Clostridium sp. bacterium and use thereof

Abstract

A Clostridium sp. bacterium and the use thereof. The bacterium can completely metabolize 2000μg / mL of uric acid in a culture medium within 8 hours. In vivo experiments show that the bacterium can significantly reduce the contents of uric acid in serum and urine of mice with hyperuricemia.

Description

Clostridium sp. bacteria and applications thereof TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms. Specifically, the present application relates to a new Clostridium sp. bacteria and applications thereof. BACKGROUND

[0002] Uric acid is the end product of purine metabolism in the human body and is mainly excreted by the kidneys. The stability of uric acid levels is crucial for maintaining human health. However, when the metabolism of purine substances in the human body is disturbed or the excretion function of the kidneys decreases, the concentration of uric acid in the body may increase, leading to a series of health problems such as hyperuricemia, gout, nephritis, kidney stones, and even severe diseases such as liver necrosis and liver cirrhosis. Hyperuricemia, especially when the uric acid level exceeds 420 μmol / L in men and 360 μmol / L in women, significantly increases the risk of diseases such as gout and nephritis. In addition, a uric acid level that is too low, such as less than 150 μmol / L in men and less than 89 μmol / L in women, may also indicate potential diseases such as liver necrosis or liver cirrhosis.

[0003] Current treatments for uric acid include drug therapy and non-drug therapy.

[0004] Drug therapy includes (1) uric acid generation inhibitors: such as febuxostat, allopurinol, etc. These drugs reduce blood uric acid levels by inhibiting the generation of uric acid in the body. Febuxostat, due to its high safety and relatively low risk of allergies, has become one of the commonly used drugs for reducing uric acid. However, its high price and potential cardiovascular damage from long-term use limit its widespread application. (2) Uric acid excretion promoters: such as benzbromarone, sulfinpyrazone, etc. These drugs reduce blood uric acid levels by increasing uric acid excretion. Benzbromarone can effectively maintain uric acid stability and improve gout symptoms, but long-term use may cause liver damage and is not suitable for patients with liver and kidney dysfunction. Sulfinpyrazone can promote uric acid excretion, but may affect the metabolism of other essential elements in the body, leading to problems such as low potassium and low calcium, and cannot be used for long-term use.

[0005] Non-drug therapy includes (1) dietary adjustment: by reducing the intake of high-purine foods such as animal offal, bean products, seafood, etc., increasing foods that help uric acid excretion such as cabbage, winter melon, celery, etc., and maintaining adequate water intake, which helps to reduce uric acid levels. (2) Lifestyle improvement: quitting smoking and drinking, maintaining regular work and rest, and engaging in moderate exercise such as walking, jogging, swimming, etc., which helps to promote metabolism and accelerate uric acid excretion. (3) Traditional health methods: such as using salt-eliminating (magnesium sulfate) foot baths, which help to relieve joint pain and swelling caused by high uric acid through promoting blood circulation and metabolism. However, these methods are mostly auxiliary means and cannot replace regular treatment.

[0006] With the increasing attention to intestinal flora, more and more intestinal flora are defined as intestinal probiotics, which are generally well accepted for pharmaceutical use due to their non-toxic side effects on the human body. CN114796285A discloses the application of Akkermansia muciniphila ATCC BAA-835 and / or its composition in preventing and / or treating hyperuricemia and / or gout. CN114259507A discloses the application of a small Christensenella in the preparation of a drug for treating and / or preventing hyperuricemia-related diseases. However, the selection of probiotics for uric acid diseases is limited, and the effects are not the same. Therefore, it is necessary to develop safer, more effective and economical probiotic products to better meet the clinical needs and improve the quality of life of patients. TECHNICAL PROBLEM

[0007] The purpose of the present application is to solve the above-mentioned deficiencies of the prior art and provide a new Clostridium sp. bacterium and its application. TECHNICAL SOLUTION

[0008] The present application is achieved by the following technical solutions:

[0009] The present application protects a new Clostridium strain, which has 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.

[0010] Preferably, the strain is Clostridium sp. ibiome024 strain or its daughter strain or subclone strain preserved under accession number CCTCC NO: M 20241964, which is preserved in China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei, China, on September 13, 2024.

[0011] In this specification, the term "strain" can be obtained directly from the preserved strain, or it can be a daughter strain (offspring) or a strain cultured from the original strain (subclone strain).

[0012] The present application also protects a medicine comprising the above-mentioned strain or extract, culture or processed product of the strain, and a pharmaceutically acceptable excipient.

