Lactobacillus plantarum LL20, bacterial agent, preparation method and use thereof

Abstract

The application discloses a lactobacillus plantarum LL20, a bacterial agent, a preparation method and application, and belongs to the technical field of microorganisms.The lactobacillus plantarum LL20 has been preserved in the China Center for Type Culture Collection on December 4, 2023, has a preservation number of CCTCC NO: M 20232427, and has a preservation address of Wuhan, China, Wuhan University.The application solves the problem that traditional probiotics have limited effects on reducing blood uric acid levels and improving related inflammation.The lactobacillus plantarum LL20 has the ability to directly degrade uric acid, has strong intestinal colonization ability and probiotic properties, can improve blood uric acid, creatinine and urea levels in the body, protect the liver and kidneys, improve intestinal flora and body metabolism levels, can significantly reduce uric acid, and has the effect of preventing gout.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Lactobacillus plantarum LL20, its inoculum, preparation method, and uses. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disorder caused by purine metabolism disorder, resulting in a blood urate concentration exceeding normal levels. HUA is defined as a fasting blood uric acid level >420 umol / L for men and >360 umol / L for women on two separate occasions under normal purine diet conditions. The saturation concentration of urate in the blood is 420 umol / L (regardless of gender). Exceeding this value can cause urate crystals to precipitate and directly adhere to and deposit in joints and surrounding soft tissues, renal tubules, and blood vessels. Simultaneously, the interaction between various immune cells and crystals releases inflammatory cytokines, leading to a series of clinical manifestations such as arthritis, subcutaneous gouty tophi, kidney stones or gouty nephropathy, and vascular endothelial damage.

[0003] High blood pressure, blood sugar, and cholesterol (HUA) have become the "fourth highest" disease associated with affluence. HUA is not only a major risk factor for gout, but it can also lead to various complications such as high blood sugar, high cholesterol, high blood pressure, obesity, cardiovascular disease, diabetes, chronic kidney disease, and kidney stones. These coexisting diseases increase the complexity of HUA and gout management and may even lead to premature death in patients. Effective prevention and treatment of HUA and gout have attracted significant attention.

[0004] Existing research indicates that uric acid is the final product of purine metabolism in the liver. Human uric acid originates from two sources: (1) endogenous uric acid (approximately 80%), generated from nucleic acids and other purine compounds produced by the breakdown and metabolism of nucleoproteins in human cells through the action of certain enzymes; and (2) exogenous uric acid (approximately 20%), generated from purine compounds (nucleic acids and nucleoproteins) in food after digestion and absorption through the action of certain enzymes. Two-thirds of the endogenously formed uric acid is excreted through the kidneys, while the remaining one-third is excreted through the intestines. Some of the excreted uric acid is reabsorbed by the intestines via transport proteins. Therefore, reducing uric acid formation in the liver and reabsorption in the kidneys, and increasing the breakdown and excretion of uric acid in the intestines, is a feasible way to lower blood uric acid levels.

[0005] Currently, uric acid-lowering drugs on the market are mainly divided into two categories: xanthine oxidase (XOD) inhibitors, such as allopurinol (AP), and uricosuric agents, such as benzbromarone. These drugs relieve the symptoms of hyperuricemia by reducing uric acid production or promoting uric acid excretion. However, these traditional methods have problems such as low tolerability, drug dependence, significant side effects, and difficulty in dietary control. In addition, only about 20% of patients with hyperuricemia eventually develop gout, so the balance between the risks and benefits of drug treatment remains controversial for most patients. Therefore, it is urgent to develop safe, effective, and side-effect-free methods for the prevention and relief of hyperuricemia and gout.

[0006] The human gut microbiota, composed of at least 1,000 different types of bacteria, archaea, eukaryotic microorganisms, and viruses, with a total of over 3 million genes, possesses powerful metabolic capabilities and plays a crucial role in nutrient absorption, energy acquisition, inflammation regulation, and immune response. Numerous studies have shown that specific probiotics can alleviate hyperuricemia (HUA) and related inflammatory responses to varying degrees through antioxidant mechanisms, enhanced intestinal barrier function, regulation of immune responses, and improvement of gut microbiota composition. Further research has revealed significant differences between the gut microbiota of HUA patients and healthy individuals, indicating that gut microbiota plays a key role in the occurrence and development of HUA and metabolic diseases such as gout. Microbial intervention, as a cost-effective and low-side-effect treatment, has gradually gained acceptance. However, traditional probiotics have limited effectiveness in lowering serum uric acid levels and improving related inflammation.

[0007] Therefore, it is of great significance to screen for lactic acid bacteria from food that can degrade uric acid in vitro, are safe, have strong probiotic capabilities, have strong intestinal colonization capabilities, and can also improve blood uric acid, creatinine, and urea levels in vivo in the HUA model, similar to allopurinol, protect the liver and kidneys, and improve intestinal flora and body metabolism.

[0008] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0009] The purpose of this invention is to provide a *Lactobacillus plantarum* LL20, its preparation method, and its uses, which solves the problem that traditional probiotics have limited effectiveness in lowering blood uric acid levels and improving related inflammation. The *Lactobacillus plantarum* LL20 of this invention has the ability to directly degrade uric acid, has strong intestinal colonization ability and probiotic properties, and can also improve blood uric acid, creatinine, and urea levels in vivo, protect the liver and kidneys, improve intestinal flora and body metabolism, significantly reduce uric acid, and has the effect of preventing gout.

