Compound lactic acid bacteria and application thereof in preparation of product for relieving hyperuricemia

Abstract

The invention belongs to the technical field of functional microorganisms and application thereof, and relates to compound lactic acid bacteria and application thereof in preparation of products for relieving hyperuricemia. The compound lactobacillus is prepared from lactobacillus plantarum PL4-9, pediococcus acidilactici FB1-3 and lactobacillus plantarum sc3-2, and the preservation numbers of the lactobacillus plantarum PL4-9, the pediococcus acidilactici FB1-3 and the lactobacillus plantarum sc3-2 are CGMCC (China General Microbiological Culture Collection Center) NO. 34075, CGMCC NO. 34076 and The compound lactic acid bacteria formed by mixing the raw materials according to the volume ratio of 1: 1: 1 can obviously reduce the serum uric acid and urea nitrogen level of a mouse with hyperuricemia, and also has an obvious relieving effect on abnormal increase of serum xanthine oxidase activity; the generation of uric acid can be reduced by inhibiting the activity of liver xanthine oxidase, and meanwhile, the liver and kidney injury induced by hyperuricemia can be remarkably relieved.

Description

Technical Field

[0001] This invention belongs to the field of functional microorganisms and their application technology, and specifically relates to a compound lactic acid bacteria and its application in the preparation of products to relieve hyperuricemia. Background Technology

[0002] Hyperuricemia is a metabolic disorder characterized by abnormally high levels of uric acid in the serum. It is a chronic metabolic disease caused by purine metabolism disorders and is closely related to a variety of health problems such as gout, kidney stones, and cardiovascular diseases.

[0003] Currently, the treatment of hyperuricemia mainly involves three approaches: first, promoting the oxidation and decomposition of uric acid by using uricase to oxidize and decompose uric acid into allantoin and hydrogen peroxide, which are then excreted from the body; second, inhibiting xanthine oxidase activity to reduce uric acid production; and third, reducing the body's absorption of nucleosides by degrading or absorbing nucleosides, thereby controlling the increase in uric acid levels.

[0004] While oral chemical drugs can reduce uric acid production or promote its excretion, drug therapy has numerous adverse reactions, leading to poor patient tolerance and treatment adherence. Therefore, exploring safe, effective, and non-toxic methods to lower uric acid is of great significance. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a compound lactic acid bacteria strain comprising: *Lactobacillus plantarum* PL4-9, *Pediococcus lactis* FB1-3, and *Lactobacillus plantarum* SC3-2. This invention also provides its application in preparing products for alleviating hyperuricemia using this compound lactic acid bacteria. The compound lactic acid bacteria product of this invention, composed of three strains, compared to oral chemical drugs mentioned in the background art for reducing uric acid, achieves excellent uric acid-lowering effects while being safe, non-toxic, and free of side effects, exhibiting numerous adverse reactions and high patient acceptability.

[0006] The technical solution of this invention is: A compound lactic acid bacteria, composed of Lactobacillus plantarum PL4-9, Pediococcus lactis FB1-3, and Lactobacillus plantarum sc3-2; The aforementioned *Lactobacillus plantarum* PL4-9, *Pediococcus lactis* FB1-3, and *Lactobacillus plantarum* sc3-2 were deposited on April 2, 2025, at the China General Microbiological Culture Collection Center, located at Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] The preservation number for *Lactobacillus plantarum* PL4-9 is CGMCC No. 34075, and its classification name is *Lactobacillus plantarum*. Lactiplantibacillusplantarum ; The preservation number for *Pediococcus lactis* FB1-3 is CGMCC No. 34076, and its classification name is *Pediococcus lactis*. Pediococcus acidilactici ; The preservation number for *Lactobacillus plantarum* sc3-2 is CGMCC No. 34077, and its classification name is *Lactobacillus plantarum*. Lactiplantibacillusplantarum .

