WO2019209086A1 - Souche de leuconostoc mesenteroides et son utilisation - Google Patents

Souche de leuconostoc mesenteroides et son utilisation Download PDF

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WO2019209086A1
WO2019209086A1 PCT/KR2019/005145 KR2019005145W WO2019209086A1 WO 2019209086 A1 WO2019209086 A1 WO 2019209086A1 KR 2019005145 W KR2019005145 W KR 2019005145W WO 2019209086 A1 WO2019209086 A1 WO 2019209086A1
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strain
mksr
leuconostoc mesenteroides
cholesterol
probiotic
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Korean (ko)
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김미숙
이새롬
정윤화
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Industry Academic Cooperation Foundation of Dankook University
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Industry Academic Cooperation Foundation of Dankook University
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Priority claimed from KR1020180049234A external-priority patent/KR101977255B1/ko
Priority claimed from KR1020190046715A external-priority patent/KR20200123617A/ko
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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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; 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
    • C12N1/20—Bacteria; Culture media therefor
    • 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; 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to a strain of Leuconostoc mesenteroides MKSR (Leukonostoc mesenteroides MKSR) and the use thereof deposited with the accession number KCTC13842BP, in particular a novel leukonostock mesenteroid strain, health for preventing or improving diabetes containing the strain Functional foods, health functional foods for improving cholesterol containing the strains, pharmaceutical compositions for preventing or treating diabetes containing the strains, pharmaceutical compositions for preventing or treating hyperlipidemia containing the strains, and food additives.
  • MKSR Leukonostoc mesenteroides MKSR
  • Hyperlipidemia is the maintenance of high levels of cholesterol in the blood. Increased serum cholesterol and triglycerides are the most common causes of hyperlipidemia. Accumulation of excess fat causes blood circulation disorders and microcirculation failure.
  • Cholesterol is an important class of molecules in animal lipids and is a precursor of steroid hormones, bile acids and vitamin D. Since cholesterol is insoluble in water, it is transported as a complex with proteins in plasma. The complex is a lipoprotein, and is classified into very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) according to the density. Increased levels of LDL-cholesterol increase the risk of atherosclerosis or coronary heart disease.
  • VLDL very low density lipoprotein
  • IDL intermediate density lipoprotein
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • Increased levels of LDL-cholesterol increase the risk of atherosclerosis or coronary heart disease.
  • HDL cholesterol lower the risk of atherosclerosis and coronary heart disease.
  • Increases in non-HDL cholesterol and LDL levels may be due to food intake, obesity, genetic factors (eg, patients with hereditary hyperlipidemia due to LDL receptor mutations), diabetes and hypothyroidism.
  • diabetes is the most representative disease of endocrine disorders caused by the malfunction of insulin. Globally, diabetes is estimated at 374 million, or about 5% of the world's population, and about 3.4 million people die from fasting hyperglycemia. Moreover, diabetes is accompanied by long-term complications such as arteriosclerosis, hypertension, cerebrovascular infarction, diabetic nephropathy, diabetic retinopathy, pyoderma, eczema, necrosis and oral disease. Is expected to be ranked seventh in mortality. According to the American Diabetes Association, health care costs for people with diabetes reach $ 132 billion (2002) annually. Type 2 diabetes, which accounts for 90% of people with diabetes, is rapidly increasing due to weight gain and lack of exercise. .
  • diabetes In Korea, about 3.5 million people are estimated to be diabetic patients in 2010 due to socio-economic development and the increase in diabetes due to overeating and lack of exercise. In addition, in Korea, non-insulin-dependent diabetes mellitus has emerged as a serious problem in economic, social and health care. According to the 2013 statistics, diabetes is among the top 10 most frequently diagnosed patients with age 65 and older, and annually reported health care expenses amount to 22.192 billion won. Diabetes treatment drugs are mainly used for blood sugar management and delayed complications. Oral glucose enhancers such as insulin, sulfonate, biguanide, and alpha-glucosidase inhibitors are used. Problems include the limitations of treatment.
