Microbiome composition of fermentation culture supernatant of halophilic Bacillus veresensis strain KMU01 with anti-obesity efficacy

Abstract

The present invention relates to a microbiome composition of the fermentation culture supernatant of the halophilic Bacillus polyfermenticus KMU01 strain, which has anti-obesity effects. It has been confirmed that the fermentation culture supernatant of the strain deposited under accession number KCTC11751BP inhibits fat production and accumulation and reduces blood cholesterol levels. Therefore, the composition can be usefully 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 invention relates to a microbiome composition of the fermentation culture supernatant of the halophilic Bacillus velezensis KMU01 strain, which has anti-obesity effects. [Background technology]

[0002] Obesity is the most typical disease among the diseases that have been rapidly changing to those of developed countries as a result of recent improvements in living standards, improved sanitary conditions, Westernized diets, and longer average life expectancies. As a result, adult diseases have emerged as the biggest medical issue today, and obesity, which is the main cause of these diseases, is also rapidly increasing.

[0003] Obesity is a disease caused by an imbalance between food intake and energy use, and refers to a state of excessive adipose tissue. Sustained obesity can lead to a variety of diseases, including high blood pressure, elevated blood cholesterol, kidney disease, stroke, arteriosclerosis, fatty liver, arthritis, cancer, sleep apnea, and diabetes. Among these, the accumulation of visceral fat within abdominal fat can cause insulin resistance and increased fat synthesis in the liver, leading to glucose and lipid metabolism disorders, high blood pressure, and coronary artery disease, making obesity treatment particularly important.

[0004] Anti-obesity drugs are generally divided into three categories: appetite suppressants, body energy metabolism promoters, and digestion and absorption inhibitors. Reductil is a representative anti-obesity drug that uses the pharmacological mechanism of appetite suppression. TM , Evoto, USA), and a representative obesity treatment that uses a pharmacological mechanism to promote internal energy is Exorise TM , Arcopharma, France), and a representative obesity treatment that uses a pharmacological mechanism to inhibit the digestion and absorption of fat is Xenical TM , Roche Pharmaceuticals, Switzerland).

[0005] With the recent development of next-generation sequencing (NGS) technology, numerous studies on the microorganisms present in the human body have been conducted, including active research aimed at proving the relationship between obesity and the gut microbiome. The gut microbiome is closely related to the host's diet, and obesity has been reported to lead to changes in the gut microbial community and function, resulting in gut microbial imbalance (dysbiosis). Indeed, transplantation of gut microbes from obese mice into normal or germ-free mice resulted in weight gain and metabolic disorders, suggesting that gut microbes are involved in the regulation of dietary energy utilization and fatty acid metabolism in adipose and liver tissues. Therefore, modulation of the gut microbial community may be a potential therapeutic strategy for improving obesity without toxic effects on metabolic disorders and is safe.

[0006] Probiotics regulate the intestinal flora, preventing the entry of pathogenic microorganisms and providing effective treatment and prevention of immune disorders. However, they can be unsafe, have issues with reaching the intestine, and can cause side effects from excessive intake. Cell-free supertanant (CFS) is gaining attention as a new alternative that overcomes these safety and functionality barriers. Recently, cultures of Bifidobacterium bifidum DS0908 and Bifidobacterium bifidum DS0905 have been shown to promote thermogenesis and reduce obesity in obese mice. Short-chain fatty acids (SCFAs) have also been reported to regulate intestinal hormones and prevent high-fat diet-induced obesity. Thus, extensive research into the physiological activities of probiotic cultures is currently underway. Summary of the Invention [Problem to be solved by the invention]

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

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

[0009] A further object of the present invention is to provide a food composition for preventing or alleviating obesity.

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

[0011] It is yet another object of the present invention to provide a method for preventing or treating obesity. [Means for solving the problem]

[0012] To achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating obesity, 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.

[0013] The present invention also provides a functional health food composition for preventing or improving obesity, which contains as an active ingredient the fermentation culture supernatant of the strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof.

[0014] The present invention also provides a food composition for preventing or improving obesity, which contains as an active ingredient a fermentation culture supernatant of the strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof.

