Bifidobacterium longum NBM 7-1 strain exhibiting activity of preventing or treating metabolic diseases

Abstract

The present invention relates to a use of Bifidobacterium longum NBM 7-1 strain, having accession number KCTC 14325BP, for preventing and treating liver diseases. By using the composition comprising the Bifidobacterium longum NBM 7-1 strain of the present invention, liver diseases can be effectively prevented, alleviated and treated.

Description

Bifidobacterium longum NBM 7-1 strain having metabolic disease prevention or treatment activity

[0001] The present invention was made under the support of the Ministry of Trade, Industry and Energy of the Republic of Korea under the project number 1415181659, and the research management specialized institution of the project is the Korea Institute of Industrial Technology Planning and Evaluation, the research project name is "Material and Component Technology Development Project", the research project name is "Establishment of an Industrialization Base through Development of High Value-Added Health Functional Food Materials and Compound Formulations Using Tisochrysis lutea and Phaeodactylum tricornutum", the main institution is Micro RGS Co., Ltd., and the research period is from August 1, 2022 to December 31, 2024.

[0002] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0167334, filed with the Korean Intellectual Property Office on November 27, 2023, the disclosure of which is incorporated herein by reference.

[0003] The present invention relates to the use of Bifidobacterium longum NBM 7-1 strain, accession number KCTC 14325BP, for preventing and treating liver diseases.

[0004]

[0005] The domestic liver disease mortality rate is very high at 23.5 per 100,000 people, and it is the leading cause of death for people in their 40s (41.1 per 100,000 people), the second leading cause of death for people in their 50s (72.4 per 100,000 people), and the third leading cause of death for people in their 30s (10 per 100,000 people), making it a major cause of death for middle-aged people in Korea.

[0006] Among the above liver diseases, fatty liver refers to a phenomenon in which neutral fat, which does not exist in normal cells, is abnormally excessively deposited within liver cells. A normal liver is composed of approximately 5% adipose tissue, and the main components of fat are neutral fat, fatty acids, phospholipids, cholesterol, and cholesterol esters. However, once fatty liver occurs, most of the components are replaced by neutral fat, and if the amount of neutral fat exceeds 5% of the liver weight, it is diagnosed as fatty liver. If fatty liver worsens and the fat lumps within the liver cells increase, important components of the cell, including the nucleus, are pushed to one side, and the function of the liver cells is impaired. In addition, the swollen liver cells caused by the accumulated fat compress the microvessels and lymph nodes between the liver cells, disrupting the circulation of blood and lymph within the liver. This prevents the liver cells from receiving adequate oxygen and nutrients, resulting in a decline in liver function.

[0007] Nonalcoholic fatty liver disease (NAFLD), in particular, has increased rapidly worldwide across all age groups due to the impact of metabolic syndrome on liver metabolism. NAFLD includes nonalcoholic steatohepatitis (NASH), which can progress from pure fatty steatosis (fatty infiltration of more than 5% of hepatocytes) to liver cirrhosis, liver failure, and hepatocellular carcinoma (HCC).

[0008] Alcoholic fatty liver disease is also caused by excessive drinking. While individual genetics and gender differences may exist, those who consume more than 80 grams of alcohol per day are more likely to develop liver diseases such as alcoholic fatty liver disease. Women are more likely to develop alcoholic liver disease even with lower amounts. Most patients with the mildest form of alcoholic fatty liver disease are asymptomatic, but those with mild hepatomegaly (a condition in which the liver is larger than normal) may complain of mild tenderness in the upper right abdomen.

[0009] Meanwhile, the human gut microbiota is a complex ecosystem comprised of diverse bacteria with a total mass of approximately 1 to 2 kg. Recent advances in knowledge and technology regarding the human gut microbiota have transformed our perspectives on human disease and its treatment. Gut microbes maintain a close relationship with the host and play a crucial role in vitamin production, the intestinal mucosal immune system, and bacterial translocation. The gut and liver are directly connected via the hepatic portal vein, and the liver is the primary metabolic organ for gut products, which comprise important dietary nutrients and microbial-related components. These two organs are collectively referred to as the gut-liver axis. Therefore, disruption of the gut microbiota can lead to liver diseases, including NAFLD. However, existing treatments for liver diseases are often ineffective due to poor compliance, lack of efficacy, or changes in diet and lifestyle. Therefore, there is an urgent need to research strains that can effectively treat liver diseases.

[0010]

[0011] The present inventors have diligently sought to discover a strain that can effectively treat liver disease. As a result, they discovered that the Bifidobacterium longum NBM 7-1 strain (accession number KCTC 14325BP) is effective in preventing, improving, and treating liver disease, thereby completing the present invention.

[0012] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating liver disease comprising Bifidobacterium longum NBM 7-1 strain having the accession number KCTC 14325BP.

