A type of Weissella WWD-1 and its application

Abstract

This invention relates to a strain of *Westernella* WWD-1 and its applications, belonging to the field of biotechnology. To address the lack of probiotics in the existing technology capable of simultaneously addressing the coexistence of hyperuricemia and hyperlipidemia, this invention provides a strain of *Westernella* WWD-1. This strain possesses multiple metabolic regulatory functions, specifically lowering uric acid, cholesterol, and triglycerides, and exhibits good probiotic properties and safety. The *Westernella* WWD-1 provided by this invention can be used in the preparation of fermented and probiotic products, and can be used to prevent or improve hyperuricemia, hyperlipidemia, and other concurrent metabolic diseases, showing significant application potential.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a Weissella var. wWD-1 and its applications. Background Technology

[0002] Hyperuricemia (HUA) and hyperlipidemia (HLP) are two common metabolic diseases, and their incidence rates have been rising steadily in recent years. More importantly, hyperuricemia and hyperlipidemia often coexist clinically—approximately 60%-80% of patients with hyperuricemia also have varying degrees of hyperlipidemia, and vice versa. They interact and influence each other, jointly exacerbating metabolic disorders and significantly increasing the risk of cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease, and stroke, as well as metabolic syndrome. This has become a serious public health problem threatening public health.

[0003] Currently, traditional interventions for hyperuricemia and hyperlipidemia mainly involve drug therapy and lifestyle interventions. Commonly used uric acid-lowering drugs (allopurinol, febuxostat, benzbromarone) and lipid-lowering drugs (statins, fibrates) have clear efficacy, but all have varying degrees of side effects and tolerability issues. For example, allopurinol can cause hypersensitivity reactions, while statins can cause muscle damage and abnormal liver function. Long-term combined use can also easily lead to drug dependence and poor adherence. Regarding dietary control, since foods rich in purines (meat, organ meats, seafood) are also rich in fat, simply restricting diet is insufficient to simultaneously lower uric acid and lipids, and can easily lead to nutritional imbalances. Therefore, seeking safe, long-term suitable new strategies that can simultaneously intervene in both metabolic abnormalities has become a current research hotspot.

[0004] Secondly, it is emphasized that the direct causative factor of hyperuricemia is uric acid itself, not inosine or guanosine. Uric acid is the final oxidation product of purine metabolism. Its molecular structure is trioxopurine, which does not contain glycosidic bonds and is chemically extremely stable. Under physiological conditions, it exists as a urate anion with extremely low solubility (approximately 0.4 mM). It easily forms urate crystals in tissues such as joints and kidneys, leading to gouty arthritis and kidney damage. The degradation of uric acid requires the participation of uricase, an enzyme that has been inactivated during human evolution. Therefore, the human body cannot further decompose uric acid into water-soluble allantoin. Compared with inosine and guanosine, uric acid has higher chemical stability and is more difficult to degrade. Probiotic strains capable of directly degrading uric acid are extremely rare in nature.

[0005] In existing patents for uric acid-lowering probiotics, the vast majority use the degradation ability of inosine and guanosine as screening indicators for strains. While these strains can degrade uric acid precursors, their ability to directly degrade uric acid remains unclear. Even if some strains exhibit high degradation rates of inosine and guanosine in vitro, their effectiveness in lowering elevated blood uric acid levels after entering the human body remains uncertain. In other words, degradation precursors are not the same as degradation end products—a crucial distinction often overlooked in current research.

[0006] More importantly, hyperuricemia and hyperlipidemia often coexist, sharing multiple metabolic pathways. Simple uric acid-lowering interventions are insufficient to improve lipid metabolism disorders, and vice versa. In clinical practice, patients with both hyperuricemia and hyperlipidemia typically require simultaneous use of both uric acid-lowering and lipid-lowering drugs, which not only increases the metabolic burden on the liver and kidneys but also raises the risk of drug interactions. Therefore, screening for a multifunctional probiotic that can directly and efficiently degrade uric acid while simultaneously lowering cholesterol and triglycerides is of significant scientific and practical value for achieving synergistic intervention for hyperuricemia and hyperlipidemia, meeting the comprehensive needs of patients, and filling functional gaps in existing products. Summary of the Invention

[0007] This invention addresses the lack of strains in the prior art capable of simultaneously addressing the coexistence of hyperuricemia and hyperlipidemia by providing a Weissella var. wWD-1 strain and its applications.

[0008] One of the objectives of this invention is to provide a Weissella WWD-1, wherein the Weissella WWD-1 ( Weissella cibaria The accession number for WWD-1 is CCTCC M 2026148, and it is classified as follows: Weissella cibaria WWD-1 is deposited at the China Center for Type Culture Collection on January 16, 2026.

[0009] A second objective of this invention is to provide a microbial agent containing the aforementioned Weissella WWD-1.

