Lipopolysaccharide-controlled intestinal bacteria and their uses

Abstract

The present invention provides a combination of the following (1) and (2): (1) comprising a naturally isolated Bacteroides vulgaris ( Bacteroides popular ) of live bacteria; (2) comprising naturally isolated Bacteroides domitilis ( Bacteroides dorei ) of the composition of live bacteria.

Description

Technical Field

[0001] The present invention relates to lipopolysaccharide (hereinafter, sometimes abbreviated as "LPS")-controlling (regulatory) intestinal bacteria and their uses. More specifically, the present invention relates to: a combination of two specific bacteria belonging to the genus Bacteroides (i.e., Bacteroides vulgatus and Bacteroides dorei) that can control the production of LPS by intestinal bacteria and its transfer to the blood; a drug comprising the combination, in particular, a preventive and / or ameliorating agent for diseases in which LPS is involved (e.g., circulatory system diseases, inflammatory diseases, metabolic diseases); a diagnostic method for the above-mentioned diseases using the amount of the bacteria present in the intestinal flora as an indicator, etc. Background Art

[0002] By improving the understanding of the intestinal flora and its role in the metabolism and immunity of the host, unprecedented attention has been paid to the development of diagnostic and therapeutic targets related to the intestinal flora for a variety of diseases. The popularity of next-generation sequencing technology or comprehensive omics analysis has dramatically expanded our understanding of the microbial world. More and more evidence suggests a close relationship between intestinal flora and cardiovascular disease. For example, it is known that the metabolites of dietary phosphatidylcholine produced by intestinal flora, namely trimethylamine (TMA) and trimethylamine-N-oxide (TMAO), are related to the progression of cardiovascular disease, particularly atherosclerosis.

[0003] However, recent clinical trial results have shown contradictory results: fish consumption can cause a transient but dramatic increase in blood TMAO levels, highlighting the current limited understanding of the relationship between diet and the gut microbiota in the field.

[0004] It is known that the genus Bacteroides includes several intestinal bacteria that are dominant in the human body and plays an important role in maintaining a healthy intestinal ecosystem. Individuals classified as intestinal type 3 characterized by low levels of Bacteroides have a high incidence of symptomatic atherosclerosis (non-patent document 1). Moreover, it has been found that the presence of Bacteroides is reduced in patients with arteriosclerotic ischemic cerebral infarction and transient ischemic attack (non-patent document 2). Consistent with these observations, the inventor's previous study using terminal restriction fragment length polymorphism (T-RFLP) analysis confirmed that compared with patients with risk factors for arteriosclerosis but without arteriosclerotic disease or healthy volunteers, the presence of Bacteroidetes in patients with coronary artery disease (CAD) is low (non-patent documents 3, 4). Moreover, by intestinal bacterial metagenomic analysis based on shotgun sequencing technology, it is also shown that the reduction of Bacteroidetes species in CAD patients is reduced (non-patent document 5), and these cognitions strongly suggest the relationship between Bacteroidetes and CAD.

[0005] However, it remains unclear which specific Bacteroides species are involved and what their true role is in CAD.

[0006] Lipopolysaccharide (LPS), a component of the outer membrane of the cell wall of Gram-negative bacteria, is known to activate immune cells such as macrophages, and metabolic endotoxemia is associated with chronic inflammation or cardiovascular-metabolic diseases such as diabetes and arteriosclerosis (Non-Patent Document 6). The present inventors have previously discovered that the formation of arteriosclerotic lesions in the aortic sinus is suppressed in germ-free ApoE-deficient mice compared to the same mice raised under conventional conditions, and that the LPS level in the blood or the inflammatory cytokine levels in macrophages and the aorta also show low values ​​(Non-Patent Document 7).

[0007] On the other hand, Patent Document 1 discloses a capsule composition for oral administration, which contains components (e.g., LPS) derived from three Bacteroides bacteria including Bacteroides vulgatus, and describes that the composition can be used for the treatment and onset delay of various inflammatory diseases or autoimmune diseases, etc. However, it has not been confirmed that these compositions actually have such efficacy.

[0008] Furthermore, Non-Patent Document 8 states that LPS derived from Bacteroides dorei has a different structure from LPS derived from Escherichia coli, has a weak ability to stimulate Toll-like receptor 4 (TLR4), and is less likely to induce innate immunity.

[0009] However, there are no reports that Bacteroides bacteria have an overall effect on LPS production in the intestine, including other intestinal bacteria.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: Japanese Patent Application No. 2013-527240;

[0013] Non-patent literature

[0014] Non-patent document 1: Karlsson, FH et al., Nat Commun. 3, 1245 (2012);

[0015] Non-patent document 2: Yin, J. et al., JAm Heart Assoc. 4, E002699 (2015);

[0016] Non-patent document 3: Emoto, T. et al., J. Atheroscler Thromb. 23, 908-921 (2016);

[0017] Non-patent document 4: Emoto, T. et al., Heart Vessels. 32, 39-46 (2017);

[0018] Non-patent document 5: Jie, Z. et al., Nat Commun. 18, 845 (2017);

[0019] Non-patent document 6: Patel, PN et al., Arterioscler. Thromb. Vasc. Biol. 35, 525-534 (2015);

[0020] Non-patent document 7: Kasahara, K. et al., J Lipid Res. 58, 519-528 (2017);

[0021] Non-patent document 8: Vatanen, T. et al., Cell. 165, 842-853 (2016). Summary of the invention

[0022] Problems to be solved by the invention

[0023] Despite the popularity of statin therapy over the past decade, CAD remains one of the leading causes of death worldwide. The present invention aims to identify novel and inexpensive specific bacterial species of the intestinal flora that can be targeted as preventive and / or therapeutic strategies for CAD.

[0024] Means for solving problems

[0025] In order to achieve the above-mentioned purpose, the inventors firstly performed random sequencing of 16S ribosomal RNA (rRNA) genes and compared the intestinal flora in CAD patients and non-CAD patients. As a result, it was found that common Bacteroides and Bacteroides dorotheli were reduced in CAD patients. Moreover, the inventors clarified the most fundamental mechanism of action linking these Bacteroides species to arteriosclerosis through a series of analyses in ApoE-deficient mice that showed a tendency to arteriosclerosis. That is, it was found that: these Bacteroides species significantly inhibited the production of LPS by intestinal bacteria while strengthening the tight junctions of the intestinal barrier, thereby reducing the permeability (permeability) of the barrier, inhibiting the transfer of LPS to the blood (i.e., inducing endotoxemia), thereby inhibiting the inflammation-induced immune response and hindering the formation of arteriosclerotic lesions.

[0026] Based on these findings, the inventors conducted further repeated studies and came to the following conclusions: the intake of Bacteroides vulgaris and Bacteroides doreae is effective in preventing and / or improving circulatory diseases represented by arteriosclerosis, inflammatory diseases, and metabolic diseases. In addition, the above-mentioned diseases can be diagnosed using the presence of this Bacteroides species in the intestinal flora as an indicator, thereby completing the present invention.

[0027] That is, the present invention provides the following contents.

[0028] [1] A combination of (1) and (2) below:

[0029] (1) A composition comprising live bacteria of Bacteroides vulgaris isolated from nature; and

[0030] (2) A composition comprising live bacteria of Bacteroides dornoni isolated from nature.

[0031] [2] The combination described in [1], wherein the composition of (1) and the composition of (2) are the same substance.

[0032] [3] The combination described in [1] or [2], which is a drug or a drug additive.

[0033] [4] The combination described in [1] or [2], which is a feed or a feed additive.

[0034] [5] The combination according to any one of [1] to [4], which is used for preventing and / or ameliorating a disease associated with an elevated level of lipopolysaccharide in the blood or in the intestine.

[0035] [6] The combination described in [5], wherein the disease is a circulatory system disease, an inflammatory disease or a metabolic disease.

[0036] [7] The combination described in [5], wherein the above-mentioned disease is a circulatory system disease selected from atrial fibrillation, heart failure, ischemic heart disease, myocardial infarction, angina pectoris, hypertension, arteriosclerosis, aortic aneurysm, aortic dissection, occlusive arteriosclerosis and aortic valve stenosis.

[0037] [8] The combination described in [5], wherein the above-mentioned disease is an inflammatory disease selected from hepatitis, non-alcoholic steatohepatitis, fatty liver, liver cancer, intestinal inflammation, irritable bowel syndrome (irritable bowel syndrome), gastritis, collagen disease, chronic rheumatoid arthritis, chronic nephritis, IgA nephropathy, bronchial asthma, interstitial pneumonia, drug-induced lung injury, eosinophilic pulmonary infiltration syndrome, atypical acid-resistant bacteria disease, allergic rhinitis, atopic dermatitis and sepsis.

[0038] [9] The combination described in [5], wherein the disease is a metabolic disease selected from the group consisting of diabetes, obesity, metabolic syndrome, lifestyle diseases, dyslipidemia and osteoporosis.

[0039]

[10] A method for preventing and / or improving a disease associated with an elevated level of lipopolysaccharide in the blood or intestine, the method comprising: orally ingesting live bacteria of naturally isolated Bacteroides vulgaris and live bacteria of naturally isolated Bacteroides doreae.

[0040]

[11] The method described in

[10] , wherein the disease is a circulatory system disease, an inflammatory disease or a metabolic disease.

[0041]

[12] A method for examining a disease associated with elevated levels of lipopolysaccharide in the blood or intestine, the method comprising: measuring the amount of Bacteroides vulgaris and Bacteroides domitilis in the intestinal flora of a subject.

[0042]

[13] The method described in

[12] , wherein the disease is a circulatory system disease, an inflammatory disease or a metabolic disease.

[0043]

[14] The method of

[12] or

[13] , comprising: detecting the 16S rRNA gene of (1) and (2) below:

[0044] (1) a 16S rRNA gene comprising a nucleotide sequence having 98% or more homology to the nucleotide sequence represented by SEQ ID NO: 1 or 2; and

[0045] (2) A 16S rRNA gene comprising a nucleotide sequence having 98% or more homology to the nucleotide sequence represented by SEQ ID NO: 3 or 4.

[0046]

[15] A reagent for testing the risk of developing a disease associated with elevated levels of lipopolysaccharide in the blood or intestine, the reagent comprising: a primer set or a probe capable of detecting the 16S rRNA gene of Bacteroides vulgaris and Bacteroides doreae.

[0047]

[16] The agent described in

[15] , wherein the disease is a circulatory system disease, an inflammatory disease or a metabolic disease.

[0048]

[17] The reagent of

[15] or

[16] , wherein the 16S rRNA gene comprises the nucleotide sequences of (1) and (2) below:

[0049] (1) a nucleotide sequence having 98% or more homology with the nucleotide sequence represented by SEQ ID NO: 1 or 2; and

[0050] (2) A nucleotide sequence having 98% or more homology with the nucleotide sequence represented by SEQ ID NO: 3 or 4.

[0051] Effects of the Invention

[0052] According to the present invention, by oral ingestion of microorganisms (probiotics) that can be fermented and produced in large quantities, circulatory diseases represented by arteriosclerosis or diseases accompanied by chronic inflammation, metabolic diseases accompanied by metabolic abnormalities such as glucose and lipid metabolism can be prevented and / or improved, thereby providing a cheap and simple control strategy for these diseases, and also contributing to the medical economy in an aging society. In addition, according to the present invention, the diagnosis / risk prediction of the above-mentioned diseases can be performed by analyzing the microbiome in feces, providing a non-invasive clinical examination method. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] [ Figure 1 ] is a graph showing changes in intestinal microorganisms in CAD patients or non-CAD patients. The V3-V4 region of bacterial 16S rRNA was sequenced in stool samples from 30 controls (who did not develop CAD but had risk factors for coronary artery) and 30 CAD patients. (a) Participants were classified into three clusters based on the presence of genera in the intestinal microbiota. (b) The distribution of each major genera between clusters. In order to compare the inter-cluster distribution of each genus, one-way ANOVA was used. (c) The distribution of controls and CAD patients in each cluster. In order to compare the three clusters, a chi-square test was used. (d) The relative presence of Bacteroides in controls and CAD patients (percentage of the total intestinal microbiota). (e) The relative presence of Bacteroides species in controls and CAD patients (percentage relative to the total Bacteroides). (f) The relative presence of Bacteroides vulgaris and Bacteroides vulgaris (percentage of the total intestinal microbiota). *P<0.05, **P<0.01, ***P<0.001. To determine statistical significance, the Mann-Whitney U test was used (b, d, f). Data are shown as median ± interquartile range (1st to 3rd interquartile range) (b, d, f).

[0054] [ Figure 2 ] is a graph showing the effects of gastric tube feeding with live Bacteroides vulgaris and Bacteroides doreae on the development of atherosclerosis and plaque inflammation. T Bacteroides dorothelii DSM17855 T 6-week-old female Apoe - / -Mice were killed at 16 weeks of age and lesions caused by atherosclerosis were evaluated. (a) Representative microscopic photographs and quantitative analysis of Oil Red O staining of atherosclerotic lesion areas in the aortic sinus (8-9 samples in each group). Black bars represent 200 μm. (b) Representative microscopic photographs and quantitative analysis of Oil Red O staining of atherosclerotic plaque areas in the aorta (7-9 samples in each group). White bars represent 1.5 mm. (c) Representative fluorescent staining of macrophages in the aortic sinus and quantitative analysis of MOMA-2 positive staining (7 samples in each group). Black bars represent 100 μm. (d) CD4 + Representative sections and quantitative analysis of T cells (8-9 samples per group). Black bars represent 50 μm. (e) mRNA levels in atherosclerotic aorta. Data were normalized to the housekeeping GAPDH gene and shown as mean values ​​(4-5 samples per group). (f) Body weight changes throughout the experiment (8-9 samples per group). (g) Comparison of plasma lipid profiles (8-9 samples per group). LB: lysine monophosphate (BMP) cultured with live Bacteroides vulgaris ATCC8482 T Bacteroides dorothelii DSM 17855 T strains were treated; T-Cho: total cholesterol; TG: triglycerides; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol. *P<0.05, Student's two-sided t-test. Data are shown as mean ± standard error of the mean.

[0055] [ Figure 3 ] is a graph showing changes in the immune response of the intestine and the production of lipopolysaccharide (LPS) by intestinal microorganisms. T Bacteroides dorothelii DSM 17855 T 16-week-old female Apoe - / -Colon and mesenteric lymph nodes (MLNs) were resected from mice. (a) mRNA expression levels of immune cell markers and chemokines / chemokine receptors in the colon (7-8 samples per group). (b) mRNA expression levels of inflammation-inducing cytokines in the colon (7-8 samples per group). (c) Flow cytometric analysis of specific cell markers in MLN (4-5 samples per group). (d) mRNA expression levels of cytokines in MLN (5 samples per group). (e) Quantification of fecal LPS levels by Limulus amebocyte lysate (lysate) assay (7-8 samples per group). (f) Quantification of plasma LPS levels by Limulus amebocyte lysate assay (5 samples per group). (g) Quantification of plasma cytokine levels using cytokine / bead array (4-5 samples per group). LB: Quantification of fecal LPS levels by Limulus amebocyte lysate assay (5 samples per group). T Bacteroides dorothelii DSM 17855 T strains were treated; MFI: mean fluorescence intensity. To detect significant differences between the two groups, Student's two-sided t-test was used. Data are expressed as mean ± standard error of the mean (ag). *P < 0.05, **P < 0.01.

