Paracasei subsp postbiotic for relieving high uric acid and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a high uric acid relieving Paracasei postbiotic and application thereof. Lacticaseibacillus paracasei The Paracasei (Probio-37) has a preservation number of CGMCC No. 18638. The postbiotic prepared by the strain stimulates the intestinal uric acid excretion pathway through the "gut-kidney axis", reduces the SUA level, adjusts the immune homeostasis, inhibits excessive inflammatory response, enhances the intestinal barrier integrity, effectively relieves local inflammatory response caused by urate crystals, and thus improves clinical symptoms such as joint swelling and pain, and has obvious therapeutic effect on hyperuricemia.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Lactobacillus paracasei postbiotics for alleviating hyperuricemia and their applications. Background Technology

[0002] The kidneys are vital excretory and endocrine organs in the human body. Through functions such as filtering metabolic waste, regulating water and electrolyte balance, and secreting active substances, they play an irreplaceable and crucial role in maintaining homeostasis. With significant changes in modern lifestyles, such as increased intake of high-purine diets, lack of exercise, and irregular sleep patterns, the incidence of metabolic-related chronic diseases continues to rise, with kidney disease affecting an expanding population. This not only impacts patients' quality of life but also places a heavy burden on the healthcare system.

[0003] Hyperuricemia, a common metabolic disease, is characterized by an imbalance in uric acid metabolism. This imbalance arises from excessive uric acid production due to abnormal purine metabolism, or reduced uric acid excretion due to dysfunction of excretory organs such as the kidneys, ultimately resulting in persistently elevated uric acid levels in the blood. When blood uric acid concentration is too high, it exceeds its solubility limit in body fluids, leading to the formation of insoluble urate crystals. These crystals circulate in the bloodstream and deposit in various tissues and organs, with the kidneys being a primary affected organ, in addition to common joint sites. Long-term urate deposition continuously increases the metabolic burden on the kidneys, causing progressive damage to kidney tissue. Initially, this may manifest as renal tubular dysfunction; as the disease progresses, it can induce chronic kidney disease, and in severe cases, even kidney failure, posing a serious threat to the patient's health. Therefore, effectively controlling and lowering blood uric acid levels is crucial for preventing kidney damage, slowing the progression of kidney disease, and avoiding its development into end-stage renal disease.

[0004] In current clinical practice, the main treatments for hyperuricemia and related kidney diseases include drug therapy, dialysis, and kidney transplantation. Drug therapy is the fundamental and commonly used approach, but some medications may cause a series of side effects with long-term use, placing an additional burden on other organs. While dialysis can alleviate symptoms in patients with end-stage renal disease, it presents challenges such as inconvenience, the need for regular procedures, and a high risk of infection. Kidney transplantation faces challenges including a severe shortage of donor kidneys, high surgical risks, and the need for long-term immunosuppressant therapy post-operatively. These limitations not only affect treatment outcomes but also prevent the full fulfillment of the clinical needs of some patients, restricting their widespread application in clinical practice.

[0005] Probiotics, as a class of live microorganisms beneficial to the host, have seen significant progress in research on gut health and immune regulation in recent years. With in-depth research, a close connection between the gut and kidneys has been gradually discovered, with gut microbiota and its metabolites influencing kidney physiological function and pathological conditions. Preliminary studies suggest that specific probiotic strains may indirectly affect kidney function by regulating the composition and metabolism of the gut microbiota. However, most existing probiotic products target gut function, and research on probiotics with clearly defined kidney-protective functions and their preparation methods is limited. Therefore, developing a probiotic that effectively protects the kidneys and its preparation method has significant clinical and application value. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a strain of *Lactobacillus paracasei* (… Lacticaseibacillus paracasei Probio-37, with accession number CGMCC No. 18638, was deposited at the China General Microbiological Culture Collection Center on June 5, 2019. This strain, after cultivation, fermentation, and freeze-drying, yields a bacterial powder and postbiotic products (including inactivated Probio-37 cells and / or metabolites), which can effectively intervene in kidney failure and damage.

[0007] The technical solution of this invention is as follows:

[0008] On one hand, the present invention provides the application of Lactobacillus paracasei postbiotic in the preparation of a product used in combination with febuxostat to relieve hyperuricemia, wherein the Lactobacillus paracasei preservation number is CGMCC No.18638.