[0013] In the present specification, the term "culture" refers to a population of microorganism strains suspended in a culture medium under conditions suitable for the survival and / or growth of the microorganism strains. As will be apparent to those of ordinary skill in the art, in some aspects, the terms as used herein relate to a combination comprising a population of microorganism strains and a culture medium in which the population is suspended. In another aspect, the terms as used herein also relate to the culture supernatant and culture components after completion of culturing of the microorganism strains of the present application. In the present application, the culture includes, but is not limited to, a bacterial solution, culture supernatant, or bacterial culture medium obtained by inoculating or transplanting the microorganism strains of the present application into a culture medium in any form (liquid or solid).

[0014] In the present specification, the term "processed product" refers to a product obtained by processing such as concentration, gelatinization, spray drying, freeze drying, vacuum drying, drum drying, liquefaction, dilution, pulverization, etc. of the culture, without particular limitation as long as it is derived from the culture. In these processes, known methods can be appropriately used.

[0015] In the present application, the microorganism strains of the present application can be viable or dead in the culture or in the processed product.

[0016] Further, the medicine also includes pharmaceutically acceptable excipients, and the classification of excipients in preparations is various, which can be classified from sources, functions and uses, administration routes, etc. According to the sources, they can be classified into natural products, semi-synthetic products and synthetic products. According to the functions and uses of the excipients in preparations, there are 65 kinds of classification, which are pH adjuster, chelating agent, inclusion agent, coating agent, protective agent, humectant, disintegrant, surfactant, virus inactivator, supplement, precipitant, film-forming material, flavoring agent, lyophilization excipient, carbon dioxide adsorbent, foaming agent, fragrance, preservative, excipient, drying agent, solidifying agent, buffer, controlled release material, adhesive, flavoring agent, antioxidant, antioxidant synergist, anti-adhesion agent, air replacement agent, condensing agent, ointment base, gel material, polishing agent, propellant, solvent, softener, emulsifier, ointment base, soft capsule material, lubricant, wetting agent, penetration enhancer, osmotic pressure regulator, suppository base, sweetener, filler, core, stabilizer, adsorbent, absorbent, diluent, antifoaming agent, flocculating agent, ethanol modifier, plaster base, ink, thickening agent, solubilizer, plasticizer, adhesive, traditional Chinese medicine processing excipient, filter aid, solubilizer, suspending agent, coloring agent.

[0017] The excipient includes at least one of adjuvant, stabilizer or protective agent, bacteriostatic agent, excipient, solubilizer, flavoring agent, diluent, buffer.

[0018] Adjuvant: is a substance mixed with one or more components of a vaccine antigen to enhance [e.g., strengthen, speed up, prolong, and / or possibly direct] its specific immune response and clinical effects of the vaccine.

[0019] Stabilizer or protective agent: is a substance used to stabilize or protect the active ingredients of a biological product, prevent its degradation or loss of activity.

[0020] Bacteriostatic agent: is a substance used to inhibit the growth of microorganisms, prevent microbial contamination. Excipient: is a substance used in lyophilized products to shape the drug, act as a support. Solubilizing agent: is a substance used to increase the solubility of the drug. Flavoring agent: is a substance used to improve the taste of oral drugs. Diluent, buffer: is a solvent used to dissolve, dilute the product, adjust the pH of the product, such as water for injection, sodium chloride injection, phosphate buffered physiological sodium chloride solution (PBS), etc.

[0021] Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (monohydrogen), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinylpyrrolidone, citric acid, cross-linked povidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, povidone, pregelatinized starch, propyl paraben, retinol palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium glycolate starch, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0022] The drug can be prepared in the form of an injection preparation or an oral preparation. The injection preparation is classified according to the state of matter, including liquid injection, injection powder, injection tablet; according to the injection part, including intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, intraspinal injection; the solvent of the preferred injection preparation includes water for injection or physiological saline.

[0023] Formulations for oral use include tablets containing the active ingredient in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers (for example, sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (for example, cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginic acid, or alginates); binding agents (for example, sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (for example, magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil, or talc). Formulations for oral use can also be in the form of chewable tablets, or in the form of hard gelatin capsules, where the active ingredient is mixed with inert solid diluents (for example, potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or in the form of soft gelatin capsules, where the active ingredient is mixed with water or an oil medium (for example, peanut oil, liquid paraffin, or olive oil). Powders, granulates, and pellets can be prepared using the ingredients mentioned above under tablets or capsules in a conventional manner using, for example, a mixer, a fluid bed apparatus, or a spray-drying apparatus.

[0024] Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, colorants, flavorings, plasticizers, humectants, and buffering agents. Formulations for oral use can also be in the form of chewable tablets, or in the form of hard gelatin capsules, where the active ingredient is mixed with inert solid diluents (for example, potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or in the form of soft gelatin capsules, where the active ingredient is mixed with water or an oil medium (for example, peanut oil, liquid paraffin, or olive oil). Powders, granulates, and pellets can be prepared using the ingredients mentioned above under tablets or capsules in a conventional manner using, for example, a mixer, a fluid bed apparatus, or a spray-drying apparatus.

