A fermentation preparation with uric acid-lowering function, its preparation method and application

Abstract

A fermentation preparation with uric acid-lowering function, its preparation method and application. The fermentation preparation is prepared by fermenting Bacillus sp. XRK-1 on a traditional Chinese medicine fermentation substrate. The traditional Chinese medicine fermentation substrate includes the following components in parts by weight: 90-100 parts of purified water, 2-4 parts of Smilax glabra, 2-4 parts of sunflower disc, 3-5 parts of Pueraria lobata, 3-5 parts of Poria cocos, 1-3 parts of edible sucrose and 1-3 parts of edible yeast powder. The present invention also discloses the preparation method of the fermentation preparation and its application in the preparation of drugs for preventing and treating hyperuricemia-related diseases. The traditional Chinese medicines in the fermentation preparation are used as fermentation substrates to promote the growth of Bacillus sp. XRK-1 and its secretion of uricase; Smilax glabra and Pueraria lobata can effectively inhibit the activity of xanthine oxidase, and the alkaloids in the sunflower disc can dissolve uric acid stones, and Poria cocos promotes urination. At the same time, the fermentation process of Bacillus sp. XRK-1 can effectively promote the overflow of active ingredients in traditional Chinese medicines. Microorganisms and traditional Chinese medicines promote each other and complement each other, and then synergistically and efficiently degrade uric acid and reduce the uric acid level in the body.

Description

Technical Field

[0001] The present invention relates to the technical field of biological agents, and in particular to a fermentation preparation with the function of reducing uric acid, its preparation method and application. Background Art

[0002] In recent years, using food materials homologous to medicine and food to condition hyperuricemia has been a highly recommended method. The traditional Chinese medicine formula has obvious effects and few adverse reactions, and the prices of Chinese herbal medicines are reasonable, and the acceptance rate of patients is high. In recent years, studies have confirmed that traditional Chinese medicine in the treatment of gout has the characteristics of multi-pathway, multi-level and multi-target, and the curative effect is exact. However, the disadvantages of traditional Chinese medicine decoction, such as cumbersome process, inconvenient to carry, bad smell and taste, and low bioavailability, limit the compliance of patients. Most of the current related studies focus on the efficacy of the biochemical components of the food materials themselves, while the efficacy of the symbiotic bacteria of the food materials is ignored.

[0003] The present invention combines probiotics with Chinese medicinal materials homologous to medicine and food, adopts a microbial fermentation process, and utilizes the characteristics that probiotics 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. It combines with the advantages of traditional Chinese medicine preparations, making it have the advantages of improved bioavailability of active ingredients, good treatment effect, better taste, convenient use, and high compliance rate of patients. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: to overcome the deficiencies of the prior art, to provide a fermentation preparation with the function of reducing uric acid, which has simple components, and each component can synergistically promote uric acid degradation and excretion, and has a significant uric acid reduction effect, and a preparation method of the fermentation preparation, and the application of the fermentation preparation with the function of reducing uric acid in the preparation of drugs for preventing and treating diseases related to hyperuricemia.

[0005] One of the technical solutions adopted by the present invention to solve its technical problems is:

[0006] A fermentation preparation with the function of reducing uric acid is prepared by fermenting Bacillus sp. XRK-1 on a traditional Chinese medicine fermentation substrate. The traditional Chinese medicine fermentation substrate includes the following components in parts by weight: 90-100 parts of purified water, 2-4 parts of Smilax glabra, 2-4 parts of sunflower disc, 3-5 parts of Pueraria lobata, 3-5 parts of Poria cocos, 1-3 parts of edible sucrose, and 1-3 parts of edible yeast powder.

[0007] For the fermentation preparation with the function of reducing uric acid of the present invention, the efficacy and active ingredients of each traditional Chinese medicine fermentation substrate:

[0008] Smilax glabra: can inhibit the activity of xanthine oxidase, reduce the level of blood uric acid, and at the same time has a diuretic effect, and can reduce the uric acid concentration in the body by promoting uric acid excretion.

[0009] Sunflower flower head: It can lower blood sugar and blood pressure, treat headache, dizziness, and tinnitus, clear heat and detoxify, cool blood and stop bleeding, and resolve phlegm and relieve cough. The sunflower flower head is rich in alkaloids, which can specifically bind to uric acid to form a sunflower alkali-uric acid complex. After being metabolized by the liver, it is excreted from the body through the kidneys, quickly reducing uric acid and having a therapeutic effect on gout.

[0010] Kudzu root: It can promote eruption and relieve diarrhea, remove vexation and quench thirst, treat typhoid fever, warm febrile disease, headache and stiff neck, vexation and thirst, diarrhea, dysentery, unsmooth eruption of maculae, hypertension, angina pectoris, and deafness. Puerarin in kudzu root can effectively reduce the serum uric acid level of HUA rats by inhibiting the activity of xanthine oxidase, a key enzyme in purine metabolism, and promoting its excretion by increasing the solubility of uric acid in urine.

[0011] Poria cocos: It can promote diuresis and percolate dampness, strengthen the spleen, and treat dysuria, edema and fullness, cough and counterflow of phlegm-fluid, vomiting and retching, diarrhea, stranguria and turbid urine, palpitation, and amnesia.

