A lactobacillus and its application in preventing and controlling bacterial infection in livestock and poultry

Abstract

The present application relates to the field of microorganisms, and specifically discloses a lactic acid bacterium and its application in preventing and controlling bacterial infections in livestock and poultry. The lactic acid bacterium is produced by fermentation of Lactococcus lactis. The strain is named Lactococcus lactis 5019 and was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on January 13, 2021. The deposit address is: Beijing, China, and the deposit number is CGMCC NO.21607. The lactic acid bacterium produced by Lactococcus lactis in the present application can be used to prevent and control bacterial infections in livestock and poultry, 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 infections in livestock and poultry. Background Art

[0002] Due to the excessive and improper use of antibiotics in humans and animals, bacterial multidrug resistance is becoming increasingly serious. In addition to hospital intensive care units becoming a breeding ground for super-resistant bacteria, there are also many reports of multidrug-resistant bacteria being isolated from animal-derived foods. In order to address the above problems, scientific research teams around the world are developing new antibacterial drugs in order to either synergistically enhance the bactericidal effect of antibiotics or replace antibiotics to achieve the effect of bactericidal treatment of bacterial infectious diseases. Today, when human public health is of great significance, the use of antibiotics requires particularly rigorous scientific guidance and strict legal supervision. Therefore, new antibacterial ingredients need to be developed in animal breeding, food sources, food storage and other links to ensure public health safety and the effectiveness of food preservation.

[0003] In the food industry, a class of lactic acid bacteria (LAB) plays an important role. This is a general term for a class of spore-free, Gram-positive bacteria that can ferment sugars to produce lactic acid. Lactic acid bacteria are a class of probiotics that can activate immune cells such as macrophages and NK cells to eliminate harmful bacteria and cancer cells in the body. Research data show that lactic acid bacteria can promote animal growth, regulate normal gastrointestinal flora, maintain microecological balance, thereby improving gastrointestinal function, increasing food digestibility and biological potency, lowering serum cholesterol, controlling endotoxins, inhibiting the growth of putrefactive bacteria in the intestine, and improving the body's immunity. However, there are also certain problems with probiotic live bacteria. For example, lactic acid bacteria are extremely unstable, have poor stress resistance, are not resistant to storage, have low gastrointestinal survival rates, are difficult to colonize, and consume energy for proliferation.

[0004] Studies have found that the inanimate bacteria and (or) their metabolites of probiotics also have obvious probiotic effects, and the probiotic functions of different components are different. Metabolites such as organic acids, special enzymes, and lactobacilli produced by lactic acid bacteria through fermentation also have certain bactericidal and antibacterial effects. 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 livestock and poultry.

[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, wherein the lactic acid bacterium 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, with the deposit address: Beijing, China, 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 at a pH of 2.0; and it has high temperature resistance, and its potency is not significantly affected after being treated at a temperature of 150°C for 5 minutes. It can be combined with actual production to develop preparations suitable for various application scenarios, which can prevent the breeding end from being infected by pathogens.

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

[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, etc., and can lyse a variety of Gram-negative bacteria, such as Escherichia coli, Salmonella, Riemerella anatipestifer, Pasteurella, Vibrio parahaemolyticus, Haemophilus paragallinarum, etc. In view of the lysis effect of lactobacillus on a variety of pathogens, the present application has developed more application fields, such as preventing and controlling the horizontal spread of a variety of pathogens in farms, assisting or replacing antibiotics to sterilize and prevent bacterial infections, and promoting the growth performance of broilers as a feed additive, thereby reducing the accumulation of antibiotics in food-borne animals and reducing 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, wherein the lactobacillus is used for preventing and controlling bacterial infection in livestock and poultry.

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

[0026] Optionally, the lactobacillus is added to livestock and poultry feed in an amount of 50-200 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 at a pH of 2.0; and it has high temperature resistance. After being treated at 150°C for 5 minutes, its potency is not significantly affected. It can be combined with actual production to develop preparations suitable for various application scenarios, which can prevent the breeding end from being infected by pathogens.