[0025] In some embodiments, the administration includes intramuscular, intravenous (e.g., in a sterile solution and in a solvent system suitable for intravenous use), intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, transdermal, intratumoral, transperitoneal, subcutaneous, subconjunctival, intracapsular, transmucosal, intrapericardial, intraumbilical, intraocular, oral (e.g., tablets, capsules, caplets, caplets or syrup), topical (e.g., in the form of a cream, gel, lotion or ointment), local, by inhalation, by injection or by infusion (e.g., continuous infusion in the form of a cream or lipid composition, local perfusion directly soaking target cells, catheterization, lavage) of the drugs described herein.

[0026] The present application also protects a pharmaceutical composition comprising the above-mentioned microbial strain or extract, culture or processed product of the strain, and a combination drug, preferably, the combination drug is another substance that synergizes with the microbial strain.

[0027] The dosage form of the drug described in the present application is granules, capsules, tablets, powders, oral liquids, suspensions or emulsions, etc.

[0028] The effective dose of the present application is that the total number of viable bacteria contained in the live bacterial preparation prepared by using the above-mentioned microbial strain as the main pharmaceutically active ingredient is 10 6 -10 14 CFU.

[0029] The administration cycle is subject to the effect, including but not limited to 1-3 times a day, 3-7 days a week, etc., and is also related to the concentration of the specific preparation.

[0030] The present application also protects a food, a ferment, a functional bacterial agent or a nutritional composition comprising the above-mentioned microbial strain or extract, culture or processed product of the strain.

[0031] The ferment or functional bacterial agent includes the bacterial liquid prepared by the above-mentioned microbial strain, or the powder or granules obtained by further processing; the ferment can further contain one or more non-antagonistic microbial agents selected from one or more of Christensenella minuta, Parabacteroides distasonii, Akkermansia muciniphila and Bacteroides thetaiotaomicron.

[0032] The effective concentration of the bacterial agent and the number of viable bacteria are 10 6 -10 14 CFU.

[0033] The ferment or functional bacterial agent can also be used as a functional food or a nutritional product.

[0034] A nutritional composition comprising the above-mentioned microbial strain, or a culture or processed product of the microbial strain. Preferably, the nutritional composition is a food, a nutraceutical, a supplement, a probiotic or a symbiotic.

[0035] The food comprises the above-mentioned microbial strain, or a culture or processed product of the microbial strain, and an auxiliary substance for achieving the function of the food, and the form of presentation includes, but is not limited to, "dietary supplement", "fermented food", etc.

[0036] The dietary supplement comprises a culture or processed product of the above-mentioned microbial strain, and further comprises cellulose, vitamins, minerals, and other nutrients.

[0037] The fermented food includes dairy products, soy products, or fruit and vegetable products, etc. The dairy products are milk, sour cream, or cheese, etc. The soy products are soy milk, tempeh, or miso, etc. The fruit and vegetable products are cucumber, carrot, beet, celery, or Chinese cabbage products, etc.

[0038] The probiotic refers to a live microorganism that is beneficial to the health of the host organism when it is provided in an appropriate amount.

[0039] The symbiotic refers to a food containing a mixture of prebiotics and probiotics. They usually contain prebiotic components that are beneficial to growth and / or metabolic activity, and in general, the effect of probiotics such as, but not limited to, the above-mentioned microbial strain combined with fructooligosaccharides or galactooligosaccharides.

[0040] The present application further protects the use of the above-mentioned strain or an extract, culture or processed product of the strain, or the above-mentioned medicament, or the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing, treating or adjuvant therapy of hyperuricemia, gout, gouty arthritis, uric acid nephropathy and kidney stones, cardiovascular disease and / or metabolic syndrome.

[0041] Hyperuricemia can cause various complications, such as:

[0042] 1. Gouty arthritis (Gout)

[0043] Mechanism: The supersaturation of uric acid in the blood forms urate crystals, which are needle-shaped crystals deposited in the joints and their surrounding tissues.

[0044] Symptoms: It causes severe pain, redness, fever and limited joint movement. The first onset is usually in the joint of the big toe (podagra), but it can also occur in the joints of the ankles, knees, wrists, fingers, etc. The onset is often sudden and the pain is unbearable.

[0045] Development: It can occur occasionally at the beginning, then the frequency of onset increases, the number of affected joints increases, and finally it can turn into chronic gout, causing permanent damage and deformity of the joints.

