Pediococcus acidilactici PA3-7 with uric acid degradation and kidney protection effects and application thereof

Abstract

The invention provides pediococcus acidilactici PA3-7 with uric acid degradation and kidney protection effects and application of the pediococcus acidilactici PA3-7. Specifically, the invention provides an application of pediococcus acidilactici PA3-7 in the aspects of uric acid degradation, kidney protection and the like, and also provides a composition with the effects of uric acid degradation and kidney protection, including a medicine and the like.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedicine and food. Specifically, the present invention relates to Pediococcus acidilactici PA3-7 with uric acid degradation and kidney protection effects and its applications. Background Art

[0002] Drug treatments for hyperuricemia mainly focus on two aspects: one is to inhibit the production of uric acid, such as corticosteroids, non-steroidal anti-inflammatory drugs, febuxostat, etc.; the other is to enhance the excretion of uric acid, such as probenecid, benzbromarone, sulfinpyrazone, etc.

[0003] However, drugs for treating hyperuricemia or gout have certain side effects and cause certain damage to the liver and kidney functions. Therefore, it is safer to use edible probiotics or natural substances without toxic side effects to effectively relieve or assist in the treatment of hyperuricemia and gout.

[0004] Therefore, there is an urgent need in this field to develop new, safe and effective probiotic drugs for degrading uric acid and having a kidney protection effect. Summary of the Invention

[0005] The present invention provides new, safe and effective Pediococcus acidilactici for degrading uric acid and having a kidney protection effect and its applications.

[0006] In a first aspect of the present invention, there is provided a Pediococcus acidilactici, which is Pediococcus acidilactici PA3-7 ( Pediococcus acidilactici PA3-7), and the Pediococcus acidilactici PA3-7 has the effects of degrading uric acid and protecting the kidney; wherein, the preservation number of the Pediococcus acidilactici PA3-7 is CCTCC NO. M 2025250.

[0007] In another preferred example, the 16S rRNA gene sequence of the Pediococcus acidilactici PA3-7 has at least 99.5% identity with SEQ ID NO: 1 as shown in SEQ ID NO: 1.

[0008] In another preferred example, the Pediococcus acidilactici PA3-7 is isolated from fermented cereal sour soup.

[0009] In a second aspect of the present invention, there is provided a composition having the effects of degrading uric acid and protecting the kidney, and the composition comprises: (a) a safe and effective amount of the Pediococcus acidilactici described in the first aspect of the present invention and / or its metabolites; and (b) a pharmaceutically acceptable carrier.

[0010] In another preferred example, the composition is selected from pharmaceutical compositions.

[0011] In another preferred example, the composition is an oral preparation.

[0012] In another preferred embodiment, the composition further contains an additional uric acid-degrading component.

[0013] In another preferred embodiment, the composition further contains an additional kidney-protecting component.

[0014] The dosage form of the composition is: (i) liquid preparation; (ii) solid preparation; (iii) semi-solid preparation.

[0015] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: powder, powder for external use, tablet, sugar-coated tablet, capsule, granule, suspension, solution, syrup, drop, and sublingual tablet.

[0016] In another preferred embodiment, the liquid preparation is selected from the group consisting of: solution product or suspension product.

[0017] In another preferred embodiment, the composition contains 1×10 - 1×10 20 cfu / mL or cfu / g of PA3-7 ， Preferably 1×10 4 -1×10 15 cfu / mL or cfu / g of PA3-7, based on the total volume or total weight of the composition.

[0018] In another preferred embodiment, in the composition, it contains 0.0001 - 99 wt%, preferably 0.1 - 90 wt% of the Lactiplantibacillus plantarum and / or its metabolites, based on the total weight of the composition.

[0019] In another preferred embodiment, the composition is in unit dosage form (one tablet, one capsule or one vial), and the mass of the composition in each unit dosage form is 0.05, preferably 0.1.

[0020] In another preferred embodiment, the composition further contains other probiotics and / or prebiotics.

[0021] In another preferred embodiment, the probiotics are selected from the group consisting of: lactic acid bacteria, Bifidobacterium, Lactobacillus acidophilus, or a combination thereof.

[0022] In another preferred embodiment, the prebiotic is selected from the group consisting of: fructooligosaccharide (FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), lactulose (LACT), soybean oligosaccharide (SOS), inulin, or a combination thereof.

[0023] In another preferred embodiment, the composition further comprises: (c) glycerol.

[0024] In another preferred embodiment, the composition further comprises: (d) glucose.

[0025] In another preferred embodiment, the content of the glucose is 5 g / L.

[0026] In another preferred embodiment, the dosage form of the composition is a solid powder.

[0027] In another preferred embodiment, the dosage form of the composition is a freeze-dried powder.

[0028] In the third aspect of the present invention, there is provided the use of the Pediococcus acidilactici described in the first aspect of the present invention, or the composition described in the second aspect of the present invention, for preparing a drug or a preparation, and the drug or the preparation is used for one or more uses selected from the following group: (a) directly degrading uric acid; (b) protecting the kidneys; (c) preventing and / or treating hyperuricemia.

[0029] In the fourth aspect of the present invention, there is provided a method for degrading uric acid in vitro, which comprises co-culturing the Pediococcus acidilactici described in the first aspect of the present invention or the composition described in the second aspect of the present invention in a culture medium containing uric acid and being rich in nutrients.

[0030] In another preferred example, the method is non-diagnostic and non-therapeutic.

[0031] In a fifth aspect of the present invention, there is provided a method for preparing a composition as described in the second aspect of the present invention, comprising the steps of: Mixing the Pediococcus acidilactici described in the first aspect of the present invention and / or its metabolites with a pharmaceutically acceptable carrier to form the composition described in the second aspect of the present invention.

[0032] In a sixth aspect of the present invention, there is provided a production method, comprising the steps of: (a) Culturing the Pediococcus acidilactici described in the first aspect of the present invention under suitable culture conditions to obtain a culture; (b) Optionally, separating the Pediococcus acidilactici cells and / or its metabolites from the culture product; and (c) Optionally, mixing the Pediococcus acidilactici cells and / or other metabolites separated in the previous step with a pharmaceutically acceptable carrier to prepare a composition.

