KR102577596B1 - Pediococcus pentosaceus KI62 and uses thereof - Google Patents

Abstract

The present invention relates to the Pediococcus pentosaceus KI62 KACC 81120BP strain having blood sugar lowering activity, and more specifically to the Pediococcus pentosaceus KI62 KACC 81120BP strain as an active ingredient. Since diabetes can be prevented, improved or treated by including it, it can be used as a food composition for preventing or improving diabetes, and further as a health functional food or pharmaceutical composition, feed composition, or pharmaceutical composition for animals.

Description

Pediococcus pentosaceus KI62 and uses thereof {Pediococcus pentosaceus KI62 and uses thereof}

The present invention relates to Pediococcus pentosaceus KI62 and its uses. In particular, it has alpha-amylase or alpha-glucosidase inhibitory activity, has acid resistance, bile resistance, and intestinal adhesion ability, has excellent short-chain fatty acid production ability, and inhibits glucose absorption and insulin secretion. It relates to a Pediococcus pentosaceus KI62 strain capable of simultaneously controlling stimulation and insulin resistance and its use.

The incidence of various lifestyle-related adult diseases is increasing due to westernized eating habits and lack of exercise. In particular, adult diseases such as diabetes, obesity, and hyperlipidemia are increasing due to changes in dietary habits toward excessive fat intake.

Diabetes is a metabolic disorder caused by impaired secretion and insufficient action of insulin secreted by pancreatic cells. It involves overproduction of glucose, decomposition of body fat and waste of protein, and abnormally increases secretion of glucagon, causing metabolic confusion. do. In the case of diabetes, characteristic symptoms of high blood sugar appear due to abnormal physiological metabolic control functions such as carbohydrate, protein, lipid, and electrolyte metabolism due to hormonal imbalance including insulin. If these hyperglycemic symptoms persist, microvascular complications such as retina, kidney, and nerves may occur. It causes macrovascular complications such as stroke, angina, 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 deficiency in the secretion of insulin, a hormone that regulates glucose in the blood, and type 2 diabetes is caused by impaired insulin secretion from pancreatic beta cells and defects in insulin action in target cells. (Insulin resistance) is observed in all cases. Insulin resistance refers to a state in which insulin action is reduced in the absence of insulin deficiency.

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

However, because it is difficult to reach the target blood sugar level with the above drugs alone, in many cases, combination therapy or combination therapy with hypoglycemic agents is performed. These diabetes treatment drugs often have side effects such as hypoglycemia, loss of appetite, nausea, vomiting, diarrhea, skin rash, lactic acidosis, liver damage, and gastrointestinal tract damage. In consideration of the above-mentioned problems, natto bacteria such as Bacillus subtilis, which have alpha-glucosidase inhibitory activity, have been developed. However, Bacillus subtilis is an aerobic bacterium and has difficulty inhabiting the intestines, so it must be supplied through separate intake. In order to receive a sufficient supply, a large amount must be consumed, and since it only acts on one mechanism, there is a problem in that sufficient blood sugar control effects cannot be obtained.

Therefore, there is an urgent need to develop substances that can prevent or treat diabetes using natural products that are edible and have few side effects.

Patent Document 1. Republic of Korea Patent Publication No. 10-2019-0068078

The present invention was created to solve the above problems, and the object of the present invention is to develop Pediococcus pentosaceus KI62 KACC, which has excellent blood sugar lowering activity and can prevent the occurrence and risk of recurrence of diabetes. The intention is to provide 81120BP.

The purpose of the present invention is to provide a probiotic composition containing Pediococcus pentosaceus KI62 KACC 81120BP or a culture thereof as an active ingredient.

The purpose of the present invention is to provide a food composition for improving postprandial blood sugar containing Pediococcus pentosaceus KI62 KACC 81120BP or a culture thereof as an active ingredient.

The purpose of the present invention is to provide a food composition for preventing or improving diabetes containing Pediococcus pentosaceus KI62 KACC 81120BP or a culture thereof as an active ingredient.

The purpose of the present invention is to provide a feed composition for preventing or improving diabetes containing Pediococcus pentosaceus KI62 KACC 81120BP or a culture thereof as an active ingredient.

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes containing Pediococcus pentosaceus KI62 KACC 81120BP or a culture thereof as an active ingredient.

In order to achieve the above object, the present invention provides Pediococcus pentosaceus KI62 KACC 81120BP, which has blood sugar lowering activity.

The Pediococcus pentosaceus KI62 KACC 81120BP may have a 16s rRNA base sequence represented by SEQ ID NO: 1.

The Pediococcus pentosaceus KI62 KACC 81120BP may have alpha-amylase or alpha-glucosidase inhibitory activity.

The Pediococcus pentosaceus KI62 KACC 81120BP may have acid resistance, bile resistance, and intestinal adhesion ability.

The Pediococcus pentosaceus KI62 KACC 81120BP may have a blood sugar lowering and anti-diabetic effect.

In order to achieve the above other objects, the present invention provides a probiotic composition containing Pediococcus pentosaceus KI62 KACC 81120BP or a culture thereof as an active ingredient.

In order to achieve the above other object, the present invention provides a food composition for improving postprandial blood sugar containing Pediococcus pentosaceus KI62 KACC 81120BP or a culture thereof as an active ingredient.

In order to achieve the above other object, the present invention provides a food composition for preventing or improving diabetes containing Pediococcus pentosaceus KI62 KACC 81120BP or a culture thereof as an active ingredient.

In order to achieve the above other object, the present invention provides a feed composition for preventing or improving diabetes containing Pediococcus pentosaceus KI62 KACC 81120BP or a culture thereof as an active ingredient.

In order to achieve the above other object, the present invention provides a pharmaceutical composition for preventing or treating diabetes containing Pediococcus pentosaceus KI62 KACC 81120BP or a culture thereof as an active ingredient.

The Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention has excellent acid resistance, bile resistance, and intestinal adhesion ability, and has excellent effects in lowering blood sugar, promoting insulin secretion, and improving insulin resistance, thereby preventing and improving diabetes. Alternatively, 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, Pediococcus pentosaceus KI62 KACC 81120BP strain has antibacterial activity against pathogenic microorganisms and food poisoning bacteria, so it can also be used as a probiotic composition.

Figure 1 shows a systematic diagram of the Pediococcus pentosaceus KI62 strain according to the present invention.
Figure 2 is a graph showing the results of analyzing the ability to produce short-chain fatty acids when K99, K263, and KI62 strains were cultured in MRS broth.
Figure 3 is a graph showing the results of analyzing the short-chain fatty acid production ability of K99, K263, and KI62 strains when cultured in MRS broth supplemented with 3% indigestible polysaccharide (maltodextrin).
Figure 4 shows Pediococcus pentosaceus KI62 in MRS liquid medium with 0.3% oxgall (with oxgall) and medium without oxgall (without oxgall). This is a growth curve according to the time each strain was cultured.
Figure 5 is a graph showing the survival rate measured after 3 hours when the Pediococcus pentosaceus KI62 strain was inoculated in an HCl solution.
Figure 6 is a graph showing the intestinal adhesion ability of Pediococcus pentosaceus KI62 strain in HT-29 cells.
Figure 7 is a graph showing the survival rate of RIN-m5F cells when treated with Pediococcus pentosaceus KI62 strain at different concentrations.
Figure 8 is a graph showing the amount of insulin secretion measured when Pediococcus pentosaceus KI62 strain was treated with RIN-m5F cells at different concentrations.
Figure 9 is a graph showing the expression level of GLP-1R, a gene involved in insulin secretion, when Pediococcus pentosaceus KI62 strain was treated with RIN-m5F cells at different concentrations. .
Figure 10 is a graph showing the expression level of IRS-2, a gene involved in insulin secretion, when Pediococcus pentosaceus KI62 strain was treated with RIN-m5F cells at different concentrations. .
Figure 11 is a graph showing the survival rate of C1C12 cells when Pediococcus pentosaceus KI62 strain was treated at different concentrations.
Figure 12 is a graph showing the glucose uptake of C1C12 cells when Pediococcus pentosaceus KI62 strain was treated at different concentrations.
Figure 13 shows the measurement of the expression level of proteins involved in sugar metabolism and utilization (p-AKT/AKT) when Pediococcus pentosaceus KI62 strain was treated with C2C12 cells at different concentrations. This is the Western blot result.
Figure 14 is a graph showing the results of Figure 13 in numbers.
Figure 15 is a Western blot result measuring the expression level of a protein involved in sugar metabolism and utilization (GLUT4) when Pediococcus pentosaceus KI62 strain was treated with C2C12 cells at different concentrations. .
Figure 16 is a graph showing the results of Figure 15 in numbers.
Figure 17 is a graph showing the weight of the normal group, control group, experimental group (KI62), and positive control over time.
Figure 18 is a graph showing AUC (Area under the curve) for blood sugar levels according to an oral glucose tolerance test.
Figure 19 is a graph showing the measurement of fructosamine in the serum of the normal group, control group, experimental group (KI62), and positive control.
Figure 20 is a graph showing the measurement of glucose concentration in the serum of the normal group, control group, experimental group (KI62), and positive control.
Figure 21 is a graph showing the measurement of insulin concentration in the serum of the normal group, control group, experimental group (KI62), and positive control.
Figure 22 is a graph showing the concentration of glycated hemoglobin in the blood of the normal group, control group, experimental group (KI62), and positive control.
Figure 23 is a graph showing the concentration of C-peptide in the serum of the normal group, control group, experimental group (KI62), and positive control.
Figure 24 shows the results of measuring insulin present in pancreatic islets of the normal group, control group, experimental group (KI62), and positive control using immunohistochemical staining.

Below, we will look at various aspects and various implementations of the present invention in more detail.

One aspect of the present invention relates to Pediococcus pentosaceus KI62 KACC 81120BP, which has hypoglycemic activity.

