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

Abstract

The present invention provides Pediococcus acidilactici PA3-7 with uric acid degradation and kidney protection effects and its application. Specifically, the present invention provides Pediococcus acidilactici PA3-7 ( Pediococcus acidilactici PA3-7) in the degradation of uric acid and kidney protection, and also provides a composition having the effects of degrading uric acid and protecting the kidney, including medicines and the like.

Description

Technical Field

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

[0002] Drug treatment for hyperuricemia mainly focuses 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, sulfapyridine, etc.

[0003] Drugs for treating hyperuricemia or gout have certain side effects and cause certain damage to liver and kidney function. Therefore, it is safer to use edible and non-toxic probiotics or natural substances to effectively relieve or assist in the treatment of hyperuricemia and gout.

[0004] Therefore, there is an urgent need in the art to develop new, safe and effective probiotic drugs for degrading uric acid and protecting the kidneys. Summary of the Invention

[0005] The present invention provides novel, safe and effective Pediococcus acidilactici for degrading uric acid and protecting the kidneys and applications thereof.

[0006] In the first aspect of the present invention, a Pediococcus acidilactici strain is provided, wherein the Pediococcus acidilactici strain is Pediococcus acidilactici strain PA3-7 ( Pediococcus acidilactici M 2025250).

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

[0008] In another preferred embodiment, the Pediococcus acidilactici PA3-7 is isolated from pulp water.

[0009] In a second aspect of the present invention, a composition having the effects of degrading uric acid and protecting the kidney is provided, the composition comprising:

[0010] (a) a safe and effective amount of Pediococcus acidilactici and / or its metabolites according to the first aspect of the present invention; and

[0011] (b) a pharmaceutically acceptable carrier.

[0012] In another preferred embodiment, the composition is selected from a pharmaceutical composition.

[0013] In another preferred embodiment, the composition is an oral preparation.

[0014] In another preferred embodiment, the composition further contains an additional uric acid-degrading component.

[0015] In another preferred embodiment, the composition further contains additional kidney-protecting ingredients.

[0016] The dosage form of the composition is: (i) liquid; (ii) solid preparation; (iii) semi-solid preparation.

[0017] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of powders, powders, tablets, sugar-coated tablets, capsules, granules, suspensions, solutions, syrups, drops, and sublingual tablets.

[0018] In another preferred embodiment, the liquid preparation is selected from the following group: a solution preparation or a suspension preparation.

[0019] In another preferred embodiment, the composition contains 1×10-1×10 20 cfu / mL or cfu / gPA3-7 ， Preferably 1×10 4 -1×10 15 cfu / mL or cfu / gPA3-7, based on the total volume or weight of the composition.

[0020] In another preferred embodiment, the composition contains 0.0001-99 wt%, preferably 0.1-90 wt% of the Lactobacillus plantarum and / or its metabolites, based on the total weight of the composition.

[0021] In another preferred embodiment, the composition is in unit dosage form (a tablet, a capsule or a vial), and the mass of the composition in each unit dosage form is 0.05, preferably 0.1.

[0022] In another preferred embodiment, the composition further contains other probiotics and / or prebiotics.

[0023] In another preferred embodiment, the probiotics are selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof.

[0024] In another preferred embodiment, the prebiotic is selected from the following group: HYPERLINK "http: / / baike.haosou.com / doc / 5410056.html" \t "http: / / baike.haosou.com / doc / _blank"fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), HYPERLINK "http: / / baike.haosou.com / doc / 6453240.html" \t "http: / / baike.haosou.com / doc / _blank"xylooligosaccharides (XOS), HYPERLINK "http: / / baike.haosou.com / doc / 6752346.html" \t "http: / / baike.haosou.com / doc / _blank"lactulose (LACT), HYPERLINK "http: / / baike.haosou.com / doc / 5385196.html" \t "http: / / baike.haosou.com / doc / _blank"Soy oligosaccharides (SOS), inulin, or a combination thereof.

[0025] In another preferred embodiment, the composition further comprises: (c) glycerol.

[0026] In another preferred embodiment, the composition further comprises: (d) glucose.

[0027] In another preferred embodiment, the glucose content is 5 g / L.

[0028] In another preferred embodiment, the composition is in the form of a solid powder.

[0029] In another preferred embodiment, the composition is in the form of a lyophilized powder.

[0030] In a third aspect of the present invention, there is provided a use of the Pediococcus acidilactici according to the first aspect of the present invention or the composition according to the second aspect of the present invention for preparing a medicament or preparation, wherein the medicament or preparation is used for one or more uses selected from the group consisting of:

[0031] (a) Direct degradation of uric acid;

[0032] (b) protect kidneys;

[0033] (c) Prevention and / or treatment of hyperuricemia.

[0034] In a fourth aspect of the present invention, a method for degrading uric acid in vitro is provided, wherein the Pediococcus acidilactici described in the first aspect of the present invention or the composition described in the second aspect of the present invention is co-cultured with a nutrient-rich culture medium containing uric acid.

[0035] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0036] In a fifth aspect of the present invention, there is provided a method for preparing the composition according to the second aspect of the present invention, comprising the steps of:

[0037] The Pediococcus acidilactici and / or its metabolites described in the first aspect of the present invention are mixed with a pharmaceutically acceptable carrier to form the composition described in the second aspect of the present invention.

