Lactobacillus reuteri JNL0037 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus reuteri JNL0037 and application thereof. The invention provides lactobacillus reuteri JNL0037, which is preserved in the China General Microbiological Culture Collection Center on December 27, 2024, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO: 33218. The lactobacillus reuteri JNL0037 can be used for preparing the lactobacillus reuteri JNL0037. The Lactobacillus reuteri JNL0037 provided by the invention has nucleoside degradation, anti-inflammatory and bacteriostatic capabilities, can be used for preparing drugs for treating hyperuricemia, gout and uric acid nephropathy, has extremely high development value and practical value, and provides a new idea for development and application of related products.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a strain of Limosilactobacillus reuteri JNL0037 and its application. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily to be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Hyperuricemia is a metabolic disease caused by purine metabolism disorders and uric acid excretion imbalance. It not only directly causes gout, but also leads to a series of serious health problems such as kidney and cardiovascular diseases. Currently, clinically, when the fasting blood uric acid of adults is > 420 µmol / L (micromoles per liter) on two non-consecutive days, it is defined as hyperuricemia. When the concentration of urate in the blood exceeds its saturation in the blood, urate will precipitate out of crystals and deposit in joints, soft tissues, cartilage, kidneys and other parts. These crystals can induce an inflammatory response, thus triggering gout. Up to 36% of hyperuricemia patients will develop gout. It has been found that among individuals with a blood uric acid level ≥ 600 μmol / L (about 10 mg / dL), approximately 50% of people will develop clinically obvious gout within 15 years. Therefore, hyperuricemia is the pathogenesis basis of gout. In addition, hyperuricemia also leads to a series of serious health problems such as kidney and cardiovascular diseases. Currently, hyperuricemia has become the second largest metabolic disease in the world. With the continuous increase in the number of hyperuricemia patients, the global demand for uric acid-lowering products is rapidly expanding.

[0004] Currently, clinically, drug treatment combined with strict diet management is generally used to regulate blood uric acid levels. However, more than 30% of gout patients are difficult to achieve the ideal uric acid control target through current drugs (including febuxostat and allopurinol, etc.). Moreover, these drugs have large side effects and are not suitable as a daily regimen for long-term control of uric acid levels. On the other hand, a strict low-purine diet plan will significantly reduce the quality of life of patients, resulting in poor patient compliance. In view of this, the market urgently needs a new, safe and effective uric acid-lowering product to meet the broad health needs.

[0005] Studies have shown that the gut microbiota of gout patients is significantly different from that of healthy people in terms of composition and functional structure. These different microbiota structures can induce chronic systemic inflammation, leading to increased purine synthesis and weakened uric acid metabolism, thus causing elevated blood uric acid and gout attacks.

[0006] Recent studies have shown that probiotics can reduce blood uric acid levels through multiple pathways: ① inhibiting the activity of xanthine oxidase (XOD), a key enzyme in uric acid synthesis; ② competing with intestinal epithelial cells for the absorption of nucleoside substances, degrading purine substances in the intestine through the purine nucleosidase produced by itself, and reducing the production of uric acid; ③ contributing to the restoration of the balance of the intestinal flora, increasing the number of beneficial bacteria and reducing harmful bacteria, thereby reducing the inflammatory response and the production of uric acid; ④ increasing the excretion of uric acid by promoting the expression of urate excretion transporters and inhibiting the expression of urate reabsorption proteins, thereby reducing the uric acid level; ⑤ enhancing the intestinal barrier, reducing intestinal permeability, preventing harmful substances from entering the blood, and thus reducing the production of inflammatory factors. At the same time, in the prior art, probiotics are mostly associated with the treatment of metabolic functions such as obesity and diabetes in the treatment or prevention of hyperuricemia. The effect of reducing blood uric acid level is not high. Summary of the Invention

[0007] In view of this, the present invention provides a strain of Limosilactobacillus reuteri JNL0037 and its applications. It has the functions of treating hyperuricemia, gout and uric acid nephropathy, and at the same time, has the function of directly degrading uric acid.

[0008] To achieve the above object, the present invention provides the following technical solutions: As the first aspect of the present invention, it lies in providing a strain of Limosilactobacillus reuteri ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037, which was deposited at the China General Microbiological Culture Collection Center on December 27, 2024. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO: 33218.

[0009] As the second aspect of the present invention, it lies in providing a microbial inoculum containing the Limosilactobacillus reuteri ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 or its ferment or its metabolite.

