Bacteroides eggerthii KGMB05336 strain and use thereof

Abstract

The present invention relates to a Bacteroides eggerthii KGMB05336 strain and a use thereof. Through the analysis of the microbiome in patients with non-alcoholic fatty liver disease that is caused by obesity, the Bacteroides eggerthii strain was identified as a significant strain. Oral administration of the Bacteroides eggerthii strain to animal models of obesity and non-alcoholic fatty liver disease caused by obesity resulted in body weight reduction, liver weight reduction, decrease of fat accumulation in visceral and liver tissues, and reduction in serum levels of AST (U / L), ALT (U / L), cholesterol (mg / dL), and glucose (mg / dL), thus demonstrating that the Bacteroides eggerthii strain can serve as a novel means for the prevention, improvement, or treatment of obesity and diseases caused by obesity, such as fatty liver, hepatomegaly, non-alcoholic fatty liver disease, and diabetes.

Description

Bacteroides egerti KGMB05336 strain and its use

[0001] The present invention relates to Bacteroides eggerthii KGMB05336 strain and its use.

[0002] Obesity, once a rare condition, is now a global epidemic. The World Health Organization estimates that 1.4 billion adults worldwide are overweight. Of these, 200 million men and 300 million women are borderline obese. Obesity is associated with a constellation of physiological disorders, including insulin resistance, type 2 diabetes, hypertension, dyslipidemia, cardiovascular disease, and metabolic syndrome. In the United States, obesity-related healthcare costs are estimated at $147 billion annually. Safe and effective interventions to address the health problems and costs associated with obesity are urgently needed.

[0003] In addition, according to the [2019 Cause of Death Statistics] by Statistics Korea, the number of deaths due to liver disease in one year was 6,496, ranking 8th. This report proves that liver disease is the 5th most common cause of death, following cancer, heart disease, pneumonia, and cerebrovascular disease. In Koreans, liver disease is the most important cause of death among the socioeconomically active middle-aged and older generation in their 40s and 50s. In the 40s, the mortality rate due to liver disease is 10.7 per 100,000 people, ranking 3rd after cancer and suicide. Viral and alcoholic diseases are known causes of liver disease, but non-alcoholic fatty liver disease is rapidly increasing due to the increase in obese patients due to Western-style eating habits. Liver disease caused by obesity can progress to cirrhosis. Once cirrhosis develops, the liver is difficult to restore to its original state, even with treatment. It has been reported that 1-3% of cirrhotic patients develop liver cancer each year. Therefore, the best way to prevent cirrhosis and liver cancer is to identify the underlying cause in the asymptomatic stage of obesity or non-alcoholic fatty liver disease and prevent the disease from worsening.

[0004] Meanwhile, the microbiome is a term that refers to the ecosystem of all microorganisms that coexist in our bodies. It is known to perform various functions in the body, such as nutrient breakdown, metabolite production, and maintaining immune homeostasis. The microbiome has a significant impact on health and disease, and is sometimes referred to as the "second genome." Recently, diagnostic methods using the microbiome are being developed. Furthermore, some studies have reported that patients' responses to treatment may vary depending on the composition of the baseline microbiome. Ninety-five percent of all microorganisms in the human body reside in the digestive tract, including the large intestine. Changes in the intestinal microbiome have been reported to contribute to liver damage and fibrosis through increased intestinal permeability, increased levels of intestinal bacteria-derived lipopolysaccharide (LPS), and stimulation of Toll-like receptors (TLRs) in hepatocytes, which activates Kupffer cells, increases inflammatory cytokines such as TNF-α, and changes in bile acids.

[0005] The inventors of the present invention discovered a significant microbiome by performing and analyzing 16S rRNA sequencing in the feces of normal and obese non-alcoholic fatty liver disease patients, and verified the effects of obesity and fatty liver disease in animal models to provide probiotic microorganisms that exhibit therapeutic effects on obesity and diseases caused by obesity.

[0006] [Prior Art Literature]

[0007] [Patent Document]

[0008] Korean Patent Publication No. 10-2015-0118084 (announced on October 21, 2015)

[0009] Korean Patent Publication No. 10-2020-0093165 (announced on August 5, 2020)

[0010] The purpose of the present invention is to newly discover a probiotic strain that exhibits a treatment and improvement effect on obesity and diseases caused by obesity, and to provide a new means for preventing, improving, or treating obesity and diseases caused by obesity, such as fatty liver, hepatomegaly, non-alcoholic fatty liver disease, and diabetes, using the probiotic strain.

[0011] The present invention provides a pharmaceutical composition for preventing or treating obesity or a disease caused by obesity, comprising the Bacteroides eggerthii KGMB05336 strain, a culture or dried product of the strain.

[0012] In addition, the present invention provides a probiotic composition for preventing or improving obesity or a disease caused by obesity, comprising the Bacteroides eggerthii KGMB05336 strain, a culture or dried product of the strain.

[0013] In addition, the present invention provides a health functional food composition for preventing or improving obesity or a disease caused by obesity, comprising the Bacteroides eggerthii KGMB05336 strain, a cultured product or dried product of the strain.