In the present invention, the term “hypoglycemic activity” refers to the effect of lowering blood sugar, which is a value representing the concentration of glucose in the blood. It is desirable for the human body to maintain blood sugar levels within a certain range to maintain homeostasis. Hyperglycemia refers to a condition in which blood sugar levels are abnormally high. High blood sugar that appears temporarily after a meal is a natural phenomenon and is called physiological hyperglycemia. However, if the blood sugar level increases beyond this range or the hyperglycemia state persists, it is highly likely that diabetes has developed, is likely to progress to diabetes, or has diabetes but is undetected. According to the U.S. Food and Drug Administration regulations, normal blood sugar levels are defined as fasting blood sugar of less than 100 mg/dl and blood sugar 2 hours after a meal of less than 140 mg/dl, and fasting blood sugar of more than 126 mg/dl and blood sugar 2 hours after a meal of more than 200 mg/dl. If this occurs, it is diagnosed as diabetes. Meanwhile, WHO defines a fasting blood sugar level of less than 110 mg/dl as normal. Even if it is below the level diagnosed as diabetes, if hyperglycemia, which is higher than normal, continues, it can cause fatty liver disease, increase the risk of progressing to severe liver disease, and cause cardiovascular disease and its complications. In the body, glucagon, adrenaline, insulin, and thyroid hormones work to maintain normal blood sugar levels. Hyperglycemia may occur if there is an abnormality in the secretion and/or activity of these hormones. Additionally, excessive sugar intake, lack of exercise, and/or stress can also cause high blood sugar. Pediococcus pentosaceus KI62 KACC 81120BP of the present invention has alpha-amylase (α-amylase) inhibitory activity, alpha-glucosidase (α-glucosidase) inhibitory activity, and increases sugar metabolism and utilization (promotes glucose absorption) ), it has multiple blood sugar lowering effects based on various mechanisms of promoting insulin secretion and improving insulin resistance, so it can have preventive and improving effects on diseases caused by hyperglycemia.

Specifically, Pediococcus pentosaceus KI62 KACC 81120BP, a novel strain useful for blood sugar lowering activity, blood sugar control, and/or diabetes prevention and treatment of the present invention, has the base sequence of 16S rRNA represented by SEQ ID NO: 1. It may be to have.

As a result of 16S rRNA base sequence analysis for identification and classification of Pediococcus pentosaceus KI62 KACC 81120BP of the present invention, it was confirmed that no bacteria with 100% homology were found. Therefore, the microorganism of the present invention was named Pediococcus pentosaceus KI62 KACC 81120BP strain and was deposited with the Korea Center for Microbial Conservation (KCCM) on June 25, 2020, and assigned the deposit number KACC 81120BP. received.

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

Accession number: KACC 81120BP

Trust date: 2020.05.14

The Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention is a pure isolate of 394 strains from kimchi and feces, and has alpha-amylase (α-amylase) inhibitory activity and alpha-glucosidase (α). -glucosidase) inhibitory activity was finally selected by measuring enzyme activity.

The Pediococcus pentosaceus KI62 KACC 81120BP strain used in the present invention has alpha-amylase or alpha-glucosidase inhibitory activity and the ability to produce short-chain fatty acids. Excellent, promotes glucose absorption, regulates insulin secretion, improves insulin resistance, increases p-AKT/AKT, GLUT4, GLP-1R and IRS-2 expression, improves glucose metabolism and utilization, lowers blood sugar and anti-glycemic effect based on improved glucose metabolism and utilization. Because it provides anti-diabetic effects, it has the effect of improving various mechanisms simultaneously. Therefore, the novel Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention can exhibit excellent anti-diabetic effects even when administered alone, and in some cases, may be administered together with commercially available diabetes treatments. . When administered together, blood sugar levels can be rapidly reduced.

Moreover, the Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention can reduce side effects such as gastrointestinal disorders caused by oral hypoglycemic agents such as flatulence by inhibiting the growth of harmful microorganisms in the intestines.

The Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention has excellent acid resistance, bile resistance, and intestinal adhesion, and can safely survive through the oral cavity to the intestines. Since it has antibacterial activity that inhibits the growth of intestinal pathogenic microorganisms or food poisoning bacteria, it can provide advantageous efficacy as a prebiotic material.

In addition, the Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention has antibiotic resistance to vancomycin, ampicillin, and polymyxin B, and the pediococcus mentioned in SCAN Common antibiotics for Coccus strains (ampicillin, chloramphenicol, gentamycin, kanamycin, streptomycin, erythromycin, clindamycin, tetracyclin) ) has a better antibiotic resistance effect than the resistance value for.

In addition, the Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention contains lipase (C14), leucine arylamidase, and cystine arylamidase. ), acid phosphatase, naphtol-AS-BI-phosphohydrolase, beta-galactosidase, beta-glucosidase ( β-glucosidase) and N-acetyl-beta-glucosanidase (N-acetyl-β-glucosaminidase). In particular , the Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention has enzyme activity against beta-glucuronidase, a oxidizing factor that converts benzopyrene into a carcinogenic substance. It is a very stable strain that does not have any

Through the following examples, when the Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention is administered orally, an excellent blood sugar lowering effect was shown by promoting glucose absorption, promoting insulin secretion, and improving insulin resistance. It was confirmed that this was equivalent to or superior to the positive control group (POS CON) administered metformin, a conventional diabetes treatment.

Therefore, the Pediococcus pentosaceus KI62 KACC 81120BP strain according to the present invention has a hypoglycemic and/or anti-diabetic effect as well as a prebiotic composition, so it can be used as a food to prevent, improve or treat diabetes, It can be used as feed or pharmaceutical composition.

The Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention may be used directly as the strain itself, or may be a culture obtained by culturing the strain. The culture may be a culture medium of the strain and/or a material containing the cultured strain. The culture may be a culture solution obtained by cultivating a strain and/or a material containing the cultured strain itself. The culture medium may be the culture medium itself, or any one selected from culture medium concentrate and dried culture medium. The culture solution concentrate means concentrating the culture solution, and the dried product of the culture solution means removing the water from the culture solution. The bacteria may be live bacteria.

The Pediococcus pentosaceus KI62 KACC 81120BP strain according to the present invention can be grown by general culture methods, recovered and concentrated through separation processes such as centrifugation, and dried (e.g., freeze-drying). It can be manufactured and used in the form of a probiotic.

Another aspect of the present invention relates to a probiotic composition containing Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient.

The Pediococcus pentosaceus KI62 KACC 81120BP strain is as described above.

In the present invention, the term "culture" is not limited to culture medium and/or material containing the cultured strain of Pediococcus pentosaceus KI62 KACC 81120BP strain, but Pediococcus pentosaceus ( Pediococcus pentosaceus) KI62 KACC 81120BP refers to the entire medium containing the strain, its metabolites, and extra nutrients obtained by culturing the strain for a certain period of time in a medium that supplies nutrients to enable the strain to grow and survive in vitro. , This also includes the culture solution from which the strain was removed after culturing the strain. The formulation of the culture is not limited, and may specifically be liquid or solid.

In the present invention, the term "probiotics" can be used interchangeably with 'probiotics' and is a general term for living bacteria that are beneficial to the human body when consumed in an appropriate amount, meaning bacteria that are beneficial to our body.

The Pediococcus pentosaceus KI62 KACC 81120BP 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 and reaches the small intestine, where it proliferates and settles. can do. In particular, this strain shows antibacterial activity against pathogenic microorganisms and food poisoning bacteria, so it can be expected to have beneficial effects on health within the colon, such as suppressing harmful microorganisms and normalizing the intestinal flora. Therefore, since it significantly satisfies all of the above conditions that must be met as a probiotic composition, it can be usefully used as a probiotic composition.

The probiotic composition according to the present invention may include not only conventional foods, functional foods, health supplements, and food additives, but also those approved as pharmaceuticals.

The composition of the present invention may be prepared according to conventional probiotic composition preparation methods, and may generally be in lyophilized or encapsulated form, a culture suspension, or in the form of a dry powder. In addition, the active ingredient of the probiotic composition , Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture, may be supplemented with one or two or more conventional pharmaceutically acceptable carriers or one or two types of carriers. It can be prepared into a conventional formulation by selecting one or more additives.

The carrier may be any one or more selected from the group consisting of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives, and the additives may be selected from the group consisting of flavoring agents, vitamins and antioxidants. You can select one or more to use.

Any pharmaceutically acceptable carrier and additive can be used. Specifically, diluents include lactose monohydrate, trehalose, corn starch, soybean oil, and microcrystalline cellulose. (microcrystalline cellulose) or mannitol (D-mannitol) are preferred, magnesium stearate or talc is preferred as a lubricant, and polyvinylpyrrolidone (PVP: polyvinyipyrolidone) or hydroxypropyl as a binder. It is preferable to select 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, or white lead. (white bee's wax) or xanthan gum. Preservatives include methyl p-hydroxy benzoate (methlparaben), propyl p-hydroxybenzoate (propylparaben), or potassium sorbate ( It is preferable to choose among potassium sorbate.

In addition, the Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention can be used as a starter to produce various lactic acid bacteria fermented milk and fermented products, where the fermented milk foods include yogurt, calpis, cheese, and butter. and the like, and fermented products include tofu, soybean paste, fermented soybean paste, jelly, kimchi, etc., but are not necessarily limited thereto. The fermented milk and fermented products can be easily produced by replacing only the strain in the conventional production method with the strain of the present invention. As described above, the composition of the present invention may be a food or a food additive, and the food is preferably a fermented food. Additionally, the probiotic composition of the present invention may be in the form of powder or granules, and the powder or granule form can be easily manufactured by a conventional method in the art.

Another aspect of the present invention relates to a food composition for lowering blood sugar containing Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient.

The Pediococcus pentosaceus KI62 KACC 81120BP strain is as described above.

According to an embodiment of the present invention , the Pediococcus pentosaceus KI62 KACC 81120BP strain simultaneously increases blood sugar levels through a complex mechanism that promotes glucose uptake in cells, promotes insulin secretion, and improves insulin resistance. It has a lowering effect. Therefore, it can effectively reduce fasting or postprandial blood sugar levels, and it has been confirmed that diabetes can be prevented or treated through a blood sugar lowering effect in actual diabetic animal models.

In addition, according to the present invention, it was confirmed that the Pediococcus pentosaceus KI62 KACC 81120BP strain significantly reduces the average glycated hemoglobin concentration in a diabetic animal model, preventing diabetes through a blood sugar lowering effect. Or, you can see that it can be treated.

Therefore, the Pediococcus pentosaceus KI62 KACC 81120BP strain according to the present invention can be used as an active ingredient in functional foods that can suppress or lower the increase in fasting or postprandial blood sugar levels in diabetic patients.

The Pediococcus pentosaceus KI62 KACC 81120BP strain of the present invention showed no toxicity at all even when treated with a very large amount of cells.

In the present invention, the term "lowering blood sugar" refers to the effect of lowering blood sugar, which is a value representing the concentration of glucose in the blood.

The 'food composition' includes Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture as an active ingredient, as well as ingredients described in the Food Standards and Specifications ('Food Code of Conduct') commonly used in food production. Includes food ingredients that can be used as food and food additives listed in the Food Additives Code.

