Microbiome composition of fermentation culture supernatant of halophilic bacillus velezensis KMU01 strain with anti-obesity efficacy

Abstract

The present invention relates to a microbiome composition of a fermentation culture supernatant of a halophilic Bacillus polyfermentus KMU01 strain having an anti-obesity effect, and a method for preparing the microbiome composition of the fermentation culture supernatant of the halophilic Bacillus polyfermentus KMU01 strain having the anti-obesity effect. The fermentation culture supernatant of the strain preserved with preservation number KCTC11751BP has been confirmed to inhibit fat production and accumulation and to reduce the content of blood cholesterol, and thus be used as a composition for preventing, treating or ameliorating obesity, or as a composition for reducing body fat (visceral fat) or blood cholesterol.

Description

Technical Field

[0001] The present disclosure relates to a microbiome composition of a fermentation culture supernatant of a halophilic Bacillus velezensis KMU01 strain having anti-obesity efficacy. Background Art

[0002] Due to the improved sanitary environment brought about by the rising living standards and the Westernized diet that extends the average life expectancy, obesity is the most representative disease among those diseases in which the disease pattern is rapidly shifting towards the disease pattern of developed countries. Therefore, adult diseases have become the greatest medical challenge today, and obesity, which is the main cause of these adult diseases, is also increasing rapidly.

[0003] Obesity is a disease caused by an imbalance between food intake and energy expenditure, and refers to a condition in which adipose tissue is excessively increased. Persistent obesity causes various diseases such as hypertension, elevated blood cholesterol, kidney disease, stroke, arteriosclerosis, fatty liver, arthritis, cancer, sleep apnea, and diabetes. Among them, the accumulation of visceral fat in the abdominal region leads to insulin resistance or increased hepatic lipid synthesis, which in turn causes abnormal glucose and lipid metabolism, hypertension, and coronary artery disease, making the importance of obesity treatment increasingly emphasized.

[0004] The treatment of obesity is generally divided into three categories, including appetite suppressants, body energy metabolism stimulants, and digestion and absorption inhibitors. Representative obesity treatments that inhibit appetite using pharmacological mechanisms include Reductil TM (Abbott, USA), representative obesity treatments that promote body energy metabolism using pharmacological mechanisms include Exorise TM (Aco Pharma, France), and representative obesity treatments that inhibit fat digestion and absorption using pharmacological mechanisms include Xenical TM (Roche, Switzerland).

[0005] Recently, with the development of next-generation sequencing (NGS) technology, many studies on the microorganisms present in the human body are being carried out, and studies confirming the correlation between obesity and the gut microbiome are also actively underway. The gut microbiome is closely related to the host diet, and it has been reported that obesity can lead to gut microbiota dysbiosis by causing changes in the colonies and functions of gut microorganisms. In fact, when gut microbiota are transplanted from obese mice to normal or germ-free mice, weight gain and metabolic disorders occur, and some studies have pointed out that gut microbiota are related to the regulation of dietary energy utilization and fatty acid metabolism in adipose tissue and liver tissue. Therefore, modulating the gut microbiota colonies may be a non-toxic, safe, and potential treatment for improving metabolic disorders in obesity.

[0006] Although probiotics have the effect of preventing and treating immune diseases by regulating the gut microbiota and blocking the invasion of pathogenic microorganisms, they are unstable and have a weak ability to reach the gut. Therefore, cell-free supernatant (CFS) is attracting attention as a new alternative material, which may overcome the safety and functional problems caused by side effects due to excessive intake. Recently, the culture media of Bifidobacterium bifidum DS0908 and Bifidobacterium bifidum DS0905 have been shown to reduce obesity by promoting thermogenesis in obese mice, and it has been reported that short-chain fatty acids (SCFAs) prevent high-fat diet-induced obesity by regulating gut hormones. In view of this, various studies on the physiological activities of strain culture media are underway. Summary of the Invention Technical Problem

[0007] One object of the present disclosure is to provide a pharmaceutical composition for preventing or treating obesity.

[0008] Another object of the present disclosure is to provide a health functional food composition for preventing or improving obesity.

[0009] Another object of the present disclosure is to provide a food composition for preventing or improving obesity.

[0010] Another object of the present disclosure is to provide a health functional food composition for reducing body fat or blood cholesterol.

[0011] Another object of the present disclosure is to provide a method for preventing or treating obesity. Technical Solution

[0012] To achieve the above object, the present disclosure provides a pharmaceutical composition for preventing or treating obesity, which comprises a fermentation culture supernatant of a Bacillus velezensis strain, its concentrate, its dried product, its fermentation metabolite, or a mixture thereof as an active ingredient.

[0013] In addition, the present disclosure provides a health functional food composition for preventing or improving obesity, which comprises the fermentation culture supernatant of the strain, its concentrate, its dried product, its fermentation metabolite, or a mixture thereof as an active ingredient.

