Application of plant lactobacillus FBL002 in degradation of purine nucleoside

Abstract

The invention relates to application of plant lactobacillus FBL002 in degradation of purine nucleoside, and belongs to the technical field of microorganisms. The plant lactobacillus FBL002 provided by the invention is preserved in Guangdong Microbial Culture Collection Center on March 13, 2025, and the preservation number is GDMCC No.66015. The strain is separated from healthy infant intestinal flora, and can effectively degrade various purine nucleosides including inosine, guanosine, adenosine, guanine and adenine, so that the plant lactobacillus FBL002 is used for developing products for degrading purine nucleosides. In addition, the strain is also suitable for preparing oral hyperuricemia treatment medicines, and the contents of uric acid, xanthine oxidase and adenosine deaminase in serum can be remarkably reduced. The invention provides a safe and efficient method for managing purine metabolism abnormality related diseases by utilizing the biodegradation activity of the plant lactobacillus FBL002, and is particularly suitable for preventing and treating hyperuricemia.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the application of *Lactobacillus plantarum* FBL002 in the degradation of purine nucleosides. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disorder caused by purine metabolism disturbances, characterized by abnormally high levels of uric acid in the blood. This may be due to excessive uric acid production, reduced excretion, or both. Long-term hyperuricemia can lead to the deposition of urate crystals in joints and other tissues, causing gout and other related diseases such as kidney stones and kidney damage. With changes in modern lifestyles and dietary structures, the incidence of hyperuricemia is rising annually and has become a global public health problem.

[0003] Traditional treatments include medication and dietary control. Medication primarily lowers blood uric acid levels by inhibiting uric acid production or promoting uric acid excretion, but it often comes with side effects such as allergic reactions and liver or kidney damage. Dietary control requires patients to restrict their intake of high-purine foods, which is not only difficult to strictly control and maintain long-term, but also affects patients' quality of life and nutritional balance.

[0004] In recent years, with in-depth research on gut microbiota, utilizing probiotics to regulate host metabolism and immune function has become an emerging strategy for treating hyperuricemia. By employing specific bacterial strains capable of ingesting and utilizing purine nucleosides from food, these strains convert purine nucleosides in the gut into purine bases that are more difficult for the body to absorb or other harmless substances. This reduces the body's absorption and utilization of purines and the subsequent production of uric acid, thereby lowering blood uric acid levels. Furthermore, some strains with these functionalities can also further regulate the host's metabolic health by improving intestinal barrier function and inhibiting the growth of harmful bacteria.

[0005] Researchers in this field have screened various lactic acid bacteria capable of degrading purine nucleotides, such as *Lactobacillus brevis*, *Lactobacillus rhamnosus*, and *Lactobacillus plantarum*. These strains have shown highly efficient degradation activity in in vitro experiments and demonstrated their potential to lower serum uric acid levels in animal models and clinical trials. However, existing studies have shown that the most efficient strains for degrading purine nucleosides are mostly *Lactobacillus fermentans* derived from Lanzhou rice water. Development of strains from other sources and species remains insufficient and requires further exploration. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention isolated a strain of *Lactiplantibacillus plantarum* with highly efficient purine nucleoside degradation capabilities from the intestinal flora of healthy infants in Wuhan, Hubei Province. Named FBL002 (and deposited by the Guangdong Provincial Center for Microbial Culture Collection on March 13, 2025, with accession number GDMCC No. 66015), this strain can efficiently degrade various purine nucleosides, including inosine, guanosine, adenosine, guanine, and adenine, in vitro. Validated in a mouse model, it can also reduce serum uric acid levels, xanthine oxidase (XOD) levels, and adenosine deaminase (ADA) levels, thereby reducing uric acid synthesis.

[0007] The first objective of this invention is to provide the application of *Lactobacillus plantarum* FBL002 in the degradation of purine nucleosides, wherein the preservation number of *Lactobacillus plantarum* FBL002 is GDMCC NO.66015.

[0008] Furthermore, the purine nucleosides include inosine, guanosine, adenosine, guanine, and adenine.

[0009] A second objective of this invention is to provide a bacterial agent for degrading purine nucleosides, the product comprising *Lactobacillus plantarum* FBL002, the preservation number of which is GDMCCNO.66015.

[0010] Furthermore, the microbial agent can be used in the preparation of food, health products, or pharmaceuticals.

[0011] Furthermore, the purine nucleosides include inosine, guanosine, adenosine, guanine, and adenine.

[0012] Furthermore, the bacterial agent comprises live, inactivated, broken, or lysed cells of *Lactobacillus plantarum* FBL002.

