Animal combined lactobacillus D27 as well as screening method and application thereof

Abstract

The invention discloses animal combined lactobacillus D27 as well as a screening method and application thereof, and relates to the technical field of microorganisms, and the key points of the technical scheme are as follows: the animal combined lactobacillus D27 is preserved in China General Microbiological Culture Collection Center (CGMCC) on May 9, 2024, and the preservation number is CGMCC No.30561. The animal combined lactobacillus D27 has the advantages that the animal combined lactobacillus D27 can be used for screening the animal combined lactobacillus D27; the animal combined lactobacillus D27 provided by the invention is a strain with excellent performance screened from the digestive tract of the calf, has the characteristics of stronger acid resistance, cholate resistance, strong adhesiveness, high lactic acid production and the like, and provides an excellent strain resource for the development of probiotics for preventing and treating calf diarrhea.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and more specifically to an animal-associated lactobacillus D27 and a screening method and application thereof. Background Art

[0002] In the dairy farming industry, the health and development of calves is crucial to the entire farming process. The calf period is a critical stage in the life of dairy cows. Because their digestive and immune systems are not fully mature, they are less adaptable to environmental changes and stress responses. This makes calves susceptible to digestive system problems, especially diarrhea. According to statistics, the incidence of digestive system diseases in calves is as high as 60%, which seriously affects their growth and development and overall health.

[0003] Diarrhea not only leads to poor nutrient absorption and delayed growth and development, but in severe cases may also lead to the death of calves. Therefore, preventing and managing calf diarrhea has become one of the important challenges in animal husbandry. In order to meet this challenge, the application of probiotics has gradually become a research hotspot, and lactic acid bacteria occupy an important position among them. Lactic acid bacteria are a type 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 feed microorganisms allowed for use. A total of 34 microorganisms are allowed for use, of which as many as 17 are lactic acid bacteria. This shows that lactic acid bacteria have great potential for application in animal husbandry.

[0004] Given that lactic acid bacteria strains from different sources have different probiotic properties, researchers are committed to screening out lactic acid bacteria strains that are effective for calves. These strains are believed to be useful as probiotic additives, effectively preventing and reducing the occurrence of diarrhea by improving the intestinal health and immune function of calves. To this end, the development and utilization of excellent lactic acid bacteria resources suitable for calves has become one of the important goals in current animal husbandry research and practice. In addition, recent studies have also shown that specific lactic acid bacteria strains are not only beneficial to calves, but their potential probiotic properties may also have a positive impact on other livestock and human health. Therefore, exploring the multiple probiotic functions of lactic acid bacteria and developing multi-purpose lactic acid bacteria products are hot topics in current scientific research and industrial applications. Through these research and development, not only can the production efficiency of animal husbandry be improved, but also sustainable and healthy agricultural development can be promoted.

[0005] Therefore, the present invention aims to screen a lactic acid bacterium with good performance from calf, explore its physiological function and probiotic properties, and provide excellent strain resources for the development of probiotics for preventing calf diarrhea. Summary of the invention

[0006] The invention aims to provide an animal-associated lactobacillus Ligilactobacillus anima lis D27 and a screening method and application thereof. The animal-associated lactobacillus is obtained by screening strains with excellent performance, and has the characteristics of strong acid resistance, bile salt resistance, strong adhesion, high lactic acid production and the like, thereby providing excellent strain resources for the development of probiotics for preventing and treating calf diarrhea.

[0007] The above-mentioned technical purpose of the present invention is achieved through the following technical scheme: an animal-associated lactobacillus Ligilactobacillus animalis D27, which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC for short) on May 9, 2024, and its deposit number is: CGMCC No.30561, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), the postal code is 100101, and the telephone number is 010-64807355.

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

[0009] Another object of the present invention is to provide a use of the animal-associated Lactobacillus D27 as described above in the preparation of an animal feed additive.

[0010] Another object of the present invention is to provide a use of the animal-associated lactobacillus D27 as described above in preventing and treating 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] Serial dilution: dilute fresh stool sample with sterile saline;

[0013] Plate coating: Spread the diluted bacterial solution on MRS solid screening medium containing cysteine ​​and mupirocin lithium salt;

[0014] Anaerobic culture: Incubate the cells in an anaerobic incubator at 37°C for 24 to 72 hours until bacterial colonies grow.

