Lactobacillus animalis D27 and screening method and application thereof

Abstract

This invention discloses a *Ligilactobacillus animalis* D27 strain, its screening method, and its applications, relating to the field of microbial technology. The key technical points are: the *Ligilactobacillus animalis* D27 strain of this invention was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 30561. The *Ligilactobacillus animalis* D27 provided by this invention is a high-performance strain screened from the digestive tract of calves, exhibiting strong acid and bile salt resistance, high adhesion, and high lactic acid production, providing an excellent strain resource for the development of probiotics for the prevention and treatment of calf diarrhea.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to an animal-associated lactobacillus D27, its screening method, and its application. Background Technology

[0002] In dairy farming, the health and development of calves are crucial to the entire breeding process. The calf stage is a critical phase in a dairy cow's life, as their digestive and immune systems are not yet fully mature, making them less adaptable to environmental changes and stress responses. This makes calves susceptible to digestive problems, especially diarrhea. Statistics show that the morbidity rate of digestive system problems in calves is as high as 60%, which seriously affects their growth, development, and overall health.

[0003] Diarrhea not only leads to poor nutrient absorption and delayed growth, but in severe cases, it can also cause calf death. Therefore, preventing and managing calf diarrhea has become a significant challenge in animal husbandry. To address this challenge, the application of probiotics has gradually become a research hotspot, with lactic acid bacteria playing a crucial role. Lactic acid bacteria are a class of bacteria that can ferment carbohydrates in animals to produce lactic acid. According to the "Catalogue of Feed Additives (2013)" implemented in my country in February 2014, lactic acid bacteria are listed as one of the permitted feed microorganisms, with a total of 34 microorganisms approved for use, of which 17 are lactic acid bacteria. This indicates the enormous potential of lactic acid bacteria in animal husbandry.

[0004] Given the different probiotic properties of lactic acid bacteria strains from various sources, researchers are dedicated to screening for lactic acid bacteria strains effective for calves. These strains are considered potential probiotic additives that can effectively prevent and reduce diarrhea by improving calves' gut health and immune function. Therefore, developing and utilizing superior lactic acid bacteria strains suitable for calves has become a key objective in current livestock research and practice. Furthermore, recent studies have shown that specific lactic acid bacteria strains are not only beneficial to calves, but their potential probiotic properties may also have positive impacts on the health of other livestock and humans. Therefore, exploring the multiple probiotic functions of lactic acid bacteria and developing multi-purpose lactic acid bacteria products is a current hot topic in scientific research and industrial application. Through these research and development efforts, not only can the production efficiency of livestock farming be improved, but sustainable and healthy agricultural development can also be promoted.

[0005] Therefore, the present invention aims to screen a high-performance lactic acid bacteria strain derived from calves, explore its physiological functions and probiotic characteristics, and provide excellent strain resources for the development of probiotics to prevent calf diarrhea. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of *Ligilactobacillus animalis* D27, its screening method, and its application. This *Ligilactobacillus animalis* is a strain selected through screening that exhibits excellent performance, strong acid and bile salt resistance, strong adhesion, and high lactic acid production. It provides a superior strain resource for the development of probiotics for the prevention and treatment of calf diarrhea.

[0007] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a strain of *Ligilactobacillus animalis* D27, which was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 30561. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), postal code 100101, and telephone number 010-64807355.

[0008] The present invention is further configured to have a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO.1.

[0009] Another object of the present invention is to provide the application of *Lactobacillus anthracis* D27 as described above in the preparation of animal feed additives.

[0010] Another object of the present invention is to provide the application of the animal-associated lactobacillus D27 as described above in the prevention and treatment of calf diarrhea.

[0011] Another object of the present invention is to provide a method for screening the above-mentioned animal-associated lactobacillus D27, comprising the following steps:

[0012] Gradual dilution: Fresh stool samples were diluted with sterile saline.

[0013] Plate plating: The diluted bacterial solution was plated on MRS solid selection medium containing cysteine ​​and mupirocin lithium salt;

[0014] Anaerobic culture: Incubate anaerobically at 37°C in an anaerobic incubator for 24 to 72 hours until colonies grow;

[0015] Single colony selection: Select single colonies with a yellow outer ring and transfer them to MRS medium containing cysteine ​​and bromocresol purple;

[0016] Purification again: Repeat the picking and culturing steps until a pure single colony is obtained;

[0017] Culture and preservation: After enriching the single colonies, add 50% glycerol and store at -80℃.

[0018] By adopting the above technical solution, the *Ligilactobacillus sanimalis* D27 strain provided by this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30561 and deposit date of May 9, 2024. The strain contains a 16S rRNA gene sequence that is at least 95% identical to SEQ ID NO. 1, ensuring its stability and consistency at the gene level, which is helpful for classification and identification, and improves the reliability of research and application. This gene sequence provides a reliable basis for further molecular biology research and helps to understand the function and application potential of this strain.