[0029] 2. The lactobacillus of the present application has a strong broad spectrum, and can lyse Gram-positive bacteria, such as Staphylococcus aureus, Clostridium perfringens, etc., and can lyse a variety of Gram-negative bacteria, such as Escherichia coli, Salmonella, Riemerella anatipestifer, Pasteurella, Vibrio parahaemolyticus, Haemophilus paragallinarum, etc.

[0030] 3. The lactobacilli of the present application have a lysis effect on a variety of pathogens. The present application has developed more application fields, such as preventing and controlling the horizontal spread of a variety of pathogens in farms, assisting or replacing antibiotics in sterilization and preventing bacterial infections, and serving as a feed additive to promote the growth performance of broilers, thereby reducing the accumulation of antibiotics in food-borne animals and reducing the public health and safety risks caused by super-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] Figure 2 This is a cell morphology diagram of Lactococcus lactis 5019 in Example 1 of the present application.

[0033] Figure 3 This is a comparison chart of the antibacterial effect of lactobacillus after high temperature treatment in Example 4 of the present application.

[0034] Figure 4 This is a diagram showing the antibacterial effect of lactobacillus on Escherichia coli, Staphylococcus aureus and ML bacteria after high temperature treatment (110°C, 120°C) in Example 4 of the present application.

[0035] Figure 5 The antibacterial effect of lactobacillus on Staphylococcus after high temperature treatment (130°C, 140°C, 150°C) in Example 4 of the present application.

[0036] Figure 6 This is a comparison chart of liver lesion scores in Example 5 of the present application.

[0037] Figure 7 This is a comparison chart of the bacterial load in the liver of each chicken in Example 5 of the present application.

[0038] Figure 8 This is a comparison chart of the average daily weight gain of broiler chickens in each group in Example 7 of the present application.

[0039] Fig. 9 This is a comparison chart of the average daily feed intake of broiler chickens in each group in Example 7 of the present application.

[0040] Fig.10 This is a comparison chart of the feed-to-meat ratio of each group of broiler chickens in Example 7 of the present application.

[0041] Fig.11This is a comparison chart of serum IgA content in each group of broiler chickens in Example 7 of the present application.

[0042] Fig.12 This is a comparison chart of serum IgG content in each group of broiler chickens in Example 7 of the present application.

[0043] Fig.13 This is a comparison chart of serum IgM content in each group of broiler chickens in Example 7 of the present application.

[0044] Fig.14 This is a comparison chart of serum SOD content in each group of broiler chickens in Example 7 of the present application.

[0045] Fig.15 This is a comparison chart of serum MDA content in each group of broiler chickens in Example 7 of the present application. DETAILED DESCRIPTION

[0046] The present application is further described in detail below with reference to the accompanying drawings and embodiments.

[0047] Example 1: Isolation, culture and biological characteristics of Lactococcus lactis

[0048] 1. Isolation and cultivation of Lactococcus lactis

[0049] 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.

[0050] 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; after adding distilled water to dissolve, adjust the pH to 7.0, add 1.5% (by weight) agar powder to the solid culture medium and sterilize at 115°C for 30 minutes.

[0051] 2. Screening and identification of Lactococcus lactis

[0052] The obtained strain was biochemically identified and found to have the following morphological characteristics and physiological and biochemical properties:

[0053] (1) Macroscopic form: Figure 1As 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.

[0054] (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.

[0055] (3) The main physiological and biochemical characteristics are shown in Table 1:

[0056] Table 1 Physiological and biochemical characteristics of strains

[0057]

[0058]

[0059] Explanation of symbols: “+”, positive; “-”, negative.

[0060] (4) Molecular biological identification results:

[0061] 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.

[0062] 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.

[0063] Example 2: Preparation of lactobacillus

[0064] A lactobacillus is prepared by the following method:

[0065] S1. Preparation of lactic acid bacteria seeds

[0066] 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.

