Goose source Weissella cibaria F11 and application thereof

Abstract

The invention relates to the technical field of probiotic development and utilization, in particular to a goose source Weissella cibaria F11 and application thereof. The Weissella cibarius F11 is preserved in the China General Microbiological Culture Collection Center on July 11, 2025, and the preservation number is CGMCC NO.35211. The Weissella cibarius F11 has the characteristics of good acid resistance and cholate resistance, and can be used for preparing the acid-resistant and cholate-resistant strain. And the bacillus subtilis has a good inhibition effect on pathogenic bacteria such as escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and salmonella typhimurium, and also has stable acid production capacity and good safety. The Weissella sinus F11 provided by the invention meets the probiotic characteristic standard, the antibiotic resistance spectrum of the Weissella sinus F11 meets the feed probiotic safety standard, the growth of goslings can be remarkably promoted, the death rate and diarrhea occurrence rate of the goslings can be remarkably reduced, and the weissella sinus F11 can be used for preparing feed probiotics. The development potential in the fields of preparation of bacteriostatic and antibacterial drugs, probiotics for poultry feed, feed additives, poultry feed and the like is huge.

Description

Technical Field

[0001] This invention relates to the field of probiotic development and utilization technology, specifically to a strain of goose-derived Weissella esculenta F11 and its application. Background Technology

[0002] In goose farming, improving the survival rate of goslings during the brooding stage is crucial for increasing farming efficiency and expanding the scale of operations. Young goslings, due to their underdeveloped digestive systems and poor adaptability to the external environment, are susceptible to various diseases, leading to high mortality rates and irreparable economic losses for goose farms. Diarrhea is one of the leading causes of gosling mortality. Therefore, it is necessary to find an economical and effective method to promote gosling growth and development, enhance their disease resistance, and reduce diarrhea and mortality rates.

[0003] Probiotics are generally considered one of the best feed additives for improving animal production performance. Due to their low cost, safety, reliability, and ability to improve poultry growth performance and gut health, they are widely used in livestock farming. For example, Chinese invention patent application number 202510446077.X discloses a probiotic-active fusion strain, Weissella syncytiophylla DY12, and experiments have confirmed that this bacterium can effectively regulate animal immunity, improve antioxidant capacity, increase lysozyme content and alkaline phosphatase activity, and increase daily weight gain, ultimately improving animal growth performance. However, current research and applications on using probiotics to reduce diarrhea and mortality rates in goslings are still limited. Therefore, screening for a strain with good probiotic characteristics that can significantly improve gosling production performance and reduce diarrhea and mortality rates is of great significance to the goose farming industry.

[0004] Based on this, the present invention isolates a strain of *Westernella esculenta* F11 with probiotic characteristics from the rectum of healthy geese, aiming to solve the above problems and provide technical support for the preparation of probiotic preparations for poultry feed. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Westernella esculenta* F11 derived from geese and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The goose-derived Weissella cibaria of this invention is named Weissella cibaria F11 and was deposited at the China General Microbiological Culture Collection Center on July 11, 2025, with accession number CGMCC NO.35211.

[0007] Preferably, the nucleotide sequence of *Westernella esculenta* F11 of the present invention is shown in SEQ ID NO.1.

[0008] The application of *Westernella tamariscina* F11 in the preparation of antibacterial drugs according to the present invention is characterized in that: the antibacterial drug includes *Westernella tamariscina* F11 or its fermentation broth; the antibacterial drug can inhibit *Escherichia coli*, *Pseudomonas aeruginosa*, *Staphylococcus aureus* and *Salmonella typhi*.

[0009] Preferably, in the application of the *Westernella esculenta* F11 in the preparation of antibacterial and antimicrobial drugs according to the present invention, the fermentation broth of *Westernella esculenta* F11 is obtained by inoculating *Westernella esculenta* F11 into MRS liquid culture medium and culturing it.

[0010] The application of the *Westernella esculenta* F11 described in this invention in promoting animal growth.

[0011] The application of the *Westernella esculenta* F11 described in this invention in the preparation of probiotic formulations that promote animal growth.