[0014] According to the present invention, as a result of analyzing the microbiome of patients with non-alcoholic fatty liver disease, a disease caused by obesity, the Bacteroides eggerthii strain was identified as a significant strain, and as a result of orally administering the Bacteroides eggerthii strain to an animal model of obesity and non-alcoholic fatty liver disease caused by obesity, body weight was reduced, liver weight was reduced, the degree of fat accumulation in the intestines and liver was improved, and serum AST (U / L), ALT (U / L), cholesterol (mg / dL), and glucose (mg / dL) levels were improved, thereby confirming that the Bacteroides eggerthii strain can be provided as a new means for preventing, improving, or treating fatty liver, hepatomegaly, non-alcoholic fatty liver disease, and diabetes, which are diseases caused by obesity and obesity.

[0015] Figure 1 shows the results of species richness analysis based on the microbiome results of the normal control group (NL) and non-alcoholic fatty liver disease (NAFLD) group.

[0016] Figure 2 shows the results of species richness analysis based on the microbiome results of the normal control group (NL) and the obese group (OB).

[0017] Figure 3 is a schematic diagram of an experiment to establish an animal model of non-alcoholic fatty liver disease caused by obesity and to evaluate the effect of a Bacteroides eggerthii strain.

[0018] Figure 4 shows the results of measuring the change in body weight of a normal diet group (ND) that was fed a normal diet according to the present invention, a negative control group (WD+PBS) that was fed only a Western diet, and an experimental group (WD+B.eggerttii) that was fed a Western diet while being administered Bacteroides eggerthii.

[0019] Figure 5 shows the results of measuring the liver weight of a normal diet group (ND) that was fed a normal diet according to the present invention, a negative control group (WD+PBS) that was fed only a Western diet, and an experimental group (WD+B.eggerttii) that was fed a Western diet while being administered Bacteroides eggerthii.

[0020] Figure 6 shows the results of measuring the degree of visceral fat accumulation in a normal diet group (ND) that was fed a normal diet according to the present invention, a negative control group (WD+PBS) that was fed only a Western diet, and an experimental group (WD+B.eggerttii) that was fed a Western diet while being administered Bacteroides eggerthii.

[0021] Figure 7 shows the results of measuring the degree of liver fat accumulation in a normal diet group (ND) that was fed a normal diet according to the present invention, a negative control group (WD+PBS) that was fed only a Western diet, and an experimental group (WD+B.eggerttii) that was fed a Western diet while being administered Bacteroides eggerthii.

[0022] Figure 8 shows the results of evaluating the pathological changes in liver tissue of a normal diet group (ND) that was fed a normal diet according to the present invention, a negative control group (WD+PBS) that was fed only a Western diet, and an experimental group (WD+B.eggerttii) that was fed a Western diet while being administered Bacteroides eggerthii.

[0023] Figure 9 shows the results of blood tests of a normal diet group (ND) that was fed a normal diet according to the present invention, a negative control group (WD+PBS) that was fed only a Western diet, and an experimental group (WD+B.eggerttii) that was fed a Western diet while being administered Bacteroides eggerthii.

[0024] Figure 10 is a heatmap analysis result analyzing the correlation between genes whose expression decreased upon administration of Bacteroides eggerthii among genes whose expression increased in the Western diet group compared to the normal diet group according to the present invention and the expression of stool microbiome mRNA.

[0025] Figure 11 is a heatmap analysis result analyzing the correlation between genes whose expression increased upon administration of Bacteroides eggerthii among genes whose expression decreased in the Western diet group compared to the normal diet group according to the present invention and the expression of stool microbiome mRNA.

[0026] Figure 12 is a result of analyzing a network of metabolites that showed a significant correlation with the microbiome, which increased in proportion in the Western diet group compared to the normal diet group according to the present invention, but decreased upon administration of Bacteroides eggerthii.

[0027] Figure 13 is a result of analyzing a network of metabolites that showed a significant correlation with the microbiome, which was decreased in proportion in the Western diet group compared to the normal diet group according to the present invention, but increased upon administration of Bacteroides eggerthii.

[0028] The terms used in this specification have been selected from widely used, current terms, taking into account the functions of the present invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their inherent meanings and the overall content of the present invention.

[0029] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0030] Hereinafter, the present invention will be described in more detail.

[0031] The present invention provides a pharmaceutical composition for preventing or treating obesity or a disease caused by obesity, comprising the Bacteroides eggerthii KGMB05336 strain, a culture or dried product of the strain.

[0032] The disease caused by the above obesity may be at least one selected from fatty liver, hepatomegaly, non-alcoholic fatty liver disease, and diabetes.

[0033] When the above pharmaceutical composition is administered orally, it has the effect of reducing body weight, improving fatty liver, and improving serum AST (U / L), ALT (U / L), cholesterol (mg / dL), and glucose (mg / dL) levels.

[0034] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0035] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0036] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0037] The above pharmaceutical composition may be formulated in the form of one or more external preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas.

