A solid preparation of Salmonella phage, its preparation method and application

Abstract

The present invention provides a solid preparation of Salmonella phage, a preparation method thereof and an application thereof, belonging to the technical field of bioengineering. The solid preparation is a dry powder obtained by spray-drying a biologically active phage and a protective agent and then mixing with a carrier. The phage is selected from Salmonella phage S4P and Salmonella phage S16P. Among them, the preservation number of Salmonella phage S4P is CCTCC NO: M2021765, and the preservation number of Salmonella phage S16P is CCTCC NO: M2021861. The solid preparation of Salmonella phage of the present invention can be stored at room temperature for a long time, still has a high titer level after being stored at high temperature for a certain period of time, also has good bile salt tolerance, and the adopted preparation method has the advantages of low cost, short cycle, simple process, etc. compared with freeze-drying.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a solid preparation of Salmonella phage, a preparation method thereof and an application thereof. Background Art

[0002] Salmonella is one of the important zoonotic pathogens. Animal-derived products, feces, urine, sewage, etc. are all transmission vectors of Salmonella, and can infect humans and animals through the digestive tract. At present, different types of Salmonella phages have been isolated and screened from farms, sewage ponds and feces, etc., and certain progress has been made in preventing and controlling Salmonella infection in livestock and poultry.

[0003] Salmonellosis in chickens is a general term for a class of common and frequently-occurring diseases caused by Salmonella bacteria, including pullorum disease, paratyphoid in chickens, typhoid in chickens, etc., which seriously endanger the health of chickens and often cause serious economic losses to chicken farms. Due to vertical transmission, the incidence rate is extremely high. Poultry are the main hosts of Salmonella. Pathogenic Salmonella can cause a large number of deaths of chicks, and the egg production rate and production performance of adult chickens decline. However, most diseased chickens are characterized by asymptomatic digestive tract colonization and do not cause systemic symptoms. These Salmonella in latent infections often use eggs and meat as the main vectors and are the main pathogenic bacteria causing human foodborne diseases.

[0004] At present, the main prevention and control measures for Salmonellosis are population purification and regular feeding of antibacterial drugs to chicken flocks to reduce the incidence of Salmonellosis. However, due to the long-term feeding of antibacterial drugs, some chicken flocks will develop serious drug resistance. Once infected with Salmonella, it will be difficult to achieve good treatment effects by using drug treatment again, which seriously affects the economic benefits of farmers.

[0005] Phage is a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes or spirochetes. In 1915, Frederick W. Twort and Felix d'Herelle discovered phages respectively. Phages are the most numerous organisms on earth, and their total amount can reach 10 32 individuals. Phages do not have a complete cell structure and have a relatively small genome. They strictly rely on the host for proliferation and have co-evolved with the host in nature for a long time. Compared with antibiotics, the high specificity of phages makes it possible to accurately and precisely remove pathogenic bacteria in the intestinal tract of livestock and poultry. While killing bacteria, it will not damage the normal intestinal flora. Instead, because harmful bacteria are removed, it promotes the colonization and occupation of beneficial bacteria. If we can compound phages of different types of harmful bacteria, we can broaden the antibacterial spectrum of phage preparations and better prevent and treat mixed infection of bacterial diseases.

[0006] Due to the poor temperature tolerance of phages, the storage conditions during the period from their production and preparation to actual application are crucial for maintaining the activity of phage preparations and are the key to their clinical application effectiveness. Since most of the currently commercially available phage products are liquid preparations, the storage conditions are almost entirely low-temperature refrigerated storage and are vulnerable to contamination. Slight improper storage will cause the product to become inactivated, resulting in great economic losses. To improve the temperature tolerance of phage products and make them less vulnerable to environmental pollution, not only suitable temperature-resistant protectants need to be found, but also formulation processes such as preparation processes and carrier media need to be considered. Solid powders are the main types of microbial preparations in China, which can be directly used in livestock and poultry feeds and have the characteristics of easy storage and convenient use. Currently, the common preparation of phage dry powders is mainly freeze-drying. For example, the patent with the application number CN20211152630.0 reported a phage lyophilized powder preparation and its production method, and the freeze-drying cycle exceeded 60h, with relatively high energy consumption costs; the patent with the application number CN201911213157.1 reported a pathogenic Escherichia coli phage oral preparation and its preparation method. The solid phage preparation obtained by spray drying has the characteristics of low cost, safety and high efficiency, but it targets animal diarrhea caused by Escherichia coli and is mainly applied to piglets.