[0015] Single colony picking: single colonies with yellow outer rings were selected and transferred to MRS medium containing cysteine ​​and bromocresol purple;

[0016] Re-purification: Repeat the picking and culturing steps until a pure single colony is obtained;

[0017] Culture and preservation: After enrichment and culture of the single colony, 50% glycerol was added and stored at -80°C.

[0018] By adopting the above technical scheme, the animal-associated Lactobacillusanimalis D27 strain provided by the present invention has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.30561 and a deposit date of May 9, 2024. The strain contains a 16srRNA gene sequence that is at least 95% consistent with SEQ ID NO.1, ensuring its stability and consistency at the gene level, facilitating classification and identification, and improving 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 the strain;

[0019] The animal-associated lactobacillus D27 provided by the present invention has a significant inhibitory effect on Escherichia coli, Salmonella and Staphylococcus aureus; the animal-associated lactobacillus D27 provided by the present invention is strictly separated, purified and preserved, and as a standardized experimental model, provides reliable materials for subsequent scientific research, is convenient for various experiments and verifications, and through studying the biological characteristics of the D27 strain, deeply understands its functions and mechanisms in different environments, and provides a theoretical basis for further application and development; the antibacterial property of the D27 strain provides an important reference and possibility for the development of new anti-infection drugs and biological treatment methods, reduces the use of antibiotics, reduces the generation and spread of drug-resistant bacteria, contributes to environmental protection and sustainable development, and has important ecological benefits.

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

[0021] 1. The animal-combined lactobacillus provided by the present invention is obtained by screening strains with excellent performance, and has the characteristics of strong acid resistance, bile salt resistance, strong adhesion, high lactic acid production, etc., providing excellent strain resources for the development of probiotics for preventing and treating calf diarrhea;

[0022] 2. The use of the animal-combined lactobacillus D27 provided by the present invention in the preparation of animal feed additives can improve the intestinal health of animals, enhance immunity, improve growth performance, reduce the occurrence of diseases, and improve breeding benefits; the strain has a significant inhibitory effect on Escherichia coli and Salmonella, and can effectively reduce the infection risk of these pathogens in animals, improve the health level of animals, reduce the use of antibiotics, and promote healthy breeding. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0025] Figure 3 is a line graph of the growth curve and acid production capacity of animal-combined lactobacillus D27 in Example 1 of the present invention;

[0026] Figure 4 is a bar graph of the survival rate of the artificial gastrointestinal fluid of the animal combined with Lactobacillus D27 in Example 2 of the present invention;

[0027] Figure 5 is a bar graph of bile salt survival rate of animal-associated Lactobacillus D27 in Example 2 of the present invention;

[0028] Figure 6 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; Figure C is a schematic diagram of the antibacterial test results of Staphylococcus aureus;

[0029] Figure 7 is a histogram of the self-agglutination ability and hydrophobicity of animal-combined Lactobacillus D27 in Example 3 of the present invention;

[0030] Figure 8 4 is a comparison chart of the hemolysis test results in Example 4 of the present invention; wherein, the left chart is a chart showing the results of the hemolysis test on Staphylococcus aureus; and the right chart is a chart showing the results of the hemolysis test on animals combined with Lactobacillus D27. DETAILED DESCRIPTION

[0031] The present invention provides an animal-combined lactobacillus D27 and a screening method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] In the following experiments, animal-associated Lactobacillus D27 is the animal-associated Lactobacillus D27 (Ligilact obacillus animalis) deposited in the General Microbiological Center of the China Culture Collection Administration of Microorganisms (referred to as CGMC C) on May 9, 2024, with a deposit number of CGMCC No.30561, and the deposit address is No. 3, Yard No. 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 from 27-day-old healthy calves collected by Shounong Modern Agricultural Technology Co., Ltd. in Dingzhou City, Hebei Province, which were frozen and taken back immediately after collection.

[0034] Sources of reagents: pepsin (1:10000), trypsin (1:250), Beijing Solebow Technology Co., Ltd.; MRS medium, Beijing Solebow Co., Ltd.; bifidobacterium proliferation medium, TPY medium, mupirocin lithium salt, agar powder, Beijing Luqiao Co., Ltd.; lysis buffer, Mix, universal primers 27F, 1492R, Beijing Liuhe BGI Gene Technology Co., Ltd.; penicillin, ampicillin, cefazolin, amikacin, gentamicin, erythromycin, norfloxacin, ciprofloxacin, co-trimoxazole, chloramphenicol, polymyxin B, tetracycline, amoxicillin, piperacillin, oxacillin, Hangzhou Microbiological Reagent Co., Ltd.

