Use of lactobacillus reuteri gold gut-ccfm1040 in reducing uric acid

Abstract

This invention discloses the application of *Lactobacillus reuteri* GOLDGUT-CCFM1040 in lowering uric acid, belonging to the field of biomedical technology. This strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No. 60515, on December 10, 2018, and its taxonomic name is... Lactobacillus reuteri The *Lactobacillus reuteri* GOLDGUT-CCFM1040 of this invention exhibits outstanding effects in lowering uric acid, improving renal function, regulating intestinal flora, and enhancing the intestinal barrier. It is safe, mild, and has multi-target synergistic characteristics, and can provide a highly efficient and promising microecological solution for nutritional intervention in hyperuricemia and related metabolic abnormalities.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to the application of Lactobacillus reuteri GOLDGUT-CCFM1040 in lowering uric acid. Background Technology

[0002] Hyperuricemia (HUA) has become one of the most common metabolic diseases plaguing modern society, characterized by abnormally high levels of uric acid (UA) in the blood. The imbalance between UA production and excretion is the core mechanism leading to this condition. On the one hand, it may be due to excessive purine intake or over-synthesis in the body; on the other hand, it is often due to impaired renal excretion function, which fails to effectively remove excess UA from the body. With changes in lifestyle and dietary structure, the prevalence of HUA is showing a continuous upward trend globally, gradually becoming a significant threat to public health.

[0003] Sustained high uric acid (UA) levels can lead to a range of health problems. The most typical clinical manifestation is gouty arthritis, caused by urate crystal deposition in joints and surrounding tissues. Acute attacks are characterized by severe pain, significantly impairing patients' physiological function and quality of life. Furthermore, high UA levels are closely associated with kidney damage, potentially leading to uric acid nephropathy and even renal insufficiency. Increasing research indicates that UA is an independent risk factor for various chronic diseases, including cardiovascular disease, metabolic syndrome, and type 2 diabetes. This suggests that the pathological impact of UA extends far beyond the musculoskeletal system, constituting a systemic metabolic disorder requiring systemic intervention.

[0004] Currently, clinical treatment strategies for hyperalbuminuria (HUA) mainly revolve around two aspects: first, inhibiting UA production, commonly using drugs such as allopurinol and febuxostat; and second, promoting UA excretion, such as benzbromarone. Although these drugs can control UA levels to some extent, their clinical application still has many limitations. Some patients may experience adverse reactions such as drug allergies and liver damage, and long-term use may increase the risk of cardiovascular disease. In addition, existing drugs have limited overall regulatory effects on the metabolic disorders underlying HUA, making it difficult to fundamentally solve the imbalance between UA production and excretion. These shortcomings have prompted researchers to continuously explore safer and more effective regulatory pathways.

[0005] Given the limitations of existing treatment options, probiotics, as live microorganisms that promote health by regulating the balance of the gut microbiota, are gradually becoming an emerging research direction for intervention in hyperuricemia (HUA). In recent years, as the core role of the gut microbiota in metabolic regulation has been increasingly revealed, probiotics have provided a new perspective on the prevention and treatment of HUA by influencing purine metabolism, systemic inflammation, and renal function through multiple dimensions of UA homeostasis. Preliminary studies indicate that certain probiotic strains (such as certain Lactobacillus and Bifidobacterium) not only have the ability to directly degrade UA in the gut but also competitively inhibit purine absorption, thereby reducing the production of exogenous UA. Furthermore, probiotics can alleviate the chronic inflammatory state caused by high UA and improve the overall metabolic environment by reshaping the gut microbiota structure and enhancing intestinal barrier function.

[0006] Currently, probiotics show promising application prospects in intervening in hypertrophic angina (HUA), but their effectiveness is highly dependent on the function of specific strains. Therefore, discovering more strains with clearly defined efficacy is key to realizing this potential in clinical applications.

[0007] Therefore, how to screen a strain that can alleviate hyperuricemia is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide the application of Lactobacillus reuteri GOLDGUT-CCFM1040 (hereinafter referred to as "CCFM1040" or "C1040") in lowering uric acid, so as to overcome the shortcomings of the prior art.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention seeks protection for the use of *Lactobacillus reuteri* GOLDGUT-CCFM1040 in the preparation of uric acid-lowering products. This strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No. 60515, on December 10, 2018, and its taxonomic name is... Lactobacillus reuteri .

[0011] It should be noted that this strain was known as *Lactobacillus reuteri* before April 2020. Lactobacillus reuteri Later, based on taxonomic adjustments, it was renamed *Lactobacillus reuteri* (…). Limosilactobacillus reuteri ).

[0012] Furthermore, the aforementioned products are biological products, food, or pharmaceuticals.

[0013] Furthermore, the aforementioned biological products are the bacterial cells, metabolites, and fermentation broth of *Lactobacillus reuteri* GOLDGUT-CCFM1040.

[0014] Furthermore, the aforementioned foods are classified as general foods, foods for special medical purposes, functional foods, and health foods.

[0015] Furthermore, the aforementioned health food products are solid beverages, dairy beverages, compressed candies, soy products, dairy products, or fruit and vegetable products.

[0016] Furthermore, the aforementioned drugs also include pharmaceutical excipients.

[0017] Furthermore, the aforementioned pharmaceutical excipients are at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, and release inhibitors.

[0018] Furthermore, the aforementioned pharmaceutical excipients are at least one of microcrystalline cellulose, hydroxypropyl methylcellulose, and lecithin.

[0019] Furthermore, the dosage forms of the above-mentioned drugs are granules, capsules, tablets, pills, or oral liquids.

[0020] Furthermore, the viable count of *Lactobacillus reuteri* GOLDGUT-CCFM1040 in the above products is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0021] The screening method for Lactobacillus reuteri GOLDGUT-CCFM1040, which has uric acid-lowering function, specifically includes the following steps: 1. In vitro screening of nucleoside and purine-degrading lactic acid bacteria The activated lactic acid bacteria were washed with physiological saline, resuspended in potassium phosphate buffer containing purines or nucleosides, reacted at 37°C with shaking for 60 min, and then inactivated in a boiling water bath. The supernatant was obtained by centrifugation and filtration. The peak area changes of the experimental group and the control group were compared by high performance liquid chromatography (HPLC), the degradation rate of purines or nucleosides by the strains was calculated, and strains that efficiently degrade UA to generate precursor substances were screened.

[0022] 2. Determination of the inhibitory activity of intracellular / extracellular metabolites of lactic acid bacteria on XOD Activated lactic acid bacteria were washed with PBS, incubated at 37°C for 12 h, and the supernatant was collected by centrifugation to obtain extracellular metabolites. The supernatant was then subjected to ultrasonic disruption and centrifugation to obtain intracellular metabolites. XOD inhibition rate was determined by recording the change in absorbance at 290 nm wavelength within 10 min after incubation of the sample with XOD and xanthine, and calculating the inhibition rate by comparing it with the blank reaction rate.

