KR102570432B1 - Lactobacillus plantarum K97 and uses thereof - Google Patents

Abstract

The present invention relates to the Lactobacillus plantarum K97 KACC 81119BP strain having blood sugar lowering activity, and more particularly, by including the Lactobacillus plantarum K97 KACC 81119BP strain as an active ingredient Since it can prevent, improve or treat diabetes, it can be used as a food composition for preventing or improving diabetes, furthermore, a health functional food or pharmaceutical composition, a feed composition, and a pharmaceutical composition for animals.

Description

Lactobacillus plantarum K97 and uses thereof {Lactobacillus plantarum K97 and uses thereof}

The present invention relates to Lactobacillus plantarum K97 and uses thereof. In particular, it has α-amylase or α-glucosidase inhibitory activity, acid resistance, bile resistance or intestinal adhesion, excellent ability to produce short-chain fatty acids, inhibition of glucose absorption and secretion of insulin. It relates to a Lactobacillus plantarum K97 strain capable of simultaneously promoting and controlling insulin resistance and its use.

Due to the westernized diet and lack of exercise, the incidence of various life-related adult diseases is increasing.

Diabetes mellitus is a metabolic disorder caused by impaired secretion and lack of action of insulin secreted from pancreatic cells, accompanied by excessive production of glucose, breakdown of body fat and waste of protein, and abnormal secretion of glucagon, resulting in metabolic confusion. do. In the case of diabetes, it shows the characteristic symptoms of hyperglycemia due to an abnormal physiological metabolic control function such as carbohydrate, protein, lipid and electrolyte metabolism due to an imbalance of hormones including insulin. It causes macrovascular complications such as stroke, angina pectoris, myocardial infarction, and peripheral vascular disease, and in severe cases, it can lead to death.

Diabetes is largely classified into two types. Type 1 diabetes is caused by a lack of secretion of insulin, a hormone that regulates blood glucose, and type 2 diabetes is a defect in insulin secretion in pancreatic beta cells and a defect in insulin action in target cells. (insulin resistance) are all observed. Insulin resistance refers to a state in which insulin action is reduced in the absence of insulin deficiency.

The most important point in diabetes treatment is to control blood sugar levels as close to normal as possible. Treatment methods include drug therapy, diet therapy, and exercise therapy. Drugs include hypoglycemic agents (alpha glucosidase inhibitors - acarbose, miglitol, voglibose, etc.), insulin secretion stimulants (sulfonylurease, meglitinide, etc.), insulin sensitivity improvers (bigunaide, glitazone, etc.), Incretin agents (exenatide, liraglutide, etc. as GLP-1 analogues, sitagliptin, vildagliptin, saxagliptin, etc. as DPP-4 inhibitors) and insulin therapy are known.

However, since it is difficult to reach the target blood glucose level with the above drugs alone, in many cases, combination therapy or combination therapy with hypoglycemic agents is performed. These diabetes treatment drugs are often accompanied by side effects such as hypoglycemia, anorexia, nausea, vomiting, diarrhea, skin rash, lactic acidosis, liver damage, and gastrointestinal damage. In consideration of the above-mentioned problems, natto bacteria such as Bacillus subtilis having alpha-glucosidase inhibitory activity have been developed, but since Bacillus subtilis is an aerobic bacterium and is difficult to inhabit in the intestine, it must be supplied through separate intake, In order to be sufficiently supplied, a large amount must be consumed, and since it acts only on one mechanism, there is a problem in that a sufficient blood sugar control effect cannot be obtained.

Therefore, there is an urgent need to develop a substance capable of preventing or treating diabetes using a natural product that is edible and has few side effects.

Patent Document 1. Korean Patent Publication No. 10-2019-0068078

The present invention has been made to solve the above problems, and an object of the present invention is Lactobacillus plantarum K97 KACC 81119BP having excellent hypoglycemic activity, which can prevent the occurrence and recurrence of diabetes is to provide

An object of the present invention is to provide a probiotic composition comprising Lactobacillus plantarum K97 KACC 81119BP or a culture thereof as an active ingredient.

An object of the present invention is to provide a food composition for improving postprandial blood sugar containing a Lactobacillus plantarum K97 KACC 81119BP strain or a culture thereof as an active ingredient.

An object of the present invention is to provide a food composition for preventing or improving diabetes comprising Lactobacillus plantarum K97 KACC 81119BP or a culture thereof as an active ingredient.

An object of the present invention is to provide a feed composition for preventing or improving diabetes comprising Lactobacillus plantarum K97 KACC 81119BP or a culture thereof as an active ingredient.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes comprising Lactobacillus plantarum K97 KACC 81119BP or a culture thereof as an active ingredient.

In order to achieve the above object, the present invention provides Lactobacillus plantarum K97 KACC 81119BP having hypoglycemic activity.

The Lactobacillus plantarum K97 KACC 81119BP may have a base sequence of 16s rRNA represented by SEQ ID NO: 1.

The Lactobacillus plantarum K97 KACC 81119BP may have α-amylase or α-glucosidase inhibitory activity.

The Lactobacillus plantarum K97 KACC 81119BP may have acid resistance, bile resistance and intestinal adhesion.

The Lactobacillus plantarum K97 KACC 81119BP may improve insulin resistance.

The present invention provides a probiotic composition comprising Lactobacillus plantarum K97 KACC 81119BP or a culture thereof as an active ingredient in order to achieve the above other object.

In order to achieve the above another object, the present invention provides a food composition for improving postprandial blood sugar containing a Lactobacillus plantarum K97 KACC 81119BP strain or a culture thereof as an active ingredient.

In order to achieve the above another object, the present invention provides a food composition for preventing or improving diabetes comprising Lactobacillus plantarum K97 KACC 81119BP or a culture thereof as an active ingredient.

In order to achieve the above another object, the present invention provides a feed composition for preventing or improving diabetes comprising Lactobacillus plantarum K97 KACC 81119BP or a culture thereof as an active ingredient.

In order to achieve the above another object, the present invention provides a pharmaceutical composition for preventing or treating diabetes comprising Lactobacillus plantarum K97 KACC 81119BP or a culture thereof as an active ingredient.

The Lactobacillus plantarum K97 KACC 81119BP strain of the present invention has excellent acid resistance, bile resistance, and intestinal adhesion, and has excellent effects on lowering blood sugar, promoting insulin secretion, and improving insulin resistance, so that it can prevent, improve or improve diabetes. It can be used as a food composition for treatment, furthermore, a health functional food, a feed composition for preventing or improving diabetes, a pharmaceutical composition for preventing or treating diabetes, or a pharmaceutical composition for animals for preventing or treating diabetes.

In addition, since the Lactobacillus plantarum K97 KACC 81119BP strain also has antibacterial activity against pathogenic microorganisms and food poisoning bacteria, it can be used as a probiotic composition.

1 shows a phylogenetic diagram of the Lactobacillus plantarum K97 strain according to the present invention.
Figure 2 is a medium (with oxgall) in which 0.3% oxgall was added to the MRS broth and a medium without oxgall (without oxgall) Lactobacillus plantarum (La ctobacillus plantarum) K97 strain It is a growth curve according to the time of culturing each.
Figure 3 is a graph showing the survival rate measured after 3 hours when the Lactobacillus plantarum K97 strain was inoculated in an HCl solution.
4 is a graph showing the intestinal adhesion ability of the Lactobacillus plantarum K97 strain in HT-29 cells.
5 is a graph showing the survival rate of RIN-m5F cells measured when the Lactobacillus plantarum K97 strain was treated by concentration.
6 is a graph showing the amount of insulin secretion measured when RIN-m5F cells were treated with Lactobacillus plantarum K97 strain at different concentrations.
7 is a graph showing the expression level of GLP-1R, a gene involved in insulin secretion, measured when RIN-m5F cells were treated with Lactobacillus plantarum K97 strain at different concentrations.
8 is a graph showing the expression level of IRS-2, a gene involved in insulin secretion, measured when RIN-m5F cells were treated with Lactobacillus plantarum K97 strain at different concentrations.
9 is a graph showing the survival rate of C1C12 cells measured when the Lactobacillus plantarum K97 strain was treated by concentration.
10 is a graph showing the measurement of glucose uptake of C1C12 cells when the Lactobacillus plantarum K97 strain was treated by concentration.
Figure 11 is a western measurement of the expression level of proteins (p-AKT / AKT) involved in sugar metabolism and utilization when C2C12 cells were treated with Lactobacillus plantarum K97 strain at different concentrations This is the blot result.
12 is a graph showing the numerical results of the results of FIG. 11 .
13 is a Western blot result of measuring the expression level of a protein (GLUT4) involved in glucose metabolism and utilization when C2C12 cells were treated with Lactobacillus plantarum K97 strain at different concentrations.
14 is a graph showing the numerical results of FIG. 13 .
15 is a graph showing the body weight of a normal group, a control group, an experimental group (K97), and a positive control over time.
16 is a graph showing AUC (Area under the curve) for blood glucose levels according to an oral glucose tolerance test.
17 is a graph showing the measurement of fructosamine in serum of a normal group, a control group, an experimental group (K97), and a positive control group.
18 is a graph showing the concentration of C-peptide measured in the serum of a normal group, a control group, an experimental group (K97), and a positive control group.
19 is a result of measuring insulin present in pancreatic islets of a normal group, a control group, an experimental group (K97), and a positive control group by immunohistochemical staining.
20 is a result of measuring glucagon present in pancreatic islets of a normal group, a control group, an experimental group (K97), and a positive control group by immunohistochemical staining. am.

In the following, various aspects and various embodiments of the present invention will be described in more detail.

One aspect of the present invention relates to Lactobacillus plantarum K97 strain KACC 81119BP having hypoglycemic activity.

As used herein, the term "hypoglycemic activity" refers to an effect of lowering blood sugar, which is a value representing the concentration of glucose in the blood. In order to maintain homeostasis, the human body preferably maintains a blood sugar level within a certain range. Hyperglycemia refers to an abnormally high blood sugar level, and hyperglycemia that appears temporarily after a meal is a natural phenomenon and is referred to as physiological hyperglycemia. However, if the blood glucose level increases beyond this range or the hyperglycemic state continues, it is highly likely that diabetes has occurred, there is a possibility of progressing to diabetes, or diabetes has developed but has not been detected. According to the U.S. Food and Drug Administration regulations, normal blood sugar level is defined as fasting blood sugar less than 100 mg/dl and 2-hour postprandial blood sugar less than 140 mg/dl, and fasting blood sugar of 126 mg/dl or more and 2-hour postprandial blood sugar of 200 mg/dl or more. Diagnosis is diagnosed as diabetes. Meanwhile, WHO defines a fasting blood sugar level of less than 110 mg/dl as a normal level. Even if it is less than the level diagnosed as diabetes, when the hyperglycemic state is higher than the normal state, it induces fatty liver, increases the risk of progressing to severe liver disease, and may cause cardiovascular disease and its complications. Glucagon, adrenaline, insulin, and thyroid hormones work to maintain normal blood sugar levels in the body. An abnormality in the secretion and/or activity of these hormones can cause hyperglycemia. Excessive sugar intake, lack of exercise, and/or stress also cause hyperglycemia. Lactobacillus plantarum K97 strain KACC 81119BP of the present invention has alpha-amylase inhibitory activity and alpha-glucosidase inhibitory activity, increased sugar metabolism and utilization (promotes glucose absorption) ), it exhibits multiple hypoglycemic effects based on various mechanisms of promoting insulin secretion and improving insulin resistance, so it can have preventive and ameliorative effects on diseases caused by hyperglycemia.

Specifically, Lactobacillus plantarum K97 strain KACC 81119BP, a novel strain having blood glucose lowering activity, blood sugar control, glucose absorption promotion, insulin secretion promotion and insulin resistance improvement effects, is useful for preventing and treating diabetes can be utilized The Lactobacillus plantarum K97 strain KACC 81119BP of the present invention may have a 16S rRNA base sequence represented by SEQ ID NO: 1.

As a result of 16S rRNA sequencing analysis for the identification and classification of La ctobacillus plantarum K97 KACC 81119BP of the present invention, it was confirmed that bacteria having completely 100% homology were not searched. Therefore, while naming the microorganism of the present invention as Lactobacillus plantarum K97 KACC 81119BP strain, it was deposited with the National Academy of Agricultural Sciences (KACC) of the Rural Development Administration on June 25, 2020, and was given the deposit number KACC 81119BP received.

