Salmonella bacteriophage and composition and application thereof

Abstract

The invention discloses salmonella bacteriophage as well as a composition and application thereof, and belongs to the technical field of microorganisms. The bacteriophage provided by the invention is wide in splitting spectrum and strong in splitting capacity, and is remarkably characterized in that the bacteriophage has a good sterilization effect on salmonella of different serotypes in a low-temperature (4 DEG C) environment, does not contain soluble source, drug-resistant and virulence genes, has efficient replication capacity and splitting activity, is strong in high-temperature resistance and acid and alkali resistance, and can be used for preparing salmonella. And a conventionally used disinfectant has no influence on the disinfectant. The salmonella bacteriophage provided by the invention has great significance for preventing and treating salmonella infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Salmonella phage, its composition and application. Background Art

[0002] Salmonella is a Gram-negative bacillus parasitizing in the intestines of humans and animals. There are more than 2,000 serotypes. Some are specifically pathogenic to humans, some are only pathogenic to animals, and some are zoonotic pathogens. Only a few are pathogenic to humans, such as Salmonella typhi causing typhoid fever, Salmonella paratyphi A and B. Other Salmonella species causing diseases in livestock and poultry (such as Salmonella typhimurium, Salmonella enteritidis, Salmonella choleraesuis, Salmonella anatum, etc.) can sometimes cause human gastroenteritis (food poisoning) or septicemia through contaminated foods such as meat, eggs, and milk. In severe cases, it can lead to shock and renal failure and death. According to statistics, Salmonella often ranks first among the bacterial causes of food poisoning in various countries around the world and is one of the important foodborne pathogens, posing a major threat to human life safety.

[0003] Salmonella is 0.6-1.0×2-4 μm in size, generally without a capsule or spores, and has low nutritional requirements. It is an intracellular parasite. The acid tolerance response gene mediates its ability to grow and reproduce in the acidic environment within phagocytes. At the same time, the bacterium can produce catalase and superoxide dismutase to protect the bacteria from the killing of intracellular bactericidal mechanisms. Salmonella is a mesophilic bacterium and grows best under conditions of 37°C, neutral pH, low salt, and high water activity. It can survive in water for 2-3 weeks, in the refrigerator for 3-4 months, and in feces in the natural environment for 1-2 months. Currently, low-temperature storage of food is an effective means to limit the large-scale reproduction of Salmonella, but it cannot effectively reduce the number of existing bacteria. Although antibiotics are effective, they will cause antibiotic residues in food, and long-term use will also cause the problem of bacterial drug resistance. Therefore, finding alternative effective preparations to control the number of Salmonella is crucial for preventing and controlling bacterial infections and food poisoning caused by Salmonella.

[0004] Bacteriophage, as the "natural killer" of bacteria, has a large number and high antibacterial activity, and has been increasingly used in the clinical treatment of infections caused by multi-drug resistant bacteria in recent years. Bacteriophage is a type of virus that uses specific microorganisms such as bacteria, fungi, actinomycetes, or spirochetes as hosts. Similar to other viruses, the head of the bacteriophage is composed of a protein capsid that encloses the genetic material. Tailed bacteriophages have "tails" of different lengths and various shapes, which are used to specifically recognize host bacteria and inject genetic material into the host cell.

[0005] Compared with traditional antibiotic drugs, phages have the advantages of strong bactericidal ability, high specificity, high safety, etc. Moreover, their bactericidal ability does not depend on bacterial drug resistance. Therefore, phages show great application potential in preventing and controlling bacterial infections and ensuring food safety. However, compared with traditional broad-spectrum antibiotics, phages also face the bottlenecks of strong specificity, narrow bactericidal spectrum, and easy bacterial tolerance.

[0006] Although there have been some reports on Salmonella phages, and phages are specific, most can only function at room temperature. Therefore, there is an urgent need to develop phages with a broad lysis spectrum, strong lysis ability, and active even at low temperatures against Salmonella, which is of great significance for preventing and controlling Salmonella infections. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a Salmonella phage with a broad lysis spectrum and strong lysis ability. Its remarkable feature is that it has a good bactericidal effect on Salmonella of different serotypes at low temperature (4°C). Moreover, according to the whole-genome map of this phage, it can be seen that the phage (CP-p-S-24008) provided by the present invention does not contain lysogenic, drug-resistant, and virulence genes, has high replication ability and lysis activity, is highly resistant to high temperature and acid-base, and is not affected by commonly used disinfectants.

[0008] Based on the above invention purpose, the present invention provides the following technical solutions:

[0009] In the first aspect, the present invention provides a Salmonella phage, characterized in that the phage has the morphological characteristics of the Siphoviridae family of long-tailed viruses and has at least one of the following characteristics:

[0010] a1) The whole-genome size is 43 - 44 kbp;

[0011] a2) It does not contain lysogenic, drug-resistant, and virulence genes;

[0012] a3) It has temperature stability at 4 - 60°C;

[0013] a4) It has acid-base stability at pH 3 - 11.

[0014] In some embodiments of the present invention, the phage is CP-p-S-24008, belonging to the Siphoviridae family of long-tailed viruses, and was deposited at the China Center for Type Culture Collection on January 14, 2025, with the deposit number CCTCC M2025135.

[0015] Specifically, the whole genome of the phage CP-p-S-24008 is 43,155 bp, and the specific nucleotide sequence is shown in SEQ ID NO:1.

[0016] Furthermore, the Salmonella phage further includes phage mutants, which are characterized in that the phage mutants have an average nucleotide identity of ≥96% in the genome with the phage CP-p-S-24008 (SEQ ID NO:1).

[0017] Even further, the phage mutants have at least 96%, 97%, 98%, 99%, 100% average nucleotide identity in the genome with the phage CP-p-S-24008 (SEQ ID NO:1).

[0018] Optionally, the RFLP DNA profile of the phage mutants is substantially equivalent to the RFLP DNA profile of the phage CP-p-S-24008.

[0019] The phage CP-p-S-24008 of the present invention includes mutant strains with a homology higher than 90% and maintaining substantially the same bactericidal activity obtained by point mutation, deletion mutation or addition mutation according to conventional methods. Since phages are very prone to mutation during replication, phage mutants are also within the scope of protection claimed in the present invention. For those skilled in the art, it does not require creative labor to screen phage mutants with extremely similar traits to the phages provided by the present invention.

[0020] In practical applications, in order to further broaden the phage lysis spectrum, give full play to the differences in the lysis spectra of different phages, and carry out complementary advantages, the phage CP-p-S-24008 can be combined with other phages for use. The present inventors unexpectedly found that the formed phage combination has a better bactericidal effect and can improve the tolerance of bacteria to the phage CP-p-S-24008.

