A composite probiotic postbiotic for improving hyperuricemia and its application

Abstract

The present invention proposes a composite probiotic postbiotic for improving hyperuricemia and its application, wherein the composite probiotic postbiotic comprises heat-inactivated Lactobacillus rhamnosus and animal Bifidobacterium lactis subspecies, the preservation number of Lactobacillus rhamnosus is CGMCC NO: 30878, and the preservation number of animal Bifidobacterium lactis subspecies is CGMCC NO: 30902. The composite probiotic postbiotic comprises heat-inactivated Lactobacillus rhamnosus and animal Bifidobacterium lactis subspecies. The production cost of these strains is relatively low, especially in the fermentation process, and the raw materials used are relatively economical. Unlike many modern drugs, probiotics and their metabolites are generally considered to be safe and have fewer side effects. The composite probiotic postbiotic is a probiotic treated by heat inactivation, which means that the live bacteria are inactivated, reducing the risk of infection and the impact on the immune system. The inactivated strain still has the ability to regulate the immune system, as a drug or health product for improving hyperuricemia and hyperuricemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a composite probiotic postbiotic for improving hyperuricemia and an application thereof. Background Art

[0002] Hyperuricemia refers to the level of uric acid in the blood that is higher than the solubility limit (about 6.8 mg / L). It is a common biochemical problem that indicates that the extracellular fluid is oversaturated, usually due to excessive uric acid production or the kidneys not excreting enough uric acid. This imbalance in uric acid levels increases the likelihood of gout in patients with hyperuricemia. Generally speaking, male patients are more likely to develop gout than female patients, and the patients tend to be younger. In addition to gout, they also have complications such as chronic kidney disease, obesity, diabetes, and hypertension.

[0003] Modern medicine believes that hyperuricemia is mainly related to excessive intake of purine, fructose and protein in the diet, which causes purine metabolism disorders, leading to disorders and imbalances in the final product of uric acid metabolism, and accumulation and increase in the blood, leading to a series of adverse reactions and damage to the kidneys and other organs.

[0004] Typically, treatments for hyperuricemia include drug therapy and diet therapy, among which diet therapy cannot adequately control the uric acid level in the body, while drug treatment for hyperuricemia is hampered by toxic side effects and the high price of marketed drugs. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a composite probiotic postbiotic for improving hyperuricemia and its application, aiming to solve the problem that therapeutic drugs for hyperuricemia are expensive and have great toxic and side effects.

[0006] To solve the above technical problems, the present invention is implemented as follows: a composite probiotic postbiotic for improving hyperuricemia, comprising heat-inactivated Lactobacillus rhamnosus and Bifidobacterium animalis lactis subsp. lactis, wherein the preservation number of the Lactobacillus rhamnosus is CGMCC NO: 30878, and the preservation number of the Bifidobacterium animalis lactis subsp. lactis is CGMCC NO: 30902.

[0007] The present invention also provides a method for preparing a composite probiotic postbiotic for improving hyperuricemia, which is used to prepare the composite probiotic postbiotic. The steps of the preparation method include:

[0008] S1. Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis are placed in Demann-Rogosa-Sharp medium and enhanced Clostridium medium respectively, and cultured in a constant temperature anaerobic environment;

[0009] S2, collecting by centrifugation, freezing, drying, and crushing in sequence to obtain Lactobacillus rhamnosus powder and Bifidobacterium animalis subsp. lactis powder, transferring the Lactobacillus rhamnosus powder and the Bifidobacterium animalis subsp. lactis powder to a Deman-Rogosa-Sharp agar plate, and counting the number of viable cells;

[0010] S3, placing the Lactobacillus rhamnosus powder and the Bifidobacterium animalis subsp. lactis powder in a low temperature environment, and suspending them in physiological saline to obtain a strain suspension;

[0011] S4, placing the strain suspension in a high temperature and high pressure environment for heat inactivation treatment, and then transferring it to a de Man-Rogosa-Sharp agar plate to confirm that no living cells remain, to obtain the composite probiotic postbiotic, wherein the composite probiotic postbiotic comprises a heat-inactivated Lactobacillus rhamnosus strain and a heat-inactivated animal Bifidobacterium lactis subsp. lactis strain;

[0012] S5. Encapsulating the composite probiotic postbiotic.

[0013] In some embodiments of the present invention, in step S1, the temperature of the anaerobic environment is 36-38°C.

[0014] In some embodiments of the present invention, in step S3, the temperature of the low-temperature environment is -18 to -22°C, and the concentration of the physiological saline is 0.85 to 0.95%.

[0015] In some embodiments of the present invention, in step S4, the temperature of the high temperature and high pressure environment is 90-100° C., and the treatment time of the thermal inactivation treatment is 5-15 min.

[0016] In some embodiments of the present invention, step S5 includes:

[0017] S5.1, mixing the composite probiotic postbiotic and polylactic acid-co-glycolic acid to obtain a mixed solution;

[0018] S5.2, adding oligofructose or inulin to the mixed solution, and then preparing sustained-release microspheres by spray drying, wherein the particle size of the sustained-release microspheres is 100-300 nm;

[0019] S5.3, adding the sustained-release microspheres into the sodium alginate solution by titration to obtain sustained-release microspheres coated with sodium alginate;

[0020] S5.4. Soak the sustained-release microspheres coated with sodium alginate in a chitosan solution, and obtain double-layer microspheres by spray drying, wherein the particle size of the double-layer microspheres is 1-10 um.

