A clostridium bacterium and use thereof

Abstract

This invention relates to the field of microbiology. Specifically, this invention relates to a novel genus of Clostridium (…). Clostridium sp. The bacteria and their applications. These bacteria can completely metabolize nearly 2000 μg / mL of uric acid in a culture medium within 8 hours, and in vivo experiments have also shown that they can significantly reduce the uric acid content in the serum and urine of hyperuricemic mice. Therefore, this invention provides a novel *Clostridium* species (…). Clostridium sp. Bacteria have the potential to replace or partially replace existing compound drugs, and have broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbiology. Specifically, this invention relates to a novel genus of Clostridium (…). Clostridium sp. Bacteria and their applications. Background Technology

[0002] Uric acid is the final product of purine metabolism in the human body and is mainly excreted through the kidneys. Stable uric acid levels are crucial for maintaining human health. However, when purine metabolism is disrupted or kidney excretion function declines, uric acid concentrations in the body may rise, leading to a range of health problems such as hyperuricemia, gout, nephritis, kidney stones, and even serious diseases like liver necrosis and Wilson's disease. Hyperuricemia, especially when uric acid levels exceed 420 μmol / L in men and 360 μmol / L in women, significantly increases the risk of gout and nephritis. Furthermore, excessively low uric acid levels, such as below 150 μmol / L in men and below 89 μmol / L in women, may also indicate underlying conditions like liver necrosis or Wilson's disease.

[0003] Currently, the treatment methods for uric acid include drug therapy and non-drug therapy.

[0004] Drug therapy includes (1) drugs that inhibit uric acid production: such as febuxostat and allopurinol. These drugs reduce blood uric acid levels by inhibiting the production of uric acid in the body. Febuxostat has become one of the commonly used drugs for lowering uric acid due to its high safety and relatively low risk of allergies. However, its high price and the cardiovascular damage that may result from long-term use limit its widespread application. (2) Drugs that promote uric acid excretion: such as benzbromarone and sulfinpyrazone. These drugs reduce blood uric acid levels by increasing the excretion of uric acid. Benzbromarone can effectively maintain stable uric acid and improve gout symptoms, but long-term use may cause liver damage and is not suitable for patients with liver and kidney dysfunction. Although sulfinpyrazone can promote uric acid excretion, it may affect the metabolism of other essential elements in the body, leading to problems such as hypokalemia and hypocalcemia, and it cannot be used for a long time.

[0005] Non-pharmacological treatments include (1) dietary adjustments: reducing the intake of high-purine foods, such as animal organs, soy products, and seafood, and increasing the intake of foods that help excrete uric acid, such as cabbage, winter melon, and celery, while maintaining adequate water intake, which helps lower uric acid levels. (2) lifestyle improvements: quitting smoking and alcohol, maintaining regular sleep patterns and moderate exercise, such as walking, jogging, and swimming, helps promote metabolism and accelerate uric acid excretion. (3) traditional health care methods: such as soaking feet in magnesium sulfate (e.g., using estradiol) to promote blood circulation and metabolism, which helps relieve joint pain and swelling caused by high uric acid. However, these methods are mostly auxiliary means and cannot replace regular treatment.

[0006] With increasing attention paid to gut microbiota, more and more gut bacteria are being defined as probiotics, and their use in pharmaceuticals is generally well-received due to their lack of toxic side effects on humans. CN114796285A discloses *Akkermansia proteoglycans* (…). Akkermansia muciniphila The use of ATCC BAA-835 and / or combinations thereof in the prevention and / or treatment of hyperuricemia and / or gout. CN114259507A discloses the use of *Synthia spp.* in the preparation of drugs for the treatment and / or prevention of hyperuricemia-related diseases. However, there are few selectable probiotics for uric acid diseases, and their effects vary. Therefore, it is necessary to develop safer, more effective, and more economical probiotic products to better meet clinical needs and improve patients' quality of life. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a new type of Clostridium ( ). Clostridium sp. Bacteria and their applications.

[0008] This invention is achieved through the following technical solution: This invention protects a new genus of Clostridium ( Clostridium The strain having a 16S rRNA sequence that is at least 98.65%, 99%, 99.5%, 99.9%, or 100% identical to the sequence shown in SEQ ID NO: 1.

[0009] Preferably, the strain is a Clostridium species preserved with accession number CCTCC NO: M 20241964. Clostridium sp. ) ibiome024 strain or its progeny strains or subclones, the Clostridium ( Clostridium sp. The strain ibiome024 is deposited at the China Center for Type Culture Collection, located at Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on September 13, 2024.

[0010] In this specification, the term "strain" can refer to a strain obtained directly from a preserved strain, a progeny strain (offspring), or a strain cultured from the original strain (subclone strain).

