KR20230174987A - Pharmaceutical composition comprising the culture metabolites of leuconostoc mesenteroides subsp. cremoris as an effective component for prevention and treatment of diabetes and health functional food comprising the same - Google Patents

Abstract

The present invention relates to an anti-diabetic composition containing metabolites obtained by culturing Leuconostoc mesenteroides strain, and more specifically, Leuconostoc mesenteroides cremoris KACC 12313 ( Leuconostoc mesenteroides subsp. cremoris KACC 12313) A pharmaceutical product for the prevention or treatment/improvement of diabetes through alpha-glucosidase inhibition and antioxidant activity containing metabolites obtained by microfiltration of strain culture as active ingredients. It relates to compositions and health functional foods. Metabolites obtained from the culture medium of Leuconostoc mesenteroides cremoris KACC 12313 ( Leuconostoc mesenteroides subsp. cremoris KACC 12313) strain as an active ingredient in the pharmaceutical composition and health functional food for the prevention or treatment of diabetes of the present invention are , As proven through the examples of this specification, it has excellent effects that can be used as medicine and health functional food for preventing or treating/improving diabetes through alpha-glucosidase inhibition and antioxidant activity. . In addition, the metabolites obtained from the strain culture medium of the present invention have excellent thermal stability and do not show loss of α-glucosidase inhibitory activity or antioxidant activity even under acidic conditions of pH 2 and in plasma, so they can be used in liquid, powder, pills, tablets, etc. It can be easily processed into various forms and can be very useful in the pharmaceutical and food industries.

Description

Pharmaceutical composition and health functional food for the prevention or treatment of diabetes containing metabolites of Leuconostoc mesenteroides cremoris strain as an active ingredient {PHARMACEUTICAL COMPOSITION COMPRISING THE CULTURE METABOLITES OF LEUCONOSTOC MESENTEROIDES SUBSP. CREMORIS AS AN EFFECTIVE COMPONENT FOR PREVENTION AND TREATMENT OF DIABETES AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention relates to an anti-diabetic composition containing metabolites obtained by culturing Leuconostoc mesenteroides strain, and more specifically, Leuconostoc mesenteroides cremoris KACC 12313 ( Leuconostoc mesenteroides subsp. cremoris KACC 12313) A pharmaceutical product for the prevention or treatment/improvement of diabetes through alpha-glucosidase inhibition and antioxidant activity containing metabolites obtained by microfiltration of strain culture as active ingredients. It relates to compositions and health functional foods.

Diabetes, one of the most common diseases in modern people, is a type of metabolic disease caused by insufficient secretion of insulin or failure to function normally, and is characterized by high glucose concentration in the blood. Hyperglycemia causes various symptoms and signs. Diabetes is divided into type 1 and type 2. Type 1 diabetes is caused by the inability to produce insulin at all due to genetic influences, and type 2 diabetes is caused by a decrease in insulin function (insulin resistance). ) is known to be the cause. In particular, type 2 diabetes is known to be largely influenced by environmental factors such as high-calorie, high-fat, and high-protein diets due to westernization of eating habits, lack of exercise, and stress. For the treatment of diabetes, insulin injections are essential in the case of type 1 diabetes, and lifestyle correction and drug treatment are necessary in the case of type 2 diabetes. Representative examples include insulin secretion stimulants (e.g., repaglinide, mitigl). Nide) and agents that delay carbohydrate absorption in the small intestine [Glucova: ingredient name - acarbose, Basin: ingredient name - voglibose], etc. are used.

Meanwhile, lactic acid bacteria are Gram-positive, acid-resistant cocci and bacilli, and have the characteristic of producing lactic acid (lactic acid) by fermenting sugars. The lactic acid bacteria strain itself is recognized as GRAS (Generally Recognized As Safe) and promotes positive health for the human body. It is known to have an impact. In fact, lactic acid bacteria are used in the fermentation of various animal and plant food raw materials such as dairy products, kimchi, and brewed foods, and recently, the strains themselves or their cultures are being consumed as probiotics.

Lactic acid bacteria produce not only flavor components but also various organic acids and antibacterial bacteriocin substances during fermentation. These organic acids and antibacterial substances increase the storability of food by suppressing or killing the growth of harmful microorganisms, and enhance the sensory and nutritional properties through fermentation. It is known to enhance digestion, absorption, intestinal peristalsis, and intestinal function. Additionally, as lactic acid bacteria are known to produce various anti-diabetic active substances during the fermentation process of sugars, lactic acid fermented products with diabetes prevention and improvement effects are being developed.