There is no need to specifically limit it, but examples include proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. The carbohydrates include monosaccharides, such as glucose, fructose, etc.; Disaccharides such as maltose, sugar, lactose, etc.; Oligosaccharides or polysaccharides such as dextrin, starch syrup, cyclodextrin, etc.; Sugar alcohols such as xylitol, sorbitol, erythritol, etc. can be used. The flavoring agent may be a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) or a synthetic flavoring agent (saccharin, aspartame, etc.).

When preparing a food composition using the Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture as an active ingredient, there is no need to specifically limit the content as long as it exhibits blood sugar-lowering activity, but the amount of 1 to 500 in the composition It may be included at a concentration of ㎍/㎖, preferably at a concentration of 1 to 300 ㎍/㎖, more preferably at a concentration of 1 to 100 ㎍/㎖, even more preferably at a concentration of 1 to 30 ㎍/㎖, most preferably at 1 to 30 ㎍/㎖. It may be included at a concentration of from 10 μg/ml.

The Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture, which is the active ingredient in the above food composition, varies depending on the condition, body weight, and presence, degree, and duration of the disease of the consumer, but may be determined appropriately by a person skilled in the art. can be selected. For example, the daily dosage 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. There is no need to specifically limit the number of administrations, but a person skilled in the art can adjust it within the range of three times a day to once a week. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.

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

In addition, the Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture can be added to general foods to induce blood sugar-lowering and blood sugar-lowering activities. Foods that can be added include, in particular, There is no need to limit it, but for example, confectionery, bread or rice cake, processed cocoa products or chocolate, processed meat or egg products, processed fish meat products, and tofu as exemplified in the food standards and specifications ('Food Code') under Article 7 of the Food Sanitation Act. Alternatively, it can be added to jelly, noodles, tea, coffee, beverages, special-purpose foods, sauces, seasoned foods, dressings, kimchi, salted seafood, 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 as exemplified in the livestock product processing standards and ingredient specifications ('Livestock Product Code') pursuant to Article 4 of the Livestock Products Sanitation Management Act.

Meanwhile, a food composition containing the Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture as an active ingredient can be used alone as a “health functional food that helps lower blood sugar levels.”

The above ‘health functional food’ refers to food manufactured (including processing) in accordance with legal standards using raw materials or ingredients with functional properties useful to the human body (Article 3, Paragraph 1 of the Health Functional Food Act). The terminology or scope of the above 'health functional food' may vary depending on the country, but it is also called 'Dietary Supplement' in the United States, 'Food Supplement' in Europe, 'Health Functional Food' or 'Health Functional Food' in Japan. It may be classified as ‘Food for Special Health Use (FoSHU)’ or ‘Health Food’ in China.

The above food composition or health functional food may additionally contain food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions of the ‘Food Additive Code’ and general test methods, etc., unless otherwise specified. Follow.

In addition, the health functional food includes the Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture, which is notified as a 'functional raw material' used in "health functional food that helps lower blood sugar levels." Raw materials or individually recognized raw materials include distilled pine needle concentrate, soybean fermentation extract, albumin, freeze-dried silkworm powder, upper crustal leaf extract mixture, Somoktae peptide complex, ginseng hydrolyzed concentrate, tagatose, hydroxypropylmethylcellulose, upper leaf extract, L -arabinose, silk protein enzyme hydrolyzate, pinitol, Rhodiola rhododendron complex extract, cinnamon extract powder, Ceriphoria lacerata mycelium culture, cypolinol Ecklonia cava alcohol 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 hydrolyzate, oat dietary fiber, indigestible maltodextrin, soy 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 containing Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient.

The food composition of the present invention can prevent or improve diabetes by containing Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient. In addition, the food composition of the present invention contains Pediococcus pentosaceus KI62 KACC 81120BP strain as an active ingredient to increase sugar metabolism or sugar utilization, inhibit the rise in blood sugar by alpha-glucosidase and alpha-amylase, and Diabetes can be prevented or improved by improving one or more aspects of normalizing blood insulin secretion.

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, increased thirst and hunger. If this is not treated, other complications may occur and lead to death.

In the present invention, diabetes includes insulin-dependent diabetes (type 1 diabetes) and non-insulin-dependent diabetes (type 2 diabetes), and furthermore, diabetes that occurs due to damage to the pancreas due to other diseases, such as hypothyroidism. This includes hyperactivity disorder, hyperadrenocorticism, diabetes caused by hypergrowth hormone hyperactivity or catecholamine hyperactivity, and gestational diabetes.

In the present invention, the term "prevention" refers to any action that inhibits or delays diabetes by administering a composition containing Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient.

In the present invention, the term "improvement" refers to the improvement of parameters related to diabetes, such as the degree of diabetes symptoms, by ingestion of a composition containing Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture as an active ingredient. It means any action that reduces

In the present invention, the term "treatment" refers to any action in which symptoms of diabetes are improved or completely cured by administration of a composition containing Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture as an active ingredient. .

The 'food composition' includes Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture as an active ingredient, as well as ingredients described in the Food Standards and Specifications ('Food Code of Conduct') commonly used in food production. Includes food ingredients that can be used as food and food additives listed in the Food Additives Code.

There is no need to specifically limit it, but examples include proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. The carbohydrates include monosaccharides, such as glucose, fructose, etc.; Disaccharides such as maltose, sugar, lactose, etc.; Oligosaccharides or polysaccharides such as dextrin, starch syrup, cyclodextrin, etc.; Sugar alcohols such as xylitol, sorbitol, erythritol, etc. can be used. The flavoring agent may be a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) or a synthetic flavoring agent (saccharin, aspartame, etc.).

When manufacturing a food composition using the Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture as an active ingredient, there is no need to specifically limit the content as long as it shows the effect of preventing or improving diabetes, but 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. there is.

The Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture, which is the active ingredient in the above food composition, varies depending on the condition, body weight, and presence, degree, and duration of the disease of the consumer, but may be determined appropriately by a person skilled in the art. can be selected. For example, the daily dosage 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. There is no need to specifically limit the number of administrations, but a person skilled in the art can adjust it within the range of three times a day to once a week. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.

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

In addition, the Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture can be added to general foods to prevent or improve diabetes. Foods that can be added do not need to be specifically limited, but examples include For example, confectionery, bread or rice cake, processed cocoa products or chocolate, processed meat or egg products, processed fish meat products, tofu or jelly, noodles, tea, It can be added to coffee, beverages, special-purpose foods, sauces, seasoned foods, dressings, kimchi, salted seafood, 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 as exemplified in the livestock product processing standards and ingredient specifications ('Livestock Product Code') pursuant to Article 4 of the Livestock Products Sanitation Management Act.

Meanwhile, the food composition containing the Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture as an active ingredient can be used alone as a "health functional food that helps prevent or improve diabetes." .

The above ‘health functional food’ refers to food manufactured (including processing) in accordance with legal standards using raw materials or ingredients with functional properties useful to the human body (Article 3, Paragraph 1 of the Health Functional Food Act). The terminology or scope of the above 'health functional food' may vary depending on the country, but it is also called 'Dietary Supplement' in the United States, 'Food Supplement' in Europe, 'Health Functional Food' or 'Health Functional Food' in Japan. It may be classified as ‘Food for Special Health Use (FoSHU)’ or ‘Health Food’ in China.

The above food composition or health functional food may additionally contain food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions of the ‘Food Additive Code’ and general test methods, etc., unless otherwise specified. Follow.

In addition, the health functional food contains the Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture as a 'functional raw material' used in "health functional food that helps prevent or improve diabetes." Notified raw materials or individually approved raw materials include distilled pine needle concentrate, soybean fermentation extract, albumin, nopa; Methyl cellulose, leaf extract, L-arabinose, silk protein enzyme hydrolyzate, pinitol, Rhodiola rhododendron complex extract, cinnamon extract powder, Ceriphoria axerrata mycelium culture, cypolinol Ecklonia root extract, chitooligosaccharide, guava leaf extract, Banaba leaf extract, evening primrose seed extract, guar gum/guar gum hydrolyzate, oat dietary fiber, indigestible maltodextrin, soybean dietary fiber, wheat dietary fiber, corn bran dietary fiber, inulin/chicory extract, fenugreek seed dietary fiber, etc. It can be used in combination with health functional food ingredients related to diabetes.

The present invention relates to a feed composition for preventing or improving diabetes comprising Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient.

The 'feed composition' contains, as an active ingredient, Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture, as well as food raw materials and food additives that can be used as food as described in the Food Standards and Specifications ('Food Code'). Food additives listed in can be used, and even if they are not food raw materials or food additives that can be used as food, raw materials that fall within the scope of sweet feed in Annex Table 1 of 'Standards and Specifications for Feed, etc.', and raw materials that fall within the scope of supplementary feed in Annex Table 2 Raw materials can be used.

The 'feed composition' may be an extractant among supplementary feeds according to 'Standards and specifications for feed, etc.', or may be a compound feed containing the supplementary feed.

When preparing a feed composition using the Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture as an active ingredient, there is no need to specifically limit the content as long as it is effective in preventing or improving diabetes, but 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. there is.

The active ingredient in the feed composition , Pediococcus pentosaceus KI62 KACC 81120BP strain or its culture, varies depending on the condition, body weight, and presence, degree, and period of disease of the ingesting animal, but may be prepared by a person skilled in the art. can be selected appropriately. For example, based on the daily dosage, 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. There is no need to specifically limit the number of administrations, but a person skilled in the art can adjust it within the range of three times a day to once a week. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.

The present invention relates to a pharmaceutical composition for preventing or treating diabetes containing Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient.

The present invention also relates to an oral pharmaceutical composition for animals for preventing or treating diabetes, comprising Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient.

Additionally, 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 new use of Pediococcus pentosaceus KI62 KACC 81120BP or a culture thereof for producing 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, depending on the desired method. Parenteral administration methods include external dermal application, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or There are methods such as intrathoracic injection, but oral administration is most preferable.

The 'pharmaceutical composition', 'medicine', 'pharmaceutical composition for animals' or 'medication for animals' includes Pediococcus pentosaceus KI62 KACC 81120BP or its culture as an active ingredient, as well as pharmaceutical compositions, etc. It may further include appropriate carriers, excipients, and diluents commonly used in manufacturing.

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 target compound, but also dissolves the compound.

