A strain of Lactobacillus paracasei JNL0025 and its application

Abstract

This invention belongs to the field of microbial technology and relates to a strain of *Lactobacillus paracasei* JNL0025 and its applications. Compared with the commercial strain *Lactobacillus casei* Shirota, the *Lactobacillus paracasei* JNL0025 provided by this invention has an increased guanine nucleoside conversion rate, comparable inhibition rate of XOD in the supernatant, and can also inhibit PNP activity; it shows a tendency to reduce pro-inflammatory M1 macrophages while increasing the proportion of anti-inflammatory M2 macrophages, and inhibits the secretion of pro-inflammatory cytokines TNF-α and IL-6. Moreover, *Lactobacillus paracasei* JNL0025 has a better anti-inflammatory effect than *Lactobacillus rhamnosus* LGG. Animal experimental results show that the *Lactobacillus paracasei* JNL0025 provided by this invention can significantly reduce serum uric acid levels in hyperuricemia model mice, alleviate renal inflammation, and alleviate DSS-induced colitis, demonstrating significant application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Lactobacillus paracasei JNL0025 and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Hyperuricemia is a metabolic disease caused by purine metabolism disorders and imbalanced uric acid excretion. It not only directly causes gout, but also leads to a series of serious health problems such as kidney and cardiovascular diseases. With the increasing number of patients with hyperuricemia, the global demand for uric acid-lowering products is rapidly expanding. At present, drug therapy combined with strict dietary management is commonly used in clinical practice to regulate blood uric acid levels, but more than 30% of gout patients find it difficult to achieve the ideal uric acid control target through current drugs (including febuxostat and allopurinol, etc.), and 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 patient's quality of life and lead to poor patient compliance.

[0004] Inflammatory bowel disease is a chronic, nonspecific intestinal inflammatory disease with an unknown etiology, mainly including two subtypes: Crohn's disease and ulcerative colitis. Currently, the first-line treatment drugs in clinical practice mainly include aminosalicylic acid drugs, corticosteroids, biological agents and immunosuppressants. Not all patients with inflammatory bowel disease can benefit from these treatments, and these drugs also have a considerable risk of side effects.

[0005] Therefore, the inventors envisioned obtaining a new, safe and effective product that can play a role in both lowering uric acid and treating inflammatory bowel disease to meet a wide range of health needs. Studies have shown that the intestinal flora of gout patients is significantly different from that of healthy people in terms of composition and functional structure. These different flora structures can induce chronic systemic inflammation, leading to increased purine synthesis and weakened uric acid metabolism, thereby causing increased blood uric acid and gout attacks; changes in the intestinal microecology of patients with inflammatory bowel disease are mainly manifested as dysbiosis, including changes in flora diversity, species and abundance.

[0006] Recent studies have shown that probiotics can lower blood uric acid levels in a variety of ways: ① Inhibiting the activity of xanthine oxidase (XOD), a key enzyme in uric acid synthesis; ② Probiotics compete with intestinal epithelial cells for the absorption of nucleosides, and degrade purine substances in the intestine through the purine nucleosidase produced by themselves, thereby reducing the production of uric acid; ③ Probiotics help restore the balance of intestinal flora, increase the number of beneficial bacteria, and reduce harmful bacteria, thereby reducing inflammatory responses and uric acid production; ④ Probiotics increase uric acid excretion by promoting the expression of urate excretion transporters and inhibiting the expression of urate reabsorption proteins, thereby reducing uric acid levels; ⑤ Probiotics can strengthen the intestinal barrier, reduce intestinal permeability, and prevent harmful substances from entering the blood, thereby reducing the production of inflammatory factors.

[0007] Probiotics can be used as a supplement to drug and dietary therapy, by regulating the balance of intestinal microecology and improving metabolic status, thereby improving the overall treatment effect. Moreover, compared with traditional drug treatment, probiotics are safer and more economical in the treatment of hyperuricemia and gout, and have no obvious side effects, making them suitable for long-term use. Therefore, probiotics have multiple mechanisms and significant advantages in the treatment of hyperuricemia and gout, providing patients with a new treatment option. At the same time, probiotics can effectively regulate the imbalance of intestinal flora, improve the microecological environment, enhance the intestinal mucosal barrier function, and regulate local and systemic immune responses, providing new options for the treatment of diseases such as inflammatory bowel disease. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention aims to provide a strain of Lactobacillus paracasei JNL0025 and its application. The strain is isolated from fermented yogurt collected in Inner Mongolia, and the 16S rRNA gene sequence identified by the 16S rRNA method (16S ribosomal RNA gene sequencing technology) is SEQ ID NO: 1. The strain can significantly reduce the blood uric acid level of hyperuricemia model mice, showing great application value.