[0010] To achieve the above objectives, the present invention provides a *Lactobacillus plantarum* (… Lactiplantibacillusplantarum Lactobacillus plantarum LL20 was deposited on December 4, 2023, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20232427 and address: Wuhan University, Wuhan, China.

[0011] A second objective of this invention is to provide a microbial agent containing the aforementioned *Lactobacillus plantarum* LL20.

[0012] Preferably, the microbial agent is a liquid, emulsion, suspension, granule, powder, wettable powder, or water dispersible agent. In the microbial agent, *Lactobacillus plantarum* LL20 can exist as live bacteria or in the form of a fermentation broth containing live bacteria.

[0013] Preferably, the microbial agent also includes pharmaceutically, food-grade, or feed-grade excipients.

[0014] More preferably, the excipient is selected from at least one of trehalose, hydroxymethyl cellulose, dextrin, soluble starch, fructooligosaccharides, isomaltooligosaccharides and stachyose.

[0015] More preferably, the *Lactobacillus plantarum* LL20 is mixed with a pharmaceutically, food-grade, or feed-grade acceptable soluble starch excipient at a mass ratio of 4:1 to prepare a powder, with each gram of powder containing 1.0 × 10⁻⁶ viable bacteria. 8 CFU.

[0016] A third objective of this invention is to provide a method for preparing the aforementioned microbial agent, the method comprising: inoculating the seed culture of Lactobacillus plantarum LL20 into MRS liquid culture medium, fermenting in an anaerobic environment at 36-38°C for 30-40 hours, centrifuging after fermentation, collecting the precipitate, and drying it to prepare the microbial agent.

[0017] Preferably, the viable count in the bacterial agent is 1.0 × 10⁻⁶. 8 CFU or above.

[0018] Preferably, the MRS liquid culture medium comprises: 10 g / L casein peptone, 5 g / L yeast extract, 10 g / L beef extract, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate, 0.2 g / L magnesium sulfate, 0.2 g / L manganese sulfate, 1.0 mL / L Tween 80, 20 g / L glucose, and 20 g / L agar, with a pH of 6.8.

[0019] Preferably, the dried precipitate is prepared into a corresponding microbial agent with excipients.

[0020] More preferably, the dried precipitate is mixed with a pharmaceutically, food-, or feed-acceptable soluble starchy excipient to prepare a bacterial powder; or, the dried precipitate, skim milk powder, and solvent are formulated into a concentrated bacterial solution.

[0021] A fourth object of the present invention is to provide a microbial agent containing the aforementioned *Lactobacillus plantarum* LL20, or the use of the aforementioned microbial agent in the preparation of drugs for treating hyperuricemia or gout, or in the preparation of functional foods that promote gut microbiota. The use of *Lactobacillus plantarum* LL20 of the present invention in the preparation of drugs for treating hyperuricemia or gout is significantly superior to allopurinol.

[0022] Preferably, it is selected from at least one of the following: (1) Use in the preparation of a medicine for reducing at least one of uric acid, creatinine and urea in the serum of patients or animals with hyperuricemia or gout; (2) Use in the preparation of a medicine for relieving at least one injury to the kidney or liver in patients with hyperuricemia or gout or diseased animals; (3) Use in the preparation of drugs for restoring metabolism in patients or animals with hyperuricemia or gout; (4) Use in the preparation of drugs that improve the intestinal colonization capacity and probiotic properties of patients or animals with hyperuricemia or gout.

[0023] More preferably, the probiotic properties include the ability to produce acid, improve the intestinal flora structure, increase the abundance of beneficial bacteria, and inhibit the proliferation of pathogenic bacteria; and / or, the restoration of metabolism in patients or animals with hyperuricemia or gout includes downregulating pro-inflammatory metabolites and upregulating anti-inflammatory and antioxidant metabolites. More preferably, the *Lactobacillus plantarum* LL20 increases the proportion of Firmicutes, increases the abundance of beneficial bacteria containing the Lachnospiraceae NK4A136 group, and reduces the abundance of potentially harmful bacteria containing Desulfobacterota; and / or, the *Lactobacillus plantarum* LL20 downregulates uric acid and related purine (purine, xanthine) metabolites, and upregulates L-tryptophan derivatives and bile acids.

[0024] The present invention relates to Lactobacillus plantarum LL20, its bacterial agent, preparation method, and uses, which solves the problem that traditional probiotics have limited effectiveness in lowering blood uric acid levels and improving related inflammation, and has the following advantages: (1) The *Lactobacillus plantarum* LL20 of this invention can directly degrade uric acid in vitro. Through multiple safety evaluations, including antibiotic sensitivity testing, it can inhibit the growth of various pathogens and exhibits good intestinal colonization and probiotic properties. Furthermore, in a hyperuricemia model, oral administration of *Lactobacillus plantarum* LL20 to rats significantly improved their serum uric acid, creatinine, and urea levels, with improvements comparable to those of positive control drugs. Simultaneously, *Lactobacillus plantarum* LL20 has a protective effect on the liver and kidneys of rats in a hyperuricemia model and can improve the intestinal flora and metabolic levels of rats in this model, with both protective and improvement effects superior to those of the positive control drug, allopurinol. Therefore, it can serve as a novel measure to alleviate hyperuricemia and treat gout, and has broad application prospects in the prevention and adjuvant treatment of hyperuricemia.