[0021] The present invention also proposes an application of a composite probiotic postbiotic in the preparation of a drug or health product for improving hyperuricemia, wherein the composite probiotic postbiotic is prepared by the above-mentioned preparation method.

[0022] In some embodiments of the present invention, the composite probiotic postbiotic improves hyperuricemia by reducing uric acid levels in blood, urine and feces.

[0023] In some embodiments of the present invention, the compound probiotic postbiotic improves hyperuricemia by increasing the expression of ABCG2 gene and inhibiting the expression of GLUT9 gene, thereby promoting the excretion of uric acid through the intestine and urine.

[0024] In some embodiments of the present invention, the composite probiotic postbiotic reduces the inflammatory response caused by hyperuricemia by regulating inflammatory factors.

[0025] Compared with the prior art, the composite probiotic postbiotic for improving hyperuricemia and its application in the present invention have the following beneficial effects:

[0026] Compound probiotic postbiotics include heat-killed Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis. The production cost of these strains is relatively low, especially during the fermentation process, and the raw materials used are relatively economical. Unlike many modern drugs, probiotics and their metabolites are generally considered safe and have fewer side effects. Compound probiotic postbiotics are probiotics that have been treated by heat inactivation, which means that the live bacteria are inactivated, reducing the risk of infection and the impact on the immune system. The inactivated strains still have the ability to regulate the immune system, and their cell wall components can stimulate the host's immune system, regulate the structure of the intestinal flora, enhance the effects of beneficial bacteria, inhibit the growth of harmful bacteria, help control purine metabolism, reduce uric acid production, and thus reduce serum uric acid levels, improve hyperuricemia and chronic intestinal inflammation caused by hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a graph showing a change trend of uric acid levels in blood in one embodiment of the present invention;

[0028] Figure 2 is a graph showing a change trend of uric acid levels in urine in one embodiment of the present invention;

[0029] Figure 3 is a graph showing the changing trends of the levels of glucose, triglycerides, insulin, interleukin-1β, interleukin-10 and lipopolysaccharide in serum in one embodiment of the present invention;

[0030] Figure 4 is a graph showing the changing trend of the enzyme activity of xanthine oxidase in serum and tissues in one embodiment of the present invention;

[0031] Figure 5 is a graph showing the changing trend of the expression levels of the ABCG2 gene and the GLUT9 gene in one embodiment of the present invention;

[0032] Figure 6 is a graph showing a trend of uric acid levels in feces in one embodiment of the present invention;

[0033] Figure 7 is a schematic diagram of a rat experiment timeline in one embodiment of the present invention;

[0034] Figure 8 It is an enlarged view of a rat kidney tissue section in one embodiment of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0036] The invention provides a composite probiotic postbiotic for improving hyperuricemia, comprising heat-inactivated lactobacillus rhamnosus and bifidobacterium animalis lactis subsp. lactis, wherein the preservation number of the lactobacillus rhamnosus is CGMCC NO: 30878, and the preservation number of the bifidobacterium animalis lactis subsp. lactis is CGMCC NO: 30902.

[0037] There is an important connection between hyperuricemia and changes in intestinal flora. Hyperuricemia affects the integrity of the intestinal epithelium and the balance of intestinal microorganisms. Intestinal microbiota and its metabolites play an important role in human uric acid metabolism; intestinal flora can participate in the absorption of purines, the synthesis of uricase, and affect the intestinal mucosal barrier, thereby directly or indirectly participating in the production and excretion of uric acid. Sustained high uric acid levels can induce chronic intestinal inflammation, leading to changes in the types, quantity and distribution of bacterial flora. Changes in intestinal microbiota can aggravate chronic intestinal inflammation, lead to damage to the intestinal mucosa, changes in intestinal permeability, hinder the normal absorption and transportation of uric acid, and ultimately cause an increase in blood uric acid levels. In addition, intestinal bacteria also play a role in the decomposition of purines, and the metabolites they produce can affect the activity of proteins in the intestine responsible for transporting uric acid, ultimately controlling the level of uric acid in the blood. In addition, intestinal bacteria have the ability to convert uric acid into highly soluble allantoin, and can also control uric acid metabolism by producing SCFAs. Therefore, whether there is an imbalance in the intestinal microbiota may affect the excretion of uric acid, leading to increased serum uric acid levels and ultimately causing hyperuricemia.

[0038] Although the heat-killed strains have lost their activity, they still retain their cell walls, intracellular components and metabolites. These components can regulate the structure of intestinal flora, enhance the effects of beneficial bacteria, inhibit the growth of harmful bacteria, help control purine metabolism, reduce the production of uric acid, and thereby lower serum uric acid levels.

[0039] The inactivated strains still have the ability to regulate the immune system, and their cell wall components can stimulate the host's immune system and reduce inflammatory responses, especially chronic intestinal inflammation caused by hyperuricemia. In addition, inactivated probiotics can enhance intestinal barrier function and reduce intestinal permeability, thereby reducing the reabsorption of uric acid.

[0040] The inactivation process retains the metabolites and functional components of the bacteria, such as short-chain fatty acids (SCFAs) and allantoin, which help promote the metabolism and excretion of uric acid. Compared with live bacteria, inactivated bacteria are more stable, have a longer shelf life, do not require special storage conditions, and avoid the potential risks brought by live bacteria, such as the risk of infection in immunocompromised individuals.