[0013] A third objective of this invention is to provide the application of *Lactobacillus plantarum* FBL002 in the preparation of drugs for the prevention or treatment of hyperuricemia, wherein the preservation number of *Lactobacillus plantarum* FBL002 is GDMCC NO.66015.

[0014] Furthermore, the aforementioned drugs for the prevention or treatment of hyperuricemia have any of the following effects:

[0015] (1) Reduce serum uric acid levels;

[0016] (2) Reduce serum xanthine oxidase levels;

[0017] (3) Reduce serum adenosine deaminase levels.

[0018] The fourth objective of this invention is to provide a drug for the prevention or treatment of hyperuricemia, characterized in that: the drug for the prevention or treatment of hyperuricemia comprises *Lactobacillus plantarum* FBL002, the preservation number of *Lactobacillus plantarum* FBL002 being GDMCC NO.66015.

[0019] Furthermore, it also includes pharmaceutically acceptable excipients.

[0020] Furthermore, the medication for the prevention or treatment of hyperuricemia is administered orally.

[0021] The beneficial effects of this invention are:

[0022] The *Lactobacillus plantarum* FBL002 screened in this invention can efficiently degrade inosine, guanosine, adenosine, guanine, and adenine, and can colonize the intestine, thereby reducing purine accumulation in the body. It can be used to prepare drugs for the prevention or treatment of hyperuricemia. This drug, administered orally, can safely lower serum uric acid levels while inhibiting the activity of key enzymes such as xanthine oxidase and adenosine deaminase. This not only helps alleviate the symptoms of hyperuricemia but also prevents complications such as gout. Furthermore, the purine nucleoside degradation products and drugs for the prevention or treatment of hyperuricemia prepared based on *Lactobacillus plantarum* FBL002 are diverse in form, easy to produce and apply, have high biocompatibility and low risk of side effects, and provide an innovative and practical biotherapy strategy for purine metabolism disorders.

[0023] Preservation of biological materials

[0024] Lactiplantibacillus plantarum FBL002 was deposited on March 13, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. It is classified as Lactiplantibacillus plantarum and has the accession number GDMCC NO.66015. Attached Figure Description

[0025] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0026] Figure 1 This is a characterization of the colony morphology of *Lactobacillus plantarum* FBL002 grown on MRS solid plates in Example 1 of the present invention.

[0027] Figure 2 Morphological characteristics of *Lactobacillus plantarum* FBL002 under a 100x optical microscope in Example 1 of this invention;

[0028] Figure 3 The ability of *Lactobacillus plantarum* FBL002 to degrade purine nucleosides in vitro in Example 3 of the present invention is given by: a, where a is the in vitro degradation rate of purine nucleosides and b is the upper limit of in vitro degradation of inosine and guanosine.

[0029] Figure 4 The intestinal colonization ability of *Lactobacillus plantarum* FBL002 in Example 3 of the present invention was determined, where a is the hydrophobicity test result and b is the self-aggregation test result.

[0030] Figure 5 This is a determination of the intestinal colonization ability of *Lactobacillus plantarum* FBL002 in Example 3 of the present invention, where a is the result of acid resistance test and b is the result of bile salt resistance test.

[0031] Figure 6 The results of the test on the resistance of *Lactobacillus plantarum* FBL002 to artificial gastric and intestinal fluids in Example 3 of this invention;

[0032] Figure 7 The results of the hemolytic activity assay of *Lactobacillus plantarum* FBL002 in Example 4 of this invention;

[0033] Figure 8 The results of the drug resistance test of *Lactobacillus plantarum* FBL002 in Example 4 of this invention;

[0034] Figure 9 The results of the effect of *Lactobacillus plantarum* FBL002 on hyperuricemic mice in Example 5 of this invention are shown, where a is the change in the renal organ coefficient of mice, b is the change in the serum uric acid level of mice, c is the change in the serum xanthine oxidase content of mice, and d is the serum adenosine deaminase content of mice.

[0035] Figure 10 The variation of *Lactobacillus plantarum* FBL002 content in mouse fecal flora in Example 5 of this invention;

[0036] Figure 11 This invention relates to the effect of *Lactobacillus plantarum* FBL002 on kidney damage in hyperuricemic mice in Example 5 of this invention. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0038] The culture media and reagents involved in the examples are shown below:

[0039] MRS broth medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, pH approximately 5.7, add distilled water to a final volume of 1000 mL, sterilize at 115°C for 15 min;

[0040] MRS solid plates: Add 15 g / L agar to MRS broth medium and sterilize at 115°C for 15 min. After sterilization, add bromocresol purple solution at a ratio of 0.5% (v / v).

[0041] Bromocresol purple solution: Dissolve 1 g of bromocresol purple in 50 mL of 20% ethanol to prepare a 20 g / L stock solution. Add 0.5% (v / v) to sterilized, slightly cooled MRS solid medium for the identification of acid-producing colonies.