[0038] In a sixth aspect of the present invention, there is provided a production method comprising the steps of:

[0039] (a) culturing the Pediococcus acidilactici described in the first aspect of the present invention under conditions suitable for cultivation, thereby obtaining a culture;

[0040] (b) optionally, isolating Pediococcus acidilactici cells and / or metabolites thereof from the culture product; and

[0041] (c) Optionally, the Pediococcus acidilactici cells and / or other metabolites isolated in the previous step are mixed with a pharmaceutically acceptable carrier to prepare a composition.

[0042] The present invention utilizes a combined feed of uric acid and potassium oxonate, with rats allowed to freely consume the feed, to establish a relatively stable hyperuricemia model. By first establishing the model and then administering the strain, or by simultaneously administering the strain with the combined feed of uric acid and potassium oxonate, the strain's ability to directly degrade uric acid in vivo can be demonstrated.

[0043] In a seventh aspect of the present invention, a method for preventing and treating hyperuricemia is provided, comprising: administering the Pediococcus acidilactici described in the first aspect of the present invention or the composition described in the second aspect of the present invention to a subject in need thereof.

[0044] In another preferred embodiment, the administration comprises oral administration.

[0045] In another preferred embodiment, the administration dosage is 0.01 / body weight / day, preferably, 0.1 / body weight / day.

[0046] In another preferred embodiment, the subject includes mammals, such as humans.

[0047] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Figure 3 shows 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.

[0049] Figure 2 The isolation and screening of the strain PA3-7 of the present invention and its morphology are shown: (A) Schematic diagram of the isolation and screening plate; (B) Colony morphology of the strain PA3-7; (C) Microscopic (1000×) morphology of the purified strain PA3-7.

[0050] Figure 3 Shown is the Neighbor-Joining phylogenetic tree of strain PA3-7 constructed based on the 16S rRNA gene sequence alignment results with Lactobacillus delbrueckii DSM 20074 (M58814) as the outer branch.

[0051] Figure 4 The results show 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.

[0052] Figure 5 Figures A and B 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.

[0053] Figure 6 The results show the effect of different glucose concentrations on the degradation of uric acid in BHI medium.

[0054] Figure 7 The degradation effect of uric acid by adding different concentrations of glucose to 1 / 2 MRS is shown.

[0055] Figure 8 Shown is the effect of glycerol on the in vitro degradation of uric acid by strain PA3-7.

[0056] Figure 9 Figure 2 shows the uric acid levels in rat serum. (A) Uric acid levels in rat serum over 28 days; (B) Trend of uric acid levels in rat serum over 28 days.

[0057] Figure 10 The body weights of the three groups of rats are shown.

[0058] Figure 11 Shown are the results of kidney pathological section scoring of the three groups of rats.

[0059] Figure 12 Shown are the kidney pathological sections of the three groups of rats on the 28th day of intervention (H&E, 40×).

[0060] Figure 13 The uric acid levels in the serum of rats in different groups at 21 days are shown.

[0061] Figure 14 A, B, and C show the uric acid levels in the serum of rats in different groups in the oral uric acid test. DETAILED DESCRIPTION

[0062] After extensive and in-depth research and extensive screening, the inventors unexpectedly obtained a strain of Pediococcus acidilactici PA3-7 ( Pediococcus acidilactici PA3-7). Experiments conducted by the present invention demonstrate that the Pediococcus acidilactici can effectively reduce uric acid levels in a complex environment close to intestinal components. Furthermore, the addition of glycerol or a specific amount of glucose significantly enhances the uric acid-lowering effect of the present invention. Animal experiments also demonstrate that the present invention significantly degrades uric acid in rats and has a significant protective effect on the kidneys. This is the basis for the completion of the present invention.

[0063] the term

[0064] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0065] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 and between.

[0066] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0067] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range and, where appropriate, fractional values ​​thereof (e.g., tenths and hundredths of an integer).

[0068] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0069] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a pharmaceutical agent that confers a therapeutic effect on the treated subject at 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 the effect). Specifically, an "effective amount" refers to an amount of a therapeutic drug that effectively treats, ameliorates, or prevents a desired disease or condition, or exhibits a detectable therapeutic or preventive 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 disease symptoms.

[0070] The effective amount is typically administered in a dosage regimen comprising a plurality of unit doses. For any particular therapeutic agent, the effective amount (and / or the appropriate unit dose in an effective dosage regimen) may vary, for example, 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 specific drug being used; the specific ingredients used; the patient's age, weight, general health, and diet; the time of administration, the route of administration; the duration of treatment; and similar factors well known in the medical field.

[0071] As used herein, the term "treatment" refers to any administration of a substance (e.g., Pediococcus acidilactici PA3-7 of the present invention) that partially or completely alleviates, improves, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the frequency, incidence, or severity of one or more specific diseases, disorders, and / or conditions (e.g., cancer). This treatment can be treatment of a subject who does not show signs of the relevant disease, disorder, and / or condition, and / or treatment of a subject who only shows early signs of the disease, disorder, and / or condition. Optionally or additionally, this treatment can be treatment of a subject who shows confirmed signs of one or more relevant diseases, disorders, and / or conditions. In some embodiments, a subject who has been diagnosed with a relevant disease, disorder, and / or condition can be treated. The treatment can be part of a "therapeutic method," which can include diagnosis or selection of a patient / individual and therapeutic intervention. The selection of a patient can include testing the patient's suitability for therapeutic intervention, which can include testing to determine whether the patient's cancer has a related defect in a protein or encoding nucleic acid.