[0010] As the third aspect of the present invention, it lies in providing the application of the Limosilactobacillus reuteri ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 described in the first aspect or the microbial inoculum described in the second aspect in the preparation of products for treating and / or preventing hyperuricemia-related diseases.

[0011] Furthermore, the present invention provides the application of the Limosilactobacillus reuteri ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 described in the first aspect or the microbial inoculum described in the second aspect in the preparation of drugs for treating and / or preventing hyperuricemia and / or gout and / or uric acid nephropathy and / or kidney inflammation.

[0012] Preferably, the product is a drug.

[0013] Furthermore, the drug contains pharmaceutically acceptable excipients and / or adjuvants.

[0014] In some specific embodiments of the present invention, in the said product, the above-mentioned Lactobacillus reuteri Limosilactobacillus reuteri subsp. reuteri JNL0037 is alive or dead or intermittently sterilized, or in the form of lysates and / or extracts, or in the form of bacterial products or in the form of supernatant or in derivative form, and the derivative form includes: metabolites, metabiomass, probiotics, cell walls and their components, exopolysaccharides, and compounds containing immunogenic components, including: supernatant, inactivated bacteria.

[0015] Furthermore, the drug has at least one of the following functions (1) to (10): (1) Degrading guanosine; (2) Degrading uric acid; (3) Protecting renal function; (4) Inhibiting the synthesis of uric acid; (5) Inhibiting the reabsorption of uric acid by the kidney; (6) Inhibiting kidney tissue inflammation; (7) Inhibiting the activity of xanthine oxidase (XOD); (8) Inhibiting the activity of purine nucleoside phosphorylase (PNP); (9) Inhibiting the gene expression of glucose facilitative transporter-9 (GLUT9) and urate transporter 1 (URAT1); (10) Reducing the transcriptional levels of pro-inflammatory cytokines IL-6 and NF-kB in kidney tissue.

[0016] Uric acid is the end product of human purine metabolism, and guanosine, as a kind of purine substance, plays an important role in purine metabolism. Probiotics can change the metabolic pathway of guanosine in the intestine by degrading guanosine, making the generated substances less likely to be absorbed by the intestine, thus reducing the source of blood uric acid. In the intestine, probiotics compete with intestinal epithelial cells for the absorption of purine substances such as guanosine. If probiotics have a high conversion rate and removal rate of guanosine, it indicates that they have more advantages in the competition, can utilize and transform guanosine, thereby reducing the absorption of guanosine by the intestine and indirectly reducing the blood uric acid level.

[0017] Guanosine and guanine are representative purine substances. There are significant differences in the absorption rates of guanosine and guanine by the intestine. The solubility of guanosine in water is significantly higher than that of guanine, which makes the dissolution rate of guanosine in the intestine faster and it is easier to form a solution state, thus facilitating its absorption by intestinal epithelial cells. While the solubility of guanine is relatively low and its dissolution rate in the intestine is slow, which limits its absorption rate. Moreover, in Caco-2 cell monolayer experiments and animal experiments, the apparent permeability coefficient of guanosine is significantly higher than that of guanine, indicating that the intestinal absorption rate of guanosine is faster; the serum uric acid content of mice gavaged with guanosine is significantly higher than that of mice gavaged with guanine.

[0018] In one embodiment of the present invention, the guanosine conversion rate of Lactobacillus mucosae JNL0037 is 100%, indicating that it can completely degrade guanosine; the guanosine removal rate of Lactobacillus mucosae JNL0037 is 44.61%, indicating that after co-incubation of guanosine with probiotics, 55.39% is converted into guanine, and 44.61% of guanosine is degraded into substances other than guanine, which may be further degraded into downstream substances of guanine. It can be seen that Lactobacillus mucosae JNL0037 can effectively reduce the source of blood uric acid.

[0019] In one embodiment of the present invention, the uric acid degradation rate of Lactobacillus mucosae JNL0037 is 2.68%. It can be seen that this strain can directly degrade uric acid.

[0020] Approximately one-third of the uric acid produced by the body is excreted into the intestine, and two-thirds is excreted through the kidneys via urine. The present invention provides intestinal bacteria with the ability to degrade uric acid, which can degrade uric acid in the intestine, reduce uric acid accumulation and reabsorption.

[0021] The results of the embodiments of the present invention show that Lactobacillus mucosae JNL0037 has the effect of protecting renal function, can inhibit the synthesis of uric acid in the liver of mice, inhibit the reabsorption of uric acid by the kidneys of mice, and inhibit kidney tissue inflammation.

