A strain of Salmonella phage vB-SenS-JZ291 and its application

Abstract

The invention discloses a strain of Salmonella phage vB-SenS-JZ291 and its application, belonging to the field of biotechnology. The Salmonella phage vB-SenS-JZ291 can lyse different serotypes of Salmonella such as Typhimurium, Typhimurium 1,4,[5],12:i:-unidirectional variant, Enteritis, Pullorum, Thompson and Mbandaka, and the lysis rates of Enteritis serotype, Typhimurium 1,4,[5],12:i:-unidirectional variant and Typhimurium serotype Salmonella are 94.7%, 96.2% and 88.6% respectively. The phage can effectively remove the content of mixed contamination of different serotypes of drug-resistant Salmonella in multiple foods within 12 hours under the conditions of 4°C and 25°C. The invention also provides an effective combination of a phage and a preservative, and the removal rate of mixed contamination of multiple drug-resistant Salmonella in food reaches 96.8%.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a Salmonella phage vB-SenS-JZ291 and application thereof. Background Art

[0002] Salmonella, a genus of Gram-negative bacteria in the family Enterobacteriaceae, is a major cause of foodborne illness worldwide, posing a serious challenge to public health. This pathogen, which includes multiple serotypes within its main pathogen, Salmonella enterica, has been of great concern due to its ability to induce a range of human infections, ranging from self-limited gastroenteritis to severe systemic disease. Salmonella includes two species: Salmonella bongori and Salmonella Enteritidis, the latter being the main pathogen causing human infections. Within S. Enteritidis, more than 2,500 serotypes have been identified, each with distinct characteristics that are often associated with a specific host or environment. This diversity complicates the epidemiology of Salmonella.

[0003] The pathogenesis of Salmonella is a complex interaction between bacterial virulence factors and the complex immune response of the host. The initial stages include adhesion and invasion of intestinal epithelial cells, which are promoted by various virulence factors including riming membranes and adhesins. Once inside the host cells, Salmonella adopts clever strategies to evade immune detection and survive inside macrophages, a key step in establishing systemic infection. Modulation of host cell signaling pathways further highlights the adaptability and resilience of Salmonella in the face of immune defenses. The consequences of Salmonella infection vary, ranging from mild gastroenteritis to severe systemic diseases such as typhoid fever. Gastroenteritis, characterized by symptoms such as diarrhea, abdominal cramps and fever, is usually self-limited but can be more severe in susceptible populations such as the elderly and immunocompromised. In contrast, systemic infections are usually caused by specific serotypes (such as Salmonella Typhi and Salmonella Paratyphi) and can be life-threatening if not treated promptly. According to the World Health Organization (WHO), foodborne diseases, led by Salmonella, cause approximately 600 million illnesses and 420,000 deaths worldwide each year.

[0004] Salmonella is sensitive to heat sources. Traditional methods often use heat treatment to kill Salmonella, but food that has been heat-sterilized will cause changes in its quality and sensory perception. Therefore, domestic and foreign countries have begun to study new sterilization technologies to replace traditional heat sterilization methods. However, each method has its scope of application. For example, ultrasonic sterilization technology is easily affected by other substances; although low-temperature plasma sterilization technology is simple to operate, the process flow is very complicated and difficult to achieve under general conditions; although chemical methods have good sterilization effects, they are prone to produce drug residues; although ultraviolet sterilization takes a short time, it is difficult to achieve complete sterilization and can only cover the surface of food.

[0005] Phage is a virus that uses bacteria as a host. It has strict host specificity and can kill host cells without developing drug resistance. It has the advantages of exponential proliferation, wide distribution, and simple structure. Salmonella phage uses a variety of mechanisms to fight bacterial hosts. It can attach to specific receptors on the surface of bacteria, inject its genetic material, replicate in the host, and eventually cause cell lysis and release new phage particles. Phages not only have good bactericidal effects, but also have excellent biosafety, with no side effects or only very mild side effects on animals and humans. Phages have made great progress in the prevention and control of foodborne pathogenic bacteria infections, controlling the spread of pathogens, and treating drug-resistant bacterial infections. In the case of serious multidrug resistance, Salmonella, as an important zoonotic and foodborne pathogen, urgently needs to adopt more efficient methods to prevent and control Salmonella infections. Therefore, it is of great significance to explore the disinfection of food by bacteriophages. Summary of the invention

[0006] The purpose of the present invention is to provide a Salmonella phage vB-SenS-JZ291 and its application to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a Salmonella phage (Salmonella sp. phage) vB-SenS-JZ291, wherein the Salmonella phage vB-SenS-JZ291 was deposited in the China Center for Type Culture Collection on April 29, 2024, with a deposit address of Wuhan University, Wuhan, China, and a deposit number of CCTCC M 2024806.

[0009] The Salmonella phage vB-SenS-JZ291 of the present invention is relatively stable in that the titer decreases from the initial 9.72log PFU / mL to 9.64log PFU / mL and 9.67log PFU / mL respectively under the conditions of 40°C and 50°C within 60 minutes; the titer remains basically unchanged in the range of pH=4-12, and has good tolerance to changes in pH; and an MOI of 0.1 is an optimal multiplicity of infection for achieving optimal phage proliferation efficiency.

[0010] The present invention also provides a microbial preparation, comprising the Salmonella phage vB-SenS-JZ291.

[0011] The present invention also provides use of the Salmonella phage vB-SenS-JZ291 or the microbial preparation in preparing medicines for preventing and / or treating diseases caused by Salmonella.

[0012] Preferably, the Salmonella includes Enteritidis Salmonella, Salmonella Typhimurium and colistin-resistant mcr-1-positive 1,4,[5],12:i:-variant Salmonella Typhimurium.

[0013] The present invention also provides application of the Salmonella phage vB-SenS-JZ291 or the microbial preparation in preparing a food antibacterial agent.

[0014] The present invention also provides a bactericidal composition for preventing and controlling Salmonella, comprising an effective amount of the Salmonella phage vB-SenS-JZ291 or the microbial preparation.

[0015] The present invention also provides a cleaning agent or disinfectant, comprising the Salmonella phage vB-SenS-JZ291, the microbial preparation or the bactericidal composition.