[0038] The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starches, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinyl pyrrolidone; 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. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media.

[0039] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs. In the case of parenteral administration, it may be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, etc.

[0040] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, age, sex, health status, dietary constitution, nature of the formulation, severity of the disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art. For example, the dosage may range from about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day in divided doses.

[0041] 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 may vary depending on the formulation method, administration method, administration time, and / or administration route of the pharmaceutical composition, and a person skilled in the art can easily determine and prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0042] In addition, the present invention provides a probiotic composition for preventing or improving obesity or a disease caused by obesity, comprising the Bacteroides eggerthii KGMB05336 strain, a culture or dried product of the strain.

[0043] In the present invention, the probiotic composition can be prepared and administered in various formulations and methods according to methods known in the art. For example, it can be prepared and administered in the form of powders, liquids and solutions, tablets, capsules, syrups, suspensions, or granules by mixing with commonly used carriers. The carriers may include, but are not limited to, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, and flavoring agents. In addition, the administration dosage can be appropriately selected depending on the absorption rate of the active ingredient in the body, the inactivation rate, the excretion rate, the age, sex, livestock species, condition, and severity of the disease of the recipient, etc.

[0044] The method for culturing the strain of the present invention can be performed according to a method commonly used in the art, and is not limited to a specific method.

[0045] When the strain or the culture of the strain or the concentrate of the culture solution of the strain obtained in the step of culturing the strain of the present invention is used as an additive, the strain or the culture of the strain or the concentrate of the culture solution of the strain may be added as is or used together with other additives, and may be used appropriately according to a conventional method. The mixing amount of the effective ingredient may be appropriately determined depending on the purpose of use.

[0046] When the probiotics of the present invention are used as food additives, the probiotics may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient mixed may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the probiotics of the present invention are added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the mixed amount may be below the above range, and since there is no problem in terms of safety, the mixed amount may also be used in an amount exceeding the above range.

[0047] There are no specific restrictions on the types of foods mentioned above. Examples of foods to which probiotics can be added include dairy products, including bread, candy, snacks, confectionery, gum, and ice cream, as well as various soups, beverages, tea, drinks, and vitamin complexes. These foods include all health foods in the conventional sense.

[0048] In addition, the present invention provides a health functional food composition for preventing or improving obesity or a disease caused by obesity, comprising the Bacteroides eggerthii KGMB05336 strain, a cultured product or dried product of the strain.

[0049] The present invention can be generally used as a commonly used food.

[0050] The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients with functional properties beneficial to the human body, as defined by the Health Functional Food Act. "Functionality" refers to ingestion for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients for the structure and functions of the human body or physiological effects.

[0051] The food composition of the present invention may include conventional food additives, and its suitability as the "food additive" is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0052] Items listed in the above "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0053] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc.

[0054] For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a composition according to the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be manufactured by mixing the composition according to the present invention with additives such as excipients and filling it into a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as needed.

[0055] The definitions of terms for the above excipients, binders, disintegrants, lubricants, flavoring agents, etc. are described in literature known in the art and include those with identical or similar functions. There are no specific restrictions on the type of food, and all health functional foods in the conventional sense are included.

[0056] As used herein, the term "prevention" refers to any action that suppresses or delays the onset of a disease by administering a composition according to the present invention. The term "treatment" refers to any action that improves or beneficially alters the symptoms of a disease by administering a composition according to the present invention. As used herein, "improvement" refers to any action that improves the worsening condition of a disease by administering or ingesting a composition according to the present invention to a subject.

[0057] In the present invention, the treatment means an approach to obtain a beneficial or desirable clinical outcome, and for the purposes of the present invention, beneficial or desirable clinical outcomes may include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (i.e., not worsening) of the disease state, delay or reduction in the rate of disease progression, improvement or palliation and alleviation (partial or total), whether detectable or undetectable, of the disease state, and may also mean an increase in survival compared to the expected survival rate in the absence of treatment. Treatment refers to both therapeutic treatments and preventative or preventative measures. The treatments include treatment required for disorders that have already occurred as well as the disorder being prevented. "Palliating" a disease means a reduction in the extent and / or undesirable clinical signs of the disease state and / or a slowing or lengthening of the time course of progression compared to the absence of treatment.

[0058] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0059]

[0060] Example 1. Discovery of Microbiome Markers

[0061] 1-1. Target audience

[0062] To analyze the microbiome associated with obesity and diseases caused by obesity, subjects for microbiome analysis were selected from among patients diagnosed with obesity and those with non-alcoholic fatty liver disease, a type of disease caused by obesity.

[0063] From October 12, 2018, to July 29, 2021, we obtained fecal and clinical data from patients with non-alcoholic fatty liver disease and healthy controls who visited Ajou University Hospital. Adults aged 19 years or older who visited the outpatient clinic of the Department of Gastroenterology or who visited the clinic through a recruitment notice agreed to the collection of blood and fecal samples (information obtained) and conducted the experiment under research approval (AJOUIRB-SUR-2019-096). Subjects who did not meet the following exclusion criteria were included.