[0021] In a second aspect, the present invention provides a phage composition, which is characterized in that the composition is selected from at least two of the Salmonella phages and their mutants shown in b1)-b8):

[0022] b1) Salmonella phage CP-p-S-24008, with the deposit number CCTCC M 2025135;

[0023] b2) Salmonella phage CP-p-S-24002, with the deposit number CCTCC M 2025130;

[0024] b3) Salmonella phage CP-p-S-24003, deposit number CCTCC M 2025131;

[0025] b4) Salmonella phage CP-p-S-24006, deposit number CCTCC M 2025133;

[0026] b5) Salmonella phage CP-p-S-24007, deposit number CCTCC M 2025134;

[0027] b6) Salmonella phage CP-p-S-24009, deposit number CCTCC M 2025136;

[0028] b7) Salmonella phage CP-p-S-24010, deposit number CCTCC M 2025137;

[0029] b8) Salmonella phage CP-p-S-24012, deposit number CCTCC M 2025132.

[0030] The "at least two" includes any two, any three, any four, any five, any six, any seven or eight of the Salmonella phages shown in b1)-b8) and their mutants.

[0031] In a specific embodiment of the present invention, the phage composition is selected from the combination formed by at least one of the Salmonella phage CP-p-S-24008 and its mutants described in the first aspect of the present invention and the Salmonella phages shown in b2)-b8) and their mutants.

[0032] In some specific embodiments of the present invention, the composition is a combination formed by any one of the phage CP-p-S-24008 and its mutants and the Salmonella phages shown in b2)-b8) and their mutants.

[0033] In some specific embodiments of the present invention, the composition is a combination formed by any two of the phage CP-p-S-24008 and its mutants and the Salmonella phages shown in b2)-b8) and their mutants.

[0034] In some specific embodiments of the present invention, the composition is a combination formed by any three of the phage CP-p-S-24008 and its mutants and the Salmonella phages shown in b2)-b8) and their mutants.

[0035] In some specific embodiments of the present invention, the composition is a combination formed by any four of bacteriophage CP-p-S-24008 and its mutants and the Salmonella phages shown in b2)-b8) and their mutants.

[0036] In some specific embodiments of the present invention, the composition is a combination formed by any five of bacteriophage CP-p-S-24008 and its mutants and the Salmonella phages shown in b2)-b8) and their mutants.

[0037] In some specific embodiments of the present invention, the composition is a combination formed by any six of bacteriophage CP-p-S-24008 and its mutants and the Salmonella phages shown in b2)-b8) and their mutants.

[0038] In some specific embodiments of the present invention, the composition is a combination formed by bacteriophage CP-p-S-24008 and its mutants and the Salmonella phages shown in b2)-b8) and their mutants.

[0039] The proportional relationship between bacteriophage CP-p-S-24008 and its mutants and other phages can be preferably determined by those skilled in the art in combination with the actual application field and common general knowledge in the art.

[0040] In a third aspect, the present invention provides a biological agent, characterized in that the biological agent contains an effective dose of the phage according to the first aspect of the present invention or the phage composition according to the second aspect of the present invention.

[0041] Furthermore, the biological agent further includes pharmaceutically acceptable excipients well-known to those skilled in the art for forming the agent. The "pharmaceutically acceptable excipients" are carriers or diluents that do not cause significant irritation to organisms and do not eliminate the biological activity of the phage. Exemplarily, excipients suitable for liquid preparations include saline, sterile water, buffer solutions, glucose solutions, glycerol, etc. And those skilled in the art can add other excipients as needed, such as antioxidants, bacteriostatic agents, dispersants, surfactants, binders or lubricants.

[0042] Optionally, the biological agent includes, but is not limited to, pharmaceutical preparations, disinfectants, cleaners, preservatives, food preservatives, feed additives, drinking water additives.

[0043] In some embodiments of the present invention, the biological agent is selected from pharmaceutical preparations, and the active ingredient in the pharmaceutical preparation is selected from the phage according to the first aspect of the present invention or the phage composition according to the second aspect of the present invention.

[0044] Optionally, the pharmaceutical preparation further comprises other antibacterial or bactericidal active ingredients, including but not limited to antibiotics and chemical antibacterial agents.

[0045] The dosage form of the pharmaceutical preparation is selected from injections, oral dosage forms (such as aqueous solutions, suspensions, emulsions, pills, capsules, granules) or other intermediate dosage forms (such as lyophilized agents).

[0046] In some embodiments of the present invention, the biological preparation is selected from disinfectants or detergents, and the active ingredient in the disinfectant or detergent is selected from the phage described in the first aspect of the present invention or the phage composition described in the second aspect of the present invention. The disinfectant or detergent is usually applied in the form of liquid soaking, spraying, combined use with an aqueous carrier, etc. to achieve disinfection and decontamination of the environment, utensils, facilities, etc.

[0047] In some embodiments of the present invention, the biological preparation is selected from preservatives or food preservatives, and the active ingredient in the preservative or food preservative is selected from the phage described in the first aspect of the present invention or the phage composition described in the second aspect of the present invention. By spraying the preservative or food preservative on the surface of the food or soaking the food in a solution containing the preservative or food preservative, the growth and reproduction of Salmonella in the food are inhibited, achieving the purpose of food preservation.

[0048] In some embodiments of the present invention, the biological preparation is selected from feed additives or drinking water additives, and the active ingredient in the feed additive or drinking water additive is selected from the phage described in the first aspect of the present invention or the phage composition described in the second aspect of the present invention. By adding the above feed additive or drinking water additive to the feed or water and mixing, the feed and drinking water of animals are disinfected and sterilized, and effective prevention and treatment of Salmonella disease are achieved after feeding the animals.

[0049] In the present invention, the "active ingredient" and "effective dose" refer to the main ingredient and its dose that play a therapeutic, alleviating or preventive purpose. In the present invention, the active ingredient includes the phage CP-p-S-24008 capable of killing bacteria or preventing and treating diseases caused by bacteria or a phage composition containing the phage CP-p-S-24008.

[0050] Fourthly, the present invention provides an application of the phage described in the first aspect of the present invention, the phage composition described in the second aspect of the present invention or the biological preparation described in the third aspect of the present invention in at least one of the following:

[0051] c1) Application in the preparation of a drug for preventing and / or treating a bacterial infection caused by Salmonella infection;

[0052] c2) Application in the preparation of an antibacterial product for preventing and treating Salmonella;

[0053] c3) Use in the preparation of feed additives.

[0054] The Salmonella is selected from one or a combination of two or more of Salmonella enteritidis, Salmonella choleraesuis or Salmonella typhimurium.

[0055] In a specific embodiment of the present invention, the Salmonella enteritidis is selected from Salmonella enteritidis BNCC192094 or Salmonella enteritidis ATCC13076.

[0056] In a specific embodiment of the present invention, the Salmonella choleraesuis is selected from Salmonella choleraesuis ATCC10708, BNCC186354 or Salmonella choleraesuis CVCC3383.

[0057] In a specific embodiment of the present invention, the Salmonella typhimurium is selected from Salmonella typhimurium ATCC25241 or Salmonella typhimurium ATCC13311.

