JP5646155B2 - Method for producing an extract having an antioxidative effect derived from sake lees - Google Patents

Description

  The present invention relates to a method for producing an extract having an antioxidant effect derived from sake lees and a method for extracting the same.

Antioxidants (components) are known as components effective for suppressing active oxygen produced in vivo.
The active oxygen produced in the living body is used for maintaining the homeostasis of the living body such as biological regulation, antibacterial activity, and antiviral activity, if appropriate, while the active oxygen produced by stress, ultraviolet exposure, etc. If it is excessive, it will cause nucleic acid degradation, protein denaturation, lipid peroxidation, etc., which will damage cells and contribute to the induction of various pathological conditions. Diseases induced or promoted by the production of excessive active oxygen in the body include cardiovascular diseases, cranial nervous system diseases, digestive system diseases, kidney diseases, respiratory diseases, metabolic / endocrine diseases Allergic diseases, eye diseases, aging / senile diseases and the like are considered (Non-patent Document 1).

Sake lees (also called “sake squeezed lees”) are used as a raw material containing the antioxidant component.
For example, as a method for obtaining an extract containing an anti-oxidant substance (also referred to as an “antioxidant component”) from sake lees in sake, that is, an extract having an anti-oxidant effect, for example, sake lees are made from 70% ethanol or more. A method in which an extract obtained by room temperature extraction with an aqueous solution is dissolved in water and oil is removed with ethyl acetate or the like to obtain a water-soluble active fraction (Patent Document 1); A method of stirring for a period of time and boiling to 90 ° C. to obtain a hot water extraction active fraction and a method of obtaining a water-containing ethanol extraction active fraction by extracting sake lees at room temperature with a 60% aqueous ethanol solution (Patent Document 2); A method (Patent Document 3) is known in which hexane extraction active fraction is obtained by extraction at room temperature with hexane which is an organic solvent.
Moreover, as a method of obtaining the extract which has the antioxidant effect | action from shochu liquor, for example, the distillation residue of rice shochu (patent document 2), shochu liquor or barley shochu liquor is extracted at room temperature with hexane, and extracted with hexane. A method for obtaining an active fraction (Patent Document 4) is known.
However, the present situation is that an extract containing a substance having an antioxidant effect derived from sake lees has not been obtained efficiently.

JP 2002-284632 A JP-A-5-310590 JP 2009-13110 A Japanese Laid-Open Patent Publication No. 2009-13279

Itakura Hiroshige, Eiyo-Hyouka To Tiryo. Vol. 19, No. 3, 293-298, 2002

Although extraction with a hydrophobic organic solvent such as hexane can produce an extract with a strong antioxidant activity from sake lees, hydrophobic organic solvents are not suitable for industrial mass production because they are difficult to handle such as solvent removal. Not. On the other hand, in the extraction method using water-soluble solvent and water such as alcohol generally used for industrial mass production from the viewpoint of safety and work efficiency, an extract having a strong antioxidant action is obtained from sake lees. There was a problem that it was not possible.
Therefore, in view of such circumstances, the present invention relates to providing an efficient method for producing an extract having an antioxidant effect derived from sake lees or an extraction method thereof.

  The present inventors have studied a method for producing an extract having an antioxidant action derived from sake lees. Surprisingly, the sake lees are made into a fluid in a supercritical state (hereinafter also referred to as “supercritical fluid”). Extract, it can efficiently obtain an extract having a strong active oxygen inhibitory action, and it has been found that such a supercritical fluid extract derived from sake lees is effective as a pharmaceutical, food, etc. exhibiting an antioxidant effect. It was.

That is, this invention provides the invention which concerns on the following (1)-(6).
(1) A method for producing an extract having an antioxidant action, comprising a step of bringing a supercritical fluid into contact with sake lees.
(2) The production method as described above, wherein the supercritical fluid is supercritical carbon dioxide.
(3) The production method as described above, wherein the supercritical carbon dioxide is under conditions of a pressure of 7 MPa or more and a temperature of 31 ° C. or more.
(4) The said manufacturing method whose extraction time is 1 minute-24 hours.
(5) A method for extracting an extract having an antioxidant action, wherein extraction is performed by bringing a supercritical fluid into contact with sake lees.
(6) The extraction method as described above, wherein the supercritical fluid is supercritical carbon dioxide.

