A strain of Weissella sinusoidalis X1 with the function of regulating metabolism and lowering uric acid and its application

Abstract

The present invention discloses a strain of Weissella cibaria X1 with the function of regulating metabolism and lowering uric acid and its application. 49 strains of lactic acid bacteria were screened from samples of fermented vegetables, healthy human oral cavity and piglet feces, and 10 strains of lactic acid bacteria were screened based on the ability to regulate metabolism and inhibit obesity and through PCA analysis; the 10 strains were evaluated for safety and the ability to lower uric acid, and finally strain X1 was screened and identified as Weissella cibaria; the X1 strain itself and its metabolites have the function of lowering uric acid, and can promote the excretion of uric acid by regulating the expression levels of OAT1, UOX and HNF4A in the body and regulate the expression of HPRT to prevent excessive uric acid production and thus reduce the level of uric acid. The present invention provides a new way to develop a method for lowering uric acid without side effects.

Description

Technical field:

[0001] The invention belongs to the field of biotechnology, and specifically relates to a strain of Weissella sinusoidalis X1 with the functions of regulating metabolism and reducing uric acid and an application thereof. Background technology:

[0002] In recent years, due to poor diet and lifestyle, the prevalence of gout has increased significantly, becoming a serious problem that plagues the health of modern people. Gout is an inflammatory arthritis caused by the deposition of urate crystals in the joints, and hyperuricemia can induce gout. Hyperuricemia (HUA) refers to excessive production or less excretion of uric acid (UA) in the body, resulting in higher than normal uric acid levels in the body, which can cause gouty acute arthritis, tophi deposition, tophi chronic arthritis, joint deformities, and uric acid kidney stones and chronic interstitial nephritis. In order to alleviate hyperuricemia and avoid gout attacks, it is necessary to control the uric acid content in the body at a normal level. At present, some drugs treat hyperuricemia by promoting uric acid excretion (such as probenecid, benzbromarone) and inhibiting the synthesis of uric acid (such as febuxostat, allopurinol, etc.). However, the use of these drugs can produce allergic reactions, cause liver necrosis and other side effects, and there are also unknown risks of drug interactions for gout patients with other diseases. Therefore, there is an urgent need to develop methods to alleviate hyperuricemia with low / no side effects.

[0003] Some studies have found that plant extracts and probiotics have the potential to be used as alternative medicines for the treatment of hyperuricemia. Among them, lactic acid bacteria (LAB) have been used in the prevention and treatment of diseases including constipation, inflammatory bowel disease, non-alcoholic fatty liver disease and obesity due to their generally recognized as safe (GRAS) and different functional properties.

[0004] Current studies have found that diseases such as insulin resistance, hyperinsulinemia, impaired glucose tolerance, hypertriglyceridemia and hypertension are all related to metabolic disorders. Existing studies on the screening of uric acid-lowering lactic acid bacteria are based on the ability to reduce xanthine oxidase or the ability to reduce purine content in vitro, and have not screened lactic acid bacteria that have both the ability to improve metabolism and the ability to lower uric acid from the perspective of regulating metabolism. Summary of the invention:

[0005] The present invention provides a strain of Weissella cibaria X1 with metabolism regulating and uric acid lowering functions and an application thereof. Firstly, lactic acid bacteria with metabolic regulation functions are screened out according to indicators such as regulating fat metabolism, regulating sugar metabolism and inhibiting obesity, and then the uric acid lowering ability of the strain is comprehensively evaluated through an in vitro uric acid lowering experiment and a hyperuricemia zebrafish model experiment, and the strain X1 is further screened out and identified as Weissella cibaria. The strain not only has a metabolic regulation function, but also has the ability to significantly reduce uric acid levels, thereby providing a new approach for developing uric acid lowering treatment methods without side effects.

[0006] The present invention is achieved through the following technical solutions:

[0007] A strain of Weissella cibaria X1 with the function of regulating metabolism and lowering uric acid was deposited in the China General Microbiological Culture Collection Center (CGMCC) on July 15, 2024, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Collection Number: CGMCC NO:31306.

[0008] The present invention also protects the use of the Weissella cibaria X1 in preparing products with the functions of regulating metabolism and lowering uric acid.

[0009] Preferably, the metabolism regulation includes regulating lipid metabolism, regulating sugar metabolism, and inhibiting obesity.

[0010] Furthermore, the regulation of lipid metabolism includes lowering cholesterol and lowering triglycerides.

[0011] Preferably, the active ingredient of the product having the function of regulating metabolism and lowering uric acid comprises the bacteria of Weissella cibaria X1 or its metabolites.

[0012] The present invention also protects the use of the Weissella cibaria X1 in preparing products for preventing and treating hyperuricemia.

[0013] The present invention also provides a product with the function of regulating metabolism and reducing uric acid, which comprises the bacteria of Weissella cibaria X1 or its metabolites as active ingredients.

[0014] The present invention also provides a product for preventing and treating hyperuricemia, which comprises the bacteria of Weissellacibaria X1 or its metabolites as active ingredients.