[0056] [ Figure 4 ] are graphs showing the effect of live Bacteroides treatment on tight junction formation. (a) Determination of fluorescein isothiocyanate (FITC)-labeled dextran concentration in serum 4 hours after oral gastric tube feeding of mice (4-5 samples per group). (b) mRNA expression levels of tight junction genes in mouse colon (7-8 samples per group). (c) Tight junction protein ZO-1 visualized by immunofluorescence staining (4-5 samples per group). White bars represent 250 μm. (d) mRNA levels of tight junction genes in HT29 cells stimulated with fecal supernatant (5 samples per group). (e) Western blot analysis of ZO-1 expression in HT29 cells stimulated with fecal supernatant (normalized to β-actin) (8 samples per group). LB: treated with live Bacteroides vulgaris ATCC 8482 T Bacteroides dorothelii DSM 17855 T strains were treated; LPS: lipopolysaccharide; MFI: mean fluorescence intensity. *P<0.05, **P<0.01, Student's two-sided t-test. Data are expressed as mean ± standard error of the mean.

[0057] [ Figure 5] are graphs showing intestinal microbial LPS production in patients with or without CAD. (a) Fecal LPS levels were quantified by horseshoe crab amebocyte lysate assay in the same samples as those used in 16SrRNA gene sequencing (29-30 samples per group). To determine significance, Student's two-sided t-test was used. *P<0.05. Data are shown as median ± interquartile range (1st to 3rd interquartile range). (b) Regarding the statistical correlation between fecal LPS levels and relative amounts of Bacteroides vulgaris and Bacteroides dodecene, simple linear correlation was calculated by determining Pearson's correlation coefficient (59 samples; 29 controls and 30 CAD). CAD: patients with coronary artery disease.

[0058] [ Figure 6 ] is a graph showing the distribution of intestinal microbiota in CAD patients or non-CAD patients. In order to compare the distribution of intestinal microbiota, principal component analysis at the species level was performed. The more important species are shown in the principal components.

[0059] [ Figure 7 ] is a graph showing the effect of tube feeding with live Bacteroides vulgaris and Bacteroides doxorubicin on the intestinal microbiota of mice prone to atherosclerosis. T Bacteroides dorothelii DSM 17855 T Female Apoe - / - Feces were collected from mice. The V3-V4 region of bacterial 16S rRNA was sequenced. (a) Relative abundance of the 15 most abundant microbial genera before and after treatment. (b) Principal component analysis (PCA) score plot at the genus level. (c) Diversity of intestinal microorganisms determined from the perspective of the Shannon-Wiener index. (d) Ratio of Gram-positive strains to Gram-negative strains after treatment. (e) Relative increase in the abundance of Bacteroides vulgaris and Bacteroides domitilis DNA in feces after treatment (n=3 per group). LB: with live Bacteroides vulgaris ATCC 8482 T Bacteroides dorothelii DSM 17855 T strains by gastric tube nutrition; before: before the experiment; after: 10 weeks after gastric tube nutrition.

[0060] [ Figure 8 ] is a graph showing the inhibition of cell proliferation and cytokine production caused by in vitro stimulation with mouse fecal supernatant. T Bacteroides dorothelii DSM17855 TRAW264.7 cells were stimulated with fecal supernatants from mice treated with either the strain or vehicle (control). (a) Representative flow cytometry histograms (left) and quantitative analysis of Ki-67 expression (right) (5 samples per group). (b) RAW264.7 cells transfected with control siRNA or TLR4 siRNA were stimulated with fecal supernatants from mice in each group. Cytokine levels were quantified by cytokine / bead array (7 samples per group). LB: LB with live Bacteroides vulgaris ATCC 8482 T Bacteroides dorothelii DSM17855 T strains were treated; MFI: mean fluorescence intensity. To detect significant differences between the two groups, Student's two-sided t-test (a) or Mann-Whitney's U test (b) was used. Data are expressed as mean ± standard error of the mean (a) or median ± interquartile range (1st to 3rd interquartile range) (b). *P < 0.05, **P < 0.01.

[0061] [ Fig. 9 ] is a graph showing treatment with live Bacteroides and immune responses in the spleen. T Bacteroides dorothelii DSM 17855 T 16-week-old Apoe - / - Splenocytes were collected from mice. (a) The percentage of immune cells expressing CD11c in splenocytes (4-5 samples per group). (b) CD11c high Expression of immune cell markers in antigen-presenting cells (4-5 samples per group). (c) CD4 + T cell count. (d) CD4 + Immune cell markers in T cell counts (5 samples per group). (e) CD4 + CD25 + Foxp3 + CTLA4 expression in Tregs (5 samples per group). LB: live Bacteroides vulgaris ATCC 8482 T Bacteroides dorothelii DSM 17855 T strains were treated; Tregs: regulatory T cells; MFI: mean fluorescence intensity. *P<0.05. To detect significant differences between the two groups, Student's two-sided t-test was used. Data are presented as mean ± standard error of the mean.

[0062] [ Fig.10 ] is a live Bacteroides vulgaris ATCC 8482 T Bacteroides dorothelii DSM 17855 TThe effect of single administration of the strain on ApoE-deficient mice. The control group only orally ingested the vehicle. The upper left shows the size of the aortic sinus atherosclerotic lesions, and the upper right shows the LPS concentration in the intestine (in feces). The upper left of the lower section shows the expression of tight junction-related genes in the large intestine, the upper right of the lower section shows the expression of cytokine genes in the large intestine, and the lower left of the lower section shows the cytokine concentration in the blood.

[0063] [ Fig.11 ] is a display of Bacteroides vulgaris ATCC 8482 T The 16S rRNA gene of strains was similar to that of Bacteroides dorotheliensis JCM 13471 T The alignment of 16S rRNA genes of strains.

[0064] [ Fig.12 ] is a display of Bacteroides vulgaris (ATCC 8482 T strains (v) and NTZ002 strain (Nv)) and Bacteroides dorothelioides (DSM17855 T strain (d) and NTZ001 strain (Nd)) on the improvement effect of fecal LPS level in the atherosclerosis mouse model.

[0065] [ Fig.13 ] is shown to be given to Bacteroides vulgaris ATCC 8482 T Bacteroides dorothelii DSM 17855 T The left and right sides of the figure show the LPS levels in the feces and cecum of mice given Bacteroides, respectively, and the center shows the LPS level in the feces of mice given antibiotics (AVNM).

[0066] [ Figure 14-1 ] Figure 14-1 and Figure 14-2 Bacteroides vulgaris ATCC 8482 T Bacteroides dorothelii DSM17855 T The improvement of various symptoms of obesity in the obese mouse model with live bacteria of Bacteroides NTZ002 (B) or Bacteroides NTZ002 and Bacteroides NTZ001 (NB). A schematically shows the administration regimen of each group. B and C show the changes in body weight and weight gain of mice, respectively. D shows the results of the OGTT test (from left to right: blood glucose level, AUC of blood glucose level and insulin resistance (HOMA-IR)).

[0067] [ Figure 14-2]E shows HE, MAC3 and Sirius red staining images of epididymal fat specimens (left), and shows the size of adipocytes, the number of coronary structures per unit cell, and the proportion of fibrotic areas from the top (right). F shows the mRNA expression of inflammatory cell markers or macrophage markers in the fat around the epididymis (left) and the mRNA expression of cytokines IFNγ and TNFα.

[0068] [ Figure 15-1 ] Figure 15-1 and Figure 15-2 Bacteroides vulgaris ATCC 8482 T Bacteroides dorothelii DSM17855 T The above figure shows the HE staining image of liver sections of 17-week-old mice. The middle figure shows the NAFLD activity score (NAS), and the lower figure shows the blood AST level and AST / ALT ratio from the left.

[0069] [ Figure 15-2 ] shows the whole-body image of the resected liver. DETAILED DESCRIPTION

[0070] 1. Lipopolysaccharide (LPS)-controlled intestinal bacteria

[0071] The present invention provides a combination of Bacteroides vulgaris and Bacteroides doreae, which are bacteria belonging to the genus Bacteroides (hereinafter, also referred to as "the combination of the present invention"). Here, "combination" refers to a substance in the state of a kit that can exist as a mixture of both, or can exist as separate compositions, but is used in combination for a certain purpose (e.g., medicine, feed or feed additive). The combination of the present invention can reduce the LPS level in the intestine by coexisting in the intestine of humans or other animals, strengthen the tight junctions of the intestinal barrier to reduce the LPS level in the blood, and inhibit the inflammatory response / immune response in the intestine, blood vessels and organs (in this specification, these effects are sometimes collectively referred to as "lipopolysaccharide (LPS) control).

[0072] More specifically, the “combination of the present invention” is formed by combining the following (1) and (2):

[0073] (1) A composition comprising live bacteria of Bacteroides vulgaris isolated from nature; and

[0074] (2) A composition comprising live bacteria of Bacteroides dornoni isolated from nature.

[0075] Bacteroides is a Gram-negative, obligately anaerobic, non-spore-forming bacillus classified as a true bacterium of the phylum Bacteroidetes, class Bacteroidetes, order Bacteroidetes, and family Bacteroidetes. Currently, there are 92 known species and 5 subspecies. Bacteroides includes several intestinal bacteria that predominate in the human body and are believed to play an important role in maintaining a healthy intestinal ecosystem.

[0076] Bacteroides vulgatus is the most common bacterium isolated from the human digestive tract and is generally considered to be a beneficial commensal bacterium. Bacteroides dorei was isolated from human feces in 2006 and identified as a new species closest to Bacteroides vulgatus (with about 96% homology) by 16S rRNA gene sequencing (Int JSys Evol Microbiol. 56, 1639-1643, 2006).

[0077] In this specification, "isolated from nature" means separation by artificial manipulation to make the density of bacteria higher than that in nature, including not only the case of complete purification (isolation and purification) but also the case of containing other impurities. On the other hand, when only natural products such as feces or cecal contents are collected, they are not included in the case of "isolated from nature".

[0078] As the active ingredients of the combination of the present invention, Bacteroides vulgaris and Bacteroides doraeformis can be provided by any method as long as they are naturally isolated bacteria, for example, bacteria newly isolated from natural separation sources (for example, feces, digestive tract contents such as cecum, soil, water) can be used. As the separation source, feces of humans or other mammals is preferred. For example, a fecal sample can be suspended in sterile phosphate buffered saline (PBS) or the like, and the resulting suspension can be inoculated with a platinum inoculation loop (platinum loop) or the like in a plate culture medium suitable for culturing Bacteroides bacteria, and the colonies of Bacteroides vulgaris and Bacteroides doraeformis are selected according to the mycological characteristics (morphological characteristics, biochemical characteristics, etc.) of the colonies that appear. Below, representative mycological characteristics of the two species are shown.

[0079] (1) Macroscopic characteristics (1-1) Round, white, convex colonies are formed on Eggerth Gagnon (EG) agar medium supplemented with 5% horse blood.

[0080] (1-2) Microscopic characteristics: The bacilli are not motile and do not form spores.

[0081] (2) Growth conditions (2-1) Temperature: Grows at 25-40°C. The optimum temperature is 37°C;

[0082] (2-2) Obligate anaerobic;

[0083] (3) Physiological / biochemical characteristics (3-1) Gram staining: negative;

[0084] (3-2) Other biochemical characteristics are shown in Table 1 [Table 1 shows that Bacteroides dorotheliensis JCM13471 T strains, 2 shows Bacteroides dorotheliensis JCM13472 strain, 3 shows Bacteroides vulgaris JCM5826 T Biochemical tests were performed using API 20A and API rapid ID32A test strips (bioMerieux). ](Extracted from Int J Sys Evol Microbiol. 56, 1639-1643, 2006).

[0085] [Table 1]

[0086]

[0087] Whether the bacteria isolated from the isolation source belongs to the strain of Bacteroides vulgaris or Bacteroides dorothelii can be determined, for example, by using the genomic DNA extracted from the strain as a template, performing PCR amplification on all or part of the 16S rRNA gene, determining the nucleotide sequence of the amplified fragment, comparing it with the sequence data of known Bacteroides vulgaris and Bacteroides dorothelii, and performing phylogenetic analysis. The phylogenetic analysis and the method for preparing the phylogenetic tree can be performed, for example, in the following order.

[0088] First, genomic DNA as a template is extracted by bacteria. The method of extracting DNA by bacteria is known, and any method can be adopted. The following methods are usually adopted: a method using cell wall decomposing enzymes such as lysozyme to treat cells, a method using glass beads to carry out physical destruction, a treatment method of repeated freezing and thawing, etc. In addition, commercially available DNA extraction reagents can also be used. Genomic DNA may not be extracted in a complete state. Therefore, the possibility of sample contamination is low, the operation is simple, and the method that can be carried out quickly can be appropriately selected.

[0089] Next, the target DNA encoding 16S rRNA is amplified by polymerase chain reaction (PCR). The sequence of the primers used in PCR can be appropriately designed so that the target DNA encoding 16S rRNA of at least all known bacteria belonging to Bacteroides vulgaris and / or Bacteroides doreae is amplified, and primers consisting of sequences preserved beyond biological species are generally used (universal primers; for example, a primer set of 27F and 357R for amplifying about 350 bases in the V1-V2 region, or a primer set of 342F and 806R for amplifying about 460 bases in the V3-V4 region used in the embodiments described later, etc.). In addition, there is no particular limitation on the conditions for PCR, and they can be appropriately selected within the range commonly used. Commercially available PCR reagents can be used to react according to the accompanying instructions.

[0090] The DNA fragment amplified by PCR is purified using a spin column or the like as necessary, and then its nucleotide sequence is determined. The nucleotide sequence can be determined according to conventional methods.

[0091] The determined nucleotide sequence can be extracted by using an appropriate gene sequence database and homology search program to perform a homology search with a known bacterial 16S ribosomal DNA sequence. For example, BLAST or FASTA can be used through the homepage of the DNA Database of Japan (DDBJ). If blastn or fasta is selected as the program, the determined nucleotide sequence is used as the sequence to be searched (Query), and 16S rRNA (Prokaryotes) is selected as the search object database for search, then the known sequence showing high homology can be extracted and output. As long as the data set of the nucleotide sequence of the 16SrRNA gene of the bacteria is included, any other gene sequence database (for example, RDP (http: / / rdp.cme.msu.edu), Silva (http: / / www.arb-silva.de), etc.) can also be used. In addition, a homology search program known to itself other than the above can also be used.

