Application of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 in lowering uric acid

Abstract

This invention discloses the application of *Bifidobacterium animalis* subsp. *lactamase* GOLDGUT-BB21 in lowering uric acid, belonging to the field of biomedical technology. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29347, on December 18, 2023, and is taxonomically named *Bifidobacterium animalis* subsp. *lactamase*. Bifidobacterium animalis subsp.lactis The present invention, Bifidobacterium lactis subspecies GOLDGUT-BB21, 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 biomedicine, and more specifically to the application of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 in lowering uric acid. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disease caused by an imbalance between the production and excretion of uric acid (UA), clinically defined as a fasting serum uric acid level >420 μmol / L on two separate occasions. Its pathogenesis mainly involves two aspects: excessive UA production (e.g., purine metabolism enzyme deficiency, high-purine diet) and reduced excretion (e.g., decreased glomerular filtration rate, abnormal uric acid transporter function). Early-stage patients are often asymptomatic, but long-term high UA can lead to gouty arthritis, uric acid kidney stones, and chronic kidney damage, and is significantly associated with an increased risk of metabolic syndromes such as hypertension and insulin resistance. With the surge in high-purine food intake, rising obesity rates, and the increase in metabolic diseases such as diabetes, the global prevalence of HUA continues to climb.

[0003] High-purine uric acid (HUA) is easily overlooked in its early stages due to the lack of typical symptoms, but its harm is insidious and cumulative. For prevention, the intake of high-purine foods such as animal organs and shellfish, as well as fructose-containing beverages, should be strictly limited. For treatment, asymptomatic individuals primarily rely on dietary and exercise interventions. When serum uric acid levels exceed 540 μmol / L, or if the individual has hypertension or diabetes, medication intervention is necessary. It is particularly important to note that long-term medication use can have adverse effects on the body, potentially leading to liver and kidney toxicity, uric acid stones, and severe gastrointestinal reactions. Regular monitoring of liver and kidney function and uric acid crystal deposition is required.

[0004] The concept of probiotics was clearly defined in 1974 by British microbiologist Roy Fuller as "active microbial preparations that exert health benefits by regulating the balance of the host's gut microbiota." Over nearly 50 years of development, its scope has expanded from basic microbiology to the interdisciplinary fields of clinical medicine and precision nutrition. Recent studies have found that specific probiotic strains can alleviate human urinary tract infection (HUA) by intervening in urogenital atrophy (UA) metabolism through multiple targets. Its mechanisms include: degrading 60%-80% of purine precursors in food and inhibiting the activity of xanthine oxidase (XOD) in the liver and intestine through its metabolites, thereby reducing UA production; activating the expression of ABCG2 transporter protein in intestinal epithelial cells, promoting intestinal excretion of UA; improving intestinal barrier function and regulating intestinal flora homeostasis; inhibiting the activation of renal NLRP3 inflammasomes by reducing serum levels of inflammatory factors such as IL-1β and TNF-α, thereby alleviating UA reabsorption and reducing renal inflammatory damage; regulating intestinal flora composition, inhibiting the excessive proliferation of endotoxin-producing bacteria, and promoting the abundance of beneficial bacteria that produce short-chain fatty acids; promoting mucin secretion, repairing tight junction proteins, and enhancing intestinal barrier function. These multi-dimensional effects of probiotics in restoring intestinal flora homeostasis, strengthening barrier function, reducing inflammation, and regulating renal excretion constitute the core link of the bidirectional communication of the "gut-kidney axis," explaining its network mechanism for regulating UA homeostasis.

[0005] 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

[0006] In view of this, the purpose of the present invention is to provide the application of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 (hereinafter referred to as "BB21") in lowering uric acid, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention seeks protection for the use of *Bifidobacterium animalis* subsp. *lactamase* GOLDGUT-BB21 in the preparation of uric acid-lowering products. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29347, on December 18, 2023, and is taxonomically named *Bifidobacterium animalis* subsp. *lactamase*. Bifidobacterium animalis subsp .lactis .

[0009] Furthermore, the aforementioned products are biological products, food, or pharmaceuticals.

[0010] Furthermore, the aforementioned biological products are the bacterial cells, metabolites, and fermentation broth of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21.

[0011] Furthermore, the aforementioned foods are classified as general foods, foods for special medical purposes, functional foods, and health foods.

[0012] Furthermore, the aforementioned health food products are solid beverages, dairy beverages, compressed candies, soy products, dairy products, or fruit and vegetable products.

[0013] Furthermore, the aforementioned drugs also include pharmaceutical excipients.

[0014] 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.