[0010] A third objective of this invention is to provide the application of the aforementioned Weissella WWD-1 or its agent in the preparation of a drug, wherein the drug has the following functions: (a) Degrades uric acid and alleviates hyperuricemia; (b) It degrades triglycerides and cholesterol, alleviating hyperlipidemia; (c) Improves the composition of gut microbiota, increases the abundance of beneficial bacteria, and inhibits opportunistic pathogens; (d) Reduces the level of renal inflammatory factors and alleviates oxidative stress damage.

[0011] Compared with the prior art, the beneficial effects of the present invention are: the present invention provides a strain of *Westbacterium wiltii* WWD-1, and a bacterial agent containing this strain, wherein *Westbacterium wiltii* WWD-1 ( Weissella cibaria The accession number for WWD-1 is CCTCC M2026148.

[0012] In vitro experiments showed that the Weissella oryzae WWD-1 provided by this invention has a high efficiency in simultaneously degrading uric acid, cholesterol and triglycerides, with degradation rates of 88.68%, 95.52% and 95.88%, respectively. In an animal model of hyperuricemia combined with hyperlipidemia, this strain can significantly reduce serum uric acid, total cholesterol and triglyceride levels, achieving synergistic intervention in purine and lipid metabolism disorders.

[0013] The Weissella javanica WWD-1 strain provided by this invention not only possesses good intestinal environment adaptability and colonization potential, but its self-aggregation ability and cell surface hydrophobicity endow it with effective intestinal adhesion ability. It also exhibits strong antioxidant activity, capable of scavenging DPPH and ABTS free radicals and alleviating oxidative stress response caused by metabolic disorders. More importantly, in animal experiments, this strain can significantly reduce the level of pro-inflammatory factors in the kidneys and increase the content of short-chain fatty acids such as butyric acid and caproic acid in feces, thereby exerting a comprehensive regulatory effect of improving intestinal microecology, enhancing intestinal barrier function, and reducing systemic inflammation.

[0014] The microbial preparation containing Weissella WWD-1 provided by this invention has the following advantages: (1) It exerts multiple metabolic regulatory effects of lowering uric acid, cholesterol and triglycerides directly in the form of live bacteria; (2) The preparation is available in various forms, making it easy to carry and store, and suitable for long-term daily use; (3) It can effectively colonize the intestine, improve the intestinal microecology, enhance the intestinal barrier function, and reduce the level of systemic inflammation; (4) It has no drug-related side effects, is highly safe, and is suitable for long-term adjuvant intervention for people with hyperuricemia and hyperlipidemia.

[0015] Uric acid is the final oxidation product of purine metabolism. Its molecular structure is trioxopurine, lacking glycosidic bonds, and it is chemically extremely stable. Under physiological conditions, it mainly exists as the urate anion, with extremely low solubility (approximately 0.4 mM). Furthermore, human evolution has deactivated the urate oxidase gene, preventing further breakdown of uric acid into water-soluble allantoin. Therefore, strains capable of directly degrading uric acid are extremely rare in nature, and screening for strains capable of directly degrading uric acid is far more difficult than screening for strains that degrade uric acid precursors. Currently, most publicly disclosed patents for uric acid-lowering strains target uric acid precursors such as inosine and guanosine, or xanthine oxidase (XOD). These precursors contain glycosidic bonds in their molecular structure, have low chemical stability, and are relatively easy to degrade. Inhibiting XOD enzyme activity only reduces uric acid production, not directly eliminating existing uric acid. In other words, "degrading precursors" or "inhibiting enzyme production" is not equivalent to "directly degrading uric acid."

[0016] The Weissella WWD-1 strain provided by this invention was obtained through targeted screening using uric acid as the sole carbon and nitrogen source, and its ability to degrade cholesterol and triglycerides was also verified. Furthermore, it possesses multiple metabolic regulatory functions, providing a new strain resource for developing microbial preparations that combine uric acid-lowering, cholesterol-lowering, and triglyceride-lowering functions. It can be used to prevent or improve hyperuricemia, hyperlipidemia, and other concurrent metabolic diseases, and has significant application prospects.

[0017] [Biological Preservation Information]: The preservation number for *Weisseria gonorrhoeae* WWD-1 is CCTCC M 2026148, and its classification name is... Weissella cibaria WWD-1 is deposited at the China Center for Type Culture Collection on January 16, 2026. Attached Figure Description

