Puppy-derived lactobacillus plantarum Z1 and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to puppy-derived lactobacillus plantarum and application thereof. The puppy-derived lactobacillus plantarum provided by the invention is preserved in the China General Microbiological Culture Collection Center on June 19, 2024, and the preservation number of the puppy-derived lactobacillus plantarum is CGMCC NO. 31010. The puppy-derived lactobacillus plantarum provided by the invention is screened from faeces of healthy puppies, has relatively strong acid resistance and cholate resistance, is extremely strong in intestinal tract adhesion capacity, has a good inhibition effect on common pathogenic bacteria, is sensitive to various antibiotics, has good probiotic potential, and can be used for preparing a feed additive for the puppies. The invention provides a new candidate strain for pet food, health care and treatment products or probiotic preparations, and has wide application prospects in the aspects of preparation of pet food, health care products, medicines and the like.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a Lactobacillus plantarum Z1 derived from puppies and its application. Background Art

[0002] In recent years, the development of pet probiotics has become a research hotspot in the field of animal health. It improves pet health through mechanisms such as regulating the balance of intestinal flora, inhibiting the proliferation of pathogenic bacteria, and enhancing the host immunity. Currently, commercially available pet probiotics mostly come from foods (such as yogurt, pickles) or mammalian milk sources. Due to poor host adaptability of such exogenous strains, there are significant limitations in intestinal colonization efficiency and functional stability. In addition, probiotics need to tolerate the harsh environments of gastric acid (pH 2.0 - 3.0) and bile salts (0.3% - 1.0%) to survive and play their roles. However, scarce candidate resources with strong acid tolerance, bile salt tolerance, and high adhesion ability exist among existing strains, severely restricting their application effects.

[0003] Lactobacillus plantarum, as an important type of lactic acid bacteria, has been proven to inhibit the proliferation of intestinal pathogenic bacteria (such as Escherichia coli, Salmonella) by secreting metabolites such as antibacterial peptides and organic acids. However, due to the lack of host specificity of traditional Lactobacillus plantarum isolated from fermented foods, its colonization efficiency and functional expression in the intestines of puppies are difficult to reach the ideal level. Research shows that host-derived strains can significantly improve the stability of probiotic functions due to their high adaptability to the intestinal microenvironment. Therefore, screening new strains of Lactobacillus plantarum with host adaptability from the intestinal environment of healthy puppies has become the key direction to break through the bottleneck of existing technologies.

[0004] In addition, the safety assessment of probiotics needs to meet strict antibiotic sensitivity standards to avoid the risk of transmission of drug-resistant genes. The application of some existing strains is limited because they carry drug-resistant genes or show tolerance to common antibiotics (such as ampicillin, erythromycin), and there is an urgent need to develop strain resources with broad-spectrum antibacterial activity and antibiotic-sensitive characteristics.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The object of the present invention is to provide a Lactobacillus plantarum Z1 derived from puppies and its application. This strain is a strain with excellent probiotic properties selected after isolation, screening, and identification from the feces of healthy puppies, providing a new candidate strain for canine milk replacers, pet foods, and health treatment products.

[0007] In the first aspect of the present invention, a Lactobacillus plantarum Z1 derived from puppies is provided. The preservation number of this Lactobacillus plantarum derived from puppies is CGMCC NO. 31010, which is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation date is June 19, 2024, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0008] The complete gene sequence of the Lactobacillus plantarum Z1 derived from puppies in the present invention is shown in SEQ ID NO: 01.

[0009] Furthermore, this Lactobacillus plantarum is isolated from the feces of healthy puppies. Extracting probiotic groups from puppy feces has significant advantages. The strain itself is derived from the pet intestine and can better adapt to the host intestinal environment compared to strains from other sources, and can precisely regulate the balance of the puppy intestinal flora. Such probiotics can tolerate the gastrointestinal environment, have a close symbiotic relationship with puppies, are stably colonized, and are highly safe without toxic side effects and rarely cause allergies, making them an excellent choice for promoting the healthy growth of puppies.