[0025] (2) The Lactobacillus plantarum LL20 of the present invention is derived from Lanzhou fermented vegetable juice, a natural food. It is a recognized safe edible fungus with no obvious toxic side effects and will not produce additional adverse reactions to the human body. It can meet the long-term intervention needs of patients with hyperuricemia and gout. Attached Figure Description

[0026] Figure 1 This invention relates to the detection of the ability of Lactobacillus plantarum LL20 to directly degrade uric acid in vitro.

[0027] Figure 2 This is a colony morphology diagram of Lactobacillus plantarum LL20, which is the present invention.

[0028] Figure 3 This invention relates to the detection of antibiotic resistance in Lactobacillus plantarum LL20.

[0029] Figure 4 This invention relates to the effect of Lactobacillus plantarum LL20 on the activity of human colorectal cancer cells HT-29.

[0030] Figure 5 The present invention is used to detect the intestinal colonization ability of Lactobacillus plantarum LL20 compared with other test strains; (A) is used to evaluate the acid resistance of the strain; (B) is used to evaluate the self-aggregation ability of the strain.

[0031] Figure 6 This invention aims to determine the acid-producing capacity of Lactobacillus plantarum LL20 compared to other test strains.

[0032] Figure 7 This is a flowchart illustrating the administration of Lactobacillus plantarum LL20 and the positive control drug allopurinol to a rat model of hyperuricemia.

[0033] Figure 8 The present invention describes the effects of Lactobacillus plantarum LL20 and the positive control drug allopurinol on the serum uric acid (A), creatinine (B), and urea (C) in a rat model of hyperuricemia on days 7, 14, and 21 via the tail vein.

[0034] Figure 9 The image shows the effects of *Lactobacillus plantarum* LL20 and the positive control drug allopurinol on the histological lesions of the kidneys in a rat model of hyperuricemia. The top image is a 100x magnified image, and the bottom image is a 400x magnified image.

[0035] Figure 10 The effects of Lactobacillus plantarum LL20 and the positive control drug allopurinol on liver biochemical indicators in a rat model with hyperuricemia on days 7, 14 and 21 are shown in (A) for ALT content and (B) for AST content.

[0036] Figure 11 The image shows the effects of *Lactobacillus plantarum* LL20 and the positive control drug allopurinol on the histological lesions of the liver in a rat model of hyperuricemia. The top image is a 100x magnified image, and the bottom image is a 400x magnified image.

[0037] Figure 12 The effects of Lactobacillus plantarum LL20 and the positive control drug allopurinol on the fecal microbial 16sα index of a rat model with hyperuricemia are presented in this invention. (A) is the ACE index; (B) is the Chao1 index; (C) is the Simpson index; and (D) is the Shannon index.

[0038] Figure 13 The effects of Lactobacillus plantarum LL20 and the positive control drug allopurinol on the fecal microbial 16sβ index of a rat model with high uric acid are shown in the figure. (A) is a graph of PCA analysis results; (B) is a graph of beta index distance.

[0039] Figure 14 The effects of Lactobacillus plantarum LL20 and the positive control drug allopurinol on the fecal microbial structure of a rat model with hyperuricemia are presented in this invention; (A) is a phylum; (B) is a genus.

[0040] Figure 15 The effects of Lactobacillus plantarum LL20 and the positive control drug allopurinol on the serum metabolome of rats with hyperuricemia are presented in this invention; (A) is PCA analysis; (B) is PLS-DA analysis; (C) is PLS-DA Splot analysis.

[0041] Figure 16 This invention relates to the effects of *Lactobacillus plantarum* LL20 and the positive control drug allopurinol on the serum metabolome structure of a rat model with hyperuricemia. (A) Comparative analysis between the model group and the control group; (B) Comparative analysis between allopurinol and the control group; (C) Comparative analysis between *Lactobacillus plantarum* LL20 and the control group; (D) Comparative analysis between allopurinol and the model group; (E) Comparative analysis between *Lactobacillus plantarum* LL20 and the model group; (F) Comparative analysis between *Lactobacillus plantarum* LL20 and allopurinol. Detailed Implementation

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] It should be noted that: Unless otherwise specified in the examples, conditions should be followed according to standard conditions or the manufacturer's recommendations. Instruments whose manufacturers are not specified are all commercially available products. Raw materials and reagents whose manufacturers are not specified are all commercially available goods or can be prepared using known methods.

[0044] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0045] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0046] Fermented vegetable juice (jiangshui) is a traditional fermented food, rich in probiotics, primarily lactic acid bacteria. These bacteria produce organic acids and antimicrobial peptides, which inhibit putrefactive and pathogenic bacteria. Due to differences in region, climate, and production processes, the lactic acid bacteria communities in different fermented vegetable juice products vary significantly. Some of these lactic acid bacteria may possess potential probiotic functions, contributing to human health. This invention provides a *Lactobacillus plantarum* LL20 derived from fermented vegetable juice. Studies have shown that this strain has significant uric acid-lowering and anti-inflammatory effects. By regulating the intestinal flora structure, inhibiting uric acid production, and promoting uric acid metabolism, it effectively reduces blood uric acid levels and alleviates inflammatory responses caused by hyperuricemia, providing a safe and efficient microbial intervention for the prevention and treatment of hyperuricemia.

[0047] The following examples provide a detailed description of the Lactobacillus plantarum LL20, the inoculant, and its uses provided by the present invention.

[0048] The culture media used in the following examples are as follows: MRS solid culture medium, prepared as follows: 10 g / L casein peptone, 5 g / L yeast extract, 10 g / L beef extract, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate, 0.2 g / L magnesium sulfate, 0.2 g / L manganese sulfate, 1.0 mL / L Tween 80, 20 g / L glucose, and 20 g / L agar, pH=6.8, 1.0 L ultrapure water, pH=6.8, sterilization conditions: 121℃, 15 min.