[0018] Figure 1 This is a graph showing the screening results of uric acid-degrading strains; the vertical axis represents the UA degradation rate, which is the uric acid degradation rate. Figure 2 This is a graph showing the screening results of triglyceride-degrading strains; the vertical axis represents the TRIG degradation rate, which is the triglyceride degradation rate. Figure 3 This is a graph showing the screening results of cholesterol-degrading strains; the vertical axis represents the cholesterol degradation rate (TC degradation rate). Figure 4 Electron micrograph of Weissella WWD-1 colony; Figure 5 This is a colony morphology diagram of Weissella WWD-1. Figure 6 Phylogenetic tree of Weissella WWD-1; Figure 7 This is a diagram of the indole test results; from left to right: positive control E. coli, negative control Negative, and Weissella javanica WWD-1. Figure 8 This is a graph showing the results of a hemolysis test. Figure 9 This is a statistical graph of DPPH free radical scavenging capacity; the vertical axis represents DPPH free radical scavenging rate; the horizontal axis represents ascorbic acid. Figure 10 This is a statistical graph of ABTS radical scavenging capacity; the vertical axis represents ABTS radical scavenging rate. Figure 11 For automatically aggregated statistical charts; y-axis Auto aggregation represents the automatic aggregation rate; the horizontal axis, Time, represents time. Figure 12 This is a statistical graph of cell surface hydrophobicity measurement; the vertical axis represents cell surface hydrophobicity; Xylene represents xylene; Ethyl acetate represents ethyl acetate; Chloroform represents chloroform. Figure 13 This is a statistical chart of acid resistance test results; viable counts represents the number of viable bacteria; survival rate represents the survival rate. Figure 14 This is a statistical chart of bile salt tolerance test results; Figure 15 This is a statistical graph of nitrite degradation experiments; the vertical axis represents the nitrite degradation rate. Figure 16 This is a graph showing the in vitro degradation rate of Weissella javanica WWD-1; the vertical axis represents the degradation rate. Figure 17 This is a statistical graph showing the IL-1β content in the livers of different groups of mice; the vertical axis represents the liver IL-1β content. 1β content; Figure 18 This is a statistical graph showing the IL-1β content in the kidneys of different groups of mice; the vertical axis represents the renal IL-1β content. 1β content. Detailed Implementation

[0019] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0021] The Weissella strain used in the following examples is Weissella WWD-1, with accession number CCTCC M 2026148, and classified as follows: Weissella cibaria WWD-1 is deposited at the China Center for Type Culture Collection on January 16, 2026.

[0022] The reagents used in the following examples are: (1) The preparation method of MRS liquid culture medium is as follows: 10g peptone, 10g beef extract powder, 10g yeast extract powder, 20g glucose, 1.0 mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.1g magnesium sulfate, 0.05g manganese sulfate, fully dissolved in 1000 mL distilled water, and the pH value is adjusted to 6.0. Sterilize at 121℃ and 0.1MPa for 20 min.

[0023] (2) The preparation method of MRS solid culture medium is as follows: 10g peptone, 10g beef extract powder, 10g yeast extract powder, 20g glucose, 1.0 mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 15g agar powder, and 1000 mL distilled water are fully dissolved and the pH value is adjusted to 6.0. Sterilize at 121℃ and 0.1 MPa for 20 min.

[0024] (3) The preparation method of MSM liquid culture medium is as follows: 17.1g disodium hydrogen phosphate, 3.0g potassium dihydrogen phosphate, 0.5g sodium chloride, 0.01g calcium chloride, 0.5g magnesium sulfate, 0.336g uric acid, 15g agar, 1000 mL distilled water, sterilized at 0.1 MPa for 20 min.

[0025] (4) The preparation method of peptone water culture medium is as follows: 10 g peptone, 5 g sodium chloride, 2 g disodium hydrogen phosphate, 2 g dipotassium hydrogen phosphate, 5 g glucose, 1000 mL distilled water, sterilized at 0.1 MPa for 20 min.

[0026] (5) The preparation method of Columbia blood agar medium is as follows: 5 g sodium chloride, 2 g glucose, 5 g peptone, 10 g tryptone, 5 g yeast extract, 5 g beef extract, 15 g agar, 1000 mL distilled water, and 60 mL defibrinated sheep blood.

[0027] (6) The method for preparing simulated gastric juice is as follows: prepare 100 mL of sodium chloride with a mass concentration of 0.5%, add 0.35 g of pepsin, vortex mix, adjust the pH with concentrated hydrochloric acid, and filter with a sterile 0.22 μm filter membrane for sterilization.

[0028] (7) The method for preparing simulated intestinal fluid is as follows: Take 1.216 g of potassium dihydrogen phosphate and 0.3 g of porcine bile salt, add 100 mL of water to dissolve them, adjust the pH value to 7.8 with 0.1 mol / L sodium hydroxide solution, then add 0.1 g of trypsin, and filter with a sterile 0.22 μm filter membrane to remove bacteria.

[0029] Example 1: Isolation, screening and identification of Weissella WWD-1 1. Isolation and Screening of Strains: Homemade pickled vegetable brine from Xiangfang District, Harbin City, Heilongjiang Province was selected as the sample. 1 mL of the brine was added to 20 mL of MRS liquid medium and incubated at 37℃ until turbidity was observed, and no wrinkled material covered the surface of the medium. Typical colonies with a milky white or white color, smooth surface, and regular edges that could grow on solid MRS medium for 24-48 hours were selected. Strains showing catalase activity negativity and Gram staining positivity were obtained using catalase activity detection and Gram staining methods. The preliminarily identified colonies were repeatedly streaked on MRS solid medium plates for further purification until uniform, morphologically consistent colonies appeared. Figure 4-5 As shown.