[0022] As the fourth aspect of the present invention, it lies in providing a drug, comprising Lactobacillus mucosae ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 described in the first aspect or the microbial inoculum described in the second aspect.

[0023] Compared with the prior art, the beneficial effects of the present invention are: 1. Lactobacillus mucosae JNL0037 screened in the present invention has the effect of significantly reducing the blood uric acid content and can be used for the prevention and / or treatment of hyperuricemia, as well as gout, gout complications and kidney damage caused by hyperuricemia.

[0024] 2. The Lactobacillus reuteri JNL0037 provided by the present invention has the function of directly degrading uric acid and has a more significant effect on reducing higher concentrations of uric acid.

[0025] 3. The treatment of hyperuricemia with Lactobacillus reuteri JNL0037 has a kidney protection function. Compared with traditional drug treatment, it has higher safety and economy in the treatment of hyperuricemia, gout, uric acid nephropathy, and kidney inflammation, and has no obvious side effects, making it suitable for long-term use and providing a new treatment option for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0027] Figure 1 It is a phylogenetic tree diagram of Lactobacillus reuteri JNL0037 in Example 1; Figure 2 It is a morphological diagram of the strain Lactobacillus reuteri JNL0037 in Example 1; Figure 3 It is an analysis diagram of the hemolytic property of the strain Lactobacillus reuteri JNL0037 in Test Example 4; among them, (a) is the α-hemolysis of Aerococcus viridans, (b) is the β-hemolysis of Streptococcus pyogenes CICC10373, and (c) is the γ-hemolysis of Lactobacillus reuteri JNL0037; Figure 4 It is an effect diagram of Lactobacillus reuteri JNL0037 in reducing the blood uric acid level of hyperuricemia model mice in Test Example 5; among them, (a) is the body weight, (b) is the serum uric acid content, * represents p < 0.05, and *** represents p < 0.001; Figure 5 It is an effect diagram of Lactobacillus reuteri JNL0037 in improving the renal function of hyperuricemia model mice in Test Example 5; among them, (a) is the serum creatinine content, (b) is the serum urea content, and **** represents p < 0.0001; Figure 6 It is an effect diagram of Lactobacillus reuteri JNL0037 in reducing the activities of purine nucleoside phosphorylase (PNP) and xanthine oxidase (XOD) in the liver of hyperuricemia model mice in Test Example 5; among them, (a) is the PNP activity, (b) is the XOD activity, * represents p < 0.05, and ** represents p < 0.01; Figure 7 It is an effect diagram of Lactobacillus reuteri JNL0037 in reducing the transcriptional levels of XOD and PNP genes in the liver of hyperuricemia model mice in Test Example 5; among them, (a) is the XOD transcriptional level, (b) is the PNP transcriptional level, and * represents p < 0.05; Figure 8 Effect diagram of Lactobacillus mucosae JNL0037 reducing the transcriptional levels of GLUT9 and URAT1 genes in the kidney tissues of hyperuricemia model mice in Test Example 5; among them, (a) is the transcriptional level of GLUT9, (b) is the transcriptional level of URAT1, * indicates p < 0.01, and *** indicates p < 0.001; Figure 9 Effect diagram of Lactobacillus mucosae JNL0037 reducing the transcriptional levels of IL-6 and NF-κB genes in the kidney tissues of hyperuricemia model mice in Test Example 5; among them, (a) is the transcriptional level of IL-6, (b) is the transcriptional level of NF-κB, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001; Figure 10 Effect diagram of Lactobacillus mucosae JNL0037 increasing the contents of acetic acid and propionic acid in the colonic contents of hyperuricemia model mice in Test Example 5; among them, (a) is the content of acetic acid, (b) is the content of propionic acid, and * indicates p < 0.05. Detailed implementation manners

[0028] The present invention discloses Lactobacillus mucosae JNL0037 and its applications. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0029] It should be pointed out that the following detailed descriptions are all exemplary and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. Unless otherwise stated, the experimental methods disclosed in the present invention all adopt conventional techniques in the art, and the reagents and raw materials used in the examples can be purchased from the market.

[0030] The composition of the MRS medium used in the examples is as follows: Peptone 10.0 g / L, beef powder 8.0 g / L, yeast powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, ammonium citrate dibasic 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, pH value 5.7 ± 0.2 (25 °C).

[0031] In the first typical embodiment of the present invention, a strain of Lactobacillus mucosae Roy ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 is provided. This strain was deposited with the China General Microbiological Culture Collection Center on December 27, 2024. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO: 33218.