Studies on the anti-diabetic activity of lactic acid bacteria include the anti-diabetic effect of chive extract and fermented lactic acid bacteria in type 2 diabetes disease model db/db mice (Baejin Kim et al. 2015, Journal of Korean Food Storage and Distribution 22: 134-144), and salted fish in salted fish. Anti-obesity and anti-diabetic effects through fermentation of red pepper using lactic acid bacteria using isolated Lactobacillus plantarum (Lee Jun-hyung et al., 2019, Journal of Life Sciences 29: 354-361), L. rhamnosus BHN-LAB 76 Effect of white-throated mushroom using lactic acid bacteria Anti-obesity and anti-diabetic effects through fermentation (Lee Jun-hyung et al., 2019, Journal of Life Sciences 29: 470-477), Physiological activity following garlic solid fermentation using Lactic acid bacteria (Lee Jung-bok et al., 2016, Journal of Life Sciences 26: 446- 452), anti-diabetic effect of mulberry leaf extract fermented with Lactobacillus plantarum (Jisoo Choi et al., 2020, Korean Journal of Food Science and Technology 52: 191-199), hypoglycemic effect of diet supplemented with fermented milk containing mushroom extract in diabetic rats (Jaeyoung Cha et al., 2004) , Journal of Life Sciences 14: 676-682), Blood sugar lowering effect by L. mesenteroides EH-1 fermentation product (Traisaeng S. et al., 2020, Sci. Rep. 10(1):7928. doi: 10.1038/s41598-020 -64916-2) is known. However, to date, there are no known research reports on the anti-diabetic and antioxidant activities of Leuconostoc mesenteroides subsp. cremoris KACC 12313.

In addition, as a patent on the anti-diabetic activity of lactic acid bacteria, Republic of Korea Patent No. 10-2097509 describes Lactobacillus rhamnosus isolated from human feces [New lactic acid bacteria and fermented lactic acid bacteria using the same with anti-obesity and anti-diabetic effects] Manufacturing method], No. 10-2007980, a composition containing Lactobacillus reuteri elf and 5-aminolevulinic acid [Composition for preventing and treating degenerative brain disease using new lactic acid bacteria], No. 10-1462160, Lactobacillus acido [Anti-diabetic composition using fermented citrus lactic acid bacteria containing lactic acid bacteria cultures of Lactobacillus acidophilus, Lactobacillus plantarum , Lactobacillus casei and Lactobacillus rhamnosus ], No. 10-1722584 discloses [lactic acid bacterial fermentation extract of chives and anti-diabetic composition containing the same], and the Korean published patent No. 10-2018-0116052 discloses Leuconostoc mesenteroides [Antioxidant and anti-diabetic composition containing lactic acid bacteria fermented bitter melon extract], which contains fermented extract of bitter melon ( Momordica charantia L.) fermented using a strain as an active ingredient, No. 10-2019-0051669, contains [miR-296 expressing Antidiabetic use of recombinant lactic acid bacteria], No. 10-2022-0015615, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus plantarum, Leuconostoc lactis and Streptococcus thermophilus, Bifidus probiotics Fermented with [Functional beverage containing active ingredients of probiotic lactic acid bacteria and Goji berry extract], No. 10-2021-0126864 [Manufacturing method of fermented lactic acid bacteria for meal replacements and resulting fermented lactic acid bacteria for meal replacements], No. 10-2012- No. 0021349 discloses [Functional food and pharmaceutical composition for treating and preventing diabetes of pork potatoes using lactic acid bacteria fermentation]. However, to date, there are no known patents on the anti-diabetic and antioxidant activities of Leuconostoc mesenteroides subsp. cremoris KACC 12313.

KR 10-2097509 B

Traisaeng S. et al., 2020, Sci. Rep. 10(1):7928. doi: 10.1038/s41598-020-64916-2

The present invention was created to solve the problems of the prior art as described above, and the problem to be solved by the present invention is to effectively use the metabolite of Leuconostoc mesenteroides cremoris KACC 12313 ( Leuconostoc mesenteroides subsp. cremoris KACC 12313). The aim is to provide pharmaceutical compositions and health functional foods for preventing or treating/improving diabetes through alpha-glucosidase inhibition and antioxidant activity contained as ingredients.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating diabetes containing metabolites of the Leuconostoc mesenteroides subsp. cremoris KACC 12313 (Leuconostoc mesenteroides subsp. cremoris KACC 12313) strain as an active ingredient. Pharmaceutical compositions are provided.

