Application of antibacterial preparation in preparation of product for inhibiting escherichia coli in broiler chicken

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of an antibacterial preparation in preparation of a product for inhibiting escherichia coli in broiler chickens. The method comprises the following steps: producing the lytic bacteriophage provided by the invention by utilizing escherichia coli, when the bacteriophage is accumulated to a certain titer, inoculating the bacillus licheniformis capable of producing antibacterial lipopeptide capable of inhibiting escherichia coli provided by the invention, simultaneously changing fermentation parameters, and feeding and supplementing carbon and nitrogen sources, so that the bacillus licheniformis rapidly grows, and the bacillus licheniformis can be used for producing the antibacterial lipopeptide capable of inhibiting escherichia coli. And the accumulated antibacterial lipopeptide can quickly kill redundant escherichia coli remaining in a fermentation system, so that the synergistic anti-escherichia coli preparation of the bacteriophage coupled antibacterial lipopeptide is prepared. According to the method, the safety problem of residual pathogenic escherichia coli in the coliphage culture process is solved, meanwhile, production of the antibacterial lipopeptide preparation is also considered, the double-effect anti-escherichia coli preparation is produced at lower cost, the antibacterial effect is better and more prominent, and the method has wide application prospects in the broiler chicken breeding industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and more specifically, relates to the application of an antibacterial preparation in the preparation of a product for inhibiting Escherichia coli in broilers. Background Art

[0002] Broiler pathogenic Escherichia coli (APEC) is the most common pathogen in broiler farming, causing colibacillosis in broilers worldwide. It is an important subtype of extraintestinal pathogenic Escherichia coli, causing various local and systemic infections in young broiler chickens. Broiler diseases caused by APEC include hepatitis, tracheitis, pericarditis, and other syndromes such as ovum peritonitis, salpingitis, colic granuloma, omphalitis, and cellulitis, collectively referred to as "colibacillosis". APEC causes broiler diseases through specific virulence factors, such as adhesins, invasins, protectins, iron acquisition systems, and toxins. These virulence factors promote APEC invasion and its escape from host immune responses, leading to extraintestinal infections, which seriously affect the development of the broiler farming industry. In order to improve the quality of broiler products and reduce the risk of pathogenic E. coli infection, antibiotics are widely used in broiler and other poultry farming fields. However, the abuse of antibiotics will greatly disrupt the balance of the natural microecological flora in the intestines of animals, which can easily lead to serious consequences such as the outbreak of multi-drug resistant pathogens or antibiotic residues in products. The Ministry of Agriculture and Rural Affairs' "Pilot Work Plan for Reducing the Use of Veterinary Antimicrobial Drugs (2018-2021)" pointed out that zero drug addition to feed should be achieved by the end of 2020, and encourages the active exploration of alternatives to veterinary antibiotics, gradually reducing the types and amounts of growth-promoting veterinary antibiotics used, and improving the level of healthy farming. Therefore, under the current background of "banning antibiotics" and "reducing antibiotics", how to effectively avoid the massive reproduction of pathogenic microorganisms in the breeding environment through green and safe methods is the focus of preventing and controlling such diseases.

[0003] Bacteriophages are viruses that infect, inhibit and kill susceptible bacteria. Lytic bacteriophages (hereinafter referred to as phages) can specifically infect host bacteria and lyse them. This property can be used to apply phages to antibacterial treatment. In recent years, with the increase of bacterial resistance to antibiotics, phage therapy has attracted widespread attention in the livestock and poultry farming industry as an effective alternative to antibiotics.

[0004] However, there are few reports on the large-scale preparation of phages, and the preparation of most phages is still limited to laboratory scale. There are three main reasons for this: (1) The host infection spectrum of phages themselves is relatively narrow, and the screening of high-titer phages is relatively difficult. Moreover, when phages are used as the sole selection pressure, host bacteria that are not eliminated by phages in time are prone to mutation and become resistant to phages. (2) Since the growth of phages depends on the reproduction of host bacteria, from the perspective of ensuring their own survival and evolution, phages will not completely disinfect host bacteria, which results in phage preparations sometimes not having as thorough an inhibitory effect on host bacteria as antibiotics. (3) During the production process, phages need to be fermented and cultured with pathogenic host bacteria. Therefore, during the preparation of phages, when the phage titer reaches the maximum, a certain amount of pathogenic host bacteria will often remain in the fermentation broth, which seriously increases the subsequent biosafety and separation and purification costs, resulting in the lack of popularization of large-scale promotion of phage preparations.

[0005] Therefore, if we can find biological agents that can be used in combination with bacteriophages to enhance the antibacterial effect of bacteriophage products within a unit concentration, and if we can innovate in the bacteriophage preparation process to reduce the impact of residual pathogenic host bacteria during the phage production process, it will greatly promote the popularization of bacteriophage-related products in the broiler farming industry. Summary of the invention

[0006] The purpose of the present invention is to provide a method for producing a dual-effect antibacterial preparation by coupling E. coli phage and antibacterial lipopeptide in one step, in order to solve the problems that the residual pathogenic host bacteria cells in the preparation of E. coli phage affect the subsequent purification and application, the antibacterial effect of unilateral E. coli phage preparation is unstable, pathogenic bacteria are easy to mutate, and the effect of E. coli phage preparation is deteriorated. The present invention takes into account the preparation of E. coli phage and antibacterial lipopeptide, can greatly reduce the residual amount of host pathogenic bacteria in the process of preparing E. coli phage, reduce the subsequent purification cost, improve biosafety, and produce more efficient phage and antibacterial lipopeptide composite biological preparation products that inhibit pathogenic E. coli at a lower preparation cost, and has broad market prospects for application in the field of broiler breeding.

