Strain composition with uric acid-lowering effect and application thereof

Abstract

The present application provides a strain composition with the function of reducing uric acid and application thereof, the strain composition comprises Lactobacillus helveticus, Lacticaseibacillus paracasei and Lactiplantibacillus plantarum. The composition can degrade uric acid, degrade inosine, degrade guanosine and inhibit xanthine oxidase.

Description

Technical Field

[0001] This invention relates to a bacterial strain composition with uric acid-lowering effects and its applications. Background Technology

[0002] Hyperuricemia is a metabolic disease characterized by abnormal uric acid accumulation due to purine metabolism disorders, which significantly increases the risk of gout, diabetes, cardiovascular disease, and chronic kidney disease. Due to changes in modern lifestyles and related genetic factors, the incidence of hyperuricemia has been rising year by year in recent years.

[0003] Currently, the most commonly used uric acid-lowering drugs are allopurinol, febuxostat, and benzbromarone. While they can effectively reduce serum uric acid levels, they may cause adverse reactions such as allergic reactions, kidney damage, liver toxicity, and gastrointestinal discomfort. Therefore, finding effective and low-side-effect-prone solutions to alleviate hyperuricemia has become a current research hotspot.

[0004] The formation pathway of uric acid is complex. Inosine and guanosine, two nucleosides, are among the precursors for uric acid production. Related research indicates that reducing nucleoside levels helps decrease uric acid levels in the host body. Xanthine oxidase is a key enzyme in uric acid production, and many current drug developments target it, aiming to reduce uric acid production by inhibiting xanthine oxidase activity. The challenge remains how to efficiently lower uric acid levels. Summary of the Invention

[0005] To address the above problems, this invention provides a bacterial strain composition with uric acid-lowering effects and its applications.

[0006] The objective of this invention is achieved through the following method: a bacterial strain composition with uric acid-lowering effect, comprising *Lactobacillus helveticus*, *Lactaseibacillus paracasei*, and *Lactiplantibacillus plantarum*.

[0007] Lactobacillus helveticus INM3107 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 28, 2024, with accession number GDMCC NO:64575.

[0008] Lacticaseibacillus paracasei in m25-LPC was deposited on February 25, 2019, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 17263.

[0009] Lactiplantibacillus plantarum in m28-LP was deposited on June 18, 2020, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.20101.

[0010] Furthermore, the colony count ratio of *Lactobacillus paracasei*: *Lactobacillus helveticus*: *Lactobacillus plantarum* was 2:1:1.

[0011] Application of a bacterial strain composition with uric acid-lowering effect in the preparation of uric acid-lowering products.

[0012] Application of a uric acid-lowering bacterial strain composition in degrading inosine.

[0013] Application of a uric acid-lowering bacterial strain composition in degrading guanosine.

[0014] Application of a bacterial strain composition with uric acid-lowering effect in inhibiting xanthine oxidase.

[0015] Application of a bacterial strain composition with uric acid-lowering effect in the preparation of fermented milk that inhibits xanthine oxidase.

[0016] Application of a uric acid-lowering bacterial strain composition in the preparation of probiotic products.

[0017] Furthermore, probiotic products include functional dairy products, functional beverages, starter cultures, probiotic powders, and bacteriocins.

[0018] Advantages of the present invention: The composition can degrade uric acid with a high degradation rate of 53.07%, the composition can degrade inosine, the composition can degrade guanosine, and the composition can inhibit xanthine oxidase;

[0019] Lactobacillus helveticus exhibits a 100% degradation rate for both guanosine and inosine.

[0020] The inhibition rate of xanthine oxidase by Lactobacillus paracasei was 44.05%.

[0021] The inhibition rate of xanthine oxidase by Lactobacillus plantarum was 37.61%. Attached Figure Description

[0022] Figure 1 This is a microscopic image of Lactobacillus helveticus.

[0023] Figure 2 This is a microscopic image of Lactobacillus paracasei.

[0024] Figure 3 This is an image of *Lactobacillus plantarum* observed under a microscope.

