JP2010285357A - Manufacturing method for antioxidative component derived from sake lees - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antioxidative component exhibiting excellent inhibiting actions on active oxygen. <P>SOLUTION: The manufacturing method for the antioxidative component comprises subjecting sake lees to extraction with an alcohol to obtain an alcohol-soluble fraction and washing the thus-obtained alcohol-soluble fraction with water to recover a water-insoluble fraction. An antioxidative agent or an antioxidative stress-ameliorating agent can be obtained by the manufacturing method. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing an antioxidant component derived from sake lees.

Antioxidant components are known as effective components 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 known as sake squeezed lees) is known as one of the raw materials for obtaining this antioxidant component, and since this liquor is produced in large quantities as a by-product in the stage of producing sake, it is cheap and stable supply It is regarded as a promising raw material for antioxidant components. However, since sake lees are not sufficiently effectively used and are currently discarded, various methods for obtaining antioxidant components have been studied.
As an antioxidant component from sake lees and a method for producing the same, for example, an extract obtained by extracting sake lees at room temperature with an ethanol solution of 70% or more is dissolved in water, oil is removed with ethyl acetate or the like, and water-soluble A method for obtaining an active fraction (Patent Document 1): a method for adding sake lees to water and stirring at 45 ° C. for 10 hours, followed by boiling to 90 ° C. to obtain a hot water extraction active fraction and a sake lees with a 60% aqueous ethanol solution A method for obtaining a water-containing ethanol-extracted active fraction by extraction at room temperature (Patent Document 2); A method for obtaining a hexane-extracted active fraction by extracting sake lees with hexane, which is a hydrophobic organic solvent, is known (Patent Document 3). .

JP 2002-284632 A JP-A-5-310590 JP 2009-13110 A

However, according to Patent Document 3, a water extraction fraction, a 100 volume% ethanol extraction fraction, and a 50 volume% ethanol aqueous mixture extraction fraction derived from sake lees are considered to have a weak active oxygen suppression action, while hexane Although the extracted fraction has a strong active oxygen suppression effect, hydrophobic organic solvents are not very suitable for industrial mass production because they are difficult to handle such as solvent removal.
In view of such circumstances, the present invention is a method for efficiently producing an anti-oxidant component derived from sake lees having an active oxygen suppressing action, and an anti-oxidant or anti-oxidative stress comprising the anti-oxidant component obtained thereby as an active ingredient It relates to providing an improving agent.

  As a result of various studies on the production method of antioxidant components derived from sake lees, the present inventors have found that alcohol-soluble fractions obtained by extraction from sake lees with alcohols (hereinafter referred to as “alcohol extraction of sake lees”). In spite of the fact that no active oxygen inhibitory action was observed, the water-insoluble fraction obtained by washing this alcoholic alcohol extract with water was unexpectedly Since the active oxygen inhibitory action was strongly expressed, it was found that such a water-insoluble fraction is effective as a pharmaceutical, food or the like that exhibits an antioxidant effect.

That is, the present invention provides the following inventions.
(1) A method for producing an antioxidant component, wherein an alcohol-soluble fraction is obtained by extraction with alcohol from sake lees, and the water-insoluble fraction is recovered by washing the obtained alcohol-soluble fraction. .
(2) The method for producing an antioxidant component according to the above (1), wherein the alcohol concentration at the time of alcohol extraction is 80% by volume or more in the solvent.
(3) The method for producing an antioxidant component according to the above (1) or (2), wherein the liquid temperature during alcohol extraction is 4 to 50 ° C.
(4) The method for producing an antioxidant component according to any one of the above (1) to (3), wherein the treatment time for alcohol extraction is 10 minutes to 24 hours.
(5) The method for producing an antioxidant component according to any one of (1) to (4), wherein the alcohol is ethanol.
(6) The method for producing an antioxidant component according to any one of (1) to (5) above, wherein the liquid temperature during water washing is 40 to 110 ° C.
(7) The manufacturing method of the antioxidant component in any one of said (1)-(6) whose processing time in the case of water washing is 3 minutes-24 hours.
(8) Any one of the above (1) to (7), wherein the amount of water used in the water washing is 10 to 200 parts by mass with respect to 1 part by mass of the alcohol-soluble fraction in terms of dry matter. The manufacturing method of the antioxidant component of description.