[0007] In order to achieve the above-mentioned object of the invention, the present invention is implemented by the following technical solutions:

[0008] The present invention provides an application of an antibacterial preparation in preparing a product for inhibiting Escherichia coli in broilers. The antibacterial preparation is an antibacterial preparation obtained by secondary fermentation of Escherichia coli phage with Bacillus licheniformis WS-2 after Escherichia coli induced fermentation. The Escherichia coli phage is enterotoxigenic Escherichia coli lytic phage PES-1, the deposit number of the enterotoxigenic Escherichia coli lytic phage PES-1 is CGMCC No.13382, and the deposit number of the Bacillus licheniformis WS-2 is CGMCC No.12813.

[0009] In a preferred embodiment, the titer of enterotoxigenic Escherichia coli lytic bacteriophage PES-1 in the antibacterial preparation is 1×10 8 ~10 10 PFU / mL, the number of spores of Bacillus licheniformis WS-2 is 1×10 9 ~10 10 cfu / mL, residual amount of E. coli host bacteria ≤10 2 cfu / mL.

[0010] In the preferred embodiment, in the above application, the method for preparing the Escherichia coli phage-coupled antibacterial lipopeptide liquid (antibacterial preparation) comprises the following steps:

[0011] S1. Inoculate Escherichia coli as a host bacterium into a fermentation medium to obtain a fermentation broth; the components of the fermentation medium include a carbon source, a nitrogen source, an inorganic salt and water, the inoculation amount of the host bacterium is 1% to 3% (v / v), and the concentration of the host bacterium in the fermentation broth is 1×10 6 ~10 8 cfu / mL;

[0012] S2, inoculating the enterotoxigenic Escherichia coli lytic phage PES-1 into the fermentation liquid for culture, to obtain the Escherichia coli phage culture liquid; the infection multiplicity of the enterotoxigenic Escherichia coli lytic phage PES-1 is 0.02-0.1, and the host bacteria concentration in the phage culture liquid does not exceed 1×10 4 cfu / mL;

[0013] S3, inoculating Bacillus licheniformis WS-2 into the phage culture solution to obtain an antibacterial preparation; the inoculation amount of Bacillus licheniformis WS-2 is 3-5%, and the host bacteria concentration in the antibacterial preparation does not exceed 2×10 2 cfu / mL.

[0014] In a preferred embodiment, the components of the fermentation medium include 4-10 g / L yeast extract, 10-20 g / L soy peptone, 10-20 g / L glucose, 30-40 g / L soluble starch, 5-12 g / L sodium chloride, 0.1-0.4 g / L magnesium sulfate, 1-4 mg / L manganese sulfate, 1-4 g / L dipotassium hydrogen phosphate, 1-4 g / L potassium dihydrogen phosphate, 0.5-1 g / L calcium carbonate, 0.1-0.3 g / L zinc sulfate, 0.2-1 mg / L ferrous sulfate, 1-3 mg / L manganese sulfate, 1-5 mg / L copper sulfate and water.

[0015] In a preferred embodiment, the culture conditions of steps S1 to S2 are pH 6 to 7, dissolved oxygen content 5 to 10%, and temperature 30 to 37°C.

[0016] In the preferred embodiment, the culture conditions of step S3 are to add 10-20 g / L glycerol to the E. coli phage culture medium, adjust the pH to 7-8, the temperature to 35-40° C., and the dissolved oxygen content to not less than 20%.

[0017] The second aspect of the present invention provides a method for preparing an antibacterial preparation based on Escherichia coli phage-coupled antibacterial lipopeptide, comprising the following steps:

[0018] S1. Inoculating pathogenic Escherichia coli as a host bacterium into a fermentation medium to obtain a fermentation broth; the fermentation medium comprises a carbon source, a nitrogen source, an inorganic salt and water;

[0019] S2, inoculating the Escherichia coli phage into the fermentation liquid to obtain a phage culture liquid;

[0020] S3. Inoculate Bacillus licheniformis WS-2 into phage culture fluid to obtain an antibacterial preparation.

[0021] In a preferred embodiment, the Escherichia coli phage is the enterotoxigenic Escherichia coli lytic phage PES-1, the deposit number of the enterotoxigenic Escherichia coli lytic phage PES-1 is CGMCC No.13382, and the deposit number of the Bacillus licheniformis WS-2 that produces antagonistic Escherichia coli antibacterial lipopeptides is CGMCC No.12813.

[0022] In a preferred embodiment, the components of the fermentation medium include 4-10 g / L yeast extract, 10-20 g / L soy peptone, 10-20 g / L glucose, 30-40 g / L soluble starch, 5-12 g / L sodium chloride, 0.1-0.4 g / L magnesium sulfate, 1-4 mg / L manganese sulfate, 1-4 g / L dipotassium hydrogen phosphate, 1-4 g / L potassium dihydrogen phosphate, 0.5-1 g / L calcium carbonate, 0.1-0.3 g / L zinc sulfate, 0.2-1 mg / L ferrous sulfate, 1-3 mg / L manganese sulfate, 1-5 mg / L copper sulfate and water.

