A Bacillus that can degrade uric acid, a biological preparation and a preparation method thereof

Abstract

A uric acid-degrading Bacillus, a biological agent and its preparation method. The uric acid-degrading Bacillus is Bacillus sp. XRK-1, with the preservation number: CCTCC M 20232477, the preservation date: December 06, 2023, and the preservation unit: China Center for Type Culture Collection. The present invention also discloses a microbial agent produced by fermenting the Bacillus sp. XRK-1 strain and its preparation method. The uric acid-degrading Bacillus sp. XRK-1, isolated from sunflower discs, has the ability to produce urate oxidase to degrade urea, with the urate oxidase activity as high as 0.103 U / ml, and can efficiently degrade uric acid in vitro. Bacillus sp. XRK-1 grows rapidly, has strong stress resistance and high biological safety; the microbial agent prepared by its fermentation, as well as the preparation method of the microbial agent, can be used for reducing uric acid and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Bacillus capable of degrading uric acid in vitro, a biological preparation and a preparation method thereof. Background Art

[0002] Hyperuricemia is a disease of abnormal uric acid metabolism, and the main causes of the disease include two aspects: abnormal metabolism of purine substances in the body and abnormal excretion of uric acid. Due to the change of people's living conditions, the dietary structure has changed significantly, and foods with a high proportion of purines are getting closer to people's daily lives. With the emergence of a high-purine diet, the number of patients with hyperuricemia is increasing year by year. Hyperuricemia has become the "fourth high" disease, ranking alongside hypertension, hyperglycemia, and hyperlipidemia. According to statistics, about 10% of patients with hyperuricemia will further develop into gout.

[0003] 30% - 40% of the uric acid in the human body is excreted through the intestinal and biliary tracts. The uric acid in the intestine comes from blood, liver, food, etc. This part of uric acid can be decomposed by intestinal flora or directly excreted. The uric acid excreted through the intestine can be decomposed by urate oxidase produced by microorganisms in the large intestine into allantoin with high solubility (5 - 10 times that of uric acid). For example, a Lactobacillus plantarum strain producing urate oxidase and inhibiting xanthine oxidase and its application disclosed in CN114149947A, the Lactobacillus plantarum BLCC2-0296 can produce urate oxidase and has the ability to directly degrade uric acid; at the same time, it also has the inhibitory activity of xanthine oxidase. Intestinal microorganisms can also assist in reducing uric acid by directly intervening in the absorption of purine components in food. For example, a Bacillus coagulans strain, method and application with uric acid-lowering and antioxidant abilities derived from shrimp paste disclosed in CN114651983A, the Bacillus coagulans GH1-1 has strong purine metabolism and xanthine oxidase XOD inhibitory abilities. Some bacteria can colonize in the host intestine, degrade nucleosides into purine bases that are relatively more difficult to absorb in the intestine, reduce the absorption of nucleosides in the intestine, and thus reduce the serum uric acid content.

[0004] Most of the existing technologies use probiotics such as Lactobacillus casei, Lactobacillus gasseri, and Lactobacillus fermentum to biodegrade adenosine, guanosine, adenosine monophosphate, guanosine monophosphate, adenine, and guanine to achieve the purpose of reducing the production of uric acid. Lactobacillus has incomparable advantages over drug treatment in reducing uric acid synthesis and treating hyperuricemia, such as no drug adverse reactions, no need for additional dietary restrictions, high patient compliance, and alleviating inflammation during renal function injury. However, the stress resistance of Lactobacillus is poor and it is not conducive to preservation, which limits its curative effect in preventing and treating hyperuricemia.

[0005] Bacillus has the advantages of a broad antibacterial spectrum, rapid growth, strong stress resistance, and high biosafety. Therefore, screening for Bacillus strains that can degrade uric acid has important research significance and application value. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a Bacillus sp. XRK-1 with high uricase production and the ability to degrade uric acid, as well as a biological preparation fermented from Bacillus sp. XRK-1. The present invention also optimizes the culture conditions of Bacillus sp. XRK-1 and the preparation method of the biological preparation.