The Leuconostoc mesenteroides subsp. cremoris KACC 12313 ( Leuconostoc mesenteroides subsp. cremoris KACC 12313) strain is preferably cultured in a medium containing 1% by weight or less of glucose.

The metabolites are preferably obtained by filtration or centrifugation-sterilization of the strain culture medium.

The metabolites preferably contain less than 0.2% by weight of glucose.

In addition, the present invention provides a health functional food for preventing or improving diabetes containing metabolites of the Leuconostoc mesenteroides subsp. cremoris KACC 12313 strain.

The Leuconostoc mesenteroides subsp. cremoris KACC 12313 ( Leuconostoc mesenteroides subsp. cremoris KACC 12313) strain is preferably cultured in a medium containing 1% by weight or less of glucose.

The metabolites are preferably obtained by filtration or centrifugation-sterilization of the strain culture medium.

The metabolites preferably contain less than 0.2% by weight of glucose.

Metabolites obtained from the culture medium of Leuconostoc mesenteroides cremoris KACC 12313 ( Leuconostoc mesenteroides subsp. cremoris KACC 12313) strain as an active ingredient in the pharmaceutical composition and health functional food for the prevention or treatment of diabetes of the present invention are , As proven through the examples herein, it has excellent effects that can be used as medicine and health functional food for preventing or treating/improving diabetes through alpha-glucosidase (α-glucosidase) inhibition and antioxidant activity. . In addition, the metabolites obtained from the strain culture medium of the present invention have excellent thermal stability and do not show loss of α-glucosidase inhibitory activity or antioxidant activity even under acidic conditions of pH 2 and in plasma, so they can be used in liquid, powder, pills, tablets, etc. It can be easily processed into various forms and can be very useful in the pharmaceutical and food industries.

Figure 1 shows the effects of glucose, MRS medium, and YPD medium on α-glucosidase activity.

Hereinafter, the present invention will be described in detail.

The inventor of the present invention evaluated the α-glucosidase enzyme inhibitory activity and antioxidant activity of 34 types of lactic acid bacteria isolated from a domestic strain distribution center and various commercially available kimchi products. What is unique is that the α-glucosidase enzyme was undesirably inhibited by glucose in the substrate in a concentration-dependent manner, and was also inhibited by MRS medium components. Therefore, in the present invention, the anti-diabetic activity was evaluated on the filtrate of embryo sap of 34 types of lactic acid bacteria cultured in YPD (yeast extract 0.5%, polypeptone 0.5%, dextrose 1%) medium with a glucose concentration of 1% or less. As a result, the present invention was completed by confirming the strong α-glucosidase enzyme inhibitory activity and excellent antioxidant power in the metabolites of lactic acid bacteria , Leuconostoc mesenteroides subsp. cremoris KACC 12313.

Therefore, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes containing metabolites of the Leuconostoc mesenteroides subsp. cremoris KACC 12313 strain as an active ingredient.

The Leuconostoc mesenteroides subsp. cremoris KACC 12313 ( Leuconostoc mesenteroides subsp. cremoris KACC 12313) strain is preferably cultured in a medium containing 1% by weight or less of glucose.

The metabolites are preferably obtained by filtration or centrifugation-sterilization of the strain culture medium.

The metabolites preferably contain less than 0.2% by weight of glucose.

In addition, the present invention provides a health functional food for preventing or improving diabetes containing metabolites of the Leuconostoc mesenteroides subsp. cremoris KACC 12313 strain.

The Leuconostoc mesenteroides subsp. cremoris KACC 12313 ( Leuconostoc mesenteroides subsp. cremoris KACC 12313) strain is preferably cultured in a medium containing 1% by weight or less of glucose.

The metabolites are preferably obtained by filtration or centrifugation-sterilization of the strain culture medium.

The metabolites preferably contain less than 0.2% by weight of glucose.

The lactic acid bacterium of the present invention is Leuconostoc mesenteroides subsp. cremoris KACC 12313 ( Leuconostoc mesenteroides subsp. cremoris KACC 12313) isolated from dry powder (Hansen's dried starter powder) for producing fermented beverages and stored at the National Academy of Agricultural Sciences, Korea. It can be distributed from the National Academy of Sciences (Seed Bank, Microbe Distribution, KACC 12313, http://genebank.rda.go.kr/microbeSearchView.do?rePage=microbe_search.jsp&sSearchWith=no&iNpage=1&sStrainsn=2556&sTxt1=12313&sTxt2=&), and has the same name. It is described as Leuconostoc cremosris (Knudsen and Sorensen 1929) and Betacoccus cremoris ( Knudsen and Sorensen 1929), and the type strain name is Type strain: KACC 12313 = ATCC 19254 = CCUG 21965 = CIP 103009 = DSM 20346 = LMG 6909 = NCIMB 12008 (formerly NCDO 543) = NRRL B-3252 = VKM B-1420.

