Lactobacillus mucilaginosus with uric acid reducing function and application of lactobacillus mucilaginosus

Abstract

The invention discloses fermentation mucus lactobacillus with a uric acid reducing function and application of the fermentation mucus lactobacillus. The fermentation mucus lactobacillus is preserved in the China General Microbiological Culture Collection Center on August 4, 2025, and the preservation number is CGMCC No.35505. The invention further discloses a preparation method of the fermentation mucus lactobacillus. The fermented lactobacillus mucus has excellent creatinine degradation capability, can reduce serum uric acid, creatinine and urea nitrogen levels of an individual with hyperuricemia, and alleviates liver or kidney injury caused by the individual with hyperuricemia; in addition, the fermented lactobacillus mucus can also improve the composition of intestinal flora and inhibit the abnormal change of the flora related to metabolic disorder, thereby restoring the stable state of the microecology of the intestinal tract. Therefore, the fermented lactobacillus mucilaginosus can be used for preparing uric acid reducing functional foods, health foods or microecological preparations.

Description

Technical Field

[0001] This invention belongs to the field of microecological preparations, functional foods and metabolic disease intervention technology, specifically involving a fermentable Lactobacillus mucinus with uric acid-lowering function and its application. Background Technology

[0002] Hyperuricemia is a common metabolic disorder caused by excessive uric acid production or reduced uric acid excretion in the body. It is a significant risk factor for gout, kidney damage, and various metabolic diseases. With changes in dietary structure and lifestyle, the incidence of hyperuricemia is increasing year by year, becoming a major public health issue affecting population health. Uric acid is the end product of purine metabolism, and its main sources include endogenous purine metabolism in the body and the breakdown of purines in the diet. Inosine, hypoxanthine, and xanthine are key intermediates in purine metabolism, with xanthine oxidase (XOD) playing a crucial catalytic role in uric acid production. When uric acid production increases or excretion is impaired, serum uric acid levels easily rise, further increasing the burden on the kidneys and causing tissue damage. Currently, clinical interventions to lower uric acid levels mainly include drugs that inhibit uric acid production or promote uric acid excretion. However, some drugs may cause liver and kidney dysfunction, gastrointestinal discomfort, or allergic reactions during long-term use, limiting their long-term application. Therefore, developing new uric acid-lowering strategies that are safe and suitable for long-term intervention is of great significance.

[0003] In recent years, studies have revealed the crucial role of gut microbiota in regulating host purine metabolism and uric acid homeostasis. On one hand, some gut microbes can participate in the metabolism or transformation of purines, thereby affecting uric acid production; on the other hand, gut microbiota imbalance may exacerbate inflammatory responses and intestinal barrier damage, thus affecting metabolic homeostasis. Regulating gut microbiota structure and improving the gut microecological environment is considered a promising metabolic regulatory pathway.

[0004] Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum As a common lactic acid bacterium, *Lactobacillus fermentum* possesses good acid and bile salt tolerance and intestinal adaptability, giving it certain advantages in maintaining intestinal microecological balance. Existing research largely focuses on its role in regulating intestinal function or immune homeostasis, but research on *Lactobacillus fermentum*, which simultaneously possesses the ability to degrade purine metabolic substrates in vitro, lower uric acid levels in vivo, and improve liver and kidney function and intestinal flora structure, remains relatively limited.

[0005] Therefore, it is necessary to develop a fermentable Lactobacillus mucinus with a clear function of lowering uric acid and improving intestinal health, so as to provide a new technical approach for long-term and safe intervention of hyperuricemia-related metabolic abnormalities. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a fermentable Lactobacillus mucinus with uric acid-lowering function and its application. By degrading purine precursors and regulating the intestinal microecology, it reduces serum uric acid levels and improves hyperuricemia-related metabolic status.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A fermentable *Lactobacillus fermentatus* 21J with uric acid-lowering function was deposited on August 4, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35505 and classified as *Lactobacillus fermentatus*. Limosilactobacillus fermentum ).

