Lactobacillus salivarius M1D9 and application thereof

Abstract

The invention discloses lactobacillus salivarius M1D9 and application thereof, and relates to the technical field of microorganisms, and the preservation number of the lactobacillus salivarius M1D9 is CGMCC (China General Microbiological Culture Collection Center) No.31974. The bacterial strain has broad-spectrum antibacterial ability and good antibacterial performance, can prevent and treat diarrhea and enhance the immune function of host animals, has biological safety, intestinal environment endurance ability and intestinal colonization ability, and is an excellent probiotic resource.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to Lactobacillus salivarius M1D9 and its applications. Background Technology

[0002] The balance of the gut microbiota plays a vital role in animal health, especially in young animals or under stress, where it is more easily disrupted, leading to health problems such as diarrhea and weakened immunity. In recent years, probiotics, as safe and environmentally friendly biological agents, have been widely used to maintain gut microbiota stability, regulate immune function, and assist in the prevention and treatment of digestive system diseases in animals. However, existing probiotic resources for pets, especially cats, are relatively scarce. Most products are derived from human or other animal strains, whose tolerance, survival rate, and colonization ability in the cat's gut are limited, resulting in inconsistent efficacy. Furthermore, currently available probiotics show significant differences in their effectiveness against common intestinal pathogens (such as Escherichia coli, Staphylococcus aureus, Salmonella, and Clostridium perfringens), lacking strains that combine broad-spectrum antibacterial properties with enhanced immune function.

[0003] Therefore, it is urgent to screen for superior strains that are suitable for the feline intestinal environment, have good resistance to gastric acid and bile salts, possess antibacterial properties, and can improve host immune function, in order to prepare safe and effective antidiarrheal and immunomodulatory microbial preparations or feed additives. Summary of the Invention

[0004] In view of this, the present invention provides Lactobacillus salivarius M1D9 and its applications.

[0005] To solve the above-mentioned technical problems, this application adopts the following technical solution: Lactobacillus salivarius M1D9, characterized in that the preservation number of said M1D9 is CGMCC No.31974. Another object of the present invention is to provide a microbial agent containing the above-mentioned Lactobacillus salivarius M1D9.

[0006] Another object of the present invention is to provide the use of the above-mentioned *Lactobacillus salivarius* M1D9 or the above-mentioned bacterial agent in the preparation of an antibacterial agent, wherein the antibacterial agent antagonizes one or more of the following pathogens: Escherichia coli, Staphylococcus aureus, Salmonella, Clostridium perfringens, Campylobacter, and Enterococcus faecalis.

[0007] Another object of the present invention is to provide the use of the above-mentioned Lactobacillus salivarius M1D9 or the above-mentioned bacterial agent in the preparation of products for the prevention and treatment of diarrhea.

[0008] Another object of the present invention is to provide the application of the above-mentioned Lactobacillus salivarius M1D9 or the above-mentioned bacterial agent in the preparation of products that enhance the body's immunity.

[0009] Another object of the present invention is to provide a microbial preparation or feed additive for cats, said microbial preparation or feed additive containing the above-mentioned Lactobacillus salivarius M1D9.

[0010] Beneficial effects: This invention screened a feline Lactobacillus salivarius M1D9 strain. This strain has broad-spectrum antibacterial ability and good antibacterial performance. It can prevent and treat diarrhea and enhance the immune function of host animals. It has biosafety, intestinal environment tolerance and intestinal colonization ability, making it an excellent probiotic resource. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0012] Figure 1 The colony and Gram staining morphology of strain M1D9.

[0013] Figure 2 This is a phylogenetic tree of M1D9 based on 16S rRNA.

[0014] Figure 3 The results of the hemolysis test for strain M1D9 are shown, with M1D9 on the left and the positive control strain on the right.

[0015] Figure 4 This is the growth kinetic curve of strain M1D9.

[0016] Figure 5 This is the pH change curve for strain M1D9.

[0017] Figure 6 The effect of bile salt tolerance on the survival rate of strain M1D9 was investigated. The control group was the stock culture medium group, and * indicates a statistically significant difference compared with the control group (P < 0.05).

