Akk11 strain-containing probiotic agent capable of relieving hyperuricemia, and the use thereof

Abstract

An Akk11 strain-containing probiotic agent capable of relieving hyperuricemia, and the use thereof. Strains in the probiotic agent include an Akkermansia muciniphila Akk11 strain and a Lactobacillus johnsonii LJ09 strain. It is found in the present application that the two strains possess a potential interaction and can cooperate with each other, thereby synergizing the effect of relieving hyperuricemia, which is specifically manifested in: being capable of remarkably regulating uric acid metabolism and improving renal clearance functions; being capable of effectively inhibiting the activity of a key enzyme for uric acid synthesis so as to reduce the production of uric acid, and being capable of remarkably relieving renal damage and renal dysfunctions associated with high levels of uric acid.

Description

Akk11 strain-containing probiotic agent for relieving high uric acid and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotic agents, and relates to an Akk11 strain-containing probiotic agent for relieving high uric acid and application thereof. BACKGROUND

[0002] Hyperuricemia is a common metabolic disorder characterized by elevated serum uric acid levels, which can further lead to health problems such as gout, kidney stones, and renal failure. Uric acid is a waste product of purine metabolism in the human body, mainly excreted through the kidneys and partially through the intestines. Traditional treatment methods focus on using drugs to inhibit the production or increase the excretion of uric acid, such as diuretics and uric acid inhibitors, but long-term use may be accompanied by side effects and drug resistance problems.

[0003] The intestinal microecosystem is closely related to the health of the host, and the balance of the intestinal flora has potential effects on the prevention and treatment of metabolic diseases. Probiotics, as an effective means of regulating intestinal flora, have been used to improve metabolic diseases such as obesity and diabetes. Specific probiotic strains can positively affect the generation and excretion mechanisms of uric acid by regulating the intestinal environment and metabolic pathways of the host.

[0004] However, there are relatively few strategies in the prior art for using probiotics to prevent and treat hyperuricemia. In view of this, it is of great significance to develop a probiotic-based treatment regimen that not only effectively reduces uric acid levels but also reduces the need for traditional drug therapy. SUMMARY

[0005] The present application provides an Akk11 strain-containing probiotic agent for relieving high uric acid and application thereof, specifically provides an Akk11 strain-containing probiotic agent for relieving high uric acid and application thereof in the preparation of a drug for preventing, improving, or treating hyperuricemia.

[0006] In a first aspect, the present application provides an Akk11 strain-containing probiotic agent for relieving high uric acid, wherein the strains in the probiotic agent include Akkermansia muciniphila Akk11 strain with a preservation number of CCTCC NO:M2024119 and Lactobacillus johnsonii LJ09 strain with a preservation number of CGMCC No.20123.

[0007] The preservation unit of the Akkermansia muciniphila Akk11 strain is China Center for Type Culture Collection, the preservation time is January 15, 2024, the preservation number is CCTCC NO: M2024119, and the address is: Wuhan University, Wuhan, China; the preservation unit of the Lactobacillus johnsonii LJ09 strain is China General Microbiological Culture Collection Center, the preservation time is June 22, 2020, the preservation number is CGMCC No. 20123, and the address is: No. 3, Beichen West Road, Chaoyang District, Beijing.

[0008] The present application develops a brand-new probiotic compound mode, which is to compound Akkermansia muciniphila Akk11 strain and Lactobacillus johnsonii LJ09 strain. It is found that the two strains have potential interaction and can cooperate with each other to synergistically improve the efficacy of relieving hyperuricemia, which is specifically shown in: (1) can significantly regulate uric acid metabolism and improve kidney clearance function; (2) can effectively inhibit the key enzyme activity of uric acid synthesis to reduce the production of uric acid; (3) can significantly improve kidney damage and renal dysfunction related to high uric acid level.

[0009] Under the condition of using the same amount of bacteria, compared with single Akk11 strain or single LJ09 strain, the compound of the two strains significantly improves the above-mentioned efficacy. Therefore, the probiotic agent provides a new strategy for preventing, relieving or treating hyperuricemia. Since Akkermansia muciniphila and Lactobacillus johnsonii are both probiotics, they are safe when used to prepare related efficacy products and are not prone to dependence.

[0010] Preferably, the ratio of viable bacteria of Akkermansia muciniphila Akk11 strain to Lactobacillus johnsonii LJ09 strain is 1:10-10:1, such as 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0011] Preferably, in the probiotic agent, the total number of viable bacteria is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g, such as 1×10 8CFU / mL (CFU / g), 1 x 10 9 CFU / mL (CFU / g), 5 x 10 9 CFU / mL (CFU / g), 1 x 10 10 CFU / mL (CFU / g), 5 x 10 10 CFU / mL (CFU / g), 1 x 10 11 CFU / mL (CFU / g), 1 x 10 12 CFU / mL (CFU / g), 1 x 10 13 CFU / mL (CFU / g), 1 x 10

[0012] Preferably, the dosage form of the probiotic agent comprises a solution, a lyophilized powder, a capsule, a tablet or a granule. The dosage form of the probiotic agent involved in the present application is not limited, including the most commonly used solution, lyophilized powder, or further prepared capsule, tablet or granule.