[0015] The beneficial effects of the present invention are as follows:

[0016] The present invention screened out 49 strains of lactic acid bacteria from plant-source (fermented vegetables) and animal-source (healthy human oral cavity, piglet feces) samples, and calculated the 10 lactic acid bacteria with the strongest metabolic regulation ability through principal component analysis according to the experimental results of three metabolic regulation ability indicators of the strains, namely, lipid metabolism regulation, sugar metabolism regulation and obesity inhibition ability. After evaluating their safety, the ability of the 10 lactic acid bacteria to inhibit xanthine oxidase (a key enzyme for uric acid synthesis) in vitro and their ability to reduce uric acid levels and xanthine oxidase levels in a hyperuricemia zebrafish model were further determined, and finally a strain X1 with the strongest uric acid reduction ability was screened out, and it was identified as Weissella cibaria; the strain Weissella cibaria X1 has certain probiotic properties, and its survival rate in simulated gastrointestinal fluid reaches 33.33±4.71%; its degradation rates of inosine and guanosine are 45.26% and 99.23% respectively; Exploration of the uric acid-lowering mechanism of cibariaX1 showed that it can degrade uric acid through extracellular metabolites, regulate the expression levels of organic anion transporter 1 (OAT1), urate oxidase (UOX), and hepatocyte nuclear factor (HNF4A) in the body to promote the excretion of uric acid, and regulate the expression of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to prevent excessive uric acid production and thus reduce the level of uric acid, providing a new way to develop methods with uric acid-lowering capabilities and no side effects.

[0017] Weissella cibaria X1, the strain was deposited on July 15, 2024 in the China General Microbiological Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Accession Number: CGMCC NO:31306. Description of the drawings:

[0018] Figure 1 These are the results of screening of lactic acid bacteria that regulate metabolism in vivo in Example 1, including: (a) cholesterol-lowering ability; (b) triglyceride-lowering ability; (c) α-amylase inhibition ability; (d) α-glucosidase inhibition ability; (e) Enterobacter cloacae inhibition ability; only 20 strains with stronger abilities are shown in ae; and (f) PCA analysis.

[0019] Figure 2 The figures are the results of the safety test on the 10 strains of lactic acid bacteria screened in Example 2, including (a) hemolytic ability; (b) bioamine production ability; (c) gelatinase production ability; and (d) DNA enzyme production ability.

[0020] Figure 3The figures are the results of screening the uric acid-lowering strains of the 10 selected lactic acid bacteria in Example 3, wherein (a) the ability of the strain to inhibit xanthine oxidase (XOD) in vitro; (b) the ability of the strain to reduce uric acid levels in a hyperuricemia zebrafish model; (c) the ability of the strain to inhibit xanthine oxidase (XOD) in a hyperuricemia zebrafish model.

[0021] Figure 4 These are the results of identifying the strain X1 obtained by screening in Example 4, including: (a) the colony morphology of X1; (b) the Gram staining of X1; and (c) the phylogenetic tree of X1.

[0022] Figure 5 These are the results of measuring the probiotic properties of strain Weissella cibaria X1 in Example 5, including: (a) acid tolerance; (b) bile salt tolerance; (c) Caco-2 cells; (d) adhesion of X1 to Caco-2 cells; (e) antioxidant capacity of X1; and (f) survival rate of X1 in simulated gastrointestinal fluid.

[0023] Figure 6 The results of in vitro and in vivo uric acid-lowering ability determination of strain Weissella cibaria X1 in Example 6, wherein: (a) HPLC chart of standard inosine (1.26 mmol / L); (b) HPLC chart of X1 and inosine (1.26 mmol / L) after co-incubation at 37°C for 12 h; (c) HPLC chart of standard guanosine content (1.26 mmol / L); (d) HPLC chart of X1 and guanosine (1.26 mmol / L) after co-incubation at 37°C for 12 h; (e) uric acid content in zebrafish (f) XOD content in zebrafish.

[0024] Figure 7 This is a qPCR analysis result diagram of the expression of key enzyme genes of uric acid metabolism by strain Weissella cibaria X1 in Example 6 in a zebrafish model. Specific implementation method:

[0025] It will be appreciated by those skilled in the art that the disclosed techniques in the following examples represent techniques that the inventors have found to work well in the practice of the present invention. However, many changes may be made in the disclosed specific embodiments, and still the same or similar results may be obtained without departing from the spirit and scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0026] Example 1 Screening of lactic acid bacteria with the ability to regulate metabolism in vivo

[0027] First, 49 strains of lactic acid bacteria were screened from samples of plant sources (fermented vegetables) and animal sources (healthy human oral cavity, piglet feces).

[0028] 1. Screening of lactic acid bacteria

[0029] Weigh 10g of sample into a sterile retort bag containing 90g of 0.85wt% saline, and beat the sample with a beater to mix it. Take 1ml of the mixed solution and dilute it 10 times with 0.85wt% saline. -3 , 10 -4 , 10 -5 Three dilution gradients were prepared by taking 0.1 ml of each of the dilution solutions containing 1 wt% CaCO 3 Spread on MRS agar plates and culture in a 37℃ incubator for 48h. After the culture is completed, pick a single colony with a calcium-dissolving ring and streak it on an MRS agar plate for purification. Make a preliminary judgment on the isolated bacteria based on colony morphology and Gram staining. After streaking and purifying 49 strains identified as lactic acid bacteria on MRS agar plates 2 to 3 times, mix them with an equal volume of glycerol water with a mass ratio of 1:1 and store them in a frozen state at -80℃.

[0030] 2. Determination of the ability to regulate lipid metabolism

[0031] The 49 strains obtained above were subjected to experiments on their ability to regulate lipid metabolism, regulate sugar metabolism and inhibit obesity. The experimental results were analyzed by principal component analysis to calculate the 10 strains of lactic acid bacteria with the strongest ability to regulate metabolism in the body.

[0032] (1) Ability to lower cholesterol.

