A lactobacillus and its application in preventing and controlling bacterial infection of pets

Abstract

The present application relates to the field of microorganisms, and specifically discloses a Lactococcus lactis, lactobacillus and its application in preventing and controlling bacterial infections in poultry and livestock. The lactobacillus is produced by fermentation of Lactococcus lactis. The strain is named Lactococcus lactis 5019 and was deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms on January 13, 2021. The deposit address is: Beijing, China, and the deposit number is CGMCC NO.21607. The lactobacillus produced by Lactococcus lactis in the present application can be used to prevent and control bacterial infections in pets, and it has the advantages of high temperature resistance and strong antibacterial activity.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and more specifically, to a lactic acid bacterium and its application in preventing and controlling bacterial infection of pets. Background Art

[0002] In today's severe public health situation, because the abuse of antibiotics has led to the emergence of more drug-resistant strains, the task of replacing and reducing antibiotics is urgent, and the world is in urgent need of corresponding response strategies. At present, probiotic preparations have been widely used in animal production as the main substitute for antibiotics. They have many functions such as promoting the digestion and absorption of nutrients, improving the balance of gastrointestinal flora and enhancing intestinal immunity. They can significantly improve the growth performance of livestock and poultry and prevent and treat diseases. However, live probiotics also have certain problems, such as poor stress resistance, storage intolerance, low gastrointestinal survival rate, difficulty in colonization, and energy consumption for proliferation. In addition, some probiotics have virulence factors and certain drug resistance, and may even cause infection.

[0003] Studies have found that the inanimate bacteria and / or their metabolites of probiotics also have obvious prebiotic effects, and the prebiotic functions of different components are not the same. In 2013, the Spaniard Tsilingiri formally proposed the concept of "postbiotics", which is temporarily translated into Chinese as "postbiotics." In May 2021, the International Scientific Association for Probiotics and Prebiotics (ISAPP) published a consensus statement on the definition and scope of postbiotics in the journal Nature, that is, postbiotics are "preparations of inanimate microorganisms and / or their components that are beneficial to the host." Compared with live probiotics, postbiotics have safe dosage parameters, longer shelf life, and good absorption, metabolism, distribution and excretion capabilities. At the same time, they overcome the potential risks of probiotics during use, such as bloating and flatulence, probiotic-related translocation, bacteremia and fungal diseases, and possible antibiotic resistance gene drift. It can be seen that the development of postbiotics will have a broader market prospect.

[0004] However, the fermentation metabolites of lactic acid bacteria still have the problem of significantly reduced antibacterial activity under high temperature conditions, which limits their application scope. Summary of the invention

[0005] In order to solve the above problems, the present application provides a lactic acid bacterium and its application in preventing and controlling bacterial infections in poultry and livestock.

[0006] The purpose of this application is achieved through the following technical solutions:

[0007] In the first aspect, the present application provides a lactobacillus, which adopts the following technical solution:

[0008] A lactic acid bacterium, which is produced by fermentation of Lactococcus lactis. The strain is named Lactococcus lactis 5019 and was deposited in the General Microbiology Center of China National Culture Collection of Microorganisms on January 13, 2021. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 21607.

[0009] By adopting the above technical scheme, the lactobacillus involved in the present application is a small molecule polypeptide with antibacterial activity produced by fermentation of Lactococcus lactis 5019, which can inhibit the synthesis of peptidoglycan by perforating the target cells, inhibiting protein synthesis by interacting with ribosomes or tRNA, and directly degrading the target cell DNA, thereby achieving an antibacterial effect.

[0010] The lactobacillus of the present application is acid-resistant and can maintain stable biological activity under the condition of pH 2.0; and it has high temperature resistance and can still maintain the original antibacterial potency and effect after being treated at different temperatures, high and low. Therefore, the lactobacillus of the present application can be applied to pet food as a pet food additive, solving the problem of decreased effect of lactobacillus caused by high-temperature puffing process of pet food.