[0092] Generally, for bacteria, identification at the genus level requires a 16S rRNA gene sequence homology of more than 95%, while identification at the species level requires a 16S rRNA gene sequence homology of more than 98% (Science 2005, 307, 1915-1920). Therefore, as a result of homology search, if the sequence of the 16S rRNA gene of the isolated bacteria has a homology of more than 98% with a known sequence of bacteria belonging to Bacteroides vulgaris or Bacteroides doreae, the bacteria can be identified as belonging to any of the above Bacteroides species.

[0093] In the case of Bacteroides vulgaris, for example, the known 16S rRNA gene sequence as a discrimination standard includes: T strain (SEQ ID NO: 1), derived from Bacteroides vulgaris JCM5826 registered with accession number NR_112946 T On the other hand, examples of known 16S rRNA gene sequences of Bacteroides dornonis include: T strain (SEQ ID NO: 3), a sequence derived from Bacteroides doreae strain JCM13472 registered with accession number AB242143 (SEQ ID NO: 4), etc., but are not limited to these.

[0094] Therefore, when the nucleotide sequence of the PCR amplified fragment has 98% or more homology with the nucleotide sequence represented by SEQ ID NO: 1 or 2, the isolated bacteria can be identified as bacteria belonging to Bacteroides vulgaris. In addition, when the nucleotide sequence of the amplified fragment has 98% or more homology with the nucleotide sequence represented by SEQ ID NO: 3 or 4, the isolated bacteria can be identified as bacteria belonging to Bacteroides dorothelii.

[0095] In addition, the molecular evolution phylogenetic tree can also be inferred based on the nucleotide sequence of the amplified DNA, and the taxonomic position of the specific isolated bacteria can be determined. Molecular evolution phylogenetic tree analysis software is also disclosed on the Internet, etc., and these software (CLUSTAL W, etc.) can be used. The result of the phylogenetic tree analysis, when the isolated bacteria are located in the same cluster as the bacteria belonging to common Bacteroides or Bacteroides dossii, can identify the bacteria as belonging to any Bacteroides species.

[0096] Alternatively, the Bacteroides vulgaris and Bacteroides dornii used in the combination of the present invention may be strains that have been isolated and commercially available, or stored in a depository and can be distributed. Examples of such strains include Bacteroides vulgaris, ATCC 8482 T strain, DSM 1447 T strain, JCM 5826 T strain, NBRC 14291 T strain, NCTC 11154 T strains, NTZ002 strains, etc. In the case of Bacteroides dorothelii, for example, DSM 17855 T strain, JCM 13471 Tstrain, JCM 13472 strain, NTZ001 strain, etc., but not limited to these. On January 15, 2019, the NTZ002 strain and NTZ001 strain were assigned the accession numbers NITE BP-02863 and NITE BP-02862, respectively, and were internationally deposited in the Patent Microorganism Collection Center of the Institute of Technology for Product Evaluation (Room 8122, 2-5-1, Kazusa Kamata, Kisarazu City, Chiba Prefecture, 〒292-0818, Japan).

[0097] The combination of Bacteroides vulgaris and Bacteroides doreae used in the present invention may be any combination of strains as long as they can reduce the LPS level in the intestine, strengthen the tight junction of the intestinal barrier to reduce the LPS level in the blood, and inhibit the inflammatory response / immune response in the intestine, blood vessels and organs (i.e., have LPS control properties) by coexisting in the intestine of humans or other animals. In a preferred embodiment, Bacteroides vulgaris ATCC 8482 can be cited. T strains or NTZ002 and Bacteroides dorotheliensis DSM 17855 T strain or NTZ001 strain, but is not limited thereto.

[0098] The common Bacteroides or Bacteroides douglas used in the combination of the present invention can exert LPS control when combined with Bacteroides douglas or common Bacteroides as a combined bacterium, respectively. It can be not only an isolate (wild strain) from a natural isolation source obtained by any of the above methods, but also a mutant obtained by subjecting these isolates to mutagenesis. As a mutagenesis treatment, for example, methods of exposure to chemical substances such as nitroso compounds (e.g., nitrosamines, nitrosoguanidines), alkylating agents (e.g., ethyl methanesulfonate (EMS)), or ultraviolet irradiation, radiation irradiation, etc., but are not limited to these. Regarding whether the obtained mutant strain shows LPS control when combined with a combined bacterium, for example, the mutant strain and the combined bacterium can be administered to a disease animal model (e.g., an ApoE-deficient atherosclerosis mouse model, an obese mouse model, a NASH mouse model, etc.), and when the improvement of symptoms and various parameters (e.g., blood or intestinal LPS levels, blood cytokine levels, intestinal barrier function, body weight, organ weight, pathological tissue examination, etc.) are improved, it can be determined that the mutant strain has LPS control. Alternatively, when immune cells such as macrophages are stimulated with fecal supernatant of the animal model, the mutant strain and the combined bacteria are added, and if the proliferation of the cells or the production of inflammatory cytokines is inhibited, the mutant strain can be determined to have LPS-controlling properties.

[0099] The Bacteroides vulgaris and Bacteroides doreae obtained in this way can be cultured under known culture conditions and maintained for proliferation.

[0100] As the culture medium, ATCC medium 2107 (10 g of peptone, 10 g of beef extract, 3 g of yeast extract, 5 g of glucose, 5 g of NaCl, 1 g of soluble starch, 0.5 g of L-cysteine ​​hydrochloride, 3 g of sodium acetate, 4 mL of 0.025% resazurin, 1 L of distilled water), ATCC medium 260 (15 g of tryptone, 5 g of soytone, 5 g of NaCl, 50 mL of defibrinated sheep blood, 950 mL of distilled water), EG medium supplemented with 5% horse blood, etc. can be used. These culture media can be used as liquid culture media or as solid culture media by adding, for example, 1.5% agar.

[0101] Since Bacteroides vulgaris and Bacteroides doreae are obligate anaerobic bacteria, the culture is carried out under anaerobic conditions (oxygen concentration is below 1 ppm). For example, the culture can be carried out in an anaerobic chamber in a mixed gas environment of 10% CO2, 10% H2 and 80% N2. The culture temperature is 25 to 40°C, preferably about 37°C. As the culture period, for example, 12 to 72 hours, preferably 24 to 48 hours, can be listed, but it is not particularly limited.

[0102] Bacteroides vulgaris and Bacteroides dornoni may be mixed cultured in the same medium as long as they do not adversely affect each other's growth or biological activity. In addition, Bacteroides vulgaris and Bacteroides dornoni included in the combination of the present invention may be single strains or a mixture of two or more strains.

[0103] The culture of Bacteroides vulgaris and Bacteroides doreae obtained in this way can be frozen and stored at -80°C or below in the form of GLYCEROL STOCK according to a method known per se, or lyophilized and stored at 2 to 8°C according to a method known per se, before use.

[0104] 2. Combination of the present invention

[0105] As long as Bacteroides vulgaris and Bacteroides doreae are in a living state (live bacteria), the above-mentioned culture (bacteria) can be directly mixed into the composition containing these bacteria, or in the state of "bacteria processed materials" such as wet bacteria recovered from the culture by a method known per se, such as centrifugation, filtration, magnetic separation, etc., or their washings (which can be washed with sterile water, culture medium, PBS, etc.) or their freeze-dried powders. It should be noted that in this specification, unless otherwise specified, "bacteria" or "bacteria processed materials" are used in the sense of "non-living" components that do not include dead bacteria or broken bacteria, bacterial extracts, bacterial components, etc.

[0106] Regarding the cells or processed cells of Bacteroides vulgaris and the cells or processed cells of Bacteroides dornoni, they can be prepared into preparations alone or together with additives acceptable in pharmaceutical or feed processing. Alternatively, the cells or processed cells can be mixed into a pharmaceutical composition or feed as a pharmaceutical additive or a feed additive. In the combination of the present invention, the cells or processed cells of Bacteroides vulgaris and the cells or processed cells of Bacteroides dornoni can be prepared into preparations as a single composition and then used in the form of a complete preparation, but are preferably mixed in the same composition and can be used as a single preparation.

[0107] When the cells or processed cells of Bacteroides vulgaris and the cells or processed cells of Bacteroides doreae are provided in the form of a single composition, these compositions may be inoculated simultaneously or with a time difference through the same route or different routes.

[0108] When the combination of the present invention is provided as a medicine or a medicine additive, the medicine mixed with the medicine or the additive can be prepared into preparations such as powders, granules, pills, soft capsules, hard capsules, tablets, chewable tablets, rapidly disintegrating tablets, syrups, solutions, suspensions, suppositories, injections, etc.

[0109] For example, as a composition for oral administration, solid or liquid dosage forms can be listed, specifically, tablets (including sugar-coated tablets, film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such compositions can be prepared by known methods and may contain additives commonly used in the field of preparations, such as excipients, binders, disintegrants, lubricants, etc. As excipients, for example, animal and plant oils such as soybean oil, saffron oil, olive oil, germ oil, sunflower oil, butter, sardine oil, etc.; polyols such as polyethylene glycol, propylene glycol, glycerol, sorbitol, etc.; surfactants such as dehydrated sorbitan fatty acid esters, sucrose fatty acid esters, glycerol fatty acid esters, polyglycerol fatty acid esters, etc.; purified water, lactose, starch, crystalline cellulose, D-mannitol, lecithin, gum arabic, sorbitol solution, sugar solution, etc. As binders, for example, hydroxypropyl methylcellulose, hydroxypropyl cellulose, gelatin, alpha starch, polyvinyl pyrrolidone, polyvinyl alcohol, etc. can be listed. Examples of disintegrants include carboxymethylcellulose calcium, carboxymethylcellulose sodium, cross-linked carboxymethylcellulose sodium, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, corn starch, etc. Examples of lubricants include talc, hydrogenated vegetable oils, waxes, light silicic anhydride and other natural sources and derivatives thereof; stearic acid, magnesium stearate, calcium stearate, aluminum stearate, etc.

[0110] In the above composition, sweeteners, colorants, pH regulators, spices, various amino acids, etc. may be further added. In addition, tablets and granules may be coated by known methods. If it is a liquid preparation, it may be dissolved or suspended in water or other appropriate media when taken.

[0111] As compositions for parenteral administration, for example, injections, suppositories, etc. are used. As a preparation method for injections, for example, the cells or cell processing materials of Bacteroides vulgaris and Bacteroides dornoni are suspended or emulsified in a sterile aqueous solution or oily solution commonly used for injections. As aqueous solutions for injection, for example, physiological saline, isotonic solutions containing glucose or other adjuvants, etc. are used. As oily solutions, for example, sesame oil, soybean oil, etc. are used. Suppositories for rectal administration can be prepared by mixing the cells or cell processing materials of Bacteroides vulgaris and Bacteroides dornoni in a common suppository matrix (base).

[0112] Moreover, when provided as a medicine or a medicine additive, the combination of the present invention can be used in combination with other drugs, such as anti-inflammatory drugs, anti-atherosclerotic drugs, antidiabetic drugs, etc., according to the target disease. The combination of the present invention and the combination agent can be prepared as a single composition (mixture) or provided as a single composition. When provided as a single composition, the combination of the present invention and the combination agent can be administered to the subject simultaneously or with a time difference in the same route or different routes.

[0113] When the combination of the present invention is provided as a feed or a feed additive, the feed to which the feed or the additive is mixed is not particularly limited as long as it is in an orally ingestible form such as a solution, a suspension, a powder, or a solid molded product.

[0114] The above-mentioned feed may be mixed with various nutrients, various vitamins (vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, vitamin E, vitamin K, etc.), various minerals (magnesium, zinc, iron, sodium, potassium, selenium, etc.), dietary fiber, dispersants, stabilizers such as emulsifiers, sweeteners, flavoring ingredients (citric acid, malic acid, etc.), flavors, royal jelly, propolis, agaricus, etc. as needed.

[0115] The number of viable bacteria of Bacteroides vulgaris and Bacteroides doreae contained in the combination of the present invention is, for example, 10 4 ~10 12 Colony forming units (cfu), preferably 10 6 ~10 10 cfu.

[0116] In the combination of the present invention, the thalli or thalli processed products of other effective microorganisms can also be further mixed. As such other microorganisms, for example, can be listed: lactic acid bacteria, yeast, Bacillus, Clostridium butyricum, Aspergillus, etc. belonging to Lactobacillus, Streptococcus, Leuconostoc, Pediococcus, Lactococcus, Enterococcus, Bifidobacterium, etc., but are not limited to these. These and use microorganisms as long as the effectiveness is visible, not only in the state of live bacteria, but also in the form of dead bacteria or thalli fragments, thalli extracts, thalli components, etc., mixed in the combination of the present invention.

[0117] The amount of the microorganisms to be used in combination is, for example, 10 4 ~10 12 Colony forming units (cfu), preferably 10 6 ~10 10 cfu.

[0118] 3. Uses of the Combination of the Invention

[0119] As described above, Bacteroides vulgaris and Bacteroides doreae can reduce the LPS level in the intestine by coexisting in the intestine, strengthen the tight junctions of the intestinal barrier to reduce the LPS level in the blood, and inhibit the inflammatory response / immune response in the intestine, blood vessels and organs (i.e., have LPS control), so by enriching the two species in the intestinal flora, diseases related to the hyperactivity of LPS levels in the blood or intestine can be prevented and / or improved. Therefore, the combination of the present invention can be used as a drug or drug additive for preventing and / or improving these diseases, or a feed or feed additive.

[0120] Examples of diseases associated with increased levels of LPS in the blood or intestines include circulatory system diseases such as atrial fibrillation, heart failure, ischemic heart disease, myocardial infarction, angina pectoris, hypertension, arteriosclerosis, aortic aneurysm, aortic dissection, occlusive arteriosclerosis, and aortic stenosis; or inflammatory diseases such as hepatitis, non-alcoholic steatohepatitis, fatty liver, liver cancer caused by progression of hepatitis, intestinal inflammation, allergic bowel syndrome, gastritis, collagen disease, chronic rheumatoid arthritis, chronic nephritis, IgA nephropathy, bronchial asthma, interstitial pneumonia, drug-induced lung injury, eosinophilic pulmonary infiltration syndrome, atypical acid-resistant bacteria disease, allergic rhinitis, atopic dermatitis, and sepsis; or metabolic diseases such as diabetes, obesity, metabolic syndrome, lifestyle-related diseases, lipid disorders, and osteoporosis.

[0121] The combination of the present invention can be orally administered to humans or other mammals (e.g., dogs, cats, mice, rats, hamsters, guinea pigs, rabbits, pigs, cattle, goats, horses, sheep, monkeys, etc.) once a day or divided into several times to obtain the above-mentioned daily intake. Alternatively, it can be administered rectally.

