Enterococcus faecium, biological agent and application of enterococcus faecium in prevention and / or treatment of hyperuricemia

Abstract

The invention belongs to the technical field of biological agents, and particularly relates to an enterococcus faecium Ef-13, a biological agent and application of the enterococcus faecium Ef-13 to prevention and / or treatment of hyperuricemia. When animals are fed with the enterococcus faecium Ef-13 or the biological agent, uric acid in poultry bodies can be efficiently degraded, and the level of the uric acid can be reduced by 45% or above; meanwhile, the liver and kidney metabolism burden caused by hyperuricemia can be relieved, the improvement rate of related indexes of liver injury in serum reaches 30% or above, and the effect of protecting the kidney and / or liver is achieved; and the composition of intestinal microbiota can be optimized, and the purine metabolism stability of the organism is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biological agents technology, specifically relating to a strain of Enterococcus faecalis, a biological agent, and its application in the prevention and / or treatment of hyperuricemia. Background Technology

[0002] Due to a defect in the uricase gene, uric acid becomes the primary end product of nitrogen metabolism in poultry. This innate physiological characteristic leads to hyperuricemia (HUA), a prevalent metabolic disorder in intensively farmed poultry. The core pathological mechanism is directly related to either excessive uric acid synthesis or impaired excretion. This disease widely affects chickens, geese, turkeys, and some wild birds, but is particularly prominent in intensive poultry farms. Goslings under 20 days old, older laying hens, and broiler chickens are the most susceptible groups, with an incidence rate reaching 20%–60%. High-calcium, high-protein diets, environmental stress in poultry houses, and nephrotropic viral infections can all induce or exacerbate the condition, leading to a significant increase in serum uric acid concentration, accompanied by glomerular damage, renal tubular fibrosis, inflammatory activation, and oxidative stress, causing significant economic losses to the poultry industry.

[0003] Studies known to the inventors have confirmed that the gut is the core pathway for uric acid excretion, accounting for 30% of total excretion. Gut microbiota imbalance is closely related to the occurrence and development of uric acid efflux (HUA). An effective uric acid-lowering strategy needs to simultaneously possess the synergistic functions of degrading intestinal uric acid, regulating microbiota balance, protecting the liver and kidneys, and regulating purine metabolism genes. However, existing prevention and treatment technologies have significant limitations. For example, while chemical drugs can temporarily regulate uric acid levels, they have significant side effects and lack the ability to coordinate multi-dimensional regulation of the gut, metabolism, liver, and kidneys, making it difficult to address the root causes of disease and organ damage. Reducing feed protein content can decrease purine intake, but it restricts the supply of essential nutrients to poultry and only acts on the upstream links of uric acid production, without directly regulating already produced uric acid, resulting in limited actual uric acid-lowering effects. Therefore, current research has not utilized this key excretion pathway of the gut and cannot relieve its inhibition of intestinal uric acid efflux transporters (such as ABCG2 and GLUT9) by reducing intestinal uric acid concentration.

[0004] Probiotics are a class of live microorganisms that are beneficial to the host. They play an important role in gastrointestinal health by regulating the balance of intestinal flora. If strains that regulate intestinal flora balance can also degrade intestinal uric acid, protect the liver and kidneys, and regulate purine metabolism, they will solve the problem that existing chemical drugs cannot synergistically regulate intestinal, metabolic, and liver and kidney functions. However, strains with these functions still need to be further explored in current research. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Enterococcus faecalis, a biological agent, and its application in the prevention and / or treatment of hyperuricemia. The Enterococcus faecalis Ef-13 of this invention can efficiently degrade uric acid in the body, while reducing the metabolic burden on the liver and kidneys caused by hyperuricemia, and can optimize the composition of intestinal microbiota and enhance the stability of purine metabolism in the body, thereby achieving the effect of preventing and / or treating hyperuricemia.

[0006] This invention provides a strain of Enterococcus faecalis Ef-13, the preservation number of which is CGMCC No. 37038.

[0007] The present invention also provides a biological agent comprising Enterococcus faecalis Ef-13 as described in the above technical solution.

[0008] Preferably, it includes fermentation broth and / or bacterial suspension of Enterococcus faecalis Ef-13.

[0009] Preferably, the effective viable count of Enterococcus faecalis Ef-13 in the biological agent is ≥1×10⁻⁶. 8 CFU / mL.

[0010] The present invention also provides a method for preparing the biological agent described in the above technical solution, comprising the following steps: inoculating Enterococcus faecalis Ef-13 into a fermentation medium and performing shaking culture to obtain the biological agent.

[0011] The present invention also provides the application of Enterococcus faecalis Ef-13 described in the above technical solution, or the biological agent described in the above technical solution, or the biological agent prepared by the preparation method described in the above technical solution, in the preparation of medicines for the prevention and / or treatment of hyperuricemia.

[0012] This invention also provides the application of the Enterococcus faecalis Ef-13 described in the above-described technical solutions, or the biological agent described in the above-described technical solutions, or the biological agent prepared by the preparation method described in the above-described technical solutions, in the preparation of one or more of the following products: 1) Lowering uric acid; 2) Protect the liver and / or kidneys; 3) Regulates gut microbiota; 4) Anti-pathogenic bacteria.

[0013] Preferably, the pathogenic bacteria include one or more of Staphylococcus aureus, Escherichia coli, and Salmonella.

[0014] The present invention also provides a medicine for the prevention and / or treatment of hyperuricemia, the active ingredient of which includes Enterococcus faecalis Ef-13 as described in the above technical solution, the biological agent as described in the above technical solution, or the biological agent prepared by the preparation method described in the above technical solution.

[0015] The present invention also provides the application of Enterococcus faecalis Ef-13 described in the above technical solutions, or the biological agent described in the above technical solutions, or the biological agent prepared by the preparation method described in the above technical solutions, or the drug described in the above technical solutions, in the preparation of animal feed additives and / or animal feed.

[0016] Beneficial effects: This invention provides a strain of Enterococcus faecalis Ef-13, which has been biopreserved with the accession number CGMCC No. 37038. Based on this, the invention also provides a biological agent containing the aforementioned Enterococcus faecalis Ef-13. Feeding animals with the Enterococcus faecalis Ef-13 or the biological agent can efficiently degrade uric acid in poultry, reducing uric acid levels by more than 45%; simultaneously, it can alleviate the metabolic burden on the liver and kidneys caused by high uric acid, with an improvement rate of over 30% in serum liver damage-related indicators, thus protecting the kidneys and / or liver; and it can optimize the composition of the intestinal microbiota and enhance the stability of purine metabolism in the body.