[0014] In addition, the present disclosure provides a food composition for preventing or improving obesity, which comprises the fermentation culture supernatant of the strain, its concentrate, its dried product, its fermentation metabolite, or a mixture thereof as an active ingredient.

[0015] In addition, the present disclosure provides a health functional food composition for reducing body fat or blood cholesterol, which composition contains the fermentation culture supernatant of the strain, its concentrate, its dried product, its fermentation metabolite, or a mixture thereof as an active ingredient.

[0016] In addition, the present disclosure provides a method for preventing or treating obesity, including treating a subject with the pharmaceutical composition for preventing or treating obesity. Beneficial effects

[0017] According to the present disclosure, it has been found that the fermentation culture supernatant of Bacillus velezensis strain KMU01 deposited under the accession number KCTC11751BP inhibits fat production and accumulation and reduces blood cholesterol levels, such that it can be effectively used as a composition for preventing, treating, or improving obesity, or as a composition for reducing body fat (visceral fat) or blood cholesterol. Description of the drawings

[0018] Figure 1 It is a schematic diagram showing the adipogenesis process using 3T3-L1 preadipocytes.

[0019] Figure 2 It is an image obtained by photographing the fat tissue extraction process in a mouse animal model.

[0020] Figure 3 It is a schematic diagram showing the intestinal microbiota analysis process of a mouse animal model.

[0021] Figure 4 It shows the analysis results of the cytotoxicity of the culture broth of Bacillus velezensis strain KMU01 (hereinafter referred to as KMU01) (hereinafter referred to as the sample) in adipocytes.

[0022] Figure 5 It shows the analysis results of the effect of the sample on the accumulation of fat and triglyceride (hereinafter referred to as TG).

[0023] Figure 6 It shows the analysis results of the effect of the sample on the expression of enzymes related to lipogenesis (fatty acid synthase; hereinafter referred to as FAS) and genes related to adipogenesis.

[0024] Figure 7 It shows the analysis results of the effect of the sample on the body weight and food efficiency of an animal model.

[0025] Figure 8 It shows the analysis results of the effect of the sample on the body composition of an animal model.

[0026] Figure 9The analysis results showing the effects of the sample on the organs and adipose tissues of the animal model are presented. iWAT: inguinal white adipose tissue, mWAT: mesenteric white adipose tissue, rWAT: retroperitoneal white adipose tissue, and eWAT: epididymal white adipose tissue.

[0027] Figure 10 The analysis results showing the effects of the sample on the adipose tissues of the animal model are presented.

[0028] Figure 11 The analysis results showing the effects of the sample on the liver TG of the animal model are presented.

[0029] Figure 12 The analysis results showing the effects of the sample on the expression of proteins related to lipogenesis and lipid synthesis in the liver of the animal model are presented.

[0030] Figure 13 The analysis results showing the effects of the sample on the gut microbiota of the animal model are presented.

[0031] It should be noted that at the 95% confidence level, there are statistical differences between all the treatment groups represented by different letters (such as a, b, etc.) in all the figures. For example, if there is group 1 represented as a, group 2 represented as b, and group 3 represented as ab, it indicates that there is a statistical difference between group 1 and group 3 at the 95% level, while there is no statistical difference between group 1 and group 2, and between group 2 and group 3. Detailed Description of the Invention

[0032] The present disclosure will be described in more detail below.

[0034] The present disclosure provides a pharmaceutical composition for preventing or treating obesity, which comprises a fermentation culture supernatant of a Bacillus velezensis strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof as an active ingredient.

[0035] The strain may be Bacillus velezensis KMU01 strain deposited under the accession number KCTC11751BP.

[0036] The strain name of Bacillus velezensis KMU01 when deposited was Bacillus polyfermenticus KMU01. Specifically, strain KMU01 was isolated as Bacillus amyloliquefaciens in 2010 and reclassified as Bacillus polyfermenticus in 2018 based on 16S rRNA gene sequences. Subsequently, experiments conducted to precisely identify the species of strain KMU01 showed that the gene sequence of strain KMU01 had 97.7% similarity with Bacillus velezensis, and currently strain KMU01 is identified as Bacillus velezensis (Functional Annotation Genome Unravels Potential Probiotic Bacillus velezensis Strain KMU01 from Traditional Korean Fermented Kimchi, DOI: https: / / doi.org / 10.3390 / foods10030563, published on May 9, 2021).

[0037] Recently, Bacillus polyfermenticus KMU01 strain has been renamed Bacillus velezensis (Genome Sequence of the Probiotic Strain Bacillus velezensis Variant polyfermenticus GF423, DOI: 10.1128 / MRA.01000-18, published on September 13, 2018).

[0038] The pharmaceutical composition may further comprise inactivated cells or spores of Bacillus velezensis.

[0039] The fermentation metabolite may be short-chain fatty acids (SCFAs), organic acids or amino acids.

[0040] The short-chain fatty acid may be butyric acid or propionic acid, and the amino acid may be the aromatic amino acid phenylalanine, or the branched-chain amino acid valine, but is not limited thereto.