[0013] Another object of the present invention is to provide a food composition for preventing or improving liver disease, comprising Bifidobacterium longum NBM 7-1 strain having the accession number KCTC 14325BP.

[0014] Another object of the present invention is to provide a feed composition for preventing or improving liver disease, comprising Bifidobacterium longum NBM 7-1 strain having the accession number KCTC 14325BP.

[0015] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0016]

[0017] In one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating liver disease, comprising Bifidobacterium longum NBM 7-1 strain having accession number KCTC 14325BP.

[0018] The present inventors have diligently sought to discover a strain that can effectively treat liver disease. As a result, we have discovered that Bifidobacterium longum NBM 7-1, accession number KCTC 14325BP, is effective in preventing, improving, and treating liver disease.

[0019] The Bifidobacterium longum NBM 7-1 strain of the present invention was deposited at the Korea Center for Bioscience and Biotechnology (KCTC) on October 6, 2020 and was assigned the accession number KCTC 14325BP.

[0020] The above Bifidobacterium longum NBM 7-1 strain can be obtained by isolation and identification from the feces of a healthy adult, and the cell characteristics of the strain are as follows:

[0021] It is a Gram-positive, branched, rod-shaped bacterium that forms milky white colonies and is catalase-negative. It is a facultative heterofermentative species and uses carbon sources such as D-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, D-sorbitol, arbutin, esculin, salicin, D-maltose, D-lactose, D-saccharohalose, D-trehalose, inulin, D-raffinose, and amidone as energy sources under anaerobic conditions.

[0022] In the present invention, the culture method, extraction method, separation method, concentration method, drying method, dilution method, etc. for the cells of the strain are not particularly limited. The medium for culturing the cells may be an EG medium; an LB medium; an RCM medium; and / or a medium containing one or more components selected from blood plate, maltose, sucrose, glucose, starch, mannitol, gelatin, casein, skim milk, other sugars, and yeast extract. As the culture method, various general aerobic or anaerobic methods may be appropriately used. The culture temperature of the strain may be set to 30°C to 40°C, and a neutralization culture method may be used in which an alkali such as sodium hydroxide is used during the culture to maintain the pH of the medium from neutral to acidic, for example, to about pH 5 to 6. In addition to the neutralization culture method, any culture method such as batch culture can be used, and after culture, the culture or its supernatant can be concentrated, dried, diluted, etc. as needed. In addition, the supernatant of the culture and the cells can be separated using centrifugation or membrane separation, and the cells can be recovered in a concentrated state. In addition, the cells can be subjected to ultrasonic treatment or enzyme treatment to extract components within the cells, or the culture or its supernatant, cells or their extracts, etc. can be dried. These can be used as the effective ingredients of the composition of the present invention.

[0023] As used herein, the term "prevention" refers to the prevention or protective treatment of a disease or disease state. As used herein, the term "treatment" refers to the reduction, suppression, alleviation, or eradication of a disease state.

[0024] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the like. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0025] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0026] The pharmaceutical composition of the present invention can be administered orally or parenterally, and can be administered by, for example, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, intratumoral injection, local administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0027] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. A skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the anticancer composition of the present invention is 0.0001-100 mg / kg.

[0028] In one embodiment of the present invention, the Bifidobacterium longum NBM 7-1 strain having the accession number KCTC 14325BP is selected from the group consisting of a live cell of the strain, a dead cell of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, a lysate of the strain, an extract of the strain, and a combination thereof.

[0029] In one embodiment of the present invention, the liver disease is viral liver disease, alcoholic liver disease, nonalcoholic liver disease, autoimmune liver disease, or metabolic liver disease. Viral liver disease refers to liver disease caused by viruses such as Hepatis A, B, C, D, and E. Alcoholic liver disease is a liver disease caused by excessive alcohol consumption, and refers to alcoholic fatty liver disease, alcoholic Hepatis, and alcoholic cirrhosis. Nonalcoholic liver disease is a liver disease that occurs in a condition other than alcohol, and refers to nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatis (NASH), etc. Autoimmune liver disease is a liver disease caused by an autoimmune reaction, and includes autoimmune hepatitis, primary biliary cirrhosis (PSC), primary cholecystic cirrhosis (PBC), etc. Metabolic liver disease is a liver disease caused by a metabolic disorder, and includes Gilson syndrome, hemochromatosis, etc.

[0030] In one embodiment of the present invention, the liver disease is selected from the group consisting of nonalcoholic liver disease, alcoholic liver disease, liver fibrosis, cirrhosis, and liver cancer. However, the present invention is not limited thereto, and includes any liver disease that can be treated by weight loss, liver weight loss, reduction in nonalcoholic fatty liver disease activity, reduction in liver fibrosis, reduction in blood ALT (Alanine Aminotransferase) level, reduction in blood triglyceride level, reduction in blood total cholesterol level, reduction in fasting blood glucose level, reduction in fecal endotoxin level, reduction in intestinal inflammatory cytokine expression level, or a combination thereof.