[0009] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0010] A first aspect of the present invention provides a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei ) JNL0025, the strain was deposited in the General Microbiology Center of China Culture Collection Administration on June 24, 2024 (the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), and the deposit number is CGMCC NO: 31043.

[0011] Preferably, the Lactobacillus paracasei ( Lacticaseibacillus paracasei) The culture medium components involved in the culture process of JNL0025 include: peptone 8~12 g / L, beef powder 6~10 g / L, yeast powder 2~6 g / L, glucose 18~25 g / L, dipotassium hydrogen phosphate 1~5 g / L, diammonium hydrogen citrate 1~5 g / L, sodium acetate 2~7 g / L, magnesium sulfate 0.1~0.5 g / L, manganese sulfate 0.01~0.08 g / L, Tween 80 1.0 g / L;

[0012] Further preferably, the composition of the MRS medium includes: 10.0 g / L peptone, 8.0 g / L beef powder, 4.0 g / L yeast powder, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 g / L Tween 80; the pH value of the medium is 5.7±0.2 (25°C).

[0013] The second aspect of the present invention provides a microbial agent, wherein the microbial agent contains the Lactobacillus paracasei described in the first aspect ( Lacticaseibacillus paracasei ) JNL0025 or its fermentation product or its metabolite.

[0014] The metabolites described in the present invention include extracellular metabolites and / or intracellular metabolites.

[0015] In the present invention, the term "fermented product" is used to refer to a fermented product. The corresponding fermented product can be obtained by fermenting Lactobacillus paracasei ( Lacticaseibacillus paracasei ) The liquid obtained by the process of JNL0025 can therefore also be called fermentation liquid; the liquid may contain bacteria (bacteria), but does not necessarily need to contain bacteria. The liquid preferably contains Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Metabolites produced by JNL0025.

[0016] And, in an embodiment of the present invention, the fermentation liquid or culture liquid containing the bacteria is separated from the liquid by centrifugation, filtration, sedimentation or other means known in the art, and the liquid remaining after the bacterial cells are removed is the "supernatant", and in the present invention, the supernatant contains Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Extracellular metabolites of JNL0025. In an embodiment of the present invention, the bacterial agent may also contain the supernatant. The present invention has proved through experiments that the supernatant obtained after fermentation and culture in MRS medium can effectively inhibit the activity of xanthine oxidase (XOD), significantly reduce the blood uric acid level of hyperuricemia model mice, alleviate kidney pathological damage, and achieve the effect of reducing in vitro anti-inflammatory and antioxidant levels and effectively inhibiting intestinal inflammation.

[0017] The third aspect of the present invention provides the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Use of JNL0025 or the above-mentioned microbial agents in the preparation of uric acid-lowering products;

[0018] Preferably, the product is a medicine; the dosage form of the medicine is an oral dosage form in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups or sprays, and a sterile injection solution.

[0019] The drug of the present invention may also contain an appropriate amount of commonly used carriers, excipients and diluents. The specific non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and a person of ordinary skill in the art can determine that they meet clinical standards.

[0020] Preferably, the drug has at least one of the following functions (1) to (6):

[0021] (1) Degradation of guanosine nucleoside;

[0022] (2) Inhibit the activity of urate synthase - xanthine oxidase (XOD);

[0023] (3) Inhibit the activity of urate synthase - purine nucleoside phosphorylase (PNP);

[0024] (4) Treatment of gout;

[0025] (5) Treatment of hyperuricemia;

[0026] (6) Treatment of uric acid nephropathy.

[0027] A fourth aspect of the present invention provides the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Use of JNL0025 or the above-mentioned microbial agents in the preparation of products for treating colitis;

[0028] Preferably, the product is a medicine; the dosage form of the medicine is an oral dosage form in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups or sprays, and a sterile injection solution.

[0029] The drug of the present invention may also contain an appropriate amount of commonly used carriers, excipients and diluents. The specific non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and a person of ordinary skill in the art can determine that they meet clinical standards.

[0030] Preferably, the drug has at least one of the following functions (1) to (4):

[0031] (1) Anti-inflammatory;

[0032] (2) Antioxidant;

[0033] (3) Regulate the structure of intestinal flora;

[0034] (4) Enhance the intestinal mucosal barrier function.

[0035] Beneficial technical effects of one or more of the above technical solutions:

[0036] (1) Compared with the commercial strain Lactobacillus casei Shirota Lactobacillus casei Compared with strain Shirota, the guanine nucleoside conversion rate of the Lactobacillus paracasei JNL0025 provided by the present invention is improved, the inhibition rate of the supernatant on XOD is equivalent, and the inhibition of PNP activity can be achieved.