[0011] Optionally, the antibacterial spectrum of the lactobacillus is Staphylococcus, Escherichia coli, Salmonella, Clostridium perfringens, Pasteurella, Haemophilus paragallinarum, Vibrio parahaemolyticus, Riemerella anatipestifer, Klebsiella pneumoniae, Clostridium perfringens, and Enterococcus faecium.

[0012] By adopting the above technical scheme, the lactobacillus of the present application has a strong broad-spectrum, which can lyse Gram-positive bacteria, such as Staphylococcus, Clostridium perfringens, Clostridium perfringens, Enterococcus faecium, etc., and can lyse a variety of Gram-negative bacteria, such as Escherichia coli, Salmonella, Riemerella anatipestifer, Pasteurella, Vibrio parahaemolyticus, Haemophilus paragallinarum, Klebsiella pneumoniae, etc., which can reduce the accumulation of antibiotics in food-borne animals and reduce the public health and safety risks caused by super-resistant bacteria.

[0013] In a second aspect, the present application provides a pharmaceutical preparation, which adopts the following technical solution:

[0014] A pharmaceutical preparation comprising the lactobacillus.

[0015] In a third aspect, the present application provides a method for preparing lactobacillus, using the following technical solution:

[0016] A method for preparing lactobacillus comprises the following steps:

[0017] S1. Preparation of lactic acid bacteria seeds: inoculating Lactococcus lactis 5019 into a culture medium, and culturing it statically to make it proliferate and activate, thereby obtaining a Lactococcus lactis seed solution;

[0018] S2, lactic acid bacteria amplification culture: inoculating Lactococcus lactis seed liquid into the culture medium, culturing it statically to make it proliferate and activate, and obtaining Lactococcus lactis fermentation liquid;

[0019] S3. Preparation of lactobacillus: The pH of the Lactococcus lactis fermentation broth is adjusted to 2-3 with concentrated hydrochloric acid, and then inactivated; and the supernatant is taken as lactobacillus.

[0020] By adopting the above technical scheme, the lactic acid fermentation broth is treated with concentrated hydrochloric acid in S3, which can promote the dissociation of lactobacillus from the cell wall and improve its solubility in the fermentation broth; then, after inactivation treatment, the cell wall is ruptured, which is conducive to the full release of lactobacillus, thereby improving the extraction rate of lactobacillus.

[0021] Preferably, in said S3, the conditions for the inactivation treatment are: heating at a temperature of 100-110° C. for 3-10 min.

[0022] Preferably, in S3, the supernatant is obtained by centrifugation, and the centrifugation conditions are: centrifugation at a speed of 8000-12000 rpm for 3-8 min.

[0023] In a fourth aspect, the present application provides an application of lactobacillus, using the following technical solution:

[0024] An application of lactobacillus for preventing and controlling bacterial infection in pets.

[0025] Optionally, the lactobacillus is used to lyse bacteria in pets, prevent bacterial infection in pets, and prepare pet feed.

[0026] Optionally, the lactobacillus is added to pet dog feed in an amount of 1000-1500 ppm.

[0027] In summary, this application has the following beneficial effects:

[0028] 1. The lactobacillus of the present application is acid-resistant and can maintain stable biological activity under the condition of pH 2.0; and it has high temperature resistance and can still maintain the original antibacterial potency and effect after being treated at high and low temperatures; therefore, the lactobacillus of the present application can be applied to pet food as a pet food additive, solving the problem of decreased lactobacillus effect caused by high-temperature puffing process of pet food.

[0029] 2. The lactobacillus of the present application has a strong broad spectrum, which can lyse Gram-positive bacteria, such as Staphylococcus, Clostridium perfringens, Clostridium perfringens, Enterococcus faecium, etc., and can lyse a variety of Gram-negative bacteria, such as Escherichia coli, Salmonella, Riemerella anatipestifer, Pasteurella, Vibrio parahaemolyticus, Haemophilus paragallinarum, Klebsiella pneumoniae, etc., which can reduce the accumulation of antibiotics in food-borne animals and reduce the public health safety risks caused by super-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a colony morphology diagram of Lactococcus lactis 5019 in Example 1 of the present application.

