease, 20 to 95 ° C., preferably 30 to 80 ° C., more preferably around 55 ° C., It is desirable to react for 10 to 240 minutes, preferably 20 to 120 minutes, more preferably about 60 minutes.

After the protease treatment, the protease is inactivated by heat treatment at 95 to 100 ° C. for 10 to 120 minutes, preferably at 100 ° C. for about 10 minutes.
Furthermore, the enzyme reaction solution is cooled to 0 to 10 ° C., then centrifuged at 100 to 20000 g for 1 to 60 minutes, and the supernatant is collected, thereby containing the “appetite suppressing composition” according to the present invention. Is obtained.
The solution containing this “appetite suppressing composition” can be further refined by dialysis. It can also be pulverized by concentration by freeze-drying or the like.

  The form for providing the appetite suppressing composition of the present invention may be any form as long as it contains the appetite suppressing composition. For example, it may be formulated by blending conventionally used pharmaceutical adjuvants or the like, or may be formulated in food. Further, the shape such as liquid or solid is not limited.

As a preparation, tablets, liquids, capsules and the like can be used in any form, and conventional additives such as excipients and stabilizers can be blended in preparation.
Moreover, it can also mix | blend with food-drinks, such as a functional food, health food, and snack food. Examples of the food and drink include cookie-like, flake-like, wafer-like, tablet, granule, and liquid forms, and can be used as seasonings, beverages, and the like.
In addition, when mix | blending with food-drinks, it is desirable to display and use functions, such as having an appetite suppression effect, having an obesity prevention effect, and having a diet effect.

  In order to sufficiently exert the effect of the appetite suppressing composition of the present invention, a form in which 0.08 μg to 40 g / per person of the protease-treated product of the rice saccharified koji and / or sake fermented koji of the present invention can be ingested. Although it is preferable, it is desirable that it is a form which can ingest 0.01-3 g / day more preferably.

The appetite-suppressing composition in the present invention is not particularly limited in the intake time and the number of intakes, but for example, it is desirable to take 10 minutes to 12 hours before meal, preferably 30 minutes to 6 hours before meal.
In addition, the intake for one dose may be taken at a time, or may be taken in multiple doses, but taking into account the ease of ingestion, once every 30 minutes to 6 hours before meals Ingestion is preferred.

  The appetite-suppressing composition in the present invention is, as will be described later, a rat "binding affinity test with a cholecystokinin (CCK) secretion-promoting receptor in the small intestinal epithelial mucosa component" and "food consumption measurement test". Detailed analysis shows that it has a significant appetite suppression effect.

  This small intestinal epithelial mucosa component has a receptor for certain peptides that promote the secretion of CCK, an endogenous appetite suppressive hormone, and is known to act on appetite suppression (eg, soy protein) White matter research, Vol. 6, pp. 94-99, 2003). In addition, the binding affinity between the CCK secretion promoting receptor and the peptide present in the small intestinal epithelial mucosa component varies greatly depending on the type of peptide sequence.

  The protease-treated product of rice saccharified and / or fermented sake lees according to the present invention has a strong binding affinity with the mucosal component prepared from the rat small intestinal epithelium. Therefore, the protease-treated product of the present invention includes the small intestinal epithelial mucosal component. It is suggested that a large amount of a specific peptide sequence showing a strong binding affinity with the CCK secretion promoting receptor is contained.

In addition, these findings indicate that the appetite suppressing composition of the present invention can obtain an appetite suppressing effect by oral ingestion rather than injection by infusion, etc. It is beneficial to.
It is known that mazindol, an appetite suppressant, has an appetite suppressing effect in both humans and rats.
This also indicates that the mechanism of appetite suppression between humans and rats is similar, and the results of experiments using rats can be applied to humans.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1 (Binding test with rat small intestinal mucosa component)
(1) Sample preparation A protease-treated product of rice saccharified koji and sake spirit fermented koji was prepared as follows.
Alcoholic fermented koji obtained in the sake brewing process (uses rice flour as a raw material) (product 1 to 4 of the present invention) and saccharified rice koji obtained in the vinegar manufacturing process (uses rice flour as a raw material) (implemented in the present invention) 5 to 8) were each suspended in 100 ml of water, adjusted to pH 6.4 with calcium hydroxide, 100 μl of Christase (manufactured by Daiwa Kasei), liquefying enzyme T (manufactured by HBI), liquefying enzyme 100 μg of each amylase of 6T (manufactured by HBI), Kokugen T (manufactured by Yamato Kasei Co., Ltd.) and spitase XT404 (manufactured by Nagase Chemtech) was added.
Thereafter, the saccharification treatment reaction is carried out by raising the temperature to 90 ° C. and holding it for 120 minutes. After holding the reaction at 100 ° C. for 30 minutes to deactivate amylase, the solution is cooled to 4 ° C. and further centrifuged (12000 × g, 10 minutes), the supernatant was removed, and the resulting precipitate was resuspended to 1% (w / v) in a protease treatment buffer (10 mM Tris, 2 mM calcium chloride, pH 7.2).

