KR20210041681A - A composition for lowering blood glucose level or blood pressure or for use of antioxidant comprising Rhynchosia nulubilis fermented by a yeast and a method for preparing the composition - Google Patents

Abstract

The present invention relates to a composition for lowering blood sugar, lowering blood pressure, or an antioxidant use comprising a yeast fermented product of Rhynchosia nulubilis, and a method for manufacturing the composition. The yeast fermented product of Rhynchosia nulubilis manufactured by the present invention has excellent inhibitory ability of α-glucosidase, few side effects, and is safe, thereby having high utility value for prevention and/or treatment of diabetes.

Description

A composition for lowering blood glucose level or blood pressure or for use of antioxidant comprising Rhynchosia nulubilis fermented by a yeast and a method for preparing the composition}

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.

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.

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.

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.

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 is high in fiber and contributes to intestinal activity. Fermenting Seomoktae contains more enzymes useful in the human body than any other food. ), remove gastric fever, treat paralysis, relieve blood stagnation (本草綱目), eat bean porridge, eliminate cattle thirst (thirsty due to diabetes) (經驗方), treat kidney disease, reduce energy, and eliminate all wind heat. It is also called Yakkong because it suppresses and in particular, activates blood and releases all poisons (本草綱目).

Korean Patent Application Publication No. 10-2017-0114049 Korean Patent Registration Publication No. 10-1908862B1

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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.

One aspect of the present invention,

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. 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. Saccharomyces in one example servazzii The KCCM12157P strain can 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. 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The composition may be formulated in various oral or parenteral formulations, but preferably may be formulated as a liquid oral solution.

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.

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.

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. 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. .

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.

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.

Furthermore, since it has excellent blood pressure lowering effect, it can be usefully used in the prevention or treatment of hypertensive diseases.

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.
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.
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.
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.
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.
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.
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.
8 is a graph showing the α-glucosidase inhibition rate (%) according to the concentration change of a conventionally known hypoglycemic agent, Acarbose, used as a positive control.
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.
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.
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.
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.
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.
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.
15 is a reducing power ( reducing power).

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. Necessary for fermentation Seo Mok-tae Appropriate amount

Example One: Seo Mok-Tae's leaven Fermented product 1, manufacture

(1) Sample acquisition and strain isolation

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) Taxonomic characteristics investigation

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.

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) Preparation of fermented yeast of Seomoktae

A culture of Saccharomyces servagi Ceb-kc-011 was prepared in the following manner:

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.

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.

Example 2: Seo Mok-Tae's leaven Fermented product 2, manufacture

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.

Example 3: Seo Mok-tae's leaven Fermented product 3, manufacture

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.

Example 4: Seo Mok-tae's leaven Fermented product 4, manufacture

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.

Example 5: Seo Mok-Tae's leaven Fermented product 5, manufacture

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 6: Seo Mok-tae's leaven Fermented product Manufacture of 6

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.

Comparative example 1: yeast Fermented product 1, manufacture

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.

Experimental example 1: yeast Viable cell count inspection

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.

Yeast ex.(Y) Peptone(P) Dextrose (D), a type of glucose Seo Mok-tae (R) CFU /mL Comparative Example 1
(YPD) 1.0% by weight 2.0% by weight 2.0% by weight 1.7Х10 ⁸ Example 3 (DR2) - - 2.0% by weight 2.0% by weight 2.8Х10 ⁷ Example 4 (DR2.5) - - 2.0% by weight 2.5% by weight 4.0Х10 ⁷ Example 5 (DR3.0) - - 2.0% by weight 3.0% by weight 4.3Х10 ⁷ Example 6 (DR3.5) - - 2.0% by weight 3.5% by weight 5.0Х10 ⁷ Example 2 (DR4.0) - - 2.0% by weight 4.0% by weight 5.8Х10 ⁷

(Weight% in the above table means the weight percentage of a specific component based on the total volume of the medium)

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. α- of the dried culture medium according to the composition of the medium Glucosidase Inhibitory ability

Example 7

A culture of Saccharomyces servagi Ceb-kc-011 was prepared in the following manner:

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.

