Clostridium hylemonae and use thereof

Abstract

The present application discloses a human faecal blautia strain capable of colonizing in an anaerobic environment of the intestinal tract and an application thereof, and relates to the technical field of microorganisms.The present application provides a strain with a uric acid degradation function, the human faecal blautia strain GH001, which has the effects of reducing uric acid level, preventing and treating hyperuricemia and gout, and the human faecal blautia strain GH001 also has the effect of relieving kidney damage to a certain extent.The present application provides a new probiotic or drug material for the treatment of hyperuricemia and related diseases.In addition, the strain has no obvious influence on the indicators of the sample body, which proves that the strain has the characteristics of high safety and can be widely applied in the medical field.

Description

Technical Field

[0001] This invention relates to the field of microbial pharmaceutical technology, and in particular to a human fecal Brut bacterium and its applications. Background Technology

[0002] Hyperuricemia is a metabolic disease caused by purine metabolism disorder. Epidemiological surveys show that the overall prevalence of hyperuricemia in the general population of China is 17.4%, and it is showing a trend towards affecting younger people. Hyperuricemia has become one of the most common metabolic diseases. Clinical studies have shown that hyperuricemia not only leads to the formation of uric acid crystals and causes gout, but it can also induce and aggravate diseases such as hypertension, diabetes, acute coronary syndrome, renal failure, stroke, and non-alcoholic fatty liver disease, making it a major national health problem that urgently needs to be addressed.

[0003] The Trichophyceae family is an important component of the human gut microbiota, and one of the most abundant and ubiquitous bacterial groups in the gastrointestinal tract of healthy individuals. As potential beneficial bacteria, Trichophyceae possesses diverse hydrolytic activities and can produce beneficial metabolites such as short-chain fatty acids. These characteristics enable Trichophyceae to play a crucial role in maintaining host health and participating in disease development. Although existing research has shown that Trichophyceae has multifaceted health effects on the host, due to limitations in research techniques and culture conditions, current research on how to utilize these microorganisms to regulate host uric acid levels and alleviate or treat hyperuricemia is relatively limited. Microorganisms with relevant functions urgently need to be developed.

[0004] This invention aims to fill this research gap by isolating and identifying a human fecal Brut strain with uric acid degradation capabilities from human feces, and exploring its potential application in regulating human uric acid levels and treating hyperuricemia. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a strain of *Brutella feces* with uric acid-degrading properties and its applications. This *Brutella feces* strain is *Brutella feces* GH001, which has excellent uric acid-degrading capabilities, can decompose most uric acid, reduce the body's absorption of uric acid, lower the uric acid content in the blood, and has the ability to treat or prevent metabolic diseases such as gout.

[0006] The present invention adopts the following technical solution:

[0007] A strain of *Brutella feces* GH001 that has the ability to degrade uric acid ( Blautia hominis GH001 was deposited on September 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46142.

[0008] Furthermore, the *Blaugranatum feces* GH001 is a Gram-positive bacterium. Physiological and biochemical analysis, as well as 16S rDNA sequencing, revealed that the 16S rDNA sequence of this strain is shown in SEQ ID NO.1.

[0009] On the other hand, this application also provides a formulation comprising *Brutella fecalis* GH001, *Brutella fecalis* GH001 inoculum, and / or *Brutella fecalis* GH001 dead bacterial suspension.

[0010] On the other hand, this application also provides a composition comprising the aforementioned *Brutella feces*.

[0011] Preferably, the composition includes excipients.

[0012] In a preferred embodiment, the composition contains 1 × 10⁻⁶ viable Blautrella feces. 8 CFU, 1×10 9 CFU, 1×10 10 CFU, 1×10 11 CFU, 1×10 12 CFU or any value above that.

[0013] As those skilled in the art will understand, the dosage of *Brutella feces* or the composition can be adjusted according to actual circumstances, as long as the total viable count reaches 1×10⁻⁶. 8 CFU or above is acceptable. This application only provides one example and does not impose any specific restrictions.