Name of depository institution: Center for Agricultural Genetic Resources (KACC), National Academy of Agricultural Sciences

Accession number: KACC 81119BP

Entrusted date: 2020.06.25

The Lactobacillus plantarum K97 KACC 81119BP strain of the present invention isolates pure 394 strains from kimchi and feces, and has alpha-amylase inhibitory activity and alpha-glucosidase (α- glucosidase) was finally selected by measuring the enzyme activity of inhibitory activity.

The Lactobacillus plantarum K97 KACC 81119BP strain used in the present invention has alpha-amylase or alpha-glucosidase inhibitory activity and has excellent ability to produce short-chain fatty acids Since it provides blood sugar lowering and antidiabetic effects based on promoting glucose absorption, regulating insulin secretion, improving insulin resistance, increasing expression of p-AKT/AKT, GLUT4, GLP-1R and IRS-2, and improving glucose metabolism and utilization, It has the effect of simultaneously improving various mechanisms. Therefore, the novel Lactobacillus plantarum K97 strain of the present invention can exhibit an excellent antidiabetic effect even when administered alone, and in some cases may be administered together with a commercially available antidiabetic agent. When administered together, blood glucose concentration can be rapidly reduced.

Moreover, the Lactobacillus plantarum K97 KACC 81119BP strain of the present invention can reduce side effects such as flatulence and gastrointestinal disorders caused by oral hypoglycemic agents by suppressing the growth of harmful microorganisms in the intestine.

The Lactobacillus plantarum K97 KACC 81119BP strain of the present invention has excellent acid resistance, bile resistance and intestinal adhesion, so it can survive safely through the oral cavity to the intestine. Since it has antibacterial activity to inhibit the growth of pathogenic microorganisms or food poisoning bacteria in the intestine, it can provide advantageous efficacy as a prebiotic material.

In addition, the Lactobacillus plantarum K97 KACC 81119BP strain of the present invention has excellent antibiotic resistance to vancomycin, ampicillin, and polymyxin B, and in addition to amikacin ( Amikacin), gentamycin, kanamycin, streptomycin, penicillin-G, oxacillin, bacitracin, ciprofloxacin, tetracycline ( antibiotic resistance to tetracycline).

In addition, Lactobacillus plantarum K97 KACC 81119BP of the present invention is alkaline phosphatase, esterase (Esterase (C4)), esterase lipase (Esterase Lipase (C8)), Lipase (C14) (Lipase (14)), Leucine arylamidase, Valine arylamidase, Cystine arylamidase, Acid phosphatase, Naphtol-AS-BI-phosphohydrolase, alpha-galactosidase, beta-galactosidase, alpha-glucosidase (α-glucosidase), beta-glucosidase (β-glucosidase) and N-acetyl-beta-glucosidase (N-acetyl-β-glucosaminidase) may exhibit enzymatic activity. In particular, the Lactobacillus plantarum K97 KACC 81119BP strain of the present invention has no enzymatic activity for beta-glucuronidase, a carcinogenic factor that converts benzopyrene into a carcinogenic substance. It is a very stable strain.

Through the following examples, when the Lactobacillus plantarum K97 KACC 81119BP strain of the present invention is orally administered, an excellent blood glucose lowering effect was shown by promoting glucose absorption, promoting insulin secretion and improving insulin resistance, , which was confirmed to be equal to or superior to the positive control group (POS CON) administered with metformin, a conventional diabetes treatment.

Therefore, Lactobacillus plantarum K97 according to the present invention Since the KACC 81119BP strain has a hypoglycemic and/or antidiabetic effect as well as a prebiotic composition, it can be used as a food, feed or pharmaceutical composition for preventing, improving or treating diabetes.

The Lactobacillus plantarum K97 KACC 81119BP strain of the present invention may be used directly or may be a culture obtained by culturing the strain. The culture may be a culture medium of a strain and/or a material containing the cultured strain. The culture may be a culture solution obtained by culturing the strain and/or a material containing the cultured strain itself. The culture medium may be a culture medium itself, or any one selected from a culture medium concentrate and a dried culture medium. The culture medium concentrate means that the culture medium is concentrated, and the dry matter of the culture medium means that the culture medium is dried. The cell may be a viable cell.

The Lactobacillus plantarum K97 KACC 81119BP strain according to the present invention is grown by a general culture method, can be recovered and concentrated by a separation process such as centrifugation, and dried (eg, freeze-dried) It can be prepared and used in the form of a probiotic by

Another aspect of the present invention relates to a probiotic composition comprising the Lactobacillus plantarum K97 KACC 81119BP strain or its culture as an active ingredient.

The Lactobacillus plantarum (La ctobacillus plantarum ) K97 KACC 81119BP strain is as described above.

In the present invention, the term "culture" is not limited to materials containing the culture solution and / or cultured strain of Lactobacillus plantarum K97 KACC 81119BP strain, but Lactobacillus plantarum (La ctobacillus plantarum ) K97 KACC 81119BP refers to the entire medium including the strain obtained by culturing the strain for a certain period of time in a medium supplying nutrients so that it can grow and survive in vitro, its metabolites, and extra nutrients, and the strain A culture solution from which strains are removed after cultivation is also included therein. The culture is not limited in its formulation, and may be specifically liquid or solid.

In the present invention, the term "probiotics" may be used interchangeably with 'probiotics', and refers to live bacteria that benefit the human body when ingested in an appropriate amount, and refers to bacteria that benefit the body.

The Lactobacillus plantarum K97 KACC 81119BP strain according to the present invention has excellent acid resistance, bile resistance and intestinal adhesion, so it survives gastric acid and bile acid in the body, reaches the small intestine, proliferates and settles in the intestine can In particular, this strain shows antibacterial activity against pathogenic microorganisms and food poisoning bacteria, so that beneficial effects on health can be expected in the colonized intestine, such as suppression of harmful microorganisms and normalization of intestinal flora. Therefore, since all of the above conditions to be provided as a probiotic composition are significantly satisfied, it can be usefully utilized as a probiotic composition.

The probiotic composition according to the present invention may include conventional foods, functional foods or health supplements, food additives, as well as those approved as pharmaceuticals.

The composition of the present invention may be prepared according to a conventional probiotic composition manufacturing method, and generally may be lyophilized or encapsulated form, culture suspension, or dry powder form. In addition, one or two or more pharmaceutically acceptable conventional carriers or one or two kinds of Lactobacillus plantarum K97 KACC 81119BP strain or its culture, which is an active ingredient of the probiotic composition, By selecting the above additives, it can be prepared into a composition of a conventional dosage form.

The carrier may be used by selecting at least one selected from the group consisting of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives, and the additives are selected from the group consisting of flavoring agents, vitamins and antioxidants Any one or more can be selected and used.

All pharmaceutically acceptable carriers and additives may be used, and specifically, diluents include lactose monohydrate, trehalose, corn starch, soybean oil, and microcrystalline cellulose. (microcrystalline cellulose) or mannitol (D-mannitorl) is preferable, magnesium stearate or talc is preferable as a lubricant, and polyvinylpyrrolidone (PVP: polyvinyipyrolidone) or hydroxypropyl as a binder It is preferable to select from cellulose (HPC: hydroxypropylcellulose). In addition, the disintegrant is selected from carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, polacrylin potassium, or cross-linked polyvinylpyrrolidone. Preferably, the sweetener is selected from white sugar, fructose, sorbitol, or aspartame, and the stabilizer is carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin, and white lead. It is selected from (white bee's wax) or xanthan gum, and as a preservative, methyl p-hydroxy benzoate (methlparaben), propyl p-hydroxybenzoate (propylparaben), or potassium sorbate ( potassium sorbate) is preferred.

In addition, the Lactobacillus plantarum K97 KACC 81119BP strain of the present invention can be used as a spawn for producing various lactic acid bacteria fermented milk and fermented products, wherein the fermented milk food includes yogurt, calpis, cheese, butter, etc. and fermented products include tofu, soybean paste, cheonggukjang, jelly, kimchi, etc., but are not necessarily limited thereto. The fermented milk and fermented products can be easily produced by replacing only the strain with the strain of the present invention in a conventional manufacturing method. As described above, the composition of the present invention may be a food or an additive for food, and the food is preferably a fermented food. In addition, the probiotic composition of the present invention may be in powder or granular form, and the powder or granular form can be easily prepared by a conventional method in the art.

Another aspect of the present invention relates to a food composition for lowering blood sugar comprising a Lactobacillus plantarum K97 KACC 81119BP strain or a culture thereof as an active ingredient.

The Lactobacillus plantarum (La ctobacillus plantarum ) K97 KACC 81119BP strain is as described above.

According to an embodiment of the present invention, the Lactobacillus plantarum K97 KACC 81119BP strain promotes glucose uptake in cells, promotes insulin secretion, and simultaneously lowers blood sugar through a complex mechanism that improves insulin resistance has the effect of Therefore, it was confirmed that fasting or postprandial blood glucose can be effectively reduced, and diabetes can be prevented or treated through the hypoglycemic action in an actual diabetic animal model.

In addition, according to the present invention, it was confirmed that the Lactobacillus plantarum K97 KACC 81119BP strain significantly reduces the average HbA1c concentration in a diabetic animal model, preventing or preventing diabetes through a hypoglycemic action. know that it can be treated.

Therefore, the Lactobacillus plantarum K97 KACC 81119BP strain according to the present invention can be used as an active ingredient of a functional food capable of suppressing or lowering blood sugar rise in diabetic patients on an empty stomach or after eating.

The Lactobacillus plantarum K97 KACC 81119BP strain of the present invention showed no toxicity even when treated with a very large amount of cells.

As used herein, the term "lowering blood sugar" refers to an effect of lowering blood sugar, which is a value representing the concentration of glucose in the blood.

The 'food composition' is an active ingredient, in addition to the Lactobacillus plantarum K97 KACC 81119BP strain or its culture, the food described in the standards and specifications of food commonly used in food manufacturing ('Food Code') It includes food raw materials and food additives described in the Food Additive Code that can be used as

It does not need to be particularly limited, but includes, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. The carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sugar, lactose and the like; oligosaccharides or polysaccharides such as dextrin, starch syrup, cyclodextrin and the like; Sugar alcohols such as xylitol, sorbitol, erythritol and the like can be used. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used.

When preparing a food composition using the Lactobacillus plantarum K97 KACC 81119BP strain or its culture as an active ingredient, it is not necessary to specifically limit as long as the content exhibits hypoglycemic activity, but 1 to 500 μg in the composition / ml concentration, preferably 1 to 300 μg / ml concentration, more preferably 1 to 100 μg / ml concentration, even more preferably 1 to 30 μg / ml concentration, most preferably 1 to 30 μg / ml concentration It may be included at a concentration of 10 μg/ml.

Lactobacillus plantarum, the active ingredient in the food composition, K97 KACC 81119BP strain or its culture varies depending on the condition, weight, presence or absence of disease and duration, but appropriately by a person skilled in the art can be chosen For example, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, even more preferably 20 to 800 mg, and most preferably 50 to 500 mg based on the daily dose. There is, and the number of administrations need not be particularly limited, but can be adjusted by a person skilled in the art within the range of three times a day to once a week. In the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range.

The food composition does not need to be particularly limited, but may be, for example, a powder, granule, tablet, capsule, pill, extract, jelly formulation, tea bag formulation or beverage formulation.

In addition, the Lactobacillus plantarum K97 KACC 81119BP strain or its culture may be added to general foods to induce hypoglycemic activity and hypoglycemic activity, and foods that can be added are particularly limited It is not necessary, but for example, confectionery, bread or rice cakes, cocoa products or chocolates, meat or egg products, fish meat products, tofu, or It can be added to jelly, noodles, tea, coffee, beverages, special purpose foods, pastes, seasoning foods, dressings, kimchi, salted fish, pickled foods, stewed foods, alcoholic beverages, raisins, and other foods. In addition, it can be added to dairy products, processed meat products, packaged meat, and egg products exemplified in the processing standards and ingredient specifications of livestock products ('Livestock Codex') according to Article 4 of the Livestock Products Sanitation Control Act.

On the other hand, the food composition containing the Lactobacillus plantarum K97 KACC 81119BP strain or its culture as an active ingredient can be used alone as a "health functional food that helps lower blood sugar".

The 'health functional food' refers to food manufactured (including processing) according to legal standards using raw materials or ingredients having functional properties useful for the human body (Article 3, Subparagraph 1 of the Health Functional Food Act). The term 'health functional food' may differ in terminology or scope from country to country, but 'Dietary Supplement' in the US, 'Food Supplement' in Europe, 'Health Functional Food' or 'Health Functional Food' in Japan. It may correspond to 'Food for Special Health Use (FoSHU)' or 'Health Food' in China.

