Enterococcus durans BHDY002 and application thereof in preparation of products for promoting animal growth

Abstract

The invention discloses enterococcus durans BHDY002 and application thereof in preparation of products for promoting animal growth, and belongs to the technical field of microorganisms. The strain is preserved in the Guangdong Microbial Culture Collection Center on November 21, 2025, the preservation address is the 5th floor of the building 59, No.100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 67336. According to the invention, enterococcus durans with excellent tolerance is successfully screened from excrement samples of wild birds of wild angoraeae, and the probiotic potential and safety of enterococcus durans are verified through morphological observation, physiological and biochemical characteristic analysis, molecular biological identification and comprehensive evaluation of probiotic performance. The research widens the screening source of high-quality probiotic resources, widens the understanding of biological functions of enterococcus durans, and provides theoretical basis and strain resources for developing probiotic preparations for promoting animal growth, antagonizing pathogenic bacteria and regulating intestinal microecology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a strain of Enterococcus durans BHDY002 and its application in preparing a product for promoting animal growth. BACKGROUND

[0002] In recent years, with the problem of antibiotic abuse becoming increasingly serious, the global spread of drug-resistant bacteria has become one of the important challenges in the field of public health. Especially the emergence of multi-drug resistant strains has put forward higher requirements for clinical treatment. Under this background, screening and studying new strains with special biological characteristics not only helps to expand our understanding of microbial diversity and ecological adaptability, but also provides new ideas for developing potential biological resources.

[0003] The research and development of probiotics as antibiotic substitutes have attracted much attention, and significant progress has been made in related research. Probiotics can regulate the balance of intestinal microecology, maintain intestinal health, enhance immune function and resist pathogen infection, and exhibit multiple probiotic functions. Enterococcus is a type of gram-positive coccus, often arranged in single, pairs or short chains. Some Enterococcus strains have excellent probiotic properties and are widely distributed in the intestines of humans and animals, can enhance the immune function of the host, and optimize the structure of intestinal flora. In addition, Enterococcus can secrete organic acids, hydrogen peroxide and various enterococcin, compete for nutrients with pathogenic bacteria in the intestinal tract, and exhibit obvious antagonistic effect on common pathogenic bacteria such as Escherichia coli and Salmonella, thus showing good application potential in the prevention and control of livestock diseases. Enterococcus also has strong self-aggregation and co-aggregation ability, which can interfere with the adhesion of pathogens to the intestinal epithelium, and help to maintain the health of the body. Studies have shown that probiotic Enterococcus not only can enhance the immune response of the host, but also can improve high cholesterol-related indicators, relieve clinical symptoms such as diarrhea and intestinal stress syndrome.

[0004] Enterococcus durans is a gram-positive lactic acid bacteria belonging to Firmicutes, Bacilli, Lactobacillales, Enterococcaceae and Enterococcus, which is a facultative anaerobe and is the same species as Enterococcus faecalis. It widely exists in different hosts, environments, food and gastrointestinal tract in nature. As an important intestinal symbiotic bacteria, Enterococcus durans is widely distributed in nature and shows significant dominance in certain specific environments. Studies have shown that Enterococcus durans has strong environmental adaptability and can survive in extreme conditions, such as acid and bile salt resistance, which makes it an ideal model for studying microbial ecology and resistance mechanisms. In addition, the potential application value of Enterococcus durans in the food industry, medical field and animal health management has also attracted widespread attention. Research has found that some Enterococcus durans have similar biological characteristics and physiological functions to Enterococcus faecalis. Not only do they have the functions of acid and bile salt resistance, cholesterol absorption, etc., but also some metabolites such as lactic acid and butyric acid produced in the host intestinal tract can provide energy for the intestinal tract and maintain the intestinal barrier, thereby effectively regulating the host intestinal health and immune function. In addition, it does not express obvious pathogenic genes and virulence genes, and is sensitive to most antibiotics. Therefore, compared with Enterococcus faecalis, Enterococcus durans not only has good probiotic properties, but also has high safety, and is a very promising substitute for Enterococcus faecalis probiotics.

[0005] At present, there are still many gaps in the research on Enterococcus durans, especially the systematic study of its distribution characteristics, biological characteristics and functional development is relatively less. In particular, the isolation and identification of Enterococcus durans in wild animal intestinal microbial community have not been fully valued. As an important part of the global ecosystem, migratory birds pass through various ecological environments during migration, and their intestinal flora may have screened more resilient strains during long-term coevolution to adapt to the diversity of food and potential pathogenic challenges that the host may ingest. The community structure of probiotics in the intestinal tract of wild birds is important background data for assessing their health status, which not only provides a basis for exploring the coevolution relationship between probiotics and hosts, but also lays a foundation for the screening of functional probiotics and related product development. SUMMARY

[0006] The purpose of the present application is to provide a strain of Enterococcus durans BHDY002 and its application in the preparation of products for promoting animal growth, in order to solve the problems existing in the prior art.

