A strain of lactobacillus animalis of canine origin capable of degrading oxalate and its use in improving kidney stones in dogs

Abstract

The application discloses a canine animal lactobacillus for degrading oxalic acid and application thereof in improving canine calculus, and belongs to the technical field of microorganisms. An animal lactobacillus is isolated from fresh feces of a healthy Chinese countryside dog, and animal lactobacillus Nourse22-002 is obtained through mutagenesis and breeding. The oxalic acid degradation efficiency of the bacterium is high, the survival rate of the bacterium in gastrointestinal juice is high, the intestinal colonization ability of the bacterium is strong, and the bacterium can quickly flocculate. In addition, the animal lactobacillus Nourse22-002 can enhance the immunity of a dog, inhibit the growth of harmful bacteria and reduce the ammonia odor of feces. The animal lactobacillus Nourse22-002 prepared into a postbiotic can relieve inflammation of a dog suffering from urinary calculus. The animal lactobacillus Nourse22-002 obtained through mutagenesis and breeding is preserved in the China Center Type Culture Collection Center of Wuhan University in Wuhan, China, on January 28, 2026, and the preservation number is CCTCC NO: M2026270.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a canine lactobacillus strain that degrades oxalic acid and its application in improving canine gallstones. Background Technology

[0002] Urolithiasis in dogs is a common urinary tract disease, with calcium oxalate stones having a high incidence and being difficult to treat. The formation of calcium oxalate stones is closely related to metabolic disorders of oxalate in dogs. Oxalate mainly comes from dietary intake and endogenous production in the liver, and is one of the metabolic products of dogs. After ingested oxalate is absorbed by the intestines, some is metabolized and degraded in the liver, and the remainder is excreted through the kidneys. When high concentrations of oxalate are excreted in urine, they easily combine with calcium ions in the urine to form highly insoluble calcium oxalate crystals. These crystals continuously accumulate and grow in the urinary system, eventually forming visible stones. Unlike magnesium ammonium phosphate stones, which can be dissolved by medication, calcium oxalate stones, once formed, cannot be removed by conventional litholytic therapy and usually require invasive treatment such as surgical incision or laser lithotripsy. Therefore, reducing the amount of oxalate obtained from a dog's diet is of great significance in alleviating canine urolithiasis.

[0003] Existing technology has already used probiotics to improve urinary tract health. Patent CN118872776A discloses a pet food composition that improves urinary tract health by inhibiting urinary stone formation. The oxalate decomposing agent includes various probiotics (such as Lactobacillus oxalate, Lactobacillus animalis, Lactobacillus acidophilus, etc.). Among them, Lactobacillus animalis (… Lactobacillus animalis ( ) are Gram-positive facultative anaerobic bacteria that have the functions of inhibiting pathogens, enhancing immunity and regulating intestinal microecology, and have therefore received widespread attention.

[0004] The optimal pH range for the growth of *Lactobacillus amylovorus* is 5.5–6.5 (Proposal of *Lactobacillus amylovorus* subsp. *animalis* subsp. nov. and an emended description of *Lactobacillus amylovorus*, 2024), while the pH of gastric juice in healthy dogs is approximately 2.5 (Gastric pH and serum gasstrin concentration in age-matched healthy dogs and dogs with chronic kidney disease, 2023). This highly acidic environment makes *Lactobacillus amylovorus* easily inactivated by the strong gastric acid after oral administration, preventing it from colonizing the intestines in large quantities and thus hindering its effectiveness. However, there are currently no reports on *Lactobacillus amylovorus* isolated from dogs and, through mutagenesis and selection, developed that can tolerate the canine gastrointestinal environment and colonize well in the canine intestines. Summary of the Invention

[0005] The purpose of this invention is to provide an animal lactobacillus that can tolerate the canine gastrointestinal environment, has good colonization ability, and has the function of degrading oxalic acid, and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a strain of canine lactobacillus ( Lactobacillus animalis The strain Nourse22-002 exhibits significantly improved oxalate degradation rate, gastrointestinal survival rate, and intestinal colonization ability compared to the original strain. This strain was deposited on January 28, 2026, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO: M2026270 and accession name: *Lactobacillus animalis* Nourse22-002. Lactobacillus animalis Nourse22-002, taxonomically named Lactobacillus animalis ( Lactobacillus animalis ).