The food composition or health functional food may additionally contain food additives, and the suitability as a food additive is determined according to the standards and standards for the item in accordance with the general rules and general test methods of the 'Food Additive Code' unless otherwise specified. follow

In addition, the health functional food includes the Lactobacillus plantarum K97 KACC 81119BP strain or its culture together with raw materials notified as 'functional raw materials' used in "health functional food that helps lower blood sugar" Or as individually recognized raw materials, pine needle distillation concentrate, soybean fermented extract, albumin, freeze-dried silkworm powder, crustacean leaf extract mixture, Seomoktae peptide complex, ginseng hydrolyzed concentrate, tagatose, hydroxypropylmethylcellulose, leaf leaf extract, L- arabinose, silk protein enzyme hydrolysate, pinitol, rhodiola rosea complex extract, cinnamon extract powder, three lipolia axerata mycelium culture, sea polynol Ecklonia extract, chitooligosaccharide, guava leaf extract, banaba leaf extract, evening primrose seed extract, Health functional food ingredients related to diabetes, such as guar gum/guar gum hydrolysate, oat dietary fiber, indigestible maltodextrin, soybean dietary fiber, wheat dietary fiber, corn bran dietary fiber, inulin/chicory extract, and fenugreek seed dietary fiber Can be used in combination.

Another aspect of the present invention relates to a food composition for preventing or improving diabetes comprising a Lactobacillus plantarum K97 KACC 81119BP strain or a culture thereof as an active ingredient.

The food composition of the present invention may prevent or improve diabetes by including the Lactobacillus plantarum K97 KACC 81119BP strain or its culture as an active ingredient. In addition, the food composition of the present invention contains the Lactobacillus plantarum K97 KACC 81119BP strain as an active ingredient, and has alpha-amylase inhibitory activity and alpha-glucosidase inhibitory activity Diabetes can be prevented or improved by improving any one or more of glucose metabolism and utilization rate increase (glucose absorption promotion), insulin secretion promotion and insulin resistance improvement-based inhibition of blood glucose rise and blood insulin secretion normalization.

In the present invention, the term "diabetes mellitus (DM, diabetes)" is a type of metabolic disease in which high blood sugar levels persist for a long period of time. Symptoms of high blood sugar include frequent urination, and intense thirst and hunger. If left untreated, it can lead to other complications that can lead to death.

In the present invention, diabetes is meant to include insulin-dependent diabetes (type 1 diabetes) and non-insulin-dependent diabetes (type 2 diabetes), and furthermore, diabetes caused by damage to the pancreas due to other diseases, such as thyroid function Hyperactivity, hyperadrenocortical function, diabetes due to hypergrowth hormone or catecholamine hyperactivity, and gestational diabetes are meant to be included.

In the present invention, the term "prevention" refers to all activities that inhibit or delay diabetes by administering a composition containing the Lactobacillus plantarum K97 KACC 81119BP strain or its culture as an active ingredient.

In the present invention, the term "improvement" refers to the intake of a composition containing the Lactobacillus plantarum K97 KACC 81119BP strain or its culture as an active ingredient, reducing parameters related to diabetes, for example, the degree of diabetic symptoms means all actions.

In the present invention, the term "treatment" refers to all activities in which symptoms of diabetes are improved or cured by administration of a composition containing the Lactobacillus plantarum K97 KACC 81119BP strain or its culture as an active ingredient.

The 'food composition' is an active ingredient, in addition to the Lactobacillus plantarum K97 KACC 81119BP strain or its culture, the food described in the standards and specifications of food commonly used in food manufacturing ('Food Code') It includes food raw materials and food additives described in the Food Additive Code that can be used as

It does not need to be particularly limited, but includes, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. The carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sugar, lactose and the like; oligosaccharides or polysaccharides such as dextrin, starch syrup, cyclodextrin and the like; Sugar alcohols such as xylitol, sorbitol, erythritol and the like can be used. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used.

When preparing a food composition using the Lactobacillus plantarum K97 KACC 81119BP strain or its culture as an active ingredient, it is not necessary to specifically limit as long as the content exhibits the efficacy of preventing or improving diabetes, but in the composition It may be included in a concentration of 1 to 500 μg / ml, preferably a concentration of 1 to 300 μg / ml, more preferably a concentration of 1 to 100 μg / ml, even more preferably a concentration of 1 to 30 μg / ml, most preferably Preferably, it may be included at a concentration of 1 to 10 μg/ml.

Lactobacillus plantarum, the active ingredient in the food composition, K97 KACC 81119BP strain or its culture varies depending on the condition, weight, presence or absence of disease and duration, but appropriately by a person skilled in the art can be chosen For example, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, even more preferably 20 to 800 mg, and most preferably 50 to 500 mg based on the daily dose. There is, and the number of administrations need not be particularly limited, but can be adjusted by a person skilled in the art within the range of three times a day to once a week. In the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range.

The food composition does not need to be particularly limited, but may be, for example, a powder, granule, tablet, capsule, pill, extract, jelly formulation, tea bag formulation or beverage formulation.

In addition, the Lactobacillus plantarum K97 KACC 81119BP strain or its culture may be added to general foods to prevent or improve diabetes, and foods that can be added do not need to be particularly limited, but for example Confectionery, bread or rice cakes, processed cocoa products or chocolates, processed meat or egg products, processed fish meat products, tofu or jelly, noodles, teas, coffee It can be added to beverages, special purpose foods, pastes, seasoning foods, dressings, kimchi, pickled fish, pickled foods, stewed foods, alcoholic beverages, raisins, and other foods. In addition, it can be added to dairy products, processed meat products, packaged meat, and egg products exemplified in the processing standards and ingredient specifications of livestock products ('Livestock Codex') according to Article 4 of the Livestock Products Sanitation Control Act.

On the other hand, the food composition containing the Lactobacillus plantarum K97 KACC 81119BP strain or its culture as an active ingredient can be used alone as a "health functional food that helps prevent or improve diabetes".

The 'health functional food' refers to food manufactured (including processing) according to legal standards using raw materials or ingredients having functional properties useful for the human body (Article 3, Subparagraph 1 of the Health Functional Food Act). The term 'health functional food' may differ in terminology or scope from country to country, but 'Dietary Supplement' in the US, 'Food Supplement' in Europe, 'Health Functional Food' or 'Health Functional Food' in Japan. It may correspond to 'Food for Special Health Use (FoSHU)' or 'Health Food' in China.

The food composition or health functional food may additionally contain food additives, and the suitability as a food additive is determined according to the standards and standards for the item in accordance with the general rules and general test methods of the 'Food Additive Code' unless otherwise specified. follow

In addition, the health functional food, along with the Lactobacillus plantarum K97 KACC 81119BP strain or its culture, is notified as a 'functional raw material' used in "health functional food that helps prevent or improve diabetes" Pine needle distillation concentrate, soybean fermented extract, albumin, freeze-dried silkworm powder, crustacean leaf extract mixture, Seomoktae peptide complex, ginseng hydrolyzed concentrate, tagatose, hydroxypropylmethylcellulose, leaf extract, L-arabinose, silk protein enzyme hydrolysate, pinitol, rhodiola rosea complex extract, cinnamon extract powder, three lipolia lockserata mycelium culture, sea polynol Ecklonia alcohol extract, chitooligosaccharide, guava leaf extract, banaba leaf extract, evening primrose seed Health functional foods related to diabetes, such as extract, guar gum/guar gum hydrolysate, oat dietary fiber, indigestible maltodextrin, soybean dietary fiber, wheat dietary fiber, corn bran dietary fiber, inulin/chicory extract, fenugreek seed dietary fiber, etc. A combination of materials can be used.

The present invention relates to a feed composition for preventing or improving diabetes comprising a Lactobacillus plantarum K97 KACC 81119BP strain or a culture thereof as an active ingredient.

The 'feed composition' is an active ingredient, in addition to the Lactobacillus plantarum K97 KACC 81119BP strain or its culture, food raw materials that can be used as food described in food standards and specifications ('Food Code'), food Food additives described in the Code of Additives can be used, and even if they are not food raw materials or food additives that can be used as food, they are not included in the scope of raw materials in the scope of single feed in Attached Table 1 of the 'Standards and Specifications for Feed, etc.', and in the scope of supplementary feed in Attached Table 2. Appropriate raw materials can be used.

The 'feed composition' may be an extractant in supplementary feed according to 'standards and specifications for feed, etc.', and may be a formulated feed containing the supplementary feed.

When preparing a feed composition using the Lactobacillus plantarum K97 KACC 81119BP strain or its culture as an active ingredient, it does not need to be particularly limited as long as the content exhibits the efficacy of preventing or improving diabetes, but in the composition It may be included at a concentration of 1 to 500 μg/ml, preferably at a concentration of 1 to 100 μg/ml, more preferably at a concentration of 1 to 30 μg/ml, and most preferably at a concentration of 1 to 10 μg/ml. .

Lactobacillus plantarum, the active ingredient in the feed composition, K97 KACC 81119BP strain or its culture varies depending on the condition, weight, presence or absence or degree and duration of disease of the ingested animal, but appropriate by those skilled in the art can be chosen appropriately. For example, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, still more preferably 20 to 800 mg, and most preferably 50 to 500 mg based on the daily dose. There is, and the number of administrations need not be particularly limited, but can be adjusted by a person skilled in the art within the range of three times a day to once a week. In the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range.

The present invention relates to a pharmaceutical composition for preventing or treating diabetes comprising a Lactobacillus plantarum K97 KACC 81119BP strain or a culture thereof as an active ingredient.

In addition, the present invention relates to an oral pharmaceutical composition for animals for the prevention or treatment of diabetes comprising the Lactobacillus plantarum K97 KACC 81119BP strain or its culture as an active ingredient.

In addition, the present invention provides a method for preventing or treating diabetes by orally administering the composition to humans or non-human animals.

In addition, the present invention provides a novel use of Lactobacillus plantarum K97 KACC 81119BP or its culture for the production of a medicine for preventing or treating diabetes or a medicine for animals.

The present invention can be administered orally or parenterally to mammals, including humans, according to the desired method, and parenteral administration methods include external skin application, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or There are intrathoracic injection injection methods, etc., but oral administration is most preferred.

The 'pharmaceutical composition', 'medicine', 'veterinary pharmaceutical composition' or 'veterinary medicine' is an active ingredient, in addition to Lactobacillus plantarum K97 KACC 81119BP or its culture, manufacturing a pharmaceutical composition, etc. It may further include suitable carriers, excipients and diluents commonly used in

The 'carrier' is a compound that facilitates the addition of the compound into cells or tissues. The 'diluent' is a compound diluted in water that not only stabilizes the biologically active form of the subject compound, but also dissolves the compound.

The carrier, excipient, and diluent are not particularly limited, but, for example, lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The amount of the pharmaceutical composition, drug, veterinary pharmaceutical composition, or veterinary drug used may vary depending on the age, sex, and weight of the patient or animal to be treated, and above all, the condition of the subject to be treated, a specific category of disease to be treated, or It will depend on the type, route of administration, and nature of the therapeutic used.

The pharmaceutical composition, drug, veterinary pharmaceutical composition or veterinary drug is appropriately selected according to the absorption rate of the active ingredient in the body, the rate of excretion, the age and weight, sex and condition of the patient or animal to be treated, the severity of the disease to be treated, etc. , It is generally preferred to administer 0.1 to 1,000 mg/kg, preferably 1 to 500 mg/kg, more preferably 5 to 250 mg/kg, and most preferably 10 to 100 mg/kg per day. The unit dosage formulation thus formulated can be administered several times at regular time intervals as needed.

The pharmaceutical composition, medicament, veterinary pharmaceutical composition, or veterinary medicament may be administered individually as a prophylactic or therapeutic agent, or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The pharmaceutical composition, drug, veterinary pharmaceutical composition, or veterinary drug may be formulated into oral dosage forms such as powders, granules, tablets, capsules, troches, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods and used. there is. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sugar or lactose, and gelatin in the compound. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. .

The method for preventing or treating diabetes is to orally administer the composition to a human or non-human animal, particularly a mammal, for example, orally administering the composition to a subject having diabetes.