[0072] hyperuricemia

[0073] Hyperuricemia (HUA) is a metabolic disease caused by abnormal uric acid metabolism or decreased excretion. Long-term conditions can lead to health problems such as gout, kidney disease, and cardiovascular disease. Uric acid is primarily derived from purine metabolism, synthesized in the liver, and excreted through the kidneys or intestines. Due to the lack of uricase in the human body, uric acid cannot be further degraded, leading to elevated uric acid levels.

[0074] Pediococcus acidilactici and its application

[0075] As used herein, the terms "Pediococcus acidilactici PA3-7 of the present invention", "Pediococcus acidilactici ..." and "Pediococcus acidilactici of the present invention" are Pediococcus acidilactici PA3-7", "strain PA3-7 of the present invention" and "PA3-7 of the present invention" can be used interchangeably and all refer to the Pediococcus acidilactici PA3-7 of the present invention, whose preservation number is CCTCC NO. M 2025250.

[0076] The present invention relates to a strain of Pediococcus acidilactici PA3-7 isolated from pulp water. The strain PA3-7 exhibits the following physiological characteristics: after culturing on MRS medium for 24 hours, the PA3-7 strain produces milky white, opaque colonies with protruding, moist, smooth, and shiny edges; and an optimal growth temperature of 37°C.

[0077] The present invention provides applications of strain PA3-7 for uric acid degradation and kidney protection. According to a preferred embodiment of the present invention, strain PA3-7 significantly degraded uric acid in rats after oral administration to hyperuricemia model mice and exhibited significant kidney protection. Therefore, this strain can be used to degrade uric acid and protect the kidneys, and is particularly useful for treating and / or preventing hyperuricemia.

[0078] Composition and application thereof

[0079] 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 a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, lactobacillus acidophilus, or a combination thereof; and / or a prebiotic selected from the group consisting of oligofructose (FOS), oligogalactose (GOS), oligoxylose (XOS), oligolactose (LACT), oligolactose (LACT), oligolactose (LACT), oligolactose (LACT), oligosaccharides ... "http: / / baike.haosou.com / doc / _blank"Soy oligosaccharides (SOS), inulin, or a combination thereof.

[0080] In a preferred embodiment, the composition is a liquid preparation, a solid preparation, or a semi-solid preparation.

[0081] In a preferred embodiment, the liquid preparation is selected from the following group: a solution preparation or a suspension preparation.

[0082] In a preferred embodiment, the dosage form of the composition is selected from the group consisting of powders, powders, tablets, sugar-coated tablets, capsules, granules, suspensions, solutions, syrups, drops, and sublingual tablets.

[0083] The pharmaceutical composition of the present invention can be administered in the form of tablets, injections or capsules. The pharmaceutical preparation includes excipients, pharmaceutically acceptable media and carriers, which can be selected according to the route of administration. The pharmaceutical preparation of the present invention can further include auxiliary active ingredients.

[0084] Lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil can be used as carriers, excipients or diluents of the pharmaceutical composition of the present invention.

[0085] In addition, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings. The pharmaceutical composition of the present invention can be produced as an enteric coating formulation by various well-known methods so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, can pass smoothly through the stomach without being destroyed by gastric acid.

[0086] Alternatively, the microorganisms of the present invention can be used in the form of capsules prepared by conventional methods. For example, standard excipients can be mixed with the freeze-dried microorganisms of the present invention to form pellets, which can then be filled into gelatin capsules. Furthermore, the microorganisms of the present invention can be mixed with pharmaceutically acceptable excipients such as liquid glue, cellulose, silicates, or mineral oil to form suspensions or dispersions, which can be filled into soft gelatin capsules.

[0087] The pharmaceutical composition of the present invention can be formulated into enteric-coated tablets for oral administration. The term "enteric coating" as used herein encompasses all conventional pharmaceutical coatings that are resistant to gastric acid degradation but fully decompose in the small intestine to rapidly release the microorganisms of the present invention. The enteric coating of the present invention can maintain its stability at 36°C for more than 2 hours in synthetic gastric acid, such as a pH 1 HCl solution, and preferably decomposes within 1 hour in synthetic intestinal fluid, such as a pH 7.0 buffer.

[0088] The enteric coating of the present invention is coated with about 16-30 mg per tablet, preferably 16-25 mg, and more preferably 16-20 mg. The thickness of the enteric coating of the present invention is 5-100 μm, and ideally 20-80 μm. The enteric coating ingredients are selected from conventional polymers known in the art.

[0089] The preferred enteric coating of the present invention is prepared from a copolymer of cellulose acetate phthalate polymer or trimellitate polymer and methacrylic acid (for example, a copolymer of methacrylic acid containing more than 40% and methylcellulose hydroxypropyl phthalate or its ester derivatives).

[0090] The cellulose acetate phthalate used in the casings of the present invention has a viscosity of approximately 45-90 cp, an acetyl content of 17-26%, and a phthalic acid content of 30-40%. The cellulose acetate trimellitate used in the casings has a viscosity of approximately 5-21 cs and an acetylphthalate content of 17-26%. Cellulose acetate trimellitate is manufactured by Eastman Koda and can be used as the casing material of the present invention.