[0016] The Salmonella phage vB-SenS-JZ291 provided by the present invention can be used as an effective ingredient of various products used for environmental disinfection, for example, disinfection and decontamination of water distribution systems, medical facilities, aquaculture facilities, public and private facilities or other environmental surfaces in the form of liquid immersion, spraying, and combined use with an aqueous carrier, which can effectively control the growth and activity of target bacteria; the liquid immersion and spraying forms include but are not limited to detergents, disinfectants, detergents, etc.; the aqueous carrier includes but is not limited to phosphate buffer, LB culture medium, chlorine-free water, etc.

[0017] The present invention also provides a food disinfection method, which uses the Salmonella phage vB-SenS-JZ291, the microbial preparation, the bactericidal composition or the cleaning agent or disinfectant to disinfect food.

[0018] Preferably, the food comprises eggs, cream and ham.

[0019] Preferably, the disinfection method further comprises applying the Salmonella phage vB-SenS-JZ291, the microbial preparation, the bactericidal composition or the cleaning agent or disinfectant in combination with a preservative to disinfect multiple drug-resistant Salmonella in food.

[0020] The present invention also provides the use of the Salmonella phage vB-SenS-JZ291 or the microbial preparation in the preparation of a bacterial agent for inhibiting enteritidis Salmonella, typhimurium and colistin-resistant mcr-1-positive typhimurium 1,4,[5],12:i:-variant Salmonella.

[0021] Preferably, the Salmonella includes Enteritidis Salmonella, Salmonella Typhimurium and colistin-resistant mcr-1-positive 1,4,[5],12:i:-variant Salmonella Typhimurium.

[0022] Based on the above technical solution, the present invention has the following technical effects:

[0023] The present invention isolates a strain of Salmonella phage vB-SenS-JZ291 from a fecal sample of a chicken farm in Shandong Province, which can lyse different serotypes of Salmonella such as Typhimurium, Typhimurium 1,4,[5],12:i:-unidirectional variant, Enteritis, Pullorum, Thompson and Mbandaka, and the lysis rates of Enteritis serotype, Typhimurium 1,4,[5],12:i:-unidirectional variant and Typhimurium serotype are 94.7%, 96.2% and 88.6% respectively. The present invention has stronger lysis performance, has good advantages in treating Salmonella infection diseases, has high titer, good prevention and treatment effect, short incubation period, high burst volume, strong tolerance to temperature and acid-base, and no virulence gene and drug resistance gene. The present invention also provides a method for using bacteriophages on a variety of foods. Under the conditions of 4°C and 25°C, the bacteriophages can effectively remove the content of mixed contamination of different serotypes of drug-resistant Salmonella in a variety of foods within 12 hours, and can significantly reduce the number of Salmonella within 2-6 hours. The present invention also provides an effective combination of bacteriophages and preservatives, and the removal rate of mixed contamination of multiple drug-resistant Salmonella in food reaches 96.8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is an observation diagram of the lysis spectrum of the bacteriophage vB-SenS-JZ291 of the present invention;

[0026] Figure 2 This is an electron microscope morphological observation diagram of the bacteriophage vB-SenS-JZ291 of the present invention;

[0027] Figure 3 is a multiplicity of infection diagram of bacteriophage vB-SenS-JZ291 of the present invention;

[0028] Figure 4 is a one-step growth curve diagram of the bacteriophage vB-SenS-JZ291 of the present invention;

[0029] Figure 5is an acid-base tolerance curve of the bacteriophage vB-SenS-JZ291 of the present invention;

[0030] Figure 6 is a thermal stability curve of the bacteriophage vB-SenS-JZ291 of the present invention;

[0031] Figure 7 The results of the egg liquid disinfection test of the bacteriophage vB-SenS-JZ291 of the present invention at 25°C;

[0032] Figure 8 The results of the egg liquid disinfection test of the bacteriophage vB-SenS-JZ291 of the present invention at 4°C;

[0033] Fig. 9 This is a graph showing the colony count results of bacteriophage vB-SenS-JZ291 of the present invention after disinfection of egg liquid at 4°C;

[0034] Fig.10 The results of the animal cream disinfection test of the bacteriophage vB-SenS-JZ291 of the present invention at 4°C;

[0035] Fig.11 The results of the animal cream disinfection test of the bacteriophage vB-SenS-JZ291 of the present invention at 25°C;

[0036] Fig.12 The results of the instant ham disinfection test of the bacteriophage vB-SenS-JZ291 of the present invention at 25°C;

[0037] Fig.13 This is a graph showing the colony count results of the instant ham disinfection test of the bacteriophage vB-SenS-JZ291 of the present invention at 25°C;

[0038] Fig.14 The results of the instant ham disinfection test of the bacteriophage vB-SenS-JZ291 of the present invention at 4°C;

[0039] Fig.15 The results of the test on the disinfection effect of Salmonella phage combined with preservatives on ready-to-eat ham. Group A is the phage treatment group; Group B is the sodium nitrite treatment group; Group C is the phage and sodium nitrite combination group;

[0040] Fig.16 These are the test results for the disinfection effect of Salmonella phage combined with preservatives on ready-to-eat ham. Group A is the phage treatment group; Group B is the sodium nitrite treatment group; and Group C is the phage and sodium nitrite combination group. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0047] Example 1

[0048] Isolation and identification of Salmonella

[0049] Sample collection: Use disposable sterile sampling cotton swabs to collect cloacal swabs from healthy broiler chickens at a broiler farm in Qingdao, place them in a physiological saline sampling tube, break and pinch part of the cotton swab, discard it, and cover the tube. Place it in a foam box with a refrigeration function and transport it to the laboratory for processing as soon as possible.

[0050] Sample pretreatment and enrichment: 10 μL of the saline suspension was inoculated into 1 mL of buffered peptone broth (BPW) and cultured at 37°C for 18-24 hours; 20 μL of the buffered peptone broth suspension was inoculated into a centrifuge tube containing 1 mL of selenite cystine (SC) liquid medium and cultured at 37°C for 18-24 hours;

[0051] Isolation and purification: Streak one loop of broth culture onto the second generation chromogenic medium plate of Salmonella at 37°C for 18-24 hours. Use an inoculation loop to streak the bacterial solution onto XLT-4 agar medium, culture at 37°C overnight, and observe the morphological characteristics of the colonies for preliminary screening.

