KR20220041406A - Composition For Improving, Preventing Or Treating Liver Diseases Using Human Intestinal Microbiome, And Treating Method Of Liver Diseases Using The Same - Google Patents

Abstract

The present invention relates to a composition for predicting or diagnosing risk of liver disease using intestinal microorganisms, a kit for predicting or diagnosing the risk of liver disease including the same, an information providing method for predicting or diagnosing risk of liver disease, and a method for screening liver disease preventive or therapeutic agents, so as to predict or diagnose the risk of liver disease and screen liver disease preventive or therapeutic agents through an agent capable of detecting one or more strains selected from the group consisting of strains of Bacteroides uniformis, stains of Bacteroides caccae, and strains of Parabacteroides.

Description

Composition For Improving, Preventing Or Treating Liver Diseases Using Human Intestinal Microbiome, And Treating Method Of Liver Diseases Using The Same

The present invention relates to a pharmaceutical composition for preventing or treating liver disease using intestinal microorganisms, a food composition for preventing or improving liver disease using the same, and a method for treating liver disease using the same.

With the development of next-generation sequencing techniques, it has become possible to quickly and accurately measure the types and relative amounts of microorganisms living in an individual's gut. In particular, this technology makes it possible to find out the type and relative amount of microorganisms living in an individual's intestines using the 16S ribosomal RNA gene variable region sequence, which is a conserved region common to intestinal microbes, and the information can be used as personal disease information or information. Research is being conducted to determine the association with biomarkers related to disease information.

US Patent Publication No. 2019-0127781 relates to a composition for diagnosing liver disease, Group A Dorea sp. CAG:317, Bacteroides cellulosilyticus, Bacteroides cellulosilyticus ( Bacteroides cellulosilyticus ) Bacteroides finegoldii ), Bacteroides dorei , Streptococcus parasanguinis , Clostridium symbiosum , Clostridium sp. 7_3_54FAA, and Clostridium sp. Clostridium bolteae ) is presented.

Korean Patent Application Laid-Open No. 2019-0037170 suggests that the Roseburia spp. strain is effective in relation to the prevention and improvement of alcoholic liver disease-related diseases.

Korean Patent No. 1940425 proposes a method for diagnosing liver disease through metagenome analysis using extracellular vesicles secreted from bacteria from blood or urine samples as samples.

However, the results presented in the above patents are between the high-risk group for liver disease with an alanine aminotransferase (ALT) of 40 IU/L or more and the normal group with an aminotransferase (Alanine aminotransferase, ALT) of less than 40 IU/L. Based on the microbiome obtained from 890 Koreans in the biomarker microorganisms that can distinguish There was a difference from the research results conducted by the present inventors who identified the correlation of

US Patent Publication No. 2019-0127781 Korean Patent Publication No. 2019-0037170 Korean Patent No. 1940425

The present invention relates to any one or more strains selected from the group consisting of Bacteroides uniformis species strains, Bacteroides caccae species strains and Parabacteroides genus strains, the strains of To provide a pharmaceutical composition for preventing or treating liver disease comprising a dead cell or a culture of the strain as an active ingredient.

In addition, the present invention provides any one or more strains selected from the group consisting of Bacteroides uniformis species strains, Bacteroides caccae species strains and Parabacteroides genus strains, and the strains thereof It is to provide a food composition for preventing or improving liver disease comprising the dead cells of, or a culture of the strain as an active ingredient.

In addition, the present invention is a therapeutically effective amount of a patient with liver disease Bacteroides uniformis ( Bacteroides uniformis ) species strain, Bacteroides caccae ) species strain and Parabacteroides ) Selection from the group consisting of spp. It is to provide a method for treating liver disease by administering any one or more strains, dead cells of the strain, or a culture of the strain.

The present invention relates to any one or more strains selected from the group consisting of Bacteroides uniformis species strains, Bacteroides caccae species strains and Parabacteroides genus strains, the strains of It relates to a pharmaceutical composition for preventing or treating liver disease comprising a dead cell or a culture of the strain as an active ingredient.

In addition, the present invention provides any one or more strains selected from the group consisting of Bacteroides uniformis species strains, Bacteroides caccae species strains and Parabacteroides genus strains, and the strains thereof It relates to a food composition for preventing or improving liver disease comprising a dead cell of, or a culture of the strain as an active ingredient.