[0007] Secondly, the present invention provides the application of the canine lactobacillus Nourse22-002 in canine food or feed.

[0008] Furthermore, the dog food or feed includes, but is not limited to, dog food, dog treats, or dog health food.

[0009] Furthermore, the canine lactobacillus Nourse22-002 can also be applied to other pets, such as foxes.

[0010] Furthermore, the canine lactobacillus Nourse22-002 can enhance the dog's immunity, inhibit harmful bacteria, and reduce canine fecal odor.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: In vitro studies have shown that *Lactobacillus animalis* Nourse22-002 exhibits highly efficient decomposition of potassium oxalate, with a degradation rate of 70.12%; it demonstrates good tolerance and adaptability to simulated canine gastrointestinal fluid, maintaining a survival rate of 83.24%; it significantly inhibits various common pathogenic bacteria such as *Escherichia coli*, *Salmonella*, *Staphylococcus aureus*, *Shigella*, and *Clostridium perfringens*, with an increased inhibition zone diameter compared to the original strain; and it possesses rapid and efficient cell aggregation and flocculation capabilities, which helps enhance its competitive colonization advantage in the intestinal environment.

[0012] In vivo studies have shown that *Lactobacillus animalis* Nourse22-002 can significantly reduce oxalate levels in rat urine; after 15 days of continuous feeding to dogs with calcium oxalate crystals, the calcium oxalate crystals in the urine of the dogs had completely disappeared; it exhibits strong colonization ability in dogs, and even after 7 days of cessation of feeding, a viable count of 3.52 1 g CFU / g can still be detected in the feces of dogs; the postbiotic preparation can effectively reduce the abundance of harmful bacteria in the canine intestine. Attached Figure Description

[0013] Figure 1 This is the phylogenetic tree of Lactobacillus animalis Nourse22-002 in this invention.

[0014] Figure 2 This is a scanning electron microscope image of Lactobacillus animalis Nourse22-002 in this invention.

[0015] Figure 3 These are microscopic images of urine crystals in rats fed with Lactobacillus animalis Nourse22-002 on days 0 and 7, respectively. A and C are microscopic images of the crystals before and after feeding with Lactobacillus animalis Nourse22-002 for 7 days, respectively. B and D are microscopic images of the crystals before and after feeding with PBS for 7 days, respectively.

[0016] Figure 4 The images show microscopic images of urine crystals in dogs with urinary crystals fed with Lactobacillus animalis Nourse22-002 on days 0 and 21, respectively. A and C are microscopic images of crystals before and after feeding Nourse22-002 on day 21, respectively; B and D are microscopic images of crystals before and after feeding with empty capsules on day 21, respectively. Detailed Implementation

[0017] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. It should be understood that these embodiments are merely illustrative of the invention and should not be construed as limiting the scope of protection of the invention. Those skilled in the art can make non-essential improvements and adjustments to the invention based on the above description.

[0018] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. The source, trade name, and components of the reagents used, if necessary, are indicated upon their first appearance, and subsequent use of the same reagents, unless otherwise specified, are identical to the initial indication.

[0019] This invention relates to the following culture media and reagents: The artificial gastric fluid was prepared according to the method of Li Hong et al. (2020) with slight modifications, wherein the pepsin content was adjusted to 25 U / mL and the pH value was adjusted to 2.0.

[0020] The artificial intestinal fluid was prepared according to the method of Xue Junmin (2018) with slight modifications, with the addition of pig bile powder (4 g / L) and the pH value adjusted to 8.3.

[0021] The intestinal digestion resistance screening medium was based on MRS basal medium, with the addition of porcine bile powder (4 g / L) and the pH adjusted to 8.3.

[0022] MRS basal culture medium was purchased from Oxoid, UK; porcine bile powder and pepsin were purchased from Sigma-Aldrich; and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0023] PCR identification of bacterial species: The 16S rDNA gene fragment of the strain was amplified by PCR using universal bacterial primers 27F and 1492R. The amplified products were sequenced, and the obtained sequences were compared with the NCBI database by BLAST to determine the species of the strain.