[0033] The present invention can establish a relatively stable hyperuricemia model by using a combined feed of uric acid and potassium oxonate and allowing rats to freely ingest it. By first establishing the model and then administering the strain, and simultaneously administering the strain and the combined feed of uric acid and potassium oxonate, the effect of the strain directly degrading uric acid in vivo can be reflected.

[0034] In a seventh aspect of the present invention, there is provided a method for preventing and treating hyperuricemia, comprising: administering to a subject in need the Pediococcus acidilactici described in the first aspect of the present invention or the composition described in the second aspect of the present invention.

[0035] In another preferred example, the administration includes oral administration.

[0036] In another preferred example, the administration dose is 0.01 / body weight / day, preferably 0.1 / body weight / day.

[0037] In another preferred example, the subject includes mammals such as humans.

[0038] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1Shows the in vitro degradation of uric acid by strain PA3-7 (partial): (A) BHI medium supplemented with 0.07 g / L uric acid; (B) BHI medium supplemented with 0.14 g / L uric acid. C: Blank control; JS2-3: Lactobacillus fermentum; JS11-3: Lactobacillus fermentum; JS16-2: Lactobacillus fermentum; JS10-4: Lactobacillus fermentum; Ya-1: Pediococcus pentosaceus; SG-6: Lactobacillus plantarum PC-10: Lactobacillus plantarum; np-2: Pediococcus acidilactici.

[0040] Figure 2 Shows the isolation, screening and morphology of the strain PA3-7 of the present invention: (A) Schematic diagram of the isolation and screening plate; (B) Colony morphology of strain PA3-7; (C) Microscopic (1000×) morphology of strain PA3-7 after purification.

[0041] Figure 3 Shows the Neighbor-Joining phylogenetic tree constructed with Lactobacillus delbrueckii DSM 20074 (M58814) as the outgroup based on the 16S rRNA gene sequence alignment results of strain PA3-7.

[0042] Figure 4 Shows the in vitro degradation of uric acid by strain PA3-7. C: Blank control; WK: Lactobacillus gasseri; JSSN-1; Lactobacillus fermentum; ns4-1, FS2-2, ns5-2, FSX32, JS5-3, JS11-4, sb-2: Pediococcus acidilactici.

[0043] Figure 5 A and B in show the in vitro degradation of uric acid by strain PA3-7. C: Blank control; WK: Lactobacillus gasseri; JSSN-1; Lactobacillus fermentum; SG-5: Pediococcus acidilactici.

[0044] Figure 6 Shows the effect of degrading uric acid in BHI medium with different concentrations of glucose.

[0045] Figure 7 Shows the effect of degrading uric acid with different concentrations of glucose added to 1 / 2 MRS.

[0046] Figure 8 Shows the effect of glycerol on the in vitro degradation of uric acid by strain PA3-7.

[0047] Figure 9 Shows the uric acid content in rat serum. (A) Uric acid content in rat serum at 28 days; (B) Trend of uric acid content in rat serum at 28 days.

[0048] Figure 10 Shows the body weights of three groups of rats.

[0049] Figure 11 The renal pathological section score results of three groups of rats are shown.

[0050] Figure 12 The renal pathological sections (H&E, 40×) of three groups of rats on the 28th day of intervention are shown.

[0051] Figure 13 The uric acid content in the serum of 21-day-old rats in different groups is shown.

[0052] Figure 14 A, B, and C in [specific context] show the uric acid content in the serum of rats in the intragastric uric acid test in different groups. Detailed implementation manners

[0053] Through extensive and in-depth research and a large number of screenings, the inventor of the present invention unexpectedly obtained Pediococcus acidilactici PA3-7 ( Pediococcus acidilactici PA3-7) with the effects of degrading uric acid and protecting the kidneys. Experiments of the present invention show that the Pediococcus acidilactici of the present invention can effectively reduce the uric acid content in a complex environment close to intestinal components, and after adding glycerol or a specific content of glucose, the uric acid-lowering effect of the Pediococcus acidilactici of the present invention can be significantly promoted. Animal experiments show that the Pediococcus acidilactici of the present invention has a significant effect on degrading uric acid in rats and has a significant protective effect on the kidneys. On this basis, the present invention is completed.

[0054] Terms To more easily understand the present disclosure, certain terms are first defined. As used in this application, unless otherwise clearly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0055] The term "about" may refer to a value or component within an acceptable error range of a specific value or component determined by a person of ordinary skill in the art, which will depend in part on how the value or component is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101.

[0056] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the term also includes "consisting essentially of...", or "consisting of...".

[0057] As used herein, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the said range and, where appropriate, fractional values thereof (such as one-tenth and one-hundredth of an integer).

[0058] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the related listed items.

[0059] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of an agent that confers a therapeutic effect on a subject being treated, with a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives an indication or feels an effect). Specifically, an "effective amount" refers to the amount of a therapeutic agent that is effective in treating, ameliorating, or preventing a desired disease or condition, or exhibits a detectable therapeutic or prophylactic effect, such as by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of the disease symptoms.

[0060] The effective amount is generally administered in a dosing regimen that includes multiple unit doses. For any particular therapeutic agent, the effective amount (and / or the appropriate unit dose in the effective dosing regimen) can vary, e.g., depending on the route of administration, depending on the combination with other drugs. In addition, the specific effective amount (and / or unit dose) for any particular patient may depend on a variety of factors, including the condition being treated; the severity of the condition; the activity of the particular drug being used; the particular ingredients used; the age, weight, general health, and diet of the patient; the time of administration, the route of administration; the duration of treatment; and similar factors well known in the medical arts.

