Application of a strain of Lactobacillus delbrueckii JNL0010 in the preparation of uric acid-lowering drugs

Abstract

The present invention discloses an application of Lactobacillus delbrueckii JNL0010 in the preparation of uric acid-lowering drugs, and belongs to the field of microbial technology. The Lactobacillus delbrueckii JNL0010 was deposited in the China General Microbiological Culture Collection Management Center on June 24, 2024, with a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a preservation number of CGMCC No.31042. The results of animal experiments showed that Lactobacillus delbrueckii JNL0010 can significantly reduce the blood uric acid level of hyperuricemia model mice and reduce the level of renal inflammation, showing great application value. Lactobacillus delbrueckii JNL0010 can not only degrade purines and nucleosides, reduce serum uric acid, and has a certain protective effect on the kidneys, but also has higher safety and less side effects than drugs, and has broad market prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to application of a strain of Lactobacillus delbrueckii JNL0010 in preparing a uric acid-lowering drug. Background Art

[0002] 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. Currently, 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 drugs is rapidly expanding.

[0003] Gout is caused by long-term purine metabolism disorder, which leads to high uric acid concentration in the blood, causing urate crystals to deposit in joints, cartilage and other tissues, causing tissue foreign body inflammatory reaction. Gout is the disease most closely related to hyperuricemia.

[0004] 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 drug to meet a wide range of health needs.

[0005] Studies have shown that the composition and functional structure of the intestinal flora of gout patients are significantly different from those of healthy people. 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 Inhibit 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.

[0006] 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.

[0007] The patent publication "A strain of lactic acid bacteria capable of degrading creatinine and urea and a method for screening the same" (publication number: CN103667124A) disclosed that Lactobacillus delbrueckii subspecies bulgaricus has the ability to reduce creatinine, but only provides the results of in vitro tests, and its in vivo effects and mechanisms are unknown.

[0008] There is currently no disclosure of Lactobacillus delbrueckii that is capable of directly degrading biological uric acid levels. Summary of the invention

[0009] In view of the gap in the prior art, the present invention provides a strain of Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. bulgaricus ) Application of JNL0010 in the preparation of a uric acid-lowering drug, wherein the Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. bulgaricus ) JNL0010 was deposited in the China General Microbiological Culture Collection Center 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. 31042. The classification name is Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp. bulgaricus .

[0010] Experimental studies of the present invention show that the Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. bulgaricus ) The drug prepared by JNL0010 has at least one of the following characteristics:

[0011] b1) Degradation of guanosine;

[0012] b2) Inhibit the activity of urate synthase.

[0013] The Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. bulgaricus ) The conversion rate of guanine nucleoside by JNL0010 was 100%, and the removal rate of guanine nucleoside was 54.52%.

[0014] The urate synthase is xanthine oxidase (XOD).

[0015] Experimental studies of the present invention show that the Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. bulgaricus ) The drug prepared by JNL0010 also has at least one of the following characteristics:

[0016] c1) reduce serum uric acid levels;

[0017] c2) reduce serum creatinine levels;

[0018] c3) Reduce serum urea levels.

[0019] Based on the above characteristics of the drug, the following applications may be included:

[0020] a1) Treatment of hyperuricemia;

[0021] a2) Treatment of gout;

[0022] a3) Treatment of uric acid nephropathy;

[0023] a4) Reduce kidney damage.

[0024] Preferably, the drug contains at least Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. bulgaricus )JNL0010 as the active ingredient.

[0025] In one embodiment of the present invention, the drug comprises Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. bulgaricus ) Lyophilized powder of JNL0010.

[0026] Furthermore, the medicine also includes an excipient.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention provides a new application of Lactobacillus delbrueckii JNL0010. The results of in vitro experiments show that the strain can degrade 9.33% uric acid within 2 hours, the guanine nucleoside conversion rate is 100%, and the guanine nucleoside removal rate is 54.52%. The inhibition rate of the supernatant of Lactobacillus delbrueckii JNL0010 on xanthine oxidase (XOD) activity is 12.9%. In comparison, the commercial strain Lactobacillus casei Shirota strain Lactobacillus casei strain Shirota, which does not have the ability to degrade uric acid, has a guanosine conversion rate of only 14.54% and a guanosine removal rate of only 5.9%.

[0029] (2) Animal experiment results show that the Lactobacillus delbrueckii JNL0010 provided by the present invention can significantly reduce the blood uric acid level of hyperuricemia model mice and alleviate kidney pathological damage, showing great application value.