[0012] When Bacillus sp. XRK-1 is fermented and cultured on a fermentation substrate composed of four traditional Chinese medicines, namely Smilax glabra Roxb., sunflower flower head, kudzu root, and Poria cocos, it can efficiently promote the overflow of highly effective active ingredients with high medicinal value in traditional Chinese medicines, such as glycosides, saponins, alkaloids, volatile oils of organic acids, trace elements, polysaccharides, and small peptides, significantly improving the bioavailability of traditional Chinese medicines, and at the same time promoting the absorption and utilization of active ingredients of traditional Chinese medicines by the body; at the same time, the nutritional components in traditional Chinese medicines promote the growth of Bacillus sp. XRK-1 and promote its secretion of urate oxidase. The synergistic effect of Bacillus sp. and traditional Chinese medicines improves the uric acid-lowering efficacy of the fermentation preparation of the present application.

[0013] Preferably, the viable count of Bacillus sp. XRK-1 in the fermentation preparation ≥ 1.0×10 7 cfu / ml, or the viable count of Bacillus sp. XRK-1 in the fermentation preparation ≥ 1.0×10 7 cfu / g. Preferably, the viable count of Bacillus sp. XRK-1 in the fermentation preparation is 1.0 - 5.0×10 7 cfu / ml.

[0014] The preservation number of the Bacillus sp. XRK-1 is: CCTCC NO: M 20232477, the preservation date is December 06, 2023, and the preservation unit is China Center for Type Culture Collection.

[0015] The Bacillus sp. is isolated from the sunflower disk. The 16S rDNA sequence of the Bacillus sp. XRK-1 is shown in Seq ID NO: 1 and has been uploaded to the public GenBank with the accession number: OR294314.

[0016] In a certain exemplary embodiment, the fermentation preparation further comprises a flavoring agent, and the flavoring agent comprises the following components in parts by weight: 4-5 parts of apple concentrated juice and 4-5 parts of strawberry concentrated juice.

[0017] In a certain exemplary embodiment, before the preparation of the traditional Chinese medicine fermentation substrate, the smilax glabra, sunflower disc, kudzu root and poria cocos are first pulverized into ultrafine powder, and the particle size thereof is 25-48 μm.

[0018] Preferably, the conditions for the fermentation production are as follows: the inoculation amount of bacillus XRK-1 is 1-5% (W / W) of the traditional Chinese medicine fermentation substrate, and the culture is carried out at 42-45 °C for 24-30 h.

[0019] The fermentation preparation is a fermentation solution obtained by filtering macromolecular substances from the fermentation culture solution, or a solid powder obtained by concentrating and then drying the fermentation solution obtained by filtering macromolecular substances from the fermentation culture solution. Preferably, freeze-drying is adopted for drying, which can better retain the activity of bacillus XRK-1 cells and the active ingredients of traditional Chinese medicine in the fermentation solution.

[0020] Another technical solution adopted by the present invention to solve its technical problems is:

[0021] A preparation method of a fermentation preparation with uric acid lowering function comprises the following steps:

[0022] S1. Preparation of traditional Chinese medicine fermentation substrate: Mix the smilax glabra, sunflower disc, kudzu root and poria cocos powder with edible yeast powder, sucrose and pure water according to the ratio and stir evenly to obtain a mixed slurry;

[0023] S2. High-temperature sterilize the mixed slurry prepared in S1 to obtain a sterilized solution;

[0024] S3. Preparation of fermentation seed liquid: Inoculate bacillus XRK-1 into an enriched liquid medium at an inoculation amount of 1%, and culture at 37 °C for 18-20 h to obtain a fermentation seed liquid;

[0025] S4. Inoculate the fermentation seed liquid prepared in S3 into the sterilized solution prepared in S2 at an inoculation amount of 1-5%, and carry out constant-temperature fermentation culture at 42-45 °C for 24-30 h to obtain a fermentation stock solution;

[0026] S5. Purification: Filter the undigested drug residues from the fermentation stock solution prepared in S4 to obtain a crude liquid fermentation preparation.

[0027] In S1, the smilax glabra, sunflower disc, kudzu root and poria cocos powder are ultrafine powder with a particle size of 25-48 μm obtained by first pulverizing the smilax glabra, sunflower disc, kudzu root and poria cocos with a pulverizing and stirring blade and then sieving. The stirring time is ≥20 min, and preferably the stirring time is 20-25 min.

[0028] In S2, the high-temperature sterilization means transporting the mixed slurry to a sterilization container, performing high-pressure sterilization at 121°C for 30 minutes, and then cooling to room temperature or 35°C for standby.

[0029] In a certain exemplary embodiment, the preparation method of the fermentation preparation with uric acid-lowering function further includes:

[0030] S6, Flavoring: Add 4 - 5 parts of apple concentrated juice and 4 - 5 parts of strawberry concentrated juice to every 100 - 124 parts by weight of the liquid fermentation preparation crude product, and then mix evenly to obtain a high-quality liquid fermentation preparation with excellent taste, thereby improving the taste of the fermentation preparation and the compliance of patients.

[0031] In a certain exemplary embodiment, the preparation method of the fermentation preparation with uric acid-lowering function further includes:

[0032] S7, Concentrate and dry the high-quality liquid fermentation preparation prepared in S6 to obtain a solid powder-like fermentation preparation. In addition, the solid powder-like fermentation preparation can be further tabletted or granulated to prepare a tablet, granule or capsule fermentation preparation.

[0033] Another technical solution adopted by the present invention to solve its technical problems is:

[0034] Use of a fermentation preparation with uric acid-lowering function in the preparation of a drug for preventing and treating diseases related to hyperuricemia.

[0035] The diseases related to hyperuricemia include gout, uric acid stones, acute and chronic uric acid nephropathy.

[0036] The drug is an oral drug, ointment or patch, and the oral drug is any one of live bacteria oral liquid, solid beverage granules, tablets, granules or capsules.