There is no need to specifically limit the carrier, excipient, and diluent, but for example, lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, 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, medicine, pharmaceutical composition for animals, or medicine for animals may vary depending on the age, gender, and weight of the patient or animal to be treated, and, above all, the condition of the subject to be treated, the specific category of the disease to be treated, or It will depend on the type, route of administration, and properties of the therapeutic agent used.

The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals is appropriately selected depending on the absorption rate of the active ingredient in the body, the excretion rate, the age, weight, sex, and condition of the patient or animal to be treated, and the severity of the disease to be treated. , it is generally preferred to administer 0.1 to 1,000 mg/kg per day, preferably 1 to 500 mg/kg, more preferably 5 to 250 mg/kg, and most preferably 10 to 100 mg/kg. The unit dosage form formulated in this way can be administered several times at regular time intervals as needed.

The pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals may be administered individually as a preventive or therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The pharmaceutical compositions, medicines, pharmaceutical compositions for animals, or medicines for animals can be used by formulating them into oral dosage forms such as powders, granules, tablets, capsules, troches, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods. there is. When formulated, it can 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. These solid preparations include the compound with at least one excipient, such as starch, calcium carbonate, sugar or lactose, and gelatin. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .

The method for preventing or treating diabetes involves orally administering the composition to humans or non-human animals, especially mammals, for example, orally administering the composition to an individual to be treated with diabetes.

The dosage, administration method, and frequency of administration for the treatment may refer to the dosage, administration method, and frequency of administration of the pharmaceutical composition, medicine, pharmaceutical composition for animals, or medicine for animals.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples are for illustrating 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 isolation

In order to select candidates with blood sugar 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 each was inoculated into modified MRS medium supplemented with bromcresol purple and sodium azide using the flat smear method, cultured at 37°C for 48 hours, and bacterial colonies were collected. After pure isolation on modified MRS medium, the colonies that turned yellow were selected as potential lactic acid bacteria. The selected strains were plated on modified MRS medium and purified through aerobic culture. After pure separation, culture was performed in Lactobacilli MRS broth (Difco, USA) at 37°C for 18 hours, and then cultured and stored in tryptic soy agar (Difco, USA) slant at 37°C for 18 hours. did.

<Experimental Example 2> Selection of strains with blood sugar enhancing activity

1) Measurement of alpha-amylase (α-amylase) inhibitory activity

Alpha-amylase (α-amylase) activity inhibition ability was measured by [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 used with modification. An enzyme solution was prepared by diluting alpha-amylase with distilled water to a concentration of 0.1 g/10 ml. A substrate solution was prepared by adding 0.5% soluble starch in 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, the dye was stained for 30 minutes using an iodine solution and the absorbance was measured at 660 nm, and the results were calculated using Equation 1 below. The absorbance was compared when treated with alpha-amylase alone (control group) and when the samples were mixed. Sample refers to the 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 alpha-glucosidase inhibitory activity

Alpha-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. 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 ㎕ of sample was mixed with 50 ㎕ of 0.75 unit/㎖ alpha-glucosidase enzyme solution and 50 ㎕ of 200 mM potassium phosphate buffer (pH 6.5), preincubated at 37°C for 10 minutes, and then incubated with 0.1 M 100 ㎕ 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 calculated using the following equation 2. The sample refers to the target substance to be measured, and the control group is treated with only the alpha-glucosidase enzyme solution without the 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 alpha-amylase and alpha-glucosidase inhibitory activities of the isolated strains, strain KI62 (94.86 ± 3.30% and 98.59 ± 0.52%, respectively) and strain K99 (99.95±0.67% and 95.18±5.66%, respectively) and K263 (98.00±3.06 and 98.70±0.44, respectively) showed the highest inhibitory activity. Accordingly, the above three strains were selected.

<Experimental Example 3> Identification of Pediococcus pentosaceus K162 strain

1) Sugar availability analysis

Sugar availability was confirmed for the final selected KI62 strain using the API20 STREP kit. The sugar utilization of the KI62 strain is shown in Table 1 below.

Strains KI62 Pyruvate + Hippurate + Esculin + Pyrrolidonyl-2-naphthylamide - 6-Bromo-2-naphyl-α-D-glucuronate + Naphtol AS-BI ß-D-glucuronate - 2-naphthyl-ß-D-galactopyranoside + 2-naphthyl phosphate - L-leucine-2 naphthylamide + Arginine + Ribose + L-Arabinose + Mannitol + Sorbitol + Lactose + Trehalose + Inulin - Raffinose + Starch - Glycogen -

2) Identification of 16SD rRNA

Genomic DNA was extracted from the KI62 strain and the 16s rRNA base sequence 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, 5 minutes, then 95°C, 30 seconds; 55°C, 2 min; 68°C, 1 minute and 30 seconds were performed 30 times, ending with 68°C, 10 minutes. For sequence analysis, dNTPs and reactants not participating in the reaction were removed from the PCR product using the Montage PCR Clean up kit (Millipore), and then primers for sequencing 785F 5'(GGA TTA GAT ACC CTG GTA)3' and 907R were used. 5'(CCG TCA ATT CMT TTR AGT TT)3' was used for automatic analysis using the Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSystems, USA).

As a result of 16S rRNA analysis of the finally selected KI62 strain, the 16S rRNA base sequence of the KI62 strain showed 99% homology with Pediococcus pentosaceus DSM20336, indicating that the new microorganism of the present invention is Pediococcus pen. It was confirmed that it was a strain belonging to the Pediococcus pentosaceus species. Therefore, the final selected KI62 strain was named Pediococcus pentosaceus KI62 and deposited with the National Academy of Agricultural Sciences (KACC) of the Rural Development Administration on June 25, 2020 (Accession Number: KACC 81120BP).

The base sequence of the 16S rRNA of the strain of the present invention is represented by SEQ ID NO: 1, and the phylogenetic tree for the Pediococcus pentosaceus KI62 strain was analyzed, and the results are shown in Figure 1.

<Experimental Example 4> Measurement of short-chain fatty acid production ability

We sought to confirm the ability of the final selected K162 strain to produce short chain fatty acids (SCFA). As comparison groups, K99 strain and K263 strain were used.

First, each strain was inoculated at 1% into MRS broth and MRS broth supplemented with 3% indigestible polysaccharide (maltodextrin) and cultured at 37°C for 18 hours. After culture was completed, only the supernatant of the culture was inoculated. It was separated and the contents of short-chain fatty acids propionic acid, acetic acid, and butyric acid were measured.

1) Measurement of acetic acid content

Culture supernatants of KI62, K99, and K263 strains obtained through the above process were prepared as samples. Boil 5 ml of the sample solution right before the experiment, cool it, dilute it until the color of the sample solution appears slightly, add a few drops of 1% phenolphthalein solution, and then add 0.1 N sodium hydroxide solution until the light red color persists for 30 seconds. After titration, the total acid was calculated according to Equation 3 below.

[Equation 3]

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

In equation 3,

V1 is the amount (ml) of 0.1 N sodium hydroxide solution consumed in the 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 KI62, K99, and K263 strains obtained through the above process were prepared as samples. First, acetonitrile (ACN) was added to 40 ml of sample to 4 g, and then 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 by gas chromatography/mass spectrometer (GC-MS). GC-MS analysis conditions are shown in Table 2.

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㎕ Split ratio Splitless Carrier Helium, 1.0 ㎖/min M.S. Ionization mode E.I. Electron impact mode 70 eV Selected ion(m/z) 74 ¹⁾ , 57, 45 MS ion source temperature 200℃

¹⁾ Quntiation

3) Measurement of butyric acid content

Culture supernatants of KI62, K99, and K263 strains obtained through the above process were prepared as samples. To extract butyric acid from the sample, chloroform-methanol extraction method was used. First, the sample extracted using chloroform-methanol was concentrated using an evaporator and then converted into fatty acid methyl ester. Fatty acid methyl ester derivatives were prepared as follows. 20 mg of lipid was taken into a tube, 2 ml of 0.5N NaOH/Methanol was added, the tube was capped, and the tube was hydrolyzed in a heating block (100° C.) for about 5 minutes. After the reaction was completed, it was cooled, 2 ml of 14% BF ₃ /Methanol was added and reacted for 5 minutes, then 2 ml of isooctane was added and shaken. After the reaction was completed, 2 ml of saturated saline solution 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. The dehydrated fatty acid methyl ester test solution was received and analyzed by injecting it into a gas chromatograph (HP-6890GC FID, Agilent Technologies, Santa Clara, CA, USA). Gas chromatography analysis conditions are shown in Table 3.

Instrument GC-FDI Column SP-2560 (Supelcom 100 m × 0.2 ㎜ 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㎕ Carrier gas He Column flow 1.5 ml/min Injection volume 1.0 μl Split ratio 50:1

4) Conclusion

Figure 2 is a graph showing the results of analyzing the short-chain fatty acid production ability of the K99, K263, and KI62 strains when cultured in MRS broth, and Figure 3 is a graph showing the results of analyzing the ability of the K99, K263, and KI62 strains to produce 3% indigestible polysaccharide (maltodextrin). This is a graph showing the results of analyzing the ability to produce short-chain fatty acids when cultured in MRS broth supplemented with . The results of Figures 2 and 3 are summarized in the table below. Different superscripts in the drawing mean that the values are significantly different from each other (p<0.05). NS means not significant (p<0.05).

division Propionic acid (ppm) Acetic acid (%, w/v) Butyric acid (g/kg) M.R.S. MRS+M M.R.S. MRS+M M.R.S. MRS+M K99 6.032± ^0.085NS 11.022±2.105 ^N.S. 1.190± ^0.014c 1.180± ^0.070b 2.033± ^0.041d 1.923± 0.050 ^NS K263 5.221± 0.000 10.357±2.578 1.270± 0.000 ^b 1.330± ^0.140a 1.800± ^0.050c 2.578± 0.023 KI62 5.953± 1.662 18.047±1.852 1.150± 0.000 ^d 1.120± ^0.070c 2.379± 0.022 ^b 2.193± 0.061

As shown in Figures 2, 3 and Table 4, it was confirmed that the Pediococcus pentosaceus KI62 strain had an equal or superior short-chain fatty acid production ability. In particular, it was confirmed that the KI62 strain had a propionic acid production capacity 1.63 times higher than other strains in MRS broth supplemented with 3% indigestible polysaccharide (maltodextrin).

<Experimental Example 5> Pediococcus pentosaceus ( Pediococcus pentosaceus ) Characterization of strain KI62

The antibiotic resistance, enzyme activity test, bile resistance and acid resistance test, antibacterial activity test, and intestinal adhesion of the finally selected Pediococcus pentosaceus KI62 strain were analyzed as follows. Results are expressed as mean ± standard deviation (SD), and statistical analysis was performed using Statistical Package for Social Sciences (SPSS, SPSS Inc., USA). Significant differences were statistically processed using one-way ANOVA, and significance was tested at the 5% level using Duncan's multiple range tests.