[0030] (3) The Lactobacillus delbrueckii JNL0010 provided by the present invention can not only degrade purine and nucleoside and reduce high serum uric acid, but also has a certain protective effect on the kidney. Compared with drugs, it is safer and has fewer side effects, and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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.

[0032] Figure 1 This is a graph showing the effect of Lactobacillus delbrueckii JNL0010 in reducing the serum uric acid level in hyperuricemia model mice in Example 4; wherein (a) is body weight, and (b) is serum uric acid level. ** is p <0.01.

[0033] Figure 2 This is a diagram showing the effect of Lactobacillus delbrueckii JNL0010 in improving the renal function of hyperuricemia model mice in Example 4; (a) is the serum creatinine content, and (b) is the serum urea content. *** is p <0.001, **** p <0.0001.

[0034] Figure 3 This is a diagram showing the effect of Lactobacillus delbrueckii JNL0010 in reducing the transcription level of GLUT9 gene in the renal tissue of hyperuricemia model mice in Example 4. p <0.01.

[0035] Figure 4 This is a diagram showing the effect of Lactobacillus delbrueckii JNL0010 in reducing the transcription level of inflammatory cytokines in renal tissue of hyperuricemia model mice in Example 4. * p <0.05, ** p <0.01, *** p <0.001. DETAILED DESCRIPTION

[0036] 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.

[0037] The composition of the MRS culture medium used in the embodiment is as follows:

[0038] Peptone 10.0g / L, beef powder 8.0g / L, yeast powder 4.0g / L, glucose 20.0g / L, dipotassium hydrogen phosphate 2.0g / L, diammonium hydrogen citrate 2.0g / L, sodium acetate 5.0g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 80 1.0g / L. pH value 5.7±0.2 (25℃).

[0039] The following information is provided in the prior patent application 202411101718X, which is cited in the present invention to illustrate the source of the Lactobacillus delbrueckii JNL0010.

[0040] The Lactobacillus delbrueckii JNL0010 provided by the present invention is isolated from fermented yogurt collected in Qinghai Province. The strain can degrade 9.33% of uric acid within 2 hours, with a guanine nucleoside conversion rate of 100% and a guanine nucleoside removal rate of 54.52%. The supernatant of Lactobacillus delbrueckii JNL0010 has an inhibition rate of 12.9% on xanthine oxidase (XOD) activity. In comparison, the commercial strain Lactobacillus casei Shirota strain Lactobacillus casei strain Shirota, which does not have the ability to degrade uric acid, has a guanine nucleoside conversion rate of only 14.54% and a guanine nucleoside removal rate of only 5.9%. Animal experimental results show that Lactobacillus delbrueckii JNL0010 can significantly reduce the blood uric acid level of hyperuricemia model mice and alleviate the level of kidney inflammation, showing great application value.

[0041] Separation and purification:

[0042] Prepare MRS medium according to the instructions and sterilize at 118℃ for 20min. Take an appropriate amount of yogurt sample, add physiological saline to dilute it in a gradient, evenly spread it on MRS solid medium, and culture it anaerobically at 37℃ for 48h. Pick a single clone and observe the bacterial morphology by Gram staining. Use primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') (SEQ ID NO.12 in the sequence list) and 1492R (5'-GGCTTACCTTGTTACGACTT-3') (SEQ ID NO.13 in the sequence list) to amplify bacterial 16S rRNA. The bacterial 16S rRNA gene sequence is shown in the sequence list SEQ ID NO.1:

[0043]

[0044] By comparing the NCBI database, a phylogenetic tree was constructed, and the results are as follows: L. delbrueckii subsp. bulgaricus .

[0045] The Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. bulgaricus ) JNL0010 was deposited in the China General Microbiological Culture Collection Center 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. 31042. The classification name is Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp. bulgaricus .

[0046] The Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. bulgaricus ) Analysis of the morphological characteristics of the JNL0010 strain: Single colonies were picked for Gram staining and observed under a microscope. The results showed that Lactobacillus delbrueckii JNL0010 was rod-shaped or slightly curved, 3.4~6.9μm (length) × 0.6~0.8μm (width), and was Gram-positive.