[0007] To achieve the above purpose, the present application provides the following solutions. One of the technical solutions of the present application is a strain of Enterococcus durans (BHDY002) which has the characteristics of being resistant to acid and bile salt, and has the function of promoting animal growth. Enterococcus durans) BHDY002, which was preserved in Guangdong Microbial Culture Collection Center on November 21, 2025, and the address is No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, and the preservation number is GDMCC No: 67336.

[0008] The second technical scheme of the present application is a microbial agent, which comprises the Enterococcus durans BHDY002.

[0009] The third technical scheme of the present application is the application of the Enterococcus durans BHDY002 or the microbial agent in the preparation of a product for promoting the growth of animals.

[0010] The fourth technical scheme of the present application is a product for promoting the growth of animals, which comprises the Enterococcus durans BHDY002 or the microbial agent.

[0011] The fifth technical scheme of the present application is the application of the Enterococcus durans BHDY002 or the microbial agent in the preparation of a bacteriostatic agent.

[0012] The sixth technical scheme of the present application is a bacteriostatic agent, which comprises the Enterococcus durans BHDY002 or the microbial agent; and the bacteriostatic agent is used for inhibiting Escherichia coli, Staphylococcus aureus, Salmonella pullorum and Pasteurella multocida.

[0013] Based on the above technical scheme, the present application has the following technical effects: The present application successfully screens the Enterococcus durans with excellent tolerance from wild bird feces samples of Anseriformes and Anas, verifies the probiotic potential and safety through morphological observation, physiological and biochemical property analysis, molecular biology identification and comprehensive evaluation of probiotic performance, broadens the screening source of high-quality probiotic resources, expands the understanding of the biological function of the Enterococcus durans, and provides a theoretical basis and strain resource for developing probiotic preparations for promoting animal growth, antagonizing pathogenic bacteria and regulating intestinal microecology. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is the colony morphology (A), gram staining (B) and gel electrophoresis verification (C) of the isolated bacteria BHDY002.

[0015] Figure 2 It is the growth and acid production curve of BHDY002.

[0016] Figure 3 It is the self-aggregation ability of the isolated bacteria BHDY002 and the co-aggregation ability with pathogenic bacteria. Among them, A: the self-aggregation ability of BHDY002; B: the co-aggregation ability of BHDY002 on Salmonella pullorum, pathogenic Escherichia coli, Pasteurella multocida and Staphylococcus aureus.

[0017] Figure 4 BHDY002 bacteriostatic effect diagram. Among them, A: Escherichia coli; B: Staphylococcus aureus; C: Salmonella; D: Pasteurella.

[0018] Figure 5 BHDY002 drug sensitivity test.

[0019] Figure 6 BHDY002 drug resistance gene detection results.

[0020] Figure 7 BHDY002 hemolytic bacteria isolated from the mouse. Among them, A: BHDY002; B: positive control.

[0021] Figure 8 The effect of intragastric administration of different doses of BHDY002 on the growth performance of mice. Among them, A: the body weight gain rate of mice intragastrically administered with different doses of BHDY002; B: the body weight gain change of mice intragastrically administered with different doses of BHDY002.

[0022] Figure 9 The effect of BHDY002 isolated from mice on organ index. Among them, A: heart index; B: liver index; C: kidney index; D: liver index.

[0023] Figure 10 The effect of different doses of BHDY002 on the immune organ index of mice. Among them, A: spleen index; B: thymus index.

[0024] Figure 11 The effect of two strains of probiotics on the growth performance of mice. Among them, A: the body weight gain rate of mice intragastrically administered with two strains of probiotics; B: the body weight gain change of mice intragastrically administered with two strains of probiotics.

[0025] Figure 12 The mouse intestinal microorganism statistics Veen diagram.

[0026] Figure 13 The relative abundance of bacterial phylum level in the mouse intestine.

[0027] Figure 14 The relative abundance of bacterial genus level in the mouse intestine.

[0028] Figure 15 The relative abundance of bacterial species level in the mouse intestine.

[0029] Figure 16 The mouse intestinal bacterial abundance heat map.

[0030] Figure 17 The sample dilution curve.

[0031] Figure 18This section presents multiple comparisons between groups using the Alpha diversity index. (a): ACE index; (b): Chao1 index; (c): Shannon index; (d): Simpson index.

[0032] Figure 19 For PCoA analysis.

[0033] Figure 20 For ANOSIM analysis. Detailed Implementation

[0034] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0035] This invention provides a strain of durable enterococcus ( Enterococcus durans BHDY002, this strain was deposited on November 21, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67336.

[0036] This invention also provides a microbial inoculant, including the durable enterococcus BHDY002.

[0037] This invention also provides the application of the durable enterococcus BHDY002 or the microbial agent in the preparation of products that promote animal growth.

[0038] This invention also provides a product for promoting animal growth, comprising the durable enterococcus BHDY002 or the microbial agent.

[0039] This invention also provides the application of the durable enterococcus BHDY002 or the microbial agent in the preparation of antibacterial agents.

[0040] In some specific implementations, the antibacterial agent is used to inhibit Escherichia coli, Staphylococcus aureus, Salmonella pullorum, and Pasteurella multocida.