[0041] Heat-killed probiotics avoid the safety issues that may be caused by live bacterial products. For people with weak immune systems, serious illnesses, or allergies to probiotics, it is safer to use inactivated postbiotic preparations, which will not cause potential problems such as bacterial imbalance and infection. After inactivation, the production, storage, and application of the product are more convenient, and strict cold chain transportation is not required. There is no need to worry about the ineffectiveness of live bacterial products during storage, thereby reducing production costs.

[0042] Example 1: Preparation of bacterial strains.

[0043] Lactobacillus rhamnosus (LR-BC21) and Bifidobacterium animalis subsp. lactis (BX-BC08) strains were isolated from traditional fermented dairy products in Yunnan and Inner Mongolia, respectively. The depository of these two strains is the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC, Beijing, China), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. Lactobacillus rhamnosus (Latin name: Lactobacillus rhamnosus), deposit number is CGMCC NO: 30878, Bifidobacterium animalis subsp. lactis (Latin name: Bifidobacterium animalis subsp. lactis), deposit number is CGMCC NO: 30902, and the deposit date of both strains is June 7, 2024.

[0044] For functional evaluation, Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis were placed in De Mann-Rogosa-Sharp medium (MRS, Oxoid, Hants, UK) and reinforced Clostridium medium (RCM, Oxoid, Hants, UK), respectively, and cultured in a constant temperature anaerobic environment until the end of the logarithmic growth phase. The temperature of the anaerobic environment is 36-38°C, for example, 37°C or 37.5°C.

[0045] The strains were collected by centrifugation at 4000 × g for 10 min at 4°C. After freeze-drying, they were crushed into powder and the number of viable cells was determined using the plate method.

[0046] The powder of Lactobacillus rhamnosus and the powder of Bifidobacterium animalis subsp. lactis are placed in a low-temperature environment and suspended in physiological saline to obtain a strain suspension; the temperature of the low-temperature environment is -18 to -22°C, for example, -19°C or -20°C, and the concentration of the physiological saline is 0.85 to 0.95%, for example, 0.9% or 0.91%.

[0047] In order to prepare heat-killed bacteria, the strain suspension is placed in a high temperature and high pressure environment for heat inactivation treatment, and then transferred to a de Man-Rogosa-Sharp agar plate to confirm that no living cells remain, thereby obtaining a composite probiotic postbiotic, which includes a heat-killed Lactobacillus rhamnosus strain and a heat-killed animal Bifidobacterium lactis subsp. lactis strain. The temperature of the high temperature and high pressure environment is 90-100°C, for example, 95°C, 96°C, and the treatment time of the heat inactivation treatment is 5-15min, for example, 10min, 11min.

[0048] Encapsulation of compound probiotic postbiotics. Through encapsulation, the active ingredients in compound probiotic postbiotics (such as inactivated probiotics and their metabolites) are effectively protected to avoid degradation due to external factors such as temperature, humidity, and light during storage and transportation. Especially when passing through the digestive system, the encapsulation layer can protect the postbiotics from being destroyed by gastric acid and digestive enzymes, ensuring that they remain active and intact before reaching the target area (such as the intestine).

[0049] The specific steps of encapsulating compound probiotics and postbiotics are as follows:

[0050] Example 1.1:

[0051] The composite probiotic postbiotic and poly(lactic acid-co-glycolic acid) (PLGA) are mixed and stirred to obtain a mixed solution.

[0052] By mixing and stirring the compound probiotic postbiotics with PLGA, the active ingredients (postbiotics) can be evenly dispersed in the polymer matrix. PLGA is a biodegradable polymer that can form a stable microsphere structure to protect the postbiotics from damage by the external environment (such as gastric acid, digestive enzymes, etc.) and prevent them from being inactivated during transportation or storage. PLGA has a sustained-release property and can gradually release the active ingredients through slow degradation, thereby achieving a long-term stable effect and enhancing the persistence of postbiotics.

[0053] Oligofructose or inulin is added to the mixed solution, and then the mixed solution is spray-dried to prepare sustained-release microspheres, wherein the particle size of the sustained-release microspheres is 100-300 nm.

[0054] As prebiotics, oligofructose or inulin can further enhance the effect of compound probiotics and postbiotics. They can provide nutrition for the beneficial bacteria in the intestines, promote the growth and colonization of probiotics, and thus indirectly help regulate the intestinal microbiota, improve intestinal health, and assist in reducing uric acid levels.

[0055] Through spray drying, sustained-release microspheres with a particle size of 100-300 nm are obtained, which effectively increases the specific surface area of ​​the microspheres, can be better dispersed in the intestines, enhance contact with the intestines, and improve absorption and sustained-release effects. Nano-scale microspheres have a smaller particle size, which helps to improve bioavailability and achieve more precise release control.

[0056] The sustained-release microspheres are added into the sodium alginate solution by titration to obtain the sustained-release microspheres coated with the sodium alginate.

[0057] Sodium alginate is a natural pH-sensitive polymer material that forms a stable protective layer in a gastric acid (low pH) environment to prevent the sustained-release microspheres from being released prematurely in the stomach, thereby protecting the active ingredients from being destroyed by gastric acid. In the alkaline environment of the intestine, sodium alginate will gradually dissolve, releasing the protected sustained-release microspheres to ensure that the active ingredients play a role in the intestine. Through this encapsulation, the compound probiotic postbiotic can be accurately delivered to the intestine, thereby improving the efficacy, especially for the regulation of intestinal health and uric acid metabolism.

[0058] The sustained-release microspheres wrapped with sodium alginate are immersed in a chitosan solution and subjected to spray drying to obtain double-layer microspheres, wherein the particle size of the double-layer microspheres is 1-10 um.