[0010] The Lactobacillus plantarum Z1 derived from puppies in the present invention has an inhibitory effect on Gram-positive bacteria and Gram-negative bacteria, including Escherichia coli, Staphylococcus aureus, Salmonella, Methicillin-resistant Staphylococcus aureus, and Klebsiella pneumoniae.

[0011] Furthermore, for the Lactobacillus plantarum Z1 derived from puppies in the present invention, the diameter of the inhibition zone against Methicillin-resistant Staphylococcus aureus is ≥ 12 mm; the diameter of the inhibition zone against Klebsiella pneumoniae is ≥ 14 mm; the diameter of the inhibition zone against Salmonella is ≥ 16 mm; and the diameter of the inhibition zone against Escherichia coli and Staphylococcus aureus is ≥ 20 mm.

[0012] The Lactobacillus plantarum Z1 derived from puppies in the present invention has acid tolerance, bile salt tolerance, and intestinal adhesion ability.

[0013] Furthermore, for the Lactobacillus plantarum Z1 derived from puppies in the present invention, the survival rate is ≥ 85.33% after surviving for 3 h at pH 2, ≥ 99.66% after surviving for 3 h at pH 2.5, and ≥ 100.00% after surviving for 3 h at pH 3.

[0014] Furthermore, the Lactobacillus plantarum Z1 derived from puppies of the present invention has a survival rate of ≥ 82.62% after surviving for 3 h under 0.3% bile salt conditions, a survival rate of ≥ 84.89% after surviving for 3 h under 0.1% bile salt conditions, and a survival rate of ≥ 83.17% after surviving for 3 h under 0.2% bile salt conditions.

[0015] Furthermore, the Lactobacillus plantarum Z1 derived from puppies of the present invention has extremely strong cell adhesion ability, with an adhesion rate of 40.78%, and has good probiotic potential. Adhesion and colonization in the intestine are important prerequisites for lactic acid bacteria to exert their ecological effects and physiological functions, and are one of the important bases for evaluating their probiotic potential. The adhesion of lactic acid bacteria to epithelial cells is the main basis for their probiotic functions. The adhered lactic acid bacteria can exist in the intestine and resist the invasion of pathogenic bacteria, produce metabolites such as antibacterial peptides to help effectively kill pathogenic bacteria, achieve functions such as preventing the intestine from being infected by pathogenic bacteria, and enhance the immune ability of the body.

[0016] The Lactobacillus plantarum Z1 derived from puppies of the present invention has no hemolytic property and is a safe and non-toxic strain.

[0017] The Lactobacillus plantarum Z1 derived from puppies of the present invention is sensitive to ampicillin, penicillin, chloramphenicol, and cefazolin, can avoid the problem of antibiotic resistance, prevent the imbalance of intestinal flora, and has safety.

[0018] The Lactobacillus plantarum Z1 derived from puppies of the present invention has antioxidant properties.

[0019] In the second aspect of the present invention, a bacterial agent is provided, and the bacterial agent contains Lactobacillus plantarum Z1 derived from puppies.

[0020] In the third aspect of the present invention, an animal food, health product, and medicine are provided, and the animal food, health product, and medicine contain Lactobacillus plantarum Z1 and / or its metabolites or a bacterial agent containing Lactobacillus plantarum Z1 derived from puppies.

[0021] In the fourth aspect of the present invention, the application of the Lactobacillus plantarum Z1 derived from puppies in drugs or foods for treating, preventing, or alleviating animal gastrointestinal dysfunction is provided.

[0022] Advantages of the present invention:

[0023] (1) The present invention provides a new strain of Lactobacillus plantarum Z1 from puppies, which has strong adhesion ability and good probiotic ability.

[0024] (2) The Lactobacillus plantarum Z1 from puppies provided by the present invention has no hemolytic property and high biosafety.

[0025] (3) The Lactobacillus plantarum Z1 from puppies provided by the present invention has good acid tolerance and bile salt tolerance.

