Salmonella phage as well as preparation method and application thereof

Abstract

The invention provides a salmonella bacteriophage and a preparation method and application thereof, the salmonella bacteriophage is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is September 10, 2021, and the preservation number is CGMCC No.23090. The invention also provides a preparation method of the salmonella bacteriophage, which comprises the following steps: carrying out combined fermentation on salmonella host bacteria from multiple species and salmonella bacteriophage seeds to obtain fermentation liquor, and centrifugally filtering the fermentation liquor to obtain the salmonella bacteriophage. According to the invention, multi-strain combined fermentation of bacteriophages of salmonella of different provenances and species is realized. According to the combined fermentation process, combined fermentation of multiple strains of bacteriophages of salmonella of different provenances and species is achieved, it is guaranteed that the bacteriophage of each strain of host bacteria can be successfully fermented, and the production cost and the production cycle of bacteriophage fermentation of a single strain of salmonella are reduced.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a Salmonella phage and a preparation method and application thereof. Background Art

[0002] Salmonellosis is a bacterial disease caused by Salmonella. There are more than 2,600 serotypes of Salmonella, among which pullorum, enteritis, typhoid, and typhoid serotypes have the highest frequency of infection in poultry, and the genomes are very similar. The complex serotypes and high infection rates of Salmonella are very harmful to farms.

[0003] Nowadays, the main methods for preventing and controlling Salmonella in many domestic chicken farms are to purify seed sources, conduct regular quarantine, and adopt an all-in and all-out production management model as well as self-breeding and self-feeding. However, since Salmonella can be transmitted vertically and horizontally, the above preventive measures are not ideal. Most farms mainly rely on drug control. With the increase in the abuse of antibiotics, Salmonella has become extremely resistant and has developed drug resistance. Therefore, the current measures to control Salmonella with antibiotics are becoming increasingly restrictive, and the development of new antimicrobial drugs has become a hot research direction.

[0004] Bacteriophages are natural killers of bacteria, abundant in number and widely distributed. Bacteriophages invade bacteria to reproduce themselves, lyse bacteria by releasing enzymes, and release progeny phages. Due to the development and spread of antibiotic resistance specificity and bacterial interactions in most bacterial species, phage therapy has once again attracted people's attention and has been applied to the biological control of foodborne and environmental pathogens in animals and humans. From the perspective of the safety of phages for animals and humans, studies have shown that phages are significantly more effective than antibiotics and other organic chemical additives, especially in the control of clinical medical infections, the prevention and control of veterinary clinical pathogens, and environmental pollution by food-related pathogens, and have good application prospects. Therefore, it is of great significance to develop a phage with a wide host spectrum. Summary of the invention

[0005] The present invention provides a Salmonella phage and a preparation method and application thereof, and the specific implementation methods are as follows:

[0006] A Salmonella phage, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is September 10, 2021, and the deposit number is: CGMCC No. 23090;

[0007] The phage titer is 1×10 10 PFU / ml~1×10 11 PFU / ml;

[0008] In addition, the present invention also provides a method for preparing Salmonella phage, comprising co-fermenting Salmonella host bacteria from various species with Salmonella phage seeds to obtain a fermentation liquid, and centrifugally filtering the fermentation liquid to obtain the Salmonella phage;

[0009] Furthermore, Salmonella host bacteria from multiple species are combined for recovery culture and fermentation culture before fermentation;

[0010] Furthermore, the recovery culture process of the Salmonella host bacteria includes: streaking the Salmonella host bacteria of various species on plates, picking single colonies in the culture medium, and culturing at 30-37° C. in a shaking incubator for 12 to 16 hours;

[0011] The fermentation and cultivation process of the Salmonella host bacteria includes: inoculating the Salmonella host bacteria after recovery and cultivation in a fermentation tank and fermenting and cultivating at a temperature of 37° C. and a rotation speed of 180 rpm / min;