[0019] The *Lactobacillus assemblica* D27 provided by this invention exhibits significant inhibitory effects against *Escherichia coli*, *Salmonella*, and *Staphylococcus aureus*. This *Lactobacillus assemblica* D27, after rigorous isolation, purification, and preservation, serves as a standardized experimental model, providing reliable material for subsequent scientific research and facilitating various experiments and verifications. By studying the biological characteristics of the D27 strain, its function and mechanism in different environments can be better understood, providing a theoretical basis for further application development. The antibacterial properties of the D27 strain provide important reference and possibilities for developing new anti-infective drugs and biological therapies. By reducing antibiotic use and decreasing the generation and spread of drug-resistant bacteria, it contributes to environmental protection and sustainable development, demonstrating significant ecological benefits.

[0020] In summary, the present invention has the following beneficial effects:

[0021] 1. The animal-associated lactobacillus provided by this invention is a strain with excellent performance selected through screening. It has strong acid resistance, bile salt resistance, strong adhesion, and high lactic acid production, providing excellent strain resources for the development of probiotics for the prevention and treatment of calf diarrhea.

[0022] 2. The application of the animal-associated lactobacillus D27 provided by this invention in the preparation of animal feed additives can improve animal intestinal health, enhance immunity, improve growth performance, reduce disease incidence, and improve breeding efficiency. This strain has a significant inhibitory effect on Escherichia coli and Salmonella, which can effectively reduce the risk of infection of these pathogens in animals, improve animal health, reduce antibiotic use, and promote healthy breeding. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the animal-associated Lactobacillus D27ANI analysis in Example 1 of the present invention;

[0024] Figure 2 This is a schematic diagram of animal-associated lactobacillus D27 under an oil immersion microscope in Example 1 of the present invention;

[0025] Figure 3 This is a line graph showing the growth curve and acid production capacity of *Lactobacillus assemblica* D27 in animal samples from Example 1 of this invention.

[0026] Figure 4 This is a bar chart showing the survival rate of artificial gastrointestinal fluid containing *Lactobacillus D27* in animals in Example 2 of this invention.

[0027] Figure 5 This is a bar chart showing the survival rate of bile salts in *Lactobacillus synergae* D27 in animals in Example 2 of this invention;

[0028] Figure 6 This is a schematic diagram of the antibacterial effect of animal combined with Lactobacillus D27 in Example 3 of the present invention; wherein, Figure A is a schematic diagram of the antibacterial test results of Escherichia coli; Figure B is a schematic diagram of the antibacterial test results of Salmonella; and Figure C is a schematic diagram of the antibacterial test results of Staphylococcus aureus.

[0029] Figure 7 This is a bar chart showing the self-agglutination ability and hydrophobicity of *Lactobacillus simulans* D27 in animal samples in Example 3 of this invention;

[0030] Figure 8 These are comparative diagrams of hemolysis test results in Example 4 of the present invention; the left diagram is the result of the Staphylococcus aureus hemolysis test; the right diagram is the result of the animal-associated lactobacillus D27 hemolysis test. Detailed Implementation

[0031] This invention provides an animal-associated lactobacillus D27, its screening method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0032] In the following experiments, *Ligilact obacillus animalis* D27 refers to *Ligilact obacillus animalis* D27, which was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMC C), with accession number CGMCC No. 30561. The depository address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), postal code 100101, telephone 010-64807355.

[0033] Source of materials: Fresh feces collected from healthy calves aged 27 days by ShouNong Modern Agricultural Technology Co., Ltd. in Dingzhou City, Hebei Province. The feces were frozen and brought back immediately after collection.

[0034] Reagent sources: Pepsin (1:10000), Trypsin (1:250), Beijing Solarbio Science & Technology Co., Ltd.; MRS medium, Beijing Solarbio Co., Ltd.; Bifidobacterium proliferation medium, TPY medium, mupirocin lithium salt, agar powder, Beijing Luqiao Co., Ltd.; Lysis buffer, Mix, universal primers 27F and 1492R, Beijing Liuhe Huada Gene Technology Co., Ltd.; Penicillin, ampicillin, cefazolin, amikacin, gentamicin, erythromycin, norfloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, chloramphenicol, polymyxin B, tetracycline, amoxicillin, piperacillin, oxacillin, Hangzhou Microbial Reagent Co., Ltd.

[0035] Main instruments: Nucleic acid electrophoresis imaging system, Bio-Rad; microscope, OLYMPUS; electric thermostatic incubator, Shanghai Jinghong Experimental Equipment Co., Ltd.; ELISA reader, Gene Technology Co., Ltd.; autoclave; micro-oxygen incubator, anaerobic incubator, Huayuexing Instrument Co., Ltd.; autoclave, Hefei Huatai Medical Co., Ltd.