The dosage, administration method and number of administrations for the treatment may refer to the dosage, administration method and number of administrations of the pharmaceutical composition, medicine, veterinary pharmaceutical composition or veterinary medicine.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples are intended to explain the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Example

<Experimental Example 1> Strain Separation

In order to select candidates having blood glucose enhancing activity, 394 strains derived from kimchi and feces were collected through the following process.

Kimchi and feces were collected as samples, and 0.1 ml of each was inoculated in a modified MRS medium supplemented with bromcresol purple and sodium azide by flat smear method, and incubated at 37 ° C for 48 hours. Colonies that turned yellow after pure isolation from the modified MRS medium were selected as potential lactic acid bacteria. The selected strains were plated on a modified MRS medium, then cultured aerobically to obtain pure isolation. After pure separation, it was cultured in Lactobacilli MRS broth (Difco, USA) at 37 °C for 18 hours, then cultured and stored in tryptic soy agar (Difco, USA) slant at 37 °C for 18 hours. did

<Experimental Example 2> Selection of strains having blood glucose enhancing activity

1) Measurement of α-amylase inhibitory activity

[Xiao Z, Storms R, Tsang A. A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. Anal Biochem. 2006;351:146-148] was modified and used. An enzyme solution was prepared by diluting alpha-amylase to a concentration of 0.1 g/10 ml with distilled water. A substrate solution was prepared by adding 0.5% soluble starch to distilled water. The enzyme solution or sample was mixed with the substrate solution and reacted at 25 °C for 10 minutes. After stopping the reaction with a 0.1N HCl solution, staining was performed for 30 minutes using an iodine solution, and the absorbance was measured at 660 nm, and the result was calculated through Equation 1 below. Absorbance was shown by comparing the absorbance when only α-amylase was treated (control group) and when samples were mixed and treated. A sample means a target substance to be measured.

[Equation 1]

α-Amylase inhibitory activity (%) = 1 - (A / B) × 100

In Equation 1 above,

A is the absorbance of the sample,

B is the absorbance of the control.

2) Measurement of α-glucosidase inhibitory activity

Alpha-glucosidase (α-glucosidase) inhibitory activity was measured using p-NPG (p-nitrophenyl α-glucopyranoside) as a substrate [Si MM, Lou JS, Zhou CX, Shen JN, Wu HH, Yang B, et al. al. Insulin releasing and alpha-glucosidase inhibitory activity of ethyl acetate fraction of Acorus calamus in vitro and in vivo. J Ethnopharmacol. 2010;128:154-159.] method was used. 50 μl of the sample was mixed with 50 μl of 0.75 unit/ml alpha-glucosidase enzyme solution and 50 μl of 200 mM potassium phosphate buffer (pH 6.5), preincubated at 37 ° C for 10 minutes, and then 0.1 M 100 μl of 3 mM p-NPG dissolved in phosphate buffer (pH 7.0) was added and reacted at 37° C. for 10 minutes. The reaction was stopped with 750 μl of 0.1 M Na ₂ CO ₃ , and the absorbance was measured at 405 nm, and then calculated according to Equation 2 below. The sample means a target substance to be measured, and the control group is treated with only an alpha-glucosidase enzyme solution without a sample.

[Equation 2]

α-Glucosidase inhibitory activity (%) = 1 - (A / B) × 100

In Equation 2 above,

A is the absorbance of the sample,

B is the absorbance of the control.

3) Results

As a result of measuring the α-amylase inhibitory activity and α-glucosidase inhibitory activity of the isolated strains, K97 strain (99.70±1.15% and 99.92±0.09%, respectively) and K99 strain (90.95±0.67% and 85.18±5.66%, respectively) and K263 strain (85.00±3.06 and 88.70±0.44, respectively) showed the highest inhibitory activity. Accordingly, the K97 strain was selected.

<Experimental Example 3> Short-chain fatty acid production ability measurement

The three selected strains were tested for their ability to produce short chain fatty acids (SCFA).

First, strains were inoculated into MRS medium (broth) and 1% MRS medium (broth) supplemented with 3% indigestible polysaccharide (maltodextrin), respectively, and cultured at 37 ° C for 18 hours. After separation, the contents of propionic acid, acetic acid, and butyric acid, which are short-chain fatty acids, were measured.

1) Measurement of acetic acid content

Culture supernatants of K97, K99, and K263 strains obtained through the above process were prepared as samples. Boil 5 ml of the sample solution immediately before the experiment, cool it, dilute it until the color of the sample solution is slightly visible, add a few drops of 1% phenolphthalein solution, and then dilute with 0.1 N sodium hydroxide solution until a pale red color persists for 30 seconds. It was titrated and the total acid was calculated according to the following formula 3.

[Equation 3]

Total (%, w/v) = V1 × f × 0.006 × 100 / V2

In Equation 3,

V1 is the amount (ml) of 0.1 N sodium hydroxide solution consumed for titration,

f is the titer of 0.1 N sodium hydroxide solution (1.000);

V2 is the amount (ml) of sample solution used for titration.

2) Measurement of propionic acid content

Culture supernatants of K97, K99, and K263 strains obtained through the above process were prepared as samples. First, after adding ACN (acetonitrile) to 40 ml of the sample to 4 g, the mixture was extracted for 30 minutes using a sonicator. The extracted solution was centrifuged at 1,770 g for 10 minutes to separate the supernatant. The separated supernatant was filtered through a 0.22 μM membrane filter, concentrated using a nitrogen concentrator, and then analyzed with a gas chromatograph/mass spectrometer (GC-MS). GC-MS analysis conditions are shown in Table 1.

Device Parameter Condition GC Column HP-FFAP (0.32 mm i.d × 30 m, 0.25 μM) Oven temperature program 60℃ (4 min) → 115℃ (28℃/min)
→ 240℃(20℃/min, 5min) Inlet temperature 200℃ Injector temperature 200℃ Injection volume 1 μl Split ratio Splitless Carrier Helium, 1.0 mL/min MS Ionization mode EI Electron impact mode 70eV Selected ion (m/z) 74 ¹⁾ , 57, 45 MS ion source temperature 200℃

¹⁾ Quantitation ions

3) Measurement of butyric acid content

Culture supernatants of K97, K99, and K263 strains obtained through the above process were prepared as samples. To extract butyric acid from the sample, a chloroform-methanol extraction method was used. First, a sample extracted using chloroform-methanol was concentrated using an evaporator, and then fatty acid methyl ester derivatized. Fatty acid methyl ester derivatives were prepared in the following manner. 20 mg of fat was taken in a tube, 2 ml of 0.5N NaOH/Methanol was added, and the stopper was closed, followed by hydrolysis in a heating block (100° C.) for about 5 minutes. After the reaction was completed, after cooling, 2 ml of 14% BF ₃ /Methanol was added, reacted for 5 minutes, and then 2 ml of isooctane was added and shaken. After the reaction, 2 ml of saturated saline was added to the tube containing the sample. After closing the stopper and shaking lightly for 5 seconds, only the isooctane layer was taken and dehydrated using anhydrous sodium sulfate. A dehydrated fatty acid methyl ester test solution was received and analyzed by injection into a gas chromatograph (HP-6890GC FID, Agilent Technologies, Santa Clara, CA, USA). Gas chromatography analysis conditions are shown in Table 2.

Instrument GC-FDI Column SP-2560 (Supelcom 100 m × 0.2 mm ID, 0.2 µM film) Detector Flame ionization detector Oven temperature 100 ℃ (2 min) - 4 ℃/min - 230 ℃ (20 min) Injector temperature 230℃ Detector temperature 250℃ Injection volume 1 μl Carrier gas He Column flow 1.5 mL/min Injection volume 1.0 μl Split ratio 50:1

4) Conclusion

'MRS' is a graph showing the results of analyzing short-chain fatty acid production ability when strains K99, K263 and K97 were cultured in MRS broth, and 'MRS+M' is a graph showing strains K99, K263 and K97 with 3% indigestibility. It is a graph showing the results of analyzing short-chain fatty acid production ability when cultured in MRS medium (broth) to which polysaccharide (maltodextrin) was added. The results are summarized in the table below.

division Propionic acid (ppm) Acetic acid (%, w/v) Butyric acid (g/kg) MRS MRS+M MRS MRS+M MRS MRS+M K97 4.664 ± 1.600 20.569 ±
0.559 1.35 ± 0.07 1.33 ± 0.14 1.896 ± 0.054 1.999 ± 0.0755 K99 6.032 ± 0.085 11.022±
2.105 1.190 ± 0.014 1.180 ± 0.070 2.033 ± 0.041 1.923 ± 0.050 K263 5.221 ± 0.000 10.357±2.578 1.270 ± 0.000 1.330 ± 0.140 1.800 ± 0.050 2.578 ± 0.023

As shown in Table 3, it was confirmed that the K97 strain had the best ability to produce short-chain fatty acids. In particular, it was confirmed that the K97 strain had 1.63 times higher production of propionic acid than other strains in the MRS medium to which 3% maltodextrin was added. Through this, the K97 strain was finally selected among the three selected strains.

<Experimental Example 4> Identification of Lactobacillus plantarum K97 strain

1) Sugar availability analysis

strains K97 Control - Glycerol - Erythritol - D-Arabinose - L-Arabinose + D-Ribose + D-Xylose - L-Xylose - D-Adonitol - Methyl-βD-Xylopyranoside - D-Galactose + D-Glucose + D-Fructose + D-Mannose + L-Sorbose - L-Rhamnose + Dulcitol - Inositol - D-Mannitol + D-Sorbitol + Methyl-αD-Mannopyranoside + Methyl-αD-Glucopyranoside - N-AcetylGlucosamine + Amygdalin + Arbutin + Esculin ferric citrate + Salicin + D-Celiobiose + D-Maltose + D-Lactose + D-Melibiose + D-Saccharose + D-Trehalose + Inulin - D-Melezitose + D-Raffinose + Amidon - Glycogen - Xylitol - Gentiobiose + D-Turanose + D-Lyxose - D-Tagatose + D-Fucose - L-Fucose - D-Arabitol - L-Arabitol - potassium Gluconate ± potassium 2-KetoGluconate - potassium 5-KetoGluconate -

Sugar availability was confirmed using the API20 STREP kit for the finally selected K97 strain. Sugar availability of the K97 strain is shown in Table 4 below.

2) Identification of 16S rRNA

Genomic DNA was extracted from the K97 strain and 16s rRNA sequencing was analyzed. The 16S rRNA region was amplified using the isolated DNA as a template. Specifically, for DNA sequence analysis, universal primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3') were used, and Big Dye terminator PCR was performed using cycle sequencing kit v.3.1 (Applied BioSystems, USA). The amplification process was 95 °C for 5 minutes, followed by 95 °C for 30 seconds; 55 °C, 2 min; 68 ℃, 1 minute and 30 seconds were performed 30 times, and 68 ℃, 10 minutes were finished. For sequence analysis, the PCR product was removed with Montage PCR Clean up kit (Millipore) to remove dNTPs and reactants that did not participate in the reaction, and then sequencing primers 785F 5' (GGA TTA GAT ACC CTG GTA) 3' and 907R Using 5' (CCG TCA ATT CMT TTR AGT TT) 3', it was automatically analyzed with an Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSystems, USA).

As a result of 16S rRNA analysis of the finally selected K97 strain, the 16S rRNA sequence of the K97 strain is Lactobacillus plantarum K97 strain Lactobacillus plantarum (La ctobacillus plantarum) WCFS1strain showed 99% homology with WCFS, confirming that the novel microorganism of the present invention is a strain belonging to La ctobacillus plantarum . Therefore, the final selected K97 strain was named Lactobacillus plantarum K97 and deposited with the National Academy of Agricultural Sciences (KACC) of the Rural Development Administration on June 25, 2020 (accession number: KACC 81119BP).

The nucleotide sequence of the 16S rRNA of the strain of the present invention is represented by SEQ ID NO: 1, and the phylogeny of the Lactobacillus plantarum K97 strain was analyzed, and the results are shown in FIG. 1.

<Experimental Example 5> Lactobacillus plantarum (La ctobacillus plantarum) Characterization of strain K97

Antibiotic resistance, enzyme activity test, bile and acid resistance test, antibacterial activity test, and intestinal adhesion of the finally selected Lactobacillus plantarum K97 strain were analyzed as follows. Results are expressed as mean ± standard deviation (SD), and statistical analysis was performed with Statistical Package for Social Sciences (SPSS, SPSS Inc., USA). Significant differences were statistically processed by one-way ANOVA and tested at the significance level of 5% using Duncan's multiple range tests.