[0012] The present invention relates to the application of *Westernella esculenta* F11 in the preparation of probiotic formulations for preventing diarrhea in animals.

[0013] The application of *Westernella esculenta* F11 described in this invention in the preparation of animal feed additives.

[0014] The application of *Westernella esculenta* F11 described in this invention in the preparation of animal feed.

[0015] Preferably, in the above application, the animal is a goose.

[0016] The beneficial effects of this invention are: 1. This invention provides a strain of *Weissella cibaria* F11 derived from goose. This bacterium exhibits excellent acid and bile salt resistance, and simultaneously demonstrates good inhibitory effects against *Escherichia coli*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, and *Salmonella typhi*. Furthermore, it possesses stable acid-producing capacity and good safety. The *Weissella cibaria* F11 provided by this invention meets the characteristics standards for probiotics, and its antibiotic resistance spectrum meets the safety standards for feed probiotics. It has great development potential in the fields of antibacterial and antimicrobial drugs, poultry feed probiotics, feed additives, and poultry feed.

[0017] 2. Through investigation of the effects of strain F11 on the production performance of goslings and the anti-diarrheal experiment, it was found that the experimental group with added F11 bacterial suspension had a significantly increased final weight compared with the control group without added F11 bacterial suspension. At the same time, the mortality rate and diarrhea rate of goslings decreased by 7% and 10.7%, respectively. This proves that the *Westernella esculenta* F11 provided by this invention has a significant promoting effect on the growth of goslings and can significantly reduce the mortality rate and the incidence of diarrhea in goslings.

[0018] Preservation Information: Strain name: Weissella cibaria F11; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: July 11, 2025; Accession number: CGMCC NO.35211. Attached Figure Description

[0019] Figure 1 The colony morphology of strain F11; Figure 2 Phylogenetic tree of strain F11; Figure 3 The growth curve and acid production capacity of strain F11 are shown. Figure 4 The acid resistance of strain F11; Figure 5 To assess the bile salt tolerance of strain F11; Figure 6 Hemolysis status of the strains (in the figure: A is Staphylococcus aureus; B is strain F11); Figure 7 This refers to the antibiotic resistance of lactic acid bacteria. Detailed Implementation

[0020] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0021] Example 1 Weissella cibaria F11 was deposited on July 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.35211.

[0022] Example 2 Using *Westernella tamariscina* F11 or its fermentation broth from Example 1, an antibacterial drug was prepared to inhibit *Escherichia coli*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, and *Salmonella typhi*.

[0023] The fermentation broth of *Westernella taurida* F11 was prepared as follows: the strain was inoculated into MRS liquid medium and cultured for 24 h; 2 mL of bacterial broth was taken and centrifuged at 8000 rpm for 5 min; the bacterial cells in the supernatant were filtered out using a 0.22 μm sterile filter membrane to obtain the broth.

[0024] Example 3 The bacterial suspension of Weissella esculenta F11 from Example 1 was used to feed goslings to promote their growth or prevent them from getting diarrhea.

[0025] The method for preparing the bacterial suspension of *Westernella esculenta* F11 is as follows: *Westernella esculenta* F11 is inoculated into MRS liquid medium and cultured for 20 h; the bacterial suspension is centrifuged at -4℃ and 5000 r / min for 10 min, and the supernatant is discarded; the bacterial cells are resuspended in sterile PBS buffer, and the OD of the bacterial suspension is adjusted. 600 The value is 0.8. Store in the refrigerator for later use.

[0026] Example 4 A probiotic preparation for promoting the growth of geese was prepared using the *Westernella esculenta* F11 from Example 1.

[0027] Example 5 A probiotic preparation for preventing diarrhea in goslings was prepared using the *Westernella esculenta* F11 strain from Example 1.

[0028] Example 6 A feed additive for geese was prepared using *Westernella esculenta* F11 from Example 1.

[0029] Example 7 Goose feed was prepared using *Westernella esculenta* F11 from Example 1.