[0031] In this specification, non-alcoholic liver disease refers to liver diseases that occur in conditions that are not caused by alcohol, such as non-alcoholic fatty liver disease, hepatitis, liver fibrosis, cirrhosis, cirrhosis, and liver cancer. Non-alcoholic fatty liver disease (NAFLD) is the early stage of fat accumulation in the liver, and is usually asymptomatic, either simple steatosis, or a condition in which inflammation and damaged (swollen) hepatocytes are present in addition to fat deposits. NASH refers to non-alcoholic steatohepatitis (NASH), which can progress to fibrosis, cirrhosis, and cancer.

[0032] Alcoholic Liver Disease (ALD) in this specification is an early stage disease caused by long-term excessive drinking, usually asymptomatic but can progress to more serious diseases such as alcoholic fatty liver disease, alcoholic steatohepatitis with moderate to severe inflammation and damage, which can progress rapidly and become life-threatening, or alcoholic cirrhosis, the most advanced stage of chronic ALD, in which normal tissue is replaced by extracellular matrix, impairing both structure and function.

[0033] The composition of the present invention may be particularly effective in cases where existing drugs for controlling elevated lipid levels and / or liver enzyme levels in the liver disease are resistant, have side effects, or are insufficiently effective. For example, it may be useful in cases of liver disease that has developed drug resistance; in cases of liver disease that has caused adverse effects such as muscle problems, cognitive loss, neuropathy, pancreatic dysfunction, liver dysfunction, and sexual dysfunction; and in cases of liver disease where a single lipid-lowering agent alone is insufficient to control severe fatty liver disease and combination therapy is required. In such cases, the strain of the present invention may be administered or ingested alone or in combination to solve the above-mentioned problems, and may have one or more of the following advantages: a synergistic effect, a reduction in the number of daily doses without loss of drug efficacy, improved cholesterol or triglyceride levels, and improved drug selectivity, tissue penetration, half-life, and / or metabolic stability.

[0034] In one embodiment of the present invention, the Bifidobacterium longum NBM 7-1 strain has at least one characteristic selected from the group consisting of weight loss, liver weight loss, nonalcoholic fatty liver disease activity reduction, liver fibrosis reduction, blood ALT (Alanine Aminotransferase) level reduction, blood triglyceride level reduction, blood total cholesterol level reduction, fasting blood sugar level reduction, fecal endotoxin level reduction, and intestinal inflammatory cytokine expression level reduction.

[0035] In one embodiment of the present invention, the composition comprises a pharmaceutically acceptable carrier, excipient or diluent.

[0036] In one aspect of the present invention, the present invention provides a food composition for preventing or improving liver disease, comprising Bifidobacterium longum NBM 7-1 strain having accession number KCTC 14325BP.

[0037] The above food may be a health functional food, a health food, or a food additive, and in this case, there is no particular limitation on the type of food. Examples of the above food include dairy products including drinks, meat, sausages, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, alcoholic beverages, and vitamin complexes, dairy products, and dairy products, and includes all health functional foods and health food compositions in the conventional sense, such as tablets, capsules, pills, or liquids.

[0038] The health functional food, health food and food additive composition containing the Bifidobacterium longum NBM 7-1 strain according to the present invention can be added to food as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method.

[0039] In the present invention, the amount of the composition in the health functional food, health food, or food additive may be 0.1 to 90 parts by weight based on the total food weight. However, in the case of long-term intake for the purpose of maintaining or regulating health, the amount may be below the above range.

[0040] In the present invention, there is no particular limitation on other ingredients added in addition to containing the Bifidobacterium longum NBM 7-1 strain, and various flavoring agents or natural carbohydrates, etc., may be contained as additional ingredients, as in conventional beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and conventional sugars and sugar alcohols such as xylitol, sorbitol, erythritol, etc. In addition to the above-mentioned flavoring agents, natural flavoring agents such as thaumatin and stevia extracts (such as rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the above natural carbohydrates may generally be about 1 g to 20 g, preferably about 5 g to 12 g, per 100 g of the health functional food, health food or food additive composition of the present invention.

[0041] In the present invention, the food composition containing the Bifidobacterium longum NBM 7-1 strain may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the health functional food and health food composition of the present invention may contain fruit pulp for the production of natural fruit juice and fruit juice drinks and vegetable drinks.

[0042] In one embodiment of the present invention, the Bifidobacterium longum NBM 7-1 strain having the accession number KCTC 14325BP is selected from the group consisting of a live cell of the strain, a dead cell of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, a lysate of the strain, an extract of the strain, and a combination thereof.

