A strain of Pediococcus pentosaceus JNL0053 and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a strain of Pediococcus pentosaceus JNL0053 and its application. The present invention provides a strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ) JNL0053, was deposited in the General Microbiology Center of China Microbiological Culture Collection Committee on June 24, 2024, with the deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31046. The Pediococcus pentosaceus JNL0053 in the present invention has the ability of nucleoside degradation, anti-inflammatory and antibacterial, and can be used to prepare drugs for the treatment of hyperuricemia, gout, uric acid nephropathy and colitis. It has extremely high development value and practical value, and provides new ideas for the development and application of related products.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Pediococcus pentosaceus JNL0053 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 disorder 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. At present, hyperuricemia has become the second largest metabolic disease in the world. With the increasing number of patients with hyperuricemia, the global demand for uric acid-lowering products is rapidly expanding.

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

[0005] At present, drug therapy combined with strict dietary management is commonly used in clinical practice to regulate blood uric acid levels. However, 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.). Moreover, these drugs have great 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. In view of this, the market urgently needs a new, safe and effective uric acid-lowering product to meet a wide range of health needs.

[0006] 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, thus causing elevated blood uric acid and gout attacks. Recent studies have shown that probiotics can reduce 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 reactions 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. 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.

[0007] The changes in the intestinal microecology of IBD patients are mainly manifested as dysbiosis, including changes in flora diversity, species and abundance. Probiotics can effectively regulate the imbalance of intestinal flora, improve the microecological environment, enhance the barrier function of the intestinal mucosa, regulate local and systemic immune responses, and provide new options for the treatment of diseases such as IBD. Bifidobacterium, Lactobacillus, VSL#3 probiotics, etc. have been widely used in clinical treatment.

[0008] Compared with traditional drug treatment, probiotics are safer and more economical in treating hyperuricemia, gout and enteritis, and have no obvious side effects, making them suitable for long-term use. Therefore, probiotics have multiple mechanisms and significant advantages in treating hyperuricemia, gout and enteritis, providing patients with a new treatment option. Summary of the invention

[0009] In view of this, the present invention provides a Pediococcus pentosaceus JNL0053 and its application, which has the function of treating hyperuricemia, gout, uric acid nephropathy and colitis.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] The present invention provides Pediococcus pentosaceus ( Pediococcus pentosaceus ) JNL0053, deposited on June 24, 2024 in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.31046.

[0012] In some specific embodiments of the present invention, the above-mentioned Pediococcus pentosaceus ( Pediococcus pentosaceus ) JNL0053 is alive or dead or intermittently sterilized, or in the form of a lysate and / or an extract, or in the form of a bacterial product, or in the form of a supernatant or a derivative, and the derivative form includes: metabolites, metabolic biological products, prebiotics, cell walls and their components, extracellular polysaccharides, and compounds containing immunogenic components, including: supernatants, inactivated bacteria.

[0013] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus JNL0053 in the preparation of a product for degrading uric acid; the product is a medicine, and the medicine contains acceptable excipients and / or adjuvants.

[0014] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus JNL0053 in preparing a product for degrading guanosine nucleoside; the product is a medicine, and the medicine contains acceptable excipients and / or adjuvants.

[0015] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus JNL0053 in the preparation of a product for inhibiting the activity of xanthine oxidase; the product is a medicine, and the medicine contains acceptable excipients and / or adjuvants.

[0016] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus JNL0053 in the preparation of a product for inhibiting the activity of purine nucleoside phosphorylase; the product is a medicine, and the medicine contains acceptable excipients and / or adjuvants.

[0017] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus JNL0053 in the preparation of a product for treating hyperuricemia and / or gout and / or uric acid nephropathy; the product is a medicine, and the medicine contains acceptable excipients and / or adjuvants.

[0018] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus JNL0053 in preparing a product for inhibiting GLUT9 gene expression; the product is a medicine, and the medicine contains acceptable excipients and / or adjuvants.

[0019] The present invention also provides the use of the Pediococcus pentosaceus JNL0053 in preparing a product for treating colitis; the product is a medicine, and the medicine contains acceptable excipients and / or adjuvants.

[0020] The present invention also provides a microbial agent, comprising the above-mentioned Pediococcus pentosaceus JNL0053 and acceptable adjuvants and / or auxiliary agents.

[0021] The present invention also provides a medicine comprising the above Pediococcus pentosaceus JNL0053 and / or the above microbial agent.