[0013] Preferably, the dosage form of the probiotic agent is a solution, which is prepared by the following method:

[0014] The Akk11 strain and the LJ09 strain are inoculated into the culture medium in turn for activation and fermentation culture to obtain a fermentation broth; the fermentation broth is centrifuged and resuspended with a solvent to obtain Akk11 bacterial suspension and LJ09 bacterial suspension; the Akk11 bacterial suspension and the LJ09 bacterial suspension are mixed according to the viable bacterial count ratio to obtain the probiotic agent.

[0015] Preferably, the dosage form of the probiotic agent is a lyophilized powder, which is prepared by the following method:

[0016] The Akk11 strain and the LJ09 strain are inoculated into the culture medium in turn for activation and fermentation culture to obtain a fermentation broth; the fermentation broth is centrifuged and resuspended with a solvent to obtain Akk11 bacterial suspension and LJ09 bacterial suspension; the Akk11 bacterial suspension and the LJ09 bacterial suspension are mixed according to the viable bacterial count ratio to obtain the probiotic agent.

[0017] Preferably, the protective agent is selected from any one or a combination of at least two of skimmed milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

[0018] In a second aspect, the present application provides the use of the probiotic agent of the first aspect in the preparation of a medicament for preventing, improving or treating hyperuricemia.

[0019] Preferably, the medicine further comprises an excipient selected from any one or a combination of at least two of a filler, a binder, a wetting agent, a disintegrant, an emulsifying agent, a co-solvent, a solubilizing agent, an osmotic pressure regulator, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent, or a buffer.

[0020] Preferably, the medicine further comprises a functional aid selected from any one or a combination of at least two of fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, inulin, grifolan, polydextrose, alpha-lactalbumin, or lactoferrin.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] The present application develops a brand-new probiotic compound mode, which is to compound Akkermansia muciniphila Akk11 strain and Lactobacillus johnsonii LJ09 strain, and find that the two strains have potential interaction and can cooperate with each other to synergistically improve the efficacy of relieving hyperuricemia, which is specifically shown in the following aspects: (1) can significantly regulate uric acid metabolism and improve kidney clearance function; (2) can effectively inhibit the activity of key enzymes of uric acid synthesis to reduce the production of uric acid; (3) can significantly improve kidney damage and renal dysfunction related to high uric acid level.

[0023] Under the condition of using the same amount of bacteria, compared with single Akk11 strain or single LJ09 strain, the compound of the two strains significantly improves the above-mentioned efficacy. Therefore, the probiotic agent provides a new strategy for preventing, relieving or treating hyperuricemia. Since Akkermansia muciniphila and Lactobacillus johnsonii are both probiotics, they are safe when used for preparing products with related efficacy, and are not easy to produce dependence. BRIEF DESCRIPTION OF DRAWINGS

[0024] Fig. 1 is a statistical result graph of the urine protein level in the urine of mice in each group.

[0025] Fig. 2 is a statistical result graph of the urea nitrogen level in the urine of mice in each group.

[0026] Fig. 3 is a statistical result graph of the uric acid level in the urine of mice in each group.

[0027] Fig. 4 is a statistical result graph of the uric acid level in the serum of mice in each group.

[0028] Fig. 5 is a statistical result graph of the urea nitrogen level in the serum of mice in each group.

[0029] Fig. 6 is a statistical result graph of the creatinine level in the serum of mice in each group.

[0030] Figure 7 is a graph of the statistical results of xanthine oxidase activity in the liver tissues of each group of mice. DETAILED DESCRIPTION

[0031] The technical solutions of the present application are further illustrated below by specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0032] The medium formula involved in the following examples is as follows:

[0033] MRS medium: 10 g / L of proteose peptone, 10 g / L of beef extract, 20 g / L of glucose, 2 g / L of sodium acetate, 5 g / L of yeast powder, 2 g / L of diammonium hydrogen citrate, 2.6 g / L of K2PO4·3H2O, 0.1 g / L of MgSO4·7H2O, 0.05 g / L of MnSO4, 1 mL / L of polysorbate 80 (Tween 80), and 0.5 g / L of cysteine hydrochloride.

