Probiotics with uric acid lowering activity, composite probiotics and their applications

Abstract

The present invention belongs to the technical field of probiotics and their applications, and discloses probiotics with uric acid lowering activity, a composite probiotic agent and applications thereof. A strain of Lentilactobacillus buchneri strain ZD62 was deposited in China Center for Type Culture Collection on March 14, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 2024469; a composite probiotic agent comprising Lentilactobacillus buchneri strain ZD62 and fermented mucin Bacillus ZD103; the Lactobacillus buchneri ZD62 and fermented mucin Bacillus ZD103 provided by the present invention have good uric acid lowering properties.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotics and their applications, and specifically relates to probiotics with uric acid-lowering activity, a composite probiotic agent and applications thereof. Background Art

[0002] Hyperuricemia (HUA) is a metabolic disorder characterized by elevated blood uric acid levels. Currently, approximately one-quarter of men and one-tenth of women suffer from asymptomatic or symptomatic hyperuricemia, the latter of which is known as gout. With the increasing incidence of hyperuricemia and gout, particularly among younger people, hyperuricemia is considered the fourth most important risk factor after the "three highs" of hypertension, hyperlipidemia, and diabetes. Hyperuricemia and gout are influenced by numerous factors, including genetics, age, gender, social and economic factors; excessive consumption of purine-rich foods such as meat, seafood, alcohol, and sugary beverages; and various metabolic syndromes, including hypertension, abnormal lipid / glucose metabolism, and chronic kidney disease. The pathogenesis of hyperuricemia is complex and remains unclear. It is generally believed that it is primarily caused by elevated blood uric acid levels due to increased uric acid production and / or decreased excretion. Current clinical drug treatments for hyperuricemia focus on reducing uric acid production or promoting uric acid metabolism. Corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, allopurinol, febuxostat, benzbromarone, and others are first-line drugs for the treatment of acute gout. However, long-term use of these drugs is often associated with adverse reactions. For example, during allopurinol treatment, patients often experience rashes, gout attacks, gastrointestinal symptoms, elevated liver enzymes, blood abnormalities, and drowsiness. Other commonly used clinical drugs, such as febuxostat and benzbromarone, also have serious side effects. Currently, the types of drugs used to treat hyperuricemia and gout are limited, and their adverse reactions cannot be ignored. Therefore, it is of great significance to seek a mild, non-toxic and side-effect-free method to effectively relieve or assist in the treatment of hyperuricemia.

[0003] Lactic acid bacteria are safe, non-toxic, and edible. Their efficacy and mechanisms of action against hyperuricemia have been increasingly reported in recent years. Traditional fermented foods are rich in various microorganisms, including lactic acid bacteria, yeast, and Aspergillus, representing a vast reservoir of probiotic strains. Traditional fermented foods in my country, such as jiangshui (rice soup) and kefir grains, are rich in beneficial microorganisms, particularly lactic acid bacteria, offering the potential for isolating natural probiotic strains with superior properties.

[0004] Chinese patent document CN 113215012A (application number: 202010932017.6) discloses a probiotic strain, composition and application for lowering blood uric acid. The Lactobacillus reuteri disclosed in the patent document can degrade nucleotides and nucleosides, the precursors of uric acid, and has the effect of lowering the serum uric acid level in rats with hyperuricemia, but there is no report on inhibiting the activity of xanthine oxidase.

[0005] Although there are reports in the prior art that lactic acid bacteria have the effect of lowering uric acid, the effects vary significantly depending on the strain. Therefore, it is of great significance to research and develop uric acid-lowering probiotic strains with excellent characteristics. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides probiotics with uric acid-lowering activity, a composite probiotic agent and applications thereof.

[0007] The technical solutions of the present invention are as follows:

[0008] A Lentilactobacillus buchneri strain ZD62 was deposited in the China Center for Type Culture Collection on March 14, 2024, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M2024469.

[0009] A composition comprising the above-mentioned Lentilactobacillus buchneri strain ZD62.

[0010] A composite probiotic comprising Lentilactobacillus buchneri strain ZD62 and Bacillus fermentans ZD103.

[0011] Use of the above-mentioned Lentilactobacillus buchneri strain ZD62, composition or composite probiotic in any of the following:

[0012] (1) Use in the preparation of drugs for preventing and / or treating hyperuricemia.

[0013] (2) Application in the preparation of drugs for preventing and / or treating gout.

[0014] (3) Use in the preparation of drugs for preventing and / or treating inflammation caused by hyperuricemia.

[0015] (4) Use in the preparation of drugs for preventing and / or treating liver damage caused by hyperuricemia.

[0016] (5) Use in the preparation of drugs for preventing and / or treating kidney damage caused by hyperuricemia.

[0017] (6) Application in the preparation of XOD inhibitors.

[0018] Use of Bacillus fermentans ZD103 in any of the following:

[0019] (1) Use in the preparation of drugs for preventing and / or treating hyperuricemia.

[0020] (2) Application in the preparation of drugs for preventing and / or treating gout.

[0021] (3) Use in the preparation of drugs for preventing and / or treating inflammation caused by hyperuricemia.

[0022] (4) Use in the preparation of drugs for preventing and / or treating liver damage caused by hyperuricemia.

[0023] (5) Use in the preparation of drugs for preventing and / or treating kidney damage caused by hyperuricemia.

[0024] (6) Application in the preparation of XOD inhibitors.

[0025] The invention relates to the use of the above-mentioned Lentilactobacillus buchneri strain ZD62 or fermentative mucin Bacillus ZD103 in producing 1-hydroxy-2-naphthoic acid, 3-indoleacrylic acid and / or indolepyruvic acid.

[0026] A composition comprising 1-hydroxy-2-naphthoic acid, 3-indoleacrylic acid and / or indolepyruvic acid.

[0027] Preferably according to the present invention, the above composition is a XOD inhibitor composition.

[0028] Use of 1-hydroxy-2-naphthoic acid, 3-indoleacrylic acid, indolepyruvic acid or the above combination in any of the following:

[0029] (1) Use in the preparation of drugs for preventing and / or treating hyperuricemia.

[0030] (2) Application in the preparation of drugs for preventing and / or treating gout.