[0035] Main instruments: nucleic acid electrophoresis imager, Bio-Rad; microscope, OLYMPU S; electric constant temperature incubator, Shanghai Jinghong Experimental Equipment Co., Ltd.; microplate reader, Gene Co., Ltd.; high pressure sterilizer; micro-aerobic incubator, anaerobic incubator, Huayuexing Instrument Co., Ltd.; high pressure sterilizer, Hefei Huatai Medical Co., Ltd.

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

[0037] The present invention provides a separation and purification method of the above-mentioned animal-associated lactobacillus Ligilactobacillus animalis D27, comprising the following steps: collecting fresh animal feces samples, using sterile containers to ensure that external contamination is avoided, quickly transporting the collected samples to a laboratory at 4° C., and processing them within 2 hours after collection;

[0038] Prepare sterile saline, i.e. 0.9% NaCl solution, place 1 gram of stool sample in 10 ml of sterile saline, mix thoroughly with a sterile glass rod mixer to obtain an initial suspension, ensuring that all operations are performed under sterile conditions; take 1 ml of the initial suspension and add it to 9 ml of sterile saline, mix thoroughly, and obtain 10 -1 Dilution; Repeat the above steps to make 10 -1 The diluent was diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 For isogradient dilutions, use new sterile pipette tips and sterile test tubes for each dilution level;

[0039] Prepare an MRS solid screening medium plate containing 0.05 g / L cysteine ​​and 0.05 g / L mupirocin lithium salt, and ensure that the medium is evenly distributed on the plate; take 100 μL of the gradient dilution solution and drop it on the MRS solid screening medium plate, and use a sterile coating stick to evenly spread the added dilution solution on the surface of the medium to ensure that the entire plate is covered;

[0040] Place the coated plate in an anaerobic workstation to make the oxygen concentration reach the anaerobic level, culture at a constant temperature of 37°C for 24 to 72 hours, and observe the growth of the colonies;

[0041] Observe the colony morphology on the culture medium plate, pick a single colony with typical morphology and larger size; inoculate the picked 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 line" operation on the plate to ensure the isolation of the single colony, culture at 37°C under anaerobic conditions for 24 to 48 hours, observe the color change of the colony, and pick a single colony with a yellow outer ring;

[0042] The single colony with a yellow outer ring was inoculated again on a new MRS solid medium plate and streaked repeatedly, and the above streaking and culturing steps were repeated until a single colony with uniform morphology and purity was obtained;

[0043] The purified single colony was inoculated into MRS liquid culture medium or solid culture medium containing 0.05 g / L cysteine, cultured under anaerobic conditions at 37° C., and 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: Pick a single colony and inoculate it in liquid culture medium for 24 hours, take 30μL of lysis solution in a 200μL PCR tube, shake the bacterial solution and take 5μL of lysis solution to mix. After a simple centrifugation, place it in a PCR amplification instrument at 80℃ for 15min for lysis, and the lysis program setting is: 80℃ for 20min; store at 4℃.

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

[0046] The sequencing results show that the 16srRNA gene sequence of the animal combined lactobacillus Ligilactobacillus animalis D27 strain is about 1500bp long, and the gene similarity after Blast comparison is 100%. The whole gene sequencing ANI analysis was performed, and the ANI analysis results are shown in the attached Figure 1 As shown, a comparative analysis of homologous gene sequences was performed, and the analysis results showed that it was most highly correlated with Ligilactobacillus animalis P38, with a similarity of 98.44%.

[0047] 1. Morphological observation: Pick a single colony with complete morphology and good growth in the culture medium, stain it using Gram staining, and observe it using an oil microscope;

[0048] The observation results are as follows Figure 2 As shown, the observation results show that the animal-associated Lactobacillus colonies are facultative anaerobic Gram-positive bacteria. The colonies are white colonies with smooth edges and raised surfaces. Microscopic observation shows that the strains are short straight rods, existing alone or in pairs.