In addition, the present invention is a therapeutically effective amount of a patient with liver disease Bacteroides uniformis ( Bacteroides uniformis ) species strain, Bacteroides caccae ) species strain and Parabacteroides ) Selection from the group consisting of spp. It relates to a method for treating liver disease by administering any one or more strains, dead cells of the strain, or a culture of the strain.

Alanine aminotransferase (ALT) is an enzyme present in hepatocytes, and its concentration is known to increase when hepatocytes are damaged. In the present invention, 890 Korean subjects were treated with a blood alanine aminotransferase (ALT) of 40 IU/L or higher in the liver disease risk group, and a blood Alanine aminotransferase (ALT) of less than 40 IU/L. Classify into normal group, and search for microorganisms whose average ratio of the microorganisms among the total intestinal microbes becomes smaller or larger in the liver disease group and normal group, and find a statistically significant difference in ratio between the liver disease group and the normal group among the detected microorganisms. The indicated microorganisms were specified as biomarker microorganisms for predicting or diagnosing liver disease risk.

Among the biomarker strains for predicting or diagnosing liver disease, by specifying microorganisms having an average ratio of the corresponding microorganisms among all intestinal microorganisms lower than the normal group in liver disease, and administering these microorganisms to individuals in need of liver disease improvement, prevention or treatment It was determined that the relative ratio or absolute value of these microorganisms among the total intestinal microorganisms can be adjusted similarly to that of the normal group, and through this, these microorganisms can be utilized for improvement, prevention or treatment of liver disease.

As biomarker microorganisms for predicting or diagnosing the risk of liver disease showing a statistically significant difference between the liver disease group and the normal group, Bacteroides uniformis species strain, Bacteroides caccae species Strains and Parabacteroides ( Parabacteroides ) It may be any one or a combination of two or more strains selected from the group consisting of strains.

By comparing the increase or decrease of the strain, the increase or decrease of the strain means an increase or decrease in the relative ratio of the biomarker microorganisms in the genomic DNA of the microorganism obtained from the sample derived from the test subject's intestinal tract, or liver disease group, diabetes risk group Or it may mean an increase or decrease in the number of bacteria of the biomarker microorganisms in the normal group or an absolute value indicating the same, and for this purpose, the relative ratio of the biomarker microorganisms in the liver disease group and the normal group, the number of bacteria or the absolute value range representing the same It can be stored in a database in advance.

Among the biomarker microorganisms, Bacteroides uniformis species strain, preferably SEQ ID NO: 1 to SEQ ID NO: 5 Bacteroides uniformis identified by any one sequence selected from the rRNA nucleotide sequence of SEQ ID NO: ( Bacteroides uniformis ) The species strain was significantly lower in the liver disease group than in the normal group. Therefore, when the Bacteroides uniformis species strain in the genomic DNA of the microorganism obtained from the sample derived from the test subject's intestinal tract is lower than that of the normal group, the risk of liver disease can be predicted or diagnosed as high. In addition, when the Bacteroides uniformis species strain is higher than the liver disease group, the risk of liver disease can be predicted or diagnosed as low.

Among the biomarker microorganisms, Bacteroides caccae species strain, preferably identified by any one sequence selected from the 16S rRNA sequence of SEQ ID NO: 6 and SEQ ID NO: 7 Bacteroides caccae The species strain was significantly lower in the liver disease group than in the normal group. Therefore, when the Bacteroides caccae species strain in the genomic DNA of the microorganism obtained from the sample derived from the intestinal tract of the test subject is lower than that of the normal group, the risk of liver disease can be predicted or diagnosed as high. In addition, when the Bacteroides caccae species strain is higher than the liver disease group, the risk of liver disease can be predicted or diagnosed as low.

Among the biomarker microorganisms, Parabacteroides spp. strain, preferably Parabacteroides genus strain identified by the 16S rRNA sequence of SEQ ID NO: 8, is significantly higher in the liver disease group than in the normal group. appear low. Therefore, in the genomic DNA of the microorganism obtained from the sample derived from the test subject's intestinal tract, when the parabacteroides genus strain is lower than that of the normal group, the risk of liver disease can be predicted or diagnosed as high. In addition, when the parabacteroides genus strain is higher than the liver disease group, the risk of liver disease can be predicted or diagnosed as low.