[0018] Figure 7 The survival rate of strain M1D9 under pH stress is represented. The control group was pH 6.2. ** indicates a statistically significant difference compared with the control group (P < 0.01), **** indicates a statistically significant difference compared with the control group (P < 0.0001), and ns indicates no significant difference between pH 4 and pH 3.

[0019] Figure 8The image shows the cell adhesion characteristics of strain M1D9; the left image is a microscopic image of normal HCT-15 cells, and the right image shows the adhesion of Lactobacillus salivarius M1D9 to HCT-15 cells.

[0020] Figure 9 The inhibitory effect of enzyme treatment on pathogenic bacteria in M1D9 fermentation broth.

[0021] Figure 10 The antibacterial effect of M1D9 fermentation broth with adjusted pH on pathogens.

[0022] Figure 11 The results show the phenotypic observations of mouse liver and spleen.

[0023] In this invention, each experiment has at least three independent experimental groups. The results are expressed as mean ± standard deviation. The data are analyzed by one-way ANOVA t-test using SPSS 24.0, where p>0.05 indicates no significant difference and p<0.05 indicates a significant difference. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0026] Example 1: Isolation and Identification of Strains 1. Isolation and purification of strains Three pet stores were randomly selected in Hohhot. Three healthy kittens around 40 days old were randomly selected from the stores. Before collecting feces, the kittens were placed in clean litter boxes and allowed to defecate naturally. The fresh feces were placed in 50 mL sterile centrifuge tubes, stored in foam insulated boxes with biological ice packs, and immediately sent to the laboratory.

[0027] Take an appropriate amount of fecal sample and place it in a sterile 50 mL centrifuge tube containing 20 mL of physiological saline and glass beads. Shake the centrifuge tube to thoroughly break up the fecal sample. Then centrifuge the sample (4 ℃, 3000 r / min, 5 min), and take the supernatant as the bacterial culture sample for subsequent experiments. Dilute each sample of bacterial culture (dilution gradient: original solution, 10... -1 10-2 10 -3 10 -4 10 -5 200 μL of each diluted sample was spread onto the surface of MRS solid medium. Two culture conditions were set up for each diluted sample: direct culture and anaerobic culture. For anaerobic culture, the spread plates were placed in an anaerobic incubator. All spread plates were incubated upside down in a 37 ℃ biochemical incubator for 48 h. Single colonies on the surface of culture plates with appropriate colony density were streaked for purification, with each colony undergoing at least two purification cultures. Single colonies were picked from the purified plates and inoculated onto MRS liquid medium. After incubation at 37 ℃ for 12 h, the culture was preserved, yielding 127 strains, including strain M1D9.

[0028] 2. Screening of strains The strains obtained in the above steps were incubated upside down in MSR medium at 37 ℃ for 24 h. Then, single colonies of each strain were picked into MRS liquid medium and incubated in a biochemical incubator at 37 ℃ for 48 h. The bacterial culture was centrifuged (4 ℃, 7000 r / min, 5 min), the bacterial precipitate was discarded, the supernatant was filtered through a 0.22 μm filter, and then stored in a refrigerator at 4 ℃.

[0029] The antibacterial activity of the strain was determined using the Oxford cup method. The pathogen was revived and subcultured for activation, and the bacterial concentration was adjusted to approximately 1.0 × 10⁻⁶. 8 CFU / mL was collected and 100 μL was spread onto the surface of LB, BHI, anaerobic blood agar, and MH agar plates, respectively, and allowed to be absorbed. LB plates were used to detect Escherichia coli (ATCC 47076) and Salmonella (ATCC 14028); BHI plates were used to detect Staphylococcus aureus (ATCC 2592) and Enterococcus faecalis (ATCC 29212); anaerobic blood agar plates were used to detect Clostridium perfringens (ATCC 12917); and MH plates were used to detect Campylobacter (ATCC 33560).

[0030] Place the Oxford cup on the surface of the culture medium and add 150 μL of the fermentation broth of the test bacteria (1.0 x 10⁻⁶). 8 The sample (CFU / mL) was placed in a refrigerator at 4 ℃ to promote the diffusion of antibacterial substances. The next day, it was placed in a biochemical incubator at 37 ℃ for 24 h and the diameter of the inhibition zone was measured.