[0049] MRS liquid culture medium is prepared in the same way as the MRS solid culture medium described above, except that agar is not added.

[0050] Example 1 Lactobacillus plantarum ( Lactiplantibacillus Obtaining plantarum LL20 1. Preparation of diluent Take 1 mL of fresh Lanzhou rice water and dilute it to 10 mL with sterile 0.85% saline solution. 0 10 -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 There were seven concentration gradients in total, with each gradient tested in triplicate. The samples in the dilutions of different concentration gradients were thoroughly mixed before proceeding to the next step of the experiment.

[0051] 2. Cultivation Take 100 μL of each dilution solution in ascending order of concentration and inoculate it into MRS solid culture medium plates, spreading them evenly using a sterile spreader. After absorption, place them in a 37°C incubator for anaerobic incubation for 24–48 h.

[0052] 3. Isolation, purification and preliminary identification of strains Anaerobically cultured on MRS solid medium at 35°C for 24 h. Single colonies were picked using an inoculation needle and streaked continuously on the surface of MRS solid medium plates. Plates with 30–200 colonies were selected. Using a sterile inoculation loop, white, smooth, round colonies with a diameter of 1–3 mm and regular edges, conforming to the standard morphology of lactic acid bacteria, were picked and dissolved in 200 μL of sterile PBS buffer (pH 7.2–7.4). After mixing, the mixture was streaked on the surface of MRS solid medium and anaerobically cultured at 37°C for 24 h. This picking and streaking process was repeated three times. A single colony was then dissolved in 200 μL of sterile PBS solution, mixed, and injected into 10 mL of MRS liquid medium using a disposable sterile syringe. The mixture was then anaerobically cultured overnight at 37°C and 180 rpm in a horizontal shaker.

[0053] By observing the colony characteristics, the complex colonies were white or milky white in color, round in shape, with neat edges and a moist and smooth surface. The catalase test was positive, and the bacteria were preliminarily identified as lactic acid bacteria. Twenty-eight pure culture strains were obtained for testing, namely LL1~LL28. At the same time, one strain of common Lactobacillus plantarum Lac16 was retained as a control group.

[0054] 4. Evaluation of the strain's ability to directly lower uric acid Collect the bacterial culture obtained in the previous step by centrifugation at 8000 r / min for 20 min, and resuspend the cells in sterile PBS buffer solution. Repeat this process three times. Add all the bacterial cultures to be tested to MRS liquid medium containing 1 mmol / L uric acid, and adjust the OD of all liquid media. 600 The concentration was adjusted to 0.5 (±0.05). A blank control (without bacterial cells) and a positive control (Lac16) were used. The mixture was incubated at 37 ℃ and 100 r / min on a shaker for 2–5 h, followed by centrifugation at 8000 r / min for 20 min. The supernatant was collected, and the uric acid content was determined according to the uric acid kit results, following the manufacturer's instructions. The uric acid degradation rate was calculated using the uric acid degradation rate formula. The results are as follows: Figure 1 As shown.

[0055] Uric acid degradation rate (%) = (final concentration of uric acid in standard solution - final concentration of uric acid in test solution) / final concentration of uric acid in standard solution × 100 Based on the results of the uric acid assay kit, strain LL20 exhibited the strongest in vitro uric acid-lowering ability. Considering the overall screening results, LL20 was selected as the subject of subsequent research. After activation, LL20 was cultured in 50 mL of MRS liquid medium with an initial inoculum concentration of 1.0 × 10⁻⁶. 6 CFU / mL, and after 24 hours, adjust each bacterial culture to 1.0 × 10⁻⁶ CFU / mL using MRS liquid medium. 9 After subculturing 2-3 times with CFU / mL in MRS medium, LL20 culture medium was obtained. The culture medium was then streaked onto plates, and LL20 colonies were detected. Figure 2 As shown.

[0056] 5. Safety evaluation of the strain (1) Evaluation of antibiotic resistance After activation, strain LL20 was cultured in 50 mL of MRS liquid medium with an initial inoculum concentration of 1.0 × 10⁻⁶. 6 CFU / mL, and after 24 hours, adjust each bacterial culture to 1.0 × 10⁻⁶ CFU / mL using MRS liquid medium. 9After subculturing the strain LL20 2-3 times in MRS medium at CFU / mL, the culture medium was obtained. The LL20 culture medium was inoculated at 5‰ and poured into sterile MRS solid medium at approximately 40°C without solidification. The mixture was then poured into petri dishes, and antibiotic susceptibility testing tablets (GEN: gentamicin, NAL: nalidixic acid, AMX: amoxicillin, CTR: ceftriaxone, AMP: ampicillin) were added. The dishes were then incubated overnight in an anaerobic incubator at 37°C.

[0057] The results are as follows Figure 3 As shown, strain LL20 is sensitive to common antibiotics at working concentrations.