1) Enzyme activity

Each selected strain 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/ml. The sample was incubated at 37°C for 5 hours using an API ZYM kit (API bioMerieux, Lyon, France) and then subjected to an enzyme reaction. Enzyme activity was expressed as a number from 0 to 5 by comparing the standard color table, and other than the control group, 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 enzymes The activity was measured, and the results are shown in Table 5 below.

Enzyme P. pentosaceus KI62 Alkaline phosphatase 0 Esterase(C4) 0 Esterase Lipase(C8) 0 Lipase(C14) One Leucine arylamidase 5 Valine arylamidase 4 Cystinearylamidase One Trypsin 0 α-chymotrypsin 0 Acid phosphatase 2 Naphtol-AS-BI-phosphohydrolase 3 α-galactosidase 0 β-galactosidase 2 β-glucuronidase 0 α-glucosidase 0 β-glucosidase 2 N-acetyl-β-glucosaminidase 2 α-mannosidase 0 α-fucosidase 0

As shown in Table 5, Pediococcus pentosaceus KI62 strain showed a high activity of 5 for Leucine arylamidase, and in the case of β-glucuronidase, a oxidizing factor that converts benzopyrene into a carcinogenic substance, the enzyme The activity was 0, indicating that it was a stable bacteria.

2) Biliary resistance

[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 P. pentosaceus KI62 strain cultured in MRS liquid medium at 37°C for 18 hours was cultured in MRS liquid medium containing 0.05% cysteine with 0.3% oxgall as a control. ) were inoculated at 1% each into medium without addition. They were cultured anaerobically in an incubator at 37°C for 7 hours, and at each hour, they were poured and hardened on a BCP plate count agar plate and then cultured anaerobically at 37°C for 48 hours and counted. The results are shown in Figure 4.

Figure 4 shows Pediococcus pentosaceus KI62 in MRS liquid medium with 0.3% oxgall (with oxgall) and medium without oxgall (without oxgall). This is a growth curve according to the time each strain was cultured. * means p<0.05, ** means p<0.01, *** means p<0.001.

As shown in Figure 4, the Pediococcus pentosaceus KI62 strain decreased slightly after 2 hours in the medium containing oxgall, but showed an overall survival rate of more than 91.67% compared to the control, so Pediococcus pen It can be seen that Pediococcus pentosaceus KI62 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, and 4 solutions and a pH 6.4 solution as a control, and 10 ml of the prepared pH solution was incubated in MRS liquid medium containing 0.05% cysteine at 37°C for 24 hours. After mixing 1 ㎖ of selected strains (approximately 10 ⁹ CFU/㎖) cultured for a time, anaerobically cultured at 37 ℃, the number of viable cells after 0, 1, 2, and 3 hours was poured onto a BCP plate count agar plate, solidified, and then incubated at 48 ℃ at 37 ℃. After anaerobic cultivation for a time, they were counted, and the results are shown in Figure 5.

Figure 5 is a graph showing the survival rate measured after 3 hours when the Pediococcus pentosaceus KI62 strain was inoculated in an HCl solution. * means p<0.05, ** means p<0.01, *** means p<0.001.

In order to function sufficiently 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 stage (0 hours), Pediococcus pentosaceus KI62 strain showed a 99-100% survival rate for 3 hours in all PH conditions. In other words, it can be seen that the Pediococcus pentosaceus KI62 strain is an excellent strain with significantly excellent acid resistance and a high survival rate in the stomach.

4) Antibiotic resistance

Pediococcus pentosaceus KI62 strain was inoculated into MRS liquid medium, cultured at 37°C for 18 hours, and then diluted to an appropriate concentration in 0.1% peptone solution. Tryptic soy liquid medium containing each antibiotic at each concentration was inoculated at a level of 10 ⁵ -10 ⁶ CFU/ml, cultured at 37°C for 48 hours, and then observed with the naked eye to determine growth. A two-fold dilution method was used to measure antibiotic resistance, and the anti-diabetic effect of Pediococcus pentosaceus KI62 strain, which was the lowest concentration inhibited, was determined by the MIC (Minimum inhibitory concentration) value. Antibiotics were purchased and used from Sigma (USA). The antibiotics used in the test were Amikacin, Gentamicin, Kanamycin, Streptomycin, Ampicillin, Penicillin-G, Oxacillin, Bacitracin, Polymyxin B, Ciprofloxacin, Tetracycline, Clindamycin, Erythromycin Rifampicin, Vancomycin, and Chloramphenicol. The results are shown in Table 6.

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

As shown in Table 6, the Pediococcus pentosaceus KI62 strain has the highest antibiotic resistance to vancomycin, with an MIC concentration of 4,096 ㎍/㎖ or more, while the MIC concentration to Clindamycin is 1.0 ㎍. It was confirmed that the lowest sensitivity was below /㎖. In other words, the antibiotic resistance of Pediococcus pentosaceus KI62 strain was confirmed to be higher than that specified by SCAN (The scientific committee on animal nutrition).

5) Antibacterial power

[Gilliand SE, Speck ML. Deconjugation of bile acids by intestinal lactobacilli. Appl Environ Mycobiol. Antibacterial activity was measured according to the method described in [1977;33:15-18]. The indicator bacteria consisting of Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus were all distributed from the Korea Food Research Institute, and the growth medium for the indicator bacteria was grown aerobically in nutriunt liquid medium. Cultivated at ℃ for 24 hours. The medium used for mixed culture and control was MRS liquid medium, inoculated with selection and indicator bacteria, respectively, and cultured at 37°C for 24 hours. As selection media, EMB agar for Escherichia coli , Bismuth sulfite agar for Salmonella Typhimurium , Listeria Isolation Agar containing Supplement (X123) for Listeria monocytogenes , and Baird Parker agar for Staphyloccous aureus were cultured at 37°C for 6 hours. The inhibition rate of indicator bacteria by Pediococcus pentosaceus KI62 strain was calculated using Equation 4, and the results are shown in Table 7.

[Equation 4]

Inhibition (%) = (Bacterial count CFU/mL in control group - Bacterial count CFU/mL after mixed culture)/Bacterial count CFU/mL in control group

Pathogens Growth Inhibition
(%) pathogens KI 62+pathogens CFU/ml pH CFU/ml pH Escherichia coli 6.80±0.14×10 ⁶ 6.22 4.80±0.28×10 ⁵ 4.72 29.41 Salmonella Typhimurium 3.15±0.64×10 ⁷ 6.17 1.95±0.21×10 ⁷ 4.75 38.10 Listeria monocytogenes 1.45±0.07×10 ⁵ 6.24 7.00±0.14×10 ⁴ 4.67 51.72 Staphylococcus aureus 7.13±0.75×10 ⁶ 5.24 3.53±0.60×10 ⁶ 4.67 50.47

* The initial bacterial count of Pediococcus pentosaceus KI62 strain is 3.63 ± 0.35 × 10 ⁶ CFU/ml

As shown in Table 7, as a result of confirming whether Pediococcus pentosaceus KI62 strain, as a probiotic strain, has antibacterial activity to inhibit harmful bacteria and improve intestinal flora, three types of food poisoning bacteria were identified. The antibacterial activity was significantly excellent against ( E. coli, S. Typhimurium, S. aureus ). Pediococcus pentosaceus KI62 strain was confirmed to have over 51% antibacterial activity against the pathogenic microorganism L. monocytogenesdp .

6) Intestinal fixation ability

HT-29 cells were used to evaluate intestinal colonization ability. HT-29 cells of the present invention were purchased through the Korea Cell Line Bank (Seoul, Korea), and were described in [Kim SJ, Cho SY, Kim SH, Song OJ, Shin IS, Cha DS, et al. It was performed according to the method described in [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 at 37°C. , cultured in an incubator under conditions supplied with 5% CO ² /95% air. To culture HT-29 cells, cells were dispensed into each well of a 12 well plate at a rate of 10 ⁶ cells/well, and the medium was replaced every two days. On the day before the experiment, when the cells reached 95%, serum free medium was added. ) to prevent further filling of cells. To test the intestinal colonization ability of the Pediococcus pentosaceus KI62 strain, 1 ml of the second subcultured strain was centrifuged at 12,000 rpm for 3 minutes and incubated in two serum-free medium. Washed three times. The washed strain was diluted with RPMI medium to adjust the OD _{at 600 nm} to 0.5, diluted with 0.1% peptone solution, and poured onto BCP plate count agar to measure the initial bacterial count. 100 ㎖ of bacterial cells with the correct OD value were dispensed into wells, incubated at 37°C and 5% CO ₂ for 2 hours, and non-adherent bacteria were washed five times with PBS. Cell-bacteria were removed by adding 1 ml of Trypsin-EDTA, diluted with 0.1% peptone solution, and then poured onto BCP plate count agar to measure the number of bacteria. The results were It is shown in Figure 6.

Figure 6 is a graph showing the intestinal adhesion ability of Pediococcus pentosaceus KI62 strain in HT-29 cells. According to this, Pediococcus pentosaceus KI62 strain adheres to intestinal cells. It was confirmed that the adhesion ability was significantly excellent. Lactobacillus rhamnosus GG ( L. rhamnosus GG) (LGG) (Gopal et al., 2001), which was used as a positive control, is a strain widely known to have a significantly excellent adhesion ability to intestinal cells. It can be seen that the Pediococcus pentosaceus KI62 strain of the present invention has an excellent intestinal adhesion ability equivalent to that of the LGG strain.

<Experimental Example 6> Identification of blood sugar improvement mechanism for RIN-m5F cells

1) Pretreatment of selected strains

Pediococcus pentosaceus KI62 strain was inoculated at 1% in MRS broth and then cultured at 37°C for 18 hours. The cultured strain was centrifuged at 1,500 xg for 15 minutes to collect bacterial cells. To remove the remaining MRS broth, the collected strains were washed three times with sterilized distilled water. The washed strain was freeze-dried and suspended at a concentration of 10 mg/ml. After sonicating for 50 seconds using an ultrasonicator, the sample was sterilized in an autoclave to prepare a sample for use in the experiment. Each sample was diluted by concentration according to the analysis, DW was used for dilution by concentration, and the prepared sample was stored in a refrigerator.