[0067] S2. Lactic acid bacteria amplification culture

[0068] 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.

[0069] S3. Preparation of lactobacillus

[0070] 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.

[0071] Example 3: Broad-spectrum antibacterial activity of lactobacillus

[0072] The lactobacillus extract prepared in Example 2 was used as a sample to test its antibacterial performance.

[0073] (I) Proliferation of multiple bacteria to be tested

[0074] 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.

[0075] Table 2 Bacteria to be tested and culture medium

[0076] 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

[0077] (II) Detection of cleavage spectrum of lactobacillus

[0078] The cleavage spectrum of lactobacillus was detected using the double-layer plate method.

[0079] (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.

[0080] (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.

[0081] (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.

[0082] Table 3 Antimicrobial spectrum test results of lactobacillus

[0083]

[0084]

[0085]

[0086] 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 livers 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.

[0087] Result analysis:

[0088] 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.

[0089] Example 4: Temperature resistance test of lactobacillus

[0090] (I) Reagents and consumables

[0091] Add 0.5% agar and 1% disodium hydrogen phosphate to the semi-solid medium and use an Oxford cup.

[0092] (II) Experimental strains

[0093] The experimental strains used were Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria), and Micrococcus luteus with a higher sensitivity was selected as a positive control.

[0094] (III) Experimental methods

[0095] (1) In order to facilitate heat treatment and subsequent application, the lactobacillus extract prepared in Example 2 was spray-dried to obtain lactobacillus powder, and the lactobacillus powder was used as a sample for heat treatment in this experiment. The treatment conditions were 110°C / 10 min, 120°C / 10 min, 130°C / 2 min, 130°C / 5 min, 140°C / 2 min, 140°C / 5 min, 150°C / 2 min; 150°C / 5 min.

[0096] (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.

[0097] (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.

[0098] (4) The heat-treated sample was diluted 5 times and dissolved in physiological saline. 200 μL of the diluted sample was added to the cooled and solidified Oxford cup double-layer plate, pre-diffused at 4°C for 3.5 hours, and cultured overnight at 37°C for 12 hours. The diameter of the inhibition zone was counted, and the results are shown in Tables 4 and 5.

[0099] Table 4 Diameter of inhibition zone after high temperature (110℃ / 120℃) treatment (mm)

[0100]

[0101]

[0102] Note: The above strains were isolated from diseased livestock and poultry lesions in pig and chicken farms in many parts of the country.

[0103] Table 5 Diameter of inhibition zone after high temperature (130℃, 140℃, 150℃) treatment (mm)

[0104]

[0105] Note: DB398 and DB400 are Staphylococci, isolated from the lesions of exudative dermatitis in a pig farm in Qingdao.

[0106] Result analysis:

[0107] According to the data in Table 4 and Table 5 and Figure 4-Figure 5 It can be seen that the lactobacillus of the present application still has an obvious antibacterial effect after being treated at 110°C and 120°C for 10 minutes, and the diameter of the inhibition zone is slightly lower than that of the original lactobacillus powder; after being treated at 150°C / 5 minutes, the diameter of the bacterial circle is slightly lower than that of the original lactobacillus powder, but it does not significantly affect its potency, indicating that the lactobacillus produced by Lactococcus lactis 5019 of the present application has excellent high temperature resistance, and the high temperature conditions are far beyond the high temperature conditions of feed production. Therefore, the lactobacillus in the present application can be developed and used as a feed additive.

[0108] Example 5: In vivo antibacterial experiment of lactobacillus

[0109] 1. Strains and experimental animals

[0110] The test strain was the standard strain of Salmonella CVCC533, purchased from the China Veterinary Drug Inspection Institute. The experimental animals were 60 ordinary broiler chickens, with 10 chickens in each group, for a total of 6 groups.