[0037] (2) The Lactobacillus paracasei JNL0025 provided by the present invention has a tendency to reduce the pro-inflammatory M1 macrophages, while significantly increasing the proportion of anti-inflammatory M2 macrophages, and inhibiting the secretion of pro-inflammatory cytokines TNF-α and IL-6. Moreover, the inhibitory effect of Lactobacillus paracasei JNL0025 is better than that of Lactobacillus rhamnosus LGG, and has the function of treating colitis.

[0038] (3) Animal experiment results show that the Lactobacillus paracasei JNL0025 provided by the present invention can significantly reduce the blood uric acid level of hyperuricemia model mice, alleviate kidney pathological damage, and alleviate DSS-induced colitis, showing great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use in the description of the embodiment will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0040] Figure 1 It is the phylogenetic tree diagram of Lactobacillus paracasei JNL0025;

[0041] Figure 2 This is the morphological diagram of Lactobacillus paracasei JNL0025 strain;

[0042] Figure 3 The hemolytic analysis diagram of Lactobacillus paracasei JNL0025 strain; (a) is the colony morphology of the α-hemolytic control strain, (b) is the colony morphology of the β-hemolytic control strain, and (c) is the colony morphology of the γ-hemolytic Lactobacillus paracasei JNL0025;

[0043] Figure 4 This is a graph showing the effect of Lactobacillus paracasei JNL0025 in reducing the blood uric acid level in hyperuricemia model mice in Experimental Example 6; (a) is body weight, (b) is serum uric acid level, and * means p<0.05;

[0044] Figure 5 This is a diagram showing the effect of Lactobacillus paracasei JNL0025 in improving renal function in hyperuricemia model mice in Experimental Example 6; (a) is the serum creatinine content, (b) is the serum urea content, and * means p<0.05;

[0045] Figure 6 The effect of Lactobacillus paracasei JNL0025 in Experimental Example 6 on inhibiting the transcription of GLUT9 and interleukin-6 genes in renal tissues of hyperuricemia model mice; (a) is the transcription level of GLUT9 gene, (b) is the transcription level of interleukin (IL)-6 gene, * is p<0.05, ** is p<0.01;

[0046] Figure 7 The figure shows the effect of the supernatant of Lactobacillus paracasei JNL0025 in Experimental Example 7 on inhibiting the inflammatory polarization of macrophages and the secretion of inflammatory factors; (a) is the proportion of M1 macrophages, (b) is the proportion of M2 macrophages, (c) is the level of TNF-α in the cell supernatant, and (d) is the level of IL-6 in the cell supernatant; * is p<0.05, ** is p<0.01; *** is p<0.001, **** is p<0.0001;

[0047] Figure 8 This is a graph showing the effect of Lactobacillus paracasei JNL0025 in reducing DSS-induced colitis in terms of body weight and disease activity index in Experimental Example 9; wherein (a) is body weight, and (b) is disease activity index;

[0048] Fig. 9 This is a diagram showing the effect of Lactobacillus paracasei JNL0025 in reducing DSS-induced colitis in colon tissue H&E staining in Experimental Example 9;

[0049] Fig.10 This is a graph showing the effect of Lactobacillus paracasei JNL0025 in reducing DSS-induced colitis on colorectal length in Experimental Example 9; ** indicates p<0.01;

[0050] Fig.11 This is a graph showing the effect of Lactobacillus paracasei JNL0025 in reducing DSS-induced colitis in terms of serum inflammatory factor levels in Experimental Example 9; wherein (a) is the level of serum IL-β, (b) is the level of serum IL-6, and (c) is the level of serum IL-10. * indicates p<0.05, and ** indicates p<0.01. DETAILED DESCRIPTION

[0051] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0052] In view of the fact that probiotics have multiple mechanisms and significant advantages in treating hyperuricemia and gout, and provide patients with a new treatment option, the present invention proposes a strain of Lactobacillus paracasei JNL0025 and its application in preparing a uric acid-lowering product.

[0053] Specifically, in a first typical embodiment of the present invention, a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei ) JNL0025, the strain was deposited in the General Microbiology Center of China Culture Collection Administration on June 24, 2024 (the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), and the deposit number is CGMCC NO: 31043.