[0041] In addition, the pharmaceutical composition can regulate adiponectin secretion.

[0042] In addition, the pharmaceutical composition can regulate one or more intestinal microorganisms selected from the group consisting of, but not limited to: Acetatifactor muris, Mucispirillum schaedleri and Eubacterium plexicaudatum.

[0043] In addition, the pharmaceutical composition can inhibit the expression of one or more selected from the group consisting of, but not limited to, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT / enhancer-binding protein α (C / EBPα), sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase-1 (SCD-1), and diacylglycerol acyltransferase (DGAT).

[0044] The obesity can be one or more selected from the group consisting of visceral obesity, abdominal obesity, general obesity, and local obesity, but not limited thereto.

[0045] According to a method that can be easily implemented by those of ordinary skill in the art to which the present disclosure pertains, the pharmaceutical composition of the present disclosure can be formulated using a pharmaceutically acceptable carrier to be prepared in unit dose form, or can be prepared by encapsulating it into a multi-capacity container.

[0046] The pharmaceutically acceptable carriers are commonly used in formulations, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate / ester, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present disclosure can further contain lubricants, humectants, sweeteners, flavoring agents, emulsifying agents, suspending agents, and preservatives.

[0047] In the present disclosure, the content of the additives contained in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0048] The pharmaceutical composition can be formulated in the form of one or more skin topical agents selected from the group consisting of, but not limited to, injectable preparations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes, and cataplasms.

[0049] The pharmaceutical composition of the present disclosure may comprise pharmaceutically acceptable carriers and diluents additionally present in the formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to: excipients such as starch, sugar and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethyl cellulose and hydroxypropyl cellulose; binders such as gelatin, alginate / ester and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol; disintegrants such as povidone and cross-linked povidone; and surfactants such as polysorbate, cetyl alcohol and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically friendly to the subject. Examples of diluents may include, but are not limited to, saline, water-soluble buffers, solvents and / or dispersion media.

[0050] According to the desired method, the pharmaceutical composition of the present disclosure can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically). When administered orally, it can be formulated into tablets, lozenges, troches, aqueous suspensions, oil-based suspensions, powders, granules, emulsions, hard capsules, soft capsules, syrups and elixirs. When administered parenterally, it can be formulated into injection solutions, suppositories, powders for respiratory inhalation, aerosol sprays, ointments, topical powders, oils and creams.

[0051] The dosage range of the pharmaceutical composition of the present disclosure can vary according to the following and can be appropriately selected by those skilled in the art: the patient's condition, weight, age, gender, health status, dietary structure specificity, nature of the formulation, severity of the disease, time of administration of the composition, method of administration, duration or interval of administration, excretion rate and drug dosage form. For example, the range can be from about 0.1 mg / kg to 10,000 mg / kg, but is not limited thereto, and it can be administered once or several times a day.

[0052] According to the desired method, the pharmaceutical composition can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically). The pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present disclosure can vary depending on the preparation method of the pharmaceutical composition, type of administration, time of administration and route of administration, and those of ordinary skill in the art can easily determine and prescribe an effective dose for the desired treatment. The administration of the pharmaceutical composition of the present disclosure can be administered once a day or in multiple separate doses.

[0054] In addition, the present disclosure provides a health functional food composition for preventing or improving obesity, which composition comprises a fermentation culture supernatant of a Bacillus velezensis strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof or a mixture thereof as an active ingredient.

[0055] In addition, the present disclosure provides a health functional food composition for reducing body fat or blood cholesterol, which composition comprises a fermentation culture supernatant of a Bacillus velezensis strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof as an active ingredient.

[0056] The strain may be the Bacillus velezensis KMU01 strain deposited under the accession number KCTC11751BP.

[0057] The present disclosure can be widely used as a conventionally used food.

[0058] The food composition of the present disclosure can be used as a health functional food. The term "health functional food" as used herein refers to a food processed and manufactured using raw materials or ingredients having beneficial functions for the human body in accordance with the Health Functional Food Act; and the term "function" as used herein refers to consumption for the purpose of producing beneficial effects for health purposes, such as regulating nutrients or physiological actions on the human body structure and functions, etc.

[0059] The health functional food composition may contain conventional food additives, and unless otherwise specified, the applicability as a "food additive" is determined according to the general rules and general test methods of the Korean Food Additive Codex approved by the Ministry of Food and Drug Safety of Korea, and according to the relevant standards and guidelines of the corresponding items.

[0060] Items listed in the Korean Food Additive Codex may include, for example: chemically synthesized compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sorghum pigment, and guar gum; and mixed preparations such as L-glutamate preparations, alkaline agents for noodles, preservatives, and tar colorants.

[0061] The food composition of the present disclosure can be manufactured or processed into forms such as tablets, capsules, powders, granules, liquids, and pills. For example, a hard capsule preparation among health functional foods in capsule form can be prepared by mixing the composition of the present disclosure with additives such as excipients and filling it into a conventional hard capsule, while a soft capsule preparation can be manufactured by mixing the composition of the present disclosure with additives such as excipients and then filling it into a capsule matrix such as gelatin. If necessary, the soft capsule preparation may contain plasticizers such as glycerol or sorbitol, colorants, and preservatives.