[0012] Furthermore, the preparation process of Salmonella phage seeds includes: adding sewage collected from a breeder farm to 1×SM buffer at a ratio of 1:1-2, precipitating at 2-8°C overnight, centrifuging at 8000-12000rpm for 3-5 minutes, filtering and sterilizing with a 0.22μm filter membrane, dropping the filtered filtrate onto a double-layer plate, culturing at 37°C for 4-6 hours, and after plaques grow, deducting the plaques and placing them in SM buffer, culturing at 37°C, 180-200rpm / min for 2-4 hours, and then centrifuging at 8000-12000rpm for 3-5 minutes, and then spreading on a double-layer plate. After culturing, picking single plaques, and purifying repeatedly three times to obtain Salmonella phage seeds;

[0013] Further, the combined fermentation includes inoculating the Salmonella host bacteria with the Salmonella phage seeds, and fermenting at 30-37° C. and 180-200 rpm for 6-10 hours after the inoculation;

[0014] Furthermore, according to the multiplicity of infection of 0.001-0.01, the number of Salmonella host bacteria reached 10 8 CFU / ml, inoculated with Salmonella phage seeds;

[0015] Furthermore, the host bacteria of Salmonella are Salmonella pullorum, Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurium, Salmonella from duck, Salmonella from pig or Salmonella from mink;

[0016] The Salmonella phage was named SD-SE-001;

[0017] The present invention also provides an application of the Salmonella phage in the preparation of medicines for preventing and treating various livestock and poultry diseases caused by Salmonella infection.

[0018] Due to the adoption of the above technical solution, the beneficial technical effects of the present invention are:

[0019] 1. The present invention provides a Salmonella phage combined fermentation process in a large-scale fermentation workshop, which can realize the combined fermentation of multiple strains of Salmonella phages of different sources and species. The combined fermentation process of the present invention can realize the combined fermentation of multiple strains of Salmonella phages of different sources and species, ensuring that the phages of each host strain can be successfully fermented, reducing the production cost and production cycle of the phage fermentation of a single strain of Salmonella, saving a lot of manpower and material resources, and greatly increasing the production capacity;

[0020] 2. The present invention provides an effective control product for Salmonella pullorum, Salmonella enteritidis, Salmonella typhimurium and Salmonella typhi in the breeding industry. The present invention develops a phage preparation that has strong lysis properties against Salmonella pullorum, Salmonella enteritidis, Salmonella typhimurium and Salmonella typhi. The preparation can be used alone or in combination with a cocktail, providing a safe, non-toxic and non-residual phage product for the prevention and control of common pathogenic Salmonella, and providing a phage source for the industrial production of phage preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a photo of the plaque in Example 1;

[0022] Figure 2 This is a transmission electron microscope photo of the Salmonella phage SD-SE-001 of Example 2;

[0023] Figure 3 This is a thermal stability test diagram of the Salmonella phage SD-SE-001 in Example 2, A represents 30 minutes of action, and B represents 60 minutes of action;

[0024] Figure 4 This is a graph showing the pH stability test results of the Salmonella phage SD-SE-001 of Example 2;

[0025] Figure 5 This is the one-step growth curve of the Salmonella phage SD-SE-001 in Example 2. DETAILED DESCRIPTION

[0026] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] Preparation method of Salmonella phage seeds

[0029] 1. Take a strain of Salmonella pullorum SE010 from a -80°C refrigerator, streak it on SS agar medium, and culture it in a constant temperature incubator at 37°C for 20 hours. Pick a single colony and inoculate it in LB broth medium. Culture it in a shaker at 37°C and 180 rpm for 14 hours to obtain a proliferated Salmonella pullorum culture liquid.

[0030] 2. Take 50 ml of sewage and feces from the chicken farm, add 500 μl of Salmonella pullorum solution, and then add LB broth culture medium to 200 ml, shake and culture at 37°C and 180 rpm for 15 hours, then take out 10 ml of the culture solution, centrifuge at 8000 rpm for 10 minutes, take the supernatant and filter it with a 0.22 μm filter membrane to obtain the phage stock solution, and store it at 2-8°C.

[0031] 3. Prepare the lower layer of the double-layer plate: Take the autoclaved LB agar medium, cool it to 50°C, pour 10-15 ml of the autoclaved LB agar medium into each sterile plate, cool it to room temperature and store it at 2-8°C for later use.