[0009] Specifically, the Lactobacillus paracasei metabiotic includes fermentation broth, fermentation broth supernatant, fermentation broth precipitate, dead bacteria, lyophilized powder, cell lysate, and / or bacterial suspension.

[0010] More specifically, the fermentation broth refers to the liquid in which the microbial strain is inoculated into a culture medium and cultured for a period of time.

[0011] More specifically, the supernatant of the fermentation broth refers to the clear liquid at the top after centrifugation of the fermentation broth; it contains abundant metabolic products from the bacterial growth and reproduction process, as well as some bacterial cell fragments. The acidic substances and bacteriocins secreted by the bacteria have antagonistic and killing effects on harmful bacteria. The amino acids and vitamins synthesized by the bacteria after decomposing food are also in the culture medium, as well as enzymes secreted by the bacteria that are useful to the human body. Some of the bacterial cell components also have an immune-boosting effect on the human body.

[0012] More specifically, the fermentation broth sediment refers to the liquid sediment obtained after centrifugation, which includes free protein, residual bacterial cells, broken cells, and culture medium residue, mainly protein and intracellular matrix.

[0013] More specifically, the dead bacteria are microorganisms that have lost their vitality and are unable to grow and reproduce. The production process causes the probiotics to lose their vitality, such as high-temperature treatment or excessive drying.

[0014] More specifically, the lyophilized powder is obtained by lyophilizing the aforementioned culture medium. The lyophilized powder generally also includes a lyophilization protectant. The lyophilization protectant includes, but is not limited to, pH buffers, fillers, sugars, nonionic surfactants, ligands, etc. The pH buffers include, but are not limited to, any one or more of Tris, amino acids or their salts, citric acid or its salts, and acetic acid or its salts. The fillers include, but are not limited to, any one or more of mannitol, glycine, and bovine serum albumin. The sugars can be disaccharides, such as sucrose or trehalose, any one or more. The nonionic surfactants include, but are not limited to, Tween, such as Tween-20, Tween-60, Tween-80, etc. The lyophilization protectant may also include antioxidants, etc. Specifically, the lyophilization protectant may also include albumin, polyethylene glycol, etc.

[0015] More specifically, the cell lysis buffer can be obtained by lysing the bacterial cells cultured from the aforementioned culture. The lysis can be physical or chemical. Physical lysis includes, but is not limited to, grinding and ultrasonic disruption. Chemical lysis includes, but is not limited to, lysis with chemical reagents and enzymatic hydrolysis, wherein the enzymatic hydrolysis can be with hydrolases or oxidases. Lysis can also be achieved by increasing intracellular pressure to induce spontaneous cell rupture.

[0016] More specifically, the bacterial suspension usually refers to a homogeneous liquid mixture formed by resuspending bacterial cells in a sterile buffer or protectant. It can be a direct concentrate of fermentation broth or a product made by rehydrating and activating lyophilized powder, which is convenient for conducting precise dosage experiments or preparing liquid formulations.

[0017] Specifically, the number of *Lactobacillus paracasei* cells is not less than 1.0 × 10⁻⁶. 9 CFU / g or 1.0×10 9 Cells / g.

[0018] Specifically, the *Lactobacillus paracasei* postbiotic is obtained by culturing in a culture medium; the components of the culture medium, by weight, include: 20-30 parts sucrose, 10-15 parts lactose, 10-20 parts soybean peptone, 3-8 parts yeast extract, 10-15 parts yeast peptone, 20-25 parts Na2HPO3, 1-3 parts citric acid, 0.1-1.0 parts MgSO4·7H2O, 0.1-0.5 parts MnSO4·5H2O, 0.5-1.5 parts Tween-80, and 0.1-0.5 parts L-cysteine ​​hydrochloride.

[0019] Preferably, the components of the culture medium, by weight, include: 23.5 parts sucrose, 12.0 parts lactose, 15.0 parts soybean peptone, 5.0 parts yeast powder, 12.0 parts yeast peptone, 21.0 parts Na2HPO3, 2 parts citric acid, 0.6 parts MgSO4·7H2O, 0.3 parts MnSO4·5H2O, 1.0 part Tween-80, and 0.3 parts L-cysteine ​​hydrochloride.

[0020] Specifically, the products include, but are not limited to: probiotic preparations or drugs.

[0021] Specifically, the dosage forms of the product include, but are not limited to: oral solid dosage forms, oral liquid dosage forms, or semi-solid dosage forms.