[0031] Figure 2 This is a cell morphology diagram of Lactococcus lactis 5019 in Example 1 of the present application.

[0032] Figure 3 This is a graph showing the detection of Enterobacter fecal bacterial load in Example 6 of the present application.

[0033] Figure 4 This is a graph showing the detection of lactic acid bacteria fecal load in Example 6 of the present application. DETAILED DESCRIPTION

[0034] The present application is further described in detail below with reference to the accompanying drawings and embodiments.

[0035] Example 1: Isolation, culture and biological characteristics of Lactococcus lactis

[0036] 1. Isolation and cultivation of Lactococcus lactis

[0037] Use a sterile sampler to collect Xinjiang traditional yogurt samples and transport them to the laboratory environment at low temperature. Dissolve the sample in sterile water and mix well. Take 100 μL of the sample solution for 10-fold gradient serial dilution, then take 100 μL of the dilution solution and spread it on the GM17 medium agar plate, invert the plate and culture it at 30°C for 24 hours, pick white, round, catalase-negative colonies, observe them under a microscope as spherical or ellipsoidal, and repeatedly streak to determine pure colonies.

[0038] The formula of the above GM17 medium: glucose 5g / L, peptone 5.0g / L, yeast extract 5.0g / L, polypeptone 5.0g / L, ascorbic acid 0.5g / L, beef extract 2.5g / L, β-glycerophosphate disodium 19g / L, 0.02g / L MgSO 4 7H 2 O; add distilled water to dissolve, adjust pH to 7.0, add 1.5% (by weight) agar powder to the solid culture medium, and sterilize at 115°C for 30 minutes.

[0039] 2. Screening and identification of Lactococcus lactis

[0040] The obtained strain was biochemically identified and found to have the following morphological characteristics and physiological and biochemical properties:

[0041] (1) Macroscopic form: Figure 1 As shown, the screened colonies were cultured in GM17 medium at 30°C for 24 hours. The colonies were white, round, moist, opaque, and had neat edges.

[0042] (2) Microscopic morphology: Figure 2 As shown, the screened strain was statically cultured in GM17 medium at 30°C for 24 hours. The bacteria were ellipsoidal, 0.7-0.9 μm×0.8-1.2 μm, arranged singly, in pairs or in chains, and were Gram-positive.

[0043] (3) The main physiological and biochemical characteristics are shown in Table 1:

[0044] Table 1 Physiological and biochemical characteristics of strains

[0045]

[0046] Explanation of symbols: “+”, positive; “-”, negative.

[0047] (4) Molecular biological identification results:

[0048] The genomic DNA of the isolated strain was extracted, amplified and sequenced to obtain a 16S rDNA fragment of the strain, the sequence of which is: SEQ ID NO: 1.

[0049] The 16S rDNA gene sequence was submitted to NCBI for online comparison. Combined with the morphological characteristics, physiological and biochemical identification of the strain, the screened strain was determined to be Lactococcus lactis and was named 5019.

[0050] Example 2: Preparation of lactobacillus

[0051] A lactobacillus is prepared by the following method:

[0052] S1. Preparation of lactic acid bacteria seeds

[0053] The frozen bacterial liquid of Lactococcus lactis 5019 was selected and streaked on GM17 medium in three zones, and statically cultured in a 30°C incubator for 24 hours. A single colony was selected and inoculated into 5 mL of GM17 liquid culture medium, and statically cultured in a 30°C incubator for 12 hours for proliferation and activation, to obtain a proliferated Lactococcus lactis seed liquid.