[0046] 2. Uric Acid Nephropathy & Kidney Stones

[0047] The kidneys are the primary organs for uric acid excretion, and thus are one of the sites most susceptible to damage from high uric acid levels.

[0048] 2.1 Uric Acid Nephrolithiasis: High concentrations of uric acid in the urine can directly form stones, blocking the urinary tract and causing severe flank pain, hematuria, and other symptoms.

[0049] 2.2 Chronic Uric Acid Nephropathy: Uric acid crystals deposit in the kidney tissue, leading to chronic interstitial nephritis, gradually damaging kidney function, and causing nocturia, proteinuria, and eventually kidney failure.

[0050] 2.3 Acute Uric Acid Nephropathy: Commonly seen after tumor chemotherapy (tumor lysis syndrome), uric acid levels rise sharply, and a large number of crystals block the renal tubules, leading to acute kidney injury.

[0051] 3. Cardiovascular Diseases

[0052] Hyperuricemia is an independent risk factor for cardiovascular disease and is closely related to a variety of problems:

[0053] 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.

[0054] 3.2 Atherosclerosis and Coronary Heart Disease: Uric acid crystals can deposit in the blood vessel wall, promote oxidative stress and inflammatory response, accelerate the formation of atherosclerosis, and increase the risk of myocardial infarction and angina pectoris.

[0055] 3.3 Heart Failure: High uric acid levels are associated with the occurrence, development, and poor prognosis of heart failure.

[0056] 4. Metabolic Syndrome

[0057] Hyperuricemia often "goes together" with other components of metabolic syndrome, causing and being caused by each other, forming a vicious cycle.

[0058] 4.1 Insulin Resistance and Type 2 Diabetes: High uric acid levels reduce the body's sensitivity to insulin, exacerbate insulin resistance, and greatly increase the risk of developing type 2 diabetes. Conversely, diabetic patients are also prone to hyperuricemia.

[0059] 4.2 Obesity and dyslipidemia: Patients with hyperuricemia often have abdominal obesity, hypertriglyceridemia, and low high-density lipoprotein cholesterol (HDL-C) and other problems.

[0060] Preferably, the use comprises at least one of: (1) reducing the urine uric acid level of the mammal; (2) reducing the fecal uric acid level of the mammal; (3) reducing the serum uric acid level of the mammal; (4) reducing the serum creatinine level of the mammal; (5) reducing the serum urea nitrogen level of the mammal; (6) reducing the liver xanthine oxidase activity and expression of the mammal; (7) reducing the kidney and intestinal pathological damage of the mammal; (8) reducing the expression of uric acid reabsorption protein GLUT9 in the kidney and colon of the mammal; (9) increasing the expression of uric acid transport protein ABCG2 in the kidney and colon of the mammal; (10) increasing the expression of intestinal tight junction protein in the colon of the mammal; (11) promoting the production of short-chain fatty acids in the intestine of the mammal.

[0061] The application also protects the use of the above-mentioned strain or extract, culture or processed product of the strain for preparing a food, ferment, functional bacterial agent or nutritional composition for regulating the uric acid level of a mammal for non-therapeutic purposes. Beneficial effects

[0062] The beneficial effects of the present application are:

[0063] The new Clostridium sp. bacteria provided by the present application can completely metabolize nearly 2000 μg / mL of uric acid in the culture medium within 8 hours, and in vivo experiments also show that it can significantly reduce the uric acid content in the serum of different hyperuricemia model mice, reduce the occurrence of gout, relieve pathological damage of the kidney and intestine of hyperuricemia mice, protect organ function, reduce patient pain, promote uric acid excretion, reduce serum uric acid level, maintain intestinal barrier and protect intestinal environment, and promote mouse health. Therefore, the new Clostridium sp. bacteria provided by the present application can be expected to replace or partially replace existing compound drugs, and has a wide application prospect. Biological preservation

[0064] Clostridium sp. ibiome024, preservation date September 13, 2024, preservation location China Center for Type Culture Collection, address Wuhan University, China Center for Type Culture Collection, Biaoyilu, Wuchang District, Wuhan, Hubei Province, preservation number CCTCC NO: M 20241964. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 shows the phylogenetic analysis of ibiome024;

[0066] Figure 2 shows the smear microscopy image of ibiome024 (40X) ;

[0067] Figure 3 shows the colony morphology of ibiome024 on solid medium;

[0068] Figure 4 shows the results of the detection of the ability of ibiome024 to metabolize uric acid after 24 hours of culture;

[0069] Figure 5 shows the curve of the ability of ibiome024 to metabolize uric acid during the culture process;

[0070] Figures 6-7 show that ibiome024 can reduce the uric acid content in the serum and urine of hyperuricemia mice induced by uric acid + potassium oxonate food;