[0058] In some embodiments of the present invention, the Salmonella also includes Salmonella enterica BNCC186357 or Salmonella typhi CICC10871.

[0059] In the present invention, the "prevention and treatment" includes prevention and treatment. As used herein, the term "prevention" refers to all actions including inhibiting or delaying the disease by administering the composition. As used herein, the term "treatment" refers to all actions including improving or ameliorating the disease by administering the composition.

[0060] As a treatment means, phage can solve the problem of corresponding host bacteria infection without generating drug resistance, making it have wide application value in preventing related diseases such as bacterial infection.

[0061] Fifthly, the present invention provides a method for preventing, alleviating and / or treating a disease caused by Salmonella infection, characterized in that the method comprises administering an effective dose of the phage described in the first aspect of the present invention, the phage composition described in the second aspect of the present invention or the biological agent described in the third aspect of the present invention to a subject.

[0062] The subject refers to any animal being examined, studied or treated. In some embodiments of the present invention, the subject is preferably a human or a non-human mammal, including but not limited to mouse, monkey, cow, sheep, pig, chicken, turkey, dog, cat, horse.

[0063] The administration includes administration by routes such as oral, intraocular, intramuscular, subcutaneous, parenteral, topical, intranasal, etc.

[0064] Optionally, the administration includes administering to a subject in the form of a suspension, feed, water, spray or injection.

[0065] In a fifth aspect, the present invention provides a method for disinfecting the environment, surface and / or equipment to reduce the Salmonella load, characterized in that the method comprises contacting an effective dose of the phage according to the first aspect of the present invention, the phage composition according to the second aspect of the present invention or the biological agent according to the third aspect of the present invention with the environment, surface and / or equipment.

[0066] Optionally, the contacting mode is selected from spraying, smearing, soaking and the like.

[0067] The technical solutions provided by the present invention have the following beneficial technical effects:

[0068] 1. In the present invention, 8 Salmonella phages were isolated from different environmental samples using 9 Salmonella strains. After detecting the lysis spectrum and performing cluster analysis on the 8 isolated Salmonella phages, it was found that one Salmonella phage CP-p-S-24008 had obvious lysis and application advantages relative to other phages in a low-temperature environment of 4°C.

[0069] 2. The optimal multiplicity of infection of the Salmonella phage CP-p-S-24008 provided by the present invention is 10 -3 . Therefore, during the production process of this phage, only a very low amount of mother liquor or seed liquor is required to amplify a large number of progeny phages, having a very high production efficiency. The one-step growth period of phage CP-p-S-24008 does not exceed 10 min and it can be amplified in large quantities in a short time, indicating that this phage has high lysis activity.

[0070] 3. The Salmonella phage CP-p-S-24008 provided by the present invention has good titer stability under the conditions of 4 - 60°C and pH 3 - 11, indicating that this phage is easy to store and apply.

[0071] 4. The Salmonella phage CP-p-S-24008 provided by the present invention has good bactericidal effects on zoonotic Salmonella of different serotypes such as Salmonella enteritidis BNCC192094 and ATCC13076, Salmonella choleraesuis ATCC10708 and CVCC3383, Salmonella typhimurium ATCC25241, etc. at 4°C, having a relatively wide lysis spectrum and coverage.

[0072] 5. The Salmonella phage CP-p-S-24008 provided by the present invention has stronger bactericidal ability at 4°C compared with phage CP-p-S-24002, phage CP-p-S-24003, phage CP-p-S-24006, phage CP-p-S-24007, phage CP-p-S-24009, phage CP-p-S-24010 or phage CP-p-S-24012.

[0073] 6. The Salmonella phage CP-p-S-24008 provided by the present invention can effectively kill Salmonella on the surface of salmon at 4°C and 25°C, and has broad application potential in ensuring food safety and environmental purification.

[0074] 7. The Salmonella phage composition provided by the present invention, through the synergistic interaction of phages, compared with the single phage CP-p-S-24008, not only broadens the host spectrum and lysis ability, improves the coverage rate of strains, but also has a synergistic bactericidal effect, showing better bactericidal effects than the single phage CP-p-S-24008 at 4°C, 25°C and 37°C, and is more effective in preventing bacteria from developing tolerance to phages. Phages have broad application potential in preventing Salmonella infection and environmental purification.

[0075] 8. The activity of the Salmonella phage provided by the present invention is not affected by low concentrations of food-related disinfectants BAC and CA, indicating that the use of conventional disinfectants in low concentrations of food and cold storage does not affect the application of Salmonella phages. Description of the Drawings

[0076] Figure 1 It is a purification result diagram of Salmonella phage CP-p-S-24008.

[0077] Figure 2 It is a clustering diagram after statistical analysis of the lysis spectra of 8 phages against 9 Salmonella strains in the present invention.

[0078] Figure 3 It is a bactericidal result diagram of 8 phages against corresponding host bacteria at 4°C in the present invention.

[0079] Figure 4 It is a bactericidal result diagram of phage CP-p-S-24008 against different bacteria at 4°C.

[0080] Figure 5 It is a transmission electron microscope result diagram of phage CP-p-S-24008.

[0081] Figure 6 It is a whole genome map of phage CP-p-S-24008.

[0082] Figure 7 RFLP DNA profile diagrams of 8 phages in the present invention.

[0083] Figure 8 Optimal multiplicity of infection diagram of phage CP-p-S-24008.

[0084] Figure 9 One-step growth curve diagram of phage CP-p-S-24008.

[0085] Figure 10 Temperature stability diagram of phage CP-p-S-24008.

[0086] Figure 11 pH stability diagram of phage CP-p-S-24008.

[0087] Figure 12 Sterilization test diagram of phage CP-p-S-24008 on the surface of salmon meat.

[0088] Figure 13 Synergistic bactericidal effect diagram of Salmonella phage in the present invention.

[0089] Figure 14 Detection diagram of the effect of food-related disinfectants on the activity of Salmonella phage in the present invention. Detailed implementation manners

[0090] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0091] The reagents and biological materials used in the specific embodiments of the present invention are all commercial products unless otherwise specified.

[0092] LB liquid medium (100 mL): 2.5 g of LB medium, add ddH2O to 1 L, adjust the pH to 7.4 ± 0.2, sterilize at 121 °C under high temperature and high pressure for 20 min.

[0093] 0.5% LB semi-solid medium (100 mL): 2.5 g of LB medium, 0.5 g of agar powder, add ddH2O to 1 L, adjust the pH to 7.4 ± 0.2, sterilize at 121 °C under high temperature and high pressure for 20 min, and store at 4 °C for later use. The solidified medium needs to be heated and melted before use.

[0094] 1.5% LB solid medium (100 mL): 2.5 g of LB medium, 1.5 g of agar powder, add ddH2O to 1 L, adjust the pH to 7.4 ± 0.2, autoclave at 121 °C for 20 min, then cool to 50 - 60 °C, pour into plates, after cooling and solidifying, store at 4 °C for later use.

