WO2021071159A1 - Antidiabetic, hypotensive, or antioxidant composition comprising yeast fermentate of rhynchosia nulubilis and method for preparing same composition - Google Patents
Antidiabetic, hypotensive, or antioxidant composition comprising yeast fermentate of rhynchosia nulubilis and method for preparing same composition Download PDFInfo
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- WO2021071159A1 WO2021071159A1 PCT/KR2020/013246 KR2020013246W WO2021071159A1 WO 2021071159 A1 WO2021071159 A1 WO 2021071159A1 KR 2020013246 W KR2020013246 W KR 2020013246W WO 2021071159 A1 WO2021071159 A1 WO 2021071159A1
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- seomoktae
- yeast
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Images
Classifications
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/14—Yeasts or derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
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- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/328—Foods, ingredients or supplements having a functional effect on health having effect on glycaemic control and diabetes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/76—Undefined extracts from plants
Definitions
- the present invention relates to a composition for lowering blood sugar, lowering blood pressure, or antioxidant comprising a fermented yeast of Seomoktae, and a method for preparing the composition.
- Reactive Oxygen Species or free radicals which are the causes of oxidative stress in modern people, are continuously generated in the body during human metabolism, and are also unstable and highly reactive, so they easily react with other in vivo substances. They attack macromolecules in the body to degrade cell membranes, inhibit protein synthesis, and cause mutations, cytotoxicity, and cancer.
- Natural bioactive components have been known to have a preventive function such as diabetes or cancer by inhibiting oxidation when they enter the body. Therefore, it is necessary for modern people to develop natural antioxidants that do not have side effects that can alleviate oxidative stress and have excellent antioxidant activity.
- diabetes which is a chronic disease
- hypoglycemic agents such as ⁇ -glucosidase inhibitors such as acarbose and voglibose are concerned about side effects such as tolerance, weight gain, swelling, nausea, and digestive disorders, and development of blood glucose improving agents for natural substances is required.
- Seo Mok-Tae (scientific name: Rhynchosia Nulubilis) is harvested when the black and round seeds of 5-7mm in diameter grow in oval pods with flowers blooming in July. If you sow and cultivate, the medicinal properties become more excellent. It has a lot of fiber and contributes to the intestinal activity. Fermenting Seomoktae has more enzymes useful for the human body than any other food. It is also called Yakkong because it relieves blood stagnation and relieves blood stagnation.Eating soybean porridge eliminates cattle thirst (thirst due to diabetes), controls kidney disease, reduces all wind heat, and in particular, activates blood and releases all poisons.
- One object of the present invention is to provide a novel composition using yeast and seomoktae to lower or control blood sugar, lower blood pressure, or have an antioxidant function without side effects.
- Another object of the present invention is to provide a method for preparing a composition having antioxidant, antihypertensive or antidiabetic activity.
- the yeast is cultivated in a medium containing Seomoktae that does not substantially contain an additional nitrogen source,
- It relates to a method for producing a composition for lowering blood sugar, lowering blood pressure, or antioxidant, comprising preparing a composition for lowering blood sugar, lowering blood pressure, or antioxidant from the culture obtained after the culture.
- Yeast is a microorganism used to make beer, wine, and makgeolli, and it is a collective term for single-celled organisms that do not have hyphae and do not have photosynthetic or motility.
- microorganisms of the genus Saccharomyces may be used as yeast, and specifically, Saccharomyces servage or Saccharomyces cerevisiae may be used, but is not limited thereto.
- Saccharomyces servazzii KCCM12157P strain may be used. The strain is isolated from kimchi, a traditional Korean food.
- Yeast by culturing is to be included in a concentration of 5 x 10 4 to 5 x 10 10 CFU/ml in the culture, specifically, yeast at a concentration of 5 x 10 4 to 5 x 10 9 CFU/ml, more Specifically, it is included in a concentration of 1 x 10 6 to 1 x 10 9 CFU/ml.
- the medium for culturing yeast is a medium containing Seomoktae, and substantially does not contain additional nitrogen sources other than Seomoktae.
- additional nitrogen source since an additional nitrogen source, mainly low molecular weight, is used as a nitrogen source, less decomposition by the yeast of Seomoktae than the high molecular weight Seomoktae decreases the active ingredient that expresses lowering blood sugar, antioxidants, or blood pressure. I think it is.
- additional nitrogen sources are not limited as long as they contain N in the structure, for example, an organic nitrogen source or an inorganic nitrogen source, more specifically, YPD ((yeast ext., peptone, dextrose), of which enzyme extract and peptone are nitrogen sources. ), urea, casein, soybean, bran, brown rice milk, meat extract, whole milk powder, dried yeast, ammonium salt, tryptophan, proline, asparagine, and N-containing amino acids such as glutamate.
- YPD (yeast ext., peptone, dextrose), of which enzyme extract and peptone are nitrogen sources. )
- urea casein, soybean, bran, brown rice milk, meat extract, whole milk powder, dried yeast, ammonium salt, tryptophan, proline, asparagine, and N-containing amino acids such as glutamate.
- the meaning of'substantially not included' refers to that the component is contained in an amount of 0.05% by weight or less, specifically 0.0005% by weight or less, based on the total weight of the total composition or medium.
- Seomoktae (also known as rat-eyed bean) is a type of black bean, with a black skin and much smaller in size than ordinary black beans.
- Seomoktae is used as a source of nitrogen for yeast, and it is believed that effective peptides effective for lowering blood sugar, lowering blood pressure, or antioxidant by decomposition of Seomoktae by yeast are provided.
- the content of Seomoktae is 0.2 weight. % To 4% by weight. If it exceeds 4% by weight, coagulation may occur due to protein coagulation during cultivation, so it is recommended to be less than 4% by weight. More specifically, the Seomoktae content is best in the range of 0.6% by weight to 1.0% by weight, and it is also best that it is 0.6% by weight. In the above range, the blood sugar lowering effect may be the best.
- a culture medium natural or synthetic medium may be used.
- the carbon source of the medium for example, monosaccharides or disaccharides may be used, and examples thereof may include glucose, glucose, galactose, sugar, dextrin, dextrose, glycerol, and the like. It is preferable to use monosaccharides such as glucose and glucose as a carbon source, and monosaccharides are more efficient as a carbon source than polysaccharides or disaccharides, and are active in decomposition by yeast, so there are many effective ingredients for lowering blood sugar, lowering blood pressure, or antioxidant. Because you lose.
- the content of the carbon source in the medium is 1% to 5% by weight based on the total volume of the medium, and in particular, 1% to 3% by weight, or 2% to 3% by weight in terms of lowering blood sugar, lowering blood pressure, or antioxidant effect. It is good to be.
- Seomoktae is used as a nitrogen source, and other nitrogen sources are practically not included except for Seomoktae. When other nitrogen sources are included, lowering blood sugar, lowering blood pressure, or antioxidant effects may be significantly lowered compared to cultures cultured with only Seomoktae as a nitrogen source.
- the inorganic salt contained in the medium is magnesium. Manganese, calcium. Iron, potassium, and the like may be used, but are not limited thereto.
- amino acids, vitamins, nucleic acids, and compounds related thereto may be added to the medium.
- Culture temperature conditions of the yeast strain can be cultured for 12 hours to 7 days, for example 12 hours to 3 days at a temperature range of 20 °C to 40 °C, specifically 21 °C to 30 °C.
- the composition of the present invention is obtained by adding and culturing herbal preparations such as gasiogapi, guava, golden, yellow white, gardenia, schisandra, yellow lotus, jade porridge, extracts thereof, or a mixture thereof to the culture medium, and using the culture medium. It is possible to further improve the effect of lowering blood sugar, lowering blood pressure, or antioxidant.
- it is preferable to add gasiogapi or gold to the culture medium, and the amount thereof added is preferably about 0.01% to 10% by weight, specifically 0.01% to 5% by weight, based on the weight of the medium, respectively.
- a composition for lowering blood sugar, lowering blood pressure, or antioxidant may be prepared from the culture obtained after the cultivation.
- the preparation of the composition may be to further include a pharmaceutically or food pharmaceutically acceptable carrier in the culture.
- the composition may be formulated and provided as a food or pharmaceutical product.
- the preparation of the composition may be to use the culture itself obtained after the cultivation or a culture supernatant thereof as a composition.
- the term "pharmaceutically or food pharmaceutically acceptable carrier” refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of a culture.
- the culture contains a yeast cell-containing material and a culture medium.
- the culture medium is a culture supernatant after centrifugation, excluding cells containing substances.
- the cell-containing material includes cells and may be a cell-containing component obtained by removing the culture supernatant or concentrating the residue thereafter.
- the composition of the culture medium or the cell-containing material may additionally include a component necessary for normal yeast cultivation, as well as a component synergistically acting on the growth of yeast, and the composition according to this may be determined by a person having ordinary skill in the art. Can be easily selected.
- the above aspect may further include separating the culture medium from the culture.
- the separation may include centrifuging the culture to remove the cell-containing material.
- the concentration method includes an evaporative concentration method, a freeze concentration method, a membrane concentration method, or a dry concentration method, and specifically, concentration under reduced pressure may be performed using a rotary evaporator. Drying may be ventilated drying, natural drying, spray drying, and freeze drying, but is not limited thereto, and specifically, may be freeze-dried.
- Another aspect of the present invention relates to a composition for lowering blood sugar, lowering blood pressure, or antioxidant comprising the fermented yeast of Seomoktae prepared by the method described herein as an active ingredient.
- the fermentation product may be a fermentation broth or a fermentation supernatant from which yeast cells-containing substances are removed.
- composition of the present invention exhibits remarkably improved blood sugar lowering, blood pressure lowering, or antioxidant properties than before fermentation due to the change of the medicinal properties of Seomoktae by yeast fermentation. It is thought that the various polymers of are decomposed to produce effective peptides, thereby reducing blood sugar, lowering blood pressure, or enhancing antioxidant effects.
- the composition may be a food composition or a pharmaceutical composition for lowering blood sugar, lowering blood pressure, or antioxidant.
- the composition of the present application may be useful for the treatment of cancer, inflammation, anemia, myocardial infarction, arteriosclerosis, diabetes, cerebral palsy, arthritis (eg, rheumatoid arthritis), Parkinson's disease, or autoimmune disease due to its antioxidant activity.
- One aspect of the food composition may be food, health food, health supplement food, or health functional food composition.
- One aspect of the pharmaceutical composition may be a quasi-drug or a pharmaceutical preparation.
- the composition further comprises a component having antidiabetic antihypertension or antioxidant effect, such as herbal preparations such as gasiogapi, guava, golden, yellow white, gardenia, schisandra, yellow lotus, jade porridge, extracts thereof, or mixtures thereof.
- a component having antidiabetic antihypertension or antioxidant effect such as herbal preparations such as gasiogapi, guava, golden, yellow white, gardenia, schisandra, yellow lotus, jade porridge, extracts thereof, or mixtures thereof.
- composition of the present invention may include 1% to 70% by weight, specifically 5% to 60% by weight of fermented yeast of Seomoktae based on the solid weight of the composition.
- the yeast fermentation product of Seomoktae may be in a liquid state or a dry state, and specifically may be in a dried powder state.
- composition may be formulated in various oral or parenteral formulations, but preferably may be formulated as a liquid oral solution.
- acceptable pharmaceutical or food acceptable carriers are sterilized and biocompatible, and include saline, sterile water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and these
- saline sterile water
- buffered saline albumin injection solution
- dextrose solution dextrose solution
- maltodextrin solution glycerol
- glycerol glycerol
- diluents, dispersants, surfactants, binders, and lubricants may be additionally added to form liquid formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.
- binders, emulsifiers, preservatives, etc. may be additionally added to prevent degradation of the composition, or amino acids, vitamins, enzymes, flavors, non-protein nitrogen compounds, silicates, buffers, extractants, It may further include oligosaccharides and the like.
- Formulations comprising the composition of the present invention include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs.
- a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid
- a lubricant such as magnesium, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be included, and in the case of a capsule formulation, a liquid carrier such as fatty oil may be further contained in addition to the above-mentioned substances.
- the daily dosage of the composition or the fermented yeast of Seomoktae may vary depending on the body weight, age, sex, health condition, treatment, prevention, or improvement of the subject's main symptoms, administration time, administration method, and severity. It may be from 0.0001 mg/kg to about 10 g/kg, specifically from about 0.01 mg/kg to about 500 mg/kg, and may be administered or applied once to 6 times a day, for example, 1 to 4 times a day.
- Another aspect of the present invention is to improve diabetes, comprising administering to a subject a therapeutically or prophylactically effective amount of a fermented yeast fermented from Seomoktae prepared according to the method described herein for prevention or treatment of diabetes, lowering blood pressure or antioxidant. , Prevention or treatment methods, blood pressure lowering or antioxidant methods.
- the therapeutically or prophylactically effective amount may vary depending on the subject's body weight, age, sex, health condition, major symptoms to be treated, prevented, or improved, administration time, administration method, and severity, but 1 day. It may be about 0.0001 mg/kg to about 10 g/kg, specifically about 0.01 mg/kg to about 500 mg/kg, and may be administered or applied once to 6 times a day, for example, 1 to 4 times a day. .
- the yeast fermented product of Seomoktae prepared according to the method of the present invention is excellent in the ability to inhibit ⁇ -glucosidase, has few side effects, and is safe, and has high utility value for the prevention and/or treatment of diabetes.
- 1 is a bar graph showing a change in the number of yeast bacteria according to the content of Seomoktae in a medium in a manufacturing method according to an embodiment and a comparative example of the present invention.
- FIG. 2 is a bar graph showing the difference in ⁇ -glucosidase inhibitory ability (%) according to each medium component in a manufacturing method according to an embodiment and a comparative example of the present invention.
- 3 is a bar graph showing the difference in ⁇ -glucosidase inhibitory ability (%) according to changes in carbon sources (starch, sucrose, glucose, galactose) of each culture medium in a manufacturing method according to an embodiment and a comparative example of the present invention to be.
- FIG. 4 is a bar graph showing the difference in the ⁇ -glucosidase inhibitory ability (%) according to the change in the content of Seomoktae in the manufacturing method according to an embodiment and a comparative example of the present invention.
- 5 is a bar graph showing the difference in ⁇ -glucosidase inhibitory ability (%) according to a change in the concentration of glucose, which is a carbon source, in a manufacturing method according to an embodiment and a comparative example of the present invention.
- FIG. 6 is a bar graph showing the difference in ⁇ -glucosidase inhibitory ability (%) according to the addition of a low molecular nitrogen source to a medium in a manufacturing method according to an embodiment and a comparative example of the present invention.