The 16S rRNA nucleotide sequence of SEQ ID NOs: 1 to 8 is an ASV nucleotide sequence capable of identifying each of the biomarker microorganisms, that is, an amplicon sequence variant nucleotide sequence. In particular, the ASV nucleotide sequence of SEQ ID NO: 8 was first identified as a nucleotide sequence capable of identifying a biomarker microorganism. Therefore, the biomarker microorganism, which is a Parabacteroides genus strain identified by the ASV base sequence of SEQ ID NO: 8, is a strain belonging to the genus Parabacteroides, but is molecularly biologically distinct from strains of the genus known in the prior art. It is the first strain to be identified in the gut.

In the present invention, 'cultivation' refers to all actions performed to grow microorganisms in appropriately artificially controlled environmental conditions, and in the present invention, it is a concept including 'fermentation'.

In the present invention, the term 'strain' includes not only live cells obtained from the culture medium, but also any processed form known to those skilled in the art, and includes, for example, dried products, frozen products, and the like, but is not limited thereto. In addition, the 'culture' is meant to include a 'fermented product', and the culture solution itself cultured in a liquid medium, the filtrate (centrifuged supernatant) from which the strain is removed by filtration or centrifugation of the culture medium is derived from the culture medium itself. includes artifacts.

In the present invention, the term 'dead cells' refers to cells sterilized by heating, pressurization, drug treatment, or the like. The cell component refers to a lysate obtained by disrupting cells by enzyme treatment, homogenization, sonication, or the like, or a fraction obtained from a cell wall fraction.

The pharmaceutical composition may further include appropriate carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, etc. in addition to any one or more of the strain, dead cells of the strain, or culture of the strain as an active ingredient.

The 'carrier' is a compound that facilitates the addition of the compound into a cell or tissue. The 'diluent' is a compound that is diluted in water to not only stabilize the biologically active form of the compound of interest, but also to dissolve the compound.

The carrier, excipient and diluent are not particularly limited, but for example, lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.

The amount of the pharmaceutical composition used may vary depending on the age, sex, and weight of the patient or the animal to be treated, and above all, it depends on the condition of the subject to be treated, the specific category or type of the disease to be treated, the route of administration, and the properties of the therapeutic agent used. will be dependent

The pharmaceutical composition is appropriately selected depending on the absorption of the active ingredient in the body, the excretion rate, the age and weight of the patient or the animal to be treated, sex and condition, the severity of the disease to be treated, etc., but generally 0.1 to 1,000 mg/kg per day , preferably 1 to 500 mg/kg, more preferably 5 to 250 mg/kg, most preferably 10 to 100 mg/kg. The unit dosage form formulated in this way can be administered several times at regular time intervals as needed.

The pharmaceutical composition may be administered individually as a prophylactic or therapeutic agent, or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent.

The pharmaceutical composition may be prepared in oral formulations such as powders, granules, tablets, capsules, troches, suspensions, emulsions, syrups, aerosols, etc., and sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, and freeze-dried formulations according to conventional methods, respectively. , it can be formulated and used in parenteral formulations such as suppositories.

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid preparations are at least in any one or more of the above-mentioned strain, dead cells of the strain, or culture of the strain. It may be prepared by mixing one or more excipients, for example, starch, calcium carbonate, sugar or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. .

Formulations for parenteral administration include non-aqueous solutions, and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The food composition is a food ingredient that can be used as food described in the standards and standards ('Food Code') of food commonly used in food production, in addition to any one or more of the strain, the dead cells of the strain, or the culture of the strain. , including food additives listed in the Food Additives Ordinance.

Although not particularly limited, examples thereof include proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. The carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sugar, lactose and the like; oligosaccharides or polysaccharides such as dextrin, starch syrup, cyclodextrin and the like; Sugar alcohols such as xylitol, sorbitol, erythritol and the like can be used. As the flavoring agent, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used.

In the food composition, the content of any one or more of the strain, the dead cells of the strain, or the culture of the strain does not need to be particularly limited, but when used for disease improvement, prevention or treatment, it is included as an amount to exhibit the effect may be included, for example, 0.1 to 99% by weight, 0.5 to 95% by weight, 1 to 90% by weight, 2 to 80% by weight, 3 to 70% by weight, 4 to 60% by weight, 5 to 50% by weight there is.

Any one or more of the strain as an active ingredient in the food composition, the dead cells of the strain, or the culture of the strain vary depending on the condition, weight, presence or severity and period of the ingestion, but appropriately by those of ordinary skill in the art. can be selected. For example, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, still more preferably 20 to 800 mg, and most preferably 50 to 500 mg based on the daily dose. And, the number of administration does not need to be particularly limited, but can be adjusted by those skilled in the art within the range of 3 times a day to once a week. In the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range.