  The method of the present invention can efficiently obtain an extract having a strong antioxidant action, and the obtained extract can be used for the production of a preparation. Since the preparation can suppress excessive active oxygen in the body, the prevention and treatment of symptoms and diseases induced or promoted by excessive production of active oxygen such as cardiovascular diseases, allergic diseases, aging / senile diseases, and the like It is useful as an active ingredient for pharmaceuticals, foods, etc. that exhibit an improvement effect.

The present invention includes a step of bringing a supercritical fluid into contact with sake lees. Through this process, the antioxidant component contained in the sake lees can be extracted by dissolving in the supercritical fluid.
Here, the “supercritical fluid” refers to a state of a substance at a temperature and pressure above the critical point, and has both gas diffusibility and liquid solubility.

The sake lees are by-products that remain when the mash is squeezed when producing sake (sake), shochu, etc., or by-products that remain without being distilled, and are also referred to as squeezed koji and residue.
The form of the sake lees is not particularly limited, and a processed product such as shredded, crushed or dried may be used as it is. The form may be, for example, liquid, paste, or solid, but may be powder or solid with reduced moisture content using a drying means such as freeze drying, cold air drying, hot air drying, hot air drying, or the like. The shape and the like are preferable from the viewpoint of improving the recovery rate of the antioxidant component. As the drying means, freeze-drying, cold air at 4 to 40 ° C. or hot air drying is preferable from the viewpoint of preventing alteration of the antioxidant component.
The water content of the sake lees at this time is not particularly limited, but is preferably 0.01 to 10% by mass, more preferably 0.01 to 5% by mass.

Incidentally, the production method of Japanese sake (sake) may be any general production method. For example, sake mash is made by fermenting steamed rice and rice bran with water and yeast. To obtain a clarified liquor, and further filtering and burning it.
Sake (sake) here can be divided into ordinary sake and specially named liquors (honjozo, ginjo sake, junmai sake, etc.) depending on the raw materials and production method, but it is not particularly limited. Includes everything.

Moreover, the manufacturing method of shochu should just be a general manufacturing method, for example, made the mash (primary mash) which fermented rice bran (or wheat straw) by adding water and yeast, and steamed to this. A method of obtaining distilled liquor by charging raw materials (steamed rice, barley, satsuma straw, buckwheat, sugar millet, etc.) and water, fermenting and brewing them, and then distilling the secondary mash. .
Here, the main raw material in the production of shochu may be any food containing starch such as cereals or potatoes, for example, rice, barley, wheat, satsuma rice, buckwheat, sugar millet, sesame, potato, yam, taro, carrot, Beans, chestnuts, shiso, etc. may be mentioned, and two or more may be combined. Preferred are moss and / or wheat.

As a means for bringing the supercritical fluid into contact with the sake lees, for example, one or more substances selected from water, ethane, ethylene, propane, nitrous oxide, carbon dioxide and the like are in a supercritical state (supercritical fluid), Examples include a means for bringing this into contact with sake lees in an extractable state and extracting an antioxidant component from the sake lees into a supercritical fluid.
Examples of more specific extraction means include, for example, a means for extracting by passing a supercritical fluid in sake lees; a means for extracting by stirring, shaking or immersing sake lees in the supercritical fluid. Of these, means for extracting by passing a supercritical fluid in the sake lees is preferable. Examples of other extraction means include reflux extraction means, ultrasonic extraction means, microwave extraction means, and centrifugal extraction means. These can be used alone or in combination of two or more.
Then, the process of collect | recovering the obtained extract may be included. Specifically, after bringing the supercritical fluid into contact with the sake lees, the supercritical fluid extraction containing the residue of the sake lees after extraction and the antioxidant components derived from the sake lees is appropriately performed by separation means such as centrifugation, filtration or chromatography. The extract may be collected from the product and the extract may be recovered, and the preparation may be produced using the recovered product.
Examples of the apparatus used for such extraction include a supercritical fluid extraction apparatus and a supercritical carbon dioxide extraction apparatus.

Among the supercritical fluids, supercritical carbon dioxide is preferable because it can be removed safely and easily.
Supercritical carbon dioxide means carbon dioxide in a fluid state under conditions of a pressure of 7 MPa or more or a temperature of 31 ° C. or more. The pressure conditions at this time are preferably 7 to 100 MPa, more preferably 7 to 50 MPa, and more preferably 7 to 30 MPa from the viewpoint of extraction efficiency. The temperature conditions are 31 to 100 ° C. and 31 to 70 ° C. Furthermore, it is preferable that it is 31-50 degreeC. Among these, supercritical carbon dioxide having a pressure of 7 to 50 MPa and a temperature of 31 to 100 ° C., more preferably a pressure of 7 to 30 MPa and 31 to 50 ° C. is preferable.