[0008] A microbial agent comprising the aforementioned *Lactobacillus fermentans* 21J.

[0009] The application of the above-mentioned fermented Lactobacillus mucinus 21J or the above-mentioned microbial agents in the preparation of uric acid-lowering products.

[0010] The application of the above-mentioned fermented Lactobacillus mucinus 21J or the above-mentioned microbial agents in the preparation of inosine-degrading products.

[0011] The above-mentioned fermented Lactobacillus mucinus 21J or the above-mentioned microbial agents are used in foods, health foods or functional products prepared for regulating intestinal flora and metabolic state.

[0012] The application of the above-mentioned fermented Lactobacillus mucinus 21J or the above-mentioned microbial agents in the preparation of products for the treatment, prevention or adjuvant treatment of hyperuricemia.

[0013] Furthermore, the above applications include at least one of the following functions: (1) Reduce serum uric acid, creatinine and urea nitrogen levels in individuals with hyperuricemia; (2) Reduce liver or kidney damage caused by hyperuricemia in individuals; (3) Improve the gut microbiota structure of individuals with hyperuricemia and promote the restoration of the microbiota to a healthy homeostasis.

[0014] Furthermore, the above-mentioned product contains 1×10 8 CFU / mL ~ 1×10 10 CFU / mL of fermented Lactobacillus mucinus 21J.

[0015] Furthermore, the aforementioned products include animal feed and pharmaceuticals.

[0016] Furthermore, it also includes food-grade or pharmaceutical-grade acceptable carriers or excipients.

[0017] The beneficial effects of this invention are as follows: This invention screened and obtained a strain of fermenting *Lactobacillus mucilaginosus* (…). Limosilactobacillus fermentum This fermenting *Lactobacillus mucinus* exhibits excellent creatinine degradation capabilities, reducing serum uric acid, creatinine, and urea nitrogen levels in individuals with hyperuricemia, and alleviating liver or kidney damage caused by hyperuricemia. Furthermore, this fermenting *Lactobacillus mucinus* restores intestinal microecological homeostasis by improving intestinal flora composition and inhibiting abnormal changes in flora related to metabolic disorders. It also improves renal energy metabolism and reduces the metabolic burden caused by abnormal uric acid metabolism. Therefore, the fermenting *Lactobacillus mucinus* of this invention can be used to prepare uric acid-lowering functional foods, health foods, or microecological preparations. Attached Figure Description

[0018] Figure 1 The peak diagrams are shown for inosine solution without strain 21J (A) and inosine solution with strain 21J (B).

[0019] Figure 2 Gram-stained microscopic image of Lactobacillus fermentans 21J.

[0020] Figure 3 The colony morphology of Lactobacillus fermentans 21J.

[0021] Figure 4 Phylogenetic tree of Lactobacillus fermentans 21J.

[0022] Figure 5 Serum uric acid levels in mice under different treatment groups.

[0023] Figure 6 The levels of blood urea nitrogen (BUN) and serum creatinine (CRE) in the serum of mice in different treatment groups were measured.

[0024] Figure 7 Paraffin sections of the kidneys of mice in different treatment groups.

[0025] Figure 8 Paraffin sections of mouse livers from different treatment groups.

[0026] Figure 9 The bar chart shows the species abundance at the phylum level for the model group, the blank group, and strain 21J.

[0027] Figure 10 The bar chart shows the species abundance at the genus level for the model group, the blank group, and strain 21J.

[0028] Figure 11 Abundance diagram of Bifidobacterium in the model group, blank group, and strain 21J group.

[0029] Figure 12 Volcano diagram of differentially expressed kidney metabolites between strain 21J and the control group.

[0030] Figure 13 Bubble plot of differentially metabolized KEGG in the kidneys of strain 21J and the control group.

[0031] Figure 14 This is a statistical graph showing the differences in renal metabolites between the hyperuricemia model group and the control group.

