Lactobacillus plantarum DY1 and application of lactobacillus plantarum DY1 combined with quercetin in improvement of hyperuricemia

Abstract

The invention belongs to the technical field of food and medicine microorganisms, and particularly relates to application of lactobacillus plantarum DY1 and combination of the lactobacillus plantarum DY1 and quercetin to improvement of hyperuricemia. The lactobacillus plantarum DY1 is preserved in the China General Microbiological Culture Collection Center on October 30, 2023, the preservation number is CGMCC NO.28810, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the lactobacillus plantarum DY1 is classified and named as lactobacillus plantarum. The combination of the strain and quercetin can significantly inhibit the activity of xanthine oxidase, adenosine dehydrogenase and inflammatory factors in a hyperuricemia mouse body, and regulate uric acid transport mRNA and intestinal flora and serum metabolic profiles. The lactobacillus plantarum DY1 has the potential of being applied to in-vivo adjustment of uric acid level, so that theoretical reference and guidance basis are provided for probiotic preparations for relieving hyperuricemia by utilizing the lactobacillus plantarum DY1 and quercetin.

Description

Technical Field

[0001] The present invention belongs to the technical field of food and drug microbiology, and particularly relates to the application of a Lactobacillus plantarum DY1 and its combination with quercetin in improving hyperuricemia. Background Art

[0002] Hyperuricemia (HUA) is characterized by elevated serum uric acid levels and is a metabolic disease caused by an imbalance between uric acid production and excretion. The definition of HUA is a serum uric acid level of 7.0 mg / dL in men and 6.0 mg / dL in women. In recent years, the incidence of HUA has been increasing year by year worldwide, especially in China and the United States. In addition, a large number of epidemiological studies have reported that HUA is closely related to the development of many chronic diseases, such as diabetes, hypertension, obesity, cardiovascular diseases, and kidney diseases. Treating HUA requires reducing the uric acid level in the blood. Reducing the uric acid level can be achieved by inhibiting uric acid synthesis and promoting uric acid metabolism. Currently, drugs for reducing uric acid production and promoting uric acid excretion have been widely developed and applied. For example, allopurinol, probenecid, etc. Although these drugs help reduce the uric acid level, they have side effects, such as allergic reactions, liver and kidney dysfunction, etc. Therefore, there is an urgent need for a safe and effective method to prevent or treat hyperuricemia. In recent decades, a large number of epidemiological observations have emphasized that a healthy diet and lifestyle can prevent or alleviate the occurrence of hyperuricemia, making the research on dietary intervention for hyperuricemia a hot topic. Probiotics are live bacteria with various health benefits, especially lactic acid bacteria, which can reduce the uric acid level in the body, thereby alleviating hyperuricemia. However, the effect of using single-strain preparations is not obvious. In addition, there are many natural bioactive compounds in the diet, such as the polyphenol compound quercetin, which has the potential to improve hyperuricemia. However, there are currently no reports on the combination of probiotics and quercetin to relieve hyperuricemia. Therefore, the development of products combining uric acid-lowering probiotics and active substances has important research significance and application value. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of a Lactobacillus plantarum DY1 and its combination with quercetin in improving hyperuricemia.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] A Lactobacillus plantarum DY1, which was deposited at the General Microbiological Center of the China National Center for Biotechnology Development on October 30, 2023, with the deposit number CGMCC NO.28810, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the taxonomic name is Lactobacillus plantarum.

[0006] Application of Lactobacillus plantarum DY1 in alleviating hyperuricemia.

[0007] The degradation rates of uric acid, hypoxanthine, inosine and guanosine by Lactobacillus plantarum DY1 are 93.97%, 87.98%, 67.87% and 100% respectively; the inhibition rates of its MRS fermentation supernatant, bacterial suspension and cell-free extract on xanthine oxidase are 43.07%, 77.85% and 21.53% respectively.

[0008] A bacterial agent for alleviating hyperuricemia, the bacterial agent contains the Lactobacillus plantarum DY1 described above.