[0032] In the second typical embodiment of the present invention, a microbial inoculum is provided, which contains the Lactobacillus mucosae Roy ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 provided in the first typical embodiment, or its fermentate or its metabolite.

[0033] In the present invention, the term "fermentate" is used to refer to the fermentation product. The corresponding fermentate can be the liquid obtained from the process of fermenting and culturing Lactobacillus mucosae Roy ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037. Therefore, it can also be called fermentation broth; the liquid may contain bacteria (bacterial cells), but it does not necessarily need to contain bacteria. The liquid preferably contains the metabolites produced by the Lactobacillus mucosae Roy ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 of the present invention.

[0034] Moreover, in the embodiments of the present invention, the fermentation broth or culture broth containing bacterial cells is separated from the liquid in which the bacterial cells grow in the fermentation broth or culture broth by centrifugation, filtration, sedimentation, or other means known in the art. The liquid remaining after removing the bacterial cells is the "supernatant", and in the present invention, the supernatant contains the extracellular metabolites of Lactobacillus mucosae Roy ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037. In the embodiments of the present invention, the inoculum may also contain this supernatant.

[0035] In summary, in the above-mentioned microbial inoculum, the above-mentioned Lactobacillus mucosae Roy ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 is alive or dead or subjected to intermittent sterilization, or in the form of lysates and / or extracts, or in the form of bacterial products, or in the form of supernatant, or in the form of derivatives. The derivative forms include: metabolites, metabiotic products, probiotics, cell walls and their components, exopolysaccharides, and compounds containing immunogenic components, including: supernatant, inactivated bacterial cells.

[0036] In the third typical embodiment of the present invention, the application of the above-mentioned Lactobacillus mucosae Roy ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 or the above-mentioned microbial inoculum in the preparation of products for treating and / or preventing hyperuricemia-related diseases is provided.

[0037] The hyperuricemia-related diseases include hyperuricemia, gout, or uric acid nephropathy.

[0038] In one or more embodiments of the present invention, the product is a drug, and the drug comprises acceptable excipients and / or adjuvants.

[0039] In one or more embodiments of the present invention, the dosage form of the drug is an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup or spray, and a dosage form in the form of a sterile injection solution.

[0040] In some specific embodiments of the present invention, the above excipients and / or adjuvants include nutritional fortifiers, sweetening regulators, acidity regulators, isotonicity regulators, fillers, binders, disintegrants, solubilizers, cosolvents, preservatives, flavoring agents, colorants, suspending agents, wetting agents, emulsifiers and / or surfactants.

[0041] The drug may further comprise an appropriate amount of commonly used carriers, excipients and diluents. Moreover, according to the usual methods, it can be made into oral dosage forms such as powder, granule, tablet, capsule, suspension, emulsion, syrup, spray, etc., and dosage forms in the form of sterile injection solutions for use. The non-drug active ingredients such as carriers, excipients and diluents that can be included in the present invention are well-known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

[0042] The carriers, excipients and diluents of the present invention include, but are not limited to, lactose, glucose, sorbitol, mannitol, xylitol, maltitol, starch, arabic gum, gelatin, calcium phosphate, cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, and mineral oil, etc. No specific limitation is made here.

[0043] In one or more embodiments of the present invention, the drug has at least one of the following functions (1) to (9): Furthermore, the drug has at least one of the following functions: (1) Degrading guanosine; (2) Degrading uric acid; (3) Protecting renal function; (4) Inhibiting the synthesis of uric acid; (5) Inhibiting the reabsorption of uric acid by the kidney; (6) Inhibiting kidney tissue inflammation; (7) Inhibiting the activity of xanthine oxidase (XOD); (8) Inhibiting the activity of purine nucleoside phosphorylase (PNP); (9) Inhibiting the gene expression of glucose transporter-9 (GLUT9) and urate transporter 1 (URAT1); (10) Reducing the transcriptional levels of pro-inflammatory cytokines IL-6 and NF-κB in kidney tissue.

[0044] In the fourth typical embodiment of the present invention, a drug is provided, which comprises the above-mentioned Lactobacillus mucosae JNL0037 ( Limosilactobacillus reuteri subsp. reuteri ) or the above-mentioned microbial inoculum.