[0017] Biological Preservation Certificate Enterococcus faecalis Ef-13, biologically classified as Enterococcus faecium It was deposited on December 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 37038. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 This is a diagram showing the preliminary isolation and purification results of the uric acid-producing strain in Example 1; Figure 2 This is a graph showing the screening results of uricase-producing strains in Example 1; Figure 3 The images show the MRS morphological characteristics (A) and Gram staining results (B) of the strain in Example 2, where the magnification of the image in B is 1000X. Figure 4 This is a gel electrophoresis image of strain Ef-13 in Example 2, where M represents the DL 2000 DNA marker and 1 represents the target band of strain Ef-13. Figure 5 This is the phylogenetic tree of strain Ef-13 in Example 2; Figure 6 The growth curve of Enterococcus faecalis Ef-13 in Example 2; Figure 7The acid production curve of Enterococcus faecalis Ef-13 in Example 2 is shown in Example 2. Figure 8 This is a graph showing the antibacterial effect of Enterococcus faecalis Ef-13 in Example 2; Figure 9 This is a graph showing the hemolytic results of Enterococcus faecalis Ef-13 in Example 2; Figure 10 This is a distribution diagram of the lengths of third-generation sequencing reads in Example 3; Figure 11 This is a statistical graph showing the correlation between GC content and sequencing depth in Example 3; Figure 12 This is a diagram showing the COG database annotation results in Example 3; Figure 13 This is a KEGG image of Enterococcus faecalis Ef-13 in Example 3; Figure 14 This is a graph showing the effect of Enterococcus faecalis Ef-13 in purine metabolism in Example 3; Figure 15 This is a graph showing the statistical results of CARD drug classification in Example 3; Figure 16 This is a statistical chart showing the species origin of the CARD resistance gene in Example 3; Figure 17 This is a statistical chart of toxicity factors in Example 3; Figure 18 This is the complete genome map of the Enterococcus faecalis plasmid in Example 3; Figure 19 This is the complete genome map of the Enterococcus faecalis chromosome in Example 3; Figure 20 The species sparseness curve, OTU cluster analysis diagram, and Simpson and Shanon index diagrams are shown in Example 4. Figure 21 This is a graph showing the results of principal coordinate analysis (PCoA) and hierarchical clustering analysis in Example 4; Figure 22 This is a graph showing the relative abundance of phyla at the level in Example 4; Figure 23 This is a graph showing the relative abundance results of the genera in Example 4; Figure 24 This is a graph showing the relative abundance results of the species level in Example 4; Figure 25 This is a diagram of the strains with different species levels in Example 4. Detailed Implementation

[0020] This invention provides a strain of *Enterococcus faecalis* Ef-13, whose preservation number is CGMCC No. 37038. *Enterococcus faecalis* Ef-13 of this invention was isolated from fermented tea. On MRS solid medium, the colonies are white, round, with relatively neat edges and clear outlines; Gram-positive; the bacterial morphology is spherical or oval, relatively uniform in size, with a diameter of 0.6–2.0 μm, arranged in pairs or short chains; the 16S rDNA sequencing fragment, as shown in SEQ ID NO:3, has 100% sequence similarity to *Enterococcus faecalis*, thus identifying it as *Enterococcus faecalis* and naming it *Enterococcus faecalis* Ef-13.

[0021] The Enterococcus faecalis Ef-13 described in this application is similar to Enterococcus faecium, which also belongs to the genus Enterococcus. Enterococcus faecalis Compared to the previous version, it has the following differences: 1) Different safety risks: Enterococcus faecalis usually carries more virulence factors (such as hemolysin and gelatinase), has a high clinical isolation rate, and is excluded from the Qualified Presumption of Safety (QPS) list by the European Food Safety Authority (EFSA); while Enterococcus faecalis has relatively fewer natural virulence factors, and certain strains (such as food-derived strains) have passed QPS certification, resulting in better safety evaluation.

[0022] 2) Risk of drug resistance transmission: Enterococcus faecalis has greater genomic plasticity and is more likely to acquire drug resistance genes through horizontal gene transfer, while the Enterococcus faecalis screened in this invention shows more stability in drug resistance gene screening.

[0023] The present invention also provides a biological agent comprising Enterococcus faecalis Ef-13 as described in the above technical solution.

[0024] In one embodiment, the biological agent comprises fermentation broth and / or bacterial suspension of Enterococcus faecalis Ef-13. In another embodiment, the effective viable count of Enterococcus faecalis Ef-13 in the biological agent is ≥1×10⁻⁶. 8 CFU / mL, further increased to 1×10 7 CFU / mL ~ 1×10 9 CFU / mL.

[0025] The present invention also provides a method for preparing the biological agent described in the above technical solution, comprising the following steps: inoculating Enterococcus faecalis Ef-13 into a fermentation medium and performing shaking culture to obtain the biological agent.

[0026] In one embodiment, the present invention involves inoculating Enterococcus faecalis Ef-13 onto MRS solid medium for activation; the activation temperature is 35-39°C, and more preferably 37°C; the activation time is 18-24 h, and more preferably 20-22 h.

[0027] After activation, as one embodiment, a single colony is picked and inoculated into MRS medium for seed culture to obtain a seed culture solution. As one embodiment, the seed culture temperature is 35-39℃, more preferably 37℃; the seed culture is a shaking culture with a shaking speed of 180 r / min; as one embodiment, the seed culture time is 10-14 h, more preferably 12 h.

[0028] After obtaining the seed culture medium, as one embodiment, the present invention inoculates the seed culture medium into a fermentation medium for shaking culture to obtain a fermentation culture medium. As one embodiment, the fermentation medium is MRS medium; the shaking culture temperature is 35-39℃, more specifically 37℃; the shaking culture time is 22-26 h, more specifically 24 h. As one embodiment, when the biological agent is a bacterial suspension, the preparation method further includes solid-liquid separation of the obtained fermentation culture medium and resuspending the precipitate; the specific operation steps are not particularly limited, and conventional bacterial suspension preparation steps in the art can be used.

[0029] The present invention also provides the application of Enterococcus faecalis Ef-13 described in the above technical solution, or the biological agent described in the above technical solution, or the biological agent prepared by the preparation method described in the above technical solution, in the preparation of medicines for the prevention and / or treatment of hyperuricemia.

[0030] This invention also provides the application of the Enterococcus faecalis Ef-13 described in the above-described technical solutions, or the biological agent described in the above-described technical solutions, or the biological agent prepared by the preparation method described in the above-described technical solutions, in the preparation of one or more of the following products: 1) Lowering uric acid; 2) Protect the liver and / or kidneys; 3) Regulates gut microbiota; 4) Anti-pathogenic bacteria.

[0031] In one embodiment, the pathogenic bacteria include one or more of Staphylococcus aureus, Escherichia coli, and Salmonella.

[0032] This invention also provides a medicament for the prevention and / or treatment of hyperuricemia, the active ingredient of which includes Enterococcus faecalis Ef-13 as described in the above-described technical solution, or the biological agent as described in the above-described technical solution, or the biological agent prepared by the preparation method described in the above-described technical solution. As one embodiment, the dosage form of the medicament is an oral preparation, and the specific form is not particularly limited; it can be conventionally set according to needs.

[0033] This invention also provides the application of Enterococcus faecalis Ef-13, the biological agent, or the preparation method described in the above-described technical solutions, or the drug described in the above-described technical solutions, in the preparation of animal feed additives and / or animal feed. As one embodiment, the animals include poultry, further including domestic poultry and / or wild birds, and further including but not limited to chickens, ducks, geese, or turkeys. As one embodiment, the application is performed by directly feeding the Enterococcus faecalis Ef-13, the biological agent, or the drug orally at a dosage of 6 mL / kg; or by mixing the Enterococcus faecalis Ef-13, the biological agent, or the drug into the basal diet at a dosage of 10% of the basal diet. Feeding the Enterococcus faecalis Ef-13, the biological agent, or the drug directly degrades uric acid and enhances intestinal excretion, without altering the feed's nutritional structure and avoiding the side effects of chemical drugs, thus specifically solving the problems of "nutritional conflict, significant side effects, and lack of coverage of the intestinal pathway" in existing technologies.