This suspension was then treated with the following various protease formulations.
That is, as a protease preparation, orientase 22BF (manufactured by the present invention 1, 5) which is a serine protease preparation produced by bacteria of the genus Bacillus (product of the present invention 1, 5), or biolase SP-4FG (Nagase Chemtech) Manufactured by the present invention products 2 and 6), and Sumiteam LP50D (manufactured by Shin Nippon Kagaku Kogyo Co., Ltd.) which is a serine protease preparation produced by Aspergillus fungi (invented products 3, 7), Or, using Sumiteam MP (manufactured by Shin Nippon Kagaku Kogyo Co., Ltd.) (product of the present invention 4, 8), each is added in an amount equivalent to 0.5% (w / w) per the “saccharified precipitate”, The protease treatment reaction was carried out by holding at 55 ° C. for 60 minutes.
After completion of the protease treatment reaction, the protease is inactivated by holding at 100 ° C. for 10 minutes, and the “supernatant” obtained by centrifugation (12000 × g, 10 minutes) is lyophilized and subjected to lyophilization treatment. Invention products 1 to 8 were obtained. Each treated product was given the product name of the protease used for the treatment.

In addition, a pepsin degradation product of soybean β-conglycinin peptide, which is comparative product 1, was prepared as follows.
That is, defatted soybean powder was dissolved in 30 mM Tris-HCl buffer (pH 8.0) and ultracentrifuged (19000 × g, 20 minutes). The supernatant after ultracentrifugation was adjusted to pH 6.0 and ultracentrifuged again (12000 × g, 20 minutes, 4 ° C.). The supernatant was adjusted to pH 4.8 to obtain a precipitate. This precipitate was redissolved in 35 mM calcium phosphate buffer (pH 7.6, 0.4 mM NaCl, 10 mM 2-mercaptoethanol). Then, this solution was put into an aqueous ammonium sulfate solution to obtain soybean β-conglycinin peptide.
This soybean β-conglycinin peptide was dissolved in a phosphoric acid solution, adjusted to pH 1.85, an aqueous pepsin solution (20 mg / ml) was added, and the mixture was incubated at 37 ° C. for 10 minutes. After completion of the reaction, the phosphoric acid solution was boiled to inactivate pepsin. Then, after cooling, the supernatant was collected by centrifugation (12000 × g, 20 minutes, 4 ° C.).
The supernatant was neutralized with calcium hydroxide, centrifuged (12000 × g, 20 minutes, 4 ° C.) to remove the salt and freeze-dried, so that the soybean β-conglycinin peptide was decomposed into pepsin. A product (Comparative product 1) was obtained.

(2) Preparation of rat small intestine brush border membrane The mucous membrane of the jejunum of 200-300 g SD male rats (manufactured by SLC Japan) was scraped with a glass slide and homogenized, treated with calcium chloride and potassium thiocyanate and ultracentrifuged. Brush border membrane vesicles were obtained by this method. The obtained brush border membrane vesicle was subjected to a buffer (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA) containing 0.1% (w / v) Triton X-100 (TRITON X-100 REDUCED, manufactured by SIGMA). After suspending in pH 7.4) and shaking at 4 ° C. overnight, the supernatant obtained by ultracentrifugation (100,000 g, 90 minutes, 4 ° C.) was used as a membrane solubilizing component.
Surfactant (Triton X-100) is removed from the brush border membrane solubilizing component using Extracti-Gel D affinity Pak column (Pierce) and diluted to 10 mM acetate buffer (pH 4) for amine coupling. It was immobilized on the sensor chip CM5 (BIACORE) by the method (Amine Coupling Kit, BIACORE).

