Application of lactobacillus rhamnosus B6 in preparation of product for reducing hyperuricemia
By regulating the intestinal flora through Lactobacillus rhamnosus B6, the problems of large side effects and unstable effects in the treatment of hyperuricemia are solved, the effect of continuously lowering uric acid is achieved, and it is used in the preparation of uric acid-lowering products.
Patent Information
- Application Number
- CN202511117606.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-11
- Publication Date
- 2025-10-17
AI Technical Summary
Existing drugs for the treatment of hyperuricemia have problems such as large side effects, short-lasting effects, unstable effects and high costs, and there is a lack of effective lactic acid bacteria resources for regulating intestinal flora to lower uric acid levels.
Using Lactobacillus rhamnosus B6 (CGMCC NO.13310) as the active ingredient, a liver-specific UOX gene knockout mouse model was used to demonstrate its ability to lower blood uric acid levels, regulate intestinal flora composition and urea cycle pathways, increase flora diversity and the abundance of butyrate-producing flora, and reduce the levels of pro-inflammatory factors.
It significantly reduces the blood uric acid level of hyperuricemia mice, regulates the intestinal flora structure, improves flora diversity, increases the abundance of butyrate-producing flora, reduces pro-inflammatory factors, improves the urea cycle pathway, and provides sustained and stable therapeutic effects.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of microbial technology, in particular to the application of Lactobacillus rhamnosus B6 in the preparation of a product for reducing high uric acid. BACKGROUND
[0002] Hyperuricemia is a metabolic disease caused by purine metabolism disorder, and is closely related to the occurrence and development of metabolic syndrome. With the development of social economy, the change of people's lifestyle and dietary structure, the prevalence of hyperuricemia is increasing year by year, and it is becoming younger and younger, which has become one of the important diseases threatening human health. Due to the lack of effective and stable animal models of hyperuricemia, the selection of drugs for treating hyperuricemia is still limited. At present, most of the drugs for treating hyperuricemia in clinical practice have the following risks: (1) large side effects, easy to cause gastrointestinal discomfort and loss of appetite; (2) short duration of effect, usually after taking effect, continuous taking for several courses is required to maintain the effect, and the duration of effect is poor; (3) unstable effect, the treatment mechanism is not clear enough, so the treatment effect is often different for different people, and is not stable enough; (4) high cost: long-term drug use will bring great economic pressure to patients.
[0003] Lactic acid bacteria are a special group of microorganisms that exist in the human body. They usually have multiple probiotic functions, such as promoting the absorption of nutrients by the human body, synthesizing various essential vitamins for the human body, regulating immunity, etc. In today's rapidly developing field of microbial flora research, although humans have a fairly deep understanding of the relationship between microbial flora and specific diseases of the human body, so far, there is basically no lactic acid bacteria resource used in clinical treatment of hyperuricemia. In view of this, finding a lactic acid bacteria that can regulate intestinal flora and reduce uric acid level to alleviate a series of health problems caused by hyperuricemia has become an important breakthrough direction in this field. Therefore, the present application is proposed. SUMMARY
[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide the application of Lactobacillus rhamnosus B6 in the preparation of a product for reducing high uric acid, wherein the Lactobacillus rhamnosus B6 is a Lactobacillus rhamnosus strain with the preservation number of CGMCC NO.13310.
[0005] Compared with the prior art, the present application has the following beneficial effects:
[0006] Compared with the prior art, the present application first uses a liver-specific UOX gene knockout mouse as a hyperuricemia model to evaluate the effect of Lacticaseibacillus rhamnosus strain (CGMCC NO.13310) on hyperuricemia, and exhibits a new use of Lacticaseibacillus rhamnosus strain (CGMCC NO.13310) in reducing blood uric acid level, regulating intestinal flora composition and urea cycle pathway, increasing flora diversity, increasing butyrate-producing flora abundance, and reducing pro-inflammatory factor level, which can be applied to products against hyperuricemia. BRIEF DESCRIPTION OF DRAWINGS
[0007] Figure 1 Effect of Lacticaseibacillus rhamnosus B6 on blood uric acid level of hyperuricemia mice is shown.