[0052] Select a single colony with a black round shape, smooth surface, and neat edges, dip it with an inoculation loop, streak it on the Salmonella chromogenic medium (second generation), and observe the colony morphology after culturing at 37°C for 18-24 hours. Select a single colony on the Salmonella chromogenic medium (second generation) whose colony morphology meets the standards of the Salmonella chromogenic medium (second generation) instruction manual, streak it again on the Salmonella chromogenic medium (second generation), and observe the colony morphology after culturing at 37°C for 18-24 hours.

[0053] Preservation of strains: Mix 600 μL of bacterial solution and 600 μL of 60% glycerol broth in a 1.5 mL centrifuge tube and store in a -20°C refrigerator.

[0054] The strain was identified as Salmonella by 16S rRNA and invA specific molecular identification and named as JZ291. It was identified as Enteritidis serotype by PCR and the above-mentioned Salmonella JZ291 was used as the host strain.

[0055] Example 2

[0056] Isolation, Identification and Characteristics of Salmonella Phage

[0057] (1) Screening of bacteriophage vB-SenS-JZ291

[0058] The samples used in the experiment of the present invention are fecal samples collected from a chicken farm in Shandong Province in 2022. The samples were resuspended in SM buffer, centrifuged at 10000r / min for 10min at 4°C, filtered using a 0.22μm water filter membrane, and the filtrate was placed at 4°C for use.

[0059] Take 10 mL of each sample filtrate and mix it with an equal volume of 2×LB broth, inoculate all fresh host bacteria at an inoculation rate of 1% of the total volume, mix well and place in an incubator for overnight culture at 37°C.

[0060] The culture solution was centrifuged at 4°C, 10,000 rpm / min for 10 min, and the supernatant was filtered through a 0.22 μm filter for sterilization. The filtrate was taken and the above steps were repeated for three times to obtain the phage enrichment solution.

[0061] Take 200μL of host bacteria (Salmonella JZ291) and inoculate it into 10mL LB broth and culture it to the logarithmic phase. Take 100μL of logarithmic phase bacterial solution, add 5mL LB semi-solid (about 50℃), mix well and quickly pour it onto the marked LB agar. After the agar solidifies, take 3μL of each phage enrichment solution and spot it on the agar surface with the host bacteria added; after spotting, place the agar plate in a 37℃ constant temperature incubator for overnight culture to obtain a plate with phage plaques.

[0062] (2) Phage purification

[0063] Use an inoculation loop to pick up a single clear and transparent plaque and place it in 1 mL of SM buffer. Let it stand overnight at 4°C until the phage particles fall off the agar. After filtering through a 0.22 μm filter, take 100 μL of the filtrate and dilute it tenfold. Take 100 μL of each dilution gradient and mix it with the corresponding logarithmic phase host bacterial solution. Incubate at room temperature for 10 minutes to allow the phage to complete adsorption, and then culture the plaques using the double-layer plate method.

[0064] The above double-layer plate method is as follows: add 5 mL of LB semi-solid to the mixture of phage and host bacteria, mix well and quickly pour it onto the marked LB agar, wait for the agar to solidify and culture it at 37°C overnight. Repeat the purification at least five times until plaques of consistent morphology, size and transparency appear.

[0065] Through the above purification process, phage vB-SenS-JZ291 (see Figure 1 ).

[0066] (3) Determination of phage lysis rate

[0067] The lysis rate of bacteriophage vB-SenS-JZ291 against 123 strains of Salmonella was measured. 100 μL of logarithmic phase bacterial solution was mixed with 5 mL of LB semi-solid (about 50°C) and quickly poured onto LB agar. After solidification, it was put away and the point where the phage solution was added was marked.

[0068] After the droplets dried, they were placed in a 37°C constant temperature incubator overnight. The next day, the lysis of each bacterial agar plate mark (whether there were clear plaques) was recorded and statistically analyzed. The lysis rate of phages is shown in Table 1.

[0069] Table 1 Phage lysis rate of different serotypes of Salmonella (%)

[0070]

[0071] The results showed that the phage was able to exhibit high lytic activity against different serotypes of Salmonella. The phage vB-SenS-JZ291 could lyse a variety of different serotypes of Salmonella, including Typhimurium, Typhimurium 1,4,[5],12:i:-unidirectional variants, Enteritidis, Pullorum, Thompson and Mbandaka, with an overall lysis rate of up to 89.4%. The lysis rates of vB-SenS-JZ291 against Enteritidis, Typhimurium 1,4,[5],12:i:-unidirectional variants and Typhimurium serotypes were as high as 94.7%, 96.2% and 88.6%, respectively, including cephalosporin-resistant and ESBL-producing Salmonella Typhimurium, colistin-resistant and mcr-1 gene-carrying Salmonella Typhimurium 1,4,[5],12:i:-unidirectional variants and resistant Enteritidis Salmonella.

[0072] (4) Electron microscopic observation of bacteriophage morphology

[0073] Add 20 μL of sample onto a 200-mesh copper grid used for electron microscopy, and allow to adsorb for 10 min. Cover the grid with 20 μL of 2% phosphotungstic acid negative staining solution, and allow the staining time to last for 5 min. Wait for the grid to dry at room temperature, and then use a 40 kV transmission electron microscope to observe the bacteriophage.

[0074] On solid culture medium, transparent plaques can be formed with uniform size and clear and regular edges. The phage has a basic structure including a head and a tail. The head is a regular icosahedron of about 60nm, and the tail is a 10nm*170nm structure (see Figure 2 ), according to morphological classification, it belongs to the order Caudovirales, family Longicaudaviridae.

[0075] (5) Determination of the optimal phage infection multiplicity (MOI)

[0076] The concentration of each host strain was adjusted to 10 8 CFU·mL -1 , set 8 infection multiplicity ratios to 10, 1, 0.1, 0.01, and 0.001, and adjust the phage titer to 10 according to the infection multiplicity ratio 2 -10 9 PFU·mL -1 . Take 100 μL of each dilution gradient phage and 100 μL of the corresponding host bacteria and mix them evenly. Make the volume of LB broth to 1 mL. Place it in a constant temperature shaker at 37°C, 180 rpm / min and culture for 4 hours. Then determine the phage titer. The infection multiplicity ratio with the highest titer is the optimal infection multiplicity (MOI). Set up 3 parallel groups for each group of infection multiplicity and take the average value.