[0042] PBS buffer: Na₂HPO₄ 2.16 g / L, KH₂PO₄ 0.20 g / L, NaCl 8.00 g / L, KCl 0.20 g / L. Add distilled water to a final volume of 800 mL, adjust pH to 7.4 with HCl, and then add distilled water to a final volume of 1000 mL. Sterilize at 115°C for 15 min.

[0043] Example 1: Screening, Isolation and Identification of Strains

[0044] We screened probiotic-rich raw materials from various regions across China, including kimchi, breast milk, black tea, infant feces, and goat milk, aiming to obtain a highly efficient lactic acid bacteria strain that degrades purine nucleosides. For solid samples, 1g of the sample was aseptically pulverized and mixed with 10mL of sterile physiological saline to prepare a liquid sample. Under aseptic conditions, 100μL of the liquid sample was thoroughly mixed with 900μL of sterile physiological saline. Then, 100μL of the mixture was taken and another 900μL of sterile physiological saline was added, and so on, to prepare different serial dilutions.

[0045] 100 μL of each of the different serial dilutions was evenly spread onto the surface of MR S solid medium plates containing bromocresol purple and incubated upside down at 37°C for 12–48 h to initially isolate colonies. Acid-producing colonies exhibiting a yellow discoloration zone were further isolated by streak plating to obtain single colonies. The purity, species, and phylogenetic characteristics of the isolated strains were identified by 16S rRNA gene sequencing and comparison with the NCBI database BLAST search.

[0046] This invention obtained eight lactic acid bacteria strains from different sources that exhibited good growth and acid production through the screening method described above. Among them, the lactic acid bacteria isolated from the intestinal flora of healthy infants in Wuhan, Hubei Province, was identified as *Lactiplantibacillus plantarum*, named FBL002, and its 16S rRNA sequence is shown in SEQ ID NO.1.

[0047] The colony morphology of *Lactobacillus plantarum* FBL002 on MRS solid plates is as follows: Figure 1 As shown, the morphology under a 100x optical microscope is as follows Figure 2 As shown.

[0048] Example 2: Determination of the purine nucleoside degradation ability of bacterial strains

[0049] The obtained 8 lactic acid bacteria strains were streaked onto MRS solid plates and incubated upside down at 37°C for 12-24 hours. Activated single colonies were picked and inoculated into 5 mL of MRS broth for further activation, and then incubated statically at 37°C for 12-14 hours until the bacterial suspension density reached 10⁻⁶. 6 -10 8 CFU / mL. Centrifuge at 8000 rpm for 10 min at 4°C in a centrifuge tube, collect the bacterial cells, wash 2-3 times with PBS buffer, and adjust the OD value to 0.05 with PBS buffer again. 600 =1. Take 2 mL of OD 600 A bacterial suspension with a concentration of 1 was centrifuged at 12,000 rpm for 5 min to collect bacterial cells. 750 μL of purine nucleoside reaction solution was added to the bacterial cells, vortexed to mix, and incubated at 37°C with shaking at 220 rpm for 1 h to carry out the purine nucleoside degradation reaction. After the reaction was completed, the system was boiled in a water bath for 5 min to terminate the reaction. The bacterial cells were separated by centrifugation at 12,000 rpm for 5 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm microporous membrane, and the concentration of residual purine nucleoside in the filtrate was determined by HPLC.

[0050] HPLC chromatographic detection conditions: Hypersil GOLD column was used. TM aQ C18 polar-terminated HPLC column; column temperature: 30℃; mobile phase: methanol-water = 5:95 (v / v); flow rate: 0.8 mL / min; detection wavelength: 250 nm; injection volume: 5 μL.

[0051] A purine nucleoside solution without bacterial cells was used as a blank control. A standard curve of purine nucleoside concentration versus peak area was established, and the residual purine nucleoside concentration in the reaction system after degradation was calculated. Using the purine nucleoside concentration of the blank control as the initial concentration, the degradation rate of various purine nucleosides by the strain within 1 hour was calculated.

[0052] Purine nucleoside reaction solution: Weigh a certain amount of purine nucleoside solid powder and prepare it with sterile PBS buffer at pH 7.4, then sonicate to aid dissolution. The concentrations of purine nucleosides used in the above steps are: inosine 0.337 g / L, guanosine 0.357 g / L, adenosine 0.337 g / L, adenine 0.2 g / L, and guanine 0.05 g / L.