[0024] The universal primer sequences 27F and 1492R are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0025] The following model strains were selected for the antibacterial activity test: Escherichia coli ( Escherichia coli ) and Salmonella ( Salmonella enterica ) isolated from feces of dogs with diarrhea; Staphylococcus aureus ( Staphylococcus aureus ), Shigella flexneri (Shigella flexneri ) and Clostridium perfringens ( Clostridium perfringens The strains were purchased from the China Industrial Microbial Culture Collection Center, with strain numbers CICC 21648, CICC 21534, and CICC 22949, respectively.

[0026] Human-derived animal lactobacillus ( Lactobacillus animalis Purchased from the Culture Collection Center of the University of Gothenburg, Sweden, strain number CCUG 68343. Example 1:

[0027] (1) Isolation of bacterial strains Fresh feces were collected from healthy Chinese rural dogs, resuspended in PBS buffer, and serially diluted 10-fold. 100 μL of each diluted bacterial suspension was spread onto MRS agar plates and incubated anaerobically at 39°C for 48 h. Plates with evenly distributed and well-grown colonies (less than 200 colonies per plate, with moist cells) were selected, and single colonies were picked.

[0028] (2) Initial functional screening Single colonies were picked and inoculated into MRS liquid medium containing a final concentration of 0.1% potassium oxalate, and cultured anaerobically at 39°C until the logarithmic growth phase (OD200). 600 ≈0.80). The culture medium was transferred at a 1% (v / v) inoculum and anaerobically cultured at 39℃ for 48 h. After culture, the culture was centrifuged at 4℃, 10000 ×g for 10 min, and the supernatant was collected for oxalic acid content determination by liquid chromatography to calculate the oxalic acid degradation rate. A blank control group without inoculation was also set up. The oxalic acid degradation rate was calculated according to formula (1): Equation (1) In the formula, C 空白 The blank control group contains oxalic acid in mg / mL; C 样品 The value represents the oxalic acid content in the supernatant of the bacterial culture to be tested, in mg / mL.

[0029] Strains with an oxalic acid degradation rate greater than 50% were selected for molecular identification.

[0030] (3) Molecular identification Strains with an oxalic acid degradation rate greater than 50% were subjected to 16S rRNA gene PCR amplification and sequencing, and were identified as *Lactobacillus animalis* (Lactobacillus). Lactobacillus animalis The strain was named DW-W.

[0031] (4) Mutagenesis and breeding The original strain of *Lactobacillus animalis*, DW-W, was irradiated with 150 Gy of cobalt-60 rays for 10 min. The mutagenic bacterial culture was centrifuged at 5000 ×g for 10 min at 4 °C, and the cells were collected and inoculated into MRS liquid medium. The culture was then incubated anaerobically at 39 °C for 48 h for rejuvenation. After rejuvenation, the bacterial pellet was centrifuged at 5000 ×g for 10 min at 4 °C and inoculated into enteric-resistant selection medium. The culture was continued for 7 days, with simulated gastric digestion screening performed on days 1 and 4.

[0032] After screening for gastrointestinal resistance, the bacterial culture was serially diluted 10-fold and then isolated and cultured. Twenty larger single colonies were selected and inoculated into MRS liquid medium, and cultured anaerobically at 39°C for 5 h. OD values ​​were measured before and after culture. 600 The growth rate of each strain was calculated, and the three strains with the fastest growth rates were selected. The growth rate was calculated according to formula (2): Equation (2) In the formula, V is the growth rate (h -1 ), OD 0h and OD 5h OD before and after cultivation 600 value.

[0033] Strains DW-1, DW-18, and DW-19 showed the fastest growth rate after 5 hours of culture, with growth rates increasing by 45.45%, 40.91%, and 52.27% respectively compared to the original strain DW-W. These three strains were selected for secondary screening.

[0034] Table 1 Growth rate of Lactobacillus from canine animals DW-W 0.044 / DW-1 0.064 45.45 DW-2 0.055 25.00 DW-3 0.062 40.91 DW-4 0.054 22.73 DW-5 0.050 13.64 DW-6 0.050 13.64 DW-7 0.050 13.64 DW-8 0.061 38.64 DW-9 0.053 20.45 DW-10 0.057 29.55 DW-11 0.054 22.73 DW-12 0.056 27.27 DW-13 0.054 22.73 DW-14 0.048 9.09 DW-15 0.050 13.64 DW-16 0.045 2.27 DW-17 0.054 22.73 DW-18 0.062 40.91 DW-19 0.067 52.27 DW-20 0.057 29.55 The three selected bacterial strains were enriched and cultured separately, then inoculated into MRS liquid medium containing a final concentration of 0.1% potassium oxalate, and cultured anaerobically at 39℃ for 48 h. After the culture, the supernatant was collected, and the oxalic acid content was detected by liquid chromatography to calculate the oxalic acid degradation rate. A blank control group without inoculation was also set up. The oxalic acid degradation rate was calculated according to formula (1).