[0023] The *Lactobacillus reuteri* strain GOLDGUT-CCFM1040 screened in this invention exhibits significant intervention effects in alleviating hyperinflammatory hyperallergy (HUA): this strain achieves a degradation rate of inosine up to 80.23%, effectively reducing exogenous UA sources; its intracellular and extracellular metabolites show strong inhibitory activity against xanthocyanin (XOD), with an inhibition rate as high as 68.16%. These results fully demonstrate that *Lactobacillus reuteri* GOLDGUT-CCFM1040 possesses highly efficient degradation, enzyme inhibition, and good probiotic properties, laying a solid foundation for the development of microecological preparations for the prevention and treatment of HUA.

[0024] The method for investigating the alleviating effect of Lactobacillus reuteri GOLDGUT-CCFM1040 on hyperuricemia in mice includes the following steps: 1. Establishment of the HUA animal model Six- to eight-week-old male C57BL / 6J rats (20-22 g) were randomly divided into three groups after one week of acclimatization feeding: normal control group (CMC-Na gavage), hyperuricemia model group (potassium oxonate 300 mg / kg + inosine and guanosine 375 mg / kg each), and CCFM1040 group (109 CFU of Lactobacillus reuteri GOLDGUT-CCFM1040 gavage 1 h after modeling), with continuous intervention for 4 weeks.

[0025] 2. Determination of physiological and biochemical parameters in mouse serum and tissues Commercially available kits were used to determine the concentrations of uric acid (UA), creatinine (Scr), and blood urea nitrogen (BUN) in mouse serum and urine, as well as the levels of endotoxin (LPS), interleukin-10 (IL-10), tumor necrosis factor (TNF-α), and interleukin-1β (IL-1β) in serum and kidney tissue. Simultaneously, the activities of XOD and purine nucleoside phosphorylase (PNP), key enzymes in uric acid metabolism, were measured in serum and liver tissue.

[0026] 3. Histopathology and Immunofluorescence Detection Colon and kidney tissues fixed with paraformaldehyde were dehydrated, embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) staining, Masson trichrome staining, and immunofluorescence staining (Claudin-1, Occludin-1, and ZO-1) were then performed. After microscopic image acquisition, the area of ​​collagen fibers stained with Masson in the kidneys was calculated using ImageJ software, and the immunofluorescence density of the three tight junction proteins in the colon tissue was quantified.

[0027] 4. Transcriptomics determination After RNA extraction, mRNA fragmentation, and cDNA library construction, mouse kidney tissue was sequenced on the Illumina platform. Differentially expressed genes (|log2FC|≥1.2, p<0.05) were screened by quality control, transcript assembly, and DESeq2. Enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

[0028] 5. RT-qPCR assay 20 mg of tissue sample was frozen, ground, and RNA was extracted using the chloroform-isopropanol method. cDNA was then synthesized by reverse transcription and detected by qPCR using the SYBR Green premix system. Relative quantitative analysis was achieved by comparing the CT values ​​of the target gene and the internal reference gene.

[0029] 6. Western Blot determination Tissue samples were washed with PBS, lysed and centrifuged to obtain total protein solution, denatured, then subjected to SDS-PAGE electrophoresis and transferred to a membrane. After blocking with skim milk, the membrane was incubated with primary and secondary antibodies in sequence, and developed by ECL chemiluminescence. The gray values ​​of the bands were analyzed using ImageJ.

[0030] 7. Metagenomics determination Total microbial DNA was extracted from feces, and after quality control, fragmented libraries were constructed and sequenced using Illumina. Host contamination was removed through quality control, and the DNA was assembled using MEGAHIT and predicted using Prodigal genes. Finally, a non-redundant gene set was constructed through clustering. The composition of the gut microbiota, the relative abundance of differentially expressed microbiota, and the main differentially expressed metabolic pathways in the KEGG pathway were analyzed.

[0031] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides a safer, more effective, and multi-target regulatory strategy for HUA intervention. Through systematic functional screening, specific probiotic strains are obtained that can efficiently degrade UA precursors (such as nucleosides and purines), inhibit xanthine oxidase (XOD) activity, and have excellent gastrointestinal tolerance. This provides reliable strain resources and experimental basis for the development of oral formulations or functional foods for alleviating HUA.

[0032] 2. The probiotic GOLDGUT-CCFM1040 of this invention has significant advantages in reducing serum uric acid and protecting kidney function. Animal experiments have verified that CCFM1040 can significantly reduce serum uric acid and serum creatinine levels in hyperuricemia model mice, demonstrating its superior intervention effect in improving uric acid metabolism disorders. Compared with traditional uric acid-lowering probiotics, this product works simultaneously on multiple key physiological pathways, thus possessing a more comprehensive regulatory capacity. First, CCFM1040 can downregulate the expression of ENT1 protein in intestinal epithelial cells, reducing the transmembrane absorption of uric acid precursors, making the intestine a more effective buffer for uric acid metabolism, and reducing the uric acid load in the body from the metabolic source. Second, CCFM1040 can activate the Foxo signaling pathway, promoting the expression of key proteins such as Prkag2 and Foxo3, and further enhancing the production of the anti-inflammatory factor IL-10, thereby effectively alleviating the inflammatory response in intestinal and kidney tissues. Furthermore, CCFM1040 can significantly improve the intestinal microecological structure, and its intervention improves… Akkermansia muciniphila It increased the abundance of short-chain fatty acid-producing bacteria and inhibited the overgrowth of parabacteroides, which have potential pro-inflammatory effects, thus promoting gut microbiota homeostasis. More importantly, CCFM1040 significantly increased the expression levels of tight junction proteins such as Claudin-1, Occludin-1, and ZO-1 in colonic tissue, thereby improving the structural integrity of the intestinal mucosal barrier, reducing the risk of endotoxins and inflammatory mediators entering the body through the intestinal wall, and further protecting the functional balance of the gut-kidney axis. Based on these comprehensive effects, *Lactobacillus reuteri* GOLDGUT-CCFM1040 showed outstanding effects in lowering uric acid, improving renal function, regulating gut microbiota, and strengthening the intestinal barrier. It is safe, mild, and has multi-target synergistic characteristics, providing a highly efficient and promising microecological solution for nutritional intervention in hyperuricemia and related metabolic abnormalities. Attached Figure Description

[0033] Figure 1 The effects of CCFM1040 intervention on baseline indicators in HUA mice were as follows: (A) urinary UA content; (B) serum PNP activity; (C) liver XOD activity; and (D) serum UA content.