[0023] In a preferred embodiment, the host bacteria is pathogenic Escherichia coli.

[0024] In the preferred embodiment, in step S1, the inoculation amount of the host bacteria is 1% to 3% (v / v), and the concentration of the host bacteria in the obtained fermentation liquid is 1×10 6 ~10 8 cfu / mL.

[0025] In a preferred embodiment, the culture conditions of steps S1 to S2 are pH 6 to 7, dissolved oxygen content 5 to 10%, and temperature 30 to 37°C.

[0026] In the preferred embodiment, in step S2, the infection multiplicity of the phage inoculated into the fermentation broth is 0.02 to 0.1, and the concentration of host bacteria in the obtained phage culture broth does not exceed 2×10 4 cfu / mL.

[0027] In the preferred embodiment, in step S3, the inoculation amount of Bacillus licheniformis WS-2 is 3-5%, and the concentration of host bacteria in the obtained antibacterial preparation does not exceed 10 2 cfu / mL.

[0028] In a preferred embodiment, the culture conditions of step S3 are to add 10-20 g / L glycerol to the phage culture solution, adjust the pH to 7-8, the temperature to 35-40° C., and the dissolved oxygen content to not less than 20%.

[0029] In a third aspect of the present invention, there is provided an antibacterial preparation prepared by any of the above-mentioned preparation methods, wherein the titer of enterotoxigenic Escherichia coli lytic phage PES-1 in the antibacterial preparation is 1×10 8 ~10 10 PFU / mL, the number of spores of Bacillus licheniformis WS-2 is 1×10 9 ~10 10 cfu / mL, residual amount of E. coli host bacteria ≤ 2×10 2 cfu / mL.

[0030] The fourth aspect of the present invention provides a use of the above antibacterial preparation in inhibiting Escherichia coli.

[0031] Detailed Overview of the Invention

[0032] A bacteriophage capable of lysing Escherichia coli, with the deposit number being CGMCC No.13382.

[0033] The invention discloses a Bacillus licheniformis WS-2 strain which produces antibacterial lipopeptide antagonizing Escherichia coli and has a deposit number of CGMCC No.12813.

[0034] The present invention provides a one-step production process for a novel Escherichia coli phage-coupled antimicrobial lipopeptide preparation, comprising the following steps:

[0035] S1. Inoculate Escherichia coli as the host bacteria into the fermentation medium. The medium formula is yeast extract powder 4-10g / L, soy peptone 10-20g / L, glucose 10-20g / L, soluble starch 30-40g / L, sodium chloride 5-12g / L, magnesium sulfate 0.1-0.4g / L, manganese sulfate 1-4mg / L, dipotassium hydrogen phosphate 1-4g / L, potassium dihydrogen phosphate 1-4g / L, calcium carbonate 0.5-1g / L, zinc sulfate 0.1-0.3g / L, ferrous sulfate 0.2-1mg / L, manganese sulfate 1-3mg / L, copper sulfate 1-5mg / L and water.

[0036] S2: The host bacteria concentration in the culture medium of step S1 reaches 1×10 6 ~10 8 cfu / mL, Escherichia coli phage was inoculated for phage amplification culture without changing the previous fermentation conditions.

[0037] S3. When the number of host bacteria in the phage fermentation broth is less than 2×10 4 cfu / mL, and when the residual host bacteria were not obviously lysed by the phage, Bacillus licheniformis WS-2 was inoculated and the fermentation was continued to obtain the fermentation broth for the one-step production of the Escherichia coli phage-coupled antimicrobial lipopeptide preparation.

[0038] Furthermore, the culture conditions of steps S1 to S2 are pH 6 to 7, dissolved oxygen is controlled at 5 to 10%, and the culture temperature is 30 to 37°C.

[0039] Furthermore, when the enterotoxigenic Escherichia coli lytic phage PES-1 is inoculated into the host bacteria, the infection multiplicity (MOI) of the phage is 0.02 to 0.1.

[0040] Furthermore, the inoculation amount of the Bacillus licheniformis WS-2 inoculated into the phage fermentation liquid is 3-5%.

[0041] Furthermore, after inoculation with Bacillus licheniformis WS-2, the fermentation parameters were adjusted: (1) the fermentation temperature was adjusted from 30 to 37°C to 32 to 40°C, (2) the pH was adjusted to 7 to 8 by adding 5 to 10 mol / L NaOH solution, (3) the dissolved oxygen was adjusted to ≥ 20%, and (4) 10 to 20 g / L glycerol was added.

[0042] Furthermore, the Escherichia coli host bacteria used for culturing bacteriophages can be used as an inducer for the production of antimicrobial lipopeptides by the Bacillus licheniformis WS-2 of the present invention, which can effectively enhance the antibacterial effect of the fermentation product on Escherichia coli.

[0043] Furthermore, the fermentation time of the enterotoxigenic Escherichia coli lytic phage PES-1 of the present invention is 5 to 10 hours, the fermentation time of Bacillus licheniformis WS-2 is 15 to 25 hours, the total fermentation time is 20 to 35 hours, and at the end of the fermentation, the titer of the enterotoxigenic Escherichia coli lytic phage PES-1 is 1×10 8 ~10 10 PFU / mL, the number of spores of Bacillus licheniformis WS-2 is 1×10 9 ~10 10 cfu / ml, residual host bacteria ≤ 2×10 2 cfu / mL.

