Human original strain lactobacillus rhamnosus tg032 and its application in preparing products for treating hyperuricemia, kidney injury and promoting glp-1 secretion

Abstract

This invention provides a human-derived *Lactobacillus rhamnosus* strain TG032 and its application in the preparation of products for treating hyperuricemia, kidney injury, and promoting GLP-1 secretion. The strain's preservation number is CCTCC NO: M 20251083. This strain exhibits a dual-pathway uric acid-lowering mechanism, combining degradation of purine nucleoside precursors and inhibition of hepatic xanthine oxidase (XOD) activity. The in vitro degradation rates of adenine, inosine, guanosine, and adenosine are 100%, 89.29%, 94.60%, and 100%, respectively. Based on the reduction of serum uric acid and inhibition of hepatic XOD activity, the minimum effective dose is 1 × 10⁻⁶. 8 CFU / each / day, at 1×10 8 ~1×10¹ 0 The dose-response relationship was clearly defined within the range of CFU / animal / day; and while lowering uric acid, it significantly reduced the levels of renal pro-inflammatory factors TNF-α, IL-6 and IL-1β, and improved glomerular atrophy and renal tubular damage.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a human-derived native strain of Lactobacillus rhamnosus TG032, and its application in the preparation of drugs for the prevention or treatment of hyperuricemia, kidney damage, and promotion of GLP-1 secretion. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disease caused by purine metabolism disorders or abnormal uric acid excretion, leading to elevated uric acid levels in the blood. Long-term high uric acid levels can induce gout, kidney damage, and various metabolic-related diseases. Currently, clinical practice mainly involves regulating uric acid levels with medications, such as reducing uric acid production by inhibiting xanthine oxidase (XOD) activity (e.g., allopurinol) or promoting uric acid excretion. However, long-term drug intervention may have adverse reactions and tolerability issues. Therefore, developing safe, low-side-effect, and long-term suitable uric acid-lowering interventions is of significant clinical importance.

[0003] In recent years, the strategy of using gut probiotics to regulate uric acid metabolism has received widespread attention. Studies have found that some lactic acid bacteria can participate in the regulation of uric acid metabolism through pathways such as degrading purines, regulating gut microbiota structure, and promoting the production of short-chain fatty acids (SCFAs). However, existing probiotic strains used to lower uric acid still have the following shortcomings: First, some strains have weak tolerance to the gastrointestinal environment, such as gastric acid, bile acids, and digestive enzymes, resulting in a significant reduction in the number of effective live bacteria that can reach the intestines and colonize after oral ingestion, making it difficult to fully exert their functions; Second, the application pathways of existing probiotic strains are relatively singular, mostly showing only the ability to degrade purines in vitro or regulate a single microbiota, lacking multi-target regulatory strains that can simultaneously act on uric acid precursor metabolism and uric acid production; Third, systematic studies on the effective dose, dose-response relationship, and nephroprotective effects of specific strains are still relatively lacking.

[0004] Therefore, there is an urgent need to screen a probiotic strain that can tolerate the gastrointestinal environment, effectively lower uric acid through a multi-target mechanism, and protect target organs such as the kidneys. Summary of the Invention

[0005] Technical problems to be solved This invention aims to address the aforementioned problems in the prior art by providing a novel human-derived *Lactobacillus rhamnosus* strain with excellent gastrointestinal tolerance, capable of synergistically lowering uric acid through dual pathways by inhibiting hepatic xanthine oxidase (XOD) activity and efficiently degrading various purine nucleoside precursors, exhibiting a clear dose-response relationship and renal protective effects. This invention also provides products containing this strain and its application in the preparation of products related to the prevention or treatment of hyperuricemia.

[0006] Technical solution This invention provides a human-derived native strain of Lactobacillus rhamnosus ( Lactobacillus rhamnosus TG032 (internal number TH02368) is a strain deposited at the China Center for Type Culture Collection (CCTCC) on May 16, 2025, with accession number CCTCC NO: M 20251083. Its 16S rDNA sequence is shown in SEQ ID NO: 1. Human-derived native strains refer to strains isolated from the intestinal flora of healthy human individuals and belonging to the host's own intrinsic flora.

[0007] This invention provides the use of the aforementioned human-derived native strain of Lactobacillus rhamnosus TG032 in the preparation of products for the prevention and / or treatment of hyperuricemia or hyperuricemia-related kidney injury.

[0008] In some embodiments, the product is a pharmaceutical product.

[0009] In some embodiments, the product has at least one of the following functions: (1) Lower serum uric acid levels; (2) Inhibits the activity of hepatic xanthine oxidase (XOD); (3) Degrading purine nucleoside precursors, wherein the purine nucleoside precursors are selected from at least one of adenine, guanosine, inosine and adenosine; (4) Improves glomerular atrophy and / or tubular damage caused by hyperuricemia; (5) Reduce the levels of pro-inflammatory factors TNF-α, IL-6 and / or IL-1β in kidney tissue; (6) Producing short-chain fatty acids, wherein the short-chain fatty acids are selected from at least one of butyric acid, valeric acid and hexanoic acid; (7) Restore the gut microbiota α diversity index (Chao1 index, Shannon index or Simpson index).

[0010] In some embodiments, the product is an oral formulation.

[0011] In some embodiments, the oral formulation is selected from powders, granules, capsules, tablets, or liquid formulations.

[0012] In some embodiments, the number of live human-derived *Lactobacillus rhamnosus* TG032 strains in the oral formulation is not less than 1 × 10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL.

[0013] The present invention provides a composition for the prevention or treatment of hyperuricemia or hyperuricemia-related kidney injury, comprising live human-derived native strain of Lactobacillus rhamnosus TG032 as the active ingredient, and pharmaceutically acceptable excipients.

[0014] In some embodiments, the concentration of live human-derived *Lactobacillus rhamnosus* TG032 strain in the composition is 1 × 10⁻⁶. 8 CFU / g~1×10¹¹ CFU / g or 1×10 8 CFU / mL ~ 1×10¹¹ CFU / mL.

[0015] In some embodiments, the dosage form of the composition is a powder, granules, capsules, tablets, or liquid preparation.