[0034] Figure 2 The effect of CCFM1040 intervention on the inflammatory response of HUA mice was investigated. Among them, (A) serum IL-10 inflammatory factor level; (B) serum LPS inflammatory factor level; (C) serum TNF-α inflammatory factor level; (D) serum IL-1β inflammatory factor level; (E) renal IL-10 inflammatory factor level; (F) renal LPS inflammatory factor level; (G) renal TNF-α inflammatory factor level; and (H) renal IL-1β inflammatory factor level.

[0035] Figure 3 The effect of CCFM1040 intervention on kidney injury in HUA mice; (A) HE staining of kidney (scale bar = 50 μm; blue arrows indicate inflammatory cell infiltration; red arrows indicate enlarged cavities); (B) Masson staining of kidney (scale bar = 50 μm; blue indicates collagen fibers, red indicates muscle fibers); (C) relative collagen content in kidney Masson staining; (D) Scr content; (E) BUN content.

[0036] Figure 4 The effects of CCFM1040 intervention on the renal transcriptome of HUA mice are shown in the following figures: (A) heatmap of gene correlation between different groups; (B) number of upregulated / downregulated genes in MOD group compared with CON and CCFM1040 group; (C) Venn diagram of the number of DEGs in MOD and CCFM1040; (D) KEGG pathway enrichment of DEGs in MOD vs CCFM1040.

[0037] Figure 5 The study investigated the effects of CCFM1040 intervention on the Foxo signaling pathway in the kidneys of HUA mice. The results included: (A) RT-qPCR measurement of the mRNA expression levels of related DEGs; (B) Western blot detection of the relative protein expression levels of related DEGs; (C) relative expression of Foxo3 protein; and (D) relative expression of Prkag protein.

[0038] Figure 6 The effects of CCFM1040 intervention on the gut microbiota and functional metabolism in mice were analyzed. The results included: (A) Chao 1 and Simpson indices; (B) PCoA analysis based on the Bray-Curtis distance matrix; (C) classification and composition of gut microbiota at the phylum and species levels; (D) relative abundance of relevant differentially expressed microbiota; (E) histogram of LDA value distribution (LDA score > 3) selected based on the LEfSe score; and (F) analysis of major differentially expressed metabolic pathways in the KEGG pathway.

[0039] Figure 7The effects of CCFM1040 intervention on the intestinal barrier and nucleoside transporter expression in HUA mice were investigated. The results included: (A) HE staining of the colon (scale bar = 50 μm; blue arrows indicate inflammatory cell infiltration; red arrows indicate vacuolation); (B) Immunofluorescence analysis of Claudin-1 in the colon (scale bar = 100 μm; red indicates Claudin-1 target protein, blue indicates cell nucleus); (C) Immunofluorescence analysis of Occludin-1 in the colon (scale bar = 100 μm; red indicates Occludin-1 target protein, blue indicates cell nucleus); (D) Immunofluorescence analysis of ZO-1 in the colon (scale bar = 100 μm; red indicates ZO-1 target protein, blue indicates cell nucleus); (E) Expression level of Claudin-1 in the colon; (F) Expression level of Occludin-1 in the colon; (G) Expression level of ZO-1 in the colon; (H) Western blot detection of ENT1 protein levels; and (I) Relative expression of ENT1 protein. Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] In this invention, *Lactobacillus reuteri* GOLDGUT-CCFM1040 is prior art. Its acquisition, isolation, identification, culture and preservation are referenced in the invention patent application with application number 201910131564.1, application date of February 22, 2019, applicant Jiangnan University, and invention title "A strain of *Lactobacillus reuteri* that can relieve allergic asthma and its application".

[0042] Example 1: In vitro screening of nucleoside and purine-degrading lactic acid bacteria Nucleoside reaction solution was prepared using 1.26 mmol / L inosine solution, 1.26 mmol / L guanosine solution, and 0.1 mol / L neutral potassium phosphate solution; purine reaction solution was prepared using 0.5 g / L xanthine solution, 0.5 g / L hypoxanthine solution, 0.5 g / L guanosine solution, and 0.1 mol / L neutral potassium phosphate solution to screen for lactic acid bacteria that degrade purines (xanthine, hypoxanthine, guanosine) and nucleosides (inosine, guanosine).

[0043] The following bacteria were identified: *Bifidobacterium animalis* subsp. *lactobacter* GOLDGUT-BB69, *Lactobacillus plantarum* GOLDGUT-LP101, *Lactobacillus rhamnosus* GOLDGUT-M520, *Bifidobacterium animalis* subsp. *lactobacter* GOLDGUT-BB18, *Lactobacillus plantarum* GOLDGUT-LP618, *Lactobacillus acidophilus* GOLDGUT-LA100, *Bifidobacterium longum* subsp. *longum* GOLDGUT-BL23, *Lactobacillus paracasei* GOLDGUT-LPC969, and *Pediococcus lactis* G. The following strains were selected as candidate strains: OLDGUT-PA0755, Bifidobacterium longum subsp. longum GOLDGUT-BL8178, Lactobacillus plantarum GOLDGUT-LP1024, Lactobacillus reuteri GOLDGUT-CCFM1040, Lactobacillus casei GOLDGUT-LC12345, Bifidobacterium animalis subsp. lactis GOLDGUT-BB21, Lactobacillus rhamnosus GOLDGUT-L818, and Lactobacillus reuteri GOLDGUT-LR99.

[0044] The candidate strain was passaged at a 2% inoculum and cultured in MRS (containing 0.5 g / L cysteine ​​hydrochloride, 37℃, anaerobic) liquid medium, activated three times, and then used for further processing. 2 mL of the bacterial culture was centrifuged at 5000 g for 5 min at 4℃. The cells were washed three times with physiological saline (0.9% W / V) and resuspended in 750 μL of 0.1 mol / L potassium phosphate buffer containing the target substrate (purine bases or nucleosides). The culture was incubated at 37℃ with shaking at 120 rpm for 60 min, then the reaction was terminated by boiling in a water bath for 5 min. The supernatant was collected by centrifugation at 10000 g for 2 min and filtered through a 0.22 μm filter for analysis. The control group was treated simultaneously with the same substrate buffer (without inoculation with the strain).