[0059] Chitosan is a material with good biocompatibility, which can further enhance the structural stability of the microspheres. By forming a double-layer package, the outer layer of chitosan and the inner layer of sodium alginate work together to ensure the safe delivery of the compound probiotic postbiotics and effectively avoid failure in the stomach. At the same time, chitosan can also help regulate the immune response of the intestinal mucosa, further enhancing the health effects of prebiotics. The particle size of the double-layer microspheres is controlled at 1~10um, which is suitable for intestinal targeted delivery. The increase in particle size helps to increase the retention time of the microspheres in the intestine, allowing the active ingredients to act on the intestine for a longer time, gradually releasing postbiotics and metabolites, and prolonging the therapeutic effect.

[0060] Example 1.2:

[0061] Sodium carboxymethyl cellulose (CMC-Na) was used as a sustained-release carrier to prepare a 1% CMC solution, and the compound probiotic postbiotics were mixed with the CMC solution and stirred evenly to obtain a sustained-release solution.

[0062] By mixing the compound probiotic postbiotic with 1% CMC solution, a uniform sustained-release solution can be formed to ensure the uniform dispersion of the active ingredients. As a thickener, CMC solution can increase the viscosity of the solution, help to slowly release the active ingredients, and prolong the retention time in the intestine.

[0063] β-cyclodextrin is added to the sustained-release solution, which is first homogenized and then freeze-dried to obtain inner-layer microspheres, wherein the particle size of the inner-layer microspheres is 1~5um. β-cyclodextrin can effectively encapsulate the active ingredients to form inner-layer microspheres, providing physical protection to prevent the active ingredients from degrading during storage and transportation. Through homogenization, the particle size of the inner-layer microspheres is controlled at 1~5µm, which can achieve precise release control, ensure the gradual release of the active ingredients in the intestine, and improve bioavailability.

[0064] Polyethylene glycol and polyvinyl alcohol are dissolved in a mixed solution of deionized water and ethanol (volume ratio 3:1), stirred evenly until completely dissolved, and the particles are filtered to remove the particles to obtain a protective layer solution, wherein the volume ratio of deionized water to ethanol is 3:1, and the protective layer solution includes 10% polyethylene glycol and 2% polyethylene.

[0065] The protective layer formed by the mixed solution of polyethylene glycol (PEG) and polyvinyl alcohol (PVA) can provide good biocompatibility and stability, protecting the inner layer of microspheres from the external environment. Undissolved particles are removed by filtration to ensure that the obtained protective layer solution is uniform and free of impurities, thereby improving the effect of subsequent coating.

[0066] The inner layer microspheres are slowly added to the protective layer solution to form a uniform suspension.

[0067] The inner layer microspheres are mixed with the protective layer solution to form a uniform suspension, ready for the subsequent coating step. This step ensures that the inner layer microspheres are evenly wrapped by the outer layer material to form a stable double-layer structure.

[0068] The uniform suspension is coated on a film substrate to form double-layer microspheres, wherein the particle size of the double-layer microspheres is 1-10 um.

[0069] The use of a coating machine can ensure that the protective layer is evenly covered on the surface of the inner microspheres, forming a stable outer layer of protection. This process can accurately control the thickness and uniformity of the coating, avoiding the problem of unstable release caused by uneven coating. The outer protective layer can effectively prevent the inner microspheres from being affected by mechanical shock, temperature fluctuations and humidity changes during storage and transportation, thereby improving the overall stability of the microspheres.

[0070] The freeze-drying process removes moisture from the microspheres, forming a dry and stable outer protective structure. This structure can provide long-term stability and protection, ensuring the effectiveness of postbiotics when used. The diameter of the microspheres is controlled within the range of 1-10um, which is suitable for intestinal targeted delivery, ensuring that the microspheres can be well dispersed in the intestine and achieve the desired therapeutic effect.

[0071] Example 2: Animal experiment.

[0072] Fifty-six male Sprague-Dawley rats, weighing 200–220 g (Shanghai Slake Laboratory Animal Co., Ltd., Shanghai, China), were housed under a 12-h day-night cycle at room temperature of 22–24°C with free access to food and water.

[0073] After acclimating to the feeding environment for 1 week, the rats were randomly divided into 7 groups: control group, model group (hyperuricemia HUA), AP group (allopurinol, 20 mg / kg body weight / day), LR-BC21 group (live LR-BC21, 1.5 × 10^8 CFU / day), BX-BC08 group (live BX-BC08, 1.5 × 10^8 CFU / day), LR-BC21+BX-BC08 group (a combination of live LR-BC21 and BX-BC08, 7.5 × 10^7 CFU / day for each strain), and LR-BC21D+BX-BC08D group (a combination of heat-killed LR-BC21 and BX-BC08, 7.5 × 10^7 CFU / day for each strain).

[0074] The timeline of the rat experiment in this example is as follows Figure 7As shown. To establish the hyperuricemia model, rats in all groups except the control group were given a high-purine diet and 300 mg / kg of potassium oxalate were administered daily by gavage for 3 weeks. The high-purine diet (per 100 g) contained 20 g of yeast extract and 0.1 g of adenine. Allopurinol or bacteria were suspended in 0.9% saline and administered by gavage starting 1 week before hyperuricemia induction and continued for 3 weeks. The control group and the model group were given an equal amount of saline by gavage.