[0061] As used herein, the term "treatment" refers to any administration of a substance (such as Pediococcus acidilactici PA3-7 of the present invention) that partially or completely alleviates, ameliorates, relieves, inhibits the symptoms, characteristics, and / or causes of one or more specific diseases, disorders, and / or conditions (such as cancer), delays its onset, reduces its severity, and / or reduces its frequency, incidence, or severity. Such treatment can be treatment of a subject who does not exhibit signs of the relevant disease, disorder, and / or condition, and / or treatment of a subject who exhibits only early signs of the disease, disorder, and / or condition. Optionally or additionally, such treatment can be treatment of a subject who exhibits definite signs of one or more relevant diseases, disorders, and / or conditions. In some embodiments, treatment can be performed on a subject who has been diagnosed with a relevant disease, disorder, and / or condition. The treatment can be part of a "method of treatment" which can include the diagnosis or selection of a patient / individual and a therapeutic intervention. The selection of a patient can include testing the suitability of the patient for a therapeutic intervention, which can include testing to determine whether the patient's cancer has a relevant defect in a protein or coding nucleic acid.

[0062] Hyperuricemia Hyperuricemia (HUA) is a metabolic disease caused by abnormal uric acid metabolism or reduced excretion. Persistent hyperuricemia can lead to health problems such as gout, kidney disease, and cardiovascular disease. Uric acid is mainly derived from purine metabolism, synthesized in the liver, and then excreted through the kidneys or intestines. Due to the lack of uricase in the human body, uric acid cannot be further degraded, resulting in an easy increase in uric acid levels.

[0063] Pediococcus acidilactici of the present invention and its applications As used herein, the terms "Pediococcus acidilactici PA3-7 of the present invention", "Pediococcus acidilactici of the present invention", "the Pediococcus acidilactici PA3-7 of the present invention", "strain PA3-7 of the present invention" and "PA3-7 of the present invention" are used interchangeably and all refer to Pediococcus acidilactici PA3-7 of the present invention, with the deposit number of CCTCC NO. M 2025250.

[0064] Pediococcus acidilactici PA3-7 of the present invention was isolated from pulp water. The physiological characteristics of strain PA3-7 are as follows: After culturing strain PA3-7 on MRS medium for 24 h, the colony color is milky white and opaque, the colony is raised, moist, with a flat and shiny edge; the optimum growth temperature is 37°C.

[0065] The present invention provides the applications of strain PA3-7 in aspects such as uric acid degradation and kidney protection. According to a preferred example of the present invention, after intragastric administration of strain PA3-7 to hyperuricemic model mice, the effect of degrading uric acid in rats is significant, and it has a significant protective effect on the kidneys. Therefore, the said strain can be used to degrade uric acid and protect the kidneys, and particularly can be used for the treatment and / or prevention of hyperuricemia.

[0066] Compositions and their applications The present invention also provides a composition, preferably a pharmaceutical composition. The composition comprises an effective amount of Pediococcus acidilactici PA3-7. In a preferred embodiment, the composition further comprises glycerol. In a preferred embodiment, the composition further comprises glucose. In a preferred embodiment, the composition further comprises probiotics selected from the group consisting of: lactic acid bacteria, Bifidobacterium, Lactobacillus acidophilus, or a combination thereof; and / or prebiotics selected from the group consisting of: fructooligosaccharide (FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), lactulose (LACT), soybean oligosaccharide (SOS), inulin, or a combination thereof.

[0067] In a preferred embodiment, the composition is a liquid preparation, a solid preparation, or a semi-solid preparation.

[0068] In a preferred embodiment, the liquid preparation is selected from the group consisting of: solution products or suspension products.

[0069] In a preferred embodiment, the dosage form of the composition is selected from the group consisting of: powders, powders for external use, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, and sublingual tablets.

[0070] The pharmaceutical composition of the present invention can be administered in any form of a pharmaceutical tablet, injection, or capsule. The pharmaceutical preparation comprises excipients, pharmaceutically acceptable media and carriers, and these substances can be selected according to the administration route. The pharmaceutical preparation of the present invention may further comprise auxiliary active components.

[0071] Lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methyl paraben, propyl paraben, talc, magnesium stearate or mineral oil, etc. can all be used as carriers, excipients or diluents, etc. of the pharmaceutical composition in the present invention.

[0072] In addition, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and fragrances, etc. The pharmaceutical composition of the present invention can be produced in enteric-coated preparations by a variety of well-known methods so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, can pass through the stomach smoothly without being destroyed by gastric acid.

[0073] In addition, the microorganism of the present invention can be used in the form of capsules prepared by conventional methods. For example, standard excipients and the lyophilized microorganism of the present invention are mixed to form pellets, and then the pellets are filled into gelatin capsules. In addition, the microorganism of the present invention and excipients allowed for drugs such as liquid glue, cellulose, silicate or mineral oil, etc. can be mixed to prepare suspensions or dispersions, and such suspensions or dispersions can be filled into soft gelatin capsules.

[0074] The pharmaceutical composition of the present invention can be made into enteric-coated tablets for oral use. The term - "enteric coating" in this application includes all coatings allowed for conventional drugs, which are not degraded by gastric acid but can be fully decomposed and rapidly release the microorganism of the present invention in the small intestine. The enteric coating of the present invention can be maintained in synthetic gastric acid such as HCl solution with pH = 1 for more than 2 hours at 36, and preferably decomposed within 1.0 hour in synthetic intestinal fluid such as buffer solution with pH = 7.0.

[0075] The enteric coating of the present invention is coated at about 16 - 30 mg per tablet, preferably 16 - 25 mg, more preferably 16 - 20 mg. The thickness of the enteric coating in the present invention is 5 - 100 μm, and the ideal thickness is 20 - 80 μm. The enteric coating components are selected from conventional polymers known in the art.

[0076] The preferred enteric coating of the present invention is prepared from cellulose acetate phthalate polymer or trimellitate polymer and a copolymer of methacrylic acid (for example, a copolymer of methacrylic acid containing more than 40% methacrylic acid and containing hydroxypropyl methylcellulose phthalate or its ester derivatives).

