Novel strain of lactobacillus fermentum and use thereof

Abstract

The present invention relates to a novel (Lactobacillus fermentum) strain. In addition, the present invention relates to a composition comprising the strain. In addition, the present invention relates to use of the Lactobacillus fermentum strain or the composition comprising the strain, in the treatment, alleviation, or prevention of metabolic diseases of a subject. The Lactobacillus fermentum strain of the present invention may inhibit the production of nitric oxide (NO) in a subject, increase the secretion of glucagon-like peptide-1 (GLP -1), which is a hormone that regulates blood glucose, by increasing insulin secretion and inhibiting glucagon secretion, reduce blood glucose, and suppress appetite or reduce body weight. Therefore, the strain and the composition comprising same of the present invention may be used for anti-inflammatory purposes, and may also be used for the treatment, alleviation, or prevention of metabolic diseases.

Description

Novel Lactobacillus fermentum strain and uses thereof

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0144445 filed on October 21, 2024, and all contents disclosed in the document of said Korean patent application are incorporated herein as part of this specification.

[0002] The present invention relates to a novel Lactobacillus fermentum strain. Furthermore, the present invention relates to a composition comprising said Lactobacillus fermentum strain. Additionally, the invention relates to the use of said Lactobacillus fermentum strain, or a composition comprising said strain, for treating, improving, or preventing metabolic diseases in a subject.

[0003] Metabolic diseases, including diabetes mellitus, obesity, dyslipidemia, and hypertension, are known to increase the risk of developing various adult diseases, thereby raising the incidence and mortality rates of various cardiovascular and cerebrovascular diseases, kidney diseases, or cancer. According to the Health Insurance Review and Assessment Service (HIRA), the number of people with the aforementioned metabolic diseases was confirmed to increase annually from approximately 11 million in 2019 to approximately 14 million in 2023 (HIRA, Metabolic Syndrome Statistics, Review Years 2019 to 2023). Diabetes, which is included in the aforementioned metabolic diseases, increased by an annual average of 4.9% from approximately 3 million people in 2018 to approximately 3.6 million people in 2022 (Health Insurance Review & Assessment Service, 2023 Statistics on Diseases and Medical Practices in Daily Life), and as of 2020, one in six adults aged 30 or older has diabetes, and three in ten elderly people aged 65 or older have diabetes (Korean Diabetes Association, Diabetes Fact Sheet in Korea 2022).

[0004] In addition, obesity increased by an annual average of 14.2% from approximately 15,000 people in 2018 to approximately 27,000 people in 2022, and hypertension increased by an annual average of 3.6% from approximately 6 million people in 2018 to approximately 7 million people in 2022 (Health Insurance Review & Assessment Service, 2023 Statistics on Diseases and Medical Practices in Daily Life).

[0005] Since the primary cause of such metabolic diseases is impaired blood sugar control due to factors such as weight gain, inflammation in adipose tissue, and insulin resistance, it is necessary to reduce appetite and body weight, alleviate inflammation, and lower blood sugar levels in order to treat, improve, or prevent these metabolic diseases. Accordingly, metformin-class or thiazolidinedione-class drugs that improve insulin sensitivity, or DPP-4 (dipeptidyl peptidase-4) inhibitors that induce an increase in incretin—a hormone that lowers blood sugar levels—are being used as diabetes treatments. Recently, GLP-1 receptor agonists such as semaglutide, dulaglutide, and liraglutide, which are effective for weight loss in addition to lowering blood sugar, have received approval from the U.S. Food and Drug Administration (FDA) and are being used as hypoglycemic agents or anti-obesity agents. However, since the aforementioned GLP-1 receptor agonists carry side effects such as gastrointestinal disorders like vomiting, nausea, diarrhea, indigestion, and dehydration, as well as risks of pancreatitis, pancreatic cancer, or thyroid cancer, or headaches (Drug Encyclopedia or New Drug Review of the Korea Pharmaceutical Information Center), there is still a need in the technical field to which this invention belongs to develop therapeutic agents or foods utilizing the microbiome that are effective for anti-inflammation, blood sugar reduction, appetite suppression, or weight loss, and have minimal side effects on the human body, for the treatment, prevention, or improvement of metabolic diseases.

[0006] Accordingly, the inventors conducted various studies and experimentally demonstrated that a specific strain of Lactobacillus fermentum is effective in reducing inflammation, increasing the secretion of GLP-1 (glucagon-like peptide-1), a blood sugar regulating hormone, reducing blood sugar, suppressing appetite, and reducing body weight, thereby completing the present invention.

[0007] Each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0008] Furthermore, terms not specifically defined in this specification should be understood to have the meanings commonly used in the technical field to which the present invention pertains. Additionally, unless specifically defined in the context, the singular includes the plural, and the plural includes the singular.

[0009]

[0010] The present invention provides a novel strain of Lactobacillus fermentum. The novel strain of Lactobacillus fermentum according to the present invention was deposited by the applicant with the Korea Research Institute of Biotechnology and Bioengineering on July 8, 2024, under accession number KCTC 15954BP. The Korea Research Institute of Biotechnology and Bioengineering is a patent strain deposit institution located at 181, Ipsin-gil, Jeongeup-si, Jeollabuk-do, Republic of Korea. The novel strain is a rod-shaped bacterium belonging to the species Lactobacillus fermentum of the genus Lactobacillus, and has a 16S rRNA gene containing the nucleotide sequence indicated by SEQ ID NO. 1.

[0011]

[0012] In this specification, "Lactobacillus fermentum strain" includes live cells, dead cells and cultures thereof, fermented products thereof, lysed products thereof, extracts thereof, and cytoplasmic fractions obtained by lysing the same, and also includes post-processed or post-processed products such as filtration, concentration, drying, extraction, and freezing of said cultures, fermented products, lysed products, extracts, cytoplasmic fractions, etc.

[0013] The term "culture" as used in this specification refers to the entire medium containing the Lactobacillus fermentum strain of the present invention obtained by culturing the strain of said Lactobacillus fermentum in a nutrient-supplied medium for a certain period, metabolites of said strain, or excess nutrients remaining after culturing the strain, and includes the culture medium or culture supernatant from which said strain has been removed after culturing said strain. Additionally, it includes the concentrate of said entire medium or culture medium, or the dried concentrate. Specifically, the culture of the present invention may use a medium easily selected by a person skilled in the art according to the purpose among media used for microbial culture, for example, a medium used for culturing Lactobacillus fermentum, such as MRS (de man, rogosa sharpe) medium or blood agar medium, but is not limited thereto as long as said strain can be cultured.

[0014] The Lactobacillus fermentum strain of the present invention exhibits anti-inflammatory, blood sugar reduction, appetite suppression, and weight loss effects.

[0015] As used in this specification, the term "anti-inflammatory" means inhibiting the occurrence of inflammation or eliminating or reducing inflammation.

[0016] The Lactobacillus fermentum strain of the present invention may have one or more of the following characteristics within the subject:

[0017] (a) Inhibition of nitric oxide (NO) production,

[0018] (b) Enhancement of GLP-1 (glucagon-like peptide-1) secretion,

[0019] (c) reduction in blood sugar, and

[0020] (d) Suppression of appetite or weight loss.

[0021] The term "GLP-1 (glucagon-like peptide-1)" as used in this specification is one of the incretin hormones, which is released from L-cells distributed in the terminal small intestine or large intestine and is stimulated by nutrient intake. The GLP-1 increases insulin secretion, inhibits glucagon secretion, and promotes satiety.

[0022] Accordingly, since the Lactobacillus fermentum strain of the present invention exhibits effects such as inhibiting the production of nitric oxide, enhancing the secretion of GLP-1, reducing blood sugar, suppressing appetite, or reducing body weight, the Lactobacillus fermentum strain of the present invention can be usefully used as an active ingredient in a composition for the treatment, improvement, or prevention of metabolic diseases including diabetes mellitus, obesity, dyslipidemia, and hypertension.

[0023] As used herein, the term "metabolic disease" refers to a disease resulting from a biochemical dysfunction of the body, which may be caused by the loss of function of organs such as the liver or pancreas, congenital enzyme disorders, or acquired endocrine organ disorders. Such metabolic diseases may be, for example, but are not limited to glucose metabolism disorders such as diabetes mellitus, impaired fasting glucose (IFG), and impaired glucose tolerance (IGT), obesity, hypertension, metabolic dysfunction-associated steatotic liver disease (MASLD), hyperinsulinemia, or dyslipidemia. In this specification, the term "metabolic disease" may be used interchangeably with the terms "metabolic disease," "metabolic syndrome," and "metabolic disorder."

[0024] As used in this specification, the term "diabetes" refers to a disease caused by the combined action of genetic factors and environmental factors such as obesity, aging, hyperglycemia, hypertension, dyslipidemia, infection, drugs, chemicals, lack of exercise, stress, and pregnancy, which induce insufficient insulin secretion or insulin dysfunction. Due to the aforementioned insulin problem, sugar absorbed into the body cannot be utilized, leading to hyperglycemia. This hyperglycemia causes increased fluid intake, which in turn increases urine volume and excretes glucose through urine. Furthermore, if the aforementioned hyperglycemic state persists, the risk of developing complications increases. These complications may include, but are not limited to, diabetic retinopathy, diabetic nephropathy, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, cardiovascular disease, neuropathy, cerebrovascular disease, coronary artery disease, or peripheral artery disease. The above diabetes includes type I diabetes mellitus, type II diabetes mellitus, gestational diabetes, and other types of diabetes.

[0025] As used herein, the term "type I diabetes mellitus" refers to a disease caused by the failure of pancreatic beta cells (β cells) to produce insulin, which occurs primarily in children. In this specification, the term "type I diabetes mellitus" may be used interchangeably with the term "pediatric diabetes."

[0026] As used in this specification, the term "type II diabetes mellitus" refers to a disease caused by insulin dysfunction due to insulin resistance, which may be accompanied by overweight or obesity.