[0045] The content of purine bases and nucleosides was determined by HPLC, and the degradation rate was calculated. The elution conditions were as follows: an Agilent ZORBAX SB-Aq column (250 mm × 4.6 mm × 5 μm) was used; the mobile phase was methanol (phase A) and 0.1% formic acid water (phase B); isogradient elution (3% A + 97% B); flow rate was 1.0 mL / min; column temperature was 25℃; and detection wavelength was 254 nm. The standard curve was plotted by diluting 500 μg / mL of substrate standard, and quantification was performed using the external standard method. The degradation rate of purines or nucleosides by the strains was calculated by comparing the peak area changes between the experimental group and the control group, and highly efficient strains were screened. The degradation rate was calculated according to the following formula: Degradation rate (%) = (C0 - C1) / C0 100%; Where C0 is the initial concentration of the standard solution and C1 is the remaining concentration of the standard solution after the reaction.

[0046] Nucleosides and purines are precursors of UA, and whether probiotics can degrade nucleosides and purines is key to inhibiting UA production.

[0047] Table 1. Effects of different probiotics on the degradation ability of nucleosides and purine bases.

[0048] As shown in Table 1, based on the degradation rates of inosine and guanosine by probiotics, the degradation capacity of the main lactic acid bacteria was ranked as follows: CCFM1040 > BB21 > LA100 > BL23 > LPC969 > BL8178. Based on the degradation rates of purines by probiotics, the degradation capacity was ranked as follows: CCFM1040 > BB21 > LPC969 > BL8178 > BL23 > LA100. Therefore, six strains with good degradation effects on UA ​​precursors were selected from the 16 strains: CCFM1040, BB21, LPC969, BL8178, LA100, and BL23.

[0049] Example 2: Determination of the inhibitory activity of intracellular / extracellular metabolites of lactic acid bacteria on XOD The culture medium of activated lactic acid bacteria strains (generation 3) was centrifuged at 10,000 r / min for 10 min to collect the bacterial pellet. The pellet was washed three times with sterile PBS, and the bacterial suspension concentration was adjusted to 1×10⁻⁶. 9 After incubating at 37℃ for 12 h with CFU / mL, the supernatant was collected as extracellular metabolites after centrifugation at 10000 r / min for 10 min. The bacterial suspension was then sonicated at 250 W for 5 min, centrifuged at 10000 r / min for 10 min, and the supernatant was collected as intracellular metabolites.

[0050] Add 50 μL of intracellular / extracellular metabolite sample from lactic acid bacteria to a 96-well plate, add 50 μL of XOD solution (0.2 U), shake for 10 s, incubate at 37℃ for 5 min, then add 150 μL of xanthine solution (0.2 mM). Measure the absorbance at 290 nm every 20 s and record the change in absorbance over 10 min. Use 50 μL of phosphate buffer (pH 7.5) as a blank instead of the sample solution. The XOD inhibition rate is calculated using the following formula: XOD inhibition rate (%) = (A0-A1) / A0 100%; Where A0 is the blank reaction rate and A1 is the sample reaction rate.

[0051] XOD is a key enzyme in purine metabolism, which can convert hypoxanthine and xanthine into UA, making it an important target for the treatment of HUA.

[0052] Table 2. Inhibition rates of xanthine oxidase by intracellular and extracellular metabolites of different probiotics

[0053] The results are shown in Table 2. The cell contents of all 16 strains exhibited varying degrees of inhibition on XOD activity. Combined with the in vitro degradation results, except for BL8178, probiotic strains with good degradation effects also showed strong inhibitory effects on XOD activity. The overall inhibitory effect of intracellular metabolites on XOD was as follows: BL23 > BB21 > CCFM1040 > LPC969 > LA100 > BL8178, with inhibition rates of 79.43%, 77.54%, 60.85%, 57.4%, 45.67%, and 22.06%, respectively. The overall inhibitory effect of extracellular metabolites on XOD was as follows: BB21 > CCFM1040 > BL23 > LA100 > LPC969 > BL8178, with inhibition rates of 70.94%, 68.16%, 58.35%, 43.79%, 28.67%, and 21.5%, respectively.

[0054] Example 3: Construction of a HUA mouse model Thirty 6-8 week old SPF-grade C57BL / 6J mice (20 ± 2 g) were randomly divided into a control group, a model group, and a *Lactobacillus reuteri* GOLDGUT-CCFM1040 (C1040) group (n = 10 mice / group) after one week of acclimatization. All animals were housed under specific pathogen-free (SPF) conditions, provided with standard laboratory feed, and had free access to water. The artificial light cycle was set to 12 h light / 12 h dark, with the ambient temperature maintained at 24 ± 1℃ and the relative humidity at 55 ± 5%. The modeling and intervention protocols are as follows: (1) Normal control group (CON): 0.5% sodium carboxymethyl cellulose (CMC-Na) was administered by gavage at 9:00 every day for 4 weeks; (2) Hyperuricemia model group (MOD): The hyperuricemia model was induced by gavage with potassium oxonate (300 mg / kg) and inosine (375 mg / kg) + guanosine (375 mg / kg) at 9:00 every day for 4 weeks; (3) GOLDGUT-CCFM1040 group (C1040): Hyperuricemia model was induced daily at 9:00 AM by gavage with potassium oxonate (300 mg / kg) and a combination of inosine (375 mg / kg) and guanosine (375 mg / kg). One hour later, probiotics GOLDGUT-CCFM1040 were administered by gavage. 9 CFU, lasting 4 weeks; After a 4-week intervention, all mice were euthanized. Blood was collected from the abdominal aorta and centrifuged at 4000 r / min for 15 min to separate the serum. The animals were then dissected, and liver, kidney, colon tissue, and contents of the colon and cecum were collected. Some kidney and colon tissues were fixed in tissue fixative for 24 h, while the remaining tissue and serum samples were stored at -80℃ for subsequent analysis.

[0055] Example 4: Determination of biochemical indicators in mouse serum and tissues This embodiment uses commercially available diagnostic kits to measure physiological and biochemical parameters in mouse serum, urine, and tissue samples to evaluate the effects of *Lactobacillus reuteri* CCFM1040 on hyperuricemia-related metabolic abnormalities and inflammatory responses. The measured parameters included urinary uric acid (UA), creatinine (Scr), blood urea nitrogen (BUN), and serum uric acid (SUA). Simultaneously, lipopolysaccharide (LPS), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were measured in serum and kidney tissue. Furthermore, the activities of xanthine oxidase (XOD) and purine nucleoside phosphorylase (PNP), which are involved in purine metabolism, were measured in serum and liver tissue.

[0056] Uric acid (UA) is the end product of purine metabolism, mainly existing in the blood as urate and excreted through the kidneys. Hyperuricemia (HUA) is a chronic disease caused by purine metabolism disorders, characterized by abnormally high blood uric acid levels. The UA content in mouse urine was measured on days 23-25 ​​of the experiment. Figure 1 As shown in Figure A, the results indicated that the urinary UA in the model group (MOD) mice was significantly elevated, with the MOD group (273.60 μmol / L) being higher than that in the control group (CON) (76.60 μmol / L), indicating that the HUA mouse model was successfully established.