  • probiotics are living microorganisms that, when ingested in moderation, have a health and beneficial effect on the host, and probiotic intake helps stabilize the intestinal flora, thereby maintaining balance between the intestinal bacteria and maintaining the health of the body. It is also known to help maintenance. Probiotics help the immune response, which is due to the increase in high macrophage activity and anti-inflammatory cytokine transcription regulators by the cell wall components of probiotic strains, resulting in an immune response that removes foreign substances such as infectious harmful microorganisms. Because. However, there have been no reports on strains for plant-derived multi-type probiotics that have an activity inhibitory effect on dietary carbohydrate digestive enzymes and at the same time have cholesterol improving effects and antioxidant and probiotic activity.
  • the present inventors have tried to find strains that can be used as probiotics from fermented foods and that can solve the problem of products for preventing and / or improving conventional diabetes and hyperlipidemia, and as a result, without the inhibitory effect on alpha amylase
  • the present invention has been completed by identifying strains that have an excellent inhibitory effect on alphaglucosidase, have cholesterol anabolic and cholesterol reducing effects, exhibit antioxidant effects, and can be used as probiotics.
  • the present invention is a leukonostock mesenoidoid MKSR strain deposited with the accession number KCTC13842BP is capable of functioning as a probiotics having a high survival rate for artificial intestinal fluids of high concentration, and is used for delaying the absorption of the existing sugar in the body
  • alpha amylase to resolve side effects of abdominal bloating, and it has an excellent inhibitory effect on alpha glucosidase, an enzyme that converts disaccharide maltose into glucose, and has a BSH (Bile salt hydrolase) activity and cholesterol
  • BSH Bacte salt hydrolase
  • the health functional food containing the strain, its culture or a mixture thereof as an active ingredient provides excellent diabetes prevention and improvement effect, cholesterol improvement effect, antibacterial effect and antioxidant effect.
  • the pharmaceutical composition containing the strain, its culture or a mixture thereof as an active ingredient provides a prophylactic or therapeutic effect of diabetes and hyperlipidemia.
  • the food additive containing the strain, its culture or a mixture thereof as an active ingredient provides an excellent effect of producing complex carbohydrates or oligosaccharides.
  • the present invention provides a Leuconostoc mesenteroides MKSR strain deposited with accession number KCTC13842BP.
  • the present invention also provides a Leuconostoc mesenteroides MKSR strain deposited with an accession number KCTC13842BP, a culture solution thereof, or a mixture thereof as an active ingredient, a health functional food for preventing or improving diabetes.
  • the present invention also provides a Leuconostoc mesenteroides MKSR strain deposited with Accession No. KCTC13842BP, a culture solution thereof or a mixture thereof as an active ingredient, a dietary supplement for improving cholesterol.
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising a Leuconostoc mesenteroides MKSR strain deposited with the accession number KCTC13842BP, a culture solution thereof, or a mixture thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating hyperlipidemia, which contains a Leuconostoc mesenteroides MKSR strain deposited with an accession number KCTC13842BP, a culture solution thereof, or a mixture thereof as an active ingredient.
  • the present invention provides a food additive containing a Leuconostoc mesenteroides MKSR strain deposited with the accession number KCTC13842BP, a culture or a mixture thereof as an active ingredient.
  • the present invention can be used as a probiotics with a high survival rate for artificial intestinal fluids deposited with the deposit No. KCTC13842BP can function as a probiotic, side effects of acarbose used to delay the absorption of the existing sugar in the body
  • acarbose used to delay the absorption of the existing sugar in the body
  • the health functional food containing the strain, its culture or a mixture thereof as an active ingredient provides excellent diabetes prevention and improvement effect, cholesterol improvement effect, antibacterial effect and antioxidant effect.
  • the pharmaceutical composition comprising the strain, its culture or a mixture thereof as an active ingredient provides a prevention or treatment effect of diabetes and hyperlipidemia.