[0041] This invention also provides an antibacterial agent, comprising the durable enterococcus BHDY002 or the microbial agent; the antibacterial agent is used to inhibit Escherichia coli, Staphylococcus aureus, Salmonella pullorum, and Pasteurella multocida.

[0042] Example 1 1. Materials and Methods 1.1 Materials 1.1.1 Samples, test bacteria and experimental animals Eight samples of wild duck feces were collected from Shenzhen Bay, Guangdong Province in December 2024. A small amount of feces was taken with a sterile cotton swab and placed in a 5 mL cryogenic tube containing MRS broth. The samples were then numbered and stored in a 4°C refrigerator for rapid transportation back to the laboratory. Caco-2 cells were kindly provided by Professor Li Youquan of Guangdong Ocean University; Lactobacillus acidophilus (BNCC185342) was kindly provided by Professor Chen Zhibao of Guangdong Ocean University. 5-6 week old BALB / c mice were purchased from Beijing Vito Li Hua Company.

[0043] 1.1.2 Main reagents MRS medium was purchased from Beijing Luqiao Technology Co., Ltd.; MRS broth was purchased from Feijing Biological Technology Co., Ltd.; pig bile salt was purchased from Beijing Solaybao Technology Co., Ltd. Bacterial genomic DNA extraction kit was purchased from Tiangeng Biochemical Technology (Beijing) Co., Ltd.

[0044] 1.2 Method 1.2.1 Isolation, purification and identification of strains After the fecal samples were thoroughly mixed by shaking, gradient dilution was performed (diluted to 10 -4 to 10 -6 ). 0.1 mL of bacterial solution at different dilutions was spread on the surface of MRS agar medium, and incubated at 37°C under anaerobic conditions for 24h. After picking up the smooth, moist round colonies and observing under a microscope, the strains that met the staining characteristics of lactic acid bacteria were purified and cultured.

[0045] The genomic DNA of the isolated bacteria was extracted according to the instructions of the bacterial DNA extraction kit. PCR amplification was performed using bacterial 16S rRNA universal primers (27F (SEQ ID NO. 1): 5'-AGAGTTTGATCCATGGCTCAG-3'; 1541R (SEQ ID NO. 2): 5'-AAGGAGGTGATCCAGCC-3'). The PCR reaction system was 50μL: DNA template 1μL, 10μmol / L upper and lower primers 2μL each, 2×Taq Master Mix 25μL, ddH2O to 50μL. The PCR reaction conditions were as follows: 95°C pre-denaturation for 5min; 95°C denaturation for 45s; 55°C annealing for 45s; 72°C extension for 1min; a total of 35 cycles; 72°C extension for 8min. The amplification products were detected by 1% agarose gel electrophoresis, and were sent to Shenguo Bioengineering Co., Ltd. for sequencing. The sequencing results were analyzed by BLAST on NCBI.

[0046] 1.2.2 Growth curve and acid production curve The isolated strains were subcultured at an inoculation amount of 1%, and the OD600nm absorbance of the bacterial liquid and the pH value of the culture liquid were detected every 2 h. The growth curve and acid production curve were plotted with the culture time of the bacterial liquid as the abscissa and the OD600nm absorbance and pH value as the ordinate.

[0047] 1.2.3 Strain tolerance test 1.2.3.1 Acid and bile salt tolerance test The pH of the MRS meat culture medium was adjusted to 3.0 with 0.1 mol / L HCl. The 0.3% pig bile salt MRS liquid culture medium was prepared. After activation, the bacterial liquid was inoculated into the MRS broth with pH 3.0 and 0.3% pig bile salt at an inoculation amount of 10%, and cultured anaerobically at 37°C for 2 h. The bacterial suspensions at 0 h and 2 h were counted, and the average value was calculated according to the formula to calculate the survival rate.

[0048] Survival rate (%) = N2 / N0 x 100%; In the formula, N2 is the viable count at 2 h; N0 is the viable count at 0 h, and the viable count is expressed in 1 g CFU / mL.

[0049] 1.2.4 Determination of agglutination ability and hydrophobicity of strains The strains were inoculated into MRS broth and cultured anaerobically for 24 h, and the bacterial cells were collected by centrifugation at 8000 r / min for 5 min. After washing with PBS for 2-3 times, the bacterial cells were resuspended in an equal amount of PBS. The absorbance values at 0, 2, 4, 6, 10 and 24 h were measured at a wavelength of 600 nm (OD 600 ) by spectrophotometry, and the self-agglutination rate was calculated according to the following formula: Self-agglutination rate (%) = (A0-A) / A0 x 100%; In the formula, A0 is the absorbance value at the initial time (0 h), and A is the absorbance value at each time point (t).

[0050] The hydrophobicity of the strains was determined by xylene adsorption method. 3 mL of bacterial suspension was added with 1 mL of xylene, and after standing at room temperature for 10 min, it was vortexed for 2 min and then stood at 37°C for 1 h. After the two phases were separated, the water phase was taken to measure the OD 600 , and the hydrophobicity rate was calculated according to the following formula: Hydrophobicity rate (%) = (A0-A1) / A0 x 100%; In the formula, A0 is the initial absorbance value of the bacterial suspension before reaction, and A1 is the absorbance value of the water phase after reaction.