[0036] Example 1: Isolation, screening and identification of Ligilactobacillus animalis D27

[0037] This invention provides a method for isolating and purifying the above-mentioned *Ligilactobacillus animalis* D27, comprising the following steps: collecting fresh animal fecal samples, using sterile containers to ensure that external contamination is avoided, rapidly transporting the collected samples to the laboratory at 4°C, and processing them within 2 hours after collection;

[0038] Prepare sterile saline solution, i.e., 0.9% NaCl solution. Place 1 gram of fecal sample into 10 ml of sterile saline solution and mix thoroughly using a sterile glass rod mixer to obtain an initial suspension. Ensure all operations are performed under aseptic conditions. Take 1 ml of the initial suspension and add it to 9 ml of sterile saline solution, mix thoroughly, and obtain 10 ml of the initial suspension. -1 Diluent; repeat the above steps to dilute 10 -1 Diluent is diluted sequentially to 10 -2 10 -3 10 -4 10 -5 For isochronous dilutions, each dilution requires a new sterile pipette tip and sterile test tube.

[0039] Prepare MRS solid screening medium plates containing 0.05 g / L cysteine ​​and 0.05 g / L mupirocin lithium salt, ensuring that the medium is evenly distributed on the plate; add 100 μL serial dilutions to the MRS solid screening medium plates, and use a sterile spreader to spread the added dilutions evenly on the surface of the medium, ensuring that the entire plate is covered.

[0040] Place the coated plates into the anaerobic workstation to achieve the anaerobic oxygen concentration, incubate at a constant temperature of 37°C for 24 to 72 hours, and observe the colony growth.

[0041] Observe the colony morphology on the culture medium plate and select a single colony with typical morphology and a relatively large size. Inoculate the selected single colony onto a new MRS solid culture medium plate containing 0.05 g / L cysteine ​​and 0.01 g / L bromocresol purple. Use a sterile inoculation loop to perform a "three-zone streak" operation on the plate to ensure the isolation of single colonies. Incubate under anaerobic conditions at 37°C for 24 to 48 hours and observe the color change of the colonies. Select single colonies with a yellow outer ring.

[0042] The single colonies with yellow outer rings were inoculated again onto new MRS solid medium plates and streaked repeatedly. The streaking and culture steps were repeated until uniform and pure single colonies were obtained.

[0043] The purified single colonies were inoculated into MRS liquid or solid medium containing 0.05 g / L cysteine ​​and cultured under anaerobic conditions at 37°C. The growth of the strain was observed regularly to ensure that a stable *Ligilactobacillus animalis* D27 strain was obtained.

[0044] Strain identification: DNA template extraction: A single colony was picked and inoculated into liquid culture medium for 24 h. 30 μL of lysis buffer was transferred to a 200 μL PCR tube. After shaking the bacterial culture well, 5 μL of the lysis buffer was added and mixed. After simple centrifugation, the mixture was placed in a PCR amplification instrument at 80℃ for 15 min for lysis. The lysis program was set as follows: 80℃ for 20 min; storage at 4℃.

[0045] PCR amplification: The reaction system consisted of 30 μL of the following components: DNA template: 6 μL, forward and reverse primers: 1 μL each (universal primers 27F: 5′-AGAGTTTGATCCTGGCTCA G-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′), Mix: 15 μL, and sterile water: 6.6 μL. PCR amplification conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles, with a final extension at 72℃ for 10 min, and storage at 16℃. The PCR amplification products were sequenced by Qingke Company, and the results were compared with BLAST in the NCBI database.

[0046] Sequencing results showed that the 16S rRNA gene sequence of *Ligilactobacillus animalis* D27 strain was approximately 1500 bp in length. Blast alignment showed 100% gene similarity. Whole-genome sequencing ANI analysis was performed, and the results are attached. Figure 1 As shown, homologous gene sequence comparison analysis revealed that it was most closely associated with *Ligilactobacillus animalis* P38, with a similarity of 98.44%.

[0047] 1. Morphological observation: Select single colonies with intact morphology and good growth from the culture medium, stain them using the Gram staining method, and observe them using an oil immersion microscope;

[0048] The observation results are attached. Figure 2 As shown in the results, the *Lactobacillus assemblica* colonies were found to be facultative anaerobic Gram-positive bacteria. The colonies were white with smooth edges and raised surfaces. Microscopic examination revealed that the strains were short, straight rods, existing singly or in pairs.