[0037] Advantages of a fermentation preparation with uric acid-lowering function according to the present invention:

[0038] The fermentation preparation adopts a preparation method of co-fermenting probiotics with Chinese medicinal materials homologous to medicine and food, enabling the probiotics to reach the maximum abundance, while promoting the overflow of active ingredients in the Chinese medicinal materials, providing the bioavailability of the drug, and maximizing the therapeutic effect of the drug.

[0039] The nutritional components of the Chinese medicinal materials in this fermentation preparation can promote the growth of Bacillus sp. XRK-1 and its secretion of uricase, facilitating the conversion of uric acid to allantoin and its excretion; Smilax glabra and Pueraria lobata can effectively inhibit the activity of xanthine oxidase, a key enzyme in purine metabolism, and at the same time promote its excretion by increasing the solubility of uric acid in urine; the alkaloids in sunflower heads can dissolve uric acid stones and reduce blood uric acid levels, and Poria cocos promotes diuresis. The components in the fermentation preparation promote the reduction of the body's uric acid level synergistically through different uric acid-lowering pathways. At the same time, the fermentation process of Bacillus sp. XRK-1 can effectively promote the overflow of active ingredients in Chinese medicinal materials. The microorganism and Chinese medicinal materials promote each other and complement each other, and then synergistically and efficiently degrade uric acid and reduce the body's uric acid level.

[0040] Advantages of the preparation method of a fermentation preparation with uric acid-lowering function of the present invention:

[0041] The preparation method of the fermentation preparation with uric acid-lowering function is simple to operate and convenient for large-scale production and application. It uses microbial fermentation to promote the overflow of active ingredients in Chinese medicinal materials. Compared with the existing extraction methods of active ingredients in Chinese medicinal materials, its production conditions are mild, without the need to add too many chemical agents, effectively retaining the efficacy of the active ingredients in Chinese medicinal materials and improving their bioavailability; this application proposes to combine probiotics with Chinese medicinal materials and adopt a microbial fermentation process to improve the drawbacks of traditional Chinese medicine preparations, improve the taste of the medicine, have a wide range of dosage forms, are easy to use, and have high patient compliance.

[0042] Biological material preservation

[0043] Bacillus sp. XRK-1 was deposited at the China Center for Type Culture Collection on December 06, 2023, with the deposit number CCTCC NO: M 20232477, and the deposit address is: China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Description of the drawings

[0044] Figure 1 — Colony morphology diagram (plate) of Bacillus sp. XRK-1 in a fermentation preparation with uric acid-lowering function of the present invention;

[0045] Figure 2 — Microscopic diagram of Bacillus sp. XRK-1 in a fermentation preparation with uric acid-lowering function of the present invention;

[0046] Figure 3 — Phylogenetic tree of Bacillus sp. XRK-1 that can degrade uric acid of the present invention;

[0047] Figure 4— Comparative analysis chart of urate oxidase activity in the supernatant and cell lysate of Bacillus sp. XRK-1 capable of degrading uric acid of the present invention during fermentation in enrichment medium and fermentation medium (optimized);

[0048] Figure 5 — Comparative analysis chart of the uric acid regulation of a fermentation preparation with uric acid-lowering function, benzbromarone group, and traditional Chinese medicine fermentation substrate for hyperuricemic mice of the present invention. Detailed implementation manners

[0049] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0050] Terms

[0051] The term "bacterial cells" as used in the present invention refers to live and / or dead cells of bacteria.

[0052] The term "spore" as used in the present invention refers to a highly stress-resistant dormant body formed by Bacillus under certain conditions.

[0053] The term "microbial preparation" as used in the present invention refers to preparations in various forms that use microorganisms with medical research value as raw materials, are manufactured using traditional or modern biotechnology, and act on the prevention (health care), treatment, and diagnosis of various physiological symptoms of the human body.

[0054] The "water" used in the present invention for the culture medium or fermentation culture process refers to sterile pure water obtained by filtering through a filter membrane (0.22 μm) unless otherwise specified.

[0055] The following are the culture media involved in the examples of the present invention:

[0056] Enrichment solid plate medium: 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, agar 12 g, distilled water 1 L, pH 7.0, autoclaved at 121 °C for 25 min.

[0057] Enrichment liquid medium: 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 7.5, autoclaved at 121 °C for 25 min.

[0058] Fermentation medium (optimized): 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, autoclaved at 121 °C for 25 min.

[0059] The preparation method of apple concentrated juice is as follows: select and trim the raw materials (apples), soak them briefly in boiling water at 100°C, and remove the epidermis with a brush peeling machine. Then, use a core-removing machine to remove the cores, dice them with a dicing machine, pre-cook for 5 minutes, convey them to a pulping machine to make pulp, extract the juice with a scroll press, remove oxygen with a vacuum machine at the same time, use a two-effect vacuum evaporator to concentrate the material to 36-40%, and then perform high-temperature sterilization. After cooling, apple concentrated juice is obtained and reserved for use.

[0060] The preparation method of strawberry concentrated juice is as follows: select and trim the raw materials (strawberries), soak them briefly in clean water, then dice them with a dicing machine, pre-cook for 5 minutes, convey them to a pulping machine to make pulp, extract the juice with a scroll press, remove oxygen with a vacuum machine at the same time, use a two-effect vacuum evaporator to concentrate the material to 36-40%, and then perform high-temperature sterilization. After cooling, strawberry concentrated juice is obtained and reserved for use.