[0044] Deposit information of the biological material samples involved in the present invention:

[0045] The microbial strain WS-2, classified as Bacillus licheniformis, was deposited in the General Microbiological Center of China Microorganism Culture Collection (CGMCC) on July 22, 2016, with the deposit number CGMCC No.12813, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing;

[0046] The microbial strain PES-1, classified and named as enterotoxigenic Escherichia coli lytic phage, was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on December 8, 2016, with the deposit number CGMCC No.13382, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0047] Based on the industry background and technical bottlenecks, the present invention has the following advantages and technical effects:

[0048] (1) In order to maximize the titer of conventional E. coli phages during the preparation process, pathogens are often cultured at high density, resulting in excessive residual pathogenic E. coli when the phage reaches the maximum titer, up to 10 3 cfu / mL or more, which not only increases the biosafety risk, but also increases the subsequent separation and purification costs. After the present invention is used, after culturing E. coli phage to a certain time, the Bacillus licheniformis strain producing pathogenic antimicrobial lipopeptides is introduced for secondary fermentation, which will further kill the residual pathogenic E. coli, and the residual host bacteria amount does not exceed 10 2 cfu / mL, effectively solving the problem of pathogenic bacteria residues during E. coli phage culture;

[0049] (2) The test results show that the pathogenic bacteria remaining in the process of culturing Escherichia coli phages in the present invention are themselves the biological inducers required by the antimicrobial lipopeptide-producing Bacillus licheniformis strains. By adjusting the fermentation parameters, an antimicrobial lipopeptide fermentation liquid with outstanding effects can be further obtained, killing two birds with one stone;

[0050] (3) The present invention obtains both a high-density Escherichia coli phage culture fluid and a good antimicrobial lipopeptide fermentation fluid, and prepares a dual-effect antimicrobial agent product in one step. Compared with the mixed compound after a single fermentation of the two, the energy consumption cost and time cost are greatly reduced. In particular, at a unit concentration, within a 28-hour action time, the Escherichia coli phage-coupled antimicrobial lipopeptide group has a better effect of inhibiting the growth of Escherichia coli than the Escherichia coli phage group alone, and finally the Escherichia coli can be inhibited at OD 600 The phage group alone inhibited the phage to OD at 18 h. 600 After reaching the lowest point of 0.25, as time went on, E. coli began to become "turbid again", and OD 600 Recovered to 0.5;

[0051] (4) When a single E. coli phage acts on pathogenic bacteria, the residual pathogens are prone to mutation due to the relatively single selection pressure, and then develop resistance to the phage. After the introduction of specific antimicrobial lipopeptides, the selection pressure of the pathogens increases at the same time, so that the combined preparation of E. coli phage coupled antimicrobial lipopeptides will show a better antibacterial effect, and the pathogens will be disinfected more thoroughly. After screening and optimization, the antibacterial preparation of the present invention has a better antibacterial effect, which can increase the survival rate of young broilers after E. coli attack to 80%, which is much higher than the combination of E. coli phage coupled antimicrobial lipopeptide preparations prepared by the same type of phage and Bacillus, which can only maintain the survival rate of young broilers after E. coli attack at 40% and 50%.

[0052] In summary, the method of the present invention prepares highly efficient pathogenic bacteria inhibitors by one-step production of Escherichia coli phage-coupled antimicrobial lipopeptides, which has multiple advantages and effects. Both the logical basis and practical application of the present invention show significant effects. Therefore, the application of this technology in the broiler breeding industry will have broad market prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a comparison chart of the effect of E. coli induction on the antibacterial effect of Bacillus licheniformis WS-2 fermentation broth;

[0054] Figure 2 It is a comparison chart of the changes in the number of residual E. coli host bacteria in the fermentation broth;

[0055] Figure 3 It is a graph of changes in phage titer;

[0056] Figure 4 This is the result diagram of the antibacterial curve of the bacteriophage-coupled antimicrobial lipopeptide preparation;

[0057] Figure 5 This is a graph showing the therapeutic effects of different bacteriophage-coupled antimicrobial lipopeptide preparations on young broiler chickens after infection with Escherichia coli.

[0058] Figure 6 This is a comparison chart of the therapeutic effects of phage-coupled antimicrobial lipopeptide preparations on young broilers and piglets after Escherichia coli infection. DETAILED DESCRIPTION

[0059] The embodiments of the present invention will be described clearly and completely below in conjunction with the examples. Obviously, the described examples are only some examples of the present invention, rather than all examples. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0060] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0061] The present invention supplements carbon and nitrogen sources in the Escherichia coli phage culture solution while inoculating Bacillus licheniformis, and changes the fermentation conditions for culture. The antibacterial preparation prepared by the method of the present invention has a good antibacterial effect and has broad application prospects in the broiler breeding industry.

[0062] The invention relates to the development of a one-step production process of a novel Escherichia coli phage-coupled antimicrobial lipopeptide preparation, wherein the phage is isolated from Escherichia coli as a host, and its deposit number is CGMCC No. 13382. The antimicrobial lipopeptide is produced by fermentation of Bacillus licheniformis WS-2, and its deposit number is CGMCC No. 12813.