[0035] The results are shown in Table 2. DW-19 showed the highest oxalic acid degradation rate, reaching 70.12%. Therefore, DW-19 was molecularly identified and used in subsequent experiments.

[0036] Table 2 Degradation efficiency of Lactobacillus oxalate in canine animals DW-W 55.81 DW-1 55.19 DW-18 59.25 DW-19 70.12 (5) Strain identification Using the genomic DNA of the animal lactobacillus mutant strain DW-19 as a template, the 16S rDNA of the strain was identified by PCR.

[0037] Sequencing results were compared using BLAST on the NCBI website, confirming that DW-19 is *Lactobacillus animalis*. The mutant strain DW-19 was renamed *Lactobacillus animalis* Nourse22-002, and its phylogenetic tree is as follows: Figure 1 As shown, this strain is most closely related to *Lactobacillus animalis* in terms of evolutionary relationship.

[0038] The aforementioned canine-derived *Lactobacillus animalis* is currently deposited at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China, on January 28, 2026, with accession number CCTCC NO: M2026270 and accession name *Lactobacillus animalis* Nourse 22-002. Lactobacillus animalis Nourse 22-002, the suggested classification name is *Lactobacillus animalis* (…). Lactobacillus animalis )Nourse22-002.

[0039] (6) Gastrointestinal resistance and passage stability test The original strain DW-W and the mutant strain Nourse22-002 were inoculated at 1% (v / v) into MRS liquid medium and cultured anaerobically at 39℃ for 48 h. After the culture, the bacterial cells were collected and treated in artificial gastric fluid for 1 h, and then transferred to artificial intestinal fluid for 1 h. Samples were taken before and after treatment, and the viable cell count was determined by the serial dilution plate method to calculate the survival rate. The survival rate was calculated according to formula (3): Equation (3) In the formula, P is the survival rate; n 后 The number of viable bacteria in the bacterial culture after 2 hours of treatment, expressed in CFU; n 前 The number of viable bacteria in the bacterial culture before treatment is expressed in CFU.

[0040] The results are shown in Table 3. The gastrointestinal survival rate of the mutant strain Nourse22-002 (83.24%) was significantly higher than that of the original strain (53.10%).

[0041] Table 3. Gastrointestinal survival rate of Lactobacillus from canine animals DW-W 53.10 Nourse22-002 83.24 The original strain DW-W and the mutant strain DW-19 were inoculated at 2% (v / v) in MRS liquid medium and cultured under anaerobic conditions at 39℃ for 48 h. This operation was repeated for 10 generations of subculture, and the gastrointestinal resistance of the 10th generation strain was evaluated.

[0042] The results are shown in Table 4. The 10th generation Nourse22-002 still showed good gastrointestinal resistance, with a survival rate of 83.98%.

[0043] Table 4 Results of passage stability test of Lactobacillus from canine animals Gastrointestinal tolerance survival rate (%) 52.55 83.98 (7) Flocculation and sedimentation test Take the bacterial culture that has been cultured overnight and adjust the OD. 600 To obtain an initial bacterial suspension of 1.0 μL, take 10 mL and place it in a 15 mL glass test tube. Incubate at room temperature. Carefully aspirate 1 mL of the supernatant from 1 cm below the surface at 0, 1, 2, 3, 5, 10, and 20 h, and measure the OD value. 600 The flocculation rate is calculated according to formula (4): Equation (4) Where: OD 600上清 It is to detect the OD of the supernatant. 600 Value; OD 600初始 OD of initial bacterial culture supernatant 600 Value, OD 600初始 =1 The flocculation rate test results of Nourse22-002 are shown in Table 5. According to the flocculation rate test results, the flocculation rate of the Nourse22-002 mutant strain is significantly faster than that of the original strain DW-W. After standing for 1 h, the flocculation rate of the mutant strain Nourse22-002 reached 42.5%, while that of the original strain DW-W was only 18.6%; after standing for 5 h, the flocculation rate of the mutant strain Nourse22-002 was as high as 93.8%, while that of the original strain DW-W was 72.5%; the mutant strain Nourse22-002 only needed about 5 h to reach a flocculation rate of over 90%, while the original strain DW-W required 20 h. The results indicate that the mutant strain Nourse22-002 has rapid and efficient flocculation characteristics.