The food composition is not particularly limited, but may be, for example, a powder, granules, tablets, capsules, pills, extracts, jelly formulations, tea bag formulations or beverage formulations.

In addition, any one or more of the above strains, dead cells of the strains, or cultures of the strains may be added in order to impart disease improvement, prevention or treatment functionality to general foods, and foods that can be added do not need to be particularly limited. However, for example, confectionery, bread or rice cakes, cocoa processed products or chocolates, edible meat or egg products, processed fish meat products, tofu or jelly products, noodles exemplified in the standards and specifications of food according to Article 7 of the Food Sanitation Act ('Food Code') , tea, coffee, beverages, special purpose foods, soy sauce, seasonings, dressings, kimchi, salted fish, pickled foods, stewed foods, alcoholic beverages, raisins, and other foods. In addition, it can be added to dairy products, processed meat products, packaged meat, and processed eggs exemplified in the processing standards and ingredient specifications of livestock products according to Article 4 of the Livestock Products Sanitation Control Act ('Livestock Products Code').

The food composition may additionally contain food additives, and the suitability as a food additive follows the standards and standards for the relevant item in accordance with the general rules and general test methods of the 'Food Additives Codex', unless otherwise specified.

In addition, it can be used as a food additive composition for bioconversion having acetaldehyde degrading ability and glutathione producing ability, including any one or more of the strain, dead cells of the strain, or a culture of the strain.

In the present invention, 'risk prediction' refers to determining whether a patient is likely to develop a disease, and to delay or prevent the onset of disease through special and appropriate management of a patient with a high risk of disease, or to prevent the onset of, or the most appropriate treatment It can be used clinically to make treatment decisions by choosing a modality. In addition, 'diagnosis' means confirming the presence or characteristics of a pathological condition, and for the purpose of the present invention, diagnosis may mean confirming whether or not the onset of .

As an agent capable of detecting the strain provided as a biomarker in the present invention, a protein, nucleic acid, lipid, glycolipid, glycoprotein or sugar (monosaccharide, disaccharide, oligosaccharide, etc.) specifically present in the corresponding strain in the sample, etc. Primers, probes, antisense oligonucleotides, aptamers, antibodies, and the like capable of specifically detecting the same organic molecule may be used.

For example, when the agent for detecting the strain is a primer, it is preferable that the primer specifically detects the genomic sequence (eg, 16S rRNA) of the microorganism and does not specifically bind to the genomic sequence of another strain.

In the present invention, the term 'primer' refers to 7 to 50 nucleic acid sequences capable of forming a base pair complementary to the template strand and functioning as a starting point for copying the template strand. Primers are usually synthesized but can also be used on naturally occurring nucleic acids. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but is sufficiently complementary so that it can hybridize with the template. Additional features that do not change the basic properties of the primer may be incorporated. Examples of additional features that may be incorporated include, but are not limited to, methylation, encapsulation, substitution of one or more nucleic acids with homologs, and modifications between nucleic acids.

In the present invention, '16s rRNA' is an rRNA constituting the 30S subunit of the prokaryotic ribosome, and the nucleotide sequence is largely conserved, while high nucleotide sequence diversity appears in some sections. In particular, since there is little diversity among homogeneous species while diversity appears among other species, prokaryotes can be usefully identified by comparing the sequences of 16S rRNA.

In the present invention, the primer can be used to amplify the conserved 16S rRNA sequence of the microorganism, and the presence of the microorganism can be detected by whether a desired product is generated as a result of the sequence amplification. A sequence amplification method using a primer may use various methods known in the art. For example, polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), multiplex PCR, touchdown PCR, hot start PCR, nested PCR, Booster PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction , vectorette PCR, TAIL-PCR (thermal asymmetric interlaced PCR), ligase chain reaction, repair chain reaction, transcription-mediated amplification, self-maintaining sequence cloning, selective amplification of the target sequence can be used, The scope of the present invention is not limited thereto.

Also, for example, when the agent for detecting the strain is an antibody, the microorganism can be detected using an immunological method based on an antigen-antibody reaction. Analysis methods for this include Western blot, ELISA (enzyme linked immunosorbent asay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket immunoelectrolysis. Electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip, etc., but are not limited thereto.

In addition, molecular and immunological methods widely used in the art can be used to detect the microorganisms of the present invention.

The composition comprising the agent capable of detecting the strain of the present invention may be implemented in the form of a diagnostic kit and provided as a kit for predicting or diagnosing the risk of liver disease.

