Bacteroides coprocola and uses thereof

Abstract

The application discloses Parabacteroides merdae 1-YM-FAB-03-17.mj, which has a preservation number of CGMCC No.1.32477. The Parabacteroides merdae provided by the application can significantly control body weight, improve blood lipids, relieve inflammation, reduce the level of branched-chain amino acids in the body and other symptoms or causes of atherosclerosis, and can be used for preparing medicines, health products or food for treating and / or preventing atherosclerosis, reducing blood lipids and obesity.

Description

Technical Field

[0001] This invention relates to the use of *Bacteroides fecalis* and pharmaceutical or probiotic compositions containing it in atherosclerosis or related diseases. Background Technology

[0002] Atherosclerosis (AS) is the main pathological basis for the incidence and death of CVD. Its pathogenesis is not yet clear, but it mainly involves lipid metabolism disorders, oxidative stress imbalance, immune response imbalance and chronic inflammation of the arterial wall.

[0003] Elevated serum branched-chain amino acid (BCAA) levels are closely associated with diabetes, obesity, and insulin resistance, and are early predictors of diabetes. Since elevated blood glucose and insulin resistance are often accompanied by abnormal lipid metabolism, thereby increasing the risk of cardiovascular disease, BCAAs are also a potential risk factor for cardiovascular disease.

[0004] Dysregulation of gut microbiota composition and function plays a crucial role in the development and progression of many chronic diseases, including ankylosing spondylitis (AS), and has become a new target for the prevention and treatment of chronic diseases, leading to the development of several probiotics. The development and clinical application of probiotics have undergone many years of research and exploration. The types and mechanisms of action of probiotics have gradually become clearer, and their clinical applications are becoming increasingly widespread, covering areas such as organ protection, immunity, antibacterial activity, lipid-lowering, blood sugar-lowering, and weight control.

[0005] Because the etiology and pathogenesis of AS are not fully understood, although some progress has been made in the treatment of AS, it is still at the stage of controlling or alleviating symptoms, and there is no effective treatment or cure. Therefore, there is an urgent need in this field to develop an effective, new, and non-toxic drug for the prevention and treatment of AS and related diseases. Summary of the Invention

[0006] This invention discloses *Bacteroides fecalis* (… Parabacteroides merdae 1-YM-FAB-03-17.mj, its accession number is CGMCC No. 1.32477.

[0007] Meanwhile, this invention also discloses *Bacteroides parafetus* (… Parabacteroides merdae The use of 1-YM-FAB-03-17.mj in the preparation of drugs for the prevention and / or treatment of atherosclerosis or related diseases.

[0008] Furthermore, atherosclerosis is a major cause of coronary heart disease, cerebral infarction, and peripheral vascular disease. Lipid metabolism disorders are the pathological basis of atherosclerosis, characterized by lesions starting in the intima of the affected arteries. Generally, this begins with the accumulation of lipids and complex carbohydrates, hemorrhage, and thrombosis, followed by fibrosis and calcification, and gradual degeneration and calcification of the arterial media, leading to thickening and hardening of the arterial wall and narrowing of the vascular lumen. The lesions often involve large and medium-sized muscular arteries; once they progress to the point of obstructing the arterial lumen, the tissues or organs supplied by that artery will experience ischemia or necrosis. Because the lipids accumulated in the arterial intima appear as a yellowish, porridge-like substance, it is called atherosclerosis.

[0009] According to the embodiments provided by the present invention, the *Bacteroides fecalis* and its agent can significantly control body weight, improve blood lipids, reduce the level of host inflammatory factors, reduce the level of branched-chain amino acids in the host, and alleviate the degree of atherosclerosis, and can be used in pharmaceuticals for the treatment of atherosclerosis or related diseases. Attached Figure Description

[0010] Figure 1 This is a GC-MS image of the branched-chain amino acids of Bacteroides faecalis, which are branched-chain short-chain fatty acids.

[0011] Figure 2 It is a model of atherosclerosis caused by Bacteroides feces. ApoE - / - Effects of plasma weight and lipid levels in mice.