[0007] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0008] A Bacillus strain capable of degrading uric acid, the Bacillus strain is Bacillus sp. XRK-1, and the preservation number is: CCTCC M 20232477, the CCTCC preservation date: December 06, 2023, the preservation unit: China Center for Type Culture Collection.

[0009] 1) Morphological observation of the strain

[0010] The Bacillus strain was isolated from sunflower discs. The colonies of Bacillus sp. XRK-1 on the plate are white, convex, and nearly circular. Under Gram staining, they appear blue-violet, indicating that the strain is a Gram-positive bacterium. The colony morphology in the microscopic field of view is uniform, and the cell morphology presents as short rods, arranged singly.

[0011] 2) Molecular biological identification of the strain

[0012] The above-mentioned isolated and purified Bacillus strain was identified by 16S rRNA. The 16S rRNA sequence of the strain is shown in Seq ID NO: 1 and is named Bacillus sp. XRK-1. The measured 16S rRNA sequence was compared by NCBI BLAST, and the highest homology with Bacillaceae gen.sp. in Genebank is 96.39%. Based on the 16S rRNA gene sequence alignment results, a Neighbor-Joining phylogenetic tree was constructed with Oceanobacillus halotolerans strain YIM 98839 as the outgroup (see Appendix Figure 4 ), and the strain was preliminarily identified as Bacillaceaegen. XRK-1.

[0013] The above-mentioned Bacillus sp. XRK-1 has the ability to produce uricase to degrade uric acid and can efficiently degrade uric acid in vitro.

[0014] In a certain exemplary embodiment, the uricase enzyme activity produced by the above-mentioned Bacillus XRK-1 is as high as 0.103 U / ml.

[0015] A microbial preparation is prepared by fermenting and producing the above-mentioned uric acid-degrading Bacillus XRK-1.

[0016] The above-mentioned microbial preparation includes one or more of viable Bacillus XRK-1 cells, spores, inactivated Bacillus XRK-1 cells, metabolites, and postbiotics thereof.

[0017] A method for preparing a microbial preparation includes the following steps:

[0018] S1. Inoculate the fermentation strain into a fermentation medium for fermentation culture to obtain a fermentation culture of Bacillus; wherein, the fermentation strain is obtained by seed culture of Bacillus XRK-1 strain;

[0019] S2. Centrifuge and separate the fermentation culture of Bacillus to harvest the bacterial cells and / or spores of Bacillus.

[0020] The fermentation medium is composed of the following raw materials: 20 g / L of sucrose, 10 g / L of yeast extract, 17.1 g / L of disodium hydrogen phosphate, 3 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, 0.01 g / L of anhydrous calcium chloride, 2 g of uric acid, distilled water, pH 7.0, autoclaved at 121 °C for 25 min.

[0021] In S1, the temperature of the fermentation culture is 30-45 °C, preferably 37-42 °C.

[0022] Optionally, S2 of the preparation method further includes washing the bacterial cells and / or spores of Bacillus with physiological saline to obtain pure products of the bacterial cells and / or spores of Bacillus.

[0023] The method for preparing the microbial preparation further includes:

[0024] S3. Optionally, the supernatant after centrifugation of the fermentation culture of Bacillus is inactivated at high temperature (121 °C, 30 min), centrifuged, and concentrated to obtain metabolites;

[0025] S4. Optionally, the fermentation culture of Bacillus is subjected to cell lysis, inactivated at high temperature (121 °C, 30 min), and concentrated to obtain postbiotics containing Bacillus cells and their metabolites;

[0026] S5. Freeze-dry one or several of the bacterial cells, spores, metabolites, and postbiotics of Bacillus to obtain freeze-dried powder.

[0027] Application of a microbial preparation of the present invention in the preparation of anti-hyperuricemic drugs.