The diagnostic kit includes a primer, a probe, an antisense oligonucleotide, an aptamer, and a detection agent such as an antibody for detecting the microorganisms, as well as one or more other component compositions, solutions, or devices suitable for the analysis method. can

For example, in the present invention, a diagnostic kit including a primer specific for a corresponding microorganism may be a diagnostic kit including essential elements for performing an amplification reaction such as PCR and the like. The diagnostic kit for PCR includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water. and the like.

In the present invention, the test subject's intestinal-derived sample may be preferably a fecal sample.

In order to detect microorganisms from a sample from the intestinal tract of a test subject, general amplification techniques known in the art, for example, polymerase chain reaction, reverse transcription-polymerase chain reaction, multiplex PCR, touchdown PCR, hot start PCR, four Steed PCR, Booster PCR, Real-Time PCR, Fractional Display PCR, Rapid Amplification of cDNA Ends, Inverse PCR, Vectoret PCR, TAIL-PCR, Ligase Chain Reaction, Repair Chain Reaction, transcription-mediated amplification, self-maintaining sequence cloning, A selective amplification reaction of a target sequence may be used, but the scope of the present invention is not limited thereto.

In addition, immunological methods based on general antigen-antibody reactions known in the art, for example, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, locate immunoelectrophoresis, Tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, etc. may be used, but the scope of the present invention is not limited thereto.

The present invention relates to any one or more strains selected from the group consisting of Bacteroides uniformis species strains, Bacteroides caccae species strains and Parabacteroides genus strains, the strains of It can be used as a pharmaceutical composition for preventing or treating liver disease comprising a dead cell or a culture of the strain as an active ingredient, a food composition for preventing or improving liver disease including the same, and a method of treating liver disease using the same.

1 is a graph showing the intestinal microbial distribution of 890 Koreans.
2 is a boxplot showing the relative proportion distribution of the entire strain belonging to the Bacteroides uniformis species in the liver disease group and the normal group.
3 is a boxplot showing the relative proportion distribution of the entire strain belonging to the Bacteroides caccae species in the liver disease group and the normal group.
4 is a boxplot showing the relative proportion distribution of all strains belonging to the genus Parabacteroides and the ASV of SEQ ID NO: 8 in the liver disease group and the normal group.
5 is a boxplot predicting the difference between a normal group and a liver disease group by constructing a multivariate linear model using the relative amounts of all biomarker microorganisms in Table 1.

Hereinafter, the present invention will be described in detail by way of experimental examples and examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited by the following examples.

Experimental Example 1: Research subject and sample collection

Fecal samples were collected from 890 Koreans participating in the health checkup. To minimize changes in fecal microorganisms, fecal samples were collected using the OMNIgene-GUT kit (DNA Genotek, Ontario, Canada) and stored at room temperature until DNA extraction.

In addition, during the health check-up, questionnaires about lifestyle, physical measurements, and blood tests were conducted, and blood alanine aminotransferase (ALT) concentrations were measured through blood tests.

Experimental Example 2: DNA Extraction

DNA was extracted from the fecal sample of Experimental Example 1 using the bead-beating extraction method, and DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany).

Experimental Example 3: 16S rRNA gene sequencing

A library targeting the V3-V4 hypervariable region of the 16S rRNA gene was prepared using the DNA extracted in Experimental Example 2, and the sequence of the portion was sequenced using Illumina MiSeq 2x300 (Illumina, CA, USA).

Experimental Example 4: Sequence analysis and annotation

The sequencing result in Experimental Example 3 was converted into an amplicon sequence variant (ASV) table using the QIIME2 DADA2 module. Each ASV corresponds to a partial sequence of 16S rRNA, and each can be used as an indicator for detecting a specific microorganism. To identify the microbial phylogeny, the microbial phylogeny was analyzed using the Naive Bayesian classifier of QIIME2 and the GreenGene 13.8 database, and the microbial species unit annotation was performed.

Experimental Example 5: Correlation analysis between intestinal microbes and blood sugar

For 890 health check-up subjects, those with blood alanine aminotransferase (ALT) above 40 IU/L were divided into liver disease group, and those with blood alanine aminotransferase (ALT) below 40 IU/L were classified as normal group.