[0075] Body weight and food intake were recorded weekly. Blood samples were collected from the tail vein of rats after a 12-h fast at weeks 0 and 1. At the end of the experiment, rats were sacrificed by cervical dislocation under ether anesthesia, and whole blood samples were collected by cardiac puncture. Serum was separated by centrifugation at 3000 g for 10 min. Liver, jejunum, ileum, and colon samples were collected and immediately frozen to –80°C after washing with phosphate-buffered saline (PBS). Parts of kidney tissue were fixed for hematoxylin and eosin (H&E) staining. Urine samples were collected at weeks 0, 1, and 4 (before rats were sacrificed). Fresh feces were collected before the end of the experiment. All samples were stored at –80°C until use.

[0076] Example 3: Biochemical detection.

[0077] Urine samples were centrifuged at 3000 g for 10 min, and the supernatant was collected. Liver and colon tissues were mixed with normal saline at a ratio of 1:9 on ice, shaken with TissuePrep (Tianjin Jieling Instrument Manufacturing Co., Ltd.), and then centrifuged at 8000 g for 10 min to obtain tissue homogenates. The protein concentration in the tissue homogenates was quantified using the biuret acid total protein assay kit from Beyotime Biotechnology (Shanghai, China). Glucose, triglycerides, insulin, interleukin-1β (IL-1β), interleukin-10 (IL-10), and lipopolysaccharide (LPS) levels in serum, uric acid (UA) in serum and urine, and xanthine oxidase (XOD) enzyme activity in serum and tissues were measured using commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's instructions.

[0078] Test results such as Figures 1 to 4 shown.

[0079] exist Figure 1In the study, the uric acid level in the HUA group was significantly higher than that in the control group, indicating that the hyperuricemia model was successfully established. The AP group (allopurinol group) showed a significant uric acid-lowering effect, especially after 1 and 4 weeks, indicating its significant inhibitory effect on uric acid. The improvement effects of the LR-BC21 and BX-BC08 groups on uric acid levels were relatively mild, but uric acid levels gradually decreased over time. The LR-BC21+BX-BC08 combined group showed better effects than the use of probiotics alone, and uric acid levels showed a significant downward trend. Compared with the live bacteria group, the LR-BC21D+BX-BC08D group (using inactivated probiotics) had a smaller reduction in uric acid levels, but still had a certain improvement effect, indicating that compound probiotic postbiotics (live or inactivated bacteria) have a certain effect on lowering uric acid levels, especially when used in combination with traditional drugs (such as allopurinol), which can produce a better therapeutic effect.

[0080] exist Figure 2 In the experiment, the uric acid level in the HUA group was significantly higher than that in the control group, indicating that the hyperuricemia model was successfully established. The AP group (allopurinol treatment group) showed a significant uric acid-lowering effect, especially the increase in uric acid excretion after 1 week and 4 weeks, indicating that the drug significantly promoted the excretion of uric acid. The LR-BC21 and BX-BC08 groups showed a mild decrease in uric acid levels, with an increase in uric acid excretion after 1 week and a further decrease after 4 weeks, but the effect was milder than that in the allopurinol group. The LR-BC21+BX-BC08 combined group showed better effects than the single strain, with a significant increase in uric acid excretion, especially after 4 weeks, showing a good regulatory effect. The LR-BC21D+BX-BC08D group (heat-killed strain) showed a certain increase in uric acid excretion, but the effect was slightly weaker than that in the live bacteria group. It shows that compound probiotic postbiotics (live or inactivated bacteria) can promote the excretion of uric acid to a certain extent, especially when live bacteria are used in combination.

[0081] exist Figure 3 In the study, the blood glucose in the HUA group was significantly higher than that in the control group and other treatment groups, indicating that hyperuricemia may be accompanied by hyperglycemia. The blood glucose in the AP group, LR-BC21 group, and BX-BC08 group was significantly reduced, especially the combination group and the inactivated bacteria group, which showed significant hypoglycemic effects.

[0082] The triglyceride level in the HUA group was significantly higher than that in the control group and the treatment group, indicating that hyperuricemia may be accompanied by abnormal lipid metabolism. The AP group, LR-BC21, and BX-BC08 groups showed different degrees of triglyceride reduction, especially the triglyceride level in the combined group and the inactivated bacteria group decreased significantly.

[0083] Blood glucose and triglycerides showed that hyperuricemia is accompanied by metabolic problems such as elevated blood glucose and dyslipidemia. Both probiotics and allopurinol have significant effects on improving blood glucose and blood lipids, especially when probiotics are used in combination.

[0084] The xanthine oxidase activity in the HUA group was higher, and the inhibitory effects of the AP group, LR-BC21, BX-BC08, combined group, and inactivated bacteria group on XOD activity were not significant, indicating that the regulatory effect of probiotics on xanthine oxidase was weak. The results of xanthine oxidase activity showed that probiotics and allopurinol had little direct effect on uric acid production, and probiotics improved hyperuricemia more through other pathways.

[0085] The LPS level in the HUA group was significantly increased, indicating that hyperuricemia may be accompanied by inflammation and impaired intestinal barrier function. The AP group and other probiotic groups (especially the group using LR-BC21 and BX-BC08 together) showed a significant effect in reducing LPS levels, indicating that probiotics and postbiotics have a certain effect on reducing inflammatory responses.

[0086] The IL-1β level in the HUA group was significantly increased, indicating that hyperuricemia induced a significant inflammatory response. The AP group, LR-BC21 group, BX-BC08 group and the combined group all significantly reduced the IL-1β level, especially the combined group and the inactivated bacteria group had a better anti-inflammatory effect.