[0026] (4) The Lactobacillus plantarum Z1 from puppies provided by the present invention is a strain with excellent probiotic properties selected after isolation, screening and identification from the feces of healthy puppies. It has good inhibitory effects on common pathogenic bacteria and is sensitive to a variety of antibiotics, providing a new candidate strain for pet food, health care products or probiotic preparations, and having broad application prospects in the preparation of pet food, health care products, drugs, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 is the acid tolerance test result at pH = 2 in Example 1 of the present invention;

[0029] Figure 2 is the bile salt tolerance test result at 0.3% bile salt concentration in Example 1 of the present invention;

[0030] Figure 3 is the antibacterial ability result of Lactobacillus plantarum Z1 from puppies in Example 1 of the present invention;

[0031] Figure 4 is the BLAST database annotation result in Example 2 of the present invention;

[0032] Figure 5 is the colony morphology of Lactobacillus plantarum Z1 from puppies in Example 2 of the present invention;

[0033] Figure 6It is the growth curve of Lactobacillus plantarum Z1 from puppies in Example 3 of the present invention;

[0034] Figure 7 It is the result of the acid tolerance ability in vitro of Lactobacillus plantarum Z1 from puppies in Example 3 of the present invention;

[0035] Figure 8 It is the result of the bile salt tolerance ability in vitro of Lactobacillus plantarum Z1 from puppies in Example 3 of the present invention;

[0036] Figure 9 It is the result of the hemolytic ability of Lactobacillus plantarum Z1 from puppies in Example 3 of the present invention;

[0037] Figure 10 It is the result of the adhesion experiment of Lactobacillus plantarum Z1 from puppies in Example 3 of the present invention;

[0038] Figure 11 It is the result of the DPPH free radical scavenging test of Lactobacillus plantarum Z1 from puppies in Example 3 of the present invention;

[0039] Figure 12 It is the result of the ABTS free radical scavenging test of Lactobacillus plantarum Z1 from puppies in Example 3 of the present invention. Detailed implementation mode

[0040] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. In the following examples, quantitative tests are all set with three repeated experiments, and the results are averaged.

[0041] Example 1: Screening and isolation of Lactobacillus plantarum Z1 from puppies

[0042] 1. Cultivation and isolation of strains

[0043] Select fecal samples from fresh and healthy puppies. After gradient dilution with sterilized PBS buffer (purchased from Wuhan Sevier Biotechnology Co., Ltd.), coat them on MRS solid medium containing 1% CaCO3, and culture them in an anaerobic incubator at 37°C for 48 hours. Pick single colonies that are white or milky white with obvious transparent circles for Gram staining and microscopic examination. Select strains that are rod-shaped and Gram-positive bacteria, and select strains Z1, Z2, Z3, and Z4 with better growth from numerous strains for enrichment culture in an anaerobic incubator at 37°C; mix the bacterial cells with sterilized 50% glycerol and store them in a refrigerator at -80°C for later use.

[0044] 2. Screening of strains

[0045] Under aseptic conditions, inoculate the strains of Z1, Z2, Z3, and Z4 preserved with glycerol into MRS broth medium, and anaerobically culture at 37°C for 24 h, and activate them for three consecutive generations; after centrifuging the bacterial liquid, wash the medium with sterilized PBS 2-3 times;

[0046] Conduct acid tolerance, bile salt tolerance, and antibacterial tests on the strains of Z1, Z2, Z3, and Z4.

[0047] The test procedures for acid tolerance, bile salt tolerance, and adhesion are as follows:

[0048] (1) Acid tolerance test

[0049] Resuspend the washed medium in MRS broth medium with a pH of 2, anaerobically culture at 37°C, take samples for gradient dilution at 0 h and 3 h respectively, spread them on the MRS solid medium plate, and count after anaerobic culture at 37°C for 48 h.

[0050] Survival rate of the strain (%) = lg(N1 / N0) * 100%;

[0051] In the formula: N0 is the viable count (CFU / mL) at 0 h; N1 is the viable count (CFU / mL) of the strain after 3 h of acid tolerance.

[0052] The experimental results are as Figure 1 shown. The survival rate of strain Z1 is the highest, up to 85.33%.

[0053] (2) Bile salt tolerance test

[0054] Resuspend the washed medium in MRS broth medium containing 0.3% bile salt and culture, anaerobically culture at 37°C, take samples for gradient dilution at 0 h and 3 h respectively, spread them on the MRS solid medium plate, and count after anaerobic culture at 37°C for 48 h.