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

[0016] The present invention also provides a method for preventing or treating obesity, comprising administering the pharmaceutical composition for preventing or treating obesity to an individual. [Effects of the Invention]

[0017] According to the present invention, it has been confirmed that the fermentation culture supernatant of the Bacillus veresensis KMU01 strain deposited under accession number KCTC11751BP inhibits fat production and accumulation and reduces blood cholesterol levels, and therefore it can be usefully used as a composition for preventing, treating, or ameliorating obesity; or as a composition for reducing body fat (visceral fat) or blood cholesterol. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram showing the adipogenesis process using 3T3-L1 preadipocytes. [Figure 2] Images showing the process of adipose tissue removal in a mouse animal model. [Figure 3] FIG. 1 is a schematic diagram showing the process of analyzing intestinal microorganisms in a mouse animal model. [Figure 4] The results are obtained by analyzing the cytotoxicity of the culture medium (hereinafter referred to as the sample) of Bacillus veresensis KMU01 (hereinafter referred to as KMU01) strain in adipocytes. [Figure 5] The results are from an analysis of the effects of the sample on the accumulation of fat and triglycerides (hereinafter referred to as TG). [Figure 6] The results show the analysis of the influence of the sample on the expression of adipocyte differentiation-related genes and fat synthesis-related enzymes (fatty acid synthase; hereinafter referred to as FAS). [Figure 7] The results of analyzing the effects of the sample on body weight and food efficiency in an animal model. [Figure 8] This is the result of analyzing the effect of the sample on the gymnastics of an animal model. [Figure 9]The results of analyzing the effects of the sample on organs and adipose tissues of animal models: iWAT: inguinal white adipose tissue, mWAT: mesenteric white adipose tissue, rWAT: retroperitoneal white adipose tissue, and eWAT: epididymal white adipose tissue. [Figure 10] This is the result of analyzing the effects of the sample on the adipose tissue of an animal model. [Figure 11] This shows the results of analyzing the effects of samples on TG in the liver of an animal model. [Figure 12] The results show the effects of the samples on the expression of proteins related to adipogenesis and lipogenesis in the liver of an animal model. [Figure 13] These are the results of an analysis of the effects of the samples on the intestinal microbiota of an animal model.

[0019] In all figures, treatment groups labeled with different letters (e.g., a, b, etc.) indicate that there is a statistical difference at the 95% confidence level between them. For example, if there is group 1 labeled a, group 2 labeled b, and group 3 labeled ab, this means that there is a statistical difference at the 95% confidence level between group 1 and group 3, but there is no statistical difference between group 1 and group 2; and between group 2 and group 3. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will now be described in more detail.

[0021] The present invention provides a pharmaceutical composition for preventing or treating obesity, which comprises a fermentation culture supernatant of a Bacillus veresensis strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof as an active ingredient.

[0022] The strain is also the Bacillus berezensis KMU01 strain deposited under accession number KCTC11751BP.

[0023] The strain name of the Bacillus veresensis KMU01 strain at the time of deposit was Bacillus polyfermenticus KMU01. Specifically, the KMU01 strain was isolated as Bacillus amyloliquefaciens in 2010 and reclassified as Bacillus polyfermenticus in 2018 based on the 16S rRNA gene sequence. Subsequently, experiments were conducted to accurately identify the species of the KMU01 strain, and it was confirmed that the gene sequence of the KMU01 strain showed 97.7% similarity with Bacillus velezensis, and the KMU01 strain has now been 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 2021.05.09).

[0024] Recently, the 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 September 13, 2018).

[0025] The pharmaceutical composition further comprises killed cells or spores of Bacillus veresensis.

[0026] The fermentation metabolites may be short chain fatty acids (SCFAs), organic acids or amino acids.

[0027] The short-chain fatty acid is butyric acid or propionic acid, and the amino acid is phenylalanine, an aromatic amino acid, or valine, a branched-chain amino acid, but is not limited thereto.

[0028] In addition, the pharmaceutical composition can regulate adiponectin secretion.

[0029] In addition, the pharmaceutical composition may regulate one or more intestinal microorganisms selected from the group consisting of, but not limited to, Acetatifactor muris, Mucispirillum schaedleri, and Eubacterium plexicaudatum.