[0049] 2. Growth curve and acid production capacity

[0050] Single colonies were picked and inoculated into 10 mL of solid culture medium and cultured for 12 h to prepare a seed culture. The inoculum conditions were set as follows: 3% inoculum, 37℃, pH 6.2±0.2. The OD value (nm=600) and pH value of the bacterial culture were measured every 2 h until 24 h of culture; the OD value of the bacterial culture was measured every 6 h. 600 The pH value was measured until 36 hours of incubation; the OD value of the bacterial culture was measured again after 48 hours. 600 Values ​​and pH values. With incubation time as the x-axis, OD... 600 Plot the growth curve and acid production curve with pH value on the ordinate;

[0051] The growth curve and acid production curve are attached. Figure 3As shown, the growth curve and acid production curve reveal that *Lactobacillus animalis* is in its logarithmic growth phase from 0 to 6 hours, and enters a plateau phase after 12 hours. After 12 hours of culture, the pH of the culture medium drops to 4.25, and then decreases slowly, reaching 4.04 after 48 hours.

[0052] 3.Physical and chemical properties

[0053] Physicochemical characteristics of lactic acid bacteria were identified using a complete set of biochemical tubes. The direct inoculation method was adopted, inoculating the same purified and isolated colonies from the plate with an inoculation needle into ampoules containing different substrates, and anaerobically incubating at 36±1℃ for 24h. The results were then interpreted.

[0054] As shown in Table 1 below, *Lactobacillus animalis* can utilize raffinose, inulin, sucrose, lactose, maltose, salicin, and cellobiose.

[0055] Table 1 Physicochemical properties of *Lactobacillus synergae* in animals

[0056]

[0057]

[0058] Example 2: Tolerance of *Ligilactobacillus animalis* D27

[0059] 1. Acid resistance

[0060] Single colonies were picked and inoculated into 10 mL of solid culture medium and cultured for 12 h to prepare a seed culture. A 3% inoculum was then inoculated into MRS liquid medium and cultured overnight. After centrifugation at 4000 rpm for 10 min, the supernatant was discarded, and 10 mL of PBS was added for later use. The pH of the MRS medium was adjusted to 2.0, 3.0, and 4.0 using HCl. Animal-associated lactobacillus was inoculated into different pH media at a 1% inoculum and anaerobically cultured for 3 h. The digestion solutions at 0 h and 3 h were used to detect viable bacterial counts and calculate the survival rate.

[0061] Strain survival rate (%) = A / B × 100%

[0062] In the formula: A represents the number of viable bacteria (CFU / mL) at 0h, and B represents the number of viable bacteria (CFU / mL) at 3h.

[0063] Conclusion: Under acid tolerance testing, *Lactobacillus assemblica* D27 remained viable at pH 2, with a survival rate of 70% at pH 3 and over 100% at pH 4. These results demonstrate the excellent adaptability and survival ability of *Lactobacillus assemblica* D27 to acidic environments. This characteristic allows it to maintain its activity as it passes through the animal's digestive system, thus exerting its probiotic effects in the intestines. This is of great significance for developing animal feed additives that can pass through the acidic environment of the stomach and exert their effects in the intestines.

[0064] 2. Tolerance to artificial gastrointestinal fluid

[0065] Artificial gastric fluid: Prepare a 0.5% NaCl solution, add 0.3% pepsin, adjust the pH to 2.5 and 3.0 with 1 mol / L HCl, then dissolve thoroughly and filter through a 0.22 μm microporous membrane for sterilization before use.

[0066] Artificial intestinal fluid: Prepare a 0.5% NaCl solution, add 0.1% trypsin, adjust the pH to 8.0 with 0.1 mol / L NaOH, then dissolve thoroughly and filter through a 0.22 μm microporous membrane for sterilization before use.

[0067] After overnight culture of *Lactobacillus simulans*, centrifuge at 4000 rpm for 10 min, discard the supernatant, and add 10 mL of PBS for later use. Inoculate *Lactobacillus simulans* into artificial gastric and intestinal fluids at a 1% inoculum and anaerobic culture for 3 h. Collect digestive fluids from 0 h and 3 h for viable bacterial count and calculate the survival rate.

[0068] Strain survival rate (%) = A / B × 100%

[0069] In the formula: A represents the number of viable bacteria (CFU / mL) at 0h, and B represents the number of viable bacteria (CFU / mL) at 3h.

[0070] Conclusion: In simulated gastric fluid at pH 2.5 and pH 3.0, *Lactobacillus assemblica* D27 exhibited relatively high survival rates (40.60% and 70%, respectively), as shown in the attached figure. Figure 4 As shown, this demonstrates its ability to adapt to the weakly acidic environment of gastric juice. Even under such acidic conditions, *Lactobacillus assemblica* D27 can maintain a certain level of activity, facilitating its passage through the stomach into the intestines. In simulated artificial intestinal fluid, *Lactobacillus assemblica* D27 exhibits an extremely high survival rate (96.86%), indicating that its survival environment in the intestines is more favorable. The alkaline conditions and lower acidity of the intestines provide an ideal environment for the growth and reproduction of *Lactobacillus assemblica* D27, enabling it to multiply rapidly and exert its probiotic effects.