[0071] Figure 8 shows that ibiome024 can reduce the uric acid content in the serum of hyperuricemia mice induced by yeast extract + intraperitoneal injection of potassium oxonate;

[0072] Figure 9 is a gel electrophoresis image of the validation of the uric acid-lowering gene. Embodiments of the present application

[0073] In order to better understand the present application, the present application will be further described below in conjunction with examples and drawings, and the following examples are only used to illustrate the present application and not to limit it. Example 1 Isolation and identification of the strain

[0074] 1 Isolation

[0075] Ibiome024 was isolated from a mixed fecal sample of four volunteers over 90 years old in Hefei, Anhui Province. The four volunteers had not used antibiotics or probiotics in the previous three months when the sample was collected. The physiological saline was divided into sterile 15 mL centrifuge tubes in a biological safety cabinet, and the GAM plates (purchased from Solarbio, item number LA4450) and sterile physiological saline were transferred into the anaerobic workstation 24 hours in advance.

[0076] The fecal samples of four healthy volunteers were stored with 80% volume fraction of sterile glycerol buffer, and then mixed in the anaerobic workstation. 0.5 mL of the mixed fecal stock solution was injected into the anaerobic bottle for incubation, and the mixed fecal sample incubated for 14 days was gradient diluted to 10 -10 The 0.1 mL of diluted bacterial solution was taken and spread on the GAM plate, which was cultured at 37°C in the anaerobic workstation for 72 hours. Single colonies were picked and cultured in GAM broth liquid medium.

[0077] 2 16s rRNA sequencing identification

[0078] PCR amplification of ibiome024 by 16s rRNA universal primers (upstream primer 27F (SEQ ID NO. 2): AGAGTTTG ATCCTGGCTCAG, downstream primer 1492R (SEQ ID NO. 3): GGTTA CCTTGTTACGACTT).

[0079] Experimental method:

[0080] PCR system (20 μL): 2 × Taq Master Mix: 10 μL; primer 1 (27F): 1 μL; primer 2 (1492R): 1 μL; ddH2O: 7 μL; bacterial solution: 1 μL.

[0081] PCR reaction program: 95℃ 10min; 95℃ 15s, 58℃ 30s, 72℃ 40s, step 2-4 30×; 72℃ 5min.

[0082] The PCR product of 16S rRNA gene was sequenced, and the result is shown as SEQ ID NO. 1.

[0083] By comparison, the sequence with the largest similarity is Hungatella hathewayi strain 1313, and the similarity is only 95.02%, which is lower than the generally accepted 98.65% (Kim M, et al. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence 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.).

[0084] 3 Phylogenetic analysis

[0085] The 16S rDNA sequencing results of the ibiome024 strain were searched using Blast and the relevant genus and species 16S rDNA sequences were downloaded. Homology analysis was performed using MEGA11 software, and a phylogenetic tree was constructed as shown in Figure 1. It can be seen that the species with the highest homology to ibiome024 are Hungatella hathewayi and Lacrimispora sphenoides. Tracing the naming history of the two species, it was found that they both belong to the Clostridium genus.

[0086] 4. Genome similarity analysis

[0087] The strain ibiome024 was subjected to whole genome sequencing, followed by ANI value analysis with Hungatella homologous strains. As shown in Table 1, the ANI value is distributed between 70.57% and 71.65%, indicating that they have high genomic similarity, but do not reach the typical threshold value (95-96%) for defining the same species. At the same time, the genomic DNA-DNA hybridization of strain ibiome024 was performed. As shown in Table 1, the hybridization value of ibiome024 with multiple known Hungatella species genomes ranges from 19.8% to 21%. According to the standards of microbial taxonomy, a DDH value below 70% usually indicates that the strains belong to different species. In combination with ANI value and DDH value, it is shown that ibiome024 and Hungatella strains have significant genetic differences at the genomic level, supporting their classification status as independent species.

[0088] Table 1. Genome hybridization results of ibiome024 with known Hungatella species

[0089] Reference genomeDDH (70%)ANI (70%)Hungatella_effluvii_DSM_2499520.6070.60Hungatella_effluvii_k17-0097-C122.0070.92Hungatella_hathewayi_AM39-16AC20.8070.57Hungatella_hathewayi_strain_DSM_1347920.8070.76Hungatella_hathewayi_WAL-1868021.0071.65Hungatella_hominis_NSJ-6620.6070.65Hungatella_hominis_RTP31015st1_E10_RTP31015_20111319.8070.57

[0090] 5 Microbiological characterization

[0091] 5.1 Smear microscopy

[0092] Ibiome024 was subjected to smear microscopy using a microscope at 40X, and the results are shown in Figure 2. As can be seen from the figure, ibiome024 was Gram-positive, short rod-shaped, without spores, and without flagellar movement.