[0034] The classification name of the Akk11 strain involved in the following examples is Akkermansia muciniphila, which is deposited with the China Center for Type Culture Collection on January 15, 2024, has a preservation number of CCTCC NO: M2024119, and is located at Wuhan University, Wuhan, China.

[0035] The classification name of the LJ09 strain involved in the following examples is Lactobacillus johnsonii, which is deposited with the China General Microbiological Culture Collection Center on June 22, 2020, has a preservation number of CGMCC No. 20123, and is located at No. 3, Beichen West Road, Beijing, China.

[0036] The method for preparing the bacterial suspension involved in the following examples is as follows: inoculate the desired strain in a liquid medium, activate at 37°C for 24 h, continuously activate for 2 times, and obtain an activation liquid; inoculate the activation liquid in a liquid medium at an inoculation amount of 5% (v / v), culture at 37°C for 24 h, and obtain a bacterial liquid; centrifuge the bacterial liquid at 4°C at 5000 rpm for 10 min, filter, obtain bacterial bodies, resuspend the bacterial bodies with a PBS solution, and obtain the bacterial suspension.

[0037] The test result data is statistically analyzed using the ggplot2 of R language. Compared with the control group, ### represents p<0.001; compared with the model group, *** represents p<0.001, ** represents p<0.01, * represents p<0.05, and NS. represents no significant difference.

[0038] EMBODIMENT

[0039] This example investigates the ability of the strain to improve the symptoms of a high uric acid mouse model.

[0040] (1) Test animals: Healthy SPF male ICR mice (70, body weight 20 ± 2 g, purchased from Shanghai Chengxi Biological Technology Co., Ltd.) were raised in a controlled environment, with room temperature maintained at 20-24°C, humidity at 50-60%, and a 12 h light / dark cycle. They had free access to food and water.

[0041] (2) Animal grouping: After 1 week of adaptive feeding, the mice were randomly assigned into 7 groups of 10 each: control group (CTL), high uric acid model group (MC group), Akk11 strain group (Akk11 group, denoted as S1 group), LJ09 strain group (LJ09 group, denoted as S2 group), commercially available mucinophilic Akkermansia group (BNCC341917 group, denoted as S3 group), complex bacteria group 1 (Akk11 + LJ09 group, with a live bacteria ratio of 2:1, denoted as S4 group), and complex bacteria group 2 (BNCC341917 + LJ09 group, with a live bacteria ratio of 2:1, denoted as S5 group).

[0042] (3) Animal modeling and intervention methods: The CTL group mice were first given 1% carboxymethylcellulose sodium (CMC-Na, purchased from Shanghai Shengguang Food Chemical Co., Ltd.) solution by gavage, and 1 hour later, they were given normal saline by gavage. The other groups were first given 270 mg / kg oxypurinol potassium (suspended in 1% CMC-Na solution, Shanghai Maikelin Biochemical Technology Co., Ltd.) by gavage every day, and 1 hour later, they were given normal saline (MC group) or each group of probiotic bacterial liquid (10 8 CFU / day / mouse, S1-S5 groups) by gavage, for a total of 21 days.

[0043] (4) Index analysis:

[0044] (4.1) Assessment of kidney function in high uric acid mice:

[0045] One day before the last administration, the mice's urine was collected by stimulating reflex for subsequent 24-hour urine analysis. The specific methods included: determination of urine protein (UP) content using the Coomassie brilliant blue method, detection of urine urea nitrogen (UUN) content using the urease method, and determination of uric acid (UA) content using the enzyme colorimetric method.

[0046] Results are shown in Figures 1, 2 and 3. Compared with the CTL group, the levels of urine protein and urea nitrogen in the urine of the MC group mice were significantly increased, indicating impaired kidney function and abnormal excretion of metabolic waste. Unlike urine protein and urea nitrogen, the level of uric acid in the model group was significantly decreased, which may be due to the accumulation of uric acid in the blood, reflecting the decreased ability of the kidney to excrete uric acid. However, after probiotic intervention, the levels of urine protein and urea nitrogen in the urine were significantly decreased, indicating that probiotics have a potential effect on improving kidney function damage. In addition, probiotic intervention also significantly increased the level of uric acid in the urine, especially in the S4 group, which may indicate that the probiotic agent involved in this application helps restore the kidney's uric acid excretion function and reduce the accumulation of uric acid in the blood.

[0047] (4.2) Effect on key biochemical indicators of high uric acid mice:

[0048] After the intervention, blood was collected from each group of mice by removing the eyeball 1 hour after administration. After the blood was allowed to stand for 1 hour, it was centrifuged for 10 minutes to separate the serum. The levels of uric acid, urea nitrogen and creatinine in the serum of mice were determined by enzyme-linked immunosorbent assay (ELISA) method using a kit.