[0071] The above experimental results demonstrate that *Lactobacillus assemblica* D27, upon entering the intestines from the stomach, can effectively adapt to and overcome the acidic environment of gastric juice, and exhibits good survival and growth capabilities within the intestines. These characteristics are crucial for developing oral probiotic products and ensuring their effectiveness in animals, particularly in maintaining digestive health and balancing the gut microbiota, showing potential applications.

[0072] 3. Bile salt tolerance

[0073] Bovine bile salts were weighed and added to MRS liquid medium to achieve bile salt concentrations of 0.3%, 0.5%, 1%, 3%, and 5%. The medium was then autoclaved at 121°C for 20 minutes. 0.1 mL of the activated bacterial suspension was added to 9.9 mL of medium and incubated at 37°C for 0 and 5 hours. 0.1 mL of the bacterial suspension was then serially diluted and spread onto the corresponding solid medium. Plates with colony counts between 30 and 300 were used for counting, and the average value was calculated from three replicates. The formula for calculating the bile salt tolerance of the strain is:

[0074] Bile salt tolerance (%) = Nx / N × 100%

[0075] In the formula: Nx represents the number of viable bacteria at 5h (CFU / mL); N represents the number of viable bacteria at 0h (CFU / mL).

[0076] Conclusion: The results of the bile salt tolerance test are attached. Figure 5 As shown, at a bile salt concentration of 0.3%, the survival rate of strain D27 reached an astonishing 1000%, meaning that at this concentration, not only did all strains survive, but significant proliferation also occurred. Even when the bile salt concentration was increased to 0.5%, the survival rate remained at 75%, demonstrating its strong adaptability to high bile salt environments.

[0077] Given that bile salt concentrations in the gut are typically between 0.1% and 0.3%, the high survival and proliferative capacity of D27 within this range indicates that it can stably survive and function within the gut. This is crucial for probiotics, as they need to colonize and multiply in the gut to exert their health benefits on the host.

[0078] In summary, these results strongly demonstrate that *Lactobacillus assemblica* D27 possesses excellent bile salt tolerance, enabling it to stably survive and proliferate in the high-bile-salt environment of the gut. This characteristic enhances D27's potential as a probiotic, particularly in maintaining gut health and balancing the gut microbiota. Therefore, D27 holds promise as an effective probiotic strain for improving and maintaining gut health in animals.

[0079] Example 3: Probiotic properties of *Ligilactobacillus animalis* D27

[0080] 1. Antibacterial ability

[0081] The inhibitory effect on pathogenic bacteria was tested using the perforation method. Animal-associated Lactobacillus D27 strain was inoculated at 5% into MRS liquid medium and incubated statically at 37°C for 24 hours. The culture was then centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected. Pathogenic strains of Escherichia coli, Salmonella, and Staphylococcus aureus were inoculated separately into MH medium and incubated overnight at 37°C to prepare pathogen suspensions. The bacterial suspensions were diluted with PBS to a concentration of 10. 6 CFU / mL, spread the indicator bacteria evenly on MH solid medium with a cotton swab and punch a hole, inject 200 μL of bacterial supernatant into each well, gently cover the plate and place it upright in a 37℃ constant temperature incubator, observe after 12 h, and measure the diameter of the inhibition zone with vernier calipers.

[0082] Conclusion: Animals combined with Lactobacillus D27 showed antibacterial effects against Escherichia coli, Salmonella, and Staphylococcus aureus on MH agar medium as follows: Figure 6 As shown in the figure. The results indicate that *Lactobacillus animalis* D27 can form significant inhibition zones on MH agar medium, especially showing outstanding inhibitory effects against *Escherichia coli* and *Salmonella*. The diameter of the inhibition zones was precisely measured using calipers; the diameter of the inhibition zone for *E. coli* was 19.42 ± 0.68 mm, while the diameter for *Salmonella* was 21.63 ± 0.59 mm, and the diameter for *Staphylococcus aureus* was 21.26 ± 0.49 mm, demonstrating the good antibacterial ability of *Lactobacillus animalis* D27.