[0030] The obtained strains were plated on MSM inorganic salt medium with uric acid as the sole carbon and nitrogen source, allowed to stand, and cultured at 37℃ for 3 days. Every 12 hours, 2 mL of fermentation broth was taken and centrifuged at 4℃ and 6000 r / min for 10 min. The supernatant was collected, and the uric acid content in the supernatant was detected by high performance liquid chromatography (HPLC). The degradation rates of cholesterol and triglycerides were determined using commercial kits from Nanjing Jiancheng.

[0031] like Figure 1-3As shown, lactic acid bacteria with high degradation capacity for uric acid, triglycerides and cholesterol were screened and obtained, which is the target strain WWD-1.

[0032] 2. Identification of strains DNA was extracted from the target strain *Westernella* WWD-1 using a bacterial genomic DNA extraction kit. PCR amplification was performed using universal primers 27F (nucleotide sequence shown in SEQ ID NO. 1) and 1492R (nucleotide sequence shown in SEQ ID NO. 2). The PCR amplification reaction system consisted of: 1 μL genomic DNA (20 ng / μL), 10× Buffer (containing 2.5 mM MgSO4), and... 2+ 5 μL of Taq polymerase (5 U / μL), 1 μL of dNTP (10 mM), 1.5 μL each of primers 27F and 1492R (10 μM), and ddH2O to a final volume of 50 μL; the PCR amplification reaction program was as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1.5 min, for 35 cycles; final extension at 72℃ for 7 min.

[0033] The PCR amplification products were recovered using the AxyPrep DNA gel recovery kit and sent to Shanghai Paisennong Biotechnology Co., Ltd. for sequencing. The obtained sequences were compared with the GenBank database using BLAST software for homology analysis.

[0034] The results are as follows Figure 6 As shown, the strain screened and identified in this embodiment belongs to the genus *Weissella*. Based on the comprehensive analysis of colony morphology, physiological and biochemical characteristics, 16S rDNA sequence analysis, and the construction of a phylogenetic tree using MEGA software, this strain was identified as *Weissella*. Weissella cibaria It was named Weissella WWD-1, and its 16S rDNA sequence is shown in SEQ ID NO.3.

[0035] Effect Experiment: 1. Safety evaluation This embodiment evaluates the safety of the obtained Weissella salina WWD-1. Escherichia coli (purchased from Yangling Ruizhiweikang Biotechnology Co., Ltd.) was selected as the indicator strain. Indole and hemolysis tests were performed on Weissella salina WWD-1. The specific steps are as follows: (1) Hemolysis test: Weissella WWD-1 was streaked onto Columbia blood agar plates (containing 5% defibrinated sheep blood), with Escherichia coli as a positive control and sterile physiological saline as a negative control. The plates were incubated at 35°C under anaerobic conditions for 48 h, and the presence of a hemolytic ring around the colony was observed. If a clear hemolytic ring (β-hemolysis) or a grass-green incomplete hemolytic ring (α-hemolysis) appeared around the colony, the hemolysis test result was positive. If no hemolytic ring appeared around the colony (γ-hemolysis), the result was negative.

[0036] (2) Indole test WWD-1 bacteria were inoculated into peptone water medium at a 2% (v / v) inoculation rate. Equal volumes of sterile physiological saline and Escherichia coli peptone water medium were used as negative and positive controls, respectively. The inoculated media were incubated at 37°C for 72 h. 3-6 drops of indole reagent (p-dimethylaminobenzaldehyde) were slowly added along the tube wall, and the color change at the interface between the two liquid layers was observed. If a red ring appeared at the interface, the indole test result was positive; if no red ring appeared, the result was negative.

[0037] The results are as follows Figure 7-8 As shown, the indole test and hemolysis test (γ-hemolysis) of Weissella WWD-1 were negative, indicating that Weissella WWD-1 does not produce indole or hemolyze, which meets the requirements of food safety grade microorganisms. It is safe and harmless to human health and can be further used for subsequent functional research and food fermentation applications.

[0038] 2. Determination of antioxidant capacity, self-aggregation rate, and hydrophobicity. (1) DPPH free radical scavenging ability: 100 μL of vitamin C solution (concentration of 15 µg / mL) and 100 μL of 0.4 mmol / L DPPH solution were added to a 96-well plate as the control group; 100 μL of Weissella WWD-1 and 100 μL of 0.4 mmol / L DPPH solution were added to a 96-well plate as the experimental group. The reaction was carried out at 25℃ in the dark for 30 min, and the DPPH free radical scavenging ability of lactic acid bacteria was determined based on the absorbance at 540 nm.