[0049] 2. Growth curve and acid production capacity

[0050] Pick a single colony and inoculate it in 10mL solid culture medium for 12h to make seed solution. The set conditions are 3% inoculation volume, 37℃ temperature, pH 6.2±0.2, and measure the OD value (nm=600) and pH value of the bacterial solution every 2h until 24h; measure the OD value of the bacterial solution every 6h. 600 The value and pH value were measured until 36 hours of culture; the OD value of the bacterial solution was measured again at 48 hours. 600 With the culture time as the horizontal axis, OD 600 , pH value is used as the vertical axis, and the growth curve and acid production curve are drawn;

[0051] The growth curve and acid production curve are shown in the attached Figure 3As shown in the figure, from the growth curve and acid production curve, it can be found that the animal-associated lactobacillus is in the logarithmic growth period of bacteria within 0 to 6 hours, and enters the plateau period after 12 hours. After 12 hours of culture, the pH of the culture medium dropped to 4.25, and then slowly decreased. After 48 hours, the pH of the culture solution dropped to 4.04.

[0052] 3.Physical and chemical properties

[0053] The physical and chemical properties of lactic acid bacteria were identified using a complete set of biochemical tubes. The direct inoculation method was used to pick the same purified and isolated colonies from the plate with an inoculation needle and inoculate them into ampoules containing different substrates. The culture was carried out at 36±1℃ for 24 hours and the results were interpreted.

[0054] As shown in Table 1 below, animal-associated Lactobacillus is able to utilize raffinose, inulin, sucrose, lactose, maltose, salicin, and cellobiose.

[0055] Table 1 Physicochemical properties of animal-associated lactobacilli

[0056]

[0057]

[0058] Example 2: Tolerance of Animals with Lactobacillus animalis D27

[0059] 1. Acid resistance

[0060] Pick a single colony and inoculate it in 10mL solid culture medium for 12h to make seed liquid. Inoculate 3% inoculation amount in MRS liquid culture medium overnight culture at 4000r / min, centrifuge for 10min, discard the supernatant, add 10mL PBS for standby. Use HCL to adjust the pH value of MRS culture medium to 2.0, 3.0 and 4.0, inoculate animal combined lactobacillus in different pH culture medium at 1% inoculation amount for anaerobic culture for 3h, take 0h and 3h digestion fluid for viable count detection, and calculate its survival rate.

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

[0062] Where: 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: In the acid resistance experiment, Animal-associated Lactobacillus D27 still survived at pH = 2, the survival rate reached 70% at pH = 3, and the survival rate reached more than 100% at pH = 4; the above results show the excellent adaptability and survival ability of Animal-associated Lactobacillus D27 to acidic environment, which enables it to remain active when passing through the animal's digestive system, thereby exerting its probiotic effect in the intestine, which is of great significance for the development of animal feed additives that can pass through the acidic environment of the stomach and exert their effects in the intestine.

[0064] 2. Artificial gastrointestinal fluid tolerance

[0065] Artificial gastric juice: prepare 0.5% NaCl solution, add 0.3% pepsin, adjust the pH value to 2.5 and 3.0 with 1 mol / L HCl, then filter and sterilize with a 0.22 μm microporous filter membrane after fully dissolving.

[0066] Artificial intestinal fluid: prepare 0.5% NaCl solution, add 0.1% trypsin, adjust the pH value to 8.0 with 0.1 mol / L NaOH, then filter and sterilize with a 0.22 μm microporous filter membrane after fully dissolving.

[0067] After overnight culture of animal combined lactobacillus, centrifuge at 4000r / min for 10min, discard the supernatant, and add 10mL PBS for standby use. Animal combined lactobacillus was inoculated into artificial gastric juice and artificial intestinal juice at a 1% inoculation amount for anaerobic culture for 3h, and the digestive juice at 0h and 3h was taken for viable bacterial count detection and its survival rate was calculated.

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

[0069] Where: 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 the simulated artificial gastric fluid pH = 2.5 and pH = 3.0, animal combined with Lactobacillus D27 showed a relatively high survival rate (40.60% and 70%). Figure 4 As shown, this shows that it has a certain degree of adaptability to the weakly acidic environment in gastric juice. Even under such acidic conditions, animal-associated Lactobacillus D27 can still maintain a certain amount of activity, which helps it pass through the stomach into the intestines; in simulated artificial intestinal fluid, animal-associated Lactobacillus D27 showed an extremely high survival rate (96.86%), which shows that its living environment in the intestines is more favorable. The alkaline conditions and lower acidity of the intestine provide an ideal environment for the growth and reproduction of animal-associated Lactobacillus D27, allowing it to multiply in large quantities and play the role of probiotics;

[0071] The above experimental results show that animal-derived Lactobacillus D27 can effectively adapt to and overcome the acidic environment of gastric juice when passing through the stomach of animals and entering the intestines, and has good survival and growth ability in the intestines. These characteristics are crucial for the development of oral probiotic products and ensuring their effectiveness in animals, especially in maintaining digestive tract health and balancing intestinal flora.