[0032] 4. Lay double-layer plates: First, dispense 500 μl of the enriched Salmonella pullorum solution into 10 ml sterile centrifuge tubes, then cool the autoclaved semi-solid culture medium (LB broth medium + 0.7% agar powder) to 50°C, pour 5 ml of the semi-solid culture medium into the above 10 ml centrifuge tubes, and pour the upper plate.

[0033] 5. Isolation and purification of bacteriophages: After the upper plate solidifies, absorb the filtered sewage liquid and drop it onto the plate. After the surface liquid dries, place the plate upside down in a 37°C constant temperature incubator for 8 hours. If bacteriophages are present, transparent, regular circular plaques will form on the culture medium, which are called plaques. Figure 1 As shown. Use sterile forceps to pick up a single plaque in 1×SM buffer, shake it in a constant temperature oscillator at 37°C for 5 hours, centrifuge it at 8000rpm for 5 minutes, filter the supernatant with a 0.22μm filter to obtain a phage extract, and then use a double-layer plate to spot a single plaque. Repeat this process 3 to 5 times until a round, transparent, uniformly sized plaque with a diameter of about 1 to 2nm is formed.

[0034] 6. Propagation of phage: On the double-layer culture medium where plaques are formed, use sterile tweezers to take a single plaque and place it in 1×SM buffer, shake it in a constant temperature oscillator at 37°C for 5 hours, centrifuge it at 8000rpm for 5 minutes, and filter the supernatant of the centrifugation with a 0.22μm filter to obtain the phage extract. Take 100μl of the host bacteria proliferation liquid and 100μl of the phage extract in 5ml LB broth medium, shake and culture it at 37°C and 180rpm for 9 hours, centrifuge the above culture liquid at 9000rpm for 8 minutes, take the supernatant, and filter it with a 0.22μm filter to obtain the phage seed.

[0035] Example 2

[0036] A method for preparing Salmonella phage

[0037] 1. Resuscitation culture of host bacteria: Take out 20 strains of Salmonella of different origins and species from a -80℃ refrigerator, including 4 strains of Salmonella pullorum, 4 strains of Salmonella enteritis, 2 strains of Salmonella gallinarum, 4 strains of Salmonella typhimurium, 2 strains of Salmonella duck, 2 strains of Salmonella suis, and 2 strains of Salmonella mink. Use an inoculation loop to streak on SS solid culture medium plates, culture in a 37℃ constant temperature incubator for 20 hours, pick a single colony in 500ml LB broth medium, and then culture on a 37℃ shaker at 180rpm for 15 hours.

[0038] 2. Host bacteria fermentation culture: After the 20 bacterial strains grow well, inoculate them into a 100L fermentation tank at a volume ratio of 1%, and ferment and culture at 37°C and 150rpm. Maintain stirring and maintain the speed at 150rpm / min, and control the pH value at around 7.0; start sampling and measuring the OD600 value 3 hours after inoculation, and then sample and measure the OD value every 1 hour. 600 value until the bacterial solution OD 600 Maintain at a stable value. The pH of the fermentation liquid decreased in the early stage of the fermentation process and was adjusted with ammonia water to maintain it at 7.0. The growth trend of the specific host bacteria fermentation liquid is shown in Table 1.

[0039] Table 1 Host bacteria fermentation growth OD 600 Value and bacterial count test results

[0040]

[0041] 3. Phage inoculation: According to the results of the host bacteria fermentation broth growth trend detection, when the broth was fermented for 7 hours and the OD value of the fermented broth reached 0.60, the phage seeds prepared in Example 1 were inoculated at an infection multiplicity of 0.01. The fermentation tank was 100L, the culture broth was 70L, and the inoculated phage amount was 7ml.

[0042] 4. Cultivation after inoculation of phage seeds: After inoculation of phage seeds, fermentation was carried out at 37°C and 150rpm. The phage titer was detected 1.5 hours after inoculation of phage seeds, and the titer was detected every hour thereafter. When the phage titer was the highest, the fermentation was terminated and the fermentation liquid was collected. The specific test results are shown in Table 2. It can be seen from the results that the phage titer reached the highest value 5 hours after inoculation of phage seeds, and the titer did not change much in subsequent fermentations. Therefore, the fermentation was terminated and the fermentation liquid was collected 5 hours after inoculation of phage seeds.