[0031] The results showed that 22 out of the 127 isolates (17.32%) exhibited antibacterial activity against Escherichia coli, and 16 out of 16 (12.60%) showed inhibitory activity against Staphylococcus aureus, with 8 out of 16 (6.30%) exhibiting dual antibacterial properties. The diameters of the inhibition zones are shown in Tables 1 and 2. Furthermore, the M1D9 strain obtained in this study exhibited the highest inhibition zone diameters against all pathogens (Escherichia coli: 34.14 ± 0.15 mm; Staphylococcus aureus: 31.15 ± 0.06 mm; Salmonella: 33.22 ± 0.08 mm; Clostridium perfringens: 27.31 ± 0.21 mm; Campylobacter: 21.88 ± 0.16 mm; Enterococcus faecalis: 28.32 ± 0.12 mm). Given that the M1D9 strain demonstrated the best broad-spectrum antibacterial activity and efficacy, it was established as the core strain for subsequent research.

[0032] Table 1. Diameter of inhibition zone against Escherichia coli for each strain

[0033] Table 2. Diameter of inhibition zone against Staphylococcus aureus for each strain

[0034] 3. Identification of strains (1) The M1D9 strain was revived by drawing lines in MSR medium and placed in a biochemical incubator at 37 ℃ for 24 h. The colony morphology was observed, and single colonies were picked for smear treatment. Gram staining was then performed, the surface moisture of the glass slide was dried, and the bacterial morphology was observed under an optical microscope at 1000x magnification.

[0035] As attached Figure 1 As shown, M1D9 colonies are round with regular edges, smooth surfaces, milky white color, and relatively moist texture, with a colony diameter of 1–2 mm. Microscopic observation after Gram staining revealed no capsules, spores, or flagella, indicating it is a Gram-positive bacterium with a rod-shaped morphology (see Appendix). Figure 1 ).

[0036] (2) Following the instructions for use of the biochemical reagent kit, perform biochemical tests on strain M1D9 for lactose, mannose, glucose, sucrose, maltose, trehalose, inulin, salicin, sorbitol, and gelatin. Record the experimental results and determine the results by referring to the instructions for use. Further identify *Lactobacillus salivarius* by referring to Table 16.1, Carbohydrate Reactions of *Lactobacillus*, in Bergey's Manual of Bacterial Identification (Chinese 8th Edition).

[0037] Table 3 Biochemical identification results of strain M1D9

[0038] (3) Molecular identification: Take an appropriate amount of M1D9 strain culture medium that has been cultured for 12 h, centrifuge it (25 ℃, 12000 r / min, 1 min), discard the supernatant, obtain the precipitate, and extract the bacterial DNA using a DNA extraction kit. Using M1D9 strain DNA as a template, amplify the 16S rRNA fragment of the bacteria using the universal primers for bacterial identification 27R (5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2). Amplification system: 1 μL DNA template, 12.5 μL Green Tap Mix, 1 μL each of upstream and downstream primers, and 9.5 μL sterile enzyme-free water. Amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 2 min, for a total of 30 cycles; final extension at 72℃ for 10 min. PCR products were subjected to agarose gel electrophoresis, followed by gel imaging and photographic recording. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained spliced ​​sequences were compared with the NCBI database to determine the strain species, and a phylogenetic tree of strain M1D9 was constructed based on the sequences (see Appendix). Figure 2 ).

[0039] Based on the above results, strain M1D9 was identified as Lactobacillus salivarius.

[0040] 4. Preservation of bacterial strains The M1D9 strain was deposited on September 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) and named *Lactobacillus salivarius*. Lactobacillus salivarius The accession number is CGMCCNo.31974. Example 2: Identification of the beneficial properties of the strain 1. Hemolysis test The bacterial culture solutions of *Lactobacillus salivarius* M1D9 and *Streptococcus beta-hemolyticus* (BNCC102660, as a positive control) were inoculated into MRS and LB liquid media, respectively, at a ratio of 1:100. The liquid medium for *Streptococcus beta-hemolyticus* was cultured on a shaker (200 r / min, 37 ℃) for 12 h. An appropriate amount of bacterial culture was then drawn onto the surface of blood agar solid medium using an inoculation loop. The medium was then incubated upside down in a 37 ℃ biochemical incubator for 24 h. The hemolysis was then observed.