[0061] Example 1

[0062] Reference Figures 1 to 3 A fermentation preparation with the function of reducing uric acid is prepared by fermenting Bacillus XRK-1 on a traditional Chinese medicine fermentation substrate. The traditional Chinese medicine fermentation substrate includes the following components in parts by weight: 100 parts of purified water, 2 parts of Smilax glabra, 4 parts of sunflower disc, 3 parts of Pueraria lobata, 4 parts of Poria cocos, 3 parts of edible sucrose, and 3 parts of edible yeast powder.

[0063] Among them, Bacillus XRK-1 has a preservation number of: CCTCC NO: M 20232477, the preservation date is December 06, 2023, and the preservation unit is the China Center for Type Culture Collection.

[0064] 1) Isolation of strains

[0065] Weigh 10 g of sunflower discs, put them into a non-woven fabric filter bag, inoculate them into 50 mL of enrichment medium with uric acid (uric acid content is 2 g / L) as the sole carbon source, and place them in a constant temperature incubator (37°C, 180 r / min) for shaking culture for 3-5 days for the screening of strains. Dilute the enriched bacterial liquid to 10 -1 / 10 -2 times, respectively suck 100 μL of the bacterial liquid and coat it on the enrichment solid plate medium, and incubate it upside down at 37°C for 24 h - 48 h, and observe the colony growth situation at any time. After the colonies are formed, invert the plate and place it in a 4°C refrigerator, and observe the colony growth situation and the size of the transparent circle at any time during the culture process (whether there is a transparent circle around the colony and the size of the transparent circle can judge whether the strain produces uricase and the strength of the uricase activity produced).

[0066] 2) Purification of strains

[0067] 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 into enriched medium for preliminary screening of enzyme-producing strains. Then inoculate the isolated single colonies into enriched medium, repeat streaking several times, and purify the enzyme-producing strains. Figure 1 shown.

[0068] 3) Morphological observation of strains

[0069] 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 2 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.

[0070] 4) Molecular biological identification of strains

[0071] The above-mentioned strain was subjected to 16S rDNA sequencing, and the 16S rDNA sequence of the strain is shown in Seq ID NO: 1.

[0072]

[0073]

[0074] Among them, the 16S rDNA of the strain was amplified using the universal primers of bacteria: upstream primer 27F: 5-AGAGTTTGATCCTGGCTCAG-3 downstream primer 1492R: 5-CTACGGCTACCTTGTTACGA-3. The PCR system is shown in Table 1 below.

[0075] Table 1 PCR system

[0076]

[0077] The PCR amplification program is shown in Table 2 below.

[0078] Table 2 PCR amplification program

[0079]

[0080] The PCR product after purification of the strain was taken for sequencing.

[0081] The BLAST result on NCBI showed that the strain was Bacillaceae. The measured sequence was uploaded to GenBank with the accession number: OR294314. The 16S rRNA sequence was aligned with NCBI BLAST, and the highest homology with Bacillaceae gen.sp. in Genebank was 96.39%. Based on the 16S rRNA gene sequence alignment result, a Neighbor-Joining phylogenetic tree was constructed with Oceanobacillus halotolerans strain YIM 98839 as the outgroup as shown in Figure 4 described below. The strain was preliminarily identified as Bacillus sp. XRK-1.

[0082] 5) Determination of the uric acid degrading enzyme activity of Bacillus sp. XRK-1

[0083] The enzyme activities of the strain in the enrichment liquid medium and the optimized fermentation medium were detected using a microplate reader: Single colonies were inoculated into 100 mL of the enrichment liquid medium and the optimized fermentation medium respectively, and placed in a constant temperature shaking incubator (37 °C, 180 r / min) for shaking culture for 36 h. 1.0 mL of the culture broth was taken, and after centrifugation, the supernatant was transferred to a new EP tube (5500×g, 10 min, 4 °C) for standby; The cell precipitate was added with 900 μL of PBS, sonicated on ice, and after centrifugation, the supernatant was taken for standby.

[0084] Uric acid standard solutions with concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mmol were prepared, and the absorbance values at 300 nm were measured to prepare a UA standard curve. The absorbance OD 300 = 3.978X + 0.0026. Under standard conditions, the sample amount that consumes 1.0 μmol of uric acid per minute is one enzyme activity unit.

[0085] 40 μL of the culture broth supernatant or the lysate supernatant and 160 μL of borate buffer (5 mmol H 3 BO 3, a 0.5 mmol uric acid solution prepared at pH 8.5. In a standard 96-well plate, at 37 °C, 300 nm (AU300), with oxygen for 60 min, the reduction of uric acid was monitored, and the uric acid concentration was determined by absorbance readings. The uricase activities in the fermentation supernatant and the supernatant of the cell lysate obtained by fermenting Bacillus XRK-1 in the enrichment medium were 0.022 U / ml and 0.047 U / ml respectively. Under the optimal culture conditions, the uricase activities in the fermentation supernatant and the supernatant of the cell lysate obtained by fermenting Bacillus XRK-1 in the optimized fermentation medium were 0.063 U / ml and 0.103 U / ml respectively. It can be seen that the ability of Bacillus XRK-1 cultured in the optimized fermentation medium to produce uricase is much higher than that of enrichment culture. During the fermentation process, Bacillus XRK-1 continuously synthesizes uricase, and the uricase is secreted into the fermentation supernatant outside the strain, which is convenient for rapid utilization. Moreover, the uricase in the living cells of Bacillus XRK-1 continuously overflows with the growth of the cells. When Bacillus XRK-1 is broken and dead, the uricase in its body still retains its activity. That is, whether Bacillus XRK-1 is a living bacterium or a dead cell can be used for reducing uric acid.

[0086] The fermentation medium under the optimal culture conditions used in the present invention comprises the following components: 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, 10 g of yeast extract, 20 g of sucrose, and 1 L of distilled water.