[0047] Bacterial culture and counting:

[0048] Prepare MRS medium according to the instructions for use and sterilize at 118℃ for 20min. After cooling to room temperature, inoculate Lactobacillus delbrueckii JNL0010 at a 1% inoculation rate and culture anaerobically at 37℃ for 24h. After culture, the cultured bacterial solution was gradient diluted with physiological saline, and 100μL was evenly spread on MRS solid medium and cultured anaerobically at 37℃ for 48h. The bacterial growth was counted according to the number of colonies grown. OD value of bacterial solution after 24h culture of Lactobacillus delbrueckii JNL0010 600 =0.30, the number of viable bacteria is 2.0×10 7 CFU / mL. OD of bacterial solution after 36h of culture 600 =2.30, OD of 48h bacterial solution 600 =3.05.

[0049] Bacterial supernatant separation:

[0050] The cultured 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 membrane.

[0051] Example 1, Analysis of Uric Acid Degradation Levels by Lactobacillus delbrueckii JNL0010

[0052] Lactobacillus delbrueckii JNL0010 and Lactobacillus casei Shirota strain Lactobacillus caseistrain Shirota was cultured at 37°C overnight to obtain seed solution. Centrifuged at 4°C and 6000 rpm for 5 min, the bacterial volume was about 4×10 7 CFU. Wash the bacteria twice with HEPES buffer (50mM, pH=7.0). Resuspend the bacteria in 1mL of uric acid solution (1mM, prepared in HEPES buffer), and use uric acid solution without bacteria (1mM, prepared in HEPES buffer) as a blank control. Incubate at 37℃ and 100rpm for 2h, then heat at 100℃ for 10min to terminate the reaction, centrifuge at 4℃ and 6000rpm for 5min, and use Elabscience uric acid colorimetric test kit (E-BC-K016-M) to detect the concentration of uric acid in the supernatant. The analysis results show (Table 1) that the uric acid degradation rate of Lactobacillus delbrueckii JNL0010 is 9.33%, and that of Lactobacillus casei Shirota is 9.33%. Lactobacillus casei strain Shirota has no ability to degrade uric acid.

[0053] Table 1. Uric acid degradation rate

[0054]

[0055] Example 2, Analysis of Degraded Guanosine Levels

[0056] Lactobacillus delbrueckii JNL0010 and Lactobacillus casei Shirota strain Lactobacillus casei strain Shirota was cultured at 37°C overnight to obtain seed solution. Centrifuged at 4°C and 6000 rpm for 5 min, the bacterial volume was about 4×10 7 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 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] Guanine nucleoside standards were prepared in PBS buffer at concentrations of 0.1mM, 0.25mM, 0.5mM, 0.75mM and 1mM. Guanine standards were prepared in PBS buffer at concentrations of 0.05mM, 0.1mM, 0.15mM, 0.75mM and 1mM.

[0058] Analytical instrument: Agilent 1260 Infiinity II HPLC, chromatographic column: Agilent ZORBAXSB-Aq. Chromatographic conditions: injection volume 20μL, flow rate 1mL / min, detection wavelength 254nm. The mobile phase used a 10mM ammonium formate aqueous solution, and the pH was adjusted to 3.3 with formic acid (300mL 10mM ammonium formate plus 60μL formic acid), 1% methanol was added, and it was used after ultrasonication (20min). The elution time was 25min, the guanosine peak time was 19.67min, and the guanine peak time was 15.7min.

[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 2) that the guanosine conversion rate of Lactobacillus delbrueckii JNL0010 was 100%, and the guanosine removal rate was 54.52%. Lactobacillus casei The guanosine conversion rate and guanosine removal rate of strain Shirota were 14.54% and 5.97%, respectively.

[0062] Table 2. Guanosine conversion rate and guanosine removal rate

[0063]

[0064] Example 3, Analysis of the level of inhibition of xanthine oxidase (XOD) by bacterial supernatant

[0065] The cultured bacteria were centrifuged at 12,000 rpm and 4°C for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter. Xanthine oxidase (XOD) activity was determined using a 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 3) that the inhibition rate of the supernatant of Lactobacillus delbrueckii JNL0010 on XOD was 12.90%.

[0068] Table 3. XOD inhibition rate

[0069]

[0070] Example 4: Animal experiment analysis of the therapeutic effect of probiotics on hyperuricemia model mice

[0071] 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 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 hours, the treatment drugs were gavaged and divided into the following groups: the control group and the model group were gavaged with 200 μL normal saline, the allopurinol group was gavaged with 5 mg / kg allopurinol solution (dissolved in normal saline), and the probiotic treatment group was gavaged with 2×10 9 CFU Lactobacillus delbrueckii JNL0010 bacteria, the experimental treatment time is 15 days.