[0091] The hydroxypropyl methylcellulose phthalate used in the enteric coating of 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%.

[0092] The hydroxypropyl methylcellulose phthalate used in the enteric coating of the present invention is HP50, produced by Shin-Etsu Chemidnl Co. Ltd. of Japan. HP50 contains 6-10% hydroxypropyl groups, 20-24% methoxy groups, 21-27% propyl groups, and has a molecular weight of 84,000 Daltons. Another enteric coating material is HP55, which contains 5-9% hydroxypropyl methylcellulose phthalate, 18-22% methoxy groups, 27-35% phthalic acid, and has a molecular weight of 78,000 Daltons.

[0093] The enteric coating of the present invention is prepared as follows: the enteric coating solution is sprayed onto the core using conventional methods. In this enteric coating method, all solvents are alcohols (such as ethanol), ketones (such as acetone), halogenated hydrocarbon compounds (such as dichloromethane), or combinations thereof. A softening agent such as di-n-butyl phthalate and triacetin is added to the enteric coating solution in a ratio of 1 part coating material to about 0.05 part or about 0.3 part softening agent. The spraying method is preferably carried out continuously, and the amount of material sprayed can be controlled according to the conditions used for coating. The spray pressure can be adjusted at will, and generally, the desired results can be obtained at an average pressure of 1-1.5 bar.

[0094] In the specification, "pharmaceutically effective amount" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals. For example, in the present invention, a drug containing 1×10-1×10 20 cfu / ml or cfu / g (especially, 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 Pediococcus acidilactici PA3-7 and / or its metabolites.

[0095] When used to prepare a pharmaceutical composition, the effective dose of Pediococcus acidilactici PA3-7 or its metabolites used may vary depending on the mode of administration and the severity of the disease to be treated. The dosage form suitable for oral administration comprises about 1×10-1×10 20 cfu / ml or cfu / g (especially, 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 the optimal therapeutic response. For example, as required by the exigencies of the therapeutic situation, several divided doses can be given daily, or the dose can be reduced proportionally.

[0096] The Pediococcus acidilactici PA3-7 or its metabolites can be administered orally or in combination. Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, while liquid carriers include culture media, polyethylene glycol, nonionic 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 administration method required. Adjuvants commonly used in the preparation of pharmaceutical compositions may also be advantageously included, such as flavorings, pigments, preservatives, and antioxidants such as vitamin E, vitamin C, BHT, and BHA.

[0097] From the standpoint of ease of preparation and administration, preferred pharmaceutical compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules. Oral administration is preferred.

[0098] The composition of the present invention is administered to the individual once or more per day. The dosage unit represents a dosage that can be divided in form and is suitable for humans 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 weight and severity of obesity, the supplementary active ingredients included and the microorganisms used. In addition, if possible, the administration can be separated and, if necessary, continuous. Therefore, the dosage does not limit the present invention. In addition, the "composition" in the present invention not only means a medicine but also means a functional food and a health supplement food. In a preferred embodiment, the composition includes: beverages, food, medicines, animal feed, etc.

[0099] In a preferred embodiment of the present invention, a food composition is also provided, which contains an effective amount of Pediococcus acidilactici PA3-7 and / or its metabolites, and the remainder is a food-acceptable carrier, wherein the dosage form of the food composition is selected from solid, dairy, solution, powder, or suspension products.

[0100] In a preferred embodiment, the composition is formulated as follows:

[0101] 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.

[0102] In another preferred embodiment, the composition is formulated as follows:

[0103] 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.

[0104] The main advantages of the present invention include:

[0105] (a) We found that Pediococcus acidilactici PA3-7 could significantly degrade uric acid when co-cultured with UA in brain heart infusion (BHI) medium (which is more in line with the intestinal environment) in vitro.

[0106] (b) Unlike the traditional screening method using uric acid as the only carbon source culture medium, the present invention uses the nutrient-rich brain heart infusion (BHI) culture medium, which is more in line with the intestinal nutritional environment and avoids the strains screened in the single carbon source uric acid culture medium from preferentially selecting other easily available carbon sources when the intestinal nutrient is rich.

[0107] (c) The present invention found that glycerol can promote the degradation of uric acid by PA3-7 in an in vitro uric acid degradation experiment. Glycerol is also produced in the intestine, which provides a new idea for improving the degradation effect of uric acid-lowering probiotics.

[0108] (d) In the in vivo animal experiment, PA3-7 was intervened in advance and after modeling in the hyperuric acid model of high uric acid and potassium oxonate diet. The test of serum UA content in rats proved that strain PA3-7 could significantly degrade uric acid in rats and had a significant protective effect on rat kidneys. In addition, direct gavage of uric acid also proved that strain PA3-7 could significantly degrade uric acid in rats.

[0109] (e) The present invention found that increasing glucose concentrations inhibited the uric acid degradation efficiency of strain PA3-7. Scientific data also demonstrated that microbial utilization of uric acid, under the action of a certain amount of glucose, can promote uric acid degradation, providing evidence for dietary adjustments in patients with hyperuricemia.

[0110] (f) The Pediococcus acidilactici PA3-7 of the present invention is screened from fermented food slurry and is a natural strain without genetic engineering or mutagenesis treatment, so it is safe and stable.