[0053] Implementation results are as follows Figure 3 As shown in Figure a, *Lactobacillus plantarum* FBL002 achieved degradation rates of 100%, 100%, 65.63%, 16.64%, and 8.27% for inosine, guanosine, adenosine, adenine, and guanine, respectively, within 1 hour. The degradation rates were 1.256 mmol·L⁻¹. -1 ·h -1 1.260 mmol·L -1 ·h -1 0.828 mmol·L -1 ·h -1 0.246 mmol·L -1 ·h -1 0.0274 mmol·L -1 ·h -1 It was significantly higher than other strains obtained during the same period of screening.

[0054] To verify the upper limit of the purine nucleoside degradation ability of *Lactobacillus plantarum* FBL002 of this invention, the concentrations of inosine and guanosine in the reaction solution were increased to 1.0 g / L and 0.5 g / L respectively for repeated tests. The results are as follows: Figure 3 As shown in b. Under the above conditions, the strain achieved degradation rates of 46.51% and 82.05% for 1.0 g / L inosine and 0.5 g / L guanosine, respectively, meaning the highest degradation rate could reach 0.867 mmol·L⁻¹. -1 ·OD -1 ·h -1 (232mg·L -1 ·OD -1 ·h -1 ), 0.724 mmol·L -1 ·OD -1 ·h -1 (263mg·L -1 ·OD -1 ·h -1 ).

[0055] The degradation rate of inosine and guanosine by *Lactobacillus plantarum* strain FBL002 is higher than that of *Lactobacillus plantarum* KLpl-3 described in patent CN114317308A; it is up to 60 times higher than that of all 27 strains of *Lactobacillus plantarum* described in patent CN114317308A; the degradation amount of inosine per unit cell mass within 1 hour (0.867 mmol) is 4.6 times that of the degradation amount of inosine per unit cell mass of unknown *Lactobacillus fermentum* GR-X described in patent CN117645941A within 24 hours (approximately 0.19 mmol). This indicates that the *Lactobacillus plantarum* strain FBL002 has extremely high utilization rate of purine nucleosides, possesses the ability to efficiently degrade purine nucleosides in vitro, and has application potential in the field of hyperuricemia.

[0056] Example 3: Determination of the intestinal colonization ability of bacterial strains

[0057] (1) Hydrophobicity and self-aggregation

[0058] The hydrophobicity of probiotic strains is consistent with their self-aggregation, which can be used as a preliminary characterization of the strain's ability to adhere to intestinal epithelial cells.

[0059] Lactobacillus plantarum FBL002 was streaked onto MRS solid plates and incubated upside down at 37°C for 12-24 hours. Activated single colonies were picked and inoculated into 5 mL of MRS broth for further activation, and then incubated statically at 37°C for 12-14 hours until the bacterial suspension density reached 10⁻⁶. 6 -10 8 CFU / mL. Centrifuge at 8000 rpm for 10 min at 4°C in a centrifuge tube, collect the bacterial cells, and resuspend them in distilled water to adjust the OD. 600 =1, to obtain the bacterial suspension to be tested.

[0060] Hydrophobicity assessment: Mix 3 mL of the test bacterial suspension with 1 mL of butanol and ethyl acetate, shake for 2 min, and incubate at 37 °C for 3 h. Measure the OD of the non-organic phase. 600 Calculate the hydrophobicity.

[0061] Hydrophobicity = (1-A) i / A0)×100%, where A i To process OD for 3 hours 600 A0 is the OD of the initial bacterial suspension to be tested. 600 .

[0062] The results are as follows Figure 4 As shown in a, Lactobacillus plantarum FBL002 has a hydrophobicity of 26.05% in butanol and 26.94% in ethyl acetate.

[0063] Self-aggregation assessment: The bacterial suspension to be tested was incubated at 37°C for 4 h and 24 h, respectively, and the OD values ​​of the initial bacterial suspension and the supernatant after 4 h and 24 h were measured. 600Calculate the self-aggregation rate.

[0064] Self-aggregation rate = (1-A) i / A0)×100%, where A i To process OD for 4 hours or 24 hours 600 A0 is the OD of the initial bacterial suspension to be tested. 600 .

[0065] The results are as follows Figure 4 As shown in b, *Lactobacillus plantarum* FBL002 exhibited a self-aggregation rate of 28.98% at 4 hours and 30.36% at 24 hours, demonstrating good hydrophobicity and self-aggregation properties. It showed some ability to colonize the intestinal tract and has the potential to be used as an oral probiotic supplement.

[0066] (2) Resistant to acid and bile salts

[0067] Human gastric juice is highly acidic, so probiotics must be acid-resistant. Tolerance to bile salts in the human small intestine is also an important criterion for selecting probiotics.

[0068] Strawberry strain FBL002 was streaked onto MRS solid plates and incubated upside down at 37°C for 12-24 hours.