  (9) An antioxidant or antioxidant stress improving agent comprising an antioxidant component obtainable by the above production method as an active ingredient.

  According to the method for producing an antioxidant component of the present invention, an antioxidant component derived from sake lees having an excellent active oxygen suppressing action can be obtained efficiently. Antioxidants or antioxidant stress improvers containing the obtained antioxidant component as an active ingredient can suppress excessive active oxygen in the body, so cardiovascular diseases, allergic diseases, aging / senile diseases, etc. It is useful as a pharmaceutical, food, or the like that exhibits a preventive, therapeutic and / or ameliorating effect on symptoms and diseases induced or promoted by excessive production of active oxygen.

  In the present invention, alcohol-soluble fractions are obtained by extraction from sake lees with alcohols.

Sake lees is a by-product that remains when squeezed and refined liquor when producing sake (sake), and is also referred to as squeezed lees. The production method of sake (sake) at this time may be a general production method. For example, sake moromi made by fermenting steamed rice and rice bran with water and yeast is prepared, and after aging, pressed. Thus, a clarified liquor is obtained, and this is further filtered and burned.
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.
The form of the sake lees used for extraction may be as it is after squeezing, but using a drying means such as freeze-drying, cold-air drying, hot-air drying, hot-air drying, etc. Is 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 is not particularly limited, but is preferably 0.1 to 20% by mass, and more preferably 0.5 to 10% by mass.

  As alcohol used for sake lees extraction, C1-C5 alcohol is preferable, for example, methanol, ethanol, propyl alcohol, isopropyl alcohol etc. are mentioned, Among these, handling is easy at a manufacturing process, and safety | security. Ethanol is preferable because of its high point. These solvents may be used alone or in a mixture of two or more. The alcohols may contain other organic solvents such as water and hexane, but the purity of the alcohol used is 80% by volume or more, more preferably 90 to 100% by volume, and further 95 to 100% by volume, Furthermore, it is preferable that it is 99-100 volume%.

The method for extracting the alcoholic extract of sake lees is not particularly limited. For example, the sake lees are brought into contact with the alcohol as an extraction solvent by an extraction means using a batch type or continuous batch type extractor or a Soxhlet extractor. And a method obtained by extraction under predetermined conditions.
The extraction means is not particularly limited, and examples thereof include solid-liquid extraction, liquid-liquid extraction, immersion, leaching, decoction, reflux extraction, ultrasonic extraction, microwave extraction, centrifugal extraction, and the like. A combination of more than one species can be used.
The amount of alcohol used as the extraction solvent at this time is preferably 1/5 to 10 parts by weight, more preferably 1/2 to 5 parts by weight, based on 1 part by weight of sake lees (in terms of dry matter). It is preferable that it is 1-3 mass parts.
The concentration of alcohols in the extraction solvent brought into contact with or brought into contact with sake lees during extraction is 80% by volume or more, more 90% by volume or more, more 95% by volume or more, and further 99 to 100% by volume. Further preferred.
The liquid temperature during this extraction is preferably from 0 to the boiling point of the solvent, more preferably from 5 to 40 ° C, and further preferably from 10 to 30 ° C.
The extraction period at this time is preferably 5 minutes to 1 day, more preferably 15 minutes to 5 hours.

Thus, after bringing alcohol into contact with the sake lees, the liquor is appropriately removed by centrifugation, filtration or the like, and a fraction dissolved in the alcohols, that is, an alcohol-soluble fraction can be obtained.
At this time, the sake lees removed by filtration or the like may be repeatedly extracted by the same extraction method as described above. The number of repetitions is preferably 1 to 3, and more preferably 1 from the viewpoint of cost and required time.

  The alcohol-soluble fraction obtained may be in any form of liquid, semi-solid, and solid, but from the viewpoint of workability, it is preferably solid such as powder or granule. At this time, it is desirable to remove the alcohols from the alcohol-soluble fraction by a drying device such as an evaporator or a dryer.