[0057] Both PNP and XOD are enzymes involved in purine metabolism and catalyze the production of UA. Inhibiting their activity and expression can effectively reduce UA production. Figure 1 As shown in Figure B, PNP activity in the MOD group mice was significantly increased to 201.56 ng / L, while it was 139.07 ng / L in the control group. Compared with the MOD group, PNP activity decreased to 142.03 ng / L after intervention with the probiotic CCFM1040, indicating that CCFM1040 intake can significantly reduce PNP activity (p<0.05). Figure 1As shown in Figure C, the XOD activity in the control group was 7.52 U / gprot, while in contrast, the XOD activity in the MOD group increased to 9.89 U / gprot. Compared with the MOD group, after intervention with CCFM1040, the XOD activity in mice decreased to 8.22 U / gprot, indicating that the intake of probiotic CCFM1040 can significantly reduce XOD activity in HUA mice (p<0.05). These results demonstrate that CCFM1040 has an inhibitory effect on PNP and XOD activity, which helps to improve the state of purine metabolism disorder and reduce UA production.

[0058] The detection of serum UA concentration showed ( Figure 1 The UA content in the MOD group mice was 130.08 μmol / L, significantly higher than that in the CON group. Compared with the MOD group, the serum UA level in mice decreased by 27.11% after probiotic CCFM1040 intervention, indicating that the intake of probiotic CCFM1040 can significantly reduce the serum UA level in HUA mice (p<0.05).

[0059] The large number of inflammatory factors released during the inflammatory response can interfere with uric acid (UA) metabolism through multiple pathways, thereby triggering hyperuricemia (HUA). The deposition of urate crystals produced by HUA in tissues activates the immune system to release inflammatory factors, exacerbating inflammation and creating a vicious cycle. Among these, LPS, TNF-α, and IL-1β are pro-inflammatory factors that can induce inflammation and activate inflammatory pathways, thus worsening the inflammatory response. IL-10, as an anti-inflammatory factor, can inhibit the production of pro-inflammatory factors, regulate immunity, and reduce the inflammatory response.

[0060] Depend on Figure 2 As shown in Figure A, the serum IL-10 level in the MOD group mice was 1296.95 pg / mL, significantly lower than that in the CON group (1535.71 pg / mL). Compared with the MOD group, the serum IL-10 level in mice increased to 1631.75 pg / mL after intervention with the probiotic CCFM1040.

[0061] Depend on Figure 2 The results from the BD study showed that the serum LPS level in the MOD group mice was 281.42 ng / L, which was higher than that in the control group (217.26 ng / L). Compared with the MOD group, the LPS level decreased by 56.96 ng / L after intervention with the probiotic CCFM1040. Similarly, the serum TNF-α and IL-1β levels in the MOD group mice were significantly higher than those in the CON group (p<0.05). Compared with the MOD group, CCFM1040 reduced the levels of TNF-α and IL-1β to levels close to those in the control group, while increasing the IL-10 level to 1631.75 pg / mL.

[0062] Figure 2The levels of inflammatory factors in the kidneys of mice with EH (extraneous inflammatory cytokines) showed a trend consistent with those in serum. Ingestion of the probiotic CCFM1040 increased IL-10 levels and significantly reduced LPS, TNF-α, and IL-1β levels in the kidneys of HUA (Human Abnormality and Inflammation) mice. These results indicate that the CCFM1040 strain can alleviate the inflammatory response induced by HUA.

[0063] BUN and Scr are primarily excreted through the kidneys. When the kidneys are inflamed or failing, their excretion decreases, leading to elevated blood concentrations and potentially causing problems such as hyperuricemia (HUA). Figure 3 As shown in the results, the Scr level in the MOD group mice was 27.78 μmol / L, significantly higher than that in the CON group (20.99 μmol / L), indicating that the MOD group mice had a decreased ability to excrete Scr, a decreased glomerular filtration rate, and impaired kidney function. After intervention with the probiotic CCFM1040, the Scr level significantly decreased to 22.64 μmol / L (p<0.05), approaching that of the CON group. Figure 3 As shown in Figure E, the BUN level in the MOD group mice was 8.60 mmol / L, significantly higher than that in the CON group mice (7.58 mmol / L). Therefore, the high-nucleoside diet induced renal function impairment and decreased BUN excretion in HUA mice, leading to BUN accumulation. Compared with the MOD group, the CCFM1040 group reduced BUN levels (to 6.68 mmol / L, p<0.05). These results indicate that the probiotic CCFM1040 has an ameliorative effect on renal excretion function and can alleviate the decrease in glomerular filtration rate and accumulation of metabolites caused by hyperuricemia.

[0064] Example 5: Histopathology and Immunofluorescence Detection This embodiment evaluates the effects of *Lactobacillus reuteri* CCFM1040 on the structure and function of kidney and colon tissues in HUA mice using histopathological and immunofluorescence methods. Colon and kidney tissues were removed from 4% paraformaldehyde, rinsed with running water, and dehydrated. Samples were embedded in paraffin, cut into 5 μm pieces, and stained with H&E and Masson staining. Colon tissue sections were incubated overnight at 4°C with Claudin-1, Occludin-1, and ZO-1 primary antibodies (1:500), washed three times with PBS, and then incubated for 50 min at room temperature in the dark with CY3-labeled goat anti-rabbit IgG secondary antibody (1:300). After PBS washing, the sections were counterstained with DAPI for 10 min. Autofluorescence quencher was added, and the sections were mounted with mounting medium. Images were acquired under a microscope. ImageJ software was used to quantitatively analyze the area of ​​renal collagen fibers and the immunofluorescence density of three proteins in colon tissue.

[0065] The kidneys are the core organs for the metabolism and excretion of urokinase (UA). H&E and Masson staining can directly reflect the pathological damage to the kidneys caused by UA and the effects of treatment interventions. Figure 3 As shown in Figure A, the glomerular structure in the CON group was intact, with tightly packed epithelial cells, an intact brush border, and a normal lumen. The MOD group showed localized inflammatory cell infiltration, brush border detachment, lumen dilation, and uneven epithelial cell arrangement. The probiotic CCFM1040 intervention group showed significantly reduced inflammatory cell infiltration and improved lumen dilation in the kidney tissue.