[0025] Figure 4 This is a chromatogram of inosine and guanosine standards.

[0026] Figure 5 The graph shows the degradation rates of inosine and guanosine in different strains.

[0027] Figure 6 This is a graph showing the inhibition of XOD by different strains.

[0028] Figure 7 A graph showing the changes in uric acid content in zebrafish from different strains.

[0029] Figure 8 The graph shows the changes in uric acid content in zebrafish containing the compound bacterial strain.

[0030] Figure 9 This is a colony morphology diagram of Lactobacillus helveticus.

[0031] Figure 10 This is a colony morphology diagram of Lactobacillus paracasei.

[0032] Figure 11 This is a colony morphology diagram of *Lactobacillus plantarum*. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments:

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

[0035] Example 1, see attached document Figure 1-11 A bacterial composition with uric acid-lowering effects, comprising *Lactobacillus helveticus*, *Lactaseibacillus paracasei*, and *Lactiplantibacillus plantarum*:

[0036] Lactobacillus helveticus INM3107 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 28, 2024, with accession number GDMCC NO:64575.

[0037] Lacticaseibacillus paracasei in m25-LPC was deposited on February 25, 2019, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 17263.

[0038] Lactiplantibacillus plantarum in m28-LP was deposited on June 18, 2020, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.20101.

[0039] A bacterial strain composition with uric acid-lowering effect, wherein the colony count ratio of Lactobacillus paracasei: Lactobacillus helveticus: Lactobacillus plantarum is 2:1:1.

[0040] Application of a bacterial strain composition with uric acid-lowering effect in the preparation of uric acid-lowering products.

[0041] Application of a uric acid-lowering bacterial strain composition in degrading inosine.

[0042] Application of a uric acid-lowering bacterial strain composition in degrading guanosine.

[0043] Application of a bacterial strain composition with uric acid-lowering effect in inhibiting xanthine oxidase.

[0044] Application of a bacterial strain composition with uric acid-lowering effect in the preparation of fermented milk that inhibits xanthine oxidase.

[0045] Application of a uric acid-lowering bacterial strain composition in the preparation of probiotic products.

[0046] Application of a uric acid-lowering bacterial strain composition in the preparation of probiotic products; probiotic products include functional dairy products, functional beverages, starter cultures, bacterial powders, and bacteriocins.

[0047] Materials and Methods

[0048] 1.1 Materials and Reagents

[0049] 1.1.1 Sample Information

[0050] The homemade fermented vegetable samples came from Taishun, Wenzhou, and the naturally fermented milk samples came from Gannan, Sichuan.

[0051] 1.1.2 Culture medium

[0052] MRS agar medium and MRS broth medium: Qingdao Haibo Biotechnology Co., Ltd.

[0053] 1.1.3 Reagents

[0054] Sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, glycerol: Sinopharm Chemical Reagent Co., Ltd.; Glacial acetic acid, concentrated hydrochloric acid, perchloric acid: Zhejiang Zhongxing Chemical Co., Ltd.; Xanthine oxidase, uric acid content assay kit: Beijing Solarbio Science & Technology Co., Ltd.; Inosine, guanosine, xanthine, allopurinol, taurine, 4-methyl-2-pentanone, potassium oxonate: Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Bacterial genome extraction kit, agarose, DNA polymerase: Shanghai Sangon Biotech Co., Ltd.; Antibiotic susceptibility test strips: Hunan Bickman Biotechnology Co., Ltd.; Skim milk powder: Anchor.

[0055] 1.2 Instruments and Equipment

[0056] 1260InfinityⅡ analytical high-performance liquid chromatograph, 7890B-5977 gas chromatograph-mass spectrometer, DB-WAX gas chromatograph column: Agilent Technologies, Inc.; MULTISKAN GO microplate reader: Thermo Fisher Scientific, Inc.; 3K15 refrigerated centrifuge: SIGMA GmbH, Germany; TH2-C constant temperature shaking incubator: Suzhou Peiying Laboratory Equipment Co., Ltd.; LRH-250 biochemical incubator: Shanghai Yiheng Scientific Instruments Co., Ltd.