- FIG. 7 is a graph showing the ⁇ -glucosidase inhibition rate (%) according to the concentration change of the composition (Seomoktae 0.6% by weight, glucose 2% by weight and yeast) prepared by the method according to an embodiment of the present invention.
- FIG. 8 is a graph showing the ⁇ -glucosidase inhibition rate (%) according to the concentration change of a conventionally known blood sugar lowering agent, Acarbose, used as a positive control.
- FIG. 9 is a graph showing the ACE inhibition rate (%) according to the concentration change of the composition (Seomoktae 0.6% by weight, glucose 2% by weight and yeast) prepared by the method according to an embodiment of the present invention.
- FIG. 10 is a graph showing the ACE inhibition rate (%) according to each concentration of a conventionally known blood pressure lowering agent, Lisinopril, used as a positive control.
- FIG. 11 is a graph showing the DPPH radical removal rate (%) according to the concentration change of the composition (Seomoktae 0.6% by weight, glucose 2% by weight and yeast) prepared by the method according to an embodiment of the present invention.
- FIG. 13 is a graph showing the ABTS radical removal rate (%) according to the concentration change of the composition (Seomoktae 0.6% by weight, glucose 2% by weight and yeast) prepared by the method according to an embodiment of the present invention.
- FIG. 14 is a graph showing the ABTS radical removal rate (%) according to each concentration of a conventionally known antioxidant, ascorbic acid, used as a positive control.
- composition 15 is a reducing power according to the concentration change of the composition (Seomoktae 0.6% by weight, glucose 2% by weight and yeast) prepared by the method according to an embodiment of the present invention and a conventionally known antioxidant used as a positive control, ascorbic acid ( reducing power).
- Kimchi a traditional Korean food, was collected, and the obtained sample was diluted in stages, spread on YPD (Yeast extract Peptone Dextrose) to which 1% sodium chloride was added, and incubated for 24 hours at 37°C. The dominant strain was isolated from the sample. The selected colonies were purely separated by transferring to a new medium for 3 times and culturing, and the pure cultured bacteria were stored in a medium to which 20% glycerol was added and stored at -70°C or below.
- YPD Yeast extract Peptone Dextrose
- the present inventors deposited the newly isolated Saccharomyces Servoji Ceb-kc-011 to the Korean Culture Center of Microorganisms (120-861 Hongje-2ga-gil, Seodaemun-gu, Seoul) on November 10, 2017. Deposited under the number KCCM12157P.
- a culture of Saccharomyces servagi Ceb-kc-011 was prepared in the following manner:
- Saccharomyces Serbage Ceb-kc-011 was inoculated with 0.2 to 0.4 liters aseptically and then cultured in an incubator at 25° C. for 24 to 48 hours to obtain a culture of Saccharomyces servaji Ceb-kc-011.
- the culture was separated into a cell-containing material and a culture solution by centrifugation, and the culture solution was dried at 60° C. for 12 hours to obtain a dried yeast fermentation broth of Seomoktae.
- Example 1 instead of Saccharomyces servo Ceb-kc-011, Saccharomyces servo was used in the same manner as in Example 1 to obtain fermented yeast 2 of Seomoktae.
- Example 2 the yeast fermented product 3 of Seomoktae of Example 3 was obtained in the same manner as in Example 2, except that Seomoktae was added in an amount of 2% by weight instead of 4% by weight.
- Example 2 the yeast fermented product 4 of Seomoktae of Example 4 was obtained in the same manner as in Example 2, except that Seomoktae was added in an amount of 2.5% by weight instead of 4% by weight.
- the yeast fermented product 5 of Seomoktae of Example 5 was obtained in the same manner as in Example 2, except that Seomoktae was added in an amount of 3% by weight instead of 4% by weight in Example 2.
- Example 2 the yeast fermented product 6 of Seomoktae of Example 6 was obtained in the same manner as in Example 2, except that Seomoktae was added in an amount of 3.5% by weight instead of 4% by weight.
- Example 2 Except for using the YPD medium containing 1.0% by weight of yeast extract, 2.0% by weight of peptone, and 2.0% by weight of dextrose, instead of the YPD medium from which Seomoktae was not added and yeast extract and peptone were removed in Example 2 In the same manner as in Example 2, fermented yeast 1 of Comparative Example 1 was obtained.
- a plate was prepared by adding 2% agar to YPD medium, and culture samples obtained by culturing in the manufacturing process of Examples 1 to 6 and Comparative Example 1 step-diluted with 0.85% sterile physiological saline were used. Inoculated and smeared at a time of 100 ⁇ l, and cultured for 24 to 48 hours in a 25°C incubator. After cultivation, colonies were counted and shown in Table 1 and FIG. 1.
- Weight% in the above table means the weight percentage of a specific component based on the total volume of the medium
- YPD which is a yeast culture medium
- yeast extracts and peptones which are nitrogen sources in YPD
- the content of Seomoktae was changed.
- the number of yeasts increased as the content of Seomoktae increased in the media prepared with Seomoktae and glucose.
- cultivation was performed with the addition amount of Seomoktae exceeding 4% by weight, and in this case, a lumping phenomenon due to protein coagulation was observed. Therefore, the appropriate amount of Seomoktae can be said to be 4% by weight or less.
- a culture of Saccharomyces servagi Ceb-kc-011 was prepared in the following manner:
- Saccharomyces servaji Ceb-kc-011 After adding 4% by weight of Seomoktae and 2% by weight of dextrose to 100 liters of purified water, 5 to 10 liters of Saccharomyces servaji Ceb-kc-011 were inoculated aseptically, and then incubated for 24 hours to 48 hours in a 25°C incubator. Time culture was performed to obtain a culture of Saccharomyces servage Ceb-kc-011.
- the culture was separated into a cell-containing material and a culture supernatant by centrifugation, and the culture supernatant was dried at 60° C. for 12 hours to obtain a dried yeast culture solution of Seomoktae of Example 7.
- Seomoktae powdered with 50-200 mesh (manufacturer: Digestion Farm, Yecheon-gun, Gyeongbuk) was added to 4% by weight based on the total volume of purified water, and then autoclaved at 121°C for 15 minutes. Then, after centrifugation at 3000rpm/5min, the supernatant was filtered and dried at 60°C for 12 hours to obtain a dried water-soluble protein, which was used as Comparative Example 2.
- Saccharomyces sergeant After adding YPD (1% by weight of yeast extract, 2% by weight of peptone, 2% by weight of dextrose) to 100 liters of purified water, 5 to 10 liters of Saccharomyces sergeant were inoculated aseptically, and then incubated at 25°C for 24 hours. To 48 hours to obtain a culture of Saccharomyces servage.
- YPD 1% by weight of yeast extract, 2% by weight of peptone, 2% by weight of dextrose
- the culture was separated into a cell-containing material and a culture supernatant by centrifugation, and the culture supernatant was dried at 60° C. for 12 hours to obtain a dry yeast culture solution.
- Example 7 YPD (yeast extract 1% by weight, peptone 2% by weight, dextrose 2% by weight) was used instead of dextrose in the same manner as in Example 7, and the yeast culture of Seomoktae of Comparative Example 4 Water was obtained.
- YPD yeast extract 1% by weight, peptone 2% by weight, dextrose 2% by weight
- Example 7 the lactic acid bacteria culture of Seomoktae of Comparative Example 5 was obtained in the same manner as in Example 7, except that the lactic acid bacteria Leuconostoc holzapfelii was used instead of Saccharomyces servaji Ceb-kc-011.
- Example 7 The compositions of Example 7 and Comparative Examples 2 to 5 are summarized and shown in Table 2 below:
- Weight% in the above table means the weight percentage of a specific component based on the total volume of the medium
- Each sample was dissolved in 0.1M sodium phosphate buffer (pH 6.8) to prepare 20 ⁇ l of a sample solution, reacted with 20 ⁇ l of ⁇ -glucosidase (1U/mL) at 37°C for 10 minutes, and then 20 ⁇ l substrate (5mM P-NPG) was added and reacted for 20 minutes each. Then, 50 ⁇ l of 0.1M Na 2 CO 3 was added to terminate the reaction, and the absorbance was measured at 405 nm. The degree of inhibition was calculated by [(A control -A sample )/A control ] ⁇ 100(%), where A control is the absorbance when there is no inhibitor, and A sample is the absorbance when there is an inhibitor.
- Comparative Example 4 S/R4/YPD cultured with yeast after adding Seomoktae to YPD medium exhibited the third highest inhibitory ability among all Comparative Examples, which is a high molecular protein Seomoktae, which is a source of low molecular nitrogen contained in the YPD medium.
- a high molecular protein Seomoktae which is a source of low molecular nitrogen contained in the YPD medium.
- it is difficult to use as a source of nutrient nitrogen for yeast so the degree of hydrolysis of Seomoktae is low.
- the water-soluble Seomoktae (R) of Comparative Example 2 was analyzed, but the inhibitory activity was insignificant.
- Comparative Example 3 when culturing yeast in YPD medium using yeast extracts and peptone as a nitrogen source in the absence of Seomoktae, ⁇ -glucosidase inhibitory ability was confirmed, as a result, showed the lowest inhibitory ability among all samples.
- the L/R4/D sample of Comparative Example 5 fermented with lactic acid bacteria (L. holzapfelii ) by adding Seomoktae as the sole nitrogen source showed lower inhibitory activity than the S/R4/D sample of Example 7 fermented with yeast.
- Example 7 except that starch was used instead of dextrose as a carbon source, it was carried out in the same manner as in Example 7 The yeast fermented product of Seomoktae of Example 8 was obtained.
- Example 7 except that sugar was used instead of dextrose as a carbon source, it was carried out in the same manner as in Example 7 The yeast fermented product of Seomoktae of Example 9 was obtained.
- Example 7 except that galactose was used instead of dextrose as the carbon source, it was carried out in the same manner as in Example 7 The yeast fermented product of Seomoktae of Example 10 was obtained.
- the ⁇ -glucosidase inhibitory activity of the dried supernatant obtained by adding four different types of sugars to Seomoktae and culturing them with yeast was the highest in the samples using glucose.
- Starch made of polymer showed the lowest inhibitory ability because yeast decomposes into carbon source and is not easy to use.
- Sugar, which is a disaccharide was also less efficient as a carbon source than glucose and galactose, which were monosaccharides, and glucose showed the highest effect among monosaccharides.
- Example 7 Example 11 to Example 17
- Example 7 instead of Seomoktae 4% by weight, Seomoktae 0.2% by weight (Example 11), 0.4% by weight (Example 12), 0.6% by weight (Example 13), 0.8% by weight (Example 14), 1.0
- the yeast of the Seomoktae of Examples 11 to 17 was carried out in the same manner as in Example 7, except that wt% (Example 15), 2.0 wt% (Example 16), and 3.0 wt% (Example 17) were used.
- the fermented product was prepared.
- Seo Mok-Tae's optimal amount of use was 0.6 wt%, and the carbon source glucose concentration was 1.0 wt% (Example 18, S/R0.6/D1), 2.0 wt% (Example 13, S/R0.6/D2), 3.0 wt%
- the ⁇ -glucosidase inhibitory ability of the samples prepared by differently from was analyzed.
- Example 13 in the same manner as in Example 13, except that 1.0% by weight of glucose (Example 19), or 3.0% by weight of glucose (Example 19) was used instead of 2.0% by weight of glucose, Examples 18 to The yeast fermented product of Seomoktae of Example 19 was prepared.
- the glucose content in the medium is preferably 1.5% by weight or more, or 2% by weight or more.
- ⁇ -glucosidase inhibitory ability is improved when a low-molecular nitrogen source is added to a medium containing 0.6% by weight of Seomoktae and 2% by weight of glucose.
- Example 13 as an additional low-molecular nitrogen source, 0.25% by weight of yeast extract + 0.25% by weight of peptone (Comparative Example 6, S/R0.6/Yex.0.25/Pep.0.25/D), 0.5% by weight of yeast extract (comparative) Example 7, S/R0.6/Yex.0.5/D), peptone 0.5% by weight (Comparative Example 8, S/R0.6/Pep.0.5/D), yeast extract 0.5% by weight + peptone 0.5% by weight (comparative Example 9, S/R0.6/Yex.0.5/Pep.0.5/D), yeast extract 1.0% by weight (Comparative Example 10, S/R0.6/Yex.1.0/D), peptone 1.0% by weight (Comparative Example 11, S/R0.6/Pep.1.0/D) was carried out in the same manner as in Example 13 (S/R0.6/D), except that the fermented product of Seomoktae yeast of Comparative Examples 6 to 11 Was prepared.
- ACE angiotensin I converting enzyme
- Example 13 The fermentation product of Example 13, that is, the ⁇ -glycosidase inhibitory ability of the dried supernatant obtained by culturing under optimal conditions (0.6 wt% Seomoktae, 2 wt% glucose) was measured by the method of Experimental Example 2, and ACE (angiotensin I converting enzyme) inhibitory ability and antioxidant activity (IC 50 value) were measured by the following method, and the results were respectively measured in Figs. 7 ( ⁇ -glycosidase inhibitory ability), Fig. 9 (ACE inhibitory ability), Figs. 11, 13 and 15 (antioxidant Function).
- ACE angiotensin I converting enzyme
- the degree of inhibition was calculated by [1-(S-Bs)/(C-Bi)] ⁇ 100, where S is the absorbance of the sample, Bs is the absorbance in the absence of substrate and enzyme, and Bi is the sample and enzyme. It is the absorbance when there is no, and C is the absorbance when the enzyme and substrate are included and there is no inhibitor.
- DPPH (2,2-diphenyl-1-picrylhydrazyl) radical dissolved in an organic solvent has a maximum absorbance at 515 nm. Antioxidants lose their color and become transparent by scavenging DPPH radicals.
- DPPH (2,2-diphenyl-1-picrylhydrazyl) 100 ⁇ M and a sample of the material to be treated were prepared, and the prepared sample was mixed with DPPH solution in a ratio of 1:20 and stored at 37° C. for 30 minutes. Thereafter, the sample after the reaction was measured for absorbance at 517 nm using a spectrophotometer, and ascorbic acid was used as a positive control.
- the DPPH scavenging ability was calculated by [(A control -A sample )/A control ] ⁇ 100, where A control is the absorbance when there is no scavenging material, and A sample is the absorbance when there is a scavenging material.
- ABTs+ 7mM ABTS and 2.45mM potassium persulfate were mixed, and after 16 hours incubation at room temperature, ABTs cations (ABTs+) were generated.