[0009] The bacterial agent contains the culture, cultured bacterial suspension or fermentation broth of this strain.

[0010] The strain DY1 in the bacterial agent is cultured to the logarithmic phase.

[0011] A composition for alleviating hyperuricemia, the composition contains the Lactobacillus plantarum DY1 described above.

[0012] The composition contains the Lactobacillus plantarum DY1 and quercetin.

[0013] When the composition is used, the number of viable bacteria of Lactobacillus plantarum DY1 required for each kilogram of body weight of the application object is 1×10 7 -1×10 12 CFU and 50 - 1000 mg of quercetin.

[0014] The number of viable bacteria of the above-mentioned Lactobacillus plantarum DY1 can preferably be 1×10 8 -1×10 11 CFU, more preferably it can be 1×10 8 -1×10 10 CFU, still more preferably it is 1×10 8 -1×10 9 CFU, even more preferably it is 1×10 9 CFU;

[0015] Quercetin can preferably be 50 - 500 mg; more preferably it can be 50 - 300 mg, still more preferably it is 100 - 300 mg, and even more preferably it is 200 mg;

[0016] The composition is a dosage form prepared by mixing the Lactobacillus plantarum DY1 and quercetin as active ingredients with pharmaceutically acceptable carriers and adjuvants;

[0017] The dosage form can be an oral preparation (for example, tablets, capsules, oral liquids, etc.), an injection preparation (for example, powders, suspensions, etc.).

[0018] Use of the described composition for relieving hyperuricemia, application of the composition in preparing related functional foods or drugs for preventing and / or treating hyperuricemia.

[0019] Effect of the uric acid-lowering composition in relieving hyperuricemia. The combination of Lactobacillus plantarum DY1 and quercetin has a significant function of improving hyperuricemia. The combination of Lactobacillus plantarum and quercetin can reduce the serum uric acid level of male mice with hyperuricemia after 22 days of intervention. The blood uric acid is reduced from 269.77 μmol / L to 187.64 μmol / L.

[0020] Mechanism of the uric acid-lowering composition in regulating hyperuricemia. The combination of Lactobacillus plantarum DY1 and quercetin can reduce the levels of xanthine oxidase and adenosine dehydrogenase activities, regulate the uric acid transporter genes URAT1, GLUT9, ABCG2 and OAT1, reduce the levels of inflammatory factors, and regulate the intestinal flora and serum metabolic profile, thereby playing a role in reducing the uric acid level. The importance of the intestinal flora was verified by fecal microbiota transplantation.

[0021] The single-strain Lactobacillus plantarum DY1 and quercetin involved in the present invention have good uric acid-lowering effects and can be used as excellent microbial preparations to replace traditional drugs for reducing uric acid and relieving hyperuricemia. In the present invention, a hyperuricemia mouse model was used as the object, and Lactobacillus plantarum DY1 and / or quercetin were administered respectively. Compared with the model group, the serum uric acid level of the mice could be significantly reduced. The combination scheme of Lactobacillus plantarum strain DY1 and quercetin was superior to the effect of single administration. This indicates that the combination of Lactobacillus plantarum DY1 and quercetin has a synergistic therapeutic function and has high application value. In summary, the probiotic and natural bioactive product composition provided by the present invention has excellent uric acid-lowering effects and has a very broad application prospect. Description of the Drawings

[0022] Figure 1 Colony morphology diagram of the strain of the present invention.

[0023] Figure 2 Gram staining result diagram of the strain of the present invention.

[0024] Figure 3 Phylogenetic tree of the strain of the present invention.

[0025] Figure 4 Standard curves of uric acid, hypoxanthine, inosine and guanosine in the present invention.

[0026] Figure 5 Result diagram of the strain of the present invention inhibiting xanthine oxidase.

[0027] Figure 6 Serum and liver biochemical indexes of mice in each group.

[0028] Figure 7 The mRNA levels of uric acid transporters in the kidneys of mice in each group.

[0029] Figure 8 The levels of inflammatory factors in the kidneys of mice in each group.