[0077] Under different infection multiplicity conditions, the number of progeny phages produced was very different. The results showed that when the MOI was 0.1, the phage titer was the highest, which was 1.86×10 10 PFU / mL; on the contrary, the phage titers at MOI of 10 and 0.001 were significantly lower, about 2.42×10 6 and 5.43×10 8 PFU / mL, indicating that too high or too low MOI is not conducive to the effective replication of phage. When MOI is 1 and 0.01, the phage titer is about 3.5×10 7 and 3.85×10 9 PFU / mL, although these values ​​are higher than those at MOI 10 and 0.001, they are still lower than the highest titer at MOI 0.1. MOI 0.1 is the optimal infection multiplicity for achieving optimal phage proliferation efficiency (see Figure 3 ).

[0078] (6) Determination of one-step growth curve of phage

[0079] After the phage and the bacterial solution cultured to the logarithmic growth phase were mixed evenly according to the optimal MOI, they were incubated at room temperature for 5 minutes, 200 μL was aspirated and centrifuged at 12000 r / min for 1 minute, and the supernatant was discarded to obtain a precipitate; the above precipitate was resuspended into a suspension using 5 mL LB broth, and then cultured at 37 ° C and 160 r / min for 3 hours, and the timing was set at the same time, and 200 μL was sampled each time, and samples were taken every 10 minutes during 0-1h, every 20 minutes during 1-2h, and every 30 minutes during 2-3h. The samples were centrifuged at 12000 r / min for 1 minute, and the supernatant was diluted 10 times. The titer of the phage was determined using the double-layer plate method, and the data was recorded and a one-step growth curve was drawn.

[0080] The one-step growth curve results of phage vB-SENS-JZ291 showed that the phage titer remained basically unchanged within 0 to 10 minutes of the initial infection, and it was in the latent period. The outbreak period lasted about 75 minutes, during which the number of phages increased rapidly. From 90 minutes on, the growth rate slowed down until about 120 minutes, when the phage titer no longer increased significantly and entered a stable period. The lysis amount of vB-SENS-JZ291 was 40.6 PFU / cell. The calculation formula for the phage lysis amount is phage lysis amount (PFU·cell -1 ) = phage titer at the end of lysis / host bacteria count at the beginning of infection. The one-step growth curve of phage is shown in Figure 4 shown.

[0081] (7) Phage pH stability determination

[0082] Use hydrochloric acid (1 mol / L) and sodium hydroxide (1 mol / L) to prepare solutions with different pH values ​​(pH = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) on the basis of PBS buffer. Use a pipette to transfer 100 μL of phage solution and add it to 900 μL of solution with different pH values. After incubation in a 37°C constant temperature incubator for 2 h, samples were taken and the titer was measured by the double-layer plate method after multiple dilution. The samples were incubated in a 37°C constant temperature incubator for 8 h and the phage titer was determined. The experiment was repeated 3 times and the data were recorded to make an acid-base tolerance curve.

[0083] The test results show that the phage vB-SENS-JZ291 is completely inactivated in extremely acidic (pH 2 and 3) and alkaline (pH 14) environments, and its titer cannot be measured. When the pH value rises to 13, the titer of the phage decreases by 2.05Log PFU / cell to only 6.80Log PFU / cell. The stability of the phage is significantly reduced under high alkaline conditions. In the range of pH = 4 to 12, its titer remains basically unchanged, indicating that the phage vB-SENS-JZ291 has good tolerance to changes in pH (see Figure 5 ).

[0084] (8) Phage thermal stability determination

[0085] Determine the phage PFU in advance, take 500 μL of phage proliferation liquid and incubate it in a water bath at 40℃, 50℃, 60℃, 70℃, and 80℃ for 1 hour, take 100 μL of samples at 20min, 40min, and 60min, and measure the titer using the double-layer plate method after multiple dilution. Repeat the experiment 3 times, record the data and make a thermal stability curve.

[0086] The test results showed that the titer of phage vB-SENS-JZ291 dropped significantly after only 20 minutes at 80°C, with the detected amount being only 1.69log PFU / mL, and the phage was completely inactivated after 40 minutes; at 70°C, the titer dropped significantly by 2.75log PFU / mL to 6.9731log PFU / mL after only 20 minutes, and it completely lost its activity after 60 minutes. At 40°C and 50°C, the titer dropped from the initial 9.72log PFU / mL to 9.64log PFU / mL and 9.67log PFU / mL respectively within 60 minutes, and was relatively stable (see Figure 6 ).

[0087] (9) Phage genome type determination

[0088] The genome of bacteriophage vB-SenS-SEC2 was extracted using a phage genome extraction kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0).

[0089] Take 10 μL of phage genome and digest it with nuclease DNA seⅠ, including 10 μL of phage genome, 3 μL of DEPC water, 5 μL of 10×DNA seⅠ buffer and 2 μL of DNA seⅠ. After 37℃ water bath for 30 minutes, add 2.5 μL of 0.5mEDTA and bathe at 80℃ for 2 minutes for use.

[0090] Take 10 μL of the phage genome and digest it with nuclease RNA seA, including 10 μL of the phage genome and 1 μL of RNA seA (10-fold dilution in buffer), incubate in a 37° C. water bath for 1 hour, and set aside.

[0091] Take 10 μL of the phage genome and digest it with the nuclease Mang Bean Nuclease, which includes 10 μL of the phage genome, 5 μL of buffer and 3 μL of Mang Bean Nuclease (10-fold dilution with sterile enzyme-free water), and incubate in a 37°C water bath for 10 min for use.

[0092] 0.8g agarose was mixed with 100mL TAE buffer and heated to prepare electrophoresis gel blocks, and SYBR GreenⅠ (1000×) working solution was prepared, including 1μL SYBR GreenⅠ, 1×TAE (or sterile water) and 1mL 6×loading Buffer. 4μL SYBR GreenⅠ working solution was mixed with 10μL nuclease digested phage genome and then allowed to stand for 5min. The sample was loaded in 1×TAE. The control group and DL10000 Marker were also mixed with SYBR GreenⅠ working solution and allowed to stand for 5min. The gel was run at 90V for 40min. The electrophoresis results were observed by a nucleic acid gel imager to determine that the nucleic acid type of the phage genome was a circular DNA double strand.