[0031] (3) Application in the preparation of XOD inhibitors.

[0032] The beneficial effects of the present invention include at least the following:

[0033] 1. The Lactobacillus buchneri ZD62 and the fermentative Bacillus mucinum ZD103 provided by the present invention have good uric acid-lowering properties. ZD62 and ZD103 can respectively achieve a degradation rate of 100% for inosine, guanosine, and adenosine at a concentration of 0.5 g / L at 30 min and 90 min, and degrade them into corresponding hypoxanthine, guanine, and adenine.

[0034] 2. The Lactobacillus buchneri ZD62 and Bacillus fermentans ZD103 provided by the present invention have good tolerance to artificial intestinal fluid, and their nucleoside degradation capabilities can be maintained at more than 50% under artificial intestinal fluid conditions. Among them, the ZD62 strain has better tolerance and its nucleoside degradation capability is almost unaffected.

[0035] 3. The metabolites of Lactobacillus buchneri ZD62 and Bacillus fermentans ZD103 provided by the present invention have a significant inhibitory effect on xanthine oxidase (XOD), and the effective ingredients that exert the inhibitory effect are acids present in the extracellular metabolites of the strains. Among them, the inhibition rates of 1-hydroxy-2-naphthoic acid, 3-indoleacrylic acid, and indolepyruvic acid in the metabolites on XOD are 98.66±1.63%, 53.87±1.38%, and 20.40±0.71%, respectively, which can effectively inhibit the activity of XOD and reduce the conversion of purine to uric acid.

[0036] 4. The Lactobacillus buchneri ZD62 and Bacillus fermentans ZD103 provided by the present invention have good indolepyruvate production capabilities, reaching 758±8.1 mg / L and 213±9.8 mg / L, respectively.

[0037] 5. The in vivo uric acid-lowering ability of Lactobacillus buchneri ZD62 and Bacillus fermentans ZD103 provided by the present invention was verified by constructing a hyperuricemia mouse model. The test results showed that in addition to a significant increase in serum uric acid levels in the model mice, there were also kidney damage, inflammatory response, and down-regulation of uric acid transporter gene transcription levels. After intervention treatment with ZD62 and ZD103, the serum uric acid levels of the hyperuricemia mice decreased significantly, kidney damage was alleviated, inflammatory factors were reduced, and ZD62 and ZD103 were used to treat the hyperuricemia mice. 2. ZD103 promotes uric acid excretion by regulating the expression of the ABCG2 gene. In addition, ZD62 and ZD103 also changed the intestinal microbial composition of hyperuricemia mice, thereby increasing the overall microbial abundance in the mouse cecum, increasing the relative abundance of Firmicutes at the phylum level, decreasing the relative abundance of Bacteroidetes, and greatly increasing the proportion of Lactobacillus at the genus level. Therefore, ZD62 and ZD103 can achieve the purpose of alleviating hyperuricemia by improving multiple physiological indicators.

[0038] 6. The Lactobacillus buchneri ZD62 and Bacillus fermentans ZD103 provided by the present invention are lactic acid bacteria isolated from fermented food syrup in Northwest China and kefir grain yogurt in Tibet. They are isolated from food substances and have good safety performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the liquid chromatography retention time diagram of inosine, guanosine and adenosine.

[0040] Figure 2 The standard curves of inosine (a), guanosine (b), and adenosine (c) are shown.

[0041] Figure 3 The colony and bacterial morphology of strains ZD62 and ZD103;

[0042] (a) ZD62 colony image; (b) ZD62 Gram microscopy image; (c) ZD62 scanning electron microscopy image; (d) ZD103 colony image; (e) ZD103 Gram microscopy image; (f) ZD103 scanning electron microscopy image.

[0043] Figure 4 This is a diagram of nucleoside degradation by ZD62 and ZD103;

[0044] (a) Degradation of mixed nucleosides by ZD62; (b) Degradation of mixed nucleosides by ZD103; (c) Degradation of single nucleosides by ZD62; (d) Degradation of single nucleosides by ZD103.

[0045] Figure 5 This is the peak diagram of the solution without adding probiotic nucleoside;

[0046] Figure 6 Peak diagram of adding ZD62 nucleoside solution;

[0047] Figure 7 Peak diagram of adding ZD103 nucleoside solution;

[0048] above Figure 5 、 Figure 6 、 Figure 7 Chinese: 1. Hypoxanthine 2. Inosine 3. Guanosine 4. Adenine 5. Adenosine 6. Guanine.

[0049] Figure 8 The graphs show the degradation rates of three nucleosides by ZD62 (a) and ZD103 (b) under artificial intestinal fluid conditions;

[0050] In the figure: a is the degradation diagram of three nucleosides by ZD62 under artificial intestinal fluid conditions; b is the degradation diagram of three nucleosides by ZD103 under artificial intestinal fluid conditions.

[0051] Figure 9Graph showing the effects of metabolites on XOD;

[0052] In the figure: (a) Effect of ZD62 and ZD103 metabolites on XOD; (b) Effect of intracellular and extracellular metabolites of ZD62 and ZD103 on XOD; (c) Effect of enzyme inactivation treatment of extracellular metabolites of ZD62 and ZD103 on XOD; (d) Effect of extracellular metabolites of ZD62 and ZD103 on XOD after pH adjustment.

[0053] Figure 10 are the inhibition curves of different metabolites;

[0054] In the figure: (a) 1-hydroxy-2-naphthoic acid; (b) 3-indoleacrylic acid; (c) indolepyruvic acid.

[0055] Figure 11 Figure 2 shows the serum uric acid levels of mice in different treatment groups.

[0056] Figure 12 Figure 2 is the content of urea nitrogen (BUN) and creatinine (CRE) in the serum of mice in different treatment groups.

[0057] Figure 13 The graph shows the activity of XOD in the liver of mice in different treatment groups.

[0058] Figure 14 The graph shows the changes in the concentration of inflammatory factors in the serum of mice in different treatment groups.

[0059] Figure 15 Figure 2 shows paraffin sections of the liver of mice in different treatment groups.

[0060] Figure 16 The kidney morphology and paraffin sections of mice in different treatment groups.