[0064] 1. HBV, HCV, HIV positive

[0065] 2. Those with autoimmune hepatitis, Wilson's disease, primary biliary cholangitis, etc.

[0066] 3. Those who are being treated for liver cancer or other organ cancer or have been diagnosed with it within the past 5 years.

[0067] 4. Those with an active infection or taking antibiotics

[0068] 5. Those who drink excessively (men: 30g / day, women: 20g / day)

[0069] Fecal samples and clinical information were obtained from Ajou University Hospital from 50 healthy adult normal controls (NL) and 148 patients diagnosed with nonalcoholic fatty liver disease (NAFLD). Among the 148 patients, those with a BMI ≥ 25 kg / m 2 Patients were classified as obese (OB) and recognized as patients with non-alcoholic fatty liver disease (NAFLD) caused by obesity. The subjects were provided with stool bags for stool collection and were explained about the collection method and storage. Stool collected within 1 to 3 days of outpatient visit was stored frozen at home, and stool samples (5 g) were collected from the subjects at the next outpatient visit. When collecting stool samples, clinical information such as presence of NAFLD, liver fibrosis test, body fat test, liver tissue test, gender, age, height, weight, comorbidities, dietary habits, drinking / smoking history, medication history, and blood test results (CBC, biochemical test, lipid panel, insulin level, c-peptide, HbA1C) were also collected. The collected clinical information and blood and stool samples were deposited in the Human Resources Bank and stored frozen.

[0070]

[0071] 1-2. Microbiome Analysis

[0072] To select microbiome markers associated with non-alcoholic fatty liver disease and obesity from the collected stool samples, 16S rRNA (ribosomal RNA) sequence analysis and metagenomics sequencing were used. The stool samples were diluted in 10 mL of phosphate-buffered saline for 24 hours, filtered through a cell strainer, and centrifuged at 10,000 xg for 10 minutes at 4°C to separate the pellet containing bacteria and the supernatant containing EV. The EV sample was sterilized and purified through a 0.22 μm filter to completely remove bacteria and foreign substances. The bacterial and EV samples were boiled at 100°C for 40 minutes, and the supernatant was collected after 30 minutes and centrifuged at 18,214 xg at 4°C to remove any remaining suspended particles and waste. Next, DNA was extracted from the bacterial and bacterial EV membranes using the DNeasy Power Soil kit (QIAGEN).

[0073] To analyze the microbiome from the DNA of microorganisms extracted from the specimens, amplicon sequencing was performed on the 16S rRNA of each strain using Illumina's MiSeq next-generation sequencing platform. Bacterial genomic DNA was amplified with primers targeting the V3-V4 hypervariable region of 16S rDNA (16S_V3_F 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3' SEQ ID NO: 1 and 16S_V4_R 5'-GTCTCTCGTGGGCTCGGATCCGT-3' SEQ ID NO: 2). The cycling conditions were 95°C for 3 min (35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s; 72°C for 5 min). Libraries were prepared using polymerase chain reaction (PCR) products according to the MiSeq system instructions (Illumina) and quantified using QIAxpert (QIAGEN). Each amplicon was then quantified, set to equimolar ratios, and pooled and sequenced on a MiSeq platform (Illumina) according to the manufacturer's recommendations. The gut microbiome was profiled using operational taxonomy unit (OTU) clustering and taxonomy profiling using 16S rRNA, internationally widely used tools such as UCLUST and QIIME for 16S rRNA sequence clustering, and 16S rRNA sequence databases such as GreenGenes, RDP, and Silva.

[0074]

[0075] 1-3. Microbiome Analysis Results

[0076] Alpha diversity, which represents the species richness of the sample, and beta diversity, which represents the similarity between samples, were analyzed based on the microbiome results of the normal control group (NL), non-alcoholic fatty liver disease (NAFLD), and obese group (OB). Among the alpha diversity indices, ACE estimates the species richness based on the information of the discovered species, considering the possibility that there may be species that are not found or visible in the sample. Shannon is a value that estimates the diversity of species present in the sample, with a higher value indicating greater diversity. The microbiome species diversity indices in the normal control group (NL), non-alcoholic fatty liver disease (NAFLD), and obese group (OB) were compared, and based on the comparison results, LEfSe (Linear discriminant analysis Effect Size) analysis was performed to identify statistically significant characteristics between specific groups. LEfSe (Linear discriminant analysis Effect Size) analysis was performed using the EZbioCloud program.<Kruskal-Wallis H test> We used this to find significant taxa and calculated the LDA score using the actual relative taxonomic composition value difference between groups.

[0077] As shown in Fig. 1, the results of comparing the microbiomes of the normal control group (NL) and the non-alcoholic fatty liver disease (NAFLD) group showed a lower species diversity index than the normal control group (NL). Based on the above results in the LEfSe analysis, significant taxa in the non-alcoholic fatty liver disease (NAFLD) group were selected with p-value < 0.01, LDA effect size >= 3 or higher, and taxa that were 'species' for animal model evaluation, and the microbial species shown in Table 1 below were selected.