[0027] As used in this specification, the term "insulin resistance" refers to a condition in which, despite normal insulin secretion from the pancreas, the reduction of blood glucose and the burning of glucose by cells by insulin do not occur normally due to decreased insulin function. Consequently, the liver, muscles, and adipose tissues fail to perform their normal functions, which in turn triggers increased insulin secretion, leading to a vicious cycle. In the presence of insulin resistance, blood glucose control is not properly maintained, so the subject is unable to burn the calories consumed; this increases the risk of metabolic diseases such as obesity, fatty liver, and diabetes. The primary causes of insulin resistance are environmental factors, such as high-calorie, high-fat, and high-protein diets resulting from the Westernization of dietary habits, lack of exercise, or stress.

[0028] As used herein, the term "subject" includes humans or any non-human animals, and said non-human animals may be vertebrates, such as primates, dogs, cattle, horses, pigs, rodents, such as mice, rats, guinea pigs, etc. In this specification, said "subject" is used interchangeably with "individual" and "patient." There are no restrictions on gender, age, etc. for subjects using the composition according to the present invention. Specifically, it may be used by any subject who wishes to obtain anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss effects.

[0029] As used herein, the term "gestational diabetes mellitus" refers to a condition caused by physiological dysfunction related to glucose metabolism during pregnancy, applicable when diabetes is first discovered during pregnancy or develops due to pregnancy. Such gestational diabetes mellitus may result in fetal injury, spontaneous miscarriage, macrosomia and resulting delivery injury, hyperinsulinism, stillbirth, etc.

[0030] As used herein, the term "other diabetes" refers to a disease caused by genetic disorders, surgery on the pancreas or the like, infection, or chemicals or drugs. In this specification, the term "other diabetes" may be used interchangeably with the term "secondary diabetes."

[0031] Impaired fasting glucose and impaired glucose tolerance correspond to borderline diabetes.

[0032] As used in this specification, the term "borderline diabetes" refers to a condition or disease in which blood sugar levels are higher than those of a normal person but lower than those of a diabetic patient, and corresponds to the pre-diabetic stage.

[0033] The term "impaired fasting glucose (IFG)" as used in this specification refers to a condition or disease in which basal glucose is higher than normal, and said condition in which basal glucose is higher than normal may be, for example, 100 to 125 mg / dL in humans.

[0034] As used herein, the term "impaired glucose tolerance (IGT)" refers to a condition or disease in which blood glucose levels two hours after a meal are higher than normal, and said condition in which blood glucose levels two hours after a meal are higher than normal may be, for example, 140 to 199 mg / dL in humans. In this specification, the term "impaired glucose tolerance (IGT)" may be used interchangeably with the terms "glucose intolerance disorder" and "postprandial glucose disorder."

[0035] As used herein, the term "obesity" refers to a disease characterized by an excessive accumulation of adipose tissue compared to normal levels, and is caused by an energy imbalance resulting from the excessive intake of nutrients relative to energy expenditure. For example, in the case of humans, obesity is defined as body weight (kg) divided by the square of height (m²). 2It is diagnosed when the body mass index (BMI), which is the value divided by ), is 25 or higher (30 or higher for Westerners). Appetite suppressants may be used to treat the above obesity. Obesity increases the risk of developing dyslipidemia, diabetes, insulin resistance, hyperinsulinemia, hypertension, metabolic disorders, fatty liver disease, gestational hypertension, periodontal disease, gout, hyperuricemia, cancer, musculoskeletal disorders, mental disorders, or asthma.

[0036] As used herein, the term "dyslipidemia" refers to a condition in which lipid components are excessively present in the blood, caused by obesity or increased synthesis or decreased breakdown of lipoproteins that transport cholesterol and triglycerides. The dyslipidemia includes hypertriglyceridemia, a condition in which triglycerides are excessively present in the blood; hypercholesterolemia, a condition in which cholesterol is excessively present in the blood; and low high-density lipoprotein (HDL) cholesterol, a condition in which HDL cholesterol is lower than normal. The dyslipidemia increases the risk of developing vascular diseases such as arteriosclerosis, hypertension, myocardial infarction, stroke, and cerebral infarction.

[0037] The term "hypertension" as used in this specification refers to a condition in which blood pressure is higher than normal due to genetics, smoking, aging, diabetes, obesity, hyperlipidemia, lack of exercise, stress, alcohol consumption, or the intake of appetite suppressants. For example, in the case of humans, it refers to a condition in which blood pressure is higher than normal (systolic blood pressure less than 120 mmHg, diastolic blood pressure less than 80 mmHg) (systolic blood pressure 140 mmHg or higher, diastolic blood pressure 90 mmHg or higher). The above-mentioned hypertension causes arteriosclerosis of the coronary arteries and cerebral blood vessels of the subject or organ damage, thereby causing complications such as heart failure, angina pectoris, myocardial infarction, cerebral hemorrhage, stroke, renal failure, nephrosis, uremia, and vision loss.

[0038] As used herein, the term "metabolic dysfunction-associated steatotic liver disease (MASLD)" refers to a disease characterized by an excessive accumulation of triglycerides in the liver, which may occur in association with diabetes or obesity. In this specification, the term "metabolic dysfunction-associated steatotic liver disease" may be used interchangeably with the terms "metabolic dysfunction-associated steatotic liver disease" or "non-alcoholic fatty liver disease (NAFLD)."

[0039] The term "hyperinsulinemia" as used in this specification refers to a condition in which there is an excessive amount of insulin in the blood. Insulin resistance in the subject reduces insulin responsiveness to blood glucose, and as a result, blood glucose levels do not decrease, leading to the production or secretion of more insulin and the occurrence of hyperinsulinemia. The hyperinsulinemia may progress to type 2 diabetes.

[0040]

[0041] Another aspect of the present invention provides a composition for anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss comprising a Lactobacillus fermentum strain deposited under accession number KCTC 15954BP.

[0042] In the composition according to the present invention, each term has the same meaning as described above unless specifically stated otherwise.

[0043] The Lactobacillus fermentum strain of the present invention and a composition containing the same can be usefully used for the treatment, improvement, or prevention of metabolic diseases.

[0044] As used in this specification, the term "treatment" refers to any act in which symptoms of a disease are improved or completely cured by the administration, ingestion, or application of the composition according to the present invention.

[0045] Furthermore, as used herein, the term "improvement" refers to any act in which the symptoms of a disease are at least reduced or improved or benefited by the administration, ingestion, or application of the composition according to the present invention.

[0046] Furthermore, as used herein, the term "prevention" refers to any act of suppressing or delaying the symptoms of a disease by administering, ingesting, or applying the composition according to the present invention.

[0047] As used herein, the term "administration" means the physical introduction of a composition of the present invention to a subject using any of the various methods and delivery systems known to a person skilled in the art. Routes of administration for the composition of the present invention include, but are not limited to, oral administration routes, or oral administration such as sublingual administration, administration through the oral mucosa, oral spray, oral application, or intraoral injection, inhalation, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as injection, infusion, nasal spray, or application. The number of administrations for the composition of the present invention may be, for example, one time, multiple times, and / or over one or more extended periods.

[0048] The content of the active ingredient included in the composition according to the present invention can be appropriately determined by considering various factors such as the purpose of use (prevention, improvement, or therapeutic treatment), duration of use, type of formulation, route of administration, condition of the subject, symptoms of the disease, degree of progression of symptoms, age, and gender. For example, the content of the Lactobacillus fermentum strain of the present invention may be included in an amount of about 0.0001 to 100 weight% based on the total weight of the composition.

[0049]

[0050] According to one embodiment of the present invention, a pharmaceutical composition for anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss is provided, comprising the Lactobacillus fermentum strain of the present invention.

[0051] In the pharmaceutical composition according to the present invention, each term has the same meaning as the foregoing unless specifically stated otherwise.

[0052] The above pharmaceutical composition for anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss may be a pharmaceutical composition used for the treatment or prevention of metabolic diseases.

[0053] The pharmaceutical composition of the present invention may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0054] The pharmaceutical composition according to the present invention can be formulated into various oral, intraoral, or parenteral administration forms.

[0055] Formulations for oral administration or administration within the mouth may include tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, etc., and these formulations may use one or more diluents or excipients such as fillers, extenders, humectants, disintegrants, lubricants, binders, and surfactants that are commonly used in addition to the extract according to the present invention. Agar, starch, alginic acid or its sodium salt, calcium monohydrogen phosphate anhydrous, etc. may be used as disintegrants; silica, talc, stearic acid or its magnesium or calcium salt, polyethylene glycol, etc. may be used as lubricants; and magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-substituted hydroxypropylcellulose, etc. may be used as binders. In addition, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. can be used as diluents, and in some cases, commonly known absorbents, coloring agents, flavoring agents, sweeteners, etc. can be used together.

[0056] In addition, parenteral formulations may include injections, creams, lotions, topical ointments, oils, moisturizers, gels, sprays, topicals, suppositories, aerosols, oral patches, dressing solutions, and nasal inhalers, and these formulations may use one or more commonly used carriers, stabilizers, or preservatives in addition to the active ingredient. Carriers may include water, suitable oils, saline solution, aqueous glucose, or glycol; stabilizers may include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid; and preservatives may include benzalkonium chloride, methyl-paraben, propyl-paraben, or chlorobutanol.

[0057] The above pharmaceutical composition may be sterilized or contain preservatives, stabilizers, hydrating agents or emulsification promoters, salts for osmotic pressure regulation, buffers, and other therapeutically useful substances, and may be formulated according to commonly used mixing, granulation, or coating methods.

