Composite probiotic formulation for alleviating hyperuricemia and use thereof

Abstract

The present invention relates to a composite probiotic formulation for alleviating hyperuricemia and use thereof. Strains in the composite probiotic formulation for alleviating hyperuricemia comprise Bifidobacterium animalis subsp. lactis BLa80 strain with a deposit number of CGMCC No. 15410, Lacticaseibacillus rhamnosus LRa05 strain with a deposit number of CGMCC No. 1.12734, and Bifidobacterium breve BBr60 strain with a deposit number of CGMCC No. 12915. The three strains can cooperate with each other and promote each other, and have a synergistic effect in the aspect of alleviating hyperuricemia, which is specifically manifested in: alleviating the phenomenon of uric acid elevation caused by hyperuricemia, reducing xanthine oxidase activity, and lowering serum uric acid nitrogen levels.

Description

A composite probiotic preparation for relieving hyperuricemia and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotic agents, and relates to a composite probiotic preparation for relieving hyperuricemia and application thereof. BACKGROUND

[0002] Hyperuricemia, as a major risk factor for gout, is closely related to the occurrence of hypertension, diabetes, cardiovascular disease, and kidney disease, and has become one of the public health problems endangering human life safety. Uric acid is the final product of purine metabolism, and is mainly excreted through the kidney and the intestinal tract. At present, the traditional treatment methods for hyperuricemia mainly include drug therapy, such as uric acid synthesis inhibitors and uric acid excretion promoters. However, these drugs may have side effects and adverse reactions, such as gastrointestinal discomfort, skin rash, liver and kidney function damage, etc., and some patients have different responses to the curative effect.

[0003] In recent years, with the in-depth research of intestinal microecology, scientists have found that probiotics play an important role in regulating human health and promoting uric acid metabolism, providing a new idea for the treatment of hyperuricemia. As a kind of active microorganism beneficial to human body, probiotics play an important role in maintaining intestinal flora balance, promoting nutrient absorption, and enhancing immunity. In recent years, a number of studies have shown that probiotics can affect uric acid metabolism through various mechanisms. When probiotics grow and reproduce in the intestinal tract, they can absorb purine substances for their own genetic synthesis, and degrade purine substances in the intestinal tract by producing purine nucleosidase, thereby reducing their further conversion into uric acid. Probiotics can enrich the intestinal flora, inhibit the growth of harmful bacteria, and restore the intestinal barrier function, thereby improving the intestinal environment for uric acid metabolism. Probiotics can promote the excretion of uric acid by affecting the expression of urate transport proteins, thereby reducing serum uric acid levels.

[0004] Although probiotics show great potential in relieving hyperuricemia, there are various probiotic products on the market with uneven quality. Therefore, it is of great significance to develop more effective treatment programs based on probiotics, which can not only effectively reduce uric acid levels, but also reduce the need for traditional drug therapy. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a composite probiotic preparation for relieving hyperuricemia and application thereof, specifically a composite probiotic preparation for relieving hyperuricemia and application thereof in the preparation of drugs for preventing, improving or treating hyperuricemia.

[0006] To achieve the object of the present application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides a complex probiotic preparation for relieving hyperuricemia, wherein the strains in the complex probiotic preparation for relieving hyperuricemia include the Bifidobacterium animalis subsp.lactis BLa80 strain with the preservation number of CGMCC No.15410, the Lactobacillus rhamnosus LRa05 strain with the preservation number of CGMCC No.1.12734, and the Bifidobacterium breve BBr60 strain with the preservation number of CGMCC No.12915.