[0007] Therefore, it is urgent to develop a solid phage preparation for Salmonella in livestock and poultry that can be stored long-term and stably and has low costs. Summary of the Invention

[0008] Aiming at the problems existing in the prior art, the main purpose of the present invention is to provide a solid preparation of Salmonella phage, its preparation method and application.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] In the first aspect of the present invention, a solid preparation of Salmonella phage is provided. The solid preparation is obtained by spray-drying a biologically active phage and a protectant and then mixing them with a carrier in proportion.

[0011] Preferably, the solid preparation is a dry powder.

[0012] The biologically active phage is one or two of Salmonella enterica serovar phage S4P and Salmonella enterica serovar phage S16P.

[0013] The Salmonella enterica serovar phage S4P (Salmonella enterica serovar phage S4P) is preserved in the China Center for Type Culture Collection. The preservation date is June 24, 2021, and the preservation number is CCTCC NO: M 2021765.

[0014] The Salmonella phage S16P (Salmonella enterica serovar phage S16P) is preserved in the China Center for Type Culture Collection. The preservation date is July 12, 2021, and the preservation number is CCTCC NO: M 2021861.

[0015] The cryoprotectant is one or more of sodium alginate, skim milk powder, gelatin, glycine, hydroxypropyl-β-cyclodextrin, poloxamer, and mannitol.

[0016] Preferably, the composition of the cryoprotectant is 2-5% sodium alginate, 5-15% skim milk powder, 10-20% gelatin, 0.5-2% glycine, 10-35% hydroxypropyl-β-cyclodextrin, 8-20% poloxamer, and 6-15% mannitol by weight.

[0017] More preferably, the composition of the cryoprotectant is 2.5-4% sodium alginate, 6-10% skim milk powder, 12-18% gelatin, 0.6-1.5% glycine, 15-30% hydroxypropyl-β-cyclodextrin, 10-18% poloxamer, and 8-12% mannitol by weight.

[0018] Even more preferably, the composition of the cryoprotectant is: 3% sodium alginate, 8% skim milk powder, 15% gelatin, 1% glycine, 20% hydroxypropyl-β-cyclodextrin, 15% poloxamer, and 10% mannitol by weight.

[0019] The carrier is one or more of corn starch, dextrin, montmorillonite, light calcium carbonate, silicon dioxide, sodium chloride, and medical stone.

[0020] Preferably, the carrier is one or several of corn starch, dextrin, light calcium carbonate, and medical stone.

[0021] In the second aspect of the present invention, a preparation method of a solid preparation of Salmonella phage is provided. The preparation steps include:

[0022] (1) Preparing a phage fermentation broth: inoculating the Salmonella phage seed liquid and the host bacteria into a fermentation medium in a ratio, and fermenting for 2-24 h under culture conditions to obtain a Salmonella phage fermentation broth;

[0023] (2) Removing the host bacteria: subjecting the Salmonella phage fermentation broth obtained in step (1) to a process of centrifugation by a tubular centrifuge, flocculation, and membrane filtration separation through a 0.22 μm membrane to obtain a sterile Salmonella phage clear liquid;

[0024] (3) Adding a protective agent: Dilute the sterile Salmonella phage clear liquid obtained in step (2) by 2 - 10 times, then add the protective agent and mix and dissolve it to obtain the Salmonella phage liquid to be spray - dried.

[0025] (4) Spray - drying: Transfer the Salmonella phage liquid to be spray - dried obtained in step (3) to a spray dryer using a peristaltic pump for spray - drying to obtain the spray - dried powder of Salmonella phage.

[0026] (5) Preparing a solid preparation of Salmonella phage: Mix the spray - dried powder of Salmonella phage obtained in step (4) and the carrier evenly in proportion using a single - column mixer. After packaging and sealing, obtain the solid preparation of Salmonella phage, and store it in a cool warehouse, dry and protected from light.