  Furthermore, the present invention is to wash the sake-alcohol alcohol-soluble fraction under predetermined conditions to obtain a water-soluble fraction and an insoluble fraction, remove the water-soluble fraction, and recover the water-insoluble fraction as an antioxidant component. To do.

The water used for washing is not particularly limited, and examples thereof include distilled water, ion exchange water, tap water, and well water. Although the water to be used may contain a small amount of an inorganic substance or an organic substance, distilled water and ion-exchanged water are preferred among the water used for washing.
At this time, the water added to the alcohol-soluble fraction of the sake lees may be any of cold water (0 to less than 10 ° C.), warm water (less than 10 to 40 ° C.), hot water (40 to 110 ° C.) and the like. Hot water is preferred. The temperature of this hot water is preferably 40 to 110 ° C, more preferably 70 to 100 ° C.
Moreover, it is preferable that the usage-amount of the water used for water washing is 10-100 mass parts with respect to 1 mass part of alcohol soluble fractions (dry matter conversion) of sake lees, More 20-80 mass parts, Furthermore, It is preferable that it is 40-60 mass parts.

The means for washing with water is not particularly limited, and the above-described extraction means can be used as appropriate. For example, the alcohol extract of sake lees and water may be left standing, or may be mixed, shaken, centrifuged, stirred, You may perform physical means, such as a sound wave and pressurization, individually or in combination. Of these, mixing, shaking and stirring are preferred. The stirring speed at this time is preferably 50 to 200 r / min. The pressure condition is preferably 0.5 to 2 atmospheres, and preferably atmospheric pressure (about 1 atmosphere).
Moreover, the liquid temperature in the case of water washing is although it does not specifically limit, It is preferable that it is 4-121 degreeC. When washing with water, it is preferable to adjust the liquid temperature by heating. Specifically, the liquid temperature is 40 to 110 ° C., more preferably 50 to 100 ° C., and further 70 to 100 ° C. From the viewpoint of improving the activity, it is preferable.
The treatment time in washing with water is preferably 3 minutes to 24 hours, more preferably 10 minutes to 10 hours, further 0.5 to 2 hours, and further preferably 0.5 to 1.5 hours.
Further, the pH of water at the time of washing with water is not particularly required to be adjusted, but is preferably adjusted to 6-8. A general pH adjuster may be used as appropriate.

Then, after washing with water, the water-soluble fraction and the water-insoluble fraction can be appropriately separated by centrifugation, filtration or the like, the water-soluble fraction can be removed, and the water-insoluble fraction can be recovered. When dividing into both fractions, centrifugation is preferable from the viewpoint of workability.
The water-soluble fraction is a fraction dissolved in water after washing the alcohol-soluble fraction under the above conditions, that is, a fraction extracted with water from the alcohol-soluble fraction. . The water-insoluble fraction is a fraction that has not been dissolved in water after being washed with water.
At this time, the water-insoluble fraction recovered by centrifugation or the like may be repeatedly washed with the same water washing as described above. The number of repetitions is preferably 1 to 3, and more preferably 1 from the viewpoint of cost and required time.

The obtained water-insoluble fraction is appropriately prepared in a liquid state 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. Also good. In addition, since the water-insoluble fraction can be dissolved in alcohols or the like, another solvent (for example, water or the like) may be mixed after appropriately dissolving in alcohols or the like.
In addition, the obtained water-insoluble fraction may be further purified by removing inert impurities by a known separation / purification technique, for example, liquid-liquid distribution, filtration, activated carbon treatment, adsorption resin, or the like.