[0030] In addition, the pharmaceutical composition may inhibit the expression of one or more proteins selected from the group consisting of, but not limited to, peroxisome proliferator-activated receptor γ (PPARγ), C / EBPα (CCAAT / enhancer binding protein α), 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).

[0031] The obesity may be one or more selected from the group consisting of visceral obesity, abdominal obesity, general obesity and localized obesity, but is not limited thereto.

[0032] The pharmaceutical composition of the present invention can be prepared in a unit dose form or in a multi-dose container by formulating it with a pharmaceutically acceptable carrier using a method that can be easily carried out by a person skilled in the art.

[0033] The pharmaceutically acceptable carrier is one commonly used in formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0034] In the present invention, the content of the additives contained in the pharmaceutical composition is not particularly limited, and may be appropriately adjusted within the range of the content used in conventional formulations.

[0035] The pharmaceutical composition may be formulated into one or more external skin preparations selected from the group consisting of an injectable dosage form such as an aqueous solution, suspension, or emulsion, a pill, capsule, granule, tablet, cream, gel, patch, spray, ointment, plaster, lotion, liniment, paste, and cataplasm, but is not limited thereto.

[0036] The pharmaceutical compositions of the present invention may contain additional pharmaceutically acceptable carriers and diluents for formulation. Examples of pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbates, cetyl alcohol, and glycerol. Pharmaceutically acceptable carriers and diluents are also biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffer solutions, solvents, and / or dispersion media.

[0037] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method. For oral administration, it can be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, pharmaceutical powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. For parenteral administration, it can be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for spraying, ointments, powders for application, oils, creams, etc.

[0038] The dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition, body weight, age, sex, health condition, dietary habits, properties of the formulation, severity of the disease, administration time, administration method, administration period or interval, excretion rate, and drug form, and can be appropriately selected by those skilled in the art. For example, the dosage range is about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0039] The pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention vary depending on the formulation method, administration mode, administration time, administration route, etc., of the pharmaceutical composition, and those skilled in the art can easily determine or prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or in divided doses.

[0040] The present invention also provides a functional health food composition for preventing or improving obesity, which contains as an active ingredient a fermented culture supernatant of a Bacillus veresensis strain, a concentrate thereof, a dried product thereof, a fermented metabolite thereof, or a mixture thereof.

[0041] The present invention also provides a health functional food composition for reducing body fat or blood cholesterol, which contains as an active ingredient a fermentation culture supernatant of a Bacillus veresensis strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof.

[0042] The strain is also the Bacillus berezensis KMU01 strain deposited under accession number KCTC11751BP.

[0043] The present invention is generally used as a conventional food product.

[0044] The food composition of the present invention can be used as a functional health food. The term "functional health food" refers to a food manufactured and processed using raw materials or ingredients that have functional properties beneficial to the human body as defined by the Act on Functional Health Foods. The term "functional" refers to the food being ingested for the purpose of regulating nutrients for the structure and function of the human body or obtaining beneficial health effects such as physiological effects.

[0045] The functional health food composition may contain common food additives, and unless otherwise specified, the suitability of a food additive shall be determined in accordance with the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety.

[0046] Examples of items listed in the "Food Additives Code" include chemically synthesized substances such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as indigo dye, licorice extract, crystalline cellulose, sorghum pigment, and guar gum; and mixed preparations such as monosodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.

[0047] The food composition of the present invention can be manufactured and processed into the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among capsule-type health functional foods, hard capsules can be manufactured by mixing the composition of the present invention with additives such as excipients and filling them into a conventional hard capsule, while soft capsules can be manufactured by mixing the composition of the present invention with additives such as excipients and filling them into a capsule base such as gelatin. The soft capsules can contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc. as needed.

[0048] The definitions of the terms excipient, binder, disintegrant, lubricant, flavoring agent, flavoring agent, etc. are those described in literature known to those skilled in the art, and include those having the same or similar functions, etc. The type of the food is not particularly limited, and includes any health functional food in the usual sense.

[0049] In the present invention, the term "prevention" refers to any action of suppressing or delaying obesity by administering a composition according to the present invention.