[0077] In the present invention, the viscosity of the cellulose acetate phthalate used for the casing is about 45 - 90 cp, the acetyl content is 17 - 26%, and the phthalic acid content is 30 - 40%. The viscosity of the cellulose acetate hemiphthalate used for the casing is about 5 - 21 cs, and the acetyl content is 17 - 26%. The cellulose acetate trimellitate is produced by Eastman Kodak Company and can be used as the casing material in the present invention.

[0078] The hydroxypropyl methylcellulose phthalate used for the casing in the present invention generally has a molecular weight of 20,000 - 130,000 daltons, an ideal molecular weight of 80,000 - 100,000 daltons, a hydroxypropyl content of 5 - 10%, a methoxy content of 18 - 24%, and a phthaloyl content of 21 - 35%.

[0079] The hydroxypropyl methylcellulose phthalate used for the casing in the present invention is HP50, which is produced by Shin-Etsu Chemical Co., Ltd. of Japan. HP50 contains 6 - 10% hydroxypropyl, 20 - 24% methoxy, and 21 - 27% propyl, and its molecular weight is 84,000 daltons. Another casing material is HP55, which contains 5 - 9% hydroxypropyl methylcellulose phthalate, 18 - 22% methoxy, and 27 - 35% phthalic acid, and its molecular weight is 78,000 daltons.

[0080] The casing of the present invention is prepared as follows: The casing solution is sprayed onto the core using a conventional method. All solvents in this intestinal coating method are alcohols (such as ethanol), ketones (such as acetone), halogenated hydrocarbon compounds (such as dichloromethane), or their combinations. Plasticizers such as di-n-butyl phthalate and glyceryl triacetate are added to the casing solution in a ratio of 1 part of the coating material to about 0.05 part or about 0.3 part of the plasticizer. The spraying method is preferably carried out continuously, and the amount of the sprayed material can be controlled according to the conditions of the coating. The spraying pressure can be adjusted arbitrarily. Generally, ideal results can be obtained under an average pressure of 1 - 1.5 bar.

[0081] The "medically effective amount" in the specification refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals. For example, in the present invention, a preparation containing 1×10 - 1×10 20 cfu / ml or cfu / g (specifically, it can contain 1×10 4 -1×10 15 cfu / ml or cfu / g; more specifically, it can contain 1×10 6 -1×10 11 cfu / ml or cfu / g) of Pediococcus acidilactici PA3 - 7 and / or its metabolites can be prepared.

[0082] When used for preparing a pharmaceutical composition, the effective dose of Pediococcus acidilactici PA3-7 or its metabolites used can vary with the mode of administration and the severity of the disease to be treated. The dosage forms suitable for oral administration include about 1×10-1×10 20 cfu / ml or cfu / g (specifically, it may contain 1×10 4 -1×10 15 cfu / ml or cfu / g; more specifically, it may contain 1×10 6 -1×10 11 cfu / ml or cfu / g) of active Pediococcus acidilactici PA3-7 or the active ingredient produced by fermentation. This dosage regimen can be adjusted to provide an optimal therapeutic response. For example, depending on the exigencies of the therapeutic situation, several separate doses may be administered daily, or the dose may be proportionally reduced.

[0083] The Pediococcus acidilactici PA3-7 or its metabolites described above can be administered by routes such as oral administration. Solid carriers include: starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, while liquid carriers include: culture medium, polyethylene glycol, non-ionic surfactants, and edible oils (such as corn oil, peanut oil, and sesame oil), as long as they are suitable for the characteristics of Pediococcus acidilactici PA3-7 or its metabolites and the specific mode of administration required. Adjuvants commonly used in the preparation of pharmaceutical compositions can also be advantageously included, such as flavoring agents, pigments, preservatives, and antioxidants such as vitamin E, vitamin C, BHT, and BHA.

[0084] From the standpoint of ease of preparation and administration, the preferred pharmaceutical compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules. Oral administration is preferred.

[0085] The composition of the present invention is administered to the individual once or more times a day. The dosage unit represents a dose that is formally separable and suitable for human or all other mammalian individuals. Each unit contains a pharmaceutically acceptable carrier and an effective therapeutic amount of the microorganism of the present invention. The dosage varies with the patient's body weight and severity of obesity, the additional active components included, and the microorganism used. In addition, if possible, it can be administered separately and continuously if needed. Therefore, the dosage does not limit the present invention. In addition, the "composition" in the present invention not only means a drug but also represents a functional food and a health supplement food. In a preferred example, the composition includes: beverages, foods, drugs, animal feeds, etc.

[0086] In a preferred embodiment of the present invention, there is also provided a food composition, which contains an effective amount of Pediococcus acidilactici PA3-7 and / or its metabolites, and the balance of a food acceptable carrier, and the dosage form of the food composition is selected from solids, dairy products, solution products, powder products, or suspension products.

[0087] In a preferred embodiment, the composition is formulated as follows: 1×10 - 1×10 20 cfu / mL of Pediococcus acidilactici PA3-7 and / or its metabolites; and a food or pharmaceutically acceptable carrier, and / or excipient.

[0088] In another preferred embodiment, the composition is formulated as follows: 1×10 6 -1×10 11 cfu / mL of Pediococcus acidilactici PA3-7 and / or its metabolites; and a food or pharmaceutically acceptable carrier, and / or excipient.

[0089] The main advantages of the present invention include: (a) The present invention discovers that Pediococcus acidilactici PA3-7 can significantly degrade uric acid in co-culture with UA in vitro in brain heart infusion medium (BHI) (more suitable for the intestinal environment).

[0090] (b) Different from the traditional screening method using uric acid as a single carbon source medium, the present invention selects the nutrient-rich brain heart infusion medium (BHI), which is more suitable for the intestinal nutrient environment, and avoids the strains screened out by the single carbon source uric acid medium preferentially selecting other easily utilizable carbon sources when the intestinal nutrients are rich.