[0022] Preferably, the oral solid dosage form is selected from tablets, capsules, granules, powders, chewable tablets, and effervescent tablets; the oral liquid dosage form is selected from oral suspensions, oral emulsions, and oral solutions; and the semi-solid dosage form is selected from gels and ointments.

[0023] The beneficial effects of this invention are: (1) The Probio-37 postbiotic of the present invention stimulates the intestinal uric acid excretion pathway through the "gut-kidney axis", reduces SUA level, and improves clinical symptoms such as joint swelling and pain.

[0024] (2) The Probio-37 postbiotic of the present invention effectively alleviates the local inflammatory response caused by urate crystals by regulating immune homeostasis, inhibiting excessive inflammatory response, and enhancing intestinal barrier integrity. Detailed Implementation

[0025] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0026] Example 1: Preparation of live probiotic strains of Lactobacillus paracasei Probio-37 (1) Activation of strain: The strain (Probio-37, which has been disclosed in CN111575211B) frozen at -40℃ was inoculated into MRS liquid medium sterilized at 121℃ for 15 min and cultured aerobically at 37℃ for 18-24 h. The strain was then passaged 1-2 times to obtain the activated strain.

[0027] (2) Preparation of optimized culture medium: Prepare the components of the optimized culture medium according to the proportion, mix them evenly, adjust the pH value to 6.5, and sterilize at 121℃ for 15min; The optimized culture medium composition is as follows: 23.5 kg sucrose, 12.0 kg lactose, 15.0 kg soybean peptone, 5.0 kg yeast extract, 12 kg yeast peptone, 21.0 kg Na2HPO3, 2.0 kg citric acid, 0.6 kg MgSO4·7H2O, 0.3 kg MnSO4·5H2O, 1.0 kg Tween-80, 0.3 kg L-cysteine ​​hydrochloride, and distilled water to a final volume of 1000 L.

[0028] (3) Preparation of seed liquid: Take the activated strains from step (1) and inoculate them into the optimized culture medium prepared in (2). Culturing at 37°C with aerobic culture until the pH value is 4.5-4.8 to obtain seed liquid.

[0029] (4) Inoculation and fermentation: The seed liquid from step (3) was inoculated into the optimized culture medium prepared in (2) at a ratio of 1‰, and fermented for 18 hours under controlled fermentation conditions; Fermentation conditions were controlled as follows: Initial fermentation was carried out at a constant temperature of 30℃ until the pH reached 5.0; then the fermentation temperature was adjusted to a constant temperature of 37℃, and the pH was maintained at 6.0 by adding the neutralizing agent NaOH, ensuring aerobic fermentation for a total fermentation time of 18 hours. Aerobic conditions were achieved by purging nitrogen gas every two hours.

[0030] (5) Termination of fermentation: Fermentation is terminated when the acid production of the bacteria stops (this can be determined by whether the pH stops decreasing when the neutralizing agent is stopped), resulting in a high-density fermentation broth of the strain. The viable count of the high-density fermentation broth reaches 2 × 10⁻⁶. 10 CFU / mL or higher.

[0031] (6) Freeze-drying a. Cell concentration: The high-density fermentation broth is concentrated into cells by centrifugation at 12000g. b. Add protectant: Add 3-8 times the volume of protectant solution to the bacterial concentrate; the protectant solution is composed of the following: 10-15 kg of skim milk, 8-12 kg of lactose, 1-2 kg of vitamin C, 1-2 kg of monosodium glutamate, and distilled water to a final volume of 1000 L.

[0032] c. Drying: The bacterial suspension with added preservatives was freeze-dried to obtain freeze-dried bacterial powders of the strains, with a total viable count of 5.0 × 10⁻⁶. 11 CFU / g or higher.