[0028] Advantages of the Bacillus capable of degrading uric acid in the present invention:

[0029] The Bacillus sp. XRK-1 capable of degrading uric acid in the present invention, isolated from sunflower discs, has the ability to produce urate oxidase to degrade uric acid. The enzyme activity of urate oxidase is as high as 0.103 U / ml, and it can efficiently degrade uric acid in vitro. Bacillus sp. XRK-1 grows rapidly, has strong stress resistance and high biosafety.

[0030] The present invention also discloses a microbial preparation prepared by fermenting Bacillus sp. XRK-1, and a preparation method of the microbial preparation. The microbial preparation can be used for reducing uric acid and has broad application prospects.

[0031] Biological material preservation

[0032] Bacillus sp. XRK-1 was deposited at the China Center for Type Culture Collection on December 06, 2023, with the deposit number CCTCC M 20232477, and the deposit address is: China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Brief description of the drawings

[0033] Figure 1 —A plate diagram of the isolation and screening of Bacillus sp. XRK-1 capable of degrading uric acid in the present invention;

[0034] Figure 2 —A colony morphology diagram of Bacillus sp. XRK-1 capable of degrading uric acid in the present invention;

[0035] Figure 3 —A microscopic examination result diagram of Gram staining of Bacillus sp. XRK-1 capable of degrading uric acid in the present invention;

[0036] Figure 4 —A phylogenetic tree of Bacillus sp. XRK-1 capable of degrading uric acid in the present invention;

[0037] Figure 5 —A biomass-time change diagram of Bacillus sp. XRK-1 capable of degrading uric acid in the present invention;

[0038] Figure 6 —A standard curve diagram for the determination of urate oxidase enzyme activity;

[0039] Figure 7 —A comparison diagram of the urate oxidase enzyme activities secreted by Bacillus sp. XRK-1 capable of degrading uric acid in the present invention under different fermentation media. Detailed implementation manners

[0040] The present invention will be further described below with reference to the drawings and embodiments.

[0041] Term

[0042] In the present invention, the term "bacterial cells" refers to live and / or dead cells of bacteria.

[0043] In the present invention, the term "spore" refers to a highly stress-resistant dormant body formed by Bacillus under certain conditions.

[0044] In the present invention, the term "microbial preparation" refers to preparations in various forms that are made from microorganisms with medical research value using traditional or modern biotechnology and act on the prevention (health care), treatment, and diagnosis of various physiological symptoms in the human body.

[0045] In the invention, the "water" used in the culture medium or fermentation culture process, without special designation, refers to sterile pure water obtained by filtration through a filter membrane (0.22 μm).

[0046] The following is the culture medium involved in the examples of the present invention:

[0047] Enrichment medium: 17.1 g of disodium hydrogen phosphate, 3 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.01 g of anhydrous calcium chloride, 2 g of uric acid, 1 L of distilled water, pH 7.5, autoclaved at 121 °C for 25 min.

[0048] Uric acid selection medium (liquid): 17.1 g of disodium hydrogen phosphate, 3 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.01 g of anhydrous calcium chloride, 2 g of uric acid, 1 L of distilled water, pH 7.5, autoclaved at 121 °C for 25 min.

[0049] Uric acid selection medium (plate): 17.1 g of disodium hydrogen phosphate, 3 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.01 g of anhydrous calcium chloride, 2 g of uric acid, 12 g of agar, 1 L of distilled water, pH 7.5, autoclaved at 121 °C for 25 min.

[0050] Example 1

[0051] Reference Figures 1 to 3 , a Bacillus sp. XRK-1 capable of degrading uric acid, with the preservation number: CCTCC M 20232477, preservation date: December 06, 2023, preservation unit: China Center for Type Culture Collection.