[0051] Further evaluate the ability of the strain to agglutinate pathogenic bacteria. 2 mL of the test bacteria suspension was mixed with 4 strains of pathogenic bacteria suspension in equal volume, and the OD was measured at 0, 2, 4, 6, 10 and 24 h respectively 600 , and the agglutination rate of the test bacteria on the 4 strains of pathogenic bacteria was calculated according to the following formula: Agglutination rate (%) = (1 - A mix / A0) × 100%; In the formula, A0 is the absorbance value of the mixture at 0 h, and A mix is the absorbance value of the mixture at each time point.

[0052] 1.2.5 Determination of bacteriostatic ability Centrifuge the activated bacteria liquid at 8000 r / min for 5 min at 4°C, take the supernatant and filter it through a 0.22 μm filter. Adjust the concentration of the activated E. coli (ATCC43888), S. aureus (ATCC6538), chicken white dysentery Salmonella (ATCC10398), Pasteurella (ATCC19427) indicator bacteria suspension to 1×10 6 CFU / mL with PBS, and spread on LB agar medium. After standing at room temperature for 10-20 min, punch with a 7 mm puncher, and suck 150 μL of the filtrate into the hole. After diffusion at room temperature for 2 h, cultivate anaerobically at 37°C for 4-6 h. When a clear inhibition zone appears, take it out. Measure the diameter of the inhibition zone (mm) with a vernier caliper, and the size of the inhibition zone diameter represents the bacteriostatic activity of the isolated bacteria.

[0053] 1.2.6 Drug sensitivity test Centrifuge the activated bacteria liquid at 8000 r / min for 5 min at 4°C, discard the supernatant and resuspend with PBS, adjust the concentration of the bacteria liquid to 1×10 8 CFU / mL. Dip the bacteria liquid with a sterile cotton swab and spread it evenly on MRS agar plates, stand for 3-5 min, and gently paste the drug sensitivity paper sheet on the agar surface with sterile forceps. Select 10 kinds of antibiotics including penicillins such as penicillin and ampicillin, glycopeptides such as vancomycin, macrolides such as erythromycin, tetracyclines such as tetracycline, fluoroquinolones such as ciprofloxacin, nitrofuran such as furadantin, phenylpropanol such as chloramphenicol, ansamycin such as rifampicin, and phosphomycin. The test results are determined according to the standard of the American Clinical and Laboratory Standards Institute (CLSI).

[0054] 1.2.7 Drug resistance gene detection test The isolated strain was inoculated into MRS broth and incubated anaerobically at 37°C for 24 h. The genomic DNA of the isolated strain was extracted according to the instructions of the bacterial DNA extraction kit. The kanamycin resistance gene, the gentamicin resistance gene, the tetracycline resistance gene, and the vancomycin resistance gene of the isolated strain were amplified by PCR using the extracted DNA as a template and synthesized primers (see Table 1). The PCR reaction system was as follows: 2x Taq Master Mix 12.5 μL, template DNA 1 μL, 10 μmol / L upper and lower primers 1 μL each, and ddH2O to make up to 25 μL.

[0055] Table 1: Drug resistance gene primer sequences

[0056] 1.2.8 Adhesion test of probiotics 1.2.8.1 Labeling of probiotics and preparation of probiotic bacterial suspension The freshly cultured lactic acid bacterial strain suspension was centrifuged (4°C, 8000 rpm, 5 min), washed with sterile PBS three times in succession, and finally resuspended in a FITC solution that had been placed at room temperature for 30 min. The bacterial suspension was incubated at 37°C for 2 h with shaking in the dark, then centrifuged (4°C, 8000 rpm) and washed with PBS three times to remove unbound FITC labeling solution. The bacterial suspension was resuspended in RPMI-1640 cell culture medium and adjusted to a concentration of 2x10 8 CFU / mL. The relative fluorescence intensity value (RFU) of the strain at a wavelength of 485 nm (absorption wavelength) and a wavelength of 530 nm (emission wavelength) was determined and recorded as the pre-adhesion relative fluorescence intensity value R0 of the strain.

[0057] 1.2.8.2 Determination of probiotic adhesion The Caco-2 cells were prepared for the test after being continuously passed for more than 7 generations. The cell concentration was adjusted to 5x10 5 cell / mL per well, and the cell solution was plated in a 24-well cell culture plate for further culture. After the cells in the 24-well plate were cultured to a monolayer (about 24 h), the cell culture medium was discarded, and the cells were washed once with sterile PBS. Then, 0.6 mL of the labeled probiotic bacterial suspension was added to each well, and three replicate wells were set for each strain. The plate was incubated at 37°C in a 5% CO2 incubator for 2 h in the dark. After removal, the culture medium was discarded, and the cells were washed three times with sterile PBS to remove the strains that had not adhered to the cells. Then, 0.3 mL of trypsin was added to each well to digest the cells for 5 min, and the digestion was terminated with RPMI-1640 cell culture medium. The relative fluorescence intensity value (RFU) of the cell suspension was determined under the same wavelength conditions and recorded as the post-adhesion relative fluorescence intensity value R of the strain. The adhesion rate (%) was calculated according to the following formula: Adhesion rate = R / R0 x 100% Wherein, R represents the relative fluorescence value after adhesion, and R0 represents the initial relative fluorescence value.