[0033] Cholesterol is an important basic substance for human metabolic activities. High cholesterol can lead to various cardiovascular diseases. The cholesterol-lowering ability of the strain was determined as follows: Cholesterol (CHO) dissolved in an ethanol-Tween 80 mixture (3:1, v / v) was added to the MRS broth medium to prepare the MRS-CHO medium, so that the final concentration of CHO was 0.5 mg / ml, and the ethanol concentration could not exceed 5% (v / v). The 49 selected lactic acid bacteria were inoculated into the MRS-CHO medium at a 2% (v / v) inoculation rate and cultured at 37°C for 24 hours; the MRS-CHO medium was used as a control; after the culture was completed, 0.1 ml of the bacterial supernatant was taken and 0.3 ml KOH (33%, w / v) and 3ml ethanol (95%, v / v) were fully mixed in a test tube, plugged with a stopper and heated at 60°C for 15min; after cooling to room temperature, 10ml hexane was added to mix with the lower layer; 3ml distilled water was added, plugged, and shaken for 1min; an equal amount (2ml) of the hexane layer was taken into a colorimetric tube and the organic solvent was evaporated at 70°C; 2ml of o-diphenylaldehyde reagent (0.5mg / ml prepared with glacial acetic acid) was added to each tube and the solution was fully mixed; after reacting for 10min, 1ml of concentrated sulfuric acid was added to make it flow down the tube wall, and after complete mixing for 10 to 90min, the absorbance value was read at 550nm using an ultraviolet spectrophotometer (UV-2550, Shimadzu, Japan). The absorbance value measured in the experimental group was recorded as A, and the absorbance value measured in the control group was recorded as A 0 The cholesterol-lowering ability of the strain was calculated according to the following formula:

[0034]

[0035] The results show that ( Figure 1 a) The overall cholesterol-lowering ability of the strains ranged from 3.73% to 95.65%, and the cholesterol-lowering ability of different strains varied significantly. Among them, 16 strains had a cholesterol-lowering ability greater than 50%. The top three lactic acid bacteria in terms of cholesterol-lowering rate were BF2 (95.65±0.47%), FD2 (91.97±0.82%), and X1 (89.72±0.76%), which were significantly higher than other strains (p<0.05).

[0036] (2) Ability to reduce triglycerides

[0037] Take 20ml of polyvinyl alcohol aqueous solution (2%, v / v) and 50ml of triglyceride and mix them completely. Take 3% (v / v) of the triglyceride mixture and add it to the MRS broth culture medium to prepare the triglyceride-MRS broth culture medium. After adjusting the pH to 6.5, sterilize it at 121°C. After cooling, inoculate the 49 strains of lactic acid bacteria screened above into the triglyceride-MRS broth culture medium at an inoculation rate of 3% (v / v). After culturing at 37°C for 72h, centrifuge at 4°C and 9690×g for 10min, take the supernatant after centrifugation, and use a triglyceride kit to determine the triglyceride content in the supernatant at 500nm. The absorbance value is recorded as A. The triglyceride-MRS broth culture medium without inoculation of strains is used as the control group, and the absorbance is recorded as A. CK The ability to lower triglycerides is calculated as follows:

[0038]

[0039] The results show that ( Figure 1 b) The ability of each strain to reduce triglycerides ranged from 17.51% to 94.59%, among which 10 strains of lactic acid bacteria had an ability of reducing triglycerides greater than 50%. The three lactic acid bacteria with higher triglyceride reduction rates were PS4 (degradation rate was 94.59±5.79%), PS3 (degradation rate was 88.62±3.45%), and PS2 (degradation rate was 85.82±13.06%) isolated from piglet feces, which were significantly higher than other strains (p<0.05).

[0040] 3. Regulate sugar metabolism

[0041] (1) Inhibit α-amylase activity

[0042] The 49 strains of lactic acid bacteria selected above were inoculated into MRS broth medium at an inoculation rate of 2% (v / v), cultured at 37°C for 24 hours, and centrifuged at 4°C and 9690×g for 10 minutes, and the supernatant after centrifugation was taken. 0.5 ml of α-amylase solution (1 mg / ml) was mixed with 0.5 ml of supernatant and reacted at 37°C for 20 minutes, and a soluble starch solution (1.5%, w / v) was prepared with 0.1 M PBS solution with pH 6.8. 1 ml of soluble starch solution was added to the mixture of the supernatant and α-amylase solution after the reaction, and reacted at 37°C for 30 minutes, and then 1 ml of DNS colorimetric reagent was added to terminate the reaction in a 100°C water bath for 10 minutes, and an MRS broth medium without inoculation was added as the control group. The absorbance value was then measured at 540 nm, and the experimental group was recorded as A and the control group was recorded as A CK The α-amylase inhibitory activity of the strain was calculated according to the following formula:

[0043]

[0044] The results show that ( Figure 1 c), the ability to inhibit α-amylase ranged from 1.03% to 60.13%. Among them, 7 strains of lactic acid bacteria had an ability to inhibit α-amylase greater than 50%, and the top three lactic acid bacteria in terms of α-amylase inhibition were JS10 (60.13±0.18%), FD2 (59.69±3.81%), and L2 (58.71±0.02%), which were significantly higher than other strains (p<0.05).

[0045] (2) Inhibition of α-glucosidase activity

[0046] The 49 strains of lactic acid bacteria selected above were inoculated into MRS broth at a 2% (v / v) inoculation volume, cultured at 37°C for 24 hours, centrifuged at 4°C and 9690×g for 10 minutes, and the supernatant was collected. 0.2 ml of α-glucosidase (0.1-0.25 mg / ml) was mixed with 0.2 ml of the supernatant, cultured at 37°C for 5 minutes, 0.2 ml of p-nitrophenyl-α-D-pyranoglucoside (PNPG, 1 mM) was added to initiate the reaction, cultured at 37°C for 20 minutes, and finally 0.8 ml of Na 2 CO 3 (1M) The reaction was terminated in a boiling water bath for 5 min, and the MRS broth medium without inoculation was added as the control group. After cooling to room temperature, the absorbance value was measured at 405 nm, and the amount of p-nitrophenol released by PNPG was measured to determine the inhibition of α-glucosidase activity. The absorbance value of the experimental group was recorded as A, and the absorbance of the control group was recorded as A CK The activity of the strain to inhibit α-glucosidase was calculated as follows:

[0047]

[0048] The results show that ( Figure 1 d) The ability of each strain to inhibit α-glucosidase ranged from 12.19% to 99.16%. Among them, 31 lactic acid bacteria had an ability to inhibit α-glucosidase greater than 50%. The lactic acid bacteria with higher ability to inhibit α-glucosidase were BF1 (99.16±1.63%), 8B8 (95.84±0.14%), and JS2 (89.24±2.80%), which were significantly higher than other strains (p<0.05).