[0052] 1) Isolation of the strain

[0053] Take 10g of sunflower disc, put it into a non-woven filter bag, inoculate it into 50mL of enrichment medium with uric acid (uric acid content is 2g / L) as the only carbon source, and place it in a constant temperature incubator (30℃, 180r / min) for shaking culture for 3-5 days to prepare for strain screening. Dilute the enriched bacterial solution to 10 -1 / 10 -2 Times, take 100μL of bacterial solution and spread it on the uric acid selection medium (plate), invert and culture at 30℃ for 24h-48h, and observe the growth of colonies at any time. After the colonies are formed, put the plate inverted in a 4℃ refrigerator, and observe the growth of colonies and the size of transparent circles at any time during the culture process (whether there is a transparent circle around the colony and the size of the transparent circle can determine whether the strain produces uric acid oxidase and the strength of the uric acid oxidase activity). If the transparent circle is Figure 1 shown.

[0054] 2) Purification of strains

[0055] Pick several single colonies from the above plate, try to ensure that the selected single colonies have different morphology and transparent circles, and inoculate the selected single colonies on uric acid selection medium for preliminary screening of enzyme-producing strains. Then inoculate the isolated single colonies on uric acid selection medium, repeat streaking for multiple times, and purify the enzyme-producing strains. Figure 2 shown.

[0056] 3) Morphological observation of strains

[0057] Select the colonies on the above plate, make a slide, perform Gram staining, and observe the colony morphology under a microscope. The Gram-positive microscopic examination results are as follows: Figure 3 As shown, the strain was blue-purple under Gram staining, indicating that the strain was a Gram-positive bacterium, the colony morphology was uniform, short rod-shaped, and the arrangement morphology was a single arrangement.

[0058] 4) Molecular biological identification of strains

[0059] The above strains were subjected to 16S rRNA sequencing, and the 16S rRNA sequence of the strain is shown in Seq ID NO: 1. The comparison result on NCBI was Bacillus (Bacillaceae), and the measured sequence was uploaded to GenBank with the number: OR294314.

[0060]

[0061]

[0062] Among them, the 16S rRNA of the strain was amplified using universal primers for bacteria:

[0063] Upstream primer 27F: 5-AGAGTTTGATCCTGGCTCAG-3

[0064] Downstream primer 1492R: 5-CTACGGCTACCTTGTTACGA-3

[0065] The PCR system is shown in Table 1 below.

[0066] Table 1 PCR system

[0067]

[0068] The PCR amplification program is shown in Table 2 below.

[0069] Table 2 PCR amplification program

[0070]

[0071] Take the PCR product after strain purification and perform sequencing. Align the measured 16S rRNA sequence with NCBI BLAST. The highest homology with Bacillaceae gen.sp. in Genebank is 96.39%. Based on the 16S rRNA gene sequence alignment results, a Neighbor-Joining phylogenetic tree constructed with Oceanobacillus halotolerans strain YIM 98839 as the outgroup is as attached Figure 4 It is preliminarily identified that this strain is Bacillus sp. XRK-1.

[0072] Example 2

[0073] Optimization of the fermentation culture conditions of Bacillus sp. XRK-1

[0074] (1) Effect of additional carbon sources on the uric acid degradation ability of the strain

[0075] On the basis of the uric acid selection medium (plate), add additional carbon sources (glucose, sucrose, raffinose, citric acid, oil) with a concentration of 2%, and detect the effect of adding different additional carbon sources on the uric acid degradation ability of the strain. The detection index is the ratio of the size of the clear zone to the size of the colony.

[0076] (2) Effect of additional nitrogen sources on the uric acid degradation ability of the strain

[0077] On the basis of the uric acid selection medium (plate), add additional nitrogen sources (peptone, yeast extract, potassium nitrate, ammonium nitrate, ammonium sulfate) with a concentration of 1%, and detect the effect of adding different additional nitrogen sources on the uric acid degradation ability of the strain. The detection index is the ratio of the size of the clear zone to the size of the colony.

[0078] (3) Effect of pH on the uric acid degradation ability of the strain

[0079] Based on the uric acid selective medium (plate), different pH values (6, 6.5, 7, 7.5, 8) were set to detect the effect of different pH values on the uric acid degradation ability of the strain. The detection index was the ratio of the size of the transparent circle to the size of the colony.