[0054] In one or more examples of this embodiment, the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) The culture medium components involved in the culture process of JNL0025 include: peptone 8~12 g / L, beef powder 6~10 g / L, yeast powder 2~6 g / L, glucose 18~25 g / L, dipotassium hydrogen phosphate 1~5 g / L, diammonium hydrogen citrate 1~5 g / L, sodium acetate 2~7 g / L, magnesium sulfate 0.1~0.5 g / L, manganese sulfate 0.01~0.08 g / L, Tween 80 1.0 g / L;

[0055] In one or more examples of this embodiment, the composition of the MRS medium includes: 10.0 g / L peptone, 8.0 g / L beef powder, 4.0 g / L yeast powder, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 g / L Tween 80; the pH value of the medium is 5.7±0.2 (25°C).

[0056] In a second typical embodiment of the present invention, a microbial agent is provided, wherein the microbial agent contains the Lactobacillus paracasei described in the first aspect ( Lacticaseibacillus paracasei ) JNL0025 or its fermentation product or its metabolite.

[0057] In a third typical embodiment of the present invention, the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei) Use of JNL0025 or the above-mentioned microbial agents in uric acid-lowering products;

[0058] In one or more examples of this embodiment, the product is a drug.

[0059] In one or more examples of this embodiment, the drug is in the form of an oral preparation in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups or sprays, and a sterile injection solution.

[0060] In one or more examples of this embodiment, the drug has at least one of the following functions (1) to (6):

[0061] (1) Degradation of guanosine nucleoside;

[0062] (2) Inhibit the activity of urate synthase - xanthine oxidase (XOD);

[0063] (3) Inhibit the activity of urate synthase - purine nucleoside phosphorylase (PNP);

[0064] (4) Treatment of gout;

[0065] (5) Treatment of hyperuricemia;

[0066] (6) Treatment of uric acid nephropathy.

[0067] In a fourth typical embodiment of the present invention, the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Use of JNL0025 or the above-mentioned microbial agents in the preparation of products for treating colitis;

[0068] In one or more examples of this embodiment, the product is a drug.

[0069] In one or more examples of this embodiment, the drug is in the form of an oral preparation in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups or sprays, and a sterile injection solution.

[0070] In one or more examples of this embodiment, the drug has at least one of the following functions (1) to (4):

[0071] (1) Anti-inflammatory;

[0072] (2) Antioxidant;

[0073] (3) Regulate the structure of intestinal flora;

[0074] (4) Enhance the intestinal mucosal barrier function.

[0075] The present invention is further described below in conjunction with examples. The present invention is further described below by way of examples, but the present invention is not limited to the scope of the embodiments described. Based on the embodiments in the present invention, any variation of the present invention by those skilled in the art without making a creative premise all belongs to the protection scope of the present invention. Meanwhile, in the embodiments of the present invention, unless otherwise specified, all preparation raw materials are commercially available products well known to those skilled in the art.

[0076] The composition of the MRS culture medium used in the embodiment is as follows:

[0077] 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, diammonium hydrogen citrate 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℃).

[0078] Example 1: Separation and purification

[0079] Prepare MRS medium according to the instructions and sterilize at 118℃ for 20 min. Take an appropriate amount of fermented yogurt, add physiological saline to dilute it in gradients, evenly apply it on MRS solid medium, and culture it anaerobically at 37℃ for 48 h. Pick a single clone and observe the bacterial morphology by Gram staining. Use primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') (SEQ ID NO:2) and 1492R (5'-GGCTTACCTTGTTACGACTT-3') (SEQ ID NO:3) to amplify bacterial 16S rRNA. The 16S rRNA gene sequence of the specific bacteria is shown in SEQ ID NO:1.

[0080] By comparing the NCBI database, a phylogenetic tree was constructed, such as Figure 1 As shown, the comparison result is Lactobacillus paracasei Lacticaseibacillus paracasei .

[0081] The Lactobacillus paracasei JNL0025 in the present invention was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on June 24, 2024, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC NO: 31043, and the classification name being Lactobacillus paracasei Lacticaseibacillus paracasei .

[0082] Morphological characteristics of the Lactobacillus paracasei JNL0025 strain were analyzed by picking a single colony for Gram staining and observing under a microscope. Figure 2As shown, Lactobacillus paracasei JNL0025 is rod-shaped, arranged in pairs or chains, 0.47~0.42 μm (width) × 0.92~1.38 μm (length), and Gram-positive.