[0027] Preferably, the specific preparation process of the phage fermentation broth in step (1) is as follows: Inoculate the Salmonella phage seed and the host bacteria into a 700L fermenter with a liquid - filling volume of 50 - 70%. The multiplicity of infection of the phage and the host bacteria is 0.000001 - 100, the fermentation temperature is 23 - 41°C, the stirring speed is 50 - 100 rpm / min, the pH is controlled at 5.6 - 7.2, the dissolved oxygen is 30 - 100%, and the tank pressure is 0.02 - 0.05 Mpa to obtain the Salmonella phage fermentation broth.

[0028] Preferably, the added mass of the protective agent in step (3) is 10 - 60% of the volume of the sterile Salmonella phage clear liquid (after dilution). More preferably, the added mass of the protective agent is 30% of the volume of the sterile Salmonella phage clear liquid.

[0029] Preferably, the mixing ratio of the spray - dried powder of Salmonella phage and the carrier in step (5) is 1:20 - 1:2. More preferably, the mixing ratio is 1:4.

[0030] Preferably, the conditions for spray - drying in step (4) are as follows: The flow rate of the peristaltic pump in spray - drying is 50 - 150 L / h, the inlet air temperature is 90 - 200°C, the outlet air temperature is 70 - 85°C, and the material - receiving temperature is 27 - 35°C.

[0031] Preferably, the environmental humidity for the mixing operation in step (5) is 15 - 40%, and the environmental temperature is 15 - 25°C.

[0032] Through the above - mentioned technical solution, a solid preparation of Salmonella phage that can be stored at room temperature is provided.

[0033] In the third aspect of the present invention, an application of a solid preparation of Salmonella phage is provided, and the solid preparation is used for preparing a drug or a feed additive for preventing and treating avian and livestock Salmonella infections.

[0034] Preferably, the solid preparation is a feed additive.

[0035] In summary, the present invention has the following beneficial effects:

[0036] 1. The solid preparation of Salmonella phage of the present invention can be stored at room temperature. There is no caking phenomenon after being placed at 60 °C for 10 days, and the titer is still 10 8 PFU / g after being placed at 40 °C for 30 days. Compared with the strict low-temperature storage conditions of liquid phage preparations, it can greatly reduce the transportation and storage costs of farmers, and the solid preparation is more convenient to use.

[0037] 2. The solid preparation of Salmonella phage of the present invention can be stored for a certain time under high-temperature storage conditions and still has a high titer level, has good temperature tolerance, and also has strong bile salt tolerance.

[0038] 3. The protective agent used in the solid preparation of Salmonella phage of the present invention can effectively protect the phage activity during the spray-drying process. After adding the carrier, it can stabilize the form of the spray-dried powder, reduce the caking tendency of the phage dry powder, and reduce the decline of phage activity during the storage of the solid preparation of phage.

[0039] 4. Compared with freeze-drying, the process of preparing Salmonella phage dry powder by spray-drying has the characteristics of low cost, short cycle, and simple process. Description of the Drawings

[0040] Figure 1 It is a schematic diagram of the plaque of Salmonella phage S4P;

[0041] Figure 2 It is a schematic diagram of the plaque of Salmonella phage S16P;

[0042] Figure 3 It is a process flow chart of the preparation method of the solid preparation of Salmonella phage of the present invention. Detailed Embodiments

[0043] The following will further elaborate on the above content through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter to the following examples. All technologies implemented based on the content of the present invention fall within the scope.

[0044] Example 1 Isolation, Purification and Preservation of Salmonella Phage S4P

[0045] (1) Sample treatment: Collect fecal samples of diseased chickens infected with Salmonella and plant sewage from a chicken farm in Binzhou, Shandong. Add an appropriate amount of SM buffer (dissolve 5.8 g of NaCl, MgSO 4 ·7H 250 mL of 1 mol / L Tris·HCl (pH = 7.4), 5 mL of 2% silica gel, add distilled water to make up to 1000 mL, sterilize and reserve; unless otherwise specified, the SM buffer is the same as this formula. After dilution, soak overnight in a refrigerator at 4°C, centrifuge at 8000 rpm / min for 10 min the next day, take the supernatant and filter through a 0.22 μm filter membrane to sterilize, and obtain the sample stock solution.