[0008] Figure 2 Effect of Lacticaseibacillus rhamnosus B6 on flora diversity of hyperuricemia mice is shown; a: PCA principal component analysis chart of negative control group, model group and Lacticaseibacillus rhamnosus B6; b: flora alpha diversity analysis chao1 index analysis chart.
[0009] Figure 3 Effect of Lacticaseibacillus rhamnosus B6 on intestinal flora composition of hyperuricemia mice is shown. DETAILED DESCRIPTION
[0010] The embodiments of the present application are illustrated by specific specific examples below, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure in the specification. The present application can also be implemented or applied by other different specific embodiments, and various modifications or changes can be made to the details in the specification based on different views and applications without departing from the spirit of the present application.
[0011] The present application first discovers that Lacticaseibacillus rhamnosus strain has the effect of reducing uric acid, can regulate intestinal flora composition and urea cycle pathway, increase flora diversity, increase butyrate-producing flora abundance, and reduce pro-inflammatory factor level, and can be applied to products for reducing uric acid.
[0012] The present application first provides the use of Lacticaseibacillus rhamnosus B6 in preparing a product for reducing uric acid, wherein the Lacticaseibacillus rhamnosus B6 is a Lacticaseibacillus rhamnosus strain with a preservation number of CGMCC NO.13310.
[0013] In certain embodiments of the present application, the product for reducing uric acid has one or more of the following functions:
[0014] 1) reducing uric acid level;
[0015] 2) modulating the abundance of butyrate-producing bacteria;
[0016] 3) reducing the level of pro-inflammatory factors;
[0017] 4) modulating the urea cycle pathway.
[0018] In a preferred embodiment, the product includes, but is not limited to, health products, food products, pharmaceuticals, and the like.
[0019] In a preferred embodiment, the dosage form of the product includes, but is not limited to, injections, suspensions, powders, tablets, or granules.
[0020] In certain embodiments of the present application, the product is a pharmaceutical, and the pharmaceutical further comprises a pharmaceutically acceptable carrier or excipient.
[0021] "Pharmaceutically acceptable" means they are not deleterious, allergenic, or otherwise undesirable when the pharmaceutical is properly administered to an animal or human.
[0022] "Pharmaceutically acceptable carrier or excipient" shall mean compatible with the effective ingredients, i.e., the active ingredients, and not deleterious to the subject to which it is administered in need. Specific examples of substances which can serve as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffered solutions, and the like. These substances are used as needed to aid formulation stability or to enhance the activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration.
[0023] In some embodiments, the Lacticaseibacillus rhamnosus strain of the present application can be administered in monotherapy (e.g., without concomitant administration of any additional therapeutic agent, or without concomitant administration of any additional therapy for the same disease to be treated or prevented with the drug of the present application). In some embodiments, the Lacticaseibacillus rhamnosus strain of the present application can also be administered in combination or concurrently with one or more other therapeutic agents.
[0024] In some embodiments of the present application, the product can contain a Lacticaseibacillus rhamnosus bacterial agent. Preferably, the strain in the bacterial agent comprises the Lacticaseibacillus rhamnosus strain with the accession number CGMCC NO. 13310.
[0025] In a specific embodiment, the preparation method of the bacterial agent can comprise: 1) inoculating Lacticaseibacillus rhamnosus B6 in a liquid medium for fermentation culture; 2) separating and collecting the bacterial body after culture in step 1), and centrifuging to collect the bacterial body; 3) mixing the bacterial body with skimmed milk, and freeze-drying to obtain.
[0026] Further, the inoculation amount of the Lacticaseibacillus rhamnosus B6 strain is 1.25 x 10 7 ~ 1 x 10 8 CFU / mL.
[0027] Further, the liquid medium can be MRS liquid medium.
[0028] Preferably, in step 1), the fermentation process is static culture, the fermentation temperature is 25-45℃, and the time is 12-36h.
[0029] More preferably, the temperature can be selected from 25℃, 27℃, 29℃, 31℃, 33℃, 35℃, 37℃, 39℃, 41℃, 43℃, 45℃, etc., and preferably 37℃; the time can be selected from 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, etc., and preferably 24h.
[0030] Preferably, in step 2), the centrifugation speed is 4000-12000g, and the time is 8-12min.