[0072] 3. Bile salt tolerance

[0073] Weigh ox bile salt and add it to MRS liquid culture medium to make the bile salt concentration in the culture medium 0.3%, 0.5%, 1%, 3%, 5%, sterilize at 121℃ for 20min, take 0.1mL of activated bacterial suspension, add it to 9.9mL culture medium, culture at 37℃ for 0h and 5h, draw 0.1mL of bacterial suspension, spread it on the corresponding solid culture medium after gradient dilution, select plates with colony counts between 30 and 300, and calculate the average value of 3 parallels. The formula for calculating the tolerance of the strain to bile salts is:

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

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

[0076] Conclusion: The results of the bile salt tolerance experiment are shown in the attached Figure 5 As shown in the figure, when the bile salt concentration was 0.3%, the survival rate of the D27 strain reached an astonishing 1000%, which means that at this concentration, not only did all the strains survive, but they also proliferated significantly. Even when the bile salt concentration was increased to 0.5%, the survival rate remained at 75%, demonstrating its strong adaptability to the high bile salt environment;

[0077] Considering that the bile salt concentration in the intestine is usually between 0.1% and 0.3%, the high survival rate and proliferation ability of D27 within this range means that it can survive and function stably in the intestine. This is crucial for probiotics because they need to colonize and reproduce in the intestine to exert health benefits to the host;

[0078] In summary, these results fully demonstrate that animal-derived Lactobacillus D27 has excellent bile salt tolerance, which enables it to stably survive and proliferate in a high bile salt environment such as the intestine. This property enhances the potential of D27 as a probiotic, especially in maintaining intestinal health and balancing intestinal flora. Therefore, D27 is expected to become an effective probiotic strain for improving and maintaining intestinal health in animals.

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

[0080] 1. Antibacterial ability

[0081] The punching method was used to test the inhibitory ability against pathogenic bacteria. The animal combined lactobacillus D27 strain was inoculated into MRS liquid culture medium at 5%, cultured at 37°C for 24 hours, centrifuged at 4000 rpm for 10 minutes, and the supernatant of the bacterial solution was collected. The pathogenic strains of Escherichia coli, Salmonella, and Staphylococcus aureus were inoculated into MH culture medium, cultured overnight at 37°C, and pathogenic bacteria suspensions were prepared. The bacterial suspension was diluted with PBS to a concentration of 10 6 CFU / mL, use a cotton swab to evenly spread the indicator bacteria on the MH solid culture medium and punch holes, inject 200μL of bacterial supernatant into each hole, cover the plate lightly and place it upright in a 37℃ constant temperature incubator, observe after 12h of incubation, and measure the diameter of the inhibition zone with a vernier caliper.

[0082] Conclusion: The antibacterial effect of animal combined lactobacillus D27 on Escherichia coli, Salmonella and Staphylococcus aureus on MH agar medium is as follows: Figure 6 As shown. The results showed that animal-associated lactobacillus D27 was able to form a significant inhibition zone on MH agar medium, especially in terms of the inhibition of Escherichia coli and Salmonella. The diameter of the inhibition zone was accurately measured with a vernier caliper. The diameter of the inhibition zone of Escherichia coli was 19.42±0.68mm, while the diameter of the inhibition zone of Salmonella was 21.63±0.59mm, and the diameter of the inhibition zone of Staphylococcus aureus was 21.26±0.49mm, showing the good antibacterial ability of animal-associated lactobacillus D27.

[0083] Specifically, in the antibacterial test against Escherichia coli, the animal-combined Lactobacillus D27 formed an inhibition zone with a diameter of 19.42±0.68mm, indicating that it can effectively inhibit the growth of Escherichia coli. In the antibacterial test against Salmonella, the animal-combined Lactobacillus D27 formed an inhibition zone with a diameter of 21.63±0.59mm, further proving its strong antibacterial effect on Salmonella. In the antibacterial test against Staphylococcus aureus, the animal-combined Lactobacillus D27 formed an inhibition zone with a diameter of 21.26±0.49mm, and the animal-combined Lactobacillus D27 also has a certain inhibitory effect on Staphylococcus aureus.