[0091] (c) The present invention discovers that glycerol can promote the degradation of uric acid by PA3-7 in the in vitro uric acid degradation experiment, and glycerol is also produced in the intestine, providing new ideas for improving the degradation effect of uric acid-lowering probiotics.

[0092] (d) In animal experiments, in a hyperuricemia model with high uric acid and potassium oxonate diet, PA3-7 is intervened in advance and after modeling. By testing the serum UA content of rats, it is proved that the strain PA3-7 can significantly degrade uric acid in rats and has an obvious protective effect on the kidneys of rats; in addition, it is also proved that the strain PA3-7 can significantly degrade uric acid in rats by directly gavage with uric acid.

[0093] (e) The present invention discovers that an increase in the concentration of glucose has an inhibitory effect on the uric acid degradation effect of the strain PA3-7, and it is also proved by scientific data that the utilization of uric acid by microorganisms can promote uric acid degradation under the action of a certain amount of glucose, providing evidence for the adjustment of the diet structure of hyperuricemia patients.

[0094] (f) Pediococcus acidilactici PA3-7 of the present invention is screened from fermented food pulp water, belonging to a natural strain, without genetic engineering or mutagenesis treatment, and is safe and stable.

[0095] (g) Pediococcus acidilactici PA3-7 of the present invention can reduce drug side effects and the risk of liver and kidney damage; directly degrade uric acid and improve the efficiency of uric acid metabolism; regulate the intestinal flora and enhance the intestinal uric acid excretion ability; reduce the inflammatory response and relieve gout symptoms; and can be taken for a long time.

[0096] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0097] Example 1 In vitro screening of uric acid-degrading probiotics 1.1 Isolation and purification of probiotics Pulp water is a traditional fermented food in the northwest of China, made from vegetables such as celery, rape, and mustard. Lactic acid bacteria, acetic acid bacteria, and yeasts are the main microorganisms during the fermentation of pulp water, especially facultative anaerobic lactic acid bacteria. Therefore, the present invention aims to screen microbial strains with uric acid-degrading ability from fermented pulp water to evaluate the effects of these strains on uric acid-induced hyperuricemia in a mouse model.

[0098] The pulp water samples of the present invention were collected from Gansu Province, Shaanxi Province, etc. 1 g of the sample was taken into 9 mL of sterile physiological saline, and the sample was shaken well and diluted at a 10-fold gradient. 100 μL of the dilution solutions of different gradients were each evenly spread on MRS medium (10 g of peptone, 5 g of beef extract powder, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 4 g of yeast extract powder, 0.2 g of magnesium sulfate, 2 g of ammonium citrate, 20 g of glucose, 0.05 g of manganese sulfate, 5 g of sodium acetate, 16 g of agar, 1 L of distilled water, pH 6.2 ± 0.2, sterilized at 121 °C for 25 min), and cultured under anaerobic conditions at 37 °C for 24 h to obtain different colonies, as shown in Figure 2 A shown in. Different single colonies were selected and streaked on a new MRS agar plate until single colonies with the same morphology were obtained. After repeated purification until there were no other contaminants, they were stored in glycerol at -80 °C. A total of 200 strains of probiotics were screened.

[0099] 1.2 In vitro screening of uric acid-degrading probiotics Select 200 strains of lactic acid bacteria of probiotics that have been screened, and inoculate these strains into BHI medium containing 420 µmol / L and 840 µmol / L UA to simulate the intestinal environment. Incubate overnight for 16 h at 37 °C at a speed of 200 r / min under micro-aerobic conditions (micro-aerobic: 15 ml centrifuge tube + 10 ml medium), and determine the bacterial concentration (1×10 9 cfu / mL) by measuring the OD600 value, and obtain the supernatant by centrifugation at 12,000 r / min. After filtering through a 0.22 µm filter membrane, inject 20 μL of the supernatant into an HPLC detection tube, and measure the uric acid content in the co-culture solution by HPLC.

[0100] 1.3 Comparison of the nutrient components of the BHI medium and the single-carbon-source uric acid screening medium of the present invention Formula of brain heart infusion medium (BHI): 12.5 g of dehydrated calf brain infusion powder, 5 g of dehydrated beef heart infusion powder, 10 g of peptone, 5 g of sodium chloride, 2 g of glucose, 2.5 g of disodium hydrogen phosphate, 1000 ml of deionized water, pH 7.4 ± 0.2; Single-carbon-source screening medium: 17.1 g of disodium hydrogen phosphate, 3 g of potassium dihydrogen phosphate, 0.5 g of sodium chloride, 0.5 g of magnesium sulfate, 0.01 g of calcium chloride, 2 g of uric acid, 12 g of agar powder, 1000 mL of deionized water.

[0101] It is found by comparison that the BHI medium is richer in nutrients and closer to the intestinal chyme environment (oligosaccharides and monosaccharides, polypeptides and amino acids, glycerol and fatty acids, electrolyte salts such as sodium, potassium, and chlorine, water, pH 7 ± 0.5). Screening through a medium close to intestinal components can prevent the strains screened by the single uric acid medium from preferentially selecting other easily utilizable carbon sources when the intestinal nutrition is rich.

[0102] The data results are as shown in Figure 1 A and B in, and the strain PA3-7 has the best in vitro uric acid-lowering effect, reaching 25%.

[0103] Example 2 Identification of uric acid-degrading probiotics 2.1 Morphological identification of strains After the strain PA3-7 is cultured on MRS medium for 24 h, the colony color is milky white and opaque, the colony is convex, moist, and the edge is flat and shiny ( Figure 2 B in). The Gram staining result of the strain PA3-7 is purple, and it is a positive bacterium ( Figure 2 C in).

[0104] 1.3 16S rRNA gene identification High-quality genomic DNA of the strain was obtained using an Ezup Column Bacterial Genomic DNA Purification Kit (Sangon Biotech, China) and sent to Shanghai Bioscience Co., Ltd. for sequencing. The returned sequences were used to generate sequences of potential isolates for species identification using the BLAST engine (NCBI). Nucleotide sequences in the GenBank database were aligned, and sequences with high homology were selected and adjusted using BioEdit.