[0087] A preparation method of a fermentation preparation with the function of reducing uric acid according to the present invention comprises the following steps:

[0088] S1. Preparation of the traditional Chinese medicine fermentation substrate: Stir and mix 2 parts of Smilax glabra, 4 parts of sunflower flower disk, 3 parts of Pueraria lobata, and 4 parts of Poria cocos powder with 3 parts of edible yeast powder, 3 parts of sucrose, and 100 parts of pure water for 20 min to make it uniform, and obtain a mixed slurry;

[0089] S2. Transfer the mixed slurry prepared in S1 to a sterilization container, sterilize it at 121 °C under high pressure for 30 min to obtain a sterilized solution, and then cool it to 35 °C for standby;

[0090] S3. Preparation of the fermentation seed liquid: Inoculate Bacillus XRK-1 into the enrichment liquid medium at an inoculation amount of 1%, and culture it at 37 °C for 18 h to obtain the fermentation seed liquid;

[0091] S4. Inoculate the fermentation seed liquid prepared in S3 into the sterilized solution prepared in S2 at an inoculation amount of 1%, and carry out constant-temperature fermentation culture at 42 - 45 °C for 24 h to obtain the fermentation stock solution;

[0092] S5, Purification: Filter the fermented stock solution prepared in S4 to remove undigested drug residues, and the crude liquid fermentation preparation can be obtained.

[0093] In S1, the smilax glabra, sunflower disc, kudzu root and poria cocos powder are ultrafine powders with a particle size of 25 μm obtained by first pulverizing smilax glabra, sunflower disc, kudzu root and poria cocos with a pulverizing and stirring blade and then sieving.

[0094] The number of viable Bacillus XRK-1 in the fermentation preparation is 4.0×10 7 cfu / mL.

[0095] Since viable Bacillus XRK-1 has the ability to secrete urate oxidase, and the active ingredients of smilax glabra and kudzu root can effectively inhibit the activity of xanthine oxidase, the alkaloids in sunflower disc can dissolve uric acid stones, and poria cocos promotes diuresis, which can effectively reduce the uric acid level in animals or humans. Therefore, the applicant proposes the application of this fermentation preparation with uric acid-lowering function in the preparation of drugs for preventing and treating diseases related to hyperuricemia.

[0096] The diseases related to hyperuricemia include gout, uric acid stones, acute and chronic uric acid nephropathy.

[0097] The drug is an oral medicine, an external plaster or a patch, and the oral medicine is any one of viable bacteria oral liquid, solid beverage granules, tablets, granules or capsules.

[0098] Example 2

[0099] A fermentation preparation with uric acid-lowering function in this example is prepared by fermenting Bacillus XRK-1 on a traditional Chinese medicine fermentation substrate. The traditional Chinese medicine fermentation substrate includes the following components in parts by weight: 100 parts of purified water, 2 parts of smilax glabra, 4 parts of sunflower disc, 3 parts of kudzu root, 4 parts of poria cocos, 3 parts of edible sucrose, 3 parts of edible yeast powder, 5 parts of apple concentrate and 5 parts of strawberry concentrate.

[0100] Among them, Bacillus XRK-1, the preservation number is: CCTCC NO: M 20232477, the preservation date is: December 06, 2023, and the preservation unit is: China Center for Type Culture Collection.

[0101] The number of viable Bacillus XRK-1 in the fermentation preparation is 4.5×10 7 cfu / mL.

[0102] The fermentation preparation with uric acid-lowering function in this example has a better taste and better patient compliance compared with Example 1.

[0103] A preparation method of a fermentation preparation with the function of reducing uric acid, comprising the following steps:

[0104] S1. Preparation of traditional Chinese medicine fermentation substrate: Stir and mix 2 parts of Smilax glabra, 4 parts of sunflower disc, 3 parts of Pueraria lobata, and 4 parts of Poria cocos powder with 3 parts of edible yeast powder, 3 parts of sucrose, and 100 parts of pure water for 25 minutes to make it uniform, obtaining a mixed slurry;

[0105] S2. Transfer the mixed slurry prepared in S1 to a sterilization container, perform high-pressure sterilization at 121 °C for 30 minutes to obtain a sterilized solution, and then cool it to 35 °C for standby;

[0106] S3. Preparation of fermentation seed liquid: Inoculate Bacillus sp. XRK-1 into an enriched liquid medium at an inoculation amount of 1%, and culture it at 37 °C for 20 hours to obtain a fermentation seed liquid;

[0107] S4. Inoculate the fermentation seed liquid prepared in S3 into the sterilized solution prepared in S2 at an inoculation amount of 1%, and perform constant-temperature fermentation culture at 37 °C for 24 hours to obtain a fermentation stock solution;

[0108] S5. Purification: Filter the fermentation stock solution prepared in S4 to remove undigested drug residues, and thus obtain a crude liquid fermentation preparation;

[0109] S6. Flavoring: Add 5 parts of apple concentrated juice and 5 parts of strawberry concentrated juice to every 119 parts by weight of the crude liquid fermentation preparation, and then mix evenly to obtain a high-quality liquid fermentation preparation with excellent taste, thereby improving the taste of the fermentation preparation and the compliance of patients.

[0110] In S1, the Smilax glabra, sunflower disc, Pueraria lobata, and Poria cocos powder are ultrafine powders with a particle size of 30 μm obtained by first pulverizing Smilax glabra, sunflower disc, Pueraria lobata, and Poria cocos with a pulverizing and stirring blade and then sieving.