[0087] The results of LPS levels and IL-1β levels showed that both probiotics and allopurinol had certain anti-inflammatory effects, especially the combined use of probiotics could significantly reduce inflammatory markers.

[0088] IL-10 is an anti-inflammatory cytokine. There was no significant difference in IL-10 levels among all experimental groups, indicating that under the experimental conditions, the effect of probiotics or allopurinol treatment on the anti-inflammatory response was not significant. The results of IL-10 levels showed that the anti-inflammatory factor IL-10 did not change significantly in each group, indicating that the anti-inflammatory mechanism was not significantly affected.

[0089] Overall, Figure 3 The data support that probiotics, postbiotics and their combination can regulate metabolism, reduce inflammation and improve related symptoms caused by hyperuricemia to a certain extent, especially when used in combination.

[0090] exist Figure 4In the results of the experiment, the liver XOD activity of the HUA group increased significantly, indicating that the model of hyperuricemia was successfully established. The AP group showed a significant decrease in the activity of XOD in the liver, and allopurinol reduced uric acid production by inhibiting the activity of XOD. The LR-BC21 and BX-BC08 groups had a certain inhibitory effect on the activity of XOD when used alone, especially the combined use (LR-BC21+BX-BC08) and the inactivated bacteria group (LR-BC21D+BX-BC08D) also showed good inhibitory effects.

[0091] The blood XOD activity of the HUA group increased significantly, indicating that hyperuricemia not only affects uric acid production in the liver, but also increases XOD activity in the blood. The XOD activity of the AP group decreased significantly, indicating that allopurinol can effectively inhibit XOD activity in the blood and reduce uric acid production. The LR-BC21 and BX-BC08 groups also showed the effect of reducing XOD activity in the blood, especially the combined use of probiotics and inactivated bacteria groups showed a good effect in reducing XOD activity.

[0092] The colon XOD activity in the HUA group was significantly increased, indicating that hyperuricemia also affects the XOD activity in the intestine, and the intestine may become an important site for uric acid production. The colon XOD activity in the AP group was significantly reduced, indicating that allopurinol is also effective in inhibiting intestinal uric acid production. The LR-BC21 and BX-BC08 groups, especially the combined group (LR-BC21+BX-BC08), showed the effect of inhibiting intestinal XOD activity and reducing the production of uric acid in the intestine. Although the inactivated bacteria group (LR-BC21D+BX-BC08D) was not as effective as the live bacteria group, it was still able to inhibit XOD activity, indicating that inactivated probiotics have a certain regulatory effect on intestinal uric acid production.

[0093] The hyperuricemia model group (HUA) showed significantly increased XOD activity in the liver, blood, and colon, indicating that the hyperuricemia model successfully induced overexpression of XOD in multiple tissues.

[0094] The allopurinol group (AP group) significantly inhibited XOD activity in the liver, blood and intestine, showing an effective uric acid-lowering effect.

[0095] The probiotic group (LR-BC21 and BX-BC08) had an inhibitory effect on XOD activity when used alone, while the combined use (LR-BC21+BX-BC08) showed a better effect, especially in the intestine, where it could significantly reduce XOD activity.

[0096] The inactivated probiotic group (LR-BC21D+BX-BC08D) had a slightly weaker effect compared to the live bacteria group, but it could still effectively reduce XOD activity, especially showing an inhibitory effect in the intestine and blood.

[0097] Figure 4 These results suggest that compound probiotic postbiotics can reduce uric acid production by regulating the activity of XOD in multiple tissues (liver, blood, and intestine), thereby playing a potential role in the treatment of hyperuricemia.

[0098] Figure 8 This is an enlarged view of a rat kidney tissue section.

[0099] The kidney tissue of the control group is normal in morphology, with clear tubular and glomerular structures, regular cell arrangement, and no obvious signs of inflammation or damage. This is the baseline of normal kidney tissue.

[0100] The renal tissue of HUA (hyperuricemia model group) showed obvious pathological changes, including dilated renal tubules, damaged glomerular structure, disordered cell arrangement, inflammatory cell infiltration, and possible manifestations of interstitial fibrosis. This indicates that hyperuricemia caused significant renal damage, which is closely related to the inflammation and damage caused by uric acid crystal deposition.

[0101] The renal tissue structure of the LR-BC21 group (Lactobacillus rhamnosus group) was improved compared with the HUA group, but some renal tubular dilatation and mild inflammatory infiltration were still observed. The use of probiotics alleviated the renal damage caused by hyperuricemia to a certain extent, but the effect was not as obvious as when used in combination.

[0102] The BX-BC08 group (animal Bifidobacterium group) was similar to the LR-BC21 group, with partial improvement in renal tissue, relatively intact tubular and glomerular structures, and less inflammatory cell infiltration, indicating that animal Bifidobacterium could alleviate renal damage caused by hyperuricemia to some extent.

[0103] The renal tissue structure of the LR-BC21+BX-BC08 combined group was close to that of the control group, with clear structures of renal tubules and glomeruli, significantly reduced inflammatory infiltration, and significantly improved renal damage. The combined use of probiotics has a stronger protective effect and can significantly reduce renal lesions caused by hyperuricemia.

[0104] Although the improvement effect of the LR-BC21D+BX-BC08D inactivated bacteria group was not as good as that of the live bacteria combined group, it still showed a good repair effect. The structure of the renal tubules and glomeruli was relatively intact, and the inflammatory response was significantly reduced, indicating that the inactivated probiotic postbiotic still has a certain renal protective effect.