[0105] Then, a phylogenetic tree was constructed based on the 16S rDNA sequences using the Neighbor-Joining (NJ) method with MEGA, and the results are shown in Figure 3 Figure [Figure number not provided in the original, so it's left as is].

[0106] The phylogenetic tree showed that the homology with the related strain Pediococcus acidilactici LES-NL-S-02 was 97%. PA3-7 was preliminarily identified as Pediococcus acidilactici, and its 16S rRNA gene sequence is shown in SEQ ID NO:1.

[0107] Example 3: Differences in Uric Acid Degradation by Different Pediococcus acidilactici Strains Using commercially available lactic acid bacteria WK and JSSN-1 that degrade uric acid, as well as the other 7 Pediococcus acidilactici strains screened in this experiment as controls, the uric acid degradation effect of strain PA3-7 was evaluated. The experimental procedure was the same as in Example 1.

[0108] The experimental results are as Figure 4 shown, and PA3-7 has the best effect on uric acid degradation.

[0109] Example 4: Comparison of the Uric Acid Degradation Effect of PA3-7 Using the Screening Method with a Single Carbon Source (Uric Acid) The experiment was carried out according to the existing research method for screening the uric acid-degrading strain JSSN-1.

[0110] Strains WK, JSSN-1, PA3-7, and SG-5 were inoculated into MRS liquid medium and cultured overnight for 16 h at 37 °C with a speed of 200 r / min under micro-aerobic conditions. The bacterial concentration was determined by measuring the OD600 value (1×10 9 cfu / mL). 1 mL of the bacterial solution was placed in a 1.5 mL centrifuge tube, centrifuged at 12,000 r / min to remove the medium, washed twice with physiological saline, and the supernatant was removed by centrifugation to obtain the bacterial cells. 1 mL of 10 mmol / L uric acid solution diluted with PBS was added respectively, and incubated at 37 °C under the condition of 200 r / min for 1 h. After 1 h, the supernatant was taken by centrifugation, filtered through a 0.22 µm filter membrane, and then 20 μL of the supernatant was injected into the HPLC detection tube, and the uric acid content in the co-culture solution was measured by HPLC.

[0111] Since uric acid is not soluble in PBS when preparing a 10 mmol / L uric acid solution, in Method 1, NaOH was added to dissolve it slowly, and the pH of the solution was 12 at this time; in Method 2, NaOH was added to dissolve it slowly, and the pH of the solution was 12 at this time, and then the pH was adjusted to 7 with dilute hydrochloric acid (uric acid precipitated after a period of time). The uric acid solution was prepared by these two methods.

[0112] The experimental results proved that by Method 1, strains JSSN-1, PA3-7, and WK all had the effect of uric acid degradation, and PA3-7 had a better uric acid degradation effect ( Figure 5 A in it), and the uric acid degradation effect of strain PA3-7 screened by using the sole carbon source screening medium could reach 33% after incubation for 1 h; by Method 2, all strains had no degradation effect on uric acid ( Figure 5 B in it). However, the pH of the human intestinal environment is 7±0.5, and there is no situation with a pH of 12. Therefore, the single carbon source method is not applicable to subsequent experiments.

[0113] Effect of Glucose on Uric Acid Degradation by Strain PA3-7 To investigate whether glucose would affect the uric acid degradation by strain PA3-7, 2 g / L, 5 g / L, 7 g / L, 10 g / L and 15 g / L of glucose were added to BHI medium without glucose, and cultured overnight for 16 h at 37 °C with a speed of 200 r / min under micro-aerobic conditions. The bacterial concentration (1×10 9 cfu / mL) was determined by measuring the OD600 value, and the supernatant was obtained by centrifugation at 12000 r / min. After filtration through a 0.22 µm filter membrane, 20 μL of the mixture was injected into the HPLC device, and the uric acid content in the co-culture solution was measured by HPLC to obtain the glucose concentration required for the best uric acid degradation effect of strain PA3-7 in BHI medium.

[0114] The experimental results are as Figure 6 shown. When 2 g / L of glucose was added to BHI medium without glucose, the uric acid degradation effect of strain PA3-7 was the best, and when the glucose concentration was above 7 g / L, strain PA3-7 had no degradation effect on uric acid.

[0115] Since strain PA3-7 grew slowly when inoculated into MRS medium without glucose containing 840 µmol / L UA, to improve the growth conditions of the strain, the medium was modified to 1 / 2 MRS without glucose and added with 1 g / L, 2 g / L, 5 g / L, 7 g / L, 10 g / L and 15 g / L of glucose respectively, and cultured overnight for 16 h at 37 °C with a speed of 200 r / min under micro-aerobic conditions (micro-aerobic: 15 ml centrifuge tube + 10 ml medium). The bacterial concentration was determined by measuring the OD600 value, and the supernatant was obtained by centrifugation at 12000 r / min. After filtration through a 0.22 µm filter membrane, 20 μL of the mixture was injected into the HPLC device, and the uric acid content in the co-culture solution was measured by HPLC to obtain the modified medium with the best uric acid degradation effect of strain PA3-7.

[0116] The experimental results are as Figure 7 shown. The 1 / 2 MRS medium without glucose had an obvious uric acid degradation effect when added with 5 g / L of glucose. Therefore, 1 / 2 MRS without glucose plus 5 g / L of glucose was selected as the modified medium for strain PA3-7 to degrade uric acid.