[0122] 4. Inspection method of the present invention and reagents used in the method

[0123] The present invention also provides a method for testing the risk of developing a disease associated with an elevated level of LPS in the blood or intestine (hereinafter also referred to as an "LPS-related disease") (hereinafter also referred to as the "test method of the present invention") and a reagent for the test method (hereinafter also referred to as the "test drug of the present invention"), the test method comprising measuring the amount of Bacteroides vulgaris and Bacteroides dorotheli in the intestinal flora of a subject. As shown in the examples described below, the amount of Bacteroides vulgaris and Bacteroides dorotheli in the intestinal flora of patients with coronary artery disease (CAD) is significantly reduced, and when the two strains are orally ingested by an arteriosclerosis mouse model, the symptoms of arteriosclerosis are improved while the level of LPS in the intestine is reduced, the tight junctions of the intestinal barrier are strengthened to reduce the level of LPS in the blood, and the inflammatory response / immune response in the intestine and blood vessels can be suppressed (i.e., it has LPS control). Furthermore, when the two strains are orally ingested by an obese mouse model, the symptoms of obesity are improved while the inflammatory response / immune response in the organs is suppressed. Moreover, if non-alcoholic fatty liver disease model mice are allowed to orally ingest the two strains, the inflammatory response / immune response in the organs can be suppressed while improving the symptoms of non-alcoholic fatty liver disease. Therefore, by measuring the presence of common Bacteroides and Bacteroides dodecene in the intestinal flora of the subject and comparing them with normal values, it can be determined whether the subject will develop an LPS-related disease or have a high risk of developing the disease in the future. It should be noted that in this specification, "risk of disease examination" is used in the sense of not only predicting the high possibility of future disease onset, but also including examinations for diagnosing existing disease. In addition, in this specification, the term "detection" is used in the sense of not only determining whether the target bacteria are present, but also quantitatively measuring their presence, except where such an interpretation is obviously wrong.

[0124] There is no particular limitation on the subjects to be examined by the examination method of the present invention. For example, it may also include: those suspected of having circulatory system diseases, inflammatory diseases, metabolic diseases and other LPS-related diseases based on clinical findings; or patients who are found to have suffered from these diseases. This is because: if it is known that the presence of common Bacteroides and Bacteroides dodecene in the intestinal flora is reduced, the enrichment of this species in the intestinal flora can be used as one of the treatment goals for the patient. It should be noted that as a sample derived from the subject for the examination, feces is preferred from the perspective of ease of collection, but as long as it is a sample that can reflect the intestinal flora of the subject, there is no particular limitation, for example, it can be intestinal contents (for example, cecal contents) and the like.

[0125] The detection of Bacteroides vulgaris and Bacteroides dobromites can be carried out, for example, by detecting the 16S rRNA gene possessed by the bacterial species. Specifically, in the case of Bacteroides vulgaris, it can be carried out by detecting the 16S rRNA gene comprising a nucleotide sequence having more than 98% homology with the nucleotide sequence represented by SEQ ID NO: 1 or 2; in the case of Bacteroides dobromites, it can be carried out by detecting the 16S rRNA gene comprising a nucleotide sequence having more than 98% homology with the nucleotide sequence represented by SEQ ID NO: 3 or 4.

[0126] When detecting the 16S rRNA gene of Bacteroides vulgaris and Bacteroides doreae, total DNA is first recovered from the sample. Methods for isolating / purifying DNA from a stool sample of a subject are known in the art, and can be performed, for example, by phenol / chloroform extraction, extraction using a commercially available DNA extraction reagent, or purification using a commercially available column kit.

[0127] The DNA recovered from the sample is dissolved in an appropriate buffer, for example, TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and used for detection of the 16S rRNA gene.

[0128] In one embodiment, detection of the 16S rRNA gene is carried out as follows: detection is carried out by using primers that can specifically amplify the 16S rRNA gene of Bacteroides vulgaris, i.e., a 16S rRNA gene comprising a nucleotide sequence having more than 98% homology to the nucleotide sequence represented by SEQ ID NO: 1 or 2 and / or the 16S rRNA gene of Bacteroides doreae, i.e., a 16S rRNA gene comprising a nucleotide sequence having more than 98% homology to the nucleotide sequence represented by SEQ ID NO: 3 or 4, and PCR is performed using DNA recovered from the sample as a template.

[0129] The primers can be any primers as long as they are designed to specifically PCR amplify a part or all of the 16S rRNA gene. Here, "specifically" means that the primers will PCR amplify a part or all of the 16S rRNA gene of Bacteroides vulgaris and / or Bacteroides doreae, but will not PCR amplify 16S rRNA genes other than the species.

[0130] Examples of the primers include a combination of two polynucleotides: a polynucleotide comprising a nucleotide sequence of about 15 to about 50 bases, preferably about 18 to about 30 bases, which hybridizes with a continuous partial sequence of a complementary sequence to a nucleotide sequence having 98% or more homology to a nucleotide sequence represented by SEQ ID NO: 1 or 2 (preferably a nucleotide sequence represented by SEQ ID NO: 1 or 2) and / or a continuous partial sequence of a complementary sequence to a nucleotide sequence having 98% or more homology to a nucleotide sequence represented by SEQ ID NO: 3 or 4 (preferably a nucleotide sequence represented by SEQ ID NO: 3 or 4); and a polynucleotide comprising a nucleotide sequence of about 15 to about 50 bases, preferably about 18 to about 30 bases, which hybridizes with a continuous partial sequence of the nucleotide sequence on the 3' side from the hybridization site. Examples of the polynucleotides include a pair of polynucleotides wherein the length of a nucleic acid fragment amplified by these polynucleotides is about 50 to about 1,000 bases, preferably about 100 to about 500 bases.

[0131] The primer preferably comprises: a partial sequence of 15 to 50 consecutive bases (preferably 18 to 30 bases) of a nucleotide sequence having 98% or more homology with the nucleotide sequence represented by SEQ ID NO: 1 or 2 (preferably the nucleotide sequence represented by SEQ ID NO: 1 or 2) and / or a partial sequence of 15 to 50 consecutive bases (preferably 18 to 30 bases) of a nucleotide sequence having 98% or more homology with the nucleotide sequence represented by SEQ ID NO: 3 or 4 (preferably the nucleotide sequence represented by SEQ ID NO: 3 or 4), or a partial sequence of 15 to 50 consecutive bases (preferably 18 to 30 bases) of their complementary sequences.

[0132] From the perspective of specificity, as a preferred primer set, at least one of them is a primer having the following nucleotide sequence: a nucleotide sequence located at any of the 9 variable regions (V1 to V9) in the 16S rRNA gene whose sequences differ between bacterial species in the nucleotide sequence represented by SEQ ID NO: 1 or 2 and / or the nucleotide sequence represented by SEQ ID NO: 3 or 4, or a nucleotide sequence complementary to its complementary chain sequence.

[0133] In the inspection method of the present invention, the 16S rRNA gene of Bacteroides vulgaris and the 16S rRNA gene of Bacteroides dorotheli can be distinguished for detection, or the two can be detected together without distinguishing them. As shown in the comparative example described later, even if either Bacteroides vulgaris or Bacteroides dorotheli is orally ingested by an arteriosclerosis mouse model alone, the symptoms of arteriosclerosis tend to improve, but no significant difference is observed, and the level of LPS in the intestine cannot be reduced. Therefore, it is expected that Bacteroides vulgaris and Bacteroides dorotheli can be distinguished for detection, but in reality it is difficult to imagine that the presence of only one of them in the intestinal flora changes significantly, so even if the two are detected together, it will not have a substantial impact on the determination result.

[0134] For example, if BLAST (NCBI) is used under default conditions to compare Bacteroides vulgaris ATCC 8482 T strain JCM 13471 T The 16S rRNA gene (SEQ ID NO: 3) of the strain is as follows: Fig.11 As shown (Query (query sequence) shows SEQ ID NO: 1, Sbjct (target sequence) shows SEQ ID NO: 3). The two sequences overlapped by 1493 nucleotides (12-1497 of SEQ ID NO: 1, 1-1490 of SEQ ID NO: 3; the number of gaps was 10), showing a high homology of about 97% (1449 / 1493). Therefore, the design regions of the common Bacteroides specific primers and the Bacteroides dornoni specific primers that can distinguish and amplify the 16S rRNA gene of Bacteroides common and Bacteroides dornoni are limited. For example, positions 181 to 210 of SEQ ID NO:1 have only about 73% (22 / 30) homology with the corresponding region of SEQ ID NO:3, while positions 1001 to 1028 of SEQ ID NO:1 have only about 60% (18 / 30) homology with the corresponding region of SEQ ID NO:3. Therefore, by designing any one or two primers in these regions, the 16S rRNA gene of Bacteroides vulgaris and the 16S rRNA gene of Bacteroides doreae can be distinguished and amplified.

[0135] On the other hand, when the 16S rRNA genes of Bacteroides vulgaris and Bacteroides dornoni are amplified together, for example, if a primer set recorded as a primer specific for Bacteroides vulgaris in "Molecular Ecology of Intestinal Flora" edited by Tomotsu Mitsuoka, pp. 109-121 (2000) (Bacteroides dornoni had not yet been identified at the time) is used (amplifying positions 136 to 608 of SEQ ID NO: 1 and positions 125 to 599 of SEQ ID NO: 3), the 16S rRNA genes of both bacterial species can be amplified together, and the 16S rRNA genes of other intestinal bacteria will not be amplified.

[0136] Moreover, when this primer set is used, although there is a TaqI restriction site in the amplified fragment of SEQ ID NO:1 (TCGA; positions 288 to 291 of SEQ ID NO:1), there is no TaqI restriction site in the amplified fragment of SEQ ID NO:3, so if the amplified fragment is digested with TaqI and then electrophoresed, two bands of 153bp and 320bp will be detected when it is assumed that only Bacteroides vulgaris exists, but a band of 475bp will be detected when only Bacteroides dobrosii exists. The above three bands will be detected when two bacterial species are present. Therefore, RFLP analysis can also be combined with primers that can amplify the 16S rRNA genes of Bacteroides vulgaris and Bacteroides dobrosii together to distinguish the two bacterial species for detection.

[0137] The temperature setting, reaction time and number of cycles in PCR can be appropriately set according to the amount of template DNA used, the type of primers, etc. The annealing temperature in PCR can be appropriately set according to the GC content of the primers.

[0138] The obtained PCR products are separated by electrophoresis (e.g., agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc.). After electrophoresis, the gel is stained with a known staining solution such as ethidium bromide solution, and the PCR products are detected using a transilluminator (transmitter). Then, the presence or amount of the specific PCR product is used as an indicator to determine whether or not there are common Bacteroides and / or Bacteroides doreae in the sample or the amount of the presence.

[0139] The PCR used in the inspection method of the present invention may be quantitative PCR. Quantitative PCR can be performed by known methods, and two analytical methods are known. The first method is to utilize the characteristic that the reaction product in the PCR reaction increases exponentially until it reaches a certain amount and then reaches stability, analyze the amount of the reaction product during the exponential increase, and calculate the initial template amount. The second method is to monitor the reaction product in real time and determine the PCR cycle number (Ct) at which the amount of the reaction product exceeds a certain value (threshold). Both of these analytical methods require changing the amount of DNA of known concentration to perform PCR, analyze the reaction products in each cycle number, and determine the quantitative PCR cycle number range by its kinetics. Estimate the amount of the target gene in the unknown sample based on the results. Thus, the amount of Bacteroides vulgaris and / or Bacteroides domitilis in the sample can be quantified, and when it is estimated that the test sample contains only 1 copy of the target gene, it can also be determined that the target bacteria are present.

[0140] In another embodiment, the detection of the 16S rRNA gene can be performed as follows: a probe that can specifically hybridize the 16S rRNA gene of Bacteroides vulgaris, i.e., a 16S rRNA gene containing a nucleotide sequence having 98% or more homology to the nucleotide sequence represented by SEQ ID NO: 1 or 2 and / or the 16S rRNA gene of Bacteroides dobrosii, i.e., a 16S rRNA gene containing a nucleotide sequence having 98% or more homology to the nucleotide sequence represented by SEQ ID NO: 3 or 4, is contacted with the total DNA or total RNA in the sample, and the hybrid is detected, thereby the 16S rRNA gene can be detected. The contact conditions can be appropriately set so that the probe hybridizes with the 16S rRNA or the gene encoding it to form a nucleic acid complex. Then, the complex is detected as a marker showing the presence of Bacteroides vulgaris and / or Bacteroides dobrosii.

[0141] The above-mentioned probe is a polynucleotide that hybridizes under stringent conditions with a continuous nucleotide sequence of about 15 bases or more, preferably about 18 to about 500 bases, more preferably about 18 to about 200 bases, and even more preferably about 18 to about 50 bases contained in a nucleotide sequence having 98% or more homology with the nucleotide sequence represented by SEQ ID NO: 1 or 2 (preferably the nucleotide sequence represented by SEQ ID NO: 1 or 2) and / or a nucleotide sequence having 98% or more homology with the nucleotide sequence represented by SEQ ID NO: 3 or 4 (preferably the nucleotide sequence represented by SEQ ID NO: 3 or 4), or its complementary sequence.

[0142] Hybridization can be performed according to a method known per se or a method based thereon, such as the method described in Molecular Cloning, 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). As stringent conditions, for example, there can be cited: after a hybridization reaction at 45°C in 6×SSC (sodium chloride / sodium citrate), washing once or more at 65°C in 0.2×SSC / 0.1% SDS. A person skilled in the art can easily adjust to the desired stringency by appropriately changing the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the probe length, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the temperature of the washing, etc.

[0143] The probe is preferably a nucleic acid comprising a continuous nucleotide sequence of about 15 bases or more, preferably about 18 to about 500 bases, more preferably about 18 to about 200 bases, and even more preferably about 18 to about 50 bases contained in a nucleotide sequence having 98% or more homology to the nucleotide sequence represented by SEQ ID NO: 1 or 2 (preferably the nucleotide sequence represented by SEQ ID NO: 1 or 2) and / or a nucleotide sequence having 98% or more homology to the nucleotide sequence represented by SEQ ID NO: 3 or 4 (preferably the nucleotide sequence represented by SEQ ID NO: 3 or 4), or a complementary sequence thereof.

[0144] From the perspective of specificity, the following nucleic acids can be cited as preferred probes: nucleic acids that hybridize under stringent conditions with a nucleotide sequence represented by SEQ ID NO: 1 or 2 or a nucleotide sequence represented by SEQ ID NO: 3 or 4, located at any one of the 9 variable regions (V1 to V9) in the 16S rRNA gene that differ in sequence between bacterial species, or a complementary sequence thereof. Specifically, the following nucleic acids can be cited: Fig.11 The regions exemplified as the design regions of the above primers have low homology in the comparison of SEQ ID NO: 1 and SEQ ID NO: 3 shown in the figure. Alternatively, nucleic acids containing the regions exemplified as primers that can specifically and simultaneously amplify the 16S rRNA gene of Bacteroides vulgaris and the 16S rRNA gene of Bacteroides dornoni can also be cited as probes that can specifically detect Bacteroides vulgaris and Bacteroides dornoni.

[0145] The primers or probes constituting the test drug of the present invention may contain additional sequences (nucleotide sequences that are not complementary to the polynucleotide to be detected) within a range that does not hinder specific detection.