[0050] In the present invention, the term "treatment" refers to any act of administering a composition according to the present invention to reverse or beneficially alter the symptoms of obesity.

[0051] In the present invention, the term "amelioration" refers to any action of administering or having an individual take the composition of the present invention to improve the adverse condition of obesity.

[0052] The present invention also provides a functional food comprising the functional health food composition.

[0053] The present invention also provides a food composition for preventing or improving obesity, comprising as an active ingredient a fermentation culture supernatant of a Bacillus veresensis strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof.

[0054] The strain is also the Bacillus berezensis KMU01 strain deposited under accession number KCTC11751BP.

[0055] The present invention also provides a method for preventing or treating obesity, comprising administering the pharmaceutical composition for preventing or treating obesity to an individual.

[0056] The method for preventing or treating obesity can show an effect of improving obesity or reducing visceral fat by reducing the relative abundance of Acetatifactory muris or Muscispirillum schoedlery strains in the intestine and increasing the relative abundance of Eubacterium plexicaudatum strains in the intestine.

[0057] The present invention will be described in detail below with reference to examples to aid in understanding the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

[0058] [Experimental Example 1] Sample preparation

[0059] To prepare the sample, the KMU01 (Bacillus velezensis KMU01) strain, deposited under the stock accession number KCTC11751BP and stored at a working cell bank at -70°C, was activated and subjected to primary seed culture in test tubes and flasks. Secondary seed culture was then performed in a 50L fermenter with a 20L working volume at 2% (v / v) inoculation for 6 hours. The main culture was performed in a 500L fermenter with a 350L working volume at 2% (v / v) inoculation for 12 hours, with glucose feeding once after 6 hours of culture. After the cultivation was completed, the cell slurry was removed by primary centrifugation using a disk centrifuge at 7200 rpm and 2 L / min, and the supernatant was subjected to two secondary centrifugations using a tubular centrifuge at 15000 rpm and 1.5 L / min to remove the cell cake, after which the supernatant was collected. The collected supernatant was then filtered through a 0.2 μm sterilization filter to obtain a sample from which the bacterial cells had been removed.

[0060] [Experimental Example 2] In vitro experiment

[0061] 2-1. Cell culture and differentiation

[0062] Preadipocyte 3T3-L1 (ATCC, Manassas, VA, USA) fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v / v) fetal bovine serum and 100 μg / mL penicillin-streptomycin at 37°C and 5% CO2. When the cells reached 100% confluence, as shown in Figure 1, they were induced to differentiate into adipocytes using DMEM supplemented with 10% fetal bovine serum (FBS), 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX), 1 μg / mL insulin, and 100 μg / mL penicillin-streptomycin. After 2 days of differentiation, the medium was replaced with 10% FBS medium containing 1 μg / mL insulin and the sample (the medium was replaced every other day), and on the 8th day, the effects on the proliferation and differentiation of adipocytes were analyzed.

[0063] 2-2. Cytotoxicity analysis

[0064] To confirm the cytotoxicity of the samples in adipocytes, MTT assay was performed. Adipocytes (3T3-L1) were treated with the samples at different concentrations (75, 150, and 300 μg / mL), and cell viability was measured by MTT assay.

[0065] 2-3. Analysis of fat accumulation and TG content

[0066] To confirm the effect of the sample on fat accumulation, adipocytes (3T3-L1) were treated with the sample at different concentrations (75, 150, and 300 μg / mL), and the fat accumulation rate of mature adipocytes was measured by staining the adipocytes with Oil-Red O (ORO).

[0067] To confirm the effect of the samples on TG, the TG content accumulated in the cells was measured using a TG quantification kit (Abcam, Cambridge, MA, USA), and the protein content was quantified using BCA (bicinchoninic acid) analysis. The cellular TG content was then normalized to the protein concentration and expressed as a percentage.

[0068] 2-4. Analysis of adipocyte differentiation-related genes and FAS expression

[0069] To confirm the effect of the samples on the expression of adipocyte differentiation-related genes and FAS, 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). The expression of adipocyte differentiation-related genes (PPARγ, C / EBPα, and SREB-1c) and FAS was then analyzed using the StepOnePlus Real-Time PCR (quantitative real-time PCR; qPCR) system (Applied Biosystem). Gene expression levels were normalized using the GAPDH (glycealdehyde-3-phosphate dehydrogenase) gene.