[0043] Table 2 Fermentation titer detection after phage inoculation in fermenter

[0044]

[0045] 5. Centrifugal filtration of fermentation liquid: The collected fermentation liquid is centrifuged in a disc centrifuge. The disc centrifuge is adjusted to a tank pressure of 0.4 and a speed of 6000 rpm / min for 20 minutes to remove the debris of the fermentation liquid and collect the centrifuge.

[0046] 6. Sterile filtration of the centrifuge: The centrifuge is first finely filtered using a 0.22 μm plate and frame to remove tiny particles in the centrifuge to obtain a fine filtrate, and then the fine filtrate is sterilized by filtering through a 0.22 μm filter membrane. The obtained sterile filtrate is the phage fluid.

[0047] 7. Aseptic packaging and storage of phage fluid: After sterilization and filtration, the sterile phage fluid is packaged in a local Class 100 filling environment and stored at 5°C.

[0048] 8. Phage titer test: Detect the phage titer of each host strain separately. The phage titer of each host strain used for fermentation is tested separately. The process is as follows: take 1 ml of sterile phage liquid and add it to a test tube containing 9 ml of SM buffer. After fully mixing, take 1 ml of the diluted phage suspension and add it to another centrifuge tube containing 9 ml of SM buffer. Repeat the above operation to dilute the sample concentration to 10 -2 ~10 -88 gradients. Select 2 to 3 appropriate sample dilution gradients, take 100μl of each with a sterile pipette tip, add to 100μl Salmonella pullorum proliferation solution, mix, and incubate at 37°C for 5 minutes. Add 5ml of the upper semi-solid culture medium (LB broth medium + 0.7% agar powder), mix, and pour on the lower LB solid culture medium. After solidification, invert and culture at 37°C for 10 hours. Observe the morphology of the individual and uniform plaques formed, and count the number of plaques. Select plates with 30 to 300 plaques for counting, calculate the phage titer according to the formula, and retain one significant figure in the result. Repeat 3 times and take the average result. Phage titer (PFU / ml) = number of plaques on the plate with appropriate plaque density × dilution factor × 10. The titers of 20 strains of Salmonella phage from different sources were all above 10 after fermentation. 10 PFU / ml and above, specific test results are shown in Table 3.

[0049] Table 3 The titers of 20 strains of Salmonella phage from different species after fermentation (PFU / ml)

[0050]

[0051] Example 3

[0052] A method for preparing Salmonella phage

[0053] 1. Resuscitation culture of host bacteria: Take out 30 strains of Salmonella of different origins and species from a -80℃ refrigerator, including 8 strains of Salmonella pullorum, 6 strains of Salmonella enteritis, 2 strains of Salmonella gallinarum, 7 strains of Salmonella typhimurium, 3 strains of Salmonella duck, 2 strains of Salmonella suis, and 2 strains of Salmonella mink. Use an inoculation loop to streak on SS solid culture medium plates, culture in a 37℃ constant temperature incubator for 18 hours, pick a single colony in 1000ml LB broth medium, and then culture on a 37℃ shaker at 180rpm for 16 hours.

[0054] 2. Host bacteria fermentation culture: After the above 30 strains of bacteria have grown well, inoculate them into a 200L fermentation tank at a ratio of 1.5%, and ferment them at 37°C and 180rpm. Maintain stirring and maintain the speed at 180rpm / min, and control the pH value at around 7.0; start sampling and measuring the OD600 value 3 hours after inoculation, and then sample and measure the OD value every 1 hour. 600 The pH value of the fermentation liquid decreased in the early stage of the fermentation process and was adjusted with ammonia water to maintain it at around 7.0. The specific growth trend of the fermentation liquid is shown in Table 4.

[0055] Table 4 Bacterial fermentation growth OD 600 Value and bacterial count test results

[0056]

[0057] 3. Phage inoculation: According to the optimal multiplicity of infection of 0.01, when the bacterial solution was fermented for 6.5 hours and the OD value of the fermented bacterial solution reached 0.56, the phage seeds obtained in Example 1 were inoculated at a multiplicity of infection of 0.01. The culture medium of the 200L fermenter was 140L, and the amount of inoculated phage was 14ml.