[0015] Furthermore, the aforementioned pharmaceutical excipients are at least one of microcrystalline cellulose, hydroxypropyl methylcellulose, and lecithin.

[0016] Furthermore, the dosage forms of the above-mentioned drugs are granules, capsules, tablets, pills, or oral liquids.

[0017] Furthermore, the viable count of *Bifidobacterium animalis* subsp. *lactobacter* GOLDGUT-BB21 in the above products is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0018] The screening method for the probiotic *Bifidobacterium animalis* subsp. *lactobacter* GOLDGUT-BB21, 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.

[0019] 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.

[0020] The *Bifidobacterium animalis* subspecies *GOLDGUT-BB21* screened in this invention has a significant intervention effect in alleviating hyperinflammatory hyperallergy (HUA): this strain has a degradation rate of 78.08% for inosine, effectively reducing the source of exogenous UA; its intracellular and extracellular metabolites show strong inhibitory activity against xanthocyanin-dependent oxidative stress (XOD), with an inhibition rate as high as 70.94%. These results fully demonstrate that *Bifidobacterium animalis* subspecies *GOLDGUT-BB21* possesses 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.

[0021] The method for investigating the alleviating effect of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 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: a normal control group (CMC-Na gavage), a hyperuricemia model group (potassium oxonate 300 mg / kg + inosine and guanosine 375 mg / kg each), and a BB21 group (10 mg / kg gavage 1 h after modeling). 9 CFU (Cellular Fusarium oxysporum subsp. Lactobacillus GOLDGUT-BB21) was used for continuous intervention for 4 weeks.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 6. 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.

[0027] This study confirmed that the probiotic GOLDGUT-BB21 has a significant intervention effect on HUA model mice. Results showed that compared with the MOD group, BB21 intervention significantly reduced serum UA levels (by 33.87%) and Scr levels (by 22.68%) in HUA mice. Furthermore, GOLDGUT-BB21 effectively regulated the gut microbiota structure and increased the production of short-chain fatty acid-producing bacteria. Akkermansia muciniphila The abundance of [the bacteria] and the inhibition of pro-inflammatory bacteria. Parabacteroides More importantly, BB21 can effectively improve intestinal barrier function by reducing the absorption of UA precursor substances; simultaneously activating the expression of key factors Prkag2 and FoxO3 proteins in the FoxO signaling pathway, promoting the production of the anti-inflammatory factor IL-10, and enhancing the expression of colonic tight junction proteins Claudin-1, Occludin-1, and ZO-1.

[0028] 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.

[0029] 2. The probiotic GOLDGUT-BB21 of this invention has significant advantages in reducing blood uric acid and protecting kidney function. Animal experiments have verified that GOLDGUT-BB21 can effectively reduce serum uric acid levels in HUA mice, upregulate the expression of colonic tight junction proteins Claudin-1, Occludin, and ZO-1, and increase the production of short-chain fatty acid-producing bacteria. Akkermansia muciniphila The abundance of *Bifidobacterium animalis* subsp. *lactamase* can alleviate kidney damage and intestinal barrier dysfunction through multi-target synergy. Notably, GOLDGUT-BB21 can alleviate HUA-induced kidney damage by regulating the Prkag2 / FoxO3 signaling pathway and reduce nucleoside absorption by downregulating nucleoside transporter expression. Based on these comprehensive effects, *Bifidobacterium animalis* subsp. *lactamase* GOLDGUT-BB21 shows outstanding effects in lowering uric acid, improving renal function, regulating gut microbiota, and enhancing 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

[0030] Figure 1 The effects of BB21 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.

[0031] Figure 2 The effect of BB21 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.

[0032] Figure 3The effect of BB21 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) BUN content; (E) Scr content.

[0033] Figure 4 The effects of BB21 intervention on the renal transcriptome of HUA mice are shown in the following figures: (A) heatmap of gene correlation among different groups; (B) number of upregulated / downregulated genes in the MOD group compared with the CON and BB21 groups; (C) Venn diagram of the number of DEGs in MOD, CON and BB21; (D) KEGG pathway enrichment of DEGs in MOD vs CON and MOD vs BB21.

[0034] Figure 5 To investigate the effect of BB21 intervention on the FoxO signaling pathway in the kidneys of HUA mice (RT-qPCR was used to measure the mRNA expression levels of related DEGs).

[0035] Figure 6 The effects of BB21 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.

[0036] Figure 7 The effects of BB21 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. Detailed Implementation

[0037] 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.