[0070] 2-5.Statistical analysis

[0071] All quantitative analyses were performed using triplicate measurements. Statistical analysis was performed using SPSS (SPSS Inc., USA) software. If significant differences were found by one-way analysis of variance (ANOVA) (P < 0.05), Duncan's multiple comparison test was performed to test for significant differences between treatment groups.

[0072] [Experimental Example 3] In vivo experiment

[0073] 3-1. Preparation of animal models

[0074] Five-week-old C57BL / 6J male mice were purchased from RAONBIO Inc., Republic of Korea. Upon arrival, animals were visually inspected and weighed. General symptoms were observed once daily during the acclimation period. At the end of the acclimation period, animals were weighed and their health status was assessed by monitoring general symptoms and weight changes. Animals were separated into six groups (approximately 10 mice per group) with five mice per cage, ensuring equal average weights. Individual identification was given to the animals' tails using a five-color permanent marker, and individual identification cards were attached to the cages. Animal models were housed for two weeks under conditions of 21–23°C, 40–60% relative humidity, and a 12-hour light / dark cycle (8:00 AM–8:00 PM). Diet and water were provided. Laboratory animal diet (6% fat and 45% fat) (ENVIGO, Research Diets Inc.) was used. Animal experiments were performed according to the Standard Operating Guidelines of Kookmin University, approved by the Ethics Committee for Animal Experiments at Kookmin University, Republic of Korea (KMU-2022-01).

[0075] 3-2. Analysis of body weight and food efficiency

[0076] To confirm the effect of the sample on the body weight and food efficiency of the animal model, the animal model (7 weeks old) prepared in Experimental Example 3-1 was divided into four groups as shown below, and the body weight and food intake were measured at weekly intervals for 13 weeks. The food efficiency was calculated using the following equation 1. Xenical was used as a positive control group. The sample and Xenical were orally administered intragastrically using a disposable syringe equipped with an oral administration probe once daily for 13 weeks from the start of administration.

[0077] 1) Normal diet group (NOR): 6% fat diet intake group

[0078] 2) High-fat diet group (HFD): 45% fat diet intake group

[0079] 3) Sample administration group (B. vele): A group fed a high-fat diet (HFD) and orally administered the sample (114 mg / kg / day).

[0080] 4) Positive control group (Xen): A group fed a high-fat diet (HFD) and administered Xenical (50 mg / kg / day).

[0081] [Number 1] Food efficiency ratio = weight gain (g / week) / food intake (g / week) x 100

[0082] 3-3.Analysis of gymnastics

[0083] To confirm the effect of the sample on the mobility of the animal model, changes in the mobility of the animal model were measured before sacrifice and at 13 weeks using dual energy X-ray absorptiometry (InAlyzer; Medikors Inc., Seongnam, Korea). For the analysis of mobility, the animal model was anesthetized by injection of ketamine (100 mg / kg BW) and xylazine (10 mg / kg BW), and measurements were then performed. Since using ketamine alone, which has an anesthetic effect, can cause side effects due to muscle contractions during the recovery process from anesthesia, it was used in combination with xylazine, a muscle relaxant.

[0084] 3-4. Weight analysis of organs and adipose tissue

[0085] To confirm the effects of the sample on organs and adipose tissue in the animal model, the animal model was fasted for 18 hours and then sacrificed. The animal was then dissected to remove the heart, liver, kidneys, and spleen, and the organs were weighed. The adipose tissue was separated into subcutaneous fat, mesenteric fat, retroperitoneal fat, and epididymal fat, as shown in Figure 2, and the weights were measured.