[0012] Figure 3 It is a model of atherosclerosis caused by Bacteroides feces. ApoE - / - The effect of biomarker levels in mouse plasma.

[0013] Figure 4 It is a model of atherosclerosis caused by Bacteroides feces. ApoE - / - A pathological section of the aorta of a mouse. Detailed Implementation

[0014] Through extensive and in-depth research and experimentation, the inventors discovered *Bacteroides parafetus* (… Parabacteroides merdae It has the effect of preventing and treating atherosclerosis caused by lipid metabolism disorders (atherosclerosis is the main cause of coronary heart disease, cerebral infarction, and peripheral vascular disease), and contains Bacteroides parafecosum ( Parabacteroides merdae An atherosclerosis model induced by feeding subjects with live bacteria inoculum on a high-fat diet. ApoE - / - In mice, this bacterial agent was found to inhibit weight gain, reduce blood lipids, improve inflammation, and reduce aortic plaque area, effectively alleviating the occurrence and development of atherosclerosis and improving obesity and other conditions.

[0015] This invention provides *Bacteroides fecalis* (… Parabacteroides merdae Its uses in the treatment and prevention of diseases such as atherosclerosis.

[0016] According to a preferred embodiment of the present invention, via *Bacteroides parafetus* (… Parabacteroides merdae A high-fat diet-induced atherosclerosis model treated with strains of bacteria ApoE - / - In mice, compared to untreated controls, the treatment resulted in slower weight gain, reduced blood lipids, and improved inflammatory symptoms. Therefore, the *Pseudomonas fecalis* (… Parabacteroides merdae It can be used to prevent and treat atherosclerosis and related diseases.

[0017] The microbial agents of this invention can be administered in any form, such as tablets, injections, or capsules. The pharmaceutical formulation includes excipients, pharmaceutically permissible media, and carriers, which can be selected according to the route of administration. The pharmaceutical formulation of this invention also contains auxiliary components.

[0018] Lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, or mineral oil can all be used as carriers, excipients, or diluents for the pharmaceuticals in this invention.

[0019] Furthermore, the pharmaceutical microbial agent of the present invention further includes lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings. The pharmaceutical microbial agent of the present invention can be produced as an enteric-coated formulation using various known methods, so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, can pass smoothly through the stomach without being destroyed by gastric acid.

[0020] The pharmaceutical formulation of the present invention can be made into enteric-coated tablets for oral use.

[0021] The "casing" in this invention includes all coatings permitted for use in conventional pharmaceuticals. These coatings are not degraded by gastric acid but can be fully decomposed in the small intestine, rapidly releasing the microorganisms of this invention. The casings of this invention can be maintained at 36-38°C for more than 2 hours in synthetic gastric acid, such as an HCl solution with pH=1, and preferably decompose within 1 hour in synthetic intestinal fluid, such as a buffer solution with pH=7.0. The casings of this invention are coated at approximately 16-30 mg per tablet, preferably 16-25 mg, and more preferably 16-20 mg. The casing thickness in this invention is 5-100 μm, ideally 20-80 μm, and the casing composition is a known conventional polymer.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] Example 1 Identifying beneficial bacteria for treating or preventing atherosclerosis

[0025] Aseptic technique was used. 50 mg of fresh stool from a healthy volunteer was placed in a glass test tube containing 9 mL of sterile saline solution and shaken thoroughly. Then, 0.5 mL of the mixture was transferred to a test tube containing 4.5 mL of sterile saline solution. This dilution was 10-10. -1 Repeat the above process to perform a 10-fold dilution, until 10... -6 Dilution, select 10 -4 10 -5 10 -6 For each of the three dilutions, 0.1 mL of bacterial culture was dropped onto an MRS medium plate. After spreading the plates, the plates were incubated anaerobically (5% CO2) in a 37°C incubator for 48 hours.