[0080] Table 3 Effects of different culture factors on the uric acid-lowering ability of Bacillus sp. XRK-1

[0081]

[0082] From the experimental results in Table 3, the top three carbon sources for improving the uric acid degradation ability of the strain were glucose, sucrose, and raffinose. The top three nitrogen sources for improving the uric acid degradation ability of the strain were peptone, yeast extract, and potassium nitrate. The top three pH values for improving the uric acid degradation ability of the strain were 6, 7, and 7.5.

[0083] (4) Optimizing the best culture conditions by orthogonal experiment

[0084] Based on the results of the single-factor experiment, the carbon source, nitrogen source, and pH that had a greater impact on the cultured strain were selected for a three-factor and three-level orthogonal optimization experiment. The experimental groups were designed according to the factor distribution shown in Table 4 on the uric acid selective medium (plate) medium, where the addition concentration of the additional carbon source was 2%, and the addition concentration of the additional nitrogen source was 1%. The diameter of the transparent circle and the diameter of the colony were measured by the cross-cross method, and the ratio of the diameter of the transparent circle to the diameter of the colony circle was calculated to select the best culture conditions.

[0085] Table 4 Orthogonal experiment factor table

[0086]

[0087] The experimental results in Table 5 showed that: Bacillus sp. XRK-1 had the highest efficiency in decomposing uric acid under the conditions of adding sucrose, yeast extract, and pH 7, and the ratio of the diameter of the transparent circle to the diameter of the colony was 6.11. From the range analysis, the pH had the greatest impact on the strain, and pH > additional nitrogen source > additional carbon source. Therefore, the fermentation medium of Bacillus sp. XRK-1 was screened as: sucrose 20 g, yeast extract 10 g, disodium hydrogen phosphate 17.1 g, potassium dihydrogen phosphate 3 g, magnesium sulfate 0.5 g, anhydrous calcium chloride 0.01 g, uric acid 2 g, distilled water 1 L, pH 7.0.

[0088] Table 5 Analysis of the experimental results of the effects of the components and pH of the strain fermentation medium on the uric acid-lowering ability

[0089]

[0090]

[0091] (5) Growth curve of XRK-1 under optimal culture conditions.

[0092] From the above experimental results, it can be seen that XRK-1 has the highest efficiency in decomposing uric acid on a uric acid selection medium (liquid) supplemented with 2% sucrose, 1% yeast extract, and pH 7.0. Prepare the fermentation medium with 20 g of sucrose, 10 g of yeast extract, 17.1 g of disodium hydrogen phosphate, 3 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.01 g of anhydrous calcium chloride, 2 g of uric acid, 1 L of distilled water, and pH 7.0. Then inoculate Bacillus XRK-1 at 5% of the medium, and then place it in a constant temperature shaking incubator (37 °C, 180 r / min) and shake culture for 24 h. Take the fermentation broth every 1 h and centrifuge it, and then measure the absorbance at 600 nm with a spectrophotometer. According to OD 600 Calculate the content of Bacillus XRK-1, and the results are as Figure 5 shown. From Figure 5 it can be seen that the strain Bacillus XRK-1 grows rapidly in the early stage, and the growth gradually slows down after 15 h. The content of Bacillus XRK-1 reaches the highest after 24 h of fermentation.

[0093] (6) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the uricase enzyme activity produced by the strain Bacillus XRK-1 in the uric acid selection medium and the fermentation medium, respectively.

[0094] Inoculate single colonies into 100 mL of uric acid selection medium (control group) and fermentation medium (optimized group) at an inoculation amount of 2%, and place them in a constant temperature shaking incubator (37 °C, 180 r / min) and shake culture for 36 h. Take 1.0 mL of the culture broth, centrifuge it, and transfer the supernatant to a new EP tube (5500×g, 10 min, 4 °C). Add 900 μL of PBS to the cell pellet and sonicate it on ice, and then take the supernatant for standby after centrifugation.