[0093] 5.2 Single colony photograph

[0094] Ibiome024 was subjected to single colony photographing after anaerobic culture on GAM medium for 72h, and the results are shown in Figure 3. The colony was milky white, round, with neat edges, and the surface was moist.

[0095] 5.3 Catalase test and detection of sugar alcohol fermentation biochemical reactions

[0096] Liquid-cultured ibiome024 and 1 strain each of Hungatella hathewayi (referred to as H. ha), Hungatella hominis (referred to as H. ho), and Hungatella effluvii (referred to as H. ef) from the microbial strain library of Hefei Han Microbial Technology Co., Ltd. were each inoculated on GAM solid medium and purified by streaking, and single colonies were picked for catalase test and detection of sugar alcohol fermentation biochemical reactions.

[0097] Catalase test: 2-3 drops of catalase reaction reagent (purchased from Qingdao Haibo Biology, item number HB8650) were added to each colony, and the results were determined as positive or negative according to the presence or absence of bubbles.

[0098] Detection of sugar alcohol fermentation biochemical reactions: 60 μL of bacterial liquid was inoculated into a commercial bacterial biochemical detection ampoule (purchased from Qingdao Haibo Biology, item 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), and then incubated at 37°C anaerobically for 48h. The results were determined according to the kit instructions, as shown in Table 2.

[0099] Table 2 Results of catalase test and detection of sugar alcohol fermentation biochemical reactions

[0100] Substrate ibiome024H.haH.hoH.ef Catalase -+++ Mannitol -+++ Sorbitol -+++ Salicylic acid ++++ Raffinose ++++ Pinetrate ++++ Glycerol -+++ Maltose ++++ Mannose ++++ Cellobiose -+++ L-Rhamnose ++++ Lactose ++++ Arabinose ++++ Trehalose -+++ Sucrose ++++ Aesculin ++++ Peptone water ---- Gelatin ---- Glucose ++++ Urease ---- Xylose +---

[0101] 5.4 Sodium Chloride Tolerance Test

[0102] Liquid cultured *ibiome024* and *H. ha*, *H. ho*, and *H. ef* strains were 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 a microplate 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.

[0103] 5.5 Temperature tolerance test

[0104] Liquid cultured *ibiome024* and *H. ha*, *H. ho*, and *H. ef* strains were used for temperature tolerance experiments (30℃, 37℃, and 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 values ​​were measured using a microplate reader. 600 The results showed that all four strains could grow within a temperature range of 30℃ to 42℃.

[0105] 5.6 pH tolerance test

[0106] Liquid cultured *ibiome024* and *H. ha*, *H. ho*, and *H. ef* strains were used for pH tolerance experiments (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 could grow in the pH range of 6-8, with the optimal pH being 7; H.ha and H.ef could grow in the pH range of 5-8, with the optimal pH being 7.

[0107] 5.7 Bile salt tolerance test

[0108] Take liquid culture ibiome024 and H. ha, H. ho, H. ef strains, 10% inoculation amount for bile salt tolerance experiment (0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% bile salt), respectively at 0, 8, 24, 26, 28, 30, 32, 48 and 72h take 200μL bacteria liquid using enzyme marker measures OD 600 . The results show that the four strains can tolerate 0.5% bile salt.

[0109] Therefore, according to the above identification results, the strain is a new species, named Clostridium sp. ibiome024, preserved in China Center for Type Culture Collection, the preservation address is: Wuhan University, China Center for Type Culture Collection, Wuhan, China; the preservation date is: September 13, 2024; the preservation number is: CCTCC NO: M 20241964. Example 2 In vitro verification of strain uric acid reduction

[0110] Experimental materials:

[0111] GAM broth medium (Solebo, LA4450), uric acid (Macklin)

[0112] Preparation of GAM medium containing uric acid: weigh 200 mg of uric acid, dissolve with 10 mmol / L Na2CO3, then add to GAM liquid medium (total volume of medium 100 mL), sterilize at 115℃ for 20 min.

[0113] Experimental process:

[0114] (1) Resuscitate the strain: take 50μL of glycerol bacteria and inoculate into 1mL GAM liquid medium, anaerobic culture for 24h, activate 2 generations;

[0115] (2) The activated strain was inoculated into GAM liquid medium containing 2g / L uric acid at a 10% inoculation amount, and anaerobic culture was carried out for 24h (or sampling at 0h, 4h, 6h, 8h);

[0116] (3) Sample pretreatment: centrifuge the bacteria liquid cultured in (2) at 13000rpm for 5min, take 200μL supernatant and add 800μL sterilized ddH2O, vortex on vortex instrument for 10s, centrifuge at 13000rpm for 15min, take supernatant for HPLC detection;

[0117] (4) HPLC detection of uric acid metabolism ability:

[0118] Column: Waters Symmetry® C18 5um 4.6*250mm

[0119] Mobile phase A: deionized water containing 0.025% formic acid; mobile phase B: 0.025% formic acid in acetonitrile.