[0026] The MRS culture medium consists of 10.0 g beef extract, 5.0 g yeast extract, 10.0 g peptone, 20.0 g glucose (or other sugars) (preferably sterilized separately by filtration and mixed into the medium before use), 2.0 g K₂HPO₄, 5.0 g sodium acetate, 0.2 g MgSO₄·7H₂O, 0.05 g MnSO₄·4H₂O, 1.0 g Tween 80, 2.0 g triammonium citrate, 15.0 g agar, and distilled water to a final volume of 1000 ml. The pH before sterilization is 6.2–6.6.

[0027] The four-zone streak method was used. Colonies with different morphologies were picked up with an inoculation loop and streaked onto MRS agar medium for isolation and culture. After 48 hours of culture, colonies with good isolation effect were picked from the four zones and inoculated onto MRS slant medium for pure culture. The culture was repeated 3 times and then stored at 4°C for later use.

[0028] The bacterial species was identified by 16S rRNA sequencing of the pure culture, and the sequence is shown as SEQ ID NO.1 in the sequence listing. After comparison with the NCBI database Blast, it was identified as... Parabacteroides merdaeThe corresponding Chinese name is *Bacteroides parafecosum*. This strain was deposited on May 18, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number of this strain is CGMCC No. 1.32477, and the strain name is 1-YM-FAB-03-17.mj.

[0029] Example 2

[0030] fecal parabacterium ( Parabacteroides merdae Genomic analysis and functional analysis of branched-chain amino acids in the metabolic pathway Materials and Methods: The tested strain was *Bacteroides fecalith* ( Parabacteroides merdae )1-YM-FAB-03-17.mj (abbreviated as PM), genomic analysis of PM revealed functional genes with metabolic branched-chain amino acids.

[0031] Anaerobic culture was performed in YCFA medium. After 24 hours of in vitro culture, 1 ml of bacterial solution was taken, sonicated for 5 min, centrifuged at 5000g for 5 min at room temperature, and the supernatant was derivatized. After hexane extraction, the contents of short-chain fatty acids and branched-chain amino acids in the bacterial solution were analyzed by GC-MS (Table 1).

[0032] Table 1. Content of branched-chain amino acids and branched-chain short-chain fatty acids

[0033] Results: After 24 hours of anaerobic culture in vitro, PM was found to have a significant ability to metabolize branched-chain amino acids as branched-chain short-chain fatty acids. Figure 1 ).

[0034] Example 3 Animal experiments verified

[0035] Material: The tested strain was: Bacteroides parafetus ( Parabacteroides merdae 1-YM-FAB-03-17.mj (abbreviated as PM).

[0036] The amount of PM bacteria administered via gavage was set at 0.2 mL / 30 g body weight, and the PM bacteria concentration was 1×10⁻⁶. 7 The concentration of cfu / mL was increased once daily for 30 days.

[0037] PM bacterial solutions need to be cultured in advance, activated weekly to ensure freshness, and their concentration measured separately.

[0038] ApoE - / -Mice were purchased from Beijing Vital River Laboratory Animal Co., Ltd. The environment was maintained at 20-24 degrees Celsius, constant humidity of 50-60%, 12 hours of light (8:00-20:00), soundproof, and with free access to food and water. Experiments were conducted one week after the mice acclimatized to the environment.

[0039] The total cholesterol (CHO) reagent kit (catalog number A111-2-1), low-density lipoprotein (LDL-C) reagent kit (catalog number A113-1-1), high-density lipoprotein (HDL-C) reagent kit (catalog number A112-1-1), and triglyceride (TG) reagent kit (catalog number A110-2-1) were purchased from Nanjing Jiancheng Bioengineering Institute.

[0040] The following assay kits were purchased from Beijing Huabodeyi Biotechnology Co., Ltd.: Plasma Oxidized Low-Density Lipoprotein (ox-LDL) Elisa Assay Kit (Catalog No. F7789-A), Monocyte Chemokinetic Factor-1 (MCP-1) Elisa Assay Kit (Catalog No. F7158-A), Lipopolysaccharide (LPS) Elisa Assay Kit (Catalog No. F10621-A), and High-Sensitivity C-Reactive Protein (hs-CRP) Elisa Assay Kit (Catalog No. F5411-A).