[0069] Acid tolerance assessment: Single colonies were picked and inoculated into 5 mL of MRS broth for activation, and incubated statically at 37°C for 30 h. A 10% inoculum was then added to MRS broth media at pH 6.2 (control group), 2, 3, and 4, and incubated at 37°C for 4 h. Samples were taken at 0, 1, 2, and 4 h, and the remaining viable bacteria in each group were diluted, plated, and counted. N was recorded. i The viable count is the number of bacteria in the i-th hour, which characterizes the strain's tolerance to acidic environments.

[0070] The results are as follows Figure 5 As shown in a, Lactobacillus plantarum FBL002 exhibits a certain degree of tolerance in a strongly acidic environment.

[0071] Bile salt tolerance assessment: Single colonies were picked and inoculated into 5 mL of MRS broth for activation, and incubated statically at 37°C for 9 h. Inoculum was then grown at a 1% inoculum size in MRS medium with or without 0.3% (w / v) bile salts to simulate the bile salt concentration environment in the small intestine. OD was measured at 0, 4, 12, and 24 h. 600 The growth status was used to characterize the strain's tolerance to bile salt environments.

[0072] The results are as follows Figure 5 As shown in b, *Lactobacillus plantarum* FBL002 showed a growth trend in the above-mentioned bile salt concentration environment, demonstrating good tolerance to bile salts.

[0073] (3) Resistant to artificial gastric and intestinal fluids

[0074] Artificial gastric fluid: KH2PO4 0.24g / L, NaHPO4 1.44g / L, NaCl 8.0g / L, KCl 0.20g / L, pepsin 3.0g / L, diluted to 800mL with distilled water, pH adjusted to 2.5 with HCl, and diluted to 1000mL with distilled water.

[0075] Artificial intestinal fluid: Based on PBS buffer, adjust pH to 8.0 with NaOH, add 1.0 g / L trypsin, and sterilize by filtration through a 0.22 μm filter membrane. PBS buffer preparation is the same as in Example 2.

[0076] Lactobacillus plantarum strain FBL002 was streaked onto MRS solid plates and incubated upside down at 37°C for 12–24 h. Single colonies were picked and inoculated into 5 mL of MRS broth for activation, and then incubated statically at 37°C for 24 h. The culture was then inoculated into simulated gastric fluid at a 1:10 volume ratio and incubated statically at 37°C. Samples were taken at 0, 1, 2, and 3 h, and the remaining viable bacteria in the simulated gastric fluid were diluted, spread, and counted. The bacterial culture inoculated into the simulated gastric fluid for 3 h was further inoculated into simulated intestinal fluid at a 1:10 volume ratio, and samples were taken at 4, 5, 6, 7, and 8 h, and viable bacteria were counted as above to characterize the strain's tolerance to the simulated gastrointestinal environment. N was recorded as N. i The number of viable bacteria in the i-th hour is given. The N4-N8 count in the intestinal fluid should be multiplied by 10 to balance the transfer dilution rate.

[0077] The results are as follows Figure 6 As shown, *Lactobacillus plantarum* FBL002 was tested and found to have an initial viable count of 4.8 × 10⁻⁶. 7 Based on CFU / mL, the number of viable bacteria remaining after gastric juice treatment within 0-3 hours was 9.4 × 10⁻⁶. 6 CFU / mL, with a residual viable count of 1.18 × 10⁻⁶ CFU / mL after 4-8 hours in intestinal fluid tolerance. 6 With a concentration of CFU / mL, it showed good tolerability in an artificial in vitro simulated gastrointestinal environment and possessed the excellent characteristics necessary for probiotics, making it suitable as an oral probiotic supplement for related fields.

[0078] Example 4: Safety determination of the strain

[0079] (1) Hemolytic

[0080] Based on the Columbia blood agar plate method, *Lactobacillus plantarum* FBL002 was streaked onto the surface of Columbia blood agar plates and incubated at 37°C inverted for 12-24 hours to observe hemolysis.

[0081] Its hemolytic activity is determined by the following indicators:

[0082] a. β-hemolysis: Completely transparent hemolytic zone, complete lysis of red blood cells, strong hemolytic activity.

[0083] b. α-Hemolysis: grass-green hemolysis zone, partial hemolysis.

[0084] c. Gamma-hemolysis: No hemolytic reaction.

[0085] Columbia blood agar medium: 23.0 g / L special peptone, 1.0 g / L soluble starch, 5.0 g / L NaCl, 15 g / L agar, pH 7.3. Sterilize at 115°C for 15 min, cool slightly to about 50°C, and add 5-10% (v / v) sterile defibrinated sheep blood.

[0086] The results are as follows Figure 7 As shown, *Lactobacillus plantarum* FBL002 showed no hemolytic reaction, indicating γ-hemolysis. It did not exhibit hemolytic activity and met the safety evaluation criteria.