The amount of supercritical carbon dioxide used in the extraction is preferably 5 to 500 parts by mass, more preferably 10 to 250 parts by mass, and further 15 to 150 parts by mass with respect to 1 part by mass of sake lees (in terms of dry matter). .
The extraction time is preferably 1 minute to 24 hours, 10 minutes to 10 hours.
In addition, the flow rate of carbon dioxide when supercritical carbon dioxide is passed through a container filled with sake lees and brought into contact with the sake lees varies depending on the capacity of the container, but with respect to 1 part by mass of sake lees (in terms of dry matter) 0.1-100 parts by mass / h, more preferably 1-25 parts by mass / h.

The obtained supercritical fluid extract is a liquid such as a stock solution, a diluted solution, a concentrated solution or a suspension; a solid state such as a dried product, a pulverized product or a powder; a semi-solid state such as a paste; It may be appropriately prepared by blending a solvent such as ethanol or drying.
Further, the obtained supercritical fluid extract appropriately removes inert impurities by a known separation / purification technique, for example, liquid-liquid extraction, solid-liquid extraction, filtration, activated carbon treatment, adsorption resin, and the like. It may be purified.

  As shown in the Examples below, when supercritical carbon dioxide is brought into contact with sake lees and extracted, a supercritical carbon dioxide extract having an antioxidant effect equivalent to that of hexane extract can be obtained. Moreover, according to the supercritical carbon dioxide extraction method, the recovery rate of the antioxidant component derived from sake lees is improved about three times that of the hexane extraction method. Therefore, if an antioxidant component is extracted from sake lees using a supercritical fluid and a supercritical fluid extract having a strong antioxidant action is recovered, an antioxidant or an oxidative stress improving agent can be produced efficiently.

Therefore, the supercritical fluid extract derived from the sake lees of the present invention can be used as an antioxidant or an oxidative stress improving agent (hereinafter referred to as “antioxidant etc.”), and further for producing this preparation. Can be used for At this time, the anti-oxidant or the like is allowed in a later-described object to be blended such as a supercritical fluid extract derived from sake lees alone or in addition to a carrier appropriately selected as necessary. Things may be used. In addition, the said formulation can be manufactured by a conventional method according to the target object which should be mix | blended.
The antioxidants are foods, pharmaceuticals for preventing, ameliorating or treating diseases induced or promoted by active oxygen in vivo, such as lifestyle-related diseases such as arteriosclerosis, diabetes, hyperlipidemia and cancer. It can be used as an active ingredient in quasi-drugs, pharmaceuticals, feeds and the like. Antioxidants and the like are also useful for bedless persons and lifestyle-related disease reserves (groups that have not yet reached lifestyle-related diseases but are close to that state (boundary region)). The food can be applied to foods, functional foods, foods for the sick, and foods for specified health use as necessary, with the concept of prevention, improvement and treatment of antioxidants, lifestyle-related diseases, etc. .
Here, the “disease induced or promoted by active oxygen in the living body” includes cardiovascular disease, cranial nervous system disease, digestive system disease, kidney disease, respiratory system disease, metabolic / endocrine disease, allergic disease, eye Examples include diseases, aging / senile diseases (Non-patent Document 1).
“Oxidative stress” refers to a state unfavorable for a living body in which the balance between the active oxygen production system and the elimination system in the living body is lost and excessive active oxygen is produced. “Improving oxidative stress” mainly refers to returning this broken balance to a normal state.

  When the antioxidant or the like of the present invention is used as an active ingredient of a pharmaceutical product, the pharmaceutical product can be administered in any dosage form. Examples of the dosage form include oral, enteral, transmucosal, transdermal, and injection. Examples of the dosage form of the preparation for oral administration include tablets, capsules, granules, powders, and syrups. Examples of parenteral administration include intravenous injections, intramuscular injections, suppositories, inhalants, transdermal absorption agents, eye drops, and nasal drops.

  In such a preparation, the antioxidant of the present invention may be used alone or in appropriate combination with a pharmaceutically acceptable carrier. Such carriers include, for example, excipients, binders, disintegrants, lubricants, diluents, osmotic pressure regulators, pH regulators, emulsifiers, preservatives, stabilizers, antioxidants, colorants, ultraviolet rays. Absorber, moisturizer, thickener, brightener, activity enhancer, anti-inflammatory agent, bactericidal agent, corrigent, flavoring agent, extender, surfactant, dispersant, buffer, preservative, fragrance, coating agent Etc.