[0041] The results are attached. Figure 3 As shown, the positive control strain, *Streptococcus pyogenes*, exhibited a clear hemolytic zone around its colonies, indicating β-hemolysis; while *Lactobacillus salivarius* M1D9 colonies did not show a hemolytic zone, indicating γ-hemolysis. Therefore, *Lactobacillus salivarius* M1D9 does not possess the ability to destroy red blood cells and is safe for use as a probiotic.

[0042] 2. Drug sensitivity test The sensitivity of experimental bacteria to various drugs was determined using the disk diffusion method, with the concentration of the activated bacterial culture adjusted to approximately 1.0 × 10⁻⁶. 8 CFU / mL, 100 μL of *Lactobacillus salivarius* M1D9 bacterial suspension was added to MRS solid medium. A small amount of autoclaved and dried glass beads were poured into the medium, and the medium was shaken to ensure even distribution of the bacterial suspension on the surface. After absorption, drug sensitivity test strips were applied to the surface of the solid medium, and the medium was incubated upside down at 37 °C for 24 h. The inhibition zone diameter was then measured using calipers. The sensitivity of *Lactobacillus salivarius* M1D9 to various drugs was assessed according to *CLSI M100 35th Edition (CLSI M100-ED35)*. Table 4 Specifications and Sensitivity Assessment Standards for Drug Sensitivity Tablets

[0043] The M1D9 strain is sensitive to many common antibiotics, but resistance also exists. It is sensitive to clindamycin, doxycycline, amoxicillin, tetracycline, cefadroxil, chloramphenicol, penicillin G, cefoxitin, cephalexin, azithromycin, neomycin, erythromycin, ofloxacin, and enrofloxacin; moderately sensitive to lincomycin, furazolidone, ceftriaxone, cefuroxime, ceftriaxone, cefotaxime, ciprofloxacin, and neomycin; and resistant to spectinomycin, gentamicin, norfloxacin, streptomycin, kanamycin, trimethoprim-sulfamethoxazole, tobramycin, polymyxin B, and amikacin.

[0044] 3. Determination of growth performance and acid production performance (1) Lactobacillus salivarius M1D9 was activated and cultured at least twice until the logarithmic growth phase. It was then inoculated into MRS liquid medium at a ratio of 1:100. Every 2 hours, an appropriate amount of bacterial culture was taken to determine the OD. 600 The pH value of the supernatant and the pH value of the supernatant were used to plot the growth curve and acid production curve based on the measurement results.

[0045] As attached Figure 4 As shown, *Lactobacillus salivarius* M1D9 is in a lag phase for the first 2 hours, in a logarithmic growth phase from 2 to 10 hours, and in a plateau phase after 10 hours. (See attached image.) Figure 5As shown, Lactobacillus salivarius M1D9 produces almost no acid in the first 2 hours. After entering the logarithmic growth phase, it produces a large amount of acid, causing the solution pH to drop rapidly. The solution pH continues to decrease from 2 to 14 hours. After 14 hours, the solution pH tends to stabilize and drops to the lowest value of 3.78.

[0046] (2) Bacterial tolerance test Preparation of artificial gastric fluid: Adjust the pH of the liquid to 2.5 with dilute hydrochloric acid, sterilize at 121 ℃ for 15 min, and when cooled to below 50 ℃, add 1 g of pepsin to every 100 mL of liquid and mix thoroughly. Preparation of artificial intestinal fluid: Dissolve 6.8 g of potassium dihydrogen phosphate in 500 mL of water, adjust the pH to 6.8 with 0.4% sodium hydroxide solution, dilute with water to 1000 mL, sterilize at 121 ℃ for 15 min, and when cooled to below 50 ℃, add 1 g of trypsin to every 100 mL of liquid and mix thoroughly.

[0047] Resuscitate *Lactobacillus salivarius* M1D9 and activate it at least twice until it reaches the logarithmic growth phase. Resuspend the bacterial pellet (obtained by centrifugation) after washing with physiological saline at a 1:10 ratio in MRS liquid medium with bile salt concentrations of 0.1%, 0.3%, and 0.5%; MRS liquid medium with pH values ​​of 5, 4, 3, and 2; artificial intestinal fluid (pH: 6.8); artificial gastric fluid (pH: 2.5); and ordinary MRS liquid medium. Incubate the bacterial suspensions containing artificial intestinal fluid and artificial gastric fluid in a shaker (37 ℃, 200 rpm) for 3 h. Incubate the other bacterial suspensions in a biochemical incubator at 37 ℃ for 3 h, manually mixing every 5 min. Dilute and spread the bacterial suspensions at two time points—at the start of inoculation and after 3 h of incubation—on MRS solid medium for viable cell count, and calculate the survival rate based on the experimental results.