[0058] 4. Cultivation after inoculation of phage seeds: After inoculation of phage seeds, fermentation was carried out at 37°C and 180rpm. The phage titer was detected from 1.5 hours after inoculation of phage, and the titer was detected every hour thereafter. When the phage titer was the highest, the fermentation was terminated and the fermentation liquid was collected. The specific test results are shown in Table 5. It can be seen from the results that the phage titer reached the highest value 5 hours after inoculation of phage fermentation, and the titer did not change much in subsequent fermentation. Therefore, the fermentation can be terminated and the fermentation liquid can be collected 5 hours after inoculation of phage fermentation.

[0059] Table 5 Fermentation titer detection after phage inoculation in fermenter

[0060]

[0061] 5. Centrifugal filtration of fermentation liquid: The collected fermentation liquid is centrifuged in a disc centrifuge. The disc centrifuge is adjusted to a tank pressure of 0.4 and a speed of 8000 rpm / min for 30 minutes to remove the fragments of the fermentation liquid and collect the centrifuge.

[0062] 6. Sterile filtration of the centrifuge: The centrifuge is first finely filtered using a 0.22 μm plate and frame to remove tiny particles in the centrifuge to obtain a fine filtrate, and then the fine filtrate is sterilized by filtering through a 0.22 μm filter membrane. The obtained sterile filtrate is the phage fluid.

[0063] 7. Aseptic packaging and storage of phage fluid: After sterilization and filtration, the sterile phage fluid is packaged in a local Class 100 filling environment and stored at 2°C.

[0064] 8. Phage titer test: Detect the phage titer of each host strain separately. The phage titer of each host strain used for fermentation is tested separately. The process is as follows: take 1 ml of sterile phage liquid and add it to a test tube containing 9 ml of SM buffer. After fully mixing, take 1 ml of the diluted phage suspension and add it to another centrifuge tube containing 9 ml of SM buffer. Repeat the above operation to dilute the sample concentration to 10 -2 ~10 -88 gradients. Select 2 to 3 appropriate sample dilution gradients, take 100μl of each with a sterile pipette tip, add to 100μl Salmonella pullorum proliferation solution, mix, and incubate at 37°C for 5 minutes. Add 5ml of the upper semi-solid culture medium (LB broth medium + 0.7% agar powder), mix, and pour on the lower LB solid culture medium. After solidification, invert and culture at 37°C for 15 hours. Observe the morphology of the individual and uniform plaques formed, and count the number of plaques. Select plates with 30 to 300 plaques for counting, calculate the phage titer according to the formula, and retain one significant figure in the result. Repeat 3 times and take the average result. Phage titer (PFU / ml) = number of plaques on the plate with appropriate plaque density × dilution factor × 10. The titers of 30 strains of Salmonella phage from different sources were all above 10 after fermentation. 10 PFU / ml and above, specific test results are shown in Table 6.

[0065] Table 6 The titers of 30 strains of Salmonella phages from different species after fermentation (PFU / ml)

[0066]

[0067]

[0068] The phage obtained in Example 2 was observed by electron microscopy: the phage was treated by phosphotungstic acid negative staining method: 100 μl of concentrated phage was taken and dropped on a paraffin sheet, and the copper mesh with the membrane side was placed on the phage droplet. After 10 minutes, the copper mesh was removed from the droplet and placed on a clean filter paper, and dried for 3 minutes. A drop of 2% phosphotungstic acid (PTA) was added to the copper mesh to stain the phage for 10 minutes, and then the staining liquid was absorbed from the side with filter paper. After the sample was dried, the phage morphology was observed using a transmission electron microscope. Figure 2 It can be seen that the head of the phage is a regular polyhedron structure with a diameter of about 54nm and a tail with a length of about 117nm. According to the classification standards of the ninth report of the International Committee on Taxonomy of Viruses, the phage can be determined to belong to the Long-tailed Bacteriophage family and named SD-SE-001.