[0034] The *Enterococcus faecium* Ef-13 described in this invention can directly degrade intestinal uric acid and regulate the expression of key purine metabolism proteins (XOD, UOX, etc.) to reduce uric acid production. Simultaneously, it enriches beneficial bacteria such as *Ackermania myxophilus*, inhibits the proliferation of harmful bacteria, and alleviates liver and kidney inflammation and damage. This addresses the core issues of "significant side effects and insufficient synergistic regulation," achieving safe and efficient prevention and treatment of avian hyperuricemia.

[0035] Experiments have revealed that, compared with existing technologies, the advantages of the Enterococcus faecalis Ef-13 described in this invention are specifically manifested as follows: 1) Highly targeted and efficient uric acid reduction: Enterococcus faecium Ef-13 achieved a uric acid degradation rate of 77.41% in vitro over 12 hours. In in vivo experiments, the serum uric acid level of 21-day-old chicks decreased to 178.65 μmol / L, a reduction of more than 45% compared to the model group, and was superior to commercially available positive control products such as Xunsuanqing. The core reason is that Enterococcus faecium Ef-13 can express key enzymes in the purine metabolism pathway, which can target and inhibit the activity of the rate-limiting enzyme in uric acid production, guide the conversion of purine metabolism to non-uric acid pathways, and directly degrade intestinal uric acid, thereby strengthening the intestinal excretion pathway (accounting for 30% of the total excretion). 2) Significant liver and kidney protection with high safety: Enterococcus faecalis Ef-13 does not cause hemolysis, has no adverse effects on poultry immune organs, can improve serum liver damage-related indicators by more than 30%, and kidney function indicators (CRE, BUN) are close to those of the healthy control group. It can also alleviate liver and kidney inflammation and fibrosis induced by hyperuricemia and avoid the liver and kidney toxicity of chemical drugs. 3) Outstanding ability to regulate gut microbiota: It can significantly enrich beneficial bacteria such as Akkermansia myxophilus, increasing the proportion of intestinal verrucous bacteria from 0.20% to 3.53%, correcting the imbalance of "decreased Bacteroidetes and increased Firmicutes", and enhancing the stability of the body's purine metabolism. This effect comes from the synergistic interaction between the strain and its metabolites and the gut microbiota. 4) Excellent probiotic properties and suitable for breeding scenarios: The strain is acid and bile salt resistant (survival rate of 14.16% in 3 hours at pH 4.0 and 43.87% in 0.3% bile salt environment), and the inhibition zone diameter of pathogenic bacteria such as Salmonella, Staphylococcus aureus and Escherichia coli reaches 17.30~22.80 mm; culture only requires conventional MRS medium and microbial equipment, can be refrigerated at 4℃, and can be directly administered or mixed with feed when applied. No special preparation processing is required. It is suitable for poultry of all ages, and the operation is simple and the cost is controllable.

[0036] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1 Isolation and screening of uric acid-degrading strains 1. Materials 1.1 Experimental Animals 1-day-old broiler chicks (weighing 35-38 g, purchased from Zhengzhou Ruixiang Poultry Co., Ltd.)

[0038] 1.2 Source of strain isolation samples Plant-derived fermented sample: Fermented tea. The preparation method is as follows: by weight percentage, mix 1% black tea, 5% yellow rock sugar and 94% water, boil and let cool. Add the previously fermented tea and ferment at room temperature for 3-5 days to obtain fermented tea.

[0039] 1.3 Culture Media and Reagents Uric acid culture medium: magnesium sulfate 0.5 g / L, sodium chloride 0.1 g / L, dipotassium hydrogen phosphate 0.5 g / L, potassium dihydrogen phosphate 0.5 g / L, uric acid 2 g / L and agar 20 g / L; Uric acid liquid culture medium: The formula is the same as that of uric acid medium, but without agar; LB medium: NaCl 10.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L. For solid medium, add 2% agar powder, sterilize at 121℃ for 20 min, and use as is.

[0040] 2. Test Methods 2.1 Initial screening and isolation / purification of uric acid-degrading strains in vitro Fermented tea was used as a sample, diluted with physiological saline, and then the solution was evenly spread on a solid culture medium with uric acid as the sole carbon and nitrogen source (final uric acid concentration of 11 mmol / L). The medium was cultured at 37°C for 48 h, and the strains with good growth were labeled and purified.

[0041] 2.2 Secondary screening of the strain's ability to degrade uric acid in vitro The initially screened strains were inoculated into LB liquid medium and placed in a constant temperature shaker at 37℃ and 180 r / min for 12 h for overnight culture. 1 mL of the cultured bacterial solution was transferred to a 1.5 mL sterile centrifuge tube and centrifuged at 8000 r / min for 5 min. After centrifugation, the supernatant was discarded, and the bacterial culture was resuspended in uric acid liquid medium. Uric acid liquid medium without inoculation was used as a blank control. The culture was incubated at 37℃ and 180 r / min for 12 h, and the remaining uric acid content was measured. The uric acid degradation rate (%) was calculated as (Cblank - Csample) / Cblank × 100%, where C represents the uric acid (UA) concentration.

[0042] 2.3 In vivo screening of highly efficient uric acid-lowering strains Sixty one-day-old chicks were randomly divided into four groups according to their weight: control group, model group, positive drug group, and strain intervention group (10 groups), with 15 chicks in each group. The experimental period was 21 days. The detailed treatment methods are shown in Table 1. In Table 1, LB represents LB liquid culture medium, and Xun Suan Qing is Nutrition Care Xun Suan Qing probiotic (NC Xun Suan Qing probiotic), which was purchased from Taobao. The same applies below and will not be described in detail.

[0043] Table 1. In vivo screening grouping of highly efficient uric acid-lowering strains

[0044] 3. Experimental Results 3.1 Initial screening of uric acid-degrading strains Using uric acid as the sole nitrogen and carbon source, a uric acid-degrading bacterial strain screening medium was used as the uric acid solid medium. The samples were inoculated and incubated at 37°C for 48 hours. The presence or absence of colonies was observed. Figure 1 As shown, a total of 13 strains capable of degrading and utilizing uric acid were screened.

[0045] 3.2 Rescreening of strains' ability to degrade uric acid Screening for uricase activity was conducted on strains that could initially grow on uric acid medium. Two out of 13 strains exhibited uricase oxidase activity, forming a distinct clear zone around the strain. The results are as follows: Figure 2 As shown, the H / C values ​​of strains Ef-13 and Ef-12 are 5.38 and 3.25, respectively.

[0046] The uric acid-lowering ability of the two uric acid oxidase-producing strains was determined. After co-incubation with uric acid for 12 h, the degradation rates of Ef-13 and Ef-12 reached 77.41% and 59.69%, respectively, as shown in Table 2. Finally, strain Ef-13 was selected for subsequent experiments.