[0008] The full-length 16S rDNA sequence of Lactobacillus plantarum PL4-9 is shown in SEQ ID No:1; The full-length 16S rDNA sequence of Pediococcus lactis FB1-3 is shown in SEQ ID No:2; The full-length 16S rDNA sequence of Lactobacillus plantarum sc3-2 is shown in SEQ ID No:3.

[0009] Further preferably, the upstream and downstream primers for amplifying the nucleotide sequence shown in SEQ ID NO:1 are as follows: 5'-GAGAGTTTGATCCTGGCTCAG-3'; 5'-AAGGAGGTGATCCAGCCGCA-3'; The upstream and downstream primers for amplifying the nucleotide sequence shown in SEQ ID NO:2 are as follows: 5'-GAGAGTTTGATCCTGGCTCAG-3', 5'-AAGGAGGTGATCCAGCCGCA-3'; The upstream and downstream primers for amplifying the nucleotide sequence shown in SEQ ID NO:3 are as follows: 5'-GAGAGTTTGATCCTGGCTCAG-3', 5'-AAGGAGGTGATCCAGCCGCA-3'.

[0010] The preparation method of the above-mentioned compound lactic acid bacteria includes the following steps: (1) Lactobacillus plantarum PL4-9, Pediococcus lactis FB1-3 and Lactobacillus plantarum sc3-2 were stored in glycerol to obtain three glycerol bacteria; Lactobacillus plantarum PL4-9, Pediococcus lactis FB1-3, and Lactobacillus plantarum sc3-2 were activated in MRS liquid medium and cultured at 37℃ for 24 h to obtain three types of bacterial suspensions. (2) Take the three bacterial cultures from (1) and inoculate them into MRS liquid medium at a volume ratio of 3%, and incubate them at 37°C for 24 hours. (3) Take the cultured bacterial solution from (2) and perform the following operations: After washing with sterile physiological saline, the bacteria were resuspended in sterile physiological saline, and the bacterial concentration in the suspension was adjusted to 1×10⁻⁶. 9 CFU / mL; (4) Take the bacterial suspension with adjusted concentration from (3) and mix them in a volume ratio of 1:1:1 to obtain compound lactic acid bacteria.

[0011] This invention provides the application of compound lactic acid bacteria in the preparation of products that alleviate hyperuricemia.

[0012] This invention also provides the application of compound lactic acid bacteria in the preparation of any one of the following: products that remove nucleosides, products that inhibit xanthine oxidase activity, and products that remove uric acid.

[0013] This invention also provides at least one of the following applications: The application of Lactobacillus plantarum PL4-9 in the preparation of products for removing uric acid.

[0014] Application of Pediococcus lactis FB1-3 in the preparation of products that inhibit xanthine oxidase activity; Application of Lactobacillus plantarum sc3-2 in the preparation of nucleoside-free products.

[0015] The treatment of hyperuricemia is achieved by reducing serum uric acid and urea nitrogen levels, alleviating the increase in serum xanthine oxidase activity, significantly inhibiting hepatic xanthine oxidase activity, reducing uric acid production, and significantly inhibiting renal β-N-acetylglucosidase activity, thereby alleviating kidney damage.