[0022] In some specific embodiments of the present invention, the above-mentioned auxiliary materials or auxiliary agents include nutritional enhancers, sweeteners, acidity regulators, isotonicity regulators, fillers, binders, disintegrants, solubilizers, solubilizers, preservatives, flavoring agents, colorants, suspending agents, wetting agents, emulsifiers and / or surfactants.

[0023] The medicine may also contain an appropriate amount of commonly used carriers, excipients and diluents. Moreover, according to the usual method, it can be made into a dosage form in the form of an oral agent such as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, and sterile injection solutions. The non-pharmaceutical active ingredients such as carriers, excipients and diluents that may 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.

[0024] The carrier, excipient and diluent of the present invention include, but are not limited to, lactose, glucose, sorbitol, mannitol, xylitol, maltitol, starch, gum arabic, gelatin, calcium phosphate, cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, and mineral oil, etc. No specific limitation is made here.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. The present invention provides a strain of Pediococcus pentosaceus JNL0053. The results of in vitro experiments show that the supernatant of Pediococcus pentosaceus JNL0053 has an inhibition rate of 43.2% on xanthine oxidase (XOD) activity and 49.6% on purine nucleoside phosphorylase (PNP). Lactobacillus casei The inhibition rate of the supernatant of strain Shirota on XOD was only 27%, and it could not inhibit the activity of PNP. The results of animal experiments showed that Pediococcus pentosaceus JNL0053 could significantly reduce the blood uric acid level of hyperuricemia model mice, and at the same time reduce the level of kidney inflammation. It has the function of treating hyperuricemia, gout, and uric acid nephropathy, showing great application value.

[0027] 2. The Pediococcus pentosaceus JNL0053 of the present invention significantly inhibited the secretion of proinflammatory cytokines TNF-α and IL-6, reduced the levels of cytokines IL-1β and IL-6 in the serum of colitis mice, and significantly increased the level of IL-10 in the serum, thus having the function of treating colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0029] Figure 1is a phylogenetic tree diagram of Pediococcus pentosaceus JNL0053 in Example 1;

[0030] Figure 2 This is a morphological diagram of the Pediococcus pentosaceus JNL0053 strain in Example 1;

[0031] Figure 3 This is a hemolytic analysis chart of Pediococcus pentosaceus JNL0053 strain in Test Example 5;

[0032] Figure 4 This is a graph showing the effect of Pediococcus pentosaceus JNL0053 in reducing blood uric acid in hyperuricemia model mice in Experimental Example 6; wherein (a) is body weight, (b) is serum uric acid content, and ** is p<0.01;

[0033] Figure 5 This is a diagram showing the effect of Pediococcus pentosaceus JNL0053 in improving the renal function of hyperuricemia model mice in Experimental Example 6; wherein (a) is the serum creatinine content, (b) is the serum urea content, * is p<0.05, ** is p<0.01, *** is p<0.001;

[0034] Figure 6 This is the effect diagram of Pediococcus pentosaceus JNL0053 in reducing the transcription level of GLUT9 gene in renal tissue of hyperuricemia model mice in Experimental Example 6, *** means p<0.001;

[0035] Figure 7 The figure is a diagram showing the effect of the supernatant of Pediococcus pentosaceus JNL0053 in Experimental Example 7 on inhibiting the inflammatory polarization of macrophages and the secretion of inflammatory factors; wherein (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;

[0036] Figure 8 This is a graph showing the effect of Pediococcus pentosaceus JNL0053 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;

[0037] Fig. 9 This is a diagram showing the effect of Pediococcus pentosaceus JNL0053 in reducing DSS-induced colitis in colon tissue H&E staining in Experimental Example 9;

[0038] Fig.10 This is a graph showing the effect of Pediococcus pentosaceus JNL0053 in reducing DSS-induced colitis on colorectal length in Experimental Example 9; where * means p<0.05, and ** means p<0.01;

[0039] Fig.11 This is a graph showing the effect of Pediococcus pentosaceus JNL0053 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, (c) is the level of serum IL-10, * is p<0.05, ** is p<0.01. DETAILED DESCRIPTION

[0040] The present invention discloses Pediococcus pentosaceus JNL0053 and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine 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.

[0041] 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 of ordinary skill in the art to which the present invention belongs. Unless otherwise specified, 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 all be purchased from the market.

[0042] The composition of the MRS culture medium used in the embodiment is as follows:

[0043] 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 5.7±0.2 (25℃).