[0086] 3-5.Blood biochemical analysis

[0087] To assess the effects of the sample on blood glucose, aspartate aminotransferase (GOT; hereafter referred to as AST), alanine aminotransferase (GPT; hereafter referred to as ALT), blood urea nitrogen (BUN), and cholesterol in the animal model, the animal model was sacrificed, and blood was collected via the heart and immediately centrifuged (2000 × g, 10 min) to separate plasma. The plasma was stored in a deep freezer at −80°C until analysis. Blood glucose, AST, ALT, and BUN were measured using a chemical analyzer (Fuji DRI-CHEM 3500i, Fuji Photo Film, Ltd., Tokyo, Japan). Total cholesterol and high-density lipoprotein (HDL)-cholesterol (HDL-C) were measured using LabAssay. TM Cholesterol was measured using a cholesterol kit (Wako, Osaka, Japan), and low-density lipoprotein (LDL) cholesterol (hereinafter referred to as LDL-C) was calculated using the following formula 2. TG was analyzed using a TG assay kit (Abcam, Cambridge, MA).

[0088] [Number 2] LDL-C = Total cholesterol - {(HDL-C) + (TG / 5)}

[0089] 3-6.Histological analysis

[0090] To confirm the effects of the test on adipose tissue in the animal model, epididymal white adipose tissue (eWAT) and liver sections were fixed in 10% formaldehyde, paraffin blocks were prepared, and H&E (Hematoxylin & Eosin) staining was performed. Adipocyte size was calculated by averaging the area of ​​15 adipocytes in the center of representative images using KFBIO Slide Manager (KFBIO, Ningbo, China).

[0091] 3-7. Analysis of TG content in the liver

[0092] To confirm the effect of the sample on TG in the liver of the animal model, the liver tissue of the animal model was crushed, TG was extracted, and then analyzed using a TG assay kit (Abcam).

[0093] 3-8. Analysis of hepatic adipocyte differentiation and lipogenesis-related protein expression

[0094] To assess the effects of the samples on hepatic adipocyte differentiation and lipogenesis-related protein expression in animal models, liver tissue was homogenized using a bullet blender (Next Advance, Troy, NY, USA) in radioimmunoprecipitation assay (RIPA) buffer containing 1% protease inhibitor and 1% kinase inhibitor, and then used in the experiments. The homogenized tissue was incubated at 4°C for 50 minutes and then centrifuged at 4°C and 15,000 x g for 15 minutes to obtain the supernatant. Equal amounts of protein were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST, 0.1%) blocking buffer, sections were incubated overnight at 4°C with antibodies against ACC, p-ACC, FAS, C / EBPα, PPARγ, SCD-1, SREBP-1c, DGAT, and β-actin. After incubation with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature, sections were washed four times with TBST buffer. Protein bands were detected using enhanced chemiluminescence detection kits (BioRad, Hercules, CA, USA), and band intensity was quantified using Image Lab software 5.1 (BioRad) after normalization with β-actin protein.

[0095] 3-9. Analysis of intestinal microorganisms

[0096] To confirm the effect of the samples on the gut microbiota of the animal model, the animal model was sacrificed, the cecum was removed, and analysis was performed using 16s rRNA metagenome sequencing, as shown in Figure 3.

[0097] 3-10.Statistical analysis

[0098] Statistical analysis was performed using GraphPad Prism 9.4.0 (GraphPad Software Inc., San Diego, CA, USA) and SPSS Statistics V.26 (SPSS Inc., Chicago, IL, USA). Significant differences (p < 0.05) between treatment groups were analyzed using one-way analysis of variance (ANOVA) and Duncan and Newman-Keuls multiple comparison tests.

[0099] [Example 1] In vitro experiment

[0100] 1-1. Cytotoxicity analysis

[0101] As a result of analyzing the cytotoxicity of the sample in adipocytes in Experimental Example 2-2, as shown in Figure 4, it was confirmed that there was no significant change in cell viability up to a treatment concentration of 300 μg / mL of the sample (B. vele).

[0102] 1-2. Analysis of fat accumulation and TG content

[0103] The effects of the sample on fat and TG accumulation were analyzed in Experimental Example 2-3. As a result, it was confirmed that the sample (B. vele) significantly suppressed fat and TG accumulation in a concentration-dependent manner, and that the 300 μg / mL treatment group reduced intracellular fat and TG accumulation by approximately 20% and 39%, respectively, as shown in Figure 5. Furthermore, these results confirmed that the KMU01 strain has an anti-obesity effect through the suppression of fat accumulation.