[0063] The development of a one-step production process for an Escherichia coli phage-coupled antimicrobial lipopeptide preparation comprises the following steps:

[0064] S1. Inoculate the host bacteria into a fermentation medium, wherein the medium formula is 4-10 g / L yeast extract, 10-20 g / L soy peptone, 10-20 g / L glucose, 30-40 g / L soluble starch, 5-12 g / L sodium chloride, 0.1-0.4 g / L magnesium sulfate, 1-4 mg / L manganese sulfate, 1-4 g / L dipotassium hydrogen phosphate, 1-4 g / L potassium dihydrogen phosphate, 0.5-1 g / L calcium carbonate, 0.1-0.3 g / L zinc sulfate, 0.2-1 mg / L ferrous sulfate, 1-3 mg / L manganese sulfate, 1-5 mg / L copper sulfate and water, pH value is 6-7, dissolved oxygen is controlled at 5-10%, and the culture temperature is 30-37°C.

[0065] S2: The host bacteria concentration in the culture medium of step S1 reaches 1×10 6 ~10 8 cfu / mL, enterotoxigenic Escherichia coli lytic phage PES-1 was inoculated for phage amplification culture without changing the previous fermentation conditions.

[0066] S3. When the number of host bacteria in the phage fermentation broth is less than 2×10 4 cfu / mL, and when the residual host bacteria were not obviously lysed by the phage, Bacillus licheniformis WS-2 was inoculated and the fermentation was continued to obtain a fermentation broth preparation produced by one step of Escherichia coli phage-coupled antimicrobial lipopeptide.

[0067] When the enterotoxigenic Escherichia coli lytic phage PES-1 is inoculated into the host bacteria, the infection multiplicity (MOI) of the phage is 0.02-0.1.

[0068] In step S3, the inoculation amount of Bacillus licheniformis WS-2 is 3-5%.

[0069] After inoculation with Bacillus subtilis DMZ-SG01, the fermentation parameters were adjusted as follows:

[0070] (1) The fermentation temperature was adjusted from 30-37°C to 32-40°C;

[0071] (2) adjusting the pH to 7-8 by adding 5-10 mol / L NaOH solution;

[0072] (3) Adjust dissolved oxygen ≥ 20%;

[0073] (4) Supplement glycerol at 10-20 g / L.

[0074] The E. coli host bacteria used to cultivate the enterotoxigenic E. coli lytic phage PES-1 can be used as an inducer for Bacillus licheniformis WS-2 to produce antimicrobial lipopeptides, which can effectively improve the antibacterial effect of the fermentation product on E. coli.

[0075] The fermentation time of enterotoxigenic Escherichia coli lytic bacteriophage PES-1 is 5 to 10 hours, and the fermentation time of Bacillus licheniformis WS-2 is 15 to 25 hours. The total fermentation time is 20 to 35 hours. At the end of the fermentation, the titer of enterotoxigenic Escherichia coli lytic bacteriophage PES-1 is 1×10 8 ~10 10 PFU / mL, the number of spores of Bacillus licheniformis WS-2 is 1×10 9 ~10 10 cfu / mL, residual host bacteria ≤ 2×10 2 cfu / mL.

[0076] Example 1: Preparation of a dual-effect antibacterial agent produced by one-step production of Escherichia coli phage-coupled antibacterial lipopeptide

[0077] 1. Fermentation of E. coli phage stage:

[0078] The seed solution of enterotoxigenic Escherichia coli lytic phage PES-1 was divided into 2 mL sterile centrifuge tubes and stored in a 4°C refrigerator for later use. The titer was about 2×10 9 pfu / mL. Escherichia coli was inoculated into the fermentation medium as the host bacteria. The medium formula was 5g / L yeast powder, 10g / L peptone, 10g / L glucose, 15g / L sucrose, 20g / L soluble starch, 8g / L sodium chloride, 0.1g / L magnesium sulfate, 1mg / L manganese sulfate, 1g / L dipotassium hydrogen phosphate, 1g / L potassium dihydrogen phosphate, 0.3g / L calcium carbonate, 0.1g / L zinc sulfate, 2mg / L ferrous sulfate, 1mg / L manganese sulfate, 1mg / L copper sulfate, pH 6, dissolved oxygen was controlled at 5%. The culture temperature was 30°C.

[0079] When the host bacteria concentration in the fermentation broth reaches 1×10 6 cfu / mL, the seed solution of enterotoxigenic Escherichia coli lytic phage PES-1 was inoculated with an infection multiplicity (MOI) of 0.01, and the phage lysis solution was fermented under the same fermentation conditions. When the phage titer in the phage fermentation solution reached 1×10 9 PFU / mL or above, and the number of host bacteria is less than 2×10 4 cfu / ml, the phage fermentation stage is stopped and the antimicrobial lipopeptide preparation stage is ready to begin.

[0080] 2. Fermentation of antimicrobial lipopeptides stage:

[0081] After the above phage fermentation stage is completed, Bacillus licheniformis WS-2 is inoculated in situ to continue fermentation, wherein the inoculation amount of Bacillus licheniformis WS-2 is 5%, and the fermentation parameters are adjusted to:

[0082] (1) The fermentation temperature was adjusted from 30°C to 37°C;

[0083] (2) adjusting the pH to 7.0 by adding 10 mol / L NaOH solution;

[0084] (3) Adjust dissolved oxygen ≥ 20%;

[0085] (4) Supplement glycerol at 20 g / L.

[0086] The fermentation time reaches 35h, the licheniformis WS-2 transsporulation rate reaches more than 90%, and the residual amount of Escherichia coli is ≤2×10 2 cfu / mL, and the fermentation was stopped, and subsequent phage titer and antibacterial effect tests were performed.