[0049] Results are shown in Figures 4, 5 and 6. Compared with the CTL group, the levels of uric acid, urea nitrogen and creatinine in the serum of the MC group mice were significantly increased, indicating that the model successfully replicated hyperuricemia and its related kidney damage. However, after probiotic intervention, the levels of uric acid, urea nitrogen and creatinine in the serum of mice were significantly decreased, tending to the levels of the normal control group, especially in the S4 group. This result not only verifies the potential effect of the probiotic agent involved in this application in regulating uric acid levels, but also suggests its positive role in protecting kidney function and promoting the excretion of metabolic waste.

[0050] (4.3) Effect on xanthine oxidase activity in the liver of high uric acid mice:

[0051] The liver is one of the organs with the highest xanthine oxidase (XOD) activity, making it an ideal tissue for assessing uric acid production and purine metabolism. By measuring the activity of xanthine oxidase in the liver, the status of whole body uric acid production can be accurately assessed, which is of key significance for studying the physiological and pathological mechanisms of uric acid-related diseases.

[0052] After the intervention, the mice were sacrificed and dissected, and the intact liver was removed. After washing with physiological saline and drying, the liver homogenate of each group of mice was prepared, and the supernatant was obtained. The activity of xanthine oxidase (XOD) in the liver of mice was determined by enzyme-linked immunosorbent assay (ELISA) method.

[0053] The results are shown in Figure 7. Compared with the CTL group, the xanthine oxidase (XOD) activity in the liver of the MC group mice was significantly increased, reflecting an increase in uric acid production in vivo, which is a key marker of hyperuricemia and its related metabolic disorders. After probiotic intervention, the xanthine oxidase activity in the liver of the mice was significantly reduced, indicating that probiotics have a potential regulatory effect on reducing uric acid production, especially in the S4 group. This significant reduction in activity may be related to the effect of probiotics on the intestinal microbial community and its metabolites, thereby indirectly regulating the metabolic function of the liver and reducing the production of uric acid.

[0054] The applicant declares that the technical solutions of the present application are illustrated by the above examples, but the present application is not limited to the above examples, that is, the present application does not mean that it must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement on the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the present application.

[0055] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments. Within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0056] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A probiotic agent for relieving hyperuricemia, comprising Akk11 strains, wherein the strains comprise Akkermansia muciniphila Akk11 strain with a preservation number of CCTCC NO: M2024119 and Lactobacillus johnsonii LJ09 strain with a preservation number of CGMCC No. 20123.

2. The probiotic agent of claim 1, wherein, The ratio of viable cell counts of the Akkermansia muciniphila Akk11 strain to the Lactobacillus johnsonii LJ09 strain is 1:10-10:

1.

3. The probiotic agent of claim 1, wherein, In the probiotic agent, the total number of viable bacteria is not less than 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g.

4. The probiotic agent of claim 1, wherein, The dosage form of the probiotic agent comprises a solution, a lyophilized powder, a capsule, a tablet or a granule.

5. The probiotic agent of claim 4, wherein, The dosage form of the probiotic agent is a solution, which is prepared by the following method: The Akk11 strain and the LJ09 strain are inoculated into culture media respectively for activation and fermentation culture in sequence to obtain fermentation broth; the fermentation broth is centrifuged respectively, resuspended with a solvent to obtain Akk11 bacterial suspension and LJ09 bacterial suspension; the Akk11 bacterial suspension and the LJ09 bacterial suspension are mixed according to the ratio of viable cell counts to obtain the probiotic agent.

6. The probiotic agent of claim 4, wherein, The dosage form of the probiotic agent is a lyophilized powder, which is prepared by the following method: The Akk11 strain and the LJ09 strain are inoculated into culture media respectively for activation and fermentation culture in sequence to obtain fermentation broth; the fermentation broth is centrifuged respectively, mixed with a protective agent and then freeze-dried to obtain Akk11 bacterial powder and LJ09 bacterial powder; the Akk11 bacterial powder and the LJ09 bacterial powder are mixed according to the ratio of viable cell counts to obtain the probiotic agent.

7. The probiotic agent of claim 6, wherein, The protective agent is selected from any one or a combination of at least two of skimmed milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol. 8.The probiotic agent of any one of claims 1-7 for use in the preparation of a medicament for preventing, improving or treating hyperuricemia.

9. Use according to claim 8, wherein, The medicament further comprises an auxiliary material selected from any one or a combination of at least two of a filler, a binder, a wetting agent, a disintegrant, an emulsifying agent, a co-solvent, a solubilizing agent, an osmotic pressure regulator, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

10. Use according to claim 8, wherein, The medicament further comprises a functional aid selected from any one or a combination of at least two of oligofructose, oligogalactose, oligoxylan, inulin, grifolan, polydextrose, α-lactalbumin or lactoferrin.

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