[0083] Example 2: Cultivation of strains

[0084] (1) Bacterial culture and counting

[0085] MRS medium was prepared according to the instructions and sterilized at 118°C for 20 min. After cooling to room temperature, Lactobacillus paracasei JNL0025 was inoculated at 1% inoculation and cultured anaerobically at 37°C for 24 h. The cultured bacterial solution was diluted with physiological saline gradients, and 100 μL was evenly spread on MRS solid medium and cultured anaerobically at 37°C for 4 to 8 h. The bacterial growth was counted according to the number of colonies grown. After 24 h of culture of Lactobacillus paracasei JNL0025, the OD600 of the bacterial solution was 1.58, and the number of viable bacteria was 1.6×10 9 CFU / mL, OD600 of bacterial solution was 3.72 after 36 h of culture.

[0086] (2) Bacterial supernatant separation

[0087] The bacterial culture liquid cultured in step (1) was centrifuged at a speed of 12000 rpm and centrifuged at 4°C for 10 min. The supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the obtained product.

[0088] Experimental Example 1: Analysis of Degraded Guanosine Nucleoside Levels

[0089] Experimental process: (1) Lactobacillus paracasei JNL0025 and Lactobacillus casei Shirota strain Lactobacillus casei strain Shirota was cultured overnight at 37°C to obtain seed solution. The cells were centrifuged at 4°C and 6000 rpm for 5 min and the bacterial volume 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 in PBS), and use the guanosine solution without cells as a blank control. Incubate at 37°C, 100 rpm for 2 h, then centrifuge at 6000 rpm for 5 min. Filter the supernatant with a 0.22 μm filter membrane and transfer to a new centrifuge tube for testing.

[0090] (2) Guanosine nucleoside standards were prepared in PBS buffer at concentrations of 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM and 1 mM. Guanine standards were prepared in PBS buffer at concentrations of 0.05 mM, 0.1 mM, 0.15 mM, 0.75 mM and 1 mM.

[0091] Analytical instrument: Agilent 1260 Infiinity II HPLC, chromatographic column: Agilent ZORBAXSB-Aq. Chromatographic conditions: injection volume 20 μL, flow rate 1 mL / min, detection wavelength 254 nm. The mobile phase used a 10 mM ammonium formate aqueous solution, and the pH was adjusted to 3.3 with formic acid (300 mL of 10 mM ammonium formate plus 60 μL formic acid), 1% methanol was added, and it was used after ultrasonication (20 min). The elution time was 25 min, the guanosine peak time was 19.67 min, and the guanine peak time was 15.7 min.

[0092] Guanosine conversion rate = (1-residual guanosine in supernatant / original guanosine in supernatant) * 100%

[0093] Guanosine removal rate = [1-(residual guanosine + guanine in supernatant) / original guanosine in supernatant]*100%

[0094] The results are shown in Table 1. The guanosine conversion rate of Lactobacillus paracasei JNL0025 was 16.41%, and the guanosine removal rate was 4.23%. Lactobacillus casei The guanosine conversion rate and guanosine removal rate of strain Shirota were 14.54% and 5.97%, respectively.

[0095] Table 1 Guanosine conversion rate and guanosine removal rate

[0096]

[0097] Experimental Example 2: Analysis of the level of inhibition of xanthine oxidase (XOD) by bacterial supernatant

[0098] Experimental process: The bacterial culture solution after culture in Example 2 was centrifuged at 12000 rpm and 4°C for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter. A 10 U / mL XOD solution was prepared, and a 50 μL sample reaction system (5 μL XOD solution + 25 μL culture medium / bacterial supernatant + 20 μL PBS) was used. No XOD was added as a negative control. XOD activity was determined using a xanthine oxidase (XOD) colorimetric test kit (Elabscience, E-BC-K805-M).

[0099] XOD inhibition rate = (1-XOD activity of sample group / XOD activity of positive control) * 100%

[0100] The results are shown in Table 2. The inhibition rate of the supernatant of Lactobacillus paracasei JNL0025 on XOD was 26.5%, which was comparable to the inhibition rate of the supernatant of Lactobacillus casei Shirota strain on XOD (about 27%).

[0101] Table 2 XOD inhibition rate

[0102]

[0103] Experimental Example 3: Analysis of the level of inhibition of purine nucleoside phosphorylase (PNP) by bacterial supernatant

[0104] Experimental process: The bacterial culture solution after culture in Example 2 was centrifuged at 12000 rpm and 4°C for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter. An 80 U / mL PNP solution was prepared, and a 50 μL sample reaction system (PNP 25 μL + culture medium / sample 25 μL) was used to determine the PNP activity using an alkaline phosphatase detection kit (Biyuntian, P0321S), and the reaction was carried out at 37°C for 10 min.

[0105] PNP inhibition rate = (1-PNP activity of sample group / PNP activity of positive control) * 100%

[0106] The results are shown in Table 3. The inhibition rate of the supernatant of Lactobacillus paracasei JNL0025 on PNP was 36.4%, while the supernatant of Lactobacillus casei Shirota strain had no inhibitory effect on PNP.