The average ratio of the total intestinal microorganisms in the liver disease group and the normal group was obtained for each ASV nucleotide sequence representing a specific microbial species, and microorganisms whose average ratio increased or decreased in the liver disease group and the normal group were selected. When the ratio of the selected microorganisms showed a statistically significant difference between the liver disease group and the normal group, it was defined as a biomarker microorganism for prediction or diagnosis of liver disease risk. One-way ANOVA was used for statistical analysis, and a significant difference was shown between the liver disease group and the normal group by comparing box plots as in FIGS. 2 to 4 .

Experiment result

The distribution of intestinal microbes in 890 Koreans is shown in FIG. 1 . Intestinal microorganisms Bacteroides, Prevotella, Picalibacterium, Unclassified Lachnospiraceae, Unclassified Luminococci, Oscillospira, Luminococcus, Parabacteroides, Suterella, Coprococcus and Classified as other and shown by arranging from left to right from subjects with high relative ratio of Bacteroides to low subjects.

As biomarker microorganisms for predicting or diagnosing the risk of liver disease showing a statistically significant ratio difference between the liver disease group and the normal group, the Bacteroides uniformis species strain, Bacteroides kakae ( Bacteroides uniformis ) in Table 1 below Bacteroides caccae ) species strains and Parabacteroides genus strains were selected.

division system between the liver disease group and the normal group
P-value Bacteroides uniformis
( Bacteroides uniformis ) Bell 0.022526 Bacteroides caccae Bell 0.038371 Parabacteroides ASV inside 0.044137

In the liver disease group and normal group of FIG. 2, the relative ratio distribution of the entire strain belonging to the Bacteroides uniformis species identified by any one ASV sequence selected from the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 5 Looking at the, Bacteroides uniformis ( Bacteroides uniformis ) In the case of the strain, the liver disease group showed a significantly lower ratio than the normal group. Therefore, when the ratio of the Bacteroides uniformis species strain in the genomic DNA of the microorganism obtained from the sample derived from the intestinal tract of the test subject is lower than that of the normal group, the risk of liver disease can be predicted or diagnosed as high. In addition, when the ratio of the Bacteroides uniformis species strain is higher than that of the liver disease group, the risk of liver disease can be predicted or diagnosed as low.

In the liver disease group and normal group of Figure 3, the relative ratio distribution of the entire strain belonging to the Bacteroides caccae species identified by any one ASV sequence selected from the nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 7 Looking at the distribution , Bacteroides caccae ( Bacteroides caccae ) In the case of the strain, the liver disease group showed a significantly lower ratio than the normal group. Therefore, when the ratio of the Bacteroides caccae species strain in the genomic DNA of the microorganism obtained from the sample derived from the intestinal tract of the test subject is lower than that of the normal group, the risk of liver disease can be predicted or diagnosed as high. In addition, when the ratio of the Bacteroides caccae species strain is higher than that of the liver disease group, the risk of liver disease can be predicted or diagnosed as low.

4, Parabacteroides in the liver disease group and normal group, Parabacteroides identified by the ASV of SEQ ID NO: 8 and the entire strain belonging to the genus Parabacteroides Looking at the relative ratio distribution of the genus strain, Parabacteroides ( Parabacteroides ) The relative ratio of all strains belonging to the genus does not have a significant difference for each group, but in the case of Parabacteroides genus strain identified by ASV of SEQ ID NO: 8, a significantly lower ratio in the liver disease group than in the normal group indicates Therefore, if the parabacteroides genus strain identified by the ASV of SEQ ID NO: 8 in the genomic DNA of the microorganism obtained from the sample derived from the intestinal tract of the subject is lower than that of the normal group, the risk of liver disease can be predicted or diagnosed. . In addition, when the parabacteroides spp. strain identified by the ASV of SEQ ID NO: 8 is high compared to the liver disease group, it can be predicted or diagnosed as having a low risk of liver disease.

The results of predicting the difference between the normal group and the liver disease group when a multivariate linear model for predicting the liver disease group was constructed by combining the entire biomarker microorganisms in Table 1 is shown in FIG. 5 . 5 is a configuration of a Bayesian Ridge model for predicting a liver disease group from the relative amounts of the three types of biomarker microorganisms. The Bayesian Ridge model is a type of linear model, and variables that have no meaning among the variables constituting the linear model are automatically excluded. way to do it In the above method, the BayesianRidge function of the Python package scikit-learn was used. According to FIG. 5 , it can be confirmed that the risk of diabetes can be predicted or diagnosed more clearly when the three types of biomarker microorganisms are combined (P value = 1.8 X 10 ^-3 ).