[0095] Example 1 Isolation of Salmonella Phage

[0096] The sewage sample was collected from a certain environmental sewage in Shanghai. Take 20 mL of the sewage sample and centrifuge at 4 °C and 12,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was reserved. Take 9 mL of the supernatant filtrate, add 1 mL of 10×LB liquid medium, and at the same time add 0.1 mL of the logarithmic-phase host bacteria liquid, and place it in a shaker at 37 °C overnight. The next day, centrifuge at 4 °C and 12,000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane to sterilize, forming a stock solution containing phage, that is, a phage suspension.

[0097] Inoculate different Salmonella strains by streaking on 1.5% LB solid medium. After culturing overnight at 37 °C, pick monoclonal colonies and inoculate them into 5 mL of LB liquid medium, and culture with shaking at 37 °C for 8 h as the host bacteria culture for later use. Pipette 0.3 mL of the above-prepared host bacteria culture and mix it evenly with 3 mL of 0.5% LB semi-solid medium preheated to about 55 °C, then spread it evenly on 1.5% LB solid medium. After it dries, divide the agar plate into 2 areas. Drop 10 μL of the above phage suspension on one area, and drop an equal amount of PBS on the other area as a control. After natural drying, place it in an incubator at 37 °C for culturing, and observe whether there are plaques formed in the area where the phage is dropped. If plaques are formed, it proves the existence of phage.

[0098] Example 2 Purification of Salmonella Phage

[0099] Test method: Pick the phage plaque in Example 1 into a sterile EP tube containing 0.5 mL of PBS, place it at 4 °C overnight to release the phage, and obtain a phage leaching solution. Using the double-layer plate method, continuously dilute the phage leaching solution by 10 times. Take 0.1 mL of the 10 -2 、10 -4 、10 -6 dilution solutions and add 0.3 mL of the logarithmic-phase host bacteria liquid, add 5 mL of 0.5% LB semi-solid medium at about 55 °C, spread evenly on the pre-prepared solid LB plate, and culture in an inverted position at 37 °C for 5 - 10 h, and then obtain a double-layer plate with plaques formed again. Pick a single, clear, uniform in size, and regular in edge plaque into a sterile EP tube containing 0.5 mL of PBS to obtain a phage leaching solution. Repeat the above steps 3 times to obtain a purified phage suspension for later use.

[0100] Test results: As Figure 1 shown, Figure 1 it is the purification result diagram of Salmonella phage CP-p-S-24008 in the present invention. It can be seen that phage CP-p-S-24008 can form transparent plaques in the agar medium, with a diameter of about 1 mm, clear and regular edges, a halo around it, and a total diameter of about 3 mm, which is a typical lytic phage.

[0101] Example 3 Lytic spectrum and cluster analysis of Salmonella phage and bacteria

[0102] According to the lysis correspondence relationship between phages and hosts, cluster analysis was performed on phages and bacteria respectively. There is great similarity between different objects aggregated into the same cluster, and great differences between objects in different clusters. The specific analysis process is as follows:

[0103] 1. Detect the lytic spectra of 8 Salmonella phages isolated from sewage with different sources against 9 Salmonella strains with different serotypes, and perform cluster analysis. The specific operations are as follows:

[0104] 1.1 Take 0.3 mL of Salmonella logarithmic-phase bacterial liquid respectively, add 5 mL of 0.5% LB semi-solid medium at about 55 °C, and spread it evenly on the pre-prepared solid 1.5% LB solid medium;

[0105] 1.2 Then divide each plate into two equal areas on average. Take 2 μL of the phage reserved in Example 2 and add it dropwise to one area, and add an equal amount of PBS dropwise to the other area as a control. After the droplets are dried, invert and culture at 37 °C for 12 h. Plaques are the clear spots formed by phage lysis on the plate where bacteria grow. According to the presence or absence of plaques, it is divided into presence (+) and absence (blank).

[0106] 2. Count the results of the lytic spectra, and classify them according to the plaque morphology into large and transparent (LC), small and transparent (SC), large and turbid (LO), small and turbid (SO), and no plaque (NP);

[0107] 3. Convert the symbols of the transparency degree of plaques into numerical values (0 for no plaque, 1 for fuzzy plaque, 2 for transparent plaque);

[0108] 4. Use the functions of the R package loaded by the RStudio software for drawing;

[0109] 5. Analyze the generated cluster results.

[0110] Test results: The lysis results (lytic spectra) of 8 Salmonella phages against 9 Salmonella strains are shown in Table 1. Cluster analysis was performed based on the lytic spectra, and the cluster results are as Figure 2As shown, the bacteria are horizontal and the phages are vertical. 0 represents no plaque, shown in pink; 1 represents a fuzzy plaque, shown in blue; 2 represents a clear plaque, shown in red. Among them, Salmonella phage CP-p-S-24008 can produce plaques on 5 strains of Salmonella, namely Salmonella enteritidis BNCC192094, Salmonella enteritidis ATCC13076, Salmonella choleraesuis ATCC10708, Salmonella choleraesuis CVCC3383, and Salmonella typhimurium ATCC25241, with a coverage rate of 55.6%. These bacteria are all zoonotic pathogens that are likely to cause human food poisoning.

[0111] Table 1 Statistical table of the lysis spectrum of Salmonella phages

[0112]

[0113] Note: "+" represents that the bacteria can be lysed by the phage, and a blank space represents that the bacteria cannot be lysed by the phage.

[0114] Example 4 Large-scale cultivation of Salmonella phages

[0115] Test method: Take 40 mL of LB liquid medium, add 0.4 mL of the host bacterial liquid in the logarithmic phase at the same time, place it in a shaker at 37°C and 200 rpm for 1 - 2 h until the logarithmic phase, then add 0.05 mL of the purified phage suspension in Example 2, and continue to place it in a shaker at 37°C and 200 rpm until the liquid in the test tube becomes clear. After centrifugation and filtration, it is reserved for use.

[0116] Detecting the phage titer by the double-layer plate method: Dilute the above phage suspension by 10-fold gradients. Take 0.1 mL of each gradient of phage dilution and mix it well with 0.3 mL of the host bacterial liquid, spread it on a double-layer agar plate, and incubate it at 37°C for 5 - 10 h. Count the plaques on each agar plate, select the plate with about 30 - 300 plaques, and calculate the initial concentration of the phage, that is, the phage titer, according to the dilution factor.

[0117] Phage titer (PFU / mL) = number of plaques × dilution factor × 10

[0118] Test results: The amplification titer of Salmonella phage CP-p-S-24008 reached 1.0 × 10^10 PFU / mL, which is a typical lytic phage.

[0119] The other 7 Salmonella phages, CP-p-S-24002, CP-p-S-24003, CP-p-S-24006, CP-p-S-24007, CP-p-S-24009, CP-p-S-24010, and CP-p-S-24012, were also easily amplified to a relatively high titer and were all deposited at the China Center for Type Culture Collection on January 14, 2025. The deposit information is shown in Table 2.