1) Enzyme activity

Each of the selected strains was cultured in MRS liquid medium at 37 °C for 18 hours, diluted with physiological saline, and then prepared as a sample at a level of 10 ⁵ to 10 ⁶ CFU/㎖. The sample was incubated at 37 ° C. for 5 hours using the API ZYM kit (API bioMerieux, Lyon, France) and then enzymatically reacted. Enzyme activity was expressed as a value of 0 to 5 by comparing standard color charts, and alkaline phosphatase, eterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, β-fucosidase Activity was measured, and the results are shown in Table 5 below.

Enzymes K97 Alkaline phosphatase 2 Esterase (C4) One Esterase Lipase (C8) One Lipase (C14) One Leucine arylamidase 5 Valine arylamidase 4 Cystinearylamidase 2 Trypsin 0 α-chymotrypsin 0 Acid phosphatase 4 Naphtol-AS-BI-phosphohydrolase 2 α-galactosidase 2 β-galactosidase 5 β-glucuronidase 0 α-glucosidase 5 β-glucosidase 5 N-acetyl-β-glucosaminidase 5 α-mannosidase 0 α-fucosidase 0

As shown in Table 5, the Lactobacillus plantarum K97 strain showed high activity of 5 against Leucine arylamidase, β-galactosidase, α-glucosidase, β-glucosidase and N-acetyl-β-glucosaminidase, In the case of β-glucuronidase, an inducing factor that converts benzopyrene into a carcinogenic substance, the enzymatic activity was zero, indicating that the bacteria are stable.

2) cholestasis

[Gilliland SE, Walker DK. Factors to consider when selecting a culture of Lactobacillus acidophilus as a dietary adjunct to produce a hypocholesterolemic effect in humans. J Dairy Sci. 1990;73:905-911]. First, the Lactobacillus plantarum K97 strain cultured at 37 ° C. for 18 hours in MRS liquid medium was added with 0.3% oxgall in MRS liquid medium containing 0.05% cysteine. As a control, 1% was inoculated into a medium without the addition of oxgall. It was anaerobically cultured in an incubator at 37 ° C. for 7 hours, poured and solidified on a BCP plate count agar plate for each hour, and then anaerobically cultured at 37 ° C. for 48 hours and counted. The results are shown in FIG. 4 .

Figure 2 is a medium (with oxgall) in which 0.3% oxgall was added to the MRS broth and a medium without oxgall (without oxgall) Lactobacillus plantarum (La ctobacillus plantarum) K97 strain It is a growth curve according to the time of culturing each. * is p<0.05, ** is p<0.01, *** is p<0.001.

As shown in Figure 2, the Lactobacillus plantarum K97 strain slightly decreased after 4 hours in the medium supplemented with oxgall, but showed a survival rate of 91.32% or more compared to the control even after 7 hours. It can be seen that the Bacillus plantarum K97 strain has excellent bile tolerance.

3) acid resistance

[Clark PA, Cotton LN, Martin JH. Selection of bifidobacteria for use as dietary adjuncts in cultured dairy foods: II-Tolerance to simulated pH of human stomachs. Cult Dairy Prod J. 1993;28:11-14]. First, 37% HCl was mixed with distilled water to prepare pH 2, 3, 4 solutions and pH 6.4 solutions as a control. After mixing 1 ml of time-cultivated selected strains (about 10 ⁹ CFU/ml), and anaerobically culturing at 37 ° C, viable cell counts after 0, 1, 2, and 3 hours were poured on a BCP plate count agar plate and solidified. Anaerobic culture was followed by counting, and the results are shown in FIG. 3 .

Figure 3 is a graph showing the survival rate measured after 3 hours when the Lactobacillus plantarum K97 strain was inoculated in an HCl solution. * is p<0.05, ** is p<0.01, *** is p<0.001.

In order to exhibit sufficient function as a probiotic composition, it must survive to the small intestine when passing through the gastrointestinal tract under low PH conditions. As shown in Figure 5, when compared to the initial (0 hour), the Lactobacillus plantarum K97 strain showed a 99-100% survival rate for 3 hours in all PH conditions. That is, it can be seen that the Lactobacillus plantarum K97 strain is remarkably excellent in acid resistance and is an excellent strain having a high survival rate in the stomach.

4) antibiotic resistance

Lactobacillus plantarum K97 strain was inoculated into the MRS liquid medium, incubated at 37 ° C. for 18 hours, and then diluted in a 0.1% peptone solution to an appropriate concentration. Each antibiotic was inoculated at a level of 10 ⁵ -10 ⁶ CFU/ml in a tryptic soy liquid medium containing each concentration, incubated at 37 °C for 48 hours, and then visually observed to determine growth. Antibiotic resistance was measured using a two-fold dilution method, and the antidiabetic effect of the Lactobacillus plantarum K97 strain, which is the lowest inhibited concentration, was determined as a MIC (minimal inhibitory concentration) value. Antibiotics were purchased from Sigma (USA) and used. Amikacin, Gentamicin, Kanamycin, Streptomycin, Ampicillin, Penicillin-G, Oxacillin, Bacitracin, Polymyxin B, Ciprofloxacin, Tetracycline, Clindamycin, Erythromycin, Rifampicin, Vancomycin, and Chloramphenicol were used in the test. The results are shown in Table 6.

Antimicrobal agents MIC (μg/ml) Amikacin 16 Gentamycin 4 Kanamycin 64 Streptomycin 128 Ampicillin >2048 Penicillin-G 64 Oxacillin 8 Bacitracin 128 Polymyxin B >512 Ciprofloxacin 256 Tetracycline 16 Clindamycin One Erythromycin 0.5 Rifampicin 2 Vancomycin >4096 Chloramphenicol 4

As shown in Table 6, the Lactobacillus plantarum K97 strain had the highest antibiotic resistance against vancomycin with a MIC concentration of 4,096 μg/ml or more, while the MIC concentration against Clindamycin was 1.0 μg/ml. It was confirmed that the sensitivity was the lowest with less than ㎖.

5) Antibacterial activity

[Gilliand SE, Speck ML. Deconjugation of bile acids by intestinal lactobacilli. Appl Environ Micobiol. 1977;33:15-18], the antibacterial activity was measured. Indicator bacteria consisting of Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes and Staphylococcus aureus were all purchased from the Korea Food Research Institute, and the growth medium of the indicator bacteria was aerobically 37 in Nutriunt liquid medium. ℃, and incubated for 24 hours. The medium used for the mixed culture and control was MRS liquid medium, inoculated with selected strains and indicator bacteria, respectively, and incubated at 37 ° C. for 24 hours. As a selective medium, EMB agar was used for Escherichia coli , Bismuth sulfite agar was used for Salmonella Typhimurium , Listeria Isolation Agar containing Supplement (X123) was used for Listeria monocytogenes , and Baird parker agar was used for Staphyloccous aureus . The inhibition rate of the indicator by the Lactobacillus plantarum K97 strain was calculated through Equation 4, and the results are shown in Table 7.

[Equation 4]

Inhibition (%) = (number of bacteria in control CFU/mL-number of bacteria after mixed culture CFU/mL)/number of bacteria in control group CFU/mL

Pathogens Growth Inhibition
(%) pathogens K97+pathogens CFU/ml pH CFU/ml pH Escherichia coli 4.25±0.21×10 ⁵ 6.21 2.93±0.31×10 ⁵ 5.68 30.98 Salmonella Typhimurium 2.07±0.40×10 ⁷ 6.19 4.70±0.04×10 ⁶ 5.69 77.42 Listeria monocytogenes 3.10±0.57×10 ⁵ 6.20 1.95±0.64×10 ⁵ 5.50 37.10 Staphyloccous aureus 2.70±0.62×10 ⁶ 5.20 4.4±0.14×10 ⁵ 5.10 83.83

* The initial bacterial count of La ctobacillus plantarum K97 strain was 5.60 ㅁ 0.99 ㅧ 10 ⁶ CFU/㎖

As shown in Table 7, as a result of confirming whether the Lactobacillus plantarum K97 strain, as a probiotic strain, has antibacterial activity to inhibit harmful bacteria and improve the intestinal flora, three food poisoning bacteria ( E. coli, S. Typhimurium, S. aureus ) had excellent antibacterial activity of 77 to 83% or more. It was confirmed that the Lactobacillus plantarum K97 strain had an antibacterial activity of 30% or more against L. monocytogenesdp, a pathogenic microorganism.

6) Intestinal fixation ability

HT-29 cells were used to evaluate intestinal fixative capacity. The HT-29 cells of the present invention were purchased through the Korea Cell Line Bank (seoul, Korea), [Kim SJ, Cho SY, Kim SH, Song OJ, Shin IS, Cha DS, et al. Effect of microencapsulation on viability and other characteristics in Lactobacillus acidophilus ATCC 43121]. First, RPMI medium supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (P/S; Gibco, USA) was used and heated at 37 °C. , 5% CO ² / 95% air was supplied and cultured in an incubator. In order to culture HT-29 cells, the cells are divided into 10 ⁶ cells/well in each well of a 12 well plate, and the medium is replaced once every 2 days. The day before the experiment, when the cells are filled to 95%, serum free medium ) to prevent further filling of cells. To test the intestinal fixation ability of La ctobacillus plantarum strain K97, 1 ml of the second subcultured strain was taken, centrifuged at 12,000 rpm for 3 minutes, and twice using a serum free medium. Washed. The washed strain was diluted with RPMI medium to adjust the OD _600nm to 0.5, diluted with 0.1% peptone solution, and then poured into BCP plate count agar to measure the initial number of bacteria. 100 ml of the cells with the OD value matched were dispensed into wells, incubated for 2 hours at 37 °C, 5% CO ₂ , and then non-adhered bacteria were washed 5 times with PBS. 1 ml of trypsin-EDTA was added to remove cell-bacteria, diluted with 0.1% peptone solution, and then poured into BCP plate count agar to measure the number of bacteria. shown in Figure 6.

Figure 4 is a graph showing the intestinal adhesion ability of the Lactobacillus plantarum K97 strain in HT-29 cells. According to this, the Lactobacillus plantarum K97 strain has a It was confirmed that the adhesion ability was excellent. Lactobacillus rhamnosus GG (LGG) (Gopal et al., 2001) used as a positive control is a strain widely known for its ability to adhere to intestinal cells. It can be seen that the Lactobacillus plantarum K97 strain of the present invention has better intestinal adhesion than the LGG strain.

<Experimental Example 6> Identification of Mechanism of Blood Glucose Improvement for RIN-m5F Cells

1) Pretreatment of selected strains

Lactobacillus plantarum (La ctobacillus plantarum) K97 strain was inoculated in 1% MRS medium (broth) and then incubated at 37 ° C. for 18 hours, respectively. Cells were collected by centrifuging the cultured strain at 1,500 xg for 15 minutes. In order to remove the remaining MRS medium (broth), the collected strains were washed three times with sterile distilled water. The washed strain was suspended at a concentration of 10 mg/ml after freeze-drying. After ultrasonication for 50 seconds using a sonicator, samples to be used in the experiment were prepared by sterilization using an autoclave. Each sample was diluted by concentration according to the analysis, and DW was used for dilution by concentration, and the prepared samples were stored in a refrigerator.

2) Cell model preparation

RIN-m5F (pancreatic cells) for use as a cell model was prepared. RIN-m5F (pancreatic cells) was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and used. Reagents required for cell culture were purchased from Gibco (USA), and the medium was RPMI-1640 (Roswell Park Memorial Institute 1640) and 10% fetal bovine serum (FBS) and 1% DMEM (Dulbecco Modified Eagle Medium). Antibiotics-antimycotic was added and subcultured once every 2 to 3 days in a 37 ℃ cell incubator in the presence of 5% CO ₂ to prepare a cell model to be used in the experiment.

3) Cytotoxicity

The sample obtained from Experimental Example 6-1 was treated with RIN-m5F, and the cell viability according to the concentration of the sample was measured by WST-1 assay (ITSBio, Seoul, Korea). First, RIN-m5F cells were each dispensed into a 96-well plate at 5×10 ⁴ cells/well. After culturing for 24 hours, the samples were treated by concentration in each well and cultured for 24 hours. After incubation was completed, 10 μl of WST-1 reagent was added per 100 μl for reaction, and then absorbance was measured at 450 nm (Ref. 600 nm) using a microplate reader (Infinite 200, Tecan Trading AG, Switzerland). was measured. Cell viability was calculated using Equation 5.