[0039] The results are as follows Figure 9 As shown, the DPPH free radical scavenging rate of Weissella WWD-1 was 77%, which was closest to the DPPH free radical scavenging rate of the positive control vitamin C (83.29%). This indicates that the Weissella WWD-1 provided by this invention has a strong DPPH free radical scavenging ability.

[0040] (2) ABTS radical scavenging ability: At room temperature and in the dark, a 7.4 mmol / L ABTS radical solution was reacted with a 2.6 mmol / L potassium persulfate solution for 24 h to generate a stable, high concentration of ABTS radical cations (ABTS· + ) Stock solution. Before measurement, use PBS to prepare the ABTS to be tested. + The stock solution was diluted at 734 nm to achieve an absorbance of 0.70 ± 0.03. 20 μL of vitamin C solution (20 µg / mL) was added to a 96-well plate containing 180 μL of ABTS radical solution as a control group; 20 μL of *Westernella vulgaris* WWD-1 bacterial culture was added to a 96-well plate containing 180 μL of ABTS radical solution as the experimental group. The plates were reacted at 25 °C in the dark for 10 min, and the ABTS radical scavenging ability of the strains was determined based on the absorbance at 734 nm.

[0041] The results are as follows Figure 10 As shown, the ABTS free radical scavenging rate of Weissella WWD-1 was 90%, which was similar to that of the positive control vitamin C; indicating that the Weissella WWD-1 provided by the present invention has a strong ABTS free radical scavenging ability.

[0042] (3) Automatic aggregation ability determination Weissella javanica WWD-1 was cultured overnight at 37°C in MRS broth. The cells were collected by centrifugation, washed twice with sterile PBS buffer (pH 7.2), resuspended in PBS, vortexed for 30 s, and the absorbance was measured at 600 nm using a UV-Vis spectrophotometer at different times (0, 1, 2, 3, 4, 5, and 24 h). The autoaggregation rate was calculated.

[0043] Automatic aggregation capability = [1-(A t / A0)]×100%.

[0044] A0: Initial absorbance value measured at 600 nm wavelength, with a time of 0 hours (i.e., immediately after the bacterial suspension is prepared and vortexed); A t : The absorbance value of the sample taken from the upper layer of the suspension at a wavelength of 600 nm, which is t hours.

[0045] Lactic acid bacteria can form a barrier through self-aggregation, which is beneficial for intestinal colonization and inhibits pathogenic bacteria. For example... Figure 11As shown, the self-aggregation ability of *Westernella* WWD-1 exhibits a non-uniform growth rate over 2-5 hours. To determine whether the strain stably aggregated and completed the aggregation experiment, absorbance values ​​were measured at 24 hours. The results showed that the self-aggregation rate of *Westernella* WWD-1 was 27% at 24 hours, and the aggregation rate gradually slowed down with little difference each hour thereafter. This indicates that the *Westernella* WWD-1 provided by this invention possesses a strong self-aggregation ability.

[0046] (4) Measurement of cell surface hydrophobicity The hydrophobicity of the cell surface of *Westernella* WWD-1 was determined using the microbial adhesion hydrocarbon method (MATH). After culturing *Westernella* WWD-1 for 24 h, the cells were centrifuged and washed twice with PBS (pH 7.2). The cells were resuspended in 5 mL of PBS buffer, and the absorbance of the cell suspension was measured at 600 nm using a UV-Vis spectrophotometer, denoted as A0. The cell suspension was then mixed with an equal volume of solvent for 5 min to obtain a suspension. Measurements were performed using ethyl acetate (alkaline solvent), chloroform (acidic solvent), or xylene (non-polar solvent). The suspension was incubated at room temperature for 30 min to achieve phase separation. The absorbance of the aqueous phase at 600 nm was measured and denoted as A. t The hydrophobicity of the cell surface of Weissella WWD-1 was obtained.

[0047] Cell surface hydrophobicity = [(A0-A t ) / A0]×100%.

[0048] A0: Initial absorbance value measured at 600 nm wavelength, with a time of 0 hours (i.e., immediately after the bacterial suspension is prepared and vortexed); A t : The absorbance value of the sample taken from the upper layer of the suspension at a wavelength of 600 nm, which is t hours.

[0049] The results are as follows Figure 12 As shown, *Westernella* WWD-1 exhibits a high adsorption rate of 69% for chloroform, indicating that its cell surface is rich in electron donors (such as amino and hydroxyl groups) and possesses Lewis basic regions. *Westernella* WWD-1 shows an adsorption rate of approximately 42% for xylene and approximately 18% for ethyl acetate, indicating that its surface electron acceptor properties are weak and its affinity for basic solvents is low. These surface characteristics facilitate the adhesion and colonization of the strain.