The metabolites described in the specification of the present invention are a general term for a collection of various low-molecular-weight or high-molecular-weight organic and inorganic substances produced and/or secreted during the growth of lactic acid bacteria. As a preferred embodiment, the Leukonostoc mesenteroides cremoris KACC 12313 metabolite of the present invention can be obtained by removing lactic acid bacteria strains from the strain culture medium grown in the medium through a separate filtration device or by centrifugation-sterilization. there is. In a preferred embodiment, the glucose content of the obtained metabolites may be 0.2% by weight or less. After obtaining the filtrate from the strain culture medium, it can be subjected to a powdering process. For example, metabolite powder can be produced through conventional processes such as reduced pressure drying, freeze drying, or spray drying. They are not decomposed by various decomposition enzymes in plasma, and remain active even when heat treated at 100°C and at pH 2 in the human stomach.

The medium may be a known medium used for culturing lactic acid bacteria. As a preferred embodiment, the culture medium of the Leukonostoc mesenteroides cremoris KACC 12313 strain of the present invention may be a medium containing 1% by weight or less of glucose or YPD medium.

The Leukonostoc mesenteroides cremoris KACC 12313 metabolite of the present invention exhibits strong inhibitory activity against α-glucosidase and antioxidant activity, preventing diabetes such as type 1 diabetes, type 2 diabetes, diabetic retinopathy, and diabetic nephropathy. It can be used as a material for pharmaceutical compositions and health functional foods related to the prevention and treatment/improvement of complications.

As a preferred embodiment, the anti-diabetic composition of the present invention can be applied as a pharmaceutical composition.

The pharmaceutical composition is formulated into various forms such as oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injections of sterile injectable solutions according to conventional methods to suit each purpose of use. It can be used orally or administered through a variety of routes, including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

These pharmaceutical compositions may additionally contain carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The effective amount of the ingredient having anti-diabetic activity in the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, and weight, and is generally 1 to 5,000 mg per body weight, preferably 100 to 3,000 mg per day or every other day. It can be administered or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intra-cerebroventricular injection. In the present invention, "administration" means providing a predetermined substance to a patient by any appropriate method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

In the present invention, “subject” includes, but is not particularly limited to, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. And, preferably, it means mammals, and more preferably, humans.

As a preferred embodiment, the anti-diabetic composition of the present invention can be applied as a health functional food.

Foods containing the active ingredient with excellent anti-diabetic activity of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, etc., and powders, granules, tablets, capsules, or beverages. It can be used in the form

The active ingredient of the present invention can generally be added at 0.01 to 15% by weight of the total food weight, and the health drink composition can be added at a rate of 0.02 to 10g, preferably 0.3 to 1g, based on 100 ml.

In addition to containing the above compounds as essential ingredients in the indicated ratio, the health functional food of the present invention may contain foodologically acceptable food additives, such as natural carbohydrates and various flavoring agents, as additional ingredients. Examples of the natural carbohydrates include common sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The flavoring agent may include thaumatin, rebaudioside A, glycyrrhizin, saccharin, and aspartame. The ratio of the flavoring agent is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the health functional food of the present invention.

In addition to the above, the health functional food of the present invention contains various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavors, colorants and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, vegetable drinks, etc. These ingredients can be used independently or in combination. The ratio of these additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active fraction of the present invention.

Hereinafter, the present invention will be described in more detail through examples. The following example is only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following example.

[Example]

Example 1. Information on 34 types of lactic acid bacteria used to evaluate anti-diabetic and antioxidant activity

12 types of lactic acid bacteria (marked and numbered as KACC) distributed from the National Institute of Agricultural Sciences (www.naas.go.kr), Republic of Korea, and 8 types of lactic acid bacteria (KRIBB) distributed from the Korea Research Institute of Bioscience and Biotechnology (www.kribb.re.kr) The strain information of 14 types of lactic acid bacteria isolated from kimchi, Schisandra chinensis, wine (red wine), rice makgeolli, etc. commercially available in Korea (marked and numbered) is shown in detail in Table 1.