[0105] Figure 8 , indicating that compound probiotic postbiotics have significant potential in preventing and alleviating renal lesions caused by hyperuricemia.

[0106] Example 4: RT-qPCR analysis of intestinal ABCG2 and GLUT9 gene expression.

[0107] Total RNA from mouse colon and jejunum tissues was extracted using Trizol reagent (Takara, Kyoto, Japan). Gene expression analysis was performed by RT-PCR using the SYBR Green Premix Pro Taq HS qPCR kit from Accurate Biotechnology, USA. All gene expression levels were based on β-actin levels. The primers used are listed in Table 1. All primers used in this study were synthesized by Tianjin Jinweizhi Biotechnology Co., Ltd. The relative mRNA levels of PCR products were calculated using the 2-∆∆Ct method with β-actin as the internal standard.

[0108] Table 1:

[0109]

[0110] The analysis results are as follows Figure 5 As shown, in Figure 5 In the results, the expression level of ABCG2 in the HUA group was lower, indicating that hyperuricemia may be accompanied by a decrease in the ability to excrete uric acid. The AP group (allopurinol group) helps to restore the expression of ABCG2 and promote the excretion of uric acid. Both the LR-BC21 and BX-BC08 groups showed the effect of increasing ABCG2 expression, especially the combined group (LR-BC21+BX-BC08), which significantly increased the expression of the ABCG2 gene and helped to increase the excretion of uric acid. The effect of the inactivated bacteria group (LR-BC21D+BX-BC08D) was slightly weaker, but it still significantly increased the expression of the ABCG2 gene.

[0111] The HUA group had a lower expression level of ABCG2 and impaired uric acid excretion. The AP group significantly increased the expression of ABCG2 in the jejunum and promoted the excretion of uric acid from the intestine. The LR-BC21, BX-BC08 and combined groups all showed significantly increased ABCG2 expression in the jejunum, especially in the combined group and the inactivated bacteria group, indicating that probiotics can significantly promote the intestinal excretion of uric acid.

[0112] The expression level of GLUT9 gene was slightly increased in the HUA group, indicating that hyperuricemia may be accompanied by increased uric acid reabsorption. The AP group significantly inhibited the expression of GLUT9 gene and reduced the reabsorption of uric acid in the intestine.

[0113] The GLUT9 gene expression levels in the LR-BC21, BX-BC08 and combination groups were significantly reduced, especially in the combination group and the inactivated bacteria group, indicating that probiotics can inhibit the reabsorption of uric acid and enhance uric acid excretion.

[0114] The expression level of GLUT9 in the HUA group was slightly increased, and uric acid reabsorption increased. The AP group significantly inhibited the expression of GLUT9 and reduced uric acid reabsorption. LR-BC21, BX-BC08 and the combined group all effectively inhibited the GLUT9 gene expression in the jejunum, especially the combined group and the inactivated bacteria group, which had a more significant inhibitory effect on uric acid reabsorption.

[0115] The expression of ABCG2 gene in the colon and jejunum was significantly increased in the probiotic intervention group, especially in the group using LR-BC21 and BX-BC08 together, indicating that probiotics can promote the excretion of uric acid and improve hyperuricemia.

[0116] Probiotics can inhibit the expression of GLUT9 gene and reduce the reabsorption of uric acid, especially in the group using LR-BC21 and BX-BC08 together and the inactivated bacteria group, indicating that probiotics play an important role in the regulation of uric acid reabsorption.

[0117] Overall, Figure 5 The data in the literature showed that compound probiotic postbiotics, especially the combined use of active and inactivated probiotics, significantly regulated uric acid metabolism genes (ABCG2 and GLUT9), and could alleviate hyperuricemia by increasing uric acid excretion and inhibiting uric acid reabsorption.

[0118] Example 5: Determination of uric acid in feces.

[0119] Uric acid analysis in feces was performed by high-performance liquid chromatography (HPLC). 50 mg of freeze-dried, pulverized rat feces was mixed with PBS solvent and homogenized by ultrasonication. Then, the extracted supernatant was obtained by centrifugation at 4,000 g, 4°C for 10 min and filtered using a 0.45 μm filter. The HPLC system consisted of an Agilent 1,200 series with a UV detector (Agilent Technologies, Waldbronn, Germany). The samples were separated under the following conditions: column, reversed-phase C18 (4.6 mm × 250 mm, 5 μm); mobile phase, 0.2% acetic acid mixed with methanol (94:6); flow rate, 1 mL / min; column temperature, 30°C; detector wavelength, 288 nm.

[0120] The results of the test are as follows Figure 6 shown.

[0121] exist Figure 6 middle,

[0122] Control group: The uric acid level in the feces was low, which is the normal excretion level.

[0123] HUA group (hyperuricemia group): The uric acid level was slightly lower than that of the control group, indicating that hyperuricemia may be accompanied by a decrease in uric acid excretion or metabolic abnormalities. The uric acid excretion in the HUA group was lower, indicating that hyperuricemia may lead to a decrease in uric acid excretion.

[0124] AP group (allopurinol treatment group): The uric acid level was slightly higher than that of the HUA group, but compared with the probiotic group, the effect of allopurinol treatment on promoting uric acid excretion was relatively mild. The allopurinol group (AP) was able to improve the excretion of uric acid to a certain extent, but the effect was not as good as that of the probiotic group.