[0078] Taxon nameTaxon rankLDA effect sizep-valueNLNAFLDBacteroides uniformisSpecies4.088980.008884.5342.3Blautia wexleraeSpecies4.046320.002312.1044.45946Alistipes putredinisSpecies3.859550.001373.1021.43649PAC001129_sSpecies3.691690.022651.0860.28919Ruminococcus gnavusSpecies3.59620.000870.231.03311PAC001654_sSpecies3.45620.000050.590.01216Bacteroides xylanisolvensgroupSpecies3.438640.001630.8940.38176Blautia hanseniigroupSpecies3.39550.028810.1860.64932Lactobacillus rogosaegroupSpecies3.385090.004440.6240.17432Bacteroides intestinalisSpecies3.359150.000620.480.0223Bacteroides eggerthiiSpecies3.356140.00950.4440.04257Bacteroides stercorisSpecies3.273780.047741.261.29932Coprococcus eutactusgroupSpecies3.273170.00140.6720.20338PAC001040_sSpecies3.240380.023210.5860.26351PAC001157_sSpecies3.204791.213E-070.380.06486Faecalimonas umbilicataSpecies3.203210.006770.0480.34054Alistipes shahiiSpecies3.199310.008250.5720.23851JPJG_sSpecies3.169010.008690.5240.23243PAC001242_sSpecies3.080470.000180.3740.04324KQ235806_s groupSpecies3.079660.007090.3760.16892PAC000195_sSpecies3.066710.000040.4220.19122Ruminococcus bromiiSpecies3.009450.0280.90.85541Roseburia intestinalisgroupSpecies3.004880.014950.7260.54662.

[0079] As shown in Fig. 2, the microbiomes of the normal control group (NL) and the obese group (OB) were compared, and the species diversity index was found to be lower in the obese group (OB) compared to the normal control group (NL). Based on the above results in the LEfSe analysis, significant taxa in the obese group (OB) were selected with a p-value < 0.01, LDA effect size >= 3 or higher, and taxa that were 'species' for animal model evaluation, and the microbial species shown in Table 2 below were selected.

[0080] Taxon nameTaxon rankLDA effect sizep-valueNLObeseBlautia wexleraeSpecies4.157360.000712.1044.95273Bacteroides uniformisSpecies3.998530.012974.5342.23818Alistipes putredinisSpecies3.936760.001723.1021.43182Ruminococcus gnavusSpecies3.67560.000530.231.24455Faecalibacterium prausnitziigroupSpecies3.656470.034075.4784.62455PAC001129_sSpecies3.579960.012071.0860.24364Bacteroides xylanisolvensgroupSpecies3.511110.005510.8940.31636PAC001654_sSpecies3.454940.000040.590.01Coprococcus eutactusgroupSpecies3.372230.005260.6720.23Bacteroides intestinalisSpecies3.357070.000830.480.01364Bacteroides eggerthiiSpecies3.338730.020610.4440.05273Lactobacillus rogosaegroupSpecies3.31760.004320.6240.18818Faecalimonas umbilicataSpecies3.269260.001150.0480.45Blautia hanseniigroupSpecies3.268180.031740.1860.53818HF545616_sSpecies3.262890.004090.390.10727PAC001242_sSpecies3.261150.00020.3740.03455JPJG_sSpecies3.250670.005990.5240.22364PAC001157_sSpecies3.240584.793E-070.380.06727Alistipes shahiiSpecies3.217180.010550.5720.25455PAC000195_sSpecies3.132471.458E-060.4220.14909PAC001040_sSpecies3.12820.024160.5860.3Ruminococcus bromiiSpecies3.117970.011630.90.87636Bacteroides cellulosilyticusSpecies3.100140.00060.3220.09455PAC001187_sSpecies3.095940.006530.2880.04364Gemmiger formicilisgroupSpecies3.084540.021890.6660.42182KQ235806_s groupSpecies3.079060.020570.3760.21545Eubacterium eligensgroupSpecies3.050392.059E-070.3460.13909PAC001034_sSpecies3.017350.045260.2580.09909.

[0081] In the above results, the strains commonly selected as significant strains in the non-alcoholic fatty liver disease (NAFLD) and obesity (OB) groups compared to the normal control group (NL) were Bacteroides uniformis, Blautia wexlerae, Alistipes putredinis, PAC001129_s, Ruminococcus gnavus, PAC001654_s, Bacteroides xylanisolvens group, Blautia hansenii group, Lactobacillus rogosae group, Bacteroides intestinalis, Bacteroides eggerthii, and Coprococcus. The strains were identified as Coprococcus eutactus group, PAC001040_s, PAC001157_s, Faecalimonas umbilicate, Alistipes shahii, JPJG_s, PAC001242_s, KQ235806_s group, PAC000195_s, and Ruminococcus bromii. Among these, Bacteroides eggerthii was selected as the final microbiome marker.