[0087] Example 2: Experiment on the antibacterial effect of Bacillus licheniformis WS-2 fermentation broth induced by Escherichia coli

[0088] The purpose of this example is to verify that the Bacillus licheniformis WS-2 strain provided by the present invention can induce an increase in the production of antimicrobial lipopeptides in the fermentation broth of Bacillus licheniformis WS-2 in the presence of Escherichia coli cells in the culture medium, thereby improving the anti-Escherichia coli effect of the fermentation broth.

[0089] Bacillus licheniformis WS-2 was aerobic fermented and cultured, and the culture medium formula was: 10g beef extract, 5g peptone, 5g yeast powder, 5g sodium chloride, 0.15g / L FeSO4·7H2O, 0.1g / L MgSO4·7H2O, 1L water, pH 7.0, and fermentation temperature 37°C. Under the same fermentation conditions, a control group with Escherichia coli was introduced. The specific method was that after Escherichia coli was cultured at 30°C for 12h using NA liquid culture medium, 1mL of culture solution was taken for sterilization. The sterilization conditions were 121°C for 20 minutes. 1mL of sterilized Escherichia coli culture solution was added to the above-mentioned 1L Bacillus licheniformis WS-2 fermentation system for fermentation culture. After 30h of fermentation, the fermentation supernatants of the two test groups were taken for analysis and comparison of the antibacterial effects by the inhibition zone method. The method is as follows:

[0090] (1) Escherichia coli was used as an indicator bacterium and inoculated into NA liquid culture medium. The medium was cultured at 37°C for 12 h and then stored at 4°C.

[0091] (2) Prepare NA solid agar medium plates. Pour 15 to 20 mL of sterilized NA solid agar medium into each plate. After the medium solidifies, add 0.1 mL of the above-mentioned Escherichia coli indicator culture medium to the NA solid agar medium. Evenly spread it on the NA solid agar medium. Absorb the excess bacterial solution, dry it, and then punch holes with a hole puncher. Add 25 μL of the prepared two groups of Bacillus licheniformis WS-2 fermentation supernatants into each hole. Set a blank control for each plate in which physiological saline is added instead of Bacillus licheniformis WS-2 fermentation supernatant. Incubate in a 37°C incubator for 12 h. Check the size of the inhibition zone produced by each group for analysis and comparison.

[0092] Depend on Figure 1 The results showed that the test group of Bacillus licheniformis WS-2 fermentation broth induced by Escherichia coli had a significantly larger inhibition zone than the test group without Escherichia coli. The sizes of the inhibition zones of the two were 19.2 mm for the test group induced by Escherichia coli and 17.5 mm for the test group without Escherichia coli induction. This proved that Bacillus licheniformis WS-2 can indeed produce better antibacterial effects through the induction of Escherichia coli, which also provides a more favorable process basis for the one-step production of antibacterial preparations by Escherichia coli phage-coupled antibacterial lipopeptides.

[0093] Example 3: Determination of residual E. coli in fermentation broth of one-step production process of E. coli phage-coupled antimicrobial lipopeptide

[0094] The residual amount of Escherichia coli was determined by the gradient dilution plate count method.

[0095] According to the method of Example 1, the fermentation broth of E. coli phage alone (the fermentation phage stage in Example 1) and the fermentation broth of E. coli phage coupled with antimicrobial lipopeptide were prepared respectively, and the two groups of culture broths were centrifuged at 10000 rpm for 10 min, and the centrifugal precipitate was washed twice with sterile distilled water, and then heated for 10 min. -1 ~10 -6 200 μL of the diluted sample was evenly spread on a NA solid culture medium plate for gradient dilution, and placed at 37°C overnight to count the number of colonies on the plate.

[0096] Residual E. coli cell content (cfu / mL) = colony count × sample dilution factor × 5

[0097] The residual amount of E. coli was plotted with lg (cfu / mL) as the ordinate and the culture time as the abscissa. The test results are shown in the following figure. Figure 2 As shown, from Figure 2 It can be seen that when E. coli phage is prepared by fermentation alone, the E. coli in the fermentation liquid will be lysed and utilized by the phage, but the minimum number of E. coli cells is still 2×10 3However, when Bacillus licheniformis WS-2 was introduced to continue the coupled fermentation, the residual amount of Escherichia coli cells in the fermentation broth was eventually reduced to 10 2 This result will greatly reduce the biosafety and post-processing purification cost issues caused by E. coli residues when preparing phage preparations.

[0098] Example 4: Analysis of phage titer in Escherichia coli phage-coupled antimicrobial lipopeptide preparation

[0099] Escherichia coli phage titer determination method:

[0100] Phage titer is the number of phages per unit volume of sample, and is determined by the single-layer agar plate method. Add 1 mL of logarithmic phase Escherichia coli host cell suspension to the single-layer LB solid medium, evenly spread on the solid medium, absorb the excess bacterial liquid, and dry it. The phage sample (in the present invention, the fermentation phage stage culture liquid and the fermentation antimicrobial lipopeptide stage culture liquid obtained in the method of Example 1) is diluted in a gradient manner (10 -1 ~10 -10 ), take 5 μL of each dilution gradient and drop it on the above plate. After drying, put the plate upside down in a 37°C incubator for overnight culture for 12 hours. Count the number of plaques formed and calculate the phage titer according to the following formula.