[0057] 1.3 Experimental Methods

[0058] 1.3.1 Isolation and screening of lactic acid bacteria

[0059] After the fermented vegetable sample was patted evenly, 25 mL of the juice was added to 225 mL of sterile physiological saline. After serial dilution, the sample was spread on MRS solid medium and incubated at 37°C for 48 h. Single colonies with lactic acid bacteria characteristics were picked and purified by streak plate method. The process was repeated 4 times.

[0060] Take 1g of sample and add it to 9mL of sterile physiological saline. Perform serial dilution and then perform separation and purification using the same method as above. Store the isolated strain with lactic acid bacteria characteristics in a -80°C freezer for later testing.

[0061] 1.3.2 Preparation of inosine guanosine standard curve and screening of inosine guanosine-degrading strains

[0062] The activated bacterial strain was inoculated into MRS liquid medium and cultured at 37°C for 24 h. After centrifugation for 10 min (8000 r / min, 4°C), the precipitate was collected and washed twice with sterile PBS (pH 7.0, 0.1 M). The bacterial concentration was then adjusted to 1 × 10⁻⁶. 9CFU / mL. Take 1 mL of the above bacterial culture and mix it with 750 μL of inosine-guanosine PBS buffer (inosine, 1.26 mmol / L; guanosine, 1.26 mmol / L). Incubate at 37 °C for 60 min, then centrifuge for 10 min (8000 r / min, 4 °C). Take 0.9 mL of the supernatant and mix it with 0.1 mL of perchloric acid (0.1 M). Filter the mixture through a 0.22 μm sterile filter and analyze it using high performance liquid chromatography (Agilent 1260). The phase parameters are shown in Table 1. The degradation rate is calculated using the following formula:

[0063] Degradation rate (%) = (1 - (post-reaction inosine (guanosine) concentration / (pre-reaction inosine (guanosine) concentration)) * 100

[0064] Table 1. Liquid phase parameters.

[0065]

[0066]

[0067] Inosine-Guanine Standard Curve Production

[0068] Under the conditions of this experiment, such as Figure 4 The retention times of inosine and guanosine were 32.486 min and 36.543 min, respectively. Figure 4 A), the standard curves are as follows: Ainosine = 2165.6x - 23.988, R0 2 =0.9993 ( Figure 4 B) and Aguanosine = 3422.8x - 132.28, R 2 =0.9985( Figure 4 C).

[0069] Screening of inosine guanosine-degrading strains

[0070] A total of 27 strains of lactic acid bacteria were isolated from the fermentation samples, such as Figure 5 As shown, each strain exhibits a certain ability to degrade nucleosides, and the degradation abilities of the strains for inosine and guanosine show a convergence. For example... Figure 5 As shown in Figure A, strain GQ1501 exhibited the strongest ability to degrade inosine, achieving a degradation rate of 100%, while the degradation rates of the remaining strains were all less than 50%. The degradation results of guanosine by the strains are as follows... Figure 5 As shown in B, the degradation rate of guanosine by strain GQ1501 was also 100%, which was significantly higher than that of other strains. Therefore, strain GQ1501 was selected for subsequent experiments.

[0071] 1.3.3 Screening of lactic acid bacteria that inhibit xanthine oxidase

[0072] The activated bacterial strain was inoculated into MRS liquid medium and cultured at 37°C for 24 h. After centrifugation for 10 min (8000 r / min, 4°C), the precipitate was collected and washed twice with sterile PBS (pH 7.0, 0.1 M). The bacterial concentration was then adjusted to 1 × 10⁻⁶. 9 CFU / mL. Take 1 mL of the above bacterial culture, sonicate (300 W, 40 kHz) for 10 min to disrupt the bacterial cells, then centrifuge for 10 min (8000 r / min, 4℃), and use the supernatant for the experiment. Use a 0.05 mg / mL allopurinol solution as a positive control. Add the relevant substances according to the amounts shown in the table below, and then measure the absorbance change at 295 nm.