  • the food additive containing the strain, its culture or a mixture thereof as an active ingredient provides an excellent effect of producing complex carbohydrates or oligosaccharides.
  • 1 is a diagram of the morphology of the leukonostock metheteroid MKSR.
  • Figure 2 relates to SEQ ID NO: 1 as a result of 16s rRNA analysis of the leukonostock metheteroid MKSR.
  • the 16s rRNA was identified 99% similar to the Leuconostoc mesenteroides strain ATCC 8293.
  • 3 is a diagram of a Phylogenetic tree of Leuconostoc mesenteroides MKSR.
  • FIGS. 4 and 5 are diagrams related to sugar utilization, oligosaccharides and dextran production according to the sugars added to the medium of different Leuconostoc mesenteroides species.
  • 6 is a diagram showing the inhibitory effect of alpha glucosidase of Leuconostoc mesenteroides MKSR.
  • Figure 7 is a photograph associated with the hemolytic reaction test results, (A) Leuconostoc mesenteroides MKSR, (B) Leuconostoc mesenteroides subsp. mesenteroides (LM) (C) are Leuconostoc citreum (LC) (D) for Staphylococcus aureus .
  • the present invention provides a Leuconostoc mesenteroides MKSR strain deposited with accession number KCTC13842BP.
  • the strain is a probiotic strain having an alpha glucosidase inhibitory activity, there is no inhibitory effect of alpha amylase, and can improve the abdominal bloating problem which is a side effect of acarbose used as a conventional diabetes treatment.
  • the strain has the effect of activating the BSH (Bile salt hydrolase) and the ability to assimilate the cholesterol (Cholesterol assimilation) can significantly improve blood cholesterol because it has the effect of significantly lowering blood cholesterol levels.
  • the BSH (Bile salt hydrolase) enzyme reduces bile salts and increases the conversion of cholesterol to bile acids in the liver, thereby maintaining homeostasis and ultimately lowering blood cholesterol levels. Cholesterol assimilation assimilates cholesterol. Can lower blood cholesterol levels.
  • the deposit No. KCTC13842BP can be used as a leukonostock mesenteroid MKSR probiotics deposited, characterized by having an antioxidant effect.
  • the strain is characterized by having an antimicrobial effect on food poisoning bacteria.
  • Probiotics allow ⁇ -glucan of lactic acid bacteria to decrease cholesterol absorption by controlling and inhibiting cholesterol by structural and chemical properties. Therefore, prolonged intake of probiotics can have a prophylactic effect of preventing atherosclerosis or hyperlipidemia.
  • Strains usable as probiotics may include Leuconostoc , Enterococcus , Streptococcus , Bacillus , etc. The present invention is more preferably used Leuconostoc .
  • the strain of the present invention may be a probiotic strain for preventing or improving diabetes or improving cholesterol.
  • the present invention also provides a Leuconostoc mesenteroides MKSR strain deposited with an accession number KCTC13842BP, a culture solution thereof, or a mixture thereof as an active ingredient, a health functional food for preventing or improving diabetes.
  • the strain may have an alpha glucosidase inhibitory activity without an inhibitory effect of alpha amylase, thereby improving abdominal bloating problem which is a side effect of acarbose, a conventional diabetes treatment.
  • the strain of the present invention may have an antioxidant effect to alleviate diabetic complications due to cell damage caused by free radicals, thereby effectively contributing to the prevention and / or improvement of diabetes.
  • the present invention also provides a Leuconostoc mesenteroides MKSR strain deposited with Accession No. KCTC13842BP, a culture solution thereof or a mixture thereof as an active ingredient, a dietary supplement for improving cholesterol.
  • the strain has a BSH (Bile salt hydrolase) activity and cholesterol assimilation (Cholesterol assimilation) has a significant effect on lowering the blood cholesterol level can contribute to the prevention and / or improvement of hyperlipidemia.
  • the health functional food includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof, Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like.
  • nutraceutical composition is in the form of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverages, tea, functional water, drinks, alcohol and vitamin complexes Can be.