In this study, 16S rDNA sequencing was performed to identify the self-isolated lactic acid bacteria and strains acquired for distribution. The isolated lactic acid bacteria were cultured at 30°C and 150rpm for 48 hours and then identified. DNA extraction for identification of lactic acid bacteria was performed using the G-spin ^TM Total DNA Extraction Kit (iNtRON, Korea) according to the manufacturer's manual. The extracted DNA was Takara EX Taq (Takara, Japan), and the primers were 16S rDNA 8F (forward primer, 5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO: 1) and 16S rDNA 1492R (reverse primer, 5'-TACGGYTACCTTGTTACGACTT-3', PCR was performed using SEQ ID NO: 2). The PCR reaction conditions were 1st step at 95°C for 5 minutes, 2nd step at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes, repeated 29 times, and 3rd step. It was reacted at 72°C for 10 minutes. Afterwards, purification and 16S rDNA sequencing were obtained by requesting Macrogen (Korea). The nucleotide sequence obtained through this was identified using the NCBI (https://www.ncbi.nlm.nih.gov) Blast program. Representative examples of Table 1 include strain No. 2, Lacticaseibacillus casei JY17 (SEQ ID NO: 3), strain No. 4, Lacticaseibacillus saniviri JY10 strain (SEQ ID No. 4), and strain No. 30, Leuconostoc mesenteroides subsp. The 16S rDNA base sequence of cremoris KACC 12313 (SEQ ID NO: 5) is shown in Table 2.

On the other hand, lactic acid bacteria are known to have strict nutritional requirements and grow well in media containing various nutrients in complex, such as MRS medium, but do not grow well in YPD medium and LB medium with relatively simple composition. Table 3 shows the composition of MRS medium, YPD medium, and LB medium.

[Table 1] Information on lactic acid bacteria used in the experiment

[Table 2] Strain No. 2 in Table 1 Lacticaseibacillus casei JY17, strain 4 Lacticaseibacillus sanivir Strains JY10 and 30 Leuconostoc mesenteroides subsp. cremoris 16S rDNA base sequence of KACC 12313

[Table 3] Composition table of MRS medium, YPD medium, and LB medium used in the experiment

However, the present inventors confirmed that α-glucosidase enzyme activity is inhibited in a concentration-dependent manner by glucose, an enzyme reaction product, and that α-glucosidase enzyme activity is also strongly inhibited by MRS medium components (Figure 1). α-glucosidase enzyme activity was inhibited by 3.4% by 1% glucose, 10.2% by 10% glucose, 13.5% by 20% glucose, and 11.5% by sterilized non-inoculated MRS medium. Confirmed. This is because in existing reports, a number of papers reported anti-diabetic activity by evaluating the α-glucosidase enzyme inhibitory activity of lactic acid bacteria culture medium grown in MRS medium (Choi, J. et al. 2020. Kor. J. Food Sci. Technol 52, 191-199; Kim, S. and Lim, S. D. 2019. J. Milk Sci. Biotechnol. 37, 223-236). Therefore, in the present invention, YPD medium was used, which showed somewhat lower growth of lactic acid bacteria but did not inhibit α-glucosidase enzyme activity, and the final glucose concentration of the filtrate after culturing was confirmed to be less than 0.2%.

Example 2. α-glucosidase enzyme inhibitory activity of 34 types of lactic acid bacteria culture medium

After culturing the 34 types of lactic acid bacteria in Example 1 at 37°C for 48 hours using YPD medium, the filtrate was evaluated for α-glucosidase enzyme inhibitory activity, and the results are shown in Table 4. At this time, α-glucosidase inhibitory activity was evaluated using pNPG (p-nitrophenol glucoside; Sigma Co., USA), and α-glucosidase (0.25U/ml) diluted with 2.5μl of sample and 50mM sodium acetate buffer (pH 5.6). 25 μl was mixed and subjected to primary reaction at 37°C for 10 minutes, and 25 μl of 1mM pNPG solution was added to perform secondary reaction at 60°C for 10 minutes. Afterwards, 25 μl of 1M NaOH was added to stop the reaction, and the inhibition rate was calculated by measuring the absorbance at 405 nm.

Inhibition rate (%) = [1-(enzyme activity when sample was added/enzyme activity when control was added)] x 100

[Table 4] α-glucosidase enzyme inhibitory activity of 34 types of lactic acid bacteria cultures

As shown in Table 4, the bacteria that showed α-glucosidase enzyme inhibition of more than 10% were No. 1 among 34 types of lactic acid bacteria. There were a total of 10 species of lactic acid bacteria numbered 2, 6, 9, 25, 29, 30, 31, 32, 33 and 34, and Leuconostoc sp. There are 6 strains, Leuconostoc mesenteroides strain is No. There were a total of 4 species, numbered 29 to 32 ( L. mesenteroides KACC 17875, L. mesenteroides subsp. cremoris KACC 12313, L. mesenteroides subsp. dextranicum KACC 12315, L. mesenteroides DK-5). These results indicate that the Leuconostoc mesenteroides strain is highly correlated with anti-diabetic activity, and in particular, it means that excellent anti-diabetic active substances can be produced when culturing the Leuconostoc mesenteroides strain in YPD medium.