[0066] Kidney damage and inflammation caused by HUA may induce renal interstitial fibrosis. Masson staining can stain collagen fibers blue, visually showing the abnormal deposition of collagen fibers in the colonic mucosa. Figure 3 (B). According to Masson's quantitative analysis ( Figure 3 In the C group, abnormal collagen fiber deposition in the kidneys was observed, with a relative collagen content of 26.03%, which was higher than that in the CON group (14.12%). After CCFM1040 intervention, the collagen content was significantly reduced by 16.00% (p<0.05), indicating that probiotics can improve renal interstitial fibrosis caused by HUA.

[0067] Gut microbiota dysbiosis is closely related to human uric acid (HUA), further aggravating its pathological progression through multiple mechanisms, including interfering with the activity of uric acid metabolic enzymes, affecting the function of related transport proteins, and exacerbating the body's inflammatory response. Against this backdrop, H&E staining of colonic tissue not only provides direct pathological evidence of intestinal barrier structural damage caused by microbiota imbalance but can also be used to evaluate the intestinal repair efficacy of targeted microbiota therapy. Figure 7 The H&E staining results of the colon in the CON group showed that the colon had a regular morphology, abundant and orderly arranged goblet cells, and no inflammatory infiltration. In the MOD group, the number of colonic goblet cells was reduced and their morphology was abnormal, with some areas showing disappearance, accompanied by local inflammatory cell infiltration. In the CCFM1040 intervention group, the number of colonic goblet cells recovered to near normal levels, inflammatory cell infiltration was reduced, and epithelial integrity was improved.

[0068] Figure 7Image B, D, and E respectively show the immunofluorescence analysis of Claudin-1, Occludin-1, and ZO-1 in mouse colon tissue. Claudin-1, Occludin, and ZO-1 are core proteins constituting the tight junctions (TJs) of intestinal epithelial cells, primarily involved in maintaining the integrity of the intercellular barrier and regulating substance permeability. Decreased Claudin-1 expression leads to barrier dysfunction, allowing LPS to enter the bloodstream and triggering an inflammatory response. Occludin enhances the mechanical strength of TJs, preventing the diffusion of macromolecules through paracellular pathways and participating in signal transduction. Reduced Occludin weakens the barrier's mechanical support, exacerbating tissue damage and inflammation. ZO-1 maintains the stability and dynamic regulation of TJ structure; abnormal expression leads to TJ complex depolymerization and a significant increase in barrier permeability. Figure 7 In the EG assay, compared with the CON group, the expression of Claudin-1, Occludin, and ZO-1 proteins in the colon tissue of the MOD group was significantly decreased (p<0.05), indicating impaired intestinal tight junctions. Compared with the MOD group, after intervention with probiotic CCFM1040, the expression of the three tight junction proteins was significantly increased (p<0.05), indicating that probiotics can restore colonic epithelial barrier function and enhance intercellular tight junctions.

[0069] Example 6: Measurement of mouse kidney transcriptomics This embodiment aims to evaluate the regulatory effect of *Lactobacillus reuteri* CCFM1040 on gene expression in the kidneys of HUA mice using transcriptomics analysis. After the experiment, mouse kidney tissue was thoroughly ground in liquid nitrogen, and TRIzol reagent was added to lyse cells and extract total RNA. After testing the purity and integrity of the extracted RNA, mRNA was enriched using Oligo(dT) magnetic beads, and the mRNA was randomly fragmented to approximately 300 bp fragments in a high-temperature ion-buffered saline solution. Double-stranded cDNA was synthesized using random primers. Subsequently, the cDNA underwent end repair, A-tailing, and adapter ligation, and a sequencing library was constructed by PCR amplification. The library was sequenced using the Illumina NovaSeq X Plus platform. Raw data underwent FAST quality control to remove low-quality sequences (Q<20, length <50 bp) and adapter contamination, obtaining high-quality clean reads. Transcript assembly was performed using StringTie, and differentially expressed genes (|log2FC|≥1.2, p<0.05) were screened using DESeq2, followed by functional enrichment analysis using the KEGG database.

[0070] To assess the effects of probiotics on gene expression in the kidneys, RNA-seq was performed on the kidneys of mice in the CON, MOD, and CCFM1040 groups. Pearson correlation coefficients were determined among every three biological samples for each gene expression level to assess inter-sample correlation. The correlation coefficients between samples within the same experimental group were all greater than 0.9, indicating good reproducibility and allowing for further DEGs screening. Figure 4 (A). Based on log2FC ≥ 1.2, p < 0.05, a total of 1783 differentially expressed genes (DEGs) were screened. Compared with the CON group, the MOD group had 745 upregulated DEGs and 603 downregulated DEGs. Compared with the MOD group, the CCFM1040 group had 214 upregulated DEGs and 221 downregulated DEGs. Figure 4 (Middle B). Venn diagram analysis showed that the MOD group and the CCFM1040 intervention group shared 111 common DEGs, suggesting that these genes may be related to the occurrence of HUA and the effect of probiotic intervention, which will be the focus of future research. Figure 4 (C)

[0071] KEGG pathway enrichment analysis showed that these 111 DEGs were enriched with a total of 22 signaling pathways (p<0.05). Figure 4The study found significant enrichment of metabolic pathways including Circadian rhythm, Cell cycle, Cellular senescence, Foxo signaling pathway, Oxytocin signaling pathway, Retinol metabolism, ABC transporters, and AMPK signaling pathway. The ABC transporters pathway encompasses multiple transporter families, including several transporters such as ABCG2, which are classified as ABC transporters. These proteins are expressed in the brush border of the proximal renal tubules and intestinal epithelial cells, and mediate uric acid secretion through an ATP-dependent mechanism. Abnormal function of these transporters leads to reduced uric acid excretion, resulting in elevated serum uric acid levels. Both the Foxo signaling pathway and the AMPK signaling pathway are key inflammatory regulatory pathways. In kidney atrophy (HUA), activation of the AMPK and Foxo signaling pathways can alleviate inflammatory responses and kidney damage. Notably, Foxo transcription factors (such as Foxo1 and Foxo3) in the Foxo signaling pathway are key regulators of cellular energy sensing and stress responses, regulated by upstream signals such as the insulin / PI3K / Akt pathway, the AMPK pathway, and SIRT1. These factors play crucial roles in regulating apoptosis, autophagy, antioxidative stress, and inflammatory responses. Studies have indicated that the Foxo signaling pathway plays a significant role in suppressing inflammatory responses. Foxo3 may also participate in the regulation of inflammatory responses by modulating the number and function of monocytes / macrophages or inhibiting their overactivation. Furthermore, Foxo3 is a downstream molecule of the PI3K / Akt signaling pathway. Inflammatory signals induce phosphorylation and activation of PI3K / Akt, leading to Akt binding to Foxo3 in the cell nucleus and subsequent phosphorylation. In pathogen-activated antigen-presenting cells, Foxo3 can inhibit the production of inflammatory cytokines such as TNF-α and IL-6. Therefore, Foxo3 plays an important biological role in regulating inflammatory responses. In summary, this embodiment demonstrates that the probiotic CCFM1040 can exert a protective effect on the kidneys of HUA mice by regulating the expression of renal ABC transporters and the activity of the Foxo signaling pathway, thereby affecting the expression of key inflammation and metabolism-related genes. Example 7: RT-qPCR assay This embodiment aims to verify the regulatory effect of probiotic CCFM1040 on genes related to the Foxo signaling pathway in the kidneys of HUA mice. Approximately 20 mg of kidney tissue sample was placed in RNA extraction buffer containing grinding beads and thoroughly ground. Trichloromethane was then added, and after centrifugation, the supernatant was collected. RNA was precipitated with isopropanol and washed twice with 75% ethanol to remove impurities. The extracted RNA was dissolved at 55°C, and its concentration and purity were then measured. In the reverse transcription stage, 2 μg of RNA was mixed with reverse transcription reagent, and cDNA was synthesized in a PCR instrument. The product was diluted, and its concentration was measured (<100 ng / μL). In the real-time quantification stage, the SYBR Green method was used. The diluted cDNA was mixed with the premixed system in a specific ratio, and the program was run in a qPCR instrument. Relative quantification was performed by comparing the CT values ​​of the target gene and the internal reference gene. Primer information is shown in Table 3 below.