[0008] The present application develops a brand-new probiotic compounding method and a brand-new strategy for relieving hyperuricemia, i.e., the Bifidobacterium animalis subsp.lactis BLa80 strain, the Lactobacillus rhamnosus LRa05 strain, and the Bifidobacterium breve BBr60 strain are compounded and used together, and it is found that the three strains can cooperate with each other, promote each other, and synergistically enhance the effect in relieving hyperuricemia, which is specifically manifested in relieving the phenomenon of uric acid increase caused by hyperuricemia, reducing the activity of xanthine oxidase and the level of serum uric acid nitrogen. Moreover, the three strains are probiotics, the product has high safety, and is not prone to resistance.

[0009] Preferably, the viable bacteria content in the complex probiotic preparation is not less than 1×10 8 CFU / g or 1×10 8 CFU / mL, for example, 1×10 8 CFU / g (CFU / mL), 1×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 3×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc., and other specific point values in the numerical range can be selected, which will not be repeated here.

[0010] Preferably, the ratio of viable cell number of the BLa80 strain, the LRa05 strain and the BBr60 strain is (1-10):(1-10):(1-10), wherein the specific point values in (1-10) can be independently selected as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., which will not be repeated here.

[0011] Preferably, the dosage form of the complex probiotic preparation includes a solution, a freeze-dried 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, freeze-dried powder, or further prepared capsule, tablet or granule.

[0012] Preferably, the dosage form of the complex probiotic preparation is a freeze-dried powder, which is prepared by the following method:

[0013] The BLa80 strain, the LRa05 strain and the BBr60 strain are inoculated into the culture medium respectively for activation and fermentation culture in turn to obtain fermentation broth; the fermentation broth is centrifuged respectively to obtain bacterial slurry; the bacterial slurry is mixed with a protective agent to resuspend to obtain resuspension; the resuspension is freeze-dried to obtain BLa80 bacterial powder, LRa05 bacterial powder and BBr60 bacterial powder; the BLa80 bacterial powder, the LRa05 bacterial powder and the BBr60 bacterial powder are mixed according to the ratio of viable cell number to obtain the complex probiotic preparation.

[0014] 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.

[0015] Preferably, the fermentation culture temperature is 35-40℃, for example 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc.; the time is 12-30h, for example 12h, 15h, 16h, 18h, 20h, 24h, 28h, 30h, etc.; other specific point values within the above numerical range can be selected, which will not be repeated here.

[0016] Preferably, the inoculation amount of the inoculation is 1-5%, for example 1%, 2%, 3%, 4%, 5%, etc., and other specific point values within the numerical range can be selected, which will not be repeated here.

[0017] Preferably, the culture medium is MRS culture medium.

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

[0019] Preferably, the medicament further contains an auxiliary material.

[0020] Preferably, the adjuvant comprises any one or a combination of at least two of a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent, or a buffer.

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

[0022] The present application creatively develops a brand new probiotic compound mode and a brand new strategy for relieving hyperuricemia, i.e., compound use of animal bifidobacterium lactis BLa80 strain, lactobacillus rhamnosus LRa05 strain and short bifidobacterium BBr60 strain, and it is found that the three can cooperate with each other, promote each other and synergistically enhance the effect in relieving hyperuricemia, which is specifically manifested in relieving the phenomenon of uric acid increase caused by hyperuricemia, reducing xanthine oxidase activity and serum uric acid nitrogen level.

[0023] The classification name of the BLa80 strain involved in the present application is Bifidobacterium animalis subsp.lactis, the deposit unit is China General Microbiological Culture Collection Center, the deposit time is March 5, 2018, the deposit number is CGMCC No.15410, and the address is No.3, Beichen West Road, Chaoyang District, Beijing.

[0024] The classification name of the LRa05 strain involved in the present application is Lactobacillus rhamnosus, the deposit unit is China General Microbiological Culture Collection Center, the deposit time is July 20, 2020, the deposit number is CGMCC No.1.12734, and the address is No.3, Beichen West Road, Chaoyang District, Beijing.