[Equation 5]

Cell viability assay (%) = (absorbance of sample treatment group / absorbance of control group) × 100

4) Insulin secretion regulation activity

RIN-m5F cells were divided into 12-well plates at 5.0 × 10 ⁵ cells per well, cultured in complete medium for 3 days, and then Lactobacillus plantarum K97 strain was added. and cultured for 24 hours. After culturing in a medium containing YCT, modified Kreb's ringer bicarbonate buffer (KRBB-HEPES, 134 mmol/ℓ NaCl, 4.8 mmol/ℓ KCl, 1 mmol/ℓ CaCl ₂ , 1.2 mmol/ℓ MgSO ₄ , 1.2 mmol/L KH ₂ PO ₄ , 5 mmol/L NaHCO ₃ , 10 mmol/L HEPES, 1 mg/mL BSA, pH 7.4) and washed twice with KRBB-HEPES buffer containing 20 mM glucose. After incubation for 1 hour, the supernatant was collected and centrifuged at 4 °C for 10 minutes, and the supernatant was collected and stored at -20 °C. The amount of secreted insulin was measured using a rat insulin RIA kit. By measuring the concentration of cellular protein in each well, the amount of insulin secretion per unit gram of protein was calculated.

5) Analysis of expression changes of genes involved in insulin secretion using RT-PCR (Real-time PCR) technique

Gene expression involved in insulin secretion was analyzed by real-time PCR. First, RIN-m5F cells were divided into 5×10 ⁵ cells/well in a 12-well plate, cultured for 3 days in complete medium, and then added with Lactobacillus plantarum K97 strain for 24 hours cultured for a while. Then, washed twice with modified Kreb's ringer bicarbonate buffer, changed to KRBP-HEPES buffer containing 20 mM glucose, and incubated for 1 hour, after removing the supernatant, using Trizol reagent (Sigma, T9424) Total RNA was isolated. The isolated RNA was quantified, and cDNA was synthesized from 1 μg of RNA using an oligo (Oilgo) dT primer and AMV reverse transcriptase. The synthesized cDNA was amplified and measured by a real-time PCR method using GLP-1R, IRS-2, and GADPH primers as a template. Each PCR product was electrophoresed using a 1% agarose gel, stained with ethidium bromide (EtBr, Sigma), and the expression level was confirmed under UV. Base sequences of primers used in PCR are summarized in Table 8 below.

designation order PPAR-γ sense
(SEQ ID NO: 2) 5'-TCGCTG ATG CAC TGC CTA TG-3' antisense
(SEQ ID NO: 3) 5'-GAG AGG TCC ACA GAG CTG ATT-3' SREBP-1c sense
(SEQ ID NO: 4) 5'-AGC AGC CCC ATG AAC AAA CAC-3' antisense
(SEQ ID NO: 5) 5'-CAG CAG TAG GTC TGC CTT GAT-3' GAPDH sense
(SEQ ID NO: 6) 5'-CGG-AGT-CAA-CGG-ATT-GG-TCG-TAT-3' antisense
(SEQ ID NO: 7) 5′-AGC-CTT-CTC-CAT-GGT-GGT-GAA-GAC-3′

5) Statistical analysis

Statistical analysis according to the statistical significance test between each group was performed with ANOVA (one-way analysis of variance test) Duncan post-test comparison, and it was determined that it was significant when p<0.05 (SPSS V12., SPSS Inc, Chicago, IL, USA).

6) Results

5 is a graph showing the survival rate of RIN-m5F cells measured when the Lactobacillus plantarum K97 strain was treated by concentration. Different superscripts mean that the values are significantly different from each other (p<0.05).

According to this, when the concentration of the Lactobacillus plantarum K97 strain was 1 to 10 μg/ml, the survival rate was greater than 100%, and at the concentration of 30 to 100 μg/ml, the survival rate was greater than 80%. Therefore, the Lactobacillus plantarum K97 strain is stable up to a concentration of 1 to 500 μg / ml (survival rate of 50% or more), preferably 1 to 300 μg / ml (survival rate of 70% or more), more preferably Preferably 1 to 100 μg/ml (survival rate of 75% or more), more preferably 1 to 30 μg/ml (survival rate of 85% or more), and most preferably 1 to 10 μg/ml (survival rate of 100% or more) can

6 is a graph showing the amount of insulin secretion measured when RIN-m5F cells were treated with Lactobacillus plantarum K97 strain at different concentrations. Different superscripts mean that the values are significantly different from each other (p<0.05).

As shown in Figure 6, the amount of insulin secretion in RIN-m5F cells (control) not treated with the sample was 73.67 ng / mg protein / 1h, and when treated with Lactobacillus plantarum K97 strain , It was confirmed that 85.95 ng/mg protein/1h and 92.87 ng/mg protein/1h were obtained by concentration. Lactobacillus plantarum K97 strain showed a significant increase in insulin secretion compared to the control group, and increased insulin secretion as the concentration increased. Accordingly, it can be seen that the Lactobacillus plantarum K97 strain has an excellent effect in preventing, improving or treating diabetes by improving insulin control ability.

7 is a graph showing the expression level of GLP-1R, a gene involved in insulin secretion, measured when RIN-m5F cells were treated with Lactobacillus plantarum K97 strain at different concentrations. Different superscripts mean that the values are significantly different from each other (p<0.05).

GLP-1 binds to GLP-1R, glucagon-like peptide 2 receptor (GLP-1R, glucagon-like peptide 2 receptor), a G protein-coupled receptor present on the surface of beta cells, transmits signals within cells, and stimulates glucose-dependent insulin secretion to beta cell By stimulating insulin gene expression and biosynthesis in cells, it can be seen that as GLP-1R expression increases, insulin gene expression in beta cells increases and insulin secretion is promoted. As shown in FIG. 7 , it was confirmed that the expression of GLP-1R in insulinoma cells, RIN-m5F, increased significantly by 1.2 to 1.3 times or more as the Lactobacillus plantarum K97 strain was treated. That is, it can be seen that the Lactobacillus plantarum K97 strain has a direct insulin secretion stimulating effect through the regulation of GLP-1R secretion in pancreatic beta cells. Therefore, the Lactobacillus plantarum K97 strain can exhibit excellent effects in preventing, improving or treating diabetes.

8 is a graph showing the expression level of IRS-2, a gene involved in insulin secretion, measured when RIN-m5F cells were treated with Lactobacillus plantarum K97 strain at different concentrations. Different superscripts mean that the values are significantly different from each other (p<0.05).

IRS is a mediator of insulin signaling and is a docking protein between insulin receptors and complex signaling molecules in cells that play a role in maintaining functions such as growth, survival, and metabolism of cells. Insulin receptor substrate 2 (IRS-2) is known to promote insulin secretion by enhancing the proliferation, function, and survival of insulin and insulin-like growth factor-1. Accordingly, when RIN-m5F cells were treated with La ctobacillus plantarum K97 strain at different concentrations, the effect on IRS-2 expression was analyzed.

As shown in FIG. 8, as a result of treating the Lactobacillus plantarum K97 strain, it was confirmed that IRS-2 expression was significantly increased in a concentration-dependent manner. In particular, in the experimental group treated with the Lactobacillus plantarum K97 strain, compared to the control group, it can be seen that the expression of IRS-2 increased significantly by 1.2 to 1.5 times or more.

<Experimental Example 7> Evaluation of glucose metabolism and utilization increasing activity in C2C12 cells

1) Pretreatment of selected strains

Lactobacillus plantarum (La ctobacillus plantarum) K97 strain was inoculated in 1% MRS medium (broth) and then incubated at 37 ° C. for 18 hours, respectively. Cells were collected by centrifuging the cultured strain at 1,500 xg for 15 minutes. In order to remove the remaining MRS medium (broth), the collected strains were washed three times with sterile distilled water. The washed strain was suspended at a concentration of 10 mg/ml after freeze-drying. After ultrasonication for 50 seconds using a sonicator, samples to be used in the experiment were prepared by sterilization using an autoclave. Each sample was diluted by concentration according to the analysis, and DW was used for dilution by concentration, and the prepared samples were stored in a refrigerator.

2) Cell model preparation

Mouse muscle cells (C2C12 myoblast) were prepared for use as a cell model. Mouse myoblasts (C2C12 myoblasts) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and used. Reagents required for cell culture were purchased from Gibco (USA), and the medium was RPMI-1640 (Roswell Park Memorial Institute 1640) and 10% fetal bovine serum (FBS) and 1% DMEM (Dulbecco Modified Eagle Medium). Antibiotics-antimycotic was added and subcultured once every 2 to 3 days in a 37 ℃ cell incubator in the presence of 5% CO ₂ to prepare a cell model to be used in the experiment.

3) Cytotoxicity

C2C12 cells were treated with Lactobacillus plantarum K97 strain (sample), and cell viability according to the concentration of the sample was measured by WST-1 assay (ITSBio, Seoul, Korea). First, C2C12 cells were each dispensed into a 96-well plate at 5×10 ⁴ cells/well. After culturing for 24 hours, the samples were treated by concentration in each well and cultured for 24 hours. After incubation was completed, 10 μl of WST-1 reagent was added per 100 μl for reaction, and then absorbance was measured at 450 nm (Ref. 600 nm) using a microplate reader (Infinite 200, Tecan Trading AG, Switzerland). was measured. Cell viability was calculated using Equation 5.

[Equation 5]

Cell viability assay (%) = (absorbance of sample treatment group / absorbance of control group) × 100

3) Evaluation of blood glucose absorption capacity

C2C12 cells were dispensed into a 96-well plate (5×10 ⁴ cells/well) and cultured for 24 hours at 37° C., 5% CO ₂ . After the cells grew to 70%, the medium was supplemented with 1% horse serum. , 1% P/S was added to DMEM differentiation medium, and the culture medium was replaced every 48 hours. Fully differentiated cells were cultured in serum free-low glucose medium and 100 nM 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino acid for 24 hours. ]-2-deoxy-D-glucose and Lactobacillus plantarum K97 strain were treated at different concentrations. Then, after washing three times with Cold PBS, 2-NBDG absorption capacity was measured at 485 nm and 535 nm spectral wavelengths using a fluorescence meter.

4) Analysis of the expression of proteins involved in glucose metabolism and utilization (GLUT4, Akt)

Expression of proteins (GLUT4, Akt) involved in glucose metabolism and utilization in the cell model treated with the selected strain was analyzed by Western blot. C2C12 cells were dispensed into a 12-well plate (5×10 ⁵ cells/well) and cultured for 24 hours at 37° C., 5% CO ₂ . After the cells grew to 70%, the medium was supplemented with 1% horse serum. (horse serum) and DMEM differentiation medium supplemented with 1% P/S were replaced, and the culture medium was replaced every 48 hours. Fully differentiated cells were treated with serum free-low glucose medium and Lactobacillus plantarum K97 strain at different concentrations for 24 hours. Thereafter, after washing three times with PBS, a lysis buffer was added, centrifuged at 1400 rpm, and the supernatant was taken, SDS sample buffer was added, and boiled at 100 ° C. for 10 minutes to induce protein denaturation. After separating the proteins using 10% SDS PAGE, Glut4 (1F8) mouse mAb (CST, #2213S), Phospho-Atk (Ser473) (CST, #9271), Akt Antibody (CST, #9272) Protein expression was analyzed using .

5) Statistical analysis

Statistical analysis according to the statistical significance test between each group was performed with ANOVA (one-way analysis of variance test) Duncan post-test comparison, and it was determined that it was significant when p<0.05 (SPSS V12., SPSS Inc, Chicago, IL, USA).

6) Results

9 is a graph showing the survival rate of C1C12 cells measured when the Lactobacillus plantarum K97 strain was treated by concentration. Different superscripts mean that the values are significantly different from each other (p<0.05).

As shown in FIG. 9 , when the Lactobacillus plantarum K97 strain was treated at different concentrations, it was confirmed that the C2C12 cell viability was more stable than the RIN-m5F cell viability.

10 is a graph showing the measurement of glucose uptake of C1C12 cells when the Lactobacillus plantarum K97 strain was treated by concentration. Different superscripts mean that the values are significantly different from each other (p<0.05).

Muscle is one of the peripheral tissues of the human body, and is a representative tissue that absorbs and consumes about 70% of blood sugar in the body, and is known to play a very important role in blood sugar control. In this experiment, the effect of the Lactobacillus plantarum K97 strain on the glucose absorption capacity of differentiated muscle cells (C2C12 cells) was investigated.