[0045] Example 1 Isolation and Purification Lactobacillus mucosae JNL0037 provided by the present invention was isolated from yogurt collected in Inner Mongolia. An appropriate amount of yogurt was taken, added with physiological saline for gradient dilution, evenly coated on MRS solid medium, and anaerobically cultured at 37 °C for 48 h. A single colony was picked with a sterilized inoculation loop and streaked and purified on MRS solid medium, and cultured at 37 °C for 48 h to obtain a Lactobacillus mucosae strain. A single colony was picked for Gram staining and observed under a microscope. As Figure 2 shown, Lactobacillus mucosae JNL0037 presents a short rod shape, 1.07 μm - 1.22 μm in length and 0.48 μm - 0.55 μm in width, and is Gram-positive.

[0046] The primers 27F 5’-AGAGTTTGATCCTGGCTCAG-3’ (SEQ ID NO.1) and 1492R 5’-GGCTTACCTTGTTACGACTT-3’ (SEQ ID NO.2) were used to amplify the bacterial 16S rRNA. The 16S rRNA gene sequence of the bacteria is:

[0047] By comparing with the NCBI database, a phylogenetic tree was constructed. As Figure 1 shown, the comparison result was Lactobacillus mucosae Limosilactobacillus reuteri .

[0048] The applicant named this Lactobacillus mucosae as JNL0037, which was deposited in the China General Microbiological Culture Collection Center on December 27, 2024. The deposit address was No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number was CGMCC NO: 33218, and the taxonomic name was Lactobacillus mucosae Limosilactobacillus reuteri subsp. reuteri .

[0049] Example 2 Cultivation of Strains (1)Bacterial culture and counting Prepare MRS medium according to the instructions and sterilize it at 118 °C for 20 min. After cooling to room temperature, inoculate Lactobacillus mucosae JNL0037 at an inoculation amount of 1%, and culture it anaerobically at 37 °C for 24 h. The cultured bacterial liquid was serially diluted with physiological saline. Take 100 μL and spread it evenly on MRS solid medium, and culture it anaerobically at 37 °C for 48 h. According to the number of growing colonies, the bacterial growth amount was statistically analyzed. After culturing Lactobacillus mucosae JNL0037 for 24 h, take 100 μL of the bacterial liquid in a 96-well plate, measure OD600 = 1.14, and the viable cell count was 3×10 8 CFU / mL.

[0050] (2)Separation of bacterial supernatant Centrifuge the cultured bacterial liquid in step (1) at 12000 rpm for 10 min at 4 °C, collect the supernatant, and filter the supernatant through a 0.22 μm filter membrane to obtain it.

[0051] Test Example 1 Analysis of the Level of Degrading Guanosine Lactobacillus mucosae JNL0037 and Lactobacillus casei strain Shirota Lactobacillus casei strainShirota (isolated from commercially available Yakult yogurt) was cultured overnight at 37 °C to obtain a seed solution. Centrifuge at 6000 rpm for 5 min at 4 °C respectively, and the cell mass was about 3×10 8 CFU. Wash the cells twice with PBS buffer (pH = 7.4), resuspend the cells with 1 mL of guanosine solution (0.5 mM, prepared with PBS), and the guanosine solution without cells was used as a blank control. Incubate at 37 °C with shaking at 100 rpm for 2 h, then centrifuge at 6000 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter membrane and transferred to a new centrifuge tube for testing.

[0052] Prepare guanosine standards with PBS buffer at concentrations of 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, and 1 mM in sequence. Prepare guanine standards with PBS buffer at concentrations of 0.05 mM, 0.1 mM, 0.15 mM, 0.75 mM, and 1 mM in sequence.

[0053] Analytical instrument: Agilent 1260 Infinity II high performance liquid chromatograph, chromatographic column: Agilent ZORBAX SB-Aq. Chromatographic conditions: injection volume 20 μL, flow rate 1 mL / min, detection wavelength 254 nm. The mobile phase uses an aqueous solution of 10 mM ammonium formate, adjust the pH to 3.3 with formic acid (add 60 μL of formic acid to 300 mL of 10 mM ammonium formate), add 1% methanol, and use it after ultrasonic treatment (20 min). Elution time is 25 min, the peak emergence time of guanosine is 19.67 min, and the peak emergence time of guanine is 15.7 min.

[0054] Guanosine conversion rate = (1 - residual guanosine in supernatant / original guanosine in supernatant) × 100%; Guanosine removal rate = [1 - (residual guanosine + guanine in supernatant) / original guanosine in supernatant] × 100%.

