Lactobacillus animalis from cat and its application for antagonizing escherichia coli infection
By providing a live lyophilized powder formulation of feline lactobacillus MFB-01, the problems of antibiotic resistance and intestinal flora imbalance caused by antibiotic abuse have been solved, achieving effective antagonism against Escherichia coli and maintenance of intestinal health.
Patent Information
- Application Number
- CN202611511123.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-28
- Publication Date
- 2026-11-06
Smart Images
Figure CN123042906A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a feline lactobacillus strain that antagonizes Escherichia coli infection and its applications. Background Technology
[0002] Currently, gastrointestinal diseases caused by enteric pathogens are among the most common illnesses in pets, especially kittens, with diarrhea being the most frequent clinical symptom. Antibiotics are the primary treatment for pet diseases; however, the overuse of antibiotics in treating bacterial infections leads to antibiotic resistance and disrupts the balance of the animal's gut microbiota, resulting in weakened immunity and other problems that seriously threaten pet health. Therefore, there is an urgent need to find green alternatives for the prevention and treatment of intestinal infections in pets.
[0003] Probiotics have functions such as enhancing animal immunity, maintaining intestinal flora balance, and resisting bacterial infections. Among them, feline probiotics can alleviate intestinal dysfunction caused by pathogenic Enterobacteriaceae, while improving intestinal barrier-related indicators and promoting intestinal homeostasis. For example, combinations of *Saccharomyces boulardii* and *Lactobacillus* have been shown to maintain intestinal health in cats. Probiotics can effectively prevent pathogens from colonizing the gut through various mechanisms, including secreting antibacterial active molecules (organic acids and antimicrobial peptides, etc.), inhibiting pathogens at intestinal adhesion sites, or enhancing intestinal epithelial cell function. Therefore, probiotics can serve as a safe, green, and pollution-free alternative to antibiotics to maintain the intestinal health of pet cats. Currently, there are few feline *Lactobacillus* strains that possess both antagonistic activity against *E. coli* and good probiotic properties; therefore, developing a new feline *Lactobacillus* strain that can effectively antagonize *E. coli* infection is of great significance. Summary of the Invention
[0004] This invention provides a feline lactobacillus strain with antagonistic activity against Escherichia coli infection and its applications.
[0005] In a first aspect, the present invention provides a feline animal lactobacillus strain with antagonistic activity against Escherichia coli infection. The feline animal lactobacillus is Lactobacillus animalis MFB-01, deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan, China, on May 23, 2024, with accession number CCTCC NO:M20241046, and classified as Lactobacillus animalis MFB-01.
[0006] Secondly, the present invention provides a feline animal lactobacillus agent comprising the aforementioned animal lactobacillus MFB-01.
[0007] Furthermore, the bacterial agent is a live lyophilized powder preparation.
[0008] Furthermore, the viable bacteria count in the live bacteria lyophilized powder formulation is 1×10⁻⁶. 8 CFU / g-1×10 10 CFU / g.
[0009] Furthermore, the live bacteria lyophilized powder formulation also contains a lyophilization protectant, and the mass ratio of the animal lactobacillus MFB-01 to the lyophilization protectant is 1:5-1:10.
[0010] Thirdly, the present invention provides the application of the aforementioned feline lactobacillus in the preparation of drugs antagonizing Escherichia coli infection.
[0011] Beneficial effects: 1. The animal lactobacillus MFB-01 strain of the present invention was isolated from the feces of healthy cats and has strong acid and bile salt resistance. After being cultured for 3 hours in MRS medium with pH=3.5 and 0.1% bile salt, the survival rates were 93.63±7.73% and 89.06±17.62%, respectively, indicating that the strain can successfully reach the intestine and exert its probiotic effect.
[0012] 2. The animal lactobacillus MFB-01 provided by this invention has strong adhesion properties to intestinal epithelial cells HT-29.