[0033] Example 2: Preparation of a postbiotic from Lactobacillus paracasei Probio-37 The specific method is as follows: (1) Weighing: Mix 4% whole soybean flour, 14.5% skim milk powder, 2.5% sodium citrate and the remainder water according to the required proportions; (2) Material preparation: Prepare the material at 58℃ for 15 minutes to obtain a liquid solution; (3) Homogenization: 58℃, first-stage pressure 19 MPa, second-stage pressure 5.0 MPa; (4) Sterilization: The homogenized liquid is sterilized at 93°C for 30 min; (5) Cooling: Cool the sterilized liquid to 35°C; (6) Adding bacterial strains and lactase: Add *Lactobacillus paracasei* Probio-37 to the feed solution at an inoculation rate of 6 × 10⁻⁶. 6 CFU / g (liquid solution), and add lactase (enzyme activity of 5000U / g) 0.5mL / kg (liquid solution) according to production requirements; (7) Constant temperature fermentation: Ferment at 35℃ until pH 4.5-4.6, approximately 18-19 h (total viable count ≥3.0×10⁻⁶). 9 CFU / g); (8) Inactivation: Mix 18g / kg of maltodextrin (fermentation liquid) according to production requirements, preheat to 58℃, homogenize under first-stage pressure of 19 MPa and second-stage pressure of 5.0 MPa, and then sterilize and inactivate (inactivation conditions 85℃, 15min). (9) Spray drying: The inactivated bacterial suspension was spray dried to obtain Lactobacillus paracasei Probio-37.

[0034] Example 3: Preparation of a microbial agent Weigh 0.1g of the bacterial powder from Example 1, add 0.9g of the excipient maltodextrin, mix well, and the viable count should not be less than 1.0×10⁻⁶. 10 CFU / g.

[0035] Example 4: Preparation of an epigenetic agent Weigh 0.1g of the probiotic postbiotic powder from Example 2, add 0.9g of the excipient maltodextrin, mix well, and the bacterial count should not be less than 1.0×10⁻⁶. 10 Cells / g.

[0036] Example 1: The therapeutic effect of Lactobacillus paracasei Probio-37 postbiotic in improving hyperuricemia. Example 1 1.1 General Information Ninety patients with hyperuricemia admitted to the Inner Mongolia People's Hospital from January to June 2025 were selected and randomly divided into three groups using a random number table: a probiotic combination group (Pro group, n=30), a postbiotic combination group (Post group, n=30), and a febuxostat group (control group, n=30). There were no statistically significant differences in general characteristics among the three groups. P >0.05), which is comparable, see Table 1.

[0037] Table 1 Comparison of General Data

[0038] 1.2 Diagnostic Criteria Inclusion criteria: (1) diagnosed with hyperuricemia; (2) patients with normal cognitive and visual functions; (3) good compliance; (4) patients or their families signed informed consent forms.

[0039] Exclusion criteria: (1) patients allergic to the drugs used in this study; (2) patients with malignant tumors; (3) patients with severe liver and kidney dysfunction; (4) patients with mental illness; (5) patients with serious complications.

[0040] 1.3 Test Methods All three groups of patients received basic dietary guidance after enrollment, maintained acid-base balance, and were strictly prohibited from smoking and drinking alcohol, ensuring adequate water intake and avoiding the use of uric acid-raising drugs. The control group received febuxostat tablets (Fuxing Wanbang (Jiangsu) Pharmaceutical Group Co., Ltd.), 40mg orally once daily; the Pro group received Lactobacillus paracasei Probio-37 probiotics (Example 3, 10 billion CFU / day) in addition to the control group's treatment; the Post group received Lactobacillus paracasei Probio-37 postbiotics (Example 4, 10 billion Cells / day) in addition to the control group's treatment. All three groups were treated for 8 weeks.

[0041] 1.4 Observation Indicators (1) Clinical efficacy: Cured: SUA returns to normal and joint symptoms completely disappear; Significantly effective: SUA decreases significantly and approaches the normal range and joint symptoms basically disappear or are significantly relieved; Effective: SUA decreases to some extent and joint symptoms are relieved to a certain degree; Ineffective: SUA does not decrease and joint symptoms do not improve significantly. Total effective rate = (cured + significantly effective + effective) / total number of cases × 100%.

[0042] (2) Kidney function indicators: Three groups of blood samples were collected and prepared before and after treatment, and the SUA level was measured using a fully automated biochemical analyzer.

[0043] (3) Inflammatory markers: Before and after treatment, 3 ml of venous blood was collected from the elbow of the three groups of patients. The blood was stored in a vacuum blood collection tube (without anticoagulant) and left to stand for 2 hours. The serum was centrifuged at 3000 r / min for 10 min and then stored for testing. The serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and high-sensitivity C-reactive protein (hs-CRP) were detected by enzyme-linked immunosorbent assay.

[0044] 1.5 Test Results As shown in Table 2, the total effective rate in the Post group was 100.0%, which was higher than that in the Pro group (96.7%) and the control group (90.0%). This indicates that Lactobacillus paracasei Probio-37 postbiotic can not only improve clinical symptoms such as joint swelling and pain, but also synergistically lower serum uric acid levels with febuxostat, thus having a significant therapeutic effect on hyperuricemia.