[0058] 1.2.9 Inhibition of pathogenic bacteria adhesion test 1.2.9.1 Preparation of pathogenic bacteria markers and probiotic bacterial suspensions The activated E. coli and S. aureus indicator bacteria suspension was centrifuged at 4°C, 8000 rpm for 5 min, washed with PBS three times, resuspended in FITC solution, incubated at 37°C for 2 h in the dark, centrifuged at 4°C, 8000 rpm for 5 min, washed with PBS three times, and the unbound FITC marker solution was removed. The bacterial suspension was resuspended in RPMI-1640 cell culture medium and adjusted to a bacterial solution concentration of 2 x 10 8 CFU / mL. Preparation of probiotic bacterial suspensions: After continuous 3 times subculture and activation, the bacterial suspension was collected by centrifugation at 8000 rpm for 5 min at 4°C, washed with sterile PBS three times, and then adjusted to a bacterial solution concentration of 2 x 10 8 CFU / mL with RPMI-1640 cell culture medium.

[0059] 1.2.9.2 Inhibition of pathogenic bacteria adhesion test Competitive test: Set up test and blank control groups, under light-proof conditions, add 0.3 mL of probiotic bacterial suspension and 0.3 mL of labeled pathogenic bacterial suspension to the single layer of Caco-2 cells and mix well, add 0.3 mL of RPMI-1640 cell culture medium and 0.3 mL of labeled pathogenic bacterial suspension to the blank control group and mix well, set up 3 replicate wells for each probiotic strain, and incubate in the dark at 37°C in a 5% CO2 incubator for 2 h. Repulsion test: Set up test and blank control groups, under light-proof conditions, add 0.6 mL of probiotic bacterial suspension to the single layer of Caco-2 cells in the test group, and add 0.6 mL of RPMI-1640 cell culture medium to the blank control group, set up 3 replicate wells for each probiotic strain, incubate at 37°C in a 5% CO2 incubator for 1 h, and then wash with PBS; add 0.6 mL of labeled pathogenic bacterial suspension to each well, and incubate in the dark at 37°C in a 5% CO2 incubator for 1 h. Replacement test: Set up test and blank control groups, under light-proof conditions, add 0.6 mL of labeled pathogenic bacterial suspension to the single layer of Caco-2 cells in the test group, incubate in the dark at 37°C in a 5% CO2 incubator for 1 h, and then wash with PBS; add 0.6 mL of probiotic bacterial suspension and RPMI-1640 cell culture medium to the test and control groups, respectively, and incubate in the dark at 37°C in a 5% CO2 incubator for 1 h.

[0060] After the end of the test, after washing with PBS for three times, 0.3 mL trypsin was added for 5 min, 0.5 mL RPMI-1640 cell culture solution was added to terminate the digestion, the cell suspension was mixed by blowing and 100 uL of the cell suspension was taken to a 96-well plate to measure the relative fluorescence intensity value (RFU) of the cell suspension. The inhibition adhesion rate (%) of the strain to the pathogenic bacteria was calculated according to the following formula: (1-R / R0) x 100%; In the formula: R: relative fluorescence intensity value of the experimental group; R0: relative fluorescence intensity value of the blank group.

[0061] 1.2.10 Hemolytic test of lactic acid bacteria The activated strain was streaked on blood agar plates and cultured at 37°C for 18-24 h, and the hemolysis phenomenon was observed.

[0062] 1.2.11 Safety test The activated strain was centrifuged at 8000 r / min for 5 min to collect the bacterial cells, resuspended in PBS and adjusted to a concentration of 10 5 , 10 7 , 10 9 CFU / mL. 7-week-old BALB / c mice were randomly divided into 4 groups, 8 mice in each group, and the above 3 bacterial suspensions and PBS were continuously fed for 21 d, 200 uL·d -1 . The body weight of the mice was measured on day 0, 7, 14 and 21. The spleen, liver and kidney of the mice were obtained by dissection on day 21, and the organ index of the mice was calculated.

[0063] Formula: Organ index = organ weight / mouse body weight.

[0064] 1.2.12 Effect of isolated bacteria on intestinal flora of mice The frozen strain (isolated bacteria and positive control bacteria Lactobacillus acidophilus BNCC185342 in this study) was inoculated and cultured overnight, and the bacterial cells were collected by centrifugation at 8000 r / min for 5 min. The best dose was selected according to the results of the safety test in 1.2.11 to prepare the bacterial solution for gavage. 7-week-old BALB / c mice were randomly divided into 3 groups, 8 mice in each group, i.e. test bacteria group, positive control group and negative control group. Continuous gavage for 21 d, 200 uL·d -1 . The body weight of the mice was measured on day 0, 7, 14 and 21. The intestinal tract of the mice was immediately sent to Guangzhou Aozhi Biological Technology Co., Ltd. for 16S sequencing on day 21.