[0016] This invention provides the application of the aforementioned human-derived native strain of Lactobacillus rhamnosus TG032 in the preparation of products for promoting the secretion of glucagon-like peptide-1 (GLP-1).

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: The human-derived native strain of Lactobacillus rhamnosus TG032 screened in this invention has excellent tolerance to the gastrointestinal environment and exhibits high survival rates in both simulated gastric and intestinal fluids in vitro, ensuring that it can pass through the gastrointestinal tract in sufficient live bacterial form and successfully colonize the intestine.

[0018] The TG032 strain of this invention acts on different stages of uric acid metabolism through two pathways: First, its in vitro degradation rates of adenine, adenosine, guanosine, and inosine reach 100%, 100%, 94.60%, and 89.29%, respectively, covering all major intestinal absorbable precursors from the upstream (adenine, adenosine) to the downstream (inosine, guanosine) of the purine metabolic network, filling the gap in the substrate spectrum of existing strains and reducing exogenous purine absorption at the source; Second, it can significantly inhibit the activity of hepatic xanthine oxidase, reducing endogenous uric acid production. From the perspective of metabolic flux, blood uric acid levels depend on both the exogenous precursor absorption flux and the endogenous synthesis flux. The contributions of the two pathways to uric acid levels are multiplicative rather than additive. Intervention in a single pathway can lead to compensatory flux replenishment in the other pathway, while simultaneous intervention in both pathways can create a complementary blockade on uric acid production flux.

[0019] This invention, through systematic dose escalation experiments, clarified that the minimum effective dose of this strain, when used to reduce serum uric acid and inhibit hepatic XOD activity, is 1×10⁻⁶. 8 CFU / each / day, and at 1×10 8 -1×10¹ 0 The dose-response relationship was clearly observed within the CFU / animal / day dosage range, providing a reliable dosage basis for the precise development and clinical application of the product.

[0020] The strain of this invention not only effectively lowers uric acid but also significantly reduces the levels of pro-inflammatory factors (TNF-α, IL-6, IL-1β) in the kidneys, improving pathological changes such as glomerular atrophy and renal tubular epithelial cell damage caused by hyperuricemia, demonstrating a protective effect on the kidneys. Simultaneously, this strain can also regulate intestinal flora imbalance caused by hyperuricemia, enhancing flora diversity and bringing it closer to a healthy state.

[0021] The strain of this invention can promote the secretion of GLP-1 (glucagon-like peptide-1) in a diabetes model, and has promising applications in the preparation of hypoglycemic drugs. Attached Figure Description

[0022] Figure 1 : Evolutionary tree of human-derived strain Lactobacillus rhamnosus TG032 (internal number TH02368).

[0023] Figure 2 Figure: Detection results of short-chain fatty acids (butyric acid (BA), valeric acid (VA), and hexanoic acid (CA)) produced by strain TG032 (internal number TH02368).

[0024] Figure 3 Plate dilution spread diagrams of strain TG032 (internal number TH02368) before and after treatment in simulated gastrointestinal fluid, where (A) gastric fluid 0h control, (B) gastric fluid 2h treatment, (C) intestinal fluid 0h control, and (D) intestinal fluid 3h treatment.

[0025] Figure 4 : Statistical chart of the number of surviving colonies (Colony) of strain TG032 (internal number TH02368) in simulated gastrointestinal fluid.

[0026] Figure 5 Figure 1 shows the effects of different doses of TG032 (internal number TH02368) strain on serum and liver biochemical indicators in rats with hyperuricemia. (A) Serum uric acid (UA) level, (B) Serum creatinine (CREA) level, (C) Serum urea nitrogen (BUN) level, and (D) Liver xanthine oxidase (XOD) activity.

[0027] Figure 6 Figure 1 shows the effect of different doses of TG032 (internal number TH02368) strain on the levels of renal inflammatory factors (TNF-α, IL-6) in rats with hyperuricemia. (A) TNF-α concentration, (B) IL-6 concentration.

[0028] Figure 7 : H&E stained pathological sections of rat kidney tissue from each group.

[0029] Figure 8 Figure 1: Effects of different doses of TG032 (internal number TH02368) strain on the intestinal flora of rats with hyperuricemia. (A) Analysis of α-variability of flora, (B) Analysis of β-variability of flora, and (C) Analysis of genus-level composition of flora.

[0030] Figure 9 Figure 1 shows the effect of different doses of TG032 (internal number TH02368) strain on serum biochemical indicators in rats with hyperuricemia in a dose-response experiment. (A) Serum uric acid level, (B) Serum creatinine level, and (C) Serum urea nitrogen level.

[0031] Figure 10 Figure 1 shows the effect of different doses of TG032 (internal number TH02368) strain on liver XOD enzyme activity in rats with hyperuricemia in a dose-response experiment.

[0032] Figure 11 The effect of different doses of TG032 (internal number TH02368) strain on the level of renal inflammatory factors in hyperuricemic rats is shown in the figure. (A) TNF-α concentration, (B) IL-6 concentration.

[0033] Figure 12 : H&E stained pathological sections of rat kidney tissue in each group during the dose-response experiment.

[0034] Figure 13 Figure 1 shows the effect of different doses of TG032 (internal number TH02368) strain on the intestinal flora of rats with hyperuricemia in a dose-response experiment. (A) Analysis of α-variability of flora, (B) Analysis of β-variability of flora, and (C) Analysis of genus-level composition of flora.

[0035] Figure 14 Statistical chart showing how strain TG032 (internal number TH02368) promotes GLP-1 secretion. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the figures and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All modifications, equivalent substitutions, and improvements made within the principles and spirit of this invention should be included within the scope of protection of this invention.