[0032] Figure 15 This is a statistical graph showing the differences in kidney metabolites between strain 21J and the control group. Detailed Implementation

[0033] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all reagents and materials used in the following examples are commercially available products.

[0035] Example 1: Screening, Isolation and Identification of Strains This embodiment involves MRS liquid culture medium: 20g glucose, 10g tryptone, 10g beef extract, 5g yeast extract, 5g anhydrous sodium acetate, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.25g manganese sulfate, 1g Tween 80, and 1000ml distilled water. The solution was thoroughly dissolved and the pH was adjusted to 6.5. The MRS plate was prepared by adding 20g agar to the MRS liquid culture medium formulation.

[0036] Take 1 mL of fecal sample from a healthy person and add it to 10 mL of MRS liquid culture medium. Activate and culture at 37°C for 24 hours. Take 0.5 mL of the activated bacterial solution and add it to physiological saline for 10... -1 ~10 -9 Serial dilutions were performed, and the diluted solutions were spread on MRS plates. After incubation, single colonies with uniform morphology were picked and purified by streak plating. This process was repeated 2-3 times until purified strains were obtained. The isolated single colonies were then preserved in glycerol.

[0037] A single colony was inoculated into MRS liquid medium and activated at 37°C for 24 h. Then, it was transferred to fresh MRS liquid medium at a 2% (v / v) inoculation rate and cultured at 37°C for another 24 h. After culturing, 2 mL of culture was centrifuged at 8000 r / min for 5 min at 4°C to collect the bacterial cells. The obtained bacterial cells were resuspended and washed with sterile PBS buffer (pH=7.5), and centrifuged again for 5 min under the same conditions to collect the bacterial cells. This washing process was repeated twice. 750 μL of reaction solution (1 mg / mL inosine standard solution dissolved in sterile PBS (pH 7.0)) was added to the washed bacterial cells, and the mixture was thoroughly resuspended and reacted at 37°C for 1 h. At the end of the reaction, 80 μL of stop agent (0.1 mol / L perchloric acid) was added and mixed thoroughly. The mixture was then centrifuged at 12000 r / min for 2 min at 4°C, and the supernatant was collected. The supernatant was filtered through a 0.22 μm microporous membrane, and the degradation rate of inosine was calculated by chromatographic analysis. The chromatographic conditions were as follows: column Aq 4.6 × 250 mm, 5 μm, column temperature 35℃, flow rate 1.0 mL / min, injection volume 10 μL, detection wavelength 260 nm, mobile phase V(water):V(chromatographic methanol) = 95:5, pH 4.7. The degradation rate of inosine by the strain was calculated using the following formula: Degradation rate (%) = (C 母液 -C 残留 ) / C 母液 ×100%, where C is the mass concentration (mg / mL). Finally, a strain 21J with a high inosine degradation rate was screened, achieving a 100% degradation rate of 1 mg / mL inosine within 1 hour (see...). Figure 1 ).

[0038] Strain strain 21J was inoculated into MRS liquid medium and cultured at 37°C for 24 hours. After dilution and plating, Gram staining and microscopic examination were performed. Figure 2 Strain 21J is a Gram-positive bacterium; colony morphology diagram is shown below. Figure 3 Genomic DNA was extracted using a bacterial genomic DNA extraction kit. The full-length 16S rDNA gene sequence was amplified using the universal bacterial primer 27F-1492R and sequenced by Shanghai Sangon Biotech Co., Ltd. The 16S rDNA sequence of strain 21J is shown in SEQ ID NO: 1. The obtained 16S rDNA sequence was compared with known sequences on the NCBI website, and a phylogenetic tree was constructed using MEGA 7.0 software. The phylogenetic tree is shown below. Figure 4 The results showed that strain 21J was related to fermenting *Lactobacillus mucilaginosus* (…). Limosilactobacillus fermentum Based on the highest homology, this strain was identified as *Lactobacillus fermentatus*. Limosilactobacillus fermentum This strain 21J was deposited at the China General Microbiological Culture Collection Center on August 4, 2025, with accession number CGMCC No. 35505.