[0047] Table 2. Uric acid degradation capacity of bacterial cells (1 mL) over 12 h

[0048] 3.3 Effects of uric acid-degrading strains on serum uric acid in chicks The results are shown in Table 3. The hyperuricemia model was effectively constructed (uric acid levels in the model group were significantly higher than those in the control group at all time points, and approximately 1.45 times higher at 21 days compared to the control group); The uric acid levels in the intervention group were significantly lower than those in the model group. Among them, the uric acid-lowering effect of the fermented tea-derived Ef-13 strain was 178.65 μmol / L after 21 days, which was lower than that in the normal control group and better than that of the positive control drug, oxalic acid.

[0049] Table 3 Serum uric acid levels in chicks (μmol / L, x̄±s, n=5)

[0050] Note: To compare with the model group.

[0051] Example 2 Isolation, identification and probiotic properties of Enterococcus faecalis Ef-13 1. Materials LB medium, uric acid-only carbon and nitrogen source medium (uric acid medium), drug sensitivity test medium (MH agar) and MRS medium; 16S rRNA sequencing reagents: bacterial genomic DNA extraction kit, PCR amplification kit (including Taq enzyme, dNTPs, buffer, etc.), universal primers (27F / 1492R), agarose, nucleic acid dyes; Acid and bile salt resistance test reagents: 0.2 mol / L HCl solution (simulating gastric acid environment, pH 2.0~3.0), porcine bile salts (preparing 0.1% and 0.3% bile salt solutions to simulate intestinal bile salt environment), phosphate buffer (PBS, used for bacterial culture pH adjustment and washing); Antibacterial test reagents: sterile Oxford cups, sterile physiological saline, indicator bacterial suspension (0.5 McFarland turbidity), used for the determination of inhibition zones by agar diffusion method; Antibiotic susceptibility testing discs: Commonly used antibiotic susceptibility testing discs for penicillin, streptomycin, tetracycline, gentamicin, etc., are used for detecting the antibiotic susceptibility of bacterial strains.

[0052] 2. Test Methods 2.1 Morphological observation The Ef-13 strain was cultured on MRS solid plates using the streak plating method and incubated upside down at 37°C for 24 h. Its morphological characteristics were then observed and recorded. The morphology of the strain was observed under a microscope using the Gram staining method.

[0053] 2.2 Molecular biological identification The Ef-13 strain was inoculated into MRS medium and cultured at 37℃ and 200 r / min for 24 h. Genomic DNA of the strain was obtained using a bacterial genomic DNA extraction kit. Using this DNA as a template, PCR amplification was performed using universal primers 27 (5'-AGAGTTTGATCCTGGCTAG-3', SEQ ID NO:1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO:2). The PCR amplification system (50 μL) consisted of: 25 μL BGI2×Super PCR Mix, 1 μL template DNA, 1 μL forward primer, 1 μL reverse primer, and 22 μL dd H2O. The amplification conditions were: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 1 min, repeated 30 times, followed by a final extension at 72℃ for 5 min. The PCR products were identified by DNA gel electrophoresis and then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0054] 2.3 Determination of growth curve and acid production capacity curve The isolated strain was inoculated into MRS medium at a 2% inoculum and incubated at 37°C for 24 h at 150 r / min, with samples taken every 2 h. The absorbance and pH of the samples were measured using a UV-Vis spectrophotometer at 600 nm. The results were recorded, and growth curves and acid production curves were plotted.

[0055] 2.4 Antibacterial test The antibacterial activity of strain Ef-13 was determined using *Escherichia coli*, *Staphylococcus aureus*, and *Salmonella* as research subjects. The bacterial suspensions of the three pathogens were evenly spread onto LB agar plates and labeled. The strains were incubated in MRS medium at 37°C and 200 rpm for 24 h, then centrifuged at 12000 rpm for 1 min, and the supernatant was collected. 150 μL of the supernatant was added to LB agar plates using the perforation method, and after incubation at 37°C for 24 h, the diameter of the inhibition zone was recorded.

[0056] 2.5 Bile Salt Tolerance Test In MRS medium containing 0%, 0.1%, 0.2%, and 0.3% porcine bile salts (sterilized at 121℃ for 15 min and then cooled), Ef-13 bacterial suspension was inoculated at a 10% inoculum. With medium without porcine bile salts as a control, the viable bacterial counts at each concentration and time point were determined by plate counting method after incubation at 37℃ for 0, 1, and 3 h, and the survival rate was calculated.

[0057] Survival rate = (Number of viable bacteria in different treatments / Initial number of viable bacteria) × 100% 2.6 Acid Resistance Test Using the same method, Ef-13 strain was inoculated into MRS medium with different pH values ​​(2.0, 2.5, 3.0, 4.0) at an inoculation rate of 10%. After incubation at 37°C for 0, 1, and 3 h, the viable count of each concentration and time period was determined by plate counting method and the survival rate was calculated.

[0058] 2.7 Drug sensitivity testing The diameter of the inhibition zone of commonly used antibiotics on isolated strains was determined by the disk diffusion method (KB method) to assess their resistance.

[0059] 2.8 Hemolytic test To evaluate the safety of strain Ef-13, strain Ef-13 was streaked onto blood agar plates and incubated statically in a constant temperature incubator (37℃, 24 h). Staphylococcus aureus was used as a positive control strain to observe whether hemolysis occurred.

[0060] 3. Test Results 3.1 Morphological characteristics of strain Ef-13 Ef-13 colonies are white, usually round, with relatively neat edges and clear outlines. Figure 3 (A). Gram staining morphological characteristics: Gram-positive, the bacteria are spherical or oval in shape, relatively uniform in size, with a diameter of about 0.6~2.0 micrometers, and usually arranged in pairs or short chains. Figure 3 (B)

[0061] 3.2 Molecular biological identification results of strain Ef-13 Depend on Figure 4

[0062] A BLAST homology search was performed in the NCBI's GenBank database to compare with... Enterococcus faecium The 16S rDNA sequence similarity was 100%. A phylogenetic tree was constructed using the MEGA 11.0 neighbor-joining method. Figure 5 It can be seen that strain Ef-13 clustered with Enterococcus faecalis in the same branch, thus confirming strain Ef-13 as Enterococcus faecalis. Figure 5 The number 13 indicates the Ef-13 strain.

[0063] 3.3 Determination of growth curve and acid production capacity curve Enterococcus faecalis Ef-13 OD at 0-4 h 600 The slow increase in OD value indicates that the bacteria are in a delayed adaptation period to the environment, starting from around 4 hours and lasting until around 8-12 hours. 600 The value rose rapidly, showing a clear logarithmic growth trend. After 12-16 hours, OD... 600 The growth rate of the value gradually slowed down and entered a stable period, such as Figure 6 As shown.

[0064] Enterococcus faecalis ( Enterococcus faecium During the cultivation process, initially (0 h), the pH was 6.44, which was in the near-neutral range. As the cultivation time increased, the pH showed a continuous decreasing trend: the decrease was rapid in the first 18 h, gradually dropping from around 6.44 to approximately 4.27; after 18 h, the decrease slowed, and the pH remained relatively stable at around 4.14 in the subsequent 18–36 h, which was in the acidic range (e.g., ...). Figure 7 (As shown).