(3) Binding test with membrane soluble component immobilized on sensor chip In order to examine the binding ability with the membrane solubilized component prepared from small intestinal epithelium in the above-mentioned inventive products 1 to 8 and comparative product 1, Biacore (BIACORE) was used to conduct a binding test with a membrane soluble component immobilized on a sensor chip.
The analyte used in the binding test was prepared by dissolving a powder sample in HBS-E buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, pH 7.4), and carrying out the present invention products 1-8 and comparative product 1 at 500 μg / Prepared to contain ml.
An HBS-E buffer (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, pH 7.4) was used as a running buffer, and an analyte was flowed at a flow rate of 20 μl / min at a temperature of 25 ° C.
The analyte was turned on for 60 seconds. Dissociation was performed for 2.5 minutes, and regeneration was performed using 10 mM HCI for 60 seconds. A cell in which ethanolamine was fixed was used as a blank cell as a control group.
The specific binding value of each analyte to the membrane solubilizing component was determined as a value obtained by subtracting the value 10 seconds before the start of injection from the binding value (RU) of the sensor chip to which the membrane solubilizing component was bound. . The results are shown in FIG.

As a result, as shown in FIG. 1, the ability to bind to the brush border membrane component per unit weight is higher than that of a pepsin degradation product (comparative product 1) of β-conglycinin peptide of soybean as a control. It turned out that each protease processed material (this invention execution product 1-8) of sake spirit fermented rice cake is higher.
From these results, it was found that each protease-treated product of rice saccharified koji or sake spirit fermented koji has a high ability to bind to the small intestine epithelium and is higher than the pepsin degradation product of soybean β-conglycinin peptide.

Example 2 (CCK secretion ability measurement test using cholecystokinin (CCK) producing cells)
(1) Sample preparation Suspended 100 g of rice saccharified rice bran in 2 L of water, adjusted pH to 6.4 with calcium hydroxide, 2 ml of Christase (manufactured by Yamato Kasei), liquefying enzyme T (manufactured by HBI), liquefied All 2 g of each amylase of enzyme 6T (manufactured by HBI), kokugen T (manufactured by Yamato Kasei), and spitase XT404 (manufactured by Nagase Chemtech) were added.
The saccharification treatment reaction was carried out by raising the temperature to 90 ° C. and holding for 120 minutes, and then amylase was inactivated by holding at 100 ° C. for 30 minutes. After cooling to 4 ° C., the supernatant was removed by centrifugation, and the precipitate was resuspended in demineralized water, washed and collected by centrifugation.
Thereafter, the entire amount of this amylase-treated saccharified cocoon was suspended in 2 L of demineralized water, and the pH was adjusted to 7.2 with sodium hydroxide.
Next, 500 mg of orientase 22BF (manufactured by HBI) as a protease preparation was added thereto, reacted at 55 ° C. for 60 minutes, and the enzyme reaction was stopped by heat treatment at 100 ° C. for 20 minutes.
Subsequently, after cooling to 4 ° C., the pH was adjusted again to 6.5 to 7.0, and the supernatant obtained by centrifugation was freeze-dried to obtain a powder sample.
The protein content of this powder sample was measured by the Lowry method based on bovine serum albumin and found to be 85.4%.
The powder sample obtained as described above was used as the product 9 of the present invention in the following CCK secretion ability measurement test.
In addition, the pepsin degradation product of soybean β-conglycinin peptide used as a control was the one prepared in Example 1 (Comparative Product 1).

(2) CCK secretion ability measurement test using CCK-producing cells STC-1 The CCK release ability of the product 9 of the present invention and the comparative product 1 prepared in the above (1) was measured. In addition, since an ELISA kit (manufactured by Teramex) for measuring CCK also reacts with glucagon, it is difficult to measure only CCK in animal blood. On the other hand, it has been found that STC-1 cells prepared from the small intestinal epithelium do not produce glucagon, so that only CCK is produced.
Therefore, using CTC-1 cells, CCK producing ability of the product 9 of the present invention and the comparative product 1 was measured as follows.
That is, a buffer containing 20 mg Hepes, 140 mM NaCl, 4.5 mM KCl, 1.2 mM CaCl 2, 1 mg / ml or 5 mg / ml of the powder sample of Invention Product 9 or Comparative Product 1 STC-1 cells were incubated at 37 ° C. for 1 hour in 2 mM MgCl 2, 10 mM Glucose, pH 7.4), and the concentration of CCK released from the STC-1 cells into the medium was measured with a commercially available ELISA kit. .
The results are shown in FIG.