[0093] (11) Phage genome sequencing

[0094] Bacteriophage vB-SENS-JZ291 was sent to Meiji Gene Technology Co., Ltd. for next-generation sequencing. Soapnuke software (v2.0.5) was used to evaluate the quality of sequencing data and remove low-quality data. BWA software (v0.7.17) was used to align the clean reads with the Salmonella genome to remove the sequences of the host bacteria. Megahit software (v1.1.2) was used to assemble the high-quality reads of the phage into contig sequences. MetaGeneMark was used to predict the gene sequence of the phage.

[0095] The whole genome of the phage was uploaded to the online websites Resfinder 3.1 and VirulenceFinder 2.0 to analyze the presence of resistance genes and virulence genes; the Blast tool of the NCBI online website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was used to find the whole genome sequences of the phage with high similarity. After comparison, the 30 sequences with the highest similarity were selected, and the whole genome phylogenetic tree was constructed using MEGA, and visualized using TVBOT (https: / / www.chiplot.online / tvbot.html); the web page (https: / / bat.infspire.org / circoletto / ) was used to align and draw homology analysis diagrams; the web page (https: / / phastest.ca / submissions / new) was used to draw the whole genome circle diagram.

[0096] The whole genome annotation results of bacteriophage vB-SENS-JZ291 show that ( Figure 7-8 ), does not carry any virulence genes or drug resistance genes. There are a total of 62 coding sequences, of which 17 coding sequences are located on the positive strand and 45 coding sequences are located on the negative strand. Among them, a total of 25 coding sequences are predicted to be coding sequences of functional proteins, encoding tail protein (Tailprotein), phage-like protein (Phage-like protein), phage fiber protein (Fiberprotein), DNA helicase (DNA helicase), endonuclease (Endonuclease), phage pore protein (Holin), terminase (Terminase), channel protein (Portal protein), head protein (Headprotein), neck protein (Neckprotein). The remaining 35 coding sequences were identified as encoding hypothetical proteins (hypotheticalprotein) of unknown function, and their functions need further research to determine. According to the complete genome sequence of the phage, genome circle maps were made respectively, and the outer circle is the annotation result of the functional protein ( Figure 8). Whole genome analysis of bacteriophage vB-SENS-JZ291 revealed 25 sequences predicted to encode functional proteins. These proteins can be divided into four main categories: phage structural proteins, host disassembly-related proteins, DNA packaging-related proteins, and DNA replication and regulation-related proteins. These categories of proteins play a vital role in the structural construction and life cycle maintenance of phages. The interactions between them synergistically promote the adsorption and lysis of host bacteria by phages, thereby achieving their own proliferation and spread. The structural proteins of phages include tail proteins encoded by CDS1, CDS2, CDS3, CDS5, and CDS50, phage-like proteins encoded by CDS4, CDS13, CDS17, CDS26, CDS37, CDS46, CDS57, and CDS63, phage fiber proteins encoded by CDS53, head proteins encoded by CDS7, CDS10, CDS11, CDS15, and CDS16, and neck proteins encoded by CDS6. The first step for phages to infect bacteria is to adsorb to the host bacteria. This process requires the tail proteins and fiber proteins of phages to recognize and adsorb to specific receptors on the surface of bacteria. Different types of phages have different tail proteins and fiber proteins, which is one of the key factors that determine the range of phage phage spectrum. Therefore, modifying and transforming the protein coding sequence of the phage tail may expand its scope of bacterial lysis and increase the applicability of the phage. In addition, there is a multi-protein structure in the phage tail region, which mediates the key processes of phage infection of bacteria, such as phage adsorption, cell wall digestion, perforation and genome injection. The host lysis-related protein part includes the phage pore protein (Holin) encoded by CDS27. Porin is one of the characteristic proteins of lytic phages. During the process of phage infection of host bacteria, pore protein will form holes on the cell membrane of host bacteria, open channels for lysozyme (Endolysin) to enter the host bacteria, and provide important help for phage lysis of host bacteria. The DNA packaging-related protein part includes the portal protein encoded by CDS18 and the terminase encoded by CDS19 and CDS20. The terminase is the core component of the DNA packaging mechanism. It can accurately recognize and cut specific DNA sequences, bind and catalyze the accurate packaging of phage DNA into the phage head. The channel protein is part of the phage head structure and provides a dedicated channel for the entry and exit of phage DNA. This unique structural design allows the phage to efficiently inject genetic information into the host during its invasion of host bacteria, thereby initiating the infection process.The DNA replication and regulation related protein part includes the endonuclease encoded by CDS47. Bacteriophage endonuclease is a nucleic acid modification enzyme with high specificity. It can recognize specific sites and cut specific sequences. It plays a key role in the entire life cycle of bacteriophages and is often purified and expressed as a DNA modification tool in molecular biology.

[0097] In summary, the phage is a long-tailed phage with a double-stranded circular DNA, good biological properties, no drug resistance or virulence genes, and has proteins such as phage lysin. It can form transparent plaques on solid culture media with uniform shape and size and clear and regular edges.

[0098] The bacteriophage vB-SenS-JZ291 isolated by the present invention was deposited in the China Center for Type Culture Collection on April 29, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC M 2024806.

[0099] Example 3

[0100] Determination of the disinfection effect of Salmonella phage in food

[0101] (1) Determination of the disinfection effect of Salmonella phage in fresh eggs

[0102] A. Sample processing: Soak the sterile edible eggs purchased from the supermarket in a 75% alcohol solution for 30 minutes, and then irradiate them with ultraviolet light on a clean bench for 20 minutes. Mix the egg yolk and egg white in a conical flask according to the rule of three eggs per group, evenly spread them on the second-generation Salmonella chromogenic medium, and incubate them in a 37°C constant temperature incubator for 22-24 hours to verify the sterility of the eggs. The test was repeated 3 times. After that, 9.8 mL of the mixture of three eggs in each group was taken and placed in a sterile homogenization bag as an egg liquid sample group, and a total of 12 groups were prepared. Six groups were set up for each temperature condition, of which three groups were used as experimental groups, two groups were used as positive control groups, and one group was used as a blank control group.