[0125] LR-BC21 group and BX-BC08 group: The excretion of uric acid increased significantly, especially in the BX-BC08 group. The uric acid concentration in the feces was significantly higher than that in the control group and the HUA group, indicating that a single probiotic can enhance the intestinal excretion of uric acid. LR-BC21+BX-BC08 group: The group using probiotics in combination showed a more significant uric acid excretion effect, and the uric acid concentration in the feces was higher than that in the single probiotic group, indicating that the synergistic effect of the two strains on uric acid excretion is stronger. The probiotic group (LR-BC21 and BX-BC08) showed a significant effect on improving uric acid excretion, especially when probiotics were used in combination, the concentration of uric acid excreted through feces increased significantly.

[0126] LR-BC21D+BX-BC08D group (inactivated bacteria group): The uric acid excretion effect of the inactivated bacteria group was close to that of the live bacteria combined group, indicating that inactivated bacteria postbiotics can still effectively promote the excretion of uric acid. The inactivated bacteria group (LR-BC21D+BX-BC08D) also showed the effect of promoting uric acid excretion, indicating that inactivated bacteria postbiotics still have a strong effect on uric acid excretion.

[0127] Figure 6 This indicates that compound probiotics and postbiotics can significantly promote the excretion of uric acid through feces, especially the combined use of probiotics or inactivated probiotics and postbiotics, which has a significant effect on promoting uric acid metabolism and excretion. This has potential therapeutic significance for improving hyperuricemia.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A composite probiotic postbiotic for improving hyperuricemia, characterized in that: The invention comprises heat-inactivated Lactobacillus rhamnosus LR-BC21 and Bifidobacterium animalis subsp. lactis BX-BC08, wherein the preservation number of the Lactobacillus rhamnosus is CGMCC NO: 30878, and the preservation number of the Bifidobacterium animalis subsp. lactis is CGMCC NO: 30902.

2. A method for preparing a composite probiotic postbiotic for improving hyperuricemia, characterized in that: For preparing the composite probiotic postbiotic according to claim 1, the preparation method comprises the following steps: S1. Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis are placed in Demann-Rogosa-Sharp medium and enhanced Clostridium medium respectively, and cultured in a constant temperature anaerobic environment; S2, collecting by centrifugation, freezing, drying, and crushing in sequence to obtain Lactobacillus rhamnosus powder and Bifidobacterium animalis subsp. lactis powder, transferring the Lactobacillus rhamnosus powder and the Bifidobacterium animalis subsp. lactis powder to a Deman-Rogosa-Sharp agar plate, and counting the number of viable cells; S3, placing the Lactobacillus rhamnosus powder and the Bifidobacterium animalis subsp. lactis powder in a low temperature environment, and suspending them in physiological saline to obtain a strain suspension; S4, placing the strain suspension in a high temperature and high pressure environment for heat inactivation treatment, and then transferring it to a de Man-Rogosa-Sharp agar plate to confirm that no living cells remain, to obtain the composite probiotic postbiotic, wherein the composite probiotic postbiotic comprises a heat-inactivated Lactobacillus rhamnosus strain and a heat-inactivated animal Bifidobacterium lactis subsp. lactis strain; S5. Encapsulating the composite probiotic postbiotic.

3. The method for preparing a composite probiotic postbiotic for improving hyperuricemia according to claim 2, characterized in that: In step S1, the temperature of the anaerobic environment is 36-38°C.

4. The method for preparing a composite probiotic postbiotic for improving hyperuricemia according to claim 2, characterized in that: In step S3, the temperature of the low-temperature environment is -18 to -22°C, and the concentration of the physiological saline is 0.85 to 0.95%.

5. The method for preparing a composite probiotic postbiotic for improving hyperuricemia according to claim 2, characterized in that: In step S4, the temperature of the high temperature and high pressure environment is 90-100° C., and the treatment time of the thermal inactivation treatment is 5-15 minutes.

6. The method for preparing a composite probiotic postbiotic for improving hyperuricemia according to claim 2, characterized in that: The step S5 comprises: S5.1, mixing the composite probiotic postbiotic and polylactic acid-co-glycolic acid to obtain a mixed solution; S5.2, adding oligofructose or inulin to the mixed solution, and then preparing sustained-release microspheres by spray drying, wherein the particle size of the sustained-release microspheres is 100-300 nm; S5.3, adding the sustained-release microspheres into the sodium alginate solution by titration to obtain sustained-release microspheres coated with sodium alginate; S5.

4. Soak the sustained-release microspheres coated with sodium alginate in a chitosan solution, and obtain double-layer microspheres by spray drying, wherein the particle size of the double-layer microspheres is 1-10 um.

7. Use of a composite probiotic postbiotic in the preparation of a drug for improving hyperuricemia, characterized in that: The composite probiotic postbiotic is prepared by the preparation method according to any one of claims 2 to 6.

8. The use of a composite probiotic postbiotic according to claim 7 in preparing a drug for improving hyperuricemia, characterized in that: The composite probiotic postbiotic improves hyperuricemia by reducing the uric acid levels in blood, urine and feces.

9. The use of a composite probiotic postbiotic according to claim 7 in preparing a drug for improving hyperuricemia, characterized in that: The compound probiotic postbiotic promotes the excretion of uric acid through the intestine and urine, thereby improving hyperuricemia by increasing the expression of ABCG2 gene and inhibiting the expression of GLUT9 gene.

10. The use of a composite probiotic postbiotic according to claim 7 in preparing a drug for improving hyperuricemia, characterized in that: The composite probiotic postbiotic reduces the inflammatory response caused by hyperuricemia by regulating inflammatory factors.

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