[0072] 4.1. The mice were weighed and blood was collected from the posterior ocular venous plexus of the mice on days 0, 5, 10 and 15. 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 urea in mouse serum.

[0073] like Figure 1 (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 delbrueckii JNL0010 treatment significantly reduced the blood uric acid level.

[0074] Figure 2 The results in (a) showed that the serum creatinine level of mice in the allopurinol group tended to increase, suggesting that allopurinol may have caused renal damage. Figure 2 The results in (b) showed that the urea content in the serum of the Lactobacillus delbrueckii JNL0010 group was significantly lower than that of the model group and the allopurinol group, indicating that Lactobacillus delbrueckii JNL0010 has the effect of protecting renal function.

[0075] 4.2, Detection and analysis of GLUT9 and inflammatory factor gene transcription levels in mouse kidney tissue:

[0076] Renal tissues of hyperuricemia mice were collected, and total RNA from renal tissues was extracted using RNAiso Plus (Takara 9109). The mRNA in the samples was reverse transcribed into cDNA using the Evo M-MLV Reverse Transcription Premix Kit (Aikara, AG11728), and then the target gene was quantified using the SYBR Green Pro Taq HS Premix qPCR Kit (Aikara, AG11701). β-actin was used as an 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 as follows:

[0077]

[0078] Figure 3 The results showed that compared with the model group and allopurinol group, treatment with Lactobacillus delbrueckii JNL0010 significantly reduced the transcription level of GLUT9, a key gene for uric acid reabsorption in the kidney tissue of mice, indicating that the reabsorption of uric acid by the mouse kidney was inhibited.

[0079] Figure 4 The results showed that compared with the model group and allopurinol group, treatment with Lactobacillus delbrueckii JNL0010 significantly reduced the transcription levels of proinflammatory cytokines interleukin (IL)-6 (IL-6), tumor necrosis factor (TNF)-α (TNF-α), and nuclear factor-κB (NF-κB) in the kidney tissue of mice, indicating that the inflammation level of kidney tissue in hyperuricemia mice was inhibited and kidney damage was reduced, proving that it can be used to alleviate or treat uric acid nephropathy.

[0080] Example 5, a microbial preparation

[0081] The supernatant containing Lactobacillus delbrueckii JNL0010 prepared in Example 3 can be used to prepare drugs.

[0082] As an embodiment, in the medicine, Lactobacillus delbrueckii JNL0010 is the only effective active ingredient.

[0083] The supernatant may be freeze-dried to prepare a lyophilized powder.

[0084] It can also be combined with solvents or other excipients to treat hyperuricemia, gout and uric acid nephropathy. When combined with solvents, injections can be prepared. When combined with other solid excipients, other conventional dosage forms such as tablets and capsules can be prepared.

[0085] Alternatively, as another embodiment, Lactobacillus delbrueckii JNL0010 is combined with other active ingredients to treat hyperuricemia, gout and uric acid nephropathy.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. bulgaricus ) Use of JNL0010 in the preparation of drugs for treating hyperuricemia, gout and uric acid nephropathy, characterized in that: The Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. bulgaricus ) JNL0010 was deposited in the China General Microbiological Culture Collection Center on June 24, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.31042.

2. The Lactobacillus delbrueckii according to claim 1 ( Lactobacillus delbrueckii subsp. bulgaricus ) Use of JNL0010 in the preparation of drugs for treating hyperuricemia, gout and uric acid nephropathy, characterized in that: The medicine contains at least Lactobacillus delbrueckii JNL0010 as an active ingredient.

3. The Lactobacillus delbrueckii according to claim 1 ( Lactobacillus delbrueckii subsp. bulgaricus ) Use of JNL0010 in the preparation of drugs for treating hyperuricemia, gout and uric acid nephropathy, characterized in that: The medicine comprises freeze-dried powder of Lactobacillus delbrueckii JNL0010.

4. The Lactobacillus delbrueckii according to claim 1 ( Lactobacillus delbrueckii subsp. bulgaricus ) Use of JNL0010 in the preparation of drugs for treating hyperuricemia, gout and uric acid nephropathy, characterized in that: The medicine also contains excipients.

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