[0058] (2) CCK-8 cell viability detection Human colorectal cancer cell line (HT-29) was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HT-29 cells were incubated in RPMI 1640 (HyClone, UT, USA) medium at 37°C in a humidified environment of 5% CO2. The medium contained 10% fetal bovine serum (FBS) (Gibco, Grand Island, NE, USA) and 1% antibiotics (100 mg / mL streptomycin and 100 U / mL penicillin). The concentration of HT-29 intestinal cells was adjusted to 2 × 10⁶ cells / mL using RPMI 1640 cell culture medium. 5 Cells were seeded at 1 × 10⁻⁶ cells / mL in 24-well cell culture plates. When HT-29 cells were in the exponential growth phase, the cells were diluted to 1 × 10⁻⁶ cells / mL. 5 Cells were inoculated at 100 μL / well in 96-well plates and pre-cultured at 37 °C and 5% CO2 for 24 h. Then, 1 mL of LL20 treatment solution was inoculated into each well. This treatment solution was prepared by activating strain LL20 and then culturing it in 50 mL of MRS liquid medium at an initial inoculation concentration of 1.0 × 10⁻⁶ cells / mL. 6 CFU / mL, and after 24 hours, adjust each bacterial culture to 1.0 × 10⁻⁶ CFU / mL using MRS liquid medium. 9 After subculturing 2-3 times in MRS medium with CFU / mL, strain LL20 culture medium was obtained. This culture medium was used as the stock solution, and the strains were divided into the following groups: control group, 10... 1 CFU / mL LL20 treatment group, 10 2 CFU / mL LL20 treatment group, 10 3 CFU / mL LL20 treatment group, 10 4 CFU / mL LL20 treatment group, 10 5 CFU / mL LL20 treatment group, 10 6 CFU / mL LL20 treatment group, 10 7CFU / mL LL20 treatment group, 10 8 CFU / mL LL20 treatment group and 10 9 The CFU / mL LL20 treatment group was prepared by corresponding dilution of the stock solution in antibiotic-free RPMI 1640 cell culture medium to obtain different concentrations of LL20 treatment solutions. 10 μL of each concentration of LL20 treatment solution was used to treat cells for 24 h. After culture, the supernatant was discarded, and the cells were washed three times with sterile PBS. 10 μL of CCK-8 solution was added to each well, and the cells were pre-cultured for 4 h. The absorbance was measured at 450 nm using a microplate reader. The cell proliferation-toxicity activity calculation formula is as follows:

[0059] Cell viability = (A with added drug - A blank) / (A0 with added drug - A blank) × 100% In the formula: Adrug is the absorbance of the pore containing cells, CCK-8 solution and drug solution; A0 is the absorbance of the pore containing cells and CCK-8 solution but without drug solution; Ablank is the absorbance of the pore containing culture medium and CCK-8 solution but without cells.

[0060] The results are as follows Figure 4 As shown, treatment with different concentrations of LL20 did not significantly inhibit the activity (toxicity) of HT-29 intestinal cells. These results demonstrate the strong safety profile of LL20.

[0061] 5. Intestinal colonization ability of the strain The selected colonies of LL12, LL16, LL17, LL18, and LL20 were activated and cultured in 50 mL of MRS liquid medium with an initial inoculum concentration of 1.0 × 10⁻⁶. 6 CFU / mL, and after 24 hours, adjust each bacterial culture to 1.0 × 10⁻⁶ CFU / mL using MRS liquid medium. 9 After subculturing in MRS medium for 2-3 times at CFU / mL, culture media of the test strains LL12, LL16, LL17, LL18 and LL20 were obtained.

[0062] (1) Strain acid resistance After activation, the test strain was anaerobically cultured overnight at 37°C, centrifuged at 4000 rpm for 20 min, and the supernatant was discarded. This step was repeated twice in PBS buffer. The strain was centrifuged again at 4000 rpm for 20 min, the supernatant was discarded, and the precipitate was resuspended in acidic MRS (pH=3) (using 4M HCl to adjust the ordinary MRS liquid medium) and ordinary MRS liquid medium, and incubated at 37°C for 3 h. After incubation, 0.1M PBS (pH 6.2) was added to neutralize the acidic culture, and the culture was serially diluted 10-fold before plating. Colony counting was performed after incubation at 37°C for 48 h. Acid tolerance is expressed as the percentage of viable cells relative to the control after culture in MRS broth (pH 3.0), and calculated using the following equation:

[0063] Acid resistance (%) = (Log colony count after 3 hours of exposure) / (Log initial colony count) × 100.

[0064] In the formula, the colony count is expressed in CFU / mL.

[0065] The results are as follows Figure 5 As shown in Figure A, it can be seen that the five tested strains all had a survival rate of over 90% after 3 hours in a medium with pH=3.0 and over 85% after 3 hours in a medium with pH=2.0. Among them, LL20 showed the strongest acid resistance, with survival rates of 96% and 91% after 3 hours in mediums with pH=3.0 and pH=2.0, respectively, demonstrating strong acid resistance.

[0066] (2) Adhesion ability of strains The activated bacterial strain was anaerobically cultured overnight at 37°C, centrifuged at 4000 rpm for 20 min, and the supernatant was discarded. The suspension was then resuspended in PBS buffer, and this step was repeated twice. After centrifugation at 4000 rpm for 20 min again, the supernatant was discarded, and the precipitate was resuspended in PBS solution until the absorbance at 600 nm reached 0.5 ± 0.02. 1 mL of the bacterial suspension was vortexed for 10 s and incubated at 37°C. The supernatant was carefully aspirated at 12 h and 24 h, and its absorbance at 600 nm was measured. The self-aggregation formula is:

[0067] Self-aggregation rate (%) = (1 − absorbance after incubation / initial absorbance) × 100 The results are as follows Figure 5 As shown in Figure B, all five tested strains exhibited strong self-aggregation abilities at 12 hours, with LL18 and LL20 showing the best self-aggregation abilities. Even after 24 hours, the self-aggregation rate did not significantly decrease. These experimental results indicate that LL20 possesses strong acid resistance and self-aggregation capabilities, suggesting a strong potential for intestinal colonization.