  Of these dosage forms, oral administration is preferable, and the content of the supercritical fluid extract of the present invention in the preparation when used as an active ingredient of an oral administration agent is usually 0% of the total weight of the preparation in terms of dry matter. It is preferably 0.0001 to 20% by mass, more preferably 0.0002 to 5% by mass, and still more preferably 0.001 to 1% by mass.

  The dosage of the above preparation may vary according to the patient's condition, body weight, sex, age or other factors, but the daily dosage per adult (60 kg) in the case of oral administration is usually converted to dry matter Thus, the supercritical fluid extract of the active ingredient is preferably 60 to 60000 mg. Moreover, although the said formulation can be administered according to arbitrary administration schedules, it is preferable to administer once to several times a day, and the administration interval is preferably 4 to 24 hours, more preferably 6 to 12 hours.

  When the antioxidant of the present invention is used as an active ingredient of food, the form of the food may be solid, semi-solid or liquid. Examples of foods include breads, noodles, confectionery, jelly, dairy products, frozen foods, instant foods, processed starch products, processed meat products, other processed foods, beverages, soups, seasonings, dietary supplements, etc. , And their raw materials. Moreover, like the said oral administration formulation, a tablet form, a pill form, a capsule form, a liquid form, a syrup form, a powder form, a granule form etc. may be sufficient.

  In order to prepare various forms of foods, the antioxidant of the present invention can be used alone or in combination with foods such as other food materials, solvents, softeners, fats and oils, emulsifiers, preservatives. Perfumes, stabilizers, colorants, antioxidants, humectants, thickeners and the like may be used in appropriate combinations.

  In addition, the content of the supercritical fluid extract of the present invention in the food is preferably 0.0001 to 20% by mass in the beverage in the case of a liquid form such as a beverage, usually as a dried product. It is more preferably 5% by mass, and further preferably 0.001 to 2% by mass. In the case of solid or semi-solid forms such as tablets and processed foods, the total composition is preferably 0.0001 to 20% by mass, more preferably 0.0002 to 5% by mass, and More preferably, it is 001-2 mass%.

Further, when the antioxidant of the present invention is used as an active ingredient of feed, the feed includes, for example, livestock feed used for cattle, pigs, chickens, sheep, horses, rabbits, rats, mice, etc. Examples include small animal feeds used for foods, fish foods used for tuna, eel, Thailand, yellowtail, shrimp, etc., pet foods used for dogs, cats, small birds, squirrels, and the like.
In addition, when producing feed, the antioxidant of the present invention alone or in addition, meat such as cattle, pigs, sheep, protein, grains, bran, potatoes, sugars, vegetables, Vitamins, minerals and other commonly used feed ingredients, and gel ingredients, shape-preserving agents, pH adjusters, seasonings, preservatives, nutritional supplements, etc., commonly used in feeds, are added as necessary. The feed can be processed and produced by conventional methods.
Moreover, although content of the supercritical fluid extract of this invention in feed changes with the usage forms, it is usually 0.0001-20 mass% normally, 0.0002-5 mass% is preferable, and it is 0.00. 001-2 mass% is more preferable.

  The present invention will be described in more detail, but the present invention is not limited thereto.

Production Example 1
A commercially available sake lees (Kobayashi Brewery: Sawa no Tsuru) was air-dried at room temperature (20-30 ° C.) to obtain dried sake lees. The moisture content of the dried sake lees was 2-3% by mass.
400g of dried sake lees were pulverized for 10 minutes using Oster Blender ST-1 (Osaka Chemical Co., Ltd.), then charged into a semi-batch supercritical carbon dioxide extraction device (Mitsubishi Chemical Corporation), 30MPa and 40 Treated with carbon dioxide at ℃ 6.5 for 6.5 hours. At this time, the amount of carbon dioxide used in the treatment was 9.2 kg. The liquid passing rate was 3.5 parts by mass / h of supercritical carbon dioxide with respect to 1 part by mass of sake lees.
The extract is separated and recovered from carbon dioxide by setting the pressure of carbon dioxide to 5.5MPa below the critical pressure and 30 ° C below the critical temperature, and 2.68g of supercritical carbon dioxide extract (recovery rate 0.67%) Got.
During recovery, carbon dioxide is vaporized by returning to a critical pressure or lower, so that workability is good and carbon dioxide is not toxic and safe.