[0117] Example 6 Effect of Glycerol on Uric Acid Degradation by Strain PA3-7 Based on Example 1, the strain PA3-7 with better in vitro uric acid degradation effect was screened out. The cryotube of strain PA3-7 for inoculation contained 500 µL of fermentation broth and 500 µL of glycerol. The monoclonal colonies of strain PA3-7 obtained by streaking were inoculated into a culture medium under micro-aerobic conditions (micro-aerobic: 15 ml centrifuge tube + 10 ml medium) at 37 °C and a speed of 200 r / min for 24 h to obtain the fermentation broth of strain PA3-7. 100 µL of the bacterial solution in the PA3-7 cryotube, 100 µL of the fermentation broth, 50 µL each of glycerol and the fermentation broth, and 50 µL of the fermentation broth were inoculated into 10 mL of BHI medium with a uric acid concentration of 420 µmol / L (0.07 g / L UA). They were cultured overnight at 37 °C and a speed of 200 r / min under micro-aerobic conditions (micro-aerobic: 15 ml centrifuge tube + 10 ml medium) for 16 h, and the OD600 value was measured to determine the bacterial concentration (1×10 9 cfu / mL), and the supernatant was obtained by centrifugation at 12000 r / min. After filtration through a 0.22 µm filter membrane, 20 μL of the supernatant was injected into an HPLC detection tube, and the uric acid content in the co-culture solution was measured by HPLC.

[0118] The experimental results are as Figure 8 shown. After the fermentation broth of strain PA3-7 was inoculated into the BHI medium in the cryotubes with added glycerol and glycerol preservation, the uric acid degradation effects of these two experimental groups were better, while the effects of inoculating the BHI medium with only different inoculation volumes of the fermentation broth of PA3-7 were worse. This indicates that glycerol has a certain promoting effect on the uric acid degradation of strain PA3-7.

[0119] Example 7: In vitro uric acid metabolism test in rats with early intervention by feeding uric acid diet Twenty-four 8-week-old male Wistar rats with a body weight of 220 - 260 g were purchased. All rats were tested after one week of adaptation. During the experiment, these rats were fed with standard commercial rat food. Four rats were placed in each cage in an environment of 22 °C ± 2 °C and 30 %–70 % relative humidity.

[0120] The rats were randomly divided into 3 groups, with 8 rats in each group, namely the blank group, the model group, and the experimental group, ensuring that there was no significant difference in body weight among the groups. Starting from D1, the rats in the blank control group were fed with normal feed, the rats in the model group were fed with 2% uric acid and 3% potassium oxonate feed every day, and after the rats in the experimental group were fed with 2 % uric acid and 3 % potassium oxonate feed, each rat was intragastrically administered 1 ml of bacterial powder (1×10 10 cfu / mL). Thereafter, the rats in the experimental group were intragastrically administered once a day for 28 days.

[0121] Rat tail vein blood was drawn and centrifuged at 12,000 rpm for 5 minutes. The upper serum was taken, diluted 10-fold with PBS, shaken well, and then centrifuged at 12,000 rpm for 5 minutes. 20 μL of the supernatant was injected into an HPLC detection tube, and the uric acid content in the serum was measured by HPLC (the normal serum uric acid concentration of male Wistar rats is 100 - 300 μmol / L).

[0122] The experimental results are shown in Figure 9 A and B in Figure 9 . After rats ingested the strain PA3-7, the serum uric acid content in their bodies decreased significantly compared with the model group ( Figure 9 B in

[0123] . In addition, the results are shown in Figure 10 . The strain PA3-7 had a significant effect on degrading uric acid in rats, which fully demonstrated that the strain PA3-7 had a good preventive effect on rats with high serum uric acid, and the body weight of the rats showed a stable growth trend.

[0124] Example 8 Histopathological Analysis of Rat Kidneys after Premature Intervention of Uric Acid Metabolism in vitro by Feeding Uric Acid Feed The rats were dissected, and their kidneys were collected and rinsed with phosphate buffered saline, and then fixed in the dark in 4% paraformaldehyde. The fixed tissues were dehydrated by an automatic dehydrator, and then subjected to paraffin embedding, sectioning, dewaxing of the sections to water, hematoxylin and eosin (H&E) staining, etc. for histopathological analysis. The kidney sections were examined using an automatic upright fluorescence microscope.

[0125] After rats were fed with 2% uric acid and 3% potassium oxonate for 4 weeks, histopathological analysis showed that the glomeruli in the blank control group had intact morphology and clear outlines; in contrast, in the model group, obvious dilation of renal tubules, flattening and even shedding of renal tubular epithelial cells (black arrows) were observed in the rat kidneys, and at the same time, there was very severe infiltration of inflammatory cells in the renal interstitium (red arrows), and the infiltrated part accounted for more than 80%, indicating severe kidney damage in the model group; in the experimental PA3-7 group, there was only slight dilation of renal tubules (black arrows) in the rat kidneys, and no shedding of renal tubular epithelial cells was seen. Although there was also infiltration of inflammatory cells in the renal interstitium (red arrows), the infiltrated part accounted for no more than 40%, indicating that the degree of kidney damage in the experimental PA3-7 group was significantly weaker than that in the model group ( Figure 12 ).

[0126] Through renal pathological section scoring (scoring basis: proportion of immune cell infiltration, 0 points: no obvious immune cell infiltration; 1 point: immune cell infiltration proportion 0-20%; 2 points: immune cell infiltration proportion 20-40%; 3 points: immune cell infiltration proportion 40-60%; 4 points: immune cell infiltration proportion 60-80%; 5 points: immune cell infiltration proportion 80-100%), the PA3-7 group had a significant protective effect compared with the model group ( Figure 11 ).

[0127] Example 9: In vitro uric acid metabolism test of rats after modeling with uric acid diet Forty-two 8-week-old male Wistar rats weighing 220-260 g were purchased. All rats were tested after one week of adaptation. During the experiment, these rats were fed with standard commercial rat food. Three mice were placed in each cage in an environment of 22°C ± 2°C and 30%–70% relative humidity. The rats were randomly divided into 4 groups: blank control group, model group, experimental group and positive drug group. There were 6 rats in the blank control group and 12 rats in each of the other groups, ensuring that there was no significant difference in body weight among the groups.