[0111] (II) In vivo antibacterial experiment of lactobacillus

[0112] This experiment set up a blank control group, a challenge control group, a (blank control + lactobacillus) group, and other lactobacillus experimental groups with different titers. The specific groups are shown in Table 6. The oral challenge dose was 2×10 8 CFU / feather, 0.2mL / feather. 0.5h after infection, each feather was gavaged with 0.2mL of lactobacillus solution.

[0113] Table 6 Grouping and treatment information of lactobacillus in vivo antibacterial experiment

[0114]

[0115] After treatment, the clinical symptoms were observed and the chickens were fed with sterile feed and water. After 7 days, the chickens in each group were dissected, the liver lesions were observed, the number of lesions was recorded and scored according to the scoring criteria in Table 7. At the same time, the bacterial load in the liver was detected and the bactericidal effect of lactobacillus in vivo was analyzed to determine the effect of lactobacillus in preventing the horizontal spread of Salmonella.

[0116] Table 7 Scoring criteria for liver lesions

[0117]

[0118] 3. Statistics of liver lesion results

[0119] The liver lesion scoring results are as follows Figure 6 The results of the bacterial load test on the liver of each chicken are shown in Table 8 and Figure 7 shown.

[0120] Table 8 Average bacterial load in liver

[0121]

[0122] Combination Figure 6-Figure 7 As can be seen from the data in Table 8, after the use of lactobacillus, the liver lesions of chickens were improved compared with the challenge group, and compared with the challenge control group, the average bacterial load in the liver of the lactobacillus stock solution test group was reduced by 87.38%, the lactobacillus 10-fold dilution test group was reduced by 76.07%, and the lactobacillus 100-fold dilution test group was reduced by 86.08%. After the blank was added with lactobacillus stock solution, there was basically no bacterial growth in the liver, which was only 2.03% of the bacterial content in the challenge group, and 4.71% lower than the bacterial content in the blank control group, indicating that lactobacillus can effectively inhibit the colonization of Salmonella in chickens and prevent its horizontal transfer.

[0123] In summary, each experimental group was able to significantly reduce the Salmonella load in chicken liver (P < 0.05), indicating that the lactobacillus of the present application can effectively inhibit the colonization and horizontal transmission of Salmonella in chickens, provide preventive protection for the breeding environment of farms, reduce economic losses caused by Salmonella contamination, and improve the economic value of farms.

[0124] Example 6: Experiment on the prevention of bacterial diseases by lactobacillus

[0125] 1. Strains and experimental animals

[0126] The test strains were Escherichia coli D18 and Clostridium perfringens 999, which were isolated from the liver and intestines of dead chickens in farms, respectively; the experimental animals were 200 ordinary AA broiler chickens, with 50 chickens in each group, for a total of 4 groups.

[0127] (II) Experiment on the prevention of bacterial infectious diseases by lactobacilli

[0128] This experiment set up a blank control group, a challenge group, a high-dose lactobacillus group, and a low-dose lactobacillus group. The specific groups are shown in Table 9. Feed was mixed according to the group sample processing requirements. At 3 days of age, the mixed lactobacillus feed and normal feed were fed according to the group. At 14 days of age, the challenge group, the high-dose lactobacillus group, and the low-dose lactobacillus group were orally administered with a challenge dose of 2×10 8 CFU / feather, 1.5mL / feather, the blank control group was orally administered with NB culture medium, 1.5mL / feather.

[0129] Table 9 Grouping and treatment information for lactobacillus preventive test

[0130]

[0131] After the infection, the clinical symptoms were observed. Abnormal conditions such as diarrhea and anal paste were considered as symptoms of disease. The number of sick animals and the number of deaths were recorded every day, and the morbidity and mortality rates were calculated. The results are recorded in Table 10.