[0043] In one embodiment of the present invention, the liver disease is selected from the group consisting of nonalcoholic liver disease, alcoholic liver disease, liver fibrosis, liver cirrhosis, and liver cancer.

[0044] In one embodiment of the present invention, the Bifidobacterium longum NBM 7-1 strain has at least one characteristic selected from the group consisting of weight loss, liver weight loss, nonalcoholic fatty liver disease activity reduction, liver fibrosis reduction, blood ALT (Alanine Aminotransferase) level reduction, blood triglyceride level reduction, blood total cholesterol level reduction, fasting blood sugar level reduction, fecal endotoxin level reduction, and intestinal inflammatory cytokine expression level reduction.

[0045] In one embodiment of the present invention, the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages.

[0046]

[0047] In one aspect of the present invention, the present invention provides a feed composition comprising Bifidobacterium longum NBM 7-1 strain having accession number KCTC 14325BP.

[0048] The above feed composition may include feed or feed additives for feeding a diet to an animal.

[0049] When the Bifidobacterium longum NBM 7-1 strain of the present invention is contained and used in a feed composition or feed additive composition, the composition may be a 20 to 90% concentrated solution or may be manufactured in the form of a powder or granules. The feed additive may additionally include one or more of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, and acid pyrophosphate; and natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid.

[0050] In the present invention, when the Bifidobacterium longum NBM 7-1 strain is used as feed, the composition can be formulated in the form of a conventional feed and can contain conventional feed ingredients together. The feed additives and feed can further include grains such as ground or crushed wheat, oats, barley, corn, and rice; plant-based protein feeds such as feeds mainly composed of rapeseed, soybeans, and sunflower; animal-based protein feeds such as blood meal, meat meal, bone meal, and fish meal; dry ingredients composed of sugars and dairy products such as various types of milk powder and whey powder, and in addition, nutritional supplements, digestion and absorption enhancers, growth promoters, etc.

[0051] In the present invention, when the Bifidobacterium longum NBM 7-1 strain is used as the feed additive, it may be administered to animals alone or in combination with other feed additives in an edible carrier. In addition, the feed additive can be easily administered to animals as a top dressing, by mixing it directly into animal feed, or in an oral formulation separate from the feed. When the feed additive is administered separately from animal feed, it can be prepared as an immediate-release or sustained-release formulation by combining it with a pharmaceutically acceptable edible carrier, as is well known in the art. Such edible carriers can be solid or liquid, for example, corn starch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the feed additive can be a top dressing in the form of a tablet, capsule, powder, troche, sugar tablet, or microdispersible form. When a liquid carrier is used, the feed additive may be in the form of a gelatin soft capsule, or a syrup, suspension, emulsion, or solution. The feed may include any protein-containing organic grain meal commonly used to satisfy the dietary needs of animals. Such protein-containing grain meal typically consists of corn, soybean meal, or a corn / soybean meal mix. The feed composition may also contain, for example, preservatives, stabilizers, wetting or emulsifying agents, and solution promoters. The feed additive composition may also be used by adding it to animal feed by immersion, spraying, or mixing.

[0052] The subject of the feed or feed additive administration of the present invention is an individual in need of prevention or improvement of liver disease, and the individual may include all animals excluding humans who have developed or may develop the liver disease, and the animal may include, but is not limited to, non-human primates such as chimpanzees, other apes, or monkey species; livestock animals such as cows, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, or cats; laboratory animals such as rodents such as rats, mice, or guinea pigs; and non-mammals such as birds or fish.

[0053] In one aspect of the present invention, the present invention provides a method for preventing or treating liver disease, comprising administering to a subject a Bifidobacterium longum NBM 7-1 strain having the accession number KCTC 14325BP.

[0054] Since the method for preventing or treating liver disease of the present invention is performed using the Bifidobacterium longum NBM 7-1 strain with the accession number KCTC 14325BP or a composition containing the same, the overlapping contents between the two inventions are commonly applied, and the description thereof is omitted to avoid excessive complexity of the present specification.

[0055] In one embodiment of the present invention, the Bifidobacterium longum NBM 7-1 strain having the accession number KCTC 14325BP is selected from the group consisting of a live cell of the strain, a dead cell of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, a lysate of the strain, an extract of the strain, and a combination thereof.

[0056] In one embodiment of the present invention, the liver disease is selected from the group consisting of nonalcoholic liver disease, alcoholic liver disease, liver fibrosis, cirrhosis, and liver cancer. However, the present invention is not limited thereto, and includes any liver disease that can be treated by weight loss, liver weight loss, reduction in nonalcoholic fatty liver disease activity, reduction in liver fibrosis, reduction in blood ALT (Alanine Aminotransferase) level, reduction in blood triglyceride level, reduction in blood total cholesterol level, reduction in fasting blood glucose level, reduction in fecal endotoxin level, reduction in intestinal inflammatory cytokine expression level, or a combination thereof.