[0069] Biological characteristics of the phage obtained in Example 2 were tested

[0070] 1. Temperature stability test of bacteriophage SD-SE-001

[0071] The centrifuge tubes containing 500 μl of phage were placed in a constant temperature water bath at 40°C, 50°C, 60°C, 70°C, and 80°C, and samples were taken after 30 minutes and 60 minutes, respectively, and the changes in phage titer were determined by the double-layer plate method. The thermal stability curve of phage SD-SE-001 was drawn with temperature as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0072] Results are visible Figure 3 , after being exposed to 40℃ and 50℃ for 1 hour, the phage SD-SE-001 basically maintained its original activity; after being exposed to 60℃ for 30 minutes, the titer dropped by 2 orders of magnitude, and after 1 hour, the titer dropped by 5 orders of magnitude; under the high temperature of 70℃ and 80℃, the phage was basically inactivated after 30 minutes. The test results show that the phage SD-SE-001 can tolerate temperatures below 50℃, and the temperature exceeding 50℃ will affect the activity of the phage.

[0073] 2. Determination of pH stability of bacteriophage

[0074] The pH values ​​of LB liquid medium were adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 with concentrated hydrochloric acid and NaOH solution. 200 μl of each pH value was taken and 200 μl of 10 11 PFU / ml of phage solution was mixed and allowed to react at 37℃ for 1 hour. 200μl of phage was taken from each tube to measure the change in phage titer. The phage pH stability curve was drawn with pH as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0075] Results are visible Figure 4 In the pH range of 4 to 9, the phage titer remained almost unchanged, all within 10 10 PFU / ml, the phage titer was the highest at pH 7.5, reaching 10 11 PFU / ml, has good stability in alkaline environment, and the titer remains at 10 at pH 12 4 PFU / ml. In the pH range of 2.0-4.0, as the pH value decreases, the phage titer decreases significantly. When the pH value is 2.0, it completely loses the ability to lyse the host. Bacteriophage SD-SE-001 is acidophobic and basophilic. This phage grows most easily in a neutral environment.

[0076] 3. Determination of the optimal phage infection multiplicity (MOI)

[0077] The concentration of host bacteria was adjusted to 1.0 × 10 8 CFU / ml, MOI was set to 0.0001, 0.001, 0.01, 0.1, 1 respectively, the same volume of phage solution and bacterial solution were mixed evenly (the volume of the two was 500μl respectively), and cultured at 37℃ constant temperature oscillator at 180rpm / min for 8-12 hours, and the mixed culture was centrifuged at 8000-10000rpm for 5-10 minutes to obtain lysate. Three replicates were made for each MOI value, and the phage titer was measured. The infection multiplicity that produced the highest titer was the phage MOI.

[0078] The results show that the MOI of phage SD-SE-001 is 0.01. Under this condition, the titer of progeny phage produced by phage infection of host bacteria is 1.8×10 11 PFU / ml, the phage titer was the highest among the 5 infection multiplicities. Detailed results are shown in Table 7.

[0079] Table 7 Detection results of optimal infection multiplicity (MOI) of bacteriophage SD-SE-001

[0080]

[0081] 4. One-step growth curve of bacteriophage

[0082] The host bacteria were cultured for 5 to 6 hours to a concentration of 1×10 8 CFU / ml, incubate the phage solution and host bacterial solution in a 37°C water bath for 5 minutes at the optimal infection multiplicity ratio to allow the phage to fully adsorb the host bacteria, centrifuge at 12000rpm for 1 minute, remove the supernatant, and use sterilized LB liquid culture medium to fully wash away the unadsorbed phage. Wash 3 times, add the washed solution to 100ml of LB liquid culture medium, mix well, and place in a 37°C constant temperature shaker for shaking culture; start sampling at 0 minutes, sample every 10 minutes, measure the phage titer, repeat 3 times, and take the average of the results. Use the infection time as the horizontal axis and the logarithm of the phage titer in the infection system as the vertical axis to draw a one-step growth curve to obtain the incubation period and outbreak period of phage SD-SE-001. The results can be seen Figure 5 The results showed that the incubation period of phage infection in host bacteria was the first 10 minutes, the lysis period was 90 minutes, and the lysis amount was about 521.

[0083] The phage obtained in Example 2 was subjected to a lysis spectrum test.