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.

Comparative example 2: Seo Mok-tae Uncultured water-soluble protein dry matter

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.

Comparative example 3: Yeast culture

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.

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.

Comparative example 4: Seo Mok-tae's Yeast culture

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.

Comparative example 5: Seo Mok-tae's Lactic acid bacteria culture

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.

The compositions of Example 7 and Comparative Examples 2 to 5 are summarized and shown in Table 2 below:

Example 7 (S/R4/D) Yeast (S) + Seomoktae (R, 4% by weight) + Dextrose (D, 2% by weight) Comparative Example 2 (R) Seo Mok-Tae (R, dried uncultured water-soluble protein) Comparative Example 3 (S/YPD) Yeast (S) + YPD (yeast ex. (1% by weight), peptone (2% by weight), dextrose (2% by weight)) Comparative Example 4 (S/R4/YPD) Yeast (S) + Seomoktae (R, 4% by weight) + YPD Comparative Example 5 (L/R4/D) Lactobacillus ( L. holzapfelii ) + Seomoktae (R, 4% by weight) + Dextrose (D, 2% by weight)

(Weight% in the above table means the weight percentage of a specific component based on the total volume of the medium)

Experimental example 2: α- Glycosidase Inhibitory ability inspection

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 inhibitory ability]

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 is [(A _control -A _sample )/A _control ]

It was calculated by 100 (%), where A _control is the absorbance when there is no inhibitor, and A _sample is the absorbance when there is an inhibitor.

[result]

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.

The results are shown in FIG. 2.

3. α- according to the type of sugar Glucosidase Inhibitory ability

Example 8

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.

Example 9

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.

Example 10

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.

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.

[result]

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. To glucose Seo Mok-tae's Α- of samples cultured with different contents Glucosidase Inhibitory ability

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%.

Example 7, Example 11 to Example 17

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.

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.

[result]

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. In Seomoktae Α- of samples cultured with different glucose content Glucosidase Inhibitory ability

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.

Example 18 and Example 19

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.

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.

[result]

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. Seo Mok-tae 0.6% by weight , Glucose 2% by weight On the badge Small molecule When additional nitrogen source is added α- glucosidase Inhibitory ability Whether to improve

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.

Comparative example 6 to Comparative example 11

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.

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.

[result]

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. Optimal conditions ( Seo Mok-tae 0.6 weight% , Glucose 2% by weight ) Of the dried supernatant obtained by culturing in α- Glycosidase Inhibitory ability , ACE ( angiotensin I converting enzyme) Inhibitory ability And Antioxidant activity Result(IC ₅₀ value)

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).

Experimental example 3: ACE Inhibitory ability Measure

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 is [1-(S-Bs)/(C-Bi)]

It was calculated by 100, where S is the absorbance of the sample, Bs is the absorbance in the absence of a substrate and enzyme, Bi is the absorbance in the absence of a sample and enzyme, and C is the absorbance in the absence of an enzyme and a substrate, and there is no inhibitor. It is the absorbance of the case.

Experimental example 4: Antioxidant activity Measure

(1) DPPH (free radical scavenging assay)

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. DPPH scavenging ability is [(A _control -A _sample )/A _control ]

It was calculated by 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)

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. ABTs+ scavenging ability is [(A _control -A _sample )/A _control ]

It was calculated by 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

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 a 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 3000

Centrifuged for 20 minutes at g. 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.

[result]

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.

α- glucosidase Inhibitory ability (IC ₅₀ ) Example 13 (S/R0.6/D) 29.5 mg/mL Acarbose 5.9 mg/mL ACE Inhibitory ability Example 13 (S/R0.6/D) 6.3 mg/mL Lisinopril 1.8 ng/mL Antioxidant activity Example 13 (S/R0.6/D) DPPH: 3.0 mg/mL
ABTS: 27.4 mg/mL Ascorbic acid DPPH: 6.8 μg/mL
ABTS: 238.6 μg/mL