[0011] The present invention also protects a drug comprising the above-described strain or extracts, cultures or processed products of said strain, and pharmaceutically acceptable excipients.

[0012] In this specification, the term "culture" refers to a population of microbial strains suspended in a culture medium under conditions suitable for the survival and / or growth of the microbial strains. As will be readily apparent to those skilled in the art, in some aspects, these terms as used herein refer to a combination comprising a population of microbial strains and a culture medium in which the population is suspended. In other aspects, these terms as used herein also refer to the culture supernatant and culture components obtained after the microbial strains of the present invention have been cultured. In the present invention, the culture includes, but is not limited to: bacterial solutions, culture supernatants, or bacterial culture media obtained by inoculating or transferring microbial strains into a culture medium of any form (liquid or solid).

[0013] In this specification, the term "processed product" refers to any product derived from a culture, without particular limitation, and can be obtained through processes such as concentration, gelatinization, spray drying, freeze drying, vacuum drying, drum drying, liquefaction, dilution, and pulverization of the culture. In these processes, well-known methods may be appropriately used.

[0014] In this invention, the microbial strains in the culture or the processed product can be live or dead bacteria.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients. Excipients in formulations can be classified in various ways, such as by source, function and use, and route of administration. By source, they can be divided into natural products, semi-synthetic products, and fully synthetic products. Based on their function and use in formulations, there are 65 types of excipients, including pH adjusters, chelating agents, inclusion agents, coating agents, protectants, humectants, disintegrants, surfactants, virus inactivators, supplements, precipitants, film-forming materials, flavoring agents, lyophilization excipients, carbon dioxide adsorbents, foaming agents, aromatics, preservatives, excipients, desiccants, curing agents, buffers, controlled-release materials, adhesives, flavoring agents, antioxidants, antioxidant synergists, anti-adhesion agents, and air displacement agents. Condensing agents, ointment bases, gel materials, polishing agents, propellants, solvents, softeners, emulsifiers, ointment bases, soft capsule materials, lubricants, wetting agents, penetration enhancers, osmotic pressure regulators, suppository bases, sweeteners, fillers, pellet cores, stabilizers, adsorbents, absorbents, diluents, defoamers, flocculants, ethanol modifiers, plaster bases, inks, thickeners, solubilizers, plasticizers, adhesives, excipients for processing traditional Chinese medicine, filter aids, solubilizers, suspending agents, and colorants.

[0016] The excipients include at least one of adjuvants, stabilizers or protectants, antibacterial agents, excipients, solubilizers, flavoring agents, diluents, and buffers.

[0017] Adjuvants: Substances that bind to a vaccine antigen to enhance (e.g., strengthen, accelerate, prolong, and / or possibly target) its specific immune response and the clinical efficacy of the vaccine. Stabilizers or Protectants: Substances used to stabilize or protect the active ingredient of a biological product, preventing its degradation or loss of activity. Antimicrobial Agents: Substances used to inhibit microbial growth and prevent microbial contamination. Excipients: Substances used in lyophilized products to shape the drug and act as a scaffold. Solubilizers: Substances used to increase the solubility of a drug. Flavoring Agents: Substances used to improve the taste of oral medications. Diluents and Buffers: Solvents used to dissolve or dilute products and adjust their pH, such as water for injection, sodium chloride injection, and phosphate-buffered saline (PBS).

[0018] Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (monohydrogen), calcium stearate, croscarmellose, croscarmellose, citric acid, croscarmellose, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium glycolate starch, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0019] The drug may be prepared in the form of an oral formulation. Formulations for oral use include tablets containing an active ingredient mixed with non-toxic, pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating agents and disintegrants (e.g., cellulose derivatives including microcrystalline cellulose, starch including potato starch, croscarmellose sodium, alginate, or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginate, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, ethyl cellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricants, flow aids, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil, or talc). Formulations for oral use may also be in the form of chewable tablets or hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or in the form of soft gelatin capsules, wherein the active ingredient is mixed with an aqueous or oil medium (e.g., peanut oil, liquid paraffin, or olive oil). Powders, granules, and pills may be prepared using the ingredients mentioned above under tablets or capsules in a conventional manner using, for example, mixers, fluidized bed equipment, or spray drying equipment.

[0020] Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, colorants, flavoring agents, plasticizers, humectants, and buffers. Formulations for oral use may also be in the form of chewable tablets, or hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or soft gelatin capsules in which the active ingredient is mixed with an aqueous or oil medium (e.g., peanut oil, liquid paraffin, or olive oil). Powders, granules, and pills may be prepared using the ingredients mentioned above under tablets or capsules in a conventional manner using, for example, mixers, fluidized bed equipment, or spray drying equipment.