[0041] Method: Female C57BL / 6J Mice, 6-8 weeks old, weighing 18-20 grams, 10 mice in total, served as the normal control group (1); females ApoE - / - Mice were 6-8 weeks old and weighed 18-20 grams. Their body weight and blood lipids were measured, and they were divided into groups according to their body weight and blood lipid levels: (2) model control group, (3) PM 1×10 7 (cfu / mL, abbreviation: PM), (4) Inactivate PM 1×10 7 cfu / mL (abbreviation: kPM), 10 animals per group ApoE - / - Mice were induced to eat a high-fat diet for 50 days, and after blood lipids were measured, they were then divided into groups for intervention for 30 days.

[0042] The model control group was given an equal volume of anaerobic PBS. Mice in each group were administered 0.2 mL / 30 g body weight of PM bacterial solution by gavage for 30 consecutive days.

[0043] During the intervention period, the body weight of mice in each group was measured. Figure 2 A).

[0044] After the last administration of PM bacteria, mice were weighed (Table 2), anesthetized, and euthanized. Blood was collected, centrifuged at 3000 rpm for 10 min at 4°C, and plasma total cholesterol (TC), low-density lipoprotein (LDL-C), and triglycerides (TG) were measured. Figure 2As shown in B-2C. Plasma oxidized low-density lipoprotein (ox-LDL), lipopolysaccharide (LPS), monocyte chemoattractant-1 (MCP-1), and high-sensitivity C-reactive protein (hs-CRP) were measured. Figure 3 As shown in A-3D.

[0045] Pick ApoE - / - The mouse aorta was quickly fixed in formalin, and tissue sections were prepared for oil red staining of the aortic root and gross sections. Figure 4 ).

[0046] result: (1) Compared with the model group, the PM strain can significantly control body weight, but the inactivated PM strain cannot significantly reduce body weight, as shown in Table 2.

[0047] Table 2. Effects of *Pseudomonas fecalis* (PM) on ApoE in atherosclerosis - / - Effect of mouse body weight

[0048] The PM group significantly reduced the incidence of high-fat diet-induced atherosclerosis in the model. ApoE - / - The mice gained weight; compared with the model group, the PM group lost 27.1% of its weight. Results are shown in Table 2. Figure 2 A.

[0049] (2) Improvement of PM strains ApoE - / - In mice, the PM group significantly reduced blood lipid levels compared to the model group, demonstrating a lower incidence of high-fat diet-induced atherosclerosis. ApoE - / - The levels of triglycerides in mouse plasma were 33.3%, cholesterol 22.9%, and low-density lipoprotein cholesterol 18.9%. The results are shown in Table 3. Figure 2 B-2C.

[0050] Table 3. Effects of *Pseudomonas fecalis* (PM) on an atherosclerosis model ApoE - / - Effects of blood lipids in mice

[0051] (3) Improvement of PM strains ApoE - / - In terms of biomarkers of atherosclerosis in mice, the PM group significantly reduced the high-fat diet-induced atherosclerosis compared to the model group. ApoE - / -The levels of oxidized low-density lipoprotein (ox-LDL) in mouse plasma were 18.5%, lipopolysaccharide (LPS) 20.2%, monocyte chemokine-1 (MCP-1) 7.9%, and high-sensitivity C-reactive protein (hs-CRP) 18.5%. The results are shown in Table 4. Figure 3 A-3D.

[0052] Table 4. Effects of Pleurobacterium feces (PM) on ApoE - / - Effects of atherosclerosis-related biomarkers in mouse plasma

[0053] (4) Improvement of PM strains ApoE - / - In mice, the aortic plaque condition was significantly reduced in the PM group compared to the model group, indicating a high-fat diet-induced atherosclerosis model. ApoE - / - The size of plaques at the root of the mouse aorta and the total area of ​​plaques as shown by gross Oil Red staining of the aorta are shown in the following figures. Figure 4 .

[0054] (5) PM strains decreased ApoE - / - Compared with the model group, PM intervention significantly reduced the levels of branched-chain amino acids in mice, thus reducing the atherosclerosis induced by a high-fat diet. ApoE - / - The levels of branched-chain amino acids in mouse feces and plasma are shown in Table 5.