[0049] 4. Ability to suppress obesity

[0050] Enterobacter cloacae is a strain that is isolated from the intestines of obese patients and produces lipopolysaccharide endotoxins. It has been proven to cause obesity in the host. Therefore, the ability of the strain to inhibit Enterobacter cloacae can be used to judge the strain's ability to inhibit obesity. The ability of the strain to inhibit Enterobacter cloacae was determined using the Oxford cup punch method. Enterobacter cloacae was inoculated into MRS agar medium at a 0.1% inoculation rate (v / v), and 200 μL of the supernatant of the 49 screened lactic acid bacteria (the supernatant was obtained in the same way as the above-mentioned experiment on regulating sugar metabolism) was added to the Oxford cup wells and placed in a 37°C incubator for 12-24 hours. The MRS broth medium without inoculation was used as a control. The size of the inhibition zone produced around the Oxford cup wells was observed and its diameter was measured. The results showed that ( Figure 1 e) The size of the inhibition zone ranged from 0.00 mm to 21.72 mm and varied significantly. Among them, the sizes of the inhibition zones of three lactic acid bacteria were greater than 20.00 mm, namely JS12 (21.72 ± 2.10 mm), S3 (20.92 ± 1.12 mm), and JS10 (20.48 ± 3.64 mm), which were significantly higher than those of other strains (p < 0.05).

[0051] 5. PCA analysis

[0052] In order to comprehensively evaluate the ability of 49 lactic acid bacteria to regulate energy metabolism, the five variables were standardized and multiplied by the component matrix to obtain a comprehensive score, and the 10 lactic acid bacteria with the highest scores were selected for subsequent experiments based on the comprehensive score.

[0053] As can be seen from the figure ( Figure 1 f), involving two principal components (PCs) accounting for 57.4% of the total variation. PC1 explained 31.30% of the variation and was characterized by the ability to inhibit Enterobacter cloacae. PC2 explained 26.10% of the variation and was characterized by the ability to reduce triglycerides. Among them, JS10, X1 and JS1 were the three lactic acid bacteria with the highest contribution and the highest correlation to PC1. The five variables were standardized and multiplied by the component matrix to obtain a comprehensive score, and the 10 lactic acid bacteria with the highest scores were selected for subsequent experiments. The results are shown in Table 1.

[0054] Table 1 PCA comprehensive scores of strains (top 10)

[0055]

[0056]

[0057] Example 2 Safety capability determination of strains

[0058] The 10 strains of lactic acid bacteria screened in Example 1 were selected, and the safe strains were selected for subsequent experiments by measuring the hemolytic activity, bioamine production ability, gelatinase production ability and DNA enzyme production ability of the lactic acid bacteria.

[0059] 1. Hemolytic

[0060] The strain was streaked on Columbia agar medium containing sheep blood and incubated at 37°C for 24 hours to evaluate the hemolysis of the strain. Staphylococcus aureus was used as a positive control. The results showed that ( Figure 2 a), the colonies of the positive control Staphylococcus aureus showed dark green around them, indicating α-hemolysis, while the colonies of the other 10 lactic acid bacteria did not show dark green around them, indicating γ-hemolysis, indicating that the 10 lactic acid bacteria screened out did not produce hemolysin and had no hemolytic property.

[0061] 2. Determination of bioamine production capacity

[0062] Biogenic amines are formed by the decarboxylation of amino acids. They have important functions in the human body, such as participating in the synthesis of proteins and nucleic acids and maintaining body temperature and blood pressure. However, excessive biogenic amines can lead to symptoms such as diarrhea, food poisoning, and accelerated carcinogenesis. Therefore, the ability of the strain to produce biogenic amines was determined to prevent it from causing excessive biogenic amines in the body. The ability of the strain to produce biogenic amines was determined by the Oxford cup punch method (the steps refer to the experiment on the ability to inhibit obesity in Example 1). The biogenic amine culture medium was composed of the following raw materials in percentage by mass: 0.5% tryptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.5% tryptophan, 0.5% histidine, 0.5% phenylalanine, 0.5% tyrosine, 0.5% lysine, and 1.8% agar powder. The pH was adjusted to 5.2±0.2. Cultured at 37°C for 24 hours. A strain of Havnia alvei that produces biogenic amines was used as a positive control. The results showed that ( Figure 2 b), the positive control was surrounded by purple-red, indicating that biogenic amines would be produced, while the other 10 lactic acid bacteria did not have purple-red surroundings, indicating that the 10 lactic acid bacteria did not produce biogenic amines.

[0063] 3. Determination of gelatinase production capacity

[0064] Gelatinase can hydrolyze collagen and cause inflammation in the human body. It is also an effective marker for early kidney disease and diabetes. The ability of the strain to produce gelatinase is determined as follows. First, prepare the gelatin medium: 120g / L gelatin, 5g / L peptone, 3g / L beef extract, pH 6.8±0.2. Inoculate the strain 5mm below the surface of the gelatin medium by puncture inoculation. Culture at 37°C for 48h and then at 4°C for 1h. If liquid appears in the tube after culture, it means that gelatinase is produced. Staphylococcus aureus was used as a positive control. The results showed that ( Figure 2 c) Liquid appeared in the gelatin culture medium inoculated with Staphylococcus aureus, indicating that gelatinase was produced, while no liquid appeared in the gelatin culture medium inoculated with 10 strains of lactic acid bacteria, indicating that the 10 strains of lactic acid bacteria did not produce gelatinase.