[0016] The present invention has the following advantages and effects compared with the prior art: (1) Different strains act on different targets and synergistically alleviate hyperuricemia. The compound lactic acid bacteria provided by this invention consists of three strains: Lactobacillus plantarum PL4-9, Pediococcus lactis FB1-3, and Lactobacillus plantarum sc3-2. These strains have excellent abilities to clear uric acid, inhibit xanthine oxidase activity, and clear nucleosides, respectively. Through the synergistic effects of these three strains on different targets, they achieve excellent relief of hyperuricemia. (2) Excellent effect of compound lactic acid bacteria in relieving hyperuricemia and supporting data As can be seen from the data in the embodiments of the present invention, the product of the present invention can significantly reduce serum uric acid (from 256.55 μmol / L to 141.86 μmol / L), urea nitrogen content (from 10.08 mmol / L to 6.72 mmol / L), and inhibit serum xanthine oxidase activity (from 12.71 U / L to 9.88 U / L) in hyperuricemic mice; and significantly inhibit liver xanthine oxidase activity (from 14.25 U / gprot to 9.68 U / gprot) and kidney β-N-acetylglucosidase activity (from 59.14 U / gprot to 25.41 U / gprot) in hyperuricemic mice, alleviating kidney damage, which further confirms the effect of the product described in (1); (3) The three strains have high safety and excellent performance. The three strains provided by this invention—Lactobacillus plantarum PL4-9, Pediococcus lactis FB1-3, and Lactobacillus plantarum sc3-2—are all non-hemolytic and exhibit high safety. Specifically, Lactobacillus plantarum PL4-9, detected by the phosphotungstic acid colorimetric method, showed an in vitro uric acid clearance rate of 27.27%. Lactobacillus plantarum sc3-2, detected by high-performance liquid chromatography, showed in vitro guanosine and inosine degradation rates of 98.32% and 98.05%, respectively. Pediococcus lactis FB1-3, detected by the uric acid formation method, showed an in vitro xanthine oxidase inhibition rate of 50.99%. Attached Figure Description

[0017] Figure 1 The hemolytic activity of three strains of Lactobacillus plantarum PL4-9, Pediococcus lactis FB1-3, and Lactobacillus plantarum sc3-2 is shown in the graph. Figure 2 Serum parameters of mice in each treatment group are shown below. A represents serum uric acid (UA) level, B represents serum creatinine (CRE) level, C represents serum blood urea nitrogen (BUN) level, and D represents serum xanthine oxidase (XOD) activity. Different superscript letters in the same column indicate significant differences (P<0.05). Figure 3 The values ​​represent liver and kidney uric acid-related indicators in mice of each treatment group. A represents liver xanthine oxidase (XOD) activity, and B represents kidney β-N-acetylglucosidase (NAG) activity. Different superscript letters in the same column indicate significant differences (P<0.05). Figure 4 The images show histopathological sections of mice in each treatment group; where A is a liver section with H&E staining, B is a kidney section with H&E staining, C is a kidney section with Masson staining, CON is the normal diet group, HUA is the high uric acid group, Mix is ​​the high uric acid + lactic acid bacteria group, and Ap is the high uric acid + allopurinol group. Detailed Implementation

[0018] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0019] Unless otherwise specified, the three strains mentioned in each embodiment of this invention are the strains that have been preserved as mentioned in the invention content of this application.

[0020] Example 1: Screening process of compound lactic acid bacteria Sample enrichment: 1g of Northeast sauerkraut, 1mL of Sichuan pickled vegetable water and infant feces were added to MRS liquid culture medium and incubated at 37℃ for 48 hours.

[0021] Lactic acid bacteria strain screening: Strains were isolated using the dilution plating method. First, the enriched bacterial suspension was serially diluted and plated onto MRS-CaCO3 medium. The plates were then incubated at 37°C for 48 hours. Based on colony morphology, color, and size, strains exhibiting a calcium-solubilizing zone were selected for three-stage streak plating for purification.

[0022] Preservation of bacterial strains: Add the activated bacterial solution and 30% glycerol in equal proportion to the preservation tube and store at -80℃.

[0023] Strain identification: Genomic DNA of the strain was extracted using the Ezup column-based bacterial genomic DNA extraction kit and amplified using specific primers for the 16S rDNA gene of lactic acid bacteria.

[0024] The forward primer 27F was 5'-GAGAGTTTGATCCTGGCTCAG-3', and the reverse primer 1541R was 5'-AAGGAGGTGATCCAGCCGCA-3'. Electrophoresis was performed on a 1% agarose gel, with the target fragment length approximately 1500 bp. The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the 16S rDNA sequence was determined for strain identification. After sequencing, BLAST alignment was performed using NCBI to confirm the strain species.