[0068] 6. Probiotic ability of bacterial strains The selected colonies of LL12, LL16, LL17, LL18, and LL20 were activated and cultured in 50 mL of MRS liquid medium with an initial inoculum concentration of 1.0 × 10⁻⁶. 6 CFU / mL, and after 24 hours, adjust each bacterial culture to 1.0 × 10⁻⁶ CFU / mL using MRS liquid medium. 9 After subculturing in MRS medium for 2–3 times at CFU / mL, culture media for the test strains LL12, LL16, LL17, LL18, and LL20 were obtained. From the start of inoculation, 5 mL samples were carefully taken from the original culture medium every 2 hours, and the pH of the fermentation broth was measured using a pH meter, with three parallel measurements performed.

[0069] The results are as follows Figure 6 As shown, the five strains have good acid production efficiency, and the pH drops below 5.0 after 6 hours. LL20 has the strongest acid production capacity, which matches its strong acid resistance and indicates that LL20 has high probiotic capacity.

[0070] 8. Strain identification (1) Morphological characteristics In liquid culture medium, it presents as a uniform turbidity with a small amount of precipitate, which disperses easily with gentle shaking. Colonies are white or milky white, round in shape, with neat edges and a moist, smooth surface.

[0071] (2) 16S rRNA sequence analysis Further 16S rRNA sequence analysis and alignment were performed on the isolated and screened strain LL20. A pair of primers was designed based on the conserved sequence of bacterial 16S rDNA and synthesized by Shanghai Sangon Biotech.

[0072] Upstream primer (P1): 5'-AGAGTTTGATCCTGGTCAGAACGAACGCT-3'; Downstream primer (P6): 5'-TACGGCTACCTTGTTACGACTTCACCCC-3'.

[0073] The selected strains were used as templates for PCR amplification. Reaction conditions: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 50 s, 52℃ annealing for 1 min, 72℃ extension for 1 min 30 s, for 30 cycles.

[0074] The target fragment was cloned and sequenced using standard methods according to *Molecular Cloning: A Laboratory Manual* (Sambrook, et al., 2001). The sequenced fragments were compared with 16S rRNA sequences registered in GenBank (accession numbers: NC_004567, NC_012984, and NC_020229, etc.), showing a gene homology of over 99%. Based on *Bergey's Mannual of Determinative Bacteriology* (Holt, et al., 1994) and *Handbook of Systematic Identification of Common Bacteria* (Dong Xiuzhu et al., 2001), strain LL20 was identified as *Lactobacillus plantarum* through morphological characteristics and 16S rRNA sequence analysis.

[0075] 10. Strain preservation Collection Name: Lactiplantibacillusplantarum LL20, namely Lactobacillus plantarum LL20, deposited at: China Center for Type Culture Collection, deposited at: Wuhan University, Wuhan, China, accession number: CCTCC NO: M20232427, deposited on December 4, 2023.

[0076] Example 2: Preparation method of Lactobacillus plantarum LL20 inoculum Lactobacillus plantarum LL20 was inoculated into MRS liquid medium at a ratio of 1% (v / v), anaerobically cultured at 35°C for 24 hours, centrifuged at 5000 rpm / min at 4°C, and the bacterial pellet was collected and dried at 50°C for 6-8 hours. It was then mixed with pharmaceutically, food-grade, or feed-grade soluble starch excipients at a ratio of 4:1 (w:w) to prepare bacterial powder. The viable count per gram of bacterial powder was 1.0 × 10⁻⁶. 8 CFU or a certain amount of skim milk powder can be added to prepare a formula containing 5.0 × 10⁻⁶ CFU per milliliter. 8 Concentrated bacterial culture of CFU (solvent is MRS liquid culture medium).

[0077] Example 3: Effects of Lactobacillus plantarum LL20 on serum uric acid, creatinine, and urea levels in a rat model of hyperuricemia. A rat model of hyperuricemia was established by gavage administration of adenine and potassium oxonate. Thirty-two male Wistar rats, 8-9 weeks old and weighing approximately 200 g (190-210 g), were used. All rats were housed separately in a controlled environment with constant temperature (20-22 ℃) and humidity (50%-85%), with free access to food. After one week of acclimatization, they were randomly divided into three groups: a normal control group (CON), a hyperuricemia model group (HUA), a positive control group (allopurinol, AP), and a Lactobacillus plantarum LL20 treatment group (LL20), with eight rats in each group. The overall experimental procedure is as follows: Figure 7 As shown, except for the control group, all other groups were administered a mixture of 300 mg / kg potassium oxonate and 100 mg / kg adenine by gavage every morning. The potassium oxonate and adenine were prepared as a 50 mg / mL suspension using 0.5% sodium carboxymethyl cellulose (CMC-Na) solution. The control group received an equal volume of 0.5% CMC-Na solution, administered by gavage at a volume of 10 mL / kg (approximately 2 mL per animal), once daily. In addition, the AP group was administered 30 mg / kg allopurinol by gavage every afternoon using 0.5% CMC-Na, the LL20 group was administered an equal volume of LL20 bacterial suspension by gavage every afternoon, and the other groups were administered an equal volume of 0.5% CMC-Na by gavage daily. The experiment lasted for 21 days. All animal experiments were approved by the Animal Care and Use Committee and the Animal Experiment Ethics Committee of Zhejiang University.