[0111] Application of the fermentation preparation with the function of reducing uric acid in this embodiment in the preparation of a drug for preventing and treating diseases related to hyperuricemia. The drug is an oral drug, and the oral drug is a live bacteria oral liquid.

[0112] Animal experiment - Analysis of the effect of the fermentation preparation with the function of reducing uric acid in this embodiment on the serum uric acid (UA) level of hyperuricemic mice

[0113] Ninety mice weighing (20±2) g were randomly divided into 9 groups of 10 mice each, and were respectively designated as the blank group, the model group, the benzbromarone group, the high, medium, and low dose groups of the Chinese herbal medicine fermentation substrate, and the high, medium, and low dose groups of the fermentation preparation. The Chinese herbal medicine fermentation substrate herein refers to the Chinese herbal medicine fermentation substrate without fermentation in this example, that is, the Chinese herbal medicine fermentation substrate includes the following components in parts by weight: 100 parts of purified water, 2 parts of Smilax glabra, 4 parts of sunflower disc, 3 parts of Pueraria lobata, 4 parts of Poria cocos, 3 parts of edible sucrose, 3 parts of edible yeast powder, 5 parts of apple concentrate, and 5 parts of strawberry concentrate.

[0114] After 5 days of adaptive feeding of the mice, except for the blank group (injected with an equal amount of normal saline), the remaining groups were subjected to modeling treatment by intraperitoneal injection of hypoxanthine at 0.3 g / kg / only and subcutaneous injection of potassium oxonate at 0.05 g / kg / only in the neck once a day for 7 consecutive days. At the same time, the benzbromarone group was given benzbromarone by gavage at 0.02 g / kg / only once, the high, medium, and low dose groups of the substrate were given the Chinese herbal medicine fermentation substrate without fermentation in this experimental example by gavage at 2 mL / kg / only, 1 mL / kg / only, and 0.5 mL / kg / only respectively once, and the high, medium, and low dose groups of the fermentation preparation were given the fermentation preparation with uric acid-lowering function in this example by gavage at 2 mL / kg / only, 1 mL / kg / only, and 0.5 mL / kg / only respectively once. The blank group and the model group were given normal saline by gavage (1 mL / kg) for 7 consecutive days. After 7 days, the tail vein blood of the mice in all groups was collected, plasma was separated by centrifugation, and serum was taken to measure the uric acid content.

[0115] The test results are shown in Table 3. Compared with the blank group, the serum UA level of the mice in the model group was significantly increased (P<0.01), indicating that the modeling treatment was successful; compared with the model group, the serum UA levels of the mice treated with benzbromarone and the high, medium, and low dose groups of the fermentation preparation were all significantly decreased (P<0.01). And combined with Figure 5 It can be seen that the fermentation preparation with uric acid-lowering function in this example can significantly reduce the serum UA level of hyperuricemia mice, and when the dose of the fermentation preparation is greater than or equal to 2 mL / kg / only, its uric acid-lowering effect is equivalent to or even better than that of gavaging benzbromarone in the prior art.

[0116] Table 3 Effects of the biological fermentation preparation on the blood uric acid of hyperuricemia mice

[0117]

[0118]

[0119] Example 3

[0120] A fermentation preparation with uric acid-lowering function in this embodiment is prepared by fermenting Bacillus XRK-1 on a traditional Chinese medicine fermentation substrate. The traditional Chinese medicine fermentation substrate includes the following components in parts by weight: 90 parts of purified water, 4 parts of Smilax glabra, 3 parts of sunflower flower disk, 5 parts of Pueraria lobata, 5 parts of Poria cocos, 2 parts of edible sucrose, 2 parts of edible yeast powder, 4 parts of apple concentrate, and 5 parts of strawberry concentrate.

[0121] Among them, Bacillus XRK-1 has a preservation number of: CCTCC NO: M 20232477, the preservation date is December 06, 2023, and the preservation unit is China Center for Type Culture Collection.

[0122] The number of viable Bacillus XRK-1 in the fermentation preparation is 5.0×10 7 cfu / mL.

[0123] A preparation method of a fermentation preparation with uric acid-lowering function according to the present invention includes the following steps:

[0124] S1. Preparation of the traditional Chinese medicine fermentation substrate: Mix 4 parts of Smilax glabra, 3 parts of sunflower flower disk, 5 parts of Pueraria lobata, 5 parts of Poria cocos powder with 2 parts of edible yeast powder, 2 parts of sucrose and 90 parts of purified water and stir for 25 min to make it uniform, obtaining a mixed slurry;

[0125] S2. Transfer the mixed slurry prepared in S1 to a sterilization container, sterilize it at 121 °C under high pressure for 30 min to obtain a sterilized solution, and then cool it to 35 °C for standby;

[0126] S3. Preparation of the fermentation seed liquid: Inoculate Bacillus XRK-1 into an enriched liquid medium at an inoculation amount of 1%, and culture it at 37 °C for 20 h to obtain a fermentation seed liquid;

[0127] S4. Inoculate the fermentation seed liquid prepared in S3 into the sterilized solution prepared in S2 at an inoculation amount of 3%, and carry out constant-temperature fermentation culture at 42 - 45 °C for 28 h to obtain a fermentation stock solution;

[0128] S5. Purification: Filter the fermentation stock solution prepared in S4 to remove undigested drug residues, and thus obtain a crude liquid fermentation preparation;

[0129] S6. Flavoring: Add 4 parts of apple concentrate and 5 parts of strawberry concentrate to every 111 parts by weight of the crude liquid fermentation preparation, and then mix evenly to obtain a high-quality liquid fermentation preparation with excellent taste, thereby improving the taste of the fermentation preparation and the compliance of patients;

[0130] S7. Carry out freeze-drying and tablet treatment on the high-quality liquid fermentation preparation prepared in S6 to obtain a tablet fermentation preparation.