[0054] S2. Lactic acid bacteria amplification culture

[0055] Take 1 mL of Lactococcus lactis seed liquid, add it into 50 mL of GM17 liquid culture medium according to 2% inoculation amount, and culture it in a 30° C. incubator for proliferation for 12 h. No ventilation is allowed during the fermentation process. Stir it once at 80 rpm every 4 h for 3 min each time to obtain Lactococcus lactis fermentation liquid.

[0056] S3. Preparation of lactobacillus

[0057] After the fermentation is completed, the pH of the Lactococcus lactis fermentation liquid is adjusted to 2.5 with concentrated hydrochloric acid, and then boiled at 100°C for 5 minutes for inactivation. After cooling, it is centrifuged at 10,000 rpm for 5 minutes to remove the bacteria, and the supernatant is the lactobacillus extract.

[0058] Example 3: Broad-spectrum antibacterial activity of lactobacillus

[0059] The lactobacillus extract prepared in Example 2 was used as a sample to test its antibacterial performance.

[0060] (I) Proliferation of multiple bacteria to be tested

[0061] Strains with different regions and multiple animal sources were selected for testing. Various Gram-positive and Gram-negative bacteria were selected and frozen and divided into three zones on their respective identification culture plates, and then cultured in a 37°C constant temperature incubator overnight. Single colonies were then selected and inoculated into 5 mL of appropriate liquid culture medium, and proliferated in a 37°C constant temperature shaking incubator overnight to obtain a single bacterial suspension. The culture media corresponding to various bacteria are shown in Table 2.

[0062] Bacteria Culture medium Bacteria Culture medium staphylococcus MSA Clostridium perfringens TSA salmonella SS Pasteurella NA Escherichia coli MAC Haemophilus paragallinarum TSA+8% serum Vibrio parahaemolyticus TCBS Riemerella anatipestifer TSA+5% serum

[0063] (II) Detection of cleavage spectrum of lactobacillus

[0064] The cleavage spectrum of lactobacillus was detected using the double-layer plate method.

[0065] (1) Melt 2% water agar and cool to 50°C, pour into a sterile culture dish, and let it stand for a few minutes to solidify to obtain the lower plate.

[0066] (2) Add 0.5% agar and 1% disodium hydrogen phosphate to a semi-solid culture medium broth (prepared by adding 0.7% agar powder to nutrient broth), sterilize under high pressure, wait until the temperature of the soft agar drops to 50°C, add 2% of the bacteria to be tested and 1% Tween-20, mix quickly, pour onto the solidified lower plate medium, and let stand for 30 minutes to obtain an upper plate.

[0067] (3) Place an Oxford cup in the center of the plate and leave it there for 20 minutes, then take 50 μL of the sample and add it to the Oxford cup. Place the plate in the refrigerator for 3.5 hours, then incubate it at 37°C for 12 hours, observe whether an inhibition zone is formed, count the diameter of the inhibition zone, and judge the lysis situation. The antibacterial spectrum results of lactobacillus are shown in Table 3.

[0068] Table 3 Antimicrobial spectrum test results of lactobacillus

[0069]

[0070]

[0071]

[0072] Note: The above-mentioned Staphylococcus strains were isolated from the skin lesions of piglets with oily skin disease in a pig farm in Shandong. Salmonella, Escherichia coli, Clostridium perfringens, Haemophilus paragallinarum, and Pasteurella were isolated from the liver and intestines of dead chickens in broiler and laying hen farms in many provinces and cities across the country. Riemerella anatipestifer was isolated from a duck farm in Shandong. Among them, ATCC25922, ATCC25923, CVCC533, and CVCC536 were purchased from the China General Microbiological Culture Collection Management Center.