[0120] Table 2 Deposit Information of Salmonella Phages

[0121] Phage number Deposit number Classification Deposit center Deposit date CP-p-S-24002 CCTCC M 2025130 Short tail CCTCC 2025.01.14 CP-p-S-24003 CCTCC M 2025131 Myovirus CCTCC 2025.01.14 CP-p-S-24006 CCTCC M 2025133 Short tail CCTCC 2025.01.14 CP-p-S-24007 CCTCC M 2025134 Long tail CCTCC 2025.01.14 CP-p-S-24008 CCTCC M 2025135 Long tail CCTCC 2025.01.14 CP-p-S-24009 CCTCC M 2025136 Short tail CCTCC 2025.01.14 CP-p-S-24010 CCTCC M 2025137 Long tail CCTCC 2025.01.14 CP-p-S-24012 CCTCC M 2025132 Myovirus CCTCC 2025.01.14

[0122] Example 5 Verification of the Bactericidal Effect of Salmonella Phages on Host Bacteria at 4°C

[0123] Test method:

[0124] (1) Take 50 μL of each of the host bacteria corresponding to the above 8 Salmonella phages cultured to the logarithmic phase and add them to a 15 mL centrifuge tube containing 5 mL of LB liquid medium. Then add 10 μL of the corresponding phage liquid in Example 4, mix well, and place it in a 4°C refrigerator. At the same time, set a control group and replace the phage liquid with 10 μL of LB liquid medium.

[0125] (2) Take 100 μL of the mixed liquid in each centrifuge tube at 24 h, 72 h, and 1 w respectively, perform 10-fold serial dilutions, and select 100 μL of the diluted solutions at 3 appropriate dilution multiples for plating and incubate overnight at 37°C.

[0126] (3) Select plates with an appropriate number of bacteria for counting (30 - 300 colonies) and perform colony counting.

[0127] Test results: As Figure 3 shown, Figure 3 is the bactericidal result graph of 8 Salmonella phages on the corresponding host bacteria at different time points at 4°C. It can be seen that at 4°C, phage CP-p-S-24008 has the best bactericidal effect on host bacteria CVCC3383, showing that the number of bacteria gradually decreases with the extension of time. The number of bacteria decreased by 2 logs at 24 h and 3 logs at 1 w. It can be seen that compared with the other 7 phages, Salmonella phage CP-p-S-24008 has a better bactericidal effect at 4°C.

[0128] Example 6 Verification of the Bactericidal Effect of Salmonella Phage CP-p-S-24008 on Different Bacteria at 4°C

[0129] Test method: (1) Take 50 μL each of Salmonella enteritidis BNCC192094, Salmonella enteritidis ATCC13076, Salmonella choleraesuis ATCC10708, Salmonella choleraesuis CVCC3383, and Salmonella typhimurium ATCC25241 cultured to the logarithmic phase, add them to a 15 mL centrifuge tube containing 5 mL of LB liquid medium, and then add 10 μL of phage CP-p-S-24008 in Example 4 to each. After mixing, place them statically in a 4 °C refrigerator.

[0130] (2) At 0 h, 24 h, 48 h, 5 d, and 7 d respectively, take 100 μL of the sample from each centrifuge tube, perform 10-fold serial dilutions, select 100 μL of the dilution solutions at 3 appropriate dilution multiples for each, spread them on plates, and incubate overnight at 37 °C.

[0131] (3) Select plates with an appropriate number of bacteria for counting (30 - 300 colonies) and perform colony counting.

[0132] Test results: As Figure 4 shown, Figure 4 is the bactericidal result diagram of Salmonella phage CP-p-S-24008 against different bacteria at 4 °C at different times. It can be seen that Salmonella phage CP-p-S-24008 has a good bactericidal effect on different bacteria in a 4 °C refrigerator.

[0133] Example 7 Transmission electron microscopy observation of Salmonella phage CP-p-S-24008

[0134] Test method: Take the phage suspension in Example 4 for electron microscopy observation. Drop 10 μL of the phage suspension on a copper grid, let it precipitate naturally for 10 min, suck off the excess liquid with filter paper, drop a drop of 2% phosphotungstic acid (PTA, 2% w / v) and stain for 1 - 2 min, and observe with a transmission electron microscope after drying at room temperature.

[0135] Test results: As Figure 5 shown, Figure 5 is the transmission electron microscopy result diagram of phage CP-p-S-24008 in the present invention. It can be seen that the head diameter of Salmonella phage CP-p-S-24008 is about 60 nm, and the tail length is about 135 nm. According to the "Virus Taxonomy - The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, phage CP-p-S-24008 belongs to the Siphoviridae family of long-tailed viruses.

[0136] Example 8 Genomic DNA extraction, sequencing and splicing analysis of Salmonella phage CP-p-S-24008

[0137] Test method:

[0138] (1) Respectively take 10 mL of Salmonella phage CP-p-S-24008 in Example 4, add DNase I (10 U / μg) and RNase A (10 U / μg) with a final concentration of 1 μg / mL, mix well, and incubate at 37 °C for 30 min;

[0139] (2) Add EDTA with a final concentration of 25 mM, and incubate at 65 °C for 10 min (inactivate DNA enzyme);

[0140] (3) Starting from this step, use the M5 λ phage genomic DNA rapid extraction kit to extract nucleic acids;

[0141] (4) After verification by 1% agarose gel nucleic acid electrophoresis, measure its concentration and purity with NanoDrop-300, and send the product to Shanghai Personal Biotechnology Co., Ltd. for sequencing;

[0142] (5) Assemble and analyze the whole-genome sequencing results of Salmonella phage CP-p-S-24008.

[0143] Test results: As Figure 6 shown, Figure 6 is the whole-genome map of phage CP-p-S-24008 in the present invention. The whole-genome sequencing results show that the complete genome size of Salmonella phage CP-p-S-24008 is 43,155 bp, and the whole-genome sequence is as shown in SEQ ID NO:1. See Figure 6 for the whole-genome map and annotation. There are no lysogenic, drug-resistant, and virulence genes in the whole genome of Salmonella phage CP-p-S-24008.

[0144] RFLP DNA profile of Salmonella phage in Example 9

[0145] Test method: (1) Take 8 strains of Salmonella phages in Example 4, extract their whole genomes respectively according to the method described in Example 8, and prepare the enzyme digestion reaction system according to Table 3; (2) Flick the tube wall to mix well, and centrifuge instantaneously to collect at the bottom of the tube; (3) Incubate at 37 °C for 30 - 60 min; (4) Verify by 1% agarose gel nucleic acid electrophoresis, set the voltage at 60 - 80 V, and the electrophoresis time at 30 - 60 min; (5) Develop and take pictures with a nucleic acid gel imager, and the results are as Figure 7 shown.