[0078] The viable count of the above LL20 bacterial solution was 10. 8 CFU / mL, prepared using the same method as in Example 1.

[0079] Twenty-one days after the experiment, blood was collected to measure the rats' blood uric acid, creatinine, and urea levels.

[0080] Experimental data showed that after 21 days of hyperuricemia modeling, the levels of serum uric acid, creatinine, and urea in rats in the HUA, AP, and LL20 groups were all higher than those in the normal group. However, the increase in serum uric acid in the AP and LL20 groups was significantly lower than that in the HUA group. In particular, the serum urea level in the LL20 group was almost similar to that in the CON group. This indicates that the serum uric acid level of LL20-treated rats was similar to that of the positive control drug AP, and it had a greater ability to improve the uric acid level than AP. This suggests that *Lactobacillus plantarum* LL20 not only reduces serum uric acid in rats but also promotes the final metabolism of uric acid (e.g., Figure 8 (As shown).

[0081] Example 4: Protective effect of Lactobacillus plantarum LL20 on the kidneys of rats with hyperuricemia. The experimental section was exactly the same as in Example 3. After 21 days of experimentation, the rats were dissected and their kidneys were harvested for pathological observation and testing of various indicators.

[0082] Experimental data showed that 21 days after the hyperuricemia model was established, the HUA group exhibited tubular dilation, interstitial edema, and inflammatory cell infiltration, with mild glomerular sclerosis or swelling. The AP group showed improvement in tubular dilation and interstitial loosening compared to the HUA group, with relatively preserved glomerular morphology but still exhibiting mild lesions. The LL20 group showed tubular and glomerular structures similar to the CON group, with reduced interstitial edema and inflammatory cell infiltration, and a more regular overall tissue structure. These results suggest that *Lactobacillus plantarum* LL20 is comparable to the positive control drug allopurinol (AP) in protecting against kidney damage, indicating that *Lactobacillus plantarum* LL20 has a protective effect on the kidneys under hyperuricemia conditions (e.g.,...). Figure 9 (As shown).

[0083] Example 5: Protective effect of Lactobacillus plantarum LL20 on the liver of rats with hyperuricemia. The experimental part was exactly the same as in Example 3. After 21 days of the experiment, blood was taken from rats to detect ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in the blood, and the livers of the rats were dissected for pathological observation.

[0084] Experimental data showed that 21 days after the hyperuricemia model was established, serum ALT and AST levels in the HUA group rats increased significantly, while those in the AP and LL20 groups showed improvement (e.g., Figure 10 (As shown). Histopathological results showed that in the CON group, the hepatocytes had regular structure, tight cell arrangement, clearly visible nuclei, and clear hepatic cords and sinusoidal structures, with no inflammatory cell infiltration or necrosis. In the HUA group, the hepatocytes were disordered, swollen, with some hepatocytes showing necrosis, sinusoidal dilation, significant inflammatory cell infiltration, and disordered liver tissue structure. In the AP group, hepatocyte swelling and necrosis were alleviated compared to the HUA group, inflammatory cell infiltration was reduced, and sinusoidal structure partially recovered, but the hepatocyte arrangement was slightly disordered. The hepatocyte structure in the LL20 group was close to that in the CON group, with significantly reduced cell swelling and necrosis, significantly alleviated inflammatory cell infiltration, normalized sinusoidal structure, and neatly arranged hepatocytes. It can be said that the liver in the HUA group showed obvious inflammation, swelling, and necrosis, while the liver lesions in the AP and LL20 groups were improved. Among them, the tissue structure and cell morphology of the LL20 group were close to those of the control group, indicating that *Lactobacillus plantarum* LL20 has a protective effect on the liver under the premise of high uric acid (e.g., Figure 11 (As shown).

[0085] Example 6: Effects of Lactobacillus plantarum LL20 on fecal microbiota in rats with hyperuricemia The experimental section was exactly the same as in Example 3. After 21 days of experimentation, the rats were dissected, and their gut microbiota was examined.

[0086] Experimental data showed that, 21 days after the hyperuricemia model was established, the HUA group exhibited significantly reduced gut microbiota diversity compared to the CON group, with decreases in ACE, Chao1, Simpson, and Shannon indices. This indicates that the HUA model led to a disruption of gut microbiota diversity (e.g., Figure 12 As shown in the figure). After intervention in the AP group, various diversity indicators recovered to some extent, but did not reach the level of the CON group. In contrast, the LL20 group showed significantly better improvement in all indicators than the AP group, especially in ACE and Chao1 index, indicating that LL20 had a stronger effect on improving gut microbial diversity. Principal component analysis (PCA) and statistical results of inter-group distance further verified this conclusion. The metabolic characteristics of the HUA group deviated significantly from those of the CON group. After intervention in the AP group, the metabolic characteristics gradually approached those of the CON group, but the LL20 group was closer to the CON group, and the inter-group distance was significantly smaller than that of the HUA and AP groups, indicating that LL20 had a better effect on improving gut microbial metabolic function than the AP group (e.g., Figure 13 (As shown). Gut microbiota composition analysis showed that the HUA group modeling led to a decrease in the proportion of Firmicutes and an increase in the proportion of Bacteroidota, while the abundance of potentially harmful bacteria such as Desulfobacterota increased (e.g., Figure 14 (As shown in the image). The AP group intervention restored the Firmicutes / Bacteroidota ratio to some extent, but the restoration effect was limited. In contrast, the LL20 group significantly increased the Firmicutes ratio and the abundance of beneficial bacteria such as the Lachnospiraceae NK4A136 group, while reducing the abundance of potentially harmful bacteria such as Desulfobacterota. This indicates that LL20 has a significant advantage in optimizing the gut microbiota structure. Overall, *Lactobacillus plantarum* LL20 can significantly improve gut microbiota diversity and metabolic dysfunction in the HUA group model rats, and its effect on regulating gut microbiota and restoring gut metabolic function is superior to that of AP.