[0104] 1-3. Analysis of adipocyte differentiation-related genes and FAS expression

[0105] Using Experimental Examples 2-4, the effects of the sample on the expression of adipocyte differentiation-related genes and FAS were analyzed. As shown in Figure 6, the sample (B.vele) suppressed the mRNA expression of PPARγ, C / EBPα, SREBP-1c, and FAS. Specifically, in the 300 μg / mL treatment group, the mRNA expression was reduced by 32%, 65%, 46%, and 53%, respectively, compared to the control group.

[0106] [Example 2] In vivo experiment

[0107] 2-1. Analysis of body weight and food efficiency

[0108] In Experimental Example 3-2, the effects of the sample on body weight and food efficiency in the animal model were analyzed. As shown in Figure 7, the high-fat diet group (HFD) showed a significant increase in body weight compared to the normal diet group (NOR), demonstrating the induction of obesity. Furthermore, the sample-administered group (B. vele) showed a significant decrease in body weight compared to the high-fat diet group (p<0.05). The high-fat diet group showed a significantly higher weight gain than the normal diet group, while the sample-administered group showed a significantly lower weight gain than the high-fat diet group (p<0.05). There was no difference in food intake between the high-fat diet and the sample-administered group, but the food efficiency was significantly lower in the sample-administered group (p<0.05). These results suggest that the sample either reduced the digestive and absorptive utilization rate of some nutrients or increased energy consumption.

[0109] 2-2.Analysis of gymnastics

[0110] In Experimental Example 3-3, the effect of the sample on the physical fitness of the animal model was analyzed. As shown in Figure 8, it was confirmed that body fat significantly increased in the high-fat diet group (HFD) compared to the normal diet group (NOR), and that body fat significantly decreased in the sample-treated group (B.vele) and the positive control group (Xen). There were no significant differences in lean mass and bone mineral content between the experimental groups. These results confirmed that the change in body weight due to the sample was due to a decrease in body fat.

[0111] 2-3. Weight analysis of organs and adipose tissue

[0112] The effects of the samples on organs and adipose tissues of the animal model were analyzed in Experimental Examples 3 and 4. As a result, no significant differences were observed in the weights of the heart, liver, and spleen between the experimental groups, as shown in Table 1. Kidney weight increased in the high-fat diet (HFD) group, but this was not statistically significant.

[0113] [Table 1]

[0114] Furthermore, as shown in Figure 9, the weight 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)] was significantly increased in the high-fat diet group (HFD) compared to the general diet group (NOR), and the sample-treated group (B.vele) showed a decrease in the weight of all adipose tissues compared to the high-fat diet group, showing a similar trend to the positive control group (Xen), in which the weight of all adipose tissues was decreased.

[0115] 2-4.Blood biochemical analysis

[0116] The effects of the samples on blood glucose, AST, ALT, BUN, and cholesterol in animal models were analyzed in Experimental Examples 3-5. As shown in Table 2, no significant difference in TG was observed between the normal diet group (NOR) and the high-fat diet group (HFD), but TG was significantly reduced in the positive control group (Xen) compared to the high-fat diet group (p<0.05). Regarding cholesterol, total cholesterol (TCHO) and HDL-C were significantly increased by the high-fat diet, and LDL-C content was significantly reduced in the sample-treated group (B.vele) compared to the high-fat diet group. No significant differences were observed between the experimental groups in indices of blood glucose, liver dysfunction (AST and ALT), and nephrotoxicity (BUN).

[0117] [Table 2]

[0118] 2-5.Histological analysis

[0119] The effects of the sample on adipose tissue in animal models were analyzed in Experimental Examples 3-6. As shown in Figure 10, in epididymal white adipose tissue (eWAT), adipocyte size was significantly increased in the high-fat diet group (HFD) compared to the normal diet group (NOR), and adipocyte size was significantly reduced in the sample-treated group (B.vele) and the positive control group (Xen) compared to the high-fat diet group, demonstrating suppression of adipocyte hypertrophy. Furthermore, in the liver, lipid droplet formation (white dots) was increased in the high-fat diet group compared to the normal diet group, and lipid droplet formation was reduced in the sample-treated group compared to the high-fat diet group.