[0082]

[0083] Example 2. Efficacy evaluation of Bacteroides eggerthii KGMB05336

[0084] 2-1. Cultivation of Bacteroides eggerthii

[0085] Based on the results of Example 1, in order to confirm whether Bacteroides eggerthii is an effective strain for obesity or non-alcoholic fatty liver disease, a commercially available Bacteroides eggerthii strain, Bacteroides eggerthii KGMB05336, was purchased from the Korean Gut Microbiome Bank (KGMB). The received Bacteroides eggerthii KGMB05336 strain was inoculated into a medium mixed with TSA (Tryptic soy agar) (MB-T0689) and 5% HBD (Horse Blood Defibrinated) (MB-H1883), and anaerobically cultured at 37°C in an anaerobic jar containing an MGC anaerobic pack. OD 600 The viable cell count was measured based on the value to determine CFU / mL. Bacteroides eggerthii KGMB05336 was deposited with the Korean Collection for Type Cultures (KCC) on August 29, 2023, and was issued the accession number KCTC15573BP.

[0086]

[0087] 2-2. Animal model of nonalcoholic fatty liver disease (NAFLD)

[0088] To determine whether Bacteroides eggerthii KGMB05336 strain improves obesity and non-alcoholic fatty liver disease (NAFLD) induced by obesity, we established an animal model of NAFLD induced by obesity (Fig. 3). Five-week-old C57BL / 6N male mice were obtained and fed a normal diet for one week to allow them to adapt to the new environment. The mice were divided into three groups: the normal diet group (ND) was fed a normal diet, and the negative control group and the experimental group were fed a Western diet (TD88137, Dooyeol biotech, Seoul, Korea) to induce obesity. The Western diet is a diet high in fat, sugar, and cholesterol, and is used to induce diseases such as obesity, non-alcoholic steatohepatitis, and diabetes in mouse animal models. The composition of the above Western diet is 42% fat, 42.7% carbohydrate, and 15% protein, and all mice were allowed to drink water freely. The normal diet group (ND) was fed only the normal diet, the negative control group (WD+PBS) was fed the Western diet and orally administered 200 μL of PBS 5 times / week for 12 weeks, and the experimental group (WD+B. eggerttii) was fed the Western diet and 10 times the Bacteroides eggerthii KGMB05336 strain. 8 The solution was diluted in PBS at 1 CFU / mouse and administered orally at 200 μL 5 times / week for 12 weeks. The body weights of the mice were measured once a week after the start of oral administration.

[0089] After the experiment was completed and before the mice were necropsied, blood from each mouse was collected by orbital blood sampling using heparinized capillary tubes. The collected blood was centrifuged at 3,000 rpm for 10 minutes to collect serum, which was stored at -80℃. On the day of necropsy, body weights were measured, laparotomy photographs were taken, and liver tissue was extracted and photographed, preserved in 10% neutral buffered formalin, and histopathological examination (H&E, Masson's trichrome staining) was performed.

[0090]

[0091] 2-3. Confirmation of the efficacy of Bacteroides eggerthii in an animal model of fatty liver disease.

[0092] As shown in Fig. 4, the body weight changes observed up to the 13th week were statistically significantly greater in the negative control group (WD+PBS) and experimental group (WD+B. eggerttii) fed a Western diet than in the normal diet group (ND) fed a normal diet. However, the experimental group (WD+B. eggerttii) fed a Western diet and administered Bacteroides eggerthii showed a significant decrease in body weight compared to the negative control group (WD+PBS) fed only a Western diet. The above results demonstrate that diet-induced obesity is treated and improved by administering Bacteroides eggerthii.

[0093] As shown in Fig. 5, the liver weight measurement results showed that the negative control group (WD+PBS) and the experimental group (WD+B.eggerttii) fed a Western diet showed a statistically significant increase in liver weight compared to the normal diet group (ND) fed a normal diet. However, the experimental group (WD+B.eggerttii) fed a Western diet and administered Bacteroides eggerthii showed a significant decrease in liver weight compared to the negative control group (WD+PBS) fed only a Western diet. The above results demonstrate that diet-induced obesity and obesity-induced hepatomegaly are treated and improved by administering Bacteroides eggerthii.

[0094] As a result of observing the fat content by cutting open the mice after the experiment was over, as shown in Fig. 6, the fat content increased significantly in the negative control group (WD+PBS) and experimental group (WD+B. eggerttii) fed a Western diet compared to the normal diet group (ND) fed a normal diet. However, in the experimental group (WD+B. eggerttii) fed a Western diet and administered Bacteroides eggerthii, the degree of fat accumulation in the intestines was confirmed to be less increased than in the negative control group (WD+PBS) fed only a Western diet. The above results demonstrate that diet-induced obesity is treated and improved by administering Bacteroides eggerthii.

[0095] In addition, as shown in Fig. 7, when the liver color of the mice was observed, the color turns yellow and enlarges as fat accumulates in the liver. Compared to the normal diet group (ND) fed a normal diet, the negative control group (WD+PBS) fed a Western diet showed a yellow color and a significant increase in size. However, in the experimental group (WD+B. eggerttii) fed a Western diet and administered Bacteroides eggerthii, the liver color was close to normal and the size was relatively similar to normal compared to the negative control group (WD+PBS) fed only a Western diet. The above results demonstrate that diet-induced obesity and obesity-induced fatty liver and hepatomegaly are treated and improved by administering Bacteroides eggerthii.