And as shown in the below-mentioned Example, the 100 volume% ethanol extract derived from sake lees and the water-soluble fraction obtained by washing this extract with water under heating conditions; The insoluble fraction and the soluble fraction obtained by washing with volume% ethanol did not show any action to suppress the active oxygen produced from leukocytes, whereas the 100 volume% ethanol extract was heated under conditions. The water-insoluble fraction obtained by washing with water, that is, the water-insoluble fraction obtained by the production method of the present invention (hereinafter also referred to as “water-insoluble fraction of the present invention”) has an excellent active oxygen inhibitory action. Admitted. Therefore, the water-insoluble fraction of the present invention is an antioxidant component derived from sake lees having excellent pharmacological activity, and is used as an antioxidant or an oxidative stress improving agent (hereinafter referred to as “antioxidant etc.”). Can also be used to produce this formulation.
The antioxidant, etc. is a food, medicine department for preventing, ameliorating or treating diseases induced or promoted by active oxygen in the living body, for example, lifestyle-related diseases such as arteriosclerosis, diabetes, hyperlipidemia and cancer. It can be used as a foreign product, medicine, feed, etc. 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)). In addition, the food can be applied to foods, functional foods, foods for the sick, and foods for specified health use with the concept of prevention, improvement and treatment of antioxidants, lifestyle-related diseases and the like.
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 a pharmaceutical, it 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 water-insoluble fraction 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 preferred, and when used as an oral administration agent, the content of the water-insoluble fraction of the present invention in the preparation is usually 0.0001 to 20 of the total mass of the preparation in terms of dry matter. It is preferable that it is mass%, it is more preferable that it is 0.0002-5 mass%, and it is still more preferable that it is 0.001-1 mass%.

  The dosage of the above formulation may vary according to the patient's condition, body weight, sex, age or other factors, but the daily dosage per adult for oral administration is usually effective on a dry matter basis The component water-insoluble fraction 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 a food, the form 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 food, the water-insoluble fraction of the present invention alone or food-acceptable with this, such as other food materials, solvents, softeners, fats and oils, emulsifiers, preservatives, A fragrance, a stabilizer, a colorant, an antioxidant, a humectant, a thickener and the like may be used in appropriate combination.

  In addition, the content of the water-insoluble fraction of the present invention in foods 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, and 0.0002 to 5 More preferably, it is mass%, and it is still more preferable that it is 0.001-2 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%.

When the antioxidant of the present invention is used as a feed, for example, a livestock feed used for cattle, pigs, chickens, sheep, horses, etc., a feed for small animals used for rabbits, rats, mice, etc., tuna, eel And fish food for use in Thailand, Hamachi, shrimp, etc., and pet food for dogs, cats, small birds, squirrels, etc.
In addition, when producing feed, in addition to the content of water-insoluble fraction, meat such as cattle, pigs, sheep, protein, grains, bran, potatoes, sugars, vegetables, vitamins, minerals In general, feed ingredients used in general, gelling agents, shape-preserving agents, pH adjusters, seasonings, preservatives, nutritional supplements, etc., which are generally used in feeds, are blended as necessary. Feed can be processed and manufactured.
In addition, the content of the water-insoluble fraction in the feed varies depending on the use form, but is usually 0.0001 to 20% by mass, preferably 0.0002 to 5% by mass, and 0.001 to 2% by mass. % Is more preferable.

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

Production Example 1
Commercially available sake lees (Kobayashi Shuzo) were air-dried at room temperature (20-30 ° C.) to obtain dry sake lees with a water content of less than 10%.
After adding 100 mL of ethanol (purity: 100% by volume) to 50 g of this dried sake lees and adjusting the alcohol concentration in the solution to be 95% by volume, sake alcohol ethanol at room temperature (liquid temperature 20-30 ° C.) for 30 minutes. The mixture was stirred. After stirring, a filtrate containing an ethanol extract (hereinafter referred to as “ethanol extract filtrate”) and a residue are obtained by filtration, and 100 L ethanol is again added to the residue, followed by stirring and filtering in the same manner as described above. An ethanol extraction filtrate was obtained. These ethanol extraction filtrates were mixed and ethanol was removed by an evaporator to obtain a dried product (sample No. 1) of an ethanol extract derived from sake lees (0.4 g).
After adding 50 mL of ion-exchanged water at 80 ° C. to 1.2 g of this ethanol extract derived from the sake lees (dried product), the mixture was stirred with heating at a liquid temperature of 80 ° C. for 1 hour (60 r / min). The product was washed with water (pH about 7).
After the ethanol extract was washed with hot water in this way, it was separated into a precipitate fraction (hereinafter referred to as “water-insoluble fraction”) and a water-soluble fraction by centrifugation (3000 rpm, room temperature, 10 minutes). The water-insoluble fraction (sample No. 2) recovered was 0.56 g and the water-soluble fraction (sample No. 3) was 0.74 g.