[0037] Example 1: Isolation, Screening and Identification of Strains The human-derived native strain of *Lactobacillus rhamnosus* TG032 (internal designation TH02368) of this invention is an acid-resistant lactobacillus obtained through targeted screening from feces of healthy donors. The specific isolation and screening method is as follows: Using modified acid-resistant BHIS liquid medium with a pH of 3.0 as the screening medium, fecal samples were inoculated and enriched under anaerobic conditions at 37°C for 6 days. The enriched solution was serially diluted, and 100 μL of each dilution was plated onto TPY solid agar plates and anaerobically cultured at 37°C until numerous single colonies grew. Subsequently, single colonies with significantly different morphologies were picked and purified by three rounds of streak plating to obtain stable pure cultures. The purified single colonies were inoculated into BHIS liquid medium and anaerobically cultured at 37°C for 72 hours. The bacterial cells were collected and sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA sequencing. The sequencing results were compared and identified in the EzBioCloud database, confirming that the strain was a human-derived native strain of *Lactobacillus rhamnosus* (…). Lactobacillus rhamnosus The strain was named *Lactobacillus rhamnosus* TG032 (internal number TH02368), and its bacterial suspension was mixed with 10% (v / v) glycerol and stored at -80°C. Simultaneously, the strain was deposited at the China Center for Type Culture Collection (CCTCC) on May 16, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251083.

[0038] Its 16S rDNA sequence is shown in SEQ ID NO:1.

[0039] SEQ ID NO:1 Two other strains of *Lactobacillus rhamnosus* were also isolated, internally numbered TH02627 and TH02370. Their 16S rDNA sequences are as follows: The TH0237016S rDNA sequence is as follows: SEQ ID NO:2: The TH0262716S rDNA sequence is as follows: SEQ ID NO:3: The phylogenetic tree of the human-derived strain *Lactobacillus rhamnosus* TG032 (internal designation TH02368) is as follows: Figure 1 As shown.

[0040] Example 2: Determination of the ability of strain TG032 (internal number TH02368) to produce short-chain fatty acids (SCFAs) 2.1 Strain activation TG032 (internal code TH02368) strain, preserved in glycerol at -80℃, was streaked onto MRS agar plates and incubated upside down in a 37℃ anaerobic workstation until single colonies grew. A single colony was picked and inoculated into 7 mL of MRS liquid medium and anaerobically cultured at 37℃ for 24 hours to obtain the first-generation culture. The first-generation culture was then transferred to a fresh 7 mL MRS liquid medium at a 2% (v / v) inoculation rate and anaerobically cultured at 37℃ for 24 hours to obtain the second-generation culture, which served as the seed culture for subsequent experiments.

[0041] 2.2 Sample Pretreatment and Detection After thawing the second-generation bacterial culture sample, vortex for 3 minutes to mix. Transfer 50 μL of sample to a 1.5 mL centrifuge tube, add 100 μL of 0.5% (v / v) phosphate solution, and vortex at 2500 r / min for 3 minutes. Then add 750 μL of methyl tert-butyl ether (MTBE) extraction reagent containing internal standard, vortex at 2500 r / min for 3 minutes, sonicate at 4℃ for 5 minutes, and centrifuge at 12000 r / min at 4℃ for 10 minutes. Transfer 200 μL of supernatant to the inner liner of a sample vial for targeted quantitative analysis using gas chromatography-tandem mass spectrometry (GC-MS / MS). Blank MRS liquid culture medium was treated in the same way as a blank control (Ctro).

[0042] 2.3 Plotting the Standard Curve A series of standard solutions with concentrations ranging from 0.005 μg / mL to 20 μg / mL were prepared and analyzed according to the method described in section 2.2 above. Standard curves for each short-chain fatty acid were plotted with the ratio of external standard to internal standard concentrations as the x-axis and the ratio of external standard to internal standard peak areas as the y-axis. The results showed that the coefficients of determination (R²) for the standard curves of butyric acid, valeric acid, and hexanoic acid were all greater than 0.99, indicating good linearity.

[0043] 2.4 Methods for detecting sample content The ratio of the integrated peak area of ​​all detected samples is substituted into the linear equation of the standard curve for calculation. Further calculations using the given formula yield the final content data of the substance in the actual sample. Note: The calculation formula has already undergone unit conversion; simply substitute the corresponding values ​​to obtain the sample content.

[0044] The content of SCFAs in the sample (pg / mL) = (C Ctro)×V×1000 / V0 C: Concentration value (μg / mL) obtained by substituting the ratio of the integrated peak area in the sample into the standard curve; Ctro: The concentration value (μg / mL) obtained by substituting the integrated peak area ratio in the blank control (pure culture medium) into the standard curve; V: Volume of extractant added during sample extraction (μL); V0: The amount of sample taken (mL).

[0045] 2.5 Experimental Results Experimental results are as follows Figure 2 As shown, strain TG032 (internal number TH02368) can promote the production of various short-chain fatty acids, particularly increasing the level of butyric acid. In addition, the levels of valeric acid and hexanoic acid were also significantly increased. Based on the standard curve, the butyric acid content in the sample was calculated to be 400 pg / mL. For specific content data, please refer to [link to relevant data]. Figure 2 This indicates that strain TG032 (internal number TH02368) has excellent fermentation acid production capacity, especially in producing butyric acid, which is crucial for intestinal barrier function.

[0046] Studies have shown that hyperuricemia may lead to intestinal barrier damage, while butyrate can effectively maintain the integrity of the intestinal mucosa. Butyrate supplementation has been shown to alleviate intestinal inflammation in hyperuricemic mouse models, reduce the levels of inflammatory factors (such as TNF-α and IL-6), thereby protecting intestinal barrier function and reducing the risk of uric acid entering the bloodstream through the damaged intestine. Furthermore, hexanoic acid, as an important SCFA, plays a significant role in regulating the gut microbiota. Hexanoic acid can promote the growth of beneficial bacteria and inhibit the proliferation of harmful bacteria, thereby improving the gut microbiota. Although valerate is a minor SCFA in the gut and its levels are low, its increasing trend suggests that strain TG032 (internal code TH02368) has the potential to promote the synthesis of diverse SCFAs in the gut, contributing to a richer intestinal metabolite profile.

[0047] The linear equations and coefficients of determination of the standard curves of the substances tested in this project are shown in Table 1 below.