[0038] In this invention, Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 is prior art. Its acquisition, isolation, identification, culture and preservation are referenced in the application application number 202410024101.6, the application date is January 5, 2024, the applicant is Shenzhen Baoshijian Biotechnology Co., Ltd., and the invention patent application is entitled "A strain of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 and its application".

[0039] 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).

[0040] 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.

[0041] 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).

[0042] 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.

[0043] Nucleosides and purines are precursors of UA, and whether probiotics can degrade nucleosides and purines is key to inhibiting UA production.

[0044] Table 1. Effects of different probiotics on the degradation capacity of nucleosides and purine bases.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] Table 2. Inhibition rates of xanthine oxidase by intracellular and extracellular metabolites of different probiotics

[0050] 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.

[0051] 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 Bifidobacterium lactis subsp. animalis GOLDGUT-BB21 (BB21) group (n = 10 mice / group) after one week of acclimatization. All animals were housed under specific pathogen-free conditions. Mice were fed a standard laboratory diet, had free access to water, and were exposed to artificial light for 12 hours daily (alternating between 12 hours of darkness and 12 hours of light). Environmental conditions were controlled at a temperature of 24 ± 1℃ and a relative humidity of 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-BB21 group (BB21): At 9:00 AM daily, a hyperuricemia model was induced by gavage with potassium oxonate (300 mg / kg) and a combination of inosine (375 mg / kg) and guanosine (375 mg / kg). One hour later, probiotic BB21 was administered by gavage. 9 CFU, lasting 4 weeks.

[0052] At the end of the experiment, all mice were euthanized, and their blood was collected and centrifuged (4000 r / min, 15 min) to obtain serum. After dissection, all liver, kidney, colon, colon contents and cecal contents were collected. Some kidney and colon tissues were fixed in tissue fixative at room temperature for 24 h. The remaining samples and serum were stored at -80℃ for later use.

[0053] Example 4: Determination of biochemical indicators in mouse serum and tissues Commercially available kits were used to determine relevant physiological and biochemical parameters in tissues and serum. Urinary UA, Scr, BUN, and SUA concentrations were measured; inflammation-related parameters, LPS, IL-10, TNF-α, and IL-1β, were measured in serum and kidney tissue; and PNP activity in serum and XOD activity in liver tissue were measured.

[0054] 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 UA value of the MOD group mice (273.60 μmol / L) was significantly higher than that of the CON group mice (76.60 μmol / L, p<0.05), indicating that the HUA mouse model was successfully established.

[0055] 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, the PNP activity in the MOD group mice was 201.56 ng / L, significantly higher than that in the CON group (139.07 ng / L, p<0.05). Compared with the MOD group, PNP activity decreased to 158.63 ng / L after probiotic BB21 intervention. Figure 1 As shown in Figure C, compared with the CON group (7.52 U / gprot), the XOD activity in the MOD group mice was significantly increased to 9.89 U / gprot (p<0.05). Compared with the MOD group, the BB21 group reduced XOD activity to 8.22 U / gprot, indicating that the intake of probiotic BB21 can significantly reduce XOD activity in HUA mice (p<0.05). The intake of probiotic BB21 can inhibit the activity of PNP and XOD, thereby effectively alleviating the metabolic imbalance of UA and reducing the probability of HUA.

[0056] Depend on Figure 1 As shown in the results, the UA content in the MOD group mice was 130.08 μmol / L, which was significantly higher than that in the CON group. Compared with the MOD group, the serum UA level in mice decreased by 33.87% after probiotic BB21 intervention, indicating that the intake of probiotic BB21 can significantly reduce the serum UA level in HUA mice (p<0.05).

[0057] 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.

[0058] 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, p<0.05). Compared with the MOD group, the serum IL-10 level in mice increased to 1430.84 pg / mL after probiotic BB21 intervention.

[0059] 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, significantly higher than that in the CON group (217.26 ng / L, p<0.05). Compared with the MOD group, the LPS level decreased by 43.01 ng / L after intervention with the probiotic BB21 (p<0.05). 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, the TNF-α and IL-1β levels in the BB21 group were significantly lower (p<0.05), but there was no significant difference compared with the CON group (p>0.05).

[0060] Figure 2 The levels of inflammatory factors in the kidneys of mice (EH) showed a trend consistent with those in serum. Ingestion of the probiotic BB21 significantly increased renal IL-10 levels and significantly decreased renal LPS, TNF-α, and IL-1β levels in HUA mice. These results indicate that BB21 can alleviate the inflammatory response induced by HUA.