[0107] Table 3 PNP inhibition rate

[0108]

[0109] Test Example 4: Bacterial Antibiotic Sensitivity Analysis

[0110] Experimental process: Lactobacillus paracasei JNL0025 was inoculated into liquid MRS medium and cultured anaerobically at 37℃ for 24 h to obtain seed liquid. The bacterial liquid was diluted to 0.5 McFarland turbidity with 0.85% saline, 100 μL of bacterial liquid was accurately aspirated and evenly spread on the surface of MRS agar medium. After the surface bacterial liquid dried, a drug-sensitive paper was placed on the surface of the culture medium. The sensitivity of Lactobacillus paracasei JNL0025 to six antibiotics, including ampicillin (10 μg / tablet), gentamicin (10 μg / tablet), kanamycin (30 μg / tablet), streptomycin (10 μg / tablet), clindamycin (2 μg / tablet) and chloramphenicol (30 μg / tablet) were detected by KB paper diffusion method. The sensitivity of each bacterial strain to each antibiotic was repeated three times. Incubate anaerobically at 37°C for 24 h.

[0111] The diameters of the inhibition zones were measured and recorded. The results are shown in Table 4.

[0112] Table 4 Antibiotic inhibition zone of Lactobacillus paracasei JNL0025

[0113]

[0114] Test Example 5: Bacterial hemolytic analysis

[0115] Experimental process:

[0116] 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℃ for 15 min. When cooled to 50℃, add 5% defibrinated sheep blood. Cool and set aside. Keep at room temperature for no more than 4 h, and store at 4℃ for no more than 7 days. Streak Lactobacillus paracasei JNL0025 and the control strain on Columbia blood agar plates. Incubate anaerobically at 37℃ for 18-24 h, and observe the colony morphology.

[0117] α-hemolysis manifests as a narrow, grass-green hemolysis ring; β-hemolysis manifests as a wide, well-defined, completely transparent hemolysis ring; γ-hemolysis is no hemolysis, that is, no hemolysis ring appears.

[0118] like Figure 3 As shown in (a), Aerococcus aureus was used as the α-hemolytic control strain;

[0119] like Figure 3 As shown in (b), S. pyogenes CICC10373 was used as a β-hemolytic control strain;

[0120] like Figure 3 As shown in (c), no hemolytic ring was observed in Lactobacillus paracasei JNL0025.

[0121] Experimental Example 6: Animal Experiment Analysis of the Therapeutic Effect of Probiotics on Hyperuricemia Model Mice

[0122] Experimental process: After 1 week of adaptive feeding, 6-8 week old male C57BL / 6J mice (weight 19-23g) were gavaged daily with hypoxanthine and potassium oxonate (the gavage dose of both drugs was 300mg / kg, dissolved in 0.5% sodium carboxymethylcellulose solution) to induce the establishment of a hyperuricemia mouse model; the control group mice were gavaged with the same volume of 0.5% sodium carboxymethylcellulose solution. After an interval of 2 hours, the treatment drugs were gavaged;

[0123] The groups were divided into the following groups: the control group and the model group were gavaged with 200 μL of normal saline, the allopurinol group was gavaged with 5 mg / kg of allopurinol solution (dissolved in normal saline), and the probiotics treatment group was gavaged with 2×10 9 CFU Lactobacillus paracasei JNL0025 bacteria, the experimental treatment time was 15 days. The mice were weighed on days 0, 5, 10 and 15, and blood was collected from the posterior ocular venous plexus of the mice. The collected blood was allowed to stand at room temperature for 2 hours, centrifuged at 3000 rpm for 15 minutes, and serum was collected. The Elabscience uric acid colorimetric test kit (E-BC-K016-M) was used to detect the concentration of uric acid in the supernatant; the Elabscience creatinine (Cr) colorimetric test kit (E-BC-K188-M) was used to detect the content of creatinine in mouse serum; and the Elabscience urea (BUN) colorimetric test kit (E-BC-K183-M) was used to detect the content of uric acid in mouse serum.

[0124] like Figure 4 (a) and (b) show that there was no significant difference in the body weight of mice in each group, but the serum uric acid level of mice in the model group was significantly higher than that in the control group on the 15th day, while the allopurinol group and Lactobacillus paracasei JNL0025 treatment significantly reduced the blood uric acid level;

[0125] like Figure 5 (a) shows that the serum creatinine level of mice in the allopurinol group has an upward trend, suggesting that allopurinol may cause renal damage. Compared with the allopurinol group, the serum creatinine level of mice in the Lactobacillus paracasei JNL0025 group was significantly reduced; Figure 5 Middle (b) shows that there is no significant difference in the urea content in the serum of the Lactobacillus paracasei JNL0025 group compared with the model group and the allopurinol group. Based on all the results, Lactobacillus paracasei JNL0025 may have the effect of protecting renal function.