[0103] B. Strain selection: The phage vB-SENS-JZ291 was selected for food disinfection testing. The three strains tested included Enteritidis Salmonella JZ291, cephalosporin-resistant and ESBL-producing Salmonella Typhimurium LY14, and colistin-resistant mcr-1-positive Salmonella Typhimurium 1,4,[5],12:i:- variant SD21L1.

[0104] C. Experimental preparation: Salmonella enterica serovar Enteritidis, Salmonella typhimurium, and colistin-resistant mcr-1-positive Salmonella typhimurium 1,4,[5],12:i:-variants were cultured to the logarithmic growth phase, and then graded diluted with saline. The concentration of the bacterial solution was adjusted to 1×10 by plate count method. 6 CFU / mL, and the three Salmonella were mixed in equal proportions. The initial titer of the phage was determined by the double-layer plate method, and the titer of the phage was adjusted to 1×10 by gradient dilution of physiological saline. 10 PFU / mL.

[0105] D. Test method: 100 μL of adjusted bacterial solution was added to each egg sample group, and then the six groups of samples were placed in an incubator at 25°C and a refrigerator at 4°C for 20 minutes to allow the bacterial solution to adapt to the growth temperature. After 20 minutes, 100 μL of pre-adjusted vB-SENS-JZ291 phage solution was added to the experimental group, and physiological saline was added to the positive control group. After sufficient mixing, they were placed in a 25°C incubator and a 4°C refrigerator again. Samples were taken at four time points: 2 hours, 4 hours, 6 hours, and 12 hours. The number of Salmonella in the samples was detected by the plate count method to evaluate the antibacterial effect of the phage.

[0106] (2) Determination of the disinfection effect of Salmonella phage in animal cream

[0107] A. Sample processing: Soak the whole box of unopened animal cream purchased from the supermarket in 75% alcohol for 30 minutes, take 200μL of animal cream stock solution and evenly spread it on the second-generation Salmonella chromogenic medium plate, and incubate it in a 37℃ constant temperature incubator for 22-24 hours to verify the sterility of the sample. The test was repeated 3 times. After that, use a pipette to draw 9.8mL of animal cream in small amounts and multiple times and place it in a 15mL sterile centrifuge tube. Under each temperature condition, 6 groups of experiments were set up, including 3 experimental groups, 2 positive control groups and 1 blank control group; a total of 12 groups of centrifuge tubes were prepared.

[0108] B. Strain selection: The phage vB-SENS-JZ291 was selected for food disinfection testing. The three strains tested included Enteritidis Salmonella JZ291, cephalosporin-resistant and ESBL-producing Salmonella Typhimurium LY14, and colistin-resistant mcr-1-positive Salmonella Typhimurium 1,4,[5],12:i:- variant SD21L1.

[0109] C. Experimental preparation: Salmonella enterica serovar Enteritidis, Salmonella typhimurium, and colistin-resistant mcr-1-positive Salmonella typhimurium 1,4,[5],12:i:-variants were cultured to the logarithmic growth phase, and gradient dilutions were performed using saline. The concentration of the bacterial solution was adjusted to 1×10 by plate count method. 6CFU / mL, and the three Salmonella were mixed in equal proportions. The initial titer of the phage was determined by the double-layer plate method, and the titer of the phage was adjusted to 1×10 by gradient dilution of physiological saline. 10 PFU / mL.

[0110] D. Test method: Add 100 μL of bacterial solution adjusted to the corresponding concentration to each animal cream sample, and then place the 6 groups of samples in an incubator at 25°C and a refrigerator at 4°C for 20 minutes to allow the bacterial solution to adapt to the growth temperature. After 20 minutes, add 100 μL of pre-adjusted vB-SENS-JZ291 phage solution to the experimental group, and add physiological saline to the blank control group. Mix well and place them in a 25°C incubator and a 4°C refrigerator again. Take samples at 2 hours, 4 hours, 6 hours, and 12 hours, and use the plate count method to detect the number of Salmonella in the samples to evaluate the antibacterial effect of the phage.

[0111] (3) Determination of the disinfection effect of Salmonella phage in ready-to-eat ham

[0112] A. Sample processing: The purchased bagged ham is first soaked in 75% alcohol for 30 minutes, then irradiated with ultraviolet light on both sides for 30 minutes each, and cut into square ham slices with a side length of 1 cm and a thickness of 0.5 cm using a sterilized knife, and placed in a sterile culture dish. Three samples are placed in each culture dish. In order to ensure the sterility of the sample, a piece of processed ham is randomly selected, 1 mL of physiological saline is added to fully grind and homogenize, 100 μL of the sample is evenly spread on the second-generation Salmonella chromogenic medium using a coating rod, and cultured in a 37°C constant temperature incubator for 22-24 hours. The sample is tested for sterility by bacterial counting method, and this process is repeated 3 times.

[0113] B. Strain selection: The phage vB-SENS-JZ291 was selected for food disinfection testing. The three strains tested included Enteritidis Salmonella JZ291, cephalosporin-resistant and ESBL-producing Salmonella Typhimurium LY14, and colistin-resistant mcr-1-positive Salmonella Typhimurium 1,4,[5],12:i:- variant SD21L1.

[0114] C. Experimental preparation: Salmonella enterica serovar Enteritidis, Salmonella typhimurium, and colistin-resistant mcr-1-positive Salmonella typhimurium 1,4,[5],12:i:-variants were cultured to the logarithmic growth phase, and gradient dilutions were performed using saline. The concentration of the bacterial solution was adjusted to 1×10 by plate count method. 6 CFU / mL, and the three Salmonella were mixed in equal proportions. The initial titer of the phage was determined by the double-layer plate method, and the titer of the phage was adjusted to 1×10 by gradient dilution of physiological saline. 10 PFU / mL.