[0073] Result analysis:

[0074] According to the data in Table 3, 120 pathogenic bacteria from different sources were used as indicator bacteria to be tested in this experiment. As for the antibacterial activity of lactobacillus, the lysis rate of the lactobacillus 5019 of the present application to 20 strains of Staphylococcus was 100%, the lysis rate to 20 strains of Salmonella was 100%, the lysis rate to 20 strains of Escherichia coli was 100%, the lysis rate to 20 strains of Vibrio parahaemolyticus was 95%, the lysis rate to 10 strains of Clostridium perfringens was 100%, the lysis rate to 10 strains of Pasteurella was 90%, the lysis rate to 10 strains of Haemophilus paragallinarum was 70%, and the lysis rate to 10 strains of Riemerella anatipestifer was 100%; the proportion of lactobacillus showing positive results for the 120 indicator bacteria was 115 / 120 (95.8%). In addition, the diameter of the inhibition zone of the lactic acid bacteria of the present application for 25 strains (83.3%) of the 30 Gram-positive indicator bacteria is greater than 25 mm, indicating that the lactic acid bacteria of the present application has extremely strong antibacterial activity against Gram-positive bacteria. The diameter of the inhibition zone of the lactic acid bacteria of the present application for 72 strains (80%) of the 90 Gram-negative indicator bacteria is greater than 15 mm, indicating that the lactic acid bacteria of the present application also has strong antibacterial activity against Gram-negative bacteria.

[0075] Example 4: Temperature resistance test of lactobacillus

[0076] (I) Reagents and consumables

[0077] A semi-solid medium, Oxford cup, and lower medium plate were added with 0.5% agar and 1% disodium hydrogen phosphate.

[0078] (II) Experimental strains

[0079] The experimental strains selected common pathogenic bacteria in pets, 10 strains each of Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae, were isolated from clinical diarrhea feces and purulent areas of pet skin. At the same time, the more sensitive Micrococcus luteus was selected as a positive control.

[0080] (III) Experimental methods

[0081] (1) In order to facilitate heat treatment and later application, the lactobacillus extract prepared in Example 2 was added to auxiliary materials and spray-dried to obtain lactobacillus powder, which was used as the sample for heat treatment in this experiment. The sample treatment groups 50°C and 60°C were treated in a water bath, and the remaining samples were treated with lactobacillus powder in a dry heat drying oven at temperatures of 110°C, 120°C, 130°C, 140°C, and 150°C, and each treatment was 2 min, 5 min, and 10 min.

[0082] (2) Mark the lower culture medium plate and divide the area according to the experimental sample groups. Place the Oxford cup vertically in the center of each area and mark it.

[0083] (3) Take the freshly cultured bacterial solution to be tested (final concentration is 10 6 CFU / mL) was added to the pre-melted semi-solid culture medium supplemented with 0.5% agar and 1% disodium hydrogen phosphate, and manually mixed and added to the lower plate with an Oxford cup, and then cooled and solidified.

[0084] (4) The heat-treated sample was diluted 5-fold and dissolved in physiological saline. 200 μL of the diluted sample was added to a cooled and solidified Oxford cup double-layer plate, pre-diffused at 4°C for 3.5 h, and cultured overnight at 37°C for 12 h.

[0085] (IV) Experimental results

[0086] The number of inhibition zones produced in each group of 10 bacteria was counted by the Oxford cup double-layer plate method, and the inhibition rate of lactobacillus on the bacteria in this group was calculated = the number of bacteria producing inhibition zones / 10×100%, and the statistical results were recorded in Table 3.

[0087] Table 3 Statistics of the thermotolerant and antibacterial rate of lactobacillus

[0088]

[0089] According to the data in Table 3, it can be seen that, excluding the measurement error, the antibacterial effect of lactobacillus was not reduced after being treated at different temperatures for different periods of time. The inhibition zone was clear and bright, and the lysis rate was almost the same. After being treated at high and low temperatures, the original antibacterial potency and effect were still maintained.

[0090] Therefore, the lactobacillus of the present application can be applied to pet food as a pet food additive, solving the problem that the effect of lactobacillus may be reduced due to the high-temperature puffing process of pet food.