Name of Depository Organization: Korea Microorganism Conservation Center

Accession number: KCCM12157P

Consignment Date: 20171110

<110> COENBIO CO., Ltd. <120> A composition for lowering blood glucose level or blood pressure or for use of antioxidant comprising Rhynchosia nulubilis fermented by a yeast and a method for preparing the composition <130> P19-0174 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1686 <212> DNA <213> Artificial Sequence <220> <223> A necleic acid sequence of Saccharomyces servazzii Ceb-kc-011 <400> 1 ttatacagtg aaactgcgaa tggctcatta aatcagttat cgtttatttg atagttcctt 60 tactacatgg tataactgtg gtaattctag agctaataca tgcttaaaat cccgaccttt 120 tggaagggat gtatttatta gataaaaaat caatgtcttc ggactctttg atgattcata 180 ataacttttc gaatcgcatg gccttgtgcc ggcgatggtt cattcaaatt tctgccctat 240 caactttcga tggtaggata gtggcctacc atggtttcaa cgggtaacgg ggaataaggg 300 ttcgattccg gagagggagc ctgagaaacg gctaccacat ccaaggaagg cagcaggcgc 360 gcaaattacc caatcctaat acagggaggt agtgacaata aataacgata cagggccctt 420 tcgggtcttg taattggaat gagtacaatg taaatacctt aacgaggaac aattggaggg 480 caagtctggt gccagcagcc gcggtaattc cagctccaat agcgtatatt aaagttgttg 540 cagttaaaaa gctcgtagtt gaactttggg cctggttggc cggtctggct ttttgccacg 600 tactggaatg caaccgggcc tttccttctg gctaacctta ggctccttgt gggtctttgg 660 cgaaccagga cttttacttt gaaaaaatta gagtggttca aagcaggcgt attgctcgaa 720 tatattagca tggaataatg gaataggacg tttggttcta ttttgttggt ttctaggacc 780 atcgtaatga ttaataggga cggtcggggg catcagtatt caattgtcag aggtgaaatt 840 cttggattta ttgaagacta actactgcga aagcatttgc caaggacgtt ttcattaatc 900 aagaacgaaa gttaggggat cgaagatgat cagataccgt cgtagtctta accataaact 960 atgccgacta gggatcgggt ggtgtttttt taatgaccca ctcggcacct tacgagaaat 1020 caaagtcttt gggttctggg gggagtatgg tcgcaaggct gaaacttaaa ggaattgacg 1080 gaagggcacc accaggagtg gagcctgcgg cttaatttga ctcaacacgg ggaaactcac 1140 caggtccaga cacaataagg attgacagat tgagagctct ttcttgattt tgtgggtggt 1200 ggtgcatggc cgttcttagt tggtggagtg atttgtctgc ttaattgcga taacgaacga 1260 gaccttaacc tactaaatag tggtgctagc atttgctggt tcttccactt cttagaggga 1320 ctatcgattt caagtcgatg gaagtttgag gcaataacag gtctgtgatg cccttagacg 1380 ttctgggccg cacgcgcgct acactgacgg agccagcgag tctaacctag gccgagaggt 1440 cctggtaatc ttgtgaaact ccgtcgtgct ggggatagag ctttgtaatt tttgctcttc 1500 aacgaggaat tcctagtaag cgcaagtcat cagcttgcgt tgattacgtc cctgcccttt 1560 gtaccaccgc ccgtcgctag taccgattga atggcttagt gaggcctcag gatctgctta 1620 gagaaggggg caactccatc tcagagcgga aaatctggtc aaacttggtc atttagagaa 1680 ctaaac 1686

Claims (13)

The yeast is cultivated in a medium containing Seomoktae that does not substantially contain an additional nitrogen source,
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 cultivation. 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. 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. The method of claim 1, wherein the yeast is Saccharomyces cervage or Saccharomyces cerevisiae. The method of claim 4, wherein the Saccharomyces service is Saccharomyces service Ceb-kc-011 deposited as KCCM12157P. 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. The method according to any one of claims 1 to 5, wherein the medium further comprises a carbon source. The production method according to claim 7, wherein the carbon source is a monosaccharide or a disaccharide. 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. 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. 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. The composition according to claim 10, wherein the composition is a pharmaceutical or food composition. 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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