[0115] D. Test method: Place the sample flat in the center of a sterile culture dish, take 10 μL of 1×10 8 CFU / mL of the three bacterial strains were added to the surface of the ham sample, and the artificial contamination of Salmonella was 1×10 6 CFU / cm 2 The ham sample was placed on a clean bench for 20 minutes to allow the Salmonella mixture to fully absorb. Then 10 μL of 1×10 10 The phage solution with PFU / mL was covered on the surface of the ham, and then placed in a disposable sterile homogenizing bag after standing for 45 minutes. The phage solution of the blank control group was replaced with physiological saline buffer. The samples of the experimental group and the control group were treated at 4°C and 25°C for 2, 4, 6, and 12 hours, respectively. The samples were taken out at the corresponding time points, 1 mL of SM buffer was added to each group of samples, and they were ground and pulverized to make a homogenate in a disposable sealed homogenizing bag. The number of Salmonella in the ham was detected by the plate count method, thereby detecting the antibacterial effect of the phage.

[0116] Measurement results:

[0117] (1) Disinfection effect of Salmonella phage in eggs:

[0118] The bacteriophage VB-SENS-JZ291 has a bactericidal effect on Salmonella in egg liquid at 25℃ and 4℃ respectively. Figure 9-10 As shown, the bactericidal effect of VB-SENS-JZ291 at 4°C is better than that at 25°C. The specific results are analyzed as follows:

[0119] At 25°C, the live bacterial count of the control group (untreated phage) continued to increase from 0 to 12 hours. The initial live bacterial count was 6.556log CFU / mL, which quickly rose to 9.903log CFU / mL after 2 hours, and reached 11.047log CFU / mL at 12 hours. In contrast, the live bacterial count of the experimental group decreased during this period. After 12 hours of action of bacteriophage VB-SENS-JZ291, the live bacterial count of the experimental group decreased by 5.093, 5.752, 6.679 and 6.746logCFU / mL respectively compared with the control group, and the bactericidal efficiencies were 51.42%, 53.89%, 59.54% and 61.06% respectively. The results showed that ( Fig. 9 ), the ability of phage to eliminate Salmonella increased with time. Especially at the 6-hour and 12-hour detection points, the Salmonella in the phage-treated group was reduced by more than 6 log CFU / mL, and the sterilization rate reached 61.06%.

[0120] At 4°C, the live count of Salmonella in the control group without phage addition remained basically unchanged within 0-12 hours. The experimental groups treated with phage showed a good bactericidal effect, and the live count was significantly lower than that of the control group ( Fig.10 ). After 6 hours of phage action at 4°C, the bacterial concentration in the phage-treated group decreased by 3.124 log CFU / mL, and the sterilization rate reached 40.15%; when the action time was extended to 12 hours, the concentration in the phage-treated group dropped significantly to 0.564 log CFU / mL, and the presence of Salmonella was no longer detected in some parallel sample groups. The average number of viable bacteria decreased by 6.519 log CFU / mL, and the sterilization rate was as high as 92.04%, indicating that almost all Salmonella can be eliminated after a long period of phage action. From a sterilization rate of 4.22% at 2 hours to 92.04% at 12 hours, phages show great potential as a biocontrol method for food safety.

[0121] (2) Disinfection effect of Salmonella phage in animal cream:

[0122] Under the experimental conditions of 25℃ and 4℃, vB-SENS-JZ291 phage was tested for sterilization of Salmonella in animal cream. Fig.11 and Fig.12 The study found that vB-SENS-JZ291 had a better effect on eliminating Salmonella at 4°C than at 25°C.

[0123] Under 25℃, in the control group without vB-SENS-JZ291 treatment, the number of live bacteria of Salmonella showed a gradual upward trend from 2 hours to 12 hours, and the live bacteria count at 12 hours reached 11.279log CFU / mL. In the experimental group treated with vB-SENS-JZ291, the number of live bacteria showed a downward trend during the same period of time. In particular, after 6 to 12 hours of treatment, the difference in live bacteria count between the experimental group and the control group was 1.962log CFU / mL and 0.860logCFU / mL, respectively, and the corresponding bactericidal rates were 17.5% and 7.62%. This result indicates that the killing effect of vB-SENS-JZ291 on Salmonella may be limited within the first 4 hours of action.

[0124] At 4°C, the live counts of Salmonella in the control group without phage treatment remained almost unchanged over 12 hours, with values ​​recorded as 6.283 log CFU / mL, 6.097 log CFU / mL, 6.283 log CFU / mL, and 6.382 log CFU / mL, respectively. In contrast, the experimental group treated with vB-SENS-JZ291 showed a significant bactericidal effect, with the live count significantly lower than that of the control group. Specifically, from 0 to 12 hours, the decrease in bacterial concentration caused by phage treatment was 0.413 log CFU / mL, 1.390 log CFU / mL, 1.299 log CFU / mL, and 0.969 log CFU / mL, respectively, with bactericidal rates of 5.99%, 22.12%, 21.31%, and 15.19%, respectively. The phages effectively reduced the number of Salmonella by specifically recognizing and infecting host cells, resulting in bacterial lysis. Even though the reduction was reduced after 12 hours, the continued activity of the phages was still considerable.

[0125] (3) Disinfection effect of Salmonella phage in ready-to-eat ham:

[0126] The clearance effect of bacteriophage vB-SENS-JZ291 on Salmonella in ham at 25℃ and 4℃ was respectively Fig.13 and Fig.14 The analysis shows that the bactericidal performance of vB-SENS-JZ291 at 4°C exceeds that at 25°C. The following is a detailed analysis of the results:

[0127] At 25°C, the live count of Salmonella in the control group continued to increase from 2 hours to 12 hours, reaching 6.531log CFU / mL, 7.564log CFU / mL, 8.528log CFU / mL and 10.145log CFU / mL, respectively. In comparison, during the same period, the test group treated with vB-SENS-JZ291 showed a significant bactericidal effect, and its live count was much lower than that of the control group. Specifically, from 0 to 12 hours, the bacterial concentration after phage treatment decreased by 2.421log CFU / mL, 2.798log CFU / mL, 3.073log CFU / mL and 3.471log CFU / mL, and the corresponding bactericidal rates were 37.07%, 36.98%, 36.04% and 34.22%.