[0044] Example 1 Separation and purification

[0045] The Pediococcus pentosaceus JNL0053 provided by the present invention is isolated from cheese collected in Inner Mongolia. An appropriate amount of fermented cheese is taken, and physiological saline is added for gradient dilution, and the mixture is evenly applied to an MRS solid medium and anaerobically cultured at 37°C for 48 hours. A single colony is picked with a sterilized inoculation loop, and the colony is streaked and purified on an MRS solid medium, and cultured at 37°C for 48 hours to separate the Pediococcus pentosaceus strain. A single colony is picked for Gram staining and observed under a microscope. Figure 2 As shown, Pediococcus pentosaceus JNL0053 is spherical, arranged in pairs or quadruplets, with a diameter of 0.47~0.67μm, and is Gram-positive.

[0046] The bacterial 16S rRNA was amplified using primers 27F 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1) and 1492R 5'-GGCTTACCTTGTTACGACTT-3' (SEQ ID NO.2). The bacterial 16S rRNA gene sequence is:

[0047]

[0048] By comparing the NCBI database, a phylogenetic tree was constructed, such as Figure 1 As shown, the alignment result is Pediococcus pentosaceus Pediococcus pentosaceus .

[0049] The applicant named the Pentosaceae Pediococcus JNL0053, and deposited it in the General Microbiological Center of the China Microbiological Culture Collection Administration on June 24, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 31046, and the classification name is Pediococcus Pentosaceae Pediococcus pentosaceus .

[0050] Example 2 Cultivation of strains

[0051] (1) Bacterial culture and counting

[0052] Prepare MRS medium according to the instructions and sterilize at 118℃ for 20 min. After cooling to room temperature, inoculate Pediococcus pentosaceus JNL0053 at a 1% inoculation rate and culture anaerobically at 37℃ for 24 h. After culture, the cultured bacterial solution was diluted with physiological saline gradient, and 100μL was evenly spread on MRS solid medium and cultured anaerobically at 37℃ for 48 h. The bacterial growth was counted according to the number of colonies grown. After 24h of culture of Pediococcus pentosaceus JNL0053, the OD600 of the bacterial solution was 1.40, and the number of viable bacteria was 1.8×10 9 CFU / mL.

[0053] (2) Bacterial supernatant separation

[0054] The bacterial culture solution cultured in step (1) was centrifuged at 12,000 rpm and 4°C for 10 min, the supernatant was collected, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the obtained product.

[0055] Experimental Example 1 Analysis of Degraded Guanosine Nucleoside Levels

[0056] Pediococcus pentosaceus JNL0053 and Lactobacillus casei Shirota Lactobacillus casei strain Shirota was cultured at 37°C overnight to obtain seed solution. Centrifuged at 4°C 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℃ with shaking at 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.

[0057] Guanosine 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.

[0058] 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 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.

[0059] Guanosine conversion rate = (1-residual guanosine in supernatant / original guanosine in supernatant) * 100%

[0060] Guanosine removal rate = [1-(residual guanosine + guanine in supernatant) / original guanosine in supernatant]*100%

[0061] The analysis results showed (Table 1) that the guanosine conversion rate of Pediococcus pentosaceus JNL0053 was 6.5% and the guanosine removal rate was 1.3%. Lactobacillus casei The guanosine conversion rate and guanosine removal rate of strain Shirota were 14.54% and 5.97%, respectively.

[0062] Table 1 Guanosine conversion rate and guanosine removal rate

[0063]

[0064] Experimental Example 2 Analysis of the level of inhibition of xanthine oxidase (XOD) by bacterial supernatant

[0065] After the culture, the bacteria were centrifuged at 12000 rpm and 4°C for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter. A 10U / 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).

[0066] XOD inhibition rate = (1-XOD activity of sample group / XOD activity of positive control) * 100%

[0067] The analysis results showed (Table 2) that the inhibition rate of the supernatant of Pediococcus pentosaceus JNL0053 on XOD was 43.2%, while the inhibition rate of the supernatant of Lactobacillus casei Shirota strain on XOD was only 27%.

[0068] Table 2 XOD inhibition rate (%)

[0069]

[0070] Experimental Example 3 Analysis of the level of inhibition of purine nucleoside phosphorylase (PNP) by bacterial supernatant

[0071] After the culture, the bacteria were centrifuged at 12000 rpm and 4°C for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter. An 80U / 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.

[0072] PNP inhibition rate = (1-PNP activity of sample group / PNP activity of positive control) * 100%

[0073] The analysis results showed (Table 3) that the inhibition rate of the supernatant of Pediococcus pentosaceus JNL0053 against PNP was 49.6%.