[0095] Prepare uric acid standard solutions with concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mmol, and measure the absorbance at 300 nm to make a UA standard curve, as Figure 6 shown. Under standard conditions, the sample amount that consumes 1.0 μmol of uric acid per minute is one enzyme activity unit.

[0096] Sample enzyme activity determination: Take 40 μL of the supernatant of the strain culture broth after fermentation in the uric acid selection medium (control group) and the fermentation medium (optimized group), or the supernatant of the strain lysate, and 160 μL of a 0.5 mmol uric acid solution prepared with borate buffer (5 mmol HBO, pH 8.5). In a standard 96-well plate, at 37 °C, 300 nm (AU300), aerobic for 60 min, monitor the reduction of uric acid, and compare Figure 6 , through OD 300Absorbance readings determine uric acid concentration.

[0097] See Figure 7 , the enzyme activity produced by Bacillus XRK-1 cultured in uric acid selection medium (control group) was 0.047 U / ml; the uricase activity produced by the strain of Bacillus XRK-1 cultured in the fermentation medium optimized by the present inventors (optimized group) was 0.103 U / ml. Therefore, the optimized fermentation medium can significantly improve the enzyme activity of uricase produced by Bacillus XRK-1.

[0098] In summary, Bacillus XRK-1 of the present invention has the ability to produce high-yield uricase, and thus can efficiently degrade urine. The microbial preparation prepared by fermenting Bacillus XRK-1 can be used for reducing uric acid, and the application of Bacillus XRK-1 in the preparation of drugs for reducing uric acid has great potential in the preparation of drugs for relieving hyperuricemia, preventing and treating gout, anti-inflammation, improving intestinal flora and other diseases.

[0099] Example 3

[0100] A microbial preparation is prepared by fermenting Bacillus XRK-1 capable of degrading uric acid in Example 1.

[0101] The microbial preparation of this example is a live powder of Bacillus XRK-1. The live powder of Bacillus XRK-1 is prepared by fermenting Bacillus XRK-1 in the fermentation medium obtained in Example 2. Its preparation method includes the following steps:

[0102] S1. Inoculate the fermentation strain into the fermentation medium and ferment at 37°C for 24 h to obtain a fermentation culture of Bacillus; wherein, the fermentation strain is obtained by seed culture of Bacillus XRK-1 strain;

[0103] S2. Centrifuge and separate the fermentation culture of Bacillus to harvest the bacterial cells and / or spores of Bacillus;

[0104] S3. Wash the bacterial cells and / or spores of Bacillus obtained in S2 with physiological saline to obtain pure products of the bacterial cells and / or spores of Bacillus, and then freeze-dry to obtain a freeze-dried powder, which is the live powder of Bacillus XRK-1.

[0105] Based on the ability of the live powder of Bacillus XRK-1 of this example to degrade uric acid in vitro, the applicant also proposes the application of the microbial preparation of this example in the preparation of drugs for reducing uric acid.

[0106] Example 4

[0107] A microbial preparation is prepared by fermenting Bacillus XRK-1 capable of degrading uric acid in Example 1.

[0108] The microbial preparation of this example is the postbiotic of Bacillus XRK-1. The postbiotic of Bacillus XRK-1 is prepared by fermenting Bacillus XRK-1 in the fermentation medium obtained in Example 2. Its preparation method includes the following steps:

[0109] S1. Inoculate the fermentation strain into the fermentation medium and ferment at 37°C for 24 h to obtain the fermentation culture of Bacillus. Among them, the fermentation strain is obtained by seed culture of Bacillus XRK-1 strain;

[0110] S2. Subject the fermentation culture of Bacillus XRK-1 to cell lysis, high-temperature inactivation (121°C, 30 min), and concentration to obtain the postbiotic containing Bacillus cells and their metabolites.

[0111] Based on the ability of the Bacillus XRK-1 cells of this example to degrade uric acid in vitro, the applicant also proposes the application of the microbial preparation of this example in the preparation of anti-hyperuricemic drugs.