[0057]

[0058] The features and advantages of the present invention are summarized as follows:

[0059] (a) The present invention provides a pharmaceutical composition for preventing or treating liver disease, comprising Bifidobacterium longum NBM 7-1 strain having accession number KCTC 14325BP.

[0060] (b) The present invention provides a food composition for preventing or improving liver disease, comprising Bifidobacterium longum NBM 7-1 strain having accession number KCTC 14325BP.

[0061] (c) The present invention provides a feed composition for preventing or improving liver disease, comprising Bifidobacterium longum NBM 7-1 strain having accession number KCTC 14325BP.

[0062] (d) When a composition containing the Bifidobacterium longum NBM 7-1 strain of the present invention is used, liver disease can be effectively prevented, improved, and treated.

[0063]

[0064] Figure 1 shows the results of mycological characterization of Bifidobacterium longum NBM 7-1 strain.

[0065] Figure 2 shows the change in body weight according to administration of Bifidobacterium longum NBM 7-1 strain.

[0066] Figure 3 shows the change in liver weight according to administration of Bifidobacterium longum NBM 7-1 strain.

[0067] Figure 4 shows the change in liver / body weight ratio (L / B ratio) according to administration of Bifidobacterium longum NBM 7-1 strain.

[0068] Figure 5 shows the change in NAS score according to administration of Bifidobacterium longum NBM 7-1 strain.

[0069] Figure 6 shows the change in fibrosis score according to administration of Bifidobacterium longum NBM 7-1 strain.

[0070] Figure 7 shows changes in hepatic neutral fat levels following administration of Bifidobacterium longum NBM 7-1 strain.

[0071] Figure 8 shows changes in ALT levels following administration of Bifidobacterium longum NBM 7-1 strain.

[0072] Figure 9 shows changes in blood triglyceride levels following administration of Bifidobacterium longum NBM 7-1 strain.

[0073] Figure 10 shows changes in total blood cholesterol levels following administration of Bifidobacterium longum NBM 7-1 strain.

[0074] Figure 11 shows the change in fasting blood sugar level according to administration of Bifidobacterium longum NBM 7-1 strain.

[0075] Figure 12 shows changes in fecal endotoxin following administration of Bifidobacterium longum NBM 7-1 strain.

[0076] Figure 13 shows changes in the level of expression of intestinal inflammatory cytokines following administration of Bifidobacterium longum NBM 7-1 strain.

[0077]

[0078] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0079]

[0080] Example

[0081] Example 1: Obtaining Bifidobacterium longum NBM 7-1 strain

[0082] 1-1. Isolation of Bifidobacterium longum NBM 7-1

[0083]

[0084] Bifidobacterium longum NBM 7-1 was isolated from the feces of a healthy adult (No. 7, Male, Seoul National University Hospital) using a selective medium. The collected fecal sample was serially diluted 10-fold in 0.85% NaCl, plated on Bifidobacterium selective medium (BSM) agar, and anaerobically cultured at 37°C for 24–48 h. Bifidobacterium longum NBM 7-1 was isolated from strains with different colony morphologies.

[0085] The finally isolated Bifidobacterium longum NBM 7-1 strain was identified by amplifying the 16S rRNA gene region and analyzing the base sequence (SEQ ID NO: 1).

[0086] The results of mycological characterization of Bifidobacterium longum NBM 7-1 strain are shown in Figure 1. The accession number of Bifidobacterium longum NBM 7-1 strain is KCTC 14325BP.

[0087]

[0088] 1-2. Cultivation and powder production of Bifidobacterium longum NBM 7-1

[0089] The seed culture was performed in flasks containing MRS or BL broth at 37°C under anaerobic conditions for 24 h. The seed culture was inoculated into the optimized medium in a fermenter and cultured in a fermenter (Bio Control & Science, MARADO-05D-PS). During fermentation, the pH was maintained at 5.5–6.0 by automatic addition of NaOH solution (25% w / v), and the fermentation was performed at 37°C for 18–20 h with stirring at 120 rpm. After completion of the culture, lyophilization for the preparation of Bifidobacterium longum NBM 7-1 powder was performed according to the manual (Cooling & Heating System, Lab-Mast 10). After lyophilization, the colony-forming unit (CFU) per 1 g of each Bifidobacterium longum NBM 7-1 powder was measured by serial dilution. The manufactured powder was suspended in sterilized phosphate buffered saline (PBS) before use and 1X10 9 The density was adjusted to CFU / mL.