[0146] The polynucleotide used as the above primer or probe may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In the case of ribonucleic acid, the thymidine residue (T) in the nucleotide sequence may be appropriately read as a uridine residue (U). In addition, it may be a DNA containing a uridine residue synthesized by replacing T at any position with U. It may also be an RNA containing a thymidine residue synthesized by replacing U at any position with T. In addition, as long as the specificity of hybridization is not reduced, point mutations such as deletion, insertion or substitution or modified nucleotides may exist in the polynucleotide.

[0147] In addition, the primers or probes may be labeled with a suitable marker (labeling agent), such as a radioisotope (e.g. 125 I. 131 I. 3 H. 14 C. 32 P. 33 P. 35 S, etc.), enzymes (e.g., β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (e.g., fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (e.g., luminol, luminol derivatives, fluorescein, lucigenin, etc.), biotin, etc.

[0148] The nucleic acid used as the primer or probe can be chemically synthesized using a general-purpose DNA synthesizer, for example. The nucleic acid can be synthesized using any other method known in the art.

[0149] The test drug of the present invention may further contain nucleic acid synthase (DNA polymerase, RNA polymerase, reverse transcriptase, etc.), other enzymes, substrates suitable for the enzyme (dNTP, rNTP, etc.), etc., as other components. In addition, it may also contain a labeled detection substance or a buffer.

[0150] In another embodiment, the detection of Bacteroides vulgaris and / or Bacteroides dornoni can also be performed by an immunological method known per se using an antibody that specifically recognizes the bacterium (hereinafter, also referred to as "the antibody of the present invention"). The antibody that specifically recognizes Bacteroides vulgaris and / or Bacteroides dornoni refers to an antibody that recognizes and binds to a surface antigen that specifically exists on the cell surface of the bacterium. Examples of such surface antigens include polysaccharides, peptidoglycans, or proteins present on the cell wall. Alternatively, it is not limited to surface antigens, and may also be an antibody that recognizes and binds to specific constituents of Bacteroides vulgaris and / or Bacteroides dornoni. Such specific antibodies can be obtained as follows: using Bacteroides vulgaris or Bacteroides dornoni or their processed bacteria as immunogens, preparing polyclonal antibodies or monoclonal antibodies by a manufacturing method known per se, and selecting clones that do not cross-react with closely related species other than Bacteroides vulgaris and / or Bacteroides dornoni from the obtained antibody clones, thereby obtaining specific antibodies. The antibodies of the present invention include not only natural antibodies but also antibody fragments having specific binding activity, specifically, for example, F(ab')2, Fab', Fab, Fv, sFv, dsFv, sdAb, etc. The class of the antibody is not particularly limited, and also includes antibodies having any isotype of IgG, IgM, IgA, IgD or IgE, etc. IgG or IgM is preferred, and IgG is more preferred in consideration of the ease of purification.

[0151] By using the antibody of the present invention, Bacteroides vulgaris and / or Bacteroides doreae can be detected and quantified by immunological methods. Examples of immunological methods include flow cytometry, radioisotope immunoassay (RIA), ELISA (Methods in Enzymol. 70: 419-439 (1980)), Western blotting, and immunohistostaining, but are not limited thereto.

[0152] When any of the above methods confirms that the presence of Bacteroides vulgaris and Bacteroides doreae in the intestinal flora of the subject is lower than the normal value (for example, a cutoff value determined based on the amount of the bacteria in the intestinal flora of healthy people (for example, mean value-2SD, etc.)), it can be determined that the subject is currently developing an LPS-related disease or has a high risk of developing the disease in the future. Conversely, when the two bacterial species are below the cutoff value, it can also be determined that the subject does not have LPS and has a low risk of developing the disease in the future.

[0153] Here, LPS-related diseases include, for example, circulatory system diseases such as atrial fibrillation, heart failure, ischemic heart disease, myocardial infarction, angina pectoris, hypertension, arteriosclerosis, aortic aneurysm, aortic dissection, occlusive arteriosclerosis, and aortic stenosis; or, inflammatory diseases such as hepatitis, non-alcoholic steatohepatitis, fatty liver, liver cancer caused by progression of hepatitis, intestinal inflammation, allergic bowel syndrome, gastritis, collagen disease, chronic rheumatoid arthritis, chronic nephritis, IgA nephropathy, bronchial asthma, interstitial pneumonia, drug-induced lung injury, eosinophilic pulmonary infiltration syndrome, atypical acid-resistant bacteria disease, allergic rhinitis, atopic dermatitis, and sepsis; or, metabolic diseases such as diabetes, obesity, metabolic syndrome, lifestyle diseases, lipid disorders, and osteoporosis.

[0154] The present invention will be described in more detail with reference to the following examples. However, the examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention in any way.

[0155] Example

[0156] Materials and methods

[0157] Recruitment of CAD patients and controls

[0158] All participants provided written informed consent after enrollment. The study was conducted in accordance with the principles of the Declaration of Helsinki. This study was approved by the Ethics Committee of Kobe University (approval number 1595) and registered in the UMIN Clinical Trial Registry (trial registration number 000015703, URL: http: / / www.umin.ac.jp / ctr / ).

[0159] Thirty CAD patients and 30 non-CAD controls with coronary risk factors were recruited from October 2014 to July 2015 through Kobe University Hospital. The CAD group included patients with stable angina and patients with previous myocardial infarction with preserved left ventricular ejection fraction (>40%) who had undergone percutaneous coronary intervention or coronary artery bypass grafting at least 6 months before the study. Patients with acute coronary syndrome were excluded. Single-vessel disease or multivessel disease was defined from the perspective of the number of major coronary arteries showing more than 75% stenosis on diagnostic coronary angiography.

[0160] Thirty patients who had coronary risk factors such as hypertension, diabetes, and / or dyslipidemia but had no current or past history of coronary artery disease or other vascular disease were recruited as age- and sex-matched controls. Past medical history of coronary artery disease or other vascular disease was defined as confirmed vascular disease, symptoms showing angina, abnormalities on electrocardiogram showing previous myocardial infarction or angina, or abnormalities on chest X-ray.

[0161] Patients with systemic diseases including liver disease, kidney disease (serum creatinine level >2.0 mg / dL), collagen disease, or cancer were excluded from the study. Patients treated with antibiotics were also excluded. Diabetes was defined as a glycated hemoglobin value >6.5% according to the National Glycated Hemoglobin Standardization Program, when oral antidiabetic drugs were used, or when insulin therapy was being administered. Hypertension was defined as a blood pressure >140 / 90 mmHg or when antihypertensive drugs were used. Dyslipidemia was defined as a low-density lipoprotein cholesterol level >140 mg / dL, a triglyceride level >150 mg / dL, or when anti-dyslipidemia drugs were used, according to the guidelines issued by the Japanese Atherosclerosis Society.

[0162] DNA extraction from stool samples

[0163] Subjects were hospitalized and fecal samples from the subjects were collected while consuming hospital meals. Fecal samples from mice were collected at 6 weeks and 16 weeks of age. Fecal samples were stored at -80°C. DNA extraction from fecal samples was performed by the Japan Gene Research Institute according to previously established procedures (Appleid and Environmental Microbiology 63, 2802-2813 (1997); Appl Environ Microbiol 71, 4153-4155 (2005); FEMS Microbiology Ecology 68, 351-362 (2009)). For each sample, cellular DNA was extracted from 0.2-0.3 g of fecal material using the G'NOMEs kit (BIO 101, La Jolla, CA) with slight changes according to the manufacturer's instructions. In brief, fecal samples were homogenized in the provided cell suspension, after which a cell lysis / denaturation solution was added and the samples were incubated at 55°C for 30 minutes. To enhance cell lysis, 750 mL of silica strain (0.1 mm diameter) was added and shaken at maximum speed in a BeadBeater apparatus (BioSpec, Bartlesville, OK) for 10 minutes. 15 mg of polyvinyl pyrrolidone was added to ensure the removal of polyphenol contamination that could inhibit subsequent quantitative PCR. The sample was vortexed and centrifuged at 20,000 g for 3 minutes, and the supernatant was recovered. The residual pellet was washed with 400 mL of TENP buffer (50 mM Tris buffer pH 8, 20 mM EDTA pH 8, 100 mM NaCl, 1% polyvinyl polypyrrolidone) and centrifuged at 20,000 g for 3 minutes. The washing step was repeated once more and the resulting supernatants were combined. An equal volume of isopropanol was added to precipitate the nucleic acids, and after storage at -20°C for 10 minutes, the pellet was centrifuged at 20,000 g for 5 minutes. The pellet was resuspended in a mixture of 400 mL of distilled water and 100 mL of a salting-out agent and incubated at 4°C for 10 minutes. The sample was centrifuged at 20,000 g for 10 minutes, and the supernatant containing DNA was transferred to a 1.5 mL clean centrifuge tube. Two volumes of 100% ethanol were added, and the DNA was precipitated by standing at room temperature for 5 minutes, followed by centrifugation at 20,000 g for 5 minutes. The DNA was redissolved in 150 mL of TE buffer, and the DNA solution was stored at -20°C until the subsequent analysis.

[0164] 16S rRNA gene amplification and pyrosequencing

[0165] A portion of the 16S rRNA gene (V3-V4 region, equivalent to positions 342 to 806 in the E. coli numbering system) was PCR amplified using the undenatured universal primer set of 342F and 806R (Table 2) (for details of the primer set and PCR conditions, refer to DNA Res. 21, 217-227 (2014)). After adding the sequencing adapter, the amplicon was sequenced by Takara Bio using the Illumina MiSeq platform (Illumina Inc., San Diego, CA) according to the manufacturer's protocol.

[0166] The bacterial composition matrix was prepared using USEARCH 10.0.240 version (Bioinformatics.26, 2460-2461 (2010)) version 0.33 (parameters: LEADING:17TRAILING:17AVGQUAL:25MINLEN:100). In addition, the high-quality 16SrRNA gene amplicon sequence generated using Trimmomatic (Bioinformatics.30, 2114-2120 (2014)) was selected using the previous protocol. The remaining reads were processed using the -fastq_mergepairs command of USEARCH (parameters: default values). Next, Tagcleaner (BMC Bioinformatics.11, 341 (2010)) version 0.16 (parameters: -tag5CTACGGGGGGCAGCAG-mm53-tag3 AGATACCCCGGTAGTCC-mm33-nomatch 3) was used to remove the sequences in the primer-free region. Afterwards, an internal python script was used to remove sequences with N. Phix reads were removed using the -filter_phix command of USEARCH. Short sequences were removed using the -sort_by_length command of USEARCH (parameter: -minseqlength300). Finally, an OTU table (Nat Meth. 10, 996-998 (2013)) was generated using the UPARSE algorithm (-fastx_unique command and otu_cluster command (parameter: -minsize1)). The representative sequence of each OTU was annotated in the genus of bacteria using RDP Classifier version 2.12 with a bootstrap value of 0.5 (Appl Environ Microbiol. 73, 5261-5267 (2007)). The representative sequences of each OTU were annotated in the reference database Silva Living Tree Project version 123 (Syst Appl Microbiol. 31, 241-250 (2008)) using BLASTN version 2.2.25 with an identity threshold of 97% and a coverage of 80%.

[0167] Analysis of short-chain fatty acids in fecal samples

[0168] Subjects were asked to take a hospital-designated diet in the hospital, and then stool samples were collected. The samples were diluted with sterile water containing 2-ethylbutyric acid as an internal control, and then labeled with 2-nitrophenylhydrazine using a YMC-Pack FA kit (#XSRFAR01, YMC, Kyoto, Japan). Short-chain fatty acids in the samples were quantified by liquid chromatography (Prominence LC-20AD; Shimadzu, Kyoto, Japan).

[0169] animal

[0170] All apolipoprotein E deficiencies (ApoE - / - ) All mice were raised in an SPF animal facility at the Kobe University Research Institute. The animals were fed a normal diet (CLEA, Japan) under a strict 12-hour light cycle and had free access to water. The 6-week-old mice were divided into three treatment groups. The control group mice were allowed to orally ingest the culture medium, and the control group mice were given 2.5×10 9 cfu / 100μl, and mice in the live Bacteroides group were orally administered live Bacteroides vulgaris and Bacteroides dornoni 5 times a week, while mice in the heat-killed Bacteroides group were orally administered heat-killed Bacteroides vulgaris and Bacteroides dornoni. All experiments were conducted in accordance with the guidelines for animal experiments in effect at the Kobe University School of Medicine (Guideline No. P160701).

[0171] Obese mouse models and nonalcoholic steatohepatitis (NASH) mouse models were prepared using C57BL / 6J mice (Charles River, Japan) according to the method described in detail below.

[0172] Cultivation and modulation of Bacteroides vulgaris and Bacteroides doreae

[0173] Bacteroides vulgaris (#8482; American Type Culture Collection, Manassas, VA and strain NTZ002 (accession number: NITE BP-02863)) and Bacteroides doxorubicin (#17855; Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany and strain NTZ001 (accession number: NITE BP-02862)) were obtained from Difco TMAnaerobic cultures were performed at 37°C in enhanced Clostridium medium (#218081; BD Bioscience, San Jose, CA). An anaerobic chamber containing 10% CO2, 10% hydrogen, and 80% nitrogen (Coy Laboratory Products, Grass Lake, MI) was used for all anaerobic microbial steps. Bacteroides vulgaris and Bacteroides doreae were heat sterilized at 121°C for 15 minutes. The heat sterilization was confirmed to have been successful by inoculating the heat-treated bacteria and their lack of growth.

[0174] Evaluation of biochemical parameters and cytokine levels in plasma

[0175] Animals were fasted overnight and blood was collected in heparin tubes by cardiac puncture under anesthesia. Blood samples were centrifuged at 4°C, 3,000 rpm for 10 minutes and stored at -80°C before measurement. The concentrations of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and triglycerides in plasma were determined enzymatically using an automated chemical analysis device (SRL). Cytometric bead array kit (BDBioscience) was used to analyze cytokine levels in plasma according to the manufacturer's instructions.

[0176] Evaluation of Atherosclerotic Lesions

[0177] The mice were anesthetized, the aorta was perfused with saline, and the section from the center of the left ventricle to the branch point of the iliac artery was removed. In order to analyze the lesions at the base of the aorta, a sample of the proximal ascending aorta to the aortic sinus was obtained. The sample was embedded in the frozen tissue section preparation sample embedding agent OCT compound (#4583, Tissue-Tek; Sakura Finetek, Tokyo). Five continuous sections (10 μm thick) covering 550 μm of the aortic sinus were collected from each mouse and stained with Oil Red O (#154-02072; Sigma-Aldrich, St. Louis, MO) or hematoxylin (#3000; Muto Chemical, Tokyo). The stained sections were captured by digital processing using an integrated fluorescence microscope (#BZ-8000; Keyence, Osaka). In order to quantitatively analyze the degree of arteriosclerosis, the total lesion area of ​​5 individual sections obtained from each mouse was calculated using ImageJ (registered trademark) software (Circulation. 120, 1996-2005 (2009)) as reported. For en face lesion analysis, aortic segments from the proximal ascending aorta to the common iliac artery branches were removed and fixed in 10% formalin buffer. After carefully removing the adventitial tissue, the aorta was cut longitudinally, stained with Oil Red O, pinned to the black wax surface, and captured with a digital camera. The stained lesion area is displayed as a percentage relative to the total area, as described in the report (Circulation. 120, 1996-2005 (2009)), using ImageJ (registered trademark) software for measurement.