[0013] 3. The animal lactobacillus MFB-01 provided by the present invention has a significant inhibitory effect on Escherichia coli, with an inhibition zone diameter of 12±2 mm. Attached Figure Description
[0014] Figure 1 This is a schematic diagram of an optical microscope image of Gram staining of Lactobacillus animalis MFB-01 according to the present invention.
[0015] Figure 2 This is a phylogenetic tree diagram of the animal lactobacillus MFB-01 of the present invention.
[0016] Figure 3 This is a graph evaluating the intestinal epithelial cell adhesion ability of the animal lactobacillus MFB-01 of the present invention.
[0017] Figure 4 This is a schematic diagram illustrating the antibacterial activity of the fermentation broth of *Lactobacillus animalis* MFB-01 against *Escherichia coli*. Detailed Implementation
[0018] To enable those skilled in the art to better understand the present invention, the present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and do not constitute a limitation on the content and scope of protection of the present invention.
[0019] Example 1: Isolation, purification and identification of Lactobacillus animalis MFB-01 1. Isolation and purification of Lactobacillus animalis strain MFB-01 Take 5g of healthy cat feces as a sample, add it to 45mL of sterile PBS solution, vortex to mix, and prepare 10 - ¹Diluent, then serially diluted 10-fold with sterile PBS to 10⁻¹⁰. -6 100 μL of each serially diluted solution was spread onto MRS agar plates containing CaCO3 and anaerobically incubated at 37°C for 48 hours. Based on morphological characteristics such as the presence of a calcified zone around the colony, colony size, shape, and color, single colonies with typical morphology were selected and inoculated into MRS liquid medium. The culture was incubated at 37°C for 24 hours. The culture was then streaked onto MRS solid medium for purification. This purification process was repeated 2-3 times until a morphologically consistent purified strain was obtained. The purified strain was inoculated into MRS liquid medium and cultured to the logarithmic growth phase. 20% glycerol was added as a protectant, and the culture was stored at -80°C for later use. The microscopic examination results of *Lactobacillus animalis* MFB-01 are shown below. Figure 1 As shown, they are Gram-positive and appear as purple rods under oil immersion, consistent with the morphological characteristics of animal lactobacillus.
[0020] 2. Identification of Lactobacillus animalis MFB-01 by 16S rDNA sequencing Lactobacillus animalis MFB-01 was cultured in MRS liquid medium. Genomic DNA was extracted using a bioengineering bacterial genomics kit. The 16S rDNA of the strain was amplified by PCR using universal primers 27F and 1492R. The primer sequences were: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR product was sent to BGI Genomics Co., Ltd. for sequencing. The sequence length was 1437 bp. NCBI BLAST alignment showed over 99% homology with Lactobacillus animalis P38, confirming it as a Lactobacillus animalis strain, named Lactobacillus animalis MFB-01. Sequencing results were compared with the GenBank database. Multiple sequence alignment of the 16S rRNA gene sequences of the target strain and reference strain was performed using MEGA 11 software, and a phylogenetic tree was constructed using the neighbor-joining method. The reliability of each branch was evaluated through 1000 repeated traditional nonparametric Bootstrap tests. Figure 2 The data shows nodes with a bootstrap support rate greater than 50%.
[0021] The 16S rDNA sequence of Lactobacillus animalis MFB-01 is as follows: Example 2: Preparation of Lactobacillus animalis MFB-01 bacterial agent 1. Preparation of fermentation broth: The activated Lactobacillus animalis strain MFB-01 was inoculated into sterile LB liquid medium and cultured at 37°C and 200 r / min for 24 hours to obtain the fermentation broth.
[0022] 2. Preparation of bacterial suspension: The fermentation broth was centrifuged at 4°C and 8000 rpm for 10 minutes, the supernatant was discarded, and the bacterial precipitate was collected. A sterile lyophilization protectant solution (trehalose) was added to the precipitate, and the mixture was shaken to resuspend the cells. The viable cell concentration in the suspension was adjusted to 1 × 10⁻⁶. 8 CFU / mL was used to obtain a bacterial suspension.