[0014] Those skilled in the art will understand that known excipients and solvents may be added to the composition without affecting the activity and efficacy of the strain.

[0015] The excipients may be appropriate solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0016] Furthermore, the composition can be prepared by common methods, such as directly mixing the bacterial strain and excipients or other components. One or more diluents or pharmaceutically acceptable carriers may also be added.

[0017] In addition, the composition may contain other known active ingredients without affecting the activity and efficacy of the microbial strain.

[0018] In a preferred embodiment, the composition may be a pharmaceutical product.

[0019] Furthermore, the dosage form of the drug is tablets, capsules, granules, suspensions, oral solutions, injections, or infusions.

[0020] On the other hand, this application also provides the use of the aforementioned *Brutella feces* or the aforementioned preparation in the preparation of uric acid degradation products.

[0021] This application is the first to use *Blaueria freundii* GH001 in human feces to degrade uric acid. *Blaueria freundii* GH001 can achieve rapid degradation of uric acid. In one specific embodiment, *Blaueria freundii* GH001OD... 600 When the value is 1.0, uric acid degradation solution with a final concentration of about 5 mM can be completely degraded within 24 hours.

[0022] On the other hand, this application also provides the use of the aforementioned *Brutella feces* or the aforementioned preparation in the preparation of products that lower uric acid levels.

[0023] Preferably, the uric acid level is the uric acid content in serum.

[0024] This application is the first to use *Blauridae frenulum* GH001 to prepare a product that lowers uric acid levels and to use it for lowering uric acid levels.

[0025] On the other hand, this application also provides the use of the aforementioned *Brutella feces* or the aforementioned preparation in the preparation of products for the prevention and / or treatment of hyperuricemia and / or gout.

[0026] Prevention and / or treatment of hyperuricemia can be achieved by degrading uric acid in the gastrointestinal tract of hyperuricemic patients, thereby reducing serum uric acid levels. While reducing the risk of hyperuricemia, this can effectively reduce the probability of hyperuricemia further developing into gout, thus playing a role in preventing and / or treating gout.

[0027] In addition, consuming the above-mentioned strains had no significant effect on the body indicators of the samples, proving that they have high safety characteristics and can be widely used in the medical field.

[0028] By adopting the above technical solution, the present invention has the following advantages compared with the prior art:

[0029] The *Brutella feces* GH001 of this invention belongs to the family Trichophytonceae and is a symbiotic bacterium in the human gut. It has good safety, strong decomposition ability, and can effectively degrade uric acid in the intestine, reduce the concentration of uric acid in the blood, and alleviate kidney damage.

[0030] The method for lowering uric acid provided by this invention is green, environmentally friendly, simple and easy to implement, and has stable effects. It not only provides a new strategy for the microbial treatment of hyperuricemia, but also provides a new perspective for a deeper understanding of the complex interaction between gut microbiota and host metabolism. Attached Figure Description

[0031] Figure 1 Figure A shows the growth of *Brutella fecalis* GH001 on a uric acid-containing medium and its effect on direct consumption of uric acid in vitro.

[0032] Figure 2 This is a schematic diagram of the morphology of *Brutella feces* GH001.

[0033] Figure 3 A graph showing serum uric acid levels in mice with hyperuricemia treated with *Blauridae fecal microbiota* GH001.

[0034] Figure 4 Comparative images of the kidneys of mice with hyperuricemia after treatment with *Brutobacterium feces* GH001. Detailed Implementation

[0035] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0036] Unless otherwise specified in the examples, the conditions shall be performed according to the standard conditions or the conditions recommended by the manufacturer.

[0037] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0038] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0039] Preservation of biological materials: A strain of *Brutella feces* ( Blautia hominis GH001 was deposited on September 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO:46142. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

[0040] The culture media and reagent components involved in the following examples are as follows:

[0041] YCFA medium: yeast extract 0.25% (w / v), tryptone 1% (w / v), dipotassium hydrogen phosphate 0.045% (w / v), potassium dihydrogen phosphate 0.045% (w / v), sodium chloride 0.09% (w / v), magnesium sulfate heptahydrate 0.009%, calcium chloride 0.009%, resazurite 0.0001% (w / v), sodium bicarbonate 0.4% (w / v), heme chloride 0.001% (w / v), glucose 0.2% (w / v), maltose 0.2% (w / v), cellobiose 0.2% (w / v), vitamin K1 0.005% (w / v).