[0062] The terms of excipients, binders, disintegrants, lubricants, flavor enhancers, and flavoring agents are described in the literature known in the art and include those having the same or similar functions. The type of food is not particularly limited and includes all health functional foods in the general sense.

[0063] As used herein, the term "prevention" refers to any effect of inhibiting or delaying obesity by administering the composition described in the present disclosure.

[0064] As used herein, the term "treatment" refers to any effect of improving or favorably altering the symptoms of obesity by administering the composition described in the present disclosure.

[0065] As used herein, the term "improvement" refers to any effect of improving the adverse conditions of obesity by administering or causing a subject to ingest the composition described in the present disclosure.

[0067] In addition, the present disclosure provides a functional food comprising the health functional food composition.

[0069] In addition, the present disclosure provides a food composition for preventing or improving obesity, which comprises a fermentation culture supernatant of Bacillus velezensis strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof as an active ingredient.

[0070] The strain may be Bacillus velezensis KMU01 strain deposited under the accession number KCTC11751BP.

[0072] In addition, the present disclosure provides a method for preventing or treating obesity, which comprises treating a subject with the pharmaceutical composition for preventing or treating obesity.

[0073] The method for preventing or treating obesity can obtain the effect of improving obesity or reducing visceral fat by: reducing the relative abundance of Acetatifactor muris or Mucispirillum schaedleri strain in the intestine, and increasing the relative abundance of Eubacterium plexicaudatum strain in the intestine. Examples

[0074] The present disclosure will be described in more detail below through examples to help understand the present disclosure. However, the following examples are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to the following examples. The examples of the present disclosure are provided to more completely explain the present disclosure to those skilled in the art.

[0076] [Experimental Example 1] Sample Preparation

[0077] To prepare the sample, the KMUO1 (Bacillus velezensis KMU01) strain preserved in the working cell bank at -70 °C with the inventory preservation number KCTC11751BP was activated and subjected to primary seed culture in test tubes and flasks. Then, 2% (v / v) of it was inoculated into a 50 L fermenter with a working volume of 20 L, followed by secondary seed culture for 6 hours. This culture was carried out for 12 hours by inoculating into a 500 L fermenter with a working volume of 350 L at 2% (v / v), and glucose was additionally supplemented once after 6 hours of this culture. After completion of the culture, the cell suspension was removed by primary centrifugation in a disk centrifuge at 7200 rpm and 2 L / min, and the supernatant was subjected to two secondary centrifugations in a tubular centrifuge at 15000 rpm and 1.5 L / min to remove cell pellet blocks and collect the supernatant. The recovered supernatant was filtered through a 0.2 μm sterile filter to obtain the final sample from which bacterial cells were removed.

[0079] [Experimental Example 2] In Vitro Experiment

[0080] 2-1. Cell Culture and Differentiation

[0081] The 3T3-L1 (ATCC, Manassas, VA, USA) fibroblasts, which are preadipocytes, were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v / v) calf serum and 100 μg / mL penicillin-streptomycin at 37 °C in the presence of 5% CO2. Thereafter, as Figure 1 shown, when the cells reached 100% confluence, differentiation into adipocytes was induced in DMEM containing 10% fetal bovine serum (FBS), 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX), 1 μg / mL insulin, and 100 μg / mL penicillin-streptomycin. Two days after differentiation, the medium was changed to 10% FBS containing 1 μg / mL insulin and the sample (the medium was changed every other day), and the effects on adipocyte proliferation and differentiation were analyzed on the 8th day.

[0083] 2-2. Cytotoxicity Assay

[0084] To identify the cytotoxicity of the sample in adipocytes, the MTT assay was performed. The sample was treated with adipocytes (3T3-L1) at different concentrations (75 μg / mL, 150 μg / mL, and 300 μg / mL), and cell viability was measured by the MTT assay.

[0086] 2-3. Analysis of Fat Accumulation and TG Content

[0087] To identify the effect of the sample on fat accumulation, the sample was used to treat adipocytes (3T3-L1) at different concentrations (75 μg / mL, 150 μg / mL, and 300 μg / mL), and the adipocytes were stained by Oil Red O (ORO) staining method to measure the fat accumulation rate of mature adipocytes.

[0088] In addition, to identify the effect of the sample on TG, a TG quantification kit (Abcam, Cambridge, MA, USA) was used to measure the content of TG accumulated in the cells, the protein content was quantified by bicinchoninic acid (BCA) assay, and then the TG content of the cells was expressed by protein concentration correction.