[0065] 4. DNA enzyme production capacity determination

[0066] DNA enzymes can hydrolyze long-chain DNA into oligonucleotide chains, which are soluble in acid. Therefore, if bacteria produce DNA enzymes, a transparent circle will appear around the colonies after adding acid to the colony plate. The strain was inoculated in DNAse agar medium and cultured at 37°C for 48 hours. After culture, observe whether there is a transparent circle around the colony. If there is a transparent circle, it means that DNA enzyme is produced. Staphylococcus aureus was used as a positive control. The results showed that ( Figure 2 d), the transparent ring appeared around the colonies of the positive control Staphylococcus aureus, indicating that DNase was produced, while no transparent ring appeared around the colonies of the other 10 lactic acid bacteria, indicating that the 10 lactic acid bacteria did not produce DNase.

[0067] The above experimental results show that the 10 selected lactic acid bacteria are all highly safe.

[0068] Example 3 Screening of uric acid-lowering lactic acid bacteria

[0069] The ability of the 10 lactic acid bacteria identified as safe to inhibit xanthine oxidase (a key enzyme in uric acid synthesis) in vitro and their ability to reduce uric acid levels and xanthine oxidase levels in a hyperuricemia zebrafish model were further tested to screen out the strain with the strongest uric acid-lowering ability.

[0070] 1. Determination of xanthine oxidase (XOD) inhibition ability.

[0071] The 10 strains of lactic acid bacteria screened above were inoculated into MRS broth medium at an inoculum size of 2% (v / v), cultured at 37°C for 24 hours, and then centrifuged at 4°C and 9690×g for 10 minutes, and the supernatant after centrifugation was taken. The bacterial supernatant (0.1 mL) of the above-identified safe strains, 0.1 M potassium phosphate buffer (pH 7.5) (0.14 mL) and 0.15 mM xanthine substrate solution (0.02 mL) were mixed, and xanthine oxidase (0.1 uint / mL) (0.02 mL) was added thereto. The reaction was carried out at 37°C for 10 minutes. An equal amount of MRS broth medium was added instead of the supernatant as a control group, and allopurinol (a xanthine oxidase inhibitor used for pain treatment) was used as a positive control. A spectrophotometer (UV-2550, Shimadzu, Japan) was used to measure the absorbance at 290 nm before and 10 minutes after the reaction, and recorded as A, B, and C, respectively. 0 and A 10 The absorbance of the control group before and after 10 min of reaction was recorded as A CK0 and A CK10 The xanthine oxidase inhibition rate (%) was calculated using the following formula:

[0072]

[0073] 2. Determination of the ability of lactic acid bacteria to reduce uric acid in vivo.

[0074] The strains with XOD inhibitory activity were further verified for their uric acid-lowering activity. 5dpf zebrafish were randomly placed in a 6-well plate (40 per well), with a capacity of 6ml per well. Four groups were set up, with 120 fish in each group, and three independent parallels. The blank group was zebrafish culture water; the model group was 200μmol / L potassium oxonate + 10μmol / L xanthine sodium salt; the experimental group was 200μmol / L potassium oxonate + 10μmol / L xanthine sodium salt + bacterial solution (final concentration of 106 / ml); the positive control group was 200μmol / L potassium oxonate + 10μmol / L xanthine sodium salt + allopurinol 2mmol / L. Except for the blank group, each group was first treated with potassium oxonate and xanthine sodium salt, and then bacterial solution or allopurinol was added 1h later. After culturing at 28.5℃ for 24h, uric acid and xanthine oxidase levels were determined using a uric acid kit and a xanthine oxidase kit.

[0075] The experimental results are as follows Figure 3 As shown, based on the results of in vitro and in vivo uric acid-lowering ability tests, a lactic acid bacteria strain X1 with the strongest uric acid-lowering effect was screened out, which was obtained by artificial screening, purification and separation from the oral cavity of a healthy human.

[0076] Example 4 Identification of strain X1

[0077] The strain X1 screened in Example 3 was subjected to morphological feature analysis, antibiotic sensitivity analysis, 16SrDNA sequence alignment and phylogenetic tree analysis.

[0078] 1. Morphological characteristics

[0079] Strain X1 was cultured on MRS agar plates for 24 h and the colony morphology was observed ( Figure 4 a), as shown in the figure, the colony is milky white, the colony is convex, the surface is smooth and opaque, and the edges are neat; strain X1 is diffusely turbid after being cultured in MRS broth for 24 hours. A single colony cultured on an MRS agar plate for 24 hours was picked and fixed on a slide for Gram staining, and then the morphological characteristics of the bacteria were observed under an optical microscope ( Figure 4 b), the bacteria are long rod-shaped and are Gram-positive.

[0080] 2. Antibiotic sensitivity

[0081] The agar disk diffusion method was used to determine the sensitivity of strain X1 to 14 clinically relevant antibiotics (gentamicin [10 mg], amikacin [30 mg], ciprofloxacin [5 mg], meprofen [10 mg], penicillin [10 UI], ampicillin [10 mg], amoxicillin [100 mg], vancomycin [30 mg], sulfonamide [300 mg], oxycillin [1 mg], erythromycin [15 mg], ceftriaxone [30 mg], cephalexin [30 mg] and cephalosporin [30 mg]). The sensitivity of the strain to the antibiotics was determined by the size of the transparent zone. The results showed (Table 2) that except for vancomycin, Weissella cibaria X1 was sensitive to the remaining 11 antibiotics, among which the inhibition zones to ampicillin, clindamycin, tetracycline, penicillin G, chloramphenicol, clarithromycin and erythromycin were greater than 15.00 mm, and the inhibition zones to gentamicin, norfloxacin, streptomycin and kanamycin were in the range of 8.00-15.00 mm.