[0042] YCFA solid plates: Yeast extract 0.25% (w / v), tryptone 1% (w / v), dipotassium hydrogen phosphate 0.045% (w / v), potassium dihydrogen phosphate 0.045% (w / v), sodium chloride 0.09% (w / v), magnesium sulfate heptahydrate 0.009%, calcium chloride 0.009%, resazurite 0.0001% (w / v), sodium bicarbonate 0.4% (w / v), heme chloride 0.001% (w / v), glucose 0.2% (w / v), maltose 0.2% (w / v), cellobiose 0.2% (w / v), vitamin K1 0.005% (w / v), agar 1.5% (w / v).

[0043] Uric acid solid plates without additional carbon source: 2.5% (w / v) uric acid, 0.1% (w / v) yeast extract, 0.045% (w / v) dipotassium hydrogen phosphate, 0.045% (w / v) potassium dihydrogen phosphate, 0.09% (w / v) sodium chloride, 0.009% (w / v) magnesium sulfate heptahydrate, 0.009% (w / v) calcium chloride, 0.0001% (w / v) resazurin, 0.4% (w / v) sodium bicarbonate, 0.001% (w / v) heme chloride, 1.5% (w / v) agar.

[0044] Columbia blood agar plate: 1% (w / v) casein pancreatic enzyme digest, 0.3% (w / v) cardiac pancreatic enzyme digest, 0.1% (w / v) corn starch, 0.5% (w / v) meat stomach enzyme digest, 0.5% (w / v) yeast extract, 0.5% (w / v) sodium chloride, 1.5% (w / v) agar, and 50-100 mL of sterile defibrinated sheep blood.

[0045] Uric acid stock solution: Weigh 0.4 g of sodium hydroxide powder, dissolve in an appropriate amount of ultrapure water, and dilute to 20 mL to obtain a 0.5 mol / L sodium hydroxide solution; weigh 300 mg of uric acid powder, add to 20 mL of 0.5 M sodium hydroxide solution, and then heat and stir with a magnetic stirrer until the uric acid powder is completely dissolved to obtain a 15 mg / L uric acid stock solution. Filter the stock solution through a 0.22 m syringe filter in a clean bench, store at room temperature, and dilute as needed for the experiment before use. Before use, neutralize the pH to 7.3-7.4 with an appropriate amount of dilute hydrochloric acid solution.

[0046] Mixed antibiotic solution: Weigh 0.025g vancomycin, 0.05g metronidazole, 0.05g ampicillin, 0.05g neomycin, and 0.25g streptomycin, dissolve them in an appropriate amount of ultrapure water, and bring the volume to 25 mL.

[0047] Example 1: Preliminary screening of *Brutella feces* strains

[0048] The human fecal solution (10 mg / ml) was filtered and diluted 10 mg / ml. 4 The bacterial culture was then inoculated with 100 μL of the diluted solution onto Columbia blood agar plates and anaerobically cultured at 37 °C. All single colonies appearing on the agar plates after 48 h of culture were selected and inoculated into 96-well plates containing 200 μL of YCFA medium per well, and incubated at 37 °C for 48 h. The bacterial pellet was then lysed with 5 μL of NaOH / SDS lysis buffer and diluted with 150 μL of deionized water. 2 μL of template was used for PCR-based 16S rRNA gene sequence amplification. PCR products were identified by sequencing. The phylogeny of all cultured isolates was determined by BLAST analysis against the NCBI 16S rRNA sequence database. The bacterial culture was then transferred to fresh YCFA medium and cultured for 24 h. Uric acid degradation capacity was then assessed after the culture became turbid.