Production Example 2
100 mL of hexane was added to 100 g of dried sake lees obtained by air-drying commercially available sake lees (Kobayashi Shuzo: Sawa no Tsuru) at room temperature (20 to 30 ° C.), and the mixture was stirred at room temperature for 1 hour. After filtration, the hexane extract was recovered to obtain 214 mg of hexane extract (recovery rate 0.21%).

  GLC analysis of these extracts (Agilent J & W GC column DB-1 (30m, 0.25mm, 0.25μm): Hold at 80 ° C for 1 minute, then heat up at 10 ° C / min and hold at 340 ° C: detection FID). In the supercritical carbon dioxide extract, three main peaks were observed (retention times of 16.9 minutes, 17.3 minutes, and 18.8 minutes), and these peaks almost coincided with the retention times of the main peaks of the hexane extract. .

Production Example 3
After 200 g of the dried sake lees obtained in Production Example 1 were pulverized for 10 minutes using Oster Blender ST-1 (Osaka Chemical Co., Ltd.), a semi-batch supercritical carbon dioxide extraction device (Mitsubishi Chemical Corporation) And treated with carbon dioxide under conditions of 30 MPa and 40 ° C. for 8 hours. At this time, the amount of carbon dioxide used in the treatment was 12.4 kg. The liquid passing rate was 7.8 parts by mass / h of supercritical carbon dioxide with respect to 1 part by mass of sake lees.
The extract is separated and recovered from carbon dioxide by setting the pressure of carbon dioxide to 5.5 MPa below the critical pressure and 30 ° C below the critical temperature, and 1.12 g of supercritical carbon dioxide extract (recovery rate 0.56%) Got.

Test example 1
Samples 1 and 2 were prepared by dissolving the supercritical carbon dioxide extract or hexane extract obtained in Production Example 1 or Production Example 2 in ethanol so that the extract was 10 mg / mL. This was measured for antioxidant activity using leukocytes isolated from rats as follows.
Animals used were SD rats (10-16 weeks old, male). Carotid artery blood was collected from the SD rat under anesthesia with Foren (Abbott Japan). This blood sample was layered on a blood cell separation reagent (manufactured by SIGMA) and centrifuged, and then the leukocyte fraction was collected.
After each sample was added to the collected leukocyte fraction to 0.01 mg / mL and reacted at room temperature for 1 hour, 10 μM of fluorescent reagent 10 μM 5,6-CM-H ₂ DCFDA (Invitrogen) was added at 10% by volume. (v / v) Fixing reagent (BECKMAN COULTER) was added and left at room temperature for 20 minutes. Thereafter, the active oxygen in leukocytes was measured with a Flowcyteometory (flow cytometer: manufactured by Becton Dickinson).
The intracellular active oxygen was measured in the same manner as described above with no sample added, and the amount of active oxygen produced without addition (control) was defined as an active oxygen production inhibition rate of 0%.

  The supercritical carbon dioxide extract had a strong antioxidation activity with an active oxygen inhibition rate of 40% when the control was an active oxygen inhibition rate of 0%. The hexane extract had an active oxygen suppression rate of 48%.

  As described above, the supercritical carbon dioxide extract was considered to have almost the same main component as the main component of the hexane extract, and had a strong antioxidant activity almost equal to that of the hexane extract. Moreover, if the supercritical carbon dioxide extraction method of the present invention is used, the recovery rate from the sake mash, which is the raw material for extraction, is about three times that in the case of the hexane extraction method. Extracts containing (oxidized substances) can be recovered safely and efficiently.

Claims (6)

  An antioxidant containing as an active ingredient an extract obtained by bringing supercritical carbon dioxide into contact with sake lees.   An oxidative stress ameliorating agent comprising as an active ingredient an extract obtained by bringing supercritical carbon dioxide into contact with sake lees. The manufacturing method of the antioxidant characterized by including the process of contacting supercritical carbon dioxide with sake lees. The manufacturing method of the oxidative stress improving agent characterized by including the process of contacting supercritical carbon dioxide with sake lees. Supercritical carbon dioxide, the production method of an anti-producing method of the oxidant or claim 4 oxidative stress improving agent according to claim 3, wherein those conditions described above or pressure 7MPa and temperature 31 ° C.. Extraction time, the manufacturing method or manufacturing process of oxidative stress improving agent of antioxidants according to any one of a 1 minute to 24 hours claims 3-5.