[0030] Figure 9 The Ace and Shannon indices of the intestinal flora of mice in each group.

[0031] Figure 10 The principal coordinate analysis (PCoA) of the intestinal flora of mice in each group

[0032] Figure 11 The heat map of the abundances of the top 25 genera in the intestinal flora of mice in each group.

[0033] Figure 12 The differential flora at the genus level in the intestinal flora of hyperuricemic mice by Lactobacillus plantarum DY1 combined with quercetin.

[0034] Figure 13 The principal coordinate analysis map of the serum untargeted metabolome of mice in each group.

[0035] Figure 14 The enrichment of differential metabolite pathways in mice of the Lactobacillus plantarum DY1 combined with quercetin group.

[0036] Figure 15 The Venn diagram of the intestinal flora of mice treated with antibiotics and control mice at the OTU level.

[0037] Figure 16 The serum uric acid levels of mice after fecal microbiota transplantation. Detailed implementation manners

[0038] The following further illustrates the detailed implementation manners of the present invention in combination with examples. It should be noted that the detailed implementation manners described herein are only for explaining and interpreting the present invention and are not limited to the present invention.

[0039] The strain DY1 of the present invention is derived from the fermented food doro. It is identified as Lactobacillus plantarum by 16S rDNA.

[0040] The present invention provides a probiotic composition suitable for people with hyperuricemia, including the Lactobacillus plantarum DY1 and quercetin, wherein the quercetin is purchased from a national pharmaceutical company.

[0041] The results of the examples of the present invention confirm that the composition has the effect of reducing serum uric acid. The present invention provides the application of the probiotic composition in the preparation of foods or drugs for preventing and / or treating hyperuricemia. The present invention will be described in detail below in combination with examples.

[0042] Example 1 Screening and Identification of Uric Acid-lowering Strains

[0043] 1.1 Isolation of Bacterial Strains

[0044] Weigh 1 g of fermented Docynia delavayi fruit and mix it thoroughly with 10 ml of PBS. Continuously dilute the mixture tenfold with sterilized distilled water, and spread the diluted solutions of different gradients on MRS solid medium containing 0.75% CaCO3. Incubate at 37°C for 48 h, and pick the colonies with clear zones around them for purification. Perform Gram staining and catalase tests on the purified strains, and select the strains that are Gram-positive and catalase-negative. Inoculate the strains into MRS liquid medium. Prepare a medium (1.71 g Na 2 HPO 4 , 0.3 g KH 2 PO 4 , 0.05 g NaCl, 0.05 g MgSO 4 .7H 2 O, 0.001 g CaCl 2 , 0.2 g UA and 1.2 g agar, per 100 mL) with uric acid as the sole carbon and nitrogen source. Transfer the strains to the new medium with uric acid as the sole carbon source. Then pick monoclonal colonies and inoculate them into MRS liquid medium with an inoculation loop, and incubate statically at 37°C. The colony morphology is as shown in Figure 1 , showing a semi-transparent circular shape. Figure 2 As shown, the Gram staining result of the strain is blue-violet, and the morphology is rod-shaped.

[0045] 1.2 Identification of Bacterial Strains

[0046] Extract its genomic DNA using a bacterial genomic DNA kit. Primers: 27f: 5'-AGAGTTTGATCCT-GGCTCAC-3', 1492r: 5'-GGTTACCTTGT-TACGACTT-3', and the Marker is DL5000. The 16S rDNA gene of strain DY1 is amplified by PCR, purified, and sequenced. The size of the 16S rDNA gene fragment obtained by PCR amplification is about 1.5 kb. The sequencing results show that the length of the 16S rDNA gene sequence of the strain is 1423 bp. Perform BLAST alignment to obtain sequences with high similarity to its 16S rDNA sequence, conduct homology analysis, and construct a Neighbor-Joinging phylogenetic tree using MEGA 7.0. The results are as shown in Figure 3 , showing that the similarity between strain DY1 and Lactobacillus plantarum is 100%.