[0058] The pharmaceutical composition of the present invention may comprise the Lactobacillus fermentum strain alone, or may further comprise a suitable carrier, excipient, or diluent commonly used in the preparation of the pharmaceutical composition. Specifically, the carrier, excipient, and diluent that may be included in the pharmaceutical composition may be, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. These may be used alone or two or more may be used in a mixture. In addition, the above pharmaceutical composition may further include other conventional additives such as antioxidants, buffers and / or bacteriostatic agents if necessary, and may additionally include dispersants, surfactants, binders, lubricants, etc. to be formulated into powders, granules, tablets, liquids, capsules, suspensions, emulsions, syrups, creams, lotions, gels, ointments, aerosols, powder sprays, creams, suppositories, pills, etc.

[0059]

[0060] According to another embodiment of the present invention, a quasi-drug composition for anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss is provided, comprising the Lactobacillus fermentum strain of the present invention.

[0061] In the composition of a quasi-drug according to the present invention, each term has the same meaning as described above unless specifically stated otherwise.

[0062] The above-mentioned quasi-drug composition for anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss may be a quasi-drug composition used for the improvement or prevention of metabolic diseases.

[0063] The quasi-drug composition according to the present invention can be manufactured in the form of general emulsion formulations and solubilized formulations, etc., using commonly known manufacturing methods. At this time, the quasi-drug composition of the present invention can be manufactured in various formulations such as patches, ointments, gels, creams, sprays, detergents, disinfectants, sanitary pads, wet wipes, masks, gauze, absorbent cotton, adhesive bandages, bandages, solid forms, liquid forms, etc., and commonly used manufacturing methods for quasi-drugs may be applied. Furthermore, the types of the above quasi-drugs are not particularly limited. Examples include toothpaste, oral spray, mouthwash, mouthwash, oral rinse, oral ointment, mouthwash, gum massage cream, cleanser, wet wipe, shower foam, soap, mask, gauze, absorbent cotton, adhesive bandage, eye mask, bandage, repellent, disinfectant, detergent, etc., and include all quasi-drugs in the conventional sense.

[0064] The quasi-drug composition according to the present invention may further include additional components in addition to the strain of the present invention to enhance efficacy. For example, there are no limitations on the additional components provided that they do not offset or reduce the efficacy of the strain according to the present invention. Optionally, carriers, excipients, diluents, etc., commonly used in the field of quasi-drugs may be further included. For example, the carrier, excipient, or diluent may be a filler, extender, binder, wetting agent, disintegrant, surfactant, lubricant, sweetener, fragrance, preservative, etc.

[0065] Optionally, ingredients typically used to add or enhance the function of the quasi-drug may also be added. For example, the quasi-drug composition may additionally include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, acacia gum, alginate, calcium phosphate, calcium carbonate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oil, injectable ester, Witepsol, macrogol, Tween 61, cocoa dough, laurize, etc.

[0066]

[0067] According to another embodiment of the present invention, a food composition for anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss is provided, comprising the Lactobacillus fermentum strain of the present invention.

[0068] In the food composition according to the present invention, each term has the same meaning as the foregoing unless specifically stated otherwise.

[0069] The above foods include all processed forms of natural materials, such as nutraceutical foods, medical foods, health functional foods, nutritional supplements, and food additives.

[0070] The above-mentioned food composition for anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss may be a food composition used for the improvement or prevention of metabolic diseases.

[0071] As used in this specification, the term "Nutraceutical food" refers to a food prepared from raw materials or ingredients that perform functions beneficial to a subject, and which improves or maintains the subject's health by maintaining normal functions or activating physiological functions.

[0072] As used herein, the term "medical food" refers to a food for special medical purposes, specifically a food in a special formulation for the specific dietary management of a subject suffering from a disease, and includes foods for special medical purposes prescribed by the Ministry of Food and Drug Safety. The medical food alleviates the subject's disease or symptoms by satisfying specific nutritional requirements that cannot be achieved by a regular diet alone, and may be consumed as part of a meal or as a meal replacement. The special formulation is a formulation that can be taken orally or via enteral nutrition, and enteral nutrition refers to the supply of nutrition by inserting a nasogastric tube into the gastrointestinal tract through the nose. In this specification, the term "medical food" may be used interchangeably with the term "medical food."

[0073] As used in this specification, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients that have functional properties useful to the human body. Herein, "functionality" means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0074] The types of the above foods are not specifically limited. Specific examples include yogurt, dairy products, meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and include all foods in the conventional sense.

[0075] The food composition according to the present invention can be manufactured by methods commonly used in the art, and during such manufacturing, raw materials and ingredients commonly added in the art may be added. The added ingredients include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. The carbohydrates include all conventional sugars such as glucose, fructose, maltose, sucrose, oligosaccharides, dextrin, cyclodextrin, etc., or sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents, natural flavoring agents such as taumatin and stevia extract, or synthetic flavoring agents such as saccharin and aspartame may be used. For example, when the food composition is manufactured as a drink, in addition to the strain according to the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, or licorice extract may be additionally included, and various natural carbohydrates, etc., may be included as additional ingredients, as in conventional beverages.

[0076] In addition, unlike general pharmaceuticals, food ingredients have the advantage of not causing side effects that may occur during long-term use of pharmaceuticals and are highly portable. Therefore, the food composition of the present invention can be used as an adjuvant to enhance or improve the preventive or therapeutic effects of metabolic diseases, and it is also possible to use it simultaneously or sequentially with the pharmaceutical composition, quasi-drug composition, and / or other composition or other therapy according to the present invention for the purpose of maximizing the above effects.

[0077] The content of the active ingredient included in the food composition of the present invention may be appropriately determined according to the purpose of use (prevention, improvement, or therapeutic treatment), the duration of use, the condition of the subject, etc. For example, when manufacturing food, the content of the strain according to the present invention may be included in the food composition in an amount of 0.001 to 20 weight%, 0.001 to 15 weight%, or 0.001 to 10 weight%. In the case of health drinks, it may be included in an amount of 0.01 to 2 g, specifically 0.02 to 2 g, and more specifically 0.3 to 1 g based on 100 ml. However, in the case of long-term consumption for the purpose of health and hygiene or health control, it may be used in an amount less than the above range. During the process of manufacturing the food composition of the present invention, the content of the active ingredient added to the food composition may be appropriately increased or decreased as needed.

[0078]

[0079] According to another embodiment of the present invention, a cosmetic composition for anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss is provided, comprising the Lactobacillus fermentum strain of the present invention.

[0080] In the cosmetic composition according to the present invention, each term has the same meaning as described above unless specifically stated otherwise.

[0081] The above anti-inflammatory cosmetic composition may be a cosmetic composition used for the improvement or prevention of metabolic diseases.

[0082] The cosmetic composition according to the present invention can be prepared in the form of a general emulsion formulation and a solubilized formulation, etc., using a commonly known manufacturing method. Specifically, it can be prepared in various formulations such as patches, ointments, skin adhesive gels, creams, packs, lotions, essences, sprays, masks, foundations, makeup bases, cleansers, water (W) type, oil (O) type, silicone (S) type, oil-in-water (O / W) type, water-in-oil (W / O) type, silicone-in-water (W / S) type, silicone-in-water (S / W) type, solid phase, liquid phase, etc. In addition, it can be prepared in the form of a foam or in the form of an aerosol containing a compressed propellant.

[0083] The cosmetic composition according to the present invention may further include additional ingredients in addition to the active ingredient. There are no limitations on the additional ingredients provided that they do not offset or reduce the anti-inflammatory, blood sugar-lowering, appetite-suppressing, or weight-loss effects of the active ingredient. Optionally, adjuvants, carriers, etc., commonly used in the field of cosmetics may be further included. For example, it may be one or more selected from fatty substances, organic solvents, solvents, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, thickeners, humectants, liquid crystal film strengtheners, pH adjusters, antimicrobial agents, water-soluble polymers, film-forming agents, amino acids, organic amines, polymer emulsions, pH adjusters, skin nutrients, antioxidants, antioxidant aids, preservatives, fragrances, oils and fats, waxes, hydrocarbon oils, higher fatty acid oils, higher alcohols, synthetic ester oils, silicone oils, fillers, metal ion chelating agents, opacifiers, anti-foaming agents, bulking agents, skin softeners, skin conditioning agents, chelating agents, preservatives, vitamins, blockers, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, and lipid vesicles. The above-mentioned adjuvants and carriers are introduced in amounts commonly used in the fields of cosmetics or dermatology.

[0084]

[0085] According to another embodiment of the present invention, a feed composition for anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss is provided, comprising the Lactobacillus fermentum strain of the present invention.

[0086] In the feed composition according to the present invention, each term has the same meaning as the foregoing unless specifically stated otherwise.

[0087] The above anti-inflammatory feed composition may be a feed composition used for the improvement or prevention of metabolic diseases.

[0088] The above feed is a substance that supplies organic or inorganic nutrients necessary for the maintenance of life and rearing of subjects other than humans, and includes both feed and feed additives.

[0089] The types of the above feed are not particularly limited. Specific examples include grains, plant-based protein feeds, animal-based protein feeds, dairy products, nutritional supplements, digestion and absorption enhancers, growth promoters, etc., and include all feed or feed additives in the conventional sense. The above grains include, for example, ground or crushed rice, wheat, oats, barley, or corn. The above plant-based protein feed includes, for example, feeds with rapeseed (rapeseed), sunflower, or soybeans as the main components. The above animal-based protein feed includes, for example, feeds with blood meal, bone meal, meat meal, or fish meal as the main components. The above dairy products include, for example, feeds with powdered milk or whey powder as the main components.