[0126] Detection and analysis of GLUT9 and inflammatory factor gene transcription levels in mouse kidney tissue: Total RNA from kidney tissue was extracted using RNA iso Plus kit (Takara·9109), and RNA was reverse transcribed into cDNA using EvoM-MILV reverse transcription premix kit (Aikara, AG11728), and then the target gene was quantified using SYBR GreenPro Taq HS premix qPCR kit (Aikara, AG11701). β-actin was used as the internal reference gene, and 2 （-△△Ct） The formula is used to calculate the expression level of other target genes relative to the internal reference gene. The primer sequences are shown in Table 5:

[0127] Table 5 Primer sequences

[0128]

[0129] The results are as follows Figure 6 (a) shows that compared with the model group and the allopurinol group, the treatment with Lactobacillus paracasei JNL0025 significantly reduced the transcription level of GLUT9 (glucose facilitative transporter-9), a key gene for uric acid reabsorption in the kidney tissue of mice, which plays an important role in the renal reabsorption of uric acid. The above results indicate that Lactobacillus paracasei JNL0025 inhibits the reabsorption of uric acid by the mouse kidney; Figure 6 Middle (b) shows that compared with the model group and allopurinol group, treatment with Lactobacillus paracasei JNL0025 significantly reduced the transcription level of the proinflammatory cytokine interleukin (IL)-6 in the kidney tissue of mice, indicating that Lactobacillus paracasei JNL0025 inhibited the level of inflammation in the kidney tissue of hyperuricemia mice.

[0130] Experimental Example 7: Analysis of the anti-inflammatory level of probiotic supernatant in vitro

[0131] Experimental procedure: RAW264.7 cells were seeded into 12-well plates (1×10 6 cells / well), and incubated in a CO2 incubator at 37°C for 12 h to adhere to the wall. Lipopolysaccharide LPS (5 μg / mL) was added to stimulate the inflammatory response of RAW264.7 cells. After 6 h, 50 μL of blank MRS medium and supernatant of Lactobacillus paracasei JNL0025 were added, respectively, and incubated in a CO2 incubator at 37°C for 24 h. The levels of inflammatory factors TNF-α and IL-6 in the cell supernatant were detected using mouse TNF-α (Biyuntian, PT512) and IL-6 (Biyuntian, PI326) ELISA kits, respectively. Cells were labeled with antibodies against mouse F4 / 80, CD206 and CD86, and cell populations were analyzed by flow cytometry. After LPS stimulation of RAW264.7 cells for 6 h, bacterial supernatants of Lactobacillus paracasei JNL0025 and Lactobacillus rhamnosus LGG were added, respectively, and incubated for 24 h.

[0132] The results of flow cytometry analysis showed that compared with the LPS-treated group, Lactobacillus paracasei JNL0025 had a tendency to reduce the pro-inflammatory M1 macrophages and significantly increased the proportion of anti-inflammatory M2 macrophages, such as Figure 7 As shown in (a) and (b);

[0133] The results of ELISA analysis showed that Lactobacillus paracasei JNL0025 significantly inhibited the secretion of pro-inflammatory cytokines TNF-α and IL-6. Figure 7 As shown in (c) and (d). Moreover, Lactobacillus paracasei JNL0025 has a better inhibitory effect on inflammation than Lactobacillus rhamnosus LGG.

[0134] Test Example 8: Analysis of Antioxidant Levels in Probiotic Supernatant in Vitro

[0135] Experimental process: Lactobacillus paracasei JNL0025 and Lactobacillus casei Shirota strain Lactobacillus casei strain Shirota and MRS medium, anaerobically cultured at 37℃ for 48 h, centrifuged at high speed, collected the bacterial suspension, and filtered through a 0.22 μm filter. The antioxidant level of the supernatant was detected by total antioxidant capacity (T-AOC) colorimetric test kit (ABTS enzyme catalysis method) (Elabscience, E-BC-K219-M), total antioxidant capacity (T-AOC) colorimetric test kit (FRAP method) (Elabscience, E-BC-K225-M) and total antioxidant capacity (T-AOC) colorimetric test kit (Elabscience, E-BC-K136-M). Blank MRS medium was used as a control.