[0146] Table 3 Nde I single enzyme digestion reaction system for Salmonella phage genomic DNA

[0147]

[0148]

[0149] Figure 7 This is the nucleic acid electrophoresis map of the genomic DNA of 8 Salmonella phages in the present invention after single digestion with Nde I. The results show that the endonuclease Nde I can cut the genomic DNA of 8 Salmonella phages into different numbers of DNA fragments. The specific RFLP DNA profiles are as Figure 7 shown. In the figure, M is the 15000bp DNA marker; 1-8 are the nucleic acid electrophoreses after single digestion of the genomic DNA of Salmonella phages CP-p-S-24002, CP-p-S-24003, CP-p-S-24006, CP-p-S-24007, CP-p-S-24008, CP-p-S-24009, CP-p-S-24010, and CP-p-S-24012 with Nde I respectively; 9 is the control: the genomic DNA of Salmonella phage CP-p-S-24008 without enzyme digestion.

[0150] Example 10 Determination of the Optimal Multiplicity of Infection (MOI) of Salmonella Phage CP-p-S-24008

[0151] The multiplicity of infection (MOI) is the ratio of the number of phages to the number of bacteria.

[0152] Test method:

[0153] (1) Transfer the host bacterium culture reserved in Example 1 to the logarithmic phase (OD600nm = 0.5) and adjust the concentration to 1×10 8 CFU / mL for standby; dilute the phage culture solution in Example 4 to 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 PFU / mL for standby;

[0154] (2) According to the multiplicities of infection of 10 1 , 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 respectively, take 100 μL of the standby phage dilution, 100 μL of the host bacterium culture, and 5 mL of LB liquid medium and mix them evenly, and culture them in a shaker at 37°C with 200 rpm for 5 h;

[0155] (3) Take 1 mL of the lysis solution and centrifuge it at 8000 rpm for 10 min at 4°C. Collect the supernatant and filter it through a 0.22 μm filter membrane.

[0156] (4) Take 100 μL of the filtrate from step (3) and perform serial 10-fold dilutions continuously.

[0157] (5) Take 100 μL of each dilution and mix it with 300 - 400 μL of the bacterial suspension respectively. After standing for 5 - 10 min, add it to the 0.5% LB semi-solid medium cooled to about 55°C, mix well, pour a double-layer plate, and incubate it overnight in a 37°C constant temperature incubator.

[0158] (6) Observe the plate and calculate the phage titer. Repeat the experiment three times and draw a bar chart.

[0159] Test results: As Figure 8 shown, Figure 8 this is the optimal multiplicity of infection graph of phage CP-p-S-24008 in the present invention. The results show that the optimal MOI of phage CP-p-S-24008 is 10 -3 , and the titer reaches 1.6×10^10 PFU / mL (the difference in titer from that obtained in Example 4 is within the allowable detection difference range).

[0160] Example 11 Determination of the one-step growth curve of Salmonella phage CP-p-S-24008

[0161] Test method: The one-step growth curve can reflect the replication characteristics of phages, including the latent period and the burst period. The specific method is as follows:

[0162] (1) Take the host bacterium culture reserved in Example 1, transfer it to a fresh medium for culture at a ratio of 1:100, and simultaneously add CaCl2 with a final concentration of 5 mmol / L. Culture the bacteria until the logarithmic phase (OD600nm = 0.5).

[0163] (2) Take 1 mL of the host bacterium cultured to the logarithmic growth phase and add the phage culture solution reserved in Example 4 at a ratio of MOI = 0.1. Mix well and incubate it in a 37°C incubator for 5 min (to allow the bacteria and phages to adsorb).

[0164] (3) Centrifuge at 4°C and 10000 rpm for 1 min, and discard the supernatant (unadsorbed phages).

[0165] (4) Take 1 mL of fresh LB liquid to resuspend the precipitate (mutually adsorbed bacteria and phage particles).

[0166] (5) Repeat steps (3) and (4), and resuspend and wash at least twice.

[0167] (6)Finally, resuspend the pellet with 10 mL of fresh LB medium. Incubate the resuspended tube in a shaker at 37 °C with a shaking speed of 200 rpm for 90 min. Take samples every 5 min (0, 5, 10, 15, 20, 25, 30 min) during the first 30 min, and then take samples every 10 min (40, 50, 60, 70, 80, 90 min). Take out 600 μL of the culture mixture each time.

[0168] (7)Centrifuge at 4 °C and 10000 rpm for 1 min, collect the supernatant, and filter it through a 0.22 μm filter membrane.

[0169] (8)Take 100 μL of the phage filtrate and perform serial 10-fold dilutions continuously.

[0170] (9)Mix 100 μL of each dilution with 300 - 400 μL of the bacterial suspension. After standing for 5 - 10 min, add it to 0.5% LB semi-solid medium cooled to about 55 °C, mix well, pour a double-layer plate, and incubate it upside down in a 37 °C constant temperature incubator overnight.

[0171] (10)Observe the plate and calculate the phage titer. Repeat the experiment three times and draw a one-step growth curve.

[0172] Test results: As Figure 9 shown, Figure 9 This is the one-step growth curve of phage CP-p-S-24008 in the present invention. The results show that the latent period of phage CP-p-S-24008 infecting the host bacterium is 10 min, the burst period is 10 min, the plateau period is 10 min, and the one-step lysis cycle period is 30 min. In addition, this phage has the ability of secondary burst. The above results indicate that phage CP-p-S-24008 can be amplified in large quantities in a short time and has high replication ability and lysis activity.

[0173] Example 12 Determination of the temperature stability of Salmonella phage CP-p-S-24008

[0174] Test method: To detect the temperature stability of the isolated phage, a total of 8 temperature values were set in this study. The specific method is as follows:

[0175] (1)Take 8 sterile 1.5 mL centrifuge tubes and label them as: 4 °C, 25 °C, 37 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C;

[0176] (2)Add 1 mL of the phage stock solution to each centrifuge tube, and place the centrifuge tubes with corresponding labels in 8 different temperature environments and let them stand for 80 min.

[0177] (3) Take 100 μL from the original phage solution at each processed temperature and perform 10-fold serial dilutions to 10 -7 ;

[0178] (4) Select dilutions with 3 appropriate dilution factors from the diluted samples at each temperature for plating, and incubate the double-layer agar plates overnight;

[0179] (5) Select plates with an appropriate number of plaques for counting (30 - 300 plaques) and calculate the phage titer. The above experiments were repeated 3 times.

[0180] Test results: As Figure 10 shown, Figure 10 this is the temperature stability graph of phage CP-p-S-24008 in the present invention. The results show that the titer of Salmonella phage CP-p-S-24008 remains stable when the temperature ≤ 60°C, and the titer gradually decreases with time when the temperature > 60°C. When the temperature rises to 70°C, part of the phage still has activity, and when the temperature rises to 80°C, the phage is completely inactivated. It can be seen that the phage CP-p-S-24008 in the present invention can tolerate a certain degree of high temperature and has good temperature stability.