[0090] The feed composition according to the present invention can be manufactured by a method commonly used in the art, and in addition to the active ingredient, the composition may further include a suitable carrier, preservative, stabilizer, wetting agent, emulsifier, solution accelerator, excipient, organic acid, phosphate, antioxidant, etc. commonly used in the art. Specifically, the carrier, preservative, stabilizer, wetting agent, emulsifier, solution accelerator, excipient, organic acid, phosphate, antioxidant that may be included in the feed composition may be one or more selected from, for example, corn starch, lactose, sucrose, soybean flakes, olive oil, sesame oil, peanut oil, propylene glycol, lactic acid, malic acid, citric acid, fumaric acid, adipic acid, sodium phosphate, potassium phosphate, polyphenol, vitamin C, green tea extract, licorice extract, tocopherol, tannic acid, and chitosan. The above carrier, preservative, stabilizer, wetting agent, emulsifier, solution accelerator, excipient, organic acid, phosphate, antioxidant, etc. are introduced in amounts commonly used in the industry.

[0091] The feed composition according to the present invention may be formulated using commonly known manufacturing methods. For example, it may be in the form of a powder, liquid, or granular formulation. The content of the active ingredient included in the feed composition of the present invention may be appropriately determined according to the purpose of use (prevention, improvement, or therapeutic treatment), duration of use, condition of the subject, etc. For example, when manufacturing the feed, the content of the strain according to the present invention may be 20 to 90 weight percent of the feed composition. The feed composition may be used by immersion, spraying, or mixing with other feed compositions.

[0092]

[0093] Another aspect of the present invention provides a method for anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss, comprising the step of administering a composition containing the Lactobacillus fermentum strain of the present invention to a subject.

[0094] In the anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss method according to the present invention, each term has the same meaning as the foregoing unless specifically stated otherwise.

[0095] According to one embodiment of the present invention, a method for treating, improving, or preventing a metabolic disease of a subject is provided, comprising the step of administering the composition to the subject.

[0096] The composition according to the present invention contains an effective amount of the Lactobacillus fermentum strain of the present invention and can be administered to a subject who requires treatment or prevention of metabolic diseases.

[0097] The above effective amount may be a "therapeutic effective amount" or a "preventive effective amount."

[0098] As used herein, the term “therapeutic effective dose” means any amount in which, when a drug or therapeutic agent is used alone or in combination with other therapeutic agents, it may result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or the prevention of damage or disability caused by disease suffering.

[0099] As used herein, the term "preventive effective dose" refers to any amount that suppresses the occurrence or recurrence of disease in a subject. The level of said effective dose may be determined based on factors including the subject's severity, age, gender, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0100] The dosage of the above composition may vary depending on the subject's age, gender, body weight, formulation method, administration route, or severity of the disease, excretion rate, administration time, pathological condition, and response sensitivity, for example, depending on the subject's symptoms, the composition of the present invention may be administered at a dose of 0.1 to 100 mg / kg once or several times a day, or at intervals of several days to several months.

[0101] In the composition for anti-inflammatory, blood glucose reduction, appetite suppression, or weight loss according to the present invention, the composition may be administered to a subject simultaneously, sequentially, or individually with other components exhibiting anti-inflammatory, blood glucose reduction, appetite suppression, or weight loss effects. The "simultaneous" administration means administering the composition and other components as a single preparation at once, or administering the composition and other components as separate preparations at once; in this case, the administration route of the composition and the administration route of the other components may differ from each other. The "sequential" administration means administering the composition and other components relatively continuously, allowing for the minimum possible time consumed in the administration interval. The "individual" administration means administering the composition and other components at regular time intervals. The method of administration of the composition and other components may be appropriately selected by an expert in the art, taking into account the efficacy and side effects on the subject.

[0102] The composition of the present invention may be administered in combination with other therapeutic agents. In this case, the composition of the present invention and the other therapeutic agents may be administered simultaneously, sequentially, or individually. The other therapeutic agents may be drugs, such as compounds or proteins, having effects of preventing, treating, and / or improving metabolic diseases, but are not limited thereto.

[0103] In addition, the above composition may be formulated to be administered simultaneously, sequentially, or individually with other therapeutic agents. For example, the strain and other therapeutic agents may be administered simultaneously as a single formulation, or they may be administered simultaneously, sequentially, or individually as separate formulations. To administer simultaneously, sequentially, or individually, the strain and other therapeutic agents included in the composition of the present invention may be formulated separately in individual containers, or formulated together in the same container. Furthermore, the pharmaceutically effective amount, administration time, administration interval, administration route, treatment period, etc., of the strain and other therapeutic agents included in the composition of the present invention may be the same or different from each other.

[0104]

[0105] Another aspect of the present invention provides the use of a Lactobacillus fermentum strain deposited under accession number KCTC 15954BP, or a composition comprising said strain, for treating, improving, or preventing metabolic diseases of a subject.

[0106] In the use of the Lactobacillus fermentum strain according to the present invention, or a composition comprising said strain, each term has the same meaning as described above unless specifically stated otherwise.

[0107] According to one embodiment of the present invention, the strain or composition may be used in the preparation or manufacture of pharmaceuticals, quasi-pharmaceuticals, cosmetics, foods, or the like for anti-inflammatory, blood sugar reduction, appetite suppression, or weight loss purposes.

[0108] The Lactobacillus fermentum strain of the present invention inhibits the production of nitric oxide (NO) in a subject, promotes the secretion of GLP-1 (glucagon-like peptide-1), a hormone that regulates blood sugar by increasing insulin secretion and inhibiting glucagon secretion, and can reduce blood sugar, suppress appetite, or reduce body weight. Therefore, the strain of the present invention and the composition containing it can be usefully utilized for anti-inflammatory, blood sugar reduction, appetite suppression, or weight reduction purposes, and can also be usefully utilized for the treatment, improvement, or prevention of metabolic diseases.

[0109] Figure 1 is a photograph showing the hemolytic result of the Lactobacillus fermentum strain of the present invention, taken after spreading the strain on a blood agar medium (Sheep blood agar) and culturing it.

[0110] Figure 2 shows the results of nitric oxide (NO) production inhibition by the Lactobacillus fermentum strain of the present invention in Raw 264.7 cells. NC 1 represents negative control group 1, which was not treated with LPS (Lipopolysaccharide), dexamethasone, and the culture supernatant of the Lactobacillus fermentum strain of the present invention as a sample; NC 2 represents negative control group 2, which was treated with LPS; PC represents positive control group, which was treated with LPS and dexamethasone; 5% represents experimental group, which was treated with LPS and 5% of the culture supernatant of the Lactobacillus fermentum strain of the present invention as a sample; DEXA represents dexamethasone; * represents P value < 0.05; ** represents P value < 0.01; and *** represents P value < 0.001.

[0111] Figure 3a shows the results of GLP-1 (glucagon-like peptide-1) secretion enhancement by the Lactobacillus fermentum strain of the present invention in STC-1 cells. NC represents a negative control group that was not treated with the culture supernatant of the Lactobacillus fermentum strain of the present invention as a sample, 6h represents experimental group 1 that was treated with 2% of the culture supernatant of the Lactobacillus fermentum strain of the present invention as a sample and cultured for 6 hours, 24h represents experimental group 2 that was treated with 2% of the culture supernatant of the Lactobacillus fermentum strain of the present invention as a sample and cultured for 24 hours, ** represents a P value < 0.01, and *** represents a P value < 0.001.

[0112] Figure 3b shows the results of GLP-1 secretion enhancement by the Lactobacillus fermentum strain of the present invention in NCI-H716 cells. NC represents a negative control group that was not treated with the culture supernatant of the Lactobacillus fermentum strain of the present invention as a sample, 2h represents experimental group 1 that was treated with 2% of the culture supernatant of the Lactobacillus fermentum strain of the present invention as a sample and cultured for 2 hours, 6h represents experimental group 2 that was treated with 2% of the culture supernatant of the Lactobacillus fermentum strain of the present invention as a sample and cultured for 6 hours, and ** represents a P value < 0.01.

[0113] Figure 4 shows the results of blood glucose reduction by the Lactobacillus fermentum strain of the present invention in Hepa1-6 cells. NC represents a negative control group that was not treated with the culture supernatant of the Lactobacillus fermentum strain of the present invention as a sample, 2% represents experimental group 1 treated with 2% of the culture supernatant of the Lactobacillus fermentum strain of the present invention as a sample, 5% represents experimental group 2 treated with 5% of the culture supernatant of the Lactobacillus fermentum strain of the present invention as a sample, and *** represents a P value < 0.001.

[0114] Figure 5 shows the results of GLP-1 secretion enhancement by the Lactobacillus fermentum strain of the present invention in a regular chow mouse model. PBS represents negative control group 1 treated with PBS (phosphate buffered saline), CPA represents negative control group 2 treated with a cryoprotective agent, KCTC 15954BP represents the experimental group treated with the freeze-dried product of the Lactobacillus fermentum strain of the present invention as a sample, 0hr after glucose ad. represents before glucose administration, 1hr after glucose ad. represents 1 hour after glucose administration, and P represents the P value.

[0115] Figure 6a shows the results of body weight loss of the Lactobacillus fermentum strain of the present invention in a high-fat diet (HFD) mouse model. Regular chow + CPA represents negative control group 1, which was fed a regular diet and administered a cryopreservation agent; 60% HFD + CPA represents negative control group 2, which was fed a high-fat diet and administered a cryopreservation agent; 60% HFD + KCTC 15954BP represents an experimental group, which was fed a high-fat diet and treated with a freeze-dried product of the Lactobacillus fermentum strain of the present invention as a sample; and Treatment represents oral administration of the cryopreservation agent to negative control group 1 and negative control group 2, and oral administration of the sample to the experimental group.

[0116] Figure 6b shows the results of the percentage change in body weight (%) of the Lactobacillus fermentum strain of the present invention in a high-fat diet mouse model. Regular chow + CPA represents negative control group 1, which was fed a regular diet and administered a cryopreservation agent; 60% HFD + CPA represents negative control group 2, which was fed a high-fat diet and administered a cryopreservation agent; 60% HFD + KCTC 15954BP represents the experimental group, which was fed a high-fat diet and treated with a freeze-dried product of the Lactobacillus fermentum strain of the present invention as a sample; Treatment represents oral administration of the cryopreservation agent to negative control group 1 and negative control group 2, and oral administration of the sample to the experimental group; and **** represents a P value < 0.0001.