[0091] Example 5: In vitro antibacterial experiment of lactobacillus

[0092] (I) Experimental groups

[0093] The concentration was prepared to be 1×10 5 CFU / mL bacterial suspension of Escherichia coli 8099, Salmonella abattoba ATCC35640, Clostridium perfringens CICC 22949, Staphylococcus aureus ATCC6538, and Enterococcus faecium ATCC8459 standard strains. The strains were provided by a third-party testing company. The plate count method was used to test the lysis effect of lactobacillus on the five standard strains. The experimental grouping information is shown in Table 4.

[0094] Table 4 Experimental group information

[0095]

[0096] (II) Experimental steps

[0097] Each bacterial suspension was diluted 5 times the volume of its corresponding enrichment solution, and 5 mL was taken from each of two tubes. 1 g of lactobacillus experimental sample and 1 g of auxiliary material control sample were added to dissolve them completely. After standing at 37°C for 24 hours, they were inoculated on TSA plates and cultured at 36°C for 24 hours, and then the plate counts were performed (Clostridium perfringens was stood and cultured in an anaerobic environment, using TSC plates).

[0098] (III) Experimental results

[0099] The experiment found that the lactobacillus of the present application can effectively inhibit harmful pathogenic bacteria such as Escherichia coli, Salmonella, Clostridium perfringens, Staphylococcus aureus, and Enterococcus faecium. After 24 hours of action, the inhibition rate of these five bacteria can reach more than 99%. The specific information of the experimental results is shown in Table 5.

[0100] Table 5 Statistical information of in vitro antibacterial results

[0101]

[0102]

[0103] Note: Inhibition rate (%) = (target bacteria concentration in control group - target bacteria concentration in experimental group) / target bacteria concentration in control group × 100%.

[0104] Example 6: The regulatory effect of lactobacillus on the intestinal tract of pets

[0105] (I) Experimental groups

[0106] Fifteen beagles were divided into three groups, with five dogs in each group. Each group was kept in the same breeding environment. A control group, a low-dose group and a high-dose group were set up. The specific groups and dosages are shown in Table 6.

[0107] The dog food and drinking water used in each group remained consistent, and the dogs were fed according to the product dosage for one month, and various indicators were observed.

[0108] Grouping Number of experimental dogs Product dosage Meals / Day Low dose group 5 1000ppm 2 High dose group 5 1500ppm 2 Blank control 5 0 2

[0109] (II) Experimental steps

[0110] 1. Fast for 12 hours and deprive water for 6 hours before the experiment.

[0111] 2. Dilute lactobacillus by 10 times with auxiliary materials as test samples. At the beginning of the experiment, feed the dogs daily according to the dosage of the product, that is, add 2g of test sample (i.e. 1000ppm) to each 200g / dog feed of the low-dose group, and add 3g of test sample (i.e. 1500ppm) to each 200g / dog feed of the high-dose group. The test sample should be evenly sprinkled on the surface of the feed, and supervise the dogs to finish eating all the food and test samples each time.

[0112] 3. The experiment was divided into 4 weeks, and the feeding was continued for 1 month before the experiment was completed.

[0113] 4. After the experiment begins, collect the first feces of each group of experimental dogs, weigh a certain weight of feces, place it in physiological saline and oscillate at 37°C for 30 minutes, dilute the extract to a suitable gradient and count the colonies on a solid culture medium plate. Set up 3 parallels for each sample. The culture medium for colony detection is shown in Table 7.

[0114] 5. Take fecal samples from each dog on the 3rd and 7th day of each week to test the content of Enterobacteriaceae and lactic acid bacteria; during the experiment, select one person to observe the fecal quality of the experimental dogs at the same time, observe whether the feces of the experimental dogs are formed, whether the fecal odor is reduced, and evaluate the product's improvement in fecal quality.

[0115] 6. Analysis of bacterial detection technology: Bacterial number = sum of the number of each plate / 3×dilution factor (CFU / g).

[0116] Detection strains Enterobacteriaceae Lactic acid bacteria Use culture medium MAC MRS Culture conditions 37℃, aerobic 37℃, anaerobic

[0117] (III) Experimental results

[0118] The results of Enterobacter fecal bacterial load are shown in Table 8 and Figure 3 .