[0065] 3.4 Antibacterial effect of Enterococcus faecalis Ef-13 against Salmonella, Staphylococcus aureus, and Escherichia coli Enterococcus faecalis Ef-13 showed varying degrees of inhibitory effects against three pathogenic bacteria. Figure 8 The left, middle, and right figures represent the inhibition zones of Staphylococcus aureus, Escherichia coli, and Salmonella, respectively. The diameters of the inhibition zones against Staphylococcus aureus, Escherichia coli, and Salmonella were 18.00±0.05 mm, 17.30±0.03 mm, and 22.80±0.08 mm, respectively. The inhibition against Salmonella was the strongest, and preliminary analysis suggests that the inhibitory effect may be related to the lactic acid content produced by the strain.

[0066] 3.5 Effect of bile salt concentration on the survival rate of Enterococcus faecalis Ef-13 Table 4 shows that the initial colony-forming units of Enterococcus faecalis Ef-13 were 8.32 × 10⁻⁶. 8At a CFU / mL level and a bile salt concentration of 0.0% (control group), the colony-forming units after 1 h and 3 h of incubation were 8.65 × 10⁻⁶. 8 CFU / mL, 8.52×10 8 At CFU / mL, the survival rates reached 105.8% and 102.40%, indicating that the strains were in a normal proliferation state. As the bile salt concentration increased to 0.1%, 0.2%, and 0.3%, the number of colony-forming units after 1 h and 3 h of incubation gradually decreased, and the survival rate also decreased accordingly. Among them, at a bile salt concentration of 0.3%, the survival rate after 1 h of incubation was 62.62%, and the survival rate after 3 h of incubation was 43.87%, showing the most significant inhibitory effect.

[0067] Table 4. Determination of bile salt tolerance of Enterococcus faecalis Ef-13

[0068] 3.6 Effect of pH on the survival rate of Enterococcus faecalis Ef-13 Table 5 shows that in the control group without acid stress, the initial colony count of Enterococcus faecalis Ef-13 was 7.50 × 10⁻⁶. 8 CFU / mL, after incubation for 1 h and 3 h, the colony count increased to 7.60 × 10⁻⁶. 8 8.08×10 8 At CFU / mL, the survival rate exceeded 100%, indicating normal proliferation. However, in acidic environments, the survival rate decreased significantly with decreasing pH and prolonged incubation time. For example, at pH 2, the survival rate was only 7.39% after 1 hour of incubation, dropping to 0.63% after 3 hours, showing a strong inhibitory effect. As the pH increased to 4, the survival rate increased to 54.67% after 1 hour of incubation, and remained at 14.16% after 3 hours, demonstrating a significant enhancement in acid tolerance. These results indicate that the acid tolerance of Enterococcus faecalis Ef-13 is pH- and time-dependent, exhibiting better tolerance in weakly acidic environments (pH 4) and significantly limited survival in strongly acidic environments (pH 2).

[0069] Table 5. Determination of acid resistance of Enterococcus faecalis Ef-13

[0070] 3.7 Results of drug resistance to Enterococcus faecalis Ef-13 The results of the drug susceptibility test of Enterococcus faecalis Ef-13 (Table 6) showed that it was sensitive to a variety of antibiotics, including ceftriaxone, ceftazidime, cefpodoxime, cefazolin, amikacin, kanamycin, gentamicin, and cefaclor (S); moderately sensitive to tetracycline, streptomycin, piperacillin, and cephalexin (I); and resistant to polymyxin B, doxycycline, minocycline, erythromycin, meropenem, vancomycin, penicillin, and ampicillin (R).

[0071] Table 6. Antimicrobial susceptibility test table for Enterococcus faecalis Ef-13

[0072] 3.8 Results of hemolytic activity of Enterococcus faecalis Ef-13 On blood agar plates, there was no obvious clear hemolytic zone around the Enterococcus faecalis Ef-13 colonies, and the red color of the medium did not fade or become clear, indicating that the strain did not exhibit hemolysis under these experimental conditions. In contrast, the control strain Staphylococcus aureus ATCC25923 showed hemolysis, exhibiting β-hemolysis. Figure 9 The left image shows the hemolytic results of Enterococcus faecalis Ef-13, and the right image shows the hemolytic results of Staphylococcus aureus.

[0073] Example 3 Genetic analysis of Enterococcus faecalis Ef-13 1. Materials The Enterococcus faecalis Ef-13 strain used in the experiment was isolated from fermented tea in our laboratory. The whole genome sequencing analysis software and major databases for Enterococcus faecalis Ef-13 are shown in Table 7.

[0074] Table 7. Whole Genome Sequencing Analysis Software

[0075] 2. Test Methods 2.1 Sample preparation and sequencing Enterococcus faecalis Ef-13 was activated and cultured for two generations in MRS medium at an inoculum of 2% (v / v) to the logarithmic phase. The cells were collected by centrifugation at 10,000 rpm / min for 5 min. The bacterial samples were sent to Shanghai Lingen Biotechnology Co., Ltd. for whole-genome sequencing. The DNA from the samples was jointly sequenced using an Illumina NovaSeq 6000 sequencing platform and a PacBio Sequence II sequencing platform.

[0076] 2.2 Genome Assembly and Analysis The genome assembly and analysis process and the software used are shown in Table 8 below.

[0077] Table 8. Whole Genome Sequencing Analysis Software

[0078] 2.3 Gene Function Annotation The protein sequence of the predicted gene is compared with general databases such as NR, Swiss-Prot, eggNOG, KEGG, and GO, as well as proprietary databases such as CARD, CAZy, and VFDB, to obtain the annotation information of the predicted gene.

[0079] 2.4 Genome-wide mapping Genome loop maps provide a clear and comprehensive view of genome annotation information, such as gene distribution on the DNA positive and negative strands, GC content, functional classification, and homology. This embodiment uses the Rcirclize package for genome loop mapping.

[0080] 3. Test Results 3.1 Statistical Analysis of TGS Third-Generation Sequencing Data Enterococcus faecalis Ef-13 was sequenced by PacBio, and detailed statistics of the data are shown in Table 9. The N50 value exceeded the average read length, meeting the standards of third-generation sequencing, thus ensuring the accuracy of gene prediction, mining, and annotation.

[0081] Table 9. Statistics of Third-Generation Sequencing Data

[0082] The length distribution of all sequencing reads was used as the main indicator for quality control. Figure 10 The length distribution chart of TGS shows that the data quality of the third-generation sequencing length distribution of this bacterium is excellent: there is a significant long read peak at 20,000 bp, highlighting the core advantages of third-generation sequencing; there are multiple peaks with high density of short reads from 0 to 5,000 bp, which can effectively correct for errors. The overall distribution is smooth and without abnormalities, the sequencing is stable, and the data is free from contamination.

[0083] 3.2 Evaluation of genome assembly results Genome assembly results Figure 11 The results showed that the GC content and average sequencing depth in the genome assembly region exhibited a concentrated and uniform distribution pattern. The GC content and sequencing depth in the core region fluctuated little, and there were very few outliers. This indicates that the genome assembly performed well in terms of GC bias control and sequencing depth uniformity, with low proportions of repetitive sequence interference and assembly gaps, and good overall assembly quality.