[0131] It should be noted that the fermentation preparation with the function of reducing uric acid in this embodiment can also be made into granules after the fine product of the liquid fermentation preparation is freeze-dried, or filled into capsules to make capsules according to the usage mode of the drug.

[0132] In S1, the powder of Smilax glabra, sunflower disc, kudzu root and Poria cocos is the ultrafine powder with a particle size of 45 μm obtained by first pulverizing Smilax glabra, sunflower disc, kudzu root and Poria cocos with a pulverizing and stirring blade and then sieving.

[0133] Application of the fermentation preparation with the function of reducing uric acid in this embodiment in the preparation of drugs for preventing and treating hyperuricemia-related diseases. The drug is an oral drug, and the oral drug is a tablet.

[0134] Example 4

[0135] A fermentation preparation with the function of reducing uric acid in this embodiment is prepared by fermenting Bacillus XRK-1 on a traditional Chinese medicine fermentation substrate. The traditional Chinese medicine fermentation substrate includes the following components in parts by weight: 99 parts of purified water, 4 parts of Smilax glabra, 2 parts of sunflower disc, 5 parts of kudzu root, 5 parts of Poria cocos, 2 parts of edible sucrose, 3 parts of edible yeast powder, 4 parts of apple concentrated juice and 4 parts of strawberry concentrated juice.

[0136] Among them, Bacillus XRK-1 has a preservation number of: CCTCC NO: M 20232477, the preservation date is December 06, 2023, and the preservation unit is the China Center for Type Culture Collection.

[0137] The number of viable Bacillus XRK-1 in the fermentation preparation is 3.5×10 7 cfu / mL.

[0138] A preparation method of a fermentation preparation with the function of reducing uric acid according to the present invention includes the following steps:

[0139] S1. Preparation of the traditional Chinese medicine fermentation substrate: Mix 4 parts of Smilax glabra, 2 parts of sunflower disc, 5 parts of kudzu root, 5 parts of Poria cocos powder with 3 parts of edible yeast powder, 2 parts of sucrose and 99 parts of purified water and stir for 25 min to make it uniform to obtain a mixed slurry;

[0140] S2. Transfer the mixed slurry prepared in S1 to a sterilization container, sterilize it at 121 °C under high pressure for 30 min to obtain a sterilized solution, and then cool it to 35 °C for standby;

[0141] S3. Preparation of the fermentation seed liquid: Inoculate Bacillus XRK-1 at an inoculation amount of 1% into an enriched liquid medium and culture it at 37 °C for 20 h to obtain a fermentation seed liquid;

[0142] S4. Inoculate the fermentation seed liquid prepared in S3 into the sterilized liquid prepared in S2 at an inoculation amount of 2%, and perform constant-temperature fermentation culture at 42 - 45 °C for 28 h to obtain the original fermentation liquid;

[0143] S5. Purification: Filter the original fermentation liquid prepared in S4 to remove the undigested drug residues, and the crude liquid fermentation preparation can be obtained;

[0144] S6. Flavoring: Add 4 parts of apple concentrated juice and 4 parts of strawberry concentrated juice to every 120 parts by weight of the crude liquid fermentation preparation, and then mix evenly to obtain the high-quality liquid fermentation preparation with excellent taste, thereby improving the taste of the fermentation preparation and the compliance of patients;

[0145] S7. Freeze-dry the high-quality liquid fermentation preparation prepared in S6 to obtain a powdered fermentation preparation.

[0146] It should be noted that for the fermentation preparation with uric acid-lowering function in this example, according to the usage method of the drug, after freeze-drying the high-quality liquid fermentation preparation, it can also be made into granules and processed into strip-shaped solid beverages for the convenience of patients to take quickly.

[0147] In S1, the smilax glabra, sunflower flower disk, kudzu root, and poria cocos powder are ultrafine powders with a particle size of 40 μm obtained by first pulverizing smilax glabra, sunflower flower disk, kudzu root, and poria cocos with a pulverizing and stirring blade and then sieving.

[0148] Application of the fermentation preparation with uric acid-lowering function in this example in the preparation of drugs for preventing and treating hyperuricemia-related diseases. The drug is an oral medicine, and the oral medicine is a granule.

[0149] Example 5

[0150] A fermentation preparation with uric acid-lowering function in this example is prepared by fermenting Bacillus XRK-1 on a traditional Chinese medicine fermentation substrate. The traditional Chinese medicine fermentation substrate includes the following components in parts by weight: 91 parts of purified water, 3 parts of smilax glabra, 3 parts of sunflower flower disk, 4 parts of kudzu root, 5 parts of poria cocos, 3 parts of edible sucrose, 1 part of edible yeast powder, 5 parts of apple concentrated juice, and 5 parts of strawberry concentrated juice.

[0151] Among them, Bacillus XRK-1, with the preservation number: CCTCC NO: M 20232477, the preservation date: December 06, 2023, and the preservation unit: China Center for Type Culture Collection.

[0152] The number of viable Bacillus XRK-1 in the fermentation preparation is 2.0×10 7 cfu / mL.