Example 3. Antioxidant activity of 34 types of lactic acid bacteria cultures

Since diabetes and oxidative stress are interrelated (Ok-Kyung Kim, 2015, Journal of the Korean Society of Petrochemicals 32: 488-496; Jae-Young Cha et al., 2005, Journal of the Korean Society of Life Sciences, 15: 809-818), the anti-diabetic activity of 34 types of lactic acid bacteria cultures The associated antioxidant activity was evaluated (Table 5). After culturing the 34 types of lactic acid bacteria in Example 1 at 37°C for 48 hours using YPD medium, the filtrate was tested for DPPH (1,1-diphenyl-2-picryl hydrazyl) anion radical scavenging activity and ABTS[2,2 -azobis (3-ethylbenzo thiazoline-6-sulfonate)] Cation radical scavenging ability, nitrite scavenging ability, and reducing power were measured.

First, in the case of DPPH scavenging ability, 380 μl of ² Absorbance was measured using Hitech, Expert96, Asys Co., Austria). DPPH radical scavenging ability was expressed as a percentage of the sample added and the sample not added.

In the case of ABTS scavenging ability, 5ml of 7mM ABTS (Sigma Co., USA) and 88ml of 140mM potassium persulfate were mixed and light was blocked for 16 hours at room temperature to form ABTS cations. This solution was then diluted with ethanol to obtain an absorbance value of 1.5 at 414nm. It was diluted with . After mixing 190 ml of the prepared diluted solution and 10 ml of the filtrate of the lactic acid bacteria culture, the mixture was reacted at room temperature for 6 minutes, the absorbance was measured at 734 nm, and the ABTS radical scavenging ability was calculated using the following equation.

ABTS radical scavenging ability (%) = [(C-S)/C] x 100,

C: Absorbance when adding solvent control DMSO, S: Absorbance when adding sample.

Meanwhile, in the case of measuring nitrite scavenging ability, the filtrate of lactic acid bacteria culture was added to the nitrite solution (1mM), 0.1N HCl was added to adjust the pH to 1.2, and reacted at 37°C for 1 hour, followed by Griess reagent (Sigma Co. , USA) was added and mixed. After leaving it at room temperature for 15 minutes, the absorbance was measured at 520 nm to measure the amount of remaining nitrite. The nitrite scavenging ability (%) was calculated using the following equation.

NSA (%) = [1-(A-C)/B] x100,

A: Absorbance after adding sample to 1mM nitrite solution and reacting for 1 hour,

B: Absorbance of 1mM nitrite solution,

C: Absorbance of filtrate sample of lactic acid bacteria culture medium.

In the above antioxidant experiment, vitamin C (Sigma Co., USA) was used as a control, and DMSO was used as a solvent control. Each activity evaluation was expressed as the average and deviation of experiments repeated three times.

Meanwhile, in the case of reducing power evaluation, the method of Oyaizu et al. was modified (Ahn Seon-mi et al., 2011. J. Life Sci. 21: 576-583). Add 2.5 ml of 0.2M sodium phosphate buffer (pH 6.6) and 2.5 ml of 10% potassium ferricyanide to 2.5 ml of filtrate of lactic acid bacteria culture, react at 50°C for 20 minutes, and then add 2.5 ml of 10% trichloroacetic acid for reaction. After completion, the supernatant was recovered by centrifugation at 4000 rpm for 10 minutes. The recovered supernatant was diluted two-fold with distilled water, mixed with freshly prepared 0.1% ferric chloride solution in a 5:1 (v/v) ratio, and evaluated by measuring absorbance at 700 nm.

[Table 5] Antioxidant activity of 34 types of lactic acid bacteria cultures

As shown in Table 5, the lactic acid bacteria culture medium contained antioxidant substances, especially No. 1 Enterococcus faecalis KACC 11304, No. 28 Leuconostoc lactis KACC 12305 and No. 30 Leuconostoc mesenteroides subsp. It was confirmed that cremoris KACC 12313 exhibits both scavenging and reducing power against active radicals.

Therefore, ultimately, Leuconostoc mesenteroides subsp, which is excellent in both α-glucosidase inhibitory activity and antioxidant activity. cremoris KACC 12313 was selected as the final anti-diabetic active lactic acid bacteria strain.