[0023] 3. Freeze-drying: The bacterial suspension was dispensed into freeze-drying trays and placed in a vacuum freeze dryer. It was pre-frozen at -40°C for 4 hours, then sublimated and dried at a vacuum of ≤10Pa and a cold trap temperature of -50°C for 24 hours, followed by desorption and drying for 6 hours to obtain the live Lactobacillus animalis MFB-01 freeze-dried powder preparation.
[0024] 4. Finished product inspection: The obtained freeze-dried powder had a moisture content of 5% and a viable bacteria count of 1×10⁻⁶. 8 CFU / g, freeze-dried survival rate reached 93.24%.
[0025] Example 3: Evaluation of the tolerance performance of Lactobacillus animalis MFB-01 1. Evaluation of the acid resistance of Lactobacillus animalis MFB-01 Lactobacillus animalis MFB-01 was inoculated into MRS liquid medium at a 5% inoculum and cultured anaerobically at 37°C for 24 hours. The cultured bacterial solution was centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, and the bacterial pellet was collected for later use.
[0026] MRS liquid culture media with pH values of 1.5, 2.5, 3.5, 4.5, and 5.5 were prepared using sterile 1 mol / L hydrochloric acid solution. The collected bacterial precipitates were resuspended in MRS liquid culture media of each pH value, and inoculated into the corresponding pH MRS liquid culture media at a 1% inoculum rate. The media were then anaerobically incubated at 37°C for 3 hours.
[0027] Samples were taken at 0h and 3h, and the viable bacterial count was determined using the plate count method, following the national standard GB4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count". The survival rate was calculated using the following formula: Survival rate (%) = (Number of viable bacteria at 3h / Number of viable bacteria at 0h) × 100%. The isolated *Lactobacillus animalis* MFB-01 maintained a high survival rate of 93.63% after culturing in MRS medium at pH 3.5.
[0028] The survival rate of Lactobacillus animalis MFB-01 in simulated gastric acid solution is shown in Table 1.
[0029] Table 1. Survival rate of Lactobacillus animalis MFB-01 at different pH values
[0030] This shows that Lactobacillus animalis MFB-01 has strong acid resistance and can tolerate the acidic environment of the stomach.
[0031] 2. Evaluation of bile salt tolerance of Lactobacillus animalis MFB-01 Lactobacillus animalis MFB-01 was inoculated into MRS liquid medium at a 5% inoculum and cultured anaerobically at 37°C for 24 hours. The cultured bacterial solution was centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, and the bacterial pellet was collected for later use.
[0032] MRS liquid culture media with bile salt concentrations of 0% (control group), 0.05%, 0.1%, 0.2%, and 0.4% were prepared using sterile bile salt solution. The collected bacterial precipitates were resuspended in sterile physiological saline and inoculated into MRS liquid culture media with different bile salt concentrations at an inoculation rate of 1%, and then anaerobically incubated at 37°C for 3 hours.
[0033] Samples were taken at 0h and 3h, and the viable bacterial count was determined using the plate count method, following the national standard GB4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count". The survival rate was calculated using the following formula: Survival rate (%) = (Number of viable bacteria at 3h / Number of viable bacteria at 0h) × 100%. The isolated *Lactobacillus animalis* MFB-01 maintained a high survival rate of 89.06% after culturing in MRS medium with a bile salt concentration of 0.1%.
[0034] The survival rate of *Lactobacillus animalis* MFB-01 in intestinal bile salt simulated solution is shown in Table 2.
[0035] Table 2. Survival rate of Lactobacillus animalis MFB-01 at different bile salt concentrations
[0036] Example 4: Evaluation of the adhesion properties of Lactobacillus animalis MFB-01 to intestinal epithelial cells Lactobacillus animalis MFB-01 was inoculated into MRS liquid medium at a 5% inoculum and cultured anaerobically at 37°C for 24 hours. The cultured bacterial solution was centrifuged at 4000 r / min for 10 min, and the bacterial pellet was collected. The bacterial pellet was washed twice with sterile PBS buffer and set aside for later use.