[0048] Survival rate =

[0049] in, The number of bacteria that survived at 0 h. The number of bacteria that survive for 3 hours.

[0050] As attached Figure 6 As shown, the survival rates of *Lactobacillus salivarius* M1D9 after culturing for 3 h in MRS liquid medium with bile salt concentrations of 0.1%, 0.3%, and 0.5% were 108.48 ± 0.20%, 56.59 ± 0.15%, and 34.80 ± 0.30%, respectively. (See attached image.) Figure 7As shown, the survival rates of M1D9 after culturing for 3 hours in MRS liquid medium with pH values ​​of 5, 4, 3, and 2 were 114.80 ± 0.29%, 101.90 ± 0.27%, 101.51 ± 0.16%, and 50.72 ± 0.50%, respectively. The relative survival rates of M1D9 after culturing for 3 hours in artificial intestinal fluid and artificial gastric fluid were calculated to be 99.96 ± 0.54% and 92.07 ± 0.57%, respectively. This indicates that *Lactobacillus salivarius* M1D9 exhibits good tolerance to artificial intestinal fluid and artificial gastric fluid.

[0051] (3) Cell adhesion test Human colorectal adenocarcinoma cell line HCT-15 (source: National Experimental Cell Resource Sharing Platform) was revived, passaged routinely, and seeded into 6-well plates. The cells were cultured at 37 ℃ in a 5% CO2 incubator until the cell density reached approximately 1 × 10⁻⁶ cells / well. 5 Cells / well. Resuscitate Lactobacillus salivarius M1D9, activate and passage it at least twice to the logarithmic growth phase, and adjust the bacterial suspension concentration to 1×10⁻⁶. 8 CFU / mL, wash the bacterial cells twice with PBS before use.

[0052] Aspirate the cell culture medium (RPMI-1640 for culturing HCT-15 cells) from the 6-well plate, gently wash the cells 2–3 times with sterile PBS, and then carefully aspirate dry. Add the prepared M1D9 bacterial suspension (resuspend the cells in RPMI-1640 at a concentration of 1×10⁻⁶). 8 Incubate 1 mL of (CFU / mL) cells per well in a 37 ℃ CO2 incubator for 2 h. After incubation, aspirate the bacterial suspension and gently wash the cells 2–3 times with sterile PBS to remove unbound bacteria. Add 1 mL of 0.1% Triton X-100 lysis buffer to each well to fully lyse the cells and release the attached bacteria. Dilute the lysed bacterial solution proportionally and spread it onto MRS solid medium for viable cell counting. For morphological observation, seed the cells into 6-well plates with sterile slides, following the same procedures as for adhesion culture. After washing to remove unbound bacteria, add 4% paraformaldehyde to fix the cell-bacteria complex, then wash with PBS and stain with 0.1% crystal violet. After staining, rinse thoroughly, air dry, mount, and observe bacterial adhesion under a light microscope.

[0053] Adhesion index = M / M0 M represents the number of bacteria adhering to the cell (unit: CFU), and M0 represents the number of cells (unit: CFU).

[0054] The adhesion index of *Lactobacillus salivarius* M1D9 to HCT-15 cells was 20.16 ± 1.78 CFU / cell. Microscopic observation showed that *Lactobacillus salivarius* M1D9 adhered to the cell surface, indicating that *Lactobacillus salivarius* M1D9 possesses cell adhesion ability (see Appendix). Figure 8 ).