[0128] Before the experiment, except for the blank control group, other groups were continuously fed with 2% uric acid and 3% potassium oxonate feed for 5 days to establish a hyperuricemia model in rats. The blank control group was fed with normal feed, the model group was fed with 2% uric acid and 3% potassium oxonate feed daily, after the experimental group was fed with 2% uric acid and 3% potassium oxonate feed, each rat was intragastrically administered 1 ml of bacterial powder (1e10 cfu), and the rats in the positive drug group were fed with 50 mg / kg benzbromarone according to body weight. Thereafter, the rats in the experimental group and the positive drug group were intragastrically administered once a day for 21 days.

[0129] The tail vein blood of the rats was drawn, centrifuged at 12000 rpm for 5 minutes, the upper serum was taken, diluted 10 times with PBS, shaken and mixed evenly, centrifuged at 12000 rpm for 5 minutes, 20 μL of the supernatant was injected into the HPLC detection tube, and the uric acid content in the serum was measured by HPLC (the normal serum uric acid concentration of male Wistar rats is 100 - 300 μmol / L).

[0130] The experimental results showed that when the serum uric acid in the rats was high, after intragastric administration of strain PA3-7 and the positive drug group, the serum uric acid content decreased significantly compared with the model group, and there was a highly significant difference from the model group (P < 0.01) ( Figure 13 ), which fully proved that strain PA3-7 had a good therapeutic effect on rats with high serum uric acid.

[0131] Example 10: Intragastric administration of uric acid to intervene in the uric acid metabolism test of rats Forty-two 8-week-old male Wistar rats weighing 220 - 260 g were purchased. All rats were tested after one week of adaptation. During the experiment, these rats were fed with standard commercial rat food. Three mice per cage were housed in an environment of 22°C ± 2°C and 30% - 70% relative humidity. The rats were randomly divided into 4 groups, namely the blank control group, the model group, the experimental group, and the positive drug group, with 8 rats in each group, ensuring that there was no significant difference in body mass among the groups.

[0132] The rats in the blank control group were fed a normal diet. The rats in the model group were intragastrically administered 1 g / kg uric acid and 1.5 g / kg potassium oxonate according to the body weight standard. After the rats in the experimental group were intragastrically administered 1 g / kg uric acid and 1.5 g / kg potassium oxonate according to the body weight standard, each was intragastrically administered 1 ml of bacterial powder (1e10 cfu). After the rats in the positive drug group were intragastrically administered 1 g / kg uric acid and 1.5 g / kg potassium oxonate according to the body weight standard, they were fed 50 mg / kg benzbromarone according to the body weight.

[0133] At 0 h, 3 h, and 6 h after intragastric administration, the tail vein blood of the rats was drawn, centrifuged at 12,000 rpm for 5 minutes, the upper serum was taken, diluted 10 times with PBS, shaken and mixed evenly, centrifuged at 12,000 rpm for 5 minutes, 20 μL of the supernatant was injected into the HPLC detection tube, and the uric acid content in the serum was measured by HPLC (the normal serum uric acid concentration of male Wistar rats is 100 - 300 μmol / L).

[0134] The experimental results showed that, compared with the model group, the effect of degrading uric acid in rats after taking the positive drug benzbromarone was significant, indicating that the model was successfully constructed. When the serum uric acid in rats was relatively high, after intragastric administration of PA3-7, on the 4th and 7th days, the serum uric acid content in the experimental group decreased significantly compared with the model group, and was slightly better than the effect of the positive drug benzbromarone ( Figure 14 A, B, and C in

[0135] Strain preservation The Pediococcus acidilactici Pediococcus acidilactici of the present invention, PA3-7, was deposited at the China Center for Type Culture Collection (CCTCC) (Wuhan, China) on February 19, 2025, with the deposit number: CCTCC NO. M 2025250.

[0136] All the documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A Pediococcus acidilactici, characterized in that, The Pediococcus acidilactici is Pediococcus acidilactici PA3-7( Pediococcus acidilactici PA3-7), and the Pediococcus acidilactici PA3-7 has the functions of degrading uric acid and protecting the kidneys; wherein, the preservation number of the Pediococcus acidilactici PA3-7 is CCTCC NO. M 2025250.

2. The Pediococcus acidilactici according to claim 1, characterized in that, The Pediococcus acidilactici PA3-7 described above is isolated from fermented grains extract.

3. A composition having the effects of degrading uric acid and protecting the kidneys, characterized in that, The composition comprises: (a) A safe and effective amount of the Pediococcus acidilactici described in claim 1 and / or its metabolites; and (b) A pharmaceutically acceptable carrier.

4. The composition according to claim 3, wherein The composition is a pharmaceutical composition.

5. The composition according to claim 3, characterized in that, The composition contains 1×10 - 1×10 20 cfu / mL or cfu / g of PA3-7 ， Preferably 1×10 4 -1×10 15 cfu / mL or cfu / g of PA3-7, based on the total volume or total weight of the composition.

6. The composition according to claim 3, wherein The composition further comprises: (c) Glycerol.

7. Use of the Pediococcus acidilactici according to claim 1, or the composition according to claim 3, characterized in that, For preparing a drug or a preparation, the drug or the preparation is used for one or more uses selected from the following group: (a) Directly degrading uric acid; (b) Protecting the kidneys; (c) Preventing and / or treating hyperuricemia.

8. A method for in vitro degradation of uric acid, characterized in that, Co-culture the Pediococcus acidilactici described in claim 1 or the composition described in claim 3 in a culture medium containing uric acid and being nutrient-rich.

9. A method for preparing the composition according to claim 3, characterized in that, Comprises the steps of: Mix the Pediococcus acidilactici described in claim 1 and / or its metabolites with a pharmaceutically acceptable carrier, thereby forming the composition described in claim 3.

10. A production method, characterized in that, Comprises the steps of: (a) Under suitable culture conditions, culture the Pediococcus acidilactici described in claim 1 to obtain a culture; (b) Optionally, separate the Pediococcus acidilactici cells and / or its metabolites from the culture product; and (c) Optionally, mix the Pediococcus acidilactici cells and / or other metabolites separated in the previous step with a pharmaceutically acceptable carrier to prepare the composition.

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