[0021] In some embodiments, administration of the drugs described herein includes intramuscular, intravenous (e.g., in the form of a sterile solution and in a solvent system suitable for intravenous use), intradermal, intraarticular, intraperitoneal, intralesional, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, transsurface, intratumoral, transperitoneal, subcutaneous, subconjunctival, intracystic, transmucosal, intraperitoneal, intraperitoneal, intraumbilical, intraocular, oral (e.g., tablets, capsules, capsule tablets, capsule-shaped tablets, or syrup), transsurface (e.g., in the form of cream, gel, lotion, or ointment), local, by inhalation, by injection, or by infusion (e.g., continuous infusion in the form of cream or lipid composition, local perfusion directly soaking target cells, catheterization, irrigation).

[0022] The present invention also protects a pharmaceutical composition comprising the above-described microbial strain or extracts, cultures or processed products of the strain, and a combination drug, preferably, the combination drug being other substances that synergistically interact with the microbial strain.

[0023] The dosage forms of the drugs described in this invention are granules, capsules, tablets, powders, oral liquids, suspensions, or emulsions, etc.

[0024] The effective dosage of this invention is that the total number of live bacteria in the live bacterial preparation made from the above-mentioned microbial strains as the main active pharmaceutical ingredient is 10. 6 -10 14 CFU.

[0025] The duration of medication should be determined based on the desired effect, including but not limited to 1-3 times daily, 3-7 days per week, and also depending on the specific concentration of the formulation.

[0026] The present invention also protects a food, fermentation agent, functional microbial agent or nutritional composition comprising the above-mentioned microbial strains or extracts, cultures or processed products of said strains.

[0027] Fermentation agent or functional microbial agent, including bacterial liquid prepared from the above-mentioned microbial strains, or powder or granules obtained by further processing; the fermentation agent may further contain one or more non-antagonistic microbial agents, a compound microbial agent prepared from one or more of Christensenella, Parabacterium, Akkermansia myxophilus, and Bacteroides polymorpha.

[0028] The effective bacterial concentration and viable count are 10. 6 -10 14 CFU.

[0029] Fermentation agents or functional microbial agents can also be used as functional foods and nutritional products.

[0030] The nutritional composition includes the aforementioned microbial strains, or cultures or processed products of the microbial strains. Preferably, the nutritional composition is a food, nutritional product, supplement, probiotic, or symbiotic.

[0031] The food includes the aforementioned microbial strains, or cultures or processed products of microbial strains, as well as auxiliary substances that enable the food's functions, and may take forms including but not limited to "dietary supplements" and "fermented foods."

[0032] The dietary supplement comprises a culture or processed product of the aforementioned microbial strain, further processed with added nutrients such as cellulose, vitamins, and minerals.

[0033] The fermented foods include dairy products, soy products, or fruit and vegetable products. The dairy products include milk, sour cream, or cheese. The soy products include soy milk, fermented black beans, or soy sauce. The fruit and vegetable products include products made from cucumbers, carrots, beets, celery, or cabbage.

[0034] The probiotics refer to live microorganisms that, when provided in appropriate amounts, are beneficial to the health of the host organism.

[0035] The symbiotic bacteria refer to foods containing a mixture of prebiotics and probiotics. They typically contain probiotic components that promote growth and / or metabolic activity, as well as, in general, probiotic effects in combination with, but not limited to, the aforementioned microbial strains and fructooligosaccharides or galactooligosaccharides.

[0036] The present invention further protects the use of the above-mentioned strains or extracts, cultures or processed products of the strains, or the above-mentioned drugs, or the above-mentioned drug compositions in the preparation of drugs for the prevention, treatment or adjunctive treatment of hyperuricemia, gout, gouty arthritis, uric acid nephropathy and kidney stones, cardiovascular diseases and / or metabolic syndrome.

[0037] High uric acid levels can cause various complications, such as: 1. Gouty arthritis Mechanism: Supersaturated uric acid in the blood forms urate crystals, which are deposited in the joints and surrounding tissues.

[0038] Symptoms: Causes severe pain, redness, swelling, fever, and limited joint movement. It usually first occurs in the big toe joint (gouty foot), but can also occur in the ankle, knee, wrist, fingers, and other joints. Attacks often come suddenly and are extremely painful.

[0039] Development: In the early stages, attacks may occur occasionally, but the frequency of attacks increases and more joints are affected. Eventually, it may develop into chronic gout, leading to permanent joint damage and deformity.

[0040] 2. Uric Acid Nephropathy & Kidney Stones The kidneys are the main organs for uric acid excretion, and therefore one of the most vulnerable sites to damage from high uric acid levels.

[0041] 2.1 Uric acid kidney stones: High concentration of uric acid in urine can directly form stones, block the urinary tract, and cause symptoms such as severe lower back pain and hematuria.