  • the health functional food may further include a food additive, and the suitability as a "food additive" is applied to the item in accordance with the General Regulations of the Food Additives Code and General Test Law, etc., unless otherwise specified. Determined by the relevant standards and standards.
  • Items listed in the "Food Additives Code” include, for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, cinnamon acid, natural additives such as dark blue, licorice extract, crystalline cellulose, high-lower pigment, guar gum, L Mixed preparations, such as a sodium glutamate preparation, a noodles addition alkali agent, a preservative preparation, and a tar pigment preparation, etc. are mentioned.
  • the pravastatin and antiplatelet agent added to the food in the process of manufacturing the health functional food can be appropriately added or subtracted as necessary, preferably 1 to 90 parts by weight to 100 parts by weight of the food to be added Can be.
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes, containing the strain, its culture or a mixture thereof as an active ingredient.
  • the pharmaceutical composition according to the present invention improves the side effects and treatment limits for organs such as alpha-glocosidase inhibitors, and provides excellent diabetes prevention and / or improvement effect while alleviating the side effects on organs even after continuous administration. can do.
  • organs such as alpha-glocosidase inhibitors
  • an antioxidant effect it is possible to alleviate the complications of diabetes due to cell damage caused by free radicals and to effectively contribute to the prevention and / or improvement of diabetes.
  • the present invention provides a pharmaceutical composition for preventing or treating hyperlipidemia, which contains the strain, its culture solution or a mixture thereof as an active ingredient.
  • the pharmaceutical composition according to the present invention has a BSH (Bile salt hydrolase) activity and cholesterol assimilation (Cholesterol assimilation) has a significant effect on lowering the blood cholesterol level can contribute to the prevention and / or improvement of hyperlipidemia.
  • compositions according to the invention may further comprise suitable carriers, excipients or diluents commonly used in the manufacture of pharmaceutical compositions.
  • Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.
  • compositions according to the invention can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., respectively, according to conventional methods.
  • sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., respectively, according to conventional methods.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient, for example, starch, calcium carbonate, sucrose ( Prepare by mixing sucrose or lactose, gelatin, etc.
  • Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .
  • the dosage of the pharmaceutical composition according to the present invention may vary depending on the age, sex and weight of the patient, but the amount of 0.4 mg / m2 / day to 10.0 mg / m2 / day may be administered twice a week for 1 to 4 cycles. .
  • Such dosage may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.
  • compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally, topically) according to the desired method, and the dosage is based on the weight, age, sex, health condition, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease.
  • the present invention provides a food additive containing a Leuconostoc mesenteroides MKSR strain deposited with the accession number KCTC13842BP, a culture or a mixture thereof as an active ingredient.
  • the strain is capable of producing one or more selected from the group consisting of mannitol, fructose, oligosaccharides and dextran, and having an antimicrobial activity against food poisoning bacteria, etc., can perform an excellent role as a food additive.
  • the strain can be used as a probiotic having an antioxidant effect can also obtain a whitening effect.
  • Leuconostoc mesenteroides MKSR was incubated at 25-40 ° C., more preferably 25-38 ° C. using MRS medium. It is possible to culture the microorganism in the temperature range, there is a problem that the culture of the microorganism is difficult when out of the temperature range. Microscopic observation, Gram staining, endospore formation, and catalase activity were measured for the microbiological properties of MKSR. API 50CHL and API zym (API bioMerieux, France) were used to investigate carbohydrate availability and enzyme production capacity. In addition, the nucleotide sequence of 16s rRNA of MKSR was requested to Solgent (Daejeon, Korea).
  • the cultured one colony was diluted and genomic DNA was extracted using genomic DNA prep kit.
  • Bacterial genomic DNA was used with 27F (5'-AGA GTT TGA TCC TGG CTC AB-3 ') as the primer primer and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3') as the reverse primer.
  • 27F 5'-AGA GTT TGA TCC TGG CTC AB-3 '
  • 1492R 5'-GGT TAC CTT GTT ACG ACT T-3'
  • PCR reaction was carried out for 10 seconds at 96 °C, 5 seconds at 50 °C, 4 minutes at 60 °C was repeated a total of 30 times.