[0055] Survival rate of the strain (%) = lg(N1 / N0) * 100%;

[0056] In the formula: N0 is the viable count (CFU / mL) at 0 h; N1 is the viable count (CFU / mL) of the strain after 3 h of bile salt tolerance.

[0057] The experimental results are as Figure 2 shown. The survival rate of strain Z1 is 82.62%.

[0058] (3) Determination of the antibacterial ability of Lactobacillus plantarum Z1 from puppies

[0059] Pet food contains a large amount of nutrients. In daily life, food is accompanied by spoilage and deterioration, which not only endangers the health of pets but also causes economic losses. Microbial contamination and growth and reproduction are the main reasons for food spoilage and deterioration. Therefore, the antibacterial performance of Z1 is one of the safety indicators for its application in food.

[0060] Using five common foodborne pathogenic bacteria, Escherichia coli, Staphylococcus aureus, Salmonella, Methicillin-resistant Staphylococcus aureus, and Klebsiella pneumoniae, as indicator bacteria, after activating the indicator bacteria for three generations, the Oxford cup method was used for the preliminary determination of antibacterial activity.

[0061] Using the Oxford cup method, the specific method is as follows: Take the activated strains and inoculate them into MRS broth medium at an inoculation amount of 2% (V / V), and anaerobically culture at 37°C for 24 h. Dilute the activated Escherichia coli, Staphylococcus aureus, and Salmonella suspensions by a 10-fold gradient, and select the bacterial suspension with a dilution of 10-2 for use. Add 5 mL of LB agar medium to the culture dish for bottom sealing. After cooling and solidifying, place the Oxford cups evenly. Take 100 μL of each gradient of indicator bacteria solution and add it to 15 mL of LB agar at 50-60°C, mix well and slowly pour it into the plate with the Oxford cups. After condensation, use forceps to pull out the Oxford cups to form round holes of 8×6×10. Add 200 μL of the supernatant of the bacterial strain fermentation to the round holes, diffuse in a 4°C refrigerator for 12 h, and then anaerobically culture at 37°C for 9-12 h. Observe whether there is an antibacterial circle, and measure the diameter of the antibacterial circle with a vernier caliper. Each group of experiments is repeated three times in parallel, and the results are expressed as x±s.

[0062] The experimental results are as Figure 3 shown. Bacterium Z1 has a strong inhibitory effect on both Gram-positive bacteria (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Salmonella, Klebsiella pneumoniae), and the diameter of the antibacterial circle is above 12 mm. Among them, the antibacterial effect on Escherichia coli is the strongest, and the diameter of the antibacterial circle of the supernatant is 20 mm.

[0063] From Figure 1 , Figure 2 and Figure 3 the results, it can be seen that Lactobacillus plantarum Z1 from puppies has extremely strong antibacterial ability, up to 20 mm, which is much higher than other Lactobacillus plantarum from puppies; and after surviving for 3 h under the acid-resistant condition of pH = 2 and the bile salt-resistant condition of 0.3% bile salt concentration, the survival rates reach 85.33% and 82.62% respectively, and the acid-resistant and bile salt-resistant abilities are relatively good.

[0064] Example 2: Identification of Lactobacillus plantarum Z1 from puppies

[0065] Perform whole-genome sequencing analysis on the selected Z1, and use the Pacbio (10Kb SMRT Bell library) and Illumina PE150 (350bp small fragment library) sequencing platforms to perform 16s rRNA genome sequencing on it. The sequenced sequences are shown as follows:

[0066]

[0067] The sequencing results were used for homology search in the gene bank by applying BLAST on the NCBI website. The results are as Figure 4 shown, and 100% of the genes were in line with the genus Lactobacillus plantarum.

[0068] The strain Z1 was diluted and spread on the plate, and the colony status was observed. As Figure 5 shown, combining with the morphological characteristics of the Z1 colony, the strain Z1 was identified as Lactobacillus plantarum.

[0069] Example 3: Determination of probiotic properties of Lactobacillus plantarum Z1 from puppies

[0070] 1. Determination of the growth curve of Lactobacillus plantarum Z1 from puppies

[0071] The cultured Z1 bacterial solution was activated for three generations and inoculated into MRS broth medium, placed in a 37°C incubator for anaerobic culture. The viable counts of each bacterium were measured at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h respectively;

[0072] The growth curve of Lactobacillus plantarum Z1 from puppies was plotted. The results are shown in Figure 6 the figure.