[0136] The antioxidant level of bacterial supernatant is shown in Table 6. The antioxidant level of the supernatant of Lactobacillus paracasei JNL0025 is not lower than that of the commercial strain Lactobacillus casei Shirota.

[0137] Table 6 Antioxidant levels of supernatant of Lactobacillus paracasei JNL0025

[0138]

[0139] Experimental Example 9: Animal Experimental Analysis of Probiotics Alleviating DSS-Induced Colitis

[0140] Experimental procedure: After one week of adaptive feeding, 6-8 week old male C57Bl / 6N mice were allowed to drink freely 2.5% DSS (molecular weight 36,000-50,000Da) aqueous solution to induce colitis mouse model. Lactobacillus paracasei JNL0025 cultured for 24 h was centrifuged at 5000 rpm and 4°C for 5 min, the supernatant was discarded, and the cells were resuspended in saline. Each mouse was gavaged with 2×10 9 CFU. 5-Amino Salicylic Acid (5-ASA, 100 mg / kg / d) was used as a positive drug control. The experimental period was 7 days, during which the weight, diarrhea, blood in stool and activity status of the mice were monitored daily. The mice were anesthetized and killed by cervical dislocation, and colon tissues were collected for hematoxylin-eosin (H&E) staining analysis. ELISA kits were used to detect the levels of inflammatory factors IL-β, IL-6 and IL-10 in the serum of mice (Lianke Bio, EK201B, EK206 and EK210 / 4-96).

[0141] The scoring criteria for the disease activity index (DAI) of colitis mice are shown in Table 7:

[0142] Table 7 DAI scoring criteria for colitis model mice

[0143]

[0144] The DAI score is the sum of the scores of weight loss, diarrhea, and blood in stool.

[0145] The colitis mouse model was induced by dextran sulfate sodium (DSS) aqueous solution, and 5-aminosalicylic acid (100 mg / kg / d), 2×10 9 CFU of Lactobacillus paracasei JNL0025 or 2 × 10 9 CFU Lactobacillus rhamnosus LGG.

[0146] like Figure 8 As shown in (a) and (b), Lactobacillus paracasei JNL0025 has a tendency to slow down the weight loss of colitis mice and reduces the disease activity index (DAI score) of mice; among them, Lactobacillus rhamnosus LGG and 5-aminosalicylic acid have weaker effects on improving the weight and disease activity index of mice.

[0147] like Fig. 9 and Fig.10As shown, treatment with Lactobacillus paracasei JNL0025 can improve DSS-induced colon shortening, significantly enhance intestinal barrier integrity, and inhibit inflammatory cell infiltration in colon tissue and mucosal destruction; among them, 5-aminosalicylic acid can inhibit colon inflammatory cell infiltration and protect intestinal barrier integrity, but Lactobacillus rhamnosus LGG has a weaker protective effect on intestinal tissue structure.

[0148] like Fig.11 As shown in (a), (b) and (c), the results of ELISA analysis showed that Lactobacillus paracasei JNL0025 significantly reduced the levels of cytokines IL-1β and IL-6 in the serum of colitis mice, and significantly upregulated the level of IL-10 in the serum; among them, the inhibitory effect of Lactobacillus rhamnosus LGG on intestinal inflammation was weaker than that of Lactobacillus paracasei JNL0025.

[0149] The strain sequence is as follows:

[0150]

[0151] 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 is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei ) JNL0025, the strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on June 24, 2024, with the deposit number CGMCC NO: 31043.

2. A microbial agent, characterized in that: The microbial agent contains the Lactobacillus paracasei described in claim 1 ( Lacticaseibacillus paracasei )JNL0025.

3. A Lactobacillus paracasei according to claim 1 ( Lacticaseibacillus paracasei ) Use of JNL0025 or the microbial agent described in claim 2 in the preparation of uric acid lowering products and the preparation of products for treating colitis.

4. The use according to claim 3, characterized in that The product described is a drug.

5. The use according to claim 4, characterized in that The dosage form of the drug is an oral dosage form in the form of powder, granule, tablet, capsule, suspension, emulsion, syrup or spray, and a sterile injection solution.

6. The use according to claim 3, characterized in that The drug has at least one of the following functions (1) to (10): (1) Degradation of guanosine; (2) Inhibit the activity of xanthine oxidase; (3) Inhibit the activity of purine nucleoside phosphorylase; (4) Treatment of gout; (5) Treatment of hyperuricemia; (6) Treatment of uric acid nephropathy; (7) Anti-inflammatory; (8) Antioxidant; (9) Regulate the structure of intestinal flora; (10) Enhance the intestinal mucosal barrier function.

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