[0039] Example 2

[0040] The animal model and experimental design used in this embodiment are as follows: (1) Animal model construction The experiment used male Kunming mice and established an animal model of hyperuricemia by feeding them a diet containing yeast extract (basal mouse diet + 10wt% yeast extract). Natural ventilation, free access to food and water were provided, and bedding was changed regularly.

[0041] (2) Animal experimental design All mice were acclimatized to a commercial basal diet for one week, then randomly divided into four groups of 12 mice each. During the experiment, the mice's food and water intake were not restricted in any way. During the modeling period, the first group was fed only the basal diet (control group), while the other three groups were fed a high-uric acid mouse diet containing yeast extract. Intervention treatment began in the third week. Except for the control group, which continued to be fed the basal diet, the other three groups received the following interventions: Group 2 (model group): Continue to feed high-uric acid rat diet containing yeast extract; in addition, administer potassium oxonate and hypoxanthine once a day by gavage, with potassium oxonate at a dose of 250 mg / kg and hypoxanthine at a dose of 300 mg / kg to maintain the high uric acid state.

[0042] Group 3 (strain 21J, experimental group): Continued feeding of high-uric acid rat diet containing yeast extract; in addition, daily gavage administration of potassium oxonate (250mg / kg) + hypoxanthine (300mg / kg) to maintain the high-uric acid state; while continuing modeling, daily gavage administration of 1×10 9 0.2 mL of fermentation broth of *Lactobacillus myxoidis* 21J (CFU / mL) was prepared as follows: 60 μL of glycerol-preserved strain 21J was inoculated into 3 mL of MRS liquid medium and activated at 37°C for 24 h to obtain activated bacterial solution. 60 μL of the activated bacterial solution was transferred to 3 mL of fresh MRS liquid medium and incubated at 37°C for 24 h. This was then passaged to the third generation. The resulting bacterial solution was centrifuged at 8000 r / min for 5 min at 4°C, and the bacterial pellet was collected, washed with sterile water, resuspended, and the bacterial suspension concentration was adjusted to 1 × 10⁻⁶. 9 CFU / mL, used for subsequent animal experiments.

[0043] Group 4 (allopurinol group): Continue to feed high-uric acid rat diet containing yeast extract; in addition, administer potassium oxonate (250mg / kg) + hypoxanthine (300mg / kg) by gavage daily to maintain high uric acid status; while continuing to establish the model, administer allopurinol by gavage once a day at a dose of 10mg / kg.

[0044] All gavage treatments were administered once daily for one week. Following animal ethics guidelines, blood was collected from the mice by enucleation, and the mice were euthanized. Fresh blood samples were incubated at 37°C for 1 hour, then centrifuged at 3000 rpm for 10 minutes to obtain mouse serum. Liver, kidney, intestinal, and serum samples were collected in sterile PE tubes. Physiological tissue sections were preserved in 4% paraformaldehyde, while other samples were stored at -80°C for later use. Mouse livers and kidneys fixed overnight in 4% paraformaldehyde were embedded in paraffin, stained with hematoxylin and eosin (H&E), and their morphology was observed using an optical microscope. Serum uric acid was measured using a uric acid assay kit, and serum creatinine (CRE) and blood urea nitrogen (BUN) levels were measured using a serum urea nitrogen (BUN) assay kit.

[0045] See results Figures 5-8 After treatment with strain 21J, the serum uric acid levels in mice were significantly lower than those in the model group, reaching 55.70 μmol / L, demonstrating that strain 21J has a good uric acid-lowering function. Figure 5 ).