[0061] Figure 17 Figure 2 shows the relative expression of ABCG2 transporter in the jejunum and kidney of mice in different treatment groups. DETAILED DESCRIPTION

[0062] The technical solution of the present invention will be further described below in conjunction with embodiments, but the scope of protection of the present invention is not limited thereto.

[0063] The contents not described in detail in the examples are based on the prior art in this field.

[0064] Lentilactobacillus buchneri strain ZD62 is abbreviated as Lactobacillus buchneri ZD62 or ZD62.

[0065] Fermentobacillus mucosa ZD103 can be purchased from the School of Bioengineering, Qilu University of Technology.

[0066] Example

[0067] The isolation, identification and performance test of lactic acid bacteria include the following:

[0068] 1 Materials and Methods

[0069] 1.1 Isolation of lactic acid bacteria from traditional fermented foods

[0070] After homogenizing the slurry, perform a gradient dilution. Yogurt is directly diluted in a gradient dilution. Tibetan kefir grains are inoculated into whole-fat sterilized milk at a ratio of 1:10. The mixture is allowed to stand at 25°C for 24 hours. The fermented kefir grains are then rinsed with sterile water and re-inoculated into the milk according to the appropriate ratio. Continue the incubation process as described above. Take 100 mg of kefir grains and grind thoroughly in a sterile centrifuge tube. Add 400 μL of sterile saline solution, mix thoroughly, and perform a gradient dilution.

[0071] 1.2 Screening of highly efficient nucleoside-degrading lactic acid bacteria

[0072] The preserved strain was activated in MRS liquid medium and cultured at 37°C, 180 rpm for 24 hours. The strain was adjusted to an OD600 of 1, then inoculated with a 2% inoculum in MRS liquid medium and cultured at 37°C, 180 rpm for 48 hours. Two milliliters of the culture medium were collected by centrifugation at 4°C, 8000 rpm for 5 minutes, and the collected cells were resuspended and washed with sterile 0.9% NaCl aqueous solution, and then centrifuged at 4°C, 8000 rpm for 5 minutes to collect the cells. The cells were washed twice. 750 μL of the reaction solution was added to the washed cells, resuspended, and then incubated at 37°C, 180 rpm for 1 hour. At the end of the reaction, the reaction solution was quickly heated at 100°C for 5 minutes to terminate the reaction. The supernatant was then centrifuged at 4°C, 12000 rpm for 2 minutes, and the supernatant was collected. The supernatant was filtered through a 0.22-micron microporous filter membrane, and the contents of the three nucleosides were determined by HPLC. The chromatographic conditions were as follows: column Aligent ZorbaxSB-C18, column temperature 35°C, flow rate 1 mL / min, injection volume 10 μL, detection wavelength 260 nm, mobile phase V (water): V (chromatographic methanol) = 95:5, pH 4.7.

[0073] The degradation rates of the strains for the three nucleosides were calculated according to the formula.

[0074] Degradation rate (%) = (C mother liquor - C residue) / C mother liquor × 100%

[0075] Where C is the mass concentration (g / L)

[0076] 1.3 Identification of highly efficient nucleoside-degrading strains

[0077] The strain was inoculated into MRS broth, cultured for 48 h, diluted and spread, and examined by Gram staining.

[0078] The fermentation broth was centrifuged to collect the bacteria, which were fixed with 2.5% glutaraldehyde (4°C, 4 h). After fixation, the bacteria were washed three times with physiological saline, and dehydrated with 30%, 50%, 70%, 90% and anhydrous ethanol respectively. Finally, the water was replaced with isoamyl acetate for 20 min. The treated bacteria were placed in a desiccator for drying and then placed on the sample stage of a scanning electron microscope, gold sputter-coated, and observed and analyzed under a high vacuum environment.

[0079] Preliminary identification was conducted using a combination of pentasaccharide fermentation experiments and the Bergey Manual of Bacterial Identification. The genome was extracted using a bacterial genomic DNA extraction kit, and the full-length 16S rDNA gene sequence was amplified using the universal bacterial primer 27F-1492R. Sequencing was performed by Shanghai Sangon Biotechnology Co., Ltd. The obtained 16S rDNA sequence was compared with known sequences from the NCBI website, and a phylogenetic tree was constructed using MEGA 7.0 software.

[0080] 1.4 Degradation of nucleosides by strains and determination of their degradation products

[0081] The activated strains were reacted with 1.5 g / L inosine, 1.5 g / L adenosine, and 0.7 g / L guanosine at 37°C and 180 rpm for 2 hours. Samples were taken at 10-minute intervals to determine the lactic acid bacteria's ability to degrade nucleosides. The degradation products of the three nucleosides were also determined. The maximum solubility of guanosine in water used in this experiment was 0.7 g / L. Inosine and guanosine were purchased from Shanghai MacLean Biochemical Technology Co., Ltd., and adenosine was purchased from Beijing Coolbo Technology Co., Ltd.

[0082] 1.5 Determination of the ability of strains to degrade three nucleosides under artificial intestinal fluid conditions

[0083] To prepare artificial intestinal fluid: Weigh 0.68g of potassium dihydrogen phosphate and dissolve it in 50mL of water. Adjust the pH to 6.8 with 0.1M sodium hydroxide solution. Dissolve 1g of trypsin in an appropriate amount of water. Mix the two solutions and dilute to 100mL with water. Weigh 0.5g each of inosine, guanosine, and adenosine powder and dissolve them in the artificial intestinal fluid to prepare an artificial intestinal fluid solution containing mixed nucleosides.

[0084] The activated ZD62 strain was reacted with a mixed nucleoside reaction solution dissolved in artificial intestinal fluid at 37°C and 180 rpm for 60 min, and the ZD103 strain was reacted for 120 min. After the reaction, the mixture was terminated by boiling water for 5 min, centrifuged at 4°C and 12,000 rpm for 2 min, and the supernatant was collected. The supernatant was filtered through a 0.22-μm microporous filter membrane, and the contents of the remaining three nucleosides were determined by HPLC. A nucleoside solution dissolved in ultrapure water was used as a control.