[0031] More preferably, the rotation speed can be selected from 4000g, 4500g, 5000g, 5500g, 6000g, 6500g, 7000g, 7500g, 8000g, 8500g, 9000g, 9500g, 10000g, 10500g, 11000g, 11500g, 12000g, etc., and preferably 10000g; the time can be selected from 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min, 12min, etc., and preferably 10min.
[0032] Preferably, the mass percentage of skimmed milk powder in the skimmed milk is 6-12%, for example 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, etc., and preferably 10%.
[0033] In a preferred embodiment, the viable bacterial count of the Lactobacillus rhamnosus B6 strain is not less than 1×10 9 CFU / mL, for example 1×10 9 CFU / mL, 2×10 9 CFU / mL, 3×10 9 CFU / mL, 4×10 9 CFU / mL, 5×10 9 CFU / mL, 6×10 9 CFU / mL, 7×10 9 CFU / mL, 8×10 9 CFU / mL, 9×10 9 CFU / mL, 1×10 10 CFU / mL, 2×10 10 CFU / mL, 5×10 10 CFU / mL, 8×10 10 CFU / mL, 1×10 11 CFU / mL, 5×10 11 CFU / mL, 1×10 12 CFU / mL, 1×10 13 CFU / mL, etc., and other specific point values in the above numerical range can be selected, which will not be described here one by one.
[0034] The strain preservation information of the present application is as follows:
[0035] Strain name: Lactobacillus rhamnosus Lacticaseibacillus rhamnosus
[0036] The preservation number is CGMCC NO.13310.
[0037] Deposit date: November 15, 2016;
[0038] Name of deposit unit: China General Microbiological Culture Collection Center;
[0039] Abbreviation of deposit unit: CGMCC;
[0040] Address of deposit unit: No. 1, Yiaobei Road, Beijing, China.
[0041] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection of the present application; in the specification and claims of the present application, the singular forms "a", "an" and "the" include the plural forms, unless otherwise explicitly stated in the text.
[0042] When the embodiments give numerical ranges, it should be understood that, unless otherwise stated by the present application, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art. In addition to the specific methods, devices, materials used in the embodiments, any method, device and material of the prior art similar or equivalent to those described in the embodiments of the present application can also be used to implement the present application according to the mastery of the prior art by those skilled in the art and the description of the present application.
[0043] Unless otherwise stated, the experimental methods, detection methods, preparation methods disclosed in the present application all use conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields.
[0044] The Lactocaseibacillus rhamnosus strain B6 involved in the following content is classified as Lactocaseibacillus rhamnosus, with a deposit date of November 15, 2016, a deposit number of CGMCC No. 13310, a deposit unit of China General Microbiological Culture Collection Center, and a deposit address of No. 1, Yiaobei Road, Beijing, China.
[0045] Example 1
[0046] Preparation of seeds (fermentation broth)
[0047] Lactobacillus rhamnosus B6 (CGMCC No. 13310) strain was picked up with a loop and inoculated into 10 mL of MRS liquid medium (purchased from Merck Co., Germany). The colony was uniformly dispersed in the liquid medium by using a vortex oscillator, and was cultured anaerobically at 37°C for 48 h. Then, 2% (v / v) of the culture was inoculated into MRS liquid medium, and was cultured anaerobically at 37°C for 24 h. The culture was centrifuged at 15,000 rpm for 10 min, and the supernatant was discarded. The bacterial cells were washed twice with sterile distilled water, and were suspended in the original culture volume of sterile distilled water to obtain a seed for fermentation. The bacterial concentration of the seed liquid was 2.5 x 10 9 CFU / mL.
[0048] Example 2
[0049] Preparation of the bacterial agent
[0050] Method for preparing skim milk: 10% (mass percentage) skim milk powder was mixed with distilled water, and was fully dissolved. Then, the mixture was sterilized at 125°C for 5 min, and was cooled to room temperature to obtain the required concentration of skim milk.
[0051] Method for preparing the bacterial agent:
[0052] (1) The fermentation seed liquid of Lactobacillus rhamnosus B6 (CGMCC No. 13310) was inoculated into MRS liquid medium at a seeding amount of 2% (v / v, the same below), and was cultured at 37°C for 24 h to obtain a fermentation liquid;
[0053] (2) The fermentation liquid obtained in step (1) was centrifuged at 10,000 g for 10 min, and the supernatant was discarded. The precipitate (bacterial cells) was washed twice with sterile distilled water.