[0050] 3. Determination of acid and bile salt resistance: The activated Weissella WWD-1 strain was inoculated at a rate of 2% (v / v) into MRS liquid medium and conventional MRS medium with pH values ​​of 2.0 and 4.0, respectively. The strains were incubated statically in a constant temperature incubator at 37°C. Samples were taken at 0 h and 3 h after treatment, and viable cell counts were performed using the plate pour method to investigate the survival of the strain under different acidic conditions.

[0051] The results are as follows Figure 13 As shown, after treatment at pH 4.0 for 3 h, the viable count of Weissella WWD-1 remained at 10. 5 The concentration of CFU / mL is above 1. The pH value of human gastric juice is usually around 2.0-4.0. This result indicates that Weissella javanica WWD-1 has the ability to tolerate the acidic environment of the stomach and can pass through the gastrointestinal tract with a high survival rate.

[0052] Activated Weissella oryzae WWD-1 was inoculated at a rate of 2% (v / v) into MRS liquid medium containing different concentrations of bovine bile salts (0.2% and 0.4%) and incubated statically at 37°C. Samples were taken at 0 h and 3 h for plate viable counts to assess the bile salt tolerance of the strain. The physiological concentration range of bile salts in the human small intestine is 0.2%-0.4%, and lactic acid bacteria that can grow and metabolize normally within this concentration range are considered to have the potential to survive and colonize in the intestinal environment.

[0053] The results are as follows Figure 14 As shown, after treatment with bile salt concentration ≤0.4% for 3 h, the viable count of Weissella WWD-1 remained at 10. 4 -10 5 The CFU / mL result indicates that Weissella javanica WWD-1 can tolerate the normal bile salt environment of the human gut and has the ability to survive and function in the gut.

[0054] 4. Determination of nitrite degradation capacity The activated Weissella oryzae WWD-1 was inoculated into 20 mL of MRS liquid medium (containing 10 mg / L sodium nitrite) at an inoculation rate of 0.15% (v / v) and cultured at 37°C for 24 h. Every 4 h, 2 mL of fermentation broth was taken, centrifuged, and 1 mL of supernatant was transferred to a 50 mL stoppered colorimetric tube. The sodium nitrite content was determined according to the national standard GB5009.33-2016 "National Food Safety Standard - Determination of Nitrates and Nitrites in Food".

[0055] The results are as follows Figure 15 As shown, Weissella WWD-1 has a nitrite degradation rate of over 95%.

[0056] 5. Tests for lowering uric acid, triglycerides, and cholesterol. (1) In vitro experiments Weissella javanica WWD-1 was inoculated into MSM liquid medium containing uric acid and cultured at 37°C with shaking until the logarithmic growth phase. The culture was centrifuged, and the bacterial pellet was collected. The pellet was washed three times with sterile PBS buffer to remove residual culture medium components and then resuspended in PBS to prepare a bacterial suspension. The bacterial suspension was transferred at an inoculation rate of 2% (v / v) to MSM medium containing 0.336 g / L uric acid, 1.330 g / L triglycerides, and 0.580 g / L cholesterol and cultured in a shaker at 37°C. Samples were taken at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. The culture was centrifuged, and the supernatant was collected. The supernatant was filtered through a 0.22 μm microporous membrane, and the residual concentration of uric acid in the supernatant was determined by high performance liquid chromatography (HPLC). The degradation rate at different time points was calculated based on the initial concentration.

[0057] Degradation rate (%) = [(C0-C t ) / C0]×100%.

[0058] C0: Initial concentrations of uric acid, triglycerides, and cholesterol in the culture medium after 0 hours of incubation; C t The residual concentrations of uric acid, triglycerides, and cholesterol in the supernatant after centrifugation and filtration after culturing for t hours.

[0059] The results are as follows Figure 16 As shown in Table 1, under the condition that uric acid, cholesterol and triglycerides are the only carbon and nitrogen sources, after 72 h, the degradation rate of uric acid by Weissella WWD-1 can reach 88.68%, the degradation rate of cholesterol can reach 95.52% and the degradation rate of triglycerides can reach 95.88%.

[0060] Table 1

[0061] (2) In vivo test a) Establishment and grouping of the animal model: Sixty male C57BL / 6J mice weighing 16-22 g (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were used. After 7 days of acclimatization, the mice were allowed free access to food and water. The ambient temperature was maintained at 22-25℃, the relative humidity at 50%-60%, and the light-dark cycle was 12 hours. After the acclimatization period, the mice were randomly divided into 4 groups of 15 mice each. Normal control group (Control): Normal feed + sterile water by gavage; Weissella WWD-1 group (W): ordinary feed + WWD-1 bacterial solution administered by gavage; Hyperuricemia + hyperlipidemia model group (HH): high-fat diet + gavage of potassium oxonate + uric acid; Weissella WWD-1 intervention group (HH+W): high-fat diet + oral gavage of potassium oxonate + uric acid + WWD-1 bacterial solution; Except for the normal control group, all other groups were treated by administering drugs while establishing the model. The specific modeling method was as follows: starting from day 8, the model group was given a mixed suspension of potassium oxonate (OP) and uric acid (UA) by gavage at a dose of 300 mg / kg body weight every day, while being supplemented with a high-fat rat diet and free access to water. The modeling was continued for 8 weeks, and all gavage operations were performed at fixed times every day.