[0084] 3.3 Genomic component analysis 3.3.1 Encoding Gene Analysis Table 10 shows that the genome contains 2934 coding genes with a total length of 2457831 bp and an average length of 837 bp. The average density in the genome is 1.027 kb, the GC content in intergenic regions is 38.8%, and the gene length accounts for a high proportion of the entire genome length (86.1%). Furthermore, the number of genes on the positive and negative strands are 1486 and 1448 respectively, indicating a relatively balanced distribution. In summary, this genome is characterized by a very high proportion of coding regions, high gene density, short average gene length, and a balanced distribution of genes on the positive and negative strands, consistent with the typical characteristics of a compact prokaryotic genome with few non-coding sequences.

[0085] Table 10 Gene Information Statistics Table

[0086] 3.3.2 Non-coding RNA Analysis This non-coding RNA (ncRNA) includes tRNA, 5S rRNA, 16S rRNA, 23S rRNA, and other ncRNA types. tRNA is the most numerous (68), rRNA is a core component of the ribosome, and other ncRNAs are involved in regulation. The various types of ncRNAs account for a very low proportion of the total genome, further indicating that this genome is prokaryotic, has a compact structure, and supports gene expression and regulation through multiple ncRNAs (Table 11).

[0087] Table 11 ncRNA Statistics

[0088] 3.3.3 Prephage Sequence Analysis The results (Table 12) show that five prephages were predicted in the host genome, distributed in different genomic locations, with lengths ranging from 16441 bp to 34918 bp. As phage genomes integrated into the bacterial genome, the different lengths of prephages may correspond to different phage types or functional modules. Their dispersed distribution suggests that they may play a role in expanding host genetic diversity, conferring phage resistance, or inducing lysis cycles, and have a potential impact on the host's evolutionary and survival strategies.

[0089] Table 12 Statistical analysis of prophage prediction results

[0090] 3.3.4 Gene function analysis The gene function annotation results (Table 13) show that among the total 2934 proteins, the NR database annotated the most proteins (2785), indicating that the genes have a high degree of homology matching in non-redundant protein databases; the annotations of KEGG (1490) and Swiss (1734) are beneficial for the analysis of metabolic pathways and protein functions; GO (654), CAZy (109), and CARD (82) provide specific functional annotations for gene ontology, carbohydrate active enzymes, and antibiotic resistance genes, respectively.

[0091] Table 13 Statistical Table of Gene Function Annotation

[0092] 3.3.5 COG Database Annotations COG functional classification results ( Figure 12The results indicate that the organism has the largest number of genes with unknown functions (class S, 458), suggesting a large number of gene functions that require further investigation. Genes in categories such as carbohydrate transport and metabolism (class G, 248), transcription (class K, 251), and replication / recombination / repair (class L, 249) are relatively abundant, indicating their active functions in carbon source utilization, transcriptional regulation of genetic information, and maintenance of genome stability. The distribution of genes in categories such as translation and ribosome synthesis (class J, 167), amino acid metabolism (class E, 158), and nucleotide metabolism (class F, 102) supports the basic requirements for protein synthesis and primary metabolism. RNA The number of genes in categories such as processing and modification (Category A), chromatin structure and dynamics (Category B), nuclear structure (Category Y), and cytoskeleton (Category Z) is zero, further supporting its prokaryotic nature in conjunction with the structural characteristics of prokaryotes. The extremely low number of genes related to cell motility (Category N, 20) and extracellular structure (Category W, 1) indicates a lack of complex motility or specialized extracellular structures. Meanwhile, genes in categories such as energy production and conversion (Category C, 77), coenzyme and lipid metabolism (Category H, 56; Category I, 63), and inorganic ion transport (Category P, 124) support basic material and energy metabolism. Genes in categories such as cell wall synthesis (Category M, 134), protein modification (Category O, 57), signal transduction (Category T, 53), and defense mechanisms (Category V, 59) ensure cell structural stability, functional regulation, and environmental adaptation. The presence of a large number of genes with unknown functions also provides research directions for subsequent gene function mining and metabolic network analysis.

[0093] 3.3.6 KEGG Database Annotations Annotation results from the KEGG database of Enterococcus faecalis Ef-13 ( Figure 13 As can be seen, energy metabolism accounts for as high as 50.2%, and metabolic modules such as carbohydrates and amino acids are also prominent, ensuring the efficient circulation of substances and energy in the intestinal environment; genetic information processing functions such as membrane transport (17.8%), transcription, translation and replication repair support the transmembrane transport of substances and genetic stability; modules such as cell growth and death, and prokaryotic community interaction ensure basic life activities, while environmental adaptation and immune system account for a very small proportion, reflecting that in the relatively stable habitat of the intestine, it adopts "efficient metabolism + sound genetic support" as its core survival strategy and can adapt without relying on complex environmental stress or immune mechanisms.

[0094] 3.3.7 Potential Mechanism of Enterococcus faecalis Ef-13 in Lowering Uric Acid in the Purine Metabolism Pathway In the purine metabolism pathway, uric acid is produced from hypoxanthine via xanthine oxidase and other catalytic processes. The red-highlighted parts in the figure represent the purine metabolism pathway catalytic enzymes annotated by strain Ef-13. Ef-13 can regulate the activity of these enzymes (or express them itself) to interfere with the breakdown of purine nucleotides (such as AMP and GMP), guiding their metabolic flow to non-uric acid production pathways. Simultaneously, it can target and inhibit the core rate-limiting enzymes in uric acid production, reducing the conversion of hypoxanthine and xanthine to uric acid. It may also enhance the enzyme activity of the purine salvage synthesis pathway, promoting the reuse of purine bases to reduce uric acid precursors from catabolic metabolism, ultimately lowering the level of uric acid in the body. Figure 14 (As shown).

[0095] 3.3.8 Annotation of Carbohydrate-Related Enzymes The carbohydrate-related enzyme annotation results of the whole genome sequencing of Enterococcus faecalis Ef-13 showed that among the CAZy family involved, the glycoside hydrolase (GH) family had the most members (23 species) and the most predicted genes (58), followed by glycoside transferases (GT) and carbohydrate esterases (CE), indicating that this bacterium has relatively rich enzymatic potential in carbohydrate metabolism, especially in the utilization of glycosides.

[0096] Table 14 Statistical Table of Carbohydrate Active Enzyme Annotations

[0097] 3.3.9 Annotation of antibiotic resistance genes from Figures 15-16 The CARD analysis results show that, in the drug classification statistics (A), Enterococcus faecalis has a large number of resistance genes against β-lactams (15 genes), macrolides (13 genes), and lincosamides (12 genes). At the same time, it also has 5 or more resistance genes in multiple drug categories such as cephalosporins, glycopeptides, and oxazolidinones. In the species origin statistics of resistance genes (B), its resistance genes mainly come from Enterococcus species such as Enterococcus faecalis (14 genes), Enterococcus faecium (13 genes), and Enterococcus davidianus (11 genes), and also involve a small number of other genera.

[0098] 3.3.10 Annotations on the virulence factors of bacterial pathogens The VFDB database is a database specifically used to study pathogenic bacteria, chlamydia, and mycoplasma pathogens. Enterococcus faecalis exhibits high virulence gene consistency (mostly ≥90%), covering functions such as pili-related components, cell wall anchoring proteins, metabolic enzymes, and adhesins. These genes provide the molecular support for its virulence-related phenotypes, including adhesion and colonization (Table 15).