[0048] Table 1. Standard Curve, Linear Equation, and Coefficient of Determination for the Sample

[0049] Example 3: Evaluation of the in vitro gastrointestinal environment tolerance of strain TG032 (internal number TH02368) This study used an in vitro simulated gastrointestinal fluid method to evaluate the survival ability of live bacteria TG032 (internal number TH02368) in the extreme environment of the gastrointestinal tract.

[0050] Take 1 mL of bacteria with a viable count of 5 × 10⁶ 8 Second-generation bacterial culture with CFU / mL was added to 9.0 mL of simulated gastric fluid (Regen Biosciences, catalog number CZ0213), vortexed to mix, and incubated statically in a 37°C anaerobic incubator. The culture was then incubated at pH 3.0 for 2 hours. Bacterial culture was aspirated before treatment (0 hours) and after treatment (2 hours) and serially diluted 10-fold with sterile PBS buffer to 10⁻⁶. -8 Select 50 μL of a suitable gradient dilution and spread it on MRS solid medium plates. After anaerobic incubation at 37℃ for 48 hours, colony counting is performed. Simulated gastric fluid survival rate (%) = (total number of viable bacteria in the 2h treatment group / total number of viable bacteria in the 0h control group) × 100%.

[0051] Take another 1 mL of bacterial suspension of the same concentration and add it to 9.0 mL of simulated intestinal fluid (Regen Biotech, catalog number CZ0201). Incubate in an anaerobic incubator at 37℃ and pH 6.75 for 3 hours. Samples were taken, diluted, spread, and counted before treatment (0 hours) and after treatment (3 hours). Simulated intestinal fluid survival rate (%) = (total number of viable bacteria in the 3h treatment group / total number of viable bacteria in the 0h control group) × 100%.

[0052] The results of flat plate coating are shown below. Figure 3 The colony count results are shown in [link to statistics]. Figure 4 The specific survival rate results are shown in Table 2.

[0053] Table 2 Survival rate of TG032 (internal number TH02368) in simulated gastrointestinal fluid.

[0054] Experimental results showed that strain TG032 (internal code TH02368) exhibited good tolerance in both simulated gastric and intestinal fluids. After 2 hours of treatment in simulated gastric fluid at pH 3.0, the survival rate remained at 31.65%, and after 3 hours of treatment in simulated intestinal fluid, the survival rate reached 104.14% (the survival rate of over 100% was due to the continued proliferation of bacteria in the intestinal fluid environment during treatment). This indicates that the strain can resist the erosion of digestive enzymes and has the potential to survive stably and successfully colonize in the intestine.

[0055] Example 4: Determination of the in vitro purine nucleoside degradation ability of TG032 (internal number TH02368) 4.1 Preparation of bacterial culture and preparation of standard products The second-generation bacterial suspension of strain TG032 (internal number TH02368) was prepared according to the method in Example 2.1. The bacterial suspension was centrifuged at 10,000 r / min for 10 minutes at 4°C, the supernatant was discarded, the bacterial cells were collected and washed twice with sterile PBS buffer, and finally resuspended in PBS to obtain a bacterial suspension.

[0056] Accurately weigh 20 mg of adenine and 20 mg of guanosine standards, dissolve them with a small amount of 0.1 mol / L NaOH, and then bring the volume to 50 mL with PBS to obtain a mixed standard solution containing 400 μg / mL each of adenine and guanosine. Prepare a mixed standard solution containing 400 μg / mL each of inosine and adenosine using the same method.

[0057] 4.2 Reaction System and Detection Take 0.75 mL of the above bacterial suspension and mix it with 0.75 mL of one of the two purine nucleoside mixed standard solutions mentioned above. Each reaction system should be set up in triplicate. A PBS system containing only the standard solution should be used as a control. Incubate the reaction system in a shaker at 37°C for 6 hours. Immediately after the reaction is complete, heat the reaction solution in a 90°C water bath for 10 minutes to terminate the reaction. After cooling, centrifuge at 4000 r / min for 10 minutes, collect the supernatant, filter it through a 0.22 μm filter membrane, and analyze it using high-performance liquid chromatography (HPLC).

[0058] HPLC conditions: mobile phase: 10 mM potassium dihydrogen phosphate (containing 1% methanol), pH=3.5; isocratic elution; flow rate: 1 mL / min; column temperature: 40℃; injection volume: 10 μL; detection wavelength: 254 nm; column: Waters C18 column (4.6 mm × 250 mm). The degradation rate was calculated by comparing the peak areas of the standards before and after the reaction. Degradation rate (%) = [(C0 - C1) / C0] × 100%, where C0 is the concentration of the standard without bacterial control, and C1 is the concentration of the standard after bacterial reaction.

[0059] Meanwhile, strains TH02627 and TH02370 were set up as control experiments, using the same method as strain TG032.

[0060] 4.3 Experimental Results The results are shown in Table 3.

[0061] Table 3. In vitro degradation rate of purine nucleosides by TG032

[0062] The results showed that strain TG032 exhibited a broad-spectrum and highly efficient purine nucleoside degradation ability, demonstrating strong degradation activity against four common uric acid precursors. The degradation rates for adenine, adenosine, guanosine, and inosine were 100%, 100%, 94.60%, and 89.29%, respectively, significantly reducing the intestinal absorption of purines from their source. Furthermore, strain TG032 showed significantly stronger abilities than its contemporaries TH02627 and TH02370 in adenine, guanosine, and inosine degradation.

[0063] Among the multiple *Lactobacillus rhamnosus* strains isolated under the same screening system, TH02627 exhibited degradation rates of only 7.06% and 5.41% for adenine and inosine, respectively, while TH02370 showed no degradation activity for guanosine and inosine (degradation rates of 0.14% and 0%, respectively). These figures represent orders of magnitude different from TG032 (degradation rates of 89.29%–100%). This control result indicates that even within the same species, from the same isolation source, and under the same screening conditions, different strains exhibit fundamental differences in the integrity of their purine metabolic pathways. The broad-spectrum and highly efficient degradation ability of TG032 is not a species-specific characteristic of *Lactobacillus rhamnosus*, but rather an individual attribute of this specific strain. Those skilled in the art, starting from any single strain in the prior art, cannot reasonably expect or probabilistically obtain the technical solution of this invention through conventional screening.