[0042] 2.2 Chronic uric acid nephropathy: Uric acid crystals deposit in the kidney tissue, which can lead to chronic interstitial nephritis, gradually damaging kidney function, resulting in increased nocturia, proteinuria, etc., and may eventually develop into kidney failure.

[0043] 2.3 Acute uric acid nephropathy: It is often seen after tumor chemotherapy (tumor lysis syndrome), when uric acid rises sharply and a large number of crystals block the renal tubules, leading to acute kidney injury.

[0044] 3. Cardiovascular Diseases Hyperuricemia is an independent risk factor for cardiovascular disease and is closely associated with a variety of problems: 3.1 Hypertension: Uric acid can promote vascular inflammation and endothelial dysfunction, activate the renin-angiotensin system, and lead to elevated blood pressure. Many patients with hypertension also have hyperuricemia.

[0045] 3.2 Atherosclerosis and Coronary Heart Disease: Uric acid crystals can be deposited on the blood vessel walls, promoting oxidative stress and inflammatory response, accelerating the formation of atherosclerosis, and increasing the risk of myocardial infarction and angina pectoris.

[0046] 3.3 Heart failure: High uric acid levels are associated with the occurrence, development, and poor prognosis of heart failure.

[0047] 4. Metabolic Syndrome Hyperuricemia often occurs alongside other components of metabolic syndrome, creating a vicious cycle where each component has a causal relationship with the other.

[0048] 4.1 Insulin Resistance and Type 2 Diabetes: High uric acid reduces the body's sensitivity to insulin, exacerbating insulin resistance and thus significantly increasing the risk of developing type 2 diabetes. Conversely, diabetic patients are also prone to high uric acid levels.

[0049] 4.2 Obesity and dyslipidemia: Patients with hyperuricemia often have abdominal obesity, hypertriglyceridemia and low high-density lipoprotein cholesterol (HDL-C).

[0050] Preferably, the uses include at least one of the following: (1) reducing uric acid levels in mammalian urine; (2) reducing uric acid levels in mammalian feces; (3) reducing uric acid levels in mammalian serum; (4) reducing creatinine levels in mammalian serum; (5) reducing blood urea nitrogen levels in mammalian serum; (6) reducing xanthine oxidase activity and expression in mammalian liver; (7) reducing pathological damage to mammalian kidneys and intestines; (8) reducing the expression of GLUT9, a uric acid reabsorption protein in mammalian kidneys and colon; (9) increasing the expression of ABCG2, a uric acid transport protein in mammalian kidneys and colon; (10) increasing the expression of intestinal tight junction proteins in mammalian colons; and (11) promoting the production of short-chain fatty acids in mammalian intestines.

[0051] The present invention also protects the use of the above-mentioned strain or extracts, cultures or processed products of the strain in the preparation of foods, fermentation agents, functional microbial agents or nutritional compositions for non-therapeutic purposes that regulate uric acid levels in mammals.

[0052] The beneficial effects of this invention are as follows: The novel Clostridium species provided by this invention ( Clostridium sp. The bacteria can completely metabolize nearly 2000 μg / mL of uric acid in the culture medium within 8 hours. In vivo experiments also show that it can significantly reduce serum uric acid levels in different hyperuricemia mouse models, reduce the occurrence of gout, alleviate kidney and intestinal pathological damage in hyperuricemia mice, protect organ function, reduce patient pain, promote uric acid excretion, lower serum uric acid levels, maintain the intestinal barrier and protect the intestinal environment, and promote mouse health. Therefore, the novel Clostridium species (Clostridium) provided by this invention... Clostridium sp. Bacteria have the potential to replace or partially replace existing compound drugs, and have broad application prospects. Biological Preservation

[0053] Clostridium ( Clostridium sp. ) ibiome024, deposited on September 13, 2024, at the China Center for Type Culture Collection, located at Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, accession number CCTCC NO: M 20241964. Attached Figure Description

[0054] Figure 1 The phylogenetic analysis of ibiome024 is shown; Figure 2 The microscopic image (40X) of the ibiome024 smear is shown; Figure 3 The colony morphology of ibiome024 on solid culture medium is shown; Figure 4The results show the ability of ibiome024 to metabolize uric acid after 24 hours of culture. Figure 5 The curve of ibiome024's ability to metabolize uric acid during culture is shown; Figures 6-7 The results showed that ibiome024 could reduce the uric acid content in the serum and urine of mice with uric acid + potassium oxonate diet-induced hyperuricemia; Figure 8 The results showed that ibiome024 could reduce serum uric acid levels in mice with hyperuricemia induced by yeast extract and intraperitoneal injection of potassium oxonate. Figure 9 Gel electrophoresis image for uric acid-lowering gene validation. Detailed Implementation

[0055] To better understand the present invention, the present invention will be further described below with reference to the embodiments and accompanying drawings. The following embodiments are only illustrative of the present invention and are not intended to limit it.