[0065] 1.2.13 Statistical analysis SPSS 22 statistical software was used to analyze the experimental data. P < 0.05 was considered statistically significant. All experiments were set up with 3 replicates. The values ​​are expressed as mean ± standard deviation.

[0066] 2 Results 2.1 Isolation and Identification of Durable Enterococci A characteristic strain was obtained through selective culture, gastric and intestinal fluid tolerance screening, and Gram staining identification. It exhibited typical morphological characteristics on MRS solid medium: moist-edged, uniformly colored, white, round colonies. Gram microscopy revealed that the bacterium was a Gram-positive coccus, without spores. Figure 1 (A and B). DNA was extracted from the isolates and PCR amplification was performed using universal 16S rRNA primers. The amplification products were verified by 1.0% agarose gel electrophoresis, yielding a specific band of 1500 bp, consistent with the expected fragment size. Figure 1 (C). The 16S rRNA gene sequence was determined and compared with Genebank. The isolate belonged to the same branch as Enterococcus durans (MT545074.1), with a sequence similarity of 100%. Therefore, the isolate was initially named BHDY002.

[0067] Enterococci durable ( Enterococcus durans The BHDY002 strain was deposited on November 21, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67336.

[0068] 2.2 Growth and acid production curves of *Enterococcus faecium* from wild birds The experimental results show that ( Figure 2 ), 2-8 hours is the logarithmic growth phase of BHDY002, OD 600 The pH value increases rapidly, but enters a stable period after 10 hours, with the OD600 value increasing more gradually. During the logarithmic growth phase, BHDY002 produces more acid, causing the pH value to decrease rapidly, and then shows a slow downward trend after 10 hours.

[0069] 2.3 Acid and bile salt tolerance of bird-derived durable enterococci The results of acid and bile salt tolerance tests are shown in Table 2. All isolated strains BHDY002 showed a certain degree of tolerance, with BHDY002 having a survival rate of over 80% in environments with pH = 3.0 and bile salt concentration of 0.3%.

[0070] Table 2. Acid and bile salt tolerance characteristics of the isolated bacteria

[0071] 2.4 Wild bird-derived Enterococcus durans in gastrointestinal tract tolerance The results of the simulated gastric and intestinal tolerance test of the isolated bacteria are shown in Table 3. The isolated strain BHDY002 showed good tolerance, with a survival rate of more than 90% in both gastric juice and intestinal juice.

[0072] Table 3 Gastrointestinal juice tolerance characteristics of isolated bacteria BHDY002

[0073] 2.5 Agglutination ability of wild bird-derived Enterococcus durans The self-agglutination test of the isolated bacteria was performed (see Table 6), and the results showed that the self-agglutination rate of the isolated bacteria BHDY002 steadily increased over time, with a self-agglutination rate of more than 20% after 24 h. Co-agglutination tests were performed between BHDY002 and four pathogenic bacteria, and the results showed that the isolated bacteria could agglutinate with chicken white dysentery Salmonella, Escherichia coli, Pasteurella multocida, and Staphylococcus aureus. Among them, BHDY002 had the strongest agglutination ability with Escherichia coli and Pasteurella multocida, with a co-agglutination rate of more than 40% after 24 h. Figure 3 2.6 Bacteriostatic ability of wild bird-derived Enterococcus durans

[0074] The in vitro bacteriostatic experiment of the isolated strain showed (see Table 4), that the isolated bacteria BHDY002 exhibited varying degrees of inhibition on four common pathogenic bacteria. Among them, BHDY002 showed strong inhibition on Escherichia coli, with a bacteriostatic circle diameter of 18.03 ± 0.53 mm. Figure 4 Table 4 Inhibition ability of isolated bacteria on pathogenic bacteria

[0075]

[0076] 2.7 Drug sensitivity test of wild bird-derived Enterococcus durans The results of the drug sensitivity test showed (see Table 5), that the isolated strain did not exhibit drug resistance characteristics. The strain was sensitive to tetracycline, erythromycin, chloramphenicol, penicillin, ampicillin, fosfomycin, and rifampicin, and was neutral sensitive to vancomycin, furan, and ciprofloxacin. Figure 5 Table 5 Results of drug sensitivity test of isolated bacteria

[0077]

[0078] 2.8 Drug resistance gene detection ​​​In this study, the genomic DNA of the isolated bacteria BHDY002 was used as a template for PCR amplification using gentamicin, kanamycin, tetracycline, and vancomycin primers. No drug-resistant genes were detected.

[0079] 2.9 Adhesion ability of Enterococcus durans from wild birds 2.9.1 Adhesion rate of Enterococcus durans from wild birds The adhesion experiment of the isolated bacteria to Caco-2 cells showed that the adhesion rate of the isolated bacteria BHDY002 to Caco-2 cells was 12.22%, and the adhesion rate of the positive control Lactobacillus acidophilus BNCC185342 was 14.61% (Table 6).