[0046] Blood urea nitrogen (BUN) and creatinine (CRE) are important products of protein and muscle metabolism, respectively, and are mainly excreted via glomerular filtration. Changes in their serum levels can, to some extent, reflect the degree of renal function damage caused by hyperuricemia. The serum BUN and CRE levels in the model group mice were significantly different from those in the control group. After intervention, the BUN and CRE levels in the strain 21J group were significantly reduced (P < 0.001), reaching 6.08 mmol / L and 18.40 mmol / L, respectively. Figure 6 This indicates that strain 21J caused low levels of damage to kidney function. Paraffin-embedded sections of the kidneys and livers of mice in different treatment groups are shown below. Figure 7 and Figure 8HE staining of kidney tissue showed that in the control group, the glomeruli were structurally intact, the renal tubules were regularly arranged, and no obvious inflammatory infiltration was observed in the interstitium. In the model group, the renal tubules were disordered, with some tubular morphology being irregular and accompanied by epithelial cell degeneration and swelling tendency. The interstitial area showed an increase in purple-stained cells and mild loosening, indicating tubulointerstitial damage and inflammatory response. The allopurinol group and strain 21J group showed significantly reduced pathological changes, with more intact renal tubular structure and more regular arrangement, weakened interstitial inflammatory response, and overall tissue morphology closer to the control group. HE staining of liver tissue showed that in the control group, the liver lobule structure was clear, the hepatocytes were arranged relatively regularly, the cytoplasm was uniformly stained, no obvious dilation of the hepatic sinusoids was observed, and inflammatory cell infiltration was not obvious. In the model group, the hepatocyte cytoplasm was lightly stained, with local vacuolar degeneration and swelling tendency. The hepatic cords were relatively disordered, and the hepatic sinusoidal cavities in some areas were widened with congestion tendency. Scattered or focal inflammatory cells were also observed. The morphology of hepatocytes in the allopurinol group and the strain 21J group were similar, the hepatic cord structure was more intact, and the hepatic sinusoidal dilation and inflammatory response were reduced compared with the model group, suggesting that intervention can alleviate the model-induced pathological damage to liver tissue.

[0047] Example 3

[0048] This embodiment further investigates the effects of fermented Lactobacillus mucinus 21J on the intestinal flora and kidney metabolism of mice with high uric acid.

[0049] Phylum-level microbial community analysis results showed ( Figure 9 In all three sample groups, Firmicutes (Bacillota) and Bacteroidetes (Bacteroidota) were the dominant phyla, accounting for the majority of abundance. Compared with the control group, the model group showed a slightly higher relative abundance of Firmicutes and a slightly lower relative abundance of Bacteroidetes, suggesting a certain shift in the microbial community structure induced by the model. After intervention with *Lactobacillus fermentatus* 21J (LF21J), the overall microbial community composition became closer to that of the control group, showing a return of the proportion of dominant phyla to normal levels, suggesting that the strain intervention had a certain regulatory effect on the microbial community.

[0050] The results of the genus-level microbial community analysis showed that ( Figure 10 After intervention with strain 21J, the relative abundance of various short-chain fatty acid-producing bacterial genera significantly increased. Compared with the model group, strain 21J intervention helped suppress abnormal changes in some genera related to metabolic disorders, restoring the overall microbial community structure to that of the control group. Furthermore, compared with the model group, strain 21J intervention also increased the relative abundance of *Bifidobacterium* (*Bifidobacterium*). Figure 11 ).

[0051] The effects of strain 21J on renal metabolism in hyperuricemic mice are shown in [reference needed]. Figure 12Differential metabolite analysis showed that, compared with the control group, strain 21J intervention detected a variety of significantly different metabolites, including 82 upregulated metabolites and 133 downregulated metabolites. The number of downregulated metabolites exceeded the number of upregulated metabolites, suggesting that the abnormally accumulated metabolites were effectively regulated, reflecting an overall reduction in the body's metabolic burden.

[0052] The results of KEGG enrichment analysis of renal metabolism in strain 21J showed that ( Figure 13 Compared with the control group, the metabolic pathways related to metabolic regulation underwent significant changes after intervention with strain 21J, mainly including amino acid metabolism, lipid metabolism, and antioxidant stress-related pathways. Among these, pathways closely related to renal energy metabolism and metabolic homeostasis, such as glycerophospholipid metabolism and linoleic acid metabolism signaling pathways, showed significant enrichment, suggesting that strain 21J intervention can regulate renal metabolic processes, helping to improve renal metabolic status and reduce the metabolic burden caused by abnormal uric acid metabolism. Furthermore, the regulation of tryptophan metabolism and antioxidant stress-related pathways further indicates that strain 21J has a potential positive role in alleviating metabolic stress and maintaining metabolic homeostasis.