[0073] 2. Determination of the in vitro acid and bile salt tolerance of Lactobacillus plantarum Z1 from puppies

[0074] The cultured Z1 bacterial solution was activated for three generations and inoculated into MRS broth medium with pH values of 2.5, 3 and bile salt concentrations of 0.1%, 0.2% respectively, placed in a 37°C incubator for anaerobic culture. Samples were taken at 0 h and 3 h respectively for plate counting;

[0075] The experimental results are shown in Figure 7 and Figure 8 respectively; It can be seen from Figure 7 that when the bacterium was cultured in the environment with pH 2.5 and pH 3 for 0 - 3 h, the growth of the bacterial cells was stable, and the viable counts were all above 10 8 CFU / mL, indicating strong acid tolerance.

[0076] It can be seen from Figure 8 that the bacterium grew relatively stably in the medium with a bile salt concentration of 0.1%; the survival rate decreased by 1% after 3 h of culture in the medium with a bile salt content of 0.2%, indicating strong bile salt tolerance.

[0077] 3. Determination of the antibiotic sensitivity of Lactobacillus plantarum Z1 from puppies

[0078] The excessive use of antibiotics in the pharmaceutical industry can lead to an enhanced drug resistance of pathogenic strains and an easy disorder and imbalance of the flora in the human intestine. Therefore, obtaining lactic acid bacteria sensitive to antibiotics has become one of the important indicators for their safety in food applications.

[0079] Adjust the concentrations of the fermentation broths of each strain cultured for 24 h to 10 8 CFU / mL respectively. Add 100 μL of the bacterial solution to the MRS solid plate, spread it evenly, and let it dry. Place 5 - 6 drug sensitivity test discs in each plate on average. After diffusing in a 4°C refrigerator for 12 h, culture anaerobically at 37°C for 9 - 12 h. The sensitivity of the strain to the drug is judged with reference to the instructions for using the drug sensitivity test discs.

[0080] The results are shown in Table 1. As can be seen from Table 1, Lactobacillus plantarum Z1 is extremely sensitive to ampicillin, penicillin, chloramphenicol, and cefazolin (cephalosporins), and is tolerant to gentamicin, ciprofloxacin, and norfloxacin.

[0081] Table 1 Antibiotic sensitivity test

[0082]

[0083] S: susceptible, sensitive; I: intermediate, moderately sensitive; R: resistance, tolerant

[0084] 4. Determination of the hemolytic ability of Lactobacillus plantarum Z1 from puppies

[0085] Dip a small amount of the bacterial solution with an inoculation loop and streak it on a Columbia blood agar plate. Culture anaerobically at 37°C for 24 - 36 h, and observe whether there is a hemolytic zone on the plate. According to the hemolytic phenomenon, it can be divided into α-hemolysis (greenish hemolysis), β-hemolysis (completely transparent hemolytic ring), and γ-hemolysis (non-hemolysis).

[0086] The hemolysis experiment can detect the safety of the strain and determine whether it has hemolytic property. The results are as Figure 9 shown. No hemolysis occurred on the blood plate for this strain, which was γ-hemolysis. Staphylococcus aureus was selected as the positive control, and a completely transparent hemolytic ring appeared around Staphylococcus aureus, which was β-hemolysis. The above results indicate that this Lactobacillus plantarum strain has no hemolytic property and is a safe and non-toxic strain.

[0087] 5. Adhesion test

[0088] After resuscitating Caco-2 cells (human colon cancer cells), inoculate them into a cell culture flask, add fresh DMEM complete medium (DMEM high-glucose medium containing double antibiotics purchased from Beijing Solarbio Science & Technology Co., Ltd., 20% fetal bovine serum), and culture them in an incubator at 37°C and 5% CO₂. Change the cell culture medium every two days. When the cell adhesion and growth fusion state reaches about 70%-80%, use 0.25% trypsin-EDTA (purchased from Beijing Solarbio Science & Technology Co., Ltd.) mixed digestive solution for passage. The last medium used is DMEM incomplete medium.