[0083] Specifically, in the antibacterial test against *Escherichia coli*, *Lactobacillus animalis* D27 formed an inhibition zone with a diameter of 19.42 ± 0.68 mm, indicating its effective inhibition of *E. coli* growth. In the antibacterial test against *Salmonella*, *Lactobacillus animalis* D27 formed an inhibition zone with a diameter of 21.63 ± 0.59 mm, further demonstrating its strong antibacterial effect against *Salmonella*. In the antibacterial test against *Staphylococcus aureus*, *Lactobacillus animalis* D27 formed an inhibition zone with a diameter of 21.26 ± 0.49 mm, indicating that *Lactobacillus animalis* D27 also has a certain inhibitory effect on *Staphylococcus aureus*.

[0084] Overall, these data indicate that *Lactobacillus assemblica* D27 exhibits significant antibacterial activity against *Escherichia coli*, *Salmonella*, and *Staphylococcus aureus*, and could serve as an effective bioantibacterial agent for the control and prevention of pathogenic infections.

[0085] 2. Self-cohesive force

[0086] The bacterial strain was inoculated into MRS medium and cultured overnight. After centrifugation at 4000 rpm, the bacterial culture was washed twice with PBS and the OD was adjusted. 600 =1.0. The OD of the bacterial culture was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 600 The value is denoted as (A0). The bacterial suspension is incubated statically in a 37°C incubator, and the supernatant is collected to determine the OD value. 600 The value is denoted as (Ax). The experiment is set up with three replicates, and the result is calculated according to the formula.

[0087] Self-cohesive force (%) = (1-Ax)×A0*100%

[0088] Conclusion: Self-aggregation is a phenomenon in which probiotic strains condense to form cell clusters. This phenomenon helps them gain a competitive advantage in harsh environments to resist the invasion of harmful bacteria. Self-aggregation enhances the survival ability and stability of probiotics and is one of their important characteristics.

[0089] The self-aggregation capacity of *Lactobacillus assemblica* D27 at different time points, as shown in the attached figure. Figure 7 As shown, the experimental results indicate that the self-cohesion capacity of *Lactobacillus animalis* D27 was 36.4% after 1 hour, indicating a moderate level of self-cohesion ability. After 3 hours, the self-cohesion capacity of *Lactobacillus animalis* D27 significantly increased, reaching 78.6%, demonstrating a high level of self-cohesion ability.

[0090] Lactobacillus animalis D27 began to show some self-aggregation within 1 hour, forming preliminary cell aggregates. This level of self-aggregation provided the probiotic with a certain degree of stability and survival advantage in the early stages. After extending the culture time to 3 hours, the self-aggregation of Lactobacillus animalis D27 increased significantly, reaching 78.6%, indicating that the degree of aggregation between its cells was significantly enhanced. This high self-aggregation means that Lactobacillus animalis can form more compact cell aggregates, thereby maintaining its stability and competitive advantage in adverse environments for a longer period of time.

[0091] In summary, *Lactobacillus animalis* exhibits good self-aggregation properties, showing moderate self-aggregation within 1 hour and reaching a high level of self-aggregation after 3 hours. This self-aggregation characteristic not only helps it better resist the invasion of harmful bacteria in harsh environments but also enhances its application potential as a probiotic, providing significant advantages for its practical application.

[0092] 3. Hydrophobicity

[0093] Take 2 mL of bacterial suspension, centrifuge at 10000 rpm for 1 min at room temperature, discard the supernatant, wash twice with PBS, and adjust the OD. 600=0.8, denoted as A0. Add an equal volume of xylene to the above liquid, let it stand for 1 h, 2 h, and 3 h, and measure the OD of the lower aqueous phase. 600 Absorbance, denoted as Ax. The formula for calculating hydrophobicity is as follows:

[0094] Hydrophobicity (%) = (1 - Ax / A0) × 100%

[0095] Conclusion: When evaluating the hydrophobic properties of probiotic surfaces, the adhesion ability of the strain to hydrocarbons is usually used as an important evaluation indicator. The surface hydrophobicity of a strain reflects its adaptability and survival ability in the process of interacting with the host and its environment; the animal-associated lactobacillus D27 provided by this invention achieved a hydrophobicity of 71.58% after 3 hours, indicating that it has high hydrophobicity;

[0096] In hydrophobicity tests, *Lactobacillus taeniformis* D27 showed a significantly enhanced ability to adhere to hydrocarbons after 3 hours of cultivation, exhibiting a hydrophobicity of 71.58%. Based on a hydrophobicity greater than 60%, *Lactobacillus taeniformis* D27 was classified as a highly hydrophobic strain. This high hydrophobicity indicates that the strain can better adhere to and survive in environments containing hydrocarbons, which is significant for its adaptability to different hosts and environments. High hydrophobicity not only enhances the survival ability of *Lactobacillus taeniformis* D27 but may also improve its colonization ability in complex environments such as the gut, thus contributing to its probiotic functions. Furthermore, high hydrophobicity may also affect the interaction between the strain and host cells, promoting its beneficial effects within the host.