  • the PCR was completed using the Solgent PCR purification kit to clean, and confirmed the PCR results using a 1% agarose gel.
  • the purified DNA was sequencing with an ABI 3730XL DNA analyzer (Applide Biosystems, Foster, USA) and analyzed using BLAST (Basic Local Alignment Search Tool).
  • the MEGA7 program was used to select Phylogenetic trees by selecting 16s rRNAs and strains closely related to MKSR.
  • MKSR Leuconostoc mesenteroides
  • MKSR was 0.5% (w / v) yeast extract, 0.5% (w / v) peptone, 2% (w / v) K 2 HPO 4 0.02% (w / v) MgSO 4 7H 2 O, 0.001% (w / v) for NaCl, the medium consisting of 0.001% (w / v) FeSO 4 ⁇ 7H 2 O, 0.001% (w / v) MnSO 4 ⁇ H 2 O, 0.013% (w / v) CaCl 2 ⁇ 2H 2 O
  • different sugars were added: 1% Maltose, 1% Fructose, 1% Glucose, 5% Sucrose, 1% Maltose and 5% Sucrose, 1% Fructose and 5% Sucrose, 1% Glucose and 5% Sucrose After addition and incubation for 18 hours, the sugar transfer reaction was performed after centrifugation at 10000 rpm for 5 minutes. The reaction product was
  • Dextran produced by lactic acid bacteria involved in the fermentation of kimchi, a traditional fermented food, has been approved for use as a food additive. This is a polysaccharide in which glucose, a constituent sugar, is mainly linked by ⁇ - (1 ⁇ 6) linkage among lactic acid bacteria produced outside the cell, and is actively used in the food industry such as crystallization of sucrose products and control of physical food properties.
  • the representative Leuconostoc mesenteroides producing Dextran is a biphasic fermentation strain, which is a lactic acid bacterium that produces dextran based on sucrose, a fermentable sugar, and is widely present in naturally fermented foods. MKSR cultured in the medium containing maltose produced dextran with oligosaccharides. Leuconostoc strains have been reported to play an important role in controlling the physical properties of foods while releasing dextransucrase as an extracellular enzyme, biosynthesizing the polymer dextran as a polymer of glucose, and having physicochemical properties of adhesiveness, hygroscopicity and thermal stability.
  • Leuconostoc lactobacillus produces sugar alcohol, mannitol and fructose, giving sweet and refreshing taste to fermented products.
  • Sugar alcohols produced by lactic acid bacteria have an advantage over the production method by hydrogenation under chemically high temperature and high pressure conditions, and many studies are being conducted on mannitol production by microorganisms.
  • Table 1 shows the results regarding the formation of oligosaccharides and dextran produced by the fermentation of Leuconostoc mesenteroides MKSR using different carbohydrates.
  • M 1% maltose
  • G 1% glucose, S, 1% sucrose
  • F 1% fructose
  • LMG LM medium + 1% glucose
  • LMM LM medium + 1% maltose
  • LMS LM medium + 5% sucrose
  • LMF LM medium + 1% fructose
  • LMGS LM medium + 1% glucose + 5% sucrose
  • LMMS LM medium + 1% maltose + 5% sucrose
  • LMFS LM medium + 1% fructose + 5% sucrose
  • MKSR is L. mesenteroides sp. It was similar to mesenteroides (LM), 98.8%, and there was a difference in sugar availability such as Xylose, D-lactose and Potassium gluconate.
  • MKSR Leuconostoc mesenteroides MKSR
  • LM Leuconostoc mesenteroides
  • LC Leuconostoc citreum
  • Bile acids affect the cell membranes of microorganisms composed of lipids and inhibit the growth of microorganisms. Many species of lactic acid bacteria, including Lactobacillus , produce bile acid hydrolase to hydrolyze bile acids and reduce this inhibitory activity. , 2016). In order to measure the resistance to gastric acid and bile of MKSR, the number of viable cells was measured by inoculation with time (see Table 3).