[0046] (2) Resuscitation and activation of the host bacteria: Pick a single colony of clinical Salmonella gallinarum isolated from a dead chicken in a chicken farm in Shandong and inoculate it into 10 mL of TSB liquid medium, culture overnight at 150 r / min and 37°C to obtain a bacterial suspension. The next day, take 100 μL of the bacterial suspension and transfer it again to 10 mL of TSB liquid medium, culture at 150 r / min and 37°C for 4 h to obtain the host bacterial suspension, and reserve it.

[0047] (3) Proliferation of the host bacteria: Take 10 mL of the prepared sample stock solution and add it to the Salmonella host bacterial suspension, put it into a sterile triangular conical flask, and incubate statically at 37°C overnight to obtain the Salmonella phage proliferation solution.

[0048] (4) Isolation of phages: Centrifuge the obtained Salmonella phage proliferation solution at 10000 r / min for 30 min, filter through a 0.22 μm filter membrane, and put it into a sterile EP tube. Dilute it 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 times with SM buffer. Isolate phages by the double-layer agar plate method. Take 100 μL of the host bacterial suspension and the phages diluted at each gradient and add them to 5 mL of semi-solid medium (add 0.6% agar powder to the TSB liquid medium, sterilize at 121°C for 15 min, and keep it warm at 55°C for standby), shake well, quickly spread it on the TSA solid plate, and place it in a 37°C constant temperature incubator for 4 - 6 h to observe the results. If there are Salmonella phages, transparent circular spots will appear on the upper layer of the plate (as Figure 1 shown).

[0049] (5) Purification of phages: Use a sterile pipette tip to pick a relatively large and transparent plaque, transfer it to 1 mL of SM buffer for dilution, then transfer all of it to 10 mL of TSB liquid medium, and then add 100 μL of the host bacterial suspension, and culture it with shaking at 37°C and 150 r / min for 4 - 6 h. Centrifuge the mixed culture solution at 8000 rpm / min for 10 min, take the supernatant and filter through a 0.22 μm filter membrane to sterilize to obtain the phage stock solution, and dilute it to 10 -5 、10 -6, 10 -7 , 10 -8 After multiplying by, respectively take 100 μL of the above phage dilution and 100 μL of the host bacteria suspension and add them to 5 mL of semi-solid medium, shake well, quickly spread them evenly on the TSA solid plate, and place them in a 37 °C constant temperature incubator for 4 - 6 h after solidification to observe the results. Repeat this step 3 - 5 times until the plaque morphology, size, and transparency are uniform each time, then the purified Salmonella phage stock solution is obtained, named S4P, and the titer of the purified Salmonella phage is ≥ 10 11 PFU / mL.

[0050] (7) Preservation of phage: The applicant preserved the purified Salmonella phage S4P in the China Center for Type Culture Collection (CCTCC), with the preservation number: CCTCC NO: M2021765, the preservation date: June 24, 2021, and the preservation address: Wuhan University, Wuhan, China, Postcode: 430072.

[0051] Example 2 Isolation, purification, and preservation of Salmonella phage S16P

[0052] (1) Preparation of host bacteria: Streak on phenol red brilliant green agar plates and DHL agar plates, and after culturing at 37 °C for 20 - 24 h, pick 5 suspicious colonies and streak them on nutrient agar plates for subsequent identification with pure culture. Extract genomic DNA from the purified Salmonella and identify the species through 16s rRNA sequence analysis; Screening results: A total of 22 Salmonella strains were obtained through isolation and identification, and the isolates were used as host bacteria for phage screening.

[0053] (2) Isolation of phage: Centrifuge the sewage sample at 5000 rpm, 4 °C for 10 min, take 5 mL of the supernatant and add it to a sterilized test tube, and at the same time add 5 mL of double-concentrated TSB liquid medium and 100 μL of logarithmic-phase bacterial liquid, and culture it overnight with shaking at 160 rpm at 37 °C. The next day, centrifuge the overnight-cultured suspension at 5000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm microporous membrane to obtain the phage stock solution. Mix 0.1 mL of the phage stock solution and 0.2 mL of the host bacteria liquid, let it stand at 37 °C for 10 min, then add it to the 40 °C TSB nutrient agar and mix well, and immediately pour it onto the solidified TSB culture agar plate to make a double-layer plate. After the upper layer of the medium solidifies, incubate it upside down at 37 °C for 4 - 6 h to observe the presence of plaques.