[0111] (g) The Pediococcus acidilactici PA3-7 of the present invention can reduce drug side effects and lower the risk of liver and kidney damage; directly degrade uric acid and improve uric acid metabolism efficiency; regulate intestinal flora and enhance intestinal uric acid excretion capacity; reduce inflammatory responses and alleviate gout symptoms; and can be taken long-term.

[0112] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed 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 indicated, percentages and parts are by weight.

[0113] Example 1 In vitro screening of uric acid-degrading probiotics

[0114] 1.1 Probiotic isolation and purification

[0115] Jiangshui (Jiangshui) is a traditional fermented food in northwestern China, made from vegetables such as celery, rapeseed, and mustard greens. Lactic acid bacteria, acetic acid bacteria, and yeasts are the primary microorganisms in the fermentation process, particularly facultative anaerobic lactic acid bacteria. Therefore, the present invention aims to screen microbial strains with uric acid-degrading capabilities from fermented Jiangshui to evaluate the effects of these strains on direct uric acid-induced hyperuricemia in a mouse model.

[0116] The slurry samples of the present invention were collected from Gansu Province, Shaanxi Province and other places. 1 g to 9 mL of sterile physiological saline was taken, the sample was shaken thoroughly, and diluted in a 10-fold gradient. 100 μL of each dilution in different gradients was 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 triammonium 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, such as Figure 2 As shown in Figure A. Different single colonies were selected and streaked onto new MRS agar plates until single colonies with identical morphology were obtained. After repeated purification until no other contaminants were present, the colonies were stored in glycerol at -80°C. A total of 200 probiotic strains were screened.

[0117] 1.2 In vitro screening of uric acid-degrading probiotics

[0118] Twenty strains of probiotic lactic acid bacteria were selected and inoculated into BHI medium containing 420 µmol / L and 840 µmol / L UA to simulate the intestinal environment. The culture was carried out overnight at 37°C and 200 rpm under slightly oxidized conditions (slightly oxidized: 15 ml centrifuge tube + 10 ml medium) for 16 hours. The bacterial concentration (1 × 10 9 cfu / mL) and centrifuged at 12,000 rpm to obtain the supernatant. After filtration through a 0.22 µm filter membrane, 20 µL of the supernatant was injected into an HPLC assay tube, and the uric acid content in the co-culture solution was measured by HPLC.

[0119] 1.3 Comparison of nutrient components between the BHI medium of the present invention and the single carbon source uric acid screening medium

[0120] Brain heart infusion medium (BHI) recipe: dehydrated calf brain extract powder 12.5 g, dehydrated ox heart extract powder 5 g, peptone 10 g, sodium chloride 5 g, glucose 2 g, sodium hydrogen phosphate 2.5 g, deionized water 1000 ml, pH 7.4 ± 0.2;

[0121] Single carbon source screening medium: disodium hydrogen phosphate 17.1 g, potassium dihydrogen phosphate 3 g, sodium chloride 0.5 g, magnesium sulfate 0.5 g, calcium chloride 0.01 g, uric acid 2 g, agar powder 12 g, deionized water 1000 mL.

[0122] By comparison, it was found that BHI culture 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 chloride, water, pH 7±0.5). Screening with a culture medium close to intestinal components can avoid the strains screened out with a single uric acid culture medium from giving priority to other easily available carbon sources when the intestine is rich in nutrients.

[0123] The data results are as follows Figure 1 As shown in Figures A and B, strain PA3-7 has the best in vitro uric acid-lowering effect, reaching 25%.

[0124] Example 2 Identification of Uric Acid-Degrading Probiotics

[0125] 2.1 Morphological identification of strains

[0126] After culturing PA3-7 on MRS medium for 24 h, the colonies were milky white and opaque, with raised, moist, smooth and shiny edges ( Figure 2 B in the figure). PA3-7 strain is purple in Gram stain, indicating positive bacteria ( Figure 2 C in ).

[0127] 1.3 16S rRNA gene identification

[0128] High-quality genomic DNA of the strain was obtained using the Ezup column bacterial genomic DNA purification kit (Sangon Biotech, China) and sent to Shanghai Boshang Technology Co., Ltd. for sequencing. The returned sequences were used to generate sequences of potential isolates for species identification using the BLAST engine (NCBI). The nucleotide sequences were aligned with the GenBank database, and sequences with high homology were selected and adjusted using BioEdit.

[0129] Then, MEGA was used to build a phylogenetic tree based on the neighbor-joining method (NJ) of 16S rDNA sequences. Figure 3 shown.

[0130] The phylogenetic tree showed that the homology with the closely 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.

[0131]

[0132] Example 3 Differences in uric acid degradation by different Pediococcus acidilactici

[0133] The uric acid degradation activity of strain PA3-7 was evaluated using commercially available uric acid-degrading lactic acid bacteria WK and JSSN-1, as well as seven other strains of Pediococcus acidilactici selected in this experiment, as controls. The experimental procedures were the same as those in Example 1.

[0134] The experimental results are as follows Figure 4 As shown in the figure, PA3-7 has the best degradation effect on uric acid.

[0135] Example 4 Comparison of uric acid degradation effects of PA3-7 using a single carbon source (uric acid) screening method

[0136] The experiment was conducted according to the existing research method for screening the uric acid-degrading strain JSSN-1.