[0084] Overall, these data indicate that Lactobacillus animalis D27 has significant antibacterial ability in inhibiting Escherichia coli, Salmonella and Staphylococcus aureus, and can be an effective bioantibacterial agent for the control and prevention of pathogenic bacterial infections.

[0085] 2. Self-cohesion

[0086] The strain was inoculated into MRS medium and cultured overnight. The bacterial solution was centrifuged at 4000 rpm and washed twice with PBS. The OD 600 =1.0. Measure the OD of the bacterial solution with an enzyme-labeled instrument 600 The bacterial suspension was placed in a 37°C constant temperature incubator, and the upper bacterial liquid was aspirated to measure the OD 600 The value is recorded as (Ax). The experiment was repeated three times and calculated according to the formula.

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

[0088] Conclusion: Self-aggregation is a phenomenon in which probiotic strains aggregate to form cell clusters, which helps to form a competitive advantage in a harsh environment to resist the invasion of harmful bacteria. Self-aggregation can enhance the survival ability and stability of probiotics and is one of the important characteristics of probiotics.

[0089] The self-cohesion of animal-associated Lactobacillus D27 at different time points, as shown in the attached Figure 7 As shown, the experimental results show that the self-cohesion capacity of animal-associated Lactobacillus D27 was 36.4% after 1 hour, indicating that it has a moderate degree of self-cohesion capacity. After 3 hours of the experiment, the self-cohesion capacity of animal-associated Lactobacillus D27 increased significantly to 78.6%, indicating that it has a high self-cohesion capacity;

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

[0091] In summary, animal-associated Lactobacillus has good self-aggregation properties, showing moderate self-aggregation within 1 hour and reaching a higher self-aggregation ability after 3 hours. This self-aggregation property not only helps it better resist the invasion of harmful bacteria in harsh environments, but also enhances its application potential as a probiotic, providing it with significant advantages in practical applications.

[0092] 3. Hydrophobicity

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

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

[0095] Conclusion: When measuring the hydrophobicity of the surface of probiotics, the adhesion ability of the strain to hydrocarbons is usually used as an important evaluation index. The surface hydrophobicity of the strain reflects its adaptability and survival ability in the process of interaction with the host and its environment; the hydrophobicity of the animal-combined Lactobacillus D27 provided by the present invention reached 71.58% after 3 hours, which shows that it has a high degree of hydrophobicity;

[0096] When the hydrophobicity test was conducted on the animal-associated Lactobacillus D27, after 3 hours of cultivation, its adhesion ability to hydrocarbons was significantly enhanced, showing a hydrophobicity rate of 71.58%. According to the standard of hydrophobicity rate greater than 60%, the animal-associated Lactobacillus D27 was identified as a highly hydrophobic strain. This high hydrophobicity indicates that the strain can better adhere and survive in an environment containing hydrocarbons, which is of great significance for its adaptability in different hosts and environments; high hydrophobicity not only enhances the survival ability of animal-associated Lactobacillus D27, but also may improve its colonization ability in complex environments such as the intestine, thereby contributing to its probiotic function. In addition, high hydrophobicity may also affect the interaction between the strain and the host cells, promoting its beneficial effects in the host.

[0097] Example 4: Safety of animal-derived Lactobacillus animalis D27

[0098] 1. Drug sensitivity test

[0099] The antibiotic sensitivity test of animal combined lactobacillus was carried out by paper diffusion method (KB method). Single colonies were selected and cultured in MRS liquid medium for 4 h, and the number of viable bacteria was adjusted to 10 8 CFU / mL, evenly spread on MRS solid culture medium, pick up the drug-sensitive paper with tweezers, stick it on the surface of the culture medium, and press lightly to make the paper and the culture medium firmly adhere. Place the plate in 37℃ anaerobic medium for 16 hours and then measure the diameter of the inhibition zone. Each group of tests is repeated three times. The results are judged according to the 2023 version of CLSIM100 "Implementation Standards for Antimicrobial Susceptibility Tests".