[0178] Immunohistochemical Analysis of Atherosclerotic Lesions

[0179] Immunohistochemical analysis of atherosclerotic lesions was performed on 10 μm frozen sections of the base of mouse aorta fixed with acetone or formalin using antibodies for detecting macrophages (MOMA-2, #T-2029, 1:400; BMA Biomedicals, Augst, Switzerland) and antibodies for detecting T cells (CD4, #550278, 1:100; BD Biosciences). Detection was performed using a biotinylated secondary antibody (#ab102250, 1:500; abcam, Cambridge, UK) and streptavidin-labeled horseradish peroxidase (#P0397, 1:500; Dako Co., Carpinteria, CA). The stained sections were captured by digital processing using an integrated fluorescence microscope. In MOMA-2 staining, the percentage of the stained area relative to the total atherosclerotic lesion area was calculated. Quantification of CD4-positive T cells was performed by counting the number of stained cells in each section.

[0180] Histological analysis of mouse colon

[0181] The ascending colon of each mouse was removed at 16 weeks of age and embedded in OCT compound. Detection was performed using a secondary antibody conjugated with Cy3 (#405408, 1:200; BioLegend, San Diego, CA), which had been immunofluorescently stained on acetone-fixed 10 μm frozen sections using an antibody that detects tight junction proteins (anti-ZO-1, #sc-33725, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA). Nuclear staining was performed using DAPI (#H-1200; Vector Laboratories Inc., Burlingame, CA). The stained sections were captured by digital processing using an integrated fluorescence microscope. The expression intensity of ZO-1 was analyzed using ImageJ (registered trademark) software.

[0182] Flow cytometry

[0183] Mesenteric lymph node (MLN) cells, spleen cells, and RAW264.7 cells were incubated with anti-CD16 / CD32 antibody (clone 2.4G2; #553142; BD Biosciences) to block Fc receptors. For surface antigen detection, 10 × 10 5 MLN cells or splenocytes were stained with the following antibodies (BD Biosciences or eBioscience).

[0184] Anti-CD4 (clone RM4-5; Cat 557871);

[0185] Anti-CD11b (clone M1 / 70; #557396);

[0186] Anti-CD11c (clone HL3; #558079);

[0187] Anti-CD44 (clone IM7; #553134);

[0188] Anti-CD62L (clone MEL-14; #553150);

[0189] Anti-CD69 (clone FN50; #555533);

[0190] Anti-CD80 (clone 16-10A1; #560016);

[0191] Anti-CD86 (clone GL1; #553692);

[0192] Anti-CD273 (clone TY25; #560086);

[0193] Anti-CD274 (clone MIH5; #558091);

[0194] Anti-F4 / 80 (clone BM8; #17-4801-82);

[0195] Anti-Ly6G (clone RB6-8C5; #12-5931-82);

[0196] • Anti-TLR4 (clone MTS510, #17-9924-82).

[0197] Intracellular staining was performed using Foxp3 / Transcription Factor Staining Buffer Set (#00-5523-00, eBioscience, San Diego, CA) or Fixation / Permeabilization Solution Kit (#554714, BD Biosciences), anti-Foxp3 antibody (clone FJK-16; #17-5773-82; eBioscience) and anti-CTLA-4 antibody (clone UC10; BD Biosciences; #553720) according to the manufacturer's instructions. An isotype-identical antibody was used as a control. The proliferation of RAW264.7 cells was tested using the anti-Ki67 panel (clone B56; #556027) according to the manufacturer's instructions. Flow cytometric analysis was performed using an Attune (registered trademark) Acoustic Focusing Cytometer (Life Technologies, Grand Island, NY) and FlowJo software (Tree Star, Inc., Ashland, OR).

[0198] In vivo intestinal permeability assay

[0199] After fasting overnight, mice were orally administered fluorescein isothiocyanate (FITC)-labeled dextran (FD4, 500 mg / kg body weight; Sigma-Aldrich). Four hours later, the plasma FD4 concentration was measured using a fluorescence spectrophotometer (EnSpire; PerkinElmer, Inc., Waltham, MA) at an excitation wavelength of 490 nm and a fluorescence wavelength of 520 nm.

[0200] Preparation of fecal supernatant

[0201] The same human fecal samples were used for LPS determination and 16S rRNA gene sequencing. Mouse fecal samples were collected from 16-week-old control mice and Bacteroides-treated mice, or 6-week-old antibiotic (AVNM)-administered mice 24 hours after the last oral ingestion. Fecal supernatants were obtained by slightly modifying the reported protocols (PLoS One.7, e47713 (2012); Gut.63, 1069-1080 (2014); BMC Microbiol.16, 9 (2016)). In brief, fecal samples were suspended in sterile PBS to a concentration of 50 mg / 500 μL and vortexed gently to prevent bacterial cells from breaking. After centrifugation at 3,000 rpm for 15 minutes, the supernatant was recovered, filtered with a 0.45 μm filter and then a 0.22 μm filter, sterilized, incubated at 70°C for 15 minutes to inactivate, and then stored at -80°C. In addition, mouse cecal stool samples were collected from 16-week-old control mice and Bacteroides -treated mice according to conventional methods.

[0202] Analysis of LPS levels in plasma and fecal supernatants

[0203] The LPS levels in plasma and feces were determined using the Limulus Amebocyte Lysate Assay Kit (#K50-643J; Lonza Inc., Basel, Switzerland) according to the manufacturer's instructions. Plasma was diluted 10-fold and fecal supernatant was diluted 10,000-fold in pyrogen-free water and inactivated at 70°C for 15 minutes. LPS assays were performed in pyrogen-free glass tubes, Eppendorf tubes, and plates.

[0204] In vitro fecal supernatant stimulation and siRNA assay

[0205] RAW264.7 macrophages were cultured in RPMI-1640 medium containing 10% FBS. 1×10 5 RAW264.7 cells were seeded in 24-well plates (flat bottom) (Corning Costar, Corning, NY) and cultured for 5 hours in the presence of fecal supernatant at 37°C, 5% CO2 to allow proliferation. To analyze cytokine production, TLR4 knockdown was performed using siRNA against TLR4 (#4390771; Invitrogen, Carlsbad, CA) using RNAiMAX reagent (#13778030, Invitrogen). TMNegative control No.1 siRNA (SilencerTM Select Negative Control No.1 siRNA) (#4390843, Invitrogen) was selected as a control. HT29 human intestinal epithelial cells were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium containing 10% FBS. The HT29 cells were inoculated in a 24-well plate (flat bottom) and cultured in the presence of fecal supernatant at 37°C and 5% CO2 for 24 hours.

[0206] RNA extraction and real-time PCR analysis

[0207] Using TRIzol TM Total RNA was extracted from tissue or cell samples using PrimeScript reverse transcription reagent kit (#RR037A; Takara, Shiga) using the SYBER Premix Ex Taq (#RR820; Takara) and LightCycler (registered trademark) 96 system (#05815916001; Roche, Mannheim, Germany) according to the manufacturer's instructions. Expression data were corrected using GAPDH as a control housekeeping gene and analyzed by the ΔΔT method.

[0208] [Table 2]

[0209]

[0210] *The numbers in [] show the SEQ ID NO.

[0211] Western blot analysis was performed as described in detail in Sci. Rep. 7, 12989 (2017). In brief, cells were rinsed twice with PBS and collected in ice-cold lysis buffer (20 mM HEPES, pH 7.4; 1% NP40; 1% SDS; and 150 mM NaCl). Proteins extracted from the total lysate were subjected to 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (#IB401002; Thermo Scientific) using an iBlot (registered trademark) gel transfer apparatus (Thermo Scientific). The membrane was blocked with 5% milk (#31149-75; Nacalai Tesque, Kyoto), incubated with the primary antibody at 4°C overnight, and then incubated with the secondary antibody conjugated with horseradish peroxidase at room temperature for 1 hour. After incubation with Immobilon Western HRP substrate (#WBKLS0500; Merck Millipore, Billerica, MA), the signal was detected using the V3 Western Workflow system (Bio-Rad, Hercules, CA). The following primary antibodies were used in this experiment.

[0212] ·Anti-ZO-1 (#8193,1:1,000; Cell Signaling Technology, Danvers, MA);

[0213] • Anti-β-actin (#A5441, 1:5,000; Sigma-Aldrich).

[0214] The band intensity was quantified using ImageJ (registered trademark) software.

[0215] Statistical analysis

[0216] Statistical analyses were performed using R software version 3.1.0 (http: / / www.r-project.org / ), JMP version 10 (SAS Institute, Cary, NC), and Prism version 7.0 (GraphPad software; San Diego, CA). The Shapiro-Wilk test was used to determine whether the data showed a normal distribution. The results for normally distributed data were expressed as mean ± standard error of the mean or mean ± standard deviation of normally distributed data, and the results for non-normally distributed data were expressed as median ± interquartile range (1st to 3rd interquartile range). For normally distributed data, Student's two-sided t-test was used to evaluate significant differences between the two groups, while for non-normally distributed data, Mann-Whitney U-test was used to evaluate significant differences between the two groups. Categorical variables were compared using Fisher's exact test or chi-square test. All tests considered values ​​of P < 0.05 to be statistically significant. To evaluate the statistical correlation between two parameters, single regression / single correlation was calculated using the least squares method, and the results were expressed as Pearson's correlation coefficient. One-way analysis of variance (ANOVA) was used to detect significant differences among the three groups. The q value was calculated using the Benjamini-Hochberg method, and the p value was adjusted for multiple comparisons. The data from 30 CAD patients and 30 controls were clustered at the genus level in the manner described in Arthritis Rheumatol. 68, 2646-2661 (2016).

[0217] result

[0218] Gut flora distribution in CAD patients

[0219] Thirty CAD patients and 30 non-CAD patients matched in age and gender with coronary artery risk factors such as hypertension, diabetes, or dyslipidemia were recruited as subjects. The intestinal bacterial profiles of these subjects were compared in detail using 16S rRNA gene sequencing in stool samples. The background of the subjects is shown in Table 3.

[0220] [Table 3]

[0221]

[0222] *p≤0.05; **p≤0.01; ***p≤0.001

[0223] In order to analyze the intestinal bacterial distribution patterns of the subjects, the samples were divided into three clusters at the genus level according to the order reported (Nature. 473, 174-180 (2011)) Figure 1 a), each cluster is characterized by the abundant presence of specific genera shown below ( Figure 1b).

[0224] Cluster 1: Bacteroides ;

[0225] Cluster 2: Prevotella;

[0226] Cluster 3: Faecalibacterium, Ruminococcus, or Bifidobacterium;

[0227] Non-CAD controls were more likely to be classified into cluster 1, whereas CAD patients were hardly classified into this cluster ( Figure 1 c). There were no significant differences in alpha diversity, Bacteroidetes / Firmicutes ratio, Gram-positive bacteria / Gram-negative bacteria ratio, and fecal short-chain fatty acid concentrations between the two groups. The abundance of Bacteroidetes genus was compared between the CAD group and the non-CAD group at the genus and species level. The results showed that the relative abundance of Bacteroidetes genus in CAD patients tended to be low compared with controls ( Figure 1 d). The results of principal component analysis showed that the presence of the main species of the intestinal microbiome was different in the two groups. In CAD patients, Bacteroides vulgaris and Bacteroides dodecene were relatively reduced, while Faecalibacterium prausnitzii and Prevotella copri were enriched ( Figure 6 ). Considering that the presence of Bacteroides in patients with atherosclerosis was low in previous reports, the present inventors focused on Bacteroides vulgaris and Bacteroides doreae as Bacteroides species. These species are involved in anti-inflammatory responses (Cell. 165, 842-853 (2016)) and are the most abundant species in the Bacteroides genus ( Figure 1 e), their presence is significantly lower in CAD patients ( Figure 1 f). Since these two bacteria have similar 16S rRNA sequencing patterns (Int J Syst Evol Microbiol. 56, 1639-1643 (2006)), they cannot be distinguished according to the methodology used by the present inventors. Therefore, the present inventors constructed a mouse model in which mice showing a tendency to arteriosclerosis orally ingested Bacteroides vulgaris and Bacteroides doreae, and used this model to confirm the causal relationship between these bacteria and arteriosclerosis in an attempt to clarify its basic mechanism.

[0228] Oral administration of live bacteria of Bacteroides vulgaris and Bacteroides dorotheli inhibits the formation of atherosclerotic plaques. To determine the effects of Bacteroides vulgaris and Bacteroides dorotheli on the development of atherosclerosis, 6-week-old female apolipoprotein E-deficient (ApoE - / -)) Mice were orally fed Bacteroides vulgaris ATCC 8482 5 times a week T Bacteroides dorothelii DSM 17855 T The mice were euthanized at 16 weeks of age and several analyses were performed to evaluate atherosclerosis and intestinal flora. Compared with control mice, mice that orally ingested live bacteria of the genus Bacteroides had a decrease in body weight ( Figure 2 f) or plasma cholesterol level ( Figure 2 g), but lesion size was significantly reduced in en face analysis of the aortic base and thoracic aorta ( Figure 2 a, b). There was no significant difference in the size of atherosclerotic lesions in mice that orally ingested heat-sterilized Bacteroides compared with control mice. In addition, the results of immunohistochemical staining and subsequent morphological analysis of atherosclerotic lesions in the aortic sinus showed that macrophages and CD4-positive T cells were significantly reduced in mice that orally ingested Bacteroides compared with the control ( Figure 2 c, d). Next, quantitative RT-PCR analysis was performed to evaluate the mRNA expression of immune cell markers and chemokines / chemokine receptors in the atherosclerotic aorta of mice. As a result, several atherogenic immune cell markers and chemokines / chemokine receptors were reduced in mice orally ingesting Bacteroides ( Figure 2 e) These results suggest that oral supplementation with live Bacteroides vulgaris and Bacteroides doreae may reduce plaque inflammation and thereby inhibit the formation of atherosclerotic lesions.

[0229] Results from 16S rRNA gene sequencing of mouse fecal samples showed that the composition of the intestinal flora changed significantly in response to oral ingestion of Bacteroides vulgaris and Bacteroides doreae ( Figure 7 a, b). Furthermore, the diversity of intestinal bacteria decreased in control mice but was maintained in mice that orally ingested live Bacteroides ( Figure 7 c). Surprisingly, the Gram-positive / Gram-negative ratio tended to be higher in mice that orally ingested live Bacteroides compared to control mice ( Figure 7 d). The presence of Bacteroides vulgaris and Bacteroides doreae was significantly increased in mice that orally ingested live Bacteroides species ( Figure 7 e).