[0137] Strains WK, JSSN-1, PA3-7, and SG-5 were inoculated into MRS liquid medium and cultured overnight at 37°C at 200 rpm under slightly dissolved oxygen conditions for 16 h. The bacterial concentration (1 × 10 9 cfu / mL), 1 mL of bacterial culture was placed in a 1.5 mL centrifuge tube and centrifuged at 12,000 rpm to remove the culture medium. The cells were washed twice with saline and the supernatant was removed by centrifugation to obtain the bacterial cells. 1 mL of 10 mmol / L uric acid solution diluted in PBS was added to each tube and incubated at 37°C and 200 rpm for 1 hour. After 1 hour, the supernatant was centrifuged and filtered through a 0.22 µm filter membrane. 20 μL of the supernatant was injected into an HPLC tube, and the uric acid content in the co-culture solution was measured by HPLC.

[0138] Since uric acid does not dissolve in PBS when preparing a 10 mmol / L uric acid solution, method 1 adds NaOH to slowly dissolve it, at which point the solution pH is 12; method 2 adds NaOH to slowly dissolve it, at which point the solution pH is 12, and then uses dilute hydrochloric acid to adjust the pH to 7 (uric acid precipitates after a period of time). These two methods complete the preparation of the uric acid solution.

[0139] The experimental results showed that strains JSSN-1, PA3-7 and WK all had uric acid degradation effects through method 1, and PA3-7 had a better uric acid reduction effect ( Figure 5 A in the figure), strain PA3-7 screened using the sole carbon source screening medium can reduce uric acid by 33% after 1 hour of incubation; using method 2, all strains have no effect on uric acid degradation ( Figure 5(B in the figure). However, the pH of the human intestinal environment is 7±0.5, and there is no pH of 12. Therefore, the single carbon source method is not suitable for subsequent experiments.

[0140] Example 5 Effect of Glucose on Uric Acid Degradation by Strain PA3-7

[0141] To investigate whether glucose affects the uric acid degradation of 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 the cells were cultured overnight at 37°C and 200 r / min in slightly oxygenated conditions for 16 h. The bacterial concentration (1×10 9 cfu / mL) and centrifuged at 12,000 rpm to obtain the supernatant. After filtration through a 0.22 µm membrane, 20 µL of the mixture was injected into an HPLC apparatus, and the uric acid content in the co-culture solution was measured by HPLC to determine the glucose concentration required for optimal uric acid degradation in BHI medium by strain PA3-7.

[0142] The experimental results are as follows Figure 6 As shown, the strain PA3-7 had the best uric acid degradation effect when 2 g / L glucose was added to the BHI medium without glucose, and when the glucose concentration was above 7 g / L, the strain PA3-7 had no effect on uric acid degradation.

[0143] Because strain PA3-7 grew slowly when inoculated in MRS medium containing 840 µmol / L UA and lacking glucose, to improve growth conditions, the medium was modified to glucose-free 1 / 2 MRS supplemented with 1 g / L, 2 g / L, 5 g / L, 7 g / L, 10 g / L, and 15 g / L of glucose, respectively. The medium was incubated overnight at 37°C at 200 rpm under slightly oxidized conditions (slightly oxidized: 15 ml centrifuge tube + 10 ml medium) for 16 h. Bacterial concentration was determined by measuring OD600 values, and the supernatant was obtained by centrifugation at 12,000 rpm. After filtration through a 0.22 µm filter, 20 μL of the mixture was injected into a HPLC instrument, and the uric acid content in the co-culture solution was measured by HPLC to determine the modified medium that best degraded uric acid by strain PA3-7.

[0144] The experimental results are as follows Figure 7 As shown in the figure, the 1 / 2 MRS medium without glucose had a significant effect on degrading uric acid when 5 g / L glucose was added. Therefore, the 1 / 2 MRS medium without glucose plus 5 g / L glucose was selected as the improved medium for strain PA3-7 to degrade uric acid.

[0145] Example 6 Effect of glycerol on uric acid degradation by strain PA3-7

[0146] Based on Example 1, strain PA3-7 was screened for its excellent in vitro uric acid degradation. A cryovial of strain PA3-7 was inoculated with 500 µL of fermentation broth and 500 µL of glycerol. A single PA3-7 clone was inoculated and cultured at 37°C at 200 rpm under slightly oxygenated conditions (15 mL centrifuge tube + 10 mL culture medium) for 24 hours to obtain a PA3-7 fermentation broth. 10 mL of BHI culture medium containing 420 µmol / L uric acid (0.07 g / L UA) was inoculated with 100 µL of the PA3-7 culture medium, 100 µL of the fermentation broth, 50 µL of glycerol, 50 µL of the fermentation broth, and 50 µL of the fermentation broth. Cultures were incubated overnight at 37°C at 200 rpm under slightly oxygenated conditions (15 mL centrifuge tube + 10 mL culture medium) for 16 hours. The bacterial concentration (1 × 10) was determined by measuring the OD600 value. 9 cfu / mL) and centrifuged at 12,000 rpm to obtain the supernatant. After filtration through a 0.22 µm filter membrane, 20 µL of the supernatant was injected into an HPLC assay tube, and the uric acid content in the co-culture solution was measured by HPLC.