[0117] Figure 6c shows the results of appetite suppression by the Lactobacillus fermentum strain of the present invention in a high-fat diet mouse model. Regular chow + CPA represents negative control group 1, which was fed a regular diet and administered a cryopreservative; 60% HFD + CPA represents negative control group 2, which was fed a high-fat diet and administered a cryopreservative; 60% HFD + KCTC 15954BP represents the experimental group, which was fed a high-fat diet and treated with a freeze-dried product of the Lactobacillus fermentum strain of the present invention as a sample; Treatment represents oral administration of the cryopreservative to negative control group 1 and negative control group 2, and oral administration of the sample to the experimental group; and P represents the P value.

[0118] Figure 7a shows the results of the evaluation of oral glucose tolerance of the Lactobacillus fermentum strain of the present invention in a high-fat diet mouse model. Regular chow + CPA represents negative control group 1, which was fed a regular diet and administered a cryopreservation agent; 60% HFD + CPA represents negative control group 2, which was fed a high-fat diet and administered a cryopreservation agent; 60% HFD + KCTC 15954BP represents the experimental group, which was fed a high-fat diet and treated with a freeze-dried product of the Lactobacillus fermentum strain of the present invention as a sample; RC represents the regular diet; HFD represents the high-fat diet; * represents a P value < 0.05; ** represents a P value < 0.01; *** represents a P value < 0.001; and **** represents a P value < 0.0001.

[0119] Figure 7b shows the results calculated as the area under the curve (AUC) of the graph over time for the evaluation of oral glucose tolerance of the Lactobacillus fermentum strain of the present invention in a high-fat diet mouse model. Regular chow + CPA represents negative control group 1, which was fed a regular diet and administered a cryopreservation agent; 60% HFD + CPA represents negative control group 2, which was fed a high-fat diet and administered a cryopreservation agent; 60% HFD + KCTC 15954BP represents the experimental group, which was fed a high-fat diet and treated with the freeze-dried product of the Lactobacillus fermentum strain of the present invention as a sample; ** represents a P value < 0.01; and **** represents a P value < 0.0001.

[0120] Figure 7c shows the results of fasting blood glucose reduction by the Lactobacillus fermentum strain of the present invention in a high-fat diet mouse model. Regular chow + CPA represents negative control group 1, which was fed a regular diet and administered a cryopreservation agent; 60% HFD + CPA represents negative control group 2, which was fed a high-fat diet and administered a cryopreservation agent; and 60% HFD + KCTC 15954BP represents the experimental group, which was fed a high-fat diet and treated with a freeze-dried product of the Lactobacillus fermentum strain of the present invention as a sample. P represents the P value, and **** represents a P value < 0.0001.

[0121] Figure 8a shows the results of GLP-1 secretion enhancement of the Lactobacillus fermentum strain and the Lactobacillus fermentum GB102 strain according to the present invention analyzed in STC-1 cells. NONE represents negative control group 1, which was not treated with PMA (phorbol 12-myristate 13-acetate), MRS (de man rogosa, sharpe agar) medium, and the culture supernatant of the novel strain according to the present invention and the culture supernatant of the GB102 strain as samples; PC represents positive control group treated with 1 μM of PMA; MEDIA represents negative control group 2 treated with MRS medium; KCTC 15954BP represents experimental group 1 treated with 5% (v / v) of the culture supernatant of the strain according to the present invention as a sample; GB102 represents experimental group 2 treated with 5% (v / v) of the culture supernatant of the GB102 strain as a sample; and P represents the P value.

[0122] Figure 8b shows the results of GLP-1 secretion enhancement by the Lactobacillus fermentum strain according to the present invention and the Lactobacillus fermentum GB102 strain analyzed in NCI-H716 cells. Each term has the same meaning as defined in Figure 8a.

[0123] Figure 9 shows the results of enhancing 5-HTP (5-hydroxytryptophan) expression in the Lactobacillus fermentum strain and the Lactobacillus fermentum GB102 strain according to the present invention. NONE represents negative control group 1, which was not treated with ionomycin, MRS medium, and the culture supernatant of the novel strain according to the present invention and the culture supernatant of the GB102 strain as samples; PC represents positive control group treated with 10 μM ionomycin; MEDIA represents negative control group 2 treated with MRS medium; KCTC 15954BP represents experimental group 1 treated with 5% (v / v) of the culture supernatant of the strain according to the present invention as a sample; GB102 represents experimental group 2 treated with 5% (v / v) of the culture supernatant of the GB102 strain as a sample; and P represents the P value.

[0124] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited by these examples.

[0125]

[0126] Example 1. Identification of Lactobacillus fermentum strain

[0127] 4 g of fresh fecal sample fluid collected from a healthy adult male was added to 80 mL of PBS (phosphate buffer saline) solution, vortexed, and resuspended to prepare a mixture. The homogenized mixture was serially diluted tenfold using PBS, and 10 -5 , 10 -6 , 10 -7 and 10 -8200 µl of each diluted solution was prepared. Subsequently, 200 µl of each diluted solution was plated onto MRS (de man rogosa, Sharpe agar), a selective medium for lactic acid bacteria, and cultured at 37°C under aerobic conditions for 48 hours to induce colonies. Afterward, colony PCR was performed to obtain the amplified 16S rRNA genes from each colony, and their nucleotide sequences were analyzed. Using the NCBI blast program, the nucleotide sequences were analyzed to identify closely related species of the microorganisms contained in the colonies. Closely related species refer to organisms that belong to the same genus but are of different species.

[0128] As a result of the search, it was confirmed that the microorganism of the colony had a high degree of similarity to Lactobacillus fermentum. To isolate the microorganism in its pure form, colony streaking was performed multiple times, and colony PCR was conducted to reconfirm the 16S rRNA gene sequence of the microorganism. The purely isolated microorganism was liquid cultured in MRS medium, and then 30% glycerol was added and frozen at -80°C.

[0129] Subsequently, 1 mL of the pure culture of the isolated microorganism was collected and submitted to an accredited sequencing laboratory to determine the 16S rRNA gene sequence. The determined 16S rRNA gene sequence is shown in Table 1 below, and the primer sequences used are shown in Table 2 below.

[0130] Type16S rRNA gene SEQ ID NO: MicroorganismCAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCAAATCTCTTAAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCA CGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAGCCAGCCGCCTAAGGTGGGACAGATGATTAGGGTTGAAGTCSEQ ID NO. 1

[0131]

[0132] Type Sequence Sequence Number Forward Primer 27F (for PCR) AGAGTTTGATCMTGGCTCAG Sequence Number 2 Reverse Primer 1492R (for PCR) TACGGYTACCTTGTTACGACTT Sequence Number 3 Forward Primer 785F (for Sequencing) GGATTAGATACCCTGGTA Sequence Number 4 Reverse Primer 907R (for Sequencing) CCGTCAATTCMTTTRAGTTT Sequence Number 5

[0133]

[0134] As a result, it was confirmed that the isolated microorganism exhibited 99% homology with Lactobacillus fermentum. Accordingly, the microorganism was deposited with the Korea Research Institute of Bioscience and Biotechnology (KRIBB), a patent strain depositary, on July 8, 2024, and was assigned accession number KCTC 15954BP. KRIBB is a patent strain depositary located at 181, Ipsin-gil, Jeongeup-si, Jeollabuk-do, South Korea.

[0135]

[0136] Example 2. Preparation of culture supernatant and freeze-dried product of Lactobacillus fermentum strain

[0137] 2% (v / v) of the Lactobacillus fermentum strain of the present invention was inoculated into a 50mL tube (50mL TPP Tube with 0.2㎛ Vent Cap (EF)) containing 9.8mL of MRS medium, and then cultured in an incubator at 37℃ and 0rpm for 24 hours. After culture, 1% (v / v) of the Lactobacillus fermentum strain culture solution was inoculated into a 50mL tube (50mL TPP Tube with 0.2㎛ Vent Cap (EF)) containing 19.8mL of MRS medium, and then cultured at 37℃ and 0rpm for 24 hours. Subsequently, the culture solution was centrifuged at 4℃ and 8000rpm for 10 minutes to separate the supernatant, and then filtered through a 0.22㎛ filter to prepare the culture supernatant of the Lactobacillus fermentum strain. After removing the supernatant, the remaining cell precipitate was freeze-dried to prepare a freeze-dried product of the Lactobacillus fermentum strain.

[0138]

[0139] Example 3. Hemolytic activity of Lactobacillus fermentum strain

[0140] In order to confirm the safety of the Lactobacillus fermentum strain of the present invention for the human body, hemolytic activity, which is the ability to destroy cells such as red blood cells present in the blood, was confirmed.

[0141] Specifically, Lactobacillus fermentum strains pure-cultured in MRS medium were collected using a loop, streaked onto sheep blood agar, and incubated aerobically at 37°C for 48 hours. Subsequently, hemolytic activity was determined by the formation of a clear zone around the cells.

[0142] As a result, as can be seen in Figure 1, it was confirmed that the Lactobacillus fermentum strain cultured on blood agar medium was non-hemolytic as no clear halo formed around the cell.

[0143] Through the above results, it was confirmed that the Lactobacillus fermentum strain of the present invention is harmless to the human body and has excellent safety, so it can be usefully utilized in medicines, cosmetics, or food applied to the human body.