[0055] (4) Analysis of antibacterial substances The fermentation broth was prepared as in step 2 of Example 1, with 10 mg / mL solutions of proteinase K, catalase, papain, and trypsin. Each solution was mixed with MRS liquid medium at a 1:9 ratio. The blank control group was treated with sterile water in the same proportion. The pH of the solutions was adjusted using 1 mol / L sodium hydroxide and 1 mol / L hydrochloric acid. The pH of the proteinase K solution was adjusted to 5.0, the proteinase and catalase solutions to 7.0, and the papain solution to 7.5. The blank control group was treated with sterile water in the same proportion. All five solutions were incubated at 37 °C for 2 h. After incubation, the mixture was boiled in a water bath for 10 min to stop the enzyme reaction. Once the mixture was cooled to room temperature, the pH was adjusted again to ensure that the pH of each group was the same as that of the control group. The antibacterial activity of each mixture was determined using the Oxford cup method, as in step 2 of Example 1. The pH of the fermentation broth was adjusted to 4.0 using 1 mol / L sodium hydroxide and 1 mol / L hydrochloric acid, and the pH of the MRS liquid medium was adjusted to 3.78. The control group was the fermentation broth. The antibacterial activity of each group of solutions was then determined using the Oxford cup method.

[0056] After treatment with papain, trypsin, catalase, and proteinase K, the inhibition zones against *Escherichia coli* were 29.96 ± 0.92 mm, 33.54 ± 0.88 mm, 32.47 ± 0.98 mm, and 30.99 ± 1.81 mm, respectively; and against *Staphylococcus aureus*, the inhibition zones were 29.34 ± 0.59 mm, 26.95 ± 0.84 mm, 28.00 ± 1.67 mm, and 28.79 ± 1.45 mm, respectively.

[0057] The inhibition zones of the original fermentation broth, fermentation broth at pH 4.0, and MRS liquid medium at pH 3.78 against *Escherichia coli* were 33.57 ± 0.45 mm, 27.88 ± 0.86 mm, and 21.89 ± 0.87 mm, respectively; and the inhibition zones against *Staphylococcus aureus* were 30.76 ± 1.65 mm, 25.87 ± 0.62 mm, and 21.59 ± 0.85 mm, respectively.

[0058] Compared to the effect of pH on the antibacterial activity of the fermentation broth, the effects of various enzymes on the antibacterial activity of the fermentation broth were not significant. However, the antibacterial activity of the fermentation broth was significantly stronger than that of MRS liquid medium at the same pH. Considering that lactic acid bacteria can utilize various sugars and can lower the pH of the culture medium to 3.78, it is preliminarily determined that the main antibacterial substance of the experimental strain M1D9 is organic acid. The inhibition zone data for each treatment group are attached. Figure 9 and attached Figure 10 .

[0059] Example 3 1. Effects of Lactobacillus salivarius M1D9 on growth, immune indicators, and diarrhea rate in mice. Sixty female mice of similar weight were selected and randomly divided into four groups: control group 1, control group 2, high-dose Lactobacillus salivarius M1D9 gavage group (M1D9_H), and low-dose Lactobacillus salivarius M1D9 gavage group (M1D9_L), with 15 mice in each group. Lactobacillus salivarius M1D9 bacterial suspension in the logarithmic growth phase was collected, washed twice with physiological saline, and then the bacterial precipitate was resuspended in physiological saline and the suspension was adjusted to different concentrations. The experiment consisted of two phases, lasting a total of 21 days.

[0060] In the first phase of the experiment, mice in control group 1 received no treatment, mice in control group 2 were administered 0.2 mL of physiological saline daily, and mice in the M1D9_H group were administered 0.2 mL of bacteria at a concentration of 1.0 × 10⁻⁶ daily. 8 Mice in the M1D9 group were administered 0.2 mL of Lactobacillus salivarius M1D9 bacterial suspension at a concentration of 1.0 × 10⁻⁶ CFU / mL daily by gavage. 6 CFU / mL of Lactobacillus salivarius M1D9 bacterial suspension was administered via gavage for 14 consecutive days. During the experiment, the animals were kept in an environment with a temperature of 22±3 ℃ and a relative humidity between 30% and 50%. The animals had free access to food and water. Mice were administered gavage at designated times each day, and the total food intake of each group of mice was recorded. Mice were weighed before and after gavage. Blood samples were collected on days 1 and 14 of the experiment to separate serum, which was then frozen for subsequent determination of serum immune indicators. After 14 days, 5 mice from each group were randomly selected and anesthetized. While the mice were anesthetized, they were euthanized by cervical dislocation, and the liver and spleen were removed, weighed, and their lengths were measured.