[0153] A preparation method of a fermentation preparation with the function of reducing uric acid includes the following steps:

[0154] S1. Preparation of traditional Chinese medicine fermentation substrate: Mix 3 parts of Smilax glabra, 3 parts of sunflower flower disc, 4 parts of Pueraria lobata, 5 parts of Poria cocos powder with 1 part of edible yeast powder, 3 parts of sucrose and 91 parts of pure water and stir for 25 minutes to make it uniform, obtaining a mixed slurry;

[0155] S2. Transport the mixed slurry prepared in S1 to a sterilization container, sterilize it at 121 °C under high pressure for 30 minutes to obtain a sterilized liquid, and then cool it to 35 °C for standby;

[0156] S3. Preparation of fermentation seed liquid: Inoculate Bacillus XRK-1 into an enriched liquid medium at an inoculation amount of 1%, and culture it at 37 °C for 20 hours to obtain a fermentation seed liquid;

[0157] S4. Inoculate the fermentation seed liquid prepared in S3 into the sterilized liquid prepared in S2 at an inoculation amount of 1.5%, and carry out constant-temperature fermentation culture at 42-45 °C for 24 hours to obtain a fermentation stock solution;

[0158] S5. Purification: Filter the fermentation stock solution prepared in S4 to remove undigested drug residues, and thus obtain a crude liquid fermentation preparation;

[0159] S6. Flavoring: Add 5 parts of apple concentrated juice and 5 parts of strawberry concentrated juice to every 110 parts by weight of the crude liquid fermentation preparation, and then mix evenly to obtain a high-quality liquid fermentation preparation in terms of taste, thereby improving the taste of the fermentation preparation and the compliance of patients;

[0160] S7. Freeze-dry the high-quality liquid fermentation preparation prepared in S6 to obtain a powdery fermentation preparation, and fill it into capsules to process into capsule preparations.

[0161] In S1, the Smilax glabra, sunflower flower disc, Pueraria lobata and Poria cocos powder are ultrafine powders with a particle size of 48 μm obtained by first pulverizing Smilax glabra, sunflower flower disc, Pueraria lobata and Poria cocos with a pulverizing and stirring blade and then sieving.

[0162] Application of the fermentation preparation with the function of reducing uric acid in this embodiment in the preparation of drugs for preventing and treating hyperuricemia-related diseases. The drug is an oral drug, and the oral drug is a capsule preparation.

[0163] A fermentation preparation with the function of reducing uric acid according to the present invention can, according to the usage mode of the drug, also concentrate and process the high-quality liquid fermentation preparation into a paste during preparation to make a paste preparation or a patch preparation. For the changes in the above technical features, those skilled in the art can understand and implement them through literal description, so no additional drawings will be given for illustration.

Claims

1. A fermented preparation having uric acid lowering function, characterized in that: The product is produced by fermenting Bacillus XRK-1 on a traditional Chinese medicine fermentation substrate, wherein the traditional Chinese medicine fermentation substrate is composed of the following components in parts by weight: 90-100 parts of purified water, 2-4 parts of Smilax glabra, 2-4 parts of sunflower flower discs, 3-5 parts of Pueraria root, 3-5 parts of Poria cocos, 1-3 parts of edible sucrose and 1-3 parts of edible yeast powder; the number of live bacteria of Bacillus XRK-1 in the fermentation preparation is ≥1.0×10 7 cfu / ml, or the number of viable bacteria of Bacillus XRK-1 in the fermentation preparation is ≥1.0*10 7 cfu / g; the preservation number of the Bacillus XRK-1 is: CCTCC M 20232477, the preservation date: December 6, 2023, and the preservation unit: China Center for Type Culture Collection.

2. The fermented preparation having uric acid lowering function according to claim 1, characterized in that: The Bacillus is isolated from a sunflower disk, and the 16S rDNA sequence of the Bacillus XRK-1 is shown in Seq ID NO:

1.

3. The fermented preparation having uric acid lowering function according to claim 1, characterized in that: The fermentation production conditions are as follows: the inoculation amount of Bacillus XRK-1 is 1-5% of the fermentation substrate of the traditional Chinese medicine by weight, and the culture is carried out at 30-45° C. for 24-30 hours.

4. The fermented preparation having uric acid lowering function according to any one of claims 1 to 3, characterized in that: The fermented preparation also includes seasonings, and the edible auxiliary materials for adjusting the taste include the following components in parts by weight: 4-5 parts of apple concentrated juice and 4-5 parts of strawberry concentrated juice.

5. A method for preparing a fermented preparation having uric acid lowering function according to any one of claims 1 to 4, characterized in that: The method for preparing the fermented preparation having the function of lowering uric acid comprises the following steps: S1. Preparation of Chinese herbal medicine fermentation substrate: Mix and evenly mix smilax glabra, sunflower flower disk, kudzu root and tuckahoe powder with edible yeast powder, sucrose and purified water according to a proportion to obtain a mixed slurry; S2, sterilizing the mixed slurry prepared in S1 at high temperature to obtain a sterilized liquid; S3, preparation of fermentation seed liquid: inoculate Bacillus XRK-1 into the enrichment liquid culture medium at an inoculum amount of 1%, and culture at 30-45° C. for 18 h-20 h to obtain the fermentation seed liquid; S4, inoculating the fermentation seed solution prepared in S3 into the sterilized solution prepared in S2 at an inoculation rate of 1-5%, and fermenting and culturing at a constant temperature of 30-45° C. for 24-30 hours to obtain a fermentation stock solution; S5. Purification: Filter the fermentation liquid prepared in S4 to remove the undecomposed drug residues to obtain a crude liquid fermentation preparation.

6. Use of a fermented preparation with uric acid lowering function as claimed in any one of claims 1 to 4 in the preparation of a medicament for preventing and treating hyperuricemia-related diseases, wherein the hyperuricemia-related disease is any one of gout, uric acid stones, acute and chronic uric acid nephropathy.

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