[0025] Example 2: Determination of the in vitro uric acid scavenging capacity of Lactobacillus plantarum PL4-9 strain Prepare a uric acid liquid culture medium with a uric acid concentration of 0.1 g / L. First, dissolve the bacteria thoroughly, then sterilize. Centrifuge the activated bacterial solution at 8000 rpm for 5 min, collect the bacterial precipitate, wash the bacterial cells twice with sterile PBS, adjust the OD600 to 1.5, take 1 mL of the balanced bacterial solution, centrifuge to collect the bacterial cells, resuspend the bacterial cells in 750 μL of uric acid liquid culture medium, and use blank uric acid liquid culture medium as a control. Incubate at 37℃ for 8 h. After the incubation is completed, centrifuge at 8000 rpm for 5 min, collect the supernatant for detection.

[0026] Perform sample addition and testing according to Table 1 below.

[0027] Table 1. Uric Acid Detection Sample Addition Procedure

[0028] Uric acid content C (mg / L) = (A measured - A blank) / (A standard - A blank) × 50 Uric acid clearance rate (%) = (C control - C residual) / C control × 100% In the formula, C represents the uric acid content of the control group; C residue represents the uric acid content corresponding to the experimental group.

[0029] Testing showed that *Lactobacillus plantarum* PL4-9 achieved a uric acid clearance rate of 27.27%. Morphological and molecular biological identification confirmed that this strain is *Lactobacillus plantarum*. Lactiplantibacillusplantarum The strain was named *Lactobacillus plantarum* PL4-9. *Lactobacillus plantarum* PL4-9 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 34075.

[0030] Example 3: Determination of the in vitro guanosine-inosine-lowering capacity of Lactobacillus plantarum sc3-2 strain To prepare a 100 mmol / L neutral potassium phosphate buffer solution: Weigh 4.2454 g K3PO4, dissolve it in ultrapure water, bring the volume up to 200 mL in a volumetric flask, and adjust the pH to 7.0 ± 0.1 with HCl.

[0031] To prepare a 1.26 mmol / L guanosine-1.26 mmol / L inosine-100 mmol / L neutral potassium phosphate buffer: Weigh 0.0358 g guanosine and 0.0338 g inosine. Dissolve the guanosine in 50 mL of 100 mmol / L neutral K3PO4 buffer at 40°C. After cooling slightly, add the inosine, mix well, and bring the volume to 100 mL. After filtration, dispense into containers and store at -20°C.

[0032] Prepare the mobile phase: V water : V methanol = 9 : 1. Take 900 mL of ultrapure water and 100 mL of chromatographic grade methanol, mix them evenly, filter them, and sonicate for 30 min.

[0033] Prepare the reaction terminator: Take 0.10 mol / L perchloric acid solution, take 0.571 mL HClO4 (70%), and dilute to 100 mL with ultrapure water.

[0034] The fully activated bacterial seed culture medium was inoculated into 7 mL of MRS medium at a 2% inoculum size and incubated statically for 24 h. After centrifugation at 8000 rpm for 5 min, the bacterial cells were washed twice with sterile physiological saline (0.9% NaCl solution). The OD600 of the bacterial suspension was adjusted to 1.5 with physiological saline. 1.6 mL of the adjusted bacterial suspension was transferred to a 2 mL tube, centrifuged, and the supernatant was discarded. 1 mL of buffer solution was added to the bacterial cells, mixed thoroughly, and the cells were resuspended. The mixture was then incubated at 37℃ and 220 rpm for 1 h. An equal volume of buffer solution was used as a control. After the reaction, the mixture was centrifuged at 8000 rpm for 8 min. 810 μL of the supernatant was mixed thoroughly with 90 μL of stop agent, filtered through a 0.22 μm aqueous filter membrane, and then transferred to a liquid chromatography vial for HPLC analysis to determine the contents of inosine and guanosine. Chromatographic conditions: Detection wavelength: 254 nm, column temperature: 35℃, flow rate: 0.8 mL / min, analysis time: 10 min, injection volume: 10 μL. The corresponding peak areas were then substituted into the standard curve formula to calculate the corresponding concentrations.

[0035] Degradation rate (%) = (C control - C residue) / C control × 100% In the formula, C_control represents the guanosine / inosine content of the control group; C residue represents the guanosine / inosine content corresponding to the experimental group.