[0096]

[0097] 2-4. Evaluation of pathological changes in liver tissue

[0098] The effects of Bacteroides eggerthii KGMB05336 strain on liver tissue pathology were evaluated. Liver tissues were collected from a mouse model of a control group fed a normal diet (Normal Diet, ND), an experimental group fed a Western diet (Western diet + PBS, WD), and an experimental group fed a Western diet plus the Bacteroides eggerthii KGMB05336 strain (WD + Strain), and then H&E stained and analyzed. As shown in Fig. 8, in the experimental group fed a Western diet, fat deposition within hepatocytes was confirmed to be aggravated, whereas in the experimental group fed the Bacteroides eggerthii KGMB05336 strain, fat deposition was confirmed to be improved. In addition, as a result of evaluating NAS (non-alcoholic fatty liver disease activity score), NAS increased and worsened in the experimental group fed a Western diet, but the level of NAS decreased in the experimental group fed the Bacteroides eggerthii KGMB05336 strain. In addition, as a result of comparing the degree of fibrosis, no fibrosis was observed at all in the normal diet group (ND), but in the experimental group fed a Western diet (WD), stages 1a, 1b, and 2 were observed in 10, 3, and 1 animal, respectively, whereas in the experimental group fed the Bacteroides eggerthii KGMB05336 strain (WD+Stain), stages 1a and 1b were observed in 8 and 1 animal, respectively, confirming that fibrosis was improved.

[0099]

[0100] 2-5. Blood test results

[0101] To evaluate the effects of Bacteroides eggerthii KGMB05336 strain on plasma factors, blood tests were performed to analyze changes in the levels of aspartate aminotransferase (AST), alanine transaminase (ALT), total bilirubin, glucose, cholesterol, triglyceride, high density lipoprotein (HDL) cholesterol, and low density lipoprotein (LDL) cholesterol related to liver function. As shown in Fig. 9, in the experimental group (WD) fed a Western diet, the levels of AST, ALT, cholesterol, blood glucose, triglyceride, HDL, and LDL cholesterol were significantly increased compared to the control group (ND). On the other hand, in the experimental group (WD+B) fed with the Bacteroides eggerthii KGMB05336 strain, the levels of AST, ALT, cholesterol, blood sugar, triglyceride, HDL, and LDL, which increased with a Western diet, were significantly reduced.

[0102] In non-alcoholic fatty liver disease, serum AST (U / L) and ALT (U / L) levels increase, and the risk of non-alcoholic fatty liver disease is measured by the serum AST (U / L) and ALT (U / L) levels. The results above demonstrate that the serum AST (U / L) and ALT (U / L) levels increased by a Western diet were reduced by administration of Bacteroides eggerthii, demonstrating that Bacteroides eggerthii has preventive, therapeutic, and ameliorating effects on obesity-induced non-alcoholic fatty liver disease. In addition, the risk of diabetes is measured by the cholesterol (mg / dL) and glucose (mg / dL) levels. The above results show that the levels of cholesterol (mg / dL) and glucose (mg / dL) increased by a Western diet were reduced by administering Bacteroides eggerthii, demonstrating that Bacteroides eggerthii has a preventive, therapeutic, and ameliorating effect on diabetes induced by obesity.

[0103]

[0104] 2-6. Transcriptome analysis in liver tissue

[0105] To assess the impact of Bacteroides eggerthii KGMB05336 strain on changes in the liver microbiota, we analyzed the transcriptome of liver tissue. Microbiome analysis was performed using mouse feces, and transcriptome analysis was performed using mouse liver tissue. The two omics data sets were integrated for analysis.

[0106] In the experimental group fed a Western-style diet (WD), we analyzed the correlation between candidate genes whose expression increased and then decreased upon feeding with the Bacteroides eggerthii KGMB05336 strain and microbiome mRNA expression. As shown in Fig. 10, among the 117 candidate genes whose expression increased upon feeding with a Western-style diet but decreased upon feeding with the Bacteroides eggerthii KGMB05336 strain, 82 genes were identified to show a positive or negative correlation with microbiome mRNA expression.

[0107] In addition, in the experimental group fed a Western-style diet (WD), the correlation between candidate genes whose expression decreased and then increased upon feeding with the Bacteroides eggerthii KGMB05336 strain and microbiome mRNA expression was analyzed. As shown in Fig. 11, among 105 candidate genes whose expression decreased upon feeding with a Western-style diet but increased upon feeding with the Bacteroides eggerthii KGMB05336 strain, 68 genes were identified to show a positive or negative correlation with microbiome mRNA expression.

[0108]

[0109] 2-7. Microbiome and Metabolome Network Analysis

[0110] To analyze the effects of Bacteroides eggerthii KGMB05336 strain on microbiome and metabolome changes, primary metabolites and bile acids were analyzed using feces, and then the correlation with the fecal microbiome was analyzed.