[0084] 76 strains of Salmonella that have been identified in the laboratory (18 strains of Salmonella pullorum, 15 strains of Salmonella enteritidis, 8 strains of Salmonella typhimurium, 5 strains of Salmonella gallinarum, 10 strains of Salmonella from pigs, 15 strains of Salmonella from ducks, and 5 strains of Salmonella from minks) were selected to prepare host bacterial suspensions, and the phage lysis spectrum was determined by the double-layer plate method. 200 μl of bacterial suspension was incubated with 200 μl of phage at 37°C for 5 minutes, added to the upper semi-solid culture medium, and a double-layer plate was prepared. After the upper layer solidified, it was placed in a 37°C incubator and inverted for overnight culture to observe the lysis results. The statistical results are shown in Table 8.

[0085] As can be seen from Table 8, the Salmonella phage SD-SE-001 has a lysis rate of 100% for Salmonella pullorum, Salmonella enteritidis, Salmonella typhimurium, and Salmonella gallinarum; a lysis rate of 100% for Salmonella from ducks, 80% for Salmonella from pigs, and 60% for Salmonella from minks.

[0086] Table 8 Lysis spectrum of Salmonella phage SD-SE-001 against Salmonella of different species

[0087]

[0088]

[0089]

[0090]

[0091] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A Salmonella phage, characterized in that The Salmonella phage is deposited in the General Microbiology Center of the China Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is September 10, 2021, and the deposit number is: CGMCC No. 23090.

2. A Salmonella phage according to claim 1, characterized in that: The phage titer is 1×10 10 PFU / ml~1×10 11 PFU / ml.

3. A method for preparing the Salmonella phage according to claim 1, characterized in that: The host bacteria of Salmonella from various species are co-fermented with Salmonella phage seeds to obtain a fermentation liquid, and the fermentation liquid is centrifuged to obtain the Salmonella phage.

4. The method for preparing a Salmonella phage according to claim 3, characterized in that: Before combined fermentation, Salmonella host bacteria from various species are resuscitated and fermented.

5. The method for preparing a Salmonella phage according to claim 4, characterized in that: The recovery culture process of the Salmonella host bacteria comprises: streaking the Salmonella host bacteria of various species on plates, picking single colonies in the culture medium, and culturing them in a shaking incubator at 30-37° C. for 12-16 hours; The fermentation culture process of the Salmonella host bacteria includes: the Salmonella host bacteria after recovery culture are inoculated into a fermentation tank and fermented at a temperature of 37° C. and a rotation speed of 180 rpm / min.

6. The method for preparing a Salmonella phage according to claim 3, characterized in that: The preparation process of the Salmonella phage seed comprises: adding sewage collected from a breeder chicken farm to a 1×SM buffer at a ratio of 1:1-2, precipitating overnight at 2-8°C, centrifuging at 8000-12000 rpm for 3-5 minutes, filtering and sterilizing with a 0.22 μm filter membrane, dropping the filtered filtrate onto a double-layer plate, culturing at 37°C for 4-6 hours, and after the growth of plaques, removing the plaques and placing them in an SM buffer, culturing at 37°C at 180-200 rpm / min for 2-4 hours, and then centrifuging at 8000-12000 rpm for 3-5 minutes, and then spreading on a double-layer plate. After culturing, picking a single plaque, and purifying repeatedly three times to obtain the Salmonella phage seed.

7. The method for preparing a Salmonella phage according to claim 3, characterized in that: The combined fermentation comprises the following steps: inoculating Salmonella host bacteria with Salmonella phage seeds, and fermenting the inoculated Salmonella host bacteria at 30-37° C. and a rotation speed of 180-200 rpm for 6-10 hours.

8. The method for preparing a Salmonella phage according to claim 7, characterized in that: According to the infection multiplicity of 0.001-0.01, the number of Salmonella host bacteria reaches 10 8 CFU / ml, inoculated with Salmonella phage seeds.

9. The method for preparing a Salmonella phage according to claim 3, characterized in that: The host bacteria of Salmonella are Salmonella pullorum, Salmonella gallinarum, Salmonella enteritidis of chicken, Salmonella typhimurium, Salmonella from duck, Salmonella from pig or Salmonella from mink.

10. The use of the Salmonella phage according to claim 1 or 2, characterized in that: Use in preparing medicines for preventing and treating various livestock and poultry diseases caused by salmonella infection.

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