[0056] Example 1: Isolation and Identification of Bacterial Strains

[0057] 1. Separation ibiome024 was isolated from mixed fecal samples of four elderly volunteers over 90 years old in Hefei, Anhui Province. None of the four volunteers had used antibiotics or probiotics in the three months prior to sample collection. Physiological saline was aliquoted into sterile 15mL centrifuge tubes in a biosafety cabinet. 24 hours prior to collection, GAM plates (purchased from Solarbio, catalog number LA4450) and sterile physiological saline were transferred to an anaerobic workbench.

[0058] Fecal samples were collected from four healthy volunteers and stored in 80% (v / v) sterile glycerol buffer. The samples were then homogenized in an anaerobic workstation, and 0.5 mL of the mixed fecal solution was injected into an anaerobic bottle using a sterile syringe for incubation. After 14 days of incubation, the mixed fecal samples were serially diluted to 10⁻⁶. -10 Take 0.1 mL of diluted bacterial solution and spread it on a GAM plate. Incubate at 37°C for 72 hours in an anaerobic workstation. Pick single clones and culture them in GAM broth liquid medium.

[0059] 2. 16S rRNA sequencing identification ibiome024 was amplified by PCR using universal 16S rRNA primers (upstream primer 27F (SEQ ID NO.2): AGAGTTTG ATCCTGGCTCAG, downstream primer 1492R (SEQ ID NO.3): GGTTA CCTTGTTACGACTT).

[0060] Experimental methods: PCR system (20 μL): 2 × Taq Master Mix: 10 μL; Primer 1 (27F): 1 μL; Primer 2 (1492R): 1 μL; ddH2O: 7 μL; Bacterial culture: 1 μL.

[0061] PCR reaction program: 95℃ for 10 min; 95℃ for 15 s, 58℃ for 30 s, 72℃ for 40 s, step 2-4 30×; 72℃ for 5 min.

[0062] The PCR product of the 16S rRNA gene was sequenced, and the results are shown in SEQ ID NO.1.

[0063] The comparison revealed that the sequence with the greatest similarity to this sequence is... Hungatella hathewayi strain 1313 showed a similarity of only 95.02%, lower than the generally accepted 98.65% (Kim M, et al. Towards a taxonomiccoherence between average nucleotide identity and 16S rRNA gene sequences similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology, 2014 Feb;64(Pt 2):346-351. doi:10.1099 / ijs.0.059774-0.).

[0064] 3. Phylogenetic Analysis The 16S rDNA sequencing results of strain ibiome024 were retrieved from GenBank using BLAST, and the 16S rDNA sequences of related genera and species were downloaded. Homology analysis was performed using MEGA11 software, and a phylogenetic tree was constructed, such as... Figure 1 As shown. It can be seen that the species with the highest homology to ibiome024 is... Hungatella hathewayi and Lacrimispora sphenoides Tracing the naming history of the two fungal species, it was found that they both belong to the genus Clostridium ( Clostridium ).

[0065] 4. Genome similarity analysis Whole genome sequencing was performed on strain ibiome024, followed by... HungatellaANI value analysis was performed on homologous strains. Table 1 shows that the ANI values ​​ranged from 70.57% to 71.65%, indicating high genomic similarity, but not reaching the typical threshold (95-96%) for defining the same species. Simultaneously, online genomic DNA-DNA hybridization was performed on strain ibiome 024. Table 1 shows that ibiome 024 is related to several known... Hungatella The hybridization values ​​of the genus and species range from 19.8% to 21%. According to microbial taxonomy standards, a dDDH value below 70% generally indicates that the strains belong to different species. Combining the ANI and dDDH values, ibiome024 and... Hungatella The strains exhibit significant genetic differences at the genomic level, supporting their taxonomic status as independent species.

[0066] Table 1 ibiome024 and known Hungatella Results of genome hybridization of different species of bacteria Reference genome dDDH (70%) ANI (95%) _DSM_24995 20.60 70.60 _k17-0097-C1 22.00 70.92 _AM39-16AC 20.80 70.57 _strain_DSM_13479 20.80 70.76 _WAL-18680 21.00 71.65 _NSJ-66 20.60 70.65 _RTP31015st1_E10_RTP31015_201113 19.80 70.57 5. Microbiological Characterization 5.1 Microscopic examination of smears Ibiome024 was examined under a microscope at 40X, and the results were as follows: Figure 2 The microscopic image shown shows that ibiome024 is Gram-positive, short rod-shaped, non-spore-forming, and lacks flagellar motility.

[0067] 5.2 Single colony photograph After anaerobic culture of ibiome024 on GAM medium for 72 hours, a single colony photo was taken, as shown below. Figure 3 As shown, the colonies are milky white, round, with neat edges and a moist surface.