[0089] Preparation of FITC solution: FITC (fluorescein isothiocyanate) labeling solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.): Before preparation, place the FITC stored at 4°C at room temperature for 30 min. Accurately weigh 0.5 g and dissolve it in 1 mL of dimethyl sulfoxide (DMSO) to prepare a solution with a concentration of 500 mg / mL. Dilute it 1000-fold (take 10 μL and add it to a 9.99 mL DMSO brown 15 mL EP tube) to obtain a labeling solution with a working concentration of 500 μg / mL, and store it at 4°C.

[0090] Fluorescent labeling: Take 1 mL of freshly cultured probiotic bacterial suspension and centrifuge it in a brown 1.5 mL EP tube (4°C, 4000 rpm, 5 min). Wash it continuously 3 times with sterile PBS. After the last washing, resuspend the cells with 1 mL of FITC solution that has been placed at room temperature for 30 min, and incubate them at 37°C with shaking in the dark for 2 h. After the dark incubation, centrifuge (4°C, 4000 rpm, 5 min) and wash the strain 3 times with sterile PBS to wash away the unbound FITC labeling solution, and resuspend it with 1 mL of DMEM cell culture medium. Take 200 μL and add it to a black 96-well plate to measure the relative fluorescence intensity value (RFU) of the strain under the conditions of wavelength 485 nm (absorption wavelength) and wavelength 530 nm (emission wavelength), which is recorded as the relative fluorescence intensity value R0 of the strain before adhesion.

[0091] Adhesion experiment: Passage the Caco-2 cells continuously for more than 3 generations and prepare for the experiment.

[0092] Before the experiment, the cell concentration was adjusted to 5×105 cells / mL with cell culture medium, and 1 mL of cell suspension was spread evenly in each well of a 24-well cell culture plate for further culture. After observing that the cells in the 24-well plate grew to a monolayer (about 24 h of culture), the cell culture medium was discarded. After washing three times with sterile PBS, 0.6 mL of the completed-labeled probiotic suspension was added. Three replicate wells were set for each strain, and they were incubated at a constant temperature in the dark at 37 °C and 5% CO2 for 2 h. After taking them out, the culture medium was discarded, and the strains that did not adhere to the cells were removed by washing three times with sterile PBS; 0.3 mL of trypsin was added to each well to digest the cells for 5 min, and the digestion was terminated with DMEM cell culture medium. The relative fluorescence intensity value (RFU) of the cell suspension was measured under the same wavelength conditions and recorded as the relative fluorescence intensity value R of the adhered strain.

[0093] Formula for adhesion rate (%): R / R0×100%

[0094] CFU (colony-forming unit): The total number of bacterial communities in a unit volume; when counting viable bacteria in culture, the colonies formed by single bacteria or multiple bacteria aggregated into clusters growing and multiplying on solid medium are called colony-forming units, and the number of viable bacteria is expressed by it.

[0095] The experimental results are shown in Figure 10 As shown, the adhesion rate of strain Z1 was as high as 40.78%.

[0096] 6. DPPH free radical scavenging test

[0097] The bacteria were collected and the concentration of the bacterial suspension was adjusted to 10 8 CFU / mL. A DPPH free radical scavenging ability kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.) was used, and the specific operation was carried out according to the steps in the instruction manual.

[0098] DPPH free radical scavenging rate of the sample (%) = (1 - (A measurement - A control) / A blank)×100%

[0099] The experimental results are shown in Figure 11 , and the DPPH free radical scavenging rate of the bacterial suspension of strain P4 was 57.37%, showing certain antioxidant properties.

[0100]

[0101] 7. ABTS free radical scavenging test

[0102] The bacteria were collected and the concentration of the bacterial suspension was adjusted to 10 8 CFU / mL. An ABTS free radical scavenging ability kit (Beijing Solarbio Science & Technology Co., Ltd.) was used, and the specific operation was carried out according to the steps in the instruction manual.

[0103] Sample ABTS radical scavenging rate (%) = (1 - (A measured - A control) / A blank) × 100%

[0104] The experimental results are shown in Figure 12 , and the ABTS radical scavenging rate of the supernatant of strain P4 is 93.53%, indicating extremely strong antioxidant activity.