[0087] (2) Drug resistance

[0088] Determining probiotic resistance is crucial for ensuring product safety, assessing its behavior in the gut microenvironment, and meeting regulatory requirements.

[0089] Strawberry strain FBL002 was streaked onto MRS solid plates and incubated upside down at 37°C for 12-24 hours. Single colonies were picked and inoculated into 5 mL of MRS broth for activation, and then incubated statically at 37°C for 12-24 hours. The bacterial suspension was then diluted to 1.5 × 10⁻⁶. 8 CFU / mL was uniformly coated onto the surface of an MRS solid plate.

[0090] Antibiotic susceptibility testing discs were placed on MRS solid plates and incubated upside down at 37°C for 24 hours. The diameter of the inhibition zone was measured to assess the strain's tolerance or susceptibility to different antimicrobial agents. The determination of strain resistance results followed the Clinical and Laboratory Standards Institute (CLSI) criteria, categorized as S (susceptible), I (intermediate), and R (resistant).

[0091] The results are as follows Figure 8 As shown, *Lactobacillus plantarum* FBL002 exhibits resistance to streptomycin, kanamycin, gentamicin, and vancomycin, which helps it survive, colonize, and antagonize harmful bacteria. It is sensitive to penicillin, erythromycin, chloramphenicol, cephalexin, and ampicillin, which can prevent overgrowth of the strain, maintain relative stability of the intestinal flora, ensure that the overall treatment effect is not affected by drug resistance in clinical treatment, and reduce the risk of drug resistance transmission.

[0092] Example 5: Effects of Lactobacillus plantarum FBL002 on a mouse model of hyperuricemia

[0093] Live bacterial suspension of *Lactobacillus plantarum* FBL002 was administered by gavage to mice with hyperuricemia. The effects of the strain on lowering serum uric acid levels in vivo were observed, as detailed below:

[0094] (1) Experimental Design

[0095] Phase 1: Adaptation Period for Mice

[0096] Male Kunming mice were randomly assigned to cages and acclimatized for one week using standard laboratory animal feed and free access to water. Each mouse was ear-tagged and randomly numbered. Experiments were conducted after the mice showed no obvious abnormalities. Natural ventilation, free access to food and water, and regular bedding changes were implemented.

[0097] Phase Two: Experimental Intervention and Modeling

[0098] During the eighth day of modeling, mice were divided into four groups of ten mice each. One group was randomly selected and fed a basal diet, while the other three groups were fed a high-uric acid diet. After two weeks of continuous modeling, serum uric acid levels were measured. Successful modeling was indicated by a significant increase in serum uric acid levels in the other three groups compared to the first group. Intervention treatment began in the fourth week. The first group (Control group) was administered 0.5 mL of physiological saline by gavage, the second group (Model group) was administered 0.5 mL of physiological saline by gavage, and the third group (FBL002 group) was administered 0.5 mL of live *Lactobacillus plantarum* FBL002 solution (1×10⁻⁶) by gavage. 8 Group 4 (Therapy group) received allopurinol (5 mg / kg) via gavage. All gavage treatments were administered once daily. Approximately 1 g of fresh feces was collected from mice in a sterile environment every other day and on the last day using a forced collection method. The intervention lasted from day 8 to day 31.

[0099] High uric acid rat food: Add 4g of potassium oxonate, 4g of uric acid, 1g of inosine, 1g of guanosine, and 1g of adenosine to 100g of basic rat food.

[0100] Phase Three: Animal Custody

[0101] All mice were anesthetized by isoflurane inhalation. Blood was collected from the mice by enucleation, and the mice were euthanized by cervical dislocation. Fresh blood samples were allowed to stand at 37°C for 1 hour, then centrifuged at 3000 rpm for 10 minutes to obtain mouse serum. Internal organ samples were obtained by abdominal dissection. All samples were collected under sterile conditions.

[0102] (2) Effects of strain FBL002 on body weight and organ coefficient in hyperuricemic mice

[0103] After the experimental mice were euthanized, the abdominal cavity was dissected to remove both kidneys. The surface fat and fascia were removed, the surface blood was dried, and the wet weight was accurately measured. The organ coefficient = (organ weight / mouse body weight) × 1000‰.