[0064] Analysis of the purine metabolism network revealed that the degradation of inosine and guanosine primarily depends on purine nucleoside phosphorylase (PNP) activity, an activity reported in some lactic acid bacteria. Adenosine degradation requires adenosine deaminase (ADA) activity, while adenine degradation requires adenine deaminase activity; both of these enzyme activities are extremely rare in lactic acid bacteria. The TG032 strain achieved complete degradation of adenine and adenosine within 6 hours (100% degradation rate), suggesting that this strain simultaneously and completely expresses multiple purine metabolism enzyme systems, including PNP, ADA, and adenine deaminase, demonstrating a rare "full-node" purine precursor clearance ability. In contrast, control strains TH02627 and TH02370 showed only partial or no degradation activity for inosine and guanosine, further confirming the scarcity of such complete enzyme combinations in *Lactobacillus rhamnosus*. Furthermore, TG032 exhibited significantly higher degradation rates for all four substrates than the control strains, indicating that its enzyme expression levels or substrate affinity were also dominant. This broad-spectrum degradation ability can directly reduce the absorption flux of four purine precursors from the intestine into the blood via intestinal epithelial nucleoside transporters, which is the structural basis for TG032 to lower uric acid at the source.

[0065] Example 5: Study on the minimum effective dose of TG032 (internal number TH02368) strain in rats with hyperuricemia This example aims to explore the minimum effective dose of TG032 (internal number TH02368) that effectively reduces serum uric acid in hyperuricemic rats.

[0066] 5.1 Grouping and treatment of experimental animals 36 male SD rats (6-8 weeks old), weighing approximately 190-220 g, with individual body weights within ±20% of the average body weight, were obtained from Speifu (Suzhou) Biotechnology Co., Ltd., with the experimental animal production license number: SCXK (Beijing) 2024-0001. After one week of adaptive feeding, they were randomly divided into 6 groups, with 6 rats in each group: Normal control group (CON group): Administer an equal volume of vehicle by gavage daily.

[0067] Model group (MOD group): Administer 500 mg / kg of hypoxanthine by gavage daily and inject 300 mg / kg of potassium oxonate intraperitoneally for 14 consecutive days.

[0068] Positive drug group (allopurinol (ALL) group): On the basis of modeling, administer 15 mg / kg of allopurinol by gavage daily.

[0069] Low-dose group (TH02368-10 7 group): On the basis of modeling, administer 1×10 7 CFU / rat of TG032 (internal number TH02368) bacterial solution by gavage daily.

[0070] Medium-dose group (TH02368-10 8 group): On the basis of modeling, administer 1×10 8 CFU / rat of TG032 (internal number TH02368) bacterial solution by gavage daily.

[0071] High-dose group (TH02368-10 9 group): On the basis of modeling, administer 1×10 9 CFU / rat of TG032 (internal number TH02368) bacterial solution by gavage daily.

[0072] Gavage of the drug or bacterial solution is performed 1 hour after daily modeling, and the experimental period is 2 weeks.

[0073] 5.2 Detection indicators and methods At the end of the experiment, blood was collected from the abdominal aorta of rats under anesthesia, and serum was separated to detect uric acid (UA), creatinine (CREA), and blood urea nitrogen (BUN) levels. After euthanizing the rats, liver and kidney tissues were immediately separated. Part of the liver tissue was used to detect xanthine oxidase (XOD) activity; part of the kidney tissue was used to detect levels of inflammatory factors such as TNF-α and IL-6, and pathological sections were prepared for H&E staining observation. Cecal contents of the rats were collected for 16S rDNA gene sequencing analysis of the intestinal flora structure.

[0074] The experimental instruments and materials used in this embodiment are shown in Tables 3.1 and 3.2, respectively.

[0075] Table 3.1 Experimental Apparatus

[0076] Table 3.2 Experimental Materials

[0077] 5.3 Experimental Results Serum and liver biochemical indicators: Results are as follows Figure 5 As shown in Table 4. Serum uric acid (UA), creatinine (CREA), and blood urea nitrogen (BUN) levels, as well as liver xanthine oxidase (XOD) activity, were measured in each group of rats using a kit. SPSS 26.0 software was used to analyze the mean, standard deviation, and differences between groups. The results are shown in Table 4. With the CON group as the control, UA, CREA, and BUN were significantly increased in the MOD group (UA P<0.001; CREA P<0.05; BUN P<0.01), indicating successful establishment of the HUA model. Simultaneously, the MOD group was used as a control, TH02368-10 8 The levels of UA and XOD in the group were significantly lower than those in the MOD group (UA P<0.01; XOD P<0.05), indicating that TG032 (internal code TH02368) can regulate uric acid production by inhibiting XOD activity, and the minimum effective dose is 1×10⁻⁶. 8 CFU / only / d.

[0078] Table 4. Effects of strain TG032 (internal number TH02368) on serum and liver biochemical parameters in rats.

[0079] Note: "#" indicates a significant difference compared to group CON, #P<0.05 indicates a significant difference, ##P<0.01 indicates an extremely significant difference, and ###P<0.001 indicates a highly significant difference; "This indicates a significant difference compared to the MOD group." P < 0.05 indicates a significant difference. P < 0.01 indicates a highly significant difference. P < 0.001 indicates a highly significant difference.

[0080] The concentrations of pro-inflammatory factors TNF-α and IL-6 in rat kidney homogenate were detected using an ELISA kit. SPSS 26.0 software was used to analyze the means, standard deviations, and differences between groups. The results are shown in Table 5. Figure 6 As shown, the TNF-α concentration in the MOD group was 274.09±12.75 pg / mL, significantly higher than that in the CON group (156.93±21.94 pg / mL, P<0.001); the IL-6 concentration in the MOD group was 123.47±12.15 pg / mL, significantly higher than that in the CON group (73.40±16.23 pg / mL, P<0.001), indicating a severe inflammatory response in the MOD group rats. After treatment with different doses of TG032 (internal code TH02368), the concentrations of pro-inflammatory factors in each treatment group were reduced to varying degrees. 7 Group and TH02368-10 9 The TNF-α concentration in the group of rats was significantly lower than that in the MOD group (P<0.001, P<0.01); TH02368-10 7 Group, TH02368-10 8 Group and TH02368-10 9 The IL-6 concentration in the group of rats was significantly lower than that in the MOD group (P<0.05, P<0.05, P<0.001). The results indicate that different doses of TG032 (internal code TH02368) have different degrees of regulatory effect on the inflammatory response in rats.