[0074] Table 3 PNP inhibition rate (%)

[0075]

[0076] Test Example 4 Analysis of bacterial antibiotic sensitivity

[0077] Pediococcus pentosaceus JNL0053 was inoculated into liquid MRS medium and cultured anaerobically at 37℃ for 24 h to obtain seed solution. The bacterial solution was diluted to 0.5 McFarland turbidity with 0.85% saline, and 100 μL of bacterial solution was accurately aspirated and evenly spread on the surface of MRS agar medium. After the surface bacterial solution dried, a drug-sensitive paper was placed on the surface of the culture medium. The sensitivity of Pediococcus pentosaceus JNL0053 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), was detected by KB paper diffusion method. The sensitivity of each bacterial strain to each antibiotic was repeated three times. Cultured anaerobically at 37℃ for 24 h. The diameters of the inhibition zones were measured and recorded. The results are shown in Table 4.

[0078] Table 4 Antibiotic inhibition zone of Pediococcus pentosaceus JNL0053

[0079]

[0080] Test Example 5 Bacterial hemolytic analysis

[0081] 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. Leave at room temperature for no more than 4 h, and store at 4°C for no more than 7 d. Streak Pediococcus pentosaceus JNL0053 and the control strain on Columbia blood agar plates. Incubate anaerobically at 37°C for 18–24 h, and observe the colony morphology.

[0082] α-hemolysis is manifested as a narrow, grass-green hemolysis ring; β-hemolysis is manifested as a wide, well-defined, completely transparent hemolysis ring; γ-hemolysis is non-hemolysis, that is, no hemolysis ring appears. Figure 3 (a) Figure 3 (b) and Figure 3 As shown in (c), Aerococcus aureus was used as an α-hemolytic control strain, Streptococcus pyogenes CICC10373 was used as a β-hemolytic control strain, and Pediococcus pentosaceus JNL0053 did not show a hemolytic ring.

[0083] Experimental Example 6 Animal Experiment Analysis of the Therapeutic Effect of Probiotics on Hyperuricemia Model Mice

[0084] After 1 week of adaptive feeding, 6-8 week-old male C57BL / 6J mice (weight 19-23 g) were gavaged daily with hypoxanthine and potassium oxonate (the gavage dose of both drugs was 300 mg / kg, dissolved in 0.5% sodium carboxymethylcellulose solution) to induce hyperuricemia mouse model. The control group mice were gavaged with the same volume of 0.5% sodium carboxymethylcellulose solution. After an interval of 2 h, the treatment drugs were gavaged and 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 probiotic treatment group was gavaged with 2×10 9 CFU Pediococcus pentosaceus JNL0053 bacteria, the experimental treatment time was 15 days. The mice were weighed on the 0th day, 5th day, 10th day and 15th day, and the blood of the posterior venous plexus of the mice was collected. After the experiment, the kidney tissue of the mice was collected and stored at -80℃ for future use.

[0085] The collected blood was left to stand at room temperature for 2 h, then centrifuged at 3000 rpm for 15 min to collect serum. 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 urea in mouse serum.

[0086] like Figure 4 As shown in (a) and (b), 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 Pediococcus pentosaceus JNL0053 treatment significantly reduced the blood uric acid level; Figure 5 As shown in (a), the serum creatinine level of mice in the allopurinol group tended to increase, suggesting that allopurinol may have caused renal damage. Compared with the allopurinol group, the serum creatinine level of mice in the Pediococcus pentosaceus JNL0053 group was significantly reduced; Figure 5 (b) in the figure shows that the urea content in the serum of the Pediococcus pentosaceus JNL0053 group was significantly lower than that of the model group and the allopurinol group, indicating that Pediococcus pentosaceus JNL0053 has the effect of protecting renal function.

[0087] Total RNA from renal 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). 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 expression levels of other target genes relative to the internal reference gene were calculated using the formula. The primer sequences are shown in Table 5.

[0088] Table 5 Primer sequences

[0089]

[0090] The results are as follows Figure 6 As shown in the figure, compared with the model group and allopurinol group, the treatment of Pediococcus pentosaceus JNL0053 significantly reduced the transcription level of GLUT9, a key gene for uric acid reabsorption, in the kidney tissue of mice. GLUT9 (glucose transporter-9) plays an important role in the reabsorption of uric acid by the kidney. The above results indicate that JNL0053 can inhibit the reabsorption of uric acid by the mouse kidney.