[0030] To further verify the reliability of the present invention and select the optimal solution, the inventors conducted a series of experiments, as follows: 1. Experimental Materials and Methods 1.1 Experimental Materials MRS broth was purchased from Qingdao Haibo Biotechnology Co., Ltd.; porcine bile saline solution was purchased from Beijing Solarbio Science & Technology Co., Ltd.; Ezup column-based bacterial genomic DNA extraction kit and SanPrep column-based DNA gel recovery kit were purchased from Sangon Biotech (Shanghai) Co., Ltd.; Taq Plus DNA polymerase, agarose, and 4S Red Plus nucleic acid staining agent (10,000X aqueous solution) were purchased from BBI Life Sciences Co., Ltd.; GeneRuler DNA Ladder Mix was purchased from ThermoScientific; and antimicrobial susceptibility testing tablets were purchased from Hangzhou Microbial Reagent Co., Ltd. Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, and Escherichia coli were preserved in our laboratory.

[0031] 1.2 Test Methods 1.2.1 Sample Preparation Aseptically collect about 1 g of fecal sample from the rectum of a healthy goose and place it in a centrifuge tube. Add 9 mL of sterile physiological saline and vortex for 5 min to mix the fecal sample with the physiological saline.

[0032] 1.2.2 Isolation, purification, preservation, and morphological observation of bacterial strains Take 100 μL and serially dilute to 10 -4 10 -5 and 10 -6 The diluted solution was evenly spread onto MRS solid medium containing 1% calcium carbonate, and incubated overnight at 37°C. Single colonies with different morphologies and calcium dissolution zones were picked, purified, and stored for later use. The purified bacterial solution was spread onto MRS solid medium, and the size, color, and edge characteristics of the pure culture colonies were observed. Gram staining and physiological and biochemical identification were performed.

[0033] 1.2.3 Molecular biological identification of the strain The bacterial strain was activated on MRS medium, and DNA was extracted according to the instructions of the bacterial DNA extraction kit. The PCR reaction system is shown in Table 1.

[0034] The primers for amplifying the 16S rDNA gene fragment were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primer sequences used for amplification are as follows: 27F: AGAGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT.

[0035] ; Using the extracted genomic DNA as a template, PCR amplification was performed. The annealing temperature and extension time were optimized based on the primer melting temperature (Tm) and fragment length. The PCR reaction conditions are shown in Table 2. Electrophoresis was performed on a 1.5% agarose gel, and the PCR amplification products were sequenced. The sequencing results were uploaded to the database, and the taxonomic position of the strain was determined by BLAST sequence alignment, and the accession number was obtained. .

[0036] 1.2.4 Determination of the beneficial characteristics of the strain (1) Determination of growth performance and acid production performance The selected strains were activated and inoculated into 5 mL of MRS liquid medium at a 1% inoculum size. The cultures were incubated at 37°C, and OD values ​​were measured at 0, 2, 4, 6, 8, 12, 16, 24, 36, and 48 h. 600 The absorbance and pH values ​​were used to plot growth curves and acid production curves, with the bacterial culture time as the x-axis and the OD value and pH as the y-axis, respectively.

[0037] (2) Determination of acid resistance and bile salt resistance The pH of MRS liquid medium was adjusted to 2.5, 3.0, 4.0, and 6.0, respectively, and the bile salt concentrations were 0, 1, 2, and 3 g / L, respectively. The lactic acid bacteria to be tested were inoculated at a 1% inoculation rate into the MRS liquid medium with the adjusted pH and bile salt concentrations, and anaerobic cultured at 37℃ for 24 h. The OD values ​​of each bacterial culture were then measured. 600 Values; plot acid tolerance curves and bile salt tolerance curves with pH and bile salt concentration on the x-axis and OD value on the y-axis, respectively.