[0054] (3) Purification of phage: After the phage plaques grew out, select the phage plaques with larger diameter and higher transparency. Cut out the phage plaques and place them in 1 mL of physiological saline. Soak them at 37 °C for 30 min, then centrifuge at 10,000 rpm for 5 min. Then, spread a double-layer plate according to the method described in step (2). After cultivation, pick single phage plaques and purify them repeatedly 5 times until the phage plaques are of the same size, that is, a biologically pure phage strain system is obtained. The obtained phage plaques are round, with a single morphology, smooth edges with a halo, clear and transparent, and the diameter is 2 - 3 mm (as Figure 2 shown).

[0055] (4) Preservation of phage: The applicant deposited the purified Salmonella phage S16P with the China Center for Type Culture Collection (CCTCC). The deposit number is: CCTCC NO: M2021861, the deposit date is: July 12, 2021, and the deposit address is: Wuhan University, Wuhan, China, Zip Code: 430072.

[0056] Example 3 A solid preparation of Salmonella phage

[0057] The solid preparation of Salmonella phage is prepared by spray-drying powder of Salmonella phage S4P according to the following steps:

[0058] (1) Preparation of phage fermentation broth: Inoculate the phage S4P seed liquid and the host bacteria into a 700 L fermentation tank medium with a loading of 70% according to a certain ratio. The multiplicity of infection of the phage and the host bacteria is 0.001. The fermentation temperature is 32 °C, the stirring speed is 100 rpm / min, the pH is controlled at 6.2 - 7.0, the dissolved oxygen is 30%, and the tank pressure is 0.02 - 0.03 Mpa. Ferment for 6 h under the culture conditions to obtain the phage fermentation broth;

[0059] (2) Removal of host bacteria: The phage fermentation broth obtained in step (1) is successively centrifuged by a tubular centrifuge, flocculated, and filtered through a 0.22 μm membrane separation process to obtain a sterile phage supernatant;

[0060] (3) Addition of protective agent: Dilute the sterile phage supernatant in step (2) by 5 times, and then add a protective agent with a mass of 30% of the volume of the diluted sterile phage supernatant, and then mix and dissolve to obtain a phage solution to be spray-dried. The protective agent composition is 3% sodium alginate, 8% skim milk powder, 15% gelatin, 1% glycine, 20% hydroxypropyl-β-cyclodextrin, 15% poloxamer, and 10% mannitol;

[0061] (4) Spray drying: Transfer the phage solution to be spray-dried in step (3) to a spray dryer using a peristaltic pump for spray drying to obtain phage dry powder; wherein the flow rate of the peristaltic pump in spray drying is 120 L / h, the inlet air temperature is 180 - 190 °C, the outlet air temperature is 70 - 85 °C, and the material receiving temperature is 30 - 35 °C;

[0062] (5) Phage solid preparation: Weigh the phage dry powder and the carrier obtained in step (4), wherein the carrier is dextrin, and the mass ratio of the phage dry powder to the carrier is 1:4. After fully mixing evenly by a single-column mixer according to the mass ratio, it is sealed and packaged to obtain a solid preparation of Salmonella phage, and stored in a cool, dry and light-proof place. The environmental humidity during the mixing operation is 15 - 40%, and the environmental temperature is 15 - 25 °C.

[0063] Example 4 A solid preparation of Salmonella phage

[0064] The solid preparation of Salmonella phage is prepared by spray-drying the Salmonella phage S16P according to the following steps to obtain the dry powder of Salmonella phage S16P:

[0065] (1) Preparation of phage fermentation broth: Inoculate the phage S16P seed liquid and the host bacteria into a 700 L fermentation tank medium with a filling volume of 70% according to a certain ratio. The multiplicity of infection of the phage and the host bacteria is 10, the fermentation temperature is 32 °C, the stirring speed is 100 rpm / min, the pH is controlled at 6.2 - 7.0, the dissolved oxygen is 30%, the tank pressure is 0.02 - 0.03 Mpa, and ferment for 6 h under the culture conditions to obtain the phage fermentation broth;

[0066] (2) Removal of host bacteria: The phage fermentation broth obtained in step (1) is centrifuged by a tubular centrifuge, flocculated, and separated by a 0.22 μm membrane filtration process to obtain a sterile phage clear liquid;