[0120] 2-6. Analysis of TG content in the liver

[0121] In Experimental Examples 3-7, the effects of the sample on TG in the liver of an animal model were analyzed. As a result, as shown in Figure 11, it was confirmed that the TG content was significantly increased in the high-fat diet group (HFD) compared to the general diet group (NOR), and that the TG content was significantly decreased in the sample-administered group (B.vele) and the positive control group (Xen) compared to the high-fat diet group.

[0122] 2-7. Analysis of hepatic adipocyte differentiation and expression of fat synthesis-related proteins

[0123] In Experimental Examples 3-8, the effects of the sample on hepatic adipocyte differentiation and the expression of lipogenesis-related proteins in an animal model were analyzed. As shown in Figure 12, the sample-treated group (B. vele) showed a significant decrease in the expression of adipocyte differentiation regulatory proteins (C / EBPα and PPARγ) and a decrease in the expression of SREBP-1c, which regulates the expression of lipogenic enzymes ACC and FAS (P<0.05). Furthermore, the expression of FAS, SCD-1, and DGAT, which are involved in the lipogenesis process, was significantly decreased (P<0.05). The phosphorylation of ACC, which inhibits ACC activation, was significantly increased, confirming that the sample contributed to the inhibition of the lipogenesis process. The positive control group (Xen) also showed a similar trend to the sample-treated group (B. vele).

[0124] 2-8. Analysis of intestinal microorganisms

[0125] In Experimental Examples 3-9, the effects of the samples on the intestinal microbiota of the animal model were analyzed. As shown in Figure 13A, α-diversity analysis, which analyzes the diversity of microorganisms present in a single sample, did not reveal significant differences in the Shannon index between the experimental groups. On the other hand, β-diversity analysis based on unweighted UniFrac principal coordinate analysis (PCA), which is frequently used to analyze microbial community patterns, revealed significant differences between the experimental groups. Sample administration altered the intestinal microbial composition to be similar to that of the general diet group (NOR).

[0126] Additionally, the relative abundance of Deferribacterium was significantly increased in the high-fat diet group (HFD) and significantly decreased in the sample-treated and positive control groups (Xen). At the phylum level, structural microbial community analysis confirmed that the high-fat diet increased the relative abundance of Firmicutes while decreasing that of Bacteroidota, resulting in an increased F / B (Firmicutes / Bacteroidota) ratio. The F / B ratio decreased in the sample-treated group. At the family level, the relative abundance of Muribaculaceae was significantly decreased in the high-fat diet group compared to the general diet group. In the sample-treated group, the relative abundance of Lachnospiraceae decreased while the relative abundance of Muribaculaceae increased. At the species level, the relative abundance of Acetatifactor muris and Mucispirillum schaedleri was significantly increased in the high-fat diet group compared to the general diet group. In the sample-administered group, the relative abundance of Acetatifactor muris and Mucispirillum schaedleri was significantly decreased compared to the high-fat diet group, while the relative abundance of Eubacterium plexicaudatum was significantly increased.

[0127] Although the specific details of the present invention have been described above, it is obvious to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention, and the substantial scope of the present invention is defined by the claims and their equivalents.

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Claims

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

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

3. The pharmaceutical composition according to claim 1, further comprising killed bacteria or spores of Bacillus veresensis.

4. The pharmaceutical composition according to claim 1, wherein the fermentation metabolite is a short-chain fatty acid (SCFA), an organic acid, or an amino acid.

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

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

7. The pharmaceutical composition according to claim 1, characterized in that it regulates one or more intestinal microorganisms selected from the group consisting of Acetatifactory muris, Mucispirillum schoedleryi, and Eubacterium plexicaudatum.

8. 2. The pharmaceutical composition according to claim 1, wherein the obesity is at least one selected from the group consisting of visceral obesity, abdominal obesity, general obesity, and localized obesity.

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

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

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

12. A method for preventing or treating obesity, comprising administering to an individual the pharmaceutical composition of claim 1.

13. The method for preventing or treating obesity according to claim 12, characterized in that the method reduces the relative abundance of Acetatifactory muris or Mucispirillum schoedlery strains in the intestine and increases the relative abundance of Eubacterium plexicaudatum strains in the intestine, thereby showing an effect of improving obesity or reducing visceral fat.

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