2) Cell model preparation

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

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 using the WST-1 assay (ITSBio, Seoul, Korea). First, RIN-m5F cells were distributed to each 96 well plate at 5×10 ⁴ cells/well. After culturing for 24 hours, samples were treated at different concentrations in each well and cultured for 24 hours. Upon completion of incubation, 10 ㎕ of WST-1 reagent was added per 100 ㎕ for reaction, and the absorbance was measured at 450 ㎚ (Ref. 600 ㎚) using a microplate reader (Infinite 200, Tecan Trading AG, Switzerland). was measured. The survival rate of cells 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 distributed in a 12- ^well plate at 5.0 Added and cultured for 24 hours. After culturing in 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 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 culturing 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. The concentration of cell protein in each well was measured, and the amount of insulin secreted per unit gram of protein was calculated.

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

The expression of genes involved in insulin secretion was analyzed through real-time PCR. First, RIN-m5F cells were distributed at 5×10 ⁵ cells/well in a 12-well plate and cultured in complete medium for 3 days, and then Pediococcus pentosaceus KI62 strain was added and incubated for 24 hours. It was cultured for some time. Afterwards, washed twice with modified Kreb's ringer bicarbonate buffer, changed to KRBP-HEPES buffer containing 20mM glucose and incubated for 1 hour, then removed the supernatant and used 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 dT primer and AMV reverse transcriptase. The synthesized cDNA was amplified and measured by real-time PCR using GLP-1R, IRS-2, and GADPH primers as a template. Each PCR product was electrophoresed using a 1% agarose gel, stained using ethidium bromide (EtBr, Sigma), and the level of expression was confirmed under UV. The 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 using ANOVA (one-way analysis of variance test) and Duncan's post hoc test comparison, and p < 0.05 was judged to be significant (SPSS V12., SPSS Inc, Chicago, IL, USA).

6) Results

Figure 7 is a graph showing the survival rate of RIN-m5F cells when treated with Pediococcus pentosaceus KI62 strain at different concentrations. Different superscripts indicate values that are significantly different from each other (p<0.05).

According to this, when the concentration of Pediococcus pentosaceus KI62 strain was 1 to 10 ㎍/㎖, the survival rate was over 100%, and at the concentration of 30 to 100 ㎍/㎖, the survival rate was over 80%. Therefore, the Pediococcus pentosaceus KI62 strain is stable up to a concentration of 1 to 500 ㎍/㎖ (survival rate of 70% or more), preferably 1 to 100 ㎍/㎖ (survival rate of 79% or more), Preferably, it may be 1 to 30 μg/ml (survival rate of 85% or more), and most preferably, it may be 1 to 10 μg/ml (survival rate of 100% or more).

Figure 8 is a graph showing the amount of insulin secretion measured when Pediococcus pentosaceus KI62 strain was treated with RIN-m5F cells at different concentrations. Different superscripts indicate values that are significantly different from each other (p<0.05).

As shown in Figure 8, the insulin secretion amount in RIN-m5F cells (control) without sample treatment was 73.67 ng/mg protein/1h, and in those treated with Pediococcus pentosaceus KI62 strain. In this case, it was confirmed that the concentration was 76.50 ng/mg protein/1h and 98.39 ng/mg protein/1h. It was confirmed that the insulin secretion amount increased as the concentration of Pediococcus pentosaceus KI62 strain increased, and it was found to increase by up to 33.5% compared to the control group.

Figure 9 is a graph showing the expression level of GLP-1R, a gene involved in insulin secretion, when Pediococcus pentosaceus KI62 strain was treated with RIN-m5F cells at different concentrations. . Different superscripts indicate values that are significantly different from each other (p<0.05).

GLP-1 transmits intracellular signals by binding to the GLP-1 receptor (GLP-1R, glucagon-like peptide 2 receptor), a G protein-coupled receptor present on the surface of beta cells, and stimulates glucose-dependent insulin secretion to It stimulates insulin gene expression and biosynthesis in cells, and as GLP-1R expression increases, insulin gene expression in beta cells increases, thereby promoting insulin secretion. As shown in Figure 9, it can be seen that GLP-1R expression significantly increases in RIN-m5F, an insulinoma cell, as the Pediococcus pentosaceus KI62 strain is treated. In other words, it can be seen that the Pediococcus pentosaceus KI62 strain has a direct insulin secretion promoting effect through regulating the amount of GLP-1R secretion in pancreatic beta cells. Therefore, Pediococcus pentosaceus KI62 strain can show excellent effects in preventing, improving, or treating diabetes.

Figure 10 is a graph showing the expression level of IRS-2, a gene involved in insulin secretion, when Pediococcus pentosaceus KI62 strain was treated with RIN-m5F cells at different concentrations. . Different superscripts indicate values that are significantly different from each other (p<0.05).

IRS is a mediator of insulin signaling and is a docking protein between the insulin receptor, which plays a role in maintaining functions such as cell growth, survival, and metabolism, and complex signaling molecules within the cell. 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, we attempted to analyze the effect on IRS-2 expression when Pediococcus pentosaceus KI62 strain was treated with RIN-m5F cells at different concentrations.

As shown in Figure 10, as a result of treating the Pediococcus pentosaceus KI62 strain, it was confirmed that IRS-2 expression was significantly increased in a concentration-dependent manner. In particular, compared to the control group, in the experimental group treated with Pediococcus pentosaceus KI62 strain, IRS-2 expression was significantly increased by 1.25-fold and 1.45-fold, respectively.

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

1) Pretreatment of selected strains

Pediococcus pentosaceus KI62 strain was inoculated at 1% in MRS broth and then cultured at 37°C for 18 hours. The cultured strain was centrifuged at 1,500 xg for 15 minutes to collect bacterial cells. To remove the remaining MRS broth, the collected strains were washed three times with sterilized distilled water. The washed strain was freeze-dried and suspended at a concentration of 10 mg/ml. After sonicating for 50 seconds using an ultrasonicator, the sample was sterilized in an autoclave to prepare a sample for use in the experiment. Each sample was diluted by concentration according to the analysis, DW was used for dilution by concentration, and the prepared sample was stored in a refrigerator.

2) Cell model preparation

Mouse muscle cells (C2C12 myoblast) were prepared to be used as a cell model. Mouse muscle cells (C2C12 myoblast) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Reagents needed for cell culture were purchased from Gibco (USA), and the medium was RPMI-1640 (Roswell Park Memorial Institute 1640) and DMEM (Dulbecco Modified Eagle Medium) with 10% fetal bovine serum (FBS) and 1% A cell model to be used in the experiment was prepared by adding antibiotics-antimycotic and subculturing once every 2 to 3 days in a cell incubator at 37°C in the presence of 5% CO ₂ .

3) Cytotoxicity

C2C12 cells were treated with Pediococcus pentosaceus KI62 strain (sample), and cell viability according to sample concentration was measured using WST-1 assay (ITSBio, Seoul, Korea). First, C2C12 cells were distributed to each 96 well plate at 5 × 10 ⁴ cells/well. After culturing for 24 hours, samples were treated at different concentrations in each well and cultured for 24 hours. Upon completion of incubation, 10 ㎕ of WST-1 reagent was added per 100 ㎕ for reaction, and the absorbance was measured at 450 ㎚ (Ref. 600 ㎚) using a microplate reader (Infinite 200, Tecan Trading AG, Switzerland). was measured. The survival rate of cells was calculated using Equation 5.

[Equation 5]

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

3) Evaluation of blood sugar absorption capacity

C2C12 cells were dispensed into a 96-well plate (5×10 ⁴ cells/well) and cultured at 37°C and 5% CO ₂ for 24 hours. After the cells had grown to 70%, the medium was added with 1% horse serum. , the culture was replaced with DMEM differentiation medium supplemented with 1% P/S, and the medium was replaced every 48 hours. Fully differentiated cells were incubated with serum free-low glucose medium and 100 nM 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino for 24 hours. ]-2-deoxy-D-glucose and Pediococcus pentosaceus KI62 strain were treated at different concentrations. Afterwards, after washing three times using Cold PBS, the 2-NBDG absorption capacity was measured at spectrum wavelengths of 485 nm and 535 nm using a fluorometer.

4) Expression analysis of proteins involved in sugar metabolism and utilization (GLUT4, Akt)

Through Western blot, we analyzed the expression of proteins involved in sugar metabolism and utilization (GLUT4, Akt) in cell models treated with selected strains. C2C12 cells were dispensed into a 12-well plate (5×10 ⁵ cells/well) and cultured at 37°C, 5% CO ₂ for 24 hours. After the cells had grown to 70%, the medium was supplemented with 1% horse serum. (horse serum) and 1% P/S were replaced with DMEM differentiation medium, and culture was performed with medium changes every 48 hours. Fully differentiated cells were treated with serum free-low glucose medium and Pediococcus pentosaceus KI62 strain at different concentrations for 24 hours. After washing three times with PBS, lysis buffer was added and centrifuged at 1400 rpm. The supernatant was taken, SDS sample buffer was added, and the mixture was 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), and Akt Antibody (CST, #9272) were used. Protein expression was analyzed using

5) Statistical analysis

Statistical analysis according to the statistical significance test between each group was performed using ANOVA (one-way analysis of variance test) and Duncan's post hoc test comparison, and p < 0.05 was judged to be significant (SPSS V12., SPSS Inc, Chicago, IL, USA).

6) Results

Figure 11 is a graph showing the survival rate of C1C12 cells when Pediococcus pentosaceus KI62 strain was treated at different concentrations. Different superscripts indicate values that are significantly different from each other (p<0.05).

As shown in Figure 11, when the Pediococcus pentosaceus KI62 strain was treated at different concentrations, it was confirmed that the survival rate of C2C12 cells showed the same pattern as the survival rate of RIN-m5F cells.

Figure 12 is a graph showing the glucose uptake of C1C12 cells when Pediococcus pentosaceus KI62 strain was treated at different concentrations. Different superscripts indicate values that 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 the body's blood sugar, and is known to play a very important role in regulating blood sugar. In this experiment, we sought to determine the effect of Pediococcus pentosaceus KI62 strain on the glucose uptake ability of differentiated muscle cells (C2C12 cells).

As shown in Figure 12, it was confirmed that the glucose uptake ability of C2C12 cells increased as the concentration of Pediococcus pentosaceus KI62 strain increased. That is, it can be seen that the Pediococcus pentosaceus KI62 strain has a significant effect in enhancing glucose consumption of C2C12 cells.