[0087] Example 7: Effects of Lactobacillus plantarum LL20 on the metabolome of a rat model of hyperuricemia The experimental section was exactly the same as in Example 3. After 21 days of the experiment, various metabolic indicators in the rats were measured.

[0088] Experimental data showed that after 21 days of hyperuricemia modeling, the metabolic characteristics of the HUA group deviated significantly from those of the CON group, indicating that HUA modeling led to metabolic disorders; the metabolic characteristics of the AP group partially recovered to near those of the CON group; and the metabolic characteristics of the LL20 group were closer to those of the CON group, showing a stronger metabolic recovery ability. In the PLS-DA loading plot, several key metabolites significantly contributed to the differences between groups, including decreased anti-inflammatory metabolites and increased pro-inflammatory metabolites, further reflecting the metabolic abnormalities induced by the HUA model and the regulatory role of LL20 (e.g., Figure 15 (As shown). The changes in differentially expressed metabolites among the groups indicated that, compared to the CON group, the HUA group exhibited significant abnormalities in multiple metabolites, including upregulated uric acid metabolism-related metabolites (such as purines and xanthines) and downregulated antioxidant metabolites (such as glutathione and L-tryptophan). Compared to the CON group, the AP group showed significant recovery in multiple metabolites, demonstrating the role of AP in regulating some metabolic disorders. In contrast, the metabolite differences between the LL20 group and the HUA and CON groups were more significant, indicating that LL20 has a broader metabolic regulatory capacity. Specifically, compared to the HUA and AP groups, LL20 significantly downregulated pro-inflammatory metabolites (such as uric acid and related purine metabolites) and significantly upregulated multiple anti-inflammatory and antioxidant metabolites (such as L-tryptophan derivatives and bile acids), demonstrating stronger anti-inflammatory and antioxidant effects (such as...). Figure 16 (As shown). The results indicate that Lactobacillus plantarum LL20 significantly improved metabolic disorders in a rat model of hyperuricemia by broadly regulating the body's metabolome. Its regulatory effect was superior to that of the traditional drug allopurinol, further validating its potential in the intervention of hyperuricemia.

[0089] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A type of Lactobacillus plantarum ( Lactiplantibacillusplantarum LL20, characterized in that, The Lactobacillus plantarum LL20 was deposited at the China Center for Type Culture Collection (CCTCC) on December 4, 2023, with accession number CCTCC NO: M20232427, at Wuhan University, Wuhan, China.

2. An inoculum containing Lactobacillus plantarum LL20 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is a liquid, emulsion, suspension, granule, powder, wettable powder, or water dispersible agent.

4. The microbial agent according to claim 2, characterized in that, The microbial agent also includes pharmaceutically, food-grade, or feed-grade excipients.

5. The microbial agent according to claim 4, characterized in that, The excipients are selected from at least one of trehalose, hydroxymethyl cellulose, dextrin, soluble starch, fructooligosaccharides, isomaltooligosaccharides, and stachyose.

6. The microbial agent according to claim 5, characterized in that, The *Lactobacillus plantarum* LL20 was mixed with a pharmaceutically, food-grade, or feed-grade soluble starch excipient at a mass ratio of 4:1 to prepare a powder, with each gram of powder containing 1.0 × 10⁻⁶ viable bacteria. 8 CFU.

7. The method for preparing the microbial agent according to any one of claims 2 to 6, characterized in that, The method includes: The seed culture of Lactobacillus plantarum LL20 was inoculated into MRS liquid medium and fermented in an anaerobic environment at 36-38℃ for 30-40 hours. After fermentation, the culture was centrifuged, the precipitate was collected and dried to prepare the inoculum.

8. The use of a microbial agent containing Lactobacillus plantarum LL20 as described in claim 1, or the use of a microbial agent as described in any one of claims 2 to 6 in the preparation of drugs for treating hyperuricemia or gout or in functional feeds, or in the preparation of functional foods that contribute to the intestinal flora.

9. The use according to claim 8, characterized in that, Selected from at least one of the following: (1) Use in the preparation of a drug or feed for reducing at least one of uric acid, creatinine and urea in the serum of patients or animals with hyperuricemia or gout; (2) Use in the preparation of drugs or feeds for relieving at least one injury to the kidney or liver of patients or animals with hyperuricemia or gout; (3) Use in the preparation of drugs or feeds for restoring metabolism in patients or animals with hyperuricemia or gout; (4) Use in the preparation of drugs or feeds that improve the intestinal colonization capacity and probiotic properties of patients or animals with hyperuricemia or gout.

10. The use according to claim 9, characterized in that, The probiotic properties include improving the intestinal flora structure, increasing the abundance of beneficial bacteria, and inhibiting the proliferation of pathogenic bacteria; Or / and, the restoration of metabolism in patients or animals with hyperuricemia or gout includes: downregulating pro-inflammatory metabolites and upregulating anti-inflammatory and antioxidant metabolites.

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