[0085] 1.6 Obtaining strain metabolites

[0086] ZD62 and ZD103 strains stored in the refrigerator were inoculated at a 2% volume ratio into 20 mL of MRS broth and cultured to an OD600 of 1. The culture was then transferred to 20 mL of MRS broth at a 2% volume ratio. 20 mL of the second-generation logarithmic growth phase culture medium was placed in a centrifuge tube and centrifuged at 8000 rpm and 4°C for 10 min. The supernatant was filtered through a 0.22 μm sterile filter to obtain extracellular metabolites of lactic acid bacteria. The cell pellet, after discarding the supernatant, was washed twice with pH 7.4 PBS buffer, resuspended in 20 mL of pH 7.4 PBS buffer, and disrupted using an ultrasonic cell disruptor. MRS broth was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0087] Ultrasonication conditions: Pulse disruption at 200 W (5 s on, 5 s off) for 20 min in an ice bath. The sonicated liquid was centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was collected and filtered through a 0.22 μm sterile filter to obtain the intracellular metabolites of lactic acid bacteria. A 100 μL sample of metabolites was transferred to an EP tube, and 400 μL of the extract (methanol:acetonitrile = 1:1 (v / v) containing an isotope-labeled internal standard mixture) was added. The mixture was vortexed for 30 s, sonicated in an ice bath for 10 min, and allowed to stand at 40°C for 1 h. The sample was centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was separated by chromatography on a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) column using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph. Phase A of the liquid chromatography was an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia water, and phase B was acetonitrile. The sample tray temperature was 4°C, and the injection volume was 2 μL.

[0088] 1.7 Determination of the inhibitory effect of metabolites on XOD

[0089] X stands for xanthine, P stands for PBS, O stands for XOD, and M stands for metabolite.

[0090] The experimental design was as follows: the blank group (XP) consisted of xanthine (800 μL) + PBS (200 μL, pH 7.4); the control group (XPO) consisted of xanthine (800 μL) + PBS (80 μL, pH 7.4) + XOD (120 μL); and the experimental group (XMO) consisted of xanthine (800 μL) + metabolites (80 μL) + XOD (120 μL). XOD and metabolites / PBS were mixed and reacted at 37°C for 30 minutes. Xanthine was then added, mixed thoroughly, and reacted at 37°C for 1 hour. After the reaction, the reaction tube was incubated at 95°C for 5 minutes to inactivate the enzyme. The remaining xanthine content in the system was determined by HPLC. To further determine the location of the metabolites and the active ingredient(s) responsible for their inhibitory effects, the metabolites were separated into intracellular and extracellular compartments, the enzyme was inactivated at high temperature, and the pH was adjusted.

[0091] 1.8 Molecular docking

[0092] Molecular docking can predict intermolecular binding patterns by studying intermolecular interactions. Therefore, it is widely used to analyze the binding mechanisms of small molecule ligands and protein interactions and to screen inhibitors. The 3D structure of XOD was downloaded from the PDB database (https: / / www.rcsb.org / ), and the structures of acidic substances were downloaded from the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform. The catalytic center of XOD consists of a molybdenum pterin center, two iron-sulfur centers, and a flavin adenine dinucleotide. The molybdenum pterin center is the key site for xanthine oxidase to catalyze the conversion of xanthine to uric acid. Using Arg880 of the molybdenum pterin center as the center, XOD was docked and scored with acids from the extracellular metabolites of Lactobacillus buchneri ZD62 and Lactobacillus mucilaginosus fermentans ZD103.

[0093] 1.9 Verification of XOD Inhibition Effect

[0094] To verify the metabolites screened by molecular docking, 1 g / L solutions of 1-hydroxy-2-naphthoic acid, 3-indoleacrylic acid, and indolepyruvic acid were prepared and diluted to different concentrations. 1-Hydroxy-2-naphthoic acid, 3-indoleacrylic acid, and indolepyruvic acid were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Referring to the method in 1.7, the metabolites were replaced with different concentrations of 1-hydroxy-2-naphthoic acid, 3-indoleacrylic acid, and indolepyruvic acid to verify the XOD inhibition effect.

[0095] Inhibition rate (%) = (experimental group - control group) / (blank group - control group) × 100%

[0096] 1.10 Determination of indolepyruvic acid content - High performance liquid chromatography (HPLC) method

[0097] Inoculate 2% by volume of ZD62, ZD103, and other lactic acid bacteria strains stored in the refrigerator into 20 mL of MRS broth. Cultivate to an OD600 of 1. Then, inoculate 2% by volume into 20 mL of MRS broth and incubate at 37°C, 180 rpm, and 48 h. Extract indolepyruvate using the same method as described in 1.6 for obtaining metabolites from these strains.

[0098] Indolepyruvic acid was measured by HPLC using an Agilent 1260 Series HPLC (Agilent Technologies, USA) equipped with a UV-visible diode array detector. The column was a ZORBAX SB-18 (5 μm, 4.6 × 150 mm), and the column oven was set at 35°C throughout the separation. The mobile phase consisted of 0.03% potassium dihydrogen phosphate solution and methanol. The injection volume was 10 μL, and the detector wavelength was set at 280 nm. 1.11 Animal Experiments

[0099] 1.11.1 Animal model construction

[0100] Because gout is primarily diagnosed in men, male Kunming mice were used in this study. A hyperuricemia model was established by feeding a diet containing 4% potassium oxonate and 4% uric acid. Natural ventilation, free access to food and water, and regular bedding changes were used.

[0101] 1.11.2 Animal Experiment Design

[0102] All mice were fed a commercial basal diet for one week and then randomly divided into seven groups of five mice each. During the experiment, the mice's food and water intake were not restricted by any factors. During the modeling period, one group was fed only the basal diet, while the other six groups were fed a high-uric acid mouse diet containing 4% potassium oxonate and 4% uric acid. Serum uric acid levels in the mice were measured after two consecutive weeks of modeling. Intervention treatment began in the third week. Except for the first group (the blank group), which continued to be fed the basal diet, the other five groups received the following interventions:

[0103] The second group (ZD62) was gavaged with 1x10 8 CFU / mL of Lactobacillus buchneri ZD62 0.5mL;

[0104] The third group (ZD103) was gavaged 1x10 8 CFU / mL of Lactobacillus fermentum ZD103 0.5mL;

[0105] The fourth group (ZD62+ZD103) was given an oral administration of 1x10 8 0.5 mL of ZD62+ZD103 bacterial suspension with CFU / mL, the ratio of ZD62 to ZD103 was 1:1;

[0106] Group 5 (allopurinol) was orally administered with allopurinol (5 mg / kg);

[0107] The sixth group (model group) was gavaged with normal saline;

[0108] All gavage treatments were performed once a day.