[0099] Statistical analysis of this virulence factor Figure 17As can be seen, adhesion bacteria accounted for the highest proportion at 64.71%, indicating that they play a key role in the adhesion and colonization of host cells; immunomodulation bacteria accounted for 20.59%, indicating that the bacteria can enhance pathogenicity by interfering with the host's immune response; stress survival bacteria accounted for 5.88%, helping it survive in the stressful environment within the host; biofilm, exoenzyme, and regulation bacteria each accounted for 2.94%, which were low proportions.

[0100] Table 15 Enterococcus faecalis virulence genes

[0101] 3.3.11 Genome Circulation Results The genome circle map ( Figures 18-19 As shown in the diagram, from the outside in, the diagram sequentially presents genome size markers, CDS distribution for different COG functional categories on the positive and negative strands, genomic localization of rRNA and tRNA, GC content (red convex and blue concave correspond to higher and lower than the genome-wide average GC content, respectively, with peak values ​​reflecting the difference from the mean), and GC skew values ​​(calculated using the (GC) / (G+C) algorithm; positive values ​​indicate that the positive strand is more inclined to transcribe CDS, and negative values ​​indicate that the negative strand is more inclined to transcribe CDS). Each layer of information system integrates genome structural features, coding region functional patterns, non-coding RNA element layout, base composition heterogeneity, and transcriptional strand bias, providing multi-dimensional visualization support for elucidating its genetic function, evolutionary background, and transcriptional regulatory mechanisms.

[0102] Example 4 Screening for the optimal dose of Enterococcus faecalis Ef-13 to lower uric acid 1. Materials One hundred and eighty one-day-old broiler roosters were cage-raised with temperature and humidity recorded using thermometers and hygrometers throughout the experiment. They had free access to feed and water. The experiment was divided into six groups: a healthy control group, a model group, probiotic (high, medium, and low dose) groups, and a positive control group (Xunsuanqing), with 18 roosters in each group. The detailed treatment methods for each group are shown in Table 16. In Table 16, LB represents LB liquid culture medium.

[0103] Table 16 Animal Experiment Treatments and Grouping

[0104] The main reagents used in this embodiment are shown in Table 17.

[0105] Table 17 Reagents and Manufacturers

[0106] 2. Test Methods 2.1 Preparation of Enterococcus faecalis Ef-13 The Ef-13 strain, which was isolated, purified, identified, and preserved in the laboratory, was activated three times in MRS medium. The activated Ef-13 was then added to the MRS medium at 2% (v / v) and cultured at 37°C for 24 h.

[0107] 2.2 Blood and tissue sample collection Twenty-one days later, fresh blood was collected from chicks anesthetized with sodium pentobarbital. After being left at room temperature for 1 hour, the blood was centrifuged (4°C, 3000 rpm) for 15 minutes to obtain serum samples. Liver, kidney, intestinal tissues and contents were placed in cryovials and flash-frozen in liquid nitrogen. Serum and tissue samples were stored at -80°C for subsequent experiments.

[0108] 2.3 Determination of key factors in uric acid metabolism in serum and liver; blood and tissue sample collection Serum uric acid, blood urea nitrogen, creatinine, and xanthine oxidase levels were measured in each group using a biochemical reagent kit. Liver tissue was ground on ice and centrifuged at low speed (8000 rpm, 10 min) to obtain a sample suspension, and the xanthine oxidase content was measured. The relevant experimental procedures were performed according to the kit instructions, with three replicates per well to avoid random errors.

[0109] 2.4 16S rDNA sequencing 2.4.1 Sample DNA Extraction According to EZNA ® The instructions for the Stool DNA kit are as follows: Extract total microbial DNA from cecal contents of 5 chicks in each group. The specific steps are as follows: (1) Sample processing: Take an appropriate amount of cecal contents sample, add the lysis buffer provided by the kit, mix thoroughly, centrifuge, and collect the supernatant; (2) DNA extraction: Follow the kit operation steps to perform lysis, binding, washing, and elution in sequence to finally obtain total microbial DNA; (3) DNA quality detection: Detect the integrity and purity of DNA by 1% agarose gel electrophoresis and observe whether there are obvious DNA bands; (4) DNA quantification: Measure the concentration and purity of DNA using a micro-ultraviolet-visible spectrophotometer.

[0110] 2.4.2 16S rDNA sequencing PCR products were purified using AMPure XT beads to remove primer dimers and other impurities. The purified PCR products were quantified using a Qubit quantitative PCR instrument. Library construction was performed according to the Illumina library construction protocol, and library quality control was conducted using an Agilent 2100 bioanalyzer. Paired-end sequencing (2×250 bp) was performed on the Illumina NovaSeq platform.

[0111] 2.5 Data Analysis The paired-end data obtained from sequencing were split, sequenced, quality-controlled, and chimeric filtered according to barcode information. High-quality data statistics were then performed to obtain a statistical table of valid sample data. Subsequently, length filtering and noise reduction were performed to obtain ASV characteristic sequences and an ASV abundance table. Based on this, row Venn diagram statistics, α-diversity analysis, and β-diversity analysis were conducted. Furthermore, species annotation was performed based on the ASV sequence files (confidence threshold of 0.7), and further diversity analysis, species classification annotation, and differential analysis were conducted based on the final ASV characteristic table and characteristic sequences.

[0112] 3. Test Results 3.1 Effects of Enterococcus faecalis Ef-13 on the growth performance of chicks The experimental results (Table 18) showed that there was no significant difference in the initial weight of chicks among the groups. p >0.05). Compared with the control group, the body weight of the 21-day-old children in the Xunsuanqing group (131.01g) and the weight of the medium-dose Ef-13 (155.67g) were lower, while the weights of the other groups were higher than those in the control group.

[0113] Table 18 Growth performance of chicks (n=18, x̄±s)

[0114] 3.2 Effects of Enterococcus faecalis Ef-13 on serum uric acid in chicks The experimental results (Table 19) showed that there was no difference in serum uric acid among the chicks in each group at day 1; in the subsequent stage, the serum uric acid in the model group was significantly higher than that in the control group. The high-dose and medium-dose Ef-13 groups could significantly reduce serum uric acid levels at day 7, which was better than the Xunsuanqing product, and maintained a stable level for 21 days.

[0115] Table 19 Serum uric acid in chicks (μmol / L, x̄±s, n=5)

[0116] Note: For comparison with the model group, # indicates comparison with the control group.

[0117] 3.3 Effects of Enterococcus faecalis Ef-13 on renal function and serum XOD in chicks This experiment investigated the effects of Enterococcus faecalis Ef-13 on renal function (CRE, BUN) and serum XOD in chicks: The levels of CRE, BUN, and XOD in the model group were significantly higher than those in the control group (#), indicating increased renal function-related indicators and enhanced XOD activity in the model group chicks; compared with the model group, CRE, BUN, and XOD levels in the cyclophosphamide group and all Ef-13 dosage groups were decreased ( Among them, the indicators of the Xunsuanqing group decreased more significantly, the BUN of the high-dose Ef-13 group was closer to that of the control group, and the XOD of each dose group was significantly lower than that of the model group. Overall, the high-dose Ef-13 group had a better regulatory effect on the renal function and XOD of chicks (Table 20).