Production Example 2
After adding 100 mL of 50% by volume ethanol aqueous solution to 100 g of dry sake lees from Production Example 1, the sake ethanol mixture was stirred for 30 minutes at room temperature (liquid temperature 20-30 ° C.) and left for 1 hour, after which it was filtered Then, an ethanol extraction filtrate was obtained, and the ethanol water mixture was removed with an evaporator to obtain a dried product (sample No. 4) of this 50% by volume ethanol water extract.
After adding 100 mL of 50 volume% ethanol aqueous solution to 50 volume% ethanol aqueous extract (dried product) derived from sake lees and stirring, the insoluble fraction and soluble fraction were separated by centrifugation (3000 rpm, room temperature, 10 minutes). Divided into. These were freeze-dried, and the recovered insoluble fraction (sample No. 5) was 0.48 g, and the soluble fraction (sample No. 6) was 0.32 g.

Test example 1
10 mg of each sample (No. 1 to No. 6) obtained above was dissolved in 1 mL of ethanol, and the antioxidant activity was measured 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 volumes of fluorescent reagent 10 μM 5,6-CM-H ₂ DCFDA (Invitrogen) was added. % (V / v) fixing reagent (BECKMAN COULTER) was added and left at room temperature for 20 minutes. Thereafter, the active oxygen in leukocytes was measured by Flowcytemetory (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%.

These results are shown in Table 1.
In the 100% EtOH extract (sample No. 1) derived from sake lees, the water-insoluble fraction which was washed with hot water, ie, 100% EtOH extract, although no active oxygen inhibitory action was observed. -It was confirmed that the water-insoluble fraction (sample No. 2) newly exhibited an active oxygen suppressing action. On the other hand, the fraction that was washed with hot water and dissolved in the washing water (water-soluble fraction), that is, 100% EtOH extract-water-soluble fraction (sample No. 3), exhibited an active oxygen inhibitory action. There wasn't.
In addition, 50% EtOH extract (sample No. 4), this was washed with the same 50% EtOH water mixture, and the soluble fraction dissolved in this was 50% EtOH extract-50% EtOH soluble fraction (sample No. 4). .6) No active oxygen inhibitory action was observed in any of the insoluble fractions not dissolved therein, ie, the 50% EtOH extract-50% EtOH insoluble fraction (sample No. 5).

Claims (9)

  A method for producing an antioxidant component, characterized in that an alcohol-soluble fraction is obtained by extraction with alcohol from sake lees and the water-insoluble fraction is recovered by washing the obtained alcohol-soluble fraction.   The method for producing an antioxidant component according to claim 1, wherein the alcohol concentration in the alcohol extraction is 80% by volume or more in the solvent.   The method for producing an antioxidant component according to claim 1 or 2, wherein the liquid temperature during alcohol extraction is 4 to 50 ° C.   The method for producing an antioxidant component according to any one of claims 1 to 3, wherein the treatment time for alcohol extraction is 10 minutes to 24 hours.   The method for producing an antioxidant component according to any one of claims 1 to 4, wherein the alcohol is ethanol.   The method for producing an antioxidant component according to any one of claims 1 to 5, wherein the liquid temperature during water washing is 40 to 110 ° C.   The method for producing an antioxidant component according to any one of claims 1 to 6, wherein the treatment time during water washing is 3 minutes to 24 hours.   The amount of water used in the water washing is 10 to 200 parts by mass with respect to 1 part by mass of the alcohol-soluble fraction in terms of dry matter, The antioxidant component according to any one of claims 1 to 7 Production method.   The antioxidant or antioxidant stress improving agent which uses as an active ingredient the antioxidant component which can be obtained with the manufacturing method of the antioxidant component of any one of Claims 1-8.