[0091] Experimental Example 7 Analysis of the anti-inflammatory level of probiotic supernatant in vitro

[0092] RAW264.7 cells were seeded into 12-well plates (1 × 10 6 cells / well), CO 2 The cells were incubated 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 Pediococcus pentosaceus JNL0053 were added respectively. CO 2 The cells were incubated at 37°C in an incubator for 24 h. Mouse TNF-α (Biyuntian, PT512) and IL-6 (Biyuntian, PI326) ELISA kits were used to detect the levels of inflammatory factors TNF-α and IL-6 in the cell supernatant, respectively. Antibodies against mouse F4 / 80, CD206, and CD86 were used to label cells, and flow cytometry was used to analyze cell populations.

[0093] After 6 h of LPS stimulation, the supernatants of Pediococcus pentosaceus JNL0053 and Lactobacillus rhamnosus LGG were added to the RAW264.7 cells and incubated for 24 h. Figure 7 As shown in (a) and (b), compared with the LPS-treated group, Pediococcus pentosaceus JNL0053 tended to reduce the proportion of pro-inflammatory M1 macrophages and significantly increased the proportion of anti-inflammatory M2 macrophages. Figure 7 As shown in (c) and (d), Pediococcus pentosaceus JNL0053 significantly inhibited the secretion of proinflammatory cytokines TNF-α and IL-6, and Pediococcus pentosaceus JNL0053 had a better inhibitory effect on inflammation than Lactobacillus rhamnosus LGG.

[0094] Experimental Example 8 Analysis of Antioxidant Levels in Probiotic Supernatant in Vitro

[0095] Experimental process: Pediococcus pentosaceus JNL0053 and Lactobacillus casei Shirota strain Lactobacillus casei strainShirota and MRS medium, cultured anaerobically at 37℃ for 48 h, centrifuged at high speed, collected the bacterial solution, and filtered through a 0.22 μm filter membrane. 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. The antioxidant level of the bacterial supernatant is shown in Table 6. The antioxidant level of the supernatant of Pediococcus pentosaceus JNL0053 is not lower than that of the commercial strain Lactobacillus casei Shirota strain.

[0096] Table 6 Antioxidant levels of the supernatant of Pediococcus pentosaceus JNL0053

[0097]

[0098] Experimental Example 9 Probiotics alleviate DSS-induced colitis

[0099] 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. Pediococcus pentosaceus JNL0053 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 physiological 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).

[0100] The scoring criteria for the disease activity index (DAI) of colitis mice are shown in Table 7.

[0101] Table 7 DAI scoring criteria for colitis model mice

[0102]

[0103] The DAI score is the sum of the scores of weight loss, diarrhea, and blood in stool.

[0104] 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 Pediococcus pentosaceus JNL0053 or 2 × 10 9 CFU Lactobacillus rhamnosus LGG. Figure 8 As shown in (a) and (b), Pediococcus pentosaceus JNL0053 has a tendency to slow down the weight loss of colitis mice and reduce the disease activity index (DAI score) of mice, while Lactobacillus rhamnosus LGG and 5-aminosalicylic acid have weaker effects on improving the weight and disease activity index of mice. Fig. 9 and Fig.10 As shown in the figure, Pediococcus pentosaceus JNL0053 treatment can improve DSS-induced colon shortening, significantly enhance intestinal barrier integrity, inhibit inflammatory cell infiltration in colon tissue and mucosal destruction, 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. ELISA analysis results are shown in the figure. Fig.11 As shown, Pediococcus pentosaceus JNL0053 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. The inhibitory effect of Lactobacillus rhamnosus LGG on intestinal inflammation was weaker than that of Pediococcus pentosaceus JNL0053.

[0105] 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 Pediococcus pentosaceus JNL0053, characterized in that: The Pediococcus pentosaceus JNL0053 was deposited in the China General Microbiological Culture Collection Center on June 24, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO: 31046, and the classification name is Pediococcus pentosaceus Pediococcus pentosaceus .

2. Use of Pediococcus pentosaceus JNL0053 according to claim 1 in preparing a product for treating colitis; The product is a drug, which contains acceptable excipients.

3. A microbial agent, characterized in that: The invention comprises the Pediococcus pentosaceus JNL0053 as claimed in claim 1 and acceptable excipients.

4. A drug, characterized in that The drug comprises the Pediococcus pentosaceus JNL0053 as claimed in claim 1 and / or the microbial agent as claimed in claim 3, and the drug has the functions of lowering uric acid and treating colitis.

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