[0109] At the conclusion of the animal experiment, in accordance with relevant animal ethics regulations, the inventors anesthetized all mice with 1% sodium pentobarbital, collected blood by enucleation, and sacrificed them simultaneously. Fresh blood samples were incubated at 37°C for 1 hour and then centrifuged at 3000 rpm for 10 minutes to obtain mouse serum. Liver, kidney, intestinal, and serum specimens were collected in sterile PE tubes. Tissue sections were preserved in 4% paraformaldehyde, and the remaining samples were stored at -80°C until further use.

[0110] 1.11.3 Analysis of mouse blood and tissue physiological indicators

[0111] Mouse liver and kidney fixed overnight in 4% paraformaldehyde were embedded in paraffin and stained with hematoxylin and eosin (H&E). The tissue morphology was observed using a light microscope.

[0112] ELISA kits were used to detect the anti-oxidative inflammation-related factors IL-18, IL-1β and TNF-α, a uric acid detection kit was used to detect the serum uric acid of mice, and serum creatinine (CRE) and blood urea nitrogen (BUN) detection kits were used to detect the levels of CRE and BUN in the mouse serum.

[0113] Freshly frozen mouse livers were weighed and homogenized with 9 volumes of normal saline at a ratio of 1:9. Liquid nitrogen was added and the mixture was uniformly ground using a handheld grinder. The supernatant was collected by low-speed centrifugation at 3000 rpm for 10 minutes. Xanthine oxidase (XOD) levels in the livers of mice in different groups were measured using an XOD assay kit produced by the Nanjing Jiancheng Bioengineering Institute, strictly following the manufacturer's instructions.

[0114] 1.11.4 Analysis of urate transporter-related gene expression levels in mouse kidney and intestine

[0115] (1) Design and synthesis of primers for uric acid transporter-related genes

[0116] Upstream and downstream primers for the ABCG2 gene encoding the urate transporter and the standard internal reference β-actin were designed using the NCBI website and used for real-time fluorescence quantitative PCR (qPCR) amplification. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0117] Table 1 Primer sequences

[0118]

[0119] (2) Extraction and detection of mRNA in mouse kidney and intestinal tissues

[0120] Mouse kidneys and intestines frozen at -80°C were used to extract RNA from the kidneys and intestines of mice in different treatment groups strictly according to the instructions of the FastPure Cell / Tissue Total RNA Isolation Kit-BOX2 Kit (Nanjing Novozymes Biotech Co., Ltd.). The A260 / A280 of the extracted RNA was measured using an ultramicro spectrophotometer to determine the concentration and purity of the RNA.

[0121] (3) RNA reverse transcription

[0122] The extracted RNA was reverse transcribed into cDNA according to the operating procedures provided by the HiScript III RT SuperMix for qPCR (+gDNAwiper) (R323) kit of Nanjing Novozymes Biotechnology Co., Ltd. The reverse transcription reaction system is as follows (see Table 2):

[0123] Table 2 Reverse transcription system

[0124]

[0125] (4) Real-time fluorescence quantitative PCR

[0126] The obtained cDNA was used as a template for qPCR amplification using the corresponding primers. The reaction system is shown in Table 3, and the qPCR reaction procedure is shown in Table 4.

[0127] Table 3 qPCR reaction system

[0128]

[0129]

[0130] Table 4 qPCR reaction procedure

[0131]

[0132] All samples were tested in triplicate, and β-actin gene was used as an internal reference. -ΔΔCt The relative expression of genes was analyzed by the method. The calculation formula is as follows: ΔCt = Ct target gene - Ct internal reference gene; ΔΔCt = ΔCt target gene in experimental group - ΔCt target gene in control group; relative expression = 2 -ΔΔCt .

[0133] 1.11.5 Analysis of mouse intestinal microbial diversity

[0134] Microbial genomic DNA from mouse cecal contents was extracted using the HiPure Soil DNA Extraction Kit (Magen, Guangzhou, China). The V3-V4 region of the 16s rDNA gene was amplified by PCR using primers 341F and 806R. Amplicon quality was assessed using a 2% agarose gel, PCR product purification was performed using AMPure XP Beads (Beckman, CA, USA), and quantification was performed using Qubit 3.0. Sequencing libraries were constructed using the Illumina DNA Prep Kit (Illumina, CA, USA). Raw data from the Illumina platform were filtered using FASTP (version 0.18.0), and clean reads were used for analysis. Primer sequences were: 341F: CCTACGGGNGGCWGCAG SEQ ID NO. 7; 806R: GGACTACHVGGGTATCTAAT SEQ ID NO. 8.

[0135] 1.11.6 Data Analysis

[0136] All experimental data were expressed as mean ± standard deviation (SD). One-way ANOVA (single-way analysis of variance) was used to analyze and compare the groups to obtain significant differences, and GraphPad software was used for plotting.

[0137] 2 Test results

[0138] 2.1 Establishment of standard curves for three nucleosides

[0139] Under the established liquid phase conditions, the retention time of inosine is 4.334 min, guanosine is 4.690 min, and adenosine is 11.705 min. The three nucleosides can be well separated under this liquid phase condition. Figure 1 , and showed a good linear relationship between the concentration of 0.1-0.5g. Figure 2 .