[0072] Table 3 RT-qPCR primer sequences

[0073] The expression levels of five DEGs enriched in the Foxo signaling pathway by renal transcriptomics were determined using RT-qPCR. Cdkn1a , Prkag2 , Pik1 , Foxo3 and Bcl6 The results showed that, compared with the MOD group, the probiotic CCFM1040 intervention group had significantly higher levels of probiotics. Bcl6 Apart from no significant difference in the relative expression levels of mRNA, Cdkn1a , Prkag2 , Pik1 and Foxo3 The relative expression levels of mRNA were significantly increased. Figure 5 The changes in gene expression levels (A) were consistent with those observed in the transcriptome. RT-qPCR validation enhanced the reliability of the transcriptome findings, demonstrating a strong correlation with sequencing data. These results indicate that RT-qPCR validated the expression changes of genes related to the Foxo signaling pathway in transcriptome studies, enhancing the reliability of transcriptome analysis results and suggesting that the probiotic CCFM1040 may exert a potential protective effect in the kidneys by regulating genes related to the Foxo signaling pathway.

[0074] Example 8: Western Blot Determination This embodiment aims to investigate the regulatory effect of probiotic CCFM1040 on the expression of key proteins in the kidneys of HUA mice. 0.1 g of kidney tissue was accurately weighed, washed with PBS, and then lysis buffer was added for tissue homogenization. After homogenization, the homogenized tube was placed on ice for 30 min, then centrifuged at 12000 r, 4℃ for 10 min. The supernatant was collected as the total protein solution. Reducing loading buffer was added, and the solution was denatured in a 95℃ metal bath for 10 min, then stored at -80℃ for later use.

[0075] Place the prepared gel solution into the electrophoresis tank, fill with electrophoresis buffer, and add 5 μL of sample. Electrophoresis is performed at a constant voltage of 200 V until the bromophenol blue is approximately 1 cm from the bottom. The gel is placed on filter paper, and then successively covered with a PVDF membrane, transfer filter paper, and sponge. Transfer is performed at a constant current of 300 mA for 30 min. After transfer, the membrane is quickly rinsed in TBST and blocked with 5% skim milk for 30 min. After discarding the blocking buffer, primary antibodies Prakag2, Foxo3, and ENT1 diluted 1:1000 are added, and the membrane is incubated overnight at 4°C. The membrane is washed three times with TBST, and then incubated with secondary antibody diluted 1:3000 for 30 min. After washing three times with TBST, the membrane is placed on the chemiluminescence analyzer tray, and ECL chemiluminescence solution is added for chemiluminescence detection. The band intensity is analyzed using ImageJ software.

[0076] Figure 5 Figure B shows the relative expression of Prkag2 and Foxo3 proteins detected by Western blot. The results showed that, compared with the MOD group, probiotic CCFM1040 intervention significantly upregulated the expression of Prkag2 and Foxo3 proteins in the kidneys. Figure 5 The results (C and D) are consistent with the relative mRNA expression levels, indicating that probiotics alleviate the inflammatory response induced by high UA by regulating the Prkag2 / Foxo3 signaling pathway. The altered expression of key target proteins (Prkag2 and Foxo3) confirmed by Western blot analysis highlights the functional relevance of these transcriptional changes in the protective effects of probiotics.

[0077] ENT1 is a balanced nucleoside transporter primarily responsible for the transmembrane transport of purine and pyrimidine nucleosides (such as adenosine, inosine, and guanosine). Its function is closely related to nucleoside uptake and metabolism, transporting exogenous nucleosides into the cell and converting them into UA during metabolism. Therefore, the expression level of ENT1 directly affects the efficiency of intracellular nucleoside uptake, and consequently, the amount of UA produced. Figure 7 The graph in H represents the relative expression of ENT1 protein detected by Western blot. (See figure.) Figure 7As shown in Figure I, the expression level of ENT1 protein in the MOD group was significantly higher than that in the CON group. The probiotic CCFM1040 downregulated ENT1 protein expression, reduced nucleoside transport, and decreased the accumulation of UA precursor substances within cells. In summary, this embodiment demonstrates that the probiotic CCFM1040 can exert a protective effect in the kidneys by upregulating the Prkag2 / Foxo3 signaling pathway and downregulating ENT1 protein expression, thereby alleviating hyperuricemia-related inflammation and UA metabolic disorders.

[0078] Example 9: Metagenomics Determination This embodiment aims to analyze the regulatory effect of probiotic CCFM1040 on the structure and function of the intestinal flora in hyperuricemic mice. Total microbial DNA was extracted from fecal samples, and the concentration and purity of the DNA were detected. Integrity was verified using 1% agarose gel electrophoresis. Qualified DNA was fragmented to 400 bp using a Covaris M220 fragmentation kit, and paired-end sequencing libraries were constructed using the NEXTFLEX® Rapid DNA-Seq Kit (Bioo Scientific), including end repair, adapter ligation, magnetic bead screening, and PCR enrichment. After library quantification, sequencing was performed on the Illumina NovaSeq platform. Raw data underwent quality shearing using fastp (v0.20.0) to remove low-quality sequences (Q<20, length <50 bp) and remove host contamination. Subsequent analysis used MEGAHIT (v1.1.2) to assemble contigs ≥300 bp, Prodigal to predict open reading frames, and CD-HIT (identity 90%, coverage 90%) to construct a non-redundant gene set.