[0073] Xanthine oxidase activity inhibition rate = (1 - (difference in absorbance before and after reaction in the experimental group) / (difference in absorbance before and after reaction in the blank group)) * 100%

[0074] Substance Addition Table (Unit: μL)

[0075] experimental group 140 20 20 20 blank 160 20 0 20

[0076] Screening of strains that inhibit xanthine oxidase activity

[0077] Depend on Figure 6 It was found that all 27 strains exhibited varying degrees of inhibitory activity against xanthine oxidase. The strongest inhibitory activity was observed in strain R2, with an inhibition rate of 44.05%, which showed no significant difference from the inhibitory activity of the positive control (0.05 mg / mL allopurinol solution) (P>0.05). Strain D1 showed the second highest inhibition rate at 37.61%, second only to strain R2, and there was no significant difference between the two (P>0.05). Therefore, strains R2 and D1 were selected for subsequent experiments. Xanthine oxidase is abbreviated as XOD.

[0078] 1.3.4 Strain Identification

[0079] DNA was extracted from the selected strains according to the instructions of the bacterial genomic DNA extraction kit. PCR was performed using universal bacterial primers (27f: AGTTTGATCMTGGCTCAG, 1492r: GGTTACCTTGTTACGACTT), and the PCR products were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0080] Blast analysis of the strain sequences on NBCI revealed that strain GQ1501, isolated from fermented milk, belongs to Lactobacillus helveticus, while strains R2 and D1, isolated from fermented vegetables, belong to Lacticaseibacillus paracasei and Lactiplantibacillus plantarum, respectively.

[0081] D1: Lactiplantibacillus plantarum inm28-LP was identified genetically, and its gene sequence is as follows:

[0082] AGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTG

[0083] CTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCC

[0084] CAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGG

[0085] TCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGAT

[0086] GGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACAT

[0087] TGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGAC

[0088] GAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGT

[0089] TAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACG

[0090] GCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGG

[0091] CGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAG

[0092] TGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGA

[0093] AATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCT

[0094] GAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGAT

[0095] GAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGC

[0096] CTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGG

[0097] AGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAAT

[0098] CTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTC

[0099] GTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCAT

[0100] TAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAA

[0101] TCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGA

[0102] ACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGC

[0103] CTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGG

[0104] CCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCT

[0105] TTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAA R2: *Lacticaseibacillus paracasei* inm25-LPC is identified through genetic identification, and its gene sequence is as follows

[0106] TGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAGTTCTCGTTGATG

[0107] ATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTA

[0108] ACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAAC

[0109] CGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTA

[0110] GCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCG

[0111] GCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCAC

[0112] AATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACT

[0113] CTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAA

[0114] AGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATT

[0115] TATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACC

[0116] GAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTA

[0117] GCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAAC

[0118] TGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGT

[0119] AAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGC

[0120] ATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAA

[0121] GCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTT

[0122] TGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCG

[0123] TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTG

[0124] CCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGA

[0125] CGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGA

[0126] GTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGC

[0127] AACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGT

[0128] TCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGC

[0129] GTAACCCTTTTAGGGAGCGAGCCGTCTAAGGTGGGACAAATGATTAGGGTGAAGTCGTAACAGGQ1501: *Lactobacillus helveticus* INM3107 has been identified through gene identification, and its gene sequence is as follows

[0130] GCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCAGAACCAGCAGAT

[0131] TTACTTCGGTAATGACGCTGGGGACGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTG

[0132] CCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCAT

[0133] GATCAGCTTATAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAG

[0134] TTGGTAAGGTAACGGCTTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCAC

[0135] ATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGG

[0136] ACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTT

[0137] GTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCA

[0138] CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTG

[0139] GGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGA

[0140] ACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGT

[0141] GGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGCAACTGACG

[0142] CTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACG

[0143] ATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCC

[0144] GCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT

[0145] GGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGTGCCA

[0146] TCCTAAGAGATTAGGAGTTCCCTTCGGGGACGCTAAGACAGGTGGTGCATGGCTGTCGTCAGC

[0147] TCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTATTAGTTGCCAGC

[0148] ATTAAGTTGGGCACTCTAATGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCA

[0149] AGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGTACAACGAGAAGC

[0150] AAGCCTGCGAAGGCAAGCGAATTCTCTGAAAGCTGTTCTCAGTTCGGACTGCAGTCTGCAACTC

[0151] GACTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGAACGCCGCGGTGAATACGTTCCCG

[0152] GGCCTTGTACACACCGCCCGTCACACCATGGAAGTCTGCAATGCCCAAAGCCGGTGGCCTAAC

[0153] CTTCGGGAAGGAGCCGTCTAAGGCAGGGCAGATGACTGGGGTGAAGTCG

[0154] 1.3.4 Tests on the acid and bile salt resistance of the strains

[0155] The culture of the selected strains was inoculated into MRS broth medium at pH 2.0 and 3.0 at a volume fraction of 5%. After incubation at 37°C for 0 h and 3 h, samples were taken for plate viable count and survival rate calculation.

[0156] The culture medium of the selected strains was inoculated at a volume fraction of 5% into MRS broth medium without bile salts and containing 0.3% bile salts, and cultured at 37°C for 3 hours. Samples were taken at 0 and 3 hours to count viable cells on plates and calculate the survival rate.

[0157] The survival rate of each strain was calculated according to formula (3): Strain survival rate / 100% = Number of viable bacteria after treatment (0h viable bacteria number) × 100. (The first application was done, then 2.0 + 3.0 were applied at 3h) 1.3.7 Antibiotic susceptibility test of strains

[0158] Spread 100 μL of bacterial suspension of the test strain (approximately 10⁷ CFU / mL) evenly onto an MRS plate. Using sterile forceps, place the antibiotic-containing test strip onto the surface of the agar medium, and then incubate the plate at 37°C for 24 h. Measure the diameter of the inhibition zone using calipers; the diameter of the inhibition zone is used to assess the antibiotic sensitivity of the strain.

[0159] To test the antibiotic susceptibility of the strains, experiments were conducted according to the CLSI antibiotic susceptibility testing standards, and the relevant results are shown in Table 2.

[0160] The GQ1501 strain is most sensitive to penicillin, with an inhibition zone diameter of 43.6 mm. It also has some sensitivity to erythromycin, ampicillin, ceftriaxone, and tetracycline, but is not sensitive to ciprofloxacin.

[0161] Except for moderate sensitivity to ciprofloxacin, strain R2 is relatively sensitive to other antibiotics. Strain D1 shows good sensitivity to erythromycin, ampicillin, ceftriaxone, and tetracycline.

[0162] Table 2 Antibiotic susceptibility of different bacterial strains. Note: S indicates sensitive; I indicates normal; R indicates insensitive.

[0163]

[0164] 1.3.9 Production of Probiotic Freeze-Dried Powder

[0165] The activated strain was inoculated into MRS liquid medium and anaerobically cultured at 37°C for 24 h. The precipitate was then centrifuged, washed with sterile physiological saline, and a freeze-drying protectant containing 12% skim milk powder and 5% glycerol was added. After pre-freezing, the mixture was freeze-dried for 24 h. The viable count was determined by plate counting. The freeze-dried powder was then stored at -20°C.

[0166] 1.3.8 Zebrafish husbandry

[0167] Wild-type AB strain zebrafish, with a water temperature of 28℃±1℃, pH of 7.5±0.5, conductivity of 500-800μS / cm, and a photoperiod of light:dark = 14h:10h.

[0168] 1.3.9 Maximum Tolerable Concentration Test in Zebrafish

[0169] Zebrafish at 120 hpf were divided into 6 groups and tested in 6-well plates, with 40 fish per well. Each group was given bacterial powder solutions of 250 g / L, 50 g / L, 10 g / L, 2 g / L, 1 g / L, and 0 g / L, respectively, and incubated in a constant temperature incubator at 28℃±1℃ for 24 h before calculating the mortality rate.