[0068] 5.3 Catalase test and detection of biochemical reactions in sugar alcohol fermentation Take ibiome024 cultured in liquid and microbial cells from the microbial bank of Hefei Han Microbial Technology Co., Ltd. Hungatella hathewayi strain (abbreviated) H.ha ), Hungatella hominis strain (abbreviated) H.ho ), Hungatella effluvii strain (abbreviated) H.ef One strain of each was purified by streaking on GAM solid medium, and single colonies were picked for catalase test and sugar alcohol fermentation biochemical reaction detection.

[0069] Catalase test: Add 2-3 drops of catalase reaction reagent (purchased from Qingdao Haibo Biotechnology, catalog number HB8650) to each colony. The result is positive or negative depending on whether there are bubbles.

[0070] Detection of biochemical reaction of sugar alcohol fermentation: 60 μL of bacterial culture was added to commercially available ampoules for bacterial biochemical detection (purchased from Qingdao Haibo Biotechnology, catalog numbers GB007, GB014, GB055, GB056, GB057, GB058, GB059, GB060, GB064, GB066, GB102-1, GB112, GB177, GB178, GB188, GB195, GB196, GB199, GB202, GB203, GS001, GB032, GS004). After inoculation, the ampoules were placed at 37℃ for anaerobic incubation for 48 h. The detection results were determined according to the kit instructions, as shown in Table 2.

[0071] Table 2 Results of catalase test and biochemical reaction detection in sugar alcohol fermentation Substrate ibiome024 catalase - + + + Mannitol - + + + Sorbitol - + + + Salicylic acid + + + + Raffinose + + + + Ginsenosides + + + + glycerin - + + + maltose + + + + Mannose + + + + Cellobiose - + + + L-Rhamnose + + + + lactose + + + + Arabic sugar + + + + Trehalose - + + + sucrose + + + + Seven-leaf spirit + + + + peptone water - - - - gelatin - - - - glucose + + + + urease - - - - Xylose + - - - 5.4 Sodium Chloride Tolerance Test Take ibiome024 cultured in liquid and H.ha , H.ho , H.ef The strain was subjected to sodium chloride tolerance tests (0.1% NaCl, 0.2% NaCl) at an inoculum size of 10%. 200 μL of bacterial culture was collected at 0, 8, 24, 26, 28, 30, 32, 48, and 72 h, and the OD values ​​were measured using an ELISA reader. 600 The results showed that the four strains could grow in NaCl concentrations of 0.1%, 0.2%, 0.2%, and 0.2%, respectively.

[0072] 5.5 Temperature tolerance test Take ibiome024 cultured in liquid and H.ha , H.ho , H.ef The strain was subjected to temperature tolerance experiments (30℃, 37℃, 42℃) at an inoculum size of 10%. 200 μL of bacterial culture was collected at 0, 8, 24, 26, 28, 30, 32, 48, and 72 h, and the OD value was measured using an ELISA reader. 600 The results showed that all four strains could grow within a temperature range of 30℃ to 42℃.

[0073] 5.6 pH tolerance test Take ibiome024 cultured in liquid and H.ha , H.ho , H.ef The strain was subjected to pH tolerance tests (pH 4, pH 5, pH 6, pH 7, pH 8) at an inoculum size of 10%. 200 μL of bacterial culture was collected at 0, 8, 24, 26, 28, 30, 32, 48, and 72 hours, and the OD values ​​were measured using a microplate reader. 600 The results showed that ibiome024 and H.ho It can grow in the pH range of 6-8, with the optimal pH being 7; H.ha and H.ef It can grow in the pH range of 5-8, with the optimal pH being 7.

[0074] 5.7 Bile salt tolerance test Take ibiome024 cultured in liquid and H.ha , H.ho , H.ef The strain was subjected to bile salt tolerance tests at a 10% inoculum (0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, and 0.5% bile salts). 200 μL of bacterial culture was collected at 0, 8, 24, 26, 28, 30, 32, 48, and 72 hours, and the OD value was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 600 The results showed that all four strains could tolerate 0.5% bile salts.

[0075] Therefore, based on the above identification results, this strain is a new species and is named Clostridium (Clostridium perfringens). Clostridium sp. ibiome024, deposited at the China Center for Type Culture Collection, located at: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China; deposit date: September 13, 2024; accession number: CCTCC NO: M20241964.

[0076] Example 2: In vitro validation of the uric acid-lowering effect of bacterial strains Experimental materials: GAM broth medium (Solepro, LA4450), uric acid (Maclean's) Preparation of GAM medium containing uric acid: Weigh 200 mg of uric acid, dissolve it in 10 mmol / L Na2CO3, add it to GAM liquid medium (total volume of medium 100 mL), and sterilize at 115℃ for 20 min.