[0111] We analyzed the network of metabolites related to the microbiome, which decreased in the experimental group fed a Western-style diet (WD), but increased when fed the Bacteroides eggerthii KGMB05336 strain. As shown in Figure 12, in Bacteroides, 2-Hydroxylsocaproic acid showed a positive correlation, 1-hexadecanol and 1-pentadecanol showed a negative correlation, and in Lactobacillus, gamma-Tocopherol, 1, 2-Propanediol, delta-Tocopherol, Linoleic acid, 1-Monomyristin, 3-Hydroxybutyric acid and phosphoric acid. 5-Nonadecylresorcinol, pyrimidine, and xylose showed representative positive correlations, and the KE159538_g strain showed positive correlations with palmitic acid, 5-heptadecylresorcinol, and phloretic acid. Bacteroides eggerthii appears to be involved in the metabolic pathway of 2-hydroxylsocaproic acid or promotes its production. This appears to be consistent with previous studies that some intestinal bacteria can produce organic acids or regulate their levels.Furthermore, the negative correlations with 1-hexadecanol and 1-pentadecanol suggest that feeding Bacteroides eggerthii may utilize or inhibit the production of these compounds. Fatty alcohols are involved in lipid metabolism, and changes in their levels may reflect changes in gut microbiota metabolism and lipid absorption.

[0112] In addition, we analyzed the network of metabolites related to the microbiome, which increased in the experimental group fed a Western-style diet (WD) but decreased when fed the Bacteroides eggerthii KGMB05336 strain. As shown in Fig. 13, KE159571_g, Mucispirillum, KE159600_g, and Alistipes strains showed positive or negative correlations with 20 fatty acids, 2 carbohydrates, and 4 other metabolites. KE159600_g showed a negative correlation with xylose, and KE159600_g or Alistipes showed a positive correlation with ferulic acid and most fatty acids. The correlation between specific gut microbes and metabolites suggests that their metabolic activity influences the production or consumption of metabolites. For example, Alistipes shows a positive correlation with various fatty acids, suggesting that it may play a key role in fatty acid metabolism.

[0113] In conclusion, the above results demonstrate that Bacteroides eggerthii has preventive, therapeutic, and ameliorating effects on diet-induced obesity and obesity-induced fatty liver, hepatomegaly, non-alcoholic fatty liver disease, and diabetes.

[0114] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0115] Numerical ranges are inclusive of the values ​​defined in the ranges above. Any maximum numerical limitation given throughout this specification includes any lower numerical limitation, as if that lower numerical limitation were explicitly stated. Any minimum numerical limitation given throughout this specification includes any higher numerical limitation, as if that higher numerical limitation were explicitly stated. Any numerical limitation given throughout this specification will include any better numerical range within the broader numerical range, as if that narrower numerical limitation were explicitly stated.

[0116] [Microbial Deposit]

[0117] Name of depositor: Korea Research Institute of Bioscience and Biotechnology

[0118] Accession number: KCTC15573BP

[0119] Date of acceptance: August 29, 2023.

Claims

1. A pharmaceutical composition for preventing or treating obesity or a disease caused by obesity, comprising the Bacteroides eggerthii KGMB05336 strain with the accession number KCTC15573BP, or a culture or dried product of the strain.

2. A pharmaceutical composition for preventing or treating obesity or a disease caused by obesity, characterized in that the disease caused by obesity in the first paragraph is at least one selected from fatty liver, hepatomegaly, non-alcoholic fatty liver disease, and diabetes.

3. A pharmaceutical composition for preventing or treating obesity or a disease caused by obesity, characterized in that the pharmaceutical composition has a weight-reducing effect when administered orally in the first paragraph.

4. A pharmaceutical composition for preventing or treating obesity or a disease caused by obesity, characterized in that the pharmaceutical composition according to claim 1 has an effect of improving fatty liver when administered orally.

5. A pharmaceutical composition for preventing or treating obesity or a disease caused by obesity, characterized in that the pharmaceutical composition according to claim 1 has an effect of improving serum AST (U / L), ALT (U / L), cholesterol (mg / dL), and glucose (mg / dL) levels when orally administered.

6. A probiotic composition for preventing or improving obesity or diseases caused by obesity, comprising the strain Bacteroides eggerthii KGMB05336, accession number KCTC15573BP, or a culture or dried product of the strain.

7. A probiotic composition for preventing or improving obesity or a disease caused by obesity, characterized in that the disease caused by obesity in paragraph 6 is at least one selected from fatty liver, hepatomegaly, non-alcoholic fatty liver disease, and diabetes.

8. A health functional food composition for preventing or improving obesity or a disease caused by obesity, comprising the strain Bacteroides eggerthii KGMB05336, accession number KCTC15573BP, or a culture or dried product of the strain.

9. A health functional food composition for preventing or improving obesity or a disease caused by obesity, characterized in that the disease caused by obesity in the 8th paragraph is at least one selected from fatty liver, hepatomegaly, non-alcoholic fatty liver disease, and diabetes.

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