[0055] The analysis results show (Table 1) that the guanosine conversion rate of Lactobacillus mucosae JNL0037 is 100%, and the guanosine removal rate is 44.61%. In comparison, the guanosine conversion rate and guanosine removal rate of the commercial strain Lactobacillus casei strain Shirota Lactobacillus casei are 14.54% and 5.97% respectively.

[0056] Table 1 Guanosine conversion rate and guanosine removal rate

[0057] Example 3, Analysis of uric acid degradation level by Lactobacillus mucosae JNL0037 Lactobacillus mucosae JNL0037 and Lactobacillus casei strain Shirota Lactobacillus casei were cultured overnight at 37 °C to obtain seed solutions. Centrifuge at 4 °C and 6000 rpm for 5 min respectively, and the cell mass is about 3×10 8CFU. Wash the bacterial cells twice with HEPES buffer (50 mM, pH = 7.0). Resuspend the bacterial cells in 1 mL of uric acid solution (1 mM, prepared with HEPES buffer), and use the uric acid solution without bacterial cells (1 mM, prepared with HEPES buffer) as a blank control. Incubate with shaking at 37 °C and 100 rpm for 2 h, then terminate the reaction by heating at 100 °C for 10 min, centrifuge at 4 °C and 6000 rpm for 5 min, and detect the concentration of uric acid in the supernatant using the Elabscience Uric Acid Colorimetric Assay Kit (E-BC-K016-M). The analysis results show (Table 2) that the uric acid degradation rate of Lactobacillus mucosae JNL0037 is 2.68%, and Lactobacillus casei strain Lactobacillus casei strain Shirota has no ability to degrade uric acid.

[0058] Table 2 Uric Acid Degradation Rate

[0059] Test Example 4 Bacterial Hemolytic Analysis Prepare Columbia blood agar plates: 23.0 g of animal tissue hydrolysate, 1.0 g of starch, 5.0 g of NaCl, 15 g of agar, 1000 mL of distilled water, pH 7.3. Sterilize at 121 °C for 15 min. When cooled to 50 °C, add 5% defibrinated sheep blood. Cool and set aside. Do not store at room temperature for more than 4 h and do not store at 4 °C for more than 7 d. Streak Lactobacillus mucosae JNL0037 and the control strains on the Columbia blood agar plates. Incubate anaerobically at 37 °C for 18 h–24 h and observe the colony morphology.

[0060] α-hemolysis is manifested as a narrow, grass-green hemolytic ring; β-hemolysis is manifested as a wide, well-defined, completely transparent hemolytic ring; γ-hemolysis is non-hemolysis, that is, no hemolytic ring appears. As shown in (a) of Figure 3 , (b) of Figure 3 and (c) of Figure 3 , Aerococcus viridans is used as an α-hemolysis control strain, Streptococcus pyogenes CICC10373 is used as a β-hemolysis control strain, and no hemolytic ring appears in Lactobacillus mucosae JNL0037.

[0061] Test Example 5 Animal Experiment Analysis of the Therapeutic Effect of Probiotics on Hyperuricemia Model Mice After 1 week of adaptive feeding, male C57BL / 6J mice (aged 6 - 8 weeks, weighing 19 g - 23 g) were intragastrically administered hypoxanthine and potassium oxonate (the intragastric dosage of both drugs was 300 mg / kg, dissolved in 0.5% sodium carboxymethylcellulose solution) daily to induce the establishment of a hyperuricemia mouse model. Mice in the control group were intragastrically administered the same volume of 0.5% sodium carboxymethylcellulose solution. After a 2-hour interval, the treatment drugs were intragastrically administered as follows: the control group and the model group were intragastrically administered 200 μL of normal saline, the allopurinol group was intragastrically administered 5 mg / kg of allopurinol solution (dissolved in normal saline), and the probiotic treatment group was intragastrically administered 1×10 9 CFU of Lactobacillus mucosae JNL0037 cells. The experimental treatment time was 15 days. The body weights of the mice were measured on days 0, 5, 10, and 15 respectively, and the blood from the retro-orbital venous plexus and kidney tissues of the mice were collected and stored at -80 °C for later use.

[0062] After the collected blood was left to stand at room temperature for 2 h, it was centrifuged at 3000 rpm for 15 min to collect the serum. The concentration of uric acid in the supernatant was detected using the Elabscience Uric Acid Colorimetric Assay Kit (E-BC-K016-M); the content of creatinine (Cr) in the mouse serum was detected using the Elabscience Creatinine (Cr) Colorimetric Assay Kit (E-BC-K188-M); the content of urea (BUN) in the mouse serum was detected using the Elabscience Urea (BUN) Colorimetric Assay Kit (E-BC-K183-M); the activity of xanthine oxidase (XOD) in the liver was measured using the Xanthine Oxidase (XOD) Colorimetric Assay Kit (Elabscience, E-BC-K805-M); the activity of purine nucleoside phosphorylase (PNP) in the liver was measured using the Alkaline Phosphatase Detection Kit (Beyotime, P0321S).