[0044] Table 5. Flocculation rate of Lactobacillus from canine animals 0 0 0 1 18.6 42.5 2 32.3 68.7 3 45.8 82.4 5 72.5 93.8 10 88.4 97.2 20 90.2 98.5 (8) Antibacterial ability test Escherichia coli, Salmonella, Staphylococcus aureus, Shigella flexneri, and Clostridium perfringens were cultured to the logarithmic growth phase. 10 µL of each bacterial culture was added to a Petri dish, and approximately 20 mL of LB agar (40–45°C) was poured in. After cooling and solidification, wells were punched in the dish. 50 µL of the fermentation broth of the test strain was added to each well, and the dish was incubated upright at 37°C under aerobic conditions for 24 h. After incubation, the diameter of the inhibition zone (DIZ) was measured using calipers. The diameter of the inhibition zone was calculated as the average of the maximum and minimum values ​​(the inner diameter of the punch was 6 mm; if there was no significant inhibitory effect, the diameter of the inhibition zone was recorded as 6 mm).

[0045] The results are shown in Table 6. The inhibition zone diameters of Nourse22-002 against Escherichia coli, Staphylococcus aureus, Salmonella, Shigella flexneri, and Clostridium perfringens were 15.0 mm, 15.0 mm, 16.5 mm, 16.0 mm, and 16.5 mm, respectively.

[0046] Table 6 Evaluation of the antibacterial activity of Lactobacillus in canine animals

[0047] (9) Evaluation of the feeding effect on rats with oxalic acid crystallization model Using SD rats as experimental animals, an oxalate crystallization model was established by feeding them a diet containing 5% oxalate and allowing them free access to water. The oxalate crystallization model was considered successfully established when calcium oxalate crystals were clearly observed in the rat urine under a microscope, and the urine pH was within the range of 5.5–6.0. Figure 3 (As shown). The experiment was divided into the Nourse22-002 group and the PBS control group. Both groups continued to be fed a diet containing 5% oxalic acid and had free access to water. The DW-19 group was given 1×10⁻⁶ oz. by oral gavage daily. 9 CFU / rat of Nourse22-002 bacterial culture was administered, while the PBS control group received an equal volume of PBS solution via gavage. Urine was collected from rats on day 0 and day 7 after gavage, and the oxalate content in the urine was detected using liquid chromatography. The oxalate content reduction rate was calculated. The oxalate content reduction rate was calculated according to formula (5): Equation (5) In the formula, C0 is the oxalic acid content in urine on day 0 after gavage, in mg / mL, and C7 is the oxalic acid content in urine on day 7 after gavage, in mg / mL.

[0048] The results are shown in Table 7. After 7 days of oral administration of Nourse22-002 bacterial solution, the oxalic acid content in the urine of rats decreased to 0.157 mg / mL, a decrease of 66.16% compared with before feeding. The number of crystals in the urine of rats in the Nourse22-002 group and the PBS group was observed under a microscope on days 0 and 7 of feeding. On day 0 of feeding, a large number of crystals were observed in the urine of rats in the Nourse22-002 group and the PBS group. Figure 3 (As shown in A and B); no crystals were observed in the urine of rats in the Nourse22-002 group on day 7 of feeding. Figure 3 As shown in Figure C), crystals were still present in the urine of rats in the PBS group. Figure 3 (As shown in D).

[0049] Table 7. Reduction rate of oxalate content in rat urine by canine lactobacillus Example 2:

[0050] Dogs with calcium oxalate urinary stones were recruited and randomly divided into two groups: the Nourse22-002 group and a control group. The experiment lasted for 21 days. The Nourse22-002 bacterial suspension was concentrated and encapsulated to prepare bacterial preparation capsules (1×10⁻⁶). 8 (CFU / capsule) In Group No. 22-002, dogs were fed one capsule of the probiotic preparation once daily; dogs in the control group were fed empty capsules without the probiotic preparation. Urine was collected from the dogs after 14 days of feeding to detect the oxalic acid content. The presence of crystals in the urine was observed under a microscope on days 0 and 21 of feeding.