[0170] The viable count of the lyophilized powder was determined by the pour plate method, and the viable count of each sample was adjusted to 1×10⁻⁶ using skim milk powder. 9 CFU / g. In the maximum tolerated concentration experiment of zebrafish, all zebrafish in the groups with administration concentrations of 250 g / L, 50 g / L, and 10 g / L died, while the mortality rate of zebrafish in the groups with administration concentrations of 2 g / L, 1 g / L, and 0 g / L was 0%. Therefore, 2 g / L and 1 g / L were selected as the high and low dose groups for the experiment.

[0171] 1.3.10 Determination of the uric acid-lowering potential of a single strain

[0172] A zebrafish hyperuricemia model was established using potassium oxonate (400 μmol / L) and xanthine (20 μmol / L). Modeling and drug administration were carried out simultaneously. The zebrafish were grouped according to Table X, with a final solution volume of 6 mL per group. After incubation at 28℃±1℃ for 24 h, the uric acid content in the zebrafish was determined according to the method described in the Solarbio Uric Acid Assay Kit.

[0173] Table 3 Dosage Design Table

[0174]

[0175] The results of the determination of the uric acid-lowering potential of lactic acid bacteria are as follows: Figure 7As shown in the figure, the uric acid content in the normal group of zebrafish was 0.156 μmol / L. Compared with the normal group, the uric acid content in the model group of zebrafish was significantly increased (P<0.05), reaching 0.323 μmol / L, indicating successful model establishment. The uric acid content in the negative control group was not significantly different from that in the model group, thus ruling out the influence of skim milk powder and its concentration on uric acid in zebrafish. The study found that the uric acid-lowering effect of probiotics is not necessarily better with higher concentrations. The group with the most significant uric acid degradation effect was group D1-1, followed by group R2-2, with degradation rates of 40.87% and 40.45% respectively. These figures were not significantly different from the positive control group (P>0.05), indicating that the uric acid-lowering effect of 1 g / L of strain D1 and 2 g / L of strain R2 may be equivalent to 2 mmol / L of allopurinol. The GQ1501-1 group also showed a degradation rate of 31.91%, which was significantly higher than that of the negative control group (P<0.05).

[0176] 1.3.11 Determination of the uric acid-lowering potential of the compound strain

[0177] The effective bacterial strains were combined in different proportions, and their uric acid-lowering potential was determined using zebrafish. Except for changing the sample concentration to 0.5 g / L, all other experimental conditions were the same as in 1.3.10.

[0178] 1.3.12 Characterization of fermentation performance of the composite strain

[0179] Prepare 12% reconstituted milk using skim milk powder, stir at 50℃ for 30 min, sterilize at 95℃ for 5 min, cool, inoculate with a compound bacterial strain at a ratio of 5%, ferment at 37℃ for 18 h, take 5 mL of fermentation broth, add 5 mL of deionized water, mix well, centrifuge at 8000 r for 10 min, filter the supernatant through a 0.45 μm membrane, and determine the XOD inhibition activity according to the method described in 1.3.3.

[0180] The flavor of fermented milk was characterized using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HSP-MS). 100 g of fermented milk sample, 50 g of distilled water, and 100 μg of internal standard solution (4-methyl-2-pentanone) were added to the HSP flask. After stirring at 55 °C for 30 min, the sample was enriched by inserting an extraction needle for 45 min before analysis. High-purity helium was used as the carrier gas in the gas chromatography at a flow rate of 1.0 mL / min. The temperature program was 35 °C for 1 min, followed by increasing the temperature at 3 °C / min to 220 °C and holding for 10 min, and finally increasing the temperature at 20 °C / min to 240 °C and holding for 5 min. The ion source temperature, interface temperature, and quadrupole temperature for mass spectrometry were 230 °C, 240 °C, and 150 °C, respectively. The electron energy was 70 eV, the ionization mode was EI, and the scan mass range was m / z 33–500. The volatile concentration was calculated using the following formula:

[0181] Volatile concentration (μg / kg) = (peak area of ​​substance × mass of internal standard) / (peak area of ​​internal standard × mass of sample)

[0182] After centrifuging the fermented milk for 10 min (8000 r / min, 4℃), the supernatant was taken and its ability to inhibit xanthine oxidase was determined according to the method in 1.3.3.