[0047] The strain Lactobacillus plantarum DY1 obtained from the above identification was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on October 30, 2023. The deposit number is CGMCC NO. 28810, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as Lactobacillus plantarum.

[0048] 1.3 Screening of uric acid-lowering lactic acid bacteria

[0049] The contents of uric acid and hypoxanthine in the reaction solution were determined by HPLC external standard method. The strain was inoculated into MRS broth medium at an inoculation amount of 2%, and activated for two generations under anaerobic conditions at 37°C. The concentration of the bacterial suspension was adjusted to 1×10 8 CFU / mL with sterile normal saline, and resuspended in 20 mmol / L (pH 7.0) neutral potassium dihydrogen phosphate solution containing 200 μg / ml uric acid, 20 mmol / L (pH 7.0)) neutral potassium dihydrogen phosphate solution containing 200 μg / ml hypoxanthine, and 20 mmol / L (pH 7.0) neutral potassium dihydrogen phosphate solution containing 600 μg / ml inosine, respectively. After culturing for 4 hours, 70% perchloric acid as a terminator was mixed evenly at a volume ratio of 9:1, and centrifuged at 4500 g for 5 minutes. The standard curve was determined based on the external standard method. The standard curve is as Figure 4 shown. The ability of the strain to degrade uric acid, hypoxanthine, and inosine was calculated according to the formula a = [(0.9C - X) / 0.9C]×100%. C: Initial amount of uric acid / hypoxanthine / inosine / guanosine (μg / mL); X: Residual amount of uric acid / hypoxanthine / inosine / guanosine (μg / mL). The degradation rates of the strain for uric acid, hypoxanthine, inosine, and guanosine were 93.97%, 87.98%, 67.87%, and 100%, respectively.

[0050] 1.4 Inhibitory ability of the strain against xanthine oxidase

[0051] The concentration of the bacterial solution was adjusted to 1×10 9 cfu / mL with sterile PBS, centrifuged at 5000 r / min for 5 min, and the supernatant was extracellular substances. The bacterial solution of 1×10 9 cfu / mL was ultrasonically broken for 5 min (200 W, working time 5 s, pause 5 s), and the obtained liquid was centrifuged at 5000 r / min for 5 min, and the supernatant was taken to obtain intracellular substances. Allopurinol (AP) at 0.2 mg / mL was used as a positive control.

[0052] Take 0.75 mL of 150 μmol xanthine substrate solution, and respectively mix it with 0.75 mL of allopurinol and the bacterial suspension of Lactobacillus plantarum DY1 (1×10 9Mix the cfu / mL), intracellular substances or extracellular substances, and finally add 0.5 mL of xanthine oxidase solution (0.5 U / mL) pre-incubated at 25 °C for 20 min to initiate the reaction, and record the change in absorbance value under ultraviolet conditions at a wavelength of 295 nm. Use a microplate reader (TECAN, infinite M200) to detect the reaction solution at a wavelength of 295 nm. Allopurinol was used as a positive control, and the calculation formula for the xanthine oxidase inhibition rate was as follows: Inhibition rate (%) = [1 - (C - D) / (A - B)]. Where A is the absorbance of the solution containing xanthine oxidase but no sample, B is the absorbance of the solution without xanthine oxidase and sample, C is the absorbance of the solution containing xanthine oxidase and sample, and D is the absorbance of the solution containing the sample but no xanthine oxidase. As Figure 5 shown, the inhibition rates of its MRS fermentation supernatant, bacterial suspension, and cell-free extract on xanthine oxidase were 77.85%, 43.07%, 21.53%, and 18.48%, respectively.

[0053] The bacterial suspension of Lactobacillus plantarum DY1 described above is the bacterial suspension prepared by diluting with normal saline in the above step 1.3.