[0055] Table 5. Effects of *Pseudomonas fecalis* (PM) on an atherosclerosis model ApoE - / - Effects of branched-chain amino acid levels in mice sequence list <110> Institute of Microbiology, Chinese Academy of Sciences <120> Bacteroides parafetus and its applications <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1495 <212> DNA <213> Parabacteroides merdae <400> 1 agagtttgat cctggctcag gatgaacgct agcgacaggc ttaacacatg caagtcgagg 60 ggcagcatga tttgtagcaa tacagattga tggcgaccgg cgcacgggtg agtaacgcgt 120 atgcaactta cctatcagag ggggatagcc cggcgaaagt cggattaata ccccataaaa 180 caggggtccc gcatgggaat atttgttaaa gattcatcgc tgatagatag gcatgcgttc 240 cattaggcag ttggcggggt aacggcccac caaaccgacg atggataggg gttctgagag 300 gaaggtcccc cacattggta ctgagacacg gaccaaactc ctacgggagg cagcagtgag 360 gaatattggt caatggccga gaggctgaac cagccaagtc gcgtgaagga agaaggatct 420 atggtttgta aacttctttt ataggggaat aaagtggagg acgtgtcctt ttttgtatgt 480 accctatgaa taagcatcgg ctaactccgt gccagcagcc gcggtaatac ggaggatgcg 540 agcgttatcc ggatttattg ggtttaaagg gtgcgtaggt ggtgatttaa gtcagcggtg 600 aaagtttgtg gctcaaccat aaaattgccg ttgaaactgg gttacttgag tgtgtttgag 660 gtaggcggaa tgcgtggtgt agcggtgaaa tgcatagata tcacgcagaa ctccgattgc 720 gaaggcagct tactaaacca taactgacac tgaagcacga aagcgtgggg atcaaacagg 780 attagatacc ctggtagtcc acgcagtaaa cgatgattac taggagtttg cgatacaatg 840 taagctctac agcgaaagcg ttaagtaatc cacctgggga gtacgccggc aacggtgaaa 900 ctcaaaggaa ttgacgggg cccgcacaag cggaggaaca tgtggtttaa ttcgatgata 960 cgcgaggaac cttacccggg tttgaacgta gtctgaccgg agtggaaaca ctttttctag 1020 caatagcaga ttacgaggtg ctgcatggtt gtcgtcagct cgtgccgtga ggtgtcggct 1080 taagtgccat aacgagcgca acccttatca ctagttacta acaggtgaag ctgaggactc 1140 tggtgagact gccagcgtaa gctgtgagga aggtggggat gacgtcaaat cagcacggcc 1200 cttacatccg gggcgacaca cgtgttacaa tggcatggac aaagggcagc tacctggtga 1260 caggatgcta atctccaaac catgtctcag ttcggatcgg agtctgcaac tcgactccgt 1320 gaagctggat tcgctagtaa tcgcgcatca gccatggcgc ggtgaatacg ttcccgggcc 1380 ttgtacacac cgcccgtcaa gccatgggag ccgggggtac ctgaagtccg taaccgcaag 1440 gatcggccta gggtaaaact ggtgactggg gctaagtcgt aacaaggtag ccgta 1495

Claims

1. Bacteroides parafetus ( Parabacteroides merdae 1-YM-FAB-03-17.mj, its accession number is CGMCC No.1.32477.

2. A microbial inoculant containing *Bacteroides parafecosum* as described in claim 1. Parabacteroides merdae 1-YM-FAB-03-17.mj.

3. The *Bacteroides parafetus* according to claim 1 ( Parabacteroides merdae Application of 1-YM-FAB-03-17.mj in converting branched-chain amino acids into branched-chain short-chain fatty acids.

4. The *Bacteroides parafetus* according to claim 1 ( Parabacteroides merdae Application of 1-YM-FAB-03-17.mj in the preparation of drugs for the prevention and / or treatment of atherosclerotic diseases.

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