[0063] Total RNA of kidney tissues was extracted using the RNA iso Plus Kit (Takara·9109), reverse transcribed into cDNA using the EvoM-MILV Reverse Transcription Premix Kit (Accurate Biotechnology, AG11728), and then the target genes were quantified using the SYBR GreenPro Taq HS Premix qPCR Kit (Accurate Biotechnology, AG11701). β-actin was used as the internal reference gene, and the relative expression levels of other target genes relative to the internal reference gene were calculated using the 2 （-△△Ct） formula. The primer sequences are shown in Table 3.

[0064] Table 3 Primer Sequences

[0065] As Figure 4As shown in (a) and (b), there were no significant differences in the body weights of each group of mice. However, the serum uric acid level of the model group mice on the 15th day (50.46 mg / L) was significantly higher than that of the control group (39.30 mg / L), while allopurinol treatment (44.55 mg / L) and Lactobacillus mucosae JNL0037 treatment (37.23 mg / L) significantly reduced the serum uric acid level; as Figure 5 As shown in (a) of , there was a tendency for the serum creatinine level of the allopurinol group mice to increase, suggesting that allopurinol might have caused renal function damage. Compared with the allopurinol group, the serum creatinine level of the Lactobacillus mucosae JNL0037 group mice was significantly reduced; as Figure 5 As shown in (b) of , the urea content in the serum of the Lactobacillus mucosae JNL0037 group was significantly lower than that of the model group and the allopurinol group, suggesting that Lactobacillus mucosae JNL0037 had an effect of protecting renal function.

[0066] As Figure 6 As shown in (a) and (b) of , compared with the model group, the activities of purine nucleoside phosphorylase (PNP) and xanthine oxidase (XOD) in the liver of the Lactobacillus mucosae JNL0037 group mice were significantly reduced; as Figure 7 As shown in (a) of , there was a tendency for the transcriptional level of the XOD gene in the liver of the Lactobacillus mucosae JNL0037 group mice to decrease; as Figure 7 As shown in (b) of , the transcriptional level of the PNP gene in the liver of the Lactobacillus mucosae JNL0037 group mice was significantly lower than that of the model group, suggesting that Lactobacillus mucosae JNL0037 inhibited the synthesis of uric acid in the liver of mice.

[0067] The results were as Figure 8 As shown in (a) and (b) of , compared with the model group and the allopurinol group, Lactobacillus mucosae JNL0037 treatment significantly reduced the transcriptional levels of the key genes GLUT9 and URAT1 for uric acid reabsorption in the kidney tissues of mice. GLUT9 and URAT1 play important roles in the process of renal reabsorption of uric acid. The above results indicate that JNL0037 can inhibit the renal reabsorption of uric acid in mice.

[0068] As Figure 9 As shown in (a) and (b) of , compared with the model group and the allopurinol group, Lactobacillus mucosae JNL0037 treatment significantly reduced the transcriptional levels of the pro-inflammatory cytokines IL-6 and NF-kB in the kidney tissues of mice, indicating that JNL0037 can inhibit kidney tissue inflammation.

[0069] Collect appropriate samples of colonic contents and weigh them precisely. Homogenize with 500 μL of water and an appropriate amount of glass beads for 1 minute at 4 °C, and centrifuge at 12000 rpm for 10 minutes at 4 °C. Take 200 μL of the supernatant and mix it with 100 μL of 15% phosphoric acid, 20 μL of 375 μg / mL 4-methylvaleric acid solution, and 280 μL of diethyl ether. After swirling for 1 minute, the sample is centrifuged again at 12000 rpm for 10 minutes at 4 °C. Then transfer the supernatant to a vial for preparation for gas chromatography-mass spectrometry (GC-MS) analysis. GC analysis is performed using a Trace1300 gas chromatograph (Thermo Fisher Scientific, USA). The chromatographic column is an Agilent HP-INNOWAX capillary column (30 m × 0.25 mm ID × 0.25 μm), the carrier gas is helium, and the flow rate is 1 mL / min. Inject the sample in a split ratio of 10:1, with an injection volume of 1 μL and an injection temperature of 250 °C. The ion source temperature is 300 °C, and the mass spectrometry transfer line temperature is 250 °C. The column temperature program starts at 90 °C, then increases at a rate of 10 °C / min to 120 °C, increases at a rate of 5 °C / min to 150 °C, and finally increases at a rate of 25 °C / min to 250 °C and is held for 2 minutes. Metabolite detection is performed on a TSQ 9000 mass spectrometer (Thermo Fisher Scientific, USA) using electron impact ionization. The single ion monitoring (SIM) mode is used, and the electron energy is 70 eV.