[0038] (3) Antibacterial performance test The antibacterial activity of the strain was determined using the perforation diffusion method. The strain was inoculated into MRS liquid medium and cultured for 24 h. 2 mL of the bacterial culture was then centrifuged at 8000 rpm for 5 min, and the bacterial cells in the supernatant were filtered out using a 0.22 μm sterile filter membrane. To reduce the influence of acid production during fermentation, the pH of the fermentation broth was adjusted to approximately 7.0 with 5 mol / L NaOH. *Escherichia coli*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, and *Salmonella typhi* were selected as pathogen indicator bacteria. After activation and culture, the indicator bacteria were plated onto LB agar plates. After the agar surface dried, wells were punched, and 100 μL of fermentation broth was added to each well. The plates were then incubated overnight. The diameter of the inhibition zone was measured using calipers, with each strain tested three times. MRS broth medium without bacterial inoculation was added to the wells as a negative control.

[0039] 1.2.5 Determination of strain safety (1) Hemolysis test All tested bacterial strains were streaked onto Columbia agar plates and incubated under anaerobic conditions for 24 h. The presence of hemolytic zones around the colonies was observed: α-hemolysis, β-hemolysis, and γ-hemolysis were recorded, with Staphylococcus aureus as a positive control.

[0040] (2) Antibiotic susceptibility testing The test strains were evenly spread on MRS solid medium. Antimicrobial susceptibility tablets for penicillin, tetracycline, streptomycin, ofloxacin, polymyxin B, erythromycin, ciprofloxacin, and tobramycin were evenly placed on the plates and incubated at 37°C for 24 h. The size of the inhibition zone was observed and recorded, and the drug susceptibility was determined based on the size of the inhibition zone.

[0041] 1.2.6 Effects of the strain on the production performance of goslings and diarrhea prevention test Sixty 7-day-old meat geese (half male and half female), all with consistent health status and a weight of (120±25) g, were selected. They were randomly divided into two groups, with three replicates per group and ten geese per replicate. The experiment consisted of two phases: weeks 0–2 (early stage) and weeks 3–4 (late stage), totaling 28 days. In the early stage, the control group was fed a complete feed, while the experimental group was fed a complete feed plus 500 μL of F11 bacterial solution (once daily). In the late stage, the control and experimental groups received the same treatment. Five geese from each group were randomly selected for fasting weighing in the morning before the start of the experiment and again at the end of the experiment. Throughout the experiment, the health status of the geese was observed, and the number of deaths and cases of diarrhea were recorded. The mortality rate and diarrhea rate of the geese were statistically analyzed.

[0042] Preparation of bacterial suspension: F11 strain was cultured in MRS liquid medium for 20 h. The bacterial suspension was centrifuged at -4℃ and 5000 r / min for 10 min, and the supernatant was discarded. The bacterial cells were resuspended in sterile PBS buffer, and the OD concentration of the bacterial suspension was adjusted. 600 The value is 0.8. Store in the refrigerator for later use.

[0043] 2. Results 2.1 Morphological and molecular biological identification Strains F11 were isolated and screened from the droppings of grey geese in Pingba. Their colony morphology is as follows: Figure 1 As shown, the strain is milky white in color, and the strain is round and slightly raised. The colony surface is smooth and moist, with neat edges, and is uniformly opaque.

[0044] Phylogenetic tree of strain F11 as follows Figure 2 As shown in the figure. Sequencing results, followed by BLAST alignment, revealed that strain F11 is *Westernella esculenta* (GenBank No: PV945939), and its nucleotide sequence is shown in SEQ ID NO.1.

[0045] SEQ ID NO.1: 2.2 Probiotic characteristics of strain F11 2.2.1 Growth curve and acid production performance of strain F11 The growth curve and acid production capacity of strain F11 are as follows: Figure 3 As shown, strain F11 entered the logarithmic growth phase 4 hours after inoculation, and remained in the stationary phase from 8 to 48 hours. The OD of strain F11 at 48 hours... 600nm The pH value reached 2.1, indicating strong growth and reproduction capabilities. The pH of strain F11 initially decreased rapidly and then stabilized, reaching 3.99 at 12 h and below 3.65 at 48 h.

[0046] 2.2.2 Acid resistance and bile salt resistance The acid resistance of strain F11 is as follows Figure 4 As shown, the growth of strain F11 basically stopped in an acidic environment with a pH of 2.5, indicating that a strongly acidic environment has an inhibitory effect on the growth of lactic acid bacteria strain F11; the growth of the strain was relatively slow at a pH of 3.0, and it could grow normally at pH 4.0 and 6.0.