  • MKSR Leuconostoc mesenteroides MKSR was passed through pepsin (pH 2) and 3% bile salt solution conditions. MKSR could be used as a probiotic as a strain suitable for survival in the intestinal environment after 100 hours of passage in 0.1% pepsin solution and 4 hours in 3% bile salt solution.
  • Table 3 is a table of the number of viable euconostoc mesenteroides MKSR cells when passed through pepsin (pH 2) and 3% bile acids.
  • the supernatant is collected by centrifugation for 5 hours at 10000rpm for 10 minutes incubated in MRS liquid medium. Some of the supernatants are adjusted to pH 5.5-6.5 with 1N NaOH and some do not adjust pH. The supernatant with pH adjustment and the supernatant without adjustment were filtered through a 0.2 ⁇ m filter. Pathogens incubated at 37 ° C. for 18 hours were treated with solid medium [BHI (Brain Heart Infusion, MB cell Co., Korea), TSA (Tryptic Soy agar, Difco Co., USA), NA (Nutrient Agar, Difco Co., USA). After spreading on the disc, the disc was raised, and the inoculum supernatant was inoculated by 20 ⁇ l, and the size of the microorganisms around the disc was measured in mm at 14 hours, 24 hours, and 48 hours.
  • BHI Brain Heart Infusion, MB cell Co., Korea
  • TSA Teryptic Soy agar, D
  • MKSR The antimicrobial activity of MKSR was found in Gram-negative bacteria E. coli, Pseudomonas aeruginosa, Shigella sonnei, S. flexneri, Klebsiella pneumoniase and Gram-positive bacteria Bacillus cereus, Listeria monocytogenes, L. innocua, Staphylococcus aureus After exposure, the cells were cultured for 14, 24, and 48 hours, and the antimicrobial activity was examined through the presence or absence of a zona pellucida formed around the disc. MKSR resulted in 14 hours Pseudomonas aeruginosa, Shigella sonnei, S. flexneri, Bacillus cereus, Listeria monocytogenes, L.
  • FRAP reagent 10 mM TPTZ (2,4,6-tripyridyl triazine) (Sigma-Aldrich Co.): 20 mM FeCl 3 ⁇ 6H 2 O: 0.3 M Sodium acetate buffer (1: 1: 10)] for 10 minutes After preheating at 37 ° C, 50 ⁇ l of Cell free supernatant or Intact Cell was added, followed by reaction in the dark for 30 minutes. The absorbance was measured at 593 nm using a spectrophotometer to determine the color change. FeSO 4 ⁇ 7H 2 O was used as a standard sample.
  • Tris-HCl buffer 50 mM tris [hydrowymethyl] amino-methane + 10 mM EDTA) (pH8.0) to 0.1 ml of cell free supernatant or Intact Cell and add 0.1 ml of 7.2 mM pyrogallol (Sigma Aldrich Co.) for 10 minutes. Left at room temperature. 50 ⁇ l of 1N HCl was added to stop the reaction, and the absorbance was measured at 420 nm using a spectrophotometer.
  • FRAP Reducing power is an important factor related to antioxidant capacity.
  • FRAP is a method of measuring the ability of reducing materials such as antioxidants to reduce the color by reducing the Fe 3+ -ferricyanide complex to Fe 2+ form. (Jeon et al. 2013).
  • the reducing power of MKSR was 3.36-3.44 FeSO 4 ⁇ 7H 2 O eq mM, and the reducing power of 1 mg / ml ascorbic acid used as a control was 4.06 FeSO 4 ⁇ 7H 2 O eq mM.
  • SOD not only radically removes free radicals by converting free radicals in the body into hydrogen peroxide, but H 2 O 2 produced by SOD produced by SOD is converted into water and oxygen molecules by peroxidase or catalase.
  • the enzyme plays an important role in protecting living organisms. Similar activity of MKSR was observed only in cell free supernatants (see Table 5). Table 5 below is related to the antioxidant activity of Leuconostoc mesenteroides MKSR.