[0067] (3) Addition of protective agent: Dilute the sterile phage clear liquid in step (2) by 5 times, and then add a protective agent with a mass of 30% of the volume of the diluted sterile phage clear liquid for mixing and dissolution to obtain the phage solution to be spray-dried; wherein the protective agent is 3% sodium alginate, 8% skim milk powder, 15% gelatin, 1% glycine, 20% hydroxypropyl-β-cyclodextrin, 15% poloxamer, and 10% mannitol;

[0068] (4) Spray drying: Transfer the phage solution to be spray-dried in step (3) to a spray dryer using a peristaltic pump for spray drying to obtain phage dry powder; wherein the flow rate of the peristaltic pump in spray drying is 120 L / h, the inlet air temperature is 180 - 190 °C, the outlet air temperature is 70 - 85 °C, and the material receiving temperature is 30 - 35 °C;

[0069] (5) Solid preparation of phage: Weigh the phage dry powder obtained in step (4) and the carrier. The carrier is dextrin, and the mass ratio of the phage dry powder to the carrier is 1:4. After fully mixing them evenly with a single-column mixer, divide them into packages and seal them to obtain the solid preparation of Salmonella phage, and store it in a cool, dry and light-proof place. The environmental humidity for the mixing operation is 15-40%, and the environmental temperature is 15-25°C.

[0070] Example 5 A solid preparation of Salmonella phage

[0071] Weigh 10% of the dry powder of Salmonella phage S4P and 10% of the dry powder of Salmonella phage S16P described in Example 3 and Example 4 respectively, and 80% of the carrier. After fully mixing them evenly with a single-column mixer, divide them into packages and seal them to obtain the solid preparation of Salmonella phage, and store it in a cool, dry and light-proof place; the carrier is dextrin and light calcium carbonate (the mass percentages are 60% and 40% respectively), the environmental humidity for the mixing operation is 15-40%, and the environmental temperature is 15-25°C.

[0072] Comparative Example 1 A solid preparation of Salmonella phage

[0073] The raw material components, dosages and preparation methods are similar to those in Example 3, except that the formulation of the protective agent is: 3% sodium alginate, 8% skimmed milk powder, 15% lactose, 1% glycine, 20% polyvinylpyrrolidone, 15% poloxamer, 10% mannitol.

[0074] Example 6 High-temperature resistance test

[0075] Conduct the high-temperature resistance test on the solid preparations of Salmonella phage obtained in Examples 3-5 and Comparative Example 1.

[0076] Weigh 10 g of the phage solid preparation and dissolve it in a sterile 100 mL buffer test tube. Divide it into 5 mL per tube with a 10 mL sterile EP tube, and place them in a water bath at 15°C, 37°C, 45°C, 60°C, 75°C for 1 h, 6 h, 24 h respectively. After cooling, detect the phage titer by the double-layer plate method, and repeat each group three times and take the average value.

[0077] Table 1 Average results of the phage high-temperature retention rate test

[0078]

[0079]

[0080] Example 7 Bile salt tolerance test

[0081] Conduct the bile salt tolerance test on the solid preparations of Salmonella phage obtained in Examples 3-5 and Comparative Example 1.

[0082] Weigh 10 g of the phage solid preparation separately and dissolve it in a sterile 100 mL buffer test tube. Aliquot 5 mL into each of 10 mL sterile EP tubes, and add bile salts at final concentrations of 0.1%, 0.3%, and 0.5% respectively. After reacting for 6 h, detect the phage titer by the double-layer plate method. Repeat the test three times for each group and take the average value.

[0083] Table 2 Average results of the 6-hour bile salt tolerance retention rate test of phages

[0084]

[0085] Example 8 Long-term stability test

[0086] Conduct a long-term stability test on the Salmonella phage solid preparations obtained in Examples 3 - 5 and Comparative Example 1.

[0087] Weigh 10 g of the phage solid preparation separately into sealed aluminum-plastic bags, and store them at 4°C, 25°C, 40°C, and 60°C respectively. Regularly take samples and detect the phage titer by the double-layer plate method. Repeat the test three times for each group and take the average value.