- a test solution was prepared by diluting so that the absorbance value at 734 nm was 0.7 or less.
- ABTs+ solution was mixed with 7mM ABTS and 2.45mM potassium persulfate at 1:1 (v/v) and incubated at room temperature for 16 hours to generate ABTs cations (ABTs+). Then, it was diluted with ethanol so that the absorbance value at 734 nm was 0.7 or less to prepare a test solution.
- ABTs+ scavenging ability was calculated by [(A control -A sample )/A control ] ⁇ 100, where A control is the absorbance when there is no scavenging material, and A sample is the absorbance when there is a scavenging material.
- IC 50 50% inhibitory concentration
- the IC 50 value of the dried culture supernatant obtained by fermenting a medium prepared with Mok-Tae Seo 0.6% by weight and 2% by weight of glucose with yeast was 29.5mg/mL for ⁇ -glucosidase inhibitory activity, 6.3mg/mL for ACE inhibitory activity, and 3 for antioxidant activity. It was found to be ⁇ 6mg/mL.
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Abstract
The present invention relates to an antidiabetic, hypotensive, or antioxidant composition comprising a yeast fermentate of Rhynchosia Nulubilis and a method for preparing the composition.
Description
본 발명은 서목태의 효모 발효물을 포함하는 혈당강하, 혈압강하 또는 항산화용 조성물 및 상기 조성물의 제조 방법에 관한 것이다.The present invention relates to a composition for lowering blood sugar, lowering blood pressure, or antioxidant comprising a fermented yeast of Seomoktae, and a method for preparing the composition.
최근 당뇨, 고혈압, 심장질환, 심혈관계 질환 등과 같은 만성질환의 유병률이 증가되면서 이에 대한 예방과 치료의 목적으로 유익 균주 및 그 생리활성 물질을 이용한 연구들이 진행되고 있다. 현대인의 산화적 스트레스의 원인인 활성산소종(Reactive Oxygen Species)이나 free radical은 체내에서 인간의 대사과정 중에 지속적으로 발생되며, 또한 불안정하고 반응성이 높아서 다른 생체 내 물질들과 쉽게 반응하게 된다. 이들은 체내 고분자들을 공격하여 세포막을 분해하고, 단백질 합성을 억제하여 돌연변이, 세포독성 및 암 등을 초래한다. Recently, as the prevalence of chronic diseases such as diabetes, hypertension, heart disease, cardiovascular disease, etc. has increased, studies using beneficial strains and their physiologically active substances are being conducted for the purpose of prevention and treatment. Reactive Oxygen Species or free radicals, which are the causes of oxidative stress in modern people, are continuously generated in the body during human metabolism, and are also unstable and highly reactive, so they easily react with other in vivo substances. They attack macromolecules in the body to degrade cell membranes, inhibit protein synthesis, and cause mutations, cytotoxicity, and cancer.
천연 생리활성 물질들(bioactive components)은 체내에 들어오면 산화를 억제하는 작용을 통해 당뇨나 암 등의 예방 기능을 가지고 있는 것으로 알려져 왔다. 따라서 현대인들에게는 산화적 스트레스를 경감시킬 수 있는 부작용이 없고 항산화 활성이 뛰어난 천연 항산화제의 개발이 필요하다. Natural bioactive components have been known to have a preventive function such as diabetes or cancer by inhibiting oxidation when they enter the body. Therefore, it is necessary for modern people to develop natural antioxidants that do not have side effects that can alleviate oxidative stress and have excellent antioxidant activity.
또한 만성질환인 당뇨병은 1970년대 발병율이 인구의 1% 미만이었던 것이 2001년 이후에는 30세 이상의 발병률이 10명 중 1명으로 10% 수준으로 증가하고 있으며, 우리나라 사망원인의 5위에 해당한다. 특히 망막증, 백내장 등 같은 합병증을 야기하므로 당뇨병 치료는 탄수화물의 소화와 흡수를 느리게 하여 식후 혈당을 저하시키면서 합병증을 예방할 수 있어야 한다. 현재 acarbose와 voglibose 등과 같은 α-glucosidase 억제제와 같은 혈당강하제는 내성과 체중증가, 부종, 오심, 소화기 장애와 같은 부작용이 우려되면서 천연 물질을 대상으로 한 혈당 개선제 개발이 요구되고 있다. In addition, the incidence rate of diabetes, which is a chronic disease, was less than 1% of the population in the 1970s. Since 2001, the incidence rate of diabetes has increased to 1 in 10 people over the age of 30, and it is the 5th cause of death in Korea. In particular, since it causes complications such as retinopathy and cataracts, diabetes treatment should be able to prevent complications while slowing the digestion and absorption of carbohydrates to lower blood sugar after meals. Currently, hypoglycemic agents such as α-glucosidase inhibitors such as acarbose and voglibose are concerned about side effects such as tolerance, weight gain, swelling, nausea, and digestive disorders, and development of blood glucose improving agents for natural substances is required.
서목태(학명: Rhynchosia Nulubilis)는 7월에 꽃이 피어 타원형의 깍지 속에 지름 5∼7mm정도의 쥐눈처럼 검고 둥근 종실이 여물면 수확하는 것으로 무농약 유기농법으로 황토에서 재배한 것이 최상의 작물이며, 유황을 뿌리고 재배하면 약성이 더욱 우수해진다. 섬유질이 많아 장의 활성에 기여하고, 서목태를 발효시키면 인체에 유용한 효소가 어느 식품보다 많은데, 그 약성에 대해서는 예로부터 속을 다스리고 관맥을 통하여 모든 약독을 제거하고, 복창을 내리고 위열을 없애며 마비증을 다스리고 어혈을 풀어내며, 콩죽을 먹으면 소갈증(당뇨로 인한 목마름)을 없애주며, 신장병을 다스리며 기를 내리어 모든 풍열을 억제하고 특히, 혈액을 활발히 하며 모든 독을 풀어주는 것이라 하여 약콩으로도 불리고 있다.Seo Mok-Tae (scientific name: Rhynchosia Nulubilis) is harvested when the black and round seeds of 5-7mm in diameter grow in oval pods with flowers blooming in July. If you sow and cultivate, the medicinal properties become more excellent. It has a lot of fiber and contributes to the intestinal activity. Fermenting Seomoktae has more enzymes useful for the human body than any other food. It is also called Yakkong because it relieves blood stagnation and relieves blood stagnation.Eating soybean porridge eliminates cattle thirst (thirst due to diabetes), controls kidney disease, reduces all wind heat, and in particular, activates blood and releases all poisons.
본 발명의 하나의 목적은 효모 및 서목태를 이용하여 부작용 없이 혈당을 저하 혹은 조절하거나 혈압을 저하시키거나 항산화 기능을 갖는 신규 조성물을 제공하는 것이다.One object of the present invention is to provide a novel composition using yeast and seomoktae to lower or control blood sugar, lower blood pressure, or have an antioxidant function without side effects.
본 발명의 다른 하나의 목적은 항산화, 항고혈압 또는 항당뇨 활성을 갖는 조성물의 제조 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preparing a composition having antioxidant, antihypertensive or antidiabetic activity.
본 발명의 일 양태는,One aspect of the present invention,
효모를, 추가의 질소원을 실질적으로 포함하지 않는 서목태 함유 배지에서 배양하고,The yeast is cultivated in a medium containing Seomoktae that does not substantially contain an additional nitrogen source,
상기 배양 후 얻어진 배양물로부터 혈당강하, 혈압강하 또는 항산화용 조성물을 제조하는 것을 포함하는, 혈당강하, 혈압강하 또는 항산화용 조성물의 제조 방법에 관한 것이다.It relates to a method for producing a composition for lowering blood sugar, lowering blood pressure, or antioxidant, comprising preparing a composition for lowering blood sugar, lowering blood pressure, or antioxidant from the culture obtained after the culture.
효모는 맥주, 포도주, 막걸리 등을 만드는 데 사용되는 미생물로서 곰팡이나 버섯 무리지만 균사가 없고 광합성능이나 운동성도 없는 단세포 생물의 총칭이다. 본원에서는 효모로 사카로마이세스 속 미생물을 이용할 수 있고, 구체적으로 사카로마이세스 서바지 또는 사카로마이세스 세레비지아에를 이용할 수 있으나 이에 제한되는 것은 아니다. 일 예에서 사카로마이세스 서바지
Saccharomyces servazzii KCCM12157P 균주가 이용될 수 있다. 상기 균주는 한국의 전통식품인 김치로부터 분리된 것이다.Yeast is a microorganism used to make beer, wine, and makgeolli, and it is a collective term for single-celled organisms that do not have hyphae and do not have photosynthetic or motility. In the present application, microorganisms of the genus Saccharomyces may be used as yeast, and specifically, Saccharomyces servage or Saccharomyces cerevisiae may be used, but is not limited thereto. In one example, Saccharomyces servazzii KCCM12157P strain may be used. The strain is isolated from kimchi, a traditional Korean food.
배양에 의해 효모는 배양물 중, 5 x 10
4 내지 5 x 10
10 CFU/ml의 농도로, 구체적으로는 효모를 5 x 10
4 내지 5 x 10
9 CFU/ml의 농도로 포함되도록 하며, 보다 구체적으로는 1 x 10
6 내지 1 x 10
9 CFU/ml의 농도로 포함되도록 한다. Yeast by culturing is to be included in a concentration of 5 x 10 4 to 5 x 10 10 CFU/ml in the culture, specifically, yeast at a concentration of 5 x 10 4 to 5 x 10 9 CFU/ml, more Specifically, it is included in a concentration of 1 x 10 6 to 1 x 10 9 CFU/ml.
효모를 배양하기 위한 배지는, 서목태를 포함하는 배지로, 서목태 외에 추가의 질소원은 실질적으로 포함하지 않는다. 추가의 질소원을 포함하는 경우, 질소원으로 고분자인 서목태 보다, 주로 저분자인 추가의 질소원을 이용하기 때문에 서목태의 효모에 의한 분해가 적게 일어나 혈당 저하, 항산화, 또는 혈압 저하를 발현하는 유효 성분이 감소하는 것으로 생각된다. 이러한 추가의 질소원으로는 N을 구조 내 포함하고 있는 것이라면 제한되지 않으며, 예를 들어 유기 질소원 또는 무기 질소원, 보다 구체적으로, YPD ((yeast ext., peptone, dextrose), 이중 효소 추출물과 펩톤이 질소원으로 사용된다), 우레아, 카제인, 대두, 밀기울, 현미유, 육류 추출물, 전지분유, 건조된 효모, 암모늄염, 트립토판, 프롤린, 아스파라긴, 글루타메이트와 같은 N 함유 아미노산 등을 들 수 있다.The medium for culturing yeast is a medium containing Seomoktae, and substantially does not contain additional nitrogen sources other than Seomoktae. In the case of including an additional nitrogen source, since an additional nitrogen source, mainly low molecular weight, is used as a nitrogen source, less decomposition by the yeast of Seomoktae than the high molecular weight Seomoktae decreases the active ingredient that expresses lowering blood sugar, antioxidants, or blood pressure. I think it is. These additional nitrogen sources are not limited as long as they contain N in the structure, for example, an organic nitrogen source or an inorganic nitrogen source, more specifically, YPD ((yeast ext., peptone, dextrose), of which enzyme extract and peptone are nitrogen sources. ), urea, casein, soybean, bran, brown rice milk, meat extract, whole milk powder, dried yeast, ammonium salt, tryptophan, proline, asparagine, and N-containing amino acids such as glutamate.
본원에서 '실질적으로 포함하지 않는'의 의미는 전체 조성물 또는 배지 전체 중량 기준, 해당 성분이 0.05 중량% 이하, 구체적으로 0.0005 중량% 이하로 포함되어 있는 것을 말한다.In the present application, the meaning of'substantially not included' refers to that the component is contained in an amount of 0.05% by weight or less, specifically 0.0005% by weight or less, based on the total weight of the total composition or medium.
서목태(일명 쥐눈이콩)는 검은콩의 일종으로 껍질이 까맣고 크기는 보통 검은콩보다 훨씬 작다. 본원에서 서목태는 효모의 질소 제공원으로 사용되며, 효모에 의한 서목태의 분해로 혈당저하, 혈압저하 또는 항산화에 효과적인 유효 펩타이드들이 제공되는 것으로 생각된다.Seomoktae (also known as rat-eyed bean) is a type of black bean, with a black skin and much smaller in size than ordinary black beans. In the present application, Seomoktae is used as a source of nitrogen for yeast, and it is believed that effective peptides effective for lowering blood sugar, lowering blood pressure, or antioxidant by decomposition of Seomoktae by yeast are provided.
배양 시, 서목태의 함량이 증가할수록 효모의 수 또한 증가하므로 서목태의 함량은 많을수록 좋지만, 배양 배지 전체 부피를 기준으로(서목태 g수 / 배지 mL수 × 100, w/v) 서목태의 함량은 0.2 중량% 내지 4 중량%가 되도록 한다. 4 중량%를 초과하면 배양시 단백질 응고에 의해 엉김 현상이 생길 수 있으므로 4 중량% 이하로 하는 것이 좋다. 보다 구체적으로 서목태 함량은 0.6 중량% 내지 1.0 중량%의 범위인 것이 가장 좋고, 0.6 중량%인 것이 또한 가장 좋다. 상기 범위에서 혈당 저하 효과가 가장 좋을 수 있다.During cultivation, as the content of Seomoktae increases, the number of yeast also increases, so the higher the content of Seomoktae, the better, but based on the total volume of the culture medium (number of grams of Seomoktae / number of ml of media × 100, w/v), the content of Seomoktae is 0.2 weight. % To 4% by weight. If it exceeds 4% by weight, coagulation may occur due to protein coagulation during cultivation, so it is recommended to be less than 4% by weight. More specifically, the Seomoktae content is best in the range of 0.6% by weight to 1.0% by weight, and it is also best that it is 0.6% by weight. In the above range, the blood sugar lowering effect may be the best.