[0090] 2-4. Analysis of genes related to adipogenesis and FAS expression

[0091] To identify the effect of the sample on genes related to adipogenesis and FAS expression, RNA was extracted using Nucleozol (Macherey-Nagel, Duren, Germany) reagent, and cDNA was synthesized using a reverse transcription kit (Applied Biosystem, Foster City, CA, USA). Subsequently, the expression of genes related to adipogenesis (PPARγ, C / EBPα, and SREB-1c) and FAS was analyzed using a StepOnePlus real-time PCR (quantitative real-time PCR; qPCR) system (Applied Biosystem), and the gene expression level was corrected using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene.

[0093] 2-5. Statistical analysis

[0094] All quantitative analyses were performed three times. Statistical analysis was performed using SPSS (SPSS Inc., USA) software, and when significant differences (P < 0.05) were observed by one-way analysis of variance (ANOVA), the Duncan multiple comparison method was implemented to test for significant differences between treatment groups.

[0096] [Experimental Example 3] In vivo experiment

[0097] 3-1. Preparation of animal model

[0098] Purchase 5-week-old male C57BL / 6J mice from RAONBIO Inc. (South Korea). After purchase, conduct an external examination of the animals and measure their body weights. During the acclimation period, observe the general symptoms once a day, and at the end of the acclimation period, measure the body weights and check the general symptoms and body weight changes to evaluate the health status of the animals. To ensure the uniformity of the average body weights of each experimental group, divide the animals into a total of 6 groups, with approximately 10 animals in each group, and house 5 animals in each cage. Make individual marks on the tails of the animals using a five-color permanent marker, and paste individual identification cards on their cages. Raise the animal models under the following conditions for 2 weeks: temperature 21 - 23 °C, relative humidity 40% - 60%, 12-hour light / dark cycle per day (8:00 AM - 8:00 PM), and provide food and drinking water. The feed used is experimental animal feed (6% fat feed and 45% fat feed) (ENVIGO, RESEARCHDIETS Inc.). The animal experiment was approved by the Institutional Animal Care and Use Committee of Konkuk University (KMU-2022-01) and was conducted in accordance with the standard operating procedures of Konkuk University.

[0100] 3-2. Analysis of Body Weight and Food Efficiency

[0101] To identify the effects of the samples on the body weights and food efficiency of the animal models, set the animal models (7-week-old) prepared in Experimental Example 3-1 into the following four groups, measure the body weights and feed intakes once a week for a total of 13 weeks, and calculate the food efficiency using the following Formula 1. Use Xenical as the positive control. Using a disposable syringe attached with an oral gavage tube, orally administer the samples and Xenical to the stomach once a day for a total of 13 weeks starting from the administration.

[0102] 1) Normal diet group (NOR): 6% fat feed intake group

[0103] 2) High-fat diet group (HFD): 45% fat feed intake group

[0104] 3) Sample administration group (B.vele): High-fat diet group (HFD) orally administered with the sample (114 mg / kg / day)

[0105] 4) Positive control group (Xen): High-fat diet group (HFD) administered with Xenical (50 mg / kg / day)

[0106] [Formula 1]

[0107] Food efficiency ratio = Increase in body weight (g / week) / Food intake (g / week) × 100

[0109] 3-3. Analysis of Body Composition

[0110] To identify the effects of the sample on the body composition of the animal model, 13 weeks before sacrifice, the changes in the body composition of the animal model were measured using dual-energy X-ray absorptiometry (InAlyzer; Medikors Inc., Seongnam, Korea). When analyzing the body composition, the animal model was anesthetized by injecting ketamine (100 mg / kg BW) and xylazine (10 mg / kg BW), and then the measurement was carried out. When only ketamine with anesthetic effect was used, side effects might occur due to muscle contraction during the recovery period from anesthesia, and thus the muscle relaxant xylazine was used together.

[0112] 3 - 4. Analysis of Organ and Adipose Tissue Weights

[0113] To identify the effects of the sample on the organs and adipose tissue in the animal model, the animal model was fasted for 18 hours, sacrificed and dissected, and then the heart, liver, kidneys, and spleen were removed to measure the weights of these organs. As Figure 2 shown, the adipose tissue was divided into inguinal white adipose tissue, mesenteric white adipose tissue, retroperitoneal white adipose tissue, and epididymal white adipose tissue to measure the weights.

[0115] 3 - 5. Blood Biochemical Analysis

[0116] To identify the effects of the sample on blood glucose, aspartate aminotransferase (GOT; hereinafter referred to as AST), alanine aminotransferase (GPT; hereinafter referred to as ALT), blood urea nitrogen (hereinafter referred to as BUN), and cholesterol in the animal model, blood was collected from the heart after sacrificing the animal model, and then immediately centrifuged (2000×g, 10 min) to separate the plasma, and the plasma was stored in a -80°C refrigerator until analysis. A chemical analyzer (Fuji DRI-CHEM 3500i, Fuji Photo Film, Ltd., Tokyo, Japan) was used to detect blood glucose, AST, ALT, and BUN, and a LabAssay TM Cholesterol Kit (Wako, Osaka, Japan) was used to detect total cholesterol and high-density lipoprotein (HDL)-cholesterol (hereinafter referred to as HDL-C), and the low-density lipoprotein (LDL)-cholesterol (hereinafter referred to as LDL-C) was calculated using the following formula 2. In addition, a TG assay kit (Abcam, Cambridge, MA) was used to analyze TG.