[0025] The classification name of the BBr60 strain involved in the present application is Bifidobacterium breve, the deposit unit is China General Microbiological Culture Collection Center, the deposit time is August 29, 2016, the deposit number is CGMCC No.12915, and the address is No.3, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0026] Fig. 1 is a statistical result graph of serum uric acid content of rats in each group on the 7th day;

[0027] Fig. 2 is a statistical result graph of serum uric acid content of rats in each group on the 28th day;

[0028] Fig. 3 is a statistical result graph of fecal uric acid content of rats in each group on the 28th day;

[0029] Figure 4 is a statistical result diagram of the fecal uric acid excretion of rats in each group on day 28;

[0030] Figure 5 is a statistical result diagram of the serum xanthine oxidase activity of rats in each group on day 28;

[0031] Figure 6 is a statistical result diagram of the serum urea nitrogen activity of rats in each group on day 28. DETAILED DESCRIPTION

[0032] The technical solutions of the present application will be further illustrated by the 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.

[0033] The medium formula involved in the following examples is as follows:

[0034] 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 Tween 80, 0.5 g / L of cysteine hydrochloride.

[0035] The BLa80 strain involved in the following examples is classified as Bifidobacterium animalis subsp. lactis, and the preservation time is March 5, 2018, and the preservation number is CGMCC No. 15410.

[0036] The LRa05 strain involved in the following examples is classified as Lactobacillus rhamnosus, and the preservation time is July 20, 2020, and the preservation number is CGMCC No. 1.12734.

[0037] The BBr60 strain involved in the following examples is classified as Bifidobacterium breve, and the preservation time is August 29, 2016, and the preservation number is CGMCC No. 12915.

[0038] The preparation method of the probiotic powder described below: seed liquids of BLa80 strain, LRa05 strain and BBr60 strain are inoculated into MRS culture medium respectively at an inoculation amount of 3% of the total mass of the culture medium, and cultured at 37°C for 18h to obtain culture liquids; the culture liquids are centrifuged to obtain bacterial bodies; the bacterial bodies are resuspended with a freeze-drying protective agent (100g / L aqueous solution of trehalose), and the mass ratio of the freeze-drying protective agent to the bacterial bodies is 2:1 to obtain a resuspension liquid; the resuspension liquid is freeze-dried by a vacuum freezing method to obtain freeze-dried powders of the three kinds of bacteria respectively. The BLa80 freeze-dried powder, the LRa05 freeze-dried powder and the BBr60 freeze-dried powder are mixed according to the ratio of the number of viable bacteria to obtain two-bacterium mixtures or three-bacterium mixtures.

[0039] The test result data is statistically analyzed using R language ggplot2. Compared with the control group, ### represents p<0.001, ## represents p<0.01, and # represents p<0.05; compared with the model group, *** represents p<0.001, ** represents p<0.01, and * represents p<0.05.

[0040] Example

[0041] This example explores the symptom improvement ability of the composite probiotic preparation on the hyperuricemia rat model:

[0042] (1) Test animals: healthy SPF level male 7-week-old SD rats (80), which are raised in a controlled environment, with room temperature maintained at 22-26°C, humidity at 50-60%, and following a 12h light / dark cycle. They can eat and drink water at will.

[0043] (2) Animal grouping: After the rats were adaptively fed for 1 week, they were randomly divided into 10 groups, 8 rats in each group: control group (CTL), high uric acid model group (MC group), allopurinol group (PG group, positive control), S1 group (combined intervention with BLa80 freeze-dried powder and LRa05 freeze-dried powder, the ratio of viable bacteria is 1:1), S2 group (combined intervention with BLa80 freeze-dried powder and BBr60 freeze-dried powder, the ratio of viable bacteria is 1:1), S3 group (combined intervention with LRa05 freeze-dried powder and BBr60 freeze-dried powder, the ratio of viable bacteria is 1:1), S4 group (combined intervention with BLa80 freeze-dried powder, LRa05 freeze-dried powder and BBr60 freeze-dried powder, the ratio of viable bacteria is 1:1:1), S5 group (combined intervention with BLa80 freeze-dried powder, LRa05 freeze-dried powder and BBr60 freeze-dried powder, the ratio of viable bacteria is 10:10:1), S6 group (combined intervention with BLa80 freeze-dried powder, LRa05 freeze-dried powder and BBr60 freeze-dried powder, the ratio of viable bacteria is 1:1:10), S7 group (combined intervention with commercially available Bifidobacterium animalis lactis ATCC700541 freeze-dried powder, commercially available Lactobacillus rhamnosus ATCC53103 freeze-dried powder and commercially available Bifidobacterium adolescentis ATCC15700 freeze-dried powder, the ratio of viable bacteria is 1:1:1).