[0120] Detection wavelength: 288 nm, detection temperature: 15°C, injection volume: 2.0 μL

[0121] Gradient elution program: 0~2.5 min 6%B, 2.5~4 min 6~95%B, 4~5.5 min 95%B, 5.5~7 min 95~6%B, 7~9 min 6%B.

[0122] Experimental results:

[0123] (1) The ability of ibiome024 to metabolize uric acid after 24 hours of culture is shown in Figure 4, which shows that ibiome024 cultured for 24 hours can completely metabolize the uric acid in the culture medium;

[0124] (2) The curve of ibiome024 metabolizing uric acid during the culture process is shown in Figure 5, which shows that ibiome024 starts to decrease the uric acid in the culture medium from 4 hours of culture, and the uric acid in the culture medium is completely metabolized at 8 hours of culture.

[0125] It is shown that the new Clostridium sp. ibiome024 can metabolize uric acid. Example 3 In vivo verification of uric acid-lowering bacteria (2% uric acid + 4% potassium oxonate food-induced hyperuricemia model)

[0126] Experimental method:

[0127] C57BL / 6J (8 weeks, male) mice, SPF level, purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd. After one week of adaptation, the mice were randomly divided into 3 groups:

[0128] (1) Normal control group (control): normal maintenance feed was given, and 0.2 mL of 0.9% saline was given orally per mouse per day;

[0129] (2) High uric acid model mice (Model): given feed containing 2% uric acid and 4% potassium oxonate (research diet), 0.2 mL of 0.9% saline was given orally per mouse per day;

[0130] (3) Experimental group (ibiome024): given feed containing 2% uric acid and 4% potassium oxonate, 0.2 mL of 5*10 8 CFU / mL ibiome024 bacterial suspension was given orally per mouse per day.

[0131] After 4 weeks of administration, urine was taken, and orbital blood was taken, and serum was separated by centrifugation at 4000 rpm for 10 min, and the uric acid content in serum and urine was detected using a uric acid detection kit (brand: Solaybao, item number: BC1365).

[0132] Experimental results:

[0133] The main clinical symptom of hyperuricemia is the increase of blood uric acid content. As shown in FIG. 6, the uric acid content in the serum of the model group of mice modeled with potassium oxonate and uric acid food was significantly increased, and the intragastric administration of Clostridium sp. ibiome024 could reduce the uric acid content in the serum.

[0134] As shown in FIG. 7, the uric acid content in the urine of the model group of mice modeled with potassium oxonate and uric acid food was increased, and the intragastric administration of Clostridium sp. ibiome024 could reduce the uric acid content in the urine. Example 4 In vivo verification of strain for reducing uric acid (hyperuricemia model induced by yeast extract powder + intraperitoneal injection of potassium oxonate)

[0135] Experimental method:

[0136] C57BL / 6J (8 weeks, male) mice, SPF level, purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd. After the mice were adapted for one week, they were randomly divided into 3 groups:

[0137] (1) Normal control group (control): normal maintenance feed was given, and 0.2 mL of 0.9% saline was intragastrically administered per mouse per day;

[0138] (2) Hyperuricemia model mice (Model): fed with 10% yeast extract powder food and intraperitoneal injection of 250 mg / kg potassium oxonate per day, and 0.2 mL of 0.9% saline was intragastrically administered per mouse per day;

[0139] (3) Experimental group (ibiome024): fed with 10% yeast extract powder food and intraperitoneal injection of 250 mg / kg potassium oxonate per day, and 0.2 mL of 5*10 8 CFU / mL ibiome024 bacterial suspension per mouse per day was intragastrically administered.

[0140] After 4 weeks of administration, orbital blood was taken, and serum was separated by centrifugation at 4000 rpm for 10 min, and the uric acid content in serum was detected using a uric acid detection kit (brand: Solaybao, item number: BC1365).

[0141] Experimental results:

[0142] As shown in Figure 8, the serum uric acid content of the model group of mice injected with yeast extract and intraperitoneal injection of potassium oxonate was significantly increased, and the serum uric acid content of the mice administered with Clostridium sp. ibiome024 was reduced. Example 5 Uric acid-lowering gene verification

[0143] According to the literature report (https: / / doi.org / 10.1038 / s41564-025-02079-4), there are eight genes involved in the process of uric acid metabolism in anaerobic bacteria (xdhAC, ygfK, ssnA, hyuA, ygeW, ygeA, ygeY and ygeX). In order to verify whether the new species has the universal uric acid-lowering performance, specific primers were designed for each gene (Table 3), and the relevant genes were amplified by PCR.