[0090]

[0091] Example 2: Verification of the liver disease treatment effect of Bifidobacterium longum NBM 7-1

[0092] Four-week-old male C57BL / 6J mice (Dooyeol Biotech, Seoul, Korea) were used as experimental animals. Liver disease was induced in the mice by feeding them a high-fat (60% kcal of fat) and high-fructose (10% in drinking water) diet for 12 weeks.

[0093] The treatment effect of Bifidobacterium longum NBM 7-1 on liver disease-induced animals was verified by administering it for 12 weeks. Bifidobacterium longum NBM 7-1 was administered at a concentration of 1X10 9CFU / day was administered, and as a positive control, Resmetirom was administered at 1.0 mg / kg BW / day.

[0094]

[0095] 2-1. Check changes in body weight and liver weight

[0096] Overweight or obesity is a major risk factor for various liver diseases, including non-alcoholic fatty liver disease (NAFLD). Furthermore, the liver weight to total body weight (L / B ratio) is generally constant in healthy individuals. However, this ratio can increase or decrease in certain disease states. Therefore, to verify the therapeutic effect of Bifidobacterium longum NBM 7-1 on liver disease, the above indicators were examined. Body weight was measured once a week. After the experiment, the body weight and liver tissue weight of each experimental animal were measured for each group to determine the liver / body weight ratio.

[0097] The results are shown in Figures 2, 3, 4, Tables 1, 2, and 3.

[0098] As shown in Figure 2 and Table 1, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), body weight was confirmed to have decreased by approximately 10% compared to the negative control group (NC), confirming a significant weight loss. In addition, a weight loss effect similar to that of the positive control group (PC) was confirmed.

[0099] Body weight changes following administration of Bifidobacterium longum NBM 7-1 strain. GroupAverageSTDEVND29.892.12NC49.531.02PC43.585.31BLO44.723.63

[0100] As shown in Fig. 3 and Table 2, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), the liver weight was confirmed to have decreased by approximately 30% compared to the negative control group (NC), confirming a significant decrease in liver weight.

[0101] Changes in liver weight following administration of Bifidobacterium longum NBM 7-1 strain. GroupAverageSTDEVND1.100.17NC2.150.27PC1.350.22BLO1.550.27

[0102]

[0103] As shown in Fig. 4 and Table 3, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), the liver / body weight ratio (L / B ratio) was significantly reduced compared to the negative control group (NC). In particular, compared to the positive control group (PC), it was confirmed that the liver / body weight ratio was restored most similarly to the untreated group.

[0104] Changes in the liver / body weight ratio (L / B ratio) according to administration of Bifidobacterium longum NBM 7-1 strain. GroupAverageSTDEVND3.660.44NC4.340.49PC3.090.30BLO3.460.39

[0105] The above results indicate that administration of Bifidobacterium longum NBM 7-1 can suppress excess weight due to fat accumulation, especially abnormal fat accumulation in the liver.

[0106]

[0107] 2-2. Check the Nonalcoholic Fatty Liver Disease Activity Score (NAS), fibrosis score, and triglyceride levels in the liver.

[0108] In some liver diseases, such as nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NAFLD), the NAFLD activity score, the degree of fibrosis, and intrahepatic triglyceride levels are important disease indicators. Therefore, to verify the therapeutic efficacy of Bifidobacterium longum NBM 7-1 in liver disease, these indicators were examined.

[0109] After administration, the liver tissues of the animals were extracted, fixed in 10% neutral formalin, embedded in paraffin, and then tissue sections were prepared. The prepared sections were stained with hematoxylin-eosin (H&E) and Sirius red to evaluate the pathological characteristics of nonalcoholic fatty liver disease, such as steatosis, inflammation, ballooning of hepatocytes, and the degree of fibrosis. The degree of disease was evaluated using the NAS (NAFLD activity score) definition, and steatosis, inflammation, and ballooning of hepatocytes were evaluated in H&E staining to create an NAS score. In addition, Sirius red staining was graded from 0 to 3 to evaluate periarterial fibrosis. All specimens were analyzed by a pathologist.

[0110] To measure triglyceride levels in the liver, the Serum Triglyceride Quantification Kit product (Cell Biolabs, Inc., San Diego, USA) was used to quantify triglyceride levels in liver tissue.

[0111] The results are shown in Fig. 5, Fig. 6, Fig. 7, Table 4, Table 5, and Table 6.

[0112] As shown in Fig. 5 and Table 4, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), the NAS score was confirmed to be approximately 40% lower than that of the negative control group (NC), confirming a significant decrease in the NAS score.

[0113]

[0114] Changes in NAS scores according to administration of Bifidobacterium longum NBM 7-1 strain GroupAverageSTDEVND0.000.00NC5.300.82PC1.251.42BLO3.271.74

[0115] As shown in Fig. 6 and Table 5, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), the fibrosis score was significantly reduced by approximately 30% compared to the negative control group (NC), confirming a significant reduction in the fibrosis score.