[0079] Gut microbiota diversity was assessed using the Chao 1 index and the Simpson index, which reflect the richness and diversity of the microbial community, respectively. Figure 6 As shown in Figure A, compared with the CON group, the Chao 1 index and Simpson index of the gut microbiota in the MOD group mice changed, but the differences were not significant (p>0.05). PCoA analysis based on the Bray-Curtis distance matrix showed that ( Figure 6 In the B group, the gut microbiota of the CON group and the MOD group were significantly separated along the PCo1 axis, indicating that the high nucleoside diet led to the disorder of the gut microbiota structure in mice. The CCFM1040 intervention group and the CON normal group clustered closely, indicating that CCFM1040 significantly reversed the imbalance of gut microbiota structure and the microbiota structure tended to normalize.

[0080] Further analysis was conducted on the composition of the gut microbiota at the phylum and species levels in each group. For example... Figure 6As shown in Figure C, at the phylum level, Bacteroidetes, Bacillota, and Verrucomicrobiota are the dominant phyla. At the species level, Muribaculum sp.、 Alistipes sp.、 Duncaniella sp. and Bacteroides acidifaciens They are the dominant bacterial species, and they all belong to Bacteroidetes.

[0081] Based on the overall characteristics of the phylum and species-level microbial community structure, we further screened for microbial groups and metabolic functions with significant differences between groups to reveal the specific regulatory targets of probiotic intervention. For example... Figure 6 As shown in D, compared with the CON group, the MOD group... Bacteroides sp.、 Bacteroides caecimuris , Paramuribaculum sp. and Duncaniella muris The relative abundance of these bacteria was significantly reduced (p<0.05), but rebounded after probiotic intervention. Bacteroides sp. and Bacteroides caecimuris Belongs to the genus Bacteroides Bacteroides , Bacteroides During metabolism, short-chain fatty acids (SCFAs) such as acetic acid and propionic acid are produced, which have anti-inflammatory and intestinal barrier-maintaining functions, and are beneficial in relieving HUA. Paramuribaculum intestinale and Paramuribaculum sp. belongs to Paramuribaculum Genus, research shows, Paramuribaculum It can produce SCFAs, such as acetic acid and propionic acid, which play an important role in maintaining intestinal barrier function and inhibiting inflammation. Prevotella sp. MGM2, Parabacteroides distasonis and Parabacteroides The relative abundance of bacteria such as *Sp.* was significantly increased in the MOD group (p<0.05), and decreased significantly after intervention with the probiotic CCFM1040. Prevotella sp. and Prevotella sp. MGM2 belongs to the genus Prevotella. Prevotella , Parabacteroides distasonis and Parabacteroides sp. belongs to the genus *Pseudomonas*. Parabacteroides , Prevotella and Parabacteroides Under certain conditions, pro-inflammatory bacteria can exacerbate inflammatory responses. In summary, intervention with the probiotic CCFM1040 can alleviate HUA-induced dysbiosis and inflammatory responses by increasing the relative abundance of beneficial bacteria and reducing the abundance of harmful bacteria.

[0082] Linear discriminant analysis (LDA) effect size (LEfSe) was used to screen for significantly enriched bacterial groups under different interventions, and their effect size distribution was displayed using a bar chart of LDA scores (Log10 transformed values). Figure 6 As shown in Figure E, a total of 19 specific bacteria were identified at the species level (LDA>3, top 5 in LDA score for a single group), including those in the MOD group. Heminiphilus faecis , Parabacteroides distasonis , Parabacteroides sp.、 Prevotella The abundance of sp. MGM2 and other species was high in the CCFM1040 intervention group. Muribaculum sp . , Akkermansia sp.、 Akkermansia muciniphila The abundance of bacteria is relatively high. Studies have shown that... Akkermansia It colonizes the human intestinal mucosa, can degrade the mucosa, thereby alleviating inflammation, and has the function of regulating the intestinal barrier and immune response. Furthermore, research indicates... Akkermansia It can effectively improve HUA in mice by inhibiting uric acid production and promoting UA secretion.

[0083] Based on metagenomic sequencing data, KEGG pathway enrichment analysis was performed to predict potential differences in the metabolic functions of the gut microbiota. For example... Figure 6 As shown in Figure F, in the MOD group, the glucagon signaling pathway, insulin signaling pathway, and p53 signaling pathway were significantly upregulated (p<0.05), while in the CCFM1040 group, these three metabolic pathways were significantly downregulated. Mechanistically, abnormalities in the insulin and glucagon signaling pathways lead to insulin resistance (IR) in the host. Studies have shown that IR increases the incidence of kidney injury, and IR in adipose tissue affects serum UA metabolism; reducing IR can lower serum UA levels. Furthermore, the JAK-STAT signaling pathway, also known as the IL-6 signaling pathway, is closely associated with many immune and inflammatory diseases due to its sustained activation. Overactivation promotes the massive release of pro-inflammatory factors (such as IL-6, TNF-α, and IL-17). Inhibiting the JAK-STAT pathway may be a promising approach to prevent and alleviate renal fibrosis. In summary, probiotic CCFM1040 intervention can maintain the stability of gut microbiota metabolic function in HUA mice to some extent.

[0084] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of *Lactobacillus reuteri* GOLDGUT-CCFM1040 in the preparation of uric acid-lowering products, characterized in that... The strain is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No. 60515, and the preservation time is December 10, 2018, and the taxonomic name is Lactobacillus reuteri .

2. Use according to claim 1, characterized in that, The product is a biological product, food, or medicine.

3. Use according to claim 2, characterized in that, The biological product is the bacterial cells, metabolites, and fermentation broth of Lactobacillus reuteri GOLDGUT-CCFM1040.

4. Use according to claim 2, characterized in that, The food products mentioned include general food, food for special medical purposes, functional food, and health food.

5. Use according to claim 4, characterized in that, The health food products mentioned are solid beverages, dairy beverages, compressed candies, soy products, dairy products, or fruit and vegetable products.

6. Use according to claim 2, characterized in that, The medicine also includes pharmaceutical excipients.

7. Use according to claim 6, characterized in that, The pharmaceutical excipients are at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, and release inhibitors.

8. Use according to claim 7, characterized in that, The pharmaceutical excipient is at least one of microcrystalline cellulose, hydroxypropyl methylcellulose, and lecithin.

9. Use according to claim 2, characterized in that, The dosage form of the medicine is granules, capsules, tablets, pills, or oral liquid.

10. The use according to claim 2, characterized in that, The viable count of *Lactobacillus reuteri* GOLDGUT-CCFM1040 in the product is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

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