[0081] Table 5. Effects of probiotics on rat inflammatory factors in Example 5.

[0082] Note: "#" indicates a significant difference compared to group CON, #P<0.05 indicates a significant difference, ##P<0.01 indicates an extremely significant difference, and ###P<0.001 indicates a highly significant difference; "This indicates a significant difference compared to the MOD group." P < 0.05 indicates a significant difference. P < 0.01 indicates a highly significant difference. P < 0.001 indicates a highly significant difference.

[0083] Kidney tissue pathological sections were prepared and H&E staining analysis was performed. For example... Figure 7 As shown, pathological analysis of rat kidney tissue revealed that the CON group rats had intact kidney tissue structure, clear glomerular boundaries, normal tubular morphology, and no obvious inflammatory cell infiltration, exhibiting a healthy kidney tissue state. Compared with the CON group, the MOD group rats showed pathological changes in the kidneys: glomerular atrophy, interstitial edema, necrosis of renal tubular epithelial cells, and obvious inflammatory cell infiltration, indicating severe kidney damage. Notably, compared with MOD, TG032 (internal code TH02368) intervention improved the glomerular and tubular structure of HUA rats to a certain extent and reduced inflammatory cell infiltration. This result suggests that TG032 (internal code TH02368) has a certain protective effect against kidney damage induced by hyperuricemia.

[0084] Gut microbiota regulation: such as Figure 8 As shown in the figure, the structure of the cecal microbiota in rats was analyzed using 16S rDNA gene sequencing technology. Regarding α-diversity, analysis using the Chao1, Shannon, and Simpson indices showed that all three indices decreased in the MOD group compared to the CON group, with a significant decrease in the Chao1 index (P<0.05), indicating that HUA modeling significantly reduced the richness and evenness of intestinal microbial species in rats. After intervention with different doses of TG032 (internal code TH02368), the rat microbiota structure showed a trend towards that of the CON group, with increased microbiota richness and evenness, indicating that TG032 (internal code TH02368) can regulate the α-diversity of the intestinal microbiota in HUA rats, promoting its transformation towards the CON group. Regarding β-diversity, there were certain differences in the intestinal microbiota between the MOD and CON groups, suggesting that HUA modeling induced changes in microbiota structure. Compared to the MOD group, the three different doses of TG032 (internal code TH02368) all improved the microbiota structure to varying degrees, with TH02368-10 showing the most significant improvement. 9 The MOD group showed a higher overlap with the CON group in terms of sample points, indicating that TG032 (internal code TH02368) has a regulatory effect on the gut microbiota structure of HUA rats, promoting the microbiota to converge with that of the CON group. Genus-level analysis showed that, compared with the CON group, the MOD group... g_Alistipes、g_ Bacteroides, g_Muribaculaceae, g_Ruminococcus, g_un_f_Oscillospiraceae Increased abundance g_[Eubacterium]siraeum_group, g_Lachnospiraceae_NK4A136_group, g_Lactobacillus Decreased abundance suggests microbial dysbiosis induced by HUA modeling; intervention with various doses of TG032 (internal code TH02368) can improve the abundance. g_[Eubacterium]_siraeum_group, g_Prevotellaceae_NK3B31_group Abundance, decrease g_ Muribaculaceae Abundance promotes the convergence of bacterial community structure towards the CON group.

[0085] Example 6: Study on the dose-effect relationship of strain TG032 (internal number TH02368) in hyperuricemic rats This example aims to expand the dose range and further clarify the complete dose-effect relationship of the uric acid-lowering effect of TG032 (internal number TH02368).

[0086] 6.1 Grouping and treatment of experimental animals In this experiment, a total of 36 SPF-grade male SD (Sprague-Dawley) rats were used, all of which were used in the experiment. The rats were 7 weeks old upon purchase, with a body weight of about 180–220 g. During grouping, the body weight of rats in each group was controlled within the range of the average body weight ±20%. The experimental animals were provided by Shanghai Shuyuan Technology Co., Ltd., and the animal production license number is SYXK (Hu) 2024-0006. 36 SD rats were randomly divided into 6 groups, 6 rats in each group: Normal control group (CON group), model group (MOD group), positive drug group (ALL group): the treatment methods are the same as those in Example 5. The modeling lasted for 17 days, and administration started from the 3rd day for 14 consecutive days.

[0087] TH02368-10 8 group, TH02368-10 9 group: the same as Example 5.

[0088] Ultra-high dose group (TH02368-10¹ 0 group): On the basis of modeling, the rats were intragastrically administered with the bacterial solution of strain TG032 (internal number TH02368) at a dose of 1×10¹ 0 CFU / rat per day.

[0089] 6.2 Detection indicators and methods The detection indicators and methods are the same as those in Example 5. The relationship between the serum uric acid level of each dose group and the dose on the 14th day of administration was analyzed with emphasis. The experimental instruments and experimental materials used in this example are shown in Table 5.1 and Table 5.2, respectively.

[0090] Table 5.1 Main instruments

[0091] Table 5.2 Main materials

[0092] 6.3 Experimental results Serum uric acid level: The results are shown in Figure 9 and Table 6. On the 14th day of administration, compared with the MOD group (363.38 μmol / L), the TH02368-10 8 group, 10 9 group and 10¹0 The serum uric acid levels in the rats decreased to 225.5 μmol / L (P<0.01), 167.13 μmol / L (P<0.001), and 157.12 μmol / L (P<0.001), respectively, showing a clear dose-dependent effect.

[0093] Table 6. Effects of TG032 (internal number TH02368) on serum biochemical parameters in rats (day 14 after drug administration)

[0094] Note: "#" indicates a significant difference compared to group CON, #P<0.05 indicates a significant difference, ##P<0.01 indicates an extremely significant difference, and ###P<0.001 indicates a highly significant difference; "This indicates a significant difference compared to the MOD group." P < 0.05 indicates a significant difference. P < 0.01 indicates a highly significant difference. P < 0.001 indicates a highly significant difference.