As shown in FIG. 2, regardless of whether the concentration of 1 mg / ml or 5 mg / ml was contained in the medium, the protease-treated product of rice saccharified rice (product 9 of the present invention) had soybean β as a control. -It was shown that CCK secretion ability was significantly higher than the sample derived from conglycinin peptide (Comparative product 1).
In particular, when 5 mg / ml of a rice saccharified protease treatment product (product 9 of the present invention) was contained in the medium, the amount of CCK secreted by STC-1 cells was determined from a sample derived from soybean β-conglycinin peptide ( Comparative product 1) It was found that the value was about twice as high as when 5 mg / ml was contained in the medium.

Example 3 (feeding amount measurement test using wild type rats)
(1) Sample preparation In the same manner as in Example 2, a powder sample of a protease-treated product of rice saccharified koji was prepared and designated as Product 10 of the present invention. The protein content of the obtained powder sample was 88% when measured by the Lowry method based on bovine serum albumin.

(2) Food intake measurement test using wild type rats Using SD male rats 12 weeks old, during the acclimation period of 10 days, food was fed only for a certain time after administration of water for injection by gastric sonde operation. I got used to the environment. During the acclimatization period, 2 ml of water for injection was administered per individual in the gastric tube operation.
Using the acclimated rats, a food intake measurement test was conducted.
As a work procedure, after 2 hours of lighting, 2 ml of “powder sample of rice saccharified koji koji protease (product of the present invention 10) dissolved in 50 mg, 200 mg or 550 mg” by gastric sonde operation, or Only 2 ml of “water” as a control was administered, and food was fed 30 minutes after that. The environment was such that it could be freely fed up to 6 hours after feeding. The water was free drinking. After that, it was under fasting.
The amount of food intake measured in each treatment group was converted to the amount of food intake per hour. In addition, as the number of samples in each administration group, 12 rats were used, the average and standard deviation were calculated, and the statistical significance was obtained. The results are shown in FIG.

  As a result, as shown in FIG. 3, in the group treated with the protease-treated product of rice saccharified rice (product of the present invention 10) compared to the group administered only with water (comparative control), any dose of 50 to 550 mg / rat was administered. Also in the group, the amount of food consumed per hour decreased with a significant difference of 5%, and the appetite suppression effect of the sample derived from rice saccharified rice was confirmed.

  From this result, when the effective dose in humans is calculated, it is estimated that the intake amount of the protease-treated product of the present invention may be up to about 40 g per person, preferably 0.08 μg per person. It was considered to be ˜40 g.

The appetite-suppressing composition of the present invention is very safe and can surely provide an appetite-suppressing effect, and is expected to be used as a functional food material and pharmaceutical preparation.
In addition, the appetite suppressing composition of the present invention also leads to effective use of rice saccharified koji or sake fermented koji, which are industrial wastes that should be discarded.
Therefore, the present invention is expected to be effectively used in the food industry and the like.

2 is a graph showing the results of a binding test between a protease-treated product and a rat small intestinal mucosa component in Example 1. It is the graph which showed the result of the CCK secretion ability measurement test using the CCK producing cell STC-1 in Example 2. 6 is a graph showing the results of a food intake measurement test using wild-type rats in Example 3.

Claims (6)

  An appetite-suppressing composition comprising a protease-treated product of rice saccharified and / or fermented sake lees as an active ingredient.   The appetite suppressing composition according to claim 1, wherein the protease is an endopeptidase.   The appetite suppressing composition according to claim 2, wherein the endopeptidase is a serine protease.   The appetite-suppressing composition according to claim 3, wherein the serine protease is produced by a microorganism belonging to the genus Bacillus and / or Aspergillus.   An appetite-suppressing composition containing 0.08 μg to 40 g of the protease-treated product according to any one of claims 1 to 4 per one intake.   In producing an appetite suppressing composition, a method for producing an appetite suppressing composition is characterized in that rice saccharified and / or fermented sake lees are treated with a protease.

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