[0144]

[0145] Example 4. Anti-inflammatory effect of Lactobacillus fermentum strain

[0146] To determine whether the Lactobacillus fermentum strain of the present invention can treat, improve, or prevent metabolic diseases through anti-inflammatory activity, the inhibitory effect of the strain on nitric oxide (NO) production was confirmed. NO is known to induce oxidative stress and promote cell damage and inflammation (Roman Fischer et al. (2015), Interrelation Oxidative Stress and Inflammation in Neurodegenerative Disease: Role of TNF, Oxidative Medicine and Cellular Longevity). Furthermore, inflammation occurs as a result of obesity and may play a causal role in causing insulin resistance, insulin secretion defects, and the disruption of energy homeostasis; it is also known that obesity-induced inflammation affects metabolic homeostasis by entailing tonic activation of the innate immune system (Alan R. Saltiel and Jerrold M. Olefsky (2017), Inflammatory mechanisms linking obesity and metabolic disease, The Journal of Clinical Investigation, 127(1):1-4).

[0147] Specifically, Raw 264.7 mouse macrophages were placed in a 96-well cell culture plate at a rate of 5 x 10⁶ 4The cells were dispensed at a concentration of cells / well and cultured for 24 hours at 37°C and 5% CO2. Afterward, the samples were washed with PBS, and LPS, dexamethasone, and the culture supernatant of the Lactobacillus fermentum strain according to Example 2 were treated according to the control and experimental group conditions below, followed by culture at 37°C and 5% CO2 for 24 hours. At this time, the samples were added to DMEM (Dulbecco modified Eagle Medium) that did not contain FBS (fetal bovine serum) for treatment.

[0148] (1) Negative Control 1 (NC 1): LPS, dexamethasone, and no sample treatment

[0149] (2) Negative control group 2 (NC 2): Treated with LPS 200 ng / mL

[0150] (3) Positive control (PC): Treated with LPS 200 ng / mL and dexamethasone 1 μM

[0151] (4) Experimental group (5%): Treated with 200 ng / mL of LPS and 5% of the culture supernatant of Lactobacillus fermentum strain as a sample.

[0152] After the culture was completed, the amount of NO was measured and shown as a graph in Figure 2.

[0153] As a result, as can be seen in Figure 2, the amount of NO in the experimental group (5%) was reduced by about 58% compared to the negative control group 2 (NC 2), which is similar to the level of the positive control group (PC).

[0154] Based on the above results, the Lactobacillus fermentum strain of the present invention exhibits anti-inflammatory activity by reducing NO, and this effect suggests that the strain may exhibit therapeutic, improving, or preventive effects against metabolic diseases.

[0155]

[0156] Example 5. Effect of Lactobacillus fermentum strain on GLP-1 (glucagon-like peptide-1) secretion enhancement

[0157] To determine whether the Lactobacillus fermentum strain of the present invention can treat, improve, or prevent metabolic diseases, the effect of the strain on enhancing GLP-1 secretion was confirmed. GLP-1 is an important hormone for regulating nutrient metabolism; it is released from L-cells distributed in the terminal small intestine or large intestine and is stimulated by nutrient intake. GLP-1 increases the secretion of glucose-stimulating insulin, inhibits glucagon secretion, and promotes satiety. Therefore, increasing the levels of active GLP-1 in the circulatory system is utilized as a basic principle for antidiabetic agents (Jeffrey Gagnon and Patricia L. Brubaker (2015), NCI-H716 cells, The impact of food bioactives on health, p.221-228).

[0158]

[0159] Example 5-1

[0160] STC-1 cells were used to confirm the GLP-1 secretion-enhancing effect of the Lactobacillus fermentum strain of the present invention. STC-1 cells are a murine intestinal secretin tumor cell line that expresses and secretes intestinal hormones related to hunger suppression, food intake regulation, and satiety, such as GLP-1. Therefore, the STC-1 cells are used as a screening platform to identify foods or compounds that regulate gastrointestinal hormone secretion in vitro (Triona McCarthy et al. (2015), STC-1 Cells, The impact of food bioactives on health, p. 211-220).

[0161] Specifically, 5 x 10 STC-1 cells in a 96-well cell culture plate 4 The cells were dispensed at a concentration of cells / well and cultured for 24 hours at 37°C and 5% CO2. Afterward, the samples were washed with PBS, and 2% of the culture supernatant of the Lactobacillus fermentum strain according to Example 2 was added as a sample according to the control and experimental group conditions below, followed by culture at 37°C and 5% CO2 for 6 or 24 hours. At this time, the samples were added to DMEM that did not contain FBS.

[0162] (1) Negative control (NC): Sample not treated

[0163] (2) Experimental group 1 (6h): 2% of the culture supernatant of Lactobacillus fermentum strain was treated as a sample and cultured for 6 hours.

[0164] (3) Experimental group 2 (24h): 2% of the culture supernatant of Lactobacillus fermentum strain was treated as a sample and cultured for 24 hours.

[0165] After culture, the supernatant from each well was collected and the amount of GLP-1 (pg / mL) was measured using a Multispecies GLP-1 ELISA kit. Then, the average GLP-1 expression levels (Fold change) of experimental group 1 (6h) and experimental group 2 (24h) relative to the negative control group (NC) were graphed in Fig. 3a.

[0166] As a result, as can be seen in Figure 3a, the amount of GLP-1 in experimental group 1 (6h) and experimental group 2 (24h) increased in a time-dependent manner and significantly increased compared to the negative control group (NC).

[0167] Based on the above results, the Lactobacillus fermentum strain of the present invention exhibits the effect of lowering blood sugar by increasing GLP-1 secretion to promote insulin secretion and inhibiting glucagon secretion, and this effect suggests that the strain may exhibit therapeutic, improving, or preventive effects against metabolic diseases.

[0168]

[0169] Example 5-2

[0170] To confirm the GLP-1 secretion-enhancing effect of the Lactobacillus fermentum strain of the present invention, NCI-H716 cells were used. NCI-H716 cells are a cell line derived from ascites fluid of colorectal adenocarcinoma in Caucasian males and are a human model that can be used for in vitro studies related to GLP-1 regulation (Jeffrey Gagnon and Patricia L. Brubaker (2015), NCI-H716 Cells, The impact of food bioactives on health, p.221-228).

[0171] Specifically, after coating a 96-well cell culture plate with 0.5 mg / mL of Matrigel, 1 x 10⁶ NCI-H716 cells were 5 The cells were dispensed at a concentration of cells / well and cultured for 24 hours at 37°C and 5% CO2. Afterward, the samples were washed with PBS, and 2% of the culture supernatant of the Lactobacillus fermentum strain according to Example 2 was added as a sample according to the control and experimental group conditions below, followed by incubation for 2 or 6 hours at 37°C and 5% CO2. At this time, the samples were added to a Krebs buffer solution containing 0.2% w / v BSA (bovine serum albumin) and 0.03% w / v bile bovine.

[0172] (1) Negative control (NC): Sample not treated

[0173] (2) Experimental group 1 (2h): 2% of the culture supernatant of Lactobacillus fermentum strain was treated as a sample and cultured for 2 hours.

[0174] (3) Experimental group 2 (6h): 2% of the culture supernatant of Lactobacillus fermentum strain was treated as a sample and cultured for 6 hours.

[0175] After culture, the supernatant from each well was collected and the amount of GLP-1 (pg / mL) was measured using a Multispecies GLP-1 ELISA kit. Then, the average GLP-1 expression levels (Fold change) of experimental group 1 (2h) and experimental group 2 (6h) relative to the negative control group (NC) were graphed in Fig. 3b.

[0176] As a result, as can be seen in Figure 3b, the amount of GLP-1 in experimental group 1 (2h) and experimental group 2 (6h) increased in a time-dependent manner and significantly increased compared to the negative control group (NC).

[0177] Based on the above results, the Lactobacillus fermentum strain of the present invention increases GLP-1 secretion to promote insulin secretion and inhibits glucagon secretion, thereby lowering blood sugar, and these effects suggest that the strain may exhibit therapeutic, improving, or preventive effects against metabolic diseases.

[0178]

[0179] Example 6. Blood glucose-lowering effect of Lactobacillus fermentum strain

[0180] In order to determine whether the Lactobacillus fermentum strain of the present invention can treat, improve, or prevent metabolic diseases, the blood glucose-reducing effect of the strain was confirmed using Hepa1-6 cells, a mouse liver cancer cell line.

[0181] Specifically, 7 x 10⁶ Hepa1-6 cells in a 96-well cell culture plate 4The cells were dispensed at a concentration of cells / well and cultured for 24 hours at 37°C and 5% CO2. Afterward, the samples were washed with PBS, and the culture supernatant of the Lactobacillus fermentum strain according to Example 2 was treated according to the control and experimental group conditions below and cultured for 24 hours at 37°C and 5% CO2. At this time, the samples were added to DMEM that did not contain FBS for treatment.

[0182] (1) Negative control (NC): Sample not treated

[0183] (2) Experimental group 1 (2%): 2% of the culture supernatant of Lactobacillus fermentum strain was treated as a sample.

[0184] (3) Experimental group 2 (5%): 5% of the culture supernatant of Lactobacillus fermentum strain was treated as a sample.

[0185] After incubation, 5 µl of the supernatant from each well was loaded onto a blood glucose meter (Accu-chek active, Roche, Switzerland) to measure the amount of glucose (mg / dL) in the medium, which was then shown as a graph in Figure 4.

[0186] As a result, as can be seen in Figure 4, the amount of glucose in the medium of experimental group 1 (2%) and experimental group 2 (5%) was significantly reduced compared to the negative control group (NC).

[0187] Based on the above results, the Lactobacillus fermentum strain of the present invention regulates blood glucose by reducing blood glucose concentration, and this effect suggests that the strain may exhibit therapeutic, improving, or preventive effects against metabolic diseases.

[0188]

[0189] Example 7. Effects of Lactobacillus fermentum strain on GLP-1 secretion enhancement and blood glucose reduction in a regular chow mouse model

[0190] To confirm the effects of GLP-1 secretion enhancement and glucose reduction in mice on a standard diet (normal mice), 8-week-old male C57BL / 6 mice (specific pathogen-free, Coatec Co., Ltd.) were purchased. These mice underwent a one-week acclimatization period and were divided into three groups after body weight measurement. All mice were housed under conditions of a temperature of 22±1℃, humidity of 55±10%, and a 12-hour light / dark cycle, and were provided with free access to water and feed. All animal experiments were conducted in accordance with the ethical standards of the Institutional Animal Care and Use Committee (IACUC) and were carried out after the review and approval of the animal experiment protocol (IACUC Approval No. KLSIACUC20240412-2-01).