[0095] Liver XOD activity: such as Figure 10 As shown in Table 7, compared with the MOD group, the liver XOD activity of all TG032 (internal number TH02368) intervention groups was significantly inhibited (P<0.01 or P<0.001), and the inhibitory effect showed a dose-dependent trend.

[0096] Table 7. Effects of strain TG032 (internal number TH02368) on biochemical parameters of rat liver.

[0097] Note: "#" indicates a significant difference compared to group CON, #P<0.05 indicates a significant difference, ##P<0.01 indicates an extremely significant difference, and ###P<0.001 indicates a highly significant difference; "This indicates a significant difference compared to the MOD group." P < 0.05 indicates a significant difference. P < 0.01 indicates a highly significant difference. P < 0.001 indicates a highly significant difference.

[0098] Kidney protective effects: as shown in Table 8 and Figure 11As shown, the levels of TNF-α and IL-6 in the kidneys of rats in the MOD group were significantly higher than those in the CON group (P<0.001, P<0.001, P<0.01), indicating that the model successfully induced a local inflammatory response in the kidneys. After allopurinol treatment, the levels of TNF-α and IL-6 in the kidneys of the ALL group showed a decreasing trend compared to the MOD group, but there was no statistically significant difference (P>0.05); after treatment with TG032 (internal code TH02368), TH02368-10... 9 Group, TH02368-10 10 The renal TNF-α level in the TG032 (internal number TH02368) group was significantly lower than that in the MOD group (P<0.05, P<0.001); the renal IL-6 level in rats in each TG032 (internal number TH02368) dose group was significantly lower than that in the MOD group (IL-6: P<0.05, P<0.05, P<0.01). These results suggest that the TG032 (internal number TH02368) strain can significantly alleviate the inflammatory state of kidney disease associated with hyperuricemia.

[0099] Table 8. Effects of strain TG032 (internal number TH02368) on rat inflammatory factors in Example 6.

[0100] Note: "#" indicates a significant difference compared to group CON, #P<0.05 indicates a significant difference, ##P<0.01 indicates an extremely significant difference, and ###P<0.001 indicates a highly significant difference; "This indicates a significant difference compared to the MOD group." P < 0.05 indicates a significant difference. P < 0.01 indicates a highly significant difference. P < 0.001 indicates a highly significant difference.

[0101] Figure 12 Pathological sections confirmed that the kidneys of the CON group rats had normal structure, regular glomerular morphology, and uniformly sized renal tubules without dilation or atrophy. In contrast, the MOD group rats showed significant glomerular atrophy, renal tubular epithelial cell damage, and sloughing. The ALL group rats also exhibited glomerular atrophy. It is noteworthy that TH02368-10... 8 The glomerular atrophy in the rats of the TH02368-10 group was improved compared with that in the MOD group; 9 With TH02368-10 10The glomeruli of the rats in the TG032 group were more regular in morphology, showing a more significant improvement compared to the MOD group. These results suggest that TG032 (internal code TH02368) has a certain ameliorative effect on glomerular atrophy caused by hyperuricemia.

[0102] Gut microbiota regulation: such as Figure 13 As shown, regarding α-diversity, analysis using Chao1, Shannon, and Simpson indices revealed that, compared to the CON group, the Shannon and Simpson indices in the MOD group decreased, with the Simpson index showing a significant decrease (P<0.05). This suggests that HUA modeling reduces the species richness of the rat gut microbiota and weakens the dominance of the dominant species. After intervention with various doses of TG032 (internal code TH02368), the structure of the rat gut microbiota showed a trend towards that of the CON group, indicating that TG032 (internal code TH02368) can regulate the α-diversity of the gut microbiota in HUA rats and promote its transformation towards the CON group. In terms of β-diversity, the intestinal microbiota structure of the MOD group and the CON group rats was significantly separated, suggesting that HUA modeling induced changes in microbiota structure. Compared with the MOD group, the three different doses of TG032 (internal code TH02368) groups all improved the microbiota structure to varying degrees, showing a trend towards the CON group, indicating that TG032 (internal code TH02368) has a regulatory effect on the intestinal microbiota structure of HUA rats and can promote the microbiota to converge with the CON group. Genus-level analysis showed that, compared with the CON group, the MOD group... g_Blautia, g_Lachnospiraceae_ NK4A136_group, g_Lactobacillus, g_un_c_Bacilli, g_un_f_Oscillospiraceae Increased abundance g_Clostridia_UCG-014, g_Muribaculaceae, g_Romboutsia Decreased abundance suggests microbial dysbiosis induced by HUA modeling; intervention with various doses of TG032 (internal code TH02368) can improve... g_Muribaculaceae Abundance, decrease g_Blautia, g_Lachnospiraceae_NK4A136_group, g_Lactobacillus Abundance promotes the convergence of bacterial community structure towards the CON group.

[0103] The above results indicate that TG032 (internal designation TH02368) at 1×10 8 CFU / each / day up to 1×10¹ 0 Within the dose range of CFU / animal / day, a clear dose-response relationship was observed in the uric acid-lowering and related kidney-protective and gut microbiota-regulating effects.

[0104] Determining the in vivo effective dose is a key step that distinguishes oral probiotic products from in vitro functional evaluations. After oral administration, probiotics must sequentially pass through the gastric acid and bile salt environment and colonize the intestines. The actual number of live bacteria performing metabolic functions is significantly lower than the ingested amount. Simultaneously, the intestinal flora also participates in purine metabolism, and exogenous bacteria must reach a certain abundance to alter the net metabolic balance of purines in the intestines. The 1×10⁻⁶ dose determined in this invention... 8The minimum effective dose of CFU / vial / day, combined with the 31.65% survival rate of this strain in simulated gastric fluid as demonstrated in Example 3, allows for the calculation of the actual number of live bacteria reaching the intestines to exert their effect, thus establishing a complete quantitative causal chain between the ingested dose, the number of effective live bacteria in the intestines, and the uric acid-lowering effect. In Example 6, 10 8 10 9 10¹ 0 Serum uric acid levels in the three dosage groups decreased progressively with increasing dosage (to approximately 62%, 46%, and 43% of the model group, respectively), and the improvement in hepatic xanthine oxidase activity and renal inflammatory factor levels also showed a synchronous dose-dependent trend. This indicates that the dose-response relationship is a holistic pattern of multi-target action of the strain rather than a random fluctuation of a single indicator. No existing research has provided such a complete dose characterization of the uric acid-lowering effect of *Lactobacillus rhamnosus*, and the dosage parameters of this invention constitute a technical contribution that distinguishes it from existing technologies.