배양의 배지로는 천연배지 또는 합성배지를 사용할 수 있다. 배지의 탄소원으로는 예를 들어, 단당류, 또는 이당류를 사용할 수 있으며, 예로는, 글루코오스, 포도당, 갈락토스, 설탕, 덱스트린, 덱스트로스, 글리세롤 등이 사용될 수 있다. 바람직하게는 탄소원으로 포도당, 글루코스와 같은 단당류를 사용하는 것이 좋으며, 단당류는 다당류 또는 이당류에 비해 탄소원으로서 이용 효율이 높아, 효모에 의한 분해가 활발하여 혈당저하, 혈압저하 또는 항산화에 효과적인 성분이 많아지기 때문이다. 상기 탄소원의 배지 중 함량은 배지 전체 부피를 기준으로 1 중량% 내지 5 중량%이고, 특히 혈당저하, 혈압저하, 또는 항산화 효과 측면에서는 1 중량% 내지 3 중량%, 또는 2 중량% 내지 3 중량%인 것이 좋다.As a culture medium, natural or synthetic medium may be used. As the carbon source of the medium, for example, monosaccharides or disaccharides may be used, and examples thereof may include glucose, glucose, galactose, sugar, dextrin, dextrose, glycerol, and the like. It is preferable to use monosaccharides such as glucose and glucose as a carbon source, and monosaccharides are more efficient as a carbon source than polysaccharides or disaccharides, and are active in decomposition by yeast, so there are many effective ingredients for lowering blood sugar, lowering blood pressure, or antioxidant. Because you lose. The content of the carbon source in the medium is 1% to 5% by weight based on the total volume of the medium, and in particular, 1% to 3% by weight, or 2% to 3% by weight in terms of lowering blood sugar, lowering blood pressure, or antioxidant effect. It is good to be.
질소원으로는 서목태를 사용하며, 서목태 외에 다른 질소원은 실질적으로 포함하지 않는다. 다른 질소원을 포함하는 경우, 혈당저하, 혈압저하, 또는 항산화 효과는 서목태만을 질소원으로 포함하여 배양된 배양물에 비해 현저히 저하될 수 있다. 배지에 포함되는 무기염으로는 마그네슘. 망간, 칼슘. 철, 칼륨 등이 사용될 수 있으나, 이들에 한정되는 것은 아니다. 상기 탄소원, 질소원 및 무기염의 성분 이외에 아미노산, 비타민, 핵산 및 그와 관련된 화합물들이 배지에 첨가될 수 있다. Seomoktae is used as a nitrogen source, and other nitrogen sources are practically not included except for Seomoktae. When other nitrogen sources are included, lowering blood sugar, lowering blood pressure, or antioxidant effects may be significantly lowered compared to cultures cultured with only Seomoktae as a nitrogen source. The inorganic salt contained in the medium is magnesium. Manganese, calcium. Iron, potassium, and the like may be used, but are not limited thereto. In addition to the components of the carbon source, nitrogen source, and inorganic salt, amino acids, vitamins, nucleic acids, and compounds related thereto may be added to the medium.
효모 균주의 배양온도 조건은 20℃내지 40℃의 온도 범위, 구체적으로는 21℃내지 30℃에서 12시간 내지 7일간, 예컨대 12시간 내지 3일간 배양할 수 있다. 일 예에서, 배양 배지에 가시오가피, 구아바, 황금, 황백, 치자, 오미자, 황련, 옥죽 등의 생약 제제, 이의 추출물, 또는 이의 혼합물을 첨가하여 배양하고, 상기 배양물을 사용함으로써 본원 발명의 조성물의 혈당 저하, 혈압 저하 또는 항산화의 효과를 보다 개선할 수 있다. 특히, 배양 배지에 가시오가피 또는 황금을 첨가하는 것이 좋으며, 첨가되는 이의 양은, 각각 배지 중량을 기준으로 0.01 중량% 내지 10 중량%, 구체적으로는 0.01 중량% 내지 5 중량% 정도가 좋다. Culture temperature conditions of the yeast strain can be cultured for 12 hours to 7 days, for example 12 hours to 3 days at a temperature range of 20 ℃ to 40 ℃, specifically 21 ℃ to 30 ℃. In one example, the composition of the present invention is obtained by adding and culturing herbal preparations such as gasiogapi, guava, golden, yellow white, gardenia, schisandra, yellow lotus, jade porridge, extracts thereof, or a mixture thereof to the culture medium, and using the culture medium. It is possible to further improve the effect of lowering blood sugar, lowering blood pressure, or antioxidant. In particular, it is preferable to add gasiogapi or gold to the culture medium, and the amount thereof added is preferably about 0.01% to 10% by weight, specifically 0.01% to 5% by weight, based on the weight of the medium, respectively.
이후, 상기 배양 후 얻어진 배양물로부터 혈당강하, 혈압강하 또는 항산화용 조성물을 제조할 수 있다. 상기 조성물 제조는 일예에서 상기 배양물에 약학적으로 혹은 식품학적으로 허용 가능한 담체를 추가로 포함하는 것일 수 있다. 이후 상기 조성물은 제제화되어 식품 혹은 의약품으로 제공될 수 있다. 또는 상기 조성물 제조는 상기 배양 후 얻어진 배양물 자체 또는 이의 배양 상등액을 조성물로 이용하는 것일 수 있다.Thereafter, a composition for lowering blood sugar, lowering blood pressure, or antioxidant may be prepared from the culture obtained after the cultivation. In one example, the preparation of the composition may be to further include a pharmaceutically or food pharmaceutically acceptable carrier in the culture. Thereafter, the composition may be formulated and provided as a food or pharmaceutical product. Alternatively, the preparation of the composition may be to use the culture itself obtained after the cultivation or a culture supernatant thereof as a composition.
본 발명에서 용어, "약학적으로 혹은 식품학적으로 허용 가능한 담체"란 생물체를 자극하지 않고 배양물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. In the present invention, the term "pharmaceutically or food pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of a culture.
상기 배양물은 효모 균체 함유 물질과 배양액을 포함한다. 상기 배양 배지에서 배양된 효모 균체를 포함하는 배양물을 원심분리하면 균체 함유 물질과 배양액으로 각각 분리할 수 있다. 따라서 배양액은 원심분리 후 균체 함유 물질을 제외한 배양 상등액이다. 균체 함유 물질은 균체를 포함하며 배양 상등액을 제거하거나 이후 잔류물을 농축한 균체 함유 성분일 수 있다. 상기 배양액 또는 균체 함유 물질의 조성은 통상의 효모 배양에 필요한 성분뿐 아니라, 효모의 생장에 상승적으로 작용하는 성분을 추가적으로 포함할 수 있으며, 이에 따른 조성은 당업계의 통상의 기술을 가진 자에 의하여 용이하게 선택될 수 있다. The culture contains a yeast cell-containing material and a culture medium. When the culture containing the yeast cells cultured in the culture medium is centrifuged, it can be separated into a cell containing material and a culture solution, respectively. Therefore, the culture medium is a culture supernatant after centrifugation, excluding cells containing substances. The cell-containing material includes cells and may be a cell-containing component obtained by removing the culture supernatant or concentrating the residue thereafter. The composition of the culture medium or the cell-containing material may additionally include a component necessary for normal yeast cultivation, as well as a component synergistically acting on the growth of yeast, and the composition according to this may be determined by a person having ordinary skill in the art. Can be easily selected.
상기 양태는 배양물로부터 배양액을 분리하는 것을 추가로 포함할 수 있다. 이 경우 상기 분리는 배양물을 원심분리하여 균체 함유 물질을 제거하는 것을 포함할 수 있다. The above aspect may further include separating the culture medium from the culture. In this case, the separation may include centrifuging the culture to remove the cell-containing material.
다음으로, 상기 배양물 또는 배양액을 농축 및/또는 건조하는 것을 추가로 포함할 수 있다. 농축 방법에는 증발농축법, 냉동농축법, 막농축법, 또는 건조농축법 등이 있으며, 구체적으로는 회전식 증발기를 사용하여 감압농축할 수 있다. 건조는 통풍건조, 자연건조, 분무건조 및 동결건조가 가능하지만, 이에 제한되는 것은 아니며, 구체적으로는 동결건조할 수 있다.Next, it may further include concentrating and/or drying the culture or culture solution. The concentration method includes an evaporative concentration method, a freeze concentration method, a membrane concentration method, or a dry concentration method, and specifically, concentration under reduced pressure may be performed using a rotary evaporator. Drying may be ventilated drying, natural drying, spray drying, and freeze drying, but is not limited thereto, and specifically, may be freeze-dried.
본 발명의 다른 양태는, 본원에 기술된 방법으로 제조된 서목태의 효모 발효물을 유효성분으로 포함하는 혈당 저하, 혈압 저하 또는 항산화용 조성물에 관한 것이다. 상기 발효물은 일 예에서 발효물 중 효모 균체 함유 물질이 제거된 발효액 또는 발효 상등액일 수 있다. Another aspect of the present invention relates to a composition for lowering blood sugar, lowering blood pressure, or antioxidant comprising the fermented yeast of Seomoktae prepared by the method described herein as an active ingredient. In one example, the fermentation product may be a fermentation broth or a fermentation supernatant from which yeast cells-containing substances are removed.
본원 발명의 조성물은 효모 발효에 의해 서목태의 약성이 변화되어 발효 전보다 현저히 개선된 혈당 저하, 혈압 저하 또는 항산화 특성을 나타내게 되며, 이는 특정 이론에 구속되는 것은 아니지만, 효모가 제공하는 여러 효소에 의해 서목태의 여러 고분자가 분해되어 유효 펩타이드가 생성되고, 이로써 혈당 저하, 혈압 저하 또는 항산화 효과가 증진되는 것으로 생각된다.The composition of the present invention exhibits remarkably improved blood sugar lowering, blood pressure lowering, or antioxidant properties than before fermentation due to the change of the medicinal properties of Seomoktae by yeast fermentation. It is thought that the various polymers of are decomposed to produce effective peptides, thereby reducing blood sugar, lowering blood pressure, or enhancing antioxidant effects.
상기 조성물은 혈당 저하, 혈압 저하, 또는 항산화를 위한 식품 조성물 또는 의약 조성물일 수 있다. 본원의 조성물은 항산화 활성으로 인해 암, 염증, 빈혈, 심근경색, 동맥경화증, 당뇨, 뇌성마비, 관절염(예:류마티즘 관절염), 파킨슨씨 병, 또는 자기면역질환 치료에 유용할 수 있다. 식품 조성물의 일 양태로는 식품, 건강식품, 건강보조식품, 또는 건강기능성 식품 조성물일 수 있다. 의약 조성물의 일 양태로는 의약외품 또는 의약적 제제일 수 있다. The composition may be a food composition or a pharmaceutical composition for lowering blood sugar, lowering blood pressure, or antioxidant. The composition of the present application may be useful for the treatment of cancer, inflammation, anemia, myocardial infarction, arteriosclerosis, diabetes, cerebral palsy, arthritis (eg, rheumatoid arthritis), Parkinson's disease, or autoimmune disease due to its antioxidant activity. One aspect of the food composition may be food, health food, health supplement food, or health functional food composition. One aspect of the pharmaceutical composition may be a quasi-drug or a pharmaceutical preparation.
일 예에서, 상기 조성물은 가시오가피, 구아바, 황금, 황백, 치자, 오미자, 황련, 옥죽 등의 생약 제제, 이의 추출물, 또는 이의 혼합물 등과 같이 항당뇨 항고혈압 또는 항산화 효과가 있는 성분을 추가로 포함할 수 있다.In one example, the composition further comprises a component having antidiabetic antihypertension or antioxidant effect, such as herbal preparations such as gasiogapi, guava, golden, yellow white, gardenia, schisandra, yellow lotus, jade porridge, extracts thereof, or mixtures thereof. I can.
본 발명의 조성물은 조성물의 고형 중량을 기준으로 1 중량% 내지 70 중량%, 구체적으로 5 중량% 내지 60 중량%의 서목태의 효모 발효물을 포함할 수 있다. The composition of the present invention may include 1% to 70% by weight, specifically 5% to 60% by weight of fermented yeast of Seomoktae based on the solid weight of the composition.
서목태의 효모 발효물은 액상상태 또는 건조상태일 수 있으며, 구체적으로는 건조된 분말 상태일 수 있다.The yeast fermentation product of Seomoktae may be in a liquid state or a dry state, and specifically may be in a dried powder state.
상기 조성물은 경구 또는 비경구의 여러가지 제형으로 제제화될 수 있으나, 바람직하게는 액상 경구 용액으로 제형화될 수 있다. The composition may be formulated in various oral or parenteral formulations, but preferably may be formulated as a liquid oral solution.
액상 용액으로 제제화되는 경우, 허용되는 약제학적 혹은 식품학적 허용 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 완충식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 액상 제형, 환약, 캡슐, 과립 또는 정제로 제제화 할 수 있다. 또한 조성물의 품질 저하를 방지하기 위하여 결착제, 유화제, 보존제 등을 추가로 첨가할 수 있고, 또는, 아미노산제, 비타민제, 효소제, 향미제, 비단백질태질소화합물, 규산염제, 완충제, 추출제, 올리고당 등을 추가로 포함할 수 있다. When formulated as a liquid solution, acceptable pharmaceutical or food acceptable carriers are sterilized and biocompatible, and include saline, sterile water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and these One or more of the components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to form liquid formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. In addition, binders, emulsifiers, preservatives, etc. may be additionally added to prevent degradation of the composition, or amino acids, vitamins, enzymes, flavors, non-protein nitrogen compounds, silicates, buffers, extractants, It may further include oligosaccharides and the like.
본 발명의 조성물을 포함하는 제형으로는, 예를 들어 정제, 트로키제, 로렌지, 수용성 또는 유성현탁액, 조제분말 또는 과립, 에멀젼, 하드 또는 소프트 캡슐, 시럽 또는 엘릭시르제를 들 수 있다. 정제 및 캡슐 등의 제형으로 제제화 하기 위해, 락토오스, 사카로오스, 솔비톨, 마니톨, 전분, 아밀로펙틴, 셀룰로오스 또는 젤라틴과 같은 결합제, 디칼슘포스페이트와 같은 부형제, 옥수수 전분 또는 고구마 전분과 같은 붕해제, 스테아르산 마그네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌글리콜 왁스와 같은 윤활제를 포함할 수 있으며, 캡슐 제형의 경우 상기 언급한 물질 외에도 지방유와 같은 액체 담체를 더 함유할 수 있다. Formulations comprising the composition of the present invention include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs. For formulation into tablets and capsules, etc., a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid A lubricant such as magnesium, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be included, and in the case of a capsule formulation, a liquid carrier such as fatty oil may be further contained in addition to the above-mentioned substances.
상기 조성물 또는 상기 서목태의 효모 발효물의 1일 투여량은 대상체의 체중, 연령, 성별, 건강상태, 치료, 예방, 또는 개선하고자 하는 주요 증상, 투여시간, 투여 방법, 중증도에 따라 달라질 수 있으나, 약 0.0001mg/kg 내지 약 10g/kg, 구체적으로는 약 0.01 mg/kg 내지 약 500mg/kg일 수 있으며, 하루 1회 내지 6회, 예컨데, 하루 1회 내지 4회 투여 혹은 적용할 수 있다. The daily dosage of the composition or the fermented yeast of Seomoktae may vary depending on the body weight, age, sex, health condition, treatment, prevention, or improvement of the subject's main symptoms, administration time, administration method, and severity. It may be from 0.0001 mg/kg to about 10 g/kg, specifically from about 0.01 mg/kg to about 500 mg/kg, and may be administered or applied once to 6 times a day, for example, 1 to 4 times a day.