[0082] Table 2 Antibiotic sensitivity of X1

[0083]

[0084]

[0085] - for insensitivity

[0086] 3. Molecular biological characteristics

[0087] The strain X1 selected above was streaked on an MRS agar plate. The DNA of the target strain was extracted using a DNA extraction kit. The extracted DNA was amplified by PCR using universal primers 27F and 1492R. The PCR product was sent to Shanghai Bioengineering Company for sequencing. The 16SrDNA sequence was submitted to a public database for comparison, and a phylogenetic tree was constructed ( Figure 4 c).

[0088] Strain X1 was identified as Weissella cibaria through morphological characteristics, antibiotic sensitivity analysis, 16S rDNA sequence alignment and phylogenetic tree analysis.

[0089] The screened strain X1 was named Weissella cibaria X1. The strain was deposited in the China General Microbiological Culture Collection (CGMCC) on July 15, 2024. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Accession Number: CGMCC NO: 31306.

[0090] The 16S rDNA sequence of strain X1 is shown in SEQ ID NO: 1:

[0091]

[0092] Example 5 Probiotic ability of strain Weissella cibaria X1

[0093] The acid and bile resistance, adhesion, antibacterial properties, in vitro antioxidant capacity, and artificial gastrointestinal fluid tolerance of Weissella cibaria X1 were further tested.

[0094] 1. Acid and bile salt resistance.

[0095] The pH value of the culture medium was adjusted with 1 mol / L HCl, and the strain Weissella cibaria X1 was inoculated at a 2% (v / v) inoculation amount into MRS broth culture medium with pH values ​​of 2.5, 3, and 4 and containing 0.1%, 0.2%, and 0.3% (w / v) ox bile salts and ordinary MRS broth culture medium (control group), and cultured at 37°C. The bacterial liquid was taken at 0h and 4h of culture for coating and counting, and the strain's tolerance to acid was compared. The survival rate of the strain was calculated as follows:

[0096]

[0097] The results are as follows Figure 5 a and Figure 5 As shown in b, the survival rate of Weissella cibaria X1 after 4 hours of culture under pH 2.5 and bile salt concentrations of 0.2% and 0.3% was 0, and the survival rates after 4 hours of culture under pH 3, pH 4 and bile salt concentrations of 0.1% were 88.33±5.14%, 104.33±3.21% and 97.10±0.90%, respectively, which were significantly decreased compared with the control group (p<0.05). This indicates that Weissella cibaria X1 has a higher survival rate under acidic conditions and the presence of bile salts, and has certain probiotic properties.

[0098] 2. Adhesion

[0099] Intestinal epithelial cells Caco-2 were inoculated in 6-well plates. The bacterial cells of bacteria X1 cultured in MRS broth for 24 h were washed twice with PBS and the number of colonies was 10 8 Bacteria X1 at CFU / mL were added to the 6-well plate inoculated with Caco-2, cultured at 37°C for 2 h, washed three times with PBS, and then Gram-stained. The plates were observed and images were taken under a 100x oil immersion lens. Figure 5 c is Caco-2 cells. It can be observed that the cells are in good growth condition and arranged closely, with few particles in the cells and clear edges. After incubation of X1 and Caco-2 for 2 hours, a large number of bacteria gathered in the cells around the edge of Caco-2 ( Figure 5d), similar to Lactobacillus rhamnosus, which has good prebiotic properties, indicating that X1 has the potential to adhere to the intestine.

[0100] 3. Antibacterial properties.

[0101] The inhibitory ability of Weissella cibaria X1 against Escherichia coli and Staphylococcus aureus was determined by the Oxford cup punch method (refer to the obesity inhibition ability experiment in Example 1 for the steps), and MRS broth medium without X1 was added to the Oxford cup well as a control group. The results showed that there was an obvious transparent ring around Weissella cibaria X1 (Table 3), while there was no clear ring around the control group, indicating that Weissella cibaria X1 had a significant inhibitory effect on Escherichia coli and Staphylococcus aureus.

[0102] Table 3 Antibacterial properties of X1

[0103]

[0104] 4. In vitro antioxidant capacity

[0105] (1) DPPH scavenging ability

[0106] The strain X1 was inoculated into MRS broth at a 2% (v / v) inoculation rate, cultured at 37°C for 24 hours, and centrifuged at 4°C and 9690×g for 10 minutes. The supernatant was collected. 1 ml of the bacterial supernatant was mixed with 1 ml of DPPH-ethanol (0.2 mM), reacted at room temperature and in the dark for 30 minutes, and the absorbance value measured at 517 nm was recorded as A. The MRS broth was used as a blank control, and the absorbance value measured was recorded as A. 0 , with 1 mg / ml vitamin C (Vc) as the positive control. The DPPH scavenging ability was calculated as follows:

[0107]

[0108] (2) Hydroxyl radical scavenging ability

[0109] Inoculate strain X1 into MRS broth at a 2% (v / v) inoculation volume, culture at 37°C for 24 h, centrifuge at 4°C, 9690 × g for 10 min, and take the supernatant. Take 0.5 ml of the bacterial supernatant and mix with 0.5 ml of 1,10-phenanthroline (2.5 mM), 0.5 ml of FeSO 4 (2.5 mM), 0.5 ml H 2 O 2(2.5mM) and 1ml PBS (20mM, pH7.4) were mixed and reacted at 37℃ in the dark for 60min. After the reaction, the absorbance value was measured at 510nm and recorded as A. The absorbance value measured by adding an equal amount of PBS as a blank control was recorded as A CK , H 2 O 2 The blank group was replaced with PBS, and the absorbance value was recorded as A 0 1 mg / ml of Vc was used as a positive control. The hydroxyl radical scavenging ability was calculated as follows:

[0110]

[0111] (3)ABTS + Free radical scavenging ability

[0112] Inoculate strain X1 into MRS broth at a 2% (v / v) inoculation volume, culture at 37°C for 24 hours, centrifuge at 4°C, 9690×g for 10 minutes, and take the supernatant. Take 5 ml of ABTS solution (7 mM) and 0.088 ml of potassium persulfate solution (140 mM) and react at room temperature in the dark for 12 to 16 hours to generate ABTS + . Dilute ABTS with ethanol + Solution, so that the absorbance measured at 734nm is 0.7±0.05. Take 1ml bacterial supernatant and 3ml diluted ABTS + The solution mixture was reacted at room temperature in the dark for 30 minutes, and the absorbance value was measured at 734 nm and recorded as A. An equal amount of MRS broth medium was added as a blank control, and the absorbance was measured and recorded as A CK 1 mg / ml Vc was used as a positive control. ABTS + The free radical scavenging capacity is calculated as follows:

[0113]

[0114] (4) Superoxide anion scavenging ability

[0115] Inoculate strain X1 into MRS broth at a 2% (v / v) inoculation rate, culture at 37°C for 24 hours, centrifuge at 4°C, 9690×g for 10 minutes, and take the supernatant after centrifugation. Mix 4.5 ml Tris-HCl buffer solution (0.05M, pH 8.0), 0.2 ml bacterial supernatant, and 0.3 ml pyrogallic acid solution (3 mM), react at 25°C for 20 minutes, and quickly add two drops of 8 mM HCl solution to terminate the reaction. The absorbance value measured at 320 nm is recorded as A. Instead of adding an equal amount of MRS broth as a blank control, the absorbance value measured is recorded as A CK . 1mg / ml Vc solution was used as positive control. The superoxide anion scavenging capacity was calculated as follows:

[0116]

[0117] By measuring the DPPH free radical, hydroxyl free radical, ABTS + The antioxidant function of Weissella cibariaX1 was evaluated by its ability to scavenge free radicals and superoxide anions. Figure 5 As shown in e, the scavenging abilities of Weissella cibaria X1 and 1 mg / ml Vc on DPPH free radicals were 69.83±0.39% and 96.40±0.73%, on hydroxyl free radicals were 20.16±3.86% and 98.88±0.66%, and on ABTS + The free radical scavenging abilities were 90.64±0.25% and 100.00±0.00%, respectively, and the superoxide anion scavenging abilities were 92.44±0.69% and 98.56±0.88%, respectively, indicating that Weissella cibaria X1 has good antioxidant capacity.

[0118] 5. Artificial gastrointestinal fluid tolerance

[0119] 0.8wt% NaCl, 0.02wt% KH 2 PO 4 and 0.115wt%NaHPO 4Prepare PBS buffer solution, add pepsin to PBS buffer solution at a ratio of 10000:1 (w / v) to prepare simulated gastric fluid, adjust the pH to 2.0 with 1M HCL, and filter the pepsin solution with a 0.22mm filter membrane. Adjust the pH of PBS buffer solution to 8.0 with 1M NaOH and sterilize. Add trypsin (250:1, v / w) to PBS buffer solution to prepare simulated intestinal fluid, and filter the trypsin solution with a 0.22mm filter membrane. Inoculate strain X1 into MRS broth medium at an inoculum of 2% (v / v), culture at 37°C for 24h, centrifuge at 4°C and 2500×g for 10min, collect the bacteria and resuspend them with sterile saline to make the absorbance at 595nm 0.8-1.0, inoculate into simulated gastric fluid at an inoculum of 2% (v / v) and culture at 37°C for 4h. The strain tolerance in the stomach was evaluated by counting the spread at 0 and 4 hours and calculating the survival rate of the strain. After culturing in simulated gastric fluid at 37°C for 4 hours, 1 ml of the bacterial solution was added to 9 ml of simulated intestinal fluid and cultured at 37°C for 4 hours. The strain tolerance in the small intestine was evaluated by counting the spread at 0 and 4 hours. The survival rate of the strain in simulated gastric fluid and simulated intestinal fluid was calculated as follows:

[0120]

[0121] The results are as follows Figure 5 f. After 4 h of culture in simulated gastric fluid, the viable cell count of Weissella cibaria X1 ranged from 2.99 to 5.37 log CFU / ml, and the survival rate was 55.87±3.86%. After 4 h of continuous culture in simulated intestinal fluid, the viable cell count of Weissella cibaria X1 ranged from 0.43 to 0.43 log CFU / ml, and the survival rate was 33.33±4.71%.

[0122] According to the acid and bile resistance, adhesion, survival rate in simulated gastrointestinal fluid, in vitro antioxidant capacity and antibacterial activity of Weissella cibaria X1, this strain has certain probiotic properties.

[0123] Example 6 Potential Mechanism of Weissella cibaria X1 in Lowering Uric Acid

[0124] To explore the uric acid-lowering mechanism of Weissella cibaria X1, its ability to degrade inosine and guanosine (precursors of uric acid) in vitro, its ability to degrade uric acid in zebrafish with hyperuricemia, its ability to inhibit XOD, and its effects on the expression levels of key enzyme genes in uric acid metabolism, such as organic anion transporter 1 (OAT1), urate oxidase (UOX), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hepatocyte nuclear factor 4A (HNF4A), were determined.