[0147] The experimental results are as follows Figure 8 As shown in the figure, strain PA3-7 fermentation broth supplemented with glycerol and inoculated with cryovials preserved with glycerol showed better uric acid degradation in both experimental groups. However, inoculation of BHI medium with only PA3-7 fermentation broth at different inoculum volumes showed poor results. This suggests that glycerol has a certain promoting effect on uric acid degradation by strain PA3-7.

[0148] Example 7 In vitro uric acid metabolism test in rats by pre-intervention with uric acid feed

[0149] Twenty-four 8-week-old male Wistar rats weighing 220–260 g were purchased. All rats were tested after a week of acclimation. During the experiment, the rats were fed a standard commercial rat chow diet. Four mice per cage were housed at 22°C ± 2°C and a relative humidity of 30%–70%.

[0150] Rats were randomly divided into 3 groups, 8 in each group, including blank group, model group and experimental group, to ensure that there was no significant difference in body weight between the groups. Starting from D1, the blank control group was fed with normal feed, the model group was fed with 2% uric acid and 3% potassium oxonate feed every day, and the experimental group was fed with 2% uric acid and 3% potassium oxonate feed. After that, each rat was gavaged with 1 ml of bacterial powder (1×10 10 cfu / mL). Thereafter, the rats in the experimental group were gavaged once a day for 28 days.

[0151] Blood was drawn from the rat tail vein and centrifuged at 12,000 rpm for 5 minutes. The upper serum layer was collected, diluted 10-fold with PBS, and then vortexed and mixed. The blood was centrifuged at 12,000 rpm for 5 minutes. 20 μL of the supernatant was injected into an HPLC test tube, and the uric acid content in the serum was measured by HPLC (the normal serum uric acid concentration of Wistar male rats is 100-300 μmol / L).

[0152] The experimental results are as follows Figure 9 As shown in A and B, after rats consumed strain PA3-7, the serum uric acid content was significantly decreased compared with the model group ( Figure 9 B in the figure); among them, there were extremely significant differences between the 7th, 17th and 21st day groups and the model group (P < 0.01) ( Figure 9 A in the figure), uric acid degradation can reach 58.98%.

[0153] In addition, the results Figure 10 As shown, strain PA3-7 has a significant effect on degrading uric acid in rats, which fully demonstrates that strain PA3-7 has a good preventive effect on rats with high serum uric acid, and the weight of rats shows a steady growth trend.

[0154] Example 8 Analysis of renal pathology after pre-intervention of uric acid metabolism in rats by ingestion of uric acid feed

[0155] Rats were dissected, and kidneys were collected and rinsed with phosphate-buffered saline before being fixed in 4% paraformaldehyde in the dark. The fixed tissues were dehydrated in an automated dehydrator and then paraffin-embedded, sectioned, dewaxed, and stained with hematoxylin and eosin (H&E) for histopathological analysis. Kidney sections were examined using an automated upright fluorescence microscope.

[0156] After 4 weeks of feeding rats with 2% uric acid and 3% potassium oxonate, histopathological analysis showed that the glomeruli of the blank control group were intact and had clear outlines. In contrast, in the model group, obvious tubular dilatation, flattened or even detached tubular epithelial cells were observed in the kidneys of the rats (black arrows). At the same time, there was very severe inflammatory cell infiltration in the renal interstitium (red arrows), and the infiltration accounted for more than 80%, indicating that the kidneys of the rats in the model group were severely damaged. In the experimental PA3-7 group, the rat kidneys had only slight tubular dilatation (black arrows), and no detachment of renal tubular epithelial cells was observed. Although there was also inflammatory cell infiltration in the renal interstitium (red arrows), the infiltration 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 ).

[0157] According to the kidney pathological section scoring (scoring basis: immune cell infiltration ratio, 0 points: no obvious immune cell infiltration; 1 point: immune cell infiltration ratio 0-20%; 2 points: immune cell infiltration ratio 20-40%; 3 points: immune cell infiltration ratio 40-60%; 4 points: immune cell infiltration ratio 60-80%; 5 points: immune cell infiltration ratio 80-100%), the PA3-7 group had a significant protective effect compared with the model group ( Figure 11 ).

[0158] Example 9: In vitro uric acid metabolism test in rats after ingestion of uric acid feed model

[0159] Forty-two 8-week-old male Wistar rats weighing 220-260 g were purchased. All rats were tested after a week of acclimation. During the experiment, the rats were fed a standard commercial rat chow diet. Three rats per cage were housed in an environment with a temperature of 22°C ± 2°C and a relative humidity of 30%–70%. The rats were randomly divided into four groups: a blank control group, a model group, an experimental group, and a positive drug group. The blank control group consisted of 6 rats, and each of the other groups consisted of 12 rats. No significant differences in body weight were observed between the groups.

[0160] Prior to the experiment, all rats, except the blank control group, were fed a diet containing 2% uric acid and 3% potassium oxonate for 5 consecutive days to establish a hyperuricemia model in rats. The blank control group was fed a normal diet, while the model group was fed a diet containing 2% uric acid and 3% potassium oxonate daily. The experimental group was fed a diet containing 2% uric acid and 3% potassium oxonate daily, followed by oral gavage of 1 ml of bacterial powder (1e10 cfu). The positive drug group was also given 50 mg / kg benzbromarone based on body weight. Thereafter, rats in the experimental and positive drug groups were gavaged once daily for 21 days.