[0036] Testing showed that *Lactobacillus plantarum* sc3-2 exhibited in vitro degradation rates of 98.32% for guanosine and 98.05% for inosine. Morphological and molecular biological identification confirmed this strain as *Lactobacillus plantarum*. (Lactiplantibacillus plantarum The strain was named *Lactobacillus plantarum* sc3-2. *Lactobacillus plantarum* sc3-2 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 34077.

[0037] Example 4: Determination of the in vitro xanthine oxidase inhibition ability of Pediococcus lactis FB1-3 strain Preparation of phosphate buffer: Weigh 5.64 mg ethylenediaminetetraacetic acid (EDTA), 13.88 g dipotassium hydrogen phosphate (K2HPO4), and 1.92 g potassium dihydrogen phosphate (KH2PO4) into a beaker, add 800 mL of water, sonicate for 20 min, adjust the pH to 7.5, and bring the volume to 1 L.

[0038] Preparation of 1 mol / L sodium hydroxide solution: Weigh 4 g of sodium hydroxide solid, dissolve it in a small amount of distilled water, wait for it to completely dissolve, cool to room temperature, and then make up to 100 mL.

[0039] Preparation of 1 mmol / L xanthine solution: Weigh 38.3 mg of xanthine into a 25 mL beaker, add 3 mL of 1 mol / L NaOH solution, sonicate for 20 min, and then bring the volume to 250 mL with the above phosphate buffer solution to obtain xanthine stock solution.

[0040] Preparation of 0.2U xanthine oxidase solution: Take 2 mg of 50 U / mg xanthine oxidase powder and add it to phosphate buffer, then make up to 500 mL to obtain 0.2U xanthine oxidase solution.

[0041] Preparation of allopurinol solution: Take 2 mg of allopurinol and dilute to 100 mL with phosphate buffer.

[0042] Take 1.6 mL of the second-generation activated bacterial suspension, centrifuge at 10000 rpm / min for 10 min to collect bacterial cells, wash the bacterial cells twice with sterile PBS (pH 7.4), and resuspend them in 1.2 mL of PBS. Adjust the OD600 to 1.5, take 1 mL of the balanced bacterial suspension and incubate at 37°C for 12 h. Centrifuge at 10000 rpm / min for 10 min, collect the supernatant, filter it through a 0.22 μm filter membrane to obtain cell metabolites, and freeze at -80°C.

[0043] Phosphate buffer, sample, and xanthine oxidase (0.2 U / mL) were added sequentially to 96-well microplates. The plates were incubated at 37°C for 3 min. Then, xanthine substrate solution (1 mmol / L) was added to start the reaction. The absorbance was measured at 295 nm using a microplate reader, with measurements taken every 30 s for a total of 10 min. Three parallel experiments were performed.

[0044] Table 2 Sample Addition Procedure for Xanthine Oxidase Inhibition Experiment Group A B C D Phosphate buffer (μL) 50 100 0 50 Sample (μL) 0 0 50 50 Xanthine oxidase (μL) 50 0 50 0 Xanthine (μL) 100 100 100 100

[0045] Inhibition rate (%) = (1 - (CD) / (AB)) × 100% After testing, the in vitro xanthine oxidase inhibition rate of *Pediococcus lactis* FB1-3 was 50.99%. Morphological and molecular biological identification confirmed that this strain is *Pediococcus lactis* (…). Pediococcus acidilactici The strain was named *Pediococcus lactis* FB1-3. *Pediococcus lactis* FB1-3 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 34076.

[0046] Example 5 Hemolysis experiment of three strains of lactic acid bacteria Sterile defibrinated sheep blood was added to sterilized Columbia blood agar medium at a ratio of 5% (V / V) to prepare blood plates.

[0047] Staphylococcus aureus (positive control), Lactobacillus plantarum PL4-9, Pediococcus lactis FB1-3, and Lactobacillus plantarum sc3-2 were spotted onto different areas of the surface of Columbia blood agar medium. After incubation at 37°C for 48 hours, changes around the colonies were observed.