본 발명의 또 다른 양태는, 본원에 기술된 방법에 따라 제조된 서목태의 효모 발효물의 치료적 또는 예방적 유효량을 당뇨 예방 또는 치료, 혈압 저하 또는 항산화를 위해 대상체에 투여하는 것을 포함하는, 당뇨 개선, 예방 또는 치료 방법, 혈압 저하 혹은 항산화 방법에 관한 것이다. 상기 치료적 또는 예방적 유효량은 전술한 바와 같이, 대상체의 체중, 연령, 성별, 건강상태, 치료, 예방, 또는 개선하고자 하는 주요 증상, 투여시간, 투여 방법, 중증도에 따라 달라질 수 있으나, 1일 약 0.0001mg/kg 내지 약 10g/kg, 구체적으로는 약 0.01 mg/kg 내지 약 500mg/kg일 수 있으며, 하루 1회 내지 6회, 예컨대, 하루 1회 내지 4회 투여 혹은 적용하는 것일 수 있다. Another aspect of the present invention is to improve diabetes, comprising administering to a subject a therapeutically or prophylactically effective amount of a fermented yeast fermented from Seomoktae prepared according to the method described herein for prevention or treatment of diabetes, lowering blood pressure or antioxidant. , Prevention or treatment methods, blood pressure lowering or antioxidant methods. As described above, the therapeutically or prophylactically effective amount may vary depending on the subject's body weight, age, sex, health condition, major symptoms to be treated, prevented, or improved, administration time, administration method, and severity, but 1 day. It may be about 0.0001 mg/kg to about 10 g/kg, specifically about 0.01 mg/kg to about 500 mg/kg, and may be administered or applied once to 6 times a day, for example, 1 to 4 times a day. .
본 발명의 방법에 따라 제조된 서목태의 효모 발효물은 α-glucosidase의 억제 능력이 우수하고 부작용이 적고 안전하여, 당뇨병의 예방 및/또는 치료에 활용 가치가 높다.The yeast fermented product of Seomoktae prepared according to the method of the present invention is excellent in the ability to inhibit α-glucosidase, has few side effects, and is safe, and has high utility value for the prevention and/or treatment of diabetes.
또한, 항산화 효능이 뛰어나므로 항산화제 및 활성산소가 과량 축적되어 유발되는 질환의 예방 또는 치료에 유용하게 사용될 수 있다.In addition, since it has excellent antioxidant efficacy, it can be usefully used for preventing or treating diseases caused by excessive accumulation of antioxidants and active oxygen.
나아가 혈압 저하 효능이 뛰어나므로 고혈압 질환의 예방 또는 치료에 유용하게 사용될 수 있다.Furthermore, since it has excellent blood pressure lowering effect, it can be usefully used in the prevention or treatment of hypertensive diseases.
도 1은 본 발명의 일 실시예 및 비교예에 따른 제조 방법에서, 배지 중 서목태의 함량에 따른 효모의 균수 변화를 나타낸 막대 그래프이다.1 is a bar graph showing a change in the number of yeast bacteria according to the content of Seomoktae in a medium in a manufacturing method according to an embodiment and a comparative example of the present invention.
도 2은 본 발명의 일 실시예 및 비교예에 따른 제조 방법에서, 각 배지 성분에 따른 α-glucosidase 저해능(%)의 차이를 보여주는 막대 그래프이다.2 is a bar graph showing the difference in α-glucosidase inhibitory ability (%) according to each medium component in a manufacturing method according to an embodiment and a comparative example of the present invention.
도 3은 본 발명의 일 실시예 및 비교예에 따른 제조 방법에서, 각 배양 배지의 탄소원(전분, 수크로스, 포도당, 갈락토스)의 변화에 따른 α-glucosidase 저해능(%)의 차이를 보여주는 막대 그래프이다.3 is a bar graph showing the difference in α-glucosidase inhibitory ability (%) according to changes in carbon sources (starch, sucrose, glucose, galactose) of each culture medium in a manufacturing method according to an embodiment and a comparative example of the present invention to be.
도 4는 본 발명의 일 실시예 및 비교예에 따른 제조 방법에서, 서목태의 함량 변화에 따른 α-glucosidase 저해능(%)의 차이를 보여주는 막대 그래프이다.4 is a bar graph showing the difference in the α-glucosidase inhibitory ability (%) according to the change in the content of Seomoktae in the manufacturing method according to an embodiment and a comparative example of the present invention.
도 5는 본 발명의 일 실시예 및 비교예에 따른 제조 방법에서, 탄소원인 포도당의 농도 변화에 따른 α-glucosidase 저해능(%)의 차이를 보여주는 막대 그래프이다.5 is a bar graph showing the difference in α-glucosidase inhibitory ability (%) according to a change in the concentration of glucose, which is a carbon source, in a manufacturing method according to an embodiment and a comparative example of the present invention.
도 6은 본 발명의 일 실시예 및 비교예에 따른 제조 방법에서, 배지에 저분자 질소원 추가에 따른 α-glucosidase 저해능(%)의 차이를 보여주는 막대 그래프이다.6 is a bar graph showing the difference in α-glucosidase inhibitory ability (%) according to the addition of a low molecular nitrogen source to a medium in a manufacturing method according to an embodiment and a comparative example of the present invention.
도 7은 본 발명의 일 실시예에 따른 방법으로 제조된 조성물(서목태 0.6 중량%, 및 포도당 2 중량% 및 효모)의 농도 변화에 따른 α-glucosidase 저해율(%)을 보여주는 그래프이다.7 is a graph showing the α-glucosidase inhibition rate (%) according to the concentration change of the composition (Seomoktae 0.6% by weight, glucose 2% by weight and yeast) prepared by the method according to an embodiment of the present invention.
도 8은 양성 대조군으로 사용된 종래 알려진 혈당 강하제, Acarbose의 농도 변화에 따른 α-glucosidase 저해율(%)을 보여주는 그래프이다.8 is a graph showing the α-glucosidase inhibition rate (%) according to the concentration change of a conventionally known blood sugar lowering agent, Acarbose, used as a positive control.
도 9는 본 발명의 일 실시예에 따른 방법으로 제조된 조성물(서목태 0.6 중량%, 및 포도당 2 중량% 및 효모)의 농도 변화에 따른 ACE 억제율(%)을 보여주는 그래프이다.9 is a graph showing the ACE inhibition rate (%) according to the concentration change of the composition (Seomoktae 0.6% by weight, glucose 2% by weight and yeast) prepared by the method according to an embodiment of the present invention.
도 10은 양성 대조군으로 사용된 종래 알려진 혈압 강하제, Lisinopril의 각 농도에 따른 ACE 억제율(%)을 보여주는 그래프이다.10 is a graph showing the ACE inhibition rate (%) according to each concentration of a conventionally known blood pressure lowering agent, Lisinopril, used as a positive control.
도 11은 본 발명의 일 실시예에 따른 방법으로 제조된 조성물(서목태 0.6 중량%, 및 포도당 2 중량% 및 효모)의 농도 변화에 따른 DPPH 라디칼 제거율(%)을 보여주는 그래프이다.11 is a graph showing the DPPH radical removal rate (%) according to the concentration change of the composition (Seomoktae 0.6% by weight, glucose 2% by weight and yeast) prepared by the method according to an embodiment of the present invention.
도 12는 양성 대조군으로 사용된 종래 알려진 항산화제, Ascorbic acid의 농도에 따른 DPPH 라디칼 제거율(%)을 보여주는 그래프이다.12 is a graph showing the DPPH radical removal rate (%) according to the concentration of ascorbic acid, a conventionally known antioxidant used as a positive control.
도 13은 본 발명의 일 실시예에 따른 방법으로 제조된 조성물(서목태 0.6 중량%, 및 포도당 2 중량% 및 효모)의 농도 변화에 따른 ABTS 라디칼 제거율(%)을 보여주는 그래프이다.13 is a graph showing the ABTS radical removal rate (%) according to the concentration change of the composition (Seomoktae 0.6% by weight, glucose 2% by weight and yeast) prepared by the method according to an embodiment of the present invention.
도 14는 양성 대조군으로 사용된 종래 알려진 항산화제, Ascorbic acid의 각 농도에 따른 ABTS 라디칼 제거율(%)을 보여주는 그래프이다.14 is a graph showing the ABTS radical removal rate (%) according to each concentration of a conventionally known antioxidant, ascorbic acid, used as a positive control.
도 15는 본 발명의 일 실시예에 따른 방법으로 제조된 조성물(서목태 0.6 중량%, 및 포도당 2 중량% 및 효모)과 양성 대조군으로 사용된 종래 알려진 항산화제, Ascorbic acid의 농도 변화에 따른 환원력(reducing power)의 차이를 보여주는 그래프이다.15 is a reducing power according to the concentration change of the composition (Seomoktae 0.6% by weight, glucose 2% by weight and yeast) prepared by the method according to an embodiment of the present invention and a conventionally known antioxidant used as a positive control, ascorbic acid ( reducing power).
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 그러나, 이들 실시 예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시 예에 국한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
1. 발효에 필요한 서목태 적정량1. Proper amount of Seomoktae required for fermentation
실시예 1: 서목태의 효모 발효물 1의 제조Example 1: Preparation of fermented yeast 1 of Seomoktae
(1) 시료 확보 및 균주 분리(1) Sample acquisition and strain isolation
한국의 전통식품인 김치를 채취하고, 확보된 시료를 단계 희석하여 1% 염화나트륨이 첨가된 YPD(Yeast extract Peptone Dextrose)에 도말 후 37℃ 조건으로 24 시간 동안 배양하였다. 상기 시료에서 우점한 균주를 분리하였다. 선별된 콜로니는 3차에 걸쳐 새로운 배지에 옮겨 배양하는 방법으로 순수 분리하였으며, 순수 배양된 균을 20% 글리세롤이 첨가된 배지에 담아 영하 70℃이하에서 보존하였다.Kimchi, a traditional Korean food, was collected, and the obtained sample was diluted in stages, spread on YPD (Yeast extract Peptone Dextrose) to which 1% sodium chloride was added, and incubated for 24 hours at 37°C. The dominant strain was isolated from the sample. The selected colonies were purely separated by transferring to a new medium for 3 times and culturing, and the pure cultured bacteria were stored in a medium to which 20% glycerol was added and stored at -70°C or below.
(2) 분류학적 특성 조사(2) Taxonomic characteristics investigation
상기 분리된 균주의 동정을 위하여, 분류학적 특성을 분석하기 위하여 18s rRNA partial sequencing에 의해 분석을 실시하였으며, 그 결과 서열번호 1의 염기서열을 가지며, 당해 분리 균주는 사카로마이세스서바지(
Saccharomyces servazzii)와 99%의 상동성을 갖는 것으로 확인되었다.For identification of the isolated strain, analysis was performed by 18s rRNA partial sequencing to analyze taxonomic characteristics, and as a result, it had the nucleotide sequence of SEQ ID NO: 1, and the isolated strain was Saccharomyces servazzii ) and 99% homology.
이에 본 발명자들은 신규 분리한 사카로마이세스서바지 Ceb-kc-011을 2017년 11월 10일 한국 미생물보존센터(Korean Culture Center of Microorganisms, 서울시 서대문구 홍제-2가-길 120-861)에 기탁번호 KCCM12157P로 기탁하였다. Accordingly, the present inventors deposited the newly isolated Saccharomyces Servoji Ceb-kc-011 to the Korean Culture Center of Microorganisms (120-861 Hongje-2ga-gil, Seodaemun-gu, Seoul) on November 10, 2017. Deposited under the number KCCM12157P.
(3) 서목태의 효모 발효물의 제조 (3) Preparation of fermented yeast of Seomoktae
사카로마이세스서바지 Ceb-kc-011의 배양물을 다음과 같은 방법으로 제조하였다:A culture of Saccharomyces servagi Ceb-kc-011 was prepared in the following manner:
정제수 100리터에, 서목태 4 중량%, yeast extract 및 펩톤 제거된 YPD 배지 (덱스트로스 2 중량% 함유, 덱스트로스는 포도당의 일종으로 본원에서는 호환되어 사용됨)를 각각 첨가 후, 사카로마이세스 서바지 Ceb-kc-011을 무균 상태에서 0.2~0.4 리터 접종한 다음 25℃ 배양기에서 24시간 내지 48시간 배양하여 사카로마이세스 서바지 Ceb-kc-011의 배양물을 수득하였다.To 100 liters of purified water, 4% by weight of Seomoktae, yeast extract and peptone-removed YPD medium (containing 2% by weight of dextrose, dextrose is a type of glucose and are used interchangeably herein), respectively, and then Saccharomyces Serbage Ceb-kc-011 was inoculated with 0.2 to 0.4 liters aseptically and then cultured in an incubator at 25° C. for 24 to 48 hours to obtain a culture of Saccharomyces servaji Ceb-kc-011.
이후, 상기 배양물을 원심분리하여 균체 함유 물질과 배양액으로 각각 분리하고, 상기 배양액을 60℃에서 12시간 건조하여 서목태의 효모 발효액 건조물을 수득하였다.Thereafter, the culture was separated into a cell-containing material and a culture solution by centrifugation, and the culture solution was dried at 60° C. for 12 hours to obtain a dried yeast fermentation broth of Seomoktae.
실시예 2: 서목태의 효모 발효물 2의 제조Example 2: Preparation of fermented yeast 2 of Seomoktae
상기 실시예 1에서 사카로마이세스서바지 Ceb-kc-011 대신, 사카로마이세스서바지를 사용한 것을 제외하고는 실시예 1과 동일하게 실시하여 서목태의 효모 발효물 2를 수득하였다.In Example 1, instead of Saccharomyces servo Ceb-kc-011, Saccharomyces servo was used in the same manner as in Example 1 to obtain fermented yeast 2 of Seomoktae.
실시예 3: 서목태의 효모 발효물 3의 제조Example 3: Preparation of fermented yeast 3 of Seomoktae
상기 실시예 2에서 서목태를 4 중량%가 아닌 2 중량%가 되는 양으로 첨가한 것을 제외하고는 실시예 2와 동일하게 실시하여 실시예 3의 서목태의 효모 발효물 3을 수득하였다.In Example 2, the yeast fermented product 3 of Seomoktae of Example 3 was obtained in the same manner as in Example 2, except that Seomoktae was added in an amount of 2% by weight instead of 4% by weight.