[0230] Treatment with live Bacteroides reduces colonic lipopolysaccharide (LPS) levels, resulting in reduced intestinal and systemic immune responses. The present inventors have previously reported that arteriosclerosis can be prevented by regulating intestinal immune responses (Circulation. 120, 1996-2005 (2009); Arterioscler Thromb Vasc Biol. 30, 2495-2503 (2010)). Therefore, in order to study the mechanism by which Bacteroides treatment reduces plaque formation, the present inventors first orally ingested Bacteroides vulgaris ATCC 8482. T Bacteroides dorothelii DSM 17855 T The results showed that the mRNA expression of certain antigen-presenting cell markers (CD11c, co-stimulatory molecule CD80) and inflammatory cytokines in the colon of mice orally ingesting Bacteroides spp. was substantially reduced compared with control mice ( Figure 3 a, b), and the mRNA expression of CCR7, which is essential for the migration of antigen-presenting cells from the intestinal lamina propria to the mesenteric lymph nodes (MLN), was significantly decreased in mice orally ingested with Bacteroides spp. ( Figure 3 a). Therefore, the inventors next conducted an experiment on the natural immune response in MLN. Through flow cytometric analysis of MLN, in mice that orally ingested Bacteroides, not only the amount of CD11c highly expressing cells was significantly reduced, but also the expression of Toll-like receptor 4 (TLR4), MHC class II, and CD80 in CD11c highly expressing cells was significantly reduced ( Figure 3 c). In addition, the expression of cytokine mRNA in MLN of mice orally ingesting Bacteroides also tended to be lower than that of control mice ( Figure 3 d) These results suggest that live Bacteroides treatment suppresses intestinal immune responses by inhibiting the activation of antigen-presenting cells associated with downregulation of TLR4 expression.

[0231] Since LPS stimulates cells through TLR4 and upregulates co-stimulatory molecules on antigen presenting cells, the inventors measured fecal LPS levels as an indicator of colonic LPS production caused by intestinal flora. Surprisingly, fecal LPS levels were dramatically reduced in mice that orally ingested Bacteroides compared to control mice ( Figure 3 e). The present inventors further used fecal supernatant to perform in vitro stimulation assays to investigate whether the reduction in colonic LPS concentration after treatment with live Bacteroides is directly reflected in the level of colonic inflammation. When RAW264.7 macrophages were stimulated with fecal supernatant, the proliferation of cells treated with fecal supernatant from mice that had orally ingested Bacteroides was significantly inhibited ( Figure 8a). In addition, in order to verify whether LPS / TLR4 signaling is the main pathway for controlling colon inflammation, siRNA was introduced to produce RAW264.7 macrophages in which TLR4 was knocked down, and the cells were stimulated with fecal supernatant. As a result, by introducing nonspecific siRNA, the secretion of inflammation-inducing cytokines IL-1b, IL-6, and TNF-α in macrophages treated with fecal supernatant from mice treated with live Bacteroides was significantly reduced compared with RAW264.7 macrophages treated with fecal supernatant from control mice. RAW264.7 macrophages introduced with TLR siRNA secreted only a very small amount of inflammation-inducing cytokines, which was significantly lower than that of RAW264.7 macrophages introduced with nonspecific siRNA ( Figure 8 b) These findings suggest that colonic LPS concentration controls colonic inflammation via LPS / TLR4-dependent signaling.

[0232] Endotoxemia is known to induce systemic inflammation, leading to the destruction of natural and adaptive immunity (acquired immunity) and the onset of atherosclerosis (Nat Commun. 7, 13436 (2016); Circulation. 133, 2434-2446 (2016); Nat Immunol. 12, 204-212 (2011)). In fact, the plasma LPS concentration was significantly reduced in mice treated with Bacteroides compared with control mice ( Figure 3 f), and the concentrations of atherogenic cytokines such as IL-2, IL-4, IL-6, IL-17A, IFN-γ and TNF-α in plasma also decreased ( Figure 3 g). The inventors further investigated the sources of these cytokines by flow cytometry of spleen cells. As a result, there was no significant difference in the proportions of CD11b-highly expressing / F4 / 80-highly expressing macrophages, CD11b-highly expressing / Ly6G-highly expressing neutrophils, and CD11c-highly expressing dendritic cells between the two groups ( Fig. 9 a), but mice orally ingesting Bacteroides showed decreased expression of MHC class II and co-stimulatory molecule CD86, and increased expression of co-inhibitory molecules programmed death ligand 1 (PD-L1) and PD-L2 on splenic CD11c-highly expressing dendritic cells ( Fig. 9 b). TLR4 expression on splenic CD11c-highly expressing dendritic cells was not significantly different between the two groups. In agreement with the high tolerogenicity of dendritic cells, the number of CD4-positive T cells was also reduced in mice treated with live Bacteroides ( Fig. 9c). In mice orally ingested with live Bacteroides, the number of effector CD44-high / CD62L-low / CD4-positive T cells was reduced, but the proportion of CD4-positive / CD25-positive / Foxp3-positive control T cells with high levels of intracellular CTLA4 was significantly high ( Fig. 9 d, e). This indicates a shift in the immune balance toward suppression. These data suggest that activation of systemic innate immune cells and suppression of Th1-driven inflammation associated with the pathogenesis of atherosclerosis are caused by the reduction in plasma LPS concentration induced by live Bacteroides treatment.

[0233] Live Bacteroides treatment strengthens the intestinal barrier in vitro and in vivo

[0234] If the composition of the intestinal flora changes following intestinal permeability, it will promote endotoxemia or arteriosclerosis (Circulation. 133, 2434-2446 (2016)), so the inventors next studied whether live Bacteroides treatment has an effect on the permeability of intestinal tight junctions. T Bacteroides dorothelii DSM 17855 T The mice of the same strain showed a significant decrease in intestinal permeability to FITC-dextran ( Figure 4 a) and a significant increase in the mRNA expression of the tight junction gene zo1 ( Figure 4 b), which is reflected by an increase in the mean fluorescence intensity of ZO-1 in the colon ( Figure 4 c). These data show that live Bacteroides treatment strengthens the tight junction barrier compared to the state in control mice. To clarify whether changes in colonic LPS concentration have an effect on the formation of tight junctions, the present inventors stimulated HT29 human colorectal adenocarcinoma cells with epithelial cell morphology with fecal supernatant. The results, consistent with the in vivo results, significantly higher expression levels of zo1 (ZO-1) were observed (at both mRNA and protein levels) in HT29 cells stimulated with fecal supernatant from mice treated with live Bacteroides compared to HT29 cells stimulated with fecal supernatant from control mice ( Figure 4 d, e). These results suggest that changes in colonic LPS concentration induced by intestinal flora may directly affect paracellular permeability mediated by tight junctions.

[0235] Fecal LPS levels are elevated in CAD patients

[0236] To further investigate the relationship between the incidence of CAD and LPS production caused by intestinal bacteria in humans, the present inventors measured LPS levels in stool samples that were the same as those used in 16S rRNA gene sequencing. As a result, LPS levels in stool of CAD patients were significantly higher than those in non-CAD controls ( Figure 5 a). Interestingly, the presence of Bacteroides species was significantly negatively correlated with fecal LPS levels (R = -0.30, P = 0.023). Moreover, a significant negative correlation was seen between fecal LPS levels and the % presence of Bacteroides vulgaris and Bacteroides doreae (R = -0.29, P = 0.027) ( Figure 5 b) These results strongly suggest that Bacteroides vulgaris and Bacteroides doreae may control LPS production induced by intestinal bacteria and that these activities may have an impact on the progression of atherosclerosis in humans.

[0237] Comparative Example: Single administration of Bacteroides vulgaris or Bacteroides donovani did not significantly improve symptoms and intestinal LPS levels in the atherosclerosis mouse model. Apo - / - Mice were orally ingested with Bacteroides vulgaris ATCC 8482 T Bacteroides dorothelii DSM 17855 T strain.

[0238] Results Fig.10 . Compared with the control (vehicle administration) group, the atherosclerotic lesions in the aortic sinus tended to improve in all the single-administration groups, but no significant differences were observed. Moreover, the LPS level in the feces (intestines) did not decrease due to the single administration of any bacterial species. Compared with the control group, the expression of tight junction-related genes in the large intestine in the group where Bacteroides dobrosii was administered alone tended to increase, and the expression of cytokine genes in the large intestine in the group where Bacteroides vulgaris was administered alone decreased compared with the control group, but there was no significant difference in the cytokine concentration in the blood among all the administration groups.

[0239] The above results suggest that Bacteroides vulgaris and Bacteroides doxorubicin act synergistically through different mechanisms when they coexist in the intestine, exerting significant LPS control.

[0240] Various combinations of Bacteroides vulgaris and Bacteroides donovani improved intestinal LPS levels in the mouse model of atherosclerosis. Apoe- / - female mice or Apoe- / - male mice were orally given various combinations of bacteria (2.5×10 9CFU / 100 μl). Apoe- / - female mice were given the drug for 10 weeks from 6 to 16 weeks of age, and LPS in feces was measured at 16 weeks of age. Apoe- / - male mice were given the drug for 7 weeks from 6 to 13 weeks of age, and LPS in feces was measured at 13 weeks of age.

[0241] Results Fig.12 Not only the standard strains (v, d) but also the NT strains (Nv, Nd) decreased the amount of LPS in feces.

[0242] Live Bacteroides treatment improves intestinal LPS levels in a mouse model of atherosclerosis

[0243] Apoe- / - female mice were orally administered Bacteroides vulgaris ATCC8482 5 times a week. T and Bacteroides dorothelii DSM17855 T A combination of bacteria (live or dead bacteria. The live bacteria count is 2.5×10 9 CFU / 100 μl). The drug was administered for 10 weeks from 6 to 16 weeks of age, and LPS in feces and cecal stool was measured at 16 weeks of age. In addition, AVNM (ampicillin, vancomycin, neomycin, metronidazole) was administered from 5 to 6 weeks of age, and LPS in feces was measured at 6 weeks of age.

[0244] Results Fig.13 The right and left sides of the figure show the LPS levels in the feces and cecum of Apoe- / - female mice treated with Bacteroides. In addition, the center of the figure shows the LPS level in the feces of Apoe- / - female mice given antibiotics. In the feces and cecum, the amount of LPS decreased when live bacteria were given, but there was no change compared with the control (NC) when dead bacteria were given.

[0245] Bacteroides treatment improves various symptoms of obesity in an obese mouse model

[0246] C57BL / 6J male mice were given 5 times a week for 12 weeks from 6 to 18 weeks of age. The NC group was given a normal diet, the Ctrl group was given a high-fat diet HFD (Oriental Yeast industrial production; containing 60% fat) and a liquid medium used for culturing Bacteroides vulgaris or Bacteroides dornoni orally at 200 μl / time, and the B group was given a high-fat diet and live bacteria Bacteroides vulgaris ATCC8482 T and live Bacteroides domitoria DSM 17855 T The NB group was given a high-fat diet and live bacteria Bacteroides vulgaris NTZ002 and Bacteroides doreae NTZ001 ( Figure 14-1 A).

[0247] As a result, the weight gain of the group given two kinds of bacteria of the standard strain (B) or two kinds of bacteria of the NT strain (NB) in addition to the high-fat diet was suppressed compared with the Ctrl group ( Figure 14-1 B, C).

[0248] In the 1 g OGTT test, the rapid increase in blood sugar levels after administration of the two bacteria of the standard strain (B) or the two bacteria of the NT strain (NB) was suppressed ( Figure 14-1 D left), the AUC value is low ( Figure 14-1 D center), low insulin resistance (HOMA-IR) ( Figure 14-1 D right), improvement in glucose tolerance was confirmed.

[0249] When HE staining, MAC3 staining or Sirius red staining of epididymal fat ( Figure 14-2 E left), in the group given two kinds of bacteria of the standard strain (B) or two kinds of bacteria of the NT strain (NB), the size of the adipocytes was small, the number of crown structures was small, and the fiber formation rate was low ( Figure 14-2 E right).

[0250] When the mRNA levels in visceral fat (epididymal fat) were measured, the mRNA expressions of MCP1, F4 / 80, iNOS, CCR5, and TLR4, which are inflammatory cell markers, were lower in the groups given two strains of the standard strain (B) or two strains of the NT strain (NB) than in the Ctrl group ( Figure 14-2 F left), the mRNA expression of Fizzi1 (considered anti-inflammatory when high in M2 macrophage markers) tended to increase ( Figure 14-2 F left). Moreover, the mRNA expression of cytokines IFNγ and TNFα was decreased ( Figure 14-2 F right).

[0251] Bacteroides treatment improved various symptoms of NASH in a NASH mouse model

[0252] The drug was administered 5 times a week to C57BL / 6J male mice between 8 and 17 weeks of age. The Ctrl group was given a very high-fat, choline-deficient, and methionine-reduced diet (a NASH model feed manufactured by EPS Yixin Co., Ltd.) as food, and the Bacteroides group was further given live Bacteroides vulgaris ATCC8482 T and live Bacteroides domitoria DSM 17855 T .

[0253] At 17 weeks of age, the livers were dissected and HE-stained. Fatty liver, inflammatory cell infiltration, and ballooning of hepatocytes were observed in the Ctrl group. However, in the groups given the two strains of the standard strain, inflammatory cell infiltration was significantly suppressed, and no ballooning was observed ( Figure 15-1In the standard strain two-bacterium administration group, the NAFLD activity score (NAS) was further improved, the blood AST and AST / ALT ratio were significantly reduced, and liver dysfunction was significantly suppressed ( Figure 15-1 Central, Lower and Figure 15-2 ).

[0254] While the present invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that variations therein may be made.

[0255] The contents of all publications described herein, including patents and patent application specifications, are incorporated herein by reference, and their entire contents are incorporated into this specification to the same extent as if expressly stated.

[0256] This application is based on Japanese Patent Application No. 2018-022578 filed in Japan on February 9, 2018, the contents of which are incorporated herein in their entirety by reference thereto.