[0088] Table 3 Average results of the long-term storage stability test of phages

[0089]

[0090]

[0091] Example 9 Clinical efficacy test

[0092] Conduct a clinical efficacy test on the Salmonella phage solid preparation obtained in Example 5.

[0093] Inject 0.5×10 7 cfu / mL of clinically isolated Salmonella enteritidis into each of 90 15-day-old SPF chickens. After all chickens show symptoms of pullorum disease, randomly divide them into three groups (30 chickens in each group): a positive control group, a treatment group with the phage solid preparation (Example 5), and a drug treatment group.

[0094] The broilers in the positive control group are given normal drinking water and feed. The treatment group with the phage solid preparation is orally administered at 0.05 g per chicken, and the drug treatment group is given amoxicillin soluble powder. Continuously observe for 72 h, record the status and mortality rate of the chicken flock, and conduct necropsy after the test to detect the detection rate of Salmonella pathogens in the intestinal segment disease materials. The results are shown in Table 4:

[0095] Table 4 Results of the clinical efficacy test of the phage solid preparation

[0096]

Claims

1. A solid preparation of Salmonella phage, characterized in that, the solid preparation is obtained by mixing the biologically active phage and a protective agent after spray drying and then mixing with a carrier; the biologically active phage is one or both of Salmonella phage S4P and Salmonella phage S16P; wherein, the preservation number of Salmonella phage S4P is CCTCC NO: M2021765, and the preservation number of Salmonella phage S16P is CCTCC NO: M2021861; the composition of the protective agent by weight is 2-5% sodium alginate, 5-15% skimmed milk powder, 10-20% gelatin, 0.5-2% glycine, 10-35% hydroxypropyl-β-cyclodextrin, 8-20% poloxamer, 6-15% mannitol; the carrier is one or several of corn starch, dextrin, montmorillonite, light calcium carbonate, silica, sodium chloride, and medical stone; The preparation method of the solid preparation of Salmonella phage, which comprises the following steps: (1) Preparation of phage fermentation broth: Inoculate the Salmonella phage seed liquid and the host bacteria into the fermentation medium in proportion, and ferment for 2-24 h under the culture conditions to obtain the Salmonella phage fermentation broth; (2) Removal of host bacteria: After centrifugation, flocculation, and 0.22 μm membrane filtration separation of the Salmonella phage fermentation broth obtained in step (1), a sterile Salmonella phage clear liquid is obtained; (3) Addition of protective agent: Dilute the sterile Salmonella phage clear liquid obtained in step (2) by 2-10 times, add the protective agent and mix and dissolve to obtain the Salmonella phage liquid to be spray dried; (4) Spray drying: Transfer the Salmonella phage liquid to be spray dried obtained in step (3) to a spray dryer with a peristaltic pump for spray drying to obtain the Salmonella phage dry powder; (5) Preparation of the solid preparation of Salmonella phage: Mix the Salmonella phage dry powder obtained in step (4) and the carrier evenly in proportion with a single-column mixer, and after sub-packaging and sealing, the solid preparation of Salmonella phage is obtained; The specific preparation process of the phage fermentation broth in step (1) is as follows: inoculate Salmonella phage seeds and host bacteria into a 700 L fermenter with a liquid filling volume of 50-70%, the multiplicity of infection of the phage and the host bacteria is 0.000001-100, the fermentation temperature is 23-41 °C, the stirring speed is 50-100 rpm / min, the pH is controlled at 5.6-7.2, the dissolved oxygen is 30-100%, and the tank pressure is 0.02-0.05 Mpa to obtain the Salmonella phage fermentation broth; the added mass of the protective agent in step (3) is 10-60% of the volume of the sterile Salmonella phage clear liquid; the mixing ratio of the Salmonella phage dry powder and the carrier in step (5) is 1:20-1:2; the conditions for spray drying in step (4) are: the peristaltic pump flow rate in spray drying is 50-150 L / h, the inlet air temperature is 90-200 °C, the outlet air temperature is 70-85 °C, and the material receiving temperature is 27-35 °C; the environmental humidity for the mixing operation in step (5) is 15-40%, and the environmental temperature is 15-25 °C.

2. Use of a Salmonella phage solid preparation as described in claim 1 in the preparation of a feed additive for preventing and treating Salmonella infections in livestock and poultry.

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