실시예 4: 서목태의 효모 발효물 4의 제조Example 4: Preparation of fermented yeast 4 of Seomoktae
상기 실시예 2에서 서목태를 4 중량%가 아닌 2.5 중량%가 되는 양으로 첨가한 것을 제외하고는 실시예 2와 동일하게 실시하여 실시예 4의 서목태의 효모 발효물 4을 수득하였다.In Example 2, the yeast fermented product 4 of Seomoktae of Example 4 was obtained in the same manner as in Example 2, except that Seomoktae was added in an amount of 2.5% by weight instead of 4% by weight.
실시예 5: 서목태의 효모 발효물 5의 제조Example 5: Preparation of fermented yeast 5 of Seomoktae
상기 실시예 2에서 서목태를 4 중량%가 아닌 3 중량%가 되는 양으로 첨가한 것을 제외하고는 실시예 2와 동일하게 실시하여 실시예 5의 서목태의 효모 발효물 5을 수득하였다.The yeast fermented product 5 of Seomoktae of Example 5 was obtained in the same manner as in Example 2, except that Seomoktae was added in an amount of 3% by weight instead of 4% by weight in Example 2.
실시예 6: 서목태의 효모 발효물 6의 제조Example 6: Preparation of fermented yeast 6 of Seomoktae
상기 실시예 2에서 서목태를 4 중량%가 아닌 3.5 중량%가 되는 양으로 첨가한 것을 제외하고는 실시예 2와 동일하게 실시하여 실시예 6의 서목태의 효모 발효물 6을 수득하였다.In Example 2, the yeast fermented product 6 of Seomoktae of Example 6 was obtained in the same manner as in Example 2, except that Seomoktae was added in an amount of 3.5% by weight instead of 4% by weight.
비교예 1: 효모 발효물 1의 제조Comparative Example 1: Preparation of yeast fermented product 1
상기 실시예 2에서 서목태를 첨가하지 않고, yeast extract 및 펩톤 제거된 YPD 배지 대신, yeast extract 1.0 중량%, 펩톤 2.0 중량%, 및 덱스트로스 2.0 중량% 로 함유하는 YPD 배지를 사용한 것을 제외하고는 실시예 2와 동일하게 실시하여 비교예 1의 효모 발효물 1을 수득하였다.Except for using the YPD medium containing 1.0% by weight of yeast extract, 2.0% by weight of peptone, and 2.0% by weight of dextrose, instead of the YPD medium from which Seomoktae was not added and yeast extract and peptone were removed in Example 2 In the same manner as in Example 2, fermented yeast 1 of Comparative Example 1 was obtained.
실험예 1: 효모 생균수 검사Experimental Example 1: Yeast viable cell count test
효모 생균수 측정 방법은 YPD 배지에 2% agar를 첨가하여 평판을 준비하고, 0.85% 멸균생리식염수로 단계 희석한 상기 실시예 1 내지 6 및 비교예 1의 제조 과정 중에서 배양하여 얻은 배양물 시료를 100㎕씩 접종 및 도말하여 25℃ 배양기에서 24~48시간 배양하였다. 배양 후 집락을 계수하여 표 1과 도 1에 나타내었다.In the method for measuring the number of viable yeast cells, a plate was prepared by adding 2% agar to YPD medium, and culture samples obtained by culturing in the manufacturing process of Examples 1 to 6 and Comparative Example 1 step-diluted with 0.85% sterile physiological saline were used. Inoculated and smeared at a time of 100 µl, and cultured for 24 to 48 hours in a 25°C incubator. After cultivation, colonies were counted and shown in Table 1 and FIG. 1.
[표 1][Table 1]
(상기 표에서 중량%는 배지 전체 부피 기준 특정 성분의 중량 백분율을 의미한다)(Weight% in the above table means the weight percentage of a specific component based on the total volume of the medium)
효모 배양 배지인 YPD 조성을 토대로 YPD 내 질소원인 yeast extracts 및 peptone을 배제 후 대체 질소원으로서 서목태 함량을 달리하여 효모를 배양시 서목태와 포도당으로 제조된 배지에서 서목태 함량이 증가할수록 효모 수 또한 증가하였다. 상기 실시예와는 별도로 서목태 첨가량을 4 중량% 초과로 하여 배양해 보았으며, 이 경우 단백질 응고에 의한 엉김 현상이 나타났다. 따라서 서목태 적정량은 4 중량% 이하라 할 수 있다.Based on the composition of YPD, which is a yeast culture medium, yeast extracts and peptones, which are nitrogen sources in YPD, were excluded, and the content of Seomoktae as an alternative nitrogen source was changed.When culturing yeast, the number of yeasts increased as the content of Seomoktae increased in the media prepared with Seomoktae and glucose. Separately from the above example, cultivation was performed with the addition amount of Seomoktae exceeding 4% by weight, and in this case, a lumping phenomenon due to protein coagulation was observed. Therefore, the appropriate amount of Seomoktae can be said to be 4% by weight or less.
2. 배지 조성에 따른 배양액 건조물의 α-글루코시다제 저해능2. α-glucosidase inhibitory ability of the dried culture medium according to the composition of the medium
실시예 7Example 7
사카로마이세스서바지 Ceb-kc-011의 배양물을 다음과 같은 방법으로 제조하였다:A culture of Saccharomyces servagi Ceb-kc-011 was prepared in the following manner:
정제수 100리터에 서목태 4 중량%, 덱스트로스 2 중량%가 되도록 각각 첨가 후, 사카로마이세스 서바지 Ceb-kc-011을 무균 상태에서 5~10 리터 접종한 다음 25℃ 배양기에서 24시간 내지 48시간 배양하여 사카로마이세스 서바지 Ceb-kc-011의 배양물을 수득하였다.After adding 4% by weight of Seomoktae and 2% by weight of dextrose to 100 liters of purified water, 5 to 10 liters of Saccharomyces servaji Ceb-kc-011 were inoculated aseptically, and then incubated for 24 hours to 48 hours in a 25°C incubator. Time culture was performed to obtain a culture of Saccharomyces servage Ceb-kc-011.
이후, 상기 배양물을 원심분리하여 균체 함유 물질과 배양 상등액으로 각각 분리하고, 상기 배양 상등액을 60℃에서 12시간 건조하여 실시예 7의 서목태의 효모 배양액 건조물을 수득하였다.Thereafter, the culture was separated into a cell-containing material and a culture supernatant by centrifugation, and the culture supernatant was dried at 60° C. for 12 hours to obtain a dried yeast culture solution of Seomoktae of Example 7.
비교예 2: 서목태 미배양 수용성 단백질 건조물Comparative Example 2: Seomoktae uncultured water-soluble protein dried product
50~200mesh로 분말화시킨 서목태(제조원: 경북 예천군 소화농장)를 정제수 전체 부피에 대하여 4중량%가 되도록 첨가한 후 121℃에서 15분간 습열멸균(autoclave)하였다. 그 다음 3000rpm/5min 원심분리 후 상등액을 여과하였고, 60℃에서 12시간 건조하여 수용성 단백질 건조물을 수득하고 이를 비교예 2로 사용하였다.Seomoktae powdered with 50-200 mesh (manufacturer: Digestion Farm, Yecheon-gun, Gyeongbuk) was added to 4% by weight based on the total volume of purified water, and then autoclaved at 121°C for 15 minutes. Then, after centrifugation at 3000rpm/5min, the supernatant was filtered and dried at 60°C for 12 hours to obtain a dried water-soluble protein, which was used as Comparative Example 2.
비교예 3: 효모의 배양물Comparative Example 3: Yeast culture
정제수 100리터에 YPD(효모 추출물 1 중량%, 펩톤 2 중량%, 덱스트로스 2 중량%)를 첨가 후, 사카로마이세스 서바지를 무균 상태에서 5~10 리터 접종한 다음 25℃ 배양기에서 24시간 내지 48시간 배양하여 사카로마이세스 서바지의 배양물을 수득하였다.After adding YPD (1% by weight of yeast extract, 2% by weight of peptone, 2% by weight of dextrose) to 100 liters of purified water, 5 to 10 liters of Saccharomyces sergeant were inoculated aseptically, and then incubated at 25°C for 24 hours. To 48 hours to obtain a culture of Saccharomyces servage.
이후, 상기 배양물을 원심분리하여 균체 함유 물질과 배양 상등액으로 각각 분리하고, 상기 배양 상등액을 60℃에서 12시간 건조하여 효모 배양액 건조물을 수득하였다.Thereafter, the culture was separated into a cell-containing material and a culture supernatant by centrifugation, and the culture supernatant was dried at 60° C. for 12 hours to obtain a dry yeast culture solution.
비교예 4: 서목태의 효모 배양물Comparative Example 4: Yeast culture of Seomoktae
상기 실시예 7에서, 덱스트로스 대신 YPD (효모 추출물 1 중량%, 펩톤 2 중량%, 덱스트로스 2 중량%)를 사용한 것을 제외하고는 실시예 7과 동일하게 실시하여 비교예 4의 서목태의 효모 배양물을 수득하였다.In Example 7, YPD (yeast extract 1% by weight, peptone 2% by weight, dextrose 2% by weight) was used instead of dextrose in the same manner as in Example 7, and the yeast culture of Seomoktae of Comparative Example 4 Water was obtained.
비교예 5: 서목태의 유산균 배양물Comparative Example 5: Lactobacillus culture of Seomoktae
상기 실시예 7에서, 사카로마이세스서바지 Ceb-kc-011 대신 유산균인
Leuconostoc holzapfelii를 사용한 것을 제외하고는 실시예 7과 동일하게 실시하여 비교예 5의 서목태의 유산균 배양물을 수득하였다.In Example 7, the lactic acid bacteria culture of Seomoktae of Comparative Example 5 was obtained in the same manner as in Example 7, except that the lactic acid bacteria Leuconostoc holzapfelii was used instead of Saccharomyces servaji Ceb-kc-011.
상기 실시예 7 및 비교예 2 내지 5의 조성을 아래 표 2에 정리하여 나타내었다:The compositions of Example 7 and Comparative Examples 2 to 5 are summarized and shown in Table 2 below:
[표 2][Table 2]
(상기 표에서 중량%는 배지 전체 부피 기준 특정 성분의 중량 백분율을 의미한다)(Weight% in the above table means the weight percentage of a specific component based on the total volume of the medium)
실험예 2: α-글리코시다제 저해능 검사Experimental Example 2: α-glycosidase inhibitory ability test
상기 제조된 실시예 7 및 비교예 2 내지 비교예 5의 발효물의 α-glucosidase 저해능을 다음과 같은 방법으로 측정하였다:The α-glucosidase inhibitory ability of the fermented products of Example 7 and Comparative Examples 2 to 5 prepared above was measured by the following method:
[α-glucosidase 저해능] [α-glucosidase inhibitory ability]
각 시료를 0.1M sodium phosphate buffer(pH 6.8)에 용해시켜 시료 용액 20㎕를 제조하고, 이를 α-glucosidase(1U/mL) 20㎕와 함께 37℃에서 10분간 반응시킨 후, 여기에 20㎕ 기질 (5mM P-NPG)을 넣어 각각 20분 동안 반응시킨다. 그리고 0.1M Na
2CO
3 50㎕를 넣어 반응을 종료시킨 후 405nm에서 흡광도를 측정하였다. 저해도는 [(A
control-A
sample)/A
control]×100(%)에 의해 산출하였으며, 여기서 A
control은 저해물질이 없을 경우의 흡광도이고, A
sample은 저해물질이 있을 경우의 흡광도이다.Each sample was dissolved in 0.1M sodium phosphate buffer (pH 6.8) to prepare 20 µl of a sample solution, reacted with 20 µl of α-glucosidase (1U/mL) at 37°C for 10 minutes, and then 20µl substrate (5mM P-NPG) was added and reacted for 20 minutes each. Then, 50 µl of 0.1M Na 2 CO 3 was added to terminate the reaction, and the absorbance was measured at 405 nm. The degree of inhibition was calculated by [(A control -A sample )/A control ]×100(%), where A control is the absorbance when there is no inhibitor, and A sample is the absorbance when there is an inhibitor.
[결과][result]
동일 농도 (50mg/mL)로 처리한 시료의 α-glucosidase 저해능 분석 결과 서목태와 포도당(덱스트로스)으로 제조된 배지를 효모로 발효한 실시예 7의 S/R4/D에서 가장 높은 α-glucosidase 저해능을 나타냈다. S/R4/D 시료의 경우 서목태가 유일한 질소원으로 사용되었기 때문에 효모에 의한 가수분해 효율이 증가된 것으로 판단되었다. YPD 배지에 서목태 첨가 후 효모로 배양한 비교예 4의 시료(S/R4/YPD)는 전체 비교예 중 세 번째로 높은 저해능을 나타냈으며, 이는 고분자 단백질인 서목태가 YPD 배지 내에 포함된 저분자 질소원인 yeast extracts 및 peptone보다 상대적으로 효모의 영양 질소원으로 이용되기 어려워 서목태의 가수분해 정도가 낮아 파생된 결과라고 판단된다. 발효 전 서목태의 α-glucosidase 저해능 효과를 확인하기 위하여 비교예 2의 수용성 서목태(R)를 분석하였으나 저해능은 미미하였다. 또한, 비교예 3에서는 서목태가 없는 상태에서 yeast extracts 및 peptone을 질소원으로 하는 YPD 배지에서 효모 배양 시 α-glucosidase 저해능을 확인한 결과 전체 시료 중 가장 낮은 저해능을 나타냈다. 유일한 질소원으로서 서목태를 첨가하여 유산균 (
L. holzapfelii)으로 발효한 비교예 5의 L/R4/D 시료는 효모로 발효한 실시예 7의 S/R4/D 시료보다 낮은 저해능을 나타냈다.As a result of analysis of α-glucosidase inhibitory ability of samples treated with the same concentration (50mg/mL), the highest α-glucosidase inhibitory activity in S/R4/D of Example 7 fermented with yeast using a medium prepared with Seomoktae and glucose (dextrose) Indicated. In the case of S/R4/D samples, it was judged that the hydrolysis efficiency by yeast was increased because Seomoktae was used as the only nitrogen source. The sample of Comparative Example 4 (S/R4/YPD) cultured with yeast after adding Seomoktae to YPD medium exhibited the third highest inhibitory ability among all Comparative Examples, which is a high molecular protein Seomoktae, which is a source of low molecular nitrogen contained in the YPD medium. Compared to yeast extracts and peptones, it is difficult to use as a source of nutrient nitrogen for yeast, so the degree of hydrolysis of Seomoktae is low. In order to confirm the α-glucosidase inhibitory effect of Seomoktae before fermentation, the water-soluble Seomoktae (R) of Comparative Example 2 was analyzed, but the inhibitory activity was insignificant. In addition, in Comparative Example 3, when culturing yeast in YPD medium using yeast extracts and peptone as a nitrogen source in the absence of Seomoktae, α-glucosidase inhibitory ability was confirmed, as a result, showed the lowest inhibitory ability among all samples. The L/R4/D sample of Comparative Example 5 fermented with lactic acid bacteria (L. holzapfelii ) by adding Seomoktae as the sole nitrogen source showed lower inhibitory activity than the S/R4/D sample of Example 7 fermented with yeast.