[0045] Table 2 Comparison of clinical efficacy [cases (%)]

[0046] As shown in Table 3, compared with the control group, the Post group showed a significant decrease in serum uric acid levels and a higher rate of achieving target uric acid levels after 8 weeks of treatment, and the treatment effect was significantly better than that of the Pro group. This indicates that Lactobacillus paracasei Probio-37 postbiotic can stimulate the intestinal uric acid excretion pathway and reduce SUA levels through the gut-kidney axis.

[0047] Table 3 Comparison of serum uric acid levels (μmol / L)

[0048] Note: Compared with the control group, P <0.05, P <0.01, P <0.001; compared with before treatment, # P<0.05, ## P <0.01, ### P <0.001.

[0049] As shown in Table 4, compared with the control group, the Post group showed a decrease in the levels of all inflammatory factors after treatment, and the treatment effect was better than that of the Pro group. Lactobacillus paracasei Probio-37 postbiotic effectively alleviates local inflammatory responses caused by urate crystals by regulating immune homeostasis, inhibiting excessive inflammatory responses, and enhancing intestinal barrier integrity, thereby improving clinical symptoms such as joint swelling and pain.

[0050] Table 4 Comparison of inflammatory factors

[0051] Note: Compared with the control group, P <0.05, P <0.01, P <0.001; compared with before treatment, # P <0.05, ## P <0.01, ### P <0.001.

[0052] In summary, Lactobacillus paracasei Probio-37 postbiotic stimulates the intestinal uric acid excretion pathway through the gut-kidney axis, reducing SUA levels; by regulating immune homeostasis, inhibiting excessive inflammatory responses, and enhancing intestinal barrier integrity, it effectively alleviates local inflammatory responses caused by urate crystals, thereby improving clinical symptoms such as joint swelling and pain, and has a significant therapeutic effect on hyperuricemia.

[0053] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. The application of *Lactobacillus paracasei* postbiotic in the preparation of products used in combination with febuxostat for relieving hyperuricemia, characterized in that... The preservation number of the Lactobacillus paracasei is CGMCC No. 18638.

2. The application according to claim 1, characterized in that, The Lactobacillus paracasei metabiotic includes Lactobacillus paracasei fermentation broth, fermentation broth supernatant, fermentation broth precipitate, dead bacteria, lyophilized powder, cell lysate, and / or bacterial suspension.

3. The application according to claim 2, characterized in that, The number of *Lactobacillus paracasei* postbiotic cells is not less than 1.0 × 10⁻⁶. 9 CFU / g or 1.0×10 9 Cells / g.

4. The application according to claim 2, characterized in that, The *Lactobacillus paracasei* postbiotic is obtained by culturing in a culture medium; the components of the culture medium, by weight, include: 20-30 parts sucrose, 10-15 parts lactose, 10-20 parts soybean peptone, 3-8 parts yeast extract, 10-15 parts yeast peptone, 20-25 parts Na2HPO3, 1-3 parts citric acid, 0.1-1.0 parts MgSO4·7H2O, 0.1-0.5 parts MnSO4·5H2O, 0.5-1.5 parts Tween-80, and 0.1-0.5 parts L-cysteine ​​hydrochloride.

5. The application according to claim 4, characterized in that, The components of the culture medium, by weight, include: 23.5 parts sucrose, 12.0 parts lactose, 15.0 parts soybean peptone, 5.0 parts yeast extract, 12.0 parts yeast peptone, 21.0 parts Na2HPO3, 2 parts citric acid, 0.6 parts MgSO4·7H2O, 0.3 parts MnSO4·5H2O, 1.0 part Tween-80, and 0.3 parts L-cysteine ​​hydrochloride.

6. The application according to any one of claims 1-5, characterized in that, The product is a microecological preparation.

7. The application according to any one of claims 1-5, characterized in that, The dosage form of the product is an oral solid dosage form, an oral liquid dosage form, or a semi-solid dosage form.

8. The application according to claim 7, characterized in that, The oral solid dosage form is selected from tablets, capsules, granules, powders, chewable tablets, and effervescent tablets; the oral liquid dosage form is selected from oral suspensions, oral emulsions, and oral solutions; and the semi-solid dosage form is selected from gels and ointments.

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