[0061] In the second phase of the experiment, from days 15 to 21, the treatment of mice in each group was the same as in the first phase. On day 15 of the experiment, control group 2, M1D9_H and M1D9_L were administered 0.2 mL / mouse (1.0 × 10⁻⁶) by gavage. 9 Mice were challenged with pathogenic Escherichia coli at CFU / mL for 2 consecutive days, and the diarrhea rate of each group was observed and statistically analyzed. Blood was collected on day 21 to separate serum.

[0062] Statistical analysis showed that during the first phase of the experiment, the mice in all groups were in good spirits, with normal food and water intake. No diarrhea was observed in the experimental groups during the observation period, and the fecal characteristics were not significantly different from the control group. Visual observation revealed no significant differences between the experimental and control groups. The body weight of the M1D9-H group was significantly higher than that of the control group on day 14. During necropsy, no obvious visual lesions were found in any of the mice's tissues or organs. The organ indices of the M1D9-H group were significantly lower than those of the control group. Experimental data for each group are shown in Tables 5-6, and organ phenotypic observations are provided in the appendix. Figure 11 .

[0063] As shown in Table 7, during days 1 to 14 of the experiment, M1D9 significantly increased the levels of IgG and IL-10 in mouse serum and significantly decreased the levels of TNF-α and IL-6. During days 14 to 21 of the experiment, after mice were orally administered pathogenic Escherichia coli, the serum IgG level in the M1D9 group was still significantly higher than that in the control group, while the levels of IL-10, TNF-α, and IL-6 were significantly lower in the M1D9 group than in the control group.

[0064] As shown in Table 8, during the experimental period from day 14 to day 21, after mice were orally administered pathogenic Escherichia coli, the diarrhea rate and mortality rate of each group of mice were counted. The results showed that M1D9 significantly reduced the diarrhea rate and mortality rate of mice.

[0065] Table 5. Body weight and total food intake of mice from day 1 to 14

[0066] Note: Different lowercase letters in the superscript of data in the same column indicate statistically significant differences (P < 0.05).

[0067] Table 6 Physiological indicators of mouse liver and spleen

[0068] Note: Different lowercase letters in the superscript of data in the same column indicate statistically significant differences (P < 0.05).

[0069] Table 7 Effects of M1D9 on serum immune markers in mice

[0070] Note: Different lowercase letters in the superscript of the same data indicate statistically significant differences (P < 0.05).

[0071] Table 8. Diarrhea rate and mortality rate of mice at 14-21 days (%)

[0072] The results of this study indicate that *Lactobacillus salivarius* M1D9 exhibits good biocompatibility, causing no adverse reactions or organ damage in mice throughout the experiment, and promoting weight gain. M1D9 significantly increased serum IgG and IL-10 levels while decreasing TNF-α and IL-6 levels, demonstrating a dual effect of enhancing humoral immunity and regulating inflammatory responses. Even after challenge with pathogenic *Escherichia coli*, M1D9 maintained high immune levels and effectively reduced diarrhea and mortality rates. In summary, M1D9 possesses multiple probiotic properties, including safety, growth promotion, immune enhancement, and anti-diarrheal effects, demonstrating significant potential for development and application.

[0073] 2. Cat feeding experiment Six cats were randomly divided into a control group and an experimental group, and placed in an animal laboratory for free-range feeding and watering during designated 12-hour periods each day for 14 days. The control group received no treatment. In the experimental group, from day 1 to 7, the bacterial culture, having reached the logarithmic growth phase, was washed 2-3 times daily with sterile physiological saline, and the concentration was adjusted to 1.0 × 10⁻⁶. 9 The bacterial precipitate was mixed with the cat's feed and fed to the cats at a dose of 1.0 × 10 CFU / mL at a specified time each day. 9 The bacterial precipitate was approximately CFU. During the trial period, the cats' eating habits, defecation, and mental state were observed and recorded daily.

[0074] On the morning of the experiment's conclusion, blood samples were collected from both groups of cats to measure blood parameters including white blood cell count, neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count, neutrophil percentage, lymphocyte percentage, monocyte percentage, eosinophil percentage, basophil percentage, red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin content. Blood biochemical parameters also included albumin, total protein, globulin, albumin-to-globulin ratio, total bilirubin, aspartate aminotransferase, alanine aminotransferase, amylase, creatine kinase, creatinine, blood urea nitrogen, blood urea nitrogen / creatinine ratio, glucose, triglycerides, calcium, inorganic phosphorus, and AST-ALT ratio.