[0044] (3) Animal modeling and intervention method:

[0045] In addition to the control group, the remaining groups were fed with rat feed containing 2.5% uric acid daily and were given 750 mg / kg body weight of oxypurinol by gavage every day. On the 7th day of the experiment, the model was confirmed to be successfully established by detecting the uric acid level in the blood after using ether anesthesia to take blood from the orbital vein. Subsequently, from the 8th day, the probiotic intervention group was given 2.5x10 9 CFU / kg body weight of each group of probiotic freeze-dried powder solution (solvent is normal saline) by gavage every day, the allopurinol group was given 30 mg / kg body weight of allopurinol (solvent is normal saline) by gavage, and the control group and the model group were given the same volume of normal saline.

[0046] (4) Sample collection:

[0047] The experiment lasted until the 28th day. The rats were again anesthetized with ether, blood serum samples were obtained, and rat fecal samples were collected. The content of uric acid in the serum and feces was detected by a fully automatic biochemical analyzer, and the content of urea nitrogen in the serum was detected. The activity of xanthine oxidase in the serum was determined by a xanthine oxidase activity detection kit. The fecal uric acid excretion was calculated, and the fecal uric acid excretion = fecal uric acid concentration x fecal weight.

[0048] (5) Index analysis:

[0049] (5.1) Changes in serum uric acid levels in rats on the 7th day:

[0050] As shown in Figure 1, compared with the control group, the serum uric acid levels of the other groups of rats were significantly increased by gavage with potassium oxonate for 7 consecutive days combined with a high uric acid diet, confirming the successful establishment of the hyperuricemia model.

[0051] (5.2) Changes in serum uric acid levels of rats on day 28:

[0052] After 21 days of continuous intervention, as shown in Figure 2, the serum uric acid levels of rats in all probiotic intervention groups and allopurinol intervention groups were reduced to varying degrees compared with the high uric acid model group, and the effect of combined intervention of BLa80 freeze-dried powder, LRa05 freeze-dried powder and BBr60 freeze-dried powder was better than that of other complex formulations.

[0053] (5.3) Changes in fecal uric acid content of rats on day 28:

[0054] After 21 days of continuous intervention, as shown in Figure 3, the fecal uric acid content of rats in all probiotic intervention groups and allopurinol intervention groups was reduced to varying degrees compared with the high uric acid model group, and the effect of combined intervention of BLa80 freeze-dried powder, LRa05 freeze-dried powder and BBr60 freeze-dried powder was more significant.

[0055] (5.4) Changes in fecal uric acid excretion of rats on day 28:

[0056] After 21 days of intervention, as shown in Figure 4, the fecal uric acid excretion of rats in all probiotic intervention groups and allopurinol intervention groups was reduced to varying degrees compared with the high uric acid model group, and the combined intervention of BLa80 freeze-dried powder, LRa05 freeze-dried powder and BBr60 freeze-dried powder was more effective in reducing uric acid excretion.

[0057] By measuring the fecal uric acid content and excretion, the immediate and long-term effects of the intervention measures on uric acid metabolism were comprehensively evaluated, so as to more accurately understand the regulatory effect of the intervention measures on the uric acid excretion pathway of rats.