[0051] The results are shown in Table 8. After feeding Nourse22-002 strain for 14 days, the oxalic acid content in the dog's urine decreased to 4.55 mg / mL, which was 63.77% lower than before feeding.

[0052] Urine samples were collected from dogs on days 0, 3, 6, 9, 12, 15, 18, and 21 of feeding and observed under a microscope for crystal formation. The results are shown in Table 9. No crystals were observed in the urine of dogs fed Nourse22-002 from day 15 onwards, while crystals were observed in the control group.

[0053] Crystallization was observed in the urine of dogs in the Nourse22-002 group and the control group on days 0 and 21 of feeding. On day 0 of feeding, crystallization was observed in the urine of dogs in both the Nourse22-002 group and the control group. Figure 4 (As shown in A and B); on day 21 of feeding, no crystals were observed in the urine of dogs in group Nourse22-002 (as shown in A and B). Figure 4 As shown in Figure C), crystals were still present in the urine of the dogs in the blank control group. Figure 4 (As shown in D).

[0054] Table 8. Reduction rate of oxalate content in urine of dogs with crystallization by canine lactobacillus.

[0055] Table 9. Crystallization in dog urine

[0056] Note: ++ indicates a high number of crystals in urine; ++ indicates a moderate number of crystals in urine; + indicates a low number of crystals in urine, but they are still present; - indicates no crystals were observed in urine. Example 3:

[0057] Twenty-four healthy dogs, weighing 3-5 kg ​​and aged 1-2 years, were selected. Breeds included Chinese Rural Dogs, Poodles, Border Collies, Taihang Dogs, Shiba Inus, and Chihuahuas, with four dogs of each breed. These were evenly distributed into four groups: a control group, a human-derived group, a Nourse22-002 group, and a DW-W group, with six dogs in each group. The experiment lasted 28 days, divided into a feeding period (days 0-7) and a fasting period (days 8-28). During the feeding period, the dogs were given probiotic capsules daily. The bacterial solution was concentrated and then encapsulated to create probiotic capsules (1×10⁻⁶). 8 Dogs were given one capsule of the probiotic preparation (CFU / capsule) once daily during the feeding period. The capsules given to dogs in the human group, DW-W group, and Nourse22-002 group contained human *Lactobacillus animalis*, the original DW-W bacteria, and the selected Nourse22-002 mutant bacteria, respectively. Dogs in the control group were given empty capsules without the probiotic preparation. Fecal samples were collected from dogs before the experiment and on days 3, 7, 8, 10, 14, 21, and 28 of the experiment. *Lactobacillus animalis* plate counts were performed, and the number of colonized viable bacteria was calculated. A colonized viable bacteria count below 2.00 lg CFU / g (i.e., 100 CFU / g) was considered to indicate no *Lactobacillus animalis* colonization in the dog.

[0058] The results are shown in Table 10. The colonization ability of the Nourse22-002 mutant was better than that of the DW-W original bacteria and human-derived *Lactobacillus*. After 7 consecutive days of feeding with the bacterial preparation, the bacterial concentrations in the human group, DW-W group, and Nourse22-002 group were 3.86, 5.21, and 5.46 lg CFU / g, respectively. After 3 days of cessation of feeding, the concentrations in the human group, DW-W group, and Nourse22-002 group were 2.58, 3.89, and 4.82 lg CFU / g, respectively. The Nourse22-002 concentration was 2.24 and 0.93 lg CFU / g higher than that in the human group and DW-W group, respectively. After 7 days of cessation of feeding, no colonization was observed in the intestines of the human group, while the concentrations in the DW-W group and Nourse22-002 group were 2.78 and 3.52 lg CFU / g, respectively. Fourteen days after feeding was stopped, no colonization was observed in the canine intestines of the DW-W group, while the Nourse22-002 group still had 2.45 lg (CFU / g). Although the concentration difference between the Nourse22-002 group (5.46 lg CFU / g) and the DW-W group (5.21 lg CFU / g) was not significant at the end of feeding, the colonization maintenance capacity of the Nourse22-002 group was significantly better than that of the DW-W group after feeding was stopped. This indicates that the Nourse22-002 has an enhanced ability to permanently colonize the canine intestines, rather than just increasing the short-term peak concentration.