[0075] Statistical analysis showed that during the experimental period, the cats in both groups maintained normal appetite and mental state. Tables 9 and 10 indicate that feeding with *Lactobacillus salivarius* M1D9 significantly increased feline serum globulin levels with a decreased albumin-to-globulin ratio, suggesting enhanced humoral immunity. Simultaneously, total bilirubin and transaminase (AST, ALT) levels significantly decreased, indicating reduced liver inflammation and enterogenic toxin load, leading to improved liver function indicators. The significant decrease in blood urea nitrogen (BUN) was associated with reduced intestinal protein putrefaction and increased nitrogen metabolism efficiency. These results collectively suggest that *Lactobacillus salivarius* improves gut-liver axis homeostasis by regulating intestinal microecological balance, achieving a dual effect of immune enhancement and liver protection.

[0076] After feeding probiotics, the total white blood cell count, percentage of neutrophils, and percentage of lymphocytes in cats increased significantly, suggesting that Lactobacillus salivarius M1D9 may have a potential role in enhancing the immune function of cats; while the erythrocyte and platelet counts showed no significant changes, indicating that the probiotics did not have an adverse effect on the hematopoietic and coagulation functions of cats.

[0077] Table 9. Cat serum biochemical indicators

[0078] Note: ALB - albumin, TP - total protein, GLOB - globulin, A / G - albumin / globulin ratio, TB - total bilirubin, AST - aspartate aminotransferase, ALT - alanine aminotransferase, AMY - amylase, CK - creatine kinase, Crea - creatinine, BUN - blood urea nitrogen, BUN / CREA - blood urea nitrogen / creatinine, GLU - glucose, TG - triglycerides, Ca - calcium, PHOS - inorganic phosphorus, AST / ALT - AST / ALT ratio, Ca*PHOS - calcium-phosphorus product. Different lowercase letters in the superscript of data in the same row indicate statistically significant differences (P < 0.05).

[0079] Table 10 Changes in feline hematological parameters

[0080] Note: WBC - White blood cell count, Neu - Neutrophil count, Lym# - Lymphocyte count, Mon# - Monocyte count, Eos# - Eosinophil count, Bas# - Basophil count, Neu% - Neutrophil percentage, Lym% - Lymphocyte percentage, Mon% - Monocyte percentage, Eos% - Eosinophil percentage, Bas% - Basophil percentage, RBC - Red blood cell count, HGB - Hemoglobin concentration, HCT - Hematocrit, MCV - Mean corpuscular volume, MCH - Mean corpuscular hemoglobin content, MCHC - Mean corpuscular hemoglobin concentration, RDW - CV - Coefficient of variation of erythrocyte distribution width, RDW - SD - Standard deviation of erythrocyte distribution width, PLT - Platelet count, MPV - Mean platelet volume, PDW - Platelet distribution width, PCT - Plateletcrit. Different lowercase letters in the same row of data in the table indicate statistically significant differences (P < 0.05).

[0081] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0082] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Lactobacillus salivarius ( Lactobacillus salivarius M1D9, characterized in that, The preservation number of the M1D9 is CGMCC No.31974.

2. A microbial inoculant, characterized in that, The microbial agent contains the Lactobacillus salivarius M1D9 of claim 1.

3. Use of Lactobacillus salivarius M1D9 according to claim 1 or of the bacterial agent according to claim 2 for the preparation of an antibacterial agent, characterized in that, The bacteriostatic agent antagonizes one or more of the following pathogenic bacteria: Escherichia coli, Staphylococcus aureus, Salmonella, Clostridium perfringens, Campylobacter and Enterococcus faecalis.

4. Use of the Lactobacillus salivarius M1D9 of claim 1 or the agent of claim 2 in the preparation of a product for preventing and treating diarrhea.

5. Use of the Lactobacillus salivarius M1D9 of claim 1 or the agent of claim 2 in the preparation of a product for improving immunity.

6. A microbial preparation or feed additive for cats, characterized in that, contains the Lactobacillus salivarius M1D9 of claim 1.

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