[0191] Phosphate buffered saline (PBS), a cryoprotective agent, and a freeze-dried Lactobacillus fermentum strain according to Example 2 were orally administered to three isolated groups of mice for 5 days under the following control and experimental group conditions. Oral administration on the 5th day was performed after fasting all mice for 15 hours prior to administration. At this time, the cryoprotective agent and the sample were added to PBS and administered, and all administered products were prepared on the day of administration.

[0192] (1) Negative control group 1 (PBS): Treated with PBS

[0193] (2) Negative control group 2 (CPA): Treated with 7.4 mg / 200 µl of cryopreservative

[0194] (3) Experimental group (KCTC 15954BP): 1 x 10⁻³ freeze-dried product of Lactobacillus fermentum strain as a sample 9 Treats CFU / 200 µL

[0195] On the 5th day, 3 hours after oral administration, 200 µl of blood was collected from all mice using the facial venous blood sampling method. After collection, 2 g / kg of glucose was orally administered to all mice, and 1 hour later, 200 µl of blood was collected from all mice again using the facial venous blood sampling method. All collected blood samples were centrifuged for 15 minutes at 4°C and 5,000 rpm to separate the serum, which was stored at -80°C and used to measure blood GLP-1 levels (pg / mL) and glucose levels (mg / dL). GLP-1 was measured using a Multispecies GLP-1 ELISA kit and is shown as a graph in Figure 5.

[0196] As a result, as can be seen in Figure 5, the amount of GLP-1 decreased in negative control group 1 (PBS) and negative control group 2 (CPA) 1 hour after glucose administration, but the amount of GLP-1 increased in the experimental group (KCTC 15954BP) 1 hour after glucose administration. In addition, compared to negative control group 1 (PBS) and negative control group 2 (CPA), the amount of GLP-1 increased in the experimental group (KCTC 15954BP).

[0197] Based on the above results, the Lactobacillus fermentum strain of the present invention increases GLP-1 secretion and reduces blood glucose, and this effect suggests that the strain may exhibit therapeutic, improving, or preventive effects against metabolic diseases.

[0198]

[0199] Example 8. Effects of Lactobacillus fermentum strain on body weight, appetite suppression, alleviation of oral glucose tolerance, and reduction of fasting blood glucose in a high-fat diet (HFD) mouse model

[0200] Since obesity is a key factor in the development of metabolic disease, diet-induced obesity rodent models are frequently used in research related to metabolic disease (Silvia Moreno-Fernandez et al. (2018), High fat / high glucose diet induces metabolic syndrome in an experimental rat model, Nutrients, 10(10): 1502). Accordingly, to confirm the effects of the Lactobacillus fermentum strain of the present invention on weight reduction, suppression of food intake, and reduction of blood glucose, a normal diet mouse model and a high-fat diet mouse model were prepared, and comparative experiments were conducted on the above effects.

[0201]

[0202] Example 8-1. Body weight loss and appetite suppression effects of Lactobacillus fermentum strain in a high-fat diet mouse model

[0203] Eight-week-old male C57BL / 6 mice (specific pathogen-free, Coatec Co., Ltd.) were purchased. These mice underwent a one-week acclimatization period and were divided into three groups after body weight measurement. All mice were housed under conditions of a temperature of 22±1℃, humidity of 55±10%, and a 12-hour light / dark cycle, and were provided with free access to water and feed. All animal experiments were conducted in accordance with the ethical standards of the Institutional Animal Care and Use Committee (IACUC) and were carried out after the review and approval of the animal experiment protocol (IACUC Approval No. KLSIACUC20240412-2-01).

[0204] Three isolated groups of mice were treated with a standard diet, a 60% high-fat diet, a cryopreservative, and freeze-dried Lactobacillus fermentum strain according to Example 2 as a sample, according to the control and experimental group conditions below. At this time, the standard diet and the 60% high-fat diet (Rodent diet with 60 kcal% fat, Research diet, D21492) were fed for 6 weeks, and starting from the 4th week of feeding, the cryopreservative and sample were additionally administered orally daily for 2 weeks. The cryopreservative and sample were administered by adding them to PBS, and all administered products were prepared on the day of administration.

[0205] (1) Negative control group 1 (Regular chow + CPA): Regular diet and 7.4 mg / 200 µl of cryopreservative administered

[0206] (2) Negative control group 2 (60% HFD + CPA): 60% high-fat diet and administration of 7.4 mg / 200 µl of cryopreservative

[0207] (3) Experimental group (60% HFD + KCTC 15954BP): 60% high-fat diet and 1 x 10⁶ freeze-dried Lactobacillus fermentum strain as a sample 9 Treats CFU / 200 µL

[0208] The body weight and food intake of the mice were measured once a week for 6 weeks, starting immediately after the above-mentioned diet and during which the cryopreservation agent and sample were administered. The mouse body weight over the 6 weeks is shown in the graph in Fig. 6a, and the percentage change in mouse body weight over the 2 weeks of additional treatment with the cryopreservation agent and sample is shown in the graph in Fig. 6b. In addition, the food intake over the 6 weeks is shown in the graph in Fig. 6c.

[0209] As a result, as can be seen in Figures 6a and 6b, the body weight of mice after treatment with the Lactobacillus fermentum strain of the present invention increased in negative control group 1 (Regular chow + CPA) and negative control group 2 (60% HFD + CPA), while it decreased in the experimental group (60% HFD + KCTC 15954BP).

[0210] In addition, as can be seen in Fig. 6c, the feed intake of mice after treatment with the Lactobacillus fermentum strain of the present invention was significantly reduced in the experimental group (60% HFD + KCTC 15954BP).

[0211] Based on the above results, the Lactobacillus fermentum strain of the present invention reduces body weight by suppressing appetite, and this effect suggests that the strain may exhibit therapeutic, improving, or preventive effects against metabolic diseases.

[0212]

[0213] Example 8-2. Effects of Lactobacillus fermentum strain on normalization of fasting blood glucose and alleviation of oral glucose tolerance in a high-fat diet mouse model

[0214] To diagnose diabetes, impaired glucose tolerance, or impaired fasting glucose, to evaluate glucose metabolism, and to measure insulin resistance or the function of insulin-secreting pancreatic beta cells, an oral glucose tolerance test (oral glucose tolerance test, OGTT) was performed.

[0215] Specifically, after completing Example 8-1, all mice were fasted for 15 hours. Subsequently, blood glucose levels (mg / dL) were measured using a blood glucose meter (Accu-chek active, Roche, Switzerland) at 0, 15, 30, 60, 120, and 150 minutes after oral administration of 2 g / kg of glucose. The blood glucose levels at each time interval are graphed in Fig. 7a, and the area under the curve (AUC) of the time interval graphs is calculated and shown in Fig. 7b. Additionally, fasting blood glucose (at 0 minutes after oral glucose administration) is shown in Fig. 7c.

[0216] As a result, as can be seen in Figure 7a, blood glucose levels decreased in the experimental group (60% HFD + KCTC 15954BP) compared to negative control group 2 (60% HFD + CPA). In particular, blood glucose levels at 15, 30, and 60 minutes after oral administration of glucose were significantly reduced.

[0217] In addition, as can be seen in Figure 7b, compared to negative control group 2 (60% HFD + CPA), the area of ​​the graph for oral glucose tolerance evaluation at different time points (0, 15, 30, 60, 120, and 150 minutes after oral glucose administration) in the experimental group (60% HFD + KCTC 15954BP) was also significantly reduced.

[0218] In addition, as can be seen in Figure 7c, when comparing fasting blood glucose (0 min after oral glucose administration) after 15 hours of fasting in mice fed a high-fat diet, the fasting blood glucose of negative control group 2 (60% HFD + CPA) was significantly increased compared to negative control group 1 (Regular chow + CPA), confirming that insulin resistance was well induced in mice fed a high-fat diet. Furthermore, compared to negative control group 2 (60% HFD + CPA), the fasting blood glucose level in the experimental group (60% HFD + KCTC 15954BP) was significantly reduced.

[0219] Based on the above results, the Lactobacillus fermentum strain of the present invention helps normalize fasting blood glucose by alleviating insulin resistance induced by a high-fat diet and suppresses a rapid increase in blood glucose, and these effects suggest that the strain may exhibit therapeutic, improving, or preventive effects against metabolic diseases.

[0220]

[0221] Example 9. Differences in effects according to strains of Lactobacillus fermentum

[0222] In order to determine whether the Lactobacillus fermentum strain according to the present invention can exhibit superior effects in the treatment, improvement, or prevention of metabolic diseases, the Lactobacillus fermentum strain according to the present invention was compared with the Lactobacillus fermentum GB102 strain (KCTC 14105BP), which is of the same species but a different strain, regarding the GLP-1 secretion-enhancing effect and the 5-HTP (5-hydroxytryptophan) expression-enhancing effect.

[0223] The Lactobacillus fermentum GB102 strain was obtained from the KCTC (Korea Center for Biological Resources, Korea Research Institute of Biotechnology and Bioengineering) on ​​March 18, 2025.

[0224]

[0225] Example 9-1. GLP-1 secretion-enhancing effect

[0226] The GLP-1 secretion-enhancing effects of the strain of the present invention and the GB102 strain were compared in STC-1 cells and NCI-H716 cells. STC-1 cells are a murine intestinal secretin tumor cell line that expresses and secretes intestinal hormones related to hunger suppression, food intake regulation, and satiety, such as GLP-1, and are known to be used as a screening platform to identify foods or compounds that regulate gastrointestinal hormone secretion in vitro. NCI-H716 cells are a cell line derived from human colorectal adenocarcinoma ascites fluid and are known to be used in in vitro studies on GLP-1 regulation.