[0054] (3) The bacterial cells obtained in step (2) were resuspended in the original culture volume of skim milk (10% (mass percentage) of the plant).
[0055] Example 3
[0056] Modulatory effect of Lactobacillus rhamnosus B6 on uric acid and intestinal flora in hyperuricemic mice
[0057] Animal experiment: Five 7-week-old SPF C57BL / 6 mice (Shanghai Tenth People's Hospital) and ten 7-week-old liver-specific UOX gene knockout mice (Shanghai Tenth People's Hospital) of the same background were purchased, and were adaptively fed for 1 week. The liver-specific UOX gene knockout mice were randomly divided into two groups, with five mice in each group. The mice were fed with Lactobacillus rhamnosus B6 (B6 intervention group) and skim milk powder (model group), respectively. The mice were intragastrically administered with Lactobacillus rhamnosus B6 at a dose of 1 x 10 9CFU / mL) 0.2 mL, for four weeks. SPF C57BL / 6 mice were fed with skim milk powder as negative control group. The experimental animal information table and gavage method are shown in Table 1.
[0058] Table 1 Experimental animal information table
[0059] Group Number / group Treatment time / day Feed Gavage method Negative control group 5 28 Normal feed Skim milk B6 intervention group 5 28 Normal feed B6 bacterial suspension Model group 5 28 Normal feed Skim milk
[0060] (1) Effect on blood uric acid level
[0061] The blood of mice was collected at the start of the experiment and after 4 weeks of gavage, respectively, and the serum uric acid level of mice was detected using a kit. The results of the change of serum uric acid of mice are shown in Figure 1 Compared with the model group, B6 intervention for 4 weeks can significantly reduce the change value of serum uric acid of mice, and the experimental results show that Lactobacillus rhamnosus B6 can significantly reduce the blood uric acid level of hyperuricemia mice, and make it close to the normal mouse uric acid level.
[0062] (2) Regulation effect on intestinal flora
[0063] At the end of 4 weeks of gavage, feces of 3 mice from each of the three groups were randomly selected for 16S RNA sequencing to analyze the intestinal flora of mice. The results of intestinal flora diversity are shown in Figure 2 As shown in Figure 2 a), the flora structure of the three groups showed significant differences and were clustered in different positions, and compared with the model group, B6 can significantly improve the Chao 1 value of intestinal flora Figure 2 b), indicating that Lactobacillus rhamnosus B6 can significantly change the intestinal flora structure of hyperuricemia mice and improve the flora diversity of hyperuricemia mice.
[0064] The results of analyzing the composition of intestinal flora of mice in three groups are shown in Figure 3 As shown in Figure 3), wherein the three OTUs showing significant differences in comparison are shown in Table 2, including OTU111, OTU384, and OTU218, and the three OTUs are all classified as Lachnospiraceae, which can effectively produce butyrate. In combination with the detection of the serum pro-inflammatory factor TNFα level of the three groups of mice, it is shown that the B6 intervention group is significantly lower than the model group in the content of pro-inflammatory factor TNFα (Table 3), indicating that the Lactobacillus rhamnosus B6 can significantly increase the abundance of butyrate-producing bacteria in hyperuricemia mice, and further reduce the level of inflammatory factors. Butyrate, as a short-chain fatty acid, has the activities of regulating microbiome, anti-inflammation, anti-obesity, regulating metabolic pathways, and anti-oxidation. The typical pathological characteristics of hyperuricemia are chronic inflammation, and high uric acid can cause oxidative stress, renal endothelial dysfunction, and inflammatory response. The Lactobacillus rhamnosus B6 can further reduce inflammation by regulating the production of short-chain fatty acids by intestinal flora, and thus regulate uric acid metabolism.
[0065] Table 2 Influence of B6 on butyrate-producing bacterial population abundance (unit: %)
[0066]
[0067] Table 3 Influence on blood pro-inflammatory factor TNFα
[0068]
[0069] By using the Picrust2 software to predict the functions of the intestinal flora of the three groups of mice, the results are shown in Table 4. Compared with the model group, the B6 intervention group can significantly up-regulate the urea cycle pathway (PWY-4984), which is significantly down-regulated in gout patients caused by high uric acid. The urea cycle is a metabolic pathway that converts ammonia into products, thereby removing it from the body. Deficiency of enzymes and transport defects on this pathway are urea cycle disorders induced by hyperuricemia. The Lactobacillus rhamnosus B6 can improve hyperuricemia by regulating the urea cycle pathway.