[0100] Table 2 Determination of drug sensitivity of animal combined lactobacillus

[0101]

[0102]

[0103] Conclusion: The results are shown in Table 2 above. The animal-associated Lactobacillus D27 strain provided by the present invention is sensitive to five antibiotics, namely, cefazolin, norfloxacin, chloramphenicol, polymyxin B and oxacillin, which means that the strain is easily inhibited or killed when these antibiotics are used, indicating that these antibiotics may have a good antibacterial effect against the strain; the animal-associated Lactobacillus D27 strain is moderately sensitive to four antibiotics, namely, ampicillin, ciprofloxacin, amoxicillin and piperacillin, which means that these drugs may require higher doses or be used in combination with other antibiotics to achieve satisfactory effects against the strain;

[0104] The animal-associated Lactobacillus D27 strain provided by the present invention shows resistance to six antibiotics, including penicillin, amikacin, gentamicin, erythromycin, co-trimoxazole and tetracycline, which indicates that these antibiotics may be ineffective or have poor effects against this strain; although the animal-associated Lactobacillus is resistant to some antibiotics, it still remains sensitive to a variety of commonly used antibiotics, and this characteristic means that there are still a variety of effective antibiotic options for this strain.

[0105] 2. Hemolysis test

[0106] Pick a single colony and inoculate it on Columbia blood agar plate, incubate at 37℃ for 24h, and judge the hemolysis according to the color and morphology around the colony. Result judgment: Staphylococcus aureus ATCC 25923 is used as the positive reference strain. If a clear transparent circle appears around the colony, it is judged as positive.

[0107] Conclusion: The results of the hemolysis test are shown in the attached Figure 8 As shown, the present invention uses Staphylococcus aureus as a positive control, and observes that during its growth on the blood plate, transparent areas appear around the colonies. This is because Staphylococcus aureus has a β-hemolytic effect, which can destroy red blood cells and cause red blood cell lysis, thereby forming a transparent area; under the same experimental conditions, no transparent area appears around the animal-associated Lactobacillus, which indicates that the strain has no hemolytic effect, that is, it will not destroy red blood cells, which is an important safety indicator, meaning that the animal-associated Lactobacillus will not cause damage to the host's red blood cells and has a higher safety; since the animal-associated Lactobacillus does not show hemolytic activity on the blood plate, this indicates that when applied to animals or humans, it will not cause red blood cell rupture or related hemolytic reactions, thereby further supporting its safety and reliability as a probiotic or other biological preparation.

[0108] In summary, the present invention isolated a lactic acid bacteria strain from the feces of a 27-day-old calf and named it Animal-Associated Lactobacillus D27. Through morphological structure observation and 16s rRNA gene sequencing analysis of the strain, it was preliminarily identified as Animal-Associated Lactobacillus. This identification process includes detailed microscopic observation to determine its morphological characteristics, and gene comparison based on the whole gene sequence to confirm its taxonomic status;

[0109] In order to evaluate the probiotic properties of animal-associated Lactobacillus D27, we conducted a number of functional assays. The results showed that the strain had a certain inhibitory ability against Escherichia coli, Salmonella and Staphylococcus aureus; on MH agar medium, through the inhibition zone experiment, we observed that animal-associated Lactobacillus D27 could effectively inhibit the growth of these three pathogens, with the diameters of the inhibition zones being 19.42±0.68mm (Escherichia coli), 21.63±0.59mm (Salmonella) and 21.26±0.49mm (Staphylococcus aureus inhibition zone), respectively, showing significant antibacterial activity;

[0110] In addition, Animal-associated Lactobacillus D27 also exhibited good self-cohesion and hydrophobicity. These properties indicate that the strain has strong adhesion ability in the intestinal environment and can tightly combine with intestinal epithelial cells to colonize in the intestine, which is crucial for its role as a probiotic in maintaining the balance of 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 treating calf diarrhea

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

[0113] Experimental animals: 60 Chinese Holstein bull calves (weight 43±2Kg) that gave birth naturally, were in similar physical condition, and were successfully passively immunized were selected as experimental animals.