[0058] (5.5) Changes in serum xanthine oxidase activity of rats on day 28:

[0059] As shown in Figure 5, the detection results of serum xanthine oxidase activity showed that hyperuricemia caused a significant increase in the activity of the enzyme in the serum of rats compared with the control group. On day 28, the xanthine oxidase activity of each probiotic intervention group and allopurinol intervention group was significantly lower than that of the high uric acid model group, indicating that the probiotic preparations involved in the present application could inhibit the activity of xanthine oxidase in the serum of rats with hyperuricemia, and the effect of the complex formulation of the three strains was better than that of other complex formulations.

[0060] (5.6) Changes in serum urea nitrogen activity of rats on day 28:

[0061] As shown in Figure 6, at day 28, the serum urea nitrogen level of the rats in the high uric acid model group was significantly higher than that of the control group, indicating that hyperuricemia can cause inflammation and impaired renal function in rats. The urea nitrogen content of each probiotic intervention group was lower than that of the model group, indicating that the probiotic preparations involved in the present application help to alleviate kidney damage in rats with hyperuricemia, and the three-strain compound formula is more effective in reducing urea nitrogen.

[0062] The applicant states that the technical solutions of the present application are illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned embodiments, that is, it does not mean that the present application must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of 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. fall within the protection scope and disclosure scope of the present application.

[0063] 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-mentioned embodiments, and 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.

[0064] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A probiotic formulation for alleviating hyperuricemia, characterized by, The strains in the complex probiotic preparation for relieving hyperuricemia include a Bifidobacterium animalis subsp. lactis BLa80 strain with a preservation number of CGMCC No. 15410, a Lactobacillus rhamnosus LRa05 strain with a preservation number of CGMCC No. 1.12734, and a Bifidobacterium breve BBr60 strain with a preservation number of CGMCC No. 12915.

2. The composite probiotic formulation according to claim 1, characterized in that, The viable bacteria content in the complex probiotic preparation is not less than 1 x 10 8 CFU / g or 1 x 10 8 CFU / mL.

3. The composite probiotic formulation according to claim 1, characterized in that, The ratio of viable cell numbers of the BLa80 strain, the LRa05 strain, and the BBr60 strain is (1-10):(1-10):(1-10).

4. The composite probiotic formulation according to claim 1, wherein, The dosage form of the complex probiotic preparation includes a solution, a lyophilized powder, a capsule, a tablet, or a granule.

5. The composite probiotic formulation according to claim 4, characterized in that, The dosage form of the complex probiotic preparation is a lyophilized powder, which is prepared by the following method: The BLa80 strain, the LRa05 strain, and the BBr60 strain are inoculated into culture media respectively for activation and fermentation culture in sequence to obtain fermentation broths; the fermentation broths are centrifuged respectively to obtain bacterial slurry; the bacterial slurry is mixed with a protective agent to obtain a resuspension; the resuspension is freeze-dried to obtain BLa80 bacterial powder, LRa05 bacterial powder, and BBr60 bacterial powder; the BLa80 bacterial powder, the LRa05 bacterial powder, and the BBr60 bacterial powder are mixed according to the ratio of viable cell numbers to obtain the complex probiotic preparation.

6. The composite probiotic formulation according to claim 5, characterized in that, 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.

7. The composite probiotic formulation according to claim 5, wherein, The fermentation culture is performed at a temperature of 35-40°C for 12-30 h. Preferably, the inoculation amount is 1-5%. Preferably, the culture medium is an MRS culture medium.

8. Use of the complex probiotic preparation in any one of claims 1-7 in the preparation of a drug for preventing, improving, or treating hyperuricemia.

9. Use according to claim 8, characterized in that, The drug further contains an auxiliary material.

10. Use according to claim 9, characterized in that, The auxiliary material includes any one or a combination of at least two of a filler, a binder, a wetting agent, a disintegrating agent, 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.

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