[0061] 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 Figure D, 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, p<0.05). Therefore, the high-nucleoside diet induced renal function impairment in HUA mice, leading to decreased BUN excretion and accumulation in the body. Compared with the MOD group, the BB21 group was able to reduce BUN levels (7.31 mmol / L, p>0.05). Figure 3The results showed that 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, p<0.05), 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 probiotic BB21, the Scr level significantly decreased to 21.48 μmol / L (p<0.05), approaching that of the CON group. These results indicate that the intake of probiotic BB21 has a promoting effect on glomerular filtration in HUA mice.

[0062] Example 5: Histopathology and Immunofluorescence Detection Colon and kidney tissues were removed from 4% paraformaldehyde, rinsed with running water, and dehydrated. The 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 with CY3-labeled goat anti-rabbit IgG secondary antibody (1:300) at room temperature in the dark for 50 min. After rinsing with PBS, 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.

[0063] 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 group CON was intact, with tightly packed epithelial cells, an intact brush border, uniform brush-like structures, and normal lumen size, without dilation or stenosis. In group MOD, local inflammatory cell infiltration was observed, the brush border was detached, the lumen was dilated, and the epithelial cells were unevenly arranged. After probiotic intervention, the inflammatory cell infiltration disappeared, and the lumen dilation was somewhat alleviated.

[0064] 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, the relative collagen content in the MOD group (26.03%) was higher than that in the CON group (14.12%). After intervention with probiotic BB21, the relative collagen content in the kidneys was significantly reduced by 18.57% (p<0.05), indicating that BB21 can improve kidney damage caused by HUA.

[0065] Gut microbiota dysbiosis is closely related to human uric acid (HUA). It exacerbates the pathological process of HUA through multiple mechanisms, including interfering with the activity of uric acid metabolic enzymes, affecting the function of related transport proteins, and intensifying 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 effects of targeted microbiota therapy. Figure 7 The H&E staining results of the colon in mice in group A showed that the colon tissue in group CON was morphologically regular, with abundant and orderly arranged goblet cells, and no inflammatory cell infiltration was observed. In group MOD mice, the shape and number of goblet cells in the colon tissue were altered, and some areas showed complete disappearance of goblet cells, with local inflammatory cell infiltration. Compared with group MOD, the number of goblet cells in the colon tissue of mice in group BB21 recovered to near normal levels, inflammatory cell infiltration was reduced, and the integrity of the epithelial layer was restored.

[0066] Figure 7 Image B, D, and E respectively show the immunofluorescence analysis of Claudin-1, Occludin-1, and ZO-1 in mouse colon tissue. Claudin-1, Occludin-1, 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 mechanical support of the barrier, 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-1, and ZO-1 proteins in the colon tissue of MOD group mice was significantly reduced (p<0.05), indicating that the intestinal barrier function of the MOD group was impaired. Compared with the MOD group, after probiotic BB21 intervention, the expression of Claudin-1, Occludin-1, and ZO-1 proteins was significantly increased (p<0.05), indicating that probiotics can restore TJ protein expression and repair the intestinal barrier.

[0067] Example 6: Measurement of mouse kidney transcriptomics Mouse kidney tissue was thoroughly ground in liquid nitrogen, and cells were lysed with TRIzol reagent to extract total RNA. After testing the purity and integrity of the extracted RNA, mRNA was enriched using Oligo(dT) magnetic beads. The mRNA was randomly fragmented to approximately 300 bp in a high-temperature ion-buffered saline solution, and double-stranded cDNA was synthesized using random primers. After end repair, A-tailing, and adapter ligation, a sequencing library was constructed by PCR amplification. High-throughput sequencing of the mRNA was performed on 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, yielding high-quality clean reads. Transcript assembly was performed using StringTie, and differentially expressed genes were analyzed using DESeq2 (log2FC≥1.2, p<0.05), followed by functional enrichment analysis using the KEGG database.

[0068] Human kidney injury (HUA) is a metabolic disease caused by excessive UA production or impaired renal excretion. It is closely related to various kidney diseases, and renal function directly affects the regulation of UA levels. The results of this study on renal inflammatory factor levels and pathological analysis showed that HUA mice induced by a high-nucleoside diet exhibited an inflammatory response in their renal tissue. Probiotic intervention reduced the level of inflammation in the renal tissue and alleviated kidney damage. Therefore, further analysis of the renal transcriptome can help explore the potential mechanisms by which probiotics alleviate HUA.