[0049] In this embodiment, the uric acid degradation capacity of the strain obtained in Example 1 was determined. All experiments were conducted under anaerobic conditions, specifically as follows: The strain obtained in Example 1 was inoculated into blank YCFA medium and cultured until the logarithmic growth phase (OD2). 600 Approximately 1). Prepare a fresh YCFA medium and add uric acid stock solution (15 mg / ml). Add 74 μL of uric acid stock solution per mL of medium to achieve a final uric acid concentration of approximately 5 mM. [The remaining text appears to be incomplete and requires further context.] 600 The strain with a concentration of 1 was inoculated into a uric acid-containing culture at a ratio of 1:10, and the entire system was incubated at 37°C and 70 rpm. Subsequently, 500 μL of culture was collected at 0 h, 12 h, 24 h, and 48 h, centrifuged at 10000 rpm for 10 min, and the supernatant was collected. The uric acid concentration in the samples at each time point was detected using a uric acid detection kit.

[0050] In addition, the strains obtained in Example 1 were inoculated onto ordinary YCFA solid plates and uric acid solid plates without additional carbon sources, and the growth of the strains on the plates was photographed and recorded after 72 h.

[0051] Finally, a strain of *Brutella foetida* GH001 with high uric acid degradation capacity was obtained through screening.

[0052] The experimental results are attached. Figure 1 As shown. Figure 1 (A) clearly shows that the human fecal Brut bacterium GH001 strain can grow on a solid plate covered with uric acid and a clear degradation zone is visible around the strain. Figure 1 (B) It can be seen that after incubating the human fecal Brutella GH001 strain in a uric acid-containing medium for 24 hours, the uric acid concentration in the medium decreased from the initial average concentration of 5407.4 μmol / L to 0 μmol / L, a decrease of 100%.

[0053] (1) Colony morphology characteristics

[0054] The strain GH001 obtained in Example 2 is a Gram-positive bacterium that grows well in YCFA medium. Its optimal growth temperature is 37°C, and its optimal growth pH is 6.5. It is strictly anaerobic, does not form spores, and has no flagella. After 12 hours of shake-based culture, it enters the stationary phase. On Columbia blood agar plates, the bacterial plaques are pale yellow, round, with neat edges, opaque, and smooth. The morphology is as follows... Figure 2 Show.

[0055] (2) Molecular biological identification

[0056] In this embodiment, 16S rDNA sequencing was performed on strain GH001: A small amount of colony was picked from a Columbia blood agar plate and added to 50 μL of prepared PCR reaction solution (the composition of which is shown in Table 1). PCR amplification was performed according to the pre-set program (as shown in Table 2). Afterwards, 5 μL of the solution was run for DNA gel electrophoresis to verify the amplification, revealing a band at approximately 1400 bp. The primer sequences involved in the amplification were: 27F: 5-AGAGTTTGATCMTGGCTCAG-3; 1492R: 5-ACCTTGTTACGACTT-3. The remaining PCR reaction solution was sent for sequencing, which was performed by Xiamen Platinum Biotech Co., Ltd. The obtained 16S rDNA sequence (as shown in SEQ ID No. 1) was BLAST-aligned on NCBI and compared with the sequence information of known strains to identify the strain. The strain was ultimately identified as *Brutella feces* (…). Blautia hominisThe present invention named the strain GH001 and deposited it on September 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46142, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

[0057] Table 1 PCR reaction system

[0058] System components Volume (μL) 2×TaqPlusMasterMix 25 Primer 27F 2 Primer 1492R 2 template Single colony water 20 Overall system 50

[0059] Table 2 PCR reaction procedures

[0060]

[0061] This embodiment uses *Brutella feces* (… Blautia hominis The experiment on GH001 lowering serum uric acid in mice is detailed below:

[0062] 1. Modeling and feeding of mice with hyperuricemia

[0063] The hyperuricemic mouse model was a Uox-KO mouse model constructed using CRISPR-Cas9 technology with the uricase gene knocked out. This mouse model is characterized by a stable increase in serum uric acid, making it suitable for studying hyperuricemia and related diseases [Reference: XIE D, ZHAO H, LU J, et al. High uric acid induces liver fat accumulation via ROS / JNK / AP-1 signaling[J]. Am J Physiol Endocrinol Metab, 2021, 320(6): E1032-e1043]. Eight-week-old male Uox-KO mice were used as subjects in this experiment.