상기 결과는 도 2에 나타내었다.The results are shown in FIG. 2.
3. 당 종류에 따른 α-글루코시다제 저해능3. α-glucosidase inhibitory ability according to sugar type
실시예 8Example 8
상기 실시예 7에 있어서, 탄소원으로 덱스트로스 대신 전분을 사용한 것을 제외하고는 실시예 7과 동일하게 실시하여
실시예 8의 서목태의 효모 발효물을 수득하였다.In Example 7, except that starch was used instead of dextrose as a carbon source, it was carried out in the same manner as in Example 7 The yeast fermented product of Seomoktae of Example 8 was obtained.
실시예 9Example 9
상기 실시예 7에 있어서, 탄소원으로 덱스트로스 대신 설탕을 사용한 것을 제외하고는 실시예 7과 동일하게 실시하여
실시예 9의 서목태의 효모 발효물을 수득하였다.In Example 7, except that sugar was used instead of dextrose as a carbon source, it was carried out in the same manner as in Example 7 The yeast fermented product of Seomoktae of Example 9 was obtained.
실시예 10Example 10
상기 실시예 7에 있어서, 탄소원으로 덱스트로스 대신 갈락토스를 사용한 것을 제외하고는 실시예 7과 동일하게 실시하여
실시예 10의 서목태의 효모 발효물을 수득하였다.In Example 7, except that galactose was used instead of dextrose as the carbon source, it was carried out in the same manner as in Example 7 The yeast fermented product of Seomoktae of Example 10 was obtained.
상기 실시예 8 내지 10의 발효물 및 실시예 7의 발효물에 대해 상기 실험예 2에 기재된 방법으로 α-글리코시다제 저해능을 측정하고 그 결과를 도 3에 나타내었다.For the fermented products of Examples 8 to 10 and the fermented products of Example 7, α-glycosidase inhibitory ability was measured by the method described in Experimental Example 2, and the results are shown in FIG. 3.
[결과][result]
종류가 다른 4종의 당을 각각 서목태에 첨가 후 효모로 배양하여 얻은 상등액 건조물의 α-glucosidase 저해능은 포도당을 사용한 시료에서 가장 높게 나타났다. 고분자로 이루어진 전분은 효모가 탄소원으로 분해하여 사용하기 쉽지않아 가장 낮은 저해능이 나타났다. 이당류인 설탕도 단당류인 포도당 및 갈락토스에 비하여 탄소원으로서의 이용 효율이 낮았고, 단당류 중에서는 포도당이 가장 높은 효과를 나타냈다.The α-glucosidase inhibitory activity of the dried supernatant obtained by adding four different types of sugars to Seomoktae and culturing them with yeast was the highest in the samples using glucose. Starch made of polymer showed the lowest inhibitory ability because yeast decomposes into carbon source and is not easy to use. Sugar, which is a disaccharide, was also less efficient as a carbon source than glucose and galactose, which were monosaccharides, and glucose showed the highest effect among monosaccharides.
4. 포도당에 서목태의 함량을 달리하여 배양한 시료의 α-글루코시다제 저해능4. α-glucosidase inhibitory ability of samples cultured with different amounts of Seomoktae in glucose
최적 탄소원인 포도당에 서목태 사용량을 달리하여 배양 후 α-glucosidase 저해능이 가장 높은 서목태 최소량을 결정하기 위하여 0.2중량%, 0.4 중량%, 0.6 중량%, 0.8 중량%, 1.0 중량%, 2.0 중량%, 3.0 중량%, 4.0 중량%의 다양한 조건으로 배양하여 시료를 제조하였다.In order to determine the minimum amount of Seomoktae with the highest α-glucosidase inhibitory ability after cultivation by varying the amount of Seomoktae in the optimal carbon source, glucose, 0.2% by weight, 0.4% by weight, 0.6% by weight, 0.8% by weight, 1.0% by weight, 2.0% by weight, 3.0 Samples were prepared by culturing under various conditions of wt% and 4.0 wt%.
실시예 7, 실시예 11 내지 실시예 17Example 7, Example 11 to Example 17
상기 실시예 7에 있어서, 서목태 4 중량% 대신 서목태 0.2 중량%(실시예 11), 0.4 중량%(실시예 12), 0.6 중량%(실시예 13), 0.8 중량%(실시예 14), 1.0 중량%(실시예 15), 2.0 중량%(실시예 16), 3.0 중량%(실시예 17)를 사용한 것을 제외하고는 실시예 7과 동일하게 실시하여 실시예 11 내지 실시예 17의 서목태의 효모 발효물을 제조하였다.In Example 7, instead of Seomoktae 4% by weight, Seomoktae 0.2% by weight (Example 11), 0.4% by weight (Example 12), 0.6% by weight (Example 13), 0.8% by weight (Example 14), 1.0 The yeast of the Seomoktae of Examples 11 to 17 was carried out in the same manner as in Example 7, except that wt% (Example 15), 2.0 wt% (Example 16), and 3.0 wt% (Example 17) were used. The fermented product was prepared.
상기 실시예 7 및 실시예 11 내지 17의 발효물에 대해 상기 실험예 2에 기재된 방법으로 α-글리코시다제 저해능을 측정하고 그 결과를 도 4에 나타내었다.For the fermented products of Example 7 and Examples 11 to 17, α-glycosidase inhibitory ability was measured by the method described in Experimental Example 2, and the results are shown in FIG. 4.
[결과][result]
탄소원인 포도당 함량을 2중량%로 고정하고 질소원인 서목태 첨가량을 달리하여 제조한 시료의 α-glucosidase 저해능을 분석한 결과 서목태를 0.6중량% 처리시 가장 높은 저해활성을 나타냈다. 서목태 사용량이 0.6~1.0 중량%까지는 유의적으로 동일한 높은 저해능을 나타내었으나 2중량% 이상에서는 오히려 저해능이 낮아졌다. 그 이유는 효모의 생장에 충분하고도 남는 과잉의 질소원이 공급되어 가수분해되지 않고 남아있는 고분자 단백질 함량 비율이 상대적으로 높아 1 중량% 이하에서 제조한 시료보다 낮은 저해능을 나타낸 것으로 판단된다. 따라서, 저해능이 가장 높으면서 경제적으로도 합리적 서목태 사용 최소량은 0.6중량%가 적절하였다.As a result of analyzing the α-glucosidase inhibitory ability of the samples prepared by fixing the carbon source glucose content at 2% by weight and varying the amount of the nitrogen source Seomoktae added, the highest inhibitory activity was shown when Seomoktae was treated with 0.6% by weight. The amount of Seomoktae used up to 0.6 to 1.0% by weight showed significantly the same high inhibitory ability, but the inhibitory ability was rather lowered at 2% by weight or more. The reason for this is that an excess nitrogen source sufficient for the growth of yeast is supplied, so that the proportion of the polymer protein content remaining without hydrolysis is relatively high, and it is judged that the inhibitory activity is lower than that of the sample prepared at 1% by weight or less. Therefore, the minimum amount of use of Seomoktae, which has the highest inhibitory ability and is economically reasonable, was 0.6% by weight.
5. 서목태에 포도당의 함량을 달리하여 배양한 시료의 α-글루코시다제 저해능5. α-glucosidase inhibitory ability of samples cultured with different amounts of glucose in Seomoktae
서목태 최적 사용량인 0.6중량%에 탄소원인 포도당 농도를 1.0중량%(실시예 18, S/R0.6/D1), 2.0중량%(실시예 13, S/R0.6/D2), 3.0중량%(실시예 19, S/R0.6/D3)로 달리하여 제조한 시료의 α-glucosidase 저해능을 분석하였다.Seo Mok-Tae's optimal amount of use was 0.6 wt%, and the carbon source glucose concentration was 1.0 wt% (Example 18, S/R0.6/D1), 2.0 wt% (Example 13, S/R0.6/D2), 3.0 wt% The α-glucosidase inhibitory ability of the samples prepared by differently from (Example 19, S/R0.6/D3) was analyzed.
실시예 18 및 실시예 19 Example 18 and Example 19
상기 실시예 13에 있어서, 포도당 2.0 중량% 대신 포도당 1.0 중량%(실시예 19), 또는 포도당 3.0 중량%(실시예 19)을 사용한 것을 제외하고는 실시예 13과 동일하게 실시하여 실시예 18 내지 실시예 19의 서목태의 효모 발효물을 제조하였다.In Example 13, in the same manner as in Example 13, except that 1.0% by weight of glucose (Example 19), or 3.0% by weight of glucose (Example 19) was used instead of 2.0% by weight of glucose, Examples 18 to The yeast fermented product of Seomoktae of Example 19 was prepared.
상기 실시예 13, 실시예 18 내지 19의 발효물에 대해 상기 실험예 2에 기재된 방법으로 α-글리코시다제 저해능을 측정하고 그 결과를 도 5에 나타내었다.For the fermented products of Example 13 and Examples 18 to 19, α-glycosidase inhibitory ability was measured by the method described in Experimental Example 2, and the results are shown in FIG. 5.
[결과][result]
유일한 질소원으로서 서목태의 최적 사용량을 0.6 중량%로 고정 후 탄소원인 포도당의 사용량을 달리하여 효모로 배양한 시료의 α-glucosidase 저해능은 포도당이 2중량%일 때 가장 높게 나타났다. 3중량%인 경우도 2 중량%와 비슷한 결과가 얻어진 것으로부터 볼 때, 배지 내 포도당 함량은 1.5 중량% 이상, 또는 2 중량% 이상인 것이 바람직함을 알 수 있다.After fixing the optimal amount of Seomoktae as the only nitrogen source to 0.6% by weight, the α-glucosidase inhibitory activity of the sample cultured with yeast by varying the amount of glucose, which is a carbon source, was highest when glucose was 2% by weight. In the case of 3% by weight, it can be seen from that a result similar to that of 2% by weight is obtained, it can be seen that the glucose content in the medium is preferably 1.5% by weight or more, or 2% by weight or more.
6. 서목태 0.6중량%, 포도당 2중량%인 배지에 저분자 질소원을 추가로 투입 시 α-glucosidase 저해능 개선 여부6. Whether or not the α-glucosidase inhibitory ability is improved when a low-molecular nitrogen source is added to a medium containing 0.6% by weight of Seomoktae and 2% by weight of glucose.
서목태 이외에 추가 저분자 질소원을 투입 시 α-glucosidase 저해능이 개선되는지 여부를 확인하기 위하여 yeast extracts 및 peptone 조성을 아래와 같이 조합하여 실험하였다.In order to check whether the α-glucosidase inhibitory ability was improved when an additional low-molecular nitrogen source was added in addition to Seo Mok-tae, the composition of yeast extracts and peptone were combined and tested as follows.
비교예 6 내지 비교예 11Comparative Examples 6 to 11
상기 실시예 13에 있어서 추가의 저분자 질소원으로 효모 추출물 0.25 중량% + 펩톤 0.25 중량%(비교예 6, S/R0.6/Yex.0.25/Pep.0.25/D), 효모 추출물 0.5 중량%(비교예 7, S/R0.6/Yex.0.5/D), 펩톤 0.5 중량% (비교예 8, S/R0.6/Pep.0.5/D), 효모 추출물 0.5 중량% + 펩톤 0.5 중량%(비교예 9, S/R0.6/Yex.0.5/Pep.0.5/D), 효모 추출물 1.0 중량%(비교예 10, S/R0.6/Yex.1.0/D), 펩톤 1.0 중량% (비교예 11, S/R0.6/Pep.1.0/D)를 추가한 것을 제외하고는 실시예 13(S/R0.6/D)와 동일하게 실시하여 비교예 6 내지 비교예 11의 서목태 효모 발효물을 제조하였다.In Example 13, as an additional low-molecular nitrogen source, 0.25% by weight of yeast extract + 0.25% by weight of peptone (Comparative Example 6, S/R0.6/Yex.0.25/Pep.0.25/D), 0.5% by weight of yeast extract (comparative) Example 7, S/R0.6/Yex.0.5/D), peptone 0.5% by weight (Comparative Example 8, S/R0.6/Pep.0.5/D), yeast extract 0.5% by weight + peptone 0.5% by weight (comparative Example 9, S/R0.6/Yex.0.5/Pep.0.5/D), yeast extract 1.0% by weight (Comparative Example 10, S/R0.6/Yex.1.0/D), peptone 1.0% by weight (Comparative Example 11, S/R0.6/Pep.1.0/D) was carried out in the same manner as in Example 13 (S/R0.6/D), except that the fermented product of Seomoktae yeast of Comparative Examples 6 to 11 Was prepared.
상기 비교예 6 내지 비교예 11의 발효물에 대해 상기 실험예 2에 기재된 방법으로 α-글리코시다제 저해능을 측정하고 그 결과를 도 6에 나타내었다.For the fermented products of Comparative Examples 6 to 11, α-glycosidase inhibitory ability was measured by the method described in Experimental Example 2, and the results are shown in FIG. 6.
[결과][result]
서목태 이외의 저분자 질소원을 추가로 사용하여 효모 발효 시 α-glucosidase 저해능이 개선되는지 여부를 확인한 결과 서목태를 단독으로 사용한 시료에서 가장 높은 저해능을 나타냈다. Yeast extracts 및 peptone 모두 사용 농도가 증가할수록 저해능이 낮아졌다. 따라서 추가 질소원은 필요하지 않으며 서목태를 유일한 질소원으로 사용 시 가장 높은 저해능을 가지는 펩타이드를 생산할 수 있음을 확인하였다.As a result of confirming whether the α-glucosidase inhibitory ability was improved during yeast fermentation using an additional low-molecular nitrogen source other than Seomoktae, the highest inhibitory activity was shown in the sample using Seomoktae alone. Both yeast extracts and peptone decreased as the concentration increased. Therefore, it was confirmed that an additional nitrogen source was not required, and when Seomoktae was used as the only nitrogen source, the peptide with the highest inhibitory ability could be produced.