[0118] Table 20 Kidney function and serum XOD in chicks (n=5, x̄±s)

[0119] Note: For comparison with the model group, # indicates comparison with the control group.

[0120] 3.4 Effects of Enterococcus faecalis Ef-13 on Organ Indices in Chicks The immune organ indices in Table 21 show no significant differences among the groups, indicating that Enterococcus faecalis Ef-13 has no adverse effects on immune organs; the digestive organ indices in Table 22 show that the gizzard index was lower in the model group, while the gizzard index in the oxalic acid and high-dose Ef-13 groups was significantly higher than that in the model group. p >0.05, p >0.01).

[0121] Table 21 Immune organ index of chicks (n=5, x̄±s, g / kg)

[0122] Table 22 Digestive organ index of chicks (n=5, x̄±s, g / kg)

[0123] Note: For comparison with the model group, # indicates comparison with the control group.

[0124] 3.5 Effects of Enterococcus faecalis Ef-13 on the gut microbiota 3.5.1 Analysis of bacterial community abundance and α-diversity like Figure 20As shown in the dilution curves, the richness of the gut microbiota in each sample increases with increasing sequencing depth. The curve flattens out when the number of reads reaches 10,000, indicating that the sequencing depth at this point essentially covers all species in the sample. OTU clustering analysis was used to assess the degree of difference between the groups; compared to the control group, the number of OTUs in each group decreased to varying degrees. Simpson and Shanon indices showed no significant difference in gut microbiota diversity.

[0125] 3.5.2 β-diversity analysis like Figure 21 As shown, according to principal coordinate analysis (PCoA) and hierarchical cluster analysis, the colony composition of the control group, Ef-13 group and Xunsuanqing group all deviated significantly from that of the model group, with the Ef-13 group and Xunsuanqing group samples showing more similar colony composition.

[0126] 3.5.3 Relative abundance analysis of species at the phylum level From gate-level data ( Figure 22 In the model group, compared to the control group, the proportion of Bacteroidetes decreased from 62.87% to 47.78%, while the proportion of Firmicutes increased from 35.57% to 49.45%. The F / B ratio (Firmite / Bacteroidetes) increased from approximately 0.57% to approximately 1.03%, and the proportion of Proteobacteria also slightly increased, successfully inducing a microbial imbalance characterized by a decrease in Bacteroidetes, an increase in Firmicutes, and a slight increase in Proteobacteria. Compared to the model group, the proportion of Verrucous Microbes in the Ef-13 group significantly increased from 0.20% to 3.53%, and the proportion of Desulfobacterium thermophilum in the Xunsuanqing group increased substantially from 0.33% to 2.49%.

[0127] 3.5.4 Species relative abundance analysis at the genus level Subordinate level ( Figure 23 Compared with the control group, the relative abundance of *Cryptospira* spp. in the model group decreased significantly, while the abundance of *Brutella*, *Lactobacillus*, and *Roseidon* spp. increased. Compared with the model group, the abundance of Ef-13 and positive products *Clostridium*, *Nil*, and *Sphingospora* spp. in the intervention group increased, while the abundance of *Cryptospira*, *Brutella*, and *Lactobacillus* spp. decreased.

[0128] 3.5.5 Horizontal Species Relative Abundance Analysis From the seed level ( Figure 24Compared with the control group, the abundance of beneficial bacteria such as *Dorefus stanneri* and *Cryptospira marines* in the model group was significantly decreased, while the abundance of Bacteroides was abnormally increased, indicating a bacterial imbalance. In the Ef-l3 group, the abundance of beneficial bacteria such as *Ackermania myxophilus* (3.52%) and *N. nielssenii* (4.43%) increased, while the abundance of a certain type of Bacteroides (22.39%) remained high. In the XSQ group, the abundance of *Bacteroides polymorpha* (17.80%) and *N. nielssenii* (5.16%) recovered somewhat, while *Ackermania myxophilus* was only 0.01%. Overall, the intervention showed some improvement, but the effects were uneven.

[0129] 3.5.6 Analysis of Differential Strains in Gut Microbiota at the Species Level Results of strain differential analysis at the species level ( Figure 25 It can be seen that in the Ef-13 intervention group, *Akermansia myxophilus* (*Akermansia spp.*) was significantly enriched. Akkermansia muciniphila ) and succinic acid-producing anaerobic bacteria ( Ercella succinogenes This may be related to the uric acid-lowering effect: the former, as beneficial gut bacteria, can indirectly improve uric acid metabolism disorders by regulating intestinal barrier function and reducing endotoxemia; the latter can participate in the microenvironment regulation related to purine metabolism through succinic acid produced by metabolic pathways; while the differentially expressed strains in the control group and the commercially available product XSQ group (such as...) Sodaliphilus pleiomorphus , Bacteroides luti There are few clear reports on its function in regulating uric acid.

[0130] In summary, Enterococcus faecalis Ef-13 may exhibit uric acid-lowering therapeutic effects by enriching beneficial gut bacteria with metabolic regulatory functions.

[0131] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Enterococcus faecalis ( Enterococcus faecium The Enterococcus faecalis Ef-13 has the accession number CGMCC No. 37038.

2. A biological agent, characterized in that, Includes Enterococcus faecalis Ef-13 as described in claim 1.

3. The biological agent according to claim 2, characterized in that, This includes fermentation broth and / or bacterial suspension of Enterococcus faecalis Ef-13.

4. The biological agent according to claim 1 or 2, characterized in that, The effective viable count of Enterococcus faecalis Ef-13 in the biological agent is ≥1×10⁻⁶. 8 CFU / mL.

5. A method for preparing the biological agent according to any one of claims 2 to 4, comprising the following steps: inoculating Enterococcus faecalis Ef-13 into a fermentation medium and performing shaking culture to obtain the biological agent.

6. The use of the Enterococcus faecalis Ef-13 of claim 1, or the biological agent of any one of claims 2 to 4, or the biological agent prepared by the preparation method of claim 5, in the preparation of a medicine for the prevention and / or treatment of hyperuricemia.

7. The use of the Enterococcus faecalis Ef-13 according to claim 1, or the biological agent according to any one of claims 2 to 4, or the biological agent prepared by the preparation method according to claim 5, in the preparation of one or more of the following products: 1) Lowering uric acid; 2) Protect the liver and / or kidneys; 3) Regulates gut microbiota; 4) Anti-pathogenic bacteria.

8. The application according to claim 7, characterized in that, The pathogens include Staphylococcus aureus (Staphylococcus aureus) Staphylococcus aureus ), Escherichia coli ( Escherichia coli ) and Salmonella ( Salmonella One or more of the following.

9. A medicine for the prevention and / or treatment of hyperuricemia, characterized in that, The active ingredient includes Enterococcus faecalis Ef-13 as described in claim 1, or the biological agent as described in any one of claims 2 to 4, or the biological agent prepared by the preparation method described in claim 5.

10. The use of the Enterococcus faecalis Ef-13 of claim 1, or the biological agent of any one of claims 2 to 4, or the biological agent prepared by the preparation method of claim 5, or the drug of claim 9, in the preparation of animal feed additives and / or animal feed.

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