PIP3 induces the translocation of Akt to the plasma membrane, and PDK2 induces phosphorylation at ser473 of Akt, so that 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 flows into cells by increasing and translocating from the cytoplasm to the cell membrane by insulin stimulation. Therefore, we attempted to analyze the effect on pAkt/Akt and GLUT4 expression levels when the Pediococcus pentosaceus KI62 strain was treated with differentiated muscle cells (C2C12 cells), and the results are shown in Figures 13 to 13. Shown in 16.

Figure 13 shows the measurement of the expression level of proteins involved in sugar metabolism and utilization (p-AKT/AKT) when Pediococcus pentosaceus KI62 strain was treated with C2C12 cells at different concentrations. This is the Western blot result, and Figure 14 is a graph showing this numerically. Different superscripts indicate values that are significantly different from each other (p<0.05).

Figure 15 is a Western blot result measuring the expression level of a protein involved in sugar metabolism and utilization (GLUT4) when Pediococcus pentosaceus KI62 strain was treated with C2C12 cells at different concentrations. , Figure 16 is a graph showing this in numbers. Different superscripts indicate values that are significantly different from each other (p<0.05).

As shown in Figures 13 to 16, the expression level of proteins involved in sugar metabolism and utilization (p-AKT/AKT and GLUT4) in C2C12 cells increased as the Pediococcus pentosaceus KI62 strain was treated. You can see that it is increasing. That is, it can be seen that the Pediococcus pentosaceus KI62 strain has a significant effect in enhancing glucose consumption of C2C12 cells.

In addition, GULT4 is an insulin transport channel, and when stimulated by insulin, intracellular GULT4 moves to the plasma membrane and promotes glucose transport. In other words, GLUT4 plays a role in lowering blood sugar levels by facilitating the influx of blood glucose into cells. Therefore, the activity and expression of GLUT4, which is involved in glucose transport, increases as the Pediococcus pentosaceus KI62 strain is treated, indicating the anti-diabetic effect of the Pediococcus pentosaceus KI62 strain. indicates excellence.

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

1) Experimental animals and breeding environment

The experimental animals are specific-pathogen free (SPF) db/db mice (a strain with a mutation in the gene that produces the leptin receptor, which causes diabetes due to a problem in leptin signal transmission) purchased at 5 weeks of age and acclimatized for a week. After a period of time, it was used in the experiment. During the acclimation period, regular mouse food (Purina Lab Rodent Chow #38057, Purina Co., Seoul Korea) was fed. During the acclimation period, filtered drinking water was changed daily and allowed to be freely consumed. During the breeding period, the temperature is 23±1℃, humidity 50±5%, noise less than 60 phones, lighting hours 08:00~20:00 (12 hours a day), illuminance 150~300 Lux, ventilation 10~12 times per hour. A meeting environment was maintained. This experiment was conducted in compliance with the animal experiment ethics regulations.

2) Experiment

During the experiment period, regular solid feed (Samtako, Gyunggi, Korea) (AIN-93G) was fed to the diet, and after the experiment was completed, the blood sugar levels of the experimental animals were measured and separated using the egg mass method to ensure that the average value of blood sugar levels was uniform between each group. Afterwards, individual identification was marked using an ear punch (each group is summarized in Table 10 below). They were divided into normal group, control group, experimental group (KI62), and positive control, 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, db/+. During the experiment, the temperature was 23±1℃, humidity 50±5%, noise less than 60 phones, lighting hours 08:00~20:00 (12 hours a day), illuminance 150~300 Lux, ventilation 10~12 times per hour. A meeting environment was maintained. This experiment was conducted in compliance with the animal experiment ethics regulations.

Classification (n=8) Experiment method Jeongsang-gun
(Normal group, NOR) (Non-diabetic mouse, db/+)
Orally administer 0.2 ml of distilled water daily for 8 weeks. control group
(Control group, CON) (Diabetic mouse, db/db)
Orally administer 0.2 ml of distilled water daily for 8 weeks. positive control
(Positive control, POS CON) (Diabetic mouse, db/db)
Orally administer 0.2 ml distilled water containing 60 mg/kg of metformin hydrochloride daily for 8 weeks. experimental group
(KI62) (Diabetic mouse, db/db)
Orally administer 0.2 ml distilled water with live cells suspended at a concentration of 8 Log/CFU KI62 daily for 8 weeks.

3) Blood sugar measurement and oral glucose tolerance test

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

4) Treatment of experimental animals and sample collection

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

5) Blood analysis

Blood was collected from the abdominal vena cava, placed in a conical tube, coagulated for 30 minutes at room temperature, and then centrifuged at 3,000 rpm for 10 minutes. Blood concentrations of fructosamine, TC, TG, LDL-C, HDL-C, and blood sugar-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). The fixed pancreatic tissue was embedded in paraffin, cut into 4 ㎛ thick sections, and the deparaffinized tissue was treated with peroxidase quenching solution for 30 minutes, then ready-to-use. The reaction was performed for 15 minutes with IHC Blocking Reagent. Afterwards, treat each with blocking reagent and activated primary antibody (working primary antibody solution) (2-10 ㎕ of antibody to 1 ㎖ of Ready-To-Use IHC antibody diluent) and incubate at room temperature for 3 hours or at low temperature. After reacting overnight, the reaction was performed using an activated anti-Rabbit IHC antibody (Working anti-Rabbit IHC Antibody) and then stained with DAB substrate and 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 using Statistical Package for Social Sciences (SPSS, SPSS Inc., USA). Significant differences were statistically processed using one-way ANOVA, and significance was tested at the 5% level using Duncan's multiple range tests.

8) Results

Figure 17 is a graph showing the weight of the normal group, control group, experimental group (KI62), and positive control over time. The db/db mouse, an animal model of type 2 diabetes used in this experiment, develops resistance to leptin and cannot control its appetite, resulting in obesity and increased blood sugar levels as it grows.

As shown in Figure 17, it was confirmed that the body weight of all animal models increased significantly compared to the normal group.

Blood sugar (glucose concentration, mg/dl) of the normal group, control group, experimental group (KI62), and positive control was measured and compared over time. Different superscripts indicate values that are significantly different from each other (p<0.05).

army 0 weeks 2 weeks 3 weeks 5 weeks 6 weeks 7 weeks 8 weeks Jeongsang-gun 104.86±23.01 ^b 105.86± ^16.68c 130.33±25.66 ^c 127.63±17.85 ^c 118.38±16.32 ^b 84.43± ^12.86c 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± ^31.86a 268.75±92.00 ^b 460.50± ^46.17a 353.00±70.27 ^b 376.50± ^13.92a 398.50±43.07 ^b 395.33±12.38 ^b experimental group
KI62 164.43±19.76 ^b 267.80± ^43.65a 392.00± ^70.01a 384.75±19.56 ^b 379.75± ^10.21a 375.25±21.18 ^b 341.00±37.15 ^b

As a result, it was confirmed that the blood sugar levels of the control group, experimental group (K97), and positive control were somewhat higher than those of the normal group. However, in the positive control group administered metformin, a diabetes treatment drug, a decrease in blood sugar was confirmed at 5, 6, 7, and 8 weeks after the start of administration, and the experimental group administered the Pediococcus pentosaceus KI62 strain (KI62) It was also confirmed that blood sugar level decreased to the same level as that of the positive control group.

The oral glucose tolerance test indirectly determines the insulin secretion function by measuring the glucose concentration in the blood at a set time after sugar intake. Therefore, in order to confirm the effect of glucose tolerance by the Pediococcus pentosaceus KI62 strain of the present invention, after fasting for 12 hours in the 8th week, glucose was orally administered at a dose of 1 g/kg, 0, 30, A glucose tolerance test was performed in which blood was measured through the caudal vein at intervals of 60, 120, and 180 minutes. The results are summarized in Figure 18 and Table 11. Figure 18 is a graph showing AUC (Area under the curve) for blood sugar levels according to an oral glucose tolerance test.

army 0 minutes 30 minutes 60 minutes 120 minutes 180 minutes Jeongsang-gun 81.875± ^13.80c 173.87±23.85 ^b 165.37±27.72 ^b 137.50± ^20.09d 115.62±14.97 ^c control group 489.80± ^34.40a 600± ^0.00a 600± ^0.00a 549.25± ^25.72a 510.75± ^37.63a positive control 398.5±43.07 ^b 600± ^0.00a 600± ^0.00a 483.00±28.82 ^b 437.50±40.41 ^b experimental group
KI62 483.60± ^25.26a 600± ^0.00a 600± ^0.00a 447.00±25.41 ^c 446.50±21.09 ^b

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

As shown in Figure 18 and Table 11, a significant blood sugar reduction effect was observed in the experimental group administered Pediococcus pentosaceus KI62 strain for 8 weeks, which was equivalent to or lower than the positive control group. Able to know. In other words, it can be seen that administration of Pediococcus pentosaceus KI62 strain can affect insulin sensitivity or resistance, thereby lowering blood sugar.

Figure 19 is a graph showing the measurement of fructosamine in the serum of the normal group, control group, experimental group (KI62), and positive control. Different superscripts indicate values that are significantly different from each other (p<0.05).

Fructosamine is a compound formed by combining glucose and protein. Unlike glycated hemoglobin, which indicates the average blood glucose concentration for 8 to 12 weeks, it is an indicator that reflects the average glucose concentration for 2 to 3 weeks.

As shown in Figure 19, fructosamine in the serum of the experimental group orally administered Pediococcus pentosaceus KI62 strain for 8 weeks was confirmed to be 4.84 ± 0.64 μmol/L, The control group (CON) was found to be 7.70 ± 1.79 μmol/L, and the normal group (NOR) was found to be 3.10 ± 0.67 μmol/L. Compared to the normal group, the control group significantly increased by 2.48 times, and the experimental group administered the Pediococcus pentosaceus KI62 strain showed a significant decrease of 37.11% compared to the control group. In particular , the experimental group administered the Pediococcus pentosaceus KI62 strain was confirmed to have a level 1.4 times lower than the positive control group (6.81 ± 1.49 μmol/L). ) It was confirmed that the KI62 strain has a better blood sugar reduction effect than metformin, a conventional diabetes treatment.

Figure 20 is a graph showing the measurement of glucose concentration in the serum of the normal group, control group, experimental group (KI62), and positive control. Different superscripts indicate values that are significantly different from each other (p<0.05).