7. 최적 조건 (서목태 0.6 중량%, 포도당 2 중량%)에서 배양하여 얻은 상등액 건조물의 α-글리코시다제 저해능, ACE(angiotensin I converting enzyme) 저해능 및 항산화능 결과 (IC50값)7. α-glycosidase inhibitory activity, ACE (angiotensin I converting enzyme) inhibitory activity and antioxidant activity result of supernatant dried product obtained by culturing under optimal conditions (Seomoktae 0.6% by weight, glucose 2% by weight) (IC50 value)
실시예 13의 발효물, 즉, 최적 조건 (서목태 0.6 중량%, 포도당 2중량%)에서 배양하여 얻은 상등액 건조물의 α-글리코시다제 저해능을 상기 실험예 2의 방법으로 측정하고, ACE (angiotensin I converting enzyme) 저해능 및 항산화능(IC
50 값)은 아래 방법으로 측정하고 그 결과를 각각 도 7(α-글리코시다제 저해능), 도 9(ACE 저해능), 도 11, 도 13 및 도 15(항산화능)에 나타내었다.The fermentation product of Example 13, that is, the α-glycosidase inhibitory ability of the dried supernatant obtained by culturing under optimal conditions (0.6 wt% Seomoktae, 2 wt% glucose) was measured by the method of Experimental Example 2, and ACE (angiotensin I converting enzyme) inhibitory ability and antioxidant activity (IC 50 value) were measured by the following method, and the results were respectively measured in Figs. 7 (α-glycosidase inhibitory ability), Fig. 9 (ACE inhibitory ability), Figs. 11, 13 and 15 (antioxidant Function).
실험예 3 : ACE 저해능 측정Experimental Example 3: Measurement of ACE inhibitory ability
배양 상등액 건조물을 0.3M NaCl이 포함된 50mM sodium borate buffer(pH8.3)에 용해시킨 후 시료 용액 10㎕에 ACE (0.2U/mL, rabbit lung) 5㎕, 50mM sodium borate buffer 31㎕ 및 기질(4mM HHL) 13㎕를 넣어 37℃에서 60분 동안 반응시킨다. 그리고 200mM sodium tetraborate 100㎕, 10mM sodium sulfite 50㎕ 및 3.4mM TNBS 50㎕를 넣어 37℃에서 20분 동안 추가로 반응시켰으며, 반응 종료 후 420nm에서 흡광도를 측정하였다. 양성 대조구로는 Lisinopril을 사용하였다. 저해도는 [1-(S-Bs)/(C-Bi)]×100에 의해 산출하였으며, 여기서 S는 시료의 흡광도이고, Bs는 기질과 효소가 없을 경우의 흡광도이고, Bi는 시료와 효소가 없을 경우의 흡광도이며, C는 효소와 기질을 포함하고 저해물질이 없을 경우의 흡광도이다.After dissolving the dried culture supernatant in 50mM sodium borate buffer (pH8.3) containing 0.3M NaCl, 5µl of ACE (0.2U/mL, rabbit lung), 31µl of 50mM sodium borate buffer and substrate ( 4mM HHL) 13µl was added and reacted at 37°C for 60 minutes. Then, 100 μl of 200 mM sodium tetraborate, 50 μl of 10 mM sodium sulfite, and 50 μl of 3.4 mM TNBS were added to react at 37° C. for 20 minutes, and absorbance was measured at 420 nm after completion of the reaction. Lisinopril was used as a positive control. The degree of inhibition was calculated by [1-(S-Bs)/(C-Bi)]×100, where S is the absorbance of the sample, Bs is the absorbance in the absence of substrate and enzyme, and Bi is the sample and enzyme. It is the absorbance when there is no, and C is the absorbance when the enzyme and substrate are included and there is no inhibitor.
실험예 4: 항산화능 측정Experimental Example 4: Measurement of antioxidant activity
(1) DPPH (free radical scavenging assay)(1) DPPH (free radical scavenging assay)
유기 용매에 녹인 DPPH (2,2-diphenyl-1-picrylhydrazyl) 라디칼은 515 nm에서 최대 흡광도를 가지게 되는데, 항산화 물질은 DPPH라디칼을 소거하여 고유의 색을 잃고 투명하게 변화시킨다. DPPH (2,2-diphenyl-1-picrylhydrazyl) 100 μM과 처리할 물질의 시료 준비하고, 준비된 시료를 DPPH 용액과 1:20의 비율로 섞어 30분간, 37 ℃에서 보관하였다. 이후, 반응이 끝난 시료를 Spectrophotometer를 이용하여 517 nm에서 흡광도를 측정하였으며, 양성 대조구로는 아스코르브산(ascorbic acid)을 사용하였다. DPPH 소거능은 [(A
control-A
sample)/A
control]×100에 의해 산출하였으며, 여기서 A
control은 소거물질이 없을 경우의 흡광도이고, A
sample은 소거물질이 있을 경우의 흡광도이다.DPPH (2,2-diphenyl-1-picrylhydrazyl) radical dissolved in an organic solvent has a maximum absorbance at 515 nm. Antioxidants lose their color and become transparent by scavenging DPPH radicals. DPPH (2,2-diphenyl-1-picrylhydrazyl) 100 μM and a sample of the material to be treated were prepared, and the prepared sample was mixed with DPPH solution in a ratio of 1:20 and stored at 37° C. for 30 minutes. Thereafter, the sample after the reaction was measured for absorbance at 517 nm using a spectrophotometer, and ascorbic acid was used as a positive control. The DPPH scavenging ability was calculated by [(A control -A sample )/A control ]×100, where A control is the absorbance when there is no scavenging material, and A sample is the absorbance when there is a scavenging material.
(2) ABTS (radical cation de-colorization assay)(2) ABTS (radical cation de-colorization assay)
7mM ABTS와 2.45mM potassium persulfate를 섞고, 상온에서 16시간 incubation 후 ABTs 양이온(ABTs+)을 생성하였다. 734nm에서 흡광도의 값이 0.7 이하가 되도록 희석하여 시험용액으로 제조하였다. ABTs+ 용액은 7mM ABTS와 2.45mM potassium persulfate를 1:1(v/v)로 섞고 상온에서 16시간 incubation 후 ABTs 양이온(ABTs+)을 생성하였다. 그리고, 734nm에서 흡광도의 값이 0.7 이하가 되도록 에탄올로 희석하여 시험용액으로 제조하였다. ABTs+ 용액 143㎕에 시료 7㎕를 가하여 흡광도 값을 5분 후에 측정하였으며, 양성 대조구로는 아스코르브산(ascorbic acid)을 사용하였다. ABTs+ 소거능은 [(A
control-A
sample)/A
control]×100에 의해 산출하였으며, 여기서 A
control은 소거물질이 없을 경우의 흡광도이고, A
sample은 소거물질이 있을 경우의 흡광도이다.7mM ABTS and 2.45mM potassium persulfate were mixed, and after 16 hours incubation at room temperature, ABTs cations (ABTs+) were generated. A test solution was prepared by diluting so that the absorbance value at 734 nm was 0.7 or less. ABTs+ solution was mixed with 7mM ABTS and 2.45mM potassium persulfate at 1:1 (v/v) and incubated at room temperature for 16 hours to generate ABTs cations (ABTs+). Then, it was diluted with ethanol so that the absorbance value at 734 nm was 0.7 or less to prepare a test solution. 7 µl of a sample was added to 143 µl of the ABTs+ solution, and the absorbance value was measured after 5 minutes, and ascorbic acid was used as a positive control. The ABTs+ scavenging ability was calculated by [(A control -A sample )/A control ]×100, where A control is the absorbance when there is no scavenging material, and A sample is the absorbance when there is a scavenging material.
(3) Reducing power(3) Reducing power
0.2 M의 인산완충용액(pH 6.6) 50㎕에 페리시안화칼륨 50㎕와 시료 20 ㎕를 넣은 후, 50℃에서 20분간 반응시켰다. 반응이 완료된 후에 10% TCA 용액 50㎕를 넣고, 3000×g로 20분간 원심분리 하였다. 상층액 100㎕와 3차 증류수 100㎕, 0.1% 염화제이철(ferric chloride) 20㎕를 넣은 뒤에 700nm에서 흡광도를 측정하였으며, 양성 대조구로는 아스코르브산(ascorbic acid)을 사용하였다.50 µl of potassium ferricyanide and 20 µl of a sample were added to 50 µl of 0.2 M phosphate buffer solution (pH 6.6), and then reacted at 50° C. for 20 minutes. After the reaction was completed, 50 μl of a 10% TCA solution was added and centrifuged at 3000×g for 20 minutes. After adding 100 µl of the supernatant, 100 µl of tertiary distilled water, and 20 µl of 0.1% ferric chloride, the absorbance was measured at 700 nm, and ascorbic acid was used as a positive control.
[결과][result]
서목태를 최적조건에서 발효한 상등액 건조물을 각각 다른 농도로 제조 후 α-glucosidase 저해능, ACE 저해능 및 항산화능을 측정하였으며, 그 결과를 SPSS 통계 프로그램을 사용하여 50% 저해하는 농도(IC
50) 값으로 계산하였다. 서목태 0.6 중량% 및 포도당 2 중량%로 제조된 배지를 효모로 발효하여 얻은 배양 상등액 건조물의 IC
50 값은 α-glucosidase 저해능의 경우 29.5mg/mL, ACE 저해능은 6.3mg/mL 및 항산화능 또한 3~6mg/mL으로 확인되었다.After preparing the dried supernatant of Seomoktae fermented at different concentrations, α-glucosidase inhibitory ability, ACE inhibitory ability, and antioxidant activity were measured, and the results were calculated as 50% inhibitory concentration (IC 50 ) values using the SPSS statistical program. Calculated. The IC 50 value of the dried culture supernatant obtained by fermenting a medium prepared with Mok-Tae Seo 0.6% by weight and 2% by weight of glucose with yeast was 29.5mg/mL for α-glucosidase inhibitory activity, 6.3mg/mL for ACE inhibitory activity, and 3 for antioxidant activity. It was found to be ~6mg/mL.
[표 3][Table 3]
Claims (13)
- 효모를, 추가의 질소원을 실질적으로 포함하지 않는 서목태 함유 배지에서 배양하고,The yeast is cultivated in a medium containing Seomoktae that does not substantially contain an additional nitrogen source,상기 배양 후 얻어진 배양물로부터 혈당강하, 혈압강하 또는 항산화용 조성물을 제조하는 것을 포함하는, 혈당강하, 혈압강하 또는 항산화용 조성물의 제조 방법.A method of producing a composition for lowering blood sugar, lowering blood pressure, or antioxidant, comprising preparing a composition for lowering blood sugar, lowering blood pressure, or antioxidant from the culture obtained after the cultivation.
- 제1항에 있어서, 상기 추가의 질소원은 YPD, 우레아, 카제인, 대두, 밀기울, 현미유, 육류 추출물, 전지분유, 건조된 효모, 암모늄염, 트립토판, 프롤린, 아스파라긴, 및 글루타메이트로 이루어진 군 중 하나 이상 선택된 것인, 제조 방법.The method of claim 1, wherein the additional nitrogen source is selected from one or more of the group consisting of YPD, urea, casein, soybean, bran, brown rice milk, meat extract, whole milk powder, dried yeast, ammonium salt, tryptophan, proline, asparagine, and glutamate. That, the manufacturing method.
- 제1항에 있어서, 상기 배양은 20℃ 내지 40℃의 온도에서 12시간 내지 7일간 실시하는 제조 방법.The method of claim 1, wherein the culturing is performed at a temperature of 20°C to 40°C for 12 hours to 7 days.
- 제1항에 있어서, 상기 효모는 사카로마이세스 서바지 또는 사카로마이세스 세레비지아에인, 제조 방법.The method of claim 1, wherein the yeast is Saccharomyces cervage or Saccharomyces cerevisiae.
- 제4항에 있어서, 상기 사카로마이세스 서바지는 KCCM12157P로 기탁된 사카로마이세스 서바지 Ceb-kc-011인, 제조 방법.The method of claim 4, wherein the Saccharomyces service is Saccharomyces service Ceb-kc-011 deposited as KCCM12157P.
- 제1항 내지 제5항 중 어느 하나의 항에 있어서, 상기 배양 후 얻어진 배양물을 원심분리하고 균체 함유 물질을 제거하여 배양 상등액을 수득하는 것을 추가로 포함하는, 제조 방법.The method according to any one of claims 1 to 5, further comprising centrifuging the culture obtained after the cultivation and removing the cell-containing material to obtain a culture supernatant.
- 제1항 내지 제5항 중 어느 하나의 항에 있어서, 상기 배지가 탄소원을 추가로 포함하는, 제조 방법.The method according to any one of claims 1 to 5, wherein the medium further comprises a carbon source.
- 제7항에 있어서, 상기 탄소원이 단당류 또는 이당류인, 제조 방법.The production method according to claim 7, wherein the carbon source is a monosaccharide or a disaccharide.
- 제1항 내지 제5항 중 어느 하나의 항에 있어서, 상기 서목태가 전체 배지 부피를 기준으로 0.2 중량% 내지 4 중량%인, 제조 방법.The method according to any one of claims 1 to 5, wherein the Seomoktae is 0.2% to 4% by weight based on the total volume of the medium.
- 제1항 내지 제5항 중 어느 하나의 항에 따른 방법에 의해 제조된 서목태의 효모 발효물을 포함하는 혈당강하, 혈압강하 또는 항산화용 조성물.A composition for lowering blood sugar, lowering blood pressure, or antioxidant comprising a fermented yeast of Seomoktae prepared by the method according to any one of claims 1 to 5.
- 제10항에 있어서, 조성물 고형 중량을 기준으로 상기 서목태의 효모 발효물이 1 중량% 내지 70 중량% 로 포함되는, 조성물.The composition of claim 10, wherein the fermented yeast of Seomoktae is contained in an amount of 1% to 70% by weight based on the solid weight of the composition.
- 제10항에 있어서, 상기 조성물이 의약 또는 식품 조성물인, 조성물.The composition according to claim 10, wherein the composition is a pharmaceutical or food composition.
- 제10항에 있어서, 상기 서목태의 효모 발효물이 액상 상태 또는 건조된 분말 상태인, 조성물.The composition of claim 10, wherein the fermented yeast of Seomoktae is in a liquid state or a dried powder state.
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