[0161] Blood was drawn from the rat tail vein and centrifuged at 12,000 rpm for 5 minutes. The upper serum layer was collected, diluted 10-fold with PBS, and then vortexed and mixed. The blood was centrifuged at 12,000 rpm for 5 minutes. 20 μL of the supernatant was injected into an HPLC test tube, and the uric acid content in the serum was measured by HPLC (the normal serum uric acid concentration of Wistar male rats is 100-300 μmol / L).

[0162] The experimental results showed that when the serum uric acid level in rats was high, the serum uric acid level in the group gavaged with strain PA3-7 and positive drug was significantly lower than that in the model group, with a very significant difference from the model group (P < 0.01) ( Figure 13 ), which fully proves that strain PA3-7 has a good therapeutic effect on rats with high serum uric acid.

[0163] Example 10 Uric acid metabolism test in rats by intragastric administration of uric acid

[0164] Forty-two 8-week-old male Wistar rats weighing 220-260 g were purchased. All rats were tested after a one-week acclimatization period. During the experiment, the rats were fed a standard commercial rat chow diet. Three rats per cage were housed in an environment with a temperature of 22°C ± 2°C and a relative humidity of 30%–70%. The rats were randomly divided into four groups (8 rats per group): a blank control group, a model group, an experimental group, and a positive drug group. No significant differences in body mass were observed between groups.

[0165] Rats in the blank control group were fed a normal diet. Rats in the model group were gavaged with 1 g / kg uric acid and 1.5 g / kg potassium oxonate according to body weight. Rats in the experimental group were gavaged with 1 g / kg uric acid and 1.5 g / kg potassium oxonate according to body weight, and then 1 ml bacterial powder (1e10 cfu) was gavaged each. Rats in the positive drug group were gavaged with 1 g / kg uric acid and 1.5 g / kg potassium oxonate according to body weight, and then 50 mg / kg benzbromarone was fed according to body weight.

[0166] Blood was drawn from the rat tail vein at 0 h, 3 h, and 6 h after gavage, and the blood was centrifuged at 12,000 rpm for 5 min. The upper serum was collected, diluted 10-fold with PBS, and then mixed by vortexing. The supernatant was centrifuged at 12,000 rpm for 5 min, and 20 μL of the supernatant was injected into an HPLC detection tube. The uric acid content in the serum was measured by HPLC (the normal serum uric acid concentration of Wistar male rats is 100-300 μmol / L).

[0167] The experimental results showed that compared with the model group, rats significantly degraded uric acid after consuming the positive drug benzbromarone, indicating that the model was successfully established. When the serum uric acid level in rats was high, the serum uric acid level in the experimental group decreased significantly on the 4th and 7th days after oral administration of PA3-7 compared with the model group, which was slightly better than the positive drug benzbromarone ( Figure 14 A, B and C in the sample), which fully proves that strain PA3-7 has a good therapeutic effect on rats with high serum uric acid.

[0168] strain collection

[0169] The present invention Pediococcus acidilactici ( Pediococcus acidilactici ) PA3-7, deposited in the China Center for Type Culture Collection (CCTCC) (Wuhan, China) on February 19, 2025, with the accession number: CCTCC NO. M 2025250.

[0170] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A Pediococcus acidilactici, characterized in that The lactic acid bacteria is lactic acid bacteria PA3-7 ( Pediococcus acidilactici PA3-7), and the lactic acid Pediococcus PA3-7 has the effects of degrading uric acid and protecting the kidneys; wherein the preservation number of the lactic acid Pediococcus PA3-7 is CCTCC NO. M 2025250; And the Pediococcus acidilactici has the following characteristics: (a) the Pediococcus acidilactici effectively reduces uric acid content in a complex environment close to intestinal components; and (b) The addition of glycerol significantly promoted the uric acid-lowering effect of Pediococcus acidilactici.

2. The Pediococcus acidilactici according to claim 1, wherein The Pediococcus acidilactici PA3-7 is separated from pulp water.

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 according to claim 1; 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, wherein The composition contains 1×10-1×10 20 cfu / mL or cfu / gPA3-7 ， Based on the total volume or weight of the composition.

6. The composition according to claim 3, wherein The composition contains 1×10 4 -1×10 15 cfu / mL or cfu / gPA3-7, based on the total volume or weight of the composition.

7. The composition according to claim 3, wherein The composition further comprises: (c) glycerol.

8. The use of the Pediococcus acidilactici according to claim 1 or the composition according to claim 3, characterized in that: For use in the preparation of a medicament for one or more uses selected from the group consisting of: (a) Protect kidneys; (b) Prevention and / or treatment of hyperuricemia.

9. A method for degrading uric acid in vitro, characterized in that: The Pediococcus acidilactici according to claim 1 or the composition according to claim 3 is co-cultured with a uric acid-containing and nutrient-rich culture medium.

10. A method for preparing the composition according to claim 3, characterized in that: Including steps: The Pediococcus acidilactici according to claim 1 is mixed with a pharmaceutically acceptable carrier to form the composition according to claim 3.

11. A production method, characterized in that: Including steps: (a) culturing the Pediococcus acidilactici according to claim 1 under conditions suitable for culture to obtain a culture; (b) isolating the Pediococcus acidilactici cells from the culture product; and (c) mixing the Pediococcus acidilactici cells isolated in the previous step with a pharmaceutically acceptable carrier to prepare a composition.

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