[0054] Example 2 Effects of Lactobacillus plantarum strain DY1 and / or quercetin on hyperuricemic mice

[0055] 2.1 Experimental grouping and administration

[0056] Establish a hyperuricemic mouse model induced by hypoxanthine and potassium oxonate: Male Kunming mice were randomly divided into 6 groups, with 9 mice in each group. They were the blank control group (Control), hyperuricemia model group (Model), probiotic group (DY1), quercetin group (Que), probiotic and quercetin combination group (DY1-Que), and positive drug allopurinol group (AP). They were intragastrically administered hypoxanthine (500 mg / kg) suspended in 0.5% CMC-Na solution and intraperitoneally injected with the modeling agent potassium oxonate (250 mg / kg) for 15 days. Blood was collected from the orbital venous plexus 30 min after the modeling administration. After the modeling was successful, except for the blank group, all were given corresponding drug intervention treatments for 22 days. The probiotic group was intragastrically administered with 1×10 9 CFU of the bacterial suspension of Lactobacillus plantarum, the quercetin group with 200 mg / kg of quercetin, the probiotic and quercetin combination group with 1×10 9 CFU of the bacterial suspension of Lactobacillus plantarum and 200 mg / kg of quercetin, and the allopurinol group with 10 mg / kg of allopurinol. Mouse feces were collected, and blood was taken from the eyeballs after anesthesia with sodium pentobarbital, and the liver and kidneys were collected.

[0057] The bacterial suspension of Lactobacillus plantarum DY1 described above is the bacterial suspension prepared by diluting with normal saline in the above step 1.3.

[0058] 2.2 Determination of biochemical indexes

[0059] The activities of uric acid and xanthine oxidase in the serum of mice, and xanthine oxidase and adenosine dehydrogenase in the liver were measured strictly according to the kit instructions. The increase in UA value is the main characteristic index of hyperuricemia. The results are shown in the figure. After 15 days of intragastric administration of hypoxanthine and intraperitoneal injection of potassium oxonate in mice, as Figure 6 shown in B, the uric acid level in the serum of the model group showed a significant difference compared with that of the blank control group (P<0.05), indicating that the model was successfully established and subsequent experiments could be carried out. As Figure 6 shown in C, after 22 days of drug administration, compared with the blank control group, the uric acid value in the hyperuricemia model group increased significantly. Compared with the model group, the uric acid values in all treatment groups decreased significantly, and the combination of probiotics and quercetin had the best effect on reducing uric acid. XOD and ADA are key enzymes in uric acid metabolism. As Figure 6 shown in D-F, the XOD in the serum and liver of the model group mice was significantly increased compared with that of the control group, and was significantly decreased after drug administration compared with the model group. The trend of ADA in the liver was the same as that of XOD.

[0060] 2.3 Determination of URAT1, GLUT9, ABCG2 and OAT1 mRNA in kidney tissues

[0061] Total RNA of the kidney tissues of each group above was extracted using a tissue extraction kit according to the manufacturer's instructions. Then the extracted RNA was reverse transcribed into complementary DNA (cDNA) and amplified accordingly. Quantitative PCR analysis was carried out according to the instructions, and the results were expressed as Ct values. Normalized with the endogenous control GAPDH, and its expression level was determined by the 2-ΔΔCT method. The results are as Figure 7 shown. The mRNA levels of URAT1 and GLUT9 in the kidneys of the model group mice were significantly increased, while the mRNA levels of URAT1 and GLUT9 were down-regulated after drug administration. In addition, compared with the control group, the gene expression levels of ABCG2 and OAT1 in the model group were significantly decreased, and the gene expression levels of ABCG2 and OAT1 were significantly up-regulated after drug administration. The above results indicate that probiotics and quercetin down-regulate the expression of URAT1 and GLUT9, up-regulate the expression of ABCG2 and OAT1, and promote the excretion of uric acid in the kidneys.

[0062] 2.4 Determination of inflammatory factors in the kidneys

[0063] The kidney tissue samples of each group above were mechanically homogenized with 0.9% normal saline at 60 Hz for 1 min using a tissue grinder, and then centrifuged at 5000 rpm for 10 min to obtain the supernatant. The contents of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the tissue supernatant were measured by ELISA method. The results are as Figure 8As shown, the levels of IL-1β and TNF-α in hyperuricemia mice were significantly increased. However, after administration of allopurinol, probiotics, quercetin, or a combination of probiotics and quercetin, the level of IL-1β was significantly decreased. And only the combination of Lactobacillus plantarum DY1 and quercetin could significantly reverse the level of TNF-α.