[0064] In this experiment, mice were housed in a negative pressure barrier environment at the Experimental Animal Center, in accordance with the Chinese Regulations on the Protection of Laboratory Animals and the Management Regulations of the Experimental Animal Center of Xiamen University. Except for the fasting experiment, all animals received ample food and water and lived under a 12-hour light-dark cycle. The animal experiments were approved by the Experimental Animal Ethics Committee of Xiamen University (Ethics No. XMULAC20200122). Twenty-four hyperuricemic (Uox-KO) mice were divided into the following two groups:

[0065] Control group: fed basic feed daily, treated with 200 μL of antibiotic solution by gavage for 1 week, followed by 200 μL of blank YCFA culture medium by gavage 3 times a week for 4 weeks.

[0066] Experimental group: fed a basic diet daily, treated with 200 μL of antibiotic solution by gavage for one week, followed by gavage three times a week with 200 μL of human fecal Brut bacteria fermentation broth (with a viable count of approximately 2 × 10⁻⁶). 8 CFU), treatment lasts 4 weeks.

[0067] 2. Detection indicators and results

[0068] (1) Serum uric acid content detection

[0069] After the experimental treatment in week 5, blood was collected via ocular vein. The EP tubes containing the blood were left to stand at room temperature for 30 minutes, then centrifuged at 3000 rpm and 4°C for 15 minutes. The separated serum was carefully transferred to a new EP tube, and the uric acid content was measured according to the instructions provided with the UA assay kit (Nanjing Jiancheng, China).

[0070] The experimental results are attached. Figure 3 As shown, after treatment with human fecal Brutella GH001 strain, the serum uric acid level of mice in the experimental group decreased significantly. At the beginning of the experiment, the average serum uric acid level of mice was 432.9 μmol / L. After 4 weeks of treatment, the average serum uric acid level of mice was 300.5 μmol / L, and the serum uric acid level decreased by 30.5%.

[0071] (2) Gross results of the kidneys

[0072] After the experimental treatment in week 5, the mice underwent cardiac perfusion, and the required kidney tissue was taken out and soaked in 10% formalin fixative for 24 hours. After the tissue hardened, it was placed on a ruler for photography.

[0073] The experimental results are attached. Figure 4 As shown, after treatment with the human fecal Brut bacteria strain GH001, the kidney damage in the experimental group mice was significantly alleviated compared with the control group.

[0074] In addition, consuming this strain had no significant effect on the sample's physical indicators, proving that it has a high degree of safety and can be widely used in the medical field.

[0075] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A strain of Brauts foetida ( Blautia hominis ), characterized by: The strain code for this *Brutella feces* is GH001. *Brutella feces* GH001 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46142, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

2. The *Brutella feces* strain as described in claim 1, characterized in that: The *Brutella feces* strain is a Gram-positive bacterium, and its 16S rDNA base sequence is shown in SEQ ID NO.

1.

3. A formulation, characterized in that, The preparation comprises *Brutella feces* as described in claim 1 or a bacterial agent prepared from *Brutella feces* as described in claim 1.

4. A drug comprising the *Brutella feces* as described in claim 1.

5. The drug as described in claim 4, characterized in that, The drug also includes excipients.

6. The use of the *Brutella feces* as described in any one of claims 1-2, the preparation as described in claim 3, or the drug as described in any one of claims 4-5 in the preparation of uric acid degradation products.

7. The use of the human fecal Brut bacteria as described in any one of claims 1-2, the preparation as described in claim 3, or the drug as described in any one of claims 4-5 in the preparation of products for the prevention or treatment of hyperuricemia and / or gout.

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