[0105] Example 7: TG032 (internal number TH02368) strain promotes GLP-1 secretion Construction of a diabetes model: (1) Grouping and modeling: C57BL / 6J mice, weighing 18-22 g, were randomly divided into a normal control group and a model group after 1 week of acclimatization. The normal group was fed a normal maintenance diet throughout the process. The model group was fed a high-fat, high-sugar diet for 4 weeks to induce insulin resistance. Then, streptozotocin was injected intraperitoneally once a day for 5 consecutive days. After the last injection, the high-fat, high-sugar diet was resumed for 7 days. During this period, body weight, water intake and general condition were monitored daily. Blood was collected from the tail tip on the 7th and 11th days after fasting to measure fasting blood glucose. If the fasting blood glucose was ≥11.1 mmol / L on both occasions and the interval was ≥72 hours, the type 2 diabetes model was considered successful.

[0106] (2) Intervention measures: After the model was successfully established, the mice were randomly divided into 3 groups: normal control group (Control, CON), model control group (Model, MOD), and Lactobacillus rhamnosus group (TH02368 group). The mice in each group were given the corresponding dose of the intervention by gavage: the Lactobacillus rhamnosus group was given Lactobacillus rhamnosus TG032 (internal number TH02368) bacterial suspension (live count 1×10⁻⁶). 9 CFU / mouse / day); normal control and model control mice were administered the same volume of physiological saline by gavage. The gavage period lasted for 10 weeks, during which fasting blood glucose and related metabolic indicators were monitored regularly.

[0107] (3) Indicator detection: After the intervention period ended, the patient fasted for 12 hours, and blood was collected from the orbital cavity to separate the serum. Glucagon-like peptide-1 (GLP-1) was detected using an ELISA kit.

[0108] (4) Experimental results: like Figure 14 As shown in Table 9, regarding hormones: compared with the normal control group, the glucagon-like peptide-1 (GLP-1) level in the model group was significantly reduced, and the Lactobacillus rhamnosus group was superior to the model group.

[0109] Table 9. Effects of each intervention group on GLP-1 secretion in mice.

[0110] Note: "#" indicates a significant difference compared to group CON, #P<0.05 indicates a significant difference, ##P<0.01 indicates an extremely significant difference, and ###P<0.001 indicates a highly significant difference; "This indicates a significant difference compared to the MOD group." P < 0.05 indicates a significant difference. P < 0.01 indicates a highly significant difference. P < 0.001 indicates a highly significant difference.

[0111] Glucagon-like peptide-1 (GLP-1) is secreted by intestinal L cells, and its secretion is regulated by the intestinal metabolite environment. Studies have shown that short-chain fatty acids can stimulate GLP-1 secretion by activating free fatty acid receptors such as GPR41 / FFAR3 and GPR43 / FFAR2 on the surface of L cells. Combined with the result in Example 2 that TG032 significantly promoted butyrate production, the effect of the present invention strain in promoting GLP-1 secretion may be an extension of its fermentation acid production function at the endocrine level, i.e., a cascade mechanism of "acid production—receptor activation—GLP-1 release". It should be noted that there is no precedent in the prior art for linking the purine degradation function of *Lactobacillus rhamnosus* with the GLP-1 secretion-promoting function in the same strain. TG032 exhibits activity in two seemingly unrelated metabolic dimensions—sugar metabolism and purine metabolism—further demonstrating the integrity of the metabolic pathway and the multi-target regulatory characteristics of this strain.

[0112] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A human-derived native strain of Lactobacillus rhamnosus TG032, characterized in that, The strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251083.

2. The human-derived native strain of *Lactobacillus rhamnosus* TG032 according to claim 1, characterized in that, Its 16S rDNA sequence is shown in SEQ ID NO:

1.

3. The use of the human-derived native strain of Lactobacillus rhamnosus TG032 according to claim 1 in the preparation of products for the prevention and / or treatment of hyperuricemia or hyperuricemia-related kidney injury.

4. The application according to claim 3, characterized in that, The product in question is a pharmaceutical product.

5. The application according to claim 3 or 4, characterized in that, The product has at least one of the following functions: (1) Lower serum uric acid levels; (2) Inhibits the activity of hepatic xanthine oxidase; (3) Degrading purine nucleoside precursors, wherein the purine nucleoside precursors are selected from at least one of adenine, guanosine, inosine and adenosine; (4) Improves glomerular atrophy and / or tubular damage caused by hyperuricemia; (5) Reduce the levels of pro-inflammatory factors TNF-α, IL-6 and / or IL-1β in kidney tissue; (6) Producing short-chain fatty acids, wherein the short-chain fatty acids are selected from at least one of butyric acid, valeric acid and hexanoic acid; (7) Restore the Chao1 index, Shannon index or Simpson index of gut microbiota.

6. The application according to claim 3 or 4, characterized in that, The product is an oral preparation.

7. The application according to claim 6, characterized in that, The dosage form of the product is tablets, capsules, granules, powders, oral liquids, suspensions, or pellets.

8. The application according to claim 6, characterized in that, The oral formulation contains at least 1×10⁻⁶ live human-derived *Lactobacillus rhamnosus* TG032 strains. 8 CFU / g or 1×10 8 CFU / mL.

9. The use of the human-derived native strain of Lactobacillus rhamnosus TG032 according to claim 1 or 2 in the preparation of products for promoting glucagon-like peptide-1 secretion.

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