Example 4. Leuconostoc mesenteroides subsp. cremoris Evaluation of acid and heat stability of antidiabetic active substance of KACC 12313

The L. mesenteroides subsp. In order to evaluate the commercial applicability of the filtrate of cremoris KACC 12313 culture medium, the thermal and acid stability of the α-glucosidase inhibitory substance was confirmed. As a result, the filtrate was heat-treated at 100°C for 1 hour and pH 2 (0.01M). Even when treated with HCl for 1 hour, there was almost no decrease in α-glucosidase inhibitory activity. Therefore, the L. mesenteroides subsp. The culture medium of cremoris KACC 12313 was confirmed to contain acid- and heat-resistant α-glucosidase inhibitory substances, confirming that it has high practical usability.

<110> ANDONG NATIONAL UNIVERSITY INDUSTRY-ACADEMIN COOPERATION <120> PHARMACEUTICAL COMPOSITION COMPRISING THE CULTURE METABOLITES OF LEUCONOSTOC MESENTEROIDES SUBSP. CREMORIS AS AN EFFECTIVE COMPONENT FOR PREVENTION AND TREATMENT OF DIABETES AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME <130> YP210018 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 16S rDNA 8F <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 16S rDNA 1492R <400> 2 tacggytacc ttgttacgac tt 22 <210> 3 <211> 1182 <212> DNA <213> Unknown <220> <223> Lacticaseibacillus casei JY17 <400> 3 acaatctttg tcaccttaga cggctcgctc cctaaaaggg ttacgccacc ggcttcgggt 60 gttacaaact ctcatggtgt gacgggcggt gtgtacaagg cccgggaacg tattcaccgc 120 ggcgtgctga tccgcgatta ctagcgattc cgacttcgtg taggcgagtt gcagcctaca 180 gtccgaactg agaatggctt taagagatta gcttgacctc gcggtctcgc aactcgttgt 240 accatccatt gtagcacgtg tgtagcccag gtcataaggg gcatgatgat ttgacgtcat 300 ccccaccttc ctccggtttg tcaccggcag tcttactaga gtgcccaact aaatgctggc 360 aactagtcat aagggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct 420 gacgacaacc atgcaccacc tgtcattttg cccccgaagg ggaaacctga tctctcaggt 480 gatcaaaaga tgtcaagacc tggtaaggtt cttcgcgttg cttcgaatta aaccacatgc 540 tccaccgctt gtgcgggccc ccgtcaattc ctttgagttt caaccttgcg gtcgtactcc 600 ccaggcggaa tgcttaatgc gttagctgcg gcactgaagg gcggaaaccc tccaacacct 660 agcattcatc gtttacggca tggactacca gggtatctaa tcctgttcgc tacccatgct 720 ttcgagcctc agcgtcagtt acagaccaga cagccgcctt cgccactggt gttcttccat 780 atatctacgc atttcaccgc tacacatgga gttccactgt cctcttctgc actcaagttt 840 cccagtttcc gatgcgcttc ctcggttaag ccgagggctt tcacatcaga cttaaaaaac 900 cgcctgcgct cgctttacgc ccaataaatc cggataacgc ttgccaccta cgtattaccg 960 cggctgctgg cacgtagtta gccgtggctt tctggttgga taccgtcacg ccgacaacag 1020 ttactctgcc gaccattctt ctccaacaac agagttttac gacccgaaag ccttcttcac 1080 tcaagcggcg ttgctccatc agacttgcgt ccattggggg aagaattccc tactgctgcc 1140 tcccctaaga aatttggggc cgggtccaat ccccaagggg gg 1182 <210> 4 <211> 1089 <212> DNA <213> Unknown <220> <223> Lacticaseibacillus sanivir JY10 <400> 4 accaatctct gtcaccttag acggctagac tcctaaaggt taccccaccg gcttcgggtg 60 ttacaaactc tcagggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg 120 gcatgctgat ccgcgattac aagcgattcc aacttcgtgt aggcaagttg caccctacag 180 tccggactga aaatggcttt aaaaaattac cttaacctcg cggttttgcg actcgttgta 240 ccatccattg tagcaggtgt gtagcccagg tcataagggg catgatgatt tgacgtcatc 300 cccaccttcc tccggtttgt caccggcagt ctgattaaag tgcccaactt aaggctggca 360 actagtcata agggttgcgc tcgttgcggg acttaaccca acatctcaca acacaagctg 420 acaacaacca tgcaccacct gtcattctgt ccccaaaggg