[0104] The results are as follows Figure 9 As shown in Figure a, compared with the Control group, the Model group, Therapy group, and FBL002 group showed a significant increase in renal organ coefficient due to hyperuricemia. Specifically, the Model group's coefficient increased from 10.26‰ to 16.88‰. Compared with the Model group, the FBL002 group showed a decrease in renal organ coefficient of 4.539‰, demonstrating that gavage treatment effectively reduced the model's renal organ coefficient, decreasing the total increase in the renal coefficient by 68.55%. The Therapy group showed an increase in renal organ coefficient, indicating that allopurinol treatment exacerbated the renal burden, possibly due to the inhibition of xanthine oxidase activity leading to xanthine and hypoxanthine deposition, as well as the drug's hepatotoxicity and nephrotoxicity, failing to alleviate renal inflammation and exacerbating renal damage. Therefore, *Lactobacillus plantarum* FBL002 can effectively alleviate the problem of increased renal organ coefficient caused by hyperuricemia, with better efficacy than allopurinol treatment. Its mechanism may lie in repairing renal damage, reducing the expression of inflammatory factors, and alleviating weight loss in model mice.

[0105] (3) Effect of strain FBL002 on serum uric acid in hyperuricemic mice

[0106] Uric acid levels were determined using the uric acid ELISA kit from Shanghai Yuchun Biotechnology Co., Ltd., by detecting serum from mice in each group.

[0107] The results are as follows Figure 9 As shown in b, compared with the Control group, the serum uric acid levels in the Model group, Therapy group, and FBL002 group of hyperuricemia model mice were significantly increased. Specifically, the serum uric acid level in the Model group increased from 40.41 mmol / L to 63.85 mmol / L. In the Therapy group, due to the drug's effect, the serum uric acid level decreased to 42.49 mmol / L. The FBL002 intervention group also effectively reduced the serum uric acid level in hyperuricemia model mice to 51.55 mmol / L, significantly lower than the Model group, with an effect equivalent to 57.53% of allopurinol. This indicates that *Lactobacillus plantarum* FBL002 can alleviate hyperuricemia and act as an adjunct therapy, with no hepatotoxic or nephrotoxic side effects, and its efficacy is sustained after colonization of the intestine.

[0108] (4) The FBL002 strain inhibited the activity of xanthine oxidase and adenosine deaminase in the serum of hyperuricemic mice.

[0109] The serum of mice in each group was detected using the xanthine oxidase ELISA kit and adenosine deaminase ELISA kit from Shanghai Yuchun Biotechnology Co., Ltd. The xanthine oxidase (XOD) content was measured as follows: Figure 9 As shown in Figure c, the adenosine deaminase (ADA) content was measured as follows: Figure 9 As shown in d.

[0110] Compared with the Control group, the XOD and ADA levels in the Model group, Therapy group, and FBL002 group all showed significant increases to varying degrees. Specifically, in the Model group, XOD level increased from 0.2876 U / L to 0.5693 U / L, and ADA level increased from 4.022 U / L to 5.725 U / L. Therapy treatment significantly reduced the levels of XOD and ADA enzymes in mouse serum to 0.3637 U / L and 4.249 U / L, respectively. The FBL002 group also significantly and effectively reduced the levels of both enzymes to 0.4346 U / L and 4.743 U / L, respectively. This demonstrates that the FBL002 strain can reduce serum uric acid levels in hyperuricemia model mice to a certain extent by reducing xanthine oxidase and adenosine deaminase, with its intervention effect on XOD and ADA enzyme levels equivalent to 65.50% and 66.56% of that of allopurinol, respectively.

[0111] (5) Colonization effect of strain FBL002 in the intestine of hyperuricemic mice

[0112] Genomic DNA was extracted from mouse fecal samples collected on day 31 using the TIANamp Stool DNAKit fecal genomic DNA extraction kit. PCR products were obtained using universal bacterial primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3') and *Lactobacillus plantarum*-specific primers (LpF: 5'-GCGGCGTGCCTAATACATGC-3'; LpR: 5'-CACCGCTACACATTGAGTTCC-3'). The amplified products were verified by agarose gel electrophoresis to determine band size. The correct PCR products were cloned into a T vector and transfected into *E. coli* competent cells (DH-5α). Amplification was performed by incubation at 37°C and 120 rpm for 6 hours with constant temperature shaking.

[0113] The plasmids carrying the target fragment in the *E. coli* were extracted using the Mini Kit I (EZNA) plasmid DNA extraction kit. After purification, the plasmids were used as standard samples and serially diluted to obtain templates for qRT-PCR. A standard curve was constructed using Ct values ​​to calculate the absolute copy number of the bacterial 16S rRNA gene in different samples. The relative proportion of *Lactobacillus plantarum* FBL002 in mouse fecal flora was calculated based on the amplification results using species-specific primers.

[0114] The results are as follows Figure 10As shown, quantitative analysis of mouse fecal DNA samples on day 31 using qRT-PCR revealed that the strain accounted for 0.08‰ of the mouse intestinal flora in the FBL002 group, significantly higher than in other groups. This indicates that the FBL002 strain of this invention, after gavage intervention, can tolerate the gastrointestinal fluid and bile salt environment and successfully adhere to and colonize the mouse intestine, thereby regulating the intestinal flora and establishing a long-term effect in alleviating hyperuricemia.