[0117] [Formula 2]

[0118] LDL-C = Total cholesterol - {(HDL-C) + (TG / 5)}

[0120] 3 - 6. Histological Analysis

[0121] To identify the effect of the sample on adipose tissue in the animal model, the epididymal white adipose tissue (eWAT) and liver sections of the animal model were fixed in 10% formaldehyde and made into paraffin blocks, and then subjected to H&E (hematoxylin & eosin) staining. The areas of 15 adipocytes at the center of the representative image were obtained using the KFBIO Slide Manager (KFBIO, Ningbo, China), and the size of the adipocytes was expressed as the average value.

[0123] 3-7. Analysis of TG content in the liver

[0124] To identify the effect of the sample on liver TG in the animal model, after grinding the liver tissue of the animal model, TG was extracted for analysis using a TG assay kit (Abcam).

[0126] 3-8. Analysis of the expression of proteins related to hepatic lipogenesis and lipid synthesis

[0127] To identify the effect of the sample on the expression of proteins related to hepatic lipogenesis and lipid synthesis in the animal model, the liver tissue was homogenized using a bullet homogenizer (Next Advance, Troy, NY, USA) in a radioimmunoprecipitation assay (RIPA) buffer containing 1% protease inhibitor and 1% phosphatase inhibitor for the experiment. The homogenized tissue was placed at 4 °C for 50 min and centrifuged at 4 °C and 15000×g for 15 min to obtain the supernatant. Equal amounts of protein were separated on 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). After blocking with a blocking buffer containing 5% bovine serum albumin in Tris-buffered saline with Tween 20 (hereinafter referred to as TBST, 0.1%), it was reacted with ACC, p-ACC, FAS, C / EBPα, PPARγ, SCD-1, SREBP-1c, DGAT, and β-actin antibodies overnight at 4 °C. After reacting with a horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour, it was washed four times with TBST buffer. Protein bands were detected using an enhanced chemiluminescence detection kit (BioRad, Hercules, CA, USA), and after correction with β-actin, the band intensity was quantified using Image Lab 5.1 software (BioRad).

[0129] 3-9. Analysis of gut microbiota

[0130] To identify the effect of the sample on the gut microbiota of the animal model, the animal model was sacrificed, the cecum was removed, and as Figure 3As shown, 16S rRNA metagenomic sequencing was used for analysis.

[0132] 3 - 10. Statistical analysis

[0133] Statistical analysis was performed using GraphPad Prism 9.4.0 (GraphPad Software Inc., San Diego, CA, USA) and SPSS statistical software V.26 (SPSS Inc., Chicago, IL, USA), and one - way analysis of variance (ANOVA) and Duncan's and Newman - Keuls multiple comparison tests were used to analyze the significant differences (p < 0.05) between treatment groups.

[0135] [Example 1] In vitro experiments

[0136] 1 - 1. Cytotoxicity assay

[0137] According to Experimental Example 2 - 2, as a result of analyzing the cytotoxicity of the sample in adipocytes, as Figure 4 shown, it was found that when the treatment concentration of the sample (B.vele) was as high as 300 μg / mL, there was no significant change in cell viability.

[0139] 1 - 2. Analysis of lipid accumulation and TG content

[0140] According to Experimental Example 2 - 3, as a result of analyzing the effect of the sample on lipid and TG accumulation, as Figure 5 shown, the sample (B.vele) significantly inhibited lipid and TG accumulation in a concentration - dependent manner, and it was found that intracellular lipid and TG accumulation in the 300 μg / mL treatment group decreased by approximately 20% and 39%, respectively. In addition, the results showed that the KMUO1 strain had an anti - obesity effect by inhibiting lipid accumulation.

[0142] 1 - 3. Analysis of lipid - related genes and FAS expression

[0143] According to Experimental Example 2 - 4, as a result of analyzing the effect of the sample on lipid - related genes and FAS expression, as Figure 6 shown, the sample (B.vele) inhibited the mRNA expression of PPARγ, C / EBPα, SREBP - 1c, and FAS, and specifically, compared with the control group, the mRNA expression in the 300 μg / mL treatment group decreased by 32%, 65%, 46%, and 53%, respectively.

[0145] [Example 2] In vivo experiments

[0146] 2-1. Analysis of body weight and food efficiency

[0147] According to Experimental Example 3-2, as a result of analyzing the effects of the sample on body weight and food efficiency in an animal model, as Figure 7 shown, it was found that the body weight of the high-fat diet group (HFD) increased significantly compared to the normal diet group (NOR), indicating the induction of obesity. In addition, it was confirmed that the body weight of the sample-administered group (B.vele) was significantly lower than that of the high-fat diet group (p < 0.05). The increase in body weight of the high-fat diet group was significantly higher than that of the normal diet group, while the sample-administered group showed significantly lower body weight than the high-fat diet group (p < 0.05). There was no difference in food intake between the high-fat diet group and the sample-administered group, but the food efficiency of the sample-administered group was significantly reduced (p < 0.05). These results suggest that the sample causes a decrease in the utilization rate of digestion and absorption of some nutrients or an increase in energy consumption.