[0257] Industrial Applicability

[0258] The combination of the present invention can be used as a drug for preventing and / or improving circulatory diseases represented by arteriosclerosis or diseases accompanied by chronic inflammation, metabolic diseases accompanied by metabolic abnormalities such as glucose and lipid metabolism. In addition, the test method and test drug of the present invention can diagnose / predict the risk of the above-mentioned diseases through microbiome analysis in feces, and are therefore effective in providing a non-invasive clinical test method. <110> Nitto Pharmaceutical Industry Co., Ltd. <120> Lipopolysaccharide-controlled intestinal bacteria and their uses <130> 092848 <150> JP 2018-022578 <151> 2018-02-09 <160> 50 <170> PatentIn version 3.5 <210> 1 <211> 1510 <212> DNA <213> Bacteroides vulgaris <400> 1 tattacaatg aagagtttga tcctggctca ggatgaacgc tagctacagg cttaacacat 60 gcaagtcgag gggcagcatg gtcttagctt gctaggccg atggcgaccg gcgcacgggt 120 gagtaacacg tatccaacct gccgtctact cttggacagc cttctgaag gaagattaat 180 acaagatggc atcatgagtc cgcatgttca catgattaaa ggtattccgg tagacgatgg 240 ggatgcgttc cattagatag taggcggggt aacggcccac ctagtcttcg atggataggg 300 gttctgagag gaaggtcccc cacattggaa ctgagacacg gtccaactc ctacgggagg 360 cagcagtgag gatatttggt caatgggcga gagcctgac cagccaagta gcgtgaagga 420 tgactgccct atgggttgta aacttctttt aaaggaat aaagtcgggt atggataccc 480 gtttgcatgt actttatgaa taaggatcgg ctaactccgt gccagcagcc gcggtaatac 540 ggaggatccg agcgttatcc ggatttattg ggttaaagg gagcgtagat ggatgtttaa 600 gtcagttgtg aaagtttgcg gctcaccgt aaaattgcag ttgatactgg atatcttgag 660 tgcagttgag gcaggcggaa tcgtggtgt agcggtgaaa tgctgata tcacgaagaa 720 ctccgattgc gaaggcagcc tgctaagctg caacgacat tgaggctcga aagtgtggt 780 atcaaacagg attagatacc ctggtagtcc acacggtaaa cgatgaatac tcgctgtttg 840 cgatatactg caagcggcca agcgaaagcg ttaagtattc cacctgggga gtacgccggc 900 aacggtgaaa ctcaaaggaa ttgacggggg cccgcacaag cggaggaaca tgtggtttaa 960 ttcgatgata cgcgaggaac cttacccggg cttaaattgc agatgaatta cggtgaaagc 1020 cgtaagccgc aaggcatctg tgaaggtgct gcatggttgt cgtcagctcg tgccgtgagg 1080 tgtcggctta agtgccataa cgagcgcaac ccttgttgtc agttactaac aggttccgct 1140 gaggactctg acaagactgc catcgtaaga tgtgaggaag gtggggatga cgtcaaatca 1200 gcacggccct tacgtccggg gctacacacg tgttacaatg gggggtacag agggccgcta 1260 ccacgcgagt ggatgccaat ccccaaaacc tctctcagtt cggactggag tctgcaaccc 1320 gactccacga agctggattc gctagtaatc gcgcatcagc cacggcgcgg tgaatacgtt 1380 cccgggcctt gtacacaccg cccgtcaagc catgggagcc gggggtacct gaagtgcgta 1440 accgcgagga gcgccctagg gtaaaactgg tgactggggc taagtcgtaa caaggtagcc 1500 gtaccggaag 1510 <210> 2 <211> 1489 <212> DNA <213> Bacteroides vulgatus <400> 2 agagtttgat cctggctcag gatgaacgct agctacaggc ttaacacatg caagtcgagg 60 ggcagcatgg tcttagcttg ctaaggccga tggcgaccgg cgcacgggtg agtaacacgt 120 atccaacctg ccgtctactc ttggacagcc ttctgaaagg aagattaata caagatggca 180 tcatgagtcc gcatgttcac atgattaaag gtattccggt agacgatggg gatgcgttcc 240 attagatagt aggcggggta acggcccacc tagtcttcga tggatagggg ttctgagagg 300 aaggtccccc acattggaac tgagacacgg tccaaactcc tacgggaggc agcagtgagg 360 aatattggtc aatgggcgag agcctgaacc agccaagtag cgtgaaggat gactgcccta 420 tgggttgtaa acttctttta taaaggaata aagtcgggta tggatacccg tttgcatgta 480 ctttatgaat aaggatcggc taactccgtg ccagcagccg cggtaatacg gaggatccga 540 gcgttatccg gatttattgg gtttaaaggg agcgtagatg gatgtttaag tcagttgtga 600 aagtttgcgg ctcaccgta aaattgcagt tgatactgga tatcttgagt gcagttgagg 660 caggcggaat tcgtggtgta gcggtgaat gcttagaat cacgagaac tccgattgcg 720 aaggcagcct gctaagctgc aactgacatt gaggctcgaa agtgtgggta tcaacagga 780 ttagataccc tggtagtcca cacggtaaac gatgaatact cgctgtttgc gatatacggc 840 aagcggccaa gcgaaagcgt taagtattcc acctggggag tacgccggca acggtgaaac 900 tcaaggaat tgacggggc ccgcacaagc ggaggaacat gtggtttaat tcgatgatac 960 gcgaggaacc ttaccggggc ttaaattgca gatgaattac ggtgaaagcc gtaagccgca 1020 agcatctgt gaagtgctg catggttgtc gtcagctcgt gccgtgaggt gtcggcttaa 1080 gtgccataac gagcgcaacc cttgttgtca gttactaca ggttatgctg aggactctga 1140 caagactgcc atcgtagat gtgaggaagg tgggatgac gtcaatcag cacggccctt 1200 acgtccgggg ctacacacgt gttacaatgg gggtacaga gggccgctac cacgcgagtg 1260 gatgccaatc cccaaacct ctctcagttc ggactggagt ctgcaacccg acccacgaa 1320 gctggattcg ctagtaatcg cgcatcagcc acggcgcggt gaatacgttc ccgggccttg 1380 tacacaccgc ccgtcaagcc atgggagccg ggggtacctg aagtgcgtaa ccgcgaggag 1440 cgccctaggg taaaactggt gactggggct aagtcgtaac aaggtaacc 1489 <210> 3 <211> 1493 <212> DNA <213> Bacteroides dorei <400> 3 agagtttgat cctggctcag gatgaacgct agctacaggc ttaacacatg caagtcgagg 60 ggcagcatgg tcttagcttg ctaaggctga tggcgaccgg cgcacgggtg agtaacacgt 120 atccaacctg ccgtctactc ttggccagcc ttctgaaagg aagattaatc caggatggga 180 tcatgagttc acatgtccgc atgattaaag gtattttccg gtagacgatg gggatgcgtt 240 ccattagata gtaggcgggg taacggccca cctagtcaac gatggatagg ggttctgaga 300 ggaaggtccc ccacattgga actgagacac ggtccaaact cctacgggag gcagcagtga 360 ggaatattgg tcaatgggcg atggcctgaa ccagccaagt agcgtgaagg atgactgccc 420 tatgggttgt aaacttcttt tataaaggaa taaagtcggg tatgcatacc cgtttgcatg 480 tactttatga ataaggatcg gctaactccg tgccagcagc cgcggtaata cggaggatcc 540 gagcgttatc cggatttatt gggtttaaag ggagcgtaga tggatgttta agtcagttgt 600 gaaagtttgc ggctcaaccg taaaattgca gttgatactg gatgtcttga gtgcagttga 660 ggcaggcgga attcgtggtg tagcggtgaa atgcttagat atcacgaaga actccgattg 720 cgaaggcagc ctgctaagct gcaactgaca ttgaggctcg aaagtgtggg tatcaaacag 780 gattagatac cctggtagtc cacacggtaa acgatgaata ctcgctgttt gcgatatacg 840 gcaagcggcc aagcgaaagc gttaagtatt ccacctgggg agtacgccgg caacggtgaa 900 actcaaagga attgacgggg gcccgcacaa gcggaggaac atgtggttta attcgatgat 960 acgcgaggaa ccttacccgg gcttaaattg cactcgaatg atccggaaac ggttcagcta 1020 gcaatagcga gtgtgaaggt gctgcatggt tgtcgtcagc tcgtgccgtg aggtgtcggc 1080 ttaagtgcca taacgagcgc aacccttgtt gtcagttact aacaggtgat gctgaggact 1140 ctgacaagac tgccatcgta agatgtgagg aaggtgggga tgacgtcaaa tcagcacggc 1200 ccttacgtcc ggggctacac acgtgttaca atggggggta cagagggccg ctaccacgcg 1260 agtggatgcc aatccctaaa acccctctca gttcggactg gagtctgcaa cccgactcca 1320 cgaagctgga ttcgctagta atcgcgcatc agccacggcg cggtgaatac gttcccgggc 1380 cttgtacaca ccgcccgtca agccatggga gccgggggta cctgaagtgc gtaaccgcga 1440 ggatcgccct agggtaaaac tggtgactgg ggctaagtct aaccaaggta acc 1493 <210> 4 <211> 1490 <212> DNA <213> Bacteroides dorei <400> 4 agagtttgat cctggctcag gatgaacgct agctacaggc ttaacacatg caagtcgagg 60 ggcagcatgg tcttagcttg ctaaggctga tggcgaccgg cgcacgggtg agtaacacgt 120 atccaacctg ccgtctactc ttggccagcc ttctgaaagg aagattaatc caggatggga 180 tcatgagttc acatgtccgc atgattaaag gtattttccg gtagacgatg gggatgcgtt 240 ccattagata gtaggcgggg taacggccca cctagtcaac gatggatagg ggttctgaga 300 ggaaggtccc ccacattgga actgagacac ggtccaaact cctacgggag gcagcagtga 360 ggaatattgg tcaatgggcg atggcctgaa ccagccaagt agcgtgaagg atgactgccc 420 tatgggttgt aaacttcttt tataaaggaa taaagtcggg tatgcatacc cgtttgcatg 480 tactttatga ataaggatcg gctaactccg tgccagcagc cgcggtaata cggaggatcc 540 gagcgttatc cggatttatt gggtttaaag ggagcgtaga tggatgttta agtcagttgt 600 gaaagtttgc ggctcaaccg taaaattgca gttgatactg gatgtcttga gtgcagttga 660 ggcaggcgga attcgtggtg tagcggtgaa atgcttagat atcacgaaga actccgattg 720 cgaaggcagc ctgctaagct gcaactgaca ttgaggctcg aaagtgtggg tatcaaacag 780 gattagatac cctggtagtc cacacggtaa acgatgaata ctcgctgttt gcgatatacg 840 gcaagcggcc aagcgaaagc gttaagtatt ccacctgggg agtacgccgg caacggtgaa 900 actcaaagga attgacgggg gcccgcacaa gcggaggaac atgtggttta attcgatgat 960 acgcgaggaa ccttacccgg gcttaaattg cactcgaatg atccggaaac ggttcagcta 1020 gcaatagcga gtgtgaaggt gctgcatggt tgtcgtcagc tcgtgccgtg aggtgtcggc 1080 ttaagtgcaa cgagcgcaac ccttgttgtc agttactaac aggtgatgct gaggactctg 1140 acaagactgc catcgtaaga tgtgaggaag gtggggatga cgtcaaatca gcacggccct 1200 tacgtccggg gctacacacg tgttacaatg gggggtacag agggccgcta ccacgcgagt 1260 ggatgccaat ccctaaaacc cctctcagtt cggactggag tctgcaaccc gactccacga 1320 agctggattc gctagtaatc gcgcatcagc cacggcgcgg tgaatacgtt cccgggcctt 1380 gtacacaccg cccgtcaagc catgggagcc gggggtacct gaagtgcgta accgcgagga 1440 tcgccctagg gtaaaactgg tgactggggc taagtcgtaa caaggtaacc 1490 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gcatccacgt gttggctca 19 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 ctccagccta ctcattggga tca 23 <210> 7 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 7 ccgagagtgc tgcctggatt a 21 <210> 8 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 8 agcttgccct ggacagtcag a 21 <210> 9 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 9 ccaacttgca gctgtccatc tc 22 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 10 catcactgcc ttgggtccag 20 <210> 11 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 11 ggtggtggct ctccttgtca tt 22 <210> 12 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 12 acaccgactc gtacagggtg tagtc 25 <210> 13 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 13 agacgtgcca gtcagcatca ac 22 <210> 14 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 14 ctattccgat agcattgggt gagtg 25 <210> 15 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 15 agtttccatg tccaaggctc attc 24 <210> 16 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 16 ttgtaacggc aaggcagcaa ta 22 <210> 17 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 17 tggcatatga ccgttgtgtg tg 22 <210> 18 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 18 acgtttgagc agatggaaac tcttg 25 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 19 tctacgaggg actgtggatg 20 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 20 tcagattcag caaggagtcg 20 <210> twenty one <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty one ctttggctat gggcttccag tc 22 <210> twenty two <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty two gcaaggagga cagagtttat cgtg 24 <210> twenty three <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty three ctcatgatag tgcctgtgtc ctcaa 25 <210> twenty four <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty four agggccagca taggtgcaag 20 <210> 25 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 25 tgtgtccgtc gtggatctga 20 <210> 26 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 26 ttgctgttga agtcgcagga g 21 <210> 27 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 27 tgaacgggaa gctcactgg 19 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 28 tccaccaccc tgttgctgta 20 <210> 29 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 29 caattcacac tgaatgccag ctc 23 <210> 30 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 30 caagcagtcc gtctcgtcca 20 <210> 31 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 31 tccaggatga ggacatgagc ac 22 <210> 32 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 32 gaacgtcaca caccagcagg tta 23 <210> 33 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 33 ccacttcaca agtcggaggc ttta 24 <210> 34 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 34 gcaagtgcat catcgttgtt catac 25 <210> 35 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 35 acgcgcaaac atgagtccag 20 <210> 36 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 36 ctcagcagca gcaacagcat c 21 <210> 37 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 37 atgtccggcc gatgctctc 19 <210> 38 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 38 tttggctgct cttgggtctg tat 23 <210> 39 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 39 gggcctaaac ccagtctgtt tg 22 <210> 40 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 40 gccccggtaag gtccatgcta 20 <210> 41 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 41 aaactggtcg ggcaattctg 20 <210> 42 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 42 agggttggac acctgaatgc ta 22 <210> 43 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 43 ctacgggggg cagcag 16 <210> 44 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 44 ggactaccgg ggtatct 17 <210> 45 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 45 tgccggcata tacgagtgtg a 21 <210> 46 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 46 cccgatggca ggtattacca ag 22 <210> 47 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 47 tttttgacag ggggagtgg 19 <210> 48 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 48 tgctgcagag gtcaaagttc aag 23 <210> 49 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 49 gaatgatggt tggtatggtg cg 22 <210> 50 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 50 tcagaagtgt gtctactgtc cg 22

Claims

1. Use of the combination of (1) and (2) below in the preparation of an agent for ameliorating a disease associated with elevated levels of lipopolysaccharide in the blood or intestine: (1) A composition comprising live bacteria of Bacteroides vulgatus isolated from nature; and (2) A composition comprising live bacteria of naturally isolated Bacteroides dorei, The above-mentioned Bacteroides vulgaris is Bacteroides vulgaris ATCC8482 T strain, and the above-mentioned Bacteroides dornonis is Bacteroides dornonis DSM17855 T strain; or The above-mentioned Bacteroides vulgaris is strain NTZ002, wherein the preservation number of strain NTZ002 is NITE BP-02863, and the above-mentioned Bacteroides doreae is strain NTZ001, wherein the preservation number of strain NTZ001 is NITE BP-02862, The above-mentioned disease is a disease selected from atherosclerosis, non-alcoholic steatohepatitis, fatty liver, intestinal inflammation, obesity and diabetes.

2. The use according to claim 1, wherein the improving agent is a medicine or a medicine additive.

3. The use according to claim 1, wherein the improving agent is feed or feed additive.

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