  • MKSR showed acid phosphatase, Naphtaol-AS-BI-phosphohydrolase, ⁇ -galactosidase, ⁇ -galactosidase, ⁇ -glucosidase and ⁇ -glucosidase activity.
  • ⁇ -galactosidase is an enzyme that decomposes sugars such as raffinose and stachyose, which are present in grains such as soybean, causing gas in the colon, and thus can be expected to have a formal effect in humans and animals.
  • Table 6 above relates to the number of viable MKSR cells when passed through pepsin (pH 2) and 3% bile acid solution.
  • Oral hypoglycemic agent a treatment for type 2 diabetes, uses a substance that inhibits enzymes involved in glycolysis. Decomposes disaccharides into monosaccharides and the latter decomposes polysaccharides into small sugars. These inhibitors of sugar enzymes inhibit the decomposition of sugar and eventually inhibit the absorption of complex carbohydrates, thereby allowing glucose to be absorbed throughout the small intestine, thereby increasing the post-prandial blood sugar rise.
  • acarbose is currently used as an inhibitor of sugar enzyme, acarbose, which has high inhibitory activity of both ⁇ -glucosidase and ⁇ -amylase, has side effects such as stomach bloating and diarrhea.
  • Table 7 shows the ⁇ -Glucosidase and ⁇ -amylase inhibitory activity of Leuconostoc mesenteroides MKSR (%)
  • Table 8 below relates to the media composition of MKSR for the production of inhibitors for ⁇ -Glucosidase.
  • ⁇ -Glucosidase inhibitory activity was high when cultured in # 1, # 2, # 5, # 7, # 9, # 4 medium (see Fig. 6).
  • ⁇ -glucosidase inhibitors include Mg 2+ and Mn 2+ .
  • Mg 2+ and Mn 2+ do not add # 7 and # 8
  • the Mg 2+ and Mn 2+ mediums do not have the ⁇ -glucosidase inhibitory activity.
  • ⁇ -glucosidase inhibitory activity was confirmed.
  • the isolated Leuconostoc strain was added to 10 ⁇ l of MRS agar plate supplemented with 0.5% (w / v) sodium taurodeoxycholic acid (TDCA; Sigma Aldrich Co. LLC., St. Louis) and 0.37 g.L- 1 CaCl 2 .
  • Cells were added and screened for measurement of Bile salt hydrolase (BSH) activity. Plates were incubated in anaerobic vessels at 37 ° C. for 72 hours. At this time, the presence of bile acid precipitated around the colony or the formation of silvery opaque granular white colonies is considered a positive reaction.
  • BSH Bile salt hydrolase
  • Table 9 shows the results of BSH activity and cholesterol assimilation of MKSR and Rambnosus GG strains.
  • BSH Bact salt hydrolase
  • the reduction of bile salts increases the bile acid conversion of cholesterol in the liver, thereby maintaining homeostasis in the body and ultimately lowering blood cholesterol levels (Choi et al., 2014).
  • the MKSR strain according to the present invention decombines the bile acid in culture by measuring the BSH activity, it can be confirmed that it can reduce cholesterol by absorbing up to 59.76% cholesterol.
  • Rambnosus GG strain as a control absorbed 44.26% cholesterol in the medium, which is about 15% lower than the MKSR strain according to the present invention.
  • Lee et al. (2011) also found plantarum NR74 strain and Lact .
  • the rhamnosus GG strain has been shown to reduce about 48% cholesterol in the medium, at least about 10% lower than the MKSR strain according to the invention.
  • MKSR strain according to the present invention has a BSH activity and excellent cholesterol assimilation ability compared to other strains, can significantly reduce cholesterol in the blood can be expected to prevent or treat hyperlipidemia .
  • Bacteria cultured at 30 ° C. for 18 hours were streaking four-stroke streaking using Tryptic soy + 5% Sheep blood (Kisan Bio Co., Korea) solid medium, and then cultured in a 37 ° C. incubator. Staphylococcus aureus was used as a positive control.