[0080] Table 6 Adhesion rate of isolated bacteria

[0081] 2.9.2 Inhibition ability of Enterococcus durans from wild birds on the adhesion of pathogenic bacteria to Caco-2 cells The inhibition experiment of the isolated bacteria on the adhesion of pathogenic bacteria to Caco-2 cells showed that the isolated bacteria BHDY002 and the positive control BNCC185342 could inhibit the adhesion of Escherichia coli and Staphylococcus aureus to Caco-2 cells through competition, exclusion, and displacement. Among them, the isolated bacteria mainly inhibited the adhesion of Escherichia coli and Staphylococcus aureus through displacement of adhesion sites, and the displacement adhesion rate of BHDY002 to Escherichia coli was up to 68.22%, and to Staphylococcus aureus was up to 57.79%.

[0082] Table 7 Inhibition rate of isolated bacteria on the adhesion of pathogenic bacteria to Caco-2 cells

[0083] 2.10 Safety of isolated bacteria 2.10.1 Hemolytic test of isolated bacteria From the results, it can be seen that the colonies of Staphylococcus aureus on the blood agar plate formed completely transparent hemolytic rings (β-hemolysis), and none of the isolated bacteria showed hemolytic phenomenon on the blood agar plate, indicating that none of the isolated bacteria had hemolytic activity. Figure 7

[0084] 2.10.2 Safety evaluation in mice 2.10.2.1 Body weight growth results On the 21st day, the body weight of the low-dose group of BHDY002 increased significantly compared with the negative control group (P<0.01), and the body weight of the mice in each dose group increased steadily, with the most obvious increase in the low-dose group.

[0085] 2.10.2.2 Organ index ​After 21 days of continuous gavage, there were no significant changes in organ indices in any of the BHDY002 dose groups compared to the PBS group.

[0086] 2.10.2.3 Immune Organ Index After 21 days of continuous gavage, there were no significant changes in the thymus and spleen indices in any of the BHDY002 dose groups compared to the PBS group.

[0087] 2.11 Effects of isolated bacteria on mouse gut microbiota 2.11.1 Results of weight gain On day 21, the weight of the BHDY002 group significantly increased compared to the negative control group (P<0.01), but showed no significant increase compared to the positive control group BNCC185342. The weight gain rates of both the BHDY002 and BNCC185342 groups showed a steady upward trend, with the BHDY002 group showing a better weight gain rate than the BNCC185342 group. Figure 11 It can be seen that the weight gain rate of the negative control group showed a stable increase from 0 to 14 days and remained unchanged from 7 to 14 days.

[0088] 2.11.2 High-throughput sequencing 2.11.2.1 OTU Quantity Analysis Operational Taxonomic Units (OTUs) are standardized labels artificially assigned to a taxonomic unit (strain, genus, species, group, etc.) for ease of analysis in phylogenetic or population genetics studies. Regarding the number of species among samples, a circle on a Veen diagram represents a group. The overlap between circles visually indicates the compositional similarity of samples at the OTU level. The numbers in the non-overlapping areas represent the number of OTUs unique to that sample (group). Combining the species represented by the characteristics, common microorganisms among different samples (groups) can be identified. For example... Figure 12 As shown, in the mouse intestine, there were 608 identical OTUs across the three groups, 720 unique OTUs in the Control group, 507 unique OTUs in the BHDY002 group, and 418 unique OTUs in the positive control BNCC185342 group.

[0089] 2.11.2.2 Species composition analysis at the phylum, genus, and species levels A columnar stacking plot was created by selecting the top ten most abundant species in each group at the phylum level. Figure 13 It can be seen that the phylum Bacteroidetes with an abundance >1% in the mouse intestine is Bacteroidetes ( Bacteroidota Firmicutes ( Bacillota Proteobacteria ( Campylobacterota ), Pseudomonas ( Pseudomonadota Patellar Bacteria (Patescibacteria ), Thermo-desulfurobacteria ( Thermodesulfobacteriota ), Actinobacteria ( Actinomycetota ), Cyanobacteria ( Cyanobacteriota ), Fusobacteria ( Fusobacteriota ) and iron-depleting bacteria ( Deferribacterota Among them, each group of Bacteroidetes ( Bacteroidota Firmicutes (BHDY002) had the highest relative abundance in mice after continuous gavage. Bacillota ), Patellar Bacteria ( Patescibacteria The abundance of Proteobacteria ( ) decreased, and the abundance of Proteobacteria ( ) decreased. ‌ Campylobacterota The abundance has increased.

[0090] Depend on Figure 14 It can be seen that, at the bacterial genus level, the dominant bacteria in mice ( Lachnospiraceae_NK4A136_ group ), genus *Alternaria* ( Alistipes ) and Bacteroides ( Bacteroides The relative abundance of *Lactobacillus salivarius* (L.) was relatively high. After continuous gavage administration of BHDY002 to mice, the abundance of *Lactobacillus salivarius* (L.) was significantly increased. Ligilactobacillus ) and Lactobacillus ( Lactobacillus The abundance of the sample increased significantly compared to the positive control group BNCC185342 and was significantly higher than that of the control group.