[0181] Example 13 Determination of pH Stability of Salmonella Phage CP-p-S-24008

[0182] Test method: To detect the acid-base stability of the isolated phage, a total of 12 pH values (pH = 2 - 13) were set in this study. The specific method is as follows:

[0183] (1) Prepare solutions with pH values of 2 - 13 using sterile PBS buffer, sodium hydroxide, and concentrated hydrochloric acid, and dispense 0.9 mL of each into 1.5 mL centrifuge tubes for later use;

[0184] (2) Take 100 μL of the purified and filtered phage solution (titer adjusted to 1×10 9 PFU / mL) and add it to 0.9 mL of sterile PBS buffer with each pH value (pH values of 2 - 13), vortex and mix well, and place it in a 37°C constant temperature incubator for static incubation for 1 h;

[0185] (3) Take out the 1.5 mL centrifuge tubes containing solutions with each pH value, take 100 μL of the phage solution from each 1.5 mL centrifuge tube after treatment with each pH value and perform 10-fold serial dilutions to 10 -6 times;

[0186] (4) Select dilutions with 3 appropriate dilution factors for plating respectively, and incubate the double-layer agar plates overnight;

[0187] (5) Select a plate with an appropriate number of plaques for counting (30 - 300 plaques), count the number of plaques at different pH values, and calculate the phage titer. The above experiment was repeated 3 times.

[0188] Test results: As Figure 11 shown, Figure 11 This is the pH stability graph of phage CP-p-S-24008 in the present invention. The results show that the titer of phage CP-p-S-24008 remains stable at pH 3 - 11. When the pH value is between 2 - 3 or 11 - 13, the titer of the above phage gradually decreases. It is worth noting that phage CP-p-S-24008 still has partial activity at pH 12. Thus, it can be seen that Salmonella phage CP-p-S-24008 in the present invention has good acid-base stability and can tolerate a certain degree of strong acid and strong base.

[0189] Example 14 Application test of Salmonella phage CP-p-S-24008 on the surface of salmon

[0190] Test method:

[0191] (1) Surface disinfection treatment of the sample: Purchase a number of salmon from a supermarket in Shanghai, wipe the surface of the salmon with an alcohol cotton ball in a biosafety cabinet, place it in a sterile petri dish, spray disinfected alcohol, irradiate with ultraviolet light for 30 min, and irradiate again for 30 min after turning it over;

[0192] (2) Adjust Salmonella CVCC3383 cultured to the logarithmic phase to 5×10^5 CFU / mL, take 20 μL and drop it on the surface of the salmon, and let it air dry naturally for 20 - 30 min;

[0193] (3) Adjust Salmonella phage CP-p-S-24008 in Example 4 to 2×10^6, 2×10^7, 2×10^8 PFU / mL respectively, take 50 μL of each concentration of phage liquid and drop it on the air-dried bacteria, that is, MOI = 10, 100, 1000, and let it air dry naturally;

[0194] (4) Place each salmon block in a 4°C refrigerator and at 25°C. Take out the corresponding salmon blocks at 0 h, 2 h, 4 h, and 6 h respectively, grind them, vortex them, and perform 10-fold serial dilution. Select 100 μL of the dilution solution with 3 appropriate dilution multiples for each and plate them, and incubate overnight at 37°C;

[0195] (5) Select a plate with an appropriate number of bacteria for counting (30 - 300 colonies), and perform colony counting.

[0196] Test results: As Figure 12 shown, Figure 12This is the bactericidal test diagram of phage CP-p-S-24008 on the surface of salmon meat in the present invention. The results show that when MOI = 10 or 100 or 1000, phage CP-p-S-24008 can effectively kill Salmonella on the surface of salmon meat stored at 4°C. Among them, especially when MOI = 100 or 1000, the bactericidal effect is better. For salmon meat stored at 25°C, phage CP-p-S-24008 can effectively kill Salmonella on the food surface when MOI = 10 or 100 or 1000.

[0197] Example 15 Synergistic bactericidal effect of Salmonella phage

[0198] Taking phage CP-p-S-24008 of Salmonella and the remaining 7 phages in Example 4 as an example, the synergistic bactericidal effect of Salmonella phage was verified.

[0199] 1. Detection of the synergistic bactericidal effect of Salmonella phage at 4°C and 25°C by the solid method

[0200] Test method:

[0201] (1) Adjust Salmonella CVCC3383 cultured to the logarithmic phase to 1×10^8 CFU / mL, take 50 μL and add it to a 15 mL centrifuge tube containing 5 mL of LB liquid medium, and then add 50 μL of phage CP-p-S-24008 in Example 4 (i.e., MOI = 100); 50 μL of each of the 8 Salmonella phages in Example 4 was added to the synergistic effect group; another negative control group was set up, and 50 μL of LB liquid medium was added instead of the phage solution. After mixing, it was placed in a 4°C refrigerator and 25°C.

[0202] (2) Take 100 μL of the samples in each centrifuge tube at 0 h, 1 h, 2 h, 4 h, 6 h, and 8 h, perform 10-fold serial dilutions, and select 100 μL of the dilution solutions at 3 appropriate dilution multiples for plating, and incubate overnight at 37°C.

[0203] (3) Select the plates with an appropriate number of bacteria for counting (30 - 300 colonies) and perform colony counting.

[0204] Test results: As Figure 13 shown, Figure 13This is the graph of the synergistic bactericidal effect of Salmonella phages in the present invention. The results show that Salmonella phage CP-p-S-24008 can significantly reduce the number of Salmonella CVCC3383 in a 4°C refrigerator and at 25°C. The bactericidal effect of phage CP-p-S-24008 combined with the other 7 Salmonella phages in Example 4 is significantly better than that of phage CP-p-S-24008 alone. The above results indicate that the synergistic effect of phage CP-p-S-24008 and the other 7 Salmonella phages makes the bactericidal effect better.

[0205] 2. Detection of the synergistic bactericidal effect of Salmonella phages at 37°C by the liquid method

[0206] Test method:

[0207] (1) Adjust Salmonella CVCC3383 cultured to the logarithmic phase to 1×10^8 CFU / mL, take 10 μL and add it to a 96-well plate containing 190 μL of LB liquid medium. Then add 1 μL of phage CP-p-S-24008 adjusted to 1×10^10 PFU / mL and adjusted to 1×10^9 PFU / mL (i.e., MOI = 10 and MOI = 1) in Example 4 respectively; in the synergistic effect group, add 1 μL of each of the 8 Salmonella phages in Example 4; another negative control group was set up.

[0208] (2) Place the 96-well plate with the added samples in a Biotek microplate reader and incubate with shaking at 37°C for 24 h, and read the OD600nm value every 20 min.

[0209] (3) After the program runs to completion, integrate the bactericidal curve.