[0047] The bile salt tolerance of strain F11 is as follows Figure 5 As shown, the OD of strain F11 under bile salt conditions of 0, 1, 2, 3, and 4 g / L was... 600nm The values ​​were 0.056, 0.671, 0.994, 1.393, and 2.018, respectively. F11 could also grow under 3 g / L bile salt conditions, but stopped growing under 4 g / L conditions, indicating that strain F11 could tolerate an environment of 3 g / L bile salt.

[0048] 2.2.3 Antibacterial properties of strain F11 The antibacterial effects of strain F11 are shown in Table 3. The results show that strain F11 exhibits varying degrees of inhibitory effects against Escherichia coli, Staphylococcus aureus, Salmonella, and Pseudomonas aeruginosa, with the strongest inhibitory effect against Staphylococcus aureus. .

[0049] 2.3 Safety of strain F11 2.3.1 Hemolysis test Using Staphylococcus aureus as a positive control, the hemolytic activity of strain F11 is as follows: Figure 6 As shown in the figure (A is Staphylococcus aureus; B is strain F11), the results show that strain F11 did not exhibit hemolysis after 48 h of incubation.

[0050] 2.3.2 Susceptibility of strain F11 to antibiotics Antibiotic resistance of strain F11 as follows Figure 7As shown in the results, strain F11 is sensitive to florfenicol (FFC), doxycycline (DO), azithromycin (AZI), clindamycin (CC), cephalosporins (C), ciprofloxacin (CIP), and erythromycin (E), but not sensitive to vancomycin-resistant (VAN), norfloxacin (NOR), gentamicin (GEN), and levofloxacin (LVX).

[0051] 2.4 Effects of strain F11 on the production performance of goslings and its anti-diarrheal test The effects of strain F11 on the growth performance of meat geese are shown in Table 4, and the mortality rate and diarrhea rate during the entire experimental period are shown in Table 5. ; As shown in Table 4, after a 28-day experimental period, compared with the control group without F11 bacterial solution, the experimental group with F11 bacterial solution significantly increased the final weight of goslings, indicating that strain F11 has a significant promoting effect on the growth of goslings.

[0052] As shown in Table 5, compared with the control group without F11 bacterial solution, the mortality rate and diarrhea rate of goslings in the experimental group with added F11 bacterial solution decreased by 7% and 10.7%, respectively, proving that strain F11 can significantly reduce the mortality rate and diarrhea rate of goslings.

[0053] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A strain of *Westernella esculenta* from geese, characterized in that, The goose-derived Weissella cibaria was named Weissella cibaria F11 and was deposited on July 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.35211.

2. The *Westernella esculenta* F11 according to claim 1, characterized in that, The nucleotide sequence of the *Westernella esculenta* F11 is shown in SEQ ID NO.

1.

3. The application of *Westernella esculenta* F11 as described in claim 1 in the preparation of antibacterial and antimicrobial drugs, characterized in that: The antibacterial drug includes *Westernella esculenta* F11 or its fermentation broth; the antibacterial drug can inhibit *Escherichia coli*, *Pseudomonas aeruginosa*, *Staphylococcus aureus* and *Salmonella typhi*.

4. The application according to claim 3, characterized in that, The fermentation broth of *Westernella esculenta* F11 is obtained by inoculating *Westernella esculenta* F11 into MRS liquid medium and culturing it.

5. The application of *Westernella esculenta* F11 as described in claim 1 in promoting animal growth.

6. The use of the *Westernella esculenta* F11 as described in claim 1 in the preparation of probiotic formulations that promote animal growth.

7. The use of the *Westernella esculenta* F11 as described in claim 1 in the preparation of a probiotic formulation for preventing diarrhea in animals.

8. The use of *Westernella esculenta* F11 as described in claim 1 in the preparation of animal feed additives.

9. The use of *Westernella esculenta* F11 as described in claim 1 in the preparation of animal feed.

10. The application according to any one of claims 5-9, characterized in that, The animal in question is a goose.

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