[0119] Table 8 Enterobacterial load detection Lg (CFU / g)

[0120]

[0121] Note: The numbers with different letters are different, a, b; P<0.05.

[0122] The results of lactic acid bacteria colony counts are shown in Tables 9 and Figure 4 .

[0123] Table 9 Lactobacillus feces bacterial load detection Lg (CFU / g)

[0124]

[0125] Note: Different letters indicate differences, a, b; P<0.05

[0126] according to Figure 3 , Figure 4 As shown in Tables 8 and 9, the test results of Enterobacteriaceae and lactobacilli in feces show that feeding different doses of lactobacillus for one week can reduce the number of Enterobacteriaceae in feces of experimental dogs. After continuous use of lactobacillus for two weeks, the number of Enterobacteriaceae in feces decreased by about 90%. After three weeks, the number of Enterobacteriaceae in feces remained at 10. 7 CFU / g, which was nearly 1 titer lower than that of the control group, but there was no significant difference in the effects between the low-dose group and the high-dose group.

[0127] By testing the content of lactic acid bacteria in feces, it can be found that feeding lactobacillus can regulate the content of lactic acid bacteria in the intestine. The low-dose group can increase the lactic acid bacteria content by up to 48.55%, and the high-dose group can increase the lactic acid bacteria content by up to 39.07%. Similarly, after 2 weeks of feeding, there was a significant difference between the experimental group and the control group, but there was no significant difference between the low-dose group and the high-dose group.

[0128] Therefore, lactobacillus can be added to pet dog feed at 1000ppm, and by mixing with feed, it can improve the balance of dog intestinal flora, regulate intestinal health, and thus improve the health of dogs. At the same time, in the experiment of observing the feces of experimental dogs, it was found that lactobacillus can effectively improve the odor of feces, thereby indirectly improving the breeding environment.

[0129] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. An application of lactobacillus, characterized in that: The lactobacillus is used to prepare pet feed, and the addition amount of the lactobacillus in pet feed is 1000-1500ppm. The lactobacillus is produced by fermentation of Lactococcus lactis. The strain is named Lactococcus lactis 5019 and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on January 13, 2021. The deposit address is: Beijing, China, and the deposit number is CGMCCNO.21607.

2. The use of lactobacillus according to claim 1 in preparing pet feed, characterized in that: The antibacterial spectrum of the lactobacillus is Staphylococcus, Escherichia coli, Salmonella, Clostridium perfringens, Pasteurella, Haemophilus paragallinarum, Vibrio parahaemolyticus, Riemerella anatipestifer, Klebsiella pneumoniae, Clostridium perfringens and Enterococcus faecium.

3. The use of lactobacillus according to claim 1 in preparing pet feed, characterized in that: The preparation method of lactobacillus comprises the following steps: S1. Preparation of lactic acid bacteria seeds: inoculating the Lactococcus lactis 5019 described in claim 1 into a culture medium, culturing it statically to make it proliferate and activate, and obtaining a Lactococcus lactis seed solution; S2, lactic acid bacteria amplification culture: inoculating Lactococcus lactis seed liquid into the culture medium, culturing it statically to make it proliferate and activate, and obtaining Lactococcus lactis fermentation liquid; S3. Preparation of lactobacillus: The pH of the Lactococcus lactis fermentation broth is adjusted to 2-3 with concentrated hydrochloric acid, and then inactivated; and the supernatant is taken as lactobacillus.

4. The use of lactobacillus according to claim 3 in preparing pet feed, characterized in that: In S3, the inactivation treatment conditions are: heating at a temperature of 100-110° C. for 3-10 min.

5. The use of lactobacillus according to claim 3 in preparing pet feed, characterized in that: In S3, the supernatant is obtained by centrifugation, and the centrifugation conditions are: centrifugation at a speed of 8000-12000 rpm for 3-8 minutes.

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