[0227] Specifically, 5 x 10 STC-1 cells or NCI-H716 cells in a 96-well cell culture plate 4 The cells were seeded at a cell / well concentration. STC-1 cells were confirmed to have a confluency of 80% or higher and an even layer formation by microscopy the following day, and then washed with PBS. Subsequently, the medium was replaced with 0.2 mL of serum-free media (without penicillin and streptomycin). NCI-H716 cells were confirmed to have a confluency of 80% or higher and an even layer formation by microscopy, then washed with PBS and replaced with 0.2 mL of Krebs buffer solution. Subsequently, PMA (phorbol 12-myristate 13-acetate), MRS medium, and the culture supernatant of the strain according to the present invention and the culture supernatant of the GB102 strain were treated according to the control and experimental group conditions below. PMA is a substance known as a GLP-1 secretagogue and was used as a positive control in this example, while MRS medium is the culture medium for the strain according to the present invention and the GB102 strain. The culture supernatant of the strain according to the present invention and the culture supernatant of the GB102 strain were prepared according to Example 2.

[0228] (1) Negative control group 1 (NONE): PMA, MRS medium, and culture supernatant of the novel strain according to the present invention and culture supernatant of the GB102 strain were not treated as samples.

[0229] (2) Positive control (PC): Treated with 1 μM PMA

[0230] (3) Negative control group 2 (MEDIA): Treated with MRS medium

[0231] (4) Experimental group 1 (KCTC 15954BP): Treated with 5% (v / v) of the culture supernatant of the strain according to the present invention as a sample.

[0232] (5) Experimental group 2 (GB102): 5% (v / v) of the culture supernatant of the GB102 strain was treated as a sample.

[0233] Cell supernatant was obtained from STC-1 cells after 24 hours of reaction, and cell supernatant was obtained from NCI-H716 cells after 4 hours of reaction. Subsequently, GLP-1 expression levels were measured via ELISA and are shown in Figures 8a and 8b.

[0234] As a result, as can be seen in Figure 8a, compared to negative control group 1 (NONE), the GLP-1 secretion of experimental group 1 (KCTC 15954BP) treated with the strain according to the present invention increased statistically significantly at the P < 0.001 level. In particular, the GLP-1 secretion of experimental group 1 (KCTC 15954BP) treated with the novel strain according to the present invention increased compared to the positive control group (PC) treated with PMA, and increased statistically significantly compared to experimental group 2 (GB102) treated with the GB102 strain at the P=0.01 level.

[0235] In addition, as can be seen in Fig. 8b, compared to negative control group 1 (NONE), the GLP-1 secretion of experimental group 1 (KCTC 15954BP) treated with the strain according to the present invention increased statistically significantly at the P < 0.001 level. In particular, the GLP-1 secretion of experimental group 1 (KCTC 15954BP) treated with the novel strain according to the present invention increased to a level similar to that of the positive control group (PC) treated with PMA, and increased statistically significantly at the P=0.003 level compared to experimental group 2 (GB102) treated with the GB102 strain.

[0236] The above results suggest that compared to other Lactobacillus fermentum strains of the same species but different strains, the Lactobacillus fermentum strain of the present invention has an excellent effect of lowering blood sugar by increasing GLP-1 secretion to promote insulin secretion and suppressing glucagon secretion, and this effect suggests that the strain according to the present invention has an excellent effect in treating, improving, or preventing metabolic diseases.

[0237]

[0238] Example 9-2. Effect of promoting 5-HTP expression

[0239] The effect of enhancing 5-HTP expression in RIN-14B cells was compared for the strain of the present invention and the GB102 strain.

[0240] 5-HTP is produced from tryptophan by tryptophan hydroxylases (TPH) and generates serotonin (5-HT, 5-hydroxytryptamine) through decarboxylation. 5-HTP leads to reduced food intake and promotion of weight loss, as well as a decrease in BMI due to symptoms of anorexia and increased satiety (Massimo E Maffei (2020), 5-Hydroxytryptophan (5-HTP): Natural Occurrence, Analysis, Biosynthesis, Biotechnology, Physiology and Toxicology, International Journal of Molecular Sciences, 22(1):181).

[0241] Serotonin is a neurotransmitter produced by neurons in the central nucleus of the hindbrain, which dominates most brain regions, and plays a role in reducing food intake and increasing satiety. Serotonin is synthesized from dietary L-tryptophan through a two-step enzymatic reaction involving TPH and AADC (L-amino acid decarboxylase). Circulating serotonin synthesized in enterochromaffin cells signals serotonin receptors on hepatocytes to stimulate hepatic gluconeogenesis and can inhibit glucose absorption by the liver during fasting (Katsura Nozawa et al. (2009), Steven C. Wyler et al. (2017), Serotonergic Control of Metabolic Homeostasis, frontiers in cellular neuroscience, volume 11).

[0242] RIN-14B cells are rat-derived EC cells known to be used in experiments related to EC cell functions, such as serotonin release.

[0243] Specifically, 5 x 10 RIN-14B cells in a 96-well cell culture plate 4Inoculated at a cells / well concentration. After 72 hours, microscopic confirmation was made that a layer of more than 90% confluence had formed evenly, and the samples were washed with PBS. Subsequently, ionomycin, MRS medium, and the culture supernatant of the strain according to the present invention and the culture supernatant of the GB102 strain were treated according to the control and experimental group conditions below. Ionomycin is a substance that induces the release of serotonin through Ca2+-dependent extracellular secretion and causes NK cells to lose their ability to secrete IFN-γ; in this example, it was used as a positive control. MRS medium is the culture medium for the strain according to the present invention and the GB102 strain. The culture supernatant of the strain according to the present invention and the culture supernatant of the GB102 strain were prepared according to Example 2, and when used as samples under the control and experimental group conditions below, they were added to HBSS (Hanks Balanced Salt Solution) for treatment.

[0244] (1) Negative control 1 (NONE): Ionomycin, MRS medium, and culture supernatant of the strain according to the present invention and culture supernatant of the GB102 strain were not treated as samples.

[0245] (2) Positive control (PC): Treated with 10 μM ionomycin

[0246] (3) Negative control group 2 (MEDIA): Treated with MRS medium

[0247] (4) Experimental group 1 (KCTC 15954BP): Treated with 2% (v / v) of the culture supernatant of the strain according to the present invention as a sample

[0248] (5) Experimental group 2 (GB102): 2% (v / v) of the culture supernatant of the GB102 strain was treated as a sample.

[0249] After 1 hour of reaction, the cell supernatant was obtained and the 5-HTP expression level was measured via ELISA, and the results are shown in Figure 9.

[0250] As a result, as can be seen in Figure 9, compared to negative control group 1 (NONE), the 5-HTP expression level of experimental group 1 (KCTC 15954BP) treated with the strain according to the present invention increased statistically significantly at the P < 0.01 level. In particular, the 5-HTP expression level of experimental group 1 (KCTC 15954BP) treated with the novel strain according to the present invention increased compared to the positive control group (PC) treated with ionomycin, and increased statistically significantly compared to experimental group 2 (GB102) treated with the GB102 strain at the P=0.03 level.

[0251] The above results suggest that compared to other Lactobacillus fermentum strains of the same species but different strains, the Lactobacillus fermentum strain of the present invention has an excellent effect of promoting reduced food intake, weight loss, symptoms of loss of appetite, and a reduction in BMI due to increased satiety by increasing 5-HTP expression and promoting serotonin production, and these effects suggest that the strain according to the present invention has an excellent effect in treating, improving, or preventing metabolic diseases.

[0252] [Consignment Number]

[0253]

Claims

1. Lactobacillus fermentum strain deposited under accession number KCTC 15954BP.

2. In Paragraph 1, A strain in which the 16S rRNA gene of the above strain comprises the nucleotide sequence represented by SEQ ID NO.

1.

3. In Paragraph 1, The strain is one or more selected from the group consisting of live cells, dead cells and cultures thereof, fermented products thereof, lysed products thereof, extracts thereof and cytoplasmic fractions obtained by lysed products.

4. In Paragraph 1, The strain above is a strain having one or more of the following characteristics within a subject: (a) Inhibition of nitric oxide (NO) production, (b) Enhancement of GLP-1 (glucagon-like peptide-1) secretion, (c) reduction in blood sugar, and (d) Suppression of appetite or weight loss.

5. A composition for anti-inflammatory, blood glucose reduction, appetite suppression, or weight loss comprising a strain of Lactobacillus fermentum deposited under accession number KCTC 15954BP.

6. In Paragraph 5, A composition in which the strain is one or more selected from the group consisting of live cells, dead cells and cultures thereof, fermented products thereof, pulverized products thereof, extracts thereof, and cytoplasmic fractions obtained by pulverizing the above.

7. In Paragraph 5, The above composition is a composition used as a pharmaceutical composition, a quasi-drug composition, a food composition, a cosmetic composition, or a feed composition.

8. In Paragraph 7, The above pharmaceutical composition is a composition used for the treatment or prevention of metabolic diseases.

9. In Paragraph 8, A composition in which the above metabolic disease is selected from the group consisting of diabetes mellitus, impaired fasting glucose, impaired glucose tolerance, obesity, hypertension, metabolic dysfunction-associated steatotic liver disease, hyperinsulinemia, and dyslipidemia.

10. In Paragraph 7, The above-mentioned quasi-drug composition, food composition, cosmetic composition, or feed composition is a composition used for the improvement or prevention of metabolic diseases.

11. A method for treating, improving, or preventing a metabolic disease of a subject, comprising the step of administering a composition according to paragraph 5 to a subject.

12. Use of the Lactobacillus fermentum strain deposited under accession number KCTC 15954BP for treating, improving, or preventing metabolic diseases in subjects.

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