As shown in FIG. 10, it was confirmed that the glucose uptake capacity of C2C12 cells increased significantly as the concentration of the Lactobacillus plantarum K97 strain increased. That is, it can be seen that the Lactobacillus plantarum K97 strain has a remarkable effect on enhancing glucose consumption in C2C12 cells.

PIP3 induces the translocation of Akt to the plasma membrane, and PDK2 induces phosphorylation at ser473 of Akt, so the activated Akt bound to the cell membrane activates GLUT4 (Glucose transporter type 4) present in fat and muscle. It is known to regulate the glucose uptake process in which glucose enters the cell by translocation from the cytoplasm to the cell membrane by insulin stimulation. Therefore, when the Lactobacillus plantarum K97 strain was treated with differentiated muscle cells (C2C12 cells), the effect on the expression levels of pAkt/Akt and GLUT4 was analyzed. As a result, FIGS. 11 to 14 shown in

Figure 11 is a western measurement of the expression level of proteins (p-AKT / AKT) involved in sugar metabolism and utilization when C2C12 cells were treated with Lactobacillus plantarum K97 strain at different concentrations It is a blot result, and FIG. 12 is a graph showing it numerically. Different superscripts mean that the values are significantly different from each other (p<0.05).

13 is a Western blot result of measuring the expression level of a protein (GLUT4) involved in glucose metabolism and utilization when C2C12 cells were treated with La ctobacillus plantarum K97 strain at different concentrations, 14 is a graph showing this numerically. Different superscripts mean that the values are significantly different from each other (p<0.05).

As shown in Figures 11 to 14, the expression levels of proteins (p-AKT / AKT and GLUT4) involved in sugar metabolism and utilization in C2C12 cells as the Lactobacillus plantarum K97 strain was treated, respectively It can be seen that the increase is more than 1.27 to 1.37 times. That is, it can be seen that the Lactobacillus plantarum K97 strain has a remarkable effect on enhancing glucose consumption in C2C12 cells.

In addition, GULT4 is an insulin transport pathway, and when stimulated by insulin, intracellular GULT4 moves to the plasma membrane and promotes glucose transport. That is, GLUT4 plays a role in lowering blood glucose by facilitating the influx of blood glucose into cells. Therefore, the fact that the activity and expression of GLUT4 involved in glucose transport increases as the Lactobacillus plantarum K97 strain is treated indicates that the Lactobacillus plantarum K97 strain has an excellent antidiabetic effect will represent

<Experimental Example 8> Evaluation of blood sugar improvement efficacy using animal models

1) Experimental animals and breeding environment

Experimental animals are db/db mice in a specific-pathogen free (SPF) state (a strain with a mutation in the gene that produces the leptin receptor, and diabetes is induced due to a problem with leptin signal transduction). After a period of time, it was used in the experiment. During the acclimatization period, regular mouse feed (Purina Lab Rodent Chow #38057, Purina Co., Seoul Korea) was fed, and filtered drinking water was changed every day during the acclimatization period so that they could consume freely. During the breeding period, temperature is 23±1℃, humidity is 50±5%, noise is less than 60 phone, lighting time is 08:00~20:00 (12 hours a day), illuminance is 150~300 Lux, ventilation is 10~12 per hour. The meeting environment was maintained. This experiment was performed in compliance with the animal experimentation ethics regulations.

2) experiment

During the experiment period, normal solid feed (Samtako, Gyunggi, Korea) (AIN-93G) was fed, and after the experiment was completed, the blood sugar level of the experimental animals was measured and separated using the egg mass method so that the average value of blood sugar level between each group was uniform. Then, individual identification was marked using an ear punch (each group is summarized in Table 9 below). It is divided into normal group, control group, experimental group (K97), and positive control group, and 8 animals per group were used in the experiment. At this time, the normal group was tested in the same way as the control group with non-diabetic mice and db/+. During the experiment, the temperature was 23±1℃, humidity 50±5%, noise less than 60 phone, lighting time 08:00~20:00 (12 hours a day), illumination 150~300 Lux, ventilation 10~12 per hour The meeting environment was maintained. This experiment was performed in compliance with the animal experimentation ethics regulations.

Division (n=8) Experiment method Normal group
(Normal group, NOR) (Non-diabetic mouse, db/+)
Oral administration of 0.2 ml distilled water daily for 8 weeks control group
(Control group, CON) (diabetic mouse, db/db)
Oral administration of 0.2 ml distilled water daily for 8 weeks positive control
(Positive control, POS CON) (diabetic mouse, db/db)
Oral administration of 0.2 ml distilled water containing 60 mg/kg of metformin hydrochloride daily for 8 weeks experimental group
(K97) (diabetic mouse, db/db)
Oral administration of 0.2 ml distilled water in which probiotics were suspended at a concentration of 8 Log/CFU K97 daily for 8 weeks

3) Blood glucose measurement and oral glucose tolerance test

Weekly weight and blood glucose levels were measured for each group. Body weight and blood glucose were measured once a week at a certain time, and food and drinking water intake were measured once a week after feeding a certain amount of food and drinking water, and the remaining amount was measured the next day. Weekly blood glucose was measured using blood collected from the tail vein using a blood glucose meter at a fixed time every week. In the oral glucose tolerance test, the test animals were fasted for 12 hours in the last week of test sample administration, blood was collected from the tail vein, and the blood glucose concentration was measured during fasting. After that, glucose solution (1 g/kg BW) was orally administered, and after 30, 60, 120, and 180 minutes After that, blood sugar was measured.

4) Handling of laboratory animals and sample collection

After the experiment was completed, each group fasted for 12 hours, collected blood from the orbital plexus, put it in a centrifuge tube, left it at room temperature for about 1 hour, centrifuged at 2,500 rpm for 10 minutes, and separated serum. . The separated serum was used for analysis while being frozen at -70 °C. After blood collection, each organ tissue (liver, kidney, spleen, testis, pancreas) was extracted, washed in 0.9% saline, dried with filter paper, and weighed.

5) blood analysis

Blood was collected from the abdominal vena cava, collected in a conical tube, coagulated at room temperature for 30 minutes, and then centrifuged at 3,000 rpm for 10 minutes. Blood levels of fructosamine, TC, TG, LDL-C, HDL-C, and blood glucose-related indicators such as glucose, insulin, HbA1c, and C-peptide were measured. Insulin resistance was calculated using Homeostasis Assessment-Insuline Resistance (HOMR-IR).

6) Histological analysis

Pancreatic islet tissue analysis was measured using an immunohistochemistry kit (Accessory Kit). After embedding the fixed pancreatic tissue in paraffin, it was sectioned and cut to a thickness of 4 μm, and the deparaffinized tissue was treated with a peroxidase quenching solution for 30 minutes, ready-to-use It was reacted for 15 minutes with IHC Blocking Reagent. Afterwards, the blocking reagent and the activated primary antibody (Working Primary Antibody solution) (2-10 μl of antibody to 1 ml of Ready-To-Use IHC antibody diluent) are treated, respectively, and incubated at room temperature for 3 hours or at low temperature. After reacting overnight (overnight), activated anti-rabbit IHC antibody (Working anti-Rabbit IHC Antibody), and then stained with DAB substrate (Substrate) and hematoxylin (hematoxylin). Stained pancreatic tissue was observed and photographed using an optical microscope.

7) Statistical analysis

Results are expressed as mean±standard deviation (SD), and statistical analysis was performed with Statistical Package for Social Sciences (SPSS, SPSS Inc., USA). Significant differences were statistically processed by one-way ANOVA and tested at the significance level of 5% using Duncan's multiple range tests.

8) Results

15 is a graph showing the body weight of a normal group, a control group, an experimental group (K97), and a positive control over time. The db/db mouse, which is an animal model for type 2 diabetes used in this experiment, develops resistance to leptin, resulting in uncontrolled appetite, resulting in obesity and increased blood sugar levels as they grow.

As shown in FIG. 15, it was confirmed that the body weight of all animal models increased significantly when compared to the normal group.

Blood glucose (glucose concentration, mg/dl) of the normal group, control group, experimental group (K97), and positive control over time was measured and compared. Different superscripts mean that the values are significantly different from each other (p<0.05).

army 0 weeks 2 weeks 3 weeks 5 weeks 6 weeks 7 weeks 8 weeks Normal group 104.86± ^23.01b 105.86±16.68 ^c 130.33±25.66 ^c 127.63±17.85 ^c 118.38± ^16.32b 84.43±12.86 ^c 126.17± ^17.31c control group 198.71± ^16.33a 347.00± ^40.97a 444.40± ^77.32ab 501.50± ^50.47a 458.00± ^34.23a 500.75± ^29.66a 463.25± ^10.99a positive control 182.00±31.86 ^a 268.75± ^92.00b 460.50± ^46.17a 353.00± ^70.27b 376.50± ^13.92a 398.50± ^43.07b 395.33± ^12.38b experimental group
K97 199.67±47.03 ^a 259.80± ^16.70b 371.60± ^30.44b 340.20± ^23.65b 337.25± ^31.78b 384.75± ^21.82b 372.50± ^4.50b

As a result, it was confirmed that the blood glucose level of the control group, the experimental group (K97), and the positive control group was slightly higher than that of the normal group. However, in the positive control group administered with metformin, an antidiabetic drug, it was confirmed that blood sugar decreased at 5, 6, 7, and 8 weeks after the start of administration, and the experimental group (K97) administered with the K97 strain of Lactobacillus plantarum also confirmed It was confirmed that blood glucose reduction to a level equal to or lower than that of the positive control group appeared.

The oral glucose tolerance test measures the glucose concentration in the blood at a set time after glucose intake to indirectly determine the insulin secretion function. Therefore, in order to confirm the glucose tolerance effect by the Lactobacillus plantarum K97 strain of the present invention, after fasting for 12 hours at 8 weeks, glucose was orally administered at a dose of 1 g / kg, 0, 30, 60 , 120 and 180 minute intervals were carried out a glucose tolerance test in which blood was measured through the tail vein. The results are summarized in FIG. 16 and Table 11. 16 is a graph showing AUC (Area under the curve) for blood glucose levels according to an oral glucose tolerance test.

army 0 minutes 30 minutes 60 minutes 120 minutes 180 minutes Normal group 81.875±13.80 ^c 173.87± ^23.85b 165.37± ^27.72b 137.50±20.09 ^c 115.62±14.97 ^c control group 489.80± ^34.40a 600±0.00 ^a 600±0.00a 549.25 ± 25.72 ^a 510.75± ^37.63a positive control 398.5± ^43.07b 600±0.00 ^a 600±0.00a 483.00± ^28.82b 437.50± ^40.41b experimental group
K97 484.75 ± 21.82 ^a 600±0.00 ^a 600±0.00a 547.00± ^20.89a 497.25 ± 34.56 ^a

In Table 11, since it exceeded the measurement range of 600 mg/dL of the blood glucose meter at 30 and 60 minutes, it was indicated as the maximum value of 600 mg/dL ( ^a ). Different superscripts mean that the values are significantly different from each other (p<0.05).

As shown in FIG. 16 and Table 11, in the experimental group administered with the Lactobacillus plantarum K97 strain for 8 weeks, a significant blood sugar reduction effect was observed, which was equal to or lower than that of the positive control group. can That is, it can be seen that when the Lactobacillus plantarum K97 strain is administered, blood glucose can be lowered by affecting insulin sensitivity or resistance.

17 is a graph showing the measurement of fructosamine in serum of a normal group, a control group, an experimental group (K97), and a positive control group. Different superscripts mean that the values are significantly different from each other (p<0.05).

Fractosamine is a compound formed by combining glucose and protein, and is an indicator that reflects the average blood glucose concentration for 2-3 weeks, unlike HbA1c, which indicates the average blood glucose concentration for 8-12 weeks.

As shown in FIG. 17, fructosamine in the serum of the experimental group orally administered with the Lactobacillus plantarum K97 strain for 8 weeks was 6.74 ± 0.99 μmol / L, and it was confirmed that the control group (CON) was 7.70 ± 1.79 μmol / L, and it was confirmed that the normal group (NOR) was 3.10 ± 0.67 μmol / L. It was confirmed that the control group significantly increased 2.48 times compared to the normal group, and the experimental group administered with the Lactobacillus plantarum K97 strain significantly decreased compared to the control group. In particular, it was confirmed that the experimental group administered with the Lactobacillus plantarum K97 strain was equal to or lower than the positive control group (6.81 ± 1.49 μmol / L), and the Lactobacillus plantarum K97 strain It can be seen that has the effect of reducing the increased blood sugar.