[0064] 2.5, 16S rRNA gene sequencing of intestinal flora

[0065] Extract the total DNA of the intestinal flora in the feces of mice in each group according to the instructions of the DNA extraction kit and detect the extraction quality (gel electrophoresis), and then quantify the DNA (ultraviolet spectrophotometry). Based on 338F (5’ACTCCTACGGGAGGCAGCAG-3’)-806R (5’-GGACTACHVGGGTWTCTAAT-3’), perform PCR amplification on the V3-V4 variable region of the 16S rRNA gene. Amplification program: pre-denaturation at 95°C for 3 min, 27 cycles (denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s), then stable extension at 72°C for 10 min, and finally store at 4°C; reaction system: 4 μL of 5×TransStartFastPfu buffer, 2 μL of dNTPs (2.5 mmol / L), 0.8 μL of upstream primer (5 μmol / L), 0.8 μL of downstream primer (5 μmol / L), 0.4 μL of TransStartFastPfu DNA polymerase, 10 ng of template DNA, make up to 20 μL, with 3 replicates for each sample. Purify and recover the PCR products according to the instructions of the DNA gel recovery kit and detect (gel electrophoresis), and then quantify the recovered products (fluorescence method). Construct a library according to the instructions of the DNA rapid sequencing kit and sequence based on the Illumina Miseq PE300 platform to obtain the original sequencing sequences. Import the above sequences into Trimmomatic for quality control and splice with the Flash software (v1.2.11) to optimize the sequences. The quality control and splicing parameters are consistent with the literature reports. Use the Usearch10.0 software to cluster the optimized sequences at a similarity level of 97% to obtain operational taxonomic units (OTUs), and perform taxonomic annotation on the OTUs based on the Silva database. Use the RDP classifier Bayesian algorithm to perform species classification annotation on each sequence, set the alignment threshold to 70%, and then statistically analyze the community composition of each sample at the phylum, class, order, family, genus, and species taxonomic levels; further use the Mothur 1.30 software to analyze and evaluate the α-diversity index; use the QIIME 1 software for β-diversity analysis to compare the similarity of different samples in terms of species diversity. For statistical analysis, use SPSS25.0 software to perform differential analysis on the experimental data. The results of measurement data are expressed as x±s. One-way ANOVA is used for comparison among multiple groups, and the least significant difference method (LSD) is used for multiple comparisons between groups. P<0.05 indicates that the difference is statistically significant; all graphs are made with Prism 7.0 (GraphPad Software, La Jolla, CA, USA). P<0.05 indicates that the difference is statistically significant. Obtain 1,402,873 optimized sequences from 30 fecal samples using the 16S rRNA gene sequencing technology. As Figure 9As shown, α-diversity analysis showed that the Ace, Shannon, and Chao indices of the hyperuricemia mouse model were significantly enhanced compared with those of other groups, indicating higher diversity and abundance of the gut microbiota. β-diversity was used to elucidate the structure and function of the gut microbiota. As Figure 10 shown, principal component analysis (PCA) based on Unweightedunifrac showed a clear separation between the model group and normal mice, indicating that the gut microbiota composition of hyperuricemic mice was disturbed. As Figure 11 shown, a heatmap of the top 25 abundant flora at the genus level was presented. The differential flora between the combined administration group and the model group was further analyzed. As Figure 12 shown, at the genus level, compared with the model group, the relative abundance of Lactobacillus in the combined administration group increased significantly, while the relative abundances of Roseburia and norank_f__norank_o__Clostridia_UCG-014 decreased significantly.