[0053] Compared with the control group, the hyperuricemia model group exhibited systemic metabolic disorders characterized by elevated uric acid and abnormal nucleoside metabolism. Figure 14 The relative upregulation of uric acid in the model group indicates the presence of features characteristic of hyperuricemia, which may lead to kidney damage, inflammatory responses, and uric acid crystal deposition. Upregulation of uric acid may stem from a decreased ability of the kidneys to clear excess uric acid or excessive uric acid synthesis, thereby exacerbating renal tubular damage and immune system activation, leading to complications such as oxidative stress, apoptosis, and gout. Furthermore, the increase in other upregulated metabolites such as caffeic acid 4-O-sulfate and fasoracetam reflects the body's adaptive response to oxidative stress and cellular damage.

[0054] Differential metabolite analysis of the kidneys of strain 21J showed that ( Figure 15The upregulation of metabolites such as Lactarovilin, Neryl Arabinofuranosyl-Glucose, and Perulactone may indicate that the strain regulates hyperuricemia-induced damage through antioxidant, anti-inflammatory, and immunomodulatory effects. The downregulation of metabolites such as Leucanthoside and Pc(18:0 / 20:5_3O) may reflect inhibition of lipid metabolism or regulation of immune function. Strain 21J alone did not induce the aforementioned hyperuricemia-related metabolic abnormalities; the metabolic changes it caused were mainly concentrated in lipid metabolism, amino acid peptides, and signaling molecule-related metabolites. These results indicate that strain 21J possesses independent metabolic regulatory characteristics and does not cause hyperuricemia-like metabolic abnormalities, providing a safety basis for its application in the regulation of hyperuricemia-related metabolism.

[0055] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A fermentable Lactobacillus mucinus 21J with uric acid-lowering function, characterized in that: The *Lactobacillus fermentatus* 21J was deposited on August 4, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35505, and classified as *Lactobacillus fermentatus* (…). Limosilactobacillus fermentum ).

2. A microbial inoculant, characterized in that: The microbial agent comprises *Lactobacillus fermentans* 21J as described in claim 1.

3. The application of the fermented Lactobacillus mucinus 21J as described in claim 1 or the microbial agent as described in claim 2 in the preparation of uric acid-lowering products.

4. The application of the fermenting Lactobacillus mucinus 21J as described in claim 1 or the microbial agent as described in claim 2 in the preparation of inosine-degrading products.

5. The application of the fermented Lactobacillus mucinus 21J as described in claim 1 or the microbial agent as described in claim 2 in foods, health foods or functional products prepared for regulating intestinal flora and metabolic state.

6. The use of the fermented Lactobacillus mucinus 21J as described in claim 1 or the microbial agent as described in claim 2 in the preparation of products for the treatment, prevention or adjunctive treatment of hyperuricemia.

7. The application according to claim 6, characterized in that: The application includes at least one of the following functions: (1) Reduce serum uric acid, creatinine and urea nitrogen levels in individuals with hyperuricemia; (2) Reduce liver or kidney damage caused by hyperuricemia in individuals; (3) Improve the gut microbiota structure of individuals with hyperuricemia and promote the restoration of the microbiota to a healthy homeostasis.

8. The application according to any one of claims 3 to 6, characterized in that: The product contains 1×10 8 CFU / mL ~ 1×10 10 CFU / mL of fermented Lactobacillus mucinus 21J.

9. The application according to any one of claims 3 to 6, characterized in that: The products include feed and medicine.

10. The application according to claim 9, characterized in that: It also contains food-grade or pharmaceutical-grade acceptable carriers or excipients.

Images