As shown in Figure 20, it was confirmed that the serum glucose concentration of the normal group (NOR) was significantly increased by more than two times that of the control group (CON) (p<0.05). The positive control group (POS CON) (499.58 ± 33.74 mg/dl) showed a slight tendency to decrease compared to the control group (CON) (538.22 ± 28.35 mg/dl). On the other hand, it was confirmed that the experimental group (KI62) administered the Pediococcus pentosaceus KI62 strain was 458.22 ± 33.19 mg/dl, a significant decrease of more than 14.86% compared to the control group (CON). It was confirmed that Pediococcus pentosaceus KI62 strain has a better blood sugar reduction effect than metformin, a conventional diabetes treatment.

Figure 21 is a graph showing the measurement of insulin concentration in the serum of the normal group, control group, experimental group (KI62), and positive control. Different superscripts indicate values that are significantly different from each other (p<0.05).

If normal insulin secretion does not occur in the pancreas, blood sugar levels in the body cannot be controlled. Even if insulin secretion is normal in the pancreas, if insulin resistance occurs in peripheral tissues, sugar cannot be metabolized and hyperinsulinemia, in which insulin levels rise in the blood, may occur. Therefore, we attempted to measure the effect on diabetes control by measuring blood insulin for each group.

As shown in Figure 21, the control group (CON), experimental group (KI62), and positive control group (POS CON) were confirmed to have significantly higher blood insulin levels overall than the normal group (NOR). On the other hand, the positive control group (POS CON) administered metformin, a conventional diabetes treatment, was confirmed to have a 13.63% decrease in blood insulin levels compared to the control group (CON), and the experimental group administered Pediococcus pentosaceus KI62 strain. (KI62) was also confirmed to have decreased blood insulin levels to a similar level as the positive control group (POS CON). In other words, it can be seen that the Pediococcus pentosaceus KI62 strain exhibits an excellent insulin reducing effect similar to that of metformin, a conventional diabetes treatment drug.

Figure 22 is a graph showing the concentration of glycated hemoglobin in the blood of the normal group, control group, experimental group (KI62), and positive control. Different superscripts indicate values that are significantly different from each other (p<0.05).

When the glucose content in the blood is high, sugar is attached to the hemogulobin of red blood cells non-enzymatically, producing glycosylated hemoglobin, which is called HbA1C. Since the lifespan of red blood cells is 8-12 weeks on average, measuring HbA1C allows you to know the average blood sugar level for the past 8-12 weeks, which reflects the glucose concentration in the blood around 4-6 weeks from the present.

As shown in Figure 22, it can be seen that the glycated hemoglobin content of the experimental group administered the Pediococcus pentosaceus KI62 strain for 8 weeks was significantly reduced compared to the control group. In other words, it can be seen that the average blood sugar level over 8-12 weeks was reduced more by oral administration of Pediococcus pentosaceus KI62 strain than by metformin, a conventional diabetes treatment drug.

Figure 23 is a graph showing the concentration of C-peptide in the serum of the normal group, control group, experimental group (KI62), and positive control. Different superscripts indicate values that are significantly different from each other (p<0.05).

C-peptides are part of the large molecule before the insulin circulating in the blood is created, and are made up of 31 amino acids that connect the A and B rings of insulin. The prototype of insulin (Proinsulin) is synthesized in the beta cells of the pancreas, and it is divided into insulin and C-peptide in the blood. Insulin and C-peptide exist in the same ratio in the blood. Moreover, because 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 Figure 23, it was confirmed that the C-peptide concentration was significantly decreased in the control group (CON) compared to the normal group (NOR). On the other hand, it can be seen that the C-peptide concentration of the experimental group administered Pediococcus pentosaceus KI62 strain for 8 weeks recovered to the normal group (NOR) level. In other words, it can be seen that the Pediococcus pentosaceus KI62 strain shows a blood sugar improvement effect.

Prior to histological examination of the pancreas, the pancreas weights of the normal group, control group, experimental group (KI62), and positive control were measured and are shown in Table 12. Different superscripts indicate values that are significantly different from each other (p<0.05).

army Pancreatic tissue weight (g) Jeongsang-gun 0.215± ^0.011a control group 0.130± ^0.007c positive control 0.158± ^0.028b Experimental group KI62 0.141±0.010 ^b.c.

As shown in Table 12, it can be seen that the pancreatic tissue weight of the control group was significantly reduced compared to the normal group. On the other hand, it was confirmed that the weight of pancreatic tissue in the experimental group administered with the Pediococcus pentosaceus KI62 strain significantly increased compared to the control group, which showed that Pediococcus pentosaceus It can be seen that the KI62 strain is effective in suppressing the reduction of pancreatic tissue.

Figure 24 shows the results of measuring insulin present in pancreatic islets of the normal group, control group, experimental group (KI62), and positive control using immunohistochemical staining. Measuring the insulin present in beta cells in pancreatic tissue is an indicator of beta cell function, and insulin can be analyzed through immunohistochemical methods.

According to Figure 24, it can be seen that insulin expression in the beta cells of islets within the pancreas was significantly decreased in the control group (CON) compared to the normal group (NOR). In other words, it can be seen that the beta cells of the islets within the pancreas are significantly damaged due to diabetes, and the pancreatic insulin is not expressed properly.

On the other hand, the experimental group (KI62) administered the Pediococcus pentosaceus KI62 strain had a significantly increased expression of insulin compared to the control group, which was similar to the positive control group (POS CON) treated with metformin, a conventional diabetes treatment. It can be confirmed that it is at a similar or higher level. In other words, it can be said to strongly suggest that the Pediococcus pentosaceus KI62 strain restores the function of pancreatic islet beta cells and promotes insulin secretion.

Figure 25 shows the results of measuring glucagon present in pancreatic islets of the normal group, control group, experimental group (KI62), and positive control using immunohistochemical staining. am. As previously explained, pancreatic islet cells, also called Langerhans islets, exist in the pancreas and are a group of endocrine cells that secrete glucagon and insulin.

According to Figure 25, it can be seen that glucagon expression in islets within the pancreas was significantly reduced in the control group (CON) compared to the normal group (NOR). In other words, it can be seen that the islets within the pancreas are damaged due to diabetes, and glucagon expression in the pancreas is not performed properly.

On the other hand, the experimental group (KI62) administered the Pediococcus pentosaceus KI62 strain significantly increased the expression of glucagon compared to the control group, which was compared to the positive control group (POS CON) treated with metformin, a conventional diabetes treatment. It can be confirmed that it is at a similar level to . In other words, this indicates that the function of the pancreatic islets in the pancreas is restored by the Pediococcus pentosaceus KI62 strain and that glucagon expression is normalized.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> KOREA FOOD RESEARCH INSTITUTE <120> Pediococcus pentosaceus KI62 and uses it <130>HPC9301 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1557 <212> RNA <213> Artificial Sequence <220> <223> 16S rRNA sequence of Pediococcus pentosaceus KI62 <400> 1 tggtaccagg ttcccttttt tagttttttt actctgctca ggatgaacgc tggcggcgtg 60 cctaatacat gcaagtcgaa cgaacttccg ttaattgatt atgacgtact tgtactgatt 120 gagattttaa cacgaagtga gtggcgaacg ggtgagtaac acgtgggtaa cctgcccaga 180 agtaggggat aacacctgga aacagatgct aataccgtat aacagagaaa accgcatggt 240 tttcttttaa aagatggctc tgctatcact tctggatgga cccgcggcgt attagctagt 300 tggtgaggta aaggctcacc aaggcagtga tacgtagccg acctgagagg gtaatcggcc 360 acattgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccac 420 aatggacgca agtctgatgg agcaacgccg cgtgagtgaa gaagggtttc ggctcgtaaa 480 gctctgttgt taaagaagaa cgtgggtaag agtaactgtt tacccagtga cggtattattaa 540 ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 600 atccggattt attgggcgta aagcgagcgc aggcggtctt ttaagtctaa tgtgaaagcc 660 ttcggctcaa ccgaagaagt gcattggaaa ctgggagact tgagtgcaga agaggacagt 720 ggaactccat gtgtagcggt gaaatgcgta gatatatgga agaacaccag tggcgaaggc 780 ggctgtctgg tctgcaactg acgctgaggc tcgaaagcat gggtagcgaa caggattaga 840 taccctggta gtccatgccg taaacgatga ttactaagtg ttggagggtt tccgcccttc 900 agtgctgcag ctaacgcatt aagtaatccg cctggggagt acgaccgcaa ggttgaaact 960 caaaagaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagctacg 1020 cgaagaacct taccaggtct tgacatcttc tgacagtcta agagattaga ggttcccttc 1080 ggggacagaa tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt 1140 aagtcccgca acgagcgcaa cccttattac tagttgccag cattaagttg ggcactctag 1200 tgagactgcc ggtgacaaac cggaggaagg tggggacgac gtcaaatcat catgcccctt 1260 atgacctggg ctacacacgt gctacaatgg atggtacaac gagtcgcgag accgcgaggt 1320 taagctaatc tcttaaaacc attctcagtt cggactgtag gctgcaactc gcctacacga 1380 agtcgggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg 1440 tacacaccgc ccgtcacacc atgagagttt gtaacaccca aagccggtgg ggtaaccttt 1500 taggagctag ccgtctaagg tgggacagat gattagggtg aagtcgtaac aggggaa 1557

Claims (10)

Pediococcus pentosaceus KI62 KACC 81120BP, which has hypoglycemic and antibacterial activity,
The antibacterial activity of Pediococcus pentosaceus KI62 KACC 81120BP is characterized by inhibiting the growth of Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes and Staphyloccous aureus . According to paragraph 1,
The Pediococcus pentosaceus KI62 KACC 81120BP is characterized by having a 16s rRNA base sequence represented by SEQ ID NO: 1. According to paragraph 1,
The Pediococcus pentosaceus KI62 KACC 81120BP is a Pediococcus pentosaceus characterized by having an alpha-amylase or alpha-glucosidase inhibitory activity. ( Pediococcus pentosaceus) KI62 KACC 81120BP. According to paragraph 1,
The Pediococcus pentosaceus KI62 KACC 81120BP is characterized by acid resistance , bile resistance , and intestinal adhesion ability. According to paragraph 1,
The Pediococcus pentosaceus KI62 KACC 81120BP is a Pediococcus pentosaceus KI62 KACC 81120BP, characterized in that it has a blood sugar lowering and anti-diabetic effect. A probiotic composition comprising Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient. A food composition for improving blood sugar after a meal comprising Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient. A food composition for preventing or improving diabetes comprising Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient. A feed composition for preventing or improving diabetes comprising Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient. A pharmaceutical composition for preventing or treating diabetes comprising Pediococcus pentosaceus KI62 KACC 81120BP strain or a culture thereof as an active ingredient.