The concentration of glucose in the serum of the normal group, the control group, the experimental group (K97), and the positive control group was measured.

As a result, it was confirmed that the normal group (NOR) (215.24 ± 2941 mg/dL) had a significant increase in serum glucose concentration more than twice that of the control group (CON) (p<0.05). The positive control group (POS CON) (499.58 ± 33.74 mg/dL) showed a slight decrease compared to the control group (CON) (538.22 ± 28.35 mg/dL). On the other hand, it was confirmed that the experimental group (K97) administered with the Lactobacillus plantarum K97 strain was 513.62 ± 39.01 ㎎ / ㎗, which was significantly reduced compared to the control group (CON). It was confirmed that the Lactobacillus plantarum K97 strain has a similar blood sugar lowering effect to metformin, a conventional diabetes treatment, and the Lactobacillus plantarum K97 strain can replace metformin, a conventional diabetes treatment. It can be used as a new treatment.

Next, the concentration of glycosylated hemoglobin in the blood of the normal group, the control group, the experimental group (K97), and the positive control group was measured.

When the content of glucose in the blood is high, sugar is attached to red blood cell hemoglobin in a non-enzymatic manner to produce glycosylated hemoglobin, which is called HbA1C. Since red blood cells have an average lifespan of 8 to 12 weeks, when HbA1C is measured, it is possible to know the average blood glucose level for the past 8 to 12 weeks, which reflects the blood glucose concentration around 4 to 6 weeks from the present.

army Glycated hemoglobin content (HbA1c, mg/dl) Normal group 162.40 ± 59.87 control group 1020.83 ± 187.24 positive control 846 ± 106.07 Experimental group K97 854.17 ± 47.83

As a result, it can be confirmed that the HbA1c content of the experimental group administered with the Lactobacillus plantarum K97 strain for 8 weeks was significantly reduced compared to that of the control group. That is, it can be seen that the average blood glucose level for 8 to 12 weeks was reduced to a level similar to metformin, a conventional antidiabetic drug, by oral administration of the Lactobacillus plantarum K97 strain.

18 is a graph showing the concentration of C-peptide measured in the serum of a normal group, a control group, an experimental group (K97), and a positive control group. Different superscripts mean that the values are significantly different from each other (p<0.05).

C-peptides, which constitute part of the large molecule before insulin circulating in the blood is made, are composed of 31 amino acids that connect the A ring and B ring of insulin. Proinsulin is synthesized in the beta cells of the pancreas, and it is divided into insulin and C-peptide in the blood, and insulin and C-peptide exist in the same ratio in the blood. Moreover, since C-peptide is not affected by anti-insulin antibodies, it is used as a diabetes biomarker that reflects the function of pancreatic beta cells.

As shown in FIG. 18, it was confirmed that the C-peptide concentration significantly decreased in the control group (CON) compared to the normal group (NOR). On the other hand, it can be seen that the experimental group administered with the Lactobacillus plantarum K97 strain for 8 weeks recovered the C-peptide concentration to a higher level than the normal group (NOR). That is, it can be seen that the Lactobacillus plantarum K97 strain exhibits an excellent blood sugar improvement effect.

Prior to the histological examination of the pancreas, the weights of the pancreas of the normal group, control group, experimental group (K97), and positive control group were measured, and are shown in Table 13. Different superscripts mean that the values are significantly different from each other (p<0.05).

army Pancreatic tissue weight (g) Normal group 0.215±0.011 ^a control group 0.130± ^0.007c positive control 0.158± ^0.028b Experimental group K97 0.149± ^0.007bc

As shown in Table 13, it can be seen that the pancreatic tissue weight of the control group was significantly decreased compared to the normal group. On the other hand, it was confirmed that the experimental group administered with the Lactobacillus plantarum K97 strain significantly increased the weight of pancreatic tissue compared to the control group. It can be seen that is effective in suppressing the reduction of pancreatic tissue.

19 is a result of measuring insulin present in pancreatic islets of a normal group, a control group, an experimental group (K97), and a positive control group by immunohistochemical staining. Measuring insulin present in beta cells in pancreatic tissue is an indicator for measuring beta cell function, and insulin can be analyzed through an immunohistochemical method.

According to FIG. 19 , it can be seen that insulin expression in beta cells of islets in the pancreas was significantly decreased in the control group (CON) compared to the normal group (NOR). That is, it can be seen that beta cells of islets in the pancreas are significantly damaged due to diabetes, and insulin expression in the pancreas is not properly performed.

In contrast, the experimental group (K97) administered with the Lactobacillus plantarum K97 strain showed a marked increase in insulin expression compared to the control group, which was similar to the positive control group (POS CON) treated with metformin, a conventional diabetes treatment. or at a higher level. In other words, it can be said to strongly suggest that beta cells of pancreatic islets in the pancreas are restored to function by La ctobacillus plantarum K97 strain to promote insulin secretion.

20 is a result of measuring glucagon present in pancreatic islets of a normal group, a control group, an experimental group (K97), and a positive control group by immunohistochemical staining. am. As described above, pancreatic islet cells, also called islets of Langerhans, are a cluster of endocrine cells that are present in the pancreas and secrete glucagon and insulin.

According to FIG. 20, it can be seen that glucagon expression in islets in the pancreas was significantly reduced in the control group (CON) compared to the normal group (NOR). That is, it can be seen that islets in the pancreas are damaged due to diabetes, and glucagon expression in the pancreas is not properly performed.

On the other hand, the experimental group (K97) administered with the Lactobacillus plantarum K97 strain showed a significant increase in glucagon expression compared to the control group, which was different from the positive control group (POS CON) treated with metformin, a conventional diabetes treatment. It can be seen that they are at a similar level. That is, it indicates that glucagon expression is normalized by restoring the function of islets in the pancreas by the Lactobacillus plantarum K97 strain.

Plasma cholesterol and plasma triglyceride concentrations were measured for blood collected from the normal group, the control group, the experimental group (K97), and the positive control group. Blood cholesterol and triglyceride concentrations were measured using an enzyme-based analysis kit (Total-C: AM 202-K, Asan Pharmaceuticals, Korea, TG: AM 157S-K, Asan Pharmaceuticals, Korea), followed by reaction with enzyme reagents, Absorbance was measured and quantified, and the results are shown in Table 14 below.

army Triglycerides
(mg/dl) Total cholesterol
(mg/dl) LDL cholesterol
(mg/dl) HDL cholesterol
(mg/dl) Normal group
(NOR) 84.18± ^11.47ab 127.85± ^12.60c 34.06± ^6.68ab 82.02±3.89 ^c control group
(CON) 107.50± ^24.45a 177.06±12.86 ^a 28.64± ^14.17ab 123.61±1.5 ^a positive control
(POS CON) 97.02± ^24.92ab 152.00± ^6.32b 19.47± ^9.05b 113.71± ^6.53b Experimental group (K97) 79.28± ^8.97b 164.53± ^12.34ab 42.90± ^10.45a 109.99± ^8.77b

As shown in Table 14, it was confirmed that the experimental group (K97) to which the Lactobacillus plantarum K97 strain was administered significantly improved clinical indicators of diabetes caused by hyperglycemia such as cholesterol and triglyceride. In particular, in the experimental group (K97) administered with the Lactobacillus plantarum K97 strain, it was confirmed that the clinical indicators of diabetes caused by hyperglycemia, such as cholesterol and triglyceride, were improved to the level of the normal group, which is useful for preventing diabetes, It can be seen that it can be used as an improvement or treatment.

Having described specific parts of the present invention in detail above, it is clear that these specific descriptions are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> KOREA FOOD RESEARCH INSTITUTE <120> Lactobacillus plantarum K97 and its uses <130> HPC9370 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1522 <212> RNA <213> artificial sequence <220> <223> 16S rRNA sequence of Lactobacillus plantarum K97 strain <400> 1 tttttttatt tttgtctctg tcagacgaac gctggcggcg tgcctaatac atgcaagtcg 60 aacgaactct ggtattgatt ggtgcttgca tcatgattta catttgagtg agtggcgaac 120 tggtgagtaa cacgtgggaa acctgcccag aagcggggga taacacctgg aaacagatgc 180 taataccgca taacaacttg gaccgcatgg tccgagtttg aaagatggct tcggctatca 240 cttttggatg gtcccgcggc gtattagcta gatggtgagg taacggctca ccatggcaat 300 gatacgtagc cgacctgaga gggtaatcgg ccacattggg actgagacac ggcccaaact 360 cctacgggag gcagcagtag ggaatcttcc acaatggacg aaagtctgat ggagcaacgc 420 cgcgtgagtg aagaagggtt tcggctcgta aaactctgtt gttaaagaag aacatatctg 480 agagtaactg ttcaggtatt gacggtattt aaccagaaag ccacggctaa ctacgtgcca 540 gcagccgcgg taatacgtag gtggcaagcg ttgtccggat ttattgggcg taaagcgagc 600 gcaggcggtt ttttaagtct gatgtgaaag ccttcggctc aaccgaagaa gtgcatcgga 660 aactgggaaa cttgagtgca gaagaggaca gtggaactcc atggttagcg gtgaaatgcg 720 tagatatatg gaagaacacc agtggcgaag gcggctgtct ggtctgtaac tgacgctgag 780 gctcgaaagt atgggtagca aacaggatta gataccctgg tagtccatac cgtaaacgat 840 gaatgctaag tgttggaggg tttccgccct tcagtgctgc agctaacgca ttaagcattc 900 cgcctgggga gtacggccgc aaggctgaaa ctcaaaggaa ttgacggggg cccgcacaag 960 cggtggagca tgtggtttaa ttcgaagcta cgcgaagaac cttaccaggt cttgacatac 1020 tatgcaaatc taagagatta gacgttccct tcggggacat ggatacaggt ggtgcatggt 1080 tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttatt 1140 atcagttgcc agcattaagt tgggcactct ggtgagactg ccggtgacaa accggaggaa 1200 ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat 1260 ggatggtaca acgagttgcg aactcgcgag agtaagctaa tctcttaaag ccattctcag 1320 ttcggattgt aggctgcaac tcgcctacat gaagtcggaa tcgctagtaa tcgcggatca 1380 gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgagagt 1440 ttgtaacacc caaagtcggt ggggtaacct tttaggaacc agccgcctaa ggtggggacag 1500 atgattaggg tgaatctagg gg 1522

Claims (10)

With Lactobacillus plantarum K97 KACC 81119BP having hypoglycemic activity,
The Lactobacillus plantarum K97 strain has leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase ( Naphtol-AS-BI-phosphohydrolase), α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase ( β-glucosidase) and N-acetyl-β-glucosaminidase, Lactobacillus plantarum K97 KACC 81119BP, characterized by exhibiting enzyme activity. According to claim 1,
The Lactobacillus plantarum K97 KACC 81119BP is Lactobacillus plantarum K97 KACC 81119BP, characterized in that it has a base sequence of 16s rRNA represented by SEQ ID NO: 1. According to claim 1,
The Lactobacillus plantarum (La ctobacillus plantarum) K97 KACC 81119BP is Lactobacillus plantarum K97 KACC 81119BP, characterized by having α-amylase or α-glucosidase inhibitory activity. According to claim 1,
The Lactobacillus plantarum K97 KACC 81119BP is characterized in that it has acid resistance, bile resistance , intestinal adhesion and antibacterial activity. According to claim 1,
The Lactobacillus plantarum K97 KACC 81119BP is Lactobacillus plantarum K97 KACC 81119BP, characterized in that it improves insulin resistance. Lactobacillus plantarum (La ctobacillus plantarum ) K97 KACC 81119BP strain or a probiotic composition comprising a culture thereof as an active ingredient. Lactobacillus plantarum (La ctobacillus plantarum ) K97 KACC 81119BP strain or a food composition for postprandial blood sugar improvement comprising a culture thereof as an active ingredient. Lactobacillus plantarum (La ctobacillus plantarum ) K97 KACC 81119BP strain or a food composition for preventing or improving diabetes comprising a culture thereof as an active ingredient. A feed composition for preventing or improving diabetes comprising the Lactobacillus plantarum K97 KACC 81119BP strain or a culture thereof according to claim 1 as an active ingredient. A pharmaceutical composition for preventing or treating diabetes comprising the Lactobacillus plantarum K97 KACC 81119BP strain or a culture thereof according to claim 1 as an active ingredient.