[0062] b) Determination of serum uric acid content: After the last administration, the mice were fasted for 12 h but allowed free water. On day 68, blood was collected from the orbital venous plexus of each group of mice. The collected blood samples were placed in 1.5 mL centrifuge tubes and allowed to stand at room temperature for 1 h. Then, the samples were centrifuged at 4℃ and 3000 r / min for 15 min. The upper serum layer was carefully aspirated and transferred to a new centrifuge tube and stored at -80℃ for later use.

[0063] Serum uric acid levels were determined using a uric acid assay kit (enzyme colorimetric method) provided by Nanjing Jiancheng Bioengineering Research Institute. The procedure was strictly followed according to the kit instructions. The principle of the assay is as follows: uric acid is oxidized by uricase to produce allantoin and hydrogen peroxide. Hydrogen peroxide then reacts with 4-aminoantipyrine and TOOS under the catalysis of peroxidase to form a quinone-based red compound. The intensity of the color is directly proportional to the uric acid content. The absorbance of each well was measured at 550 nm using an ELISA reader. Three replicates were used for each sample group, and the average values ​​were statistically analyzed.

[0064] Serum uric acid concentration (μmol / L) = (A measurement - A blank) / (A standard - A blank) × C standard.

[0065] In the formula: Ameasured is the absorbance value of the sample well (serum of each group of mice); Ablank is the absorbance value of the blank well (double-distilled water); Astandard is the absorbance value of the standard well (500 μmol / L uric acid standard solution); Cstandard is the concentration of the uric acid standard, i.e., 500 μmol / L.

[0066] After a 67-day experimental period (including 7 days of acclimatization feeding and a subsequent 60 days of continuous gavage modeling and intervention), serum uric acid levels in mice of each group were measured and analyzed. The results are shown in Table 2. Compared with the model group, the serum uric acid concentration in the Weissella WWD-1 intervention group was significantly reduced, with a degradation rate of 67.83%. This indicates that the Weissella WWD-1 provided by this invention can efficiently and directly degrade uric acid in animals, exhibiting excellent uric acid-lowering activity.

[0067] Table 2

[0068] c) Blood lipid levels in mice: Elevated serum TC and TRIG levels are the main characteristics of hyperlipidemia. By analyzing the serum lipid levels of mice in each group, we evaluated the preventive effect of Weissella javanica WWD-1 on hyperlipidemia induced by a long-term high-fat diet.

[0069] Blood samples were collected from mice in each group using the orbital blood sampling method. Serum was separated by centrifugation at 4℃ and 3000 r / min for 15 min. The levels of total cholesterol (TC) and triglycerides (TRIG) in the serum were measured according to the kit instructions provided by Nanjing Jiancheng Bioengineering Research Institute.

[0070] The results are shown in Table 3. Compared with the normal control group, the serum TC and TRIG levels in the model group mice were significantly increased by 162.5% and 116.3%, respectively (p<0.05), indicating that the hyperlipidemia model was successfully established. Compared with the model group, the serum TC and TRIG levels in the Weissella jejuni WWD-1 intervention group (WWD-1) mice were significantly decreased by 58.16% and 64.66%, respectively (p<0.05), indicating that this intervention can effectively reduce TRIG levels and bring them closer to normal.

[0071] It is evident that the Weissella javanica WWD-1 provided by this invention can significantly reduce the serum TC and TRIG levels in mice induced by a high-fat diet, effectively improve lipid metabolism disorders, and thus reduce the risk of hyperlipidemia.

[0072] Table 3

[0073] 6. Measurement of the ability to improve gut microbiota and reduce inflammatory factors The levels of interleukin-1β (IL-1β) in liver and kidney homogenates of mice in the model group (HH) and the Weisslerella WWD-1 intervention group were detected using an enzyme-linked immunosorbent assay (ELISA) kit provided by Wuhan Sewell Biotechnology Co., Ltd. Simultaneously, the levels of short-chain fatty acids (SCFAs) in fecal samples from both groups of mice were quantitatively analyzed using an Agilent gas chromatography-mass spectrometry system.

[0074] Results of the detection of changes in the levels of the inflammatory factor IL-1β in the kidneys and liver are as follows: Figure 17-18As shown, compared with the model control group (HH), after intervention with Weissella WWD-1, the content of the pro-inflammatory cytokine IL-1β in mouse kidney tissue homogenate significantly decreased from 3967.672 pg / mL to 2990.82 pg / mL, a decrease of 24.62%; the content of IL-1β in mouse liver tissue significantly decreased from 151.934 pg / mL to 80.772 pg / mL, a decrease of 46.84%. It is evident that the Weissella WWD-1 provided by this invention can effectively block the activation of inflammatory pathways in the liver and kidneys, and reduce the induced inflammatory damage.