[0105]

[0106] Example 4: Application of Lactobacillus plantarum Z1 in preventing gastrointestinal dysfunction in dogs

[0107] To explore the effect of Lactobacillus plantarum Z1 on the gastrointestinal function of dogs, 10 sterilized dogs of similar body weight and about 1 year old were randomly and equally divided into a control group (fed a basic nutritional baked dog food) and an experimental group (fed a dog food based on the control group formula with an additional 1 billion CFU of Lactobacillus plantarum Z1 per kilogram of dog food), and the experiment lasted for 7 days. The formula of the basic nutritional baked dog food: 60 - 70 parts of dried chicken, 5 - 10 parts of chicken liver, 5 - 10 parts of chicken heart, 1 - 5 parts of potato flour, 1 - 5 parts of tapioca starch, 1 - 5 parts of chicken oil, 1 - 5 parts of fish oil, 1 - 5 parts of chicken liver powder, 1 - 5 parts of krill powder, 1 - 5 parts of cellulose, 1 - 5 parts of fruit and vegetable powder, 1 - 5 parts of vitamins and minerals. The fecal volume of the test dogs was collected for 7 days, and the fecal status and score were analyzed.

[0108] The scoring criteria and rules are as follows:

[0109] Score Status 1 Watery diarrhea 2 Viscous, lacking consistency, and basically unformed 3 Higher water content and unformed 4 Well-formed and cylindrical 5 Dry and hard, spherical

[0110] The results are shown in the following table. Compared with the control group, the fecal score of the dogs in the experimental group was better than that of the basic baked dog food group. Therefore, adding Lactobacillus plantarum Z1 to the baked dog food helps to improve soft stools and diarrhea in dogs and helps the feces to form. Strain Z1 plays a certain role in protecting the gastrointestinal function of dogs.

[0111]

[0112] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Lactobacillus plantarum Z1 derived from puppies, characterized in that: The preservation number of the Lactobacillus plantarum derived from puppies is CGMCC NO. 31010. It is preserved in the General Microbiology Center of the China National Center for Culture Collection of Microorganisms. The preservation date is June 19, 2024, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

2. The Lactobacillus plantarum Z1 derived from puppies according to claim 1, characterized in that: The complete gene sequence of the Lactobacillus plantarum derived from puppies is shown as SEQ ID NO:

01.

3. The Lactobacillus plantarum Z1 derived from puppies according to claim 2, characterized in that: The Lactobacillus plantarum derived from puppies has an inhibitory effect on Gram-positive bacteria and Gram-negative bacteria, including Escherichia coli, Staphylococcus aureus, Salmonella, Methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae.

4. The Lactobacillus plantarum Z1 derived from puppies according to claim 2, characterized in that: The Lactobacillus plantarum derived from puppies has acid tolerance, bile salt tolerance and intestinal adhesion ability.

5. The Lactobacillus plantarum Z1 derived from puppies according to claim 2, characterized in that: The Lactobacillus plantarum derived from puppies has no hemolytic property and is a safe and non-toxic strain.

6. The Lactobacillus plantarum Z1 derived from puppies as described in claim 2, characterized in that: The Lactobacillus plantarum derived from puppies is sensitive to ampicillin, penicillin, chloramphenicol, cefazolin, can avoid the problem of antibiotic resistance, prevent the imbalance of intestinal flora, and has safety.

7. The Lactobacillus plantarum Z1 derived from puppies according to claim 2, characterized in that: The Lactobacillus plantarum derived from puppies has antioxidant property.

8. A bacterial agent, characterized in that, It contains the Lactobacillus plantarum (Lactobacillus plantarum) Z1 described in claim 1.

9. An animal food, health product, or medicine, characterized in that, The animal food, health product, and medicine contain the Lactobacillus plantarum (Lactobacillus plantarum) Z1 described in claim 1 and / or its metabolites or the bacterial agent described in claim 8.

10. Use of the Lactobacillus plantarum (Lactobacillus plantarum) Z1 described in claim 1 or 2 in a drug or food for treating, preventing or alleviating animal gastrointestinal dysfunction.

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