[0048] Depend on Figure 1 It can be seen that Staphylococcus aureus, as a positive control, showed a clear hemolytic zone around its cells. The remaining three lactic acid bacteria strains, however, did not show hemolytic zones, indicating that they are not hemolytic.

[0049] Example 6 Preparation of compound lactic acid bacteria A method for preparing a compound lactic acid bacteria, comprising the following steps: (1) Lactobacillus plantarum PL4-9, Pediococcus lactis FB1-3 and Lactobacillus plantarum sc3-2 were stored in glycerol respectively; the above three strains were activated in MRS liquid medium and cultured at 37℃ for 24h to obtain three kinds of bacterial solutions after culture. (2) The three bacterial cultures were inoculated into MRS liquid medium at a volume ratio of 3% and cultured at 37°C for 24 hours. (3) Take the cultured bacterial solution from (2) and perform the following operations: Wash twice with sterile saline and resuspend in sterile saline to adjust the bacterial concentration to 1×10⁻⁶. 9 CFU / mL; (4) Take the bacterial suspension with adjusted concentration from (3) and mix them evenly in a volume ratio of 1:1:1. The resulting bacterial suspension is the compound lactic acid bacteria.

[0050] Example 7 In vivo experiment 7.1 Model Construction and Experiment A mouse model of hyperuricemia was established: Male KM mice aged 4-5 weeks were selected and fed a standard maintenance diet as the basal diet. A hyperuricemia model was induced in mice using a high-uric acid diet (3% uric acid + 3% potassium oxonate). The following groups were established: a control group, a hyperuricemia model group, a hyperuricemia + lactic acid bacteria group (i.e., a hyperuricemia + compound lactic acid bacteria group; unless otherwise specified, all lactic acid bacteria groups mentioned in the embodiments of this invention are compound lactic acid bacteria, prepared according to Example 6), and a hyperuricemia + allopurinol group. Eight mice were in each group. They were fed the diet shown in Table 3 below and administered the test solution by gavage once daily. The experiment lasted for 6 weeks, during which the mice had free access to food and water.

[0051] Table 3 Experimental Groups and Feeding / Gavage Treatments Processing group Gavage (0.2 mL / day / animal) Feed categories control group sterile saline Basic feed High uric acid model group sterile saline High uric acid feed High uric acid + lactic acid bacteria group <![CDATA[Bacterial suspension 10 9 CFU / mL]]> High uric acid feed High uric acid + allopurinol group Allopurinol 10 mg / kg BW High uric acid feed

[0052] During the experiment, the mice's health status and feeding status were observed daily, and weight changes were recorded weekly. After the experiment, the mice were fasted for 12 hours but allowed free water. Blood was collected from the eyeballs and serum was separated to detect three renal function parameters (uric acid UA, creatinine CRE, and blood urea nitrogen BUN) and xanthine oxidase (XOD) activity. Liver samples were collected for H&E staining analysis and xanthine oxidase (XOD) activity detection. Kidney samples were collected for H&E staining, Masson staining, and β-N-acetylglucosidase (NAG) activity detection.

[0053] 7.2 Experimental Results and Analysis Table 4 Serum markers of mice in each treatment group Group control group High uric acid model group High uric acid + lactic acid bacteria group High uric acid + allopurinol group Serum uric acid (μmol / L) <![CDATA[133.57±5.65 b ]]> <![CDATA[256.55±26.41 a ]]> <![CDATA[141.86±8.97 b ]]> <![CDATA[109.96±6.09 b ]]> Serum creatinine (μmol / L) <![CDATA[19.74±0.70 b ]]> <![CDATA[26.82±1.84 a ]]> <![CDATA[22.79±1.22 ab ]]> <![CDATA[20.72±1.65 b ]]> Serum urea nitrogen (mmol / L) <![CDATA[6.50±0.44 b ]]> <![CDATA[10.08±0.38 a ]]> <![CDATA[6.72±0.22 b ]]> <![CDATA[5.88±0.28 b ]]> Serum xanthine oxidase (U / L) <![CDATA[9.88±0.41 b ]]> <![CDATA[12.71±0.61 a ]]> <![CDATA[9.88±0.44 b ]]> <![CDATA[9.17±0.29 b ]]>

[0054] All data are expressed as mean ± standard error (SEM). Data were analyzed using one-way ANOVA (n=6) with SPSS 26.0 software (IBM, USA). Different superscript letters in the same column indicate significant differences (P<0.05).