[0183] Results of Lactic Acid Bacteria Compound in Lowering Uric Acid

[0184] Based on the results of single-strain experiments, lactic acid bacteria compound experiments were conducted according to three ratios: R2:GQ1501:D1 = 1:1:1 (combination A), R2:GQ1501:D1 = 2:1:1 (combination B), and R2:GQ1501:D1 = 3:1:1 (combination C). The drug concentration for each group was 0.5 g / L.

[0185] The effect of lactic acid bacteria compound in lowering uric acid is as follows Figure 8 As shown in the figure, among the three combinations, combination B had the highest degradation rate of 53.07%, which was significantly improved compared to the previous single-strain effect (P<0.05) and higher than the positive control group. Combination B is a combination that can effectively enhance the ability of the strain to degrade uric acid.

[0186] Characterization of fermentation performance of compound strains in skim milk system

[0187] Experimental results showed that the fermented milk prepared by the compound strain had a certain ability to inhibit xanthine oxidase, with an inhibition rate of 36.24% and an allopurinol equivalent of 13.36 μg / mL.

[0188] The fermented milk prepared by this compound bacterial strain system has good flavor characteristics, and the main flavor substances are shown in Table 4. Ketones are one of the important flavor substances in fermented milk. The most abundant flavor substance in the fermented milk prepared by the compound strain is 2-nonanone, with a retention time of about 33.05 min and a content of 0.086 mg / kg. It is a ketone that can provide fruity, floral, and sweet flavors. The second most abundant is 2-heptanone, with a retention time of 23.13 min and a content of 0.049 mg / kg. This substance has a fruit-like flavor and a low sensory threshold of 0.001-0.01 mg / kg. In addition to the above ketones, 2,3-butanedione, also known as diacetyl, was also detected in the fermented milk, with a content of 0.021 mg / kg and a sensory threshold of 0.001 mg / kg. It can impart milky and buttery flavors to the product and is an extremely important flavor substance in fermented milk, cheese, and other products. 3-Hydroxy-2-butanone, also known as acetoin, is a common substance in fermented milk that imparts a buttery flavor. Besides ketones, this fermented milk also contains a significant amount of acidic flavor compounds, such as acetic acid, butyric acid, and hexanoic acid. Acetic acid has the highest content, reaching 0.047 mg / kg, and it gives fermented milk its characteristic sour taste. Furthermore, furfural and toluene, which are also important flavor compounds in fermented milk, were detected in the fermented milk.

[0189] Table 4. Content of major flavor compounds in fermented milk

[0190]

[0191]

[0192] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, which fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention patent should be determined by the appended claims.

Claims

1. A bacterial strain composition with uric acid-lowering effect, characterized in that: The bacterial composition includes Lactobacillus helveticus ( Lactobacillus helveticus Lactobacillus paracasei ( Lacticaseibacillus paracasei Lactobacillus plantarum ( Lactiplantibacillus plantarum ): Lactobacillus helveticus ( Lactobacillus helveticus INM3107 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 28, 2024, with accession number GDMCC NO:64575; Lactobacillus paracasei ( Lacticaseibacillus paracasei Inm25-LPC was deposited on February 25, 2019, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 17263; Lactobacillus plantarum ( Lactiplantibacillus plantarum Inm28-LP was deposited on June 18, 2020, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.20101; the colony count ratio of Lactobacillus paracasei: Lactobacillus helveticus: Lactobacillus plantarum was 2:1:

1.

2. The use of a bacterial composition with uric acid-lowering effect according to claim 1 in the preparation of uric acid-lowering products.

3. The application of a uric acid-lowering bacterial strain composition according to claim 1 in the preparation of probiotic products.

4. The application of the uric acid-lowering bacterial strain composition according to claim 3 in the preparation of probiotic products, characterized in that: Probiotic products include functional dairy products, functional beverages, and starter cultures.

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