[0077] Experimental procedure: (1) Resuscitation strain: Take 50 μL of glycerol bacteria and inoculate it into 1 mL of GAM liquid medium. Culture anaerobicly for 24 h and activate for 2 generations; (2) Inoculate the activated strain into GAM liquid medium containing 2 g / L uric acid at an inoculation rate of 10% and culture anaerobically for 24 h (or take samples at 0 h, 4 h, 6 h, and 8 h of culture). (3) Sample pretreatment: Centrifuge the bacterial culture in (2) at 13000 rpm for 5 min, take 200 μL of supernatant and add it to 800 μL of sterile ddH2O, vortex for 10 s on a vortex apparatus, centrifuge at 13000 rpm for 15 min, and take the supernatant for HPLC detection. (4) HPLC detection capability for metabolizing uric acid: Column: Waters Symmetry® C18 5um 4.6*250mm Mobile phase A: Deionized water containing 0.025% formic acid; Mobile phase B: Acetonitrile containing 0.025% formic acid.

[0078] Detection wavelength: 288 nm, detection temperature: 15 °C, injection volume: 2.0 μL Gradient elution program: 0~2.5min 6%B, 2.5~4min 6~95%B, 4~5.5min 95%B, 5.5~7min 95~6%B, 7~9min 6%B.

[0079] Experimental results: (1) The ability of ibiome024 to metabolize uric acid after 24 hours of culture is as follows: Figure 4 As shown, ibiome024 can completely metabolize uric acid in the culture medium after 24 hours of culture; (2) The uric acid metabolism curve during ibiome024 culture is as follows Figure 5 As shown, the uric acid in the culture medium of ibiome024 began to decrease after 4 hours of culture, and was completely metabolized after 8 hours.

[0080] Explanation of the new species of Clostridium ( Clostridium sp. ibiome024 can metabolize uric acid.

[0081] Example 3: In vivo validation of the uric acid-lowering effect of bacterial strains (hyperuricemia model induced by 2% uric acid + 4% potassium oxonate diet) Experimental methods: C57BL / 6J (8 weeks old, male) mice, SPF grade, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. After one week of acclimatization, the mice were randomly divided into 3 groups: (1) Normal control group: fed normal maintenance diet and administered 0.2 mL of 0.9% physiological saline by gavage / animal / day; (2) Hyperuricemia model mice (Model): fed a diet containing 2% uric acid and 4% potassium oxonate (research diet), and gavaged with 0.2 mL of 0.9% physiological saline / mouse / day; (3) Experimental group (ibiome024): fed with feed containing 2% uric acid and 4% potassium oxonate, 0.2 mL was administered by gavage at a concentration of 5*10 8 CFU / mL ibiome024 bacterial suspension / bacterial / day.

[0082] Four weeks after administration, urine and blood were collected from the orbital rim. Serum was separated by centrifugation at 4000 rpm for 10 min. The uric acid content in serum and urine was detected using a uric acid test kit (brand: Solarbio, catalog number: BC1365).

[0083] Experimental results: The main clinical symptom of hyperuricemia is elevated blood uric acid levels. For example... Figure 6 As shown, the serum uric acid content of mice in the model group induced by potassium oxonate and uric acid-containing food was significantly increased, and the new species of Clostridium (…) was administered via gavage. Clostridium sp. ibiome024 can reduce the uric acid content in serum.

[0084] like Figure 7 As shown, the uric acid content in the urine of mice in the model group induced by potassium oxonate and uric acid-containing food was increased, and the new species of Clostridium (…) was administered via gavage. Clostridium sp. ibiome024 can reduce the uric acid content in urine.

[0085] Example 4: In vivo validation of the uric acid-lowering effect of the strain (yeast extract + intraperitoneal injection of potassium oxonate-induced hyperuricemia model) Experimental methods: C57BL / 6J (8 weeks old, male) mice, SPF grade, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. After one week of acclimatization, the mice were randomly divided into 3 groups: (1) Normal control group: fed normal maintenance diet and administered 0.2 mL of 0.9% physiological saline by gavage / animal / day; (2) Hyperuricemia model mice (Model): They were fed a diet containing 10% yeast extract and injected intraperitoneally with 250 mg / kg potassium oxonate daily, and gavaged with 0.2 mL of 0.9% physiological saline / mouse / day; (3) Experimental group (ibiome024): fed with feed containing 10% yeast extract and administered intraperitoneal injections of 250 mg / kg potassium oxonate daily, orally administered 0.2 mL of 5*10 solution. 8 CFU / mL ibiome024 bacterial suspension / bacterial / day.