[0149] 2-2. Body composition analysis

[0150] According to Experimental Example 3-3, as a result of analyzing the effects of the sample on body composition in an animal model, as Figure 8 shown, it was found that the body fat of the high-fat diet group (HFD) increased significantly compared to the normal diet group (NOR), while the body fat of the sample-administered group (B.vele) and the positive control group (Xen) decreased significantly. There was no significant difference in lean body mass and bone mineral content among the experimental groups. These results suggest that the change in body weight caused by the sample is attributed to the decrease in body fat.

[0152] 2-3. Analysis of organ and adipose tissue weights

[0153] According to Experimental Example 3-4, as a result of analyzing the effects of the sample on organs and adipose tissue in an animal model, as shown in Table 1, there was no significant difference in the weights of the heart, liver, and spleen among the experimental groups. The kidney weight of the high-fat diet group (HFD) increased, but it did not show statistical significance.

[0154] Table 1 Weight (mg) NOR HFD B.vele Xen Heart 146±24 150±10 151±23 157±20 Liver 1104±76 1085±92 1047±86 1121±88 Kidney 341±37 368±46 355±38 381±46 Spleen 69±12 69±7 76±13 73±13

[0155] In addition, as Figure 9As shown, compared with the normal diet group (NOR), the weights of all adipose tissues (inguinal white adipose tissue (iWAT), mesenteric white adipose tissue (mWAT), retroperitoneal white adipose tissue (rWAT), and epididymal white adipose tissue (eWAT)) in the high-fat diet group (HFD) were significantly increased, while the weights of all adipose tissues in the sample administration group (B.vele) were decreased compared with the high-fat diet group, showing a similar trend to the positive control group (Xen) in which the weights of all adipose tissues were decreased.

[0156] 2 - 4. Blood biochemical analysis

[0157] According to Experimental Examples 3 - 5, as a result of analyzing the effects of the sample on blood glucose, AST, ALT, BUN, and cholesterol in the animal model, as shown in Table 2, there was no significant difference in TG between the normal diet group (NOR) and the high-fat diet group (HFD), but the TG in the positive control group (Xen) was significantly lower than that in the high-fat diet group (p < 0.05). The significant increase in total cholesterol (TCHO) and HDL-C was attributed to the high-fat diet, while the LDL-C content in the sample administration group (B.vele) was significantly lower than that in the high-fat diet group. There was no significant difference in the indexes of blood glucose, abnormal liver function (AST and ALT), and nephrotoxicity (BUN) among the experimental groups.

[0158] Table 2

[0160] 2 - 5. Histological analysis

[0161] According to Experimental Examples 3 - 6, as a result of analyzing the effects of the sample on adipose tissues in the animal model, as Figure 10 shown, in the epididymal white adipose tissue (eWAT), the size of adipocytes in the high-fat diet group (HFD) was significantly larger than that in the normal diet group (NOR), while the size of adipocytes in the sample administration group (B.vele) and the positive control group (Xen) was significantly smaller than that in the high-fat diet group, indicating that adipocyte hypertrophy was inhibited. In addition, in the liver, the formation of lipid droplets (white dots) in the high-fat diet group was increased compared with the normal diet group, while the lipid droplets in the sample administration group were decreased compared with the high-fat diet group.

[0163] 2 - 6. Analysis of liver TG content

[0164] According to Experimental Examples 3 - 7, as a result of analyzing the effects of the sample on liver TG in the animal model, as Figure 11As shown, the TG content in the high-fat diet group (HFD) was significantly increased compared with that in the normal diet group (NOR), while the TG content in the sample administration group (B.vele) and the positive control group (Xen) was significantly decreased compared with that in the high-fat diet group.

[0166] 2-7. Analysis of the expression of proteins related to hepatic lipogenesis and lipid synthesis

[0167] According to Experimental Examples 3-8, as a result of analyzing the effects of the sample on the expression of proteins related to hepatic lipogenesis and lipid synthesis in an animal model, as Figure 12 shown, the expression of lipogenesis regulatory proteins (C / EBPα and PPARγ) in the sample administration group (B.vele) was significantly decreased, and the expression of SREBP-1c, which regulates the expression of lipid synthesis enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), showed a decrease (P<0.05). In addition, the expression of FAS, SCD-1, and DGAT, which are involved in the lipid synthesis process, was significantly decreased (P<0.05). The phosphorylation level of ACC, which inhibits ACC activation, was significantly increased, indicating that the sample helps to inhibit the lipid synthesis process. The positive control group (Xen) also showed a similar trend to the sample administration group (B.vele).