[0101] Phage titer (pfu / mL) = number of phage plaques × sample dilution factor × 200

[0102] The test results of this example are shown in Figure 3 At the end of the E. coli phage fermentation, the phage titer was 3.5×10 9 pfu / mL, after the fermentation stage of coupled antimicrobial lipopeptide, the phage titer was 3.2×10 9 pfu / mL, indicating that the titer of the E. coli phage is relatively stable throughout the preparation process and is not affected by the antimicrobial lipopeptide fermentation stage in the one-step production process of the phage-coupled antimicrobial lipopeptide of the present invention.

[0103] Example 5: Comparative analysis of the inhibitory effect of Escherichia coli phage-coupled antimicrobial lipopeptide preparations on Escherichia coli

[0104] Comparison of inhibition curves

[0105] The absorbance of E. coli culture medium was measured at 600 nm by spectrophotometry to indicate the relative number of bacterial cells.

[0106] According to the method of Example 1, the fermentation culture broth of Escherichia coli phage alone and the fermentation culture broth of phage-coupled antimicrobial lipopeptide were prepared respectively, and then Escherichia coli was cultured in 50 mL shake flasks at 37°C for 8 h using NA liquid medium, and the OD at this time was detected. 600 Then, the cells were divided into three groups. The phage fermentation broth and the phage-coupled antimicrobial lipopeptide fermentation broth with a multiplicity of infection (MOI) of 0.02 were added to the above 50 mL shake flask E. coli culture broth, and the same dose of sterile water was added as a blank control. The OD 600 As the vertical axis, the culture time as the horizontal axis, the antibacterial curve of each group against Escherichia coli was drawn. Figure 4 The results showed that within 28 hours of action, the E. coli phage-coupled antimicrobial lipopeptide group had a better inhibitory effect on the growth of E. coli than the E. coli phage group alone, and eventually E. coli could be inhibited at OD 600 The phage group alone inhibited the phage to OD at 18 h. 600 After reaching 0.25, as time goes by, E. coli begins to become turbid again, and OD 600 It rebounded to 0.5, which is also a common phenomenon when phages lyse host bacteria. The main reason is that when E. coli phages are used as the only selection pressure, some residual host bacteria cells are prone to mutation, thus becoming resistant to phages, and then continue to grow using the remaining nutrients in the culture medium, resulting in OD 600 The Escherichia coli phage-coupled antibacterial lipopeptide preparation of the present invention can effectively solve this problem. While having a more significant antibacterial curve, it also avoids the "re-turbidity" of Escherichia coli, thus having a better antibacterial effect.

[0107] Example 6: Protective effects of different combinations of E. coli phage-coupled antimicrobial lipopeptide preparations on E. coli-challenged chickens

[0108] The purpose of this example is to verify that the Escherichia coli phage-coupled antimicrobial lipopeptide preparation combination provided by the present invention is the optimal combination by comparing the protective effects of different combinations of Escherichia coli phage-coupled antimicrobial lipopeptides on young chickens challenged with Escherichia coli.

[0109] The experimental groups were the Escherichia coli phage-coupled antimicrobial lipopeptide preparation combination of the present invention (S3 group), other alternative Escherichia coli phage-coupled antimicrobial lipopeptide preparation combinations (S1 group, S2 group) and a blank control group. Each group consisted of 10 7-day-old chicks. The chicks in each experimental group were first challenged with pathogenic Escherichia coli by injection or gavage. The challenge dose was 1 mL of Escherichia coli culture fluid per chicken. On the second day after the challenge, each group of chicks was injected orally with the Escherichia coli phage-coupled antimicrobial lipopeptide preparation of each experimental group in the same manner for 2 consecutive days. The dose was 0.05 mL per chicken. After 10 days, the survival rate of the chicks in each group was counted.

[0110] The experimental results are shown in Figure 5 As shown, the E. coli phage-coupled antimicrobial lipopeptide preparation combination (S3 group) provided by the present invention has a more significant effect on the survival rate of young chickens compared to the other two alternative combinations (S1 group, S2 group). After E. coli challenge, the survival rate of young chickens in the S3 group reached 80%, which is much higher than 40% of the S1 group and 50% of the S2 group. The results show that the E. coli phage-coupled antimicrobial lipopeptide preparation provided by the present invention in the S3 group can more effectively protect young chickens from infection and damage by E. coli. Among them, the phages and strains of the E. coli phage-coupled antimicrobial lipopeptide preparation combination of the S1 group and the S2 group are selected from the same type of phage and bacillus as the present invention.

[0111] Example 7: Comparison of the protective effects of the Escherichia coli phage-coupled antimicrobial lipopeptide preparation of the present invention on Escherichia coli infection in different farmed animals

[0112] In livestock and poultry farming, young chickens and pigs are susceptible to E. coli infection and serious diseases. The purpose of this example is to investigate and compare whether the E. coli phage-coupled antimicrobial lipopeptide preparation of the present invention has similar protective effects on other farmed animals infected with E. coli pathogens, thereby defining the boundaries that the content of the present invention can cover.

[0113] The experiment selected 7-day-old chicks and piglets as the subjects of investigation, and the groups were as follows: A0: a blank control group in which the chicks were challenged with E. coli but not treated with the present invention; A1: an experimental group in which the chicks were challenged with E. coli and then treated with the present invention; B0: a blank control group in which the piglets were challenged with E. coli but not treated with the present invention; B1: an experimental group in which the piglets were challenged with E. coli and then treated with the present invention. The attack and treatment methods refer to Example 6 of the present invention, and the survival rates of the chicks and piglets in each group were counted after the experiment was carried out for 10 days.