[0066] 2.6. Effects of Lactobacillus plantarum DY1 and quercetin on hyperuricemic mice by untargeted metabolomics

[0067] After thawing at 4℃, transfer 100 μL of mouse serum samples from each group to 1.5 ml EP tubes using a pipette. Methanol (300 μL) is used to precipitate proteins, and then 10 μL of internal standard (2-chlorophenylalanine at 2.9 mg / mL) is added. After vortex mixing for 30 seconds, the samples are centrifuged at 12,000 rpm for 15 minutes at 4℃; 200 μL of the supernatant is transferred to a sample vial for measurement. The instrument analysis platform is the UHPLC-Q Exactive HF-X system of Thermo Fisher Scientific, an LC-MS system. The samples are ionized by electrospray ionization, and mass spectrometry signals are collected using positive and negative ion scanning modes respectively. The heater temperature is 425℃; the sheath gas flow rate is 50 arb; the auxiliary gas flow rate is 13 arb; full scan mode (m / z 70 - 1050); electrospray voltage 3.5 kV (+) / 3.5 kV (-); the capillary temperature is 325℃, and the S-lens RF level is 50%. Normalized collision energy (eV) 20, 40, 60; resolution (Full MS) 60000; resolution (MS2) 7500. Quality control (QC) samples are prepared by mixing equal volumes of the extracts of all samples. The volume of each QC is the same as that of the samples and is processed and detected using the same method as the analysis samples. During the instrument analysis process, one QC sample is inserted among every 5 - 15 analysis samples to examine the stability of the entire detection process. The raw data is imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, peak alignment, etc., and finally a data matrix containing information such as retention time, mass-to-charge ratio, and peak intensity is obtained. Subsequently, the software is used for feature peak library search and identification, matching the MS and MS / MS mass spectrometry information with the metabolic database. The MS mass error is set to less than 10 ppm, and metabolites are identified according to the secondary mass spectrometry matching score. The main databases are mainstream public databases such as the KEGG pathway database (http: / / www.genome.jp / kegg / ), http: / / www.hmdb.ca / , and https: / / metlin.scripps.edu / as well as a self-built database. The metabolomics data conforms to a normal distribution. In addition, peaks with missing values in more than 80% of the samples are removed. Then, the obtained data matrix is imported into the SIMCA 14.1 software to analyze the metabolite profile by OPLS-DA and visualize the metabolic differences between the normal group and the model group.Metabolites with variable importance in projection (VIP) greater than 1 and t-test P-value less than 0.05 (P<0.05) in OPLS-DA were considered differential metabolites. Metabolic pathway analysis was completed on the MetaboAnalyst 5.0 platform. From the PCA plot ( Figure 13 ), it can be seen that in the positive and negative modes, there were obvious differences between the model mice and other groups, while the metabolic profiles of the DY1-Que group were closer to those of the normal mice. In Figure 14 , an OPLS-DA model was used to identify potential biomarkers between the control group and the model group (positive: R2Y = 0.995; Q2 = 0.893. Negative: R2Y = 1; Q2 = 0.96). A total of 162 differential metabolites were identified by this model, and these metabolites might be related to the pathogenesis of hyperuricemia mice. Among them, 130 differential metabolites could be identified by the KEGG and HMDB databases. Among them, 86 biomarkers related to HUA mice were altered in DY1-Que. Using KEGG enrichment analysis and topological analysis, the degree of participation of metabolites in biological reactions was evaluated according to the positions of metabolites in related pathways, and specific metabolic pathways involved were determined. As Figure 15 shown, through pathway enrichment analysis, it was determined that DY1-Que was mainly involved in glutathione metabolism, sphingolipid metabolism, tryptophan metabolism, and purine metabolism (impact>0, p<0.05). The differential metabolites included oxidized glutathione, indoleacetaldehyde, CAMP, sphinganine, sphinganine, indole-3-acetamide, 3-(3-indolyl)-2-oxopropionic acid, 3-indoleacetic acid, uric acid, 5-hydroxy-l-tryptophan, xanthine, 3-methylindole, ADP, adenosine monophosphate, and glutathione. Further analysis of the correlation between the differential flora and differential metabolites in the combined administration group found that there was a significant correlation between the differential flora and multiple differential metabolites.