[0070] Table 4 Influence of B6 on intestinal flora urea cycle pathway (PWY-4984)
[0071]
[0072] In summary, the Lactobacillus rhamnosus B6 can regulate the composition of intestinal flora, improve the diversity of flora, increase the abundance of butyrate-producing bacteria, reduce the level of pro-inflammatory factors, up-regulate the urea cycle pathway, and thus significantly reduce the blood uric acid level of hyperuricemia mice, making it closer to that of normal mice.
[0073] Applicant states that the application illustrates a Lactobacillus rhamnosus in preparing high uric acid reducing product by the above-mentioned embodiments, and the application is not limited to the above-mentioned embodiments, that is, it does not mean that the application must rely on the above-mentioned embodiments to be implemented. The skilled in the art should understand that any improvement of the application, equivalent replacement of each raw material of the product of the application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the application.
[0074] The above has described the basic concept, and it is obvious that the above detailed disclosure is only as an example for the skilled in the art, and does not constitute a limitation on the specification. Although it is not explicitly stated here, the skilled in the art can make various modifications, improvements and corrections to the specification. Such modifications, improvements and corrections are suggested in the specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of the specification.
[0075] At the same time, the specification uses specific words to describe the embodiments of the specification. As "one embodiment", "an embodiment", and / or "some embodiments" means a certain feature, structure or characteristic related to at least one embodiment of the specification. Therefore, it should be emphasized and noted that the "an embodiment" or "one embodiment" or "one alternative embodiment" mentioned in different places in the specification does not necessarily refer to the same embodiment. In addition, some features, structures or characteristics in one or more embodiments of the specification can be properly combined.
[0076] Finally, it should be understood that the embodiments described in the specification are only used to illustrate the principles of the embodiments of the specification. Other variations can also belong to the scope of the specification. Therefore, as an example but not limitation, alternative configurations of the embodiments of the specification can be considered as consistent with the teachings of the specification. Accordingly, the embodiments of the specification are not limited to the embodiments explicitly introduced and described in the specification.
Claims
1. The application of Lactobacillus rhamnosus B6 in the preparation of a product for reducing hyperuricemia, characterized in that: The Lactobacillus rhamnosus B6 is a Lacticaseibacillus rhamnosus strain with a preservation number of CGMCC NO.13310.
2. The use according to claim 1, characterized in that The uric acid-lowering product has one or more of the following functions: 1) Lower uric acid levels; 2) regulating the abundance of butyrate-producing bacteria; 3) Reduce the levels of pro-inflammatory factors; 4) Regulate the urea cycle pathway.
3. The use according to claim 1, characterized in that The product includes one or more of health care products, food or medicine.
4. The use according to claim 3, characterized in that The dosage form of the product includes one or more of injection, suspension, powder, tablet or granule.
5. The use according to claim 1, characterized in that The viable count of Lactobacillus rhamnosus in the product is not less than 1×10 9 CFU / g.
6. The use according to any one of claims 1 to 5, characterized in that The product contains the Lacticaseibacillus rhamnosus bacterial agent.
7. The use according to claim 6, characterized in that The preparation method of the Lactobacillus rhamnosus B6 bacterial agent comprises the following steps: 1) inoculating Lacticaseibacillus rhamnosus into a liquid culture medium for fermentation; 2) isolating and collecting the bacterial cells cultured in step 1) by centrifugation; 3) The bacterial cells obtained in step 2) are mixed with skim milk and freeze-dried.
8. The use according to claim 7, characterized in that The inoculum amount of Lactobacillus rhamnosus B6 was 1.25×10 7 ~1×10 8 CFU / mL.
9. The use according to claim 7, characterized in that The fermentation process is static culture, the fermentation temperature is 25-45° C., and the fermentation time is 12 to 36 hours.
10. The use according to claim 7, characterized in that The mass percentage of skim milk powder in the skim milk is 6 to 12%; And / or, the centrifugal speed is 4000-12000 g, and the time is 8-12 min.