[0132] Table 10 Statistics of morbidity in broiler chickens

[0133] Grouping Treatment Incidence mortality rate 1 Lactobacillus high dose group 6％ 0 2 Lactobacillus low dose group 4％ 0 3 The challenge control group 16％ 12％ 4 Blank control group 2％ 0

[0134] According to the data in Table 10, compared with the challenge control group, the incidence rates of the high-dose lactobacillus group and the low-dose lactobacillus group were reduced by 10% and 12%, respectively, while the mortality rate of the challenge control group was 12%, and that of the other groups was 0. This indicates that adding lactobacillus to feed can prevent bacterial infection, reduce the incidence and mortality of chickens after challenge, and thus play an effective protective role.

[0135] Example 7: Study on the application of lactobacillus as feed additive

[0136] 1. Experimental animals and group settings

[0137] The experimental animals were 240 AA broiler chicks, 60 chicks in each group, 4 groups in total, 3 replicates in each group, and 20 chicks in each replicate. The grouping settings and feeding methods are shown in Table 11.

[0138] Table 11 Grouping of research on the application of lactobacilli as feed additives

[0139]

[0140] (II) Experimental methods

[0141] According to the added dosage of each group, the lactobacillus powder was evenly mixed into the feed, and the pigs were allowed to eat freely during the feeding period.

[0142] The experiment was divided into two phases. In the first phase, chick feed was fed from 1 to 21 days, and in the second phase, adult feed was fed from 22 to 42 days. The entire feeding cycle was 6 weeks. Each group of broilers was weighed on an empty stomach every week, and the feed consumption of each group was recorded every day. The average daily weight gain and average feed intake were calculated, and the health and mortality of the experimental animals were recorded at the same time.

[0143] After 6 weeks, each group was weighed, weight gain was compared, and feed-to-meat ratio was calculated. At the same time, 10 chickens were randomly selected for blood collection, serum was prepared, and the contents of IgA, IgG, IgM, SOD, and MDA were tested to compare the levels of immune factors and antioxidant levels in each group.

[0144] (III) Experimental results After feeding lactobacillus mixed with feed for 6 weeks, the weight gain and feed intake data of each group of broilers were counted. After statistical analysis, when the lactobacillus addition was 50ppm, the average daily weight gain, average daily feed intake, and feed-to-meat ratio of the experimental group were significantly different from those of the control group (p < 0.05). The comparison charts of average daily weight gain, average daily feed intake, and feed-to-meat ratio are shown in Figures 8 to 10 . This shows that lactobacillus can effectively promote the growth performance of broilers and can be used as a feed additive in poultry farming.

[0145] The kit detects the content of IgA, IgG, and IgM. See the comparison chart of antibody content for details. Figures 11 to 13 , and the SOD and MDA content comparison chart is shown in Fig.14 , 15 As can be seen from the above figure, the average antibody level of the experimental group with lactobacillus was significantly higher than that of the blank control group, among which the increase in IgM was the most significant; and after adding lactobacillus, the antioxidant level of the animals was significantly improved.

[0146] The above results indicate that the lactobacilli in the present application can effectively promote growth, regulate the immune level and antioxidant level of animals, and can be used as feed additives in livestock and poultry farming to improve the farming returns of livestock and poultry.

[0147] 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. A lactobacillus, characterized in that: 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 CGMCC NO.21607.

2. A lactobacillus according to claim 1, characterized in that: The antibacterial spectrum of the lactobacillus is Staphylococcus aureus, Escherichia coli, Salmonella, Clostridium perfringens, Pasteurella, Haemophilus paragallinarum, Vibrio parahaemolyticus and Riemerella anatipestifer.

3. A pharmaceutical preparation, characterized in that: Contains the lactobacillus as claimed in claim 1.

4. The method for preparing lactobacillus according to claim 1, characterized in that: The steps include: 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.

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

6. The method for preparing lactobacillus according to claim 4, 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.

7. The use of lactobacillus according to claim 1, characterized in that: The application of the lactobacillus in preparing livestock and poultry feed.

8. The use according to claim 7, characterized in that: The lactobacillus is added in an amount of 50-200 ppm in livestock and poultry feed.

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