[0097] Example 4: Safety of Ligilactobacillus animalis D27

[0098] 1. Drug sensitivity test

[0099] Antibiotic susceptibility testing of *Lactobacillus assemblica* in animals was performed using the disk diffusion method (KB method). Single colonies were picked and incubated in MRS liquid medium for 4 hours, and the viable count was adjusted to 10⁻⁶. 8 CFU / mL, evenly spread on MRS solid medium. Use tweezers to pick up the antimicrobial susceptibility test strips and attach them to the surface of the medium, pressing gently to ensure firm adhesion. Incubate the plates in anaerobic conditions at 37°C for 16 hours, then measure the diameter of the inhibition zone. Each test is repeated three times. Result interpretation follows the 2023 version of CLSIM 100 "Standard for the Implementation of Antimicrobial Susceptibility Testing".

[0100] Table 2. Animal sensitivity assessment for combined lactobacillus drugs

[0101]

[0102]

[0103] Conclusion: The results are shown in Table 2 above. The *Lactobacillus ani* D27 strain provided by this invention showed sensitivity to five antibiotics: cefazolin, norfloxacin, chloramphenicol, polymyxin B, and oxacillin. This means that the strain was easily inhibited or killed when these antibiotics were used, indicating that these antibiotics may have a good antibacterial effect against this strain. The *Lactobacillus ani* D27 strain showed moderate sensitivity to four antibiotics: ampicillin, ciprofloxacin, amoxicillin, and piperacillin. This means that these drugs may require higher doses or be used in combination with other antibiotics to achieve satisfactory results against this strain.

[0104] The *Lactobacillus assemblica* D27 strain provided by this invention exhibits resistance to six antibiotics: penicillin, amikacin, gentamicin, erythromycin, trimethoprim-sulfamethoxazole, and tetracycline. This indicates that these antibiotics may be ineffective or have poor efficacy against this strain. Although *Lactobacillus assemblica* is resistant to some antibiotics, it remains sensitive to many commonly used antibiotics. This characteristic means that there are still a variety of effective antibiotic options available against this strain.

[0105] 2. Hemolysis test

[0106] Single colonies were picked and inoculated onto Columbia blood agar plates and incubated at 37°C for 24 hours. Hemolysis was assessed based on the color and morphology around the colonies. Result interpretation: Staphylococcus aureus ATCC 25923 was used as a positive control strain; a clear halo around the colony was considered positive.

[0107] Conclusion: The results of the hemolysis test are attached. Figure 8 As shown, this invention uses Staphylococcus aureus as a positive control and observes that during its growth on blood agar plates, a clear area appears around the colony. This is because Staphylococcus aureus has β-hemolytic activity, which can destroy red blood cells, leading to red blood cell lysis and thus forming a clear area. Under the same experimental conditions, no clear area appears around Lactobacillus animalis, indicating that this strain does not have hemolytic activity, i.e., it does not destroy red blood cells. This is an important safety indicator, meaning that Lactobacillus animalis will not damage the host's red blood cells and has high safety. Since Lactobacillus animalis did not show hemolytic activity on blood agar plates, this indicates that when applied to animals or humans, it will not cause red blood cell rupture or related hemolytic reactions, thus further supporting its safety and reliability as a probiotic or other biological agent.

[0108] In summary, this invention isolated a strain of lactic acid bacteria from the feces of 27-day-old calves and named it *Lactobacillus animalis* D27. Through morphological observation and 16S rRNA gene sequencing analysis, the strain was preliminarily identified as *Lactobacillus animalis*. This identification process included detailed microscopic observation to determine its morphological characteristics and gene alignment based on the whole genome sequence to confirm its taxonomic position.

[0109] To evaluate the probiotic properties of *Lactobacillus animalis* D27, we conducted several functional assays. The results showed that this strain exhibited inhibitory activity against *Escherichia coli*, *Salmonella*, and *Staphylococcus aureus*. On MH agar, through inhibition zone experiments, we observed that *Lactobacillus animalis* D27 effectively inhibited the growth of these three pathogenic bacteria, with inhibition zone diameters of 19.42±0.68 mm (*Escherichia coli*), 21.63±0.59 mm (*Salmonella*), and 21.26±0.49 mm (*Staphylococcus aureus*), demonstrating significant antibacterial activity.

[0110] In addition, Lactobacillus animalis D27 also exhibits good self-aggregation and hydrophobicity. These characteristics indicate that the strain has a strong adhesion ability in the intestinal environment and can bind tightly to intestinal epithelial cells, thereby colonizing the intestine. This is crucial for its role as a probiotic in maintaining the balance of the intestinal microecology and provides excellent strain resources for the development of probiotics for the prevention and treatment of calf diarrhea.