[0125] 1. The ability of Weissella cibaria X1 to degrade inosine and guanosine

[0126] Weissella cibaria X1 was resuspended in PBS (0.1 mol / L, pH 7.0) containing purine nucleosides (1.26 mmol / L inosine and 1.26 mmol / L guanosine), incubated at 37°C for 12 h, and centrifuged at 9690×g for 5 min to obtain the supernatant. The concentrations of inosine and guanosine were determined by high performance liquid chromatography (HPLC). The specific method was as follows: elution started with a constant flow isocratic elution of 2% methanol (v / v) for 5 min; then 2% to 12% methanol (v / v) was added for 5 to 10 min; 12% to 15% methanol (v / v) for 10 to 12 min; and 15% methanol (v / v) for 12-30 min, and finally monitored at 254 nm. The results are as follows Figure 6 As shown in a-6d, Weissella cibaria X1 significantly reduced the levels of inosine and guanosine at 2.210 and 2.237 points, respectively (p<0.05), with degradation rates of 45.26% and 99.23%, respectively. This is similar to the results of the current study, and the degradation rate of guanosine by X1 is higher, indicating that X1 has great potential for degrading uric acid.

[0127] 2. The uric acid-lowering effect of Weissella cibaria X1 in the zebrafish model.

[0128] In order to explore the uric acid-lowering effect of Weissella cibaria X1, the hyperuricemia zebrafish model was used to measure the uric acid-lowering ability and xanthine oxidase-lowering ability of Weissella cibaria X1 bacterial suspension and cell fragments. X1 was centrifuged at 9690×g for 5 min, the bacteria were washed 2-3 times with PBS, and then resuspended in PBS to a final OD of 595nmWhen the concentration of the bacterial suspension reaches 0.60±0.02, the bacterial suspension obtained above is crushed in an ultrasonic crusher for 10 minutes to obtain a cell rupture liquid. A hyperuricemia zebrafish model was constructed according to the method in Example 3, with the group with zebrafish culture water added as the CK group, the group with bacterial suspension added as the BS group, the group with cell rupture liquid added as the CFF group, the group with allopurinol added as the APL group, the group with probenecid (a uric acid-lowering drug) added as the P group, and the hyperuricemia model group as the HUA group. After culturing at 28.5°C for 24 hours, the uric acid and xanthine oxidase levels were measured using a uric acid kit and a xanthine oxidase kit. The results are as follows Figure 6 e and Figure 6 As shown in figure f, the results showed that both the bacterial suspension and cell fragmentation liquid of Weissella cibaria X1 could significantly reduce the levels of uric acid and xanthine oxidase in the zebrafish in the hyperuricemia zebrafish model, among which the bacterial suspension was significantly better than the cell fragmentation liquid in reducing the levels of uric acid and xanthine oxidase (p<0.05), indicating that both the intracellular and extracellular metabolism of X1 have a therapeutic effect on zebrafish with hyperuricemia, and the extracellular metabolites are significantly better than the intracellular substances in reducing uric acid and xanthine oxidase.

[0129] 3. Analysis of qPCR results of the expression of key enzyme genes in uric acid metabolism

[0130] According to the above grouping, 40 zebrafish in each group were pipetted into a 1.5 ml sterile centrifuge tube, immediately frozen in liquid nitrogen, and total RNA was extracted using an animal tissue RNA extraction kit. MightyScript Plus First Strand cDNA Synthesis Master Mix (gDNA digester) (Shanghai Biotechnology Co., Ltd, Shanghai, China) was used for cDNA synthesis. The primers used are shown in Table 4. SGExcel Fast SYBR Mixture (Shanghai Biotechnology Co., Ltd, Shanghai, China) was used for qPCR amplification. The expression data were normalized according to the expression of Actin and 2 -△△Ct The method calculated the relative quantification of the mRNA level of each gene. The results showed that ( Figure 7), the expression of HPRT in the APL group was significantly higher than that in the other three groups, because APL inhibits the production of uric acid by inhibiting the production of XOD or acting on HPRT. Compared with the HUA group, the expression of OAT1 in the P group was significantly increased (p<0.01), because probenecid mediates the production of OAT1 and promotes the excretion of uric acid. The expression levels of the four genes in the BS group were significantly higher than those in the HUA group, while only the expression level of HPRT in CFF was significantly higher than that in the HUA group (p<0.01). Comprehensive analysis shows that the role of X1 in degrading uric acid is partly due to the fact that the metabolites produced during its growth process regulate the expression levels of OAT1, UOX, and HNF4A in the body, promote the excretion of uric acid, and regulate the expression of HPRT, preventing excessive uric acid production and thus reducing the level of uric acid.

[0131] Table 4 Primer information

[0132]

Claims

1. Weissella cibaria X1, deposited with CGMCC NO:31306.

2. Use of Weissella cibaria X1 according to claim 1 in the preparation of products with the functions of regulating metabolism and lowering uric acid, wherein the metabolism regulation is selected from the group consisting of regulating lipid metabolism, regulating sugar metabolism, and inhibiting obesity.

3. The use according to claim 2, characterized in that: The regulation of lipid metabolism includes lowering cholesterol and lowering triglycerides.

4. The use according to claim 2 or 3, characterized in that: The active ingredient of the product for regulating metabolism and lowering uric acid function comprises the bacteria of Weissella cibaria X1.

5. Use of the Weissella cibaria X1 described in claim 1 in the preparation of a product for preventing and treating hyperuricemia.

6. A product with the function of regulating metabolism and lowering uric acid, characterized in that: The invention comprises the bacteria of Weissella cibaria X1 according to claim 1 as an active ingredient, wherein the metabolism regulating agent is selected from the group consisting of regulating lipid metabolism, regulating sugar metabolism, and inhibiting obesity.

7. A product for preventing and treating hyperuricemia, characterized in that: The invention comprises the bacterial cell of Weissella cibaria X1 according to claim 1 as an active ingredient.

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