[0112] Example 5

[0113] A microbial preparation is prepared by fermenting Bacillus XRK-1 capable of degrading uric acid in Example 1.

[0114] The microbial preparation of this example is a composition of the live bacteria powder of Bacillus XRK-1 and metabolites. The microbial preparation is prepared by fermenting Bacillus XRK-1 in the fermentation medium obtained in Example 2. Its preparation method includes the following steps:

[0115] S1. Inoculate the fermentation strain into the fermentation medium and ferment at 37°C for 24 h to obtain the fermentation culture of Bacillus. Among them, the fermentation strain is obtained by seed culture of Bacillus XRK-1 strain;

[0116] S2. Centrifuge and separate the fermentation culture of Bacillus, and the centrifugal precipitate is the bacterial cells and / or spores of Bacillus;

[0117] S3. Centrifuge the supernatant obtained from the fermentation culture of Bacillus in step S2, inactivate it at high temperature (121°C, 30 min), centrifuge, and concentrate to obtain metabolites;

[0118] S4. Freeze-dry the mixture of the bacterial cells / spores of Bacillus obtained in S2 and the metabolites obtained in S3 to obtain the freeze-dried powder, which is the microbial preparation of this application.

[0119] Based on the ability of Bacillus XRK-1 cells in this embodiment to degrade uric acid in vitro, the applicant also proposed the application of the microbial preparation in this embodiment in the preparation of anti-hyperuricemic drugs.

[0120] The microbial preparation prepared by fermenting a uric acid-degradable Bacillus in the present invention may also be a composition obtained by mixing the viable cell powder of Bacillus XRK-1 and postbiotics in a weight ratio of 2:1, or may be a composition obtained by mixing the spores of Bacillus XRK-1 and metabolites in a weight ratio of 3:1. The composition of the microbial preparation can adjust the ratio between the components according to the application. With the changes in the above technical features, those skilled in the art can understand and implement them through the written description, so no additional drawings will be provided for illustration.

Claims

1. A bacillus capable of degrading uric acid, characterized in that: The bacillus is Bacillus XRK-1 ( Bacillus sp. XRK-1), the deposit number is: CCTCC M 20232477, the deposit date is: December 6, 2023, the deposit unit is: China Center for Type Culture Collection, the Bacillus is isolated from a sunflower disk, the 16SrRNA sequence of the Bacillus XRK-1 is shown in Seq ID NO: 1; the urate oxidase activity produced by the Bacillus XRK-1 after fermentation for 36 hours is as high as 0.103U / ml.

2. A microbial preparation, characterized in that: The uric acid-degradable bacillus is fermented and produced according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The microbial preparation comprises one or more of live bacteria and spores of Bacillus XRK-1.

4. A method for preparing a microbial preparation, characterized in that: The preparation method of the microbial preparation comprises the following steps: S1. Inoculating the fermentation strain into a fermentation medium for fermentation culture to obtain a fermentation culture of Bacillus; wherein the fermentation strain is obtained by seed culture of the Bacillus XRK-1 strain according to claim 1; S2. Centrifuge the fermentation culture of Bacillus to harvest bacterial cells and / or spores of Bacillus.

5. The method for preparing the microbial preparation according to claim 4, characterized in that: S2 also includes washing the bacterial cells and / or spores of Bacillus with physiological saline to obtain pure bacterial cells and / or spores of Bacillus.

6. The method for preparing the microbial preparation according to claim 4, characterized in that: The preparation method of the microbial preparation also includes the following steps: S3. Freeze-dried powder obtained by freeze-drying one or more of the bacterial cells and spores of Bacillus.

7. The method for preparing the microbial preparation according to claim 4, characterized in that: In S1, the fermentation culture temperature is 30-45°C, and the fermentation medium is composed of the following raw materials: 20 g sucrose, 10 g yeast extract, 17.1 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.01 g / L anhydrous calcium chloride, 2 g uric acid, distilled water, pH 7.0, and high pressure sterilization at 121°C for 25 min.

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