[0111] Example 5: Application of Ligilactobacillus animalis D27 in calf diarrhea

[0112] Experiment location: ShouNong Modern Agriculture Technology Co., Ltd., Dingzhou City, Hebei Province.

[0113] Experimental animals: Sixty Chinese Holstein bull calves (weighing 43±2 kg) that were born naturally, had similar body conditions, and had been successfully passively immunized were selected as experimental animals.

[0114] Experimental Design: A single-factor randomized experimental design was used to randomly divide calves into four groups: control group (C group), low-dose group (L group), medium-dose group (M group), and high-dose group (H group), with 15 male calves in each group. The experimental period was 56 days. Each calf in the L group received 5 × 10⁻⁶ Lactobacillus avium assorti (5 × 10⁻⁶) as a supplement to its normal milk. 8 cfu / d); In group M, each calf was given Lactobacillus avium (5×10⁻⁶ cfu / d) as part of its normal milk. 9 cfu / d); In group H, each calf's normal milk was supplemented with Lactobacillus animalis (5×10⁻⁶ cfu / d); 10(cfu / d); an equal volume of blank culture medium was added to the normal milk of each calf in the control group. Calves were fed the bacterial culture solution starting on day 2 after collection, and all groups of calves followed the feeding and management model of Hebei Dingzhou Shou Nong Modern Agricultural Technology Co., Ltd. At 3 days of age, calves were introduced to easily digestible and absorbable pelleted starter feed, ensuring free access to food and water. The feed amount was adjusted according to the principle that uneaten feed should not exceed 10% of the total feed intake. The measured values ​​of the nutrient composition (dry matter basis) of the calf starter feed are shown in Table 3 below:

[0115] Table 3. Measured values ​​(%) of basic dry matter nutrients in starter feed

[0116]

[0117]

[0118] Indicator Measurement: During the experiment, the daily feed intake and uneaten feed amounts for calves were accurately recorded. After collecting the uneaten feed, the dry matter intake (DMI) of the calves was calculated. At days 1, 28, and 56 of age, the body weight of the experimental calves was measured before the morning feed, and the average daily gain (ADG) was calculated. Feed conversion ratio (FCR) was calculated based on DMI and ADG. The FCR calculation formula is as follows:

[0119] FCR = DMI / ADG

[0120] Starting from 3 days after birth, the fecal condition of each group of calves was photographed and recorded daily. A 4-point scale was used to evaluate the feces; the fecal scoring criteria are detailed in Table 4. The number of days and number of calves with diarrhea were also recorded. Medication administration, frequency, and duration were also recorded. The diarrhea rate was calculated using the following formula:

[0121] Diarrhea frequency (%) = (Number of calves with diarrhea × Number of days with diarrhea) / (Number of experimental calves × Number of experimental days) × 100

[0122] Table 4. Stool Scoring Criteria

[0123]

[0124]

[0125] Data were analyzed using one-way ANOVA with SPSS 25.0 software. Results are expressed as mean ± standard deviation. Diarrhea rate was analyzed using the chi-square test in SPSS 25.0 software. P < 0.01 was considered highly significant, and P < 0.05 was considered significant.

[0126] The weight changes of calves at different stages are shown in Table 5 below. The results show that the control group, medium-dose group, and high-dose group significantly increased the calf weight at 56 days and significantly increased the average daily weight gain from 0 to 56 days. The medium-dose group significantly reduced the feed conversion ratio. These results demonstrate that adding a certain dose of *Lactobacillus tamariscina* can improve the growth performance of calves.

[0127] Table 5. Effects of combined Lactobacillus in animals on growth performance of calves

[0128]

[0129] Note: Different labels on the same side indicate significant differences, and different lowercase letters indicate significant differences (P<0.05).

[0130] The results of the effects of *Lactobacillus animalis* combined with animal diarrhea frequency and diarrhea scores on calf diarrhea are shown in Table 6 below. The results show that, compared with the control group, the medium-dose and high-dose groups significantly reduced the frequency of diarrhea in calves from 0 to 28 days of age, while the low-dose and medium-dose groups significantly reduced the frequency of diarrhea from 28 to 56 days of age. This demonstrates that feeding calves with *Lactobacillus animalis* D27 can reduce the frequency of diarrhea in calves.

[0131] Table 6. Effects of combined Lactobacillus in animals on the frequency and diarrhea score of diarrhea in calves.

[0132]

[0133] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A type of animal-associated lactobacillus ( Ligilactobacillus animalis) D27, its characteristics are: This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 9, 2024, with accession number CGMCC No. 30561.

2. The use of the animal-associated lactobacillus D27 as described in claim 1 in the preparation of a feed additive for the prevention and treatment of calf diarrhea.

Images