[0116]

[0117] Changes in fibrosis scores according to administration of Bifidobacterium longum NBM 7-1 strain GroupAverageSTDEVND0.000.00NC1.000.00PC0.330.49BLO0.730.47

[0118] As shown in Fig. 7 and Table 6, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), the liver neutral fat level was significantly reduced by approximately 15% compared to the negative control group (NC).

[0119]

[0120] Changes in liver triglyceride levels following administration of Bifidobacterium longum NBM 7-1 strain. GroupAverageSTDEVND13.745.94NC33.827.29PC16.245.25BLO29.187.99

[0121] The above results indicate that Bifidobacterium longum NBM 7-1 can be effectively used to treat nonalcoholic liver diseases such as nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.

[0122]

[0123] 2-3. Check changes in blood liver function enzyme levels, triglyceride levels, total cholesterol levels, and fasting blood sugar levels.

[0124] Liver damage can lead to increased levels of alanine aminotransferase (ALT). Elevated ALT levels can be observed in certain liver diseases, including nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and acute hepatitis. Increased blood triglycerides may indicate fat accumulation in the liver, which can increase the risk of developing NAFLD and NASH. High blood cholesterol levels are a risk factor for cardiovascular disease. Some studies have suggested a link between NAFLD and high blood cholesterol levels. Chronic hyperglycemia is known to be an independent factor that increases the risk of developing NAFLD and NASH. Therefore, to verify the therapeutic efficacy of Bifidobacterium longum NBM 7-1 for liver disease, we examined changes in blood liver function enzyme levels, triglyceride levels, total cholesterol levels, and fasting blood glucose levels.

[0125] To measure the level of blood liver function enzymes, the Asan set GPT (ALT) Assay kit (ASAN Pharm. Co., LTD., Seoul, Korea) was used to measure the blood ALT concentration. To measure the level of blood triglyceride, the Serum Triglyceride Quantification Kit product (Cell Biolabs, Inc., San Diego, USA) was used to measure the blood triglyceride concentration. To measure the level of blood total cholesterol, the Asan set total cholesterol test solution (ASAN Pharm. Co., LTD., Seoul, Korea) was used to measure the blood total cholesterol concentration. To measure the level of fasting blood glucose, the Asan set glucose test solution (ASAN Pharm. Co., LTD., Seoul, Korea) was used to measure the blood glucose concentration.

[0126] The results are shown in Figs. 8, 9, 10, 11, Tables 7, 8, 9, and 10.

[0127] As shown in Fig. 8 and Table 7, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), the ALT level was significantly reduced by approximately 40% compared to the negative control group (NC), confirming a significant reduction in the ALT level.

[0128]

[0129] Changes in ALT levels following administration of Bifidobacterium longum NBM 7-1 strain. GroupAverageSTDEVND13.1912.78NC60.3811.59PC12.496.48BLO37.2523.46

[0130] As shown in Fig. 9 and Table 8, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), the blood triglyceride level was reduced by approximately 40% compared to the negative control group (NC), confirming a significant decrease in the blood triglyceride level.

[0131]

[0132] Changes in blood triglyceride levels following administration of Bifidobacterium longum NBM 7-1 strain. GroupAverageSTDEVND162.4536.22NC260.0025.27PC143.4131.96BLO147.4031.17

[0133] As shown in Fig. 10 and Table 9, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), the total blood cholesterol level was significantly reduced by approximately 30% compared to the negative control group (NC), confirming a significant reduction in the total blood cholesterol level.

[0134]

[0135] Changes in total blood cholesterol levels following administration of Bifidobacterium longum NBM 7-1 strain. GroupAverageSTDEVND117.2232.93NC249.1129.09PC177.6031.61BLO175.9073.49

[0136] As shown in Figure 11 and Table 10, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), fasting blood sugar levels were significantly reduced by approximately 20% compared to the negative control group (NC). In particular, compared to the positive control group (PC), fasting blood sugar levels were also significantly reduced by approximately 15%, demonstrating an excellent fasting blood sugar reduction effect.

[0137]

[0138] Changes in fasting blood sugar levels following administration of Bifidobacterium longum NBM 7-1 strain. GroupAverageSTDEVND97.7343.18NC222.4248.37PC211.3639.94BLO178.7554.85

[0139] 2-4. Confirmation of changes in the expression levels of fecal endotoxin and intestinal inflammatory cytokine (IL-1β)

[0140] Endotoxin refers to lipopolysaccharide (LPS) found in the outer cell membrane of Gram-negative bacteria. When the gut bacterial balance is disrupted, large amounts of endotoxin enter the bloodstream. Because the liver receives blood directly from the intestines through the portal venous system, it is susceptible to damage from exposure to these endotoxins. Furthermore, the inflammatory response occurring within the intestine promotes the production of various cytokines. Some of these cytokines may contribute to the development and progression of liver disease. Therefore, to verify the therapeutic effect of Bifidobacterium longum NBM 7-1 on liver disease, we examined changes in the expression levels of fecal endotoxin and intestinal inflammatory cytokines (IL-1β).