[0070] As Figure 10 shown in (a) and (b) of , compared with the model group, treatment with Lactobacillus mucosae JNL0037 significantly increased the concentrations of short-chain fatty acids acetic acid and propionic acid in the colon. According to literature reports, acetic acid and propionic acid have beneficial effects such as regulating intestinal motility and immunity, enhancing the intestinal barrier, inhibiting the growth of harmful bacteria, and inhibiting cholesterol synthesis, indicating that JNL0037 can exert beneficial functions by increasing the levels of short-chain fatty acids in the intestine.

[0071] Test Example 6 Analysis of the in vitro antioxidant level of probiotic supernatant Experimental procedure: Lactobacillus mucosae JNL0037 and Lactobacillus casei strain Shirota Lactobacillus caseiThe strain Shirota was anaerobically cultured in MRS medium at 37 °C for 48 h, centrifuged at high speed, the bacterial liquid was collected, and passed through a 0.22 μm filter membrane. The antioxidant levels of the supernatant were detected using the total antioxidant capacity (T-AOC) colorimetric assay kit (ABTS enzyme-catalyzed method) (Elabscience, E-BC-K219-M) and the total antioxidant capacity (T-AOC) colorimetric assay kit (FRAP method) (Elabscience, E-BC-K225-M), respectively. Blank MRS medium was used as a control. The antioxidant levels of the bacterial supernatants are shown in Table 4. The antioxidant level of the supernatant of Lactobacillus mucosae JNL0037 was better than that of the commercial strain Lactobacillus casei Shirota. The antioxidant effect of probiotics can reduce the damage caused by free radicals to biological macromolecules such as cell biomembranes, nucleic acids, and proteins, reduce oxidative stress, and maintain the integrity and stability of cells and the internal environment.

[0072] Table 4 Antioxidant levels of the supernatant of Lactobacillus mucosae JNL0037

[0073] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill in the art can modify or equivalently replace the technical solution of the present invention according to needs, without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain of Limosilactobacillus reuteri ( Limosilactobacillus reuteri subsp. reuteri ), JNL0037, characterized in that It was deposited at the China General Microbiological Culture Collection Center on December 27, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO: 33218.

2. A microbial inoculant, characterized in that, Comprising Lactobacillus reuteri ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 or its ferment or its metabolite.

3. Use of Lactobacillus reuteri ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 or the microbial inoculum according to claim 2 in the preparation of a product for treating and / or preventing hyperuricemia-related diseases.

4. Use of Lactobacillus mucosae ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 or the microbial agent according to claim 2 in the preparation of a medicament for treating and / or preventing hyperuricemia and / or gout and / or uric acid nephropathy and / or kidney inflammation.

5. The application according to claim 4, characterized in that, The drug contains pharmaceutically acceptable excipients and / or adjuvants.

6. The application according to claim 4, characterized in that, The drug has the function of degrading guanosine.

7. The application according to claim 4, wherein The drug has at least one of the following functions: (1) Degrading uric acid; (2) Inhibiting the synthesis of uric acid; (3) Inhibiting the reabsorption of uric acid by the kidney.

8. The application according to claim 4, characterized in that The drug has at least one of the following functions: (1) Protecting renal function; (2) Inhibiting kidney tissue inflammation; (3) Reducing the transcriptional levels of pro-inflammatory cytokines IL-6 and NF-kB in kidney tissue.

9. The application according to claim 4, characterized in that, The drug has at least one of the following functions: (1) Inhibiting the activity of xanthine oxidase; (2) Inhibiting the activity of purine nucleoside phosphorylase; (3) Inhibiting the gene expression of glucose transporter-9 and urate transporter 1.

10. A drug, characterized in that, Comprising Lactobacillus reuteri ( Limosilactobacillus reuteri subsp. reuteri ) JNL0037 as claimed in claim 1 or the microbial inoculum as claimed in claim 2.

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