aacttctaat ctcttaaaat 480 agcaaaaaat gtcaagacct ggtaaggttc ttcgcgttgc ttcaaattaa accacatgct 540 ccaccgctgg tgcgggcccc cgtcaattcc tttgagtttc aaccttgcgg tcgtactccc 600 caggcggaat acttaatgcg ttagctgcag cactgaaggg cggaaaccct ccaacactta 660 gtattcatcg tttacggcat ggactaccag ggtatctaat cctgttcgct acccatgctt 720 tcgagcctca gcgtcagtta cagaccaaac agccgccttc gccactggtg ttcttccata 780 tatctacgca ttccaccgct acacatggag ttccactgtc ctcttctgca ctcaagttcc 840 cagtttccga tgcacttcct cggttagagc cgaggctttc acatcacact taagaaaccc 900 gccggcgctc gctttacgcc caataaatcc ggatacgctg gcccacctac gtataccgcg 960 gctgctgaca cgtagttagc cgtgctttct gttagatacc ggtcacctac gtgacagtta 1020 ctctcacgta cgttcttctc caacacagag ttacgatccg aaaccttctt cactcacgcg 1080 gcagtgctc 1089 <210> 5 <211> 1453 <212> DNA <213> Unknown <220> <223> Leuconostoc mesenteroides subsp. cremoris KACC 12313 <400> 5 ctggcggcgt gcctaataca tgcaagtcga acgcatagcg aaaggtgctt gcacctttca 60 agtgagtggc gaacgggtga gtaacacgtg gacaacctgc ctcaaggctg gggataacat 120 ttggaaacag atgctaatac cgaataaaac ttagtgtcgc atgacacaaa gttaaaaggc 180 gcttcggcgt cacctagaga tggatccgcg gtgcattagt tagttggtgg ggtaaaggcc 240 taccaagaca atgatgcata gccgagttga gagactgatc ggccacattg ggactgagac 300 acggcccaaa ctcctacggg aggctgcagt agggaatctt ccacaatggg cgaaagcctg 360 atggagcaac gccgcgtgtg tgatgaaggc tttcgggtcg taaagcactg ttgtatggga 420 agaacagcta gaataggaaa tgattttagt ttgacggtac cataccagaa agggacggct 480 aaatacgtgc cagcagccgc ggtaatacgt atgtcccgag cgttatccgg atttattggg 540 cgtaaagcga gcgcagacgg tttattaagt ctgatgtgaa agcccggagc tcaactccgg 600 aatggcattg gaaactggtt aacttgagtg cagtagaggt aagtggaact ccatgtgtag 660 cggtggaatg cgtagatata tggaagaaca ccagtggcga aggcggctta ctggactgca 720 actgacgttg aggctcgaaa gtgtgggtag caaacaggat tagataccct ggtagtccac 780 accgtaaacg atgaacacta ggtgttagga ggtttccgcc tcttagtgcc gaagctaacg 840 cattaagtgt tccgcctggg gagtacgacc gcaaggttga aactcaaagg aattgacggg 900 gacccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960 gtcttgacat cctttgaagc ttttagagat agaagtgttc tcttcggaga caaagtgaca 1020 ggtggtgcat ggtcgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080 cgcaaccctt attgttagtt gccagcattc agatgggcac tctagcgaga ctgccggtga 1140 taaaccggag gaaggcgggg acgacgtcag atcatcatgc cccttatgac ctgggctaca 1200 cacgtgctac aatggcgtat acaacgagtt gccaacccgc gagggtgagc taatctctta 1260 aagtacgtct cagttcggat tgtagtctgc aactcgacta catgaagtcg gaatcgctag 1320 taatcgcgga tcagcacgcc gcggtgaata cgttcccggg tcttgtacac accgcccgtc 1380 acaccatggg agtttgtaat gcccaaagcc ggtggcctaa ccttttagga aggagccgtc 1440 taaggcagga cag 1453

Claims (5)

A pharmaceutical composition for the prevention or treatment of diabetes containing metabolites of the Leuconostoc mesenteroides subsp. cremoris KACC 12313 strain as an active ingredient. The pharmaceutical composition according to claim 1, wherein the Leuconostoc mesenteroides subsp. cremoris KACC 12313 strain is cultured in a medium containing 1% by weight or less of glucose. The pharmaceutical composition according to claim 1, wherein the metabolites are obtained by filtration or centrifugation-sterilization of the strain culture medium. The pharmaceutical composition according to claim 3, wherein the metabolites contain less than 0.2% by weight of glucose. A health functional food for preventing or improving diabetes containing the active ingredient according to any one of claims 1 to 5.