[0086] Four weeks after drug administration, blood was collected from the orbital cavity, and serum was separated by centrifugation at 4000 rpm for 10 min. The serum uric acid content was measured using a uric acid detection kit (brand: Solarbio, catalog number: BC1365).

[0087] Experimental results: like Figure 8 As shown, the serum uric acid level was significantly increased in mice in the model group induced by feeding them a food containing yeast extract and intraperitoneal injection of potassium oxonate. The new strain of Clostridium (…) was administered via gavage. Clostridium sp.ibiome024 can reduce the uric acid content in serum.

[0088] Example 5: Validation of Uric Acid-Lowering Genes According to literature reports (https: / / doi.org / 10.1038 / s41564-025-02079-4), a total of eight genes are involved in the uric acid metabolism process in anaerobic bacteria (xdhAC, ygfK, ssnA, hyuA, ygeW, ygeA, ygeY, and ygeX). In order to verify whether this new species has universal uric acid-lowering properties, specific primers were designed for each gene (Table 3), and the relevant genes were amplified by PCR.

[0089] Amplification system: 10 μL of 2 × Primerstar (R045Q; Takara), 1 μL of primer F, 1 μL of primer R, and liquid culture of bacterial solution (ibiome024). Hungatella hathewayi Three strains Hungatella hominis Three strains Hungatella effluvii 1 μL of 3 strains of one species or 6 strains of another species, and 7 μL of ddH2O were used for amplification. The amplification conditions were: 98℃ for 10 min; 98℃ for 10 s, 56℃ for 15 s, 72℃ for 30 s, for 30 cycles; 72℃ for 5 min. The amplification system was subjected to agarose gel electrophoresis (1% agarose, 150 V, 20 min). The specific gel image is shown below. Figure 9 As shown (the holes are, in order, Marker, ibiome024, ...), Hungatella hathewayi Three strains Hungatella hominis Three strains Hungatella effluvii (Three strains of one species and six control strains of other species). Among them, the... Hungatella The 9 strains were obtained from the microbial bank of Hefei Han Microbial Technology Co., Ltd.

[0090] Table 3 Primers for each gene

[0091] Depend on Figure 9 It can be seen that the new species of Clostridium ( Clostridium sp. Using specific primers, xdhAC, ygfK, ssnA, hyuA, ygeW, ygeA, ygeY, and ygeX all amplified bands of the expected size, while the other six control strains (belonging to different genera) did not. Bifidobacterium longum subsp. Suillum , Parabacteroides merdae , Bifidobacterium adolescentis , Bifidobacterium pseudocatenulatum The failure of the new Clostridium species to amplify relevant bands or to amplify a single band of the expected size indicates that the new species of Clostridium ( Clostridium sp.Unlike the other 6 control strains, this strain contains the uric acid-lowering genes xdhAC, ygfK, ssnA, hyuA, ygeW, ygeA, ygeY, and ygeX.

[0092] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A species of Clostridium ( Clostridium The strain of ) is characterized by It also includes the following features: （ 1) The strain has a 16S rRNA sequence that is 100% identical to the sequence shown in SEQ ID NO: 1; (2) The average nucleotide identity (ANI) between the strain and the whole genome sequence of Clostridium sp. ibiome024 strain with accession number CCTCC NO:M 20241964 is not less than 95%; (3) The strain was cultured under anaerobic conditions in a medium containing 2 g / L uric acid for 24 hours and was found by HPLC to be able to completely metabolize the uric acid in the medium.

2. The strain according to claim 1, characterized in that: The strain in question is a Clostridium species preserved under accession number CCTCC NO: M20241964. Clostridium sp. ) ibiome024 strain, the Clostridium ( Clostridium sp. The strain ibiome024 is deposited at the China Center for Type Culture Collection, located at Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on September 13, 2024.

3. A drug, characterized in that: It comprises the strain described in claim 1 or 2, and pharmaceutically acceptable excipients.

4. The drug according to claim 3, characterized in that: The excipients include at least one of adjuvants, stabilizers or protectants, antibacterial agents, excipients, solubilizers, flavoring agents, diluents, and buffers.

5. A pharmaceutical composition, characterized in that: It includes the strain and combination drug as described in claim 1 or 2.

6. The pharmaceutical composition according to claim 5, characterized in that: The combined drug is another substance that has a synergistic effect with the strain.

7. Use of the strain of claim 1 or 2, or the drug of claim 3 or 4, or the pharmaceutical composition of claim 5 or 6 in the preparation of a drug for the prevention, treatment or adjunctive treatment of hyperuricemia, gout, or uric acid kidney stones caused by excessively high concentrations of uric acid in urine.

8. The use according to claim 7, characterized in that, The use includes at least one of the following: (1) Reduces uric acid levels in mammalian urine; (2) Reduce serum uric acid levels in mammals.

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