[0115] (6) Effects of FBL002 strain on kidney damage in mice

[0116] Fresh mouse kidney tissue was fixed by immersion in 4% paraformaldehyde for 24 hours. Excess membrane tissue and fat were removed by scalpel in a fume hood. The tissue was then dehydrated using a dehydrator with the following gradient solutions:

[0117] 0-4h: 75% alcohol; 4-6h: 85% alcohol; 6-8h: 90% alcohol; 8-10h: 95% alcohol; 10-11h: anhydrous ethanol; 11-11.5h: anhydrous ethanol; 11.5-12h: benzyl alcohol; 12-12h 10min: xylene I; 12h 10min-12h 20min: xylene II; 12h 20min-12h 30min: wax I; 12h 30min-13h 30min: wax II; 13h 30min-14h 30min: wax III.

[0118] After dehydration and paraffin infiltration, kidney tissue was embedded in an embedding machine and cooled at -20°C. The paraffin blocks were removed and trimmed, sectioned (4μm) on a paraffin microtome, and spread flat on a 40°C water spreader. The sections were gently lifted onto glass slides, dried, and then dewaxed using the following gradient solutions:

[0119] 0-20 min: Xylene I; 20-40 min: Xylene II; 40-60 min: Anhydrous Ethanol I; 60-70 min: Anhydrous Ethanol II; 70-80 min: 95% ethanol; 80-85 min: 90% ethanol; 85-90 min: 80% ethanol; 90-95 min: 70% ethanol; 95-100 min: Distilled water.

[0120] After dewaxing, mouse kidney tissue sections were stained with Harris hematoxylin for 5 min, rinsed with deionized water for destaining, differentiated with 1% hydrochloric acid alcohol for about 5 seconds, rinsed with deionized water, and then blued with 0.6% ammonia solution, followed by rinsing with deionized water. They were then stained with eosin for 2 min. Dehydration and clearing were performed using the following gradient solutions:

[0121] 0-5 min: 95% ethanol I; 5-10 min: 95% ethanol II; 10-15 min: anhydrous ethanol I; 15-20 min: anhydrous ethanol II; 20-25 min: xylene I; 25-30 min: xylene II.

[0122] After dehydration and clearing, the slides were dried, mounted with neutral resin, and then examined under a microscope.

[0123] The results are as follows Figure 11 As shown in the image, kidney sections from the Model group mice revealed abnormal damage induced by hyperuricemia. The glomeruli were structurally incomplete, with significant dilation of the glomerular capsule, irregular arrangement of tubular epithelial cells, and dilated interstitial spaces. Significant inflammation, edema, and a degree of fibrosis were present in the tissues. No such abnormal damage was observed in the FBL002 treatment group or the Therapy group; their kidney tissue morphology was normal, with no obvious abnormalities or lesions.

[0124] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of *Lactobacillus plantarum* FBL002 in the degradation of purine nucleosides, characterized by: The preservation number of the *Lactobacillus plantarum* FBL002 is GDMCC NO.66015.

2. The application according to claim 1, characterized in that: The purine nucleosides include inosine, guanosine, adenosine, guanine, and adenine.

3. A bacterial agent for degrading purine nucleosides, characterized in that: The product contains *Lactobacillus plantarum* FBL002, which has the accession number GDMCC NO.66015.

4. The microbial agent according to claim 3, characterized in that: The purine nucleosides include inosine, guanosine, adenosine, guanine, and adenine.

5. The microbial agent according to claim 3, characterized in that: The product contains live, inactivated, broken, or lysed Lactobacillus plantarum FBL002 bacteria.

6. The application of *Lactobacillus plantarum* FBL002 in the preparation of drugs for the prevention or treatment of hyperuricemia, characterized in that: The preservation number of the *Lactobacillus plantarum* FBL002 is GDMCC NO.66015.

7. The application according to claim 6, characterized in that, The medications mentioned above for the prevention or treatment of hyperuricemia serve any of the following purposes: (1) Reduce serum uric acid levels; (2) Reduce serum xanthine oxidase levels; (3) Reduce serum adenosine deaminase levels.

8. A drug for the prevention or treatment of hyperuricemia, characterized in that: The drug for treating hyperuricemia contains *Lactobacillus plantarum* FBL002, which has the accession number GDMCC NO.66015.

9. The drug for the prevention or treatment of hyperuricemia according to claim 8, characterized in that: It also includes pharmaceutically acceptable excipients.

10. The drug for the prevention or treatment of hyperuricemia according to claim 8, characterized in that: The medications for the prevention or treatment of hyperuricemia are administered orally.

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