[0169] 2-8. Analysis of gut microbiota

[0170] According to Experimental Examples 3-9, as a result of analyzing the effects of the sample on gut microbiota in an animal model, as Figure 13 shown in A, in the results of α-diversity analysis, which analyzes the microbial diversity present in individual samples, no significant differences were observed in the Shannon index among the experimental groups. On the other hand, the results of β-diversity analysis based on unweighted UniFrac principal coordinate analysis (PCA), which is commonly used in microbial colony analysis, showed significant differences among the experimental groups. Sample administration changed the gut microbiota composition to be similar to that of the normal diet group (NOR).

[0171] In addition, the relative abundance of Deferribacterota was significantly increased in the high-fat diet group (HFD), while it was significantly decreased in the sample administration group and the positive control group (Xen). In the structural microbial colony analysis at the phylum level, the relative abundance of Firmicutes increased due to the high-fat diet, but the relative abundance of Bacteroidota decreased, indicating an elevated Firmicutes / Bacteroidota (F / B) ratio, while the F / B ratio in the sample administration group decreased. At the family level, the relative abundance of Muribaculaceae in the high-fat diet group was significantly lower than that in the normal diet group. The relative proportion of Lachnospiraceae in the sample administration group decreased, while the relative proportion of Muribaculaceae increased. At the species level, the relative abundances of Acetatifactor muris and Mucispirillum schaedleri in the high-fat diet group were significantly increased compared with those in the normal diet group. Compared with the high-fat diet group, the relative abundances of Acetatifactor muris and Mucispirillum schaedleri in the sample administration group were significantly decreased, while the relative abundance of Eubacterium plexicaudatum was significantly increased.

[0173] Although specific parts of the present disclosure have been described in detail above, it will be apparent to those skilled in the art that the specific description is only a preferred exemplary embodiment, but the scope of the present disclosure is not limited thereto. In other words, the substantial scope of the present disclosure is defined by the appended claims and their equivalents.

[0174] (Translation) Receipt certificate under the original deposit Recipient: Cheng Wenxi Gukmin University, 861-1, Jeongneung-dong, Seongbuk-gu, Seoul 136-702, Republic of Korea The same as the original translation above

[0175] (Translation) Certificate of change or subsequent designation of taxonomic nomenclature regarding scientific description and / or advice Recipient: Cheng Wenxi Gukmin University, 77, Jeongneung-ro, Seongbuk-gu, Seoul 02707, Republic of Korea Attachment: Notice of Changes to Scientific Descriptions and Proposed Taxonomic Nomenclature or Late Designations in accordance with Rule 8.1 The same as the original translation above

[0176] (Translation) Certificate of Changes to Scientific Descriptions and / or Proposed Taxonomic Nomenclature or Late Designations Recipient: Wenxi Cheng Gukmin University, 77 Jeongneung-ro, Seongbuk-gu, Seoul 02707, Republic of Korea Attachment: Notice of Changes to Scientific Descriptions and Proposed Taxonomic Nomenclature or Late Designations in accordance with Rule 8.1 The same as the original translation above

Claims

1. A pharmaceutical composition for preventing or treating obesity, said composition comprising a fermentation culture supernatant of a Bacillus velezensis strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein The strain is Bacillus velezensis KMU01 strain deposited under the accession number KCTC11751BP.

3. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition further comprises inactivated cells or spores of Bacillus velezensis.

4. The pharmaceutical composition according to claim 1, wherein The fermentation metabolite is short-chain fatty acids (SCFAs), organic acids or amino acids.

5. The pharmaceutical composition according to claim 4, wherein, The short-chain fatty acid is butyric acid or propionic acid.

6. The pharmaceutical composition according to claim 4, wherein, The amino acid is the aromatic amino acid phenylalanine or the branched-chain amino acid valine.

7. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition modulates one or more gut microbiota selected from the group consisting of Acetatifactor muris, Mucispirillum schaedleri and Eubacterium plexicaudatum.

8. The pharmaceutical composition according to claim 1, wherein, The obesity is one or more selected from the group consisting of visceral obesity, abdominal obesity, general obesity and local obesity.

9. A health functional food composition for preventing or improving obesity, said composition comprising a fermentation culture supernatant of a Bacillus velezensis strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof as an active ingredient.

10. A food composition for preventing or improving obesity, said composition comprising a fermentation culture supernatant of a Bacillus velezensis strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof as an active ingredient.

11. A health functional food composition for reducing body fat or blood cholesterol, said composition comprising a fermentation culture supernatant of a Bacillus velezensis strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof as an active ingredient.

12. A method for preventing or treating obesity, comprising treating a subject with the pharmaceutical composition according to claim 1.

13. The method according to claim 12, wherein, The method for preventing or treating obesity obtains the effect of improving obesity or reducing visceral fat by: reducing the relative abundance of Acetatifactor muris or Mucispirillum schaedleri strains in the gut and increasing the relative abundance of Eubacterium plexicaudatum strain in the gut.

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