[0141] To measure fecal endotoxin, feces were placed in a phosphate buffered solution, disrupted by bead beating, and centrifuged to extract the supernatant. The extracted supernatant was used as a Pierce TMEndotoxin levels were quantified using the Chromogenic Endotoxin Quant Kit product (Thermo Scientific, Massachusetts, USA).

[0142] The results are shown in Fig. 12, Fig. 13, Table 11 and Table 12.

[0143] As shown in Fig. 12 and Table 11, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), the fecal endotoxin level was significantly reduced by approximately 15% compared to the negative control group (NC). In particular, compared to the positive control group (PC), the fecal endotoxin reduction effect was also confirmed to be excellent.

[0144]

[0145] Changes in fecal endotoxins following administration of Bifidobacterium longum NBM 7-1 strain. GroupAverageSTDEVND0.760.11NC1.000.11PC0.900.06BLO0.840.17

[0146] As shown in Figure 13 and Table 12, in the Bifidobacterium longum NBM 7-1 administration group (BLO group), the level of IL-1β, an intestinal inflammatory cytokine, was reduced by approximately 30% compared to the negative control group (NC), confirming a significant decrease in the level of IL-1β, an intestinal inflammatory cytokine.

[0147]

[0148] Changes in fecal endotoxins following administration of Bifidobacterium longum NBM 7-1 strain. GroupAverageSTDEVND370.68100.16NC418.56132.61PC313.6595.85BLO296.2184.31

[0149] The above verification results confirmed that Bifidobacterium longum NBM 7-1 can have excellent therapeutic effects on various liver diseases.

[0150]

[0151]

[0152]

[0153] The present invention relates to a novel use of Bifidobacterium longum NBM 7-1 strain, which strain can be used in a pharmaceutical composition, a food composition, a feed composition for preventing, improving, or treating liver diseases, and a method for treating diseases.

[0154]

[0155] [Accession number]

[0156] Name of depositor: National Institute of Biological Resources

[0157] Accession number: KCTC 14325BP

[0158] Date of acceptance: 20201006

[0159]

[0160]

Claims

1. A pharmaceutical composition for preventing or treating liver disease, comprising Bifidobacterium longum NBM 7-1 strain having the accession number KCTC 14325BP.

2. A pharmaceutical composition for preventing or treating liver disease, wherein in paragraph 1, the Bifidobacterium longum NBM 7-1 strain having the accession number KCTC 14325BP is selected from the group consisting of a live cell of the strain, a dead cell of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, a lysate of the strain, an extract of the strain, and a combination thereof.

3. A pharmaceutical composition for preventing or treating liver disease, wherein the liver disease in paragraph 1 is selected from the group consisting of nonalcoholic liver disease, alcoholic liver disease, liver fibrosis, liver cirrhosis, and liver cancer.

4. A pharmaceutical composition for preventing or treating liver disease, wherein the composition comprises a pharmaceutically acceptable carrier, excipient or diluent in the first paragraph.

5. A food composition for preventing or improving liver disease, comprising Bifidobacterium longum NBM 7-1 strain having the deposit number KCTC 14325BP.

6. A food composition for preventing or improving liver disease, wherein in paragraph 5, the Bifidobacterium longum NBM 7-1 strain having the accession number KCTC 14325BP is selected from the group consisting of a live cell of the strain, a dead cell of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, a lysate of the strain, an extract of the strain, and a combination thereof.

7. A food composition for preventing or improving liver disease, wherein the liver disease in paragraph 5 is selected from the group consisting of nonalcoholic liver disease, alcoholic liver disease, liver fibrosis, liver cirrhosis, and liver cancer.

8. A feed composition for preventing or improving liver disease, comprising Bifidobacterium longum NBM 7-1 strain having the deposit number KCTC 14325BP.

9. A method for preventing or treating liver disease, comprising a step of administering to a subject a Bifidobacterium longum NBM 7-1 strain having the deposit number KCTC 14325BP.

10. A method for preventing or treating liver disease in claim 9, wherein the Bifidobacterium longum NBM 7-1 strain having the accession number KCTC 14325BP is selected from the group consisting of a live cell of the strain, a dead cell of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, a lysate of the strain, an extract of the strain, and a combination thereof.

11. A method for preventing or treating liver disease in claim 9, wherein the liver disease is selected from the group consisting of nonalcoholic liver disease, alcoholic liver disease, liver fibrosis, liver cirrhosis, and liver cancer.

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