[0055] Table 5. Liver and kidney uric acid-related indicators in mice of different treatment groups Group control group High uric acid model group High uric acid + lactic acid bacteria group High uric acid + allopurinol group Hepatic xanthine oxidase (U / gprot) <![CDATA[12.94±0.33 ab ]]> <![CDATA[14.25±0.34 a ]]> <![CDATA[9.68±0.97 c ]]> <![CDATA[12.33±0.51 b ]]> Renal β-N-acetylglucosidase (U / gprot) <![CDATA[41.40±2.63 b ]]> <![CDATA[59.14±2.05 a ]]> <![CDATA[25.41±3.69 c ]]> <![CDATA[25.75±0.87 c ]]>

[0056] All data are expressed as mean ± standard error (SEM). Data were analyzed using one-way ANOVA (n=6) with SPSS 26.0 software (IBM, USA). Different superscript letters in the same column indicate significant differences (P<0.05).

[0057] The experimental results showed that, compared with the control group, the serum UA, CRE, BUN levels and XOD activity of mice in the hyperuricemia model group were significantly increased, while the above indicators in the hyperuricemia + lactic acid bacteria group returned to normal levels, with no significant difference from the normal diet group. Figure 2 Compared with the control group, the liver XOD activity of mice in the hyperuricemia model group was increased to some extent but not significantly different, while the XOD activity in the hyperuricemia + lactic acid bacteria group was significantly decreased compared with the hyperuricemia model group, indicating that the lactic acid bacteria of the present invention can reduce uric acid production by inhibiting liver XOD activity. Figure 3 (A); meanwhile, liver H&E staining results directly showed an increase in inflammatory factors in the liver of mice in the high uric acid group, and a loss of cell nuclei, while the high uric acid + lactic acid bacteria group alleviated this phenomenon ( Figure 4 (A). Under the induction of a high uric acid diet, renal NAG activity was significantly increased, while NAG activity was significantly decreased in the high uric acid + lactic acid bacteria group compared with the high uric acid model group, indicating that high uric acid can induce kidney damage and the lactic acid bacteria of this invention can alleviate this damage. Figure 3(B) Meanwhile, the histopathological results of the kidney tissue showed that the kidneys of mice in the hyperuricemia model group had inflammatory cell infiltration, a large number of vacuolated renal tubular epithelial cells, and a lot of collagen deposition in the renal tubular interstitial area, while the hyperuricemia + lactic acid bacteria group significantly reduced renal inflammation and fibrosis. Figure 4 (B, C). The above results indicate that the compound lactic acid bacteria of the present invention can significantly alleviate kidney damage induced by a high uric acid diet.

[0058] In summary, the compound lactic acid bacteria in this invention is composed of *Lactobacillus plantarum* PL4-9 (CGMCC No. 34075), *Pediococcus lactis* FB1-3 (CGMCC No. 34076), and *Lactobacillus plantarum* sc3-2 (CGMCC No. 34077) in a 1:1:1 volume ratio. It can be used in the preparation of products for alleviating hyperuricemia, and its uric acid-lowering effect is significant. Detailed analysis is as follows: This compound lactic acid bacteria alleviates hyperuricemia through multi-target action, specifically by reducing serum uric acid and urea nitrogen levels, alleviating the increase in serum xanthine oxidase activity, significantly inhibiting hepatic xanthine oxidase activity, reducing uric acid production, and significantly inhibiting renal β-N-acetylglucosidase activity, alleviating kidney damage, making it more efficient and safer.