[0140] 2.2 Screening of efficient nucleoside-degrading strains

[0141] A total of 52 bacterial strains were isolated and purified from various traditional fermented foods. Six strains showed inosine degradation rates exceeding 30%, and two strains showed rates exceeding 60%. Six strains showed rates of guanosine degradation exceeding 30%, and two strains showed rates exceeding 60%. Twelve strains showed rates of adenosine degradation exceeding 30%, and three strains showed rates exceeding 60%. Among them, strain ZD62 was able to reduce the concentration of three nucleosides at a concentration of 0.5 g / L to 0 g / L in 60 minutes, achieving a degradation rate of 100%. Strain ZD103 also showed rates of over 60% for all three nucleosides at a concentration of 0.5 g / L in 60 minutes. Subsequently, strains ZD62 and ZD103, which demonstrated significant advantages in nucleoside degradation, were selected for further study.

[0142] 2.3 Identification of highly efficient nucleoside-degrading strains

[0143] On the MRS plate, ZD62 and ZD103 colonies were round, milky white, with complete edges and smooth surfaces ( Figure 3 Middle a, Figure 3 (d) ZD62 Gram staining showed purple ( Figure 3 b), ZD103 Gram staining showed blue-purple ( Figure 3 e), both strains are Gram-positive bacteria. ZD62 and ZD103 are short rods under a scanning electron microscope, so the two strains are judged to be Gram-positive rods ( Figure 3 Middle C, Figure 3(f) Sugar fermentation experiments showed that strain ZD62 could ferment maltose, sucrose, and raffinose to produce acid, while strain ZD103 could ferment maltose, sucrose, raffinose, inulin, and lactose to produce acid. Based on the Bergey's Manual of Bacterial Identification, both strains ZD62 and ZD103 were determined to be Lactobacillus species. The 16S rDNA sequence of strain ZD62 is shown in SEQ ID NO. 1, and the 16S rDNA sequence of strain ZD103 is shown in SEQ ID NO. 2. A BLAST comparison of the 16S rDNA sequences of both strains into the NCBI database revealed that strain ZD62 shared 100% homology with Lentilactobacillus buchneri strain JCM 1115. Strain ZD103 shared 99.78% homology with Limosilactobacillus fermentum strain CIP 102980. In the phylogenetic tree constructed with MEGA7 software, strain ZD62 and Lactobacillus buchneri belonged to the same branch and were most closely related. Therefore, the strain was identified as a Lactobacillus buchneri strain and named Lentilactobacillus buchneri strain ZD62, abbreviated as Lactobacillus buchneri ZD62 or ZD62. ZD103 and Lactobacillus fermentum belonged to the same branch and were most closely related. Therefore, the strain was identified as Lactobacillus fermentum and named Lactobacillus fermentum ZD103, abbreviated as ZD103.

[0144] Lentilactobacillus buchneri strain ZD62 was deposited in the China Center for Type Culture Collection on March 14, 2024, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M 2024469.

[0145] 2.4 Degradation of nucleosides by strains and determination of degradation products

[0146] Depend on Figure 4 It can be seen that Lactobacillus buchneri ZD62 can reduce the concentration of the three mixed nucleosides with a concentration of 0.5g / L to 0g / L within 30min ( Figure 4 a); fermentation of Lactobacillus mucilaginosus ZD103 can reduce the concentration of three mixed nucleosides with a concentration of 0.5g / L to 0g / L at 90min ( Figure 4(b) During the reaction between ZD62 and ZD103 and the mixed nucleoside solution, the two strains simultaneously reduced the concentrations of inosine, guanosine, and adenosine, with no order of precedence. When the concentrations of individual nucleosides were increased, it was found that Lactobacillus buchneri ZD62 could reduce the concentrations of inosine and adenosine, both at 1.5 g / L, to 0 g / L within 30 minutes, and completely degrade guanosine, at a concentration of 0.7 g / L, in just 10 minutes. Figure 4 c). The fermentation of Lactobacillus mucilaginosus ZD103 can completely degrade the concentration of inosine with a concentration of 1.5g / L in 100min, completely degrade the concentration of guanosine with a concentration of 0.7g / L in 40min, and completely degrade the concentration of adenosine with a concentration of 1.5g / L in 110min ( Figure 4 (d)

[0147] Depend on Figure 5 、 Figure 6 、 Figure 7 ( Figure 5 、 Figure 6 、 Figure 7 (In Chinese: 1. Hypoxanthine 2. Inosine 3. Guanosine 4. Adenine 5. Adenosine 6. Guanine) It can be seen that after ZD62 and ZD103 reacted with the three nucleosides respectively, three new products appeared as the peak areas of the three nucleosides decreased. HPLC verification confirmed that they were hypoxanthine, guanine, and adenine, respectively. This shows that the mechanism by which ZD62 and ZD103 reduce purine nucleosides is to convert them into the corresponding purine bases. Purine bases are less easily absorbed by the intestine than purine nucleosides. Therefore, ZD62 and ZD103 can reduce uric acid production by indirectly reducing the absorption of purine nucleosides by the intestine.

[0148] 2.5 Tolerance of highly efficient nucleoside-degrading strains to artificial intestinal fluid

[0149] The small intestine is the place where lactic acid bacteria can degrade nucleosides. The intestinal fluid in the small intestine can have an adverse effect on the activity of lactic acid bacteria. Therefore, the inventors tested the degrading nucleosides ability of strains ZD62 and ZD103 in artificial intestinal fluid. Figure 8 As shown in Figure 2, the degradation rates of inosine, guanosine, and adenosine by strain ZD62 in artificial intestinal fluid and aqueous solution were basically the same ( Figure 8 In a), the ZD62 strain can also efficiently degrade the three nucleosides in artificial intestinal fluid; the ZD103 strain has a significantly reduced ability to degrade nucleosides in artificial intestinal fluid, but the degradation rate of the three nucleosides is still maintained above 50% ( Figure 8 (b) indicates that both strains have a certain tolerance to artificial intestinal fluid, but ZD62 has better tolerance. In summary, both ZD62 and ZD103 can be used as high-quality bacterial strain resources for the development of probiotic products for the prevention and treatment of hyperuricemia.