[0114] The experimental results are shown in Figure 6As shown, the effect of the E. coli phage-coupled antibacterial lipopeptide preparation provided by the present invention on the treatment of E. coli infection in young broilers is significantly better than that in piglets. The treatment effect of young chickens is 60 percentage points higher than that of the control group, while the treatment effect of piglets is not obvious compared with the control group. This shows that the present invention is more suitable for use in broiler farming, but not in pig farming. It is speculated that it may be related to the degree of biological activity of phages in different farmed animals, and the intestines of pigs are longer and more complex than those of chickens, which leads to the fact that the preparation of the present invention is more restricted by multiple factors in the pig body. This experimental result also confirms the scope of application of the present invention, that is, it is positioned in the field of broiler farming, and can effectively prevent and control the infection of pathogenic E. coli.

[0115] The present invention provides a one-step production process for a novel Escherichia coli phage-coupled antibacterial lipopeptide preparation and its highly effective inhibitory effect on pathogenic Escherichia coli. The method is: using Escherichia coli to produce the lytic phage provided by the present invention, when the phage accumulates to a certain titer, inoculating the Bacillus licheniformis strain that can produce antibacterial lipopeptides that inhibit Escherichia coli provided by the present invention, while changing the fermentation parameters and adding a carbon source, at this time, the Bacillus licheniformis grows rapidly, and the accumulated antibacterial lipopeptides can quickly kill the excess Escherichia coli remaining in the fermentation system, thereby preparing an enhanced anti-Escherichia coli preparation of Escherichia coli phage-coupled antibacterial lipopeptides. The invention solves the safety problem of residual Escherichia coli in the process of culturing Escherichia coli phages, while taking into account the production of antibacterial lipopeptide preparations, producing dual-effect anti-Escherichia coli preparations at a lower cost, with better and more prominent antibacterial effects, and has broad application prospects in the broiler breeding industry.

[0116] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Use of an antibacterial preparation in the preparation of a product for inhibiting Escherichia coli in broilers, characterized in that: The antibacterial preparation is an antibacterial preparation obtained by secondary fermentation of Escherichia coli phage with Bacillus licheniformis WS-2 after Escherichia coli induced fermentation. The Escherichia coli phage is enterotoxigenic Escherichia coli lytic phage PES-1, the deposit number of the enterotoxigenic Escherichia coli lytic phage PES-1 is CGMCC No.13382, and the deposit number of the Bacillus licheniformis WS-2 is CGMCC No.12813.

2. The use according to claim 1, characterized in that: The titer of enterotoxigenic Escherichia coli lytic bacteriophage PES-1 in the antibacterial preparation is 1×10 8 ~10 10 PFU / mL, the number of spores of Bacillus licheniformis WS-2 is 1×10 9 ~10 10 cfu / mL, residual amount of E. coli host bacteria ≤10 2 cfu / mL.

3. The use according to claim 1, characterized in that: The preparation method of the antibacterial preparation comprises the following steps: S1. Inoculate Escherichia coli as a host bacterium into a fermentation medium to obtain a fermentation broth; the components of the fermentation medium include a carbon source, a nitrogen source, an inorganic salt and water, the inoculation amount of the host bacterium is 1% to 3% (v / v), and the concentration of the host bacterium in the fermentation broth is 1×10 6 ~10 8 cfu / mL; S2, inoculating the enterotoxigenic Escherichia coli lytic phage PES-1 into the fermentation liquid for culture, to obtain the Escherichia coli phage culture liquid; the infection multiplicity of the enterotoxigenic Escherichia coli lytic phage PES-1 is 0.02-0.1, and the host bacteria concentration in the phage culture liquid does not exceed 1×10 4 cfu / mL; S3, inoculating Bacillus licheniformis WS-2 into the phage culture solution to obtain an antibacterial preparation; the inoculation amount of Bacillus licheniformis WS-2 is 3-5%, and the host bacteria concentration in the antibacterial preparation does not exceed 2×10 2 cfu / mL.

4. The use according to claim 3, characterized in that: The components of the fermentation medium include 4-10 g / L yeast extract, 10-20 g / L soy peptone, 10-20 g / L glucose, 30-40 g / L soluble starch, 5-12 g / L sodium chloride, 0.1-0.4 g / L magnesium sulfate, 1-4 mg / L manganese sulfate, 1-4 g / L dipotassium hydrogen phosphate, 1-4 g / L potassium dihydrogen phosphate, 0.5-1 g / L calcium carbonate, 0.1-0.3 g / L zinc sulfate, 0.2-1 mg / L ferrous sulfate, 1-3 mg / L manganese sulfate, 1-5 mg / L copper sulfate and water.

5. The use according to claim 3, characterized in that: The culture conditions of steps S1 to S2 are pH 6 to 7, dissolved oxygen content 5 to 10%, and temperature 30 to 37°C.

6. The use according to claim 3, characterized in that: The culture conditions of step S3 are to add 10-20 g / L glycerol to the Escherichia coli phage culture solution, adjust the pH to 7-8, the temperature to 35-40° C., and the dissolved oxygen content to not less than 20%.

Images