  • Hemolysis is an abnormal action formed by the normal action of erythrocyte destruction and the genetic defect of erythrocytes, chemicals, poisons such as snakes, and toxic substances produced by microorganisms. If hemolysis occurs in cells, the oxygen transport function of red blood cells is lost, resulting in fatal results in vivo (Yun Hye-ju et al., 2013).
  • lactic acid bacteria was evaluated for the presence of hemolytic results obtained as shown in FIG. The red blood cells were destroyed around the cells of S. aureus , a control group, and a transparent ring was formed. All three types of lactic acid bacteria used in this study showed no clear ring due to the destruction of red blood cells around the cells. See FIG. 7).

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Abstract

La présente invention concerne une souche de Leuconostoc mesenteroides MKSR déposée sous le numéro d'accession KCTC13842BP et son utilisation. De façon spécifique, la présente invention concerne une nouvelle souche de Leuconostoc mesenteroides, un aliment fonctionnel diététique pour prévenir ou améliorer le diabète contenant la souche, un aliment fonctionnel diététique pour améliorer le cholestérol contenant la souche, une composition pharmaceutique pour prévenir ou traiter le diabète contenant la souche, une composition pharmaceutique pour prévenir ou traiter l'hyperlipidémie contenant la souche, et un additif alimentaire.
PCT/KR2019/005145 2018-04-27 2019-04-29 Souche de leuconostoc mesenteroides et son utilisation Ceased WO2019209086A1 (fr)

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KR1020180049234A KR101977255B1 (ko) 2018-04-27 2018-04-27 기탁번호 KCTC18665P로 기탁된 류코노스톡 메젠테로이드 MKSR(Leuconostoc mesenteroides MKSR)균주 , 이를 포함하는 당뇨 예방 또는 개선용 건강기능 식품 및 식품첨가제
KR10-2018-0049234 2018-04-27
KR10-2019-0046715 2019-04-22
KR1020190046715A KR20200123617A (ko) 2019-04-22 2019-04-22 류코노스톡 메젠테로이드 mksr 균주를 함유하는 콜레스테롤 개선용 건강 기능 식품, 및 고지혈증 예방 또는 치료용 약학 조성물

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WO2023282577A1 (fr) * 2021-07-06 2023-01-12 단국대학교 천안캠퍼스 산학협력단 Produit de fermentation de momordica charantia probiotique ayant une fonction antidiabétique, antidémence et antioxydante améliorée
KR20230007957A (ko) * 2021-07-06 2023-01-13 단국대학교 천안캠퍼스 산학협력단 항당뇨, 항치매 및 항산화 기능을 강화한 프로바이오틱 여주 발효물
CN117165471A (zh) * 2023-08-23 2023-12-05 湖南麦肯伟科技有限公司 一种产广谱抗菌肽的肠膜明串珠菌及其应用
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WO2023282577A1 (fr) * 2021-07-06 2023-01-12 단국대학교 천안캠퍼스 산학협력단 Produit de fermentation de momordica charantia probiotique ayant une fonction antidiabétique, antidémence et antioxydante améliorée
KR20230007957A (ko) * 2021-07-06 2023-01-13 단국대학교 천안캠퍼스 산학협력단 항당뇨, 항치매 및 항산화 기능을 강화한 프로바이오틱 여주 발효물
KR102842536B1 (ko) * 2021-07-06 2025-08-05 단국대학교 천안캠퍼스 산학협력단 항당뇨, 항치매 및 항산화 기능을 강화한 프로바이오틱 여주 발효물
CN117165471A (zh) * 2023-08-23 2023-12-05 湖南麦肯伟科技有限公司 一种产广谱抗菌肽的肠膜明串珠菌及其应用
WO2025218360A1 (fr) * 2024-04-19 2025-10-23 大江生医股份有限公司 Utilisation de leuconostoc mesenteroides et/ou de métabolites correspondants

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