[0210] Test results: As Figure 13 shown, Figure 13 This is the graph of the synergistic bactericidal effect of Salmonella phages in the present invention. The results show that Salmonella phage CP-p-S-24008 can significantly reduce the number of Salmonella CVCC3383 at 37°C, but at 8 h, Salmonella CVCC3383 develops tolerance to phage CP-p-S-24008. The bactericidal effect of phage CP-p-S-24008 combined with the other 7 Salmonella phages in Example 4 is significantly better than that of phage CP-p-S-24008 alone, and until 24 h, Salmonella CVCC3383 does not develop tolerance to the phage composition. In addition, different MOI values (MOI = 10 and MOI = 1) do not affect the bactericidal effect of the phage. The above results indicate that the synergistic effect of phage CP-p-S-24008 and the other 7 Salmonella phages makes the bactericidal effect better.

[0211] Example 16 Effect of Food-related Disinfectants on the Activity of Salmonella Phages

[0212] Test method:

[0213] (1) Take 500 μL of each of the 8 Salmonella phages in Example 4 and add them to 1.5 mL centrifuge tubes. Then add 500 μL of 0.025% benzalkonium chloride (BAC) and 0.05% chlorhexidine (CA) respectively. Additionally, set up a negative control group and add 500 μL of sterile water. After mixing evenly, let them stand at 4°C for 24 h;

[0214] (2) Take 100 μL of the samples from each centrifuge tube, perform 10-fold serial dilutions, select the dilution solutions of 3 appropriate dilution multiples, mix them evenly with the corresponding host bacterial solutions, and then plate them. Incubate the double-layer agar plates overnight;

[0215] (3) Select the plates with an appropriate number of plaques for counting (30 - 300 plaques), count the number of plaques after treatment with disinfectants at different concentrations, and calculate the phage titer.

[0216] Test results: As Figure 14 shown, Figure 14 is the phage titer diagram of 8 Salmonella phages after treatment with food-related disinfectants in the present invention. The results show that low concentrations of food-related disinfectants BAC and CA have no effect on the activity of the 8 Salmonella phages in Example 4, indicating that the use of common disinfectants in low concentrations in food and cold storage does not affect the application of Salmonella phages.

[0217] The above examples are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

[0218] The complete genome sequence (SEQ ID NO: 1) of Salmonella phage CP-p-S-24008 involved in the present invention is as follows:

[0219]

[0220] Since the total genome sequence size of Salmonella phage CP-p-S-24008 shown in SEQ ID NO:1 is 43,155 bp, which results in the inability to submit the WIPOST.26 nucleotide sequence listing, in preparing the nucleotide sequence listing of the present invention, the nucleotide sequence shown in SEQ ID NO:1 in the specification is divided into 11 sequences for submission, and are named SEQ ID NO:1-1, SEQ ID NO:1-2, SEQ ID NO:1-3, SEQ ID NO:1-4, SEQ ID NO:1-5, SEQ ID NO:1-6, SEQ ID NO:1-7, SEQ ID NO:1-8, SEQ ID NO:1-9, SEQ ID NO:1-10, and SEQ ID NO:1-11 in sequence.

Claims

1. A Salmonella phage, characterized in that The bacteriophage has morphological characteristics of the long-tailed virus Siphoviridae family and has at least one of the following characteristics: a1) The whole genome size is 43-44 kbp; a2) does not contain lysogeny, drug resistance and virulence genes; a3) Temperature stability at 4-60℃; a4) It is acid-base stable at pH 3-11.

2. The bacteriophage according to claim 1, characterized in that The bacteriophage is CP-pS-24008, which belongs to the long-tailed virus family Siphoviridae. It was deposited in the China Center for Type Culture Collection on January 14, 2025, with the deposit number CCTCC M 2025135.

3. The bacteriophage according to claim 1, characterized in that The total genome size of the bacteriophage CP-pS-24008 is 43155 bp, and the nucleotide sequence is shown in SEQ ID NO:

1.

4. The bacteriophage according to claims 1-3, characterized in that The Salmonella phage also includes phage mutants, which have an average nucleotide identity of ≥96% on the genome with phage CP-pS-24008 (SEQ ID NO: 1).

5. The bacteriophage according to claim 4, characterized in that The RFLP DNA spectrum of the phage mutant is substantially identical to the RFLP DNA spectrum of phage CP-pS-24008.

6. A bacteriophage composition, characterized in that: The composition is selected from at least two of the Salmonella phages and mutants thereof shown in b1) to b8): b1) Salmonella phage CP-pS-24008, deposit number: CCTCC M 2025135; b2) Salmonella phage CP-pS-24002, deposit number: CCTCC M 2025130; b3) Salmonella phage CP-pS-24003, deposit number: CCTCC M 2025131; b4) Salmonella phage CP-pS-24006, deposit number: CCTCC M 2025133; b5) Salmonella phage CP-pS-24007, deposit number: CCTCC M 2025134; b6) Salmonella phage CP-pS-24009, deposit number: CCTCC M 2025136; b7) Salmonella phage CP-pS-24010, deposit number CCTCC M 2025137; b8) Salmonella phage CP-pS-24012, with the deposit number of CCTCC M 2025132.

7. The bacteriophage composition according to claim 6, characterized in that The composition is selected from a combination of the Salmonella phage CP-pS-24008 and mutants thereof according to any one of claims 1 to 5 and at least one of the Salmonella phages and mutants thereof shown in b2) to b8).

8. A biological agent, characterized in that: The biological preparation contains an effective dose of the bacteriophage according to any one of claims 1 to 5 or the bacteriophage composition according to any one of claims 6 to 7.

9. The biological agent according to claim 8, characterized in that The biological preparation also includes pharmaceutically acceptable excipients well known to those skilled in the art to form the preparation.

10. The biological agent according to claim 8, characterized in that The biological preparations include pharmaceutical preparations, disinfectants, cleaning agents, preservatives, food preservatives, feed additives or drinking water additives.

11. Use of the bacteriophage according to any one of claims 1 to 5, the bacteriophage composition according to any one of claims 6 to 7, or the biological agent according to any one of claims 8 to 10 in at least one of the following: c1) Use in the preparation of a medicament for preventing and / or treating bacterial infection caused by Salmonella infection; c2) Use in the preparation of antibacterial products for preventing and controlling Salmonella; c3) Application in the preparation of feed additives.

12. A method for preventing, alleviating and / or treating diseases caused by Salmonella infection, characterized in that: The method comprises administering to the subject an effective dose of the bacteriophage of any one of claims 1 to 5, the bacteriophage composition of any one of claims 6 to 7, or the biological agent of any one of claims 8 to 10.

13. A method for disinfecting an environment, surface and / or equipment to reduce the Salmonella load, characterized in that: The method comprises contacting an effective amount of the bacteriophage of any one of claims 1 to 5, the bacteriophage composition of any one of claims 6 to 7, or the biological agent of any one of claims 8 to 10 with the environment, surface and / or equipment.

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