[0059] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A compound lactic acid bacteria, characterized in that, The compound lactic acid bacteria consists of Lactobacillus plantarum PL4-9, Pediococcus lactis FB1-3, and Lactobacillus plantarum sc3-2. The *Lactobacillus plantarum* PL4-9, *Pediococcus lactis* FB1-3, and *Lactobacillus plantarum* sc3-2 were all deposited on April 2, 2025, at the China General Microbiological Culture Collection Center, located at Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The preservation number for *Lactobacillus plantarum* PL4-9 is CGMCC No. 34075, and its classification name is *Lactobacillus plantarum*. Lactiplantibacillus plantarum ; The preservation number for *Pediococcus lactis* FB1-3 is CGMCC No. 34076, and its classification name is *Pediococcus lactis*. Pediococcus acidilactici ; The preservation number for *Lactobacillus plantarum* sc3-2 is CGMCC No. 34077, and its classification name is *Lactobacillus plantarum*. Lactiplantibacillus plantarum .

2. The compound lactic acid bacteria as described in claim 1, characterized in that, The full-length 16S rDNA sequence of Lactobacillus plantarum PL4-9 is shown in SEQ ID No:1; The full-length 16S rDNA sequence of the *Pediococcus lactis* FB1-3 is shown in SEQ ID No:2; The full-length 16S rDNA sequence of *Lactobacillus plantarum* sc3-2 is shown in SEQ ID No:

3.

3. The compound lactic acid bacteria as described in claim 2, characterized in that, The upstream and downstream primers for amplifying the nucleotide sequence shown in SEQ ID NO:1 are, in order: 5'-GAGAGTTTGATCCTGGCTCAG-3'; 5'-AAGGAGGTGATCCAGCCGCA-3'; The upstream and downstream primers for amplifying the nucleotide sequence shown in SEQ ID NO:2 are, in order: 5'-GAGAGTTTGATCCTGGCTCAG-3'; 5'-AAGGAGGTGATCCAGCCGCA-3'; The upstream and downstream primers for amplifying the nucleotide sequence shown in SEQ ID NO:3 are, in order: 5'-GAGAGTTTGATCCTGGCTCAG-3'; 5'-AAGGAGGTGATCCAGCCGCA-3'.

4. The method for preparing a compound lactic acid bacteria as described in claim 1, characterized in that, The steps include the following: (1) Take Lactobacillus plantarum PL4-9, Pediococcus lactis FB1-3 and Lactobacillus plantarum sc3-2 respectively, activate them in MRS liquid medium, culture them, and obtain three kinds of bacterial solutions after culture; (2) Take the three bacterial cultures from (1) and inoculate them into MRS liquid medium at a volume ratio of 3% respectively, and culture them separately. (3) Take the bacterial suspensions cultured in (2) and perform the following operations: wash with sterile physiological saline, resuspend them in sterile physiological saline, and adjust the bacterial concentration in the suspension to 1×10⁻⁶. 9 CFU / mL; (4) Take the bacterial suspension with adjusted concentration from (3) and mix them in a volume ratio of 1:1:1 to obtain compound lactic acid bacteria.

5. The application of the compound lactic acid bacteria according to claim 1 in the preparation of products for relieving hyperuricemia.

6. The use of the compound lactic acid bacteria according to claim 1 in the preparation of any one of the following: products that remove nucleosides, products that inhibit xanthine oxidase activity, and products that remove uric acid.

7. The application of *Lactobacillus plantarum* PL4-9 in the preparation of uric acid removal products, characterized in that... The *Lactobacillus plantarum* PL4-9 described herein has the preservation information as described in claim 1.

8. The application of *Lactobacillus plantarum* sc3-2 in the preparation of nucleoside-removed products, characterized in that... The *Lactobacillus plantarum* sc3-2 described herein has the preservation information as described in claim 1.

9. The application of *Pediococcus lactis* FB1-3 in the preparation of products inhibiting xanthine oxidase activity, characterized in that... The *Pediococcus lactis* FB1-3 described herein has the preservation information as described in claim 1.

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