[0144] Amplification system: 2 × Primerstar (R045Q; Takara) 10 μL, primer F 1 μL, primer R 1 μL, liquid cultured bacteria (ibiome024, Hungatella hathewayi strain 3, Hungatella hominis strain 3, Hungatella effluvii strain 3 or other species 6 strains) 1 μL, ddH2O 7 μL; amplification conditions: 98℃ 10 min; 98℃ 10 s, 56℃ 15 s, 72℃ 30 s, 30 cycles; 72℃ 5 min. The amplification system was subjected to agarose gel electrophoresis (1% agarose, 150 V, 20 min). The specific gel map is shown in Figure 9 (the wells are Marker, ibiome024, Hungatella hathewayi strain 3, Hungatella hominis strain 3, Hungatella effluvii strain 3, and other species 6 control strains, respectively). Among them, the 9 Hungatella strains were derived from the microbial strain library of Hefei Hanwei Microbial Technology Co., Ltd.

[0145] Table 3 Gene primers

[0146]

[0147] As shown in Fig. 9, the Clostridium sp. can amplify xdhAC, ygfK, ssnA, hyuA, ygeW, ygeA, ygeY and ygeX by using specific primers, respectively, and the bands of expected size can be amplified, while the other six control strains (belonging to Bifidobacterium longum subsp. Suillum, Parabacteroides merdae, Bifidobacterium adolescentis, Bifidobacterium pseudocatenulatum, respectively) cannot amplify the related bands or cannot amplify the single band of expected size, indicating that the Clostridium sp. is different from the other six control strains, and xdhAC, ygfK, ssnA, hyuA, ygeW, ygeA, ygeY and ygeX uric acid-reducing genes exist.

[0148] The above-described embodiments are merely intended to describe the preferred embodiments of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the claims of the present application.

Claims

1. A strain of Clostridium characterized by: The strain has 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.

2. The strain according to claim 1, characterized in that: The strain is a Clostridium sp. ibiome024 strain preserved under accession number CCTCC NO: M 20241964 or a progeny strain or a subclone strain thereof, which is preserved at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on September 13, 2024.

3. A medicament, characterized by: The strain of claim 1 or 2, or an extract, culture, or processed product of the strain, and a pharmaceutically acceptable excipient.

4. The medicament according to claim 3, characterized in that: The excipient includes at least one of an adjuvant, a stabilizer or protective agent, a bacteriostatic agent, an excipient, a solubilizing agent, a flavoring agent, a diluent, a buffer.

5. A pharmaceutical composition, characterized by: The strain of claim 1 or 2, or an extract, culture, or processed product of the strain, and a combination drug.

6. The pharmaceutical composition according to claim 5, characterized in that: The combination drug is another substance that synergizes with the strain.

7. A food, ferment, functional bacteria agent, or nutritional composition comprising the strain of claim 1 or 2, or an extract, culture, or processed product of the strain.

8. Use of the strain of claim 1 or 2, or an extract, culture, or processed product of the strain, or the drug of claim 3 or 4, or the pharmaceutical composition of claim 5 or 6 in the preparation of a drug for preventing, treating, or adjuvant treating hyperuricemia, gout, gouty arthritis, uric acid nephropathy and kidney stones, cardiovascular disease, and / or metabolic syndrome.

9. Use according to claim 8, characterized in that, The use includes at least one of the following: (1) reducing the urine uric acid level of a mammal; (2) reducing the fecal uric acid level of a mammal; (3) reducing the serum uric acid level of a mammal; (4) reducing the serum creatinine level of a mammal; (5) reducing the serum urea nitrogen level of a mammal; (6) reducing the liver xanthine oxidase activity and expression of a mammal; (7) reducing the kidney and intestinal pathological damage of a mammal; (8) reducing the expression of uric acid reabsorption protein GLUT9 in the kidney and colon of a mammal; (9) increasing the expression of uric acid transport protein ABCG2 in the kidney and colon of a mammal; (10) increasing the expression of intestinal tight junction protein in the colon of a mammal; (11) promoting the production of short-chain fatty acids in the intestine of a mammal.

10. Use of the strain of claim 1 or 2, or an extract, culture, or processed product of the strain, in the preparation of a food, ferment, functional bacteria agent, or nutritional composition for regulating the uric acid level of a mammal for non-therapeutic purposes.

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