[0150] 2.6 Determination of the inhibitory effect of metabolites on XOD

[0151] like Figure 9 As shown in Figure a, the xanthine concentration in the XPO group was significantly lower than that in the blank group containing only xanthine, indicating that XOD has good enzyme activity under these reaction conditions. The xanthine concentration in the XMO group of the ZD62 and ZD103 strains was significantly higher than that in the XPO group, indicating that the metabolites of both strains inhibited the activity of XOD; the extracellular metabolites of both strains significantly inhibited the activity of XOD, but the intracellular metabolites of both strains had no significant inhibitory effect on XOD ( Figure 9 b) indicates that the metabolites that inhibit XOD exist outside the cells of the strains; the extracellular metabolites of the two strains have an inhibitory effect on XOD regardless of whether the enzyme is inactivated ( Figure 9 c), it is inferred that the inhibition of XOD by the extracellular metabolites of the strains is not caused by enzymes; the extracellular metabolites of the two strains were neutralized with sodium hydroxide to adjust the pH to 7, and it was found that the neutralized extracellular metabolites lost their inhibitory effect on XOD ( Figure 9 In (d), we conclude that the inhibition of XOD by ZD62 and ZD103 may be due to the acids present in their extracellular metabolites. In summary, we preliminarily conclude that the inhibitory effect on XOD is due to the acids present in the extracellular metabolites of the two bacterial strains.

[0152] 2.7 Molecular docking

[0153] Non-target metabolomics analysis of the fermentation broth revealed 382 substances, including fatty acids, organic acids, amino acids, and aromatic compounds, in the fermentation broth of Lactobacillus buchneri ZD62, and 387 substances, including fatty acids, organic acids, amino acids, and aromatic compounds, in the fermentation broth of Lactobacillus mucilaginosus ZD103. Screening revealed 52 acids shared by the metabolites of the two strains. These 52 acids were docked with XOD, and three substances with strong XOD interactions (a more negative value indicates a stronger interaction) were selected for validation. Based on the docking results, 1-hydroxy-2-naphthoic acid, 3-indoleacrylic acid, and indolepyruvate were ultimately selected for further validation.

[0154] According to the interaction pattern diagram between 1-hydroxy-2-naphthoic acid and XOD, 1-hydroxy-2-naphthoic acid forms a hydrogen bond interaction with the amino acid residue GLU1261 of XOD, and forms a hydrophobic interaction with the amino acid residues LEU1014 and VAL1011. According to the interaction pattern diagram between 3-indoleacrylic acid and XOD, 3-indoleacrylic acid forms a hydrogen bond interaction and a hydrophobic interaction with the amino acid residue ALA1079 of XOD. According to the interaction pattern diagram between indolepyruvic acid and XOD, indolepyruvic acid forms a hydrogen bond interaction with the amino acid residues GLY1260 and GLN1194 of XOD, and forms a hydrophobic interaction with the amino acid residue MET1038. The chemical bonds formed between these three substances and xanthine oxidase help the small molecules to play a role, thereby producing a certain promoting or inhibiting effect on the enzyme.

[0155] 2.8 Verification of XOD Inhibition Effect

[0156] It has been verified that 1-hydroxy-2-naphthoic acid, 3-indoleacrylic acid, and indolepyruvic acid have different degrees of inhibition on XOD within the concentration range of 0.1-1g / L ( Figure 10 Among them, 1-hydroxy-2-naphthoic acid reached its maximum inhibition rate at 0.8 g / L (98.66±1.63%), showing strong XOD inhibition ability; 3-indoleacrylic acid reached its maximum inhibition rate at 0.8 g / L (53.87±1.38%), and when the concentration was higher than 0.8 g / L, the inhibition rate began to decline; the inhibition rate of indolepyruvic acid on XOD showed a trend of first increasing and then decreasing, and had no inhibitory effect on XOD at concentrations below 0.5 g / L. In the concentration range of 0.5-0.9 g / L, the inhibition effect on XOD gradually increased with increasing concentration, and reached the maximum inhibition rate at 0.9 g / L (20.40±0.71%). This provides theoretical support for the conclusion that ZD62 and ZD103 metabolites inhibit XOD activity.

[0157] 2.9 Indolepyruvate production capacity of strains ZD62 and ZD103

[0158] Fermentation was carried out according to the fermentation method described in 1.6 Obtaining metabolites of strains, and the concentration of indolepyruvic acid in the fermentation broth of each strain was detected, as shown in Table 5.

[0159] Table 5 Comparison of indolepyruvate production capacity of different lactic acid strains

[0160]

[0161]

[0162] 2.10 Analysis of mouse blood and tissue physiological indicators

[0163] like Figure 11 As shown in the results, regardless of probiotic intervention or allopurinol treatment, the serum uric acid levels of mice were significantly reduced in the high uric acid model group (model group). Among them, allopurinol, which had the best effect in lowering serum uric acid, could reduce the serum uric acid content of hyperuricemic mice to 66.67±11.09μmol / L. The results of probiotic intervention showed that the uric acid-lowering effect of ZD103 fermented mucobacterium was second only to allopurinol, reducing the serum uric acid content of hyperuricemic mice to 70.59±5.55μmol / L. The ZD62 strain alone only reduced the uric acid level of hyperuricemic mice to 117.65±10.38μmol / L, but when used in combination with ZD103, the effect of lowering serum uric acid was greatly improved, and it could be reduced to 72.19±8.32μmol / L. The participation of ZD103 strain improved the effect of lowering uric acid, but because the bacterial count of ZD103 after the two strains were mixed was lower than the bacterial count when ZD103 was used alone, the uric acid lowering effect of ZD103 alone was better than that of the combined effect of the two strains.

[0164] BUN is a metabolic product of protein in the body. After being filtered through the glomeruli, it is reabsorbed in the renal tubules. CRE is an important substance produced by human muscle metabolism and mainly excreted through the glomeruli. To a certain extent, the degree of kidney damage caused by HUA can be measured by the levels of BUN and CRE in serum.

[0165] like Figure 12 As shown in the data, there was no significant difference in the serum BUN content between the model group mice and the control group mice, but there were significant differences in the serum BUN content and CRE between the allopurinol group mice and other groups. It is speculated that the use of allopurinol caused certain damage to the mouse kidneys, thereby affecting the reabsorption of BUN and the excretion of CRE by the mouse kidneys; the serum CRE content of the model group mice was significantly higher than that of the control group (P < 0.01), indicating that the excessive uric acid level in HUA mice affected the normal excretion of CRE by the glomeruli, resulting in increased CRE content. After intervention treatment, the serum CRE content of the ZD62 + ZD103 group was significantly lower than that of the model group (P < 0.001), which indicates that the combined use of ZD62 + ZD103 strains can effectively alleviate renal damage in hyperuricemia mice.