[0069] 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 BB21 groups. Pearson correlation coefficients were determined among every three biological samples for each gene expression level to assess inter-sample correlation. Pearson correlation coefficients between samples within the same experimental group were all >0.9, indicating good reproducibility and allowing for further screening of differentially expressed genes. Figure 4 (A)

[0070] Based on log2FC ≥ 1.2 and p < 0.05, a total of 1946 differentially expressed genes (DEGs) were screened. Compared with the CON group, the MOD group had 745 upregulated DEGs and 603 downregulated DEGs. Figure 4 (B) Compared with the MOD group, 268 DEGs were upregulated and 221 DEGs were downregulated in the BB21 group. The Venn diagram shows that 117 genes are common DEGs in the comparisons between the MOD group and the CON / BB21 intervention groups. These 117 genes are likely key genes related to disease mechanisms and treatment effects, and will be the focus of future research. Figure 4(C). Enrichment analysis of 117 DEGs using the KEGG pathway database identified 22 signaling pathways (p<0.05). Figure 4 The study found significant enrichment of metabolic pathways including Circadian rhythm, Cell cycle, Cellular senescence, FoxO signaling pathway, 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. ABCG2 is expressed in the brush border of the proximal renal tubules and intestinal epithelial cells, mediating UA secretion through an ATP-dependent mechanism. Its dysfunction leads to reduced UA 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 shown that the FoxO signaling pathway plays a crucial role in suppressing inflammatory responses. FoxO3 may also participate in the regulation of inflammatory responses by modulating the number and function of monocytes and 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.

[0071] Example 7: RT-qPCR assay Approximately 20 mg of kidney tissue sample was placed in RNA extraction buffer containing grinding beads and thoroughly ground. Chloroform 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 determined. 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 determined (<100 ng / μL). For real-time quantification, 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 in the FoxO signaling pathway enriched 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 BB21 intervention group had significantly higher levels of [something]. 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 This trend is consistent with changes in gene expression levels in the transcriptome. RT-qPCR validation enhances the credibility of transcriptome research results, as it is highly correlated with sequencing data.

[0074] Example 8: Metagenomics Determination Total microbial DNA was extracted from fecal samples, and its concentration and purity were determined. Integrity was verified using 1% agarose gel electrophoresis. Qualified DNA was fragmented to 400 bp using a Covaris M220 fragmentation kit. 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.

[0075] The Chao 1 index and the Simpson index reflect the richness and diversity of the gut microbiota, respectively. For example... 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 there was no significant difference (p>0.05). Principal coordinate analysis (PCoA) visually presents the similarity or difference of the overall gut microbiota structure of different groups through dimensionality reduction visualization. PCoA analysis based on the Bray-Curtis distance matrix shows that ( Figure 6 In the BB21 intervention group (B), the gut microbiota of the CON and MOD groups showed significant segregation along the PCo1 axis, indicating that a high-nucleoside diet led to dysbiosis of the gut microbiota structure in mice. The BB21 intervention group clustered closely with the CON normal group, indicating that BB21 significantly reversed the imbalance of gut microbiota structure and normalized the microbiota structure.

[0076] Further analysis was conducted on the composition of the gut microbiota at different taxonomic levels in the CON, MOD, and BB21 groups. For example... Figure 6 As 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.

[0077] 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 6As 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. Among them, 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 probiotic BB21. 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 specific conditions, these bacteria are pro-inflammatory and can exacerbate inflammatory responses. It is noteworthy that the key bacterial groups identified in the inter-group difference analysis were also enriched in the LDA value distribution histogram based on the LEfSe method (LDA score > 3). Figure 6 The results (E) further confirm the significant changes in these microbiota during model establishment and intervention. In summary, intervention with probiotic BB21 alleviates HUA-induced dysbiosis and inflammatory responses by increasing the relative abundance of beneficial bacteria and decreasing the abundance of harmful bacteria.

[0078] KEGG pathway enrichment analysis based on metagenomic sequencing data can predict potential differences in metabolic function among different gut microbiota and infer potential molecular pathways that may affect host UA levels, providing hypotheses and directions for revealing specific mechanisms. KEGG pathway enrichment analysis of significantly different gut microbiota in three groups of samples revealed that six metabolic pathways were significantly enriched, and these pathways are known to be closely related to the occurrence and development of HUA. Figure 6(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 BB21 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 reduce 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 BB21 intervention can maintain the stability of gut microbiota metabolic function in HUA mice to some extent.

[0079] 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 Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 in the preparation of uric acid-lowering products, characterized in that, This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29347, on December 18, 2023, and is taxonomically named *Bifidobacterium animalis* subsp. *lactam*. Bifidobacterium animalis subsp .lactis .

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

3. The application according to claim 2, characterized in that, The biological products are the bacterial cells, metabolites, and fermentation broth of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21.

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

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

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

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

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

10. The application according to claim 2, characterized in that, The viable count of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 in the product is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

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