[0128] At 4°C, the live count of Salmonella in the control group without phage treatment remained almost unchanged throughout the observation period, with count values ​​of 6.847log CFU / mL, 6.721log CFU / mL, 6.785log CFU / mL and 6.909log CFU / mL, respectively. In contrast, the phage-treated test group showed excellent bactericidal ability, with live counts significantly lower than those in the control group. From 0 to 12 hours, the phage action reduced the bacterial concentration by 2.620log CFU / mL, 2.263log CFU / mL, 2.386log CFU / mL and 2.458log CFU / mL, respectively, and the bactericidal rates reached 38.2%, 33.7%, 35.2% and 35.6%, respectively. These results demonstrate that vB-SENS-JZ291 has a significant elimination effect on Salmonella in ham at 4°C, especially after 2 hours of treatment, when the bactericidal rate reached a maximum of 38.2%. This indicates that bacteriophages can quickly and effectively reduce the number of Salmonella, showing its potential for application in food safety.

[0129] Compared with animal cream, egg and ham samples showed stronger antibacterial effects, which may be due to the viscosity of animal cream and the phages in the milk suspension were captured by the interaction with protein particles through hydrophobic effects or electrostatic charges, which hindered the direct contact between phages and bacteria. In contrast, the fluidity of eggs is conducive to the dispersion of phages, while the smaller surface area of ​​ham increases the contact opportunities between phages and bacteria. Over time, the difference in viable counts between the phage-treated group and the control group continued to increase, which further confirmed the potential application of phages as biopreservatives in low-temperature stored foods and provided important data support for the development of phage-based food safety strategies.

[0130] Example 4

[0131] Determination of the disinfection effect of Salmonella phage combined with preservatives on ready-to-eat ham

[0132] A. Sample processing: Use a sterilized knife to cut the purchased bagged ham into square slices of 1 cm in length and 0.5 cm in thickness, and place them in a sterile culture dish. Soak in 75% alcohol for 10 minutes, then irradiate the front and back sides with a UV lamp for 10 minutes each, and place 3 samples in each culture dish. To ensure the sterility of the sample, randomly select a piece of processed ham, add 1 mL of saline to fully grind and homogenize, take 100 μL of the sample and evenly spread it on the XLT4 culture medium with a coating rod, and culture it in a 37°C constant temperature incubator for 22-24 hours. The sterility of the sample is tested by the bacterial counting method, and this process is repeated 3 times.

[0133] B. Strain selection: The bacteriophage vB-SENS-JZ291 was selected for food disinfection testing. The test strains included Enteritidis Salmonella JZ291, colistin-resistant mcr-1-positive typhimurium 1,4,[5],12:i:- variant Salmonella SD21L1.

[0134] C. Experimental preparation: Enteritidis Salmonella and colistin-resistant mcr-1-positive typhimurium 1,4,[5],12:i:-variant Salmonella were cultured to the logarithmic growth phase, and gradient dilutions were performed using saline. The concentration of the bacterial solution was adjusted to 1×10 by plate count method. 6 The initial titer of the phage was determined by the double-layer plate method, and the titer of the phage was adjusted to 1×10 by gradient dilution with saline. 10 PFU / mL. Prepare a 1.51 g / L sodium nitrite solution for later use.

[0135] D. Test method: Place the sample flat in the center of a sterile culture dish and drop 10 μL of 1×10 6 CFU / mL of the two strains were mixed in equal proportions to produce an artificial contamination level of 1×10 4 CFU / cm 2 The ham sample was placed on a clean bench for 30 minutes to allow the Salmonella mixture to fully adsorb on the ham surface. Then 10 μL of 1×10 10 The ham surface was covered with phage solution of 1×10 / mL, which was used as the phage treatment group; the ham to be dripped with the bacterial solution in another group was left to stand for 30 minutes, and then 10 μL of sodium nitrite solution was added, which was used as the sodium nitrite treatment group; the ham to be dripped with the bacterial solution in the last group was left to stand for 30 minutes, and then 10 μL of 1×10 10 PFU / mL of phage solution and 10μL of sodium nitrite solution were used as the combined treatment group; the phage solution covering the surface of the ham was replaced with saline as the blank control group. After standing for 45 minutes, the ham slices were transferred to a disposable sterile homogenizing bag, and 1mL of saline was added to grind and crush the ham slices to make a homogenate in a disposable sealed homogenizing bag. 100μL of the homogenate was spread on an XLT4 plate, cultured in a 37℃ incubator, and the results were statistically analyzed.

[0136] Test results: The number of colonies on the plate was counted. Fig.15 The colony count of the blank control group was 1.57×10 3 CFU / cm 2 The phage-treated group is represented as group A in the figure. The number of colonies on the ham was 1×10 2 CFU / cm 2The removal rate of mixed Salmonella reached 93.6%. The group treated with sodium nitrite is represented as group B in the figure. The number of colonies on the ham was 4.2×10 2 CFU / cm 2 The clearance rate of mixed Salmonella reached 73.2%. The combined treatment group is represented as group C in the figure. The number of colonies on the ham was 5×10 1 CFU / cm 2 The elimination rate of mixed Salmonella in this group reached 96.8%. The experimental results showed that the disinfection effect of the combination of bacteriophage and preservative on drug-resistant Salmonella was significantly better than that of the bacteriophage treatment group or the preservative treatment group (P<0.01)( Fig.16 ).

[0137] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A bactericidal composition for preventing and controlling Salmonella, characterized in that: including Salmonella bacteriophage vB-SenS-JZ291 and sodium nitrite solution; The Salmonella include Enteritidis Salmonella, Typhimurium Salmonella and mcr-1 positive 1,4,[5],12:i:-variant Salmonella; The Salmonella phage vB-SenS-JZ291 was deposited in the China Center for Type Culture Collection on April 29, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC M 2024806; The titer of the Salmonella phage vB-SenS-JZ291 is 1×10 10 PFU / mL; the concentration of the sodium nitrite solution is 1.51 g / L; The volume ratio of the Salmonella phage vB-SenS-JZ291 and the sodium nitrite solution is 1:

1.

2. A cleaning agent or disinfectant, characterized in that: The invention comprises the bactericidal composition according to claim 1.

3. A food disinfection method, characterized in that: The food is disinfected using the bactericidal composition as claimed in claim 1 or the cleaning agent or disinfectant as claimed in claim 2.

4. The food disinfection method according to claim 3, characterized in that: The food comprises eggs, cream and ham.

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