[0166] XOD in mouse liver is an important purine metabolic enzyme that forms uric acid by oxidizing xanthine and hypoxanthine. Therefore, it is necessary to detect the changes in XOD after elevated uric acid. Figure 13 As shown, except for the significantly decreased XOD activity in the liver of hyperuricemia mice in the ZD62 treatment group, there was no significant difference in XOD activity in the mice in the other treatment groups, indicating that the extracellular metabolites of the ZD62 strain can effectively inhibit the activity of XOD.

[0167] like Figure 14 As shown in the results, the levels of IL-1β (P<0.05) and TNF-α (P<0.001) in the blood of mice in the model group were significantly higher than those in the control group, and the level of IL-18 in the blood of mice in the model group was not significantly different from that in the control group, indicating that the increase in uric acid levels caused an inflammatory response in mice to a certain extent.

[0168] The combined use of ZD62 and ZD103 significantly reduced the levels of IL-1β, IL-18, and TNF-α in the serum of hyperuricemic mice. ZD103 alone reduced the levels of IL-1β and IL-18 in the serum of hyperuricemic mice. These results suggest that probiotics can alleviate the inflammatory response caused by hyperuricemia to varying degrees, and the combined use of the two strains is more effective.

[0169] like Figure 15 As shown in the data, the nuclear staining of liver cells in the model group mice deepened, indicating that a large amount of uric acid caused a certain burden on the mouse liver. The ZD62 and ZD103 strains alleviated the deepening of the nuclear staining of liver cells. This result shows that Lactobacillus buchneri ZD62 and Lactobacillus fermentans ZD103 have a certain alleviating effect on liver damage caused by high uric acid.

[0170] Renal morphology showed that the kidneys of the model group mice were atrophic. HE staining results showed that the kidneys of the model group mice had a large number of inflammatory infiltrations, loose cytoplasm, mild staining, and occasional suppurative lesions. Allopurinol group had a certain effect on alleviating the inflammatory infiltration and loose cytoplasm of the kidneys of hyperuricemia mice, but Figure 16 It can be seen that the kidneys of mice in the allopurinol group were significantly swollen, which indicates that the use of allopurinol has caused certain damage to the kidneys of mice. The ZD62, ZD103, and ZD62+ZD103 groups all significantly alleviated the inflammatory response of the kidneys of mice with high uric acid. Figure 16 It can be seen that the inflammatory infiltration of the kidneys of mice treated with ZD62, ZD103, and ZD62+ZD103 was greatly reduced, the purulent lesions disappeared, and the cytoplasmic loosening was also well relieved. Among them, the effect of the ZD103 group was the most significant. This result shows that ZD62, ZD103, ZD62+ZD103, and allopurinol treatment can relieve the renal inflammatory response of mice caused by hyperuricemia, but the use of allopurinol will cause kidney damage to mice, so the comprehensive evaluation shows that the therapeutic effect of probiotics is better than that of allopurinol.

[0171] 2.10 Analysis of ABCG2 gene transcription levels in the jejunum and kidneys of mice in each group

[0172] Compared with the control group, the relative expression of ABCG2 gene in the jejunum of the model group mice was significantly decreased (P < 0.01), indicating that the increase in serum uric acid level in the model group mice was related to the inhibition of intestinal uric acid excretion mediated by ABCG2. Figure 17 As can be seen from Figure a, after the intervention treatment with lactic acid bacteria, the expression levels of ABCG2 genes in the ZD103 group and the ZD62+ZD103 group were significantly higher than those in the model group (P < 0.05), indicating that the combined use of ZD103 strain and ZD62+ZD103 strains can improve the down-regulation of the ABCG2 gene in the jejunum of hyperuricemia mice to improve the ability of the mouse intestine to excrete uric acid, thereby reducing the uric acid level in hyperuricemia mice; Figure 17b shows that the relative expression level of ABCG2 genes in the kidneys of the model group mice was significantly lower than that of the Control group (P < 0.001), indicating that the increase in serum uric acid levels in the model group mice was partly due to the decreased expression of the transporter protein encoded by the ABCG2 gene in the kidney, which led to a decrease in the excretion of uric acid by the kidney. After different intervention treatments, the relative expression of the ABCG2 gene in the kidneys of mice in the ZD62+ZD103 group was significantly higher than that in the model group (P < 0.001). This result indicates that the combined use of ZD62 and ZD103 strains can improve the ability of the kidneys of hyperuricemia mice to excrete uric acid and achieve a therapeutic effect of lowering uric acid. The inventors also found that ZD62 and ZD103 did not significantly improve the expression of the ABCG2 gene in the kidneys of hyperuricemia mice when used alone, but the effect of combined use showed significant differences compared with either of the two groups (P < 0.001). It is inferred that the combined use of ZD62 and ZD103 is more effective in improving the excretion of uric acid in the kidneys of hyperuricemia mice.

Claims

1. One plant Lentilactobacillus buchneri strain ZD62 was deposited in the China Center for Type Culture Collection on March 14, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 2024469.

2. A composition comprising the Lentilactobacillus buchneri strain ZD62.

3. The method according to claim 1 Lentilactobacillus buchneri strain ZD62 or the use of the composition according to claim 2 in any of the following: (1) Use in the preparation of drugs for the prevention and / or treatment of hyperuricemia; (2) Use in the preparation of drugs for the prevention and / or treatment of gout; (3) Use in the preparation of drugs for preventing and / or treating inflammation caused by hyperuricemia; (4) Use in the preparation of drugs for preventing and / or treating liver damage caused by hyperuricemia; (5) Use in the preparation of drugs for preventing and / or treating kidney damage caused by hyperuricemia; (6) Application in the preparation of XOD inhibitors.

4. The method according to claim 1 Lentilactobacillus buchneri strain Application of ZD62 in producing 1-hydroxy-2-naphthoic acid, 3-indoleacrylic acid and / or indolepyruvic acid.

Images