[0037] Human colon cancer cells HT-29 in logarithmic growth phase were adjusted to a concentration of 5 × 10⁻⁶ cells using DMEM medium containing 10% fetal bovine serum. 5 Cells / mL were inoculated at a rate of 2 mL per well into a 6-well plate and cultured overnight until cell adhesion occurred. The washed cells were resuspended in serum-free DMEM medium, and the bacterial culture was added to the 6-well plate at an MOI of 100:1. The plates were incubated at 37°C and 5% CO2 for 6 h. After incubation, the medium was discarded, and the cells were washed three times with sterile PBS buffer to remove unadhered free cells. Trypsin-EDTA digestion solution was added to each well for 5 min to digest the cells. Digestion was terminated with 1 mL of DMEM medium containing 10% fetal bovine serum, and the cells were mixed thoroughly by pipetting. 15 μL of the culture was spread onto MRS agar plates and anaerobically cultured at 37°C for 48 h. Colony counts were then performed, and the count value was taken as the number of adherent viable cells. The adhesion rate was calculated using the following formula: Adhesion rate (%) = (Number of adherent viable cells / Initial inoculation number) × 100%. Three replicates of cells that were not digested with trypsin were taken, washed twice with PBS, and fixed with methanol for 20 min. The fixed cells were Gram stained and examined under a microscope. 10-20 fields of view were randomly selected to observe the adhesion performance of Lactobacillus animalis MFB-01 to HT-29 cells.
[0038] like Figure 3 As shown, the adhesion rate of Lactobacillus animalis MFB-01 to human colon cancer cells HT-29 was 90.26%, indicating that Lactobacillus animalis MFB-01 has a strong ability to adhere to intestinal epithelial cells in the intestine.
[0039] Example 5: Effect of Lactobacillus animalis MFB-01 on the antibacterial activity of Escherichia coli Lactobacillus animalis MFB-01 was inoculated into MRS liquid medium at a 5% inoculum and anaerobically cultured at 37°C for 24 hours. The cultured bacterial suspension was centrifuged at 4000 rpm for 10 minutes. The supernatant from the logarithmic fermentation phase was collected and placed in a sterile petri dish. Sterile antimicrobial susceptibility testing tablets were added and soaked for 2-3 minutes. The Escherichia coli bacterial suspension was evenly spread onto LB agar plates using sterile cotton swabs. After absorption, the tablets were placed on the plates, and the plates were incubated at 37°C for 24 hours. The antibacterial effect was observed, and the diameter of the inhibition zone was measured using calipers. The results are as follows: Figure 4As shown, the fermentation supernatant of Lactobacillus animalis MFB-01 has a strong antibacterial effect against Escherichia coli, with an inhibition zone diameter of 12±2 mm.
[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A feline lactobacillus strain with antagonistic activity against Escherichia coli infection, characterized in that, The feline-derived *Lactobacillus animalis* MFB-01 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan, China, on May 23, 2024, with accession number CCTCC NO: M20241046, and classified as *Lactobacillus animalis* MFB-01.
2. A feline animal lactobacillus inoculant, characterized in that, It contains the animal lactobacillus MFB-01 as described in claim 1.
3. The microbial agent according to claim 2, characterized in that, The bacterial agent is a live lyophilized powder preparation.
4. The microbial agent according to claim 3, characterized in that, The viable bacteria count in the live bacteria freeze-dried powder preparation is 1×10⁻⁶. 8 CFU / g-1×10 10 CFU / g.
5. The microbial agent according to claim 3, characterized in that, The live bacteria freeze-dried powder formulation also contains a freeze-drying protectant, and the mass ratio of the animal lactobacillus MFB-01 to the freeze-drying protectant is 1:5-1:
10.
6. The use of the feline animal lactobacillus according to claim 1 in the preparation of a drug antagonizing Escherichia coli infection.