<110> KOREA FOOD RESEARCH INSTITUTE <120> Composition For Improving, Preventing Or Treating Liver Diseases Using Human Intestinal Microbiome, And Treating Method Of Liver Diseases Using The Same <130> HPC9570 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 422 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides uniformis <400> 1 tgaggaatat tggtcaatgg acgagagtct gaaccagcca agtagcgtga aggatgactg 60 ccctatgggt tgtaaacttc ttttatacgg gaataaagtg aggcacgcgt gcctttttgt 120 atgtaccgta tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga 180 tccgagcgtt atccggattt attgggttta aagggagcgt aggcggacgc ttaagtcagt 240 tgtgaaagtt tgcggctcaa ccgtaaaatt gcagttgata ctgggtgtct tgagtacagt 300 agaggcaggc ggaattcgtg gtgtagcggt gaaatgctta gatatcacga agaactccga 360 ttgcgaaggc agcctgctgg actgtaactg acgctgatgc tcgaaagtgt gggtatcaaa 420 ca 422 <210> 2 <211> 422 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides uniformis <400> 2 tgaggaatat tggtcaatgg acgagagtct gaaccagcca agtagcgtga aggatgactg 60 ccctatgggt tgtaaacttc ttttatacgg gaataaagtg aggcacgcgt gcctttttgt 120 atgtaccgta tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga 180 tccgagcgtt atccggattt attgggttta aagggagcgt aggcggacgc ttaagtcagt 240 tgtgaaagtt tgcggctcaa ccgtaaaatt gcagttgata ctgggtgtct tgagtacagt 300 agaggcaggc ggaattcgtg gtgtagcggt gaaatgctta gatatcacga agaactccga 360 ttgcgaaggc agcttgctgg actgtaactg acgctgatgc tcgaaagtgt gggtatcaaa 420 ca 422 <210> 3 <211> 422 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides uniformis <400> 3 tgaggaatat tggtcaatgg acgagagtct gaaccagcca agtagcgtga aggatgactg 60 ccctatgggt tgtaaacttc ttttatacgg gaataaagtg aggcacgtgt gcctttttgt 120 atgtaccgta tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga 180 tccgagcgtt atccggattt attgggttta aagggagcgt aggcggacgc ttaagtcagt 240 tgtgaaagtt tgcggctcaa ccgtaaaatt gcagttgata ctgggtgtct tgagtacagt 300 agaggcaggc ggaattcgtg gtgtagcggt gaaatgctta gatatcacga agaactccga 360 ttgcgaaggc agcttgctgg actgtaactg acgctgatgc tcgaaagtgt gggtatcaaa 420 ca 422 <210> 4 <211> 422 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides uniformis <400> 4 tgaggaatat tggtcaatgg acgagagtct gaaccagcca agtagcgtga aggatgactg 60 ccctatgggt tgtaaacttc ttttatacgg gaataaagtg aggcacgtgt gcctttttgt 120 atgtaccgta tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga 180 tccgagcgtt atccggattt attgggttta aagggagcgt aggcggacgc ttaagtcagt 240 tgtgaaagtt tgcggctcaa ccgtaaaatt gcagttgata ctgggtgtct tgagtacagt 300 agaggcaggc ggaattcgtg gtgtagcggt gaaatgctta gatatcacga agaactccga 360 ttgcgaaggc agcctgctgg actgtaactg acgctgatgc tcgaaagtgt gggtatcaaa 420 ca 422 <210> 5 <211> 422 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides uniformis <400> 5 tgaggaatat tggtcaatgg acgagagtct gaaccagcca agtagcgtga aggatgactg 60 ccctatgggt tgtaaacttc ttttatacgg gaataaagtt aggcacgtgt gcctttttgt 120 atgtaccgta tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga 180 tccgagcgtt atccggattt attgggttta aagggagcgt aggcggatgc ttaagtcagt 240 tgtgaaagtt tgcggctcaa ccgtaaaatt gcagttgata ctgggtgtct tgagtacagt 300 agaggcaggc ggaattcgtg gtgtagcggt gaaatgctta gatatcacga agaactccga 360 ttgcgaaggc agcttgctgg actgtaactg acgctgatgc tcgaaagtgt gggtatcaaa 420 ca 422 <210> 6 <211> 422 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides caccae <400> 6 tgaggaatat tggtcaatgg acgcgagtct gaaccagcca agtagcgtga aggatgactg 60 ccctatgggt tgtaaacttc ttttatatgg gaataaagtt gtccacgtgt ggatttttgt 120 atgtaccata tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga 180 tccgagcgtt atccggattt attgggttta aagggagcgt aggcggattg ttaagtcagt 240 tgtgaaagtt tgcggctcaa ccgtaaaatt gcagttgata ctggcagtct tgagtgcagt 300 agaggtgggc ggaattcgtg gtgtagcggt gaaatgctta gatatcacga agaactccga 360 ttgcgaaggc agctcactgg agtgtaactg acgctgatgc tcgaaagtgt gggtatcaaa 420 ca 422 <210> 7 <211> 422 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides caccae <400> 7 tgaggaatat tggtcaatgg acgcgagtct gaaccagcca agtagcgtga aggatgactg 60 ccctatgggt tgtaaacttc ttttatatgg gaataaagtg gtccacgtgt ggatttttgt 120 atgtaccata tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga 180 tccgagcgtt atccggattt attgggttta aagggagcgt aggcggattg ttaagtcagt 240 tgtgaaagtt tgcggctcaa ccgtaaaatt gcagttgata ctggcagtct tgagtgcagt 300 agaggtgggc ggaattcgtg gtgtagcggt gaaatgctta gatatcacga agaactccga 360 ttgcgaaggc agctcactgg agtgtaactg acgctgatgc tcgaaagtgt gggtatcaaa 420 ca 422 <210> 8 <211> 422 <212> DNA <213> Artificial Sequence <220> <223> Parabacteroides ASV <400> 8 tgaggaatat tggtcaatgg ccgagaggct gaaccagcca agtcgcgtga aggaagaagg 60 atctatggtt tgtaaacttc ttttataggg gaataaagtg gaggacgtgt ccttttttgt 120 atgtacccta tgaataagca tcggctaact ccgtgccagc agccgcggta atacggagga 180 tgcgagcgtt atccggattt attgggttta aagggtgcgt aggtggtgat ttaagtcagc 240 ggtgaaagtt tgtggctcaa ccataaaatt gccgttgaaa ctgggttact tgagtgtgtt 300 tgaggtaggc ggaatgcgtg gtgtagcggt gaaatgcata gatatcacgc agaactccga 360 ttgcgaaggc agcttactaa accataactg acactgaagc acgaaagcgt ggggatcaaa 420 ca 422