[0114] Experimental design: The experiment used a single-factor randomized experimental design to randomly divide the calves into four groups, namely the 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. Animal combined Lactobacillus (5×10 8 cfu / d; Animal-associated Lactobacillus (5×10 9 cfu / d; in group H, animal-associated Lactobacillus (5×10 10cfu / d); an equal amount of blank culture medium was added to the normal milk of each calf in the control group. Calves were fed with bacterial liquid from the second day after collection, and each group of calves was fed and managed according to the feeding and management model of Shounong Modern Agricultural Technology Co., Ltd. in Dingzhou City, Hebei Province. At 3 days of age, calves were guided to eat easily digestible and easily absorbed granular starter feed to ensure that calves could eat and drink freely, and the feeding amount was adjusted according to the principle that the remaining feed did not exceed 10% of the feeding amount. The actual measured values ​​of the nutritional composition (dry matter basis) of calf starter feed are shown in Table 3 below:

[0115] Table 3 Measured values ​​of dry matter basic nutritional components of starter feed (%)

[0116]

[0117]

[0118] Index determination: During the test period, the amount of calf starter feed and the amount of leftover feed were accurately recorded every day, and the dry matter intake (DMI) of the calf was calculated after collecting the leftover feed. The body weight of the experimental calves was weighed before morning feeding at the 1st, 28th and 56th days of age, and the average daily weight gain (ADG) of the calves was calculated; the feed conversion efficiency (FCR) was calculated based on DMI and ADG. The FCR calculation formula is as follows:

[0119] FCR=DMI / ADG

[0120] Starting from the 3rd day of birth, take photos and record the feces of each group of calves every day. Use a 4-point system to judge the feces. See Table 4 for the feces scoring criteria. Also record the number of days of diarrhea and the number of calves with diarrhea. Also record the medication status, number of medications, and days of medication for the calves. The diarrhea rate calculation formula is as follows:

[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 Fecal scoring criteria

[0123]

[0124]

[0125] SPSS 25.0 statistical software was used for one-way analysis of variance, and the results were expressed as mean ± standard deviation. The diarrhea rate was analyzed using the chi-square test in SPSS 25.0 software. P < 0.01 was considered extremely significant, and P < 0.05 was considered significant.

[0126] The weight changes of calves at different stages are shown in Table 5. The results show that compared with the control group, the medium-dose group and the high-dose group, the weight of calves at 56 days was significantly increased, and the average daily weight gain from 0 to 56 days was significantly increased. The medium-dose group can significantly reduce the feed-to-weight ratio. The results show that adding a certain dose of animal combined lactobacillus can improve the growth performance of calves.

[0127] Table 5 Effect of animal combined with lactobacillus on growth performance of calves

[0128]

[0129] Note: The same shoulder with different marks indicates significant difference, and different lowercase letters indicate significant difference (P<0.05).

[0130] The results of the effect of Animal Combined Lactobacillus on the frequency and score of diarrhea in calves are shown in Table 6. The results show that compared with the control group, the medium-dose group and the high-dose group can significantly reduce the frequency of diarrhea in calves from 0 to 28 days, and the low-dose group and the medium-dose group can significantly reduce the frequency of diarrhea from 28 to 56 days. It is proved that feeding Animal Combined Lactobacillus D27 to calves can reduce the frequency of diarrhea in calves.

[0131] Table 6 Effect of animal combined with lactobacillus on diarrhea frequency and diarrhea score in calves

[0132]

[0133] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. An animal-associated lactobacillus Ligilactobacillus animalis D27, characterized in that: This strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on May 9, 2024, and its deposit number is: CGMCC No.30561.

2. An animal-associated lactobacillus D27 according to claim 1, characterized in that: The 16s rRNA gene sequence has a 16s rRNA gene sequence that is at least 95% identical to SEQ ID NO.

1.

3. Use of the animal-associated Lactobacillus D27 as claimed in claim 1 in the preparation of animal feed additives.

4. Use of the animal-associated lactobacillus D27 as claimed in claim 1 in preventing calf diarrhea.

5. A method for screening an animal-associated lactobacillus D27 as claimed in claim 1, characterized in that: The following steps are involved: Serial dilution: dilute fresh stool sample with sterile saline; Plate coating: Spread the diluted bacterial solution on MRS solid screening medium containing cysteine ​​and mupirocin lithium salt; Anaerobic culture: Incubate the cells in an anaerobic incubator at 37°C for 24 to 72 hours until bacterial colonies grow. Single colony picking: single colonies with yellow outer rings were selected and transferred to MRS medium containing cysteine ​​and bromocresol purple; Re-purification: Repeat the picking and culturing steps until a pure single colony is obtained; Culture and preservation: After enrichment and culture of the single colony, 50% glycerol was added and stored at -80°C.

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