[0075] Short-chain fatty acids are important metabolites produced by the fermentation of dietary fiber by gut microbiota, playing a crucial role in maintaining intestinal barrier function and regulating inflammatory responses. GC-MS analysis results are shown in Table 4. Compared with the model group, the levels of various short-chain fatty acids in the feces of mice in the *Westernella* WWD-1 intervention group were significantly increased. Compared with the model group, butyric acid content increased from 9.761 μg / g to 32.285 μg / g, an increase of 230.75%; hexanoic acid content increased from 16.903 μg / g to 40.168 μg / g, an increase of 137.64%. Butyric acid, as a major energy source for colonic epithelial cells, has anti-inflammatory effects and enhances intestinal barrier function; hexanoic acid exhibits antibacterial and anti-inflammatory properties. These results indicate that the *Westernella* WWD-1 provided in this invention can effectively regulate the metabolic activity of gut microbiota, increase the production of beneficial short-chain fatty acids, thereby improving intestinal barrier integrity, inhibiting systemic inflammation, and indirectly promoting uric acid excretion.

[0076] Table 4

[0077] Example 2: Application of Weissella WWD-1 in the preparation of microbial preparations 1. Activation and Expansion Culture of the Strains: The Weissella WWD-1 strain obtained in Example 1 was aseptically inoculated into MRS liquid medium and cultured at 37°C for 12-36 hours, for two consecutive generations of activation. Subsequently, it was transferred to expansion medium at an inoculation rate of 2% (v / v) and cultured on a shaker at 37°C for 24 hours to obtain a viable count ≥10⁻⁶. 9 Fermentation broth at CFU / mL.

[0078] 2. Cell collection: Centrifuge the fermentation broth at 4℃ and 8000 r / min for 10 minutes, collect the cell precipitate, wash 2-3 times with sterile physiological saline, and finally resuspend in sterile water to adjust the cell suspension concentration to 1×10⁻⁶. 10 -1×10 11 CFU / mL.

[0079] 3. Preparation of freeze-drying protectant: Prepare the freeze-drying protectant according to the following mass percentages: 10% skim milk powder, 5% trehalose, 1% glycerol, and the remainder is sterile water; after stirring and dissolving, sterilize at 121℃ for 20 min, and cool for later use.

[0080] 4. Mixing and dispensing: Mix the above bacterial suspension and freeze-drying protectant at a volume ratio of 1:2 until homogeneous, and immediately dispense into sterile freeze-drying trays, with the liquid thickness not exceeding 1 cm.

[0081] 5. Freeze-drying: Place the packaged samples in a -80℃ freezer for 4 hours, then transfer them to a freeze dryer and dry them at a vacuum of less than 10 Pa and a temperature of -50℃ for 24-48 hours until the moisture content is less than 3% to obtain the freeze-dried product.

[0082] 6. Grinding and Sieving: The freeze-dried product was ground under aseptic conditions and passed through an 80-mesh sieve to obtain a live WWD-1 freeze-dried powder. Testing showed that the live bacteria count in the freeze-dried powder was ≥1×10⁻⁶. 10 CFU / g.

[0083] Example 3: Application of Weissella javanica WWD-1 in the preparation of fermented kimchi Under aseptic conditions, the *Westbrook* WWD-1 obtained in Example 1 was inoculated into MRS liquid medium and incubated statically at 37°C for 12-36 hours. After two generations of activation, the culture was washed twice with ddH2O and suspended to obtain the *Westbrook* WWD-1 seed culture. 5 kg of fresh Chinese cabbage was selected as the fermentation raw material. After cleaning and cutting, 500 g of salt was added for salting for 12 hours, during which time the cabbage was compacted with a heavy object to fully soften it and remove some moisture. After being removed, it was repeatedly rinsed with clean water to remove excess salt and drained to obtain the kimchi fermentation substrate. The *Westbrook* WWD-1 seed culture was then added to a final concentration of 10 g / L in the fermentation system. 7 The fermentation substrate was inoculated at a rate of CFU / mL and fermented at 4°C for 21 days to obtain fermented kimchi.

[0084] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A type of Weissella ( Weissella cibaria WWD-1, characterized in that, The Weissella oryzae WWD-1 has the accession number CCTCC NO: M 2026148 and is classified as follows: Weissella cibaria WWD-1 is deposited at the China Center for Type Culture Collection on January 16, 2026.

2. A microbial agent, characterized in that, The bacterial agent contains WWD-1 of Weissella as described in claim 1.

3. The use of the Weissella WWD-1 of claim 1 or the bacterial agent of claim 2 in the preparation of a drug for relieving hyperuricemia and hyperlipidemia.

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