[0091] Species-level analysis of mouse gut revealed ( Figure 15 After mice were continuously administered BHDY002 via gavage, compared with the positive control group, *Lactobacillus murineis* (Lactobacillus) Lactobacillus murinus The relative abundance of *Lactobacillus murineis* (Lactobacillus) increased significantly compared to the Control group. Lactobacillus murinus The relative abundance of Escherichia coli (E. coli) increased significantly in the BHDY002 group and the positive control BNCC185342 group. Escherichia_coli The relative abundance of ) was lower than that of the Control group.

[0092] like Figure 16 As can be seen, heatmap analysis uses color intensity to visually display the abundance information of important bacterial groups in the mouse gut across different samples. A redder color indicates higher abundance of the species, while a bluer color indicates lower abundance. The figure below shows sample clustering and abundance heatmaps (genus level) for different clusters.

[0093] 2.11.2.3 Alpha Diversity Analysis 2.11.2.3.1 Dilution Curve Dilution curves can reveal the trend of microbial diversity in a sample as the sequencing volume changes. They can be used not only to compare species richness in samples with different sequencing data volumes, but also to indicate whether the sequencing data volume of a sample is reasonable. Figure 17As shown, in this experiment, when the number of sample sequences first showed rapid increase; when the sequence number continued to increase to 2000, the upward trend of the curve slowed down, and the increase speed of the number of OTUs of each sample gradually slowed down; when the number of sample sequences exceeded 40000, the curve tended to be flat. This shows that the sequence range determined in this test is extensive, the data quantity is reasonable, and it can reasonably reflect the vast majority of microbial community information in the sample, and the sequencing accuracy is high.

[0094] 2.11.2.3.2 Alpha diversity index Alpha diversity can reflect the abundance and diversity of microbial communities in a single group (sample), including evaluating the species abundance and diversity of environmental communities through a series of statistical analysis indices. Among them, the richness and diversity of species can be calculated by Chao, Ace, Shannon, Simpson, etc. In this detection, there was no significant difference in Chao1 index, Ace index, Shannon index, Simpson index and diversity index calculated based on the phylogenetic tree between groups (P>0.05).

[0095] 2.11.2.4 Beta diversity analysis 2.11.2.4.1 PCoA analysis PcoA analysis can reflect the differences of multiple groups of data on a two-dimensional coordinate graph, so as to analyze the species composition of samples and the difference and distance relationship between samples. A point in the figure represents a sample, and samples in the same group use consistent colors. The distance of the connection line of each sample point reflects the similarity between samples, and the shorter the distance, the greater the similarity. The distance of samples in the same group indicates the strength of sample repeatability, and the distance of samples in different groups reflects the difference between groups. As shown in the figure, Figure 19 As shown, the distribution of samples in the BHDY002 group is relatively dispersed, and there is a certain distance between each sample, indicating that the intestinal flora structure of mice has changed after continuous gavage of BHDY002. The distance between samples in the BHDY002 group and the positive control BNCC185342 group is relatively close, indicating that the intestinal flora structure of mice in the two groups has certain similarity. The distance between BHDY002, positive control BNCC185342 group and Control group is far, indicating that the intestinal flora composition structure of mice in the test group and Control group has certain difference.

[0096] 2.11.2.4.2 ANOSIM analysis ANOSIM analysis is a non-parametric test for comparing whether the difference between two or more groups is significantly greater than the difference within the group. Through ANOSIM analysis, the difference in community structure can be judged. As shown in the figure, Figure 20As shown, the difference between BHDY002 and Control group was <0.05, indicating that the continuous gavage of BHDY002 to mice changed the composition of intestinal flora of mice.

[0097] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can also be made by those skilled in the art. Here, all the embodiments are not required to be exhausted. Any modification, equivalent replacement and improvement within the spirit and principle of the present application should be included in the protection scope of the claims of the present application.

Claims

1. A strain of durable enterococcus ( Enterococcus durans BHDY002, characterized in that, This strain was deposited on November 21, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67336.

2. A microbial inoculant, characterized in that, Includes the durable enterococcus BHDY002 as described in claim 1.

3. The use of the durable enterococcus BHDY002 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of products that promote animal growth.

4. A product that promotes animal growth, characterized in that, Includes the durable enterococcus BHDY002 as described in claim 1 or the microbial agent as described in claim 2.

5. The use of the durable enterococcus BHDY002 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of antibacterial agents.

6. The application according to claim 5, characterized in that, The antibacterial agent is used to inhibit Escherichia coli, Staphylococcus aureus, Salmonella pullorum, and Pasteurella multocida.

7. An antibacterial agent, characterized in that, Includes the durable enterococcus BHDY002 of claim 1 or the microbial agent of claim 2; the antibacterial agent is used to inhibit Escherichia coli, Staphylococcus aureus, Salmonella pullorum, and Pasteurella multocida.

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