Claims (6)

Bacteroides uniformis ( Bacteroides uniformis ) Species strain, Bacteroides caccae ) Species strain and Parabacteroides ) Any one or more strains selected from the group consisting of genus strains, dead cells of the strain, Or a pharmaceutical composition for preventing or treating liver disease comprising a culture of the strain as an active ingredient. According to claim 1, wherein the Bacteroides uniformis ( Bacteroides uniformis ) species strain is to include any one sequence selected from the 16S rRNA nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 5, the Bacteroides kakae ( Bacteroides caccae ) species strain is to include any one sequence selected from the 16S rRNA nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 7, the Parabacteroides genus strain is the 16S rRNA sequence of SEQ ID NO: 8 A pharmaceutical composition for preventing or treating liver disease, comprising: Bacteroides uniformis ( Bacteroides uniformis ) Species strain, Bacteroides caccae ) Species strain and Parabacteroides ) Any one or more strains selected from the group consisting of genus strains, dead cells of the strain, Or a food composition for preventing or improving liver disease comprising a culture of the strain as an active ingredient. The method of claim 3, wherein the Bacteroides uniformis ( Bacteroides uniformis ) species strain is to include any one sequence selected from the 16S rRNA nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 5, the Bacteroides kakae ( Bacteroides caccae ) species strain is to include any one sequence selected from the 16S rRNA nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 7, the Parabacteroides genus strain is the 16S rRNA sequence of SEQ ID NO: 8 A food composition for preventing or improving liver disease, comprising: Any one or more selected from the group consisting of Bacteroides uniformis sp. strain, Bacteroides caccae sp . strain, and Parabacteroides sp. strain in a therapeutically effective amount for a liver disease patient A method for treating liver disease by administering a strain, a dead cell of the strain, or a culture of the strain. According to claim 5, wherein the Bacteroides uniformis ( Bacteroides uniformis ) species strain is to include any one sequence selected from the 16S rRNA nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 5, the Bacteroides kakae ( Bacteroides caccae ) species strain is to include any one sequence selected from the 16S rRNA nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 7, the Parabacteroides genus strain is the 16S rRNA sequence of SEQ ID NO: 8 A method of treating liver disease, characterized in that it comprises.

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