[0068] 2.7. Microbiota transplantation

[0069] Male Kunming mice were divided into two groups: a control group and an antibiotic group. A mouse sterile intestinal model was established by gavage of vancomycin (100 mg / kg), neomycin sulfate (200 mg / kg), metronidazole (200 mg / kg), and ampicillin (200 mg / kg) in sequence to serve as the antibiotic group. The antibiotic group received a 7-day course of antibiotic treatment. Three days after discontinuation of antibiotics, the mice underwent fecal microbiota transplantation by gavage. The antibiotic-treated mice were divided into 3 groups: a hyperuricemia model group, a fecal microbiota transplantation control (Control(FMT)) group, and a fecal microbiota transplantation DY1-Que group (DY1-Que(FMT)). The hyperuricemia model in the antibiotic group was established using the same method as in the experiment, that is, by gavage of hypoxanthine (500 mg / kg) and intraperitoneal injection of potassium oxonate (250 mg / kg), once a day, to establish a hyperuricemia model in mice. After the Control(FMT) group and the DY1-Que group defecated naturally, fecal specimens were collected in a timely manner. Subsequently, 1 mL of PBS was added to every 100 mg of feces, and centrifuged at 2000 g for 4 minutes. The obtained supernatant was used as the transplantation fluid. Except for the control group, the other three groups all established a hyperuricemia model using the previously established method. Each mouse in the Control(FMT) and DY1-Que(FMT) groups was given 200 μL of the intestinal microbiota transplantation fluid, and the experiment lasted for 5 weeks. After the last gavage administration, the mice were fasted for 12 hours, and all mice were anesthetized with 1% sodium pentobarbital, and serum and fecal samples were collected. The sequencing results are as Figure 16 shown that the OTU level of the intestinal microbiota in the antibiotic group mice decreased significantly, indicating that the mouse intestinal sterile model was successfully established. Five weeks after fecal microbiota transplantation, as Figure 16 shown, compared with the model group, the serum uric acid level in the DY1-Que(FMT) group decreased significantly, indicating that the combination of probiotics and quercetin alleviates hyperuricemia by regulating the intestinal microbiota. The experiment shows that Lactobacillus plantarum DY1 combined with quercetin can effectively alleviate hyperuricemia.

[0070] The above embodiments only describe the preferred methods of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A Lactobacillus plantarum DY1 strain, characterized in that: Lactobacillus plantarum DY1 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on October 30, 2023, with the deposit number CGMCC NO.28810, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the taxonomic name is Lactobacillus plantarum.

2. The application of the Lactobacillus plantarum DY1 according to claim 1, characterized in that: the application of the Lactobacillus plantarum DY1 in alleviating hyperuricemia.

3. A bacterial agent for alleviating hyperuricemia, characterized in that: the bacterial agent contains the Lactobacillus plantarum DY1 according to claim 1.

4. The bacterial agent for alleviating hyperuricemia according to claim 3, characterized in that: the bacterial agent contains the culture, cultured bacterial suspension or fermentation broth of the strain.

5. The bacterial agent for alleviating hyperuricemia according to claim 4, characterized in that: the strain DY1 in the bacterial agent is cultured to the logarithmic phase.

6. A composition for alleviating hyperuricemia, characterized in that: the composition contains the Lactobacillus plantarum DY1 according to claim 1.

7. The composition according to claim 6, characterized in that: the composition contains the Lactobacillus plantarum DY1 and quercetin.

8. The composition according to claim 7, characterized in that: When the composition is used, the viable count of Lactobacillus plantarum DY1 administered to the subject per kilogram of body weight is 1×10 7 -1×10 12 CFU and 50 - 1000 mg of quercetin.

9. The application of the composition for alleviating hyperuricemia according to claim 6, characterized in that: the application of the composition in the preparation of related functional foods or drugs for preventing and / or treating hyperuricemia.

Images