[0059] Table 10 Count of viable colonized Lactobacillus animalis Example 4:

[0060] Twelve dogs with weakened immune systems were selected and evenly divided into a control group and a Nourse22-002 group, with six dogs in each group. The experiment lasted for 21 days. The Nourse22-002 bacterial solution was concentrated and encapsulated to prepare bacterial preparation capsules (1×10⁻⁶). 8 Dogs were given one capsule of the probiotic preparation (CFU / capsule) once daily during the feeding period. Dogs in the control group were given empty capsules without the probiotic preparation. Blood samples were collected from dogs on days 0 and 21 to prepare serum, and IgA, IgG, and IgM assays were performed according to the instructions of the kits.

[0061] The results are shown in Table 11. After 21 days of testing, the serum levels of IgA, IgG and IgM in the Nourse22-002 group were significantly higher than those in the control group, reaching 12.81, 9.02 and 28.87 g / L, respectively, representing increases of 54.71%, 39.63% and 49.74% compared to before feeding.

[0062] Table 11 Serum Immunoglobulins Example 5:

[0063] Twelve healthy dogs were selected and evenly divided into a control group and a Nourse22-002 group, with six dogs in each group. The experiment lasted for 21 days. The Nourse22-002 bacterial solution was concentrated and encapsulated to prepare bacterial preparation capsules (1×10⁻⁶). 8 (CFU / capsule) Dogs were given one capsule of the probiotic preparation once daily during the feeding period. Dogs in the control group were given empty capsules without the probiotic preparation. Before feeding and 21 days after feeding, 10 g of feces from each dog was placed in a resealable bag. A portable gas detector probe was inserted into the bag, and the reading was recorded after it stabilized.

[0064] The results are shown in Table 12. After 21 days of feeding, the ammonia content in the feces of dogs in the Nourse22-002 group decreased significantly to 0.52 ppm, which was 52.29% lower than before feeding.

[0065] Table 12 Ammonia content in dog feces 0 1.15 1.09 21 1.08 0.52 Example 6:

[0066] Twelve dogs were recruited and randomly divided into two groups: the Nourse22-002 group and a control group. The experiment lasted for 21 days. The Nourse22-002 strain was fermented and cultured, then heat-inactivated at 95°C for 30 minutes. The absence of viable bacteria was verified by plate coating. The resulting product was then centrifuged and freeze-dried to produce a postbiotic powder. The Nourse22-002 group was fed the postbiotic powder daily at a dose of 500 mg / kg of body weight, evenly mixed into their diet. The control group was fed only their regular diet for 21 days. Fresh fecal samples were collected from the dogs at the end of the experiment to analyze the abundance of intestinal microbiota.

[0067] The abundance of harmful bacteria in the canine gut microbiota is shown in Table 13. After 21 days of feeding, compared with the control group, the total proportion of harmful bacteria in the feces of dogs in the Nourse22-002 group decreased by 65.22% compared with the control group.

[0068] Table 13 Abundance percentage of harmful bacteria in gut microbiota Peptoclostridium 0.39 0.07 Clostridium_sensu_stricto_1 0.03 0.03 Peptococcus 0.02 0.04 Peptostreptococcus 0.01 0.01 Escherichia-Shigella 0.01 0.01 Total proportion of harmful bacteria 0.46 0.16 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 strain of canine lactobacillus, characterized in that, The *Lactobacillus animalis* was deposited on January 28, 2026, at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026270 and accession name *Lactobacillus animalis*. Lactobacillus animalis )Nourse22-002.

2. The *Lactobacillus animalis* according to claim 1, characterized in that, Lactobacillus animalis Nourse22-002 possesses the following characteristics: a) It has the ability to degrade oxalic acid; b) It has the ability to tolerate canine stomach acid and bile salts; c) High number of viable bacteria colonizing the canine intestines and long colonization time; d) It can rapidly aggregate and flocculate; e) The postbiotic has the effect of inhibiting harmful bacteria in the intestine.

3. The use of a